Methods and compositions for producing copper accumulating plants to slow decomposition and enhance carbon storage

Abstract

The present disclosure relates to plant cells, plants, and plant parts comprising a promoter sequence operably linked to a Copper Transporter 1 protein and / or a Nicotianamine Synthase 1 protein. The plant cells, plants, and plant parts optionally further comprise one or more additional heterologous polynucleotides encoding additional promoter sequences operably linked to additional polynucleotide sequences encoding any one or more of an Antioxidant Protein 1 (ATX1) protein, a Heavy Metal ATPase 4 protein, a Heavy Metal ATPase 5 protein, a Heavy Metal ATPase 9 protein, and / or a Yellow Stripe Like 16 protein. The present disclosure also relates to methods for increasing copper resistance and / or accumulation in a plant cell to reduce fungal decomposition in a plant cell, plant, or plant part and to enhance carbon storage.

Description

METHODS AND COMPOSITIONS FOR PRODUCING COPPER ACCUMULATING PLANTS TO SLOW DECOMPOSITION AND ENHANCE CARBON STORAGE

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No.63 / 656,810, filed June 6, 2024, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under Grant No. DE-AR0001491 awarded by the Department of Energy (ARPA-E). The government has certain rights in the invention. FIELD

[0003] The present disclosure relates to copper accumulating and / or resistant plants and methods to increase accumulation of copper in plants to reduce susceptibility to pathogens and to slow decomposition of plants. SEQUENCE LISTING STATEMENT

[0004] This application contains a computer readable Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file was created on April 4, 2025, is named 257948000161.xml and is 405,439 bytes in size. BACKGROUND

[0005] Since the industrial revolution, greenhouse gasses, such as carbon dioxide, methane, nitrous oxide and chlorofluorocarbons, have been increasing in the atmosphere. Despite decades of conscious efforts from public and private sectors, the upward trend in atmospheric CO2level shows no sign of slowing down. Current concentrations of CO2and other greenhouse gases in the air are at levels never seen before in human history causing climate changes with profound impact on food productivity, human health, and other socioeconomic aspects, presenting the biggest challenge humans have ever faced.

[0006] To combat climate change, it is a must to reduce the level of CO2in the atmosphere. Much of the global efforts have been focused on reducing greenhouse gas emissions generated by human activities. Less attention has been paid to CO2released from wood decay despite the fact that forest trees as a whole are one of the most dominant players in the carbon cycle. Out of the total amount of carbon stored in the global forest, about 8% is retained in the deadwood. On a yearlybasis, the amount of CO2released from dead and decaying wood worldwide totals 10.9 gigatons, roughly 115% of annual fossil fuel emissions from all human activities (Seibold et al., “The Contribution of Insects to Global Forest Deadwood Decomposition,” Nature 597:77-81 (2021)). Slowing or preventing the process of CO2release from dead wood is important to retain the carbon stored in tree biomass.

[0007] Wood decay fungi are significant contributors to decomposition of tree biomass and carbon release. Wood decay fungi are classified into soft rot species, which are typically Ascomycota, and white and brown rot species, which are typically Basidiomycota. These fungi degrade the main wood cell wall components: cellulose, hemicelluloses, and lignin, a polyphenolic polymer in plant cell walls that plays a major role in the development of plants and their defense against pathogens, such as the pathogens that decompose tree biomass. Fungi, such as white-rot basidiomycetes, use the enzyme lignin peroxidase to biodegrade lignin and digest the resulting fragments, with consequential release of carbon dioxide into the atmosphere. However, lignin degrading enzymes require metal ions for their function. For example, located at the center of the catalytic region, copper serves as a cofactor for lignin degradation enzyme laccase. Excess amounts of copper, however, are toxic to the growth of white rot fungi. In fact, copper-based fungicides provide one of the most effective fungal-growth controls in horticulture. In the timber industry, pressure treatment to inject wood preservatives into harvested wood has remained as a common practice to make finished wood product decay resistant. Copper is a component of the preservative mixture. However, it is difficult to provide widespread treatments of decaying timber in forest settings, making it difficult to prevent decomposition of tree biomass and carbon release.

[0008] Improving fungal disease resistance in living trees and other plants to enhance their health and longevity and increase their ability to sequester carbon is also an important consideration. Fungal diseases pose a significant threat to plant life, often leading to widespread damage and even plant death. Current methods of preventing fungal diseases include the use of fungicides and biological controls. However, these methods present several challenges. The effectiveness of fungicides can be limited by factors such as fungal resistance, environmental conditions, and potential harm to non-target organisms, as well as the logistical difficulty of treating large organisms such as trees. Biological control methods require a thorough understanding of the interactions between organisms, which can be complex and variable. Thus, there is a pressing need for innovative solutions to improve fungal disease resistance in trees and other plants.

[0009] Additional challenges, such as metal contamination in soil from heavy metals such as copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb), make it more difficult to use land for carbon sequestration due to their toxicity to many plant species, including trees. Hyper-accumulation of metals is the process by which certain plant species are able to enhance uptake of metal ions from the soil and increase the incorporation of metal ions in their tissues, but most plants and trees are sensitive to heavy metal contamination in the soil and grow poorly in the presence of heavy metals.

[0010] Therefore, there is a need to improve copper tolerance in trees and other plants, as well as to improve fungal disease resistance and prevent decomposition of tree biomass to improve carbon sequestration.

[0011] The present disclosure is directed to overcoming these and other deficiencies in the art. SUMMARY

[0012] One aspect of the present disclosure relates to a transgenic plant cell comprising a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein. The transgenic plant cell comprises increased copper resistance and / or accumulation compared to a non-transgenic plant cell of a same species.

[0013] Other aspects of the present disclosure relate to plant cells optionally further comprising one or more additional expression cassettes comprising additional promoter sequences operably linked to additional polynucleotide sequences encoding any one or more of a Heavy Metal ATPase 4 (HMA4) protein, a Heavy Metal ATPase 5 (HMA5) protein, an Antioxidant Protein 1 (ATX1) protein, a Heavy Metal ATPase 9 (HMA9) protein, and / or a Yellow Stripe Like 16 (YSL16) protein.

[0014] The present disclosure also relates to transgenic plants and plant parts comprising the transgenic plant cell of any of the embodiments described herein.

[0015] A further aspect of the present disclosure relates to a method for increasing copper resistance, copper accumulation, and / or increasing fungal disease resistance, and / or reducing fungal decomposition in a plant or plant cell. This method involves transforming a plant or a plant cell with a first expression cassette sequence comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or transforming theplant or plant cell with a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein. The plant or plant cell transformed with the first and / or second expression cassette sequence comprises increased copper resistance and / or accumulation compared to a non-transgenic plant or plant cell of a same species.

[0016] Another aspect of the present disclosure relates to a DNA construct comprising a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein. When transformed with the DNA construct of the present disclosure, a plant cell comprising the DNA construct comprises increased copper resistance and / or accumulation in comparison to a non-transgenic plant cell of a same species.

[0017] Further aspects of the present disclosure relate to a recombinant expression vector comprising DNA construct described herein and a recombinant host cell comprising the DNA construct described herein.

[0018] The present disclosure relates to transgenic plant cells, plants, and plant parts that exhibit increased fungal disease resistance, increased copper accumulation, increased copper resistance, enhanced uptake of metal ions from the soil, reduced decomposition of biomass, and / or improved carbon sequestration. These and other benefits are achieved by the compositions and methods described herein. As shown herein, wood from engineered trees accumulated copper and showed much reduced fungal growth and decay compared to non-engineered controls without affecting growth. These results demonstrated the use of bioengineering to enhance carbon retention in the territorial system and to alleviate the climate change crisis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGs.1A-B are photographs of fungal growth on gray poplar (Populus x canescens) wood sections treated with different amounts of copper (“Cu”), nickel (“Ni”) , or Cu and Ni after 80 days incubation with fungi at 23°C and 90% relative humidity. FIG.1A shows fungal strain Phlebia tremellosa growth on wood sections; FIG.1B shows fungal strain Heterobasidion annosum growth on wood sections. Each wood section is a representative of three replicates.

[0020] FIGs.2A-D are photographs of fungal growth on Populus fremontii (FIG.2A), Populus alba (FIG.2B), Maple (FIG.2C), and Oak (FIG.2D) wood sections treated with different amounts of copper (Cu), nickel (Ni), or Cu and Ni after 80 days incubation with fungi at 23°C and90% relative humidity. FIG.2A, C, and D show fungal strain Phlebia tremellosa growth on wood sections; FIG.2B shows fungal strain Heterobasidion annosum growth on wood sections.

[0021] FIGs.3A-B are photographs of fungal growth over time on wood blocks treated with different amounts of Cu (0-20 mM, as indicated). FIG.3A shows growth of the fungal strain Heterobasidion annosum on loblolly wood and the resulting wood decay after a 105-day incubation. FIG.3B shows growth of the fungal strain Heterobasidion annosum on redwood wood and the resulting wood decay after a 105-day incubation.

[0022] FIGs.4A-F are graphs showing percentage wood mass loss due to fungi mediated decay over different concentrations of Cu, Ni, and Cu + Ni treatments after a 80-day incubation. FIGs.4A-C show results of gray poplar wood sections inoculated with fungal strain Phlebia tremellosa; FIGs.4D-F show results of gray poplar wood sections inoculated with fungal strain Heterobasidion annosum. Three replicates were used for each treatment. “*” and “**” indicate p < 0.05 and p < 0.01 compared to the control by the T-test, respectively.

[0023] FIGs.5A-B are graphs showing wood decay of loblolly wood (FIG.5A) and redwood wood (FIG.5B) caused by the fungal strain Heterobasidion annosum after a 105-day incubation. These graphs correspond to the analysis of the samples shown in the photographs of FIGs.3A and FIG.3B. FIG.5A shows percentage loblolly wood mass loss due to fungi mediated decay. Three replicates were used for each treatment. “*” and “**” indicate p < 0.05 and p < 0.01 compared to the control by the T-test, respectively. FIG.5B shows percentage redwood wood mass loss due to fungi mediated decay. Three replicates were used for each treatment. “*” and “**” indicate p < 0.05 and p < 0.01 compared to the control by the T-test, respectively.

[0024] FIGs.6A-C are schematic illustrations of engineering strategies to enrich Cu in the stems of plants. FIG.6A shows a 4-step model dividing Cu ion flow into four major steps, including root uptake (1), xylem loading (2), long distance translocation (3), and stem storage (4) of Cu. FIG. 6B is a schematic illustration of a plant showing the leaf, stem, and the root organs; FIG.6C shows Cu ion flow in a 6-step cell and organ model including root uptake (1), transportation (2), xylem loading (3), long distance translocation (4), stem storage (5), and reallocation from leaf back to the stem (6) of Cu. X, xylem; P, phloem; V, vacuole.

[0025] FIG.7 is an illustration of DNA constructs used in certain embodiments of the present disclosure. Specifically, FIG.7 shows maps of DNA constructs pLC0241-pLC0249 and DNA constructs pLC0252-pLC0257. Each of the constructs include “LB”, indicating the left border of the Agrobacterium T-DNA; a selectable marker gene expression cassette, which includes aNopaline synthase “Nos” promoter driving the expression of an NPT II selectable marker and the Nos terminator; and “RB” indicating the right border of the T-DNA. pLC0241 comprises the Squamosa promoter-binding like protein 10 related promoter (“Spl10 promoter”) driving expression of the Larrea tridentata Copper Transporter 1 gene “LtCOPT1”, followed by the Nos terminator. pLC0242 comprises the NADH Dehydrogenase (Ubiquinone) 1 Alpha / Beta Subcomplex 1 (“NDUFAB1”) promoter driving expression of LtCOPT1, followed by the Nos terminator. pLC0243 comprises the Aquaporin 1-4 Related (“PIP1-4”) promoter driving expression of LtCOPT1, followed by the Nos terminator. pLC0244 comprises the Aquaporin 2-1 Related (“PIP2-1”) promoter driving expression of LtCOPT1, followed by the Nos terminator. pLC0245 comprises the Cellulose Synthase A4 (“CesA4”) promoter driving expression of Thlaspi caerulescens Nicotianamine Synthase 1 (“TcNAS1”), followed by the Nos terminator. pLC0246 comprises the Cinnamyl Alcohol Dehydrogenase (“CAD”) promoter driving the expression of TcNAS1, followed by the Nos terminator. pLC0247 comprises the NAC Domain-Containing Protein 12 (“NAC12”) promoter driving expression of TcNAS1, followed by the Nos terminator. pLC0248 comprises the PIP1-4 promoter driving expression of the Oryza sativa Heavy Metal ATPase 4 (“OsHMA4”) gene, followed by the Cauliflower Mosaic Virus 35S (“CaMV”) terminator. pLC0249 comprises both a PIP1-4 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, and the CAD promoter driving expression of the TcNAS1 gene, followed by the Arabidopsis thaliana Heat Shock Protein (“AtHSP”) terminator. pLC0252 comprises the NDUFAB1 promoter driving expression of the Oryza sativa Heavy Metal ATPase 5 (“OsHMA5”) gene, followed by the potato Proteinase Inhibitor II (“Pin II”) terminator. pLC0253 comprises the PIP1-4 promoter driving expression of the Oryza sativa Heavy Metal ATPase 9 (“OsHMA9”) gene, followed by the Nos terminator. pLC0254 comprises both the PIP1-4 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, and the CesA4 promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0255 comprises both the NDUFAB1 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, and the CesA4 promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0256 comprises both the NDUFAB1 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, and the CAD promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0257 comprises the NDUFAB1 promoter driving expression of the OsHMA9 gene, followed by the PinII terminator.

[0026] FIG.8 is an illustration of constructs used in certain embodiments of the present disclosure. Specifically, FIG.8 shows maps of constructs pLC0250, pLC0251, and pLC0258- pLC0260. Each of the constructs include LB, the left border of the T-DNA; a selectable marker cassette, which comprises a Nopaline synthase Nos promoter driving expression of an NPT II selectable marker followed by the Nos terminator; and RB indicating the right border of the T-DNA. pLC0250 comprises the PIP1-4 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, the PIP2-1 promoter driving expression of the OsHMA5 gene, followed by the CaMV terminator, and the CAD promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0251 comprises the PIP1-4 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, the CesA4 promoter driving expression of the OsHMA4 gene, followed by the PinII terminator, the PIP2-1 promoter driving expression of the OsHMA5 gene, followed by the CaMV terminator, and the CAD promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0258 comprises the CesA4 promoter driving expression of the OsHMA4 gene, followed by the PinII terminator, the PIP2-1 promoter driving expression of the OsHMA5 gene, followed by the CaMV terminator, and the CAD promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0259 comprises the NDUFAB1 promoter driving expression of the Oryza sativa Antioxidant Protein 1 (“OsATX1”) gene, followed by the Octopine Synthase (“Ocs”) terminator, the PIP1-4 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, the CesA4 promoter driving expression of the OsHMA4 gene, followed by the PinII terminator, the PIP2-1 promoter driving expression of the OsHMA5 gene, followed by the CaMV terminator, and the CAD promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator. pLC0260 comprises the NDUFAB1 promoter driving expression of the Oryza sativa Antioxidant Protein 1 (“OsATX1”) gene, followed by the Octopine Synthase (“Ocs”) terminator, the PIP1-4 promoter driving expression of the LtCOPT1 gene, followed by the Nos terminator, the CesA4 promoter driving expression of the OsHMA4 gene, followed by the PinII terminator, the PIP2-1 promoter driving expression of the OsHMA5 gene, followed by the CaMV terminator, the CAD promoter driving expression of the TcNAS1 gene, followed by the AtHSP terminator, and the Arabidopsis thaliana Sucrose-Proton Symporter 2 (“AtSPS2”) driving expression of the Oryza sativa Yellow Stripe Like 16 (“OsYSL16”) gene, followed by the CaMV terminator.

[0027] FIG.9 is a photograph showing the poplar hydroponic culture system.

[0028] FIG.10 is a graph showing fresh weight increases in trees grown with 0.1 vs 0.2 µM CuSO4in the hydroponic culture over 2 weeks.

[0029] FIGs.11A-B are graphs comparing LtCopt1 transgenic C1 trees growing in hydroponic (blue) vs in soils (red). FIG.11A shows relative expression levels of LtCopt1; FIG.11B shows root Cu levels. “n” indicates the number of biological replicates.

[0030] FIG.12 is a graph showing root Cu concentration (ppm, dry weight based) measured from individual T0 transgenic and control trees. The red dashed line indicates the average value of wild type gray poplar control (5.3 ppm).

[0031] FIG.13 is a graph showing stem Cu concentration (ppm, dry weight based) measured from individual T0 transgenic and control trees. The red dashed line indicates the average value of wild type gray poplar control (2.7 ppm).

[0032] FIG.14 is a graph showing Cu distribution among root, stem and leaf in wild-type gray poplar and different transgenic trees transformed with the T-DNAs from constructs LC0243, LC0246, LC0249, LC0250, or LC0251. The “-” or “+” indicates absence or presence of the corresponding transgene, respectively.

[0033] FIG.15 is a graph showing stem nicotianamine contents in the wild-type (WT) control and LC0247, LC0246, and LC0245 transgenic events. At least six biological replicates were analyzed in each group.

[0034] FIG.16 is a partial sequence alignment of the Copper Transporter 1 (COPT1) proteins of SEQ ID NOs:2-30 using Clustal Omega. The Cu binding motif of MX3M (Ren et al., “X-ray Structures of the High-Affinity Copper Transporter Ctr1,” Nature Communications 10:1386 (2019), which is hereby incorporated by reference in its entirety) within the second transmembrane domain is underlined.

[0035] FIG.17 is a partial sequence alignment of the Antioxidant 1 (ATX1) proteins of SEQ ID NOs:32-61 using Clustal Omega. The key feature of Cu binding and coordinating motif “MXCXXC” (Rosenzweig et al., “Crystal Structure of the Atx1 Metallochaperone Protein at 1.02 Å Resolution,” Structure 7(6): 605-617 (1999), which is hereby incorporated by reference in its entirety) is underlined.

[0036] FIG.18 is a partial sequence alignment of the Heavy Metal ATPase 5 (HMA5) proteins of SEQ ID NOs:63-93 using Clustal Omega. The Cu binding “GMTCXXC” motif (SEQ ID NO:115) (Gitschier et al., “Solution Structure of the Fourth Metal-Binding Domain from the Menkes Copper-Transporting ATPase,” Nature Structural Biology 5:47-54 (1998), which is hereby incorporated by reference in its entirety) is underlined.

[0037] FIG.19 is a partial sequence alignment of the Heavy Metal ATPase 5 (HMA5) proteins of SEQ ID NOs:63-93 using Clustal Omega. The p-type ATPase phosphorylation domain “DKTGT” motif (SEQ ID NO:138) (Gitschier et al., “Solution Structure of the Fourth Metal- Binding Domain from the Menkes Copper-Transporting ATPase,” Nature Structural Biology 5:47- 54 (1998), which is hereby incorporated by reference in its entirety) is underlined.

[0038] FIG.20 is a partial sequence alignment of the Heavy Metal ATPase 9 (HMA9) proteins of SEQ ID NOs:95-114 using Clustal Omega. The Cu binding motif “GMTCXXC” (SEQ ID NO:115) (Gitschier et al., “Solution Structure of the Fourth Metal-Binding Domain from the Menkes Copper-Transporting ATPase,” Nature Structural Biology 5:47-54 (1998), which is hereby incorporated by reference in its entirety) is underlined.

[0039] FIG.21 is a partial sequence alignment of the Heavy Metal ATPase 9 (HMA9) proteins of SEQ ID NOs:95-114 using Clustal Omega. The p-type ATPase phosphorylation domain “DKTGT” motif (SEQ ID NO:138) (Gitschier et al., “Solution Structure of the Fourth Metal- Binding Domain from the Menkes Copper-Transporting ATPase,” Nature Structural Biology 5:47- 54 (1998), which is hereby incorporated by reference in its entirety) is underlined.

[0040] FIG.22 is a partial sequence alignment of the Heavy Metal ATPase 4 (HMA4) proteins of SEQ ID NOs:117-137 using Clustal Omega. The Cu binding motif “GMXCXXC” (SEQ ID NO:139) (Gitschier et al., “Solution Structure of the Fourth Metal-Binding Domain from the Menkes Copper-Transporting ATPase,” Nature Structural Biology 5:47-54 (1998), which is hereby incorporated by reference in its entirety) is underlined.

[0041] FIG.23 is a partial sequence alignment of the Heavy Metal ATPase 4 (HMA4) proteins of SEQ ID NOs:117-137 using Clustal Omega. The p-type ATPase phosphorylation domain “DKTGT” motif (SEQ ID NO:138) (Gitschier et al., “Solution Structure of the Fourth Metal-Binding Domain from the Menkes Copper-Transporting ATPase,” Nature Structural Biology 5:47-54 (1998), which is hereby incorporated by reference in its entirety) is underlined.

[0042] FIG.24 is a partial sequence alignment of the Yellow Strip Like 16 (YSL16) proteins of SEQ ID NOs:142-168 using Clustal Omega. Transmembrane domain (TM) 13 is underlined. (protein transmembrane domains are predicted with TMHMM-2.0).

[0043] FIG.25 is a partial sequence alignment of the Nicotianamine synthase (NAS) function domain from the proteins of SEQ ID NOs:170-184 using Clustal Omega. The whole NAS domain spans from aa 1-279 in TcNas1 (website: prosite.expasy.org / prosite_details).

[0044] FIGs.26A-H are photographs and graphs of fungal growth on gray poplar (Populus x canescens) wood sections treated with different amounts of Cu, Ni , or Cu and Ni after 1 year incubation with fungi at 23°C and 90% relative humidity. FIG.26A is a photograph showing Phlebia tremellosa (brownish fluffy staff) growth on wood blocks. FIG.26B is a photograph showing Heterobasidion annosum (snow white staff) growth on wood blocks. FIGs.26C-E are graphs showing the percentage of wood mass loss due to Phlebia tremellosa medicated decay. FIGs. 26F-H are graphs showing the percentage of wood mass loss due to Heterobasidion annosum medicated decay. Three replicates were used for each treatment. “*” and “**” indicate p < 0.05 and p < 0.01 compared to the control by the T-test, respectively.

[0045] FIG.27 is a graph showing the tissue Cu contents from wood blocked treated with different amount of Cu. Wood blocks were ground up and tissue metal was measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Each data point is represented by three replicates.

[0046] FIGs.28A-C show a comparison of Cu treatments vs pressure treatment in reducing fungal growth and wood decay on loblolly pine wood. Wood blocks were treated with either different amounts of copper or industry standard pressure treatment. Fungal strain Phlebia tremellosa was inoculated and incubated on the woods for 90 days at 23°C and 90% relative humidity. FIG.28A is a photograph showing fungal growth on wood blocks; FIG.28B is a graph of the percentage of wood mass loss due to decay for each treatment; FIG.28C is a graph showing the tissue Cu contents in the wood blocks. Each wood block is a representative of three replicates. “**” indicate p < 0.01 compared to the control by the T-test.

[0047] FIGs.29A-B illustrate the identification of a Larrea COPT1 transporter. FIG.29A shows the sequence of the Larrea tridentata Copper Transporter 1 coding sequence (SEQ ID NO:1). FIG.29 B is a model of the predicted protein structure of the Larrea Copper Transporter 1 protein (Robetta).

[0048] FIGs.30A-C show that LtCOPT1 is a root Cu uptake transporter. FIG.30A shows photographs of assays demonstrating that LtCOPT1 rescues growth of a yeast ctr1 mutant line that cannot grow under low Cu conditions and on nonfermentable carbon sources; FIG.30B shows photographs of Arabidopsis plants overexpressing LtCopt1, which led to significant growth inhibition in Arabidopsis under 50 µM Cu; FIG.30C is a graph showing that the fresh weight of LtCOPT1 over expressing plants was reduced under 50 µM Cu.

[0049] FIGs.31A-B are graphs showing that expression of various constructs in Poplar enriches Cu in the stem woody tissues. FIG.31A is a graph showing that Cu is enriched in the stems in plants with the LC0251 construct; FIG.31B is a graph showing that Cu is enriched in the stem woody tissues in plants transformed with various constructs LC0241-LC0251. Eight WT replicates and ten independent T0 events were used in these analyses.

[0050] FIGs.32A-D show that woods from engineered trees showed much reduced fungal decay. FIG.32A is a photograph showing reduced growth of fungal strain Phlebia tremellosa on the wood blocks of T0 gray poplar transgenic events engineered with the LC0251 construct; FIG.32B is a graph showing a reduced percentage of wood mass loss caused by Phlebia tremellosa infection; FIG.32C is a photograph showing reduced growth of fungal strain Heterobasidion annosum on the wood blocks of T0 gray poplar transgenic events engineered with the LC0251 construct; FIG.32D is a graph showing a reduced percentage of wood mass loss caused by Heterobasidion annosum infection. Inoculated wood blocks were incubated for 90 days at 23°C and 90% relative humidity. Ten replicates were used for genotype of woods. “*” and “**” indicate p < 0.05 and p < 0.01 compared to the control by the T-test, respectively. DETAILED DESCRIPTION

[0051] The present disclosure is directed to the development of copper enriching plants to increase copper resistance and / or accumulation in plant cells, plants, and plant parts, such as woody tissues that provides fungal growth resistance to slow decay. Compositions and methods for inducing copper uptake, copper-accumulation, and tolerance to copper within plant cells, plants, and plant parts (e.g., the stems) of non-copper accumulating plants such as trees and shrubs are disclosed. The resulting copper accumulation will increase copper storage, inhibit fungal lipid peroxidase activity, and increase rot resistance, provide fungal disease resistance and durability and longevity of plant parts, and thereby slowing fungal decomposition of biomass and carbon dioxide release into the atmosphere.

[0052] Unless otherwise indicated, the definitions and embodiments described in this and other sections of the disclosure are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art.

[0053] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or moremethods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0054] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0055] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, may be used in the claims and the specification of the present disclosure in place of the term “comprising,” or words having similar meanings, to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but excluding the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of,” may be used in the claims and the specification of the present disclosure in place of the term “comprising,” or words having similar meanings, to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. In embodiments or claims where the term “comprising” (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of” or “consisting essentially of.” The methods and / or compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0056] Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0057] A “coding region” is a portion of a nucleic acid that is transcribed and translated into a polypeptide or protein.

[0058] A “gene” refers to an assembly of nucleotides that encode a polypeptide and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific functional RNA, protein, or polypeptide, optionally including regulatory sequences preceding (5' noncoding sequences) and following (3' non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its own regulatory sequences. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “heterologous” or “exogenous” gene, polynucleotide, or nucleic acid refers to DNA not naturally located in the cell, or if naturally from the cell, the gene is “heterologous” if it is introduced into a different location than the original location in the cell. Heterologous DNA can be introduced into the host organism by gene transfer, such as transformation, transfection, or bombardment. Heterologous genes can comprise native genes inserted into a non-native organism, or chimeric genes, such as a native gene under control of a different promoter than its endogenous promoter. Heterologous DNA can include a gene or other polynucleotides such as promoters and / or terminators foreign to the cell. A “transgene” is a gene that has been introduced into the genome typically by a transformation or transfection procedure.

