Carbon immobilization with transgenic plants expressing carbonic anhydrases

Abstract

The root specific expression of carbonic anhydrase enhances the environmental carbon immobilization ability of plants. Here, a plant is genetically engineered such that it can inherit a genetic material comprising a gene encoding a transportable carbonic anhydrase protein under the control of a promoter that is active in the root cell, thereby allowing carbonic anhydrase to be transported from the root of the plant into the extracellular environment. These genetically altered plants exhibit increased levels of carbonic anhydrase activity in the extracellular environment of the plant roots, thereby enhancing carbon immobilization in the extracellular environment such as soil.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to compositions and methods for enhancing atmospheric CO sequestration in soil using plants. The present invention provides genetically modified plants that express enzymes to promote CO sequestration, as well as enzyme sequences and their use in various applications. The present invention also relates to parts of such modified plants, such as plant cells, plant parts, plant organs, fruits, seeds, embryos, germplasm, and processed plant products. Background Art

[0002] Greenhouse gas emissions from human activities have already caused global surface temperatures to rise by approximately 1°C above pre-industrial levels (Masson-Delmotte, 2018). Carbon dioxide (CO2) emissions are the primary cause of this warming. To meet the Paris Agreement's goal of limiting temperature rise to well below 2°C (ideally below 1.5°C) and mitigating the worst impacts of climate change (Horowitz, 2016), concerted efforts are needed to reduce global CO2 emissions and develop technologies to increase atmospheric CO2 sequestration (Masson-Delmotte, 2018). Summary of the Invention

[0003] The inventors have unexpectedly discovered that the carbonic anhydrase gene expressed in root cells can cause CO2 (e.g., in the form of bicarbonate) to be sequestered in the extracellular environment of the root. Thus, they provide a solution for the sequestration of atmospheric CO2 relevant to industrial biotechnology. Prior to the present invention, there has not been an overexpression of the carbonic anhydrase gene in plant root cells, nor has the carbonic anhydrase protein been exported from plant root cells, nor has the carbonic anhydrase protein been overexpressed on the plant root cell surface or in the extracellular environment. Therefore, the method used herein is novel. In general, the present invention achieves enhanced atmospheric carbon sequestration without observable negative or harmful anatomical, physiological, biochemical or developmental effects on modified plants.

[0004] The present invention provides plants comprising, or having heritable genetic material comprising, a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells, such that the carbonic anhydrase is secreted / transported from the roots of the plant into the extracellular environment. Thus, the present invention provides a plant having modified genetic material such that the modification causes the modified plant to express carbonic anhydrase or a portion thereof in at least some of the root cells of the plant. Such expression in the root cells is in addition to any endogenous carbonic anhydrase expression in the plant, particularly in plant roots, where endogenous expression would not provide secretable / transportable carbonic anhydrase or any substantial carbonic anhydrase enzymatic activity in the extracellular environment of the plant roots.

[0005] The expressed carbonic anhydrase can result in increased levels of extracellular carbonic anhydrase enzyme activity in the roots, in other words, in the extracellular environment of the plant roots. The extracellular environment of the roots can be, for example, soil or any other location where the plant may germinate, grow or be cultivated, including aeroponic and / or hydroponic growth environments.

[0006] Also provided herein are plants in which the carbonic anhydrase is overexpressed in the extracellular environment of root cells compared to plants that do not comprise, or whose heritable genetic material does not comprise, a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0007] Additionally or alternatively, provided herein are plants wherein the heritable genetic material of the plant has been modified to comprise a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells. The genetic modification results in overexpression of carbonic anhydrase in the extracellular environment of the plant root compared to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells or whose heritable genetic material has not been altered.

[0008] In some embodiments, the gene encoding the secretable / transportable carbonic anhydrase protein further comprises a signal peptide sequence for directing the secretion / transport of the carbonic anhydrase into the extracellular environment of plant roots.

[0009] Also provided herein are polynucleotide sequences comprising a sequence encoding a polypeptide sequence under the control of a suitable promoter, wherein the polypeptide sequence comprises a carbonic anhydrase sequence and a signal peptide sequence, wherein the promoter is active in plant roots and can drive the expression of the carbonic anhydrase in root cells, wherein the signal peptide sequence can direct the carbonic anhydrase to be secreted / transported to the extracellular environment of the plant roots. The signal peptide sequence of any polynucleotide sequence disclosed herein can be naturally encoded or translationally fused to the carbonic anhydrase sequence to ensure that the carbonic anhydrase is secreted / transported to the extracellular environment of the plant roots. The signal peptide sequence for directing secretion / transport can be derived from any secretory protein that is transported to the extracellular environment by a protein secretion pathway. The signal peptide sequence can also be derived from extensin, and in some embodiments, it can be derived from carrot plants or from vacuolar sorting proteins, or from pea plants.