[0059] “Operably linked” means an association between nucleic acid sequences on a single nucleic acid molecule such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0060] A “promoter” refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment. A promoter is a non-coding genomic DNA sequence, usually upstream (5′) to and operably linked to the relevant coding sequence, and its primary function is to act as a binding site for RNA polymerase to initiate transcription by the RNA polymerase. A promoter may also include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The terms “capable of controlling expression” or “initiating transcription”, refer to the primary function of a promoter. Additionally, there is “expression” of RNA, including functional RNA, or the expression of a polypeptide for operably linked encoding nucleotide sequences, as the transcribed RNA ultimately may be translated into the corresponding polypeptide. Promoters vary in their “strength” (i.e., their ability to promote transcription). The nucleotide sequence of the promoter determines the nature of the RNA polymerase binding and other related protein factors that attach to the RNA polymerase and / or promoter, and the rate of RNA synthesis.

[0061] A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. A “stem-specific” promoter is a promoter that drives expressionprimarily in stem tissues of a plant but may also be active to a lower level in other tissue cell types. A “root-specific” promoter is a promoter that drives expression primarily in root tissues of a plant but may also be active to a lower level in other tissue cell types.

[0062] A “reference sequence” means a nucleic acid or amino acid used as a comparator for another nucleic acid or amino acid, respectively, when determining sequence identity. A reference sequence can be a wildtype sequence.

[0063] “Sequence identity”, “percent identity” or “% identical” refers to the exactness of a match between a reference sequence and a sequence being compared to it when optimally aligned. For example, sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Multalin program (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucleic Acids Res.16:10881-90 (1988), which is hereby incorporated by reference in its entirety) or the Megalign®program of the LASERGENE®bioinformatics computing suite (DNASTAR®Inc., Madison, Wis.). Sequences may also be aligned using algorithms known in the art including, but not limited to, CLUSTAL V algorithm or the BLASTN or BLAST 2 sequence programs.

[0064] As used herein, the term “plant cell” includes cells, protoplasts, cell tissue cultures from which plants can be regenerated, calli, clumps, and cells that are intact in plants or parts of plants including, but not limited to, seeds, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, hypocotyls, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, nucellar tissue, ovaries, and other plant tissue or cells. In some embodiments, the plant cell is a protoplast.

[0065] “Plant parts” includes any part of a plant including, but not limited to, seeds, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, hypocotyls, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, nucellar tissue, ovaries, branches, trunks, bark, wood, and other plant tissue.

[0066] “Transformation” refers to the introduction of a nucleic acid into a host cell or a host organism. A host cell containing a transformed DNA construct or DNA fragment is referred to as a “transgenic” or “recombinant” cell. A host organism containing a transformed DNA construct or DNA fragment (or a transgenic cell) is referred to as a “transgenic” or “recombinant” organism. “Transfection” refers to the introduction of a nucleic acid, a protein, or both into a host cell or organism. Thus, isolated polynucleotides disclosed herein can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and / or replication in a host cell. Aconstruct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region, such as a regulatory region controlling inducible or constitutive, environmental regulation, developmental regulation, or tissue-specific expression, a transcription initiation start site, a ribosome binding site, a transcription termination site, and / or a polyadenylation signal.

[0067] A “vector” is a nucleic acid, plasmid or virus used to transfer coding information to a host cell. A “cloning vector” is a small piece of DNA into which a foreign DNA fragment is inserted to be transcribed. Typically, the vector contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. The DNA sequence in the expression vector is operably linked to appropriate expression control sequences, including a promoter, to direct RNA synthesis and protein expression. The expression vector can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Useful selectable markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. The vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun, Ti-mediated gene transfer, calcium phosphate transfection, DEAE-Dextran-mediated transfection, lipofection, or electroporation. Examples of vectors include, but are not limited to, viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA, such as vaccinia and adenovirus, P1-based artificial chromosomes, yeast plasmids, Bacillus vectors, and Aspergillus vectors. Examples of bacterial vectors include, but are not limited to, pQE vectors, pBluescript plasmids, pNH vectors, and lambda-ZAP vectors. Examples of eukaryotic vectors include pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 vectors.

[0068] A “gene expression cassette” or an “expression cassette” is a DNA construct that contains regulatory elements for transcription and translation of a cloned gene. The expression cassette typically includes a nucleotide sequence that initiates transcription, such as a promoter; operably linked to a coding sequence of a gene of interest or fragment thereof, which is the actual gene or genes to be expressed; and may include a terminator sequence, which signals the end oftranscription. The gene expression cassette is designed to be inserted into a host organism, where it can be transcribed and translated to produce the desired protein or other product. The specific elements and their arrangement can be varied to control the level, timing, and location of gene expression in the host organism. Exemplary expression cassettes according to embodiments of the present disclosure are illustrated in FIGs.7 and 8. For example, pLC0251 comprises an expression cassette for selectable marker NPTII, an expression cassette for LtCOPT, an expression cassette for OsHMA4, an expression cassette for OsHMA5, and an expression cassette for TcNAS1.

[0069] Preferences and options for a given aspect, feature, embodiment, or parameter of the disclosure should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the disclosure. Genes and / or Gene Products to Engineer Copper Resistant and / or Accumulating Plants and Trees with Fungal Disease Resistance and / or Reduced Wood Decomposition

[0070] One aspect of the present disclosure relates to a transgenic plant cell comprising a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein, where the transgenic plant cell has increased copper resistance and / or accumulation compared to a non-transgenic plant cell of a same species. This aspect also encompasses plant cells optionally further comprising one or more additional expression cassettes each of which encodes an additional promoter sequence operably linked to an additional polynucleotide sequence encoding any one or more of a Heavy Metal ATPase 4 (HMA4) protein, a Heavy Metal ATPase 5 (HMA5) protein, an Antioxidant Protein 1 (ATX1) protein, a Heavy Metal ATPase 9 (HMA9) protein, and a Yellow Stripe Like 16 (YSL16) protein.

[0071] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0072] Certain genes are described in various embodiments of the present disclosure that facilitate the uptake, transportation, and / or accumulation of copper in plants. As shown in FIGs.6A- C, and without being bound by any particular theory, this process can be broken down into either four steps (FIG.6A) or six steps (FIG.6C). The 4-step model includes the following steps: (1) rootuptake of Cu from the soil, (2) unloading Cu in the root xylem, (3) long distance translocation of Cu within the xylem, and (4) storage of Cu in the stem.

[0073] In the 6-step model, copper is taken up from the soil to the plant (step 1), transported to the stem (step 2), uploaded to the stem (step 3), transported up the stem (step 4), stored in vacuoles and / or the cell walls (step 5), and if transported to the leaf, reallocated back to the stem (step 6).

[0074] In step 1 of either the 4-step or 6-step model, a Copper Transporter may be used to facilitate uptake of copper from soil. Alternatively, a Nicotianamine Synthase 1 may be used by alone or in concert with the Copper Transporter to facilitate uptake of copper from soil. In steps 2 of the 4-step model or 3 of the 6-step model, Heavy Metal ATPase 5, Antioxidant Protein 1, and / or Heavy Metal ATPase 9 facilitate transport of the copper to the stem alone or in concert with each other. In step 3 of the 4-step model or 4 of the 6-step model, Nicotianamine Synthase 1 can facilitate the transport of copper to the stem. In step 4 of the 4-step model or step 5 of the 6-step model, Heavy Metal ATPase 4 facilitates the transfer of copper to the vacuole and / or cell wall. In step 6 of the 6-step model, Yellow Stripe Like 16 facilitates the reallocation of copper from the leaves to the stem. In some embodiments, one or more of these proteins may function at multiple steps. Not all steps may be necessary to achieve copper accumulation and / or resistance in plants.

[0075] In some embodiments of the present disclosure, copper accumulation and / or resistance in plants is conferred by transgenic expression of a Copper Transporter 1 (“COPT1”). Copper-accumulating plants may use specific metal transporters to absorb and transport heavy metals from the soil through their roots. In some embodiments, the plant cell comprises a heterologous polynucleotide encoding a COPT1 protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequence encoding a COPT1 protein.

[0076] An exemplary Copper Transporter is the gene Copper Transporter 1 (COPT1) from Larrea tridentata (LtCOPT1). The coding sequence for LtCOPT1 (SEQ ID NO:1) is provided in Table 1 infra. The amino acid sequence for LtCOPT1 (SEQ ID NO:2) is provided in Table 1 infra.

[0077] Other Copper Transporter 1 (COPT1) protein sequences are also suitable for use in embodiments of the present disclosure and are provided as SEQ ID NOs:3-30 (see Table 1 infra). These COPT1 sequences include Copper Transporters from Populus trichocarpa Settler14 (Accession No. PtStettler14.09G031100.1.p; SEQ ID NO:3), Populus nigra x maximowiczii (Accession No. Poman.09G030500.1.p; SEQ ID NO:4), Populus trichocarpa (Accession No.Potri.009G038700.1.p; SEQ ID NO:5), Populus deltoides (Accession No. Podel.09G038300.1.p; SEQ ID NO:6), Populus tremula x Populus alba (PtXaAlbH.09G030600.1.pSEQ ID NO:7), Populus tremula x Populus alba (PtXaTreH.09G029300.1.p; SEQ ID NO:8, Populus euphratica (GenBank Accession No. XP_011032213.1; SEQ ID NO:9), Populus tomentosa (GenBank Accession No. KAG6760903.1; SEQ ID NO:10), Populus alba x Populus berolinensis (GenBank Accession No KAJ6895552.1; SEQ ID NO:11), Populus alba (GenBank Accession No. XP_034896685.1; SEQ ID NO:12), Populus trichocarpa (GenBank Accession No. XP_002313411.1; SEQ ID NO:13), Pinus taeda (Accession No. PITA_33900; SEQ ID NO:14), Picea sitchensis (GenBank Accession No. ABK25604.1; SEQ ID NO:15), Picea sitchensis (GenBank Accession No. ABK25665.1; SEQ ID NO:16), Quercus suber (GenBank Accession No. XP_023883544.1; SEQ ID NO:17), Panicum virgatum (Accession No. Pavir.5NG518300.1.p; SEQ ID NO:18), Panicum virgatum (Accession No. Pavir.5KG503200.2.p; SEQ ID NO:19), Panicum virgatum (Accession No. Pavir.5NG518600.1.p; SEQ ID NO:20), Cannabis sativa (GenBank Accession No. XP_030506547.1; SEQ ID NO:21), Arabidopsis thaliana (Accession No. AT5G59030.1; SEQ ID NO:22), Oryza sativa (GenBank Accession No. NP_001415070 (formerly XP_015630809.1) (LOC_Os01g56420.1); SEQ ID NO:23), Zea mays (Accession No. Zm00001eb151980_P001 ; SEQ ID NO:24), Triticum urartu (Accession No. EMS68339.1; SEQ ID NO:25), Triticum aestivum (GenBank Accession No. XP_044341509.1; SEQ ID NO:26), Sorghum bicolor (Accession No. Sobic.003G310200.1.p; SEQ ID NO:27), Gossypium hirsutum (GenBank Accession No. XP_06665301.1; SEQ ID NO:28), Vitis vinifera (GenBank Accession NO. NP_001267996.1; SEQ ID NO:29), and Glycine max (GenBank Accession No. NP_001238742.2; SEQ ID NO:30) each of which is hereby incorporated by reference in its entirety.

[0078] In some embodiments, the Copper Transporter is LtCOPT1 having an amino acid sequence of SEQ ID NO:2. In some embodiments, the Copper Transporter comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of LtCOPT1 of SEQ ID NO:2. In some embodiments, the COPT1 protein is Larrea tridentata COPT1 comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the Copper Transporter is a COPT1 having an amino acid sequence of any one of SEQ ID NOs:2-30. In some embodiments, the Copper Transporter comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to theamino acid sequence of any one of SEQ ID NOs:2-30. In some embodiments, the COPT1 protein comprises an amino acid sequence of any one of SEQ ID NOs:2-30 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:2-30.

[0079] In some embodiments of the present disclosure, copper accumulation and / or resistance in plants is conferred by transgenic expression of a Nicotianamine Synthase 1 (“NAS1”). In some embodiments, the plant cell comprises a heterologous polynucleotide encoding a NAS1 protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequence encoding a NAS1 protein. An exemplary Nicotianamine Synthase is the gene Nicotianamine Synthase 1 (NAS1) from Thlaspi caerulescens (TcNAS1). The coding sequence for TcNAS1 (SEQ ID NO:169) (GenBank Accession No. AJ300446.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra. The amino acid sequence for TcNAS1 (SEQ ID NO:170) (GenBank Accession No. CAC82913.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra.

[0080] Other Nicotianamine Synthase 1 (NAS1) protein sequences are also suitable for use in embodiments of the present disclosure and are provided as SEQ ID NOs:171-184 (see Table 1 infra). These NAS1 protein sequences include NAS1 from Populus nigra x maximowiczii (Accession No. Poman.05G009400.1.p ; SEQ ID NO:171), Populus deltoides (Accession No. Podel.05G016500.1.p; SEQ ID NO:172), Populus tremula x Populus alba (Accession No. PtXaAlbH.05G009800.1.p; SEQ ID NO:173), Populus tremula x Populus alba (Accession No. PtXaTreH.05G008800.1.p; SEQ ID NO:174), Populus trichocarpa (Accession No. Potri.005G014300.1.p; SEQ ID NO:175), Populus trichocarpa Stettler14 (Accession No. PtStettler14.06G240900.1.p; SEQ ID NO:176), Pinus taeda (Accession No. PITA_14382.1.p; SEQ ID NO:177), Panicum virgatum (Accession No. Pavir.9NG676000.1.p; SEQ ID NO:178), Arabidopsis thaliana (Accession No. AT1G09240.1, SEQ ID NO:179), Zea mays (Accession No. Zm00001eb396230_P001; SEQ ID NO:180), Vitus vinifera (Accession No. VIT_214s0060g01190.1; SEQ ID NO:181), Gossypium hirsutum (Accession No. Gohir.D08G222700.1.p; SEQ ID NO:181), Triticum aestivum (Accession No. Traes_4DL_4E10A6DFB.1; SEQ ID NO:183), and Sorghum bicolor (Accession No. Sobic.001G395900.1.p; SEQ ID NO:184) each of which is hereby incorporated by reference in its entirety.

[0081] In some embodiments, the Nicotianamine Synthase is TcNAS1 having an amino acid sequence of SEQ ID NO:170. In some embodiments, the Nicotianamine Synthase comprises anamino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of TcNAS1 of SEQ ID NO:170. In some embodiments, the NAS1 protein is Thlaspi caerulescens NAS1 comprising an amino acid sequence of SEQ ID NO:170 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:170. In some embodiments, the Nicotianamine Synthase is a NAS1 having an amino acid sequence of any one of SEQ ID NOs:170- 184. In some embodiments, the Nicotianamine Synthase comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of any one of SEQ ID NOs:170-184. In some embodiments, the NAS1 protein comprises an amino acid sequence of any one of SEQ ID NOs: 170- 184 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:170-184.

[0082] In some embodiments, the plant cell comprises a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a COPT1 protein, and a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a NAS1 protein.

[0083] In some embodiments, the plant cell comprises a heterologous polynucleotide encoding the Heavy Metal ATPase 5 (“HMA5”) protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein. In some embodiments, the plant cell further comprises a third expression cassette comprising a third promoter sequence operably linked to a third polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein. An exemplary Heavy Metal ATPase 5 (HMA5) is the gene Heavy Metal Synthase 5 from Oryza sativa (OsHMA5). The coding sequence for OsHMA5 (SEQ ID NO:62) is a codon optimized version of GenBank Accession No. AB840272.1, (which is hereby incorporated by reference in its entirety) is provided in Table 1 infra. The amino acid sequence for OsHMA5 (SEQ ID NO:63) (GenBank Accession No. NP_001389338.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra.

[0084] Other Heavy Metal ATPase 5 protein sequences are also suitable for use in the embodiments of the present disclosure and are provided as SEQ ID NOs:64-93 (see Table 1 infra). These HMA5 protein sequences include HMA5 from Populus trichocarpa (Accession No. Potri.001G105800.2.p; SEQ ID NO:64), Populus trichocarpa Stettler14 (Accession No.PtStettler14.01G095200.1.p; SEQ ID NO:65), Populus tremula x Populus alba (Accession No. PtXaAlbH.03G098600.1.p; SEQ ID NO:66), Populus tremula x Populus alba (Accession No. PtXaTreH.03G103300.1.p; SEQ ID NO:67), Populus deltoides (Accession No. Podel.01G111700.1.p; SEQ ID NO:68), Populus tremula x Populus alba (Accession No. PtXaTreH.01G088000.1.p; SEQ ID NO:69), Populus nigra x maximowiczii (Accession No. Poman.01G091000.1.p; SEQ ID NO:70), Populus nigra x maximowiczii (Accession No. Poman.03G110500.1.p; SEQ ID NO:71), Populus trichocarpa (Accession No. Potri.003G125600; SEQ ID NO:72), Populus trichocarpa Stettler14 (Accession No. PtStettler14.03G113400; SEQ ID NO:73), Populus tremula x Populus alba (Accession No. PtXaAlbH.01G090100.1.p; SEQ ID NO:74), Populus deltoides (Accession No. Podel.03G134500.1.p; SEQ ID NO:75), Pinus taeda (Accession No. PITA_15838.1.p; SEQ ID NO:76), Pseudotsuga menziesii (Accession No. PSME_00009300-RA; SEQ ID NO:77), Picea sitchensis (GenBank Accession No. ABR17750.1; SEQ ID NO:78), Pinus lambertiana (Accession No. PILAhq_059364-RA; SEQ ID N:79), Quercus suber (GenBank Accession No. XP_023892768.1; SEQ ID NO:80), Panicum virgatum (Accession No. Pavir.7NG278400.1.p; SEQ ID NO:81), Panicum virgatum (Accession No. Pavir.7KG284600.1.p; SEQ ID NO:82), Panicum virgatum (Accession No. Pavir.7KG284700.1.p; SEQ ID NO:83), Panicum virgatum (Accession No. Pavir.7NG278500.1.p; SEQ ID NO:84), Cannabis sativa (GenBank Accession No. XP_030501095.1; SEQ ID NO:85), Arabidopsis thaliana (Accession No. AT1G63440.1; SEQ ID NO:86), Zea mays (Accession No. Zm00001eb074380_P001; SEQ ID NO:87), Sorghum bicolor (Accession No. Sobic.006G173700.1.p; SEQ ID NO:88), Sorghum bicolor (Accession No. Sobic.006G173800.1.p; SEQ ID NO:89), Triticum aestivum (Accession No. Traes_2DL_51FF05F66.1; SEQ ID NO:90), Vitus vinifera (Accession No. VIT_202s0025g03630.1; SEQ ID NO:91), Gossypium hirsutum (Accession No. Gohir.A08G181300.1.p; SEQ ID NO:92), and Gossypium hirsutum (Accession No. Gohir.D08G200000.1.p; SEQ ID NO:93), each of which is hereby incorporated by reference in its entirety.

[0085] In some embodiments, the Heavy Metal ATPase 5 is OsHMA5 having an amino acid sequence of SEQ ID NO:63. In some embodiments, the Heavy Metal ATPase 5 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of OsHMA5 of SEQ ID NO:63. In some embodiments, the HMA5 protein is Oryza sativa HMA5 comprising an amino acid sequence of SEQ ID NO:63 or an amino acid sequence that has at least 95% sequence identity to theamino acid sequence of SEQ ID NO:63. In some embodiments, the Heavy Metal ATPase 5 is an HMA5 having an amino acid sequence of any one of SEQ ID NOs:63-93. In some embodiments, the Heavy Metal ATPase 5 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 63-93. In some embodiments, the HMA5 protein comprises an amino acid sequence of any one of SEQ ID NOs:63-93 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:63-93.

[0086] In some embodiments, the plant cell comprises a heterologous polynucleotide encoding the Heavy Metal ATPase 4 (“HMA4”) protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein. In some embodiments, the plant cell further comprises a fourth expression cassette comprising a fourth promoter sequence operably linked to a fourth polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein. An exemplary Heavy Metal ATPase 4 (HMA4) is the gene Heavy Metal Synthase 4 from Oryza sativa (OsHMA4). The coding sequence for OsHMA4 (SEQ ID NO:116) (GenBank Accession No. KU168832.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra. The amino acid sequence for OsHMA4 (SEQ ID NO:117) (GenBank Accession No. ANQ29703.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra.

[0087] Other Heavy Metal ATPase 4 protein sequences are also suitable for use in the embodiments of the present disclosure and are provided as SEQ ID NOs:118-137 (see Table 1 infra). These HMA4 protein sequences include HMA4 from Populus trichocarpa Stettler14 (Accession No. PtStettler14.01G018300.1.p; SEQ ID NO:118), Populus trichocarpa (Accession No. Potri.001G019100.1.p; SEQ ID NO:119), Populus deltoides (Accession No. Podel.01G020800.1.p; SEQ ID NO:120), Populus nigra x maximowiczii (Accession No. Poman.01G016500.3.p; SEQ ID NO:121), Populus tremula x Populus alba (Accession No. PtXaAlbH.01G017700.3.p; SEQ ID NO:122), Populus tremula x Populus alba (Accession No. PtXaTreH.01G016200.2.p; SEQ ID NO:123), Pinus taeda (Accession PITA_32477.1.p; SEQ ID NO:124), Pinus lambertiana (Accession PILAhq_043285-RA; SEQ ID N:125), Quercus suber (GenBank Accession No. XP_050275628.1; SEQ ID NO:126), Panicum virgatum (Accession No. Pavir.1NG089700.3.p; SEQ ID NO:127), Panicum virgatum (Accession No. Pavir.1KG102777.1.p; SEQ ID NO:128), Cannabissativa (GenBank Accession No. XP_030504802.1; SEQ ID NO:129), Zea mays (Accession No. Zm00001eb236270_P001; SEQ ID NO:130), Sorghum bicolor (Accession No. Sobic.004G079900.2.p; SEQ ID NO:131), Triticum aestivum (Accession No. Traes_6DS_26C5A0A44.1; SEQ ID NO:132), Triticum aestivum (Accession No. Traes_6AS_6F306F27E.1; SEQ ID NO:133), Vitus vinifera (Accession No. VIT_206s0004g01890.1; SEQ ID NO:134), Gossypium hirsutum (Accession No. Gohir.A05G068500.1.p; SEQ ID NO:135), Gossypium hirsutum (Accession No. Gohir.D05G071500.3.p; SEQ ID NO:136), and Glycine max (Accession No. XP_003520989.1; SEQ ID NO:137), each of which is hereby incorporated by reference in its entirety.

[0088] In some embodiments, the Heavy Metal ATPase 4 is OsHMA4 having an amino acid sequence of SEQ ID NO:117. In some embodiments, the Heavy Metal ATPase 4 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of OsHMA4 of SEQ ID NO:117. In some embodiments, the HMA4 protein is Oryza sativa HMA4 comprising an amino acid sequence of SEQ ID NO:117 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:117. In some embodiments, the Heavy Metal ATPase 4 is an HMA4 having an amino acid sequence of any one of SEQ ID NOs:117-137. In some embodiments, the Heavy Metal ATPase 4 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of any one of SEQ ID NOs:117-137. In some embodiments, the HMA4 protein comprises an amino acid sequence of any one of SEQ ID NOs:117-137 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 117-137.

[0089] In some embodiments, the plant cell comprises a heterologous polynucleotide encoding the Antioxidant Protein 1 (“ATX1”) protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequence encoding an Antioxidant Protein 1 protein. In some embodiments, the plant cell further comprises a fifth expression cassette comprising a fifth promoter sequence operably linked to a fifth polynucleotide sequence encoding an Antioxidant Protein 1 (ATX1) protein. An exemplary Antioxidant Protein 1 is the gene Antioxidant Protein 1 from Oryza sativa (OsATX1). The coding sequence for OsATX1 (SEQ ID NO:31) (GenBank Accession No. XM_015792604, which is hereby incorporated by reference in its entirety) is provided in Table 1infra. The amino acid sequence for OsATX1 (SEQ ID NO:32) (GenBank Accession No. XP_015648090.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra.

[0090] Other Antioxidant 1 (ATX1) protein sequences are also suitable for use in the embodiments of the present disclosure and are provided as SEQ ID NOs:33-61 (see Table 1 infra). These ATX1 protein sequences include Antioxidant Proteins from Populus tremula x Populus alba (Accession No. PtXaTreH.10G198400.1.p; SEQ ID NO:33), Populus tremula x Populus alba (Accession No. PtXaAlbH.10G191600.1.p; SEQ ID NO:34), Populus nigra x maximowiczii (Accession No. Poman.10G199100.1.p; SEQ ID NO:35), Populus trichocarpa (Accession No. Potri.010G236500.1.p; SEQ ID NO:36), Populus trichocarpa Stettler14 (Accession No. PtStettler14.10G212000.1.p; SEQ ID NO:37), Populus deltoides (Accession No. Podel.10G243300.1.p; SEQ ID NO:38), Populus trichocarpa Stettler14 (Accession No. PtStettler14.08G019500.1.p; SEQ ID NO:39), Populus deltoides (Accession No. Podel.08G030800.1.p; SEQ ID NO:40), Populus tremula x Populus alba (Accession No. PtXaAlbH.08G017400.1.p; SEQ ID NO:41), Populus tremula x Populus alba (Accession No. PtXaTreH.08G016700.1.p; SEQ ID NO:42), Populus trichocarpa (Accession No. Potri.008G023800.1.p; SEQ ID NO:43), Populus nigra x maximowiczii (Accession No. Poman.08G017800.1.p; SEQ ID NO:44), Populus tremula x Populus alba (Accession No. PtXaAlbH.10G191600.2.p; SEQ ID NO:45), Pinus taeda (Accession No. PITA_32630; SEQ ID NO:46), Picea sitchensis (GenBank Accession No. ABK20883.1; SEQ ID NO:47), Pseudotsuga menziesii (Accession No. PSME_00000392-RA; SEQ ID NO:48), Pinus lambertiana (Accession No. PILAhq_036623-RA; SEQ ID N:49), Quercus suber (GenBank Accession No. XP_023901161.1; SEQ ID NO:50), Panicum virgatum (Accession No. Pavir.8KG208500.1.p; SEQ ID NO:51), Cannabis sativa (GenBank Accession No. XP_030489546.2; SEQ ID NO:52), Arabidopsis thaliana (Accession No. AT1G66240.1; SEQ ID NO:53), Zea mays (Accession No. Zm00001eb032340; SEQ ID NO:54), Triticum aestivum (Accession No. Traes_7DL_4B6FAFD6B.1; SEQ ID NO:55), Triticum aestivum (Accession No. Traes_7BL_956842621.1; SEQ ID NO:56), Sorghum bicolor (Accession No. Sobic.008G179500.1.p; SEQ ID NO:57), Gossypium hirsutum (Accession No. Gohir.A10G198300.1.p; SEQ ID NO:58), Gossypium hirsutum (Accession No. Gohir.D10G205400.1.p; SEQ ID NO:59), Vitus vinifera (Accession No. VIT_213s0074g00770.2;SEQ ID NO:60), and Glycine max (Accession No. XP_003536318.1; SEQ ID NO:61), each of which is hereby incorporated by reference in its entirety.

[0091] In some embodiments, the Antioxidant Protein 1 is OsATX1 having an amino acid sequence of SEQ ID NO:32. In some embodiments, the Antioxidant Protein 1 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of OsATX1 of SEQ ID NO:32. In some embodiments, the ATX1 protein is Oryza sativa ATX1 comprising an amino acid sequence of SEQ ID NO:32 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:32. In some embodiments, the Antioxidant Protein is a ATX1 having an amino acid sequence of any one of SEQ ID NOs:32-61. In some embodiments, the Antioxidant Protein comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of any one of SEQ ID NOs:32-61. In some embodiments, the ATX1 protein comprises an amino acid sequence of any one of SEQ ID NOs:32-61 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:32-61.

[0092] In some embodiments, the plant cell comprises a heterologous polynucleotide encoding a Heavy Metal ATPase 9 (“HMA9”) protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequence encoding a Heavy Metal ATPase 9 (HMA9) protein. In some embodiments, the plant cell comprises a sixth expression cassette comprising a sixth promoter sequence operably linked to a sixth polynucleotide sequence encoding the HMA9 protein. An exemplary Heavy Metal ATPase 9 (HMA9) is the gene Heavy Metal Synthase 9 from Oryza sativa (OsHMA9). The coding sequence for OsHMA9 (SEQ ID NO:94) is a codon optimized version of GenBank Accession No. NM_001421690.1 (which is hereby incorporated by reference in its entirety) and is provided in Table 1 infra. The amino acid sequence for OsHMA9 (SEQ ID NO:95) (GenBank Accession No. NP_001408619.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra.

[0093] Other Heavy Metal ATPase 9 (HMA9) protein sequences are also suitable for use in the embodiments of the present disclosure and are provided as SEQ ID NOs:96-114 (see Table 1 infra). These HMA9 sequences include HMA9 from Populus tremula x Populus alba (Accession No. PtXaAlbH.03G053700.1.p; SEQ ID NO:96), Populus nigra x maximowiczii (Accession No. Poman.03G064400.1.p; SEQ ID NO:97), Populus trichocarpa (Accession No.Potri.003G075700.1.p; SEQ ID NO:98), Populus tremula x Populus alba (Accession No. PtXaTreH.03G057600.2.p; SEQ ID NO:99), Populus trichocarpa Stettler14 (Accession No. PtStettler14.03G065900.1.p; SEQ ID NO:100), Populus deltoides (Accession No. Podel.03G078700.1.p; SEQ ID NO:101), Pseudotsuga menziesii (Accession No. PSME_00001386- RA; SEQ ID NO:102), Pinus lambertiana (Accession No. PILAhq_049730-RA; SEQ ID NO:103), Quercus suber (GenBank Accession No. XP_023885007.1; SEQ ID NO:104), Cannabis sativa (GenBank Accession No. XP_030481230.1; SEQ ID NO:105), Arabidopsis thaliana (Accession No. AT5G44790.1; SEQ ID NO:106), Zea mays (Accession No. Zm00001eb207090; SEQ ID NO:107), Sorghum bicolor (Accession No. Sobic.010G220600.1.p; SEQ ID NO:108), Triticum aestivum (Accession No. Traes_7DL_DF97DD324.2; SEQ ID NO:109), Triticum aestivum (Accession No. Traes_6AS_9321C1C5B.1; SEQ ID NO:110), Vitus vinifera (Accession No. VIT_201s0011g01360.1; SEQ ID NO:111), Gossypium hirsutum (Accession No. Gohir.D12G047100.1.p; SEQ ID NO:112), Gossypium hirsutum (Accession No. Gohir.A12G048300.1.p; SEQ ID NO:113), and Glycine max (Accession No. XP_003547418.1; SEQ ID NO:114), each of which is hereby incorporated by reference in its entirety.

[0094] In some embodiments, the Heavy Metal ATPase 9 is OsHMA9 having an amino acid sequence of SEQ ID NO:95. In some embodiments, the Heavy Metal ATPase 9 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of OsHMA9 of SEQ ID NO:95. In some embodiments, the HMA9 protein is Oryza sativa HMA9 comprising an amino acid sequence of SEQ ID NO:95 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:95. In some embodiments, the Heavy Metal ATPase 9 is an HMA9 having an amino acid sequence of any one of SEQ ID NOs:95-114. In some embodiments, the Heavy Metal ATPase 9 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 95-114. In some embodiments, the HMA9 protein comprises an amino acid sequence of any one of SEQ ID NOs:95-114 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:95-114.

[0095] In some embodiments, the plant cell comprises a heterologous polynucleotide encoding a Yellow Stripe Like 16 (YSL16) protein. In some embodiments, the plant cell comprises an expression cassette comprising a promoter sequence operably linked to a polynucleotide sequenceencoding a Yellow Stripe Like 16 (YSL16) protein. In some embodiments, the plant cell further comprises a seventh expression cassette comprising a seventh promoter sequence operably linked to a seventh polynucleotide sequence encoding a Yellow Stripe Like 16 (YSL16) protein. An exemplary Yellow Stripe Like 16 (YSL16) is the gene Yellow Stripe Like 16 from Oryza sativa (OsYSL16). The coding sequence for OsYSL16 (SEQ ID NO:141) (based on GenBank Accession No. NM_001402405.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra. The amino acid sequence for OsYSL16 (SEQ ID NO:142) (GenBank Accession No. NP_001389334.1, which is hereby incorporated by reference in its entirety) is provided in Table 1 infra.

[0096] Other Yellow Stripe Like 16 protein sequences are also suitable for use in the embodiments of the present disclosure and are provided as SEQ ID NOs:143-168 (see Table 1 infra). These YSL16 protein sequences include YSL16 from Populus tremula x Populus alba (Accession No. PtXaTreH.12G005700.2.p; SEQ ID NO:143), Populus nigra x maximowiczii (Accession No. Poman.12G006100.1.p; SEQ ID NO:144), Populus trichocarpa (Accession No. Potri.011G055224.1.p; SEQ ID NO:145), Populus trichocarpa Stettler14 (Accession No. PtStettler14.11G000700.1.p; SEQ ID NO:146), Populus tremula x Populus alba (Accession No. PtXaAlbH.12G006600.4.p; SEQ ID NO:147), Populus deltoides (Accession No. Podel.12G030200.1.p; SEQ ID NO:148), Pinus taeda (Accession No. PITA_23967; SEQ ID NO:149), Pinus taeda (Accession No. PITA_07006; SEQ ID NO:150), Pseudotsuga menziesii (Accession No. PSME_00040922-RA; SEQ ID NO:151), Picea sitchensis (GenBank Accession No. ADE76428.1; SEQ ID NO:152), Pinus lambertiana (Accession No. PILAhq_055001-RA; SEQ ID N:153), Quercus suber (GenBank Accession No. XP_023893223.1; SEQ ID NO:154), Panicum virgatum (Accession No. Pavir.7NG266200.1.p; SEQ ID NO:155), Panicum virgatum (Accession No. Pavir.7KG270400.1.p; SEQ ID NO:156), Cannabis sativa (GenBank Accession No. KAF4374227.1; SEQ ID NO:157), Cannabis sativa (GenBank Accession No. XP_030505680.1; SEQ ID NO:158), Arabidopsis thaliana (Accession No. AT5G24380.1; SEQ ID NO:159), Arabidopsis thaliana (Accession No. AT5G53550.1; SEQ ID NO:160), Zea mays (Accession No. Zm00001eb209500; SEQ ID NO:161), Sorghum bicolor (Accession No. Sobic.006G164300.1.p; SEQ ID NO:162), Triticum aestivum (Accession No. Traes_6AL_E36FCEF64.2; SEQ ID NO:163), Triticum aestivum (Accession No. Traes_1DL_7E5ED8683.2; SEQ ID NO:164), Triticum aestivum (Accession No. Traes_6DL_AA95B2246.1; SEQ ID NO:165), Gossypium hirsutum (Accession No. Gohir.D03G116400.2.p; SEQ ID NO:166), Vitus vinifera (Accession No. VIT_202s0025g02500.1;SEQ ID NO:167), and Glycine max (Accession No. XP_003523338.2; SEQ ID NO:168), each of which is hereby incorporated by reference in its entirety.

[0097] In some embodiments, the Yellow Stripe Like 16 is OsYSL16 having an amino acid sequence of SEQ ID NO:142. In some embodiments, the Yellow Stripe Like 16 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of OsYSL16 of SEQ ID NO:142. In some embodiments, the YSL16 protein is Oryza sativa YSL16 comprising an amino acid sequence of SEQ ID NO:142 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:142. In some embodiments, the Yellow Stripe Like 16 is an YSL16 having an amino acid sequence of any one of SEQ ID NOs:142-168. In some embodiments, the Yellow Stripe Like 16 comprises an amino acid sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 142-168. In some embodiments, the YSL16 protein comprises an amino acid sequence of any one of SEQ ID NOs:142-168 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:142-168.

[0098] The transgenic plant cell of the present disclosure may comprise any one or more of the first heterologous polynucleotide; the first expression cassette; the second heterologous polynucleotide; the second expression cassette; the third heterologous polynucleotide; the third expression cassette; the fourth heterologous polynucleotide; the fourth expression cassette; the fifth heterologous polynucleotide; the fifth expression cassette; the sixth heterologous polynucleotide; the sixth expression cassette; and the seventh heterologous polynucleotide, seventh promoter sequence, and seventh polynucleotide sequence in any combination thereof. In some embodiments, the transgenic plant cell comprises an expression cassette comprising a promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; an expression cassette comprising a promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein; an expression cassette comprising a promoter sequence operably linked to a third polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein; and an expression cassette comprising a promoter sequence operably linked to a fourth polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein.Promoters and Terminators

[0099] In some embodiments, each expression cassette comprises a promoter sequence operably linked to each heterologous polynucleotide (gene sequence). In some embodiments, the promoter is also heterologous to the plant cell. As shown in FIGs.7 and 8, promoters are typically located 5’ of the gene sequence in order to drive transcription.

[0100] In some embodiments, the promoter is a constitutive promoter. Suitable constitutive promoters that are functional in a plant cell include, but are not limited to, the cauliflower mosaic virus 35S (CaMV35S) promoter, a tandem 35S promoter, a cauliflower mosaic virus 19S promoter, a figwart mosaic virus 35S (FMV35S) promoter, a nopaline synthase gene promoter; an octopine synthase gene promoter, a potato or tomato protease inhibitor I or II gene promoter, an actin promoter, and a ubiquitin promoter.

[0101] In some embodiments, the promoter is an inducible promoter. Suitable inducible promoters that are functional in a plant cell may include, but are not limited to, a phenylalanine ammonia-lyase gene promoter, a chalcone synthase gene promoter, a pathogenesis-related protein gene promoter, a copper-inducible regulatory element; tetracycline and chlor-tetracycline-inducible regulatory elements.

[0102] In some embodiments, the promoter sequence comprises a tissue / organ-specific promoter such as a root-specific or stem-specific promoter. In some embodiments, the promoter is a stem-specific promoter. Stem-specific expression drives copper transport and accumulation into the tissues important for slowing wood decomposition and / or providing fungal disease resistance, while preventing negative pleiotropic effects of copper accumulation, which may occur if copper accumulates in other plant tissues, such as leaves and meristematic tissues. Some gene products, such as HMA4, are located in the vacuole membrane and transport metal ions in and out of vacuoles. For example, vacuole membrane located HMA4 facilitates the sequestration of copper in the vacuole and prevents or avoids the cytotoxicity of elevated Cu content in the cells. Stem-specific expression of these genes increases the transporters in the stem cell vacuole membrane and thus help create a pulling force to drive copper accumulation in stem tissue. In some embodiments, the stem-specific promoter is a xylem-specific promoter. One component of xylem is lignin, which is extremely resistant to digestion, and helps xylem cells facilitate the conduction of water and minerals throughout the plant. Xylem-specific promoters may include, but are not limited to, one or more of a Populus trichocarpa Cinnamyl Alcohol Dehydrogenase (CAD) promoter, Pinus taeda Cinnamyl Alcohol Dehydrogenase (CAD) promoter, Nicotiana tabacum TobRB7 promoter, Agrobacteriumrhizogenes roID promoter, Arabidopsis Tracheary Element Differentiation-Related 4 (AtTED4) promoter, 4-Coumarate:CoA ligase (4CL) promoter, Solanum lycopersicum SIREO promoter, Populus ET304 promoter, Eucalyptus grandis TIP2 promoter, and promoters of other genes associated with stem and / or xylem development.

[0103] An exemplary stem-specific promoter is the Populus trichocarpa Cinnamyl Alchohol Dehydrogenase (“CAD4”) promoter (GenBank Accession No. KX273782.1, which is hereby incorporated by reference in its entirety) (SEQ ID NO:185), provided in Table 1 infra. CAD4 is associated with xylem development . In some embodiments, the stem-specific promoter is a Populus trichocarpa CAD4 promoter having a nucleotide sequence of SEQ ID NO:185. In some embodiments, the stem-specific promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:185.

[0104] In some embodiments, the stem-specific promoter is a phloem-loading promoter. Phloem-loading refers to the process by which sugars, primarily in the form of sucrose, are actively transported into the phloem tissue of plants for long-distance transport to other parts of the plant, such as roots and fruits, where the sugars are used for metabolic processes and growth. See for example, Truernit and Sauer, “The Promoter of the Arabidopsis thaliana SUC2 Sucrose-H+ Symporter Gene Directs Expression of β-Glucuronidase to the Phloem: Evidence for Phloem Loading and Unloading by SUC2,” Planta 196.3 (1995):564-570, which is hereby incorporated by reference in its entirety.) In some embodiments, the phloem-loading promoter is the Arabidopsis thaliana Sucrose-Proton Symporter 2 (AtSuc2) promoter (SEQ ID NO:186), provided in Table 1 infra. In some embodiments, the phloem-loading promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:186.

[0105] In some embodiments, the stem-specific promoter is the Populus alba allele from the poplar 717 hybrid (Populus tremula x Populus alba) NAC domain-containing protein 12 (“NAC12”) promoter (SEQ ID NO:187), provided in Table 1 infra. In some embodiments, the stem-specific promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:187.

[0106] In some embodiments, the stem-specific promoter is the Populus alba allele from the poplar 717 hybrid (Populus tremula x Populus alba) Cellulose synthase A4 (“CesA4”) promoter(SEQ ID NO:188), provided in Table 1 infra. In some embodiments, the stem-specific promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:188.

[0107] In some embodiments, the xylem-specific promoter is Pinus taeda Cinnamyl Alchohol Dehydrogenase (CAD) (SEQ ID NO:193) (GenBank Accession No. FJ428228, which is hereby incorporated by reference in its entirety).

[0108] In some embodiments, the promoter is a root-specific promoter. Root-specific expression drives copper uptake, accumulation, and transport. An exemplary root-specific promoter includes the Populus alba allele from the poplar 717 hybrid (Populus tremula x Populus alba) NADH Dehydrogenase (Ubiquinone) 1 Alpha / Beta Subcomplex 1 (“NDUFAB1”) promoter (SEQ ID NO:189), provided in Table 1 infra. In some embodiments, the root-specific promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:189.

[0109] In some embodiments, the root-specific promoter is the Populus alba allele from the poplar 717 hybrid (Populus tremula x Populus alba) Populus alba) Aquaporin 1-4 Related (“PIP1- 4”) promoter (SEQ ID NO:190), provided in Table 1 infra. In some embodiments, the root-specific promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:190.

[0110] In some embodiments, the root-specific promoter is the Populus alba allele from the poplar 717 hybrid (Populus tremula x Populus alba) Populus alba) Aquaporin 2-1 Related (“PIP2- 1”) promoter (SEQ ID NO:191), provided in Table 1 infra. In some embodiments, the root-specific promoter comprises a nucleotide sequence that has at least 80%, 83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:191.

[0111] In some embodiments, the root-specific promoter is the Populus alba allele from the poplar 717 hybrid (Populus tremula x Populus alba) Squamosa Promoter-Binding Like Protein 10 Related (“Spl10 promoter”) promoter (SEQ ID NO:192), provided in Table 1 infra. In some embodiments, the root-specific promoter comprises a nucleotide sequence that has at least 80%,83%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% (or any number or range therein) sequence identity to the nucleotide sequence of SEQ ID NO:192.

[0112] In some embodiments, the promoter sequence is selected from any one of SEQ ID NOs:185-193. In some embodiments, the promoter sequence comprises a stem-specific promoter having a nucleotide sequence of any one of SEQ ID NOs:185, 187, 188, or 193. In some embodiments, the promoter sequence comprises a root-specific promoter having a nucleotide sequence of any one of SEQ ID NOs:189-192. In some embodiments, the promoter sequence operably linked to the polynucleotide sequence encoding the COPT1 protein is a root-specific promoter. In some embodiments, the promoter sequence operably linked to the polynucleotide sequence encoding the NAS1 protein is a stem-specific promoter.

[0113] In some embodiments, expression cassette comprises a terminator sequence operably linked to gene sequence. A 3’ terminator sequence is an untranslated regulatory element at the 3′ end of the coding region of a gene, which comprises sequences capable of affecting transcription or mRNA processing. The 3’ terminator may also comprise signals for polyadenylation of the mRNA transcript. In some embodiments, the 3' terminator is the Nopaline Synthase 3' region (Nos 3') (Fraley et al, “Expression of Bacterial Genes in Plant Cells,” Proc. Natl. Acad. Sci.80:4803-4807 (1983), which is hereby incorporated by reference in its entirety). An example of the use of different 3' non-translated regions is provided in Ingelbrecht et al., “Different 3’ End Regions Strongly Influence the Level of Gene Expression in Plant Cells,” Plant Cell 1:671-680 (1989), which is hereby incorporated by reference in its entirety). Non-limiting examples of polyadenylation signals include one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., “Tissue-Specific and Light-Regulated Expression of a Pea Nuclear Gene Encoding the Small Subunit of Ribulose-1,5- Bisphosphate Carboxylase,” EMBO J.3:1671-1679 (1984), which is hereby incorporated by reference in its entirety) and AGRtu.nos (GenBank Accession No. E01312, which is hereby incorporated by reference in its entirety). As shown in FIGs.7 and 8, terminators are located 3’ of the gene sequence in order to terminate transcription.

[0114] In some embodiments, the 3′ terminator is a terminator sequence from Populus trichocarpa GDP-mannose transporter (PtGDPMT) (GenBank Accession No. XM_024607936.1, which is hereby incorporated by reference in its entirety). In some embodiments, the 3′ terminator is a Cauliflower Mosaic Virus 35S 3′ terminator , a Nopaline Synthase 3’ terminator (GenBank accession No. MK078637.1, which is hereby incorporated by reference in its entirety), an Arabidopsis thaliana Heat Shock Protein (AtHSP) terminator (GenBank accession No. KP008108.1,which is hereby incorporated by reference in its entirety), potato Proteinase Inhibitor II (Pin II) terminator (GenBank accession No. JD589713.1, which is hereby incorporated by reference in its entirety) or other terminator sequence capable of functioning in plants. DNA Constructs and Vectors

[0115] Another aspect of the present disclosure relates to a DNA construct comprising a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein. In some embodiments, the DNA construct comprises a promoter sequence operably linked to a heterologous polynucleotide encoding a COPT1 protein. In some embodiments, the DNA construct comprises a promoter sequence operably linked to a heterologous polynucleotide encoding a NAS1 protein. In some embodiments, the DNA construct comprises a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein.

[0116] In some embodiments, a plant cell and / or transgenic plant comprising the DNA construct of the present disclosure has increased copper resistance and / or accumulation compared to a plant or plant cell of a same species that lacks the first expression cassette and / or the second expression cassette.

[0117] This aspect of the present disclosure also encompasses a vector comprising a DNA construct described herein and a recombinant host cell comprising a DNA construct described herein.

[0118] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0119] The heterologous polynucleotide sequences and / or expression cassettes of the present disclosure may be present in a DNA construct, either on one linear fragment or multiple linear fragments of DNA, or in one or more vectors such as a plasmid for use in transformation. In some embodiments, the heterologous polynucleotide sequences are present in an expression cassette comprising a promoter, a gene of interest, and a 3’ terminator. In some embodiments, a DNA construct comprises one or more expression cassettes (see, for example, the constructs of FIG.7 and FIG.8). In some embodiments, each expression cassette in a DNA construct has a differentpromoter linked to each gene. In some embodiments, one or more of the expression cassettes in a DNA construct may have the same promoter operably linked to one or more genes. In some embodiments, one or more of the expression cassettes in a DNA construct may have the same 3’ terminator operably linked to one or more genes.

[0120] In some embodiments, the vector includes left and right Agrobacterium T-DNA border sequences (Peralta and Ream, “T-DNA Border Sequences Required for Crown Gall Tumorigenesis,” Proc. Natl. Acad. Sci.82:5112-5116 (1985), which is hereby incorporated by reference in its entirety). These border sequences allow the introduction of heterologous DNA located between the left (LB) and right (RB) T-DNA border sequences into a host cell when using Agrobacterium-mediated DNA transformation. In some embodiments, the Agrobacterium is Agrobacterium tumefaciens.

[0121] Standard cloning procedures known in the art can be used to prepare the DNA construct and / or the vector, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York (2001), which is hereby incorporated by reference in its entirety.

[0122] In some embodiments, the DNA construct comprises a third expression cassette comprising a third promoter sequence operably linked to a third polynucleotide sequence encoding a protein selected from the group consisting of: an ATX1 protein, a HMA4 protein, a HMA5 protein, a HMA9 protein, and a YSL16 protein. In some embodiments, the DNA construct comprises a fourth expression cassette comprising a fourth promoter sequence operably linked to a fourth polynucleotide sequence encoding a protein selected from the group consisting of: an ATX1 protein, a HMA4 protein, a HMA5 protein, a HMA9 protein, and a YSL16 protein. In some embodiments, the DNA construct comprises a fifth expression cassette comprising a fifth promoter sequence operably linked to a fifth polynucleotide sequence encoding a protein selected from the group consisting of: an ATX1 protein, a HMA4 protein, a HMA5 protein, a HMA9 protein, and a YSL16 protein. In some embodiments, the DNA construct comprises a sixth expression cassette comprising a sixth promoter sequence operably linked to a sixth polynucleotide sequence encoding a protein selected from the group consisting of: an ATX1 protein, a HMA4 protein, a HMA5 protein, a HMA9 protein, and a YSL16 protein. In some embodiments, the DNA construct comprises a seventh expression cassette comprising a seventh promoter sequence operably linked to a seventh polynucleotide sequence encoding a protein selected from the group consisting of: an ATX1 protein, a HMA4 protein, a HMA5 protein, a HMA9 protein, and a YSL16 protein. In some embodiments,one or more of the first, second, third, fourth, fifth, six, or seventh promoter is the same promoter. In some embodiments each of the first, second, third, fourth, fifth, six, or seventh promoter is a different promoter. In some embodiments, the DNA construct comprises a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a COPT1 protein, a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a NAS1 protein, a third expression cassette comprising a third promoter sequence operably linked to a third polynucleotide sequence encoding an HMA5 protein, and a fourth expression cassette comprising a fourth promoter sequence operably linked to a fourth polynucleotide sequence encoding an HMA4 protein. Transgenic Plants and Transgenic Trees

[0123] The methods and compositions of the present disclosure can be used over a broad range of plant species. In some embodiments, a transgenic plant comprises the transgenic plant cell. In some embodiments, the transgenic plant cell or plant is selected from a tree, shrub, grass, rice, maize, wheat, sorghum, barley, oats, rye, abaca, amaranth, bamboo, big bluestem, broccoli, Brussels sprouts, buckwheat, cabbage, canola, cacao, cassava, cauliflower, coir, collard greens, cotton, elephant grass, flax, foxtail millet, giant reed, grape, hemp, hops, indigo, jute, kale, kenaf, kohlrabi, millet, miscanthus, mustard, Napier grass, pearl millet, prairie cordgrass, quinoa, ramie, reed canary grass, sisal, sorghum, spelt, sugar beet, sugar cane, switchgrass, teff, tobacco, triticale, and yerba mate. In some embodiments, the transgenic plant cell or plant is selected from a tree, shrub, or a grass.

[0124] In some embodiments, the transgenic plant is a tree. Suitable trees include, but are not limited to, an alder (Alnus spp.), Amarelão (Apuleia leiocarpa), Angico-branco (Anadenanthera colubrina), apple (Malus domestica), Araucaria (Araucaria spp.), ash (Fraxinus spp.), aspen (Populus spp.), baobab (Adansonia spp.), balsa (Ochroma pyramidale), banyan (Ficus benghalensis), basswood (Tilia americana), beech (Fagus spp.), birch (Betula spp.), boxelder (Acer negundo), buckeye (Aesculus spp.), butternut (Juglans cinerea), Canafístula (Cassia fistula), catalpa (Catalpa spp.), cedar (Cedrus spp.), Cedro-rosa (Cedrela odorata), chestnut (Castanea spp.), cherry (Prunus spp.), coffee (Coffea spp.), cypress (Cupressus spp.), dogwood (Cornus spp.), Douglas fir (Pseudotsuga menziesii), elder (Sambucus spp.), elm (Ulmus spp.), eucalyptus (Eucalyptus spp.), fig (Ficus spp.), fir (Abies spp.), gum (Eucalyptus spp.), hackberry (Celtis spp.), hawthorn (Crataegus spp.), hemlock (Tsuga spp.), hickory (Carya spp.), hornbeam (Carpinus spp.), juniper (Juniperus spp.), ironwood (Ostrya spp.), Ipê-roxo-de-bola (Handroanthus impetiginosus), Jatobá-da-mata(Hymenaea courbaril), jacaranda (Jacaranda spp.), katsura (Cercidiphyllum japonicum), kauri (Agathis australis), larch (Larix spp.), linden (Tilia spp.), liquidambar (Liquidambar styraciflua), loblolly pine (Pinus taeda), locust (Robinia spp.), magnolia (Magnolia spp.), mahogany (Swietenia spp.), maple (Acer spp.), monkey puzzle (Araucaria araucana), mulberry (Morus spp.), oak (Quercus spp.), olive (Olea europaea), osage orange (Maclura pomifera), pagoda (Styphnolobium japonicum), palm (Arecaceae spp.), Paricá (Anadenanthera peregrina), Pau-de-jucá (Libidibia ferrea), Paineira (Ceiba speciosa), paulownia (Paulownia spp.), pear (Pyrus spp.), peach (Prunus persica), persimmon (Diospyros spp.), pine (Pinus spp.), plane (Platanus spp.), plum (Prunus spp.), poplar (Populus spp.), redwood (Sequoia sempervirens), rowan (Sorbus aucuparia), sequoia (Sequoiadendron giganteum), silverbell (Halesia spp.), Sitka spruce (Picea sitchensis), spruce (Picea spp.), sycamore (Platanus spp.), sweetgum (Liquidambar styraciflua), Tamboril (Enterolobium contortisiliquum), tamarack (Larix laricina), teak (Tectona grandis), tulip tree (Liriodendron tulipifera), walnut (Juglans spp.), willow (Salix spp.), willow oak (Quercus phellos), yew (Taxus spp.), Zelkova (Zelkova spp.), coconut palm (Cocos nucifera), rubber tree (Hevea brasiliensis), mangrove (Rhizophora spp.), ebony (Diospyros spp.), and sandalwood (Santalum album).. In some embodiments, the tree is a poplar, Douglas Fir, Sitka Spruce, sugar pine, loblolly pine, or an oak tree. In some embodiments, the tree is a poplar tree.

[0125] Poplar trees include but are not limited to Populus species. In some embodiments, the poplar tree is a Populus species selected from: P. alba, P. alba×P. grandidentata, P. alba×P. tremula, P. alba×P. tremula var. glandulosa, P. alba×P. tremuloides, P. balsamifera, P. balsamifera subsp. trichocarpa, P. balsamifera subsp. trichocarpa×P. deltoides, P. ciliata, P. deltoides, P. euphratica, P. euramericana, P. kitakamiensis, P. lasiocarpa, P. laurifolia, P. maximowiczii, P. maximowiczii×P. balsamifera subsp. trichocarpa, P. nigra, P. sieboldii×P. grandidentata, P. suaveolens; P. szechuanica, P. tomentosa, P. tremula, P. tremula×P. tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis), Populus angulata, P. angustifolia, P. deltoides subsp., P. fremontii, P. grandidentata, P. heterophylla, P. sargentii, P. wislizeni, P. deltoids x P. nigra, P. angustifolia x P. deltoids, P. balsamifera x P. deltoids, and trihybrid P. deltoides x P. balsamifera x angustifolia. In some embodiments, the transgenic tree is a Populus tremula x Populus alba hybrid.