[0010] The carbonic anhydrase (including the polynucleotide or peptide encoding it itself) is transportable and in some embodiments can be secreted or secretable. Additionally, in some embodiments, the carbonic anhydrase can be attached to the exterior of the cell or the plasma membrane by a transmembrane domain, a transmembrane helix, a membrane anchor, a glycosylphosphatidylinositol (GPI) anchor, or any other biomolecule that allows the carbonic anhydrase to remain attached to the plasma membrane or cell wall after secretion from the cell.

[0011] The promoter, signal peptide sequence or carbonic anhydrase of polynucleotide disclosed herein or plant can be derived from the gene of the plant species identical with the plant expressing it or variety.Described promoter can be root-specific, and in some embodiments it can be root hair and / or root epidermis specific.The expression domain (expression domain) of promoter can also include other cells and tissues of plant, as long as expression domain includes root.

[0012] Also provided herein is a carbonic anhydrase further comprising a transmembrane domain or a membrane anchoring sequence. The carbonic anhydrase can be an α, β, γ, δ, ζ, η, θ or ι subtype carbonic anhydrase. In some embodiments, it is a monomer.

[0013] Provided herein are polypeptide sequences encoded by the polynucleotides disclosed herein. Also provided are plant cells, cell lines, or progeny thereof comprising the polynucleotide sequences or polypeptide sequences disclosed herein. The progeny may be progeny of the plant cell or progeny of the cell line.

[0014] In some embodiments, the plants disclosed herein, polynucleotides, polypeptides, cells, cell lines or offspring can be derived from plants, wherein the plants are crops. In some embodiments, the plants are row crops or cover crops. In some embodiments, the plants are selected from corn, soybean, pea, cotton, rape, camelina, potato, tomato, beet, cassava, sweet potato, alfalfa, wheat, barley, sorghum, oats, sorghum, millet, rye, teff, rice, or clover, cress, Brassica, vetch and prairie grasses. In addition, the polynucleotides disclosed herein, polypeptides, cells, cell lines or offspring can be derived from plants, wherein the plants are trees. In some embodiments, wherein the trees are poplar, spruce, pine, eucalyptus, oil palm or rubber tree.

[0015] Also provided herein are plants, polypeptides, polynucleotides, cells, cell lines or progeny disclosed herein, wherein more carbon is sequestered into the extracellular environment of the plant compared to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0016] Additionally provided are plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus tissue, cell cultures, pollen grains, or seeds derived from or obtained from a plant disclosed herein.

[0017] Provided herein are vectors comprising the polynucleotide sequences disclosed herein. The vectors are optionally plasmids. Plasmids according to the present invention may further comprise one or more additional elements selected from the group consisting of enhancers, plant selectable markers, multiple cloning sites, and recombination sites. The construction, modification, propagation, and generation of vectors or plasmids are well known to those of ordinary skill in the art.

[0018] Also provided are compositions for transforming plant cells comprising a polynucleotide, vector, or plasmid disclosed herein, optionally comprising microparticles coated with the polynucleotide or vector. The microparticles may be metal or synthetic. In some embodiments, the metal is tungsten or gold.

[0019] Also provided are bacteria comprising a polynucleotide, plasmid, or vector disclosed herein, optionally wherein the bacteria is E. coli. or Agrobacterium sp. In some embodiments, the Agrobacterium is A. tumefaciens.

[0020] Also provided herein are plants comprising a polynucleotide disclosed herein stably integrated into their genome; and in some embodiments, heritably integrated into their genome.

[0021] Provided herein are methods of producing a plant or seed comprising the steps of:

[0022] (a) providing a vector or plasmid disclosed herein;

[0023] (b) activating a promoter operably linked to the polypeptide-encoding DNA;

[0024] (c) Optionally, wherein the polypeptide is overexpressed in the extracellular environment of the cell compared to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0025] This article also provides a method for increasing the carbon sequestration capacity of plants in soil, the method comprising changing the heritable genetic material of the plant so that the carbonic anhydrase protein is transported from the root of the plant to the extracellular environment. Changing the heritable genetic material can include inserting at least one polynucleotide into the heritable genetic material of the plant cell. Changing the heritable genetic material can also include carrying out gene editing to the heritable genetic material of the plant cell.

[0026] The methods according to the present invention can employ classical and well-known gene modification techniques, including transformation methods, by which the polynucleotides of the present invention are incorporated into the plant genome. Any necessary root cell expression regulatory elements may be present. In some embodiments, such incorporation is stable and heritable, thereby allowing the modification to be introduced into a specific crop plant line; this is advantageous for the purposes of crop improvement or breeding programs. Gene editing methods can be used to incorporate or generate the polynucleotides of the present invention.

[0027] Although a variety of gene editing methods are known, including TALE nucleases (TALEN) or zinc finger nucleases (Zinc Finger), CRISPR systems can also be used. For example, a CRISPR-Cas system can be used, in which a guide RNA (gRNA) is selected to target the action of the CRISPR-Cas protein to the desired genomic site, resulting in homologous recombination (HR) events or non-homologous end joining repair, i.e., insertion-deletion of polynucleotides into the plant genome.