[0126] In some embodiments, the transgenic plant is a shrub. Exemplary shrubs include, without limitation, azalea (Rhododendron spp.), boxwood (Buxus spp.), camellia (Camellia spp.), dogwood (Cornus spp.), forsythia (Forsythia spp.), gardenia (Gardenia spp.), holly (Ilex spp.),hydrangea (Hydrangea spp.), juniper (Juniperus spp.), lilac (Syringa spp.), magnolia (Magnolia spp.), oleander (Nerium oleander), rhododendron (Rhododendron spp.), rose (Rosa spp.), spirea (Spiraea spp.), viburnum (Viburnum spp.), and yew (Taxus spp.). In some embodiments, the transgenic plant is a grass. In some embodiments, the transgenic plant is a grass. Exemplary grasses include, without limitation, big bluestem (Andropogon gerardii), bermuda (Cynodon dactylon), bluegrass (Poa spp.), bluestem (Schizachyrium scoparium), buffalo (Bouteloua dactyloides), elephant grass (Pennisetum purpureum), fescue (Festuca spp.), foxtail millet (Setaria italica), fountain (Pennisetum alopecuroides), giant reed (Arundo donax), miscanthus (Miscanthus spp.), Napier grass (Pennisetum purpureum), pampas (Cortaderia selloana), pearl millet (Pennisetum glaucum), prairie cordgrass (Spartina pectinata), reed (Phragmites australis), reed canary grass (Phalaris arundinacea), ryegrass (Lolium spp.), St. Augustine (Stenotaphrum secundatum), switchgrass (Panicum virgatum), wheatgrass (Agropyron spp.), yellow Indian grass (Sorghastrum nutans), and zoysia (Zoysia spp.). Methods of Producing Copper Resistant and / or Accumulating Plants and Trees with Fungal Disease Resistance and / or Reduced Wood Decomposition

[0127] A further aspect of the present disclosure relates to a method for increasing copper resistance, increasing copper accumulation, increasing fungal disease resistance, and / or reducing fungal decomposition in a plant or plant cell. This method involves transforming a plant or plant cell with a first expression cassette sequence comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or transforming the plant or plant cell with an expression cassette comprising a promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein. The plant or plant cell transformed with the first expression cassette sequence and / or the second expression cassette comprises increased copper resistance and / or accumulation compared to a non-transgenic plant of a same species. In some embodiments, the method comprises transforming the plant or plant cell with an expression cassette comprising a promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein and an expression cassette comprising a promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein. In some embodiments, the method further comprises transforming the plant or plant cell with an expression cassette comprising a promoter sequence operably linked to a third polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein and an expression cassette comprising a promoter sequence operably linked to afourth polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein. In some embodiments, the method comprises transforming the plant or plant cell with an expression cassette comprising a promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; an expression cassette comprising a promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein; an expression cassette comprising a promoter sequence operably linked to a third polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein; and an expression cassette comprising a promoter sequence operably linked to a fourth polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein.

[0128] This aspect of the present disclosure can be carried out with any of the embodiments disclosed herein.

[0129] In some embodiments, the one or more heterologous polynucleotides, expression cassettes, DNA constructs, and vectors of the present disclosure are introduced into cells. Methods of introducing polynucleotides into plants include transformation. Transformation refers to both stable transformation and transient transformation. A transient transformation refers to the introduction of the DNA construct into the plant cell of a host organism resulting in gene expression without genetically stable inheritance. A stable transformation refers to the introduction of the DNA construct into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleotide fragment is stably integrated in the genome of the host organism and any subsequent generation.

[0130] Selectable markers may be used to select for plants or plant cells that comprise a DNA construct. Selection of transformed cells comprising the DNA construct utilizes an antibiotic or other compound useful for selective growth as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the vector with which the host cell was transformed. The marker may encode biocide resistance, antibiotic resistance (e.g., kanamycin, Geneticin (G418), bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate, glufosinate, etc.). Examples of selectable markers include, but are not limited to, a neo gene (also known as nptII) which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulfonylurea resistance; and a methotrexate resistant DHFR gene. Multiple selectable markers are available that confer resistanceto ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin and tetracycline, etc. Examples of selectable markers are described in, e.g., U.S. Patent Nos.5,550,318; 5,633,435; 5,780,708; and 6,118,047, which are hereby incorporated by reference in their entirety.

[0131] In some embodiments, the DNA construct, or vector comprising the heterologous polynucleotide sequence, or expression cassette is introduced into a host cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a plant cell. In some embodiments, more than one DNA construct or vector can be separately introduced into a host cell. In some embodiments, each DNA construct or vector introduced into a host cell has a different selectable marker.

[0132] In some embodiments, the one or more heterologous polynucleotides are introduced into the plant cells by particle bombardment, electroporation, or Agrobacterium tumefaciens infection, to obtain transgenic plant cells. In some embodiments, Agrobacterium infection is used to transform a plant or plant cell. Bacteria species and strains in addition to Agrobacterium, can also be used to transform a plant or plant cell. Any genera within the family Rhizobiaceae can be used in place of Agrobacterium to transform a plant. For instance, members of the Phyllobacterium, Rhizobium, Sinorhizobium, and Mesorhizobium genera can be used to transform a plant according to the present disclosure. See Broothaerts et al., “Gene Transfer to Plants by Diverse Species of Bacteria,” Nature 433:629-633 (2005), which is incorporated herein by reference.

[0133] A variation of Agrobacterium transformation uses vacuum infiltration in which whole plants are used (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety). In some embodiments, transformation involves fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., “Liposome-Mediated Delivery of Tobacco Mosaic Virus RNA into Tobacco Protoplasts: A Sensitive Assay for Monitoring Liposome-Protoplast Interactions,” Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which is hereby incorporated by reference in its entirety).

[0134] In some embodiments, transformation can be accomplished by electroporation (Fromm et al., “Expression of Genes Transferred into Monocot and Dicot Plant Cells by Electroporation,” Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is hereby incorporated by reference in its entirety). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversiblypermeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.

[0135] In some embodiments, transformation can be accomplished through PEG-mediated DNA transfer, microinjection, or vacuum infiltration to provide for stable or transient expression of the DNA construct. Other methods of transformation include polyethylene-mediated plant transformation, micro-injection, physical abrasives, and laser beams (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998), which is hereby incorporated by reference in its entirety). In any of these methods, transformation can be enhanced by the use of a suitable microbe, such as a Rhizobia microbe, or an Agrobacterium, to facilitate DNA uptake by plant cells.

[0136] In some embodiments, transient or stable transformation of DNA constructs described herein is performed using particle bombardment (also known as biolistic transformation). In some embodiments, particle bombardment involves propelling inert or biologically active particles at cells. This technique is disclosed, for example, in Klein et al., “High-Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells,” Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety, and is also known as biolistic transformation of the host cell, as disclosed in U.S. Patent Nos.4,945,050; 5,036,006; and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports 14:6-12 (1995), which are hereby incorporated by reference in their entirety. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. In other embodiments, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into cells.

[0137] In some embodiments, biolistic methods use gold or tungsten particles typically of 0.5 to 2 micrometers in size and coated with DNA, RNA, or ribonucleotide particles that has been precipitated onto the particles; the particles are discharged using a “gene gun” powered by a gas at high pressure (typically hundreds to thousands of pounds per square inch) onto a plant held in an evacuated chamber. More recent biolistic methods using equipment such as the Helios® gene gun (Bio-Rad Laboratories, Inc.) use lower pressures (in the hundreds of pounds per square inch).Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or hereafter developed, can also be used.

[0138] Methods for transforming trees are known in the art, such as described in U.S. Pat. No.6,518,485, which is hereby incorporated by reference in its entirety, which discloses biolistic transformation method of gymnosperm somatic embryos, and Li et al., “Simple, Rapid and Efficient Transformation of Genotype Nisqually-1: A Basic Tool for the First Sequenced Model Tree,” Scientific Reports 7:2638 (2017), which is hereby incorporated by reference in its entirety.

[0139] Any method of transformation that results in efficient transformation of the host cell of choice is appropriate for practicing the methods of the present disclosure.

[0140] After transformation, transformed plant cells can be regenerated. Means for regeneration vary from species to species of plant, but generally a petri plate containing explants or a suspension of transformed protoplasts is first provided. Callus tissue is formed and transformation of callus tissue can also be performed. Shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, genotype, and history of the culture. Methods for transforming trees are known in the art, such as described in U.S. Pat. No.6,518,485, which is hereby incorporated by reference in its entirety, which discloses biolistic transformation method of gymnosperm somatic embryos, and Li et al., “Simple, Rapid and Efficient Transformation of Genotype Nisqually-1: A Basic Tool for the First Sequenced Model Tree,” Scientific Reports 7:2638 (2017), which is hereby incorporated by reference in its entirety. Any method of transformation that results in efficient transformation of the host cell of choice is appropriate for practicing the methods of the present disclosure.

[0141] Transformed cells may first be identified using a selection marker simultaneously introduced into the host cells along with the DNA construct or vector of the present disclosure. Suitable selection markers are described above. Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow. Other types of markers are also suitable for inclusion in the vector, such as reporter genes as described above. The selection employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers may be preferred.

[0142] Plant cells and tissues selected by means of an inhibitory agent or other selection marker are then tested for the acquisition of all or part of the heterologous polynucleotide(s), gene cassette(s), or DNA construct. In some embodiments, a transgenic cell comprises the heterologous polynucleotide(s). In some embodiments, the transgenic cell is a transgenic plant cell. In some embodiments, a transgenic plant comprises the transgenic plant cell. A transgenic plant can also comprise more than one heterologous polynucleotide sequence or expression cassette within its genome. Each heterologous polynucleotide or expression cassette may confer a different trait to the transgenic plant. Copper Accumulation, Copper Resistance, Fungal Disease Resistance & Decreased Fungal Decomposition

[0143] The transgenic plants comprising the heterologous polynucleotides of the disclosure can have a number of properties when grown in the presence of copper. In some embodiments, the transgenic plant comprises a property selected from the group consisting of: increased copper accumulation, increased copper resistance, increased fungal disease resistance, decreased fungal decomposition, and increased biomass in the presence of copper compared to a plant of the same species not comprising the transgenic plant cell.

[0144] In some embodiments, the transgenic plant has increased copper accumulation compared to a plant of the same species not comprising the transgenic plant cell. A copper “accumulating” plant is a plant with the ability to take up copper ions from the environment and accumulate copper in plant tissues at a greater level than a non-copper accumulating plant. A transgenic copper accumulating plant is a plant with the ability to take up copper ions from the environment and accumulate copper in plant tissues at a greater level than a non-transgenic or wild- type plant of the same species. Copper-accumulating plants have the ability to grow on metalliferous soil and accumulate high amounts of heavy metals in their tissues, without affecting growth or with fewer phytotoxic effects. In some embodiments, the transgenic plant has a copper concentration in the root greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 ppm, (or any number or range therein) or greater than 1000 ppm compared to a plant of the same species not comprising the transgenic plant cell. In some embodiments, the transgenic plant has a copper concentration in the root of at least 5 ppm. In some embodiments, the transgenic plant has a copper concentration in the stem greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 ppm, (or any number or range therein) or greater than 1000 ppm compared to a plant of the same species not comprising the transgenic plant cell. In some embodiments, the transgenic plant has a copper concentration in the stem of at least 5 ppm. In some embodiments, the transgenic plant has a copper concentration in the stem greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100 fold (or any number or range therein) or greater than 100 fold more than the copper concentration in the root. In some embodiments, the transgenic plant has a copper concentration in the stem greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100 fold (or any number or range therein) or greater than 100 fold more than the copper concentration in the leaf.

[0145] In some embodiments, the transgenic plant has increased copper resistance compared to a plant of the same species not comprising the transgenic plant cell. A plant with “resistance” to copper is a plant that can increase biomass and / or survive on higher levels of copper in the environment, e.g., in the soil, than a plant without resistance to copper. A transgenic copper resistant plant is a plant with the ability to increase biomass and / or survive on higher levels of copper in the environment than a non-transgenic or wild-type plant of the same species.

[0146] In some embodiments, the transgenic plant has increased biomass in the presence of copper compared to a plant not comprising the transgenic plant cell. Comparisons can be made at any time during plant growth or between any tissue type. In some embodiments, the transgenic plant produces more biomass when grown in the presence of copper compared to the biomass a control plant is capable of producing. In some embodiments, the transgenic plant produces at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (or any number or range therein) or more than 100% increased total biomass compared to a control plant in the presence of copper. In some embodiments, the transgenic plant produces at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (or any number or range therein) or more than 100% increased root biomass compared to root biomass produced from a control plant in the presence of copper. In some embodiments, the transgenic plant produces at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (or any number or range therein) or more than 100% increased stem biomass compared to stem biomass produced from a control plant in the presence of copper. In some embodiments, the transgenic plant produces at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (or any number or range therein) ormore than 100% increased leaf biomass compared to leaf biomass produced by a control plant in the presence of copper.

[0147] The development of transgenic plants or plant parts with increased fungal disease resistance could have significant benefits for forestry, agriculture, and environmental conservation. Fungal disease resistance refers to a plant's ability to defend against or repel infections caused by fungal pathogens, thereby minimizing damage and loss. Fungal disease resistance could protect plants from fungal disease damage, reduce the need for chemical fungicides, leading to more sustainable and environmentally friendly practices, and enhance the health and longevity of plants, increasing their ability to sequester carbon and contribute to climate change mitigation. In some embodiments, the transgenic plant or plant part has increased fungal disease resistance compared to a plant or plant part of the same species not comprising the transgenic plant cell. In some embodiments, increased fungal disease resistance can be determined by measuring the amount of surface area covered by a fungus on the plant or plant part. In some embodiments, the transgenic plant or plant part has increased fungal disease resistance by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any number or range therein compared to a non-transgenic plant or plant part of the same species.

[0148] In some embodiments, the method is effective for slowing or preventing fungal decomposition compared to a plant or plant part of the same species not comprising the transgenic plant cell. In some embodiments, decreased fungal decomposition can be measured in a transgenic plant or plant part, e.g., by comparing the amount of biomass (e.g., wood mass) loss in the transgenic fungal infected plant or plant part compared to a fungal infected plant or plant part of the same species not comprising the transgenic plant cell. In some embodiments, the biomass loss in the transgenic plant or plant part is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or any number or range therein compared to a plant or plant part of the same species not comprising the transgenic plant cell. In some embodiments, the wood mass loss in the transgenic plant or plant part is reduced by at least 5% compared to a plant or plant part of the same species not comprising the transgenic plant cell. Biomass loss can be measured over any period of time. In some embodiments, the amount of biomass lost is measured over 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 15 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years, or any number or range therein, or more than 10 years. In some embodiments, the method is effective for slowing or preventing fungal decomposition and / or carbon dioxide releaseinto the air. In some embodiments, the transgenic plant or plant part has decreased CO2emission into the atmosphere compared to a plant or plant part of the same species not comprising the transgenic plant cell. In some embodiments, decreased CO2emissions are estimated by measuring the amount of CO2emissions produced by decomposing plant or plant part compared to a transgenic plant or plant part as described herein. In some embodiments, the transgenic plant or plant parts as described herein store more carbon compared to a non-transgenic plant or plant part of the same species. In some embodiments, the storage of carbon is determined over 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 15 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years, or any number or range therein, or more than 10 years.

[0149] Wood decay fungi are classified into soft rot species, which are typically Ascomycota, and white and brown rot species, which are typically Basidiomycota. These fungi degrade the main wood cell wall components: cellulose, hemicelluloses, and lignin, a polyphenolic polymer in plant cell walls that plays a major role in the development of plants and their defense against pathogens, such as the pathogens that decompose tree biomass. Fungi, such as white-rot basidiomycetes, use the enzyme lignin peroxidase to biodegrade lignin and digest the resulting fragments, with consequential release of carbon dioxide into the atmosphere. However, lignin degrading enzymes such as manganese-dependent lignin peroxidase require specific metal ions such as manganese for their function. Therefore, the presence of other metal ions, such as copper, in plant tissues may affect enzyme fungal decomposition.

[0150] In some embodiments, the fungus is a soft rot fungus, a white rot fungus, or a brown rot fungus. In some embodiments, the fungus is from the genus Chaetomium, Ceratocystis, Kretzschmaria, Phialophora, Fusarium, Penicillium, Aspergillus, Trichoderma, Phanerochaete, Pleurotus, Phlebia, Trametes, Ganoderma, Stereum, Heterobasidion, Armillaria, Laetiporus, Gloeophyllum, Antrodia, Fomitopsis, Laetiporus, Meripilus, and Serpula.

[0151] Heterobasidion annosum is one of the most economically significant forest pathogens in the Northern Hemisphere. This fungus causes root and butt rot in conifers and some hardwood species, leading to significant losses in the timber industry. The disease it causes is often referred to as annosus root rot. Phlebia tremellosa is a species of white rot fungus known for its ability to degrade lignin, a complex organic polymer that provides rigidity in the cell walls of plants. By breaking down lignin, P. tremellosa and other white rot fungi play a crucial role in the carbon cycleas they help return carbon stored in wood back to the atmosphere. In some embodiments, the fungus is Heterobasidion annosum or Phlebia tremellosa.

[0152] In some embodiments, the fungus is selected from the genus Agaricus, Antrodia, Armillaria, Aspergillus, Aureobidium, Beauveria, Bjerkandera, Botryosphaeria, Botrytis, Ceratobasidium, Ceratocystis, Chaetomium, Cladosporium, Coniophora, Cryptosporiopsis, Daedalea, Dichomitus, Fibroporia, Fomes, Fomitiporia, Fomitopsis, Fusarium, Ganoderma, Gloeophyllum, Heterobasidion, Hyphodontia, Inonotus, Irpex, Kretzschmaria, Kuehneromy, Laetiporus, Lenzites, Meruliporia, Ophiostoma, Penicillium, Phanerochaete, Phellinus, Phialophora, Phlebia, Pleurotus, Polyporus, Postia, Poria, Rhizoctonia, Rhodonia, Schizophyllum, Serpula, Stereum, Trametes, Trichaptum, Trichoderma, and Xylaria.

[0153] The following examples are intended to exemplify the embodiments of the disclosure but are by no means intended to limit the scope thereof. EXAMPLES Example 1 – Increasing Copper Content in Wood Blocks Provides Fungal Resistance and Reduces Fungi-Led Wood Decay

[0154] To evaluate the effect of copper (Cu) and / or nickel (Ni) in wood tissues on fungal growth and wood decay, an experiment was performed using wood blocks prepared from greenhouse grown hybrid poplar (gray poplar, Populus alba x Populus tremula, clone 717-IB4). Wood sticks were cut into pieces of 10 mm columns with 10 mm diameter, oven dried, and weighed for initial biomass. Wood blocks were then soaked in solutions containing various metal concentrations as listed in Table 2. Wood blocks were then inoculated with or without fungi and incubated in a Percival growth chamber in the dark at 23oC and 90% relative humidity. Table 2. Concentration of Metal(s) for Wood Treatment Prior to Fungal Decay Assay Metal Amount 0

[0155] Two fungal species obtained from the American Type Culture Collection (“ATCC”) were selected for fungal growth and wood decay experiment: Phlebia tremellosa, a fungal strain heavily used in the research labs around the world due to its fast growth and high efficiency indigesting woods, and Heterobasidion annosum, the economically most important fungal species that causes widespread tree damage across the whole northern hemisphere.

[0156] After 80 days incubation, wood blocks were imaged in the biosafety hood, autoclaved, and dried in the oven before being weighed on a fine balance. As shown in FIGs.1A-B, wood blocks were pre-treated with Cu alone, Ni alone, or Cu plus Ni over a range of increasing concentrations. The Cu and Cu plus Ni treatments at higher concentrations showed no signs of fungal growth, indicating inhibitory effect of the respective treatments. Wood blocks pre-treated with Ni alone did not reduce fungal growth even when treated at very high Ni concentrations. Both fungal species showed similar growth pattern on the wood blocks pre-treated with different metal concentrations. This result indicates that Cu content in wood blocks has inhibitory effect on fungal growth. Higher concentration of Cu has more pronounced inhibitory effect. On the other hand, Ni content in wood blocks does not appear to have fungal-inhibitory effect.

[0157] Similar wood decay experiments were done on other hard woods, including Populus fremontii, Populus alba, Maple, and Oak (FIGs.2A-D), similar conclusion can be drawn that Cu content in wood blocks has inhibitory effect on fungal growth. For example, Maple wood blocks treated with 5mM copper showed no fungal growth on the wood block under the mesh. Same observation was made with P. fremontii, P. alba, and Oak wood blocks. Higher concentration of Cu has a more pronounced inhibitory effect.

[0158] Similarly, Cu-treated wood blocks from other soft wood tree species including loblolly and redwood showed inhibitory effects of Cu on fungal growth. As shown in FIGs.3A-B, loblolly and redwood wood blocks treated with just 1mM Cu had a strong inhibitory effect on fungal growth. The Cu treated wood blocks were later dried and ground for Cu content analysis via Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and the actual Cu content in the blocks are shown in Table 3. In the wood blocks treated with 1mM Cu, 102 ppm copper was detected in the wood tissues from the wood block showing fungal growth inhibition. Table 3. Cu Contents in Treated Wood Blocks Cu treatment (mM) Actual Cu content (ppm)3 731 A-B.ga n, wo eren unga s ra ns, e a reme osa an e ero as on annosum, were each tested on wood blocks originated from greenhouse grown hybrid Populus tremula × Populus alba, clone INRA 717-1B4 trees. Regarding fungal growth on wood, increasing Cu in wood tissues reduced growth of both fungal strains (FIGs.26A-B). This fungal reduction became obvious from treatments of 2.5 mM and higher Cu. At 20 mM Cu treatment, no fungal growth could be detected for either strain. Adding Ni to the wood seemed to have no distinguishable effect on the fungal growth. Both fungal stains grew well on all the wood blocks, regardless of the Ni treatments. Adding both Cu and Ni showed similar effect on fungal growth as adding Cu alone, in that fungal growth was greatly reduced when treated with 2.5 mM or more Cu. Example 2 – Increasing Copper Content in Wood Blocks Reduces Fungi-Led Wood Decay

[0160] By comparing weight changes of each individual wood block before and after the fungal incubation experiments described in Example 1, wood decay caused by fungal growth could be calculated as the % mass loss. Weight changes / wood decay after the fungal incubation was calculated as ΔW= W1-W2, and percentage mass loss was calculated as (ΔW x 100) %= (W1-W2) x100 / W1. A whole set of control wood blocks that had the same metal treatments but had no fungal inoculation were used to calibrate wood mass changes caused by any system errors other than the fungal infection.

[0161] As shown in FIG.4A, fungal species Phlebia tremellosa caused a 7.7% wood mass loss after 80 days of incubation in non-metal-treated wood blocks. Wood blocks treated with Cu from 0 to 5 mM did not show significant changes in the rate of wood decay. However, when pre- treated with higher concentration of Cu at the range of 10 – 100 mM, wood blocks showed a great reduction in decay. For example, mass loss was lowered to 2.9% on wood blocks treated with 10 mM Cu, a 62.3% reduction in decay compared to the non-metal treated control (FIG.4A). Treating wood blocks with Ni did not reduce wood decay. In fact, fungi grew well or even better on the Ni treated woods (FIGs.1A-B), higher concentration of Ni treatments even led to more wood decay and mass loss (FIG.4B). For example, in treatments with 50 µM or more Ni, wood mass loss nearly doubled compared to the non-Ni treatment control. Comparing the Cu only treatment (FIG.4A) and Cu plus Ni treatments (FIG.4C), metal treatments in both groups did not lead to significant massloss reduction until the Cu treatment reached 10 mM, suggesting that the addition of Ni did not enhance decay resistance provided by Cu treatment. In the low metal ranges (Cu at 0.5 and 1 mM), addition of Ni led to increased mass loss (FIG.4C), likely due the enhanced effect of Ni on fungal growth (FIGs.1A-B) and decay activity (FIG.4B).

[0162] The economically important tree-damaging fungal species Heterobasidion annosum was overall much slower in digesting woods compared to Phlebia tremellosa. Still, the effect of metal pre-treatment on the growth of H. annosum on the wood blocks and the resultant fungi caused wood decay was similar to that of P. tremellosa. Cu treatments at 10 mM and above inhibited the growth of H. annosum (FIG.1B, FIG.2B) and reduced wood decay (FIG.4D). Ni treatments did not inhibit H. annosum growth (FIG.1B, FIG.2B) nor wood decay (FIG.4E). Similar to the pattern shown by the P. tremellosa, the addition of Ni resulted in higher decay activities of H. annosum on woods treated with low Cu, such as 0.5 mM (FIG.4F). In the Cu only treatments (FIG.4D), higher concentration of Cu led to remarkably reduced wood decay. As much as 92% mass loss reduction was achieved with 10 mM Cu treatment.

[0163] Increasing Cu content in loblolly and redwood woods also reduced fungi caused decay. As shown in FIG.5A, 1 mM Cu treatment to the loblolly wood led to a 73% reduction in biomass loss caused by the fungi strain H. annosum. While in redwood, treatment of 1 mM Cu reduced wood decay caused by P. tremellosa by 55% (FIG.5B). These wood decay results from different tree species indicate that increasing Cu accumulation most likely is a universal and efficient strategy to inhibit fungal growth on trees and reduce wood decay.

[0164] Weight changes of every wood block were also measured and calculated after the one-year fungal infection experiment described in Example 1 as shown in FIGs.26A-H. For wood blocks inoculated with Phlebia tremellosa (FIGs.26A, and C-E), fungal infection led to a 10.6% mass loss in the negative control in which no metal was added to the wood. At 5 mM and higher Cu treatments, wood decay was sharply reduced. The 5 mM Cu treatment reduced decay by 51%, while 10 mM and 20 mM Cu treatments diminished the decay even further, by 77% and 97%, respectively (FIG.26C). Treating wood with Ni alone, low or higher amount, did not reduce wood decay (FIG. 26D). However, Ni at higher treatments range enhanced Cu generated decay reduction. Comparing to the Cu alone treatments (FIG.26C), adding Ni reduced wood decay by 83% and 94% at 5 mM and 10 mM Cu treatments, respectively (FIG.26E). Even at 2.5 mM Cu treatment, adding Ni reduced decay by 43% (FIG.26E), compared to no detectable decay reduction when wood was treated with same amount of Cu, but alone (FIG.26C).

[0165] Similar decay reduction trends were observed from wood blocks inoculated with Heterobasidion annosum (FIGs.26B and F-H). One year’s infection caused a 6.2% mass loss from the wood when no metal was added. At 5, 10, and 20 mM Cu treatments, decay was reduced by 69%, 90% and 100% when compared to the negative control, respectively (FIG.26F). Ni along did not reduce wood decay (FIG.26G). Adding both Ni and Cu caused 47%, 77%, and 82% at 2.5, 5, 10, and 20 mM Cu treatments, respectively (FIG.26H), which again, when compared to the Cu alone treatment, adding Ni appeared to make the Cu treatment more effective in reducing wood decay.

[0166] Wood blocks from these metal treatments were then tested for the actual tissue content by ICP-MS. In the control, 1.9 ppm Cu was detected in the wood. Increasing Cu treatments led to a linear Cu increase in the wood (FIG.27). For example, 0.5 mM Cu treatment led to a 151.6 ppm wood tissue Cu, while double the Cu treatment to 1 mM led to a doubled wood tissue Cu, which was 316.1 ppm. In general, this in vitro assay showed that under these conditions, tissue Cu need to be increased dramatically to obtain the goal of reducing fungi caused wood decay. As shown above, when treated with Cu alone, at least 5 mM Cu treatment was needed to reduce the decay, which is equivalent to 1418.7 ppm Cu in the wood tissues, 747 times of the amount of Cu in the control wood.

[0167] Loblolly pine is the most valuable lumber tree species in America. To reduce decay and increase shelf life, timbers are normally pressure treated before releasing to the market. In addition to poplar wood, fungal caused decay was also tested in loblolly wood treated with Cu and the effectiveness of Cu treatment vs industry standard of pressure treatment was compared. As shown in FIGs.28A-C, over a 3-month period, fungal infection led to an 8.1% mass loss in untreated loblolly wood. Treating with 1-, 2-, and 3-mM Cu led to significantly reduction in wood decay, lowered to 5.0%, 3.0%, and 2.3% mass loss, respectively. Pressure treated wood lost 1.8% mass, close to, but significantly lower than the wood from the 3 mM Cu treatment. Tissue Cu contents showed that indeed, the pressure treated wood had 2161.6 ppm Cu, much higher than the 3 mM Cu treated wood, which was 901.8 ppm, which already accumulated a thousand times higher Cu than the control.

[0168] Taken together, these results showed that increasing Cu alone in the wood of poplar and loblolly inhibited fungal growth and reduced wood decay. This Cu effect on reducing wood decay lasts over time. These results demonstrated that increasing Cu in trees could help to reduce wood decay when trees are dead or harvested.Example 3 – Engineering Design to Enhance Cu Accumulation in Tree Stems: Pathway & Gene Identification

[0169] Metal ion homeostasis regulation is complex in all living organisms. The wood decay results in Examples 1 and 2, clearly showed that engineering trees to be decay resistant by accumulating more Cu is challenging, requiring a comprehensive understanding of the Cu homeostasis and regulation in trees. To achieve this goal, a 4-step model was developed with four different pathways / steps identified to achieve the goal of substantially increasing Cu accumulation plant and tree stems. As shown in FIG.6A the 4-step model includes the following steps: (1) root uptake of Cu from the soil, (2) unloading Cu in the root xylem, (3) long distance translocation of Cu within the xylem, and (4) storage of Cu in the stem. Based on this blueprint, Cu pathways were rewired. The key genes identified to help regulate one or more of the four steps of the simplified model are shown in Table 4 and include LtCOPT1, OsATX1, OsHMA4, OsHMA5, OsHMA9, and TcNAS1. Related genes from other organisms that can also be used, include the sequences listed in Table 1.

[0170] A more complex 6-step model was also generated as shown in FIG.6C. The numbered steps in FIG.6C are as follows: (1) root uptake of copper from soil; (2) intracellular transportation; (3) xylem loading; (4) long distance xylem transportation; (5) xylem unloading and vacuole storage; and (6) leaf phloem loading and recycling. Seven genes were cloned and put into trees to help move copper in each of the six steps in order to enrich Cu in stems. The genes identified to help regulate one or more of the six steps are shown in Table 4 and include LtCOPT1, OsATX1, OsHMA4, OsHMA5, OsHMA9, TcNAS1, and OsYSL16. Related genes from other organisms that can also be used, such as the sequences listed in Table 1.

[0171] Three of these genes were synthesized for use in constructs including OsHMA5 (Andrés-Colás et al., “The Arabidopsis Heavy Metal P-type ATPase HMA5 Interacts with Metallochaperones and Functions in Copper Detoxification of Roots,” The Plant Journal 45:225-236 (2006); Kobayashi et al., “Amino Acid Polymorphism in Strictly Conserved Domains of a P-type ATPase HMA5 are Involved in the Mechanism of Copper Tolerance Variation in Arabidopsis,” Plant Physiology 148:969-980 (2008); and Deng et al., “A Member of the Heavy Metal P-type ATPase OsHMA5 is Involved in Xylem Loading of Copper in Rice,” Plant Physiology 163:1353- 1362 (2013), each of which is hereby incorporated by reference in its entirety), TcNAS1 (Pianelli et al., “Nicotianamine Over-Accumulation Confers Resistance to Nickel in Arabidopsis thaliana,” Transgenic Research 14:739-748 (2005), which is hereby incorporated by reference in its entirety),and OsHMA4 (Huang et al., “A Heavy Metal P-type ATPase OsHMA4 Prevents Copper Accumulation in Rice Grain,” Nature Communications 7:12138 (2016), which is hereby incorporated by reference in its entirety). Each of these genes was intended to enhance Cu movement / storage along each of the four steps shown in FIG.6A. Briefly, LtCOPT1 (the identification of which is described in Example 4) was used to increase Cu uptake from the environments by the roots, OsHMA5 to enhance Cu xylem unloading in the root, TcNAS1 to facilitate Cu long distance translocation in the xylem up to the shoot, and OsHMA4 to boost Cu storage in the stems. Table 4. List of Genes used in Engineering Trees to Enrich Cu in Stems and Other Tissues Genes Encoded Proteins 4-Step 6-Step Protein Origin Model Model SEQ ID

[0172] Larrea tridentata is a shrub plant that thrives in the arid North American deserts. This plant was known to accumulate high levels of heavy metals including Cu. To search for candidate genes for the engineering pipeline, the whole genome and transcriptome of Larrea was sequenced and analyzed, from which a several Cu transporter candidate genes were identified.

[0173] Root, stem and leave tissue were collected from an individual Larrea tridentata plant and DNA and RNA was isolated and sequenced by Novogene (Novogene Corporation Inc.). Genome sequencing was conducted via a 350 bp insert DNA library on the Illumina platform PE150. K-mer coverage depth was estimated at 121. After excluding the error k-mer, the genome size of L. tridentata was estimated at 570 Mb with a repeat content of 44.63%. Assembly proceeded with a SOAPdenovo workflow, which yielded a final assembly size of 790.6 Mb, with a contig N50 of 0.30kb and scaffold N50 of 0.35 kb. RNA-seq was conducted with Pacbio Sequel II. De novo assembly proceeded with Augustus, GlimmerHMM, SNAP, Geneid, and Genscan, producing a final list of 17, 722 predicted genes.

[0174] Using the six Arabidopsis copper transporter protein sequences (Copt1-6), BLASTp was conducted on a database of 17k cDNA sequences from Larrea. The top hits (with > % identity) were selected for a Reciprocal Best Hit (RBH) Blast search on Arabidopsis thaliana and the entire Rosidae non-redundant protein collection. As a result, four putative Larrea Copt transporters were identified for potential investigation.

[0175] One candidate appeared to be a homolog of the Arabidopsis Copper Transporter 1 (AtCopt1) and was named as LtCOPT1. The protein was predicted to have three transmembrane domains (FIG.29B), similar to the known homologs in yeast, animals and plants. To characterize its biochemical function, a LtCOPT1 complementation assay was performed using a ctr1 mutant yeast cell line in the BY4741 background. This cell fails to grow under low Cu and on nonfermentable carbon sources due to low cytosol Cu and nonfunctional superoxide dismutase, which is critical for the yeast metabolism with nonfermentable carbon sources and needs Cu as cofactor.

[0176] The LtCOPT1 coding sequence was amplified with Phusion PCR polymerase, with added KpnI and XbaI enzyme sites on the 5’ and 3’ end, respectively. After enzyme digestion and ligation, LtCOPT1 was cloned into the yeast expression vector pYES2, which contains the GAL1 inducible promoter. The resulting plasmid pYES2-LtCOPT1 was verified by both enzyme digestion and sequencing.

[0177] A yeast ctr1 mutant strain in the BY4741 background (YPR124W, horizondiscovery.com) was used to characterize the Cu transporter activity. After transformation with pYES2-LtCOPT1, transgenic cells were selected on SC-URA plates. Cells grew overnight in 2x YPAD medium was rinsed with H2O and diluted to OD6000.15.50 µl cells were then added to the different growth assay plates.

[0178] Though the LtCOPT1 transformant cells grew well on fermentable carbon source (FIG.30A, Glucose plate), they could not grow on the nonfermentable carbon source (FIG.30A, YPEG plate). Adding 0.01% galactose recovered cell growth from the LtCOPT1 line as well as the positive control of the AtCopt1 line (FIG30A, YPEG + 0.01% Galactose plate), but not the empty vector control, most likely due to the activation of the transgenes that were driven by the inducible Gal1 promoter. This in turn enabled cells in both the LtCOPT1 and AtCOPT1 lines to scavenge Cu from the medium to support cell growth. These results suggest that LtCOPT1 functions similarly toAtCopt1 as a high affinity root uptake Cu transporter. Indeed, Cu supplementation enabled growth of both cell lines on nonfermentable carbon (FIG.30A, YPEG + 10 µM Cu plate), further suggesting that recovery of the cell growth was due to the Cu transporter activity of both LtCOPT1 and AtCOPT1. These results demonstrated that LtCOPT1 is high affinity Cu transporter gene.

[0179] To test the biological function of LtCOPT1 in plants, overexpression of LtCOPT1 was tested to determine if it would lead to Cu toxicity and inhibition of plant growth. To do this, the LtCOPT1 coding sequence was cloned in the pMDC32 vector, the resulting construct was transformed to Arabidopsis copt1 mutant plant via Agrobacterium-mediated transformation using the floral dip method (Zhang et al., “Agrobacterium-mediated Transformation of Arabidopsis thaliana Using the Floral Dip Method,” Nature Protocol 1:641-646 (2006), which is hereby incorporated by reference in its entirety). Transgenic Arabidopsis plants were grown on ½ MS plates with 0 or 50 µM added Cu for eight days before plants were photographed and fresh weight was measured.

[0180] It was hypothesized that instead of testing LtCOPT1 in recovering plant growth from Cu deficiency, it would be easier to test Cu toxicity instead, due to the challenge of creating a deficiency condition for a micronutrient such as Cu. Both the LtCOPT1 overexpression lines and the Arabidopsis copt1 control plants were grown in half MS medium with or without supplemented Cu. When 50 µM Cu was added to the medium, growth of LtCOPT1 overexpressing plants were significantly inhibited compared to the copt1 mutant control plants (FIG.30B). When compared to growth with no added Cu, LtCOPT1 overexpressing plants accumulated only 0.7 of the fresh weight grown with 50 µM Cu, while the Arabidopsis copt1 mutant control accumulated 1.4 times of the fresh weight under same conditions, suggesting overexpression of LtCOPT1 lead to more growth inhibition. Example 5 – Discovery of Tissue-Specific Promoters to Drive Expression of Genes for Increased Cu Accumulation in Respective Tissue / Organ Types

[0181] Root- and / or stem-specific promoters were identified to drive expression of various genes in the construct designs. These promoter sequences were discovered via a combination of literature survey and database search. The most frequently used databases included the JGI phytozome genome database (Goodstein et al., “Phytozome: A Comparative Platform for Green Plant Genomics,” Nucleic Acids Research, 40 (D1): d1178-D1186 (2012), which is hereby incorporated by reference in its entirety) and the ePlant Poplar gene expression visualization database (Waese et al., “ePlant: Visualizing and Exploring Multiple Levels of Data For HypothesisGeneration in Plant Biology,” Plant Cell, 29:1806-1821 (2017), which is hereby incorporated by reference in its entirety). The major criteria for selecting the candidate promoter genes were 1) a tissue specific expression pattern that was corroborated by at least two different molecular methods (such as qRT-PCR, microarray, RNAseq, ESTseq, etc.) and 2) published promoters (e.g., Chen et al., “Transcriptional Landscape of Highly Lignified Poplar Stems at Single-Cell Resolution,” Genome Biology 22:319 (2021); Kohler et al., “The Poplar Root Transcriptome: Analysis of 7,000 Expressed Sequences,” FEBS Letters, 542:37-41 (2003), each of which is hereby incorporated by reference in its entirety). After extensive searches using these criteria, an initial list of 12 root-specific (Table 5) and 7 stem-specific expressed genes (Table 6) were chosen for promoters from the poplar genomes. Of these, 4 root-specific and 3 stem-specific promoters were cloned and built into constructs (see Table 7 in Example 6). Promoter regions were selected based on the three criteria. First, a length of 2 kb was determined to be ideal, with flexibility of 500 bp while considering additional criteria. Promoters would contain the native 5'UTR, and thus were measured immediately upstream of the start codon and would not contain any transcript from surrounding genes. Secondly, regions of low GC content would be selected, including two major regions of low-GC (>500 bp region <40% GC) if possible. Third, the promoter must be bound upstream by a ~30bp window of GC content ~50% in order for a primer to successfully amplify the region. Some consideration was given to predicting transcription factor binding sites. At least two primer pairs were designed for amplification with Q5 polymerase (New England Biolabs) and Primestar GXL (Takara) and the strongest amplicons were selected for cloning. Table 5. Poplar Root-Specific Genes with Different Expression Levels Gene SEQ ID NO: Expression Level Potri.003G128600 190 Very High Potri.001G158400 Very High Potri.004G015300 Very High Potri.016G089500 191 High Potri.004G047100 High Potri.001G340200 HighPotri.009G027200 Medium Potri.004G118800 Medium Potri.013G084500 189 Medium Potri.012G090400 Low ~ Medium Potri.013G090300 Low Potri.003G172600 192 Low Table 6. Poplar Stem-Specific Genes with Different Expression Levels Gene SEQ ID NO: Expression Level Potri.002G257900188Very HighPotri.006G181900 Very High Potri.009G095800185HighPotri.010G039700 Medium Potri.001G104800 Low ~ Medium Potri.014G448400187LowPotri.002G178700 Low Example 6 – Vector Design and Constructs for Plant Transformation

[0182] Promoters, genes and terminators were cloned into vectors using standard procedures. Gene expression constructs pLC0241-pLC0260 were made and are illustrated in FIGs.7 and 8. Additional construct designs can be made using various combinations of promoters, genes, and terminators. Enhanced copper accumulation in engineered poplar trees is expected in roots with constructs pLC0241 to pLC0244 and in stems with constructs pLC0249, pLC0250, pLC0251, pLC0254, pLC0256, and pLC0258. Enhanced copper accumulation in engineered poplar trees could also be achieved with other combinations of genes and promoters in Tables 1 and 4-7 as well as with combinations of expression cassettes in constructs.

[0183] To spatially express these genes as precisely as shown on the engineering blueprint, several novel organ and tissue specific promoters from poplar were identified and cloned (Example 5 & Tables 5-8). Among these, four were root promoters and three were stem promoters (Table 8). These promoters were predicted to have different activity levels. Ten constructs in combinations with different genes and promoters are listed in Table 9. Some of these constructs contained a single gene, such as pLC0241- pLC0244 each containing the LtCOPT1 gene but driven by different root- specific promoters, and pLC0245-pLC0247 each containing the TcNAS1 gene but driven by different stem-specific promoters. Some constructs contained two genes, such as pLC0249, three genes, such as pLC0250, and four genes, such as pLC0251. These constructs were then delivered to poplar trees via Agrobacterium-medicated transformation.

[0184] Five of the six gene coding sequences were synthesized commercially, among these, OsHMA5 and OsYSL16 were codon optimized using the P. alba codon table (Twist Bioscience). Transformation constructs were built into the pKGW destination vector following a modified LR Clonase (Thermo Fisher Scientific) reaction schema. Using a strategy similar to the Golden Gateway approach (Kirchmaier et al, “Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs,” PLoS ONE 8(10): e76117 (2013), which is hereby incorporated by reference in its entirety). Kanamycin-resistant entry vectors were constructed so that type-IIS restriction nuclease sites generated custom overhangs inside of the recombination sites, and ampicillin-resistant shuttle vectors were built with corresponding type-IIS sites so that each promoter, coding sequence, and terminator can be isolated with the same custom overhangs. This allowed individual gene expression cassettes to be cloned in a single ligation reaction directly into entry vectors, and up to four genes could be combined in a recombination reaction that builds the spectinomycin-resistant transformation construct in pKGW. The exceptions to this system were the five- and six-gene constructs. To build the five-gene construct, the fifth gene was cloned into in a kanamycin-resistant entry vector with a AtxI site. The four-gene construct was linearized with SbaI, and the AtxI cassette was introduced via ligation-free cloning with NEBuilder (New England Biolabs). Similarly, to build the six-gene construct, the sixth gene was cloned into a kanamycin- resistant entry vector, while the five gene construct was linearized with Sbf-I. The sixth expression cassette was introduced via ligation-free cloning with NEBuilder (New England Biolabs.) Sequences of all transformation vectors were confirmed via Oxford Nanopore sequencing. pKGW- based transformation vectors were transformed into Agrobacterium strain GV3101 and vector sequence was re-confirmed before stocking in glycerol.Table 7. List of Promoters Used in Constructs Promoters SEQ ID NO: Full names Species PtXaAlbH.SPL10_Pro 192 Squamosa promoter-binding Populus alba like protein 10 related allele from the promoter poplar 717 hybrid (Populus tremula x Populus alba) PtXaAlbH.Ndufab1_Pro 189 NADH dehydrogenase Same as above (ubiquinone) 1 alpha / beta subcomplex 1 (NDUFAB1) promoter PtXaAlbH.PIP2-1_Pro 191 Aquaporin 2-1 related Same as above promoter PtXaTreH.Pip1-4_Pro 190 Aquaporin 1-4 related Populus tremula promoter allele from the poplar 717 hybrid (Populus tremula x Populus alba) PtXaTreH.NAC12_Pro 187 NAC Domain-Containing Same as above Protein 12 promoter PotriCAD_Pro 185 Cinnamyl Alcohol Populus Dehydrogenase promoter trichocarpa PtXaAlH.CESA4_Pro 188 Cellulose synthase A4 Populus alba promoter allele from the poplar 717 hybrid (Populus tremula x Populus alba) AtSuc2_Pro 186 Arabidopsis thaliana sucrose- Arabidopsis proton symporter 2 thaliana

[0185] Target expression sites of various promoters used in constructs is shown in Table 8 below: Table 8. Organ-specific Promoters Identified and Cloned Target Expression Site Promoter Gene ID Root PtXaAlbH.SPL10_Pro Porti.003G172600 PtXaAlbH.Ndufab1_Pro Porti.013G084500 PtXaAlbH.PIP2-1_Pro Porti.016G089500PtXaTreH.Pip1-4_Pro Porti.003G128600 Stem PtXaTreH.NAC12_Pro Porti.001G448400 PotriCAD_Pro Porti.009G095800 PtXaAlH.CESA4_Pro Porti.002G257900

[0186] Transformation constructs are listed in Table 9 below with the engineered step referring to the 4-step model. Illustrations of constructs pLC0241-pLC0249 and pLC0252-pLC0257 are shown in FIG.7. Illustrations of larger constructs pLC0250, pLC0251, and pLC0258-pLC0260 are shown in FIG.8. Table 9. Transformation Constructs Made with Different Transcription Units Vector ID Transcription Unit(s) Engineered Step(s) (4-step model) pLC0241 PtXaAlbH.SPL10_Pro::LtCOPT1:Nos_Term 1 pLC0242 PtXaAlbH.Ndufab1_Pro::LtCOPT1:Nos_Term 1 pLC0243 PtXaTreH.Pip1-4_Pro::LtCOPT1:Nos_Term 1 pLC0244 PtXaAlbH.PIP2-1_Pro::LtCOPT1:Nos_Term 1 pLC0245 PtXaAlH.CESA4_Pro::TcNAS1:Nos_Term 1, 3 pLC0246 PotriCAD_Pro::TcNAS1:Nos_Term 1, 3 pLC0247 PtXaTreH.NAC12_Pro::TcNAS1:Nos_Term 1, 3 pLC0249 PtXaTreH.Pip1-4_Pro::LtCOPT1:Nos_Term 1, 3 PotriCAD_Pro::TcNAS1:AtHSP_Term pLC0250 PtXaTreH.Pip1-4_Pro::LtCOPT1:Nos_Term 1, 2, 3 PotriCAD_Pro::TcNAS1:AtHSP_Term PtXaAlbH.PIP2-1_Pro::OsHMA5:CaMV_Term pLC0251 PtXaTreH.Pip1-4_Pro::LtCOPT1:Nos_Term 1, 2, 3, 4 PotriCAD_Pro::TcNAS1:AtHSP_Term PtXaAlbH.PIP2-1_Pro::OsHMA5:CaMV_Term PtXaAlH.CESA4_Pro:: OsHMA4:Pill_Term Example 7 – Agrobacterium-Mediated Transformation of Plant Species Including P. alba X P. tremula

[0187] Hybrid Populus tremula × Populus alba, clone INRA 717-1B4, were grown in a greenhouse at the Hayward CA site. Temperature and light vary over seasons, but over the year the greenhouse has an average photosynthetic photon flux density (PPFD) of 30 mol / m2 / d m−2s−1, a 26°C / 23°C day / night temperature, and a humidity levels of approximately 40% relative humidity. Parental plants were shaped to allow production of axillary shoots. Cuttings made to these axillary shoots produced ramets (or sister plants). Ramets were then potted with a standard peat basedpotting mix and placed in a domed tray for rooting within a Percival growth chamber, which typically took 28-35 days. Rooted ramets were transplanted into 2-gallon pots and randomly distributed within the greenhouse.

[0188] As previously reported (Tao et al., “Enhanced Photosynthetic Efficiency for Increased Carbon Assimilation and Woody Biomass Production in Engineered Hybrid Poplar,” Forests 14(4), 827 (2023), which is hereby incorporated by reference in its entirety), stem explants isolated from in vitro propagated plants of hybrid Populus tremula × Populus alba, clone INRA 717-1B4, were used for Agrobacterium-mediated transformation. Recombinant Agrobacterium tumefaciens colonies strain GV3101 separately transformed with each plasmid (i.e., one of pLC0241-pLC0256 or pLC0258-pLC0260) were cultured for approximately 24 hours on an orbital shaker. Leaves of the hybrid Populus tremula x Populus alba, clone INRA 717-1B4 were collected and used to grow stem explants. Stem explants were swirled in the Agrobacterium suspensions for 1 hour, and then co- cultured on agar medium for 48 hours at 24°C in darkness. The explants were washed four times in 50 ml centrifuge tubes with sterile deionized water at 125 rpm at 24°C, transferred to growth media with the selection reagent kanamycin for 21 days at 24°C in darkness, and then transferred to standard light conditions 16 h light / 8 h dark to recover green calli. The green calli were transferred to agar medium containing kanamycin for regeneration and selection of transformed shoots. The regenerated shoots were excised when they were approximately 1 cm long and transferred to agar medium supplemented with kanamycin for rooting. After 5-8 weeks, the rooted and elongated shoots were propagated on the same medium. Rooted transgenic plants were transferred out of Magenta boxes in tissue culture into soil pots, acclimated, and grown under inverted diurnal cycle. Stable T0 generation transgenic events were evaluated and further propagated. After transformation, callus formation, shooting, shoot elongation, and rooting spanned approximately 4 months.

[0189] When T0 transgenic events grew up to about 2 inches in height and had sufficient root growth, they were transferred to a Percival growth chamber for acclimation. Plants were transplanted into a peat-based substrate in a domed tray and periodically misted to maintain humidity and prevent wilting. Domes were slowly removed over a 4-week acclimation period. Then the trees were transferred to 2-gallon pots. PROPHETIC Example 8 –Tissue Culture, Growth Conditions, and Genetic Transformation of Loblolly Pine

[0190] Loblolly pine can be transformed with constructs as follows: Loblolly tissue culture and growth conditions. Initiation of embryogenic callus formation was performed bysubjecting immature embryos of Pinus taeda to plant growth regulators in a DCR-based semi-solid gelled medium. Proliferating embryogenic masses are selected and subcultured onto fresh medium for continued growth in the dark at 26 °C. Cell lines are then transferred to a liquid suspension proliferation medium in 125 mL flasks and shaken orbitally in the dark at 110 rpm. Increased settled cell volume (SCV) is used as an indicator of cell proliferation in suspension cultures. Once suspension cultures reached an appropriate level of cell density, cells are plated onto rafts using a vacuum manifold. Rafts are then moved to either maturation medium for somatic embryo development or osmotic medium for genetic transformation.

[0191] Loblolly genetic transformation for increased copper content via particle bombardment. Plated suspension cells are transferred to a 0.4 M sorbitol gelled medium 24 hrs prior to bombardment for osmotic pretreatment and replaced to the dark at 26° C. Particle bombardment is performed utilizing a PDS-1000 / He Particle delivery system for cell transfection in laminar flow hood. A few hours prior to particle bombardment, gold 0.6 µM microcarriers are prepped on ice by first washing with 200 proof ethanol and subsequently coating them with a mixture of construct DNA containing copper gene expression cassette(s) and a selectable marker expression cassette, CaCl2, and 0.1 M spermidine. Prepped microcarriers are precipitated onto sterilized microcarriers in 10 µL aliquots. Macrocarriers are loaded into the PDS-1000 / He gene gun assembly along with sterilized 1550 psi rupture discs and stopping screens. The cell-containing osmotic petri dish cover is removed, and the petri dish is placed on the target shelf with a 9 cm microcarrier flight distance. The PDS-1000 / He chamber is vacuum pressurized to 27 inHg and pressurized Grade 5 Helium is subsequently utilized to burst the 1550 psi rupture discs. Post-bombardment, cultures are allowed to rest in the dark on the osmotic medium overnight before transferring them to the proliferation medium. Cell-containing rafts are moved to a proliferation medium containing an appropriate concentration (2-20 mg / L) of antibiotic for selection of transformed cells. Three biweekly subcultures onto fresh antibiotic containing medium are performed. New translucent / yellowish callus growth arising from the necrotic tissue beneath is pulled from the bombardment rafts using forceps. This new growth is deemed a transgenic event. These putative transgenic cell lines are subcultured onto fresh selection medium twice to kill any lingering, non-transgenic cells before initiating suspension cultures to bulk more tissue. Once these transgenic cell suspension cultures reached adequate density, they are plated onto rafts and transferred to either semi-solid proliferation medium for cryopreservation or maturation medium for regeneration and replaced in the dark.

[0192] Agrobacterium-mediated loblolly genetic transformation for increased copper content. Recombinant Agrobacterium harboring copper gene expression cassette(s) in binary vectors are cultured according to previously described examples. Embryogenic callus suspension cultures are initiated one week prior to transformation to ensure proliferation. Agrobacterium cultures are centrifuged at 4000 rpm for 10 mins to pellet cells. The supernatant is removed and the Agrobacterium pellet is resuspended in fresh loblolly liquid proliferation medium with the addition of acetosyringone for induction. This liquid induction medium is subsequently added to the loblolly suspension cells. The culture is then returned to the dark at 26 °C and allowed to co-cultivate for 48 hrs. After the co-cultivation period, cells are washed with antibiotic in liquid suspensions and the cells are then plated onto rafts under vacuum to remove the co-cultivation medium. Plated cells are again washed with approximately 50 mL of sterile DI under mild vacuum before transferring the rafts to gelled maintenance medium containing cefotaxime and appropriate antibiotic selection agent including kanamycin. Rafts are transferred to a fresh selection medium every two weeks and observed for emerging antibiotic-resistant transformants.