[0028] Thus, the polynucleotides disclosed herein may comprise a polynucleotide encoding a CRISPR-Cas protein, optionally further comprising a guide RNA (gRNA), wherein the gRNA directs the CRISPR-Cas protein to the locus of an endogenous carbonic anhydrase coding sequence in the genome of a plant cell, optionally wherein regulatory elements are inserted such that the carbonic anhydrase is expressed in at least some root cells of the plant.

[0029] Also provided herein are processed plant products obtained from a plant or plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus culture, pollen grain or seed disclosed herein; optionally, wherein the processed product comprises a detectable nucleic acid sequence of: (i) a transportable carbonic anhydrase downstream of a gene expression regulatory element that is active in at least some root cells of the plant, or (ii) a polynucleotide or at least a portion of a polynucleotide disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described with reference to examples and accompanying drawings.

[0031] Figure 1 Depicted is a schematic diagram of the genetic vector used for soybean transformation to express secreted soybean carbonic anhydrase in roots.

[0032] Figure 2Show that, compared with wild type (control plant), the carbonic anhydrase activity in the extracellular environment around the genetically engineered plant root is enhanced. A) Shows the carbonic anhydrase activity in the supernatant after the hairy root tissue of the control and engineered soybean releases the enzyme. Shows the standard error bar. B) Shows the carbonic anhydrase activity in the hydroponic solution grown by the control and genetically engineered plants. Shows the standard error bar. C) Shows that the genetically engineered plants have produced a measurable decrease in soil pH relative to the control pots and control plants with only soil. This proves that carbonic anhydrase has catalyzed the conversion of CO2 in the air into bicarbonate and protons in the soil. Shows the standard error bar. D) Shows that the engineered plant roots sequester CO2 from the atmosphere, while the control plant roots cannot do this. During the 30-minute measurement period, the pot containing soil and plant roots (with the above-ground tissue removed) showed CO2 sequestration. Shows the standard error bar. E) Shows the carbonic anhydrase activity in the extracellular environment of the wild type and transgenic plant roots. This shows that transgenic plants have higher carbonic anhydrase activity. F) shows no significant difference in growth phenotype between a control and a genetically engineered Arabidopsis plant that secretes carbonic anhydrase from its roots. Photos of a 5-week-old control plant (left) and an engineered plant of the present invention (right) were taken. Scale bar represents 5 cm.

[0033] Figure 3 A general schematic diagram of a DNA sequence fused to a carbonic anhydrase protein is depicted. Typical features of such constructs are the inclusion of a gene sequence encoding a protein with carbonic anhydrase activity, a promoter to ensure expression of the sequence in, for example, plant root tissue, a terminator, and a peptide sequence, such as an N-terminal signal peptide, that specifies cellular export.

[0034] Figure 4It was demonstrated that compared to the wild type (control plant), the carbonic anhydrase activity in the extracellular environment surrounding the roots of the genetically engineered plants was enhanced. A) shows that the carbonic anhydrase activity in the hydroponic solution grown by the genetically engineered soybean plants in which carbonic anhydrase is transported from the plant roots to the extracellular environment is higher than that of the control plants. Standard error bars are shown. B) shows that the genetically engineered soybean plants in which carbonic anhydrase is transported from the plant roots to the extracellular environment produced a measurable decrease in soil pH relative to the control plants. This proves that carbonic anhydrase has catalyzed the conversion of CO2 in the air into bicarbonate and protons in the soil. Standard error bars are shown. C) shows that the genetically engineered soybean plants in which carbonic anhydrase is embedded in the root surface produced a measurable decrease in soil pH relative to the control plants. This proves that carbonic anhydrase has catalyzed the conversion of CO2 in the air into bicarbonate and protons in the soil. Standard error bars are shown. D) shows that the carbonic anhydrase activity in the hydroponic solution grown by the genetically engineered soybean plants with the alternative carbonic anhydrase increases relative to the hydroponic solution grown by the control plants. Standard error bars are shown. E) shows that the carbonic anhydrase activity in the roots of the genetically engineered soybean plants increases relative to the control plants. Standard error bars are shown. F) shows no significant difference in growth phenotype between the control and genetically engineered soybean plants that secrete carbonic anhydrase from their roots. Photos of 5-week-old control plants (left) and engineered plants of the present invention (right) were taken. Scale bar represents 5 cm.

[0035] Figure 5 A+B: Confocal images of soybean protoplasts expressing fluorescent protein-tagged carbonic anhydrase. A) Conticribra weissflogii carbonic anhydrase fused to a fluorescent protein. B) Conticribra weissflogii carbonic anhydrase fused to a fluorescent protein with the PsBP80 signal peptide at the N-terminus and the PsBP80 transmembrane domain at the C-terminus. C+D: Light microscopic images of GUS-stained Arabidopsis thaliana roots. C) GUS staining of Arabidopsis roots expressing GUS under the control of the pGmPIN2b promoter. D) GUS staining of Arabidopsis roots expressing GUS under the control of the pGmEXPA7 promoter. Scale bars: A) 10 μm, B) 20 μm, C) 50 μm, D) 50 μm. The arrow in A) highlights cytoplasmic GFP expression, and the arrow in B) highlights fluorescent protein expression localized to the plasma membrane of soybean protoplasts. Arrows on the left and right panels of C) and the left panel of D) highlight GUS-stained root areas.

[0036] Figure 6It is demonstrated that carbonic anhydrase activity in the extracellular environment surrounding the roots of genetically engineered plants enhances inorganic carbon sequestration in the soil compared to wild type (control plants). A) Analysis of soil inorganic carbon content shows that soil grown in engineered soybean plants of the present invention accumulates more inorganic carbon than soil grown in control soybean plants and soil in which no plants are grown. B) Shows that after treatment with slaked lime Ca(OH)2, soil grown in engineered Arabidopsis plants of the present invention accumulates more soil inorganic carbon than soil grown in control Arabidopsis plants. C) Shows that when soil inorganic carbon content is normalized to soil organic carbon content, the results shown in B are consistent. D) Shows that soil grown in engineered soybean plants of the present invention accumulates more soil inorganic carbon than soil grown in control soybean plants. E) Shows that when soil inorganic carbon content is normalized to soil organic carbon content, the results shown in D are consistent. DETAILED DESCRIPTION

[0037] Weathering-mediated CO2 sequestration

[0038] One way CO2 is naturally sequestered from the atmosphere is through the chemical process of rock weathering. This occurs when atmospheric CO2 dissolves in rainwater to form carbonic acid (H2CO3). When the carbonic acid encounters carbonate and / or silicate rocks, a chemical reaction occurs that causes the rock to break down and release bicarbonate (HCO3 - ) and calcium (Ca 2+ ) ions. These ions are carried by surface waters to the oceans, where they precipitate and fall to the seafloor for long-term burial (i.e., hundreds of thousands of years). The ability of this process to promote long-term sequestration of atmospheric CO2 has led to proposals for methods to accelerate chemical rock weathering. These methods, known as enhanced weathering, involve mechanically crushing rocks into dust and spreading it over terrestrial ecosystems, thereby significantly increasing the surface area of ​​rock exposed to rainwater (containing dissolved CO2). This has been proposed to greatly increase the rate of rock weathering, and therefore the rate of CO2 sequestration from the atmosphere (Strefler et al., 2018).

[0039] Carbonic anhydrase

[0040] Carbonic anhydrase is a naturally occurring enzyme that catalyzes the conversion of gaseous carbon dioxide (CO2) and water (H2O) to bicarbonate (HCO3 - ) and hydrogen ions (H + ) Carbonic anhydrase is a widely distributed enzyme, with members of this enzyme family found in organisms across the tree of life, from bacteria and archaea to plants and animals. Given the activity of this enzyme, there has been interest in using carbonic anhydrase to capture CO2 from industrial flue gases in industrial settings (as discussed in Boone et al., 2013 and Steger et al., 2022). WO2015183935A2 also describes the catalytic removal of CO2 from industrial flue gases using modified carbonic anhydrases. Also disclosed is the use of modified carbonic anhydrases that are immobilized on a substrate and cross-linked with mineral ions, thereby precipitating bicarbonate and removing CO2. This process is used for desalination and in vitro removal of CO2. Similarly, WO2013159228A1 describes an industrial method in which CO2 is contacted with a carbonic anhydrase solution containing an additional absorption compound that jointly sequesters CO2.

[0041] Carbonic anhydrase (CA) is a metalloenzyme containing a metal cofactor (usually Zn 2+ , but in some cases Cd 2+ 、Co 2 + 、Fe 2+ and Mn 2+ , although a newly described class of carbonic anhydrases has been found to be active under metal-free conditions (Hirakawa et al., 2021). To date, eight classes of carbonic anhydrases have been identified (α, β, γ, δ, ζ, η, θ, and ι), with different distributions across the three domains of life. α, β, and γ carbonic anhydrases are found in eukaryotes and prokaryotes, while the other classes are found in some microalgae and protist species. A newly described ι-CA was recently identified in the microalga Thalassiosira pseudonana and the Gram-negative bacterium Burkholderia territorii. Depending on the class, carbonic anhydrases can be active as monomers, dimers, trimers, tetramers, or octamers. In addition to this formal diversity, carbonic anhydrases have a wide variety of functions across and within organisms. For example, in plants, carbonic anhydrases are present in several different plant tissues and have different subcellular localizations (DiMario et al., 2017). Regardless of their class or the organism in which they are found, all carbonic anhydrases catalyze the same biochemical reaction, namely the reaction of gaseous CO2 and water (H2O) with bicarbonate ions (HCO3 - ) and hydrogen ions (H + ) between each other.