[0193] Post-selection and bulking, transgenic cell lines are plated onto maturation medium for somatic embryo development in the dark at 26 °C. This maturation medium contains reduced levels of plant growth regulators and a high concentration (8-12 g / L) of gelling agent. Subculturing onto fresh medium is performed every 3 weeks, and after 9-12 weeks buttery yellow somatic embryos formed. Somatic embryos are then evaluated under the microscope for their shape and size, and 1 – 5 mm torpedo-shaped embryos containing cotyledons are excised from the suspensor cells and transferred to the germination medium. Approximately 30-40 somatic embryos per germination plate are then moved immediately to a lighted rack room displaying a 16 / 8 hrs diurnal photoperiod at approximately 24 °C (+ / -1 °C). Germination of the somatic embryos is observed over a 4–7-week period. Germination is considered successful with the greening up of the cotyledons, elongation, and formation of a tap root and / or subsequent secondary roots. Germinants that shows these characteristics and are ~ 1 cm long (shoot + root) are transferred to outgrowth medium and given 4 weeks to continue to develop roots before removal from the lightroom for acclimation in the greenhouse. Acclimation conditions fully shade and hourly misted for 2 weeks at 21 °C, two weeks with one, early morning misting and shade at 21 °C and then 4 weeks early with one misting and no shade at 21 °C. These 5-8 cm seedlings are then moved outside, sprayed with a pre-emergent herbicide, and allowed to grow to approximately 15 cm at ambient conditions before transfer to soil for outgrowth and analysis.Example 9 – Molecular Analysis of Transgenic Events

[0194] Leaf tissue samples were collected from transgenic plants 1-2 weeks post transplantation into soil pots. Genomic DNA was extracted from tissue samples using the NucleoSpin Plant II kit (Takara Bio Inc., Kusatsu, Japan). Total RNA was extracted from various tissue samples including leaf, petiole, and stem using the NucleoSpin RNA Plants and Fungi extraction kit (Takara Bio Inc., Kusatsu, Japan). Quantitation of genomic DNA and total RNA was performed on a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s protocol.

[0195] Genomic DNA was subjected to PCR and qPCR analysis. While PCR results provide data for the presence or absence of transgenes, qPCR analysis revealed how many copies of each transgene were inserted in the genome. For copy number analysis, 50-100 mg samples of young leaf tissue were collected for DNA extraction using the NucleoSpin 96 Plant II DNA extraction kit (Macherey-Nagel, Dueren, Germany). Multiplex qPCR assay with technical triplicate for each event was performed using CFX Opus 96 machine (Bio-Rad, Hercules, CA, USA) to obtain the Cqs for copy number estimation. Each 20 uL qPCR reaction consists of 20 ng of DNA, forward and reverse primers, probe (Table X), and 2X TaqPath ProAmp master mix (Thermo Fisher Scientific, Waltham, MA, USA). PP2A was selected as a reference gene and a single copy event was used as a calibrator. Copy number variation was determined using the 2-ΔΔCqmethod (Weaver et al., “Taking qPCR to a Higher Level: Analysis of CNV Reveals the Power of High Throughput qPCR to Enhance Quantitative Resolution,” Methods 50:271-276 (2010), which is hereby incorporated by reference in its entirety) in CopyCaller v2.1 software (Thermo Fisher Scientific, Waltham, MA, USA). Events with transgene copy numbers less than two were included in evaluation of the construct effect.

[0196] To analyze transgene expression, either stem or root tissues were collected. For each stem sample a 1 cm length of basal stem was collected and further separated into bark and woody tissues.200 mg of the bark tissue and 880 mg of the woody tissue were used for RNA extraction. 290 mg of the primary root tissue was collected from the base of the root and was used for RNA extraction. RNA extraction was carried out using the NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Dueren, Germany). Gene expression analysis was carried out using a multiplex qRT-PCR assay with technical duplicates for each event via CFX Opus96 machine (Bio-Rad, Hercules, CA, USA). Each 20 uL reaction contained 200 ng of RNA, forward and reverse primers, probe (Table 10), and 2X custom-made One-Step RT-qPCR Master Mix with a lower amount of DTT (Launchworks, Bothell, WA, USA). The 2(−∆∆Cq) method (Weaver et al., “Taking qPCR to aHigher Level: Analysis of CNV Reveals the Power of High Throughput qPCR to Enhance Quantitative Resolution,” Methods 50:271-276 (2010), which is hereby incorporated by reference in its entirety) was used to analyze the expression data.

[0197] Primers used for analyzing transgene copy number and / or gene expression are shown in Table 10. Primer probe mix was prepared in advance, with probe concentrations in the final stock at 2.5 µM (10X stock). To prepare 10x stock primer probe mix, dilution and reconstitution were performed so that the final concentration of probe is 2.5 µM and the primers are 5 µM. Probe primer stock mix was prepared in advance, stored in dark, and kept on ice during the process of reaction preparation.

[0198] Fluorescent probes (IDT Technologies, Coralville, IA) such as SEQ ID NO:202 and SEQ ID NO:205 can be labeled with Fluorescein (FAM), TEXAS RED (TexRd) or other fluorescent markers on the 5′ end and included a quencher (ZEN) and 3IABkFQ. Fluorescent labeling and sequences of specific probes and primers for each gene of various constructs are listed in Table 10. Table 10. Primers Used for Gene Expression Analysis & Transgene Copy Number Gene Forward primer Reverse primer Probe Probe targets sequence sequence sequence color CCTGAACCCTAAA GGGAATGATTCA / 5HEX / AATCAGTCT / ZEN / GGTCC PP2A TAGCAACCT GGTCATCGT CGTCCGGT / 3IABkFQ / HEX (SEQ ID NO:194) (SEQ ID NO:195) (SEQ ID NO:196) CGTTGGCTACCCGT CTCGTCAAGAAG / 5Cy5 / CCGCTTCCT / TAO / CGTGCTT Cy5 or NPTII GATATT GCGATAGAAG TACGGTAT / 3IAbRQSp / FAM (SEQ ID NO:197) (SEQ ID NO:198) (SEQ ID NO:199) / 5TexRd- CTATGCCACCAAT GACAAAGCATAC XN / TGATGGCTGGGTGCTATTCG COPT1 GCCAAAC ATCCCATGATTAG TexasRed AAGG / 3IAbRQSp / (SEQ ID NO:200) (SEQ ID NO:201) (SEQ ID NO:202)T / ZEN / T NAS1 TCTCCT GTCACATCC CTT CCA CAC CGT TG / 3IABkFQ / FAM (SEQ ID NO:203) (SEQ ID NO:204) (SEQ ID NO:205) GAAGGAGAGAAAG AACAGCTTGGCCC / 5-FAM / TG TCG ATA G / ZEN / A HMA4 ACGAGGAAG TGAA GAC GGT GGT GGC T / 3IABkFQ / TexasRed (SEQ ID NO:206) (SEQ ID NO:207) (SEQ ID NO:208) CAGCTGTAGCCGG CCTCCTCCTCAGC / 5Cy5 / AA GGA GGA G / TAO / G TGG HMA5 AGAAG ACCTA AGG AGG AGA TG / 3IAbRQSp / Cy5 (SEQ ID NO:209) (SEQ ID NO:210) (SEQ ID NO:211) HMA9 ATACCGAGCGAGA CTGCCTCCGGATC / 5-FAM / TG TGA ACG C / ZEN / T FAMTGTGAATG GAATATG ACT GTT TCT GAG GTG (SEQ ID NO:212) (SEQ ID NO:213) G / 3IABkFQ / (SEQ ID NO:214) / 5- GTGGAGTCCTTTG GTTTTCTTGCCCG FAM / ACACCAGAT / ZEN / GCCGTT ATX1 ACGTAGAC TCTTTGAA FAM CTTCAGACC / 3IABkFQ / (SEQ ID NO:215) (SEQ ID NO:216)YSL16FAM (SEQ ID NO:218) (SEQ ID NO:219) (SEQ ID NO:220)

[0199] qRTPCR preparation: Master mix was prepared in a 1.5 mL or 2 mL tube following manufacturer’s protocol and kept on ice in the dark until used. The reaction master mix was thoroughly mixed and then microfuged. Working on ice, 18 µL of the reaction master mix was first added, followed by 2 µL of normalized RNA samples (200 ng total RNA in each reaction) to each sample well. Next, 2 µL nuclease free water was used for the NTC (no template control), and 2 µL standard was added to the standard well. The reaction plate was covered with an optical adhesive cover and sealed. The plate was vortexed to mix the reaction followed by centrifugation of the plate at 150 × g for 1 minute to collect the contents at the bottom of the wells and eliminate air bubbles. The qRT-PCR protocol was executed using Bio-Rad CFX Opus 96 machine (BioRad Laboratories, Hercules, CA) or similar machine. PCR was performed with a UNG incubation step at 25oC for 2 minutes, followed by a reverse transcription step at 53oC for 10 minutes. Polymerase was activated at 95oC for 2 minutes, followed by 40 amplification cycles of 95oC for 3 seconds and 60oC for 30 seconds.

[0200] An example of expression analysis of COPT1 is shown in FIG.11A. COPT1 expression levels were normalized relative to expression of endogenous gene Potri.015G068300 (PP2A; Protein Phosphatase 2A-2). Example 10 – ICP-MS Methods for Tissue Copper Content Analysis

[0201] Wood tissue disruption. Grinding woody tissues without a powerful grinder always poses a challenge, nevertheless, a simple method has been developed by combining manual wood cutting and grinding with a GenoGrinder® (SPEX SamplePrep, Metuchen, NJ). Briefly, ten- millimeter sections of dried stem tissue (7.5 mm diameter) from ~8-week-old poplar were cut into small pieces (< 1mm thick) with a razor blade. Tissue was placed into a 2 mL tube with a single 6.35 mm stainless steel BB and shaken in the GenoGrinder® for 3 min at 1750 rpm, yielding fineparticles suitable for metals extraction and analysis. This protocol requires less than 3 min. of processing time per sample and utilizes on-site equipment.

[0202] Tissue metal extraction from grind wood tissues is largely based on the method of using strong acids in programmed heat conditions. Specific steps included: 1) weighing 250 mg of ground tissue into a 50 ml digestion vessel; 2) adding 5 ml of nitric acid (69%) and place tubes into the HotBlock (ensure that reflux lids are in place); 3) activating the heating program (10 min. ramp to 83 °C, 20 min. hold at 83 °C); 4) allow the block to cool to 50 °C, then adding 500 µl of 30% hydrogen peroxide; 5) activating the heating program (10 min. ramp to 83 °C, 20 min. hold at 83 °C); 6) repeat steps 4 & 5 and allowing the block to cool to 50 °C; 7) removing tubes from the HotBlock and allowing samples to cool to room temperature before adding 20 mL of LC-MS grade water; 8) after 8 hours of incubation at room temperature transferring the sample to ICP autosampler tubes for analysis.

[0203] ICP-MS was performed using a standard protocol. Briefly, the sample digestion protocol was adapted from EPA method 3050B. Two milliliters of concentrated nitric acid were added to 100 mg of tissue, and samples were heated to 83 °C for 40 min in an Environmental Express HotBlock. Samples were removed and allowed to cool before adding 0.5 mL of 30% hydrogen peroxide and returning the samples to the HotBlock for an additional 20 min incubation. This was performed once more for a total of 1.0 mL of hydrogen peroxide. Sample volumes were adjusted to 50 mL with deionized water (18 MΩ cm-1), and data was acquired with an Agilent 7700 inductively coupled plasma mass spectrometer (ICP-MS).

[0204] T0 trees reached around 75 cm tall over the summertime (grown 6-7 weeks) were sampled. Over winter season, trees grew more slowly and were slender in shape. Trees were not sampled until they reach around 125 cm (grown 10-12 weeks) to ensure enough biomass could be collected (especially from the roots). Four samples were collected from each tree. Samples included roots, stem woody tissue, stem bark tissue, and leaves. At the sampling time, each individual whole tree was pulled out of the 2-gallon pots, soils were carefully cleared away from the root system, then about half of the root ball was cut vertically, collected, and rinsed with DiH2O. The stem was cut at a position below which 4-5 healthy buds were formed. The basal ~20 cm part of the cut stem was then peeled, and the woody vs bark tissues were separately collected. For leaves, the #9, 10, 29, 30, 39 and 40 leaves started from the 1stopened leaf on the shoot tip were collected to cover the young, mature and senescent stages. All cleaned samples were dried in oven at about 80 °C before beinggrounded, extracted in strong acids and tested on an ICP-MS machine (Agilent 7700) for tissue Cu contents.

[0205] ICP-MS was performed using a standard protocol. Briefly, the sample digestion protocol was adapted from EPA method 3050B. Two milliliters of concentrated nitric acid were added to 100 mg of tissue, and samples were heated to 83 °C for 40 min in an Environmental Express HotBlock. Samples were removed and allowed to cool before adding 0.5 mL of 30% hydrogen peroxide and returning the samples to the HotBlock for an additional 20 min incubation. This was performed once more for a total of 1.0 mL of hydrogen peroxide. Sample volumes were adjusted to 50 mL with deionized water (18 MΩ cm-1), and data was acquired with an Agilent 7700 inductively coupled plasma mass spectrometer (ICP-MS). Example 11 – Development of a HTP Copper Content Assay System Via Hydroponic Culture

[0206] A hydroponic tank system for a high throughput (HTP) transgenic screening platform was developed. The nutrient recipe from Siebrecht et al., “Nutrient translocation in the xylem of poplar—diurnal variations and spatial distribution along the shoot axis,” Planta 217 (2003): 783-793 (2003), which is hereby incorporated by reference in its entirety), was chosen, which provided a reasonable ratio and concentration of major nutrient elements including N, P, K and Ca. It was important to maintain the amount of Cu in the assay system at a similar level to the average concentration of bioavailable Cu in the U.S. soils, thus the U.S. Geographic Survey’s soil metal database was searched (website: pubs.usgs.gov / sir / 2017 / 5118 / index). Although the U.S. soil Cu was found to be very patchy, the majority of areas had total soil Cu contents of 10 ppm (157 μM) and below. However, data of the actual bio-available Cu in the U.S. soil was not available. Our preliminary data from poplar Cu toxicity assays showed that 1 μM Cu in liquid culture already inhibited root growth. So, we chose to use Cu at a concentration of 0.1 or 0.2 μM. To minimize the impact of trace Cu content in the greenhouse water supplies, we tested and evaluated the Cu content in the greenhouse water supplies. All greenhouse water samples collected at various times during the day throughout the week contained Cu that exceeded the intended input in our assays, whereas laboratory sourced deionized (DI) water contained no Cu. Therefore, DI water was chosen for use in the hydroponic testing system.

[0207] The prototype hydroponic culture system was made using a 38 L Rubbermaid HDPE storage tub (23.875x15.875x8.75in) as the culture container, an EcoAir commercial pump to circulate and oxygenate the solution, along with 1⁄4 in internal diameter tubing and corresponding air stones to disperse air to the roots. One inch diameter sponges were used to support the stem of thetrees while growing in the hydroponic culture. This system has a capacity of growing and testing 48 plants (see FIG.9). This culture system was tested with rooted WT poplar cuttings for a 2-week period using 0.1 and 0.2 μM CuSO4, respectively. 1 mM MES buffer was added to stabilize the pH. After 2 weeks growth in the MES stabilized media, trees were harvested, and fresh weight increases in trees supplied with 0.1 vs 0.2 μM CuSO4were compared. Trees grew with both Cu concentrations looked healthy and as shown in FIG.10, trees grown with 0.2 μM CuSO4gained slightly more fresh weight. Based on this, 0.2 μM CuSO4was chosen for future assays.

[0208] To validate this hydroponic culture system, a hydroponic / soil parallel growth assay was performed with C1 plants. C1 plants are cuttings from a T0 plant. Typically, eight weeks post potting, T0 plants go through a vegetative propagation cycle using a stem cutting method frequently used in horticulture practice. Briefly, the apical meristem was removed to release the apical dominance to stimulate the formation of axillary buds to produce branches. The axillary branches were cut and inserted into rockwool cubes for support to produce roots in hydroponic conditions. To maintain the genetic lineage of transgenic lines, cuttings from the T0 plant were named C1 plants with C1.1 referring to apical cutting and C1.2 and so on referring to axillary cuttings. C1 cuttings normally established root systems after approximately two weeks in hydroponic conditions and would then be transferred to soil pots for C1 plant evaluations. Multi branches or cuttings produced from one T0 mother plant are sister C1 plants called ramets, serving as biological replicates for C1 evaluation.

[0209] The expression level of one transgene, LtCopt1, and tissue Cu content in in the roots in plants from the same T0 events were tested and compared under the two different conditions of soil and hydroponic growth. In total, 27 C1 plants from three different T0 events pLC0241.0101, pLC0242.0201, and pLC0244.0101 were growing in either hydroponic or soil. After six weeks, roots were collected for gene expression analysis and tissue Cu accumulation. On the gene expression level, trees from these three T0 events showed a similar pattern in hydroponic vs soil conditions, in that the expression of LtCopt1 was the lowest in event pLC0242.0201, higher in pLC0241.0101, and the highest in pLC0244.0101 (FIG.11A). These results show that trees originated from the same T0 events did not change the expression of the LtCopt1 transgene significantly when growing in the different conditions of soil versus hydroponic culture.

[0210] Tissue Cu accumulation followed the same pattern as that of the gene expression, in that C1 trees from the pLC0242.0201 T0 event had the lowest root Cu while C1 trees from the pLC0241.0101 had an intermediate root Cu accumulation, and C1 trees from the T0 pLC0244.0101event had the highest root Cu in both growth conditions (FIG.11 B). For each individual event, trees did accumulate different amount of tissue Cu when grown under these two different conditions, and overall soil grown trees showed less variations in tissue Cu. Results from this hydroponic / soil parallel growth assay validated the hydroponic system as a useful tool for transgenic tree analysis. Example 12 – Identification of Transgenic Events with Elevated Copper Content in Various Tissue Types

[0211] Tissue Cu content in transgenic poplar events were analyzed in comparison to wild- type controls using ICP-MS in root, stem wood, stem barks, and leaves. The non-transgenic trees went through the same tissue culture and regeneration processes, making them ideal control to the target transgenic events. Tissue Cu contents varied among events from the same construct (since expression of a transgene can be influenced by integration position in the genome in different events), as well as between events with the same gene but driven by different promoters (same gene but different construct). Compared to the wild-type non-transgenic control plants, every one of the 10 constructs tested (including LC0241-LC0247 and LC0249-LC0250) had transgenic plant events that accumulated more Cu both in roots (FIG.12) and stems (FIG.13). In the root tissues of top transgenic events of LC0241, LC0242, and LC0244 where the LtCOPT1 gene alone was driven by various promoters, Cu content was approximately 20 ppm or 400% higher than non-transgenic controls (FIG.12). This result demonstrated that LtCOPT1 alone was sufficient to increase copper uptake from the soil. The overall extend of Cu increase was similar among the T0 events that either carried one, two or three transgenes, with the median value varied between 4 and 5.6 ppm. However, T0 trees carrying the four gene construct showed a median value of 7.5 ppm Cu in the stem wood tissues, a dramatic increase compared to the control and events carrying all the other constructs. In one event, Cu in the stem wood tissues reached 23.9 ppm, about nine times of the level in the control trees (FIG.13). For these same T0 events, many also showed elevated Cu levels in the roots, though no such remarkable increase was found in the four-gene transgenic events (FIG.12).

[0212] In one LC0245 event and one LC0246 event transformed with TcNAS1, roots accumulated 400% more Cu compared to the wild-type trees (FIG.12). Because TcNAS1 gene was the only transgene in these constructs, these results indicated that TcNAS1 gene plays an important role in copper uptake from the soil. Transgenic events of constructs with LtCOPT1 as the only transgene (constructs LC0241, LC0242, LC0243, and LC0244) also showed 200% or more higher copper content in stem tissue (FIG.13), indicating some of the elevated copper in root tissue due touptake boost by LtCOPT1 was successfully uploaded to stem tissue. Events of constructs with TcNAS1 alone (LC0245, LC0246, and LC0247) also showed elevated copper content in stem tissue (FIG.13). One event of LC0245 with NcNAS1 driven by CESA4 promoter showed more than 300% higher copper content. Together with the increased Cu accumulation in root tissues (FIG.12), ectopic expression of TcNAS1 was found to be beneficial in the strategy to increase Cu accumulation in above ground tissue types.

[0213] Combinations of additional genes together with LtCOPT1 and TcNAS1, were shown to further optimize the copper uptake and transportation pathway. For example, pLC0251 was designed as a four gene construct (FIG.8). This construct includes a Pip1-4 promoter driving the LtCopt1gene to facilitate uptake of copper from soil for step 1 enhancement, a CESA4 promoter driving the OsHMA4 gene to facilitate storage of copper in the vacuole, a PIP2-1 promoter driving OSHMA5 gene to facilitate the process of copper uploading to root xylem, and a stem specific CAD promoter driving TcNAS1 to facilitate the upward transportation of copper in stem tissue. In one event of this four-gene construct, the stem accumulated Cu to a much higher level of 1200% of that of the control, reaching about 24 ppm (FIG.13). Two other events with likely different genomic insertions also showed greater than 800% Cu content in stems, indicating the increase of Cu accumulation is due to the effect of the four genes in LC0251 construct, not from the insertion disruption of endogenous gene(s). These trees were visually indistinguishable from other T0 and the control trees, indicating that 24 ppm accumulation of Cu was non-toxic to young poplar trees.

[0214] Nicotianamine is an organic molecule that is important for metal transport and tissue storage. Poplar trees transformed with the Nicotianamine Synthase gene TcNas1 driven by various promoters with increasing strength (i.e., driving increasingly greater gene expression) showed correspondingly greater increases in nicotianamine. As shown in FIG.15, the lowest amounts of nicotianamine were found in the stems of the wild-type control (WT) with increasingly more nicotianamine found in the stems of LC0247, LC0246 to LC0245 transgenic trees. Nicotianamine was measured using LC-MS. The nicotianamine in the stems of transgenic trees increased in trees with the LC0247 construct having the PtXaTreH.Pip1-4 promoter (SEQ ID NO:190) driving TcNas1 expression, to a further increase in trees with the LC0246 construct having the PotriCAD promoter (SEQ ID NO:185) driving TcNas1 expression, to the highest levels in trees with the LC0245 construct having the PtXaA.CesA4 promoter (SEQ ID NO:188) driving TcNas1 expression.

[0215] Besides root and stem wood tissues, stem barks and leaves were also measured for Cu content in many T0 events. Interestingly, compared to the control, engineering all four steps of Cuhomeostasis by transforming four genes all together not only increased the overall Cu accumulation in the stem and root, as shown above (FIGs.12 & 13), it also altered the Cu distribution patterns within the trees. Deviated from the pattern in the WT control in which leaves and roots stored more Cu than the roots, these four-gene transgenic trees accumulated more Cu in the stems (FIGs.14 & 31A). Furthermore, comparing Cu accumulation in the stem wood vs bark tissue showed that in the WT control and all the T0 events carrying one, two or three transgenes, stem wood and bark tissue showed similar Cu content, making the stem wood vs bark Cu ratio from close to 1. However, the four-gene transgenic events by average accumulated more than 3 times higher Cu in the stem wood compared to the bark tissue (FIG.31B), pointing to the promoter driving the expression of OsHMA4 as quite stem wood tissue specific, confirmed the design hypothesis and strategy. Example 13 – Strategy to Alter Copper Accumulation in an Organ-Specific Pattern

[0216] Construct design pLC0251 consisted of four gene expression cassettes (promoter:gene) including (PtXaTreH.Pip1-4 promoter:LtCopt1:Term / PtXaAIH.CesA4 promoter:OsHMA4 / PtXaAlbH.Pip2-1 promoter:OsHMA5 / PotriCAD promoter:TcNAS1; see FIG. 8). Transgenic events generated with pLC0251 not only had increased Cu accumulation across all organs (root, stem, and leaf), they also show altered Cu distribution patterns among the organs (FIG. 14) as was the desired outcome. It is known that leaf and root are the major storage sites for minerals and ions in plants, and wild-type control trees confirmed that pattern. However, when LtCOPT1 was transformed to the trees (LC0243 in FIG.14), adding this root expressed Cu transporter enhanced root Cu uptake from soils resulting in the root being the highest Cu containing organ. This result confirmed our hypothesis of the role of this transporter in root Cu uptake (Step 1 in FIG.6B), but also indicated that the bottleneck to enrich Cu in the stems would then be the Cu unloading to the xylem tissue (Steps 2-5 in FIG.6B)

[0217] Adding OsHMA5 or TcNAS1 or a combination of both was demonstrated to help Cu xylem unloading and long-distance Cu translocation upward, since a large portion of the Cu went to leaf again in transgenic trees with constructs LC0246, LC0249, and LC0250 (FIG.14). These constructs resulted in increased Cu content in all three organ types (root, stem wood, and leaf) maintaining a similar organ distribution pattern as that of the wild-type control. Further addition of another gene, the tonoplast localized Cu pump OsHMA4 driven by a stem specific promoter, resulted in alteration of Cu accumulation pattern such that stem surpassed leaf and root to become the highest Cu storage organ (LC0251 in FIG.14).

[0218] In construct pLC0260, two more expression cassettes were added. Based on the models disclosed herein, OsATX1 is expected to enhance Cu root intracellular transport and xylem unloading. OsYSL16 is expected to facilitate Cu recycling from leaves. Both could drive additional Cu accumulation in stems. Example 14 – Demonstration of Wood Decay Reduction in Transgenic Woody Tissues with Elevated Copper Content

[0219] As shown in the Examples and Figures above, the decay assays from the Cu treated wood demonstrated that potentially hundreds of times of more Cu need to be enriched in the trees to achieve the goal of reducing wood decay. The top transgene events from the work disclosed herein so far showed about nine times higher Cu accumulation in the stem wood. Wood was collected from these trees and fungal caused decay was tested.

[0220] Wood was collected from stems of three pLC0251 T0 trees that showed the highest tissue Cu levels (17.2, 17.7, and 23.9 ppm, respectively). Samples were inoculated with either one of the two fungi strains, Phlebia tremellosa or Heterobasidion annosum, and incubated for 90 days. Fungal growth was detected, and wood mass loss was measured as described above.

[0221] Reduced fungal growth of both fungi strains was seen on the wood blocks collected from the transgenic trees in comparing to those harvested from the WT control trees (FIGs.32A & C). Weight change measurements indicated that after the 90-day incubation, infection of fungi strain Phlebia tremellosa led to a 20.3% mass reduction in the control woods, while mass reduction in wood from the three transgenic trees were 19.2%, 17.6, and 18.3%, respectively (Fig.32B), representing a 5%, 13% (significant), and 10% (significant) reduction in wood decay. As shown before, fungi strain Heterobasidion annosum had a slower wood decay activity compared to Phlebia tremellosa, degraded only 12.6% mass in the control wood. Strikingly, decay by this fungal strain in wood from the three Cu enriching transgenic trees were much reduced, only 7.6%, 6.9%, and 7.9%, respectively (Fig.32D), leading to a 40%, 45%, and 37% decay reductions in the three transgenic trees, respectively. These results confirmed that this second fungi strain is more sensitive to the Cu inhibitory effects.

[0222] Overall, these results showed that 1) naturally accumulated Cu in the wood tissues by trees was much more efficient in inhibiting fungal growth and reducing wood decay compared with supplementing Cu to the wood via conventional approaches such as pressure treatment; and 2) usingbio-engineering approaches to enriching Cu in trees to reduce fungi caused wood decay is a viable approach. Prophetic Example 15 – Copper Toxicity Tolerance / Accumulation Screening for Phytoremediation

[0223] T0 transgenic events harboring DNA constructs can be produced via Agrobacterium- mediated transformation as described. Rooted events under kanamycin selection can be transferred from Magenta boxes to soil, acclimated, and grown in a controlled environment as described in Examples 7 and 8. The apical meristems of 4-5 weeks old T0 plants can be removed to induce branch formation and subsequent cutting production. Tissue samples can be collected after branch formation is induced.

[0224] Three or more cuttings from each soil grown T0 event can be produced and incubated hydroponically for root generation. Once roots are initiated, the cuttings can be transferred to buffered solutions containing high levels of Cu that are toxic to non-transgenic wild type trees. After 14 days incubation at 25oC and 75% relative humidity, the root length can be measured and compared with that of day 0 root length.

[0225] Transgenic events showing copper tolerance can be further tested for the level of tolerance at increased copper concentration at the following concentrations: 0, 20, 40, 80, 160, and 320 μM.

[0226] Selected Cu toxicity tolerant events can be grown in high Cu / metal soils (such as mine sites) to clean up the environments by phytoremediation. Prophetic Example 16 – Demonstration of Wood Pressure Treatment Avoidance

[0227] Using the wood decay assay described in Examples 1 & 2, a comparison in parallel of the success of industry standard pressure treatment versus transgenic Cu enriching in reducing fungi caused wood decay is performed. Results from both 3-month and one-year assays show that increasing Cu accumulation in wood is comparable, or in higher tissue Cu levels, more effective in inhibiting fungal growth and wood decay, pointing to the direction of eliminating the industry practice of lumber pressure treatment, which will save cost, cut carbon footprint, and make the lumber production more environmentally friendly.Prophetic Example 17 – Demonstration of increasing Cu accumulation in another plant (Arabidopsis)

[0228] Several constructs from FIGs 7 and 8 are used for transformation of Arabidopsis thaliana via flower dip transformation as described in Zhang et al., “Agrobacterium-Mediated Transformation of Arabidopsis thaliana Using the Floral Dip Method,” Nature Protocol 1, 641-646 (2006), which is hereby incorporated by reference in its entirety. T1 seeds are collected, and transgenic events are identified during germination on media containing appropriate antibiotic selection agent such as hygromycin. Transgenic plants are transplanted in soil pots and evaluation of Cu content in various tissue types are carried out. Compared to wild type (WT) Arabidopsis, plants transformed with constructs optimized for root Cu accumulation show relatively more Cu increase in roots compared to stem and leaves. Plants transformed with constructs optimized for stem Cu accumulation show higher Cu content in stem tissues than WT control. Transgenic plants can also show altered pattern of tissue Cu accumulation. Prophetic Example 18 – Elevated Copper Accumulation in Transgenic Poplar Plants Provides Fungal Disease Resistance in Living Trees

[0229] Selected transgenic poplar events with various degrees of increase in copper accumulation in roots or stems or both were vegetatively propagated such that each transgenic event has multiple ramets or sister trees. These trees, together with non-transgenic controls, are transplanted into soil pots in a controlled growth room dedicated to testing fungal disease resistance. After growth for a period of time, fungal strains including Phlebia tremellosa or Heterobasidion annosum are inoculated onto each of the testing trees including transgenic events and non-transgenic controls. Trees are kept in the testing growth room for eight weeks or longer when the control trees inoculated with fungi show disease symptoms. Trees of transgenic events show various degrees of fungal disease resistance. Trees with the highest amount of copper content show fungal disease resistance with no symptom of fungal infection, similar to that of trees not inoculated with fungal strains, transgenic or non-transgenic. Furthermore, various degrees of fungal disease symptoms can be found in non-transgenic control trees or transgenic trees with slightly increased copper content that are inoculated with fungal strains, with the worst fungal disease symptom found in inoculated non-transgenic control trees.Example 19: Exemplary Sequences for Copper Accumulation in Plants

[0230] Various sequences of the disclosure are provided in Table 1 below. To facilitate synthesis and cloning of the DNA sequences, the coding sequences of OsHMA5 and OsHMA9 were codon optimized against the Populus alba genome using its codon table. In many species, the genomes show preference to use certain codons to code amino acids, thus a codon table can be compiled from each of these species, and the nucleotide sequence adjusted accordingly. A polynucleotide sequence encoding any of the protein sequences provided in Table 1 below may be codon optimized for expression in any plant.

[0231] In cloning the OsYSL16 coding sequence, a single nucleic acid change was made in a wobble base to remove an internal enzyme cutting site, this G189A modification is consistent with optimal codon usage in Populous trichocarpa and avoids repetitive codon usage in the local region.

[0232] The Larrea tridentata Copper Transporter LtCOPT1 (SEQ ID NOs:1-2) was identified by genome sequencing and transcriptome profiling. Additional COPT transporters were identified from multiple Populus species, as well as Pinus, Pichea, Quercus, Panicum, Cannabis, Arabidopsis, Oryza, Zea, Triticum, Sorghum, Gossypium, Vitis, and Glycine species through sequence identity to the LtCOPT1 amino acid sequence (SEQ ID NO:2). The percent sequence identity to LtCOPT1 (SEQ ID NO:2) for each of SEQ ID NOs:3-30 is provided in Table 1 below and ranged from 33 to 51% amino acid sequence identity with LtCOPT1. A Copper Transporter 1 protein can be recognized by the hallmark Cu binding motif “MXXXM”, which is conserved across all plant species listed in Table 1 (see alignment in FIG.16). ATX1 homologs can be recognized by the central feature of Cu binding and coordinating motif “MXCXXC”, which is conserved (see alignment in FIG.17). In the HMA5 homologs, highly conserved domains include not only the two key features of this type of p-type ATPases, the Cu binding site “GMTCXXC” (SEQ ID NO:115; FIG.18) and the p-type ATPase phosphorylation domain “DKTGT” (SEQ ID NO:138; FIG.19), but also some transmembrane (“TM”) domains such as TM6, 7, and 8, and regions between these TMs. Similarly, in the HMA9 homologs, beside the Cu binding site “GMTCXXC” (SEQ ID NO:115; FIG. 20) and the p-type ATPase phosphorylation domain “DKTGT” (SEQ ID NO:138; FIG.21), a few TM regions, such as TM 2, 3, 5, 6, 7, and regions in between are highly conserved. Again, in the HMA4 homologs, the Cu binding motif “GMXCXXC” (SEQ ID NO:139; FIG.22) and the p-type ATPase phosphorylation domain “DKTGT” (SEQ ID NO:138; FIG.23) are conserved, the TM 3, 4, 5, 6, 7, 8 and regions in between are highly conserved. No specific functional motif has been characterized in the YSL16 homologs. However, it is known that transmembrane (TM) regions intransporters play key roles in regulating ion movement across membranes. The YSL16 homologs are predicted to have 14 TM domains. Amino acid sequences in these TM regions are very similar / identical, as exemplified by the TM13 (FIG.24). For the TcNas1 homologs, amino acid similarity in the Nicotianamine synthase (NAS) domain are relatively high, as exemplified by the part of the alignment shown in FIG.25. Table 1. Sequences for Copper Accumulation in Plants SEQ ID NO:SequenceLarrea >LtCopt1 CDS A T T G C G L M V F V F V FSEQ ID NO:6MEHGHDMPGMGGGMAPPPPMNMAGTMQHHEMMMMHMTFFWGKSVEILFSGWPGSSDKRPHMYFVALLFVFVLSILVEWLSHCQLMKPGSNHVAAGLVQTLLHALRVGLAYMVMLAIMSFNGGVFLAAV A A A A A A A A A A A A ) Y ASEQ ID NO:14MRMMMHMTFYWGMEVSILFDGWRTQTLMQYWGSLLVLFLASVFHEYVVSIRAHIRMKYNNIISRQENSYNSMESAEPQAKSMHLLPLSQRRTRCYVIKIAETLLFGVNALLGYLLMLAAMSYNGGVVL T D Q I A A V K A V Y M Y F S NOryza sativa > NP_001415070.1 (formerly XP_015630809.1 )(Oryza sativa, 41% COPT1 identities) F A L A V A V A Y M , V S A I V HOryza sativa >OsATX1 CDS Antioxidant ATGGCTGAGACTGTTGTGCTCAGGGTTGGGATGTCCTGTGAAGGTTGTGTTGGAGCTGTTAAGC C C T % T T T T TPopulus > Podel.10G243300.1.p (P.deltoidesWV94, 84% identities) deltoides MSQTVVLKVGMSCEGCVGAVKRVLGKMEGVESYDIDLKEQKVTVKGNVQPDAVLQTVSKTGKKT T T T % T T SSEQ ID NO:44 Populus tremula > PtXaAlbH.10G191600.2.p (P.tremula x P. alba HAP2, alba, 86% E E C V T T G T TCannabis sativa > XP_030489546.2(C. sativa, 74% identities) Antioxidant MAQTVVLKVGMSCQGCVGAVKRVLGKLEGVESYDIDIDQQKVTVKGNVPAETVFQTVSKTGKKT D T T C Q K K T TAntioxidant Protein 1 T T A G A T C A A T G A A G G A C G G T T A G T A C G AGAGTGCCCTTGCACAGATTGTAAGACTCGTAGAGTCAGCTCAAATGGCAAAAGCTCCTGTACAA AAGTTTGCTGATCAGATATCTAGGGTTTTTGTTCCCTTGGTCATCATTCTTTCTTTGCTTACTT T C C C G G T C G C G A C C A T G T G E K E V F A G S Q A L N V A VPopulus > Potri.001G105800.2.p (P.trichocarpa, 74% identities) trichocarpa MATKFLALACIRKESYGDLSPRPRYPSMPKYPKGVSAQETNVEGSEAKAVFCVLGMTCAACAGS R L K D L V N A A K Q A E V S R L K D L V N A A K Q A E V G C I ISEQ ID NO:66NVIESSGTSGRFKATIFPEGRGRESHRQEEIKQYYRSFLWSLVFTVPVFLISMIFMYIPGIKHALESKIVNMLSIGAILRWVLSTPVQFIIGRRFYTGSYKALRNGSPNMDVLIALGTNAAYFYSVYS G T F G A M V N V G C I I A S G T F G A M V N V S R L K D L V NENGVLHIKATRVGSESALSQIVRLVESAQMAKAPVQKFADRISKYFVPLVIILSISTWLAWFLA GKFHGYPDSWIPKSMDSFQLALQFGISVMVIACPCALGLATPTAVMVGTGVGASQGILIKGGQA K Q A E V S R L K D L V N A A K Q A E V S R L K D L V N A A K Q AEAKPEQKAEKVKELQAAGHVVAMVGDGINDSPALVAADVGMAIGAGTDIAIEAADIVLMKSNLE DVITAIDLSRKTFSRIRLNYIWALGYNLLGIPIAGGVLFPGTGFRLPPWIAGAAMAASSVSVVV G C I I A S G T F G A M V N V G C I I A S G T F G A M V N VStettler14 Heavy MATKFLALACIRKESTYGDLSPRPRYPSMPKYPKGVSVRETNVEGSEAKAVFSVMGMTCSACAG Metal ATPase 5 SVEKAVKRLPGIREAVVDVLNNKAQVLFYPSFVNEETIRETIEDAGFEATLIQEGTSDRSTQVC I I A S G T F G A M V N V S R L K D L V N A A K Q A E V G C I I ALDTKIVNMLSIGAILRWVLSTPVQFIIGRRFYTGSYKALRNGSPNMDVLIALGTNAAYFYSVYS VLRSATSPSFESADFFETSSMLISFILLGKYLEVLAKGKTSEAIAKLMDLAPGTAILLTLDDQG T F G A M V N V H K Q E W G A A S N A Q D Y G V A P M V T I S T LAKAVVEYAKSLREGSSEASRLPEAQDFVSITGHGVRARVHHKNILVGNVKLMLDSGITISEDAY DILKEVEGMARTGLLVSINEELVGIIAISDPVKPEAQDVVSILKLMKVKCIMVTGDNWGTANAI A A S G G R Y C G T Y G A I T D P G I A P M V T I S T L F I A AQuercus suber > XP_023892768.1 (Q.suber, 71% identities) Heavy Metal MAAKLIALACFRSEGYGDLSPRPHYPSMPSYPKGKGVSAQESCSVEQGSSPPEAKAATFSVFGM N E M I Y L D S G K L E I A E E D D F P I M I P Y S T I R H K I TGEDRSRIDLKIDGVLNERLIMILESSIQALPGVEDIKVDTELHKITISYKPDQTGPRDLIEVIE SATSGDVTASIYPEAEGREHHRYGEIKRYKQSFLWSLIFTIPVFLTSMVFMYIPGLKDGLDKKV A E G F S S I P T L G T E N W C T V F G E S S V F S I Y I I S A SMITGESRPVAKRKGDTVIGGTVNENGVLHVRATFVGSESALAQIVRLVESAQMAKAPVQKFADQ ISRVFVPLVILFSLLTWLAWFVAGRLHSYPNSWLPPSMDSFQLALQFGISVMVIACPCALGLAT D L V A T C T F R I S D I F I V A I V A A R F R I Y L D S G K L EVGIDSVIAEAKPEQKAEKVKELQAAGHVVAMVGDGINDSPALVAADVGMAIGAGTDIAIEAADI VLMKSNLEDVITAIDLSRKTFSRIRLNYVWALGYNLMGIPIAAGVLFPGTRFRLPPWIAGAAMA A A H K L G E E D A F M M L S G I A E L I K H K L R K E N LSorghum bicolor > Sobic.006G173800.1.p (Sorghum bicolor, 85%) Heavy Metal MLLRYAHQLDTEQTQCTCCFSRCSVASPCSLSSLSHSHTHNPPADAGLFATFYKCRRGSNQITR K G A T R V I P I A G Q K A W Q V N G L A E I V K E G N S Q L N TKVINMLSVGETLRWVLSTPVQFIIGRRFYTGSYKALRHGSANMDVLIALGTNAAYFYSVYSVLR AATSEDFKSTDFFETSSMLISFILLGKYLEVLAKGKTSDAIAKLMDLAPETAILLTLDKEGNII E G L F T G D C L C S G I K R A S C T G E S R L K H Y E G W G Y S T SNLEDVITAIHLSRKTFSRIRLNYIWALGYNILGIPIAAGALFPSTGFRLPPWIAGAAMAASSVS VVCCSLLLKNYERPKKLENLEIGGIQIE S R L K H Y E G W G Y S T S S G C T C A G T C G A T T C T T A T T AGTGCAATGATAATTACATTTGTGTTGTTTGGGAAGTATCTTGAGGTGCTTGCAAAAGGGAAAAC TTCTGATGCTATAAAGAAGCTTGTAGAATTGGTCCCTGCTACTGCTCTTTTGCTTCTGAAAGAC T T C G T T T G G A A G A A A A A A G C A G T A T A A K E V P Y D I LVWFLCGWVGAYPNSWISGTSNCFVFSLMFAIAVVVIACPCALGLATPTAVMVATGVGANHGVLV KGGDALERAQNVNYVIFDKTGTLTQGKAVVTTAKVFSGMDLGDFLTLVASAEASSEHPLAKAIV E T I R G S I F I S L A A N L R K D F G S I F I S L A A N L RKLMTESGIAIPDQVENFVVELEESAKTGVLVAFDDNIIGILGIADPLKREAAVVIEGLLKMGVK PVMVTGDNWRTARAVAKEVGIQDVRAEVMPAGKADVIQSFQKDGSIVAMVGDGINDSPALAAAD F G S I F I S L A A N L R K D F % G S I F I S L A A N L R K D FPopulus > PtStettler14.03G065900.1.p (P.trichocarpa Stettler14, 70% trichocarpa identities) G S I F I S L A A N L R K D F G S I F I S L A A N L R K D F K S EIINAIEDAGFDAELIQSGQQDKLSIMIAGMLTVEDAKFVEDMLHNLKGVREHVVDPLSAKYDIF FDPEVIGLRSIVDAIESGGDGRFKVTLQNPYTSYFSSRMDESSQMFRLFTSSLTFSVPIIFIRV T A E L A P K P V F M S I V I F T V W L V D G G I V L E L L L S NLHGALHIKATKVGGDAVLSQIISLVETAQMSKAPIQKFADFVASIFVPTVVALALLTLLGWYIG GALGAYPEQWLPEKGNHFVFALMFGISVVVIACPCALGLATPTAVMVATGVGANNGVLIKGGDA H P T I W A K E V P Y D V L L I E T I G G Q E V A Y G I V I V A DNWRTARAVAKEVGIEDVRAEVMPAGKADVIRSLQKDGSTVAMVGDGINDSPALAAADVGMAIGA GTDVAIEAADYVLMRNNLEDVITAIDLSRKTLTRIRLNYVFAMAYNVVSIPIAAGVFFPVLRVQ V L D I F L Y N C A H I R D P M V V D C A L S I H A S P V FTriticum >Traes_7DL_DF97DD324.2 (Triticum aestivum, 90% identities) aestivum Heavy MTCSACTGAVEAAVSARRGVRRVAVSLLQNRARVVFDPALVKVEDIIEAIEDAGFDAEILPDSA I F I T I V S N L R H D L E S A V L L A L G L F P T I W C G G R DSLLLLRCGPFLMGDWLKWALVSLVQFVIGKRFYIAAGRALRNGSANMDVLVALGTSASYFYSVC ALLYGAVTGFWSPTYFEASAMLITFVLLGKYLESLAKGKTSDAIKKLVELAPATALLLVKDKGG T Y G A V N G L V L S L I L A S V V E V M A A V L S L I L A S V VGASNGVLIKGGDALERAQKVQYVIFDKTGTLTQGKAKVTTVKVFSEMHRGEFLTLVASAEASSE HPLAKAIVEYARHFHFFDENSLTEDAEYSSKESPISAWLLDVAEFSAVPGRGIQCFIDGKRVLV M A A S G S D L Y E H S E H T A A V G T A G A T G A A G C TAGCTTGTATTCACCACCAAAGCAAAGAGAGGCAGAGCGCCATCATGAAATTAGGAATTACAGGA ATCAATTTCTTTGGAGTTGCCTGTTTTCTGTGCCTGTATTCATGTTCTCCATGGTTCTGCCAAT A T C T G T G G G T C T A C A T A C A C A C C T G C C G C C G G S L ASLYSPPKQREAERHHEIRNYRNQFLWSCLFSVPVFMFSMVLPMISPFGDWLFYKVCNNMTIGML LRWLLCSPVQFIIGWRFYVGAYHALKRGYSNMDVLVALGTNAAYFYSVYIVLKALTSESFEGQD Q V P F I T K R K L T K P L T S V P V P V L A L L T K P L T S VRATHVGSETALSQIVQLVEAAQLSRAPVQKLADRISKIFVPTVVIAAFITWLGWFIPGEAGLYP KHWIPKAMDRFELALQFGISVLVVACPCALGLATPTAVMVATGKGASQGVLIKGGNALQKAHKV P V L A L L T K P L T S V P V P V L A L L T K P L T S V P V P VAIDGGVAGAFAVTDPVKPEAECVISFLRSMGISSIMVTGDNWATASAIAKEVGIEKVFAETDPL GKADRIKDLQGKGMTVAMVGDGINDSPALVAADVGMAIGAGTDVAIEAADIVLIKSNLEDVVTA L L T K P L T S V P V P V L A L % L T K P L T S V P V P V L AIDLSRKTMSRIRLNYVWALGYNILGMPIAAGILYPFTGIRLPPWLAGACMAASSLSVVCSSLML QSYKKPLRVRDRAS D A H E W S A S R T D R S V F E D D T M Y L V W L V L E I A I ISEQ ID NO:126ERALQMIDGVKTAVVGLALEEAKVHFDPNLTDTDCIIEAIEDAGFGAELISSENDANKVHLTVEGVNSPEDLTIIRSSLESVVGVTHVEMNLEENKITVAYDPDLNGPRSLINCIKEAGNGSKSYNAS L F K G E D T V I K P S R V H W E D E M T S G D L H A S V K T F TQLLQRNDVIKIVPGTKVPVDGVVIKGQSHVNESMITGEARPIAKKPGDRVIGGTVNDNGCIIVK ATHVGSQTALSQIVQLVEAAQLARAPVQKLADKISKFFVPTVVVAAFLTWLGWFIPGQFHLYPK K L A G I Q N E K S M Q I H I V S G D S K S R V H W E D E M T S GALAVSDPLKPKAGQVISYLKSMGISSIMVTGDNWATAKSIAKEVGISQVFAEIDPVGKAEKIKD LQMQGLTVAMVGDGVNDSPALAAADVGMAIGAGTDVAIEAADIVLMKSNLEDVITAIDLSRKTL H S R V H W E D E M T S G D L H V I E S L L R G K D M I I K PTriticum > Traes_6AS_6F306F27E.1 (Triticum aestivum, 88% identities) aestivum Heavy MEQNGESHLKEPLLHAADGASAAAARVSPRKERTTRKVMFNVRGMSCGSWVVAGLKGVESIQVS M E K S M I S E T E V Q R V V V E T L F R G K D T V I K P S A A HGSYKASLYIPPRQRETEQHHEINNYRNQFLLSCLFSVPLFIFSMVLPMLPPFGDWLVYKIYNMF TVGLLLRWVLCTPVQFIVGRRFYKGSYHALRLKSANMDVLVAMGTNAAYFYSVYVAIKSLSSDT S V P V S T E D S S A A H F T S V P V S T E D S A A S K V L I V AISVLVVACPCALGLATPTAVMVASGMGASQGVLIKGGDALEKAHKVKIVVFDKTGTLTVGKPEV VSAVLFSEFSMEELCDMTIAVEASSEHPIAKAVAAHAKRLRQKFGSCTEEVPDVDDFEVHMGAG P M A G G A G T A A T T G G T A T G A T T G T C G ACTTTAAGACCGGCCATCTGACTCTTACAAGTCCTCGTTCAATGCTGGTCGGGCAGGTGGTTGGA ACCTTGATGGGTTGCGTCGTTGCACCCCTCACCTTCTTTCTATTCTATAAGGCATTCGATGTAG G T C C C A I G Y F M F Y G F V % T L G N K A L V L L T L GLike 16 SEQ ID TATAVLINGFHTPTGDKMARKQVHGFMKFFSLSFLWAFFQWFYSGGGEKCGFSQFPAFGLKAWK NO:144 NSFYFDFSMTYIGAGMICSHLVNLSLLLGAVLSWGLMWPLIGGLKGEWFPSTLSESSMKSLNGY V L P D G T L G N K A L V L L T L G N K A L V L L T L G NSFYFDFSMTYIGAGMICSHLVNLSLLLGAVLSWGLMWPLIGGLKGEWFPSTLPESSMKSLNGYK VFISISLILGDGLYNFLKILYFTARSMRARAKANKIKTEDKNQARDDLQRNEIFLREGIPLWVA L V L L T Q F Y F Y A L N P F D S G F S P A N M P F E S G FGAVISWGLMWPLIHNHKGDWYTAADTSMHGLQGYKVFVAIALILGDGLYNFVKIFYITVTTIYI QGKKKRSQRELPVVTGDGNEAAADAAIPYDEQRRNEMFMKDSIPFWVAGAGYVALAAISIGVIP A N M P G R P R I I S V D P L G K G K E L Q A A S W T Q N I SSVAIPYMFPHTMRWYYVLIAYVFAPVLAFCNAYGTGLTDQNLAYNYAKAALFVFAAWAGKDHGG VLVGLVACGVMKSILASASDLMHDFKTGYLTLSSPRSMFVSQVIGAAMGCIVAPLTFWLFYISF I A L Y G N K W F A R D V Y D T S F Y G F V V Y I P S V G V AFAVAANLARDLLPRRLARLVPLPMAMAVPFLVGASFAIDMCVGSLVVFVWHRLDSKKAGLLVPA VASGLICGDGIWTFPSSLLALAKIKPPICMKFTPGS V G K G K R Y A I S V G K G K R Y A I S N Y N M I C A T L WArabidopsis > AT5G53550.1 (Arabidopsis thaliana, 67% identities) thaliana Yellow MRSMMMEREGRNEIEREVIDDLEETQNEGDDFKSIPPWKEQITFRGIVASLIIGIIYSVIVMKL G T E L D G I A G M E T G G G A V K G M L V A H I M G W P C YStripe Like 16 TVIVMKIALTTGLVPTLNVSAALLSFLALRGWTRLLDRFGIVSRPFTRQENTIVQTCGVACYTI SEQ ID NO:163 AFAGGFGSTLLGLNKKTYELAGDSPGNVPGSWKEPGIGWMTGFLLACSFGGLLTLIPLRQVLVV P S Q N Q F V V F Y F M V G I A A I G K G T R Y V V S G G A FYFDFSMTYIGAGMICSHLVNLSLLLGAVLSWGVMWPLIGGLKGEWFPATLPESSMKSLNGYKVF ISIALILGDGLYNFLKILFLIARGIHTNVKVRSLKIFSHEQKQQQIDLQRNELFVRENIPIWVA L L F L N K L S L G L F P L S C V D W P G P C R C T A T T T ACAATCTTTCACAACTTTGACATCGACCCATCAGCGAACTCACTCGCTTCTCTCCTGGTTTCCTC TGATCCAGACATCTCTCAACGCATGTTCTTCCACACCGTTGACATAATGGATGTGACAGAGAGC A A G G C T K A S L F L G E F H L G E F H L G E F HPopulus tremula > PtXaTreH.05G008800.1.p (P.tremula x P. alba HAP1, tremula, 61% x Populus alba identities) L G E F H L G E F H L G E F T P S F Y L M R T G VNicotianamine MGCQDEQLVQTICDLYEKISKLESLKPSEDVNILFKQLVSTCIPPNPNIDVTKMCDRVQEIRLN Synthase 1 SEQ LIKICGLAEGHLENHFSSILTSYQDNPLHHLNIFPYYNNYLKLGKLEFDLLEQNLNGFVPKSVA S L S R V D F A L G E F S L G D F M A L G T R V D F V VNLSKLEYDLLVRYVPGLAPSRVAFVGSGPLPFSSLVLAARHLPNALFDNYDRCGAANDRARKLV RADEGLRKRMSFHTADVASLTDGLGKYDVVFLAALVGMAAEDKAKVVAHLGRHMADGAALVVRS I T G T C A T C T T T A A T A T C A A C A T A G T T G T A T C T G TATATACTCATCCATAGTGGCTTATAATTCTTCAACTCTCCACAGAAACTCCATCTCTCTCTCTT AGCCTCATTGTTTCAAGAAA T T G G T G C T T A T T A C A T T C C G T T C A G T G T T T C G C ANAC domain- >PtXaTreH.NAC12_Pro containing GCCTTTCTCGCCACTGATTTCTTAGTGGTTGCCATTTTGACTCTAGAGGAAGCGTTTTCCTTTT A A T A C G G G A T T T A A A C G A T T T A A T T A A A C T T A T T CTCTTAGACTCAAACCCACGACTTCTCAACTCACAGTTTGGTGCAGTACATCTCTCGCAGCATTA CACACACACACTCAAAGCCACAGTTGATATCAGCTTCCAACCACCTCCTCTTTAATACCCTCTC T T C G T T T G T G T A A G A A T A A T T T A A C T A A T A T T TSEQ ID NO:190TTTGGACCCCACCACCGTTCATGTCATGTCAGCCCGTTGTTATACAAGAAGGACGCTACTGTAATTTTAAGATTGTAACTGAGGCTGAATTTCTTTTTCAAAATTTGATTTTTTAGAAAAACTAATTA A T T T C G A C A A T T T G A T C G G A T T A T C T G A C G G T A A TAACTCGAGTGAATCAAGGTTAACTTGCCAAGCTCGCAATCTATGTCATATGACTGAGATAATCC AATATAAAAAAAATTAAAACAAATAATGGAACATAATTCTTAATAAATCTAATGTTGAATGATT A A A T T C A A A G T A A A A T A G T A A G T T C T A T T G A A A A GTGTCAGCATCTCGAGGTTAAGAGGTTTTAAGTTTCTGTGCTTGAGATCTGTGAACTGATTGGTG GATGTTTGTGTTTGTAGATTTGGAGATTGATTGATTTCTGCAAGGGAATATACTGGGTTAAAAA G A A T T G G G T T T C T T C A C A A

[0233] The experiments described herein relate to engineering trees for reduced wood decay caused by fungi infection, and thus enhance carbon retention in the territorial system to mitigate the climate change problem. Two proof-of-concepts (“POCs”) were tested: 1) increasing Cu alone inhibits fungal growth and reduces wood decay, and 2) Cu can be substantially increased in the tree stems. As shown herein, both POCs were demonstrated and wood from the engineered trees indeed became resistant to fungi that cause decay.