[0042] Recently, Matt et al. (2022) described the expression of a glycosylphosphatidylinositol-anchored, membrane-bound extracellular carbonic anhydrase in the calcified primary mesenchymal cells of sea urchin larvae. The extracellular CO2 hydration demonstrated in these findings suggests that the generated bicarbonate is taken up by the cells for the construction of carbonate structures that form the skeletal tissue of this organism.

[0043] Plants offer an interesting opportunity to sequester CO₂ as bicarbonate in the soil using the enzyme carbonic anhydrase and to store this bicarbonate long-term via groundwater in a manner similar to chemical weathering. Plant roots are in direct contact with the soil and the gaseous CO₂ in the soil, which is itself in equilibrium with atmospheric CO₂.

[0044] Historically, plants have been genetically engineered to express and secrete enzymes. Li et al. (2009) described soybean plants transformed with a construct containing a gene encoding a phytase from the fungus Aspergillus ficuum fused at its N-terminus to the signal peptide sequence of the carrot extensin gene. This recombinant gene was then fused to the promoter of the Arabidopsis thaliana Pky10 gene, which enabled gene expression in the roots of the transgenic soybean plants.

[0045] Here, we propose to engineer plants to express an engineered form of carbonic anhydrase in their root cells (particularly the outer cell layer, i.e., epidermal cells), which is secreted from the cells into the extracellular environment. In particular, targeting plants (e.g., crop plant species) covering large areas of the globe would provide a means to sequester atmospheric CO2 in the soil as bicarbonate ions on a large scale. Subsequently, a portion of this bicarbonate is expected to be carried to the ocean and buried there by transport through surface waters.

[0046] Bioremediation refers to any process that uses biological systems (such as plants or bacteria) to remove environmental pollutants (such as CO2) from natural or industrial environments. Bioremediation methods, such as the method proposed in this invention, offer specific advantages. Bioremediation generally requires fewer resources and less energy than industrial, man-made techniques and can be tailored through genetic engineering to meet the specific needs of a particular environment. Furthermore, these biological methods do not produce hazardous waste as byproducts, eliminating the need for expensive, specialized waste disposal.

[0047] This area needs to find a method for accelerating the long-term sequestration of CO by utilizing bioremediation methods. In addition, it is necessary to realize this by a scalable method that can be implemented quickly and globally, in order to urgently solve the problem of CO2 release and atmospheric CO2 concentration increase caused by human activities. The present invention has demonstrated that expressing carbonic anhydrase in the root of a plant can cause the extracellular carbonic anhydrase enzyme activity level to increase, thereby enhancing CO2 sequestration by converting CO2 into bicarbonate, and thus, the present invention has solved this demand. The present invention has also demonstrated that a variety of different natural or engineered carbonic anhydrase genes from different species can provide this function in plants. The inventors have unexpectedly found that when carbonic anhydrase protein is secreted from a plant and / or when carbonic anhydrase protein is anchored to the outer edge of a cell by, for example, a transmembrane domain or an anchor, this function can be provided. Alternatively, the N-terminal or C-terminal can be fused with a transmembrane domain, a transmembrane helix or a glycosylphosphatidylinositol (GPI) anchor or other biomolecules that make carbonic anhydrase remain attached to plasma membrane or cell wall after secretion from the cell. The present invention also demonstrates that the various constitutive forms of the carbonic anhydrase proteins described above can be expressed in plants using a variety of promoters that achieve the desired expression in root tissue.

[0048] The following detailed description conveys exemplary embodiments of the present invention in sufficient detail to enable one of ordinary skill in the art to practice the invention. The features or limitations of the various embodiments described do not necessarily limit other embodiments of the invention or the invention as a whole. Therefore, the following detailed description does not limit the scope of the invention, which is defined solely by the claims.

[0049] Conventional techniques in botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology, and bioinformatics used to implement the present invention are well known and mastered by those of ordinary skill in the art. Specific techniques are described in detail in the literature.

[0050] The term "genetic engineering (modification)" is used in this specification. As is familiar to those skilled in the art, this term may encompass the terms "gene modification ()" and "gene editing", each of which is considered to have different meanings and basic technical methods in the art. However, the term "modified ()" herein can be more simply understood in a non-limiting sense to mean "changed ()", "changed ()" or "different ()". The terms "changed ()", "changed ()" and "modified ()" are used interchangeably herein. The terms "increase / improve", "improve" or "enhance" are used interchangeably herein.

[0051] The present invention provides plants whose heritable genetic material comprises a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, such that the carbonic anhydrase is transported from the roots of the plant to the extracellular environment.

[0052] The plant of the present invention can be a genetically modified plant, a genetically enhanced plant, a non-natural plant and / or a genetically engineered plant. In some cases, the plant can be a transgenic plant.

[0053] Plants according to the present invention can be monocots or dicots; in some embodiments, they are crop plants, such as fruits, vegetables, grains, oilseeds, and legumes, and are generally used for food, animal feed, biofuel, or biomass production. Crop plants are any plants grown on a commercial scale for human or animal consumption or use. Plants can also be trees, particularly those commonly used in forestry and land management, carbon capture, and biofuel or biomass production. Plants can be horticultural plants. In some embodiments, the plants are cover crops, i.e., plants used to cover bare soil between harvests. Cover crops are used to reduce soil erosion, improve soil fertility and quality, conserve water, control weeds / pests, and maintain biodiversity and wildlife in agricultural ecosystems. Cover crops can be used during the fallow period of cultivated land. During the fallow period, the cultivated land is not sown with harvestable seeds for at least one or more growing cycles. This allows the cultivated land to recover. Cover crops can be sown during the fallow period to help the land recover.

[0054] Inheritable genetic material refers to any polynucleotide sequence and any relevant genetic element that can be passed from one generation to the next generation by sexual or asexual reproduction in any cell or organelle.The plant sexual breeding method can comprise breeding.The asexual reproduction (also referred to as clonal propagation) carried out by grafted plant cuttings is also considered to be that heritable genetic material is incorporated in the plant genome.Therefore, heritable genetic material can only be present in some part of plant.

[0055] The term "gene" refers to a polynucleotide sequence encoding all or part of a polypeptide, comprising introns, exons, and an open reading frame (ORF) for transcription. The term encoding refers to the ability of a nucleotide sequence as a translation template, and the arrangement of nucleotides represents codons, which further represent amino acids. A gene can also include other genetic elements, such as promoters, repressors, terminators, enhancers, and / or transcription factor binding elements. Genes can be inserted into the genome of an organism using genetic engineering methods known in the art (e.g., transformation). A gene can be exogenous (i.e., non-natural, inserted into an existing genome) or endogenous (i.e., present in the genome as a natural gene).

[0056] The polypeptide of the present invention may be a fusion protein. A fusion protein refers to a polypeptide derived from a single nucleotide sequence comprising two or more genes or gene portions, wherein the genes encode separate polypeptides that are not transcribed and translated together in nature.

[0057] Typically, the polynucleotides of the present invention comprise a promoter, a start codon, a sequence encoding an N-terminal signal peptide, a sequence encoding a protein having carbonic anhydrase activity, optionally a sequence encoding a C-terminal peptide that targets the polypeptide to a further specific location (e.g., anchoring in the plasma membrane), a stop codon, and a terminator, e.g. Figure 3 As typically shown in .

[0058] The coding sequence of the carbonic anhydrase encoded in the DNA polynucleotide sequence of the present invention can correspond to any of the polynucleotide sequences in SEQ ID NO: 1 or SEQ ID NO: 2, or to a sequence having at least 65% identity to any of said sequences; or to a sequence having at least 70% identity to any of said sequences; or to a sequence having at least 80% identity to any of said sequences, or to a sequence having at least 85%, at least 90% or at least 95% identity to any of said sequences. In some embodiments, the carbonic anhydrase will have any of the percentages of homology mentioned herein and retain the enzymatic activity of the carbonic anhydrase.

[0059] The amino acid sequence of the carbonic anhydrase encoded in the polynucleotide sequence of the present invention can correspond to any of the amino acid sequences of SEQ ID NO: 8 or SEQ ID NO: 9, or a sequence having at least 65% identity to any of said sequences; or a sequence having at least 70% identity to any of said sequences; or a sequence having at least 80% identity to any of said sequences, or a sequence having at least 85%, at least 90% or at least 95% identity to any of said sequences. In some embodiments, the carbonic anhydrase will have any of the percent homology mentioned herein and retain the enzymatic activity of the carbonic anhydrase, preferably in the extracellular environment of the root.

[0060] The polynucleotide can correspond to a full-length carbonic anhydrase or a portion thereof. The terms "part," "variant," "homologue" or other representative expressions in this area relevant to a carbonic anhydrase sequence or its functional fragment herein mean any carbonic anhydrase orthologue with different polynucleotide or peptide sequences from any species, including plants.

[0061] In terms of percent identity relative to a reference sequence (such as SEQ ID NO: 1, 2, 8 or 9), variants of carbonic anhydrase can have (in order of increasing preference) at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% overall sequence identity relative to the reference sequence.

[0062] Carbonic anhydrase catalyzes the conversion of water (H2O) and carbon dioxide (CO2) to bicarbonate (HCO3 - ) and hydrogen ions (H + ) can be reversed by enzymes as follows:

[0063]

[0064] In the absence of carbonic anhydrase enzyme activity, the pH of the aqueous environment determines whether the forward or reverse reaction is more dominant. Carbonic anhydrase is widely distributed in all organisms. Carbonic anhydrase changes the rate at which this reaction occurs and the reaction between CO2 and HCO3 - Carbonic anhydrases from different species may have minimal sequence similarity but can adopt similar tertiary structures to catalyze the reaction in very different contexts.

[0065] In some embodiments, the carbonic anhydrase can be an α, β, γ, δ, ζ, η, θ, or ι isotype carbonic anhydrase, and in some embodiments, is a monomer. Three types of carbonic anhydrase occur naturally in plants: the α, β, and γ isotypes. The β isotype is more well-characterized, including those with efficient CO2 hydration; for example, the β isotype functions as part of C4 photosynthesis in C4 plants. The β isotype is a dimer, while the α isotype is a monomer. The advantage of the monomeric isotype is that it does not need to form multimers on the cell surface.