[0234] An improved high throughput lab wood decay assay platform was designed and validated. This platform was different from the protocols published by others, which incubated fungi infected wood blocks on nutrient-rich agar media and applied metal mesh pieces to keep replicate wood blocks in position (Maynard et al., “Diversity Begets Diversity in Competition for Space,” Nature Ecology & Evolution 1:0156 (2017); Lustenhouwer et al., “A Trait-based Understanding of Wood Decomposition by Fungi,” Proceedings of the National Academy ofSciences 117(21):11551-11558 (2020), each of which is hereby incorporated by reference in its entirety), the assay described herein removed the agar media, disused the metal mesh and replaced petri dishes with 24-well plates. These changes were made based on the concerns that 1) the presence of nutrient-rich media would decouple fungal growth from wood decay. When nutrients are easily available, fungi may not need to decompose wood to acquire nutrients, leading to the observation of weak correlation between fungal growth and actual wood decay; and 2) rusted meal mesh would contaminate the wood bocks over time and complicate the “metal treatment” experiments. Preliminary data validated these concerns (not shown).

[0235] In the assays described herein, except for the small amount from the inoculation fungi column, all nutrients to sustain fungal growth over the incubation time were acquired by the fungi from decomposing the wood, making fungal growth and wood decay tightly linked and correlated. Incubating individual inoculated wood block in each well of the 24-well plates eliminated the need of using metal mesh to position replicate wood blocks, thus preventing environmental contamination. Results from these assays were highly replicable and consistent.

[0236] Decay assays from metal treated wood showed clearly that higher Cu, but not Ni, alone, inhibited fungal growth and reduced decay of wood collected from hybrid Populus tremula × Populus alba, clone INRA 717-1B4, and loblolly pine. The same conclusion were drawn from results on wood collected from other tree species, including Populus alba, Populus fremontii, maple, and oak (data not shown). These results demonstrate that Cu can be applied as a universal agent to reduce fungi caused wood decay in the absence of other chemicals and substances.

[0237] Interestingly, the two fungi strains used in this study consistently showed different responses in decomposing wood and sensitivity to the Cu treatment, with Phlebia tremellosa being very efficient in digesting wood, as reported in previous studies (Blanchette and Reid, “Ultrastructural Aspects of Wood Delignification by Phlebia (Merulius) tremellosus,” Applied and Environmental Microbiology 52:239-245 (1986); and Lustenhouwer et al., “A Trait-based Understanding of Wood Decomposition by Fungi,” Proceedings of the National Academy of Sciences 117(21):11551-11558 (2020), each of which is hereby incorporated by reference in its entirety), while Heterobasidion annosum, the fungi that is long known to cause significant damages to the forest trees (Garbelotto and Gonthier, “Biology, Epidemiology, and Control of Heterobasidion Species Worldwide,” Annual Review of Phytopathology 51:39-59 (2013), which is hereby incorporated by reference in its entirety), being more sensitive to the Cu inhibitory effect. It is worth noting that though Cu is effective in reducing wood decay, enrichment of hundreds of times higheramount is required in wood treated by surface application. How the Cu treatments play out in wood decay in a natural condition is not known, as various factors other than fungi, biotical and abiotical, also play roles in wood decomposition (Brischke and Alfredsen, “Wood-water Relationships and Their Role for Wood Susceptibility to Fungal Decay,” Applied Microbiology and Biotechnology 104:3781-3795 (2020); Seddon et al., “Understanding the Value and Limits of Nature-based Solutions to Climate Change and Other Global Challenges,” Phil. Trans. R. Soc. B 375:20190120 (2020); Fukasawa, Y., “Ecological Impacts of Fungal Wood Decay Types: A Review of Current Knowledge and Future Research Directions,” Ecological Research 36:910-931 (2021), each of which is hereby incorporated by reference in its entirety).

[0238] Though Cu is an essential nutrient in all known living organisms, tight and complex mechanisms evolved to regulate the homeostasis, as over supply causes toxicity (Palmer and Guerinot, “Facing the Challenges of Cu, Fe and Zn Homeostasis in Plants,” Nature Chemical Biology 5:333-340 (2009); Marschner, P., “Marschner’s Mineral Nutrition of Higher Plants,” 3rd edn. Academic Press, Elsevier Ltd. (2012) doi.org / 10.1016 / C2009-0-63043-9, each of which is hereby incorporated by reference in its entirety). A holistic design involving multiple Cu transport and storage steps was considered to increase Cu accumulation in trees significantly while avoiding toxicity issues. With combined approaches, a Cu transporter LtCOPT1 from Larrea tridentata was identified and characterized. Assays on yeast and Arabidopsis showed LtCOPT1 had the activity to take up Cu in the roots. Based on previous studies, three more genes were chosen to engineer to enhance Cu accumulation the stem wood. These included OsHMA5 for root xylem unloading (Andrés-Colás et al., “The Arabidopsis Heavy Metal P-type ATPase HMA5 Interacts with Metallochaperones and Functions in Copper Detoxification of Roots,” The Plant Journal 45:225-236 (2006); Kobayashi et al., “Amino Acid Polymorphism in Strictly Conserved Domains of a P-type ATPase HMA5 are Involved in the Mechanism of Copper Tolerance Variation in Arabidopsis,” Plant Physiology 148:969-980 (2008); Deng et al., “A Member of the Heavy Metal P-type ATPase OsHMA5 is Involved in Xylem Loading of Copper in Rice,” Plant Physiology 163:1353-1362 (2013), each of which is hereby incorporated by reference in its entirety), TcNAS1 for xylem long- distance translocation and cellular detoxication and storage (Pich and Scholz, “Translocation of Copper and Other Micronutrients in Tomato Plants (Lycopersicon esculentum Mill.): Nicotianamine- stimulated Copper Transport in the Xylem,” Journal of Experimental Botany 47:41-47 (1996); Pianelli et al., “Nicotianamine Over-Accumulation Confers Resistance to Nickel in Arabidopsis thaliana,” Transgenic Research 14:739-748 (2005), each of which is hereby incorporated byreference in its entirety), and OsHMA4 for stem storage (Huang et al., “A Heavy Metal P-type ATPase OsHMA4 Prevents Copper Accumulation in Rice Grain,” Nature Communications 7:12138 (2016), which is hereby incorporated by reference in its entirety).

[0239] In addition, several promoter sequences from poplar trees were identified and cloned to drive the expression of these four genes either in roots or in stems. Transgenic trees with either one, two, three, or four transgenes all showed increased Cu tissue contents. However, the four-gene transgenic trees showed the most significant Cu increase in the stems. In one event, tree accumulated about nine times higher Cu than the non-transgenic controls. Comparing the three-gene versus four-gene transgenic trees, the results pointed to the stem woody tissues expressed OsHMA4 as helpful in enriching Cu in the stem wood. Trees used in this study were tested at a young age, after growing about 12 weeks in the greenhouse. It will be interesting to see how the Cu accumulation pattern continues over time with long-term field experiments.

[0240] Besides these four genes, constructs with two additional genes have been made. These include OsATX1 that facilitate Cu transfer to the OsHMA5 pump in the root (Shin et al., “Copper Chaperone Antioxidant Protein1 is Essential for Copper Homeostasis,” Plant Physiology 159:1099-1110 (2012); Zhang et al., “OsATX1 Interacts with Heavy Metal P1B-type ATPase and Affects Copper Transport and Distribution,” Plant Physiology 178:329-344 (2018), each of which is hereby incorporated by reference in its entirety), and OsYsl16 to recycle Cu from mature leaves (Zheng et al., “YSL16 Is a Phloem-Localized Transporter of the Copper-Nicotianamine Complex That Is Responsible for Copper Distribution in Rice,” The Plant Cell 24: 3767-82 (2012); Sheng et al., “YSL3-mediated Copper Distribution is Required for Fertility, Seed Size and Protein Accumulation in Brachypodium,” Plant Physiology 186:655-676 (2021), each of which is hereby incorporated by reference in its entirety). It is likely that the six-gene transgenic trees will accumulate even higher Cu in the stem wood.

[0241] Even though the lab-based wood decay assays showed that hundreds of times higher Cu is necessary to significantly reduce wood decay when wood was soaked and treated with Cu, it was surprisingly shown that decay assays using wood from the engineered trees showed that when naturally accumulated by trees, six to nine times higher Cu already showed much reduced decay.

[0242] Cu is one of the most abundant elements in the earth’s crust. Though bioavailability of Cu is regulated by many factors, such as soil pH, organic matter, etc. (Marschner, P., “Marschner’s Mineral Nutrition of Higher Plants,” 3rd edn. Academic Press, Elsevier Ltd. (2012), which is hereby incorporated by reference in its entirety), Cu deficiency was rarely reported in forestsoils and tree nurseries. This suggest that there will be a sustained Cu supply for trees, even when trees acquire the capacity to accumulate much higher amount of Cu, as shown herein. To put these engineered trees in use in enhancing wood carbon retention, it is challenging to replacing trees from the natural forest. A more realistic perspective could be to grow these trees in the urban areas. In the US alone, urban trees draw down millions of tons of carbon annually, and the urban areas are predicted to keep expanding for the next few decades (Nowak and Greenfield, 2018). Biotechnologies such as enriching Cu in trees could play an important role by enhancing plant resistance to fungal disease, which cause significant losses to global food production and serious damages to the ecosystems (Fisher et al., “Emerging Fungal Threats to Animal, Plant and Ecosystem Health,” Nature 484:186-194 (2012); Savary et al., “The Global Burden of Pathogens and Pests on Major Food Crops,” Nature Ecology & Evolution 3:430-439 (2019); Fones et al., “Threats to Global Food Security from Emerging Fungal and Oomycete Crop Pathogens,” Nature Food 1:332-342 (2020), each of which is hereby incorporated by reference in its entirety). There are other potential uses of the engineered Cu enriching trees, including: 1) to reduce or even eliminate the use of preservation practices such as pressure treatment in the timber industry, which are costly, environmentally harmful, and increase carbon footprint (Coggins, “Trends in Timber Preservation- A Global Perspective,” Journal of Tropical Forest Science 20:262-272 (2008), which is hereby incorporated by reference in its entirety); and 2) phytoremediation of contaminated soils and groundwater from the copper mine lands (Vangronsveld et al., “Phytoremediation of Contaminated Soils and Groundwater: Lessons From the Field,” Environmental Science and Pollution Research 16:765-794 (2009), which is hereby incorporated by reference in its entirety); 3) Phytomining (Brooks et al., “Phytomining,” Trends in Plant Science 3:359-362 (1998), which is hereby incorporated by reference in its entirety). As shown herein, bioengineered trees provide valuable options to mitigate or solve challenging environmental and societal issues.

[0243] Although certain embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. A transgenic plant cell comprising: a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein, wherein the transgenic plant cell has increased copper resistance and / or accumulation compared to a non-transgenic plant cell of a same species.

2. The transgenic plant cell of claim 1, wherein the plant cell comprises the first expression cassette.

3. The transgenic plant cell of claim 1 or claim 2, wherein the COPT1 protein comprises an amino acid sequence of any one of SEQ ID NOs:2-30 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:2-30.

4. The transgenic plant cell of any one of claims 1-3, wherein the COPT1 protein comprises Larrea tridentata COPT1 comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

2.

5. The transgenic plant cell of claim 1, wherein the plant cell comprises the second expression cassette.

6. The transgenic plant cell of claim 1 or claim 5, wherein the NAS1 protein comprises an amino acid sequence of any one of SEQ ID NOs:170-184 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:170- 184.

7. The transgenic plant cell of any one of claims 1, 5, or 6, wherein the NAS1 protein comprises Thlaspi caerulescens NAS1 comprising an amino acid sequence of SEQ IDNO:170 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

170.

8. The transgenic plant cell of any one of claims 1-7, wherein the plant cell comprises both the first expression cassette and the second expression cassette.

9. The transgenic plant cell of any one of claims 1-8, wherein the plant cell further comprises a third expression cassette comprising a third promoter sequence operably linked to a third polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein.

10. The transgenic plant cell of claim 9, wherein the HMA5 protein comprises an amino acid sequence of any one of SEQ ID NOs:63-93 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:63-93.

11. The transgenic plant cell of claim 9 or claim 10, wherein the HMA5 protein comprises Oryza sativa HMA5 comprising an amino acid sequence of SEQ ID NO:63 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

63.

12. The transgenic plant cell of any one of claims 1-11, wherein the plant cell further comprises a fourth expression cassette comprising a fourth promoter sequence operably linked to a fourth polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein.

13. The transgenic plant cell of claim 12, wherein the HMA4 protein comprises an amino acid sequence of any one of SEQ ID NOs:117-137 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:117-137.

14. The transgenic plant cell of claim 12 or claim 13, wherein the HMA4 protein comprises Oryza sativa HMA4 comprising an amino acid sequence of SEQ ID NO:117 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

117.

15. The transgenic plant cell of any one of claims 1-14, wherein any one or more of the first promoter sequence, the second promoter sequence, the third promoter sequence, and the fourth promoter sequence comprises a root-specific or stem-specific promoter.

16. The transgenic plant cell of any one of claims 1-15, wherein any one or more of the first promoter sequence, the second promoter sequence, the third promoter sequence, and the fourth promoter sequence is selected from any one of SEQ ID NOs:185-193.

17. The transgenic plant cell of claim 15, wherein the first promoter sequence operably linked to the first polynucleotide sequence encoding the COPT1 protein is a root-specific promoter.

18. The transgenic plant cell of claim 15 or claim 17, wherein the root-specific promoter comprises a nucleotide sequence of any one of SEQ ID NOs:189-192.

19. The transgenic plant cell of any one of claims 1 and 5-16, wherein the second promoter sequence operably linked to the second polynucleotide sequence encoding the NAS1 protein is a stem-specific promoter.

20. The transgenic plant cell of claim 19, wherein the stem-specific promoter comprises a nucleotide sequence of any one of SEQ ID NOs: 185, 187, 188 or 193.

21. A transgenic plant comprising the transgenic plant cell of any one of claims 1-20.

22. The transgenic plant of claim 21, wherein the transgenic plant is selected from a tree, shrub, grass, rice, maize, wheat, sorghum, barley, oats, rye, abaca, amaranth, bamboo, big bluestem, broccoli, Brussels sprouts, buckwheat, cabbage, canola, cacao, cassava, cauliflower, coir, collard greens, cotton, elephant grass, flax, foxtail millet, giant reed, grape, hemp, hops, indigo, jute, kale, kenaf, kohlrabi, millet, miscanthus, mustard, Napier grass, pearl millet, prairie cordgrass, quinoa, ramie, reed canary grass, sisal, sorghum, spelt, sugar beet, sugar cane, switchgrass, teff, tobacco, triticale, and yerba mate.

23. The transgenic plant of claim 21 or claim 22, wherein the transgenic plant is a transgenic tree or shrub.

24. The transgenic plant of claim 23, wherein the transgenic plant is a transgenic tree selected from an alder, Amarelão, Angico-branco, apple, Araucaria, ash, aspen, baobab, balsa, banyan, basswood, beech, birch, boxelder, buckeye, butternut, Canafístula, catalpa, cedar, Cedro- rosa, chestnut, cherry, coffee, coconut palm, cypress, dogwood, Douglas fir, elder, elm, eucalyptus, fig, fir, gum, hackberry, hawthorn, hemlock, hickory, hornbeam, juniper, ironwood, Ipê-roxo-de- bola, Jatobá-da-mata, jacaranda, katsura, kauri, larch, linden, liquidambar, loblolly pine, locust, magnolia, mahogany, maple, mangrove, monkey puzzle, mulberry, oak, olive, osage orange, pagoda, palm, Paricá, Pau-de-jucá, Paineira, paulownia, pear, peach, persimmon, pine, plane, plum, poplar, redwood, rowan, rubber tree, sandalwood, sequoia, silverbell, Sitka spruce, spruce, sycamore, sweetgum, Tamboril, tamarack, teak, tulip tree, walnut, willow, willow oak, yew, and Zelkova.

25. The transgenic plant of any one of claims 21-24, wherein the transgenic plant comprises a property selected from the group consisting of: increased copper accumulation, increase copper resistance, decreased fungal decomposition, and increased biomass in the presence of copper compared to a non-transgenic plant of a same species.

26. The transgenic plant of claim 25, wherein the transgenic plant is a poplar tree, and wherein said poplar tree has a copper concentration in the stem of at least 5 ppm.

27. A method for increasing copper resistance, increasing copper accumulation, increasing fungal disease resistance, and / or reducing fungal decomposition in a plant or plant cell, said method comprising: transforming a plant or plant cell with a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or transforming the plant or plant cell with a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein; wherein the plant or plant cell transformed with the first expression cassette sequence and / or the second expression cassette sequence comprises increased copper resistance and / or accumulation, increased fungal disease resistance, and / or reduced fungal decomposition compared to a non-transgenic plant or plant cell of a same species.

28. The method of claim 27, wherein the transgenic plant or plant cell comprises the first expression cassette.

29. The method of claim 27 or claim 28, wherein the COPT1 protein comprises an amino acid sequence of any one of SEQ ID NOs:2-30 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:2-30.

30. The method of any one of claims 27-29, wherein the COPT1 protein comprises Larrea tridentata COPT1 comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

2.

31. The method of claim 27, wherein the transgenic plant or plant cell comprises the second expression cassette.

32. The method of claim 27 or claim 31, wherein the NAS1 protein comprises an amino acid sequence of any one of SEQ ID NOs:170-184 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:170-184.

33. The method of any one of claims 27 and claim 31-32, wherein the NAS1 protein comprises Thlaspi caerulescens NAS1 comprising an amino acid sequence of SEQ ID NO:170 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

170.

34. The method of any one of claims 27-33 further comprising: transforming the plant or plant cell with one or more additional expression cassettes, wherein each of the one or more additional expression cassettes comprises an additional one or more promoter sequence(s) each operably linked to an additional one or more polynucleotide sequence(s) encoding an HMA5 protein and / or an HMA4 protein.

35. The method of any one of claims 27-34, wherein the additional one or more promoter sequence comprises a root-specific or stem-specific promoter.

36. The method of any one of claims 27-35, wherein the additional one or more promoter sequence is selected from any one or more of SEQ ID NOs:185-193.

37. The method of any one of claims 27-30 and claims 34-36, wherein the first promoter sequence is a root-specific promoter.

38. The method of claim 37, wherein the root-specific promoter comprises a nucleotide sequence of any one of SEQ ID NOs: 185, 187, 188 or 193.

39. The method of any one of claims 27 and 31-36, wherein the second promoter sequence is a stem-specific promoter.

40. The method of claim 39, wherein the stem-specific promoter comprises a nucleotide sequence of any one of SEQ ID NOs:189-192.

41. The method of any one of claims 27-40, wherein the transgenic plant or plant cell is selected from a tree, shrub, grass, rice, maize, wheat, sorghum, barley, oats, rye, abaca, amaranth, bamboo, big bluestem, broccoli, Brussels sprouts, buckwheat, cabbage, canola, cacao, cassava, cauliflower, coir, collard greens, cotton, elephant grass, flax, foxtail millet, giant reed, grape, hemp, hops, indigo, jute, kale, kenaf, kohlrabi, millet, miscanthus, mustard, Napier grass, pearl millet, prairie cordgrass, quinoa, ramie, reed canary grass, sisal, sorghum, spelt, sugar beet, sugar cane, switchgrass, teff, tobacco, triticale, and yerba mate.

42. The method of any one of claims 27-41, wherein said transforming is carried out by any one or more of particle bombardment, electroporation, and Agrobacterium tumefaciens infection.

43. The method of any one of claims 27-42, wherein the method further comprises: culturing the plant cell transformed with the first expression cassette and / or the second expression cassette under conditions effective to obtain a transgenic plant.

44. The method of claim 43, wherein the transgenic plant is selected from atree, shrub, grass, rice, maize, wheat, sorghum, barley, oats, rye, abaca, amaranth, bamboo, big bluestem, broccoli, Brussels sprouts, buckwheat, cabbage, canola, cacao, cassava, cauliflower, coir, collard greens, cotton, elephant grass, flax, foxtail millet, giant reed, grape, hemp, hops, indigo, jute, kale, kenaf, kohlrabi, millet, miscanthus, mustard, Napier grass, pearl millet, prairie cordgrass, quinoa, ramie, reed canary grass, sisal, sorghum, spelt, sugar beet, sugar cane, switchgrass, teff, tobacco, triticale, and yerba mate.

45. The method of claim 43 or claim 44, wherein the transgenic plant is a transgenic tree or shrub.

46. The method of claim 45, wherein the transgenic plant is a transgenic tree selected from alder, Amarelão, Angico-branco, apple, Araucaria, ash, aspen, baobab, balsa, banyan, basswood, beech, birch, boxelder, buckeye, butternut, Canafístula, catalpa, cedar, Cedro-rosa, chestnut, cherry, coffee, coconut palm, cypress, dogwood, Douglas fir, elder, elm, eucalyptus, fig, fir, gum, hackberry, hawthorn, hemlock, hickory, hornbeam, juniper, ironwood, Ipê-roxo-de-bola, Jatobá-da-mata, jacaranda, katsura, kauri, larch, linden, liquidambar, loblolly pine, locust, magnolia, mahogany, maple, mangrove, monkey puzzle, mulberry, oak, olive, osage orange, pagoda, palm, Paricá, Pau-de-jucá, Paineira, paulownia, pear, peach, persimmon, pine, plane, plum, poplar, redwood, rowan, rubber tree, sandalwood, sequoia, silverbell, Sitka spruce, spruce, sycamore, sweetgum, Tamboril, tamarack, teak, tulip tree, walnut, willow, willow oak, yew, and Zelkova.

47. The method of any one of claims 43-46, wherein the transgenic plant comprises a property selected from the group consisting of: increased copper accumulation, increased copper resistance, decreased fungal decomposition, and increased biomass in the presence of copper compared to a non-transgenic plant of a same species.

48. The method of any one of claims 43-47, wherein the transgenic plant comprises increased biomass in the presence of copper compared to a non-transgenic plant of a same species.

49. The method of any one of claims 43-47, wherein the transgenic plant comprises increased copper accumulation in the roots and / or stem tissue compared to a non- transgenic plant of a same species.

50. The method of any one of claims 42-47, wherein the method is effective for slowing or preventing fungal decomposition and / or carbon dioxide release into the air in the transgenic plant compared to a non-transgenic plant of a same species.

51. The method of claim 50, wherein the fungal decomposition is by any one or more of a soft rot fungus, a white rot fungus, or a brown rot fungus.

52. A transgenic plant cell culture produced by the method of any one of claims 42-51.

53. A DNA construct comprising: a first expression cassette comprising a first promoter sequence operably linked to a first polynucleotide sequence encoding a Copper Transporter 1 (COPT1) protein; and / or a second expression cassette comprising a second promoter sequence operably linked to a second polynucleotide sequence encoding a Nicotianamine Synthase 1 (NAS1) protein.

54. The DNA construct of claim 53, wherein the DNA construct comprises the first expression cassette.

55. The DNA construct of claim 53 or claim 54, wherein the COPT1 protein comprises an amino acid sequence of any one of SEQ ID NOs:2-30 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:2-30.

56. The DNA construct of any one of claims 53-55, wherein the COPT1 protein comprises Larrea tridentata COPT1 comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

2.

57. The DNA construct of claim 53, wherein the DNA construct comprises the second expression cassette.

58. The DNA construct of claim 53 or claim 57, wherein the NAS1 protein comprises an amino acid sequence of any one of SEQ ID NOs:170-184 or an amino acid sequencethat has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:170- 184.

59. The DNA construct of any one of claims 53, 57, or 58, wherein the NAS1 protein comprises Thlaspi caerulescens NAS1 comprising an amino acid sequence of SEQ ID NO:170 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

170.

60. The DNA construct of any one of claims 53-59, wherein the DNA construct comprises both the first expression cassette and the second expression cassette.

61. The DNA construct of any one of claims 53-60, wherein the DNA construct further comprises a third expression cassette comprising a third promoter sequence operably linked to a third polynucleotide sequence encoding a Heavy Metal ATPase 5 (HMA5) protein.

62. The DNA construct of claim 61, wherein the HMA5 protein comprises an amino acid sequence of any one of SEQ ID NOs:63-93 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs:63-93.

63. The DNA construct of claim 61 or claim 62, wherein the HMA5 protein comprises Oryza sativa HMA5 comprising an amino acid sequence of SEQ ID NO:63 or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

63.

64. The DNA construct of any one of claims 53-63, wherein the DNA construct further comprises a fourth expression cassette comprising a fourth promoter sequence operably linked to a fifth polynucleotide sequence encoding a Heavy Metal ATPase 4 (HMA4) protein.

65. The DNA construct of claim 64, wherein the HMA4 protein comprises an amino acid sequence of any one of SEQ ID NOs:117-137 or an amino acid sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 117-137.

66. The DNA construct of claim 64 or claim 65, wherein the HMA4 protein comprises Oryza sativa HMA4 comprising an amino acid sequence of SEQ ID NO:117 or an aminoacid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

117.

67. The DNA construct of any one of claims 53-66, wherein any one or more of the first promoter sequence, the second promoter sequence, the third promoter sequence, and the fourth promoter sequence comprises a root-specific or stem-specific promoter.

68. The DNA construct of any one of claims 53-67, wherein any one or more of the first promoter sequence, the second promoter sequence, the third promoter sequence, and the fourth promoter sequence is selected from any one of SEQ ID NOs:185-193.

69. The DNA construct of any one of claims 53-68, wherein the first promoter sequence comprises a root-specific promoter.

70. The DNA construct of claim 69, wherein the root-specific promoter comprises a nucleotide sequence of any one of SEQ ID NOs:189-192.

71. The DNA construct of any one of claims 53-70, wherein the second promoter sequence comprises a stem-specific promoter.

72. The DNA construct of claim 71, wherein the stem-specific promoter comprises a nucleotide sequence of any one of SEQ ID NOs: 185, 187, 188 or 193.

73. A vector comprising the DNA construct according to any one of claims 53-72.

74. A host cell comprising the DNA construct according to claim any one of claims 53-72.

75. The host cell according to claim 74, wherein the host cell is a plant cell.

76. The host cell according to claim 75, wherein the host cell is a bacterium.

77. The host cell according to claim 76, wherein the bacterium is an Agrobacterium.

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