[0066] The carbonic anhydrase sequence used in the present invention can be a homologue, analogue, orthologue or paralogue of any subtype carbonic anhydrase separated from any species. Those of ordinary skill in the art will anticipate that any carbonic anhydrase will all work equivalently in the present invention, as long as it is suitable for being expressed by plant of the present invention (for example it is made up of the amino acid of naturally occurring protein form). The application's embodiments have proved that sequence identity or similarity between the carbonic anhydrase are not that the plant of the present invention shows its surprising sequestration CO2 ability necessary. As mentioned above, no matter how the sequence similarity of the carbonic anhydrase from different species, all can adopt similar tertiary structure. Therefore, the technical staff will understand that the plant of the present invention of overexpressing any type of carbonic anhydrase in the extracellular environment of root cell will all play the effect of sequestration CO2. The technical staff will understand that carbonic anhydrase is defined by its function. Therefore, any catalysis gaseous CO2 and water (H2O) to bicarbonate ion (HCO3 - ) and hydrogen ions (H + ) are contemplated as carbonic anhydrase proteins as part of the present invention.

[0067] In some embodiments, carbonic anhydrases with high catalytic efficiency in the direction of forming bicarbonate are selected so that the reverse reaction is not dominant. Carbonic anhydrases can be selected based on efficacy, such as determined by enzyme activity assays. In addition, codon usage can be changed to enhance the expression of specific plant species.

[0068] In some embodiments, in any aspect of the present invention defined herein, the promoter is a root cell specific promoter (i.e., a root cell specific promoter or a promoter expressed in root cells). A root cell active promoter is a promoter that currently causes a polynucleotide sequence to be expressed in root cells, i.e., it is sufficient to produce protein in root cells. The promoter may be active in other cells of the plant, and in some embodiments is relatively higher in root cells. Compared to a wild-type promoter, the activity of the promoter may be enhanced. The activity of the promoter may be conditionally enhanced. The promoter may be an inducible promoter. The activity of the promoter may vary, for example, seasonally, diurnally, or in response to external signals (i.e., temperature, pH, soil nutrient content). The promoter may be constitutively active.

[0069] The promoter can be a synthetic promoter containing different selected elements. For example, such a synthetic promoter can include a root cell specific transcription factor binding element upstream of the promoter element. Two or more transcription factor binding elements can be present, which can be the same or different. Multiple such transcription factor binding elements can be used to enhance the activity and / or specificity of the promoter in root cells. The promoter can be derived from non-plant organisms, such as the 35S promoter.

[0070] In other aspects, the root cell specific promoter can be derived from a gene that is preferentially or specifically expressed in plant root cells and is therefore a naturally occurring promoter. The gene can be expressed at an endogenous level in other plant cell types. Promoters can be selected based on their predicted function in a particular species. Many root cell specific promoters are well known to those skilled in the art. Root cell specific promoters can have the activity of a certain expression level in some or all other plant cells, but are highly expressed in the root.

[0071] The root-specific promoter encoded in the DNA polynucleotide sequence of the present invention can correspond to any of the polynucleotide sequences of SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence having at least 65% identity to any of said sequences; in some embodiments, a sequence having at least 70% identity to any of said sequences; and in some embodiments, a sequence having at least 80% identity to any of said sequences, or a sequence having at least 85%, at least 90%, or at least 95% identity to any of said sequences. In some embodiments, the encoded carbonic anhydrase will have any of the percent homology mentioned herein and retain the enzymatic activity of carbonic anhydrase.

[0072] The polynucleotide may correspond to a full-length root-specific promoter or a portion thereof. As noted above, one of ordinary skill in the art will anticipate that any promoter active in plant roots will function equivalently in the present invention.

[0073] "Part," "variant," "homolog," or other representative terms in the art used herein in connection with a root-specific promoter sequence or a functional fragment thereof refers to any root-specific promoter ortholog from any species (including plants) having a different polynucleotide or polypeptide sequence.

[0074] In terms of percent identity relative to a reference sequence (such as SEQ ID NO: 6 or 7), variants of the root-specific promoter may have (in order of increasing preference) at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% overall sequence identity relative to the reference sequence.

[0075] In one embodiment, a plant is provided whose heritable genetic material comprises a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, wherein the gene encoding the transportable carbonic anhydrase protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence with at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the promoter comprises SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence with at least 65% identity to SEQ ID NO: 6 or SEQ ID NO: 7.

[0076] In one embodiment, a plant is provided whose heritable genetic material comprises a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, wherein the gene encoding the transportable carbonic anhydrase protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence with at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the promoter comprises SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence with at least 65% identity to SEQ ID NO: 6 or SEQ ID NO: 7; and wherein the signal peptide sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence with at least 65% identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0077] Root cell specific promoter can have universal activity with endogenous level in all plant cells. Endogenous level of carbonic anhydrase mRNA and full-length polypeptide is lower in cells other than root cells. In any biological background in which carbonic anhydrase is expressed, the expression level of carbonic anhydrase can exceed the endogenous level. Said biological background can be responsive to environmental changes.

[0078] The term "secretion" is used to describe the transport of proteins from the interior of the cell to the exterior of the cell. It is used interchangeably with "transport" or "export". Therefore, secretable / transportable carbonic anhydrase can be secreted / transported to the extracellular environment. In some embodiments, carbonic anhydrase is transported across the plasma membrane by a secretory pathway. The secretory pathway can be conventional or unconventional. In some embodiments, carbonic anhydrase is transported by a conventional secretory pathway, resulting in carbonic anhydrase being transported across the cell membrane at the cell periphery. Unconventional transport of proteins such as carbonic anhydrase can occur by one of four pathways: direct protein translocation across the plasma membrane by membrane transporters, lysosomal secretion, exosome release, and blebbing. The transport of carbonic anhydrase can be active or passive. Secretory proteins such as carbonic anhydrase of the present invention are located in the plasma membrane (also referred to as the extracellular membrane) in cells in some embodiments. Carbonic anhydrase can contact the extracellular membrane by extracellular matrix proteins (such as glycoproteins, such as extensins) or polysaccharides. Suitable glycoprotein and polysaccharide anchors are well known in the art. Carbonic anhydrase can contact with plasma membrane by molecular anchor (as transmembrane domain, transmembrane helix or glycosylphosphatidylinositol (GPI) anchor) or other biomolecule, and described biomolecule makes carbonic anhydrase keep being attached to plasma membrane or cell wall after secretion from cell.Carbonic anhydrase can contact with plasma membrane by a part of carbonic anhydrase polypeptide of the present invention, wherein a part of described polypeptide represents transmembrane protein.One or more transmembrane domains can be present.Carbonic anhydrase can not contact with plasma membrane.Carbonic anhydrase through secretion / transshipment can be adjacent to plasma membrane or in the extracellular environment closely adjacent to plasma membrane, for example, in cell wall region.Carbonic anhydrase can keep closely adjacent to plasma membrane, but exceeds cell wall region.In some embodiments, carbonic anhydrase is secreted into the environment close to root surrounding, such as soil or alternative bedding medium, when plant is present in such medium.In some embodiments, carbonic anhydrase is not secreted in plant nodule.When plant is separated from another part of itself (i.e. cuttings), carbonic anhydrase can continue to be secreted / transshipped when in vitro / leaving strain. In case of secretion, the carbonic anhydrase secreted / transferred can diffuse into the extracellular environment or remain on the root surface. The carbonic anhydrase secreted from the root cells can diffuse into other tissues or extracellular environments, such as those adjacent to the root cells. This can include any cell that is closely adjacent to the root or rhizosphere.

[0079] Carbonic anhydrase can be released from the cell by non-secretory pathways, such as by apoptosis or any physical perturbation of the cell membrane that exposes intracellular carbonic anhydrase to the extracellular environment.

[0080] Compared to plants whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, the carbonic anhydrase may be overexpressed in the extracellular environment of the root cells.

[0081] The term "overexpression" about carbonic anhydrase refers to expression of the carbonic anhydrase protein compared to endogenous baseline. The term "production" should be construed as referring to the generation of carbonic anhydrase protein, and can be used interchangeably with the term expression. The plant whose heritable genetic material does not comprise the gene of the coding translocating carbonic anhydrase protein under the control of an active promoter in root cells will show endogenous carbonic anhydrase levels under all biological backgrounds, and these carbonic anhydrase levels will be used as the baseline comparison of plant of the present invention. The baseline level of carbonic anhydrase has generally reflected the endogenous level of carbonic anhydrase, and it can fluctuate according to the inside and external environment of plant. The carbonic anhydrase level (i.e., any amount higher than baseline) that increases will be considered to represent the overexpression of carbonic anhydrase protein. In some embodiments, compared with endogenous baseline expression, plant of the present invention expresses 0.005%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 25%, 50%, 75%, 100% or 200% extra carbonic anhydrase protein. Endogenous baseline expression can be established in a control plant, which may be a wild-type plant. Cells of the same type and morphology at the same location in the plant (ie root cells) should be compared to each other.

[0082] Plants whose heritable genetic material does not include a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells are plants that lack a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells in their genome. This plant has not been modified in the same manner as the plants of the present invention. Compared to the transformed plants of the present invention, this plant can be referred to as an untransformed plant. This plant may include other genetic modification elements related to or unrelated to carbonic anhydrase that are different from the present invention. Plants whose heritable genetic material does not include a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells can be referred to as or used as a "control" plant. Therefore, the control plant is not genetically modified to change the expression of the polynucleotides and / or polypeptides disclosed herein. This plant will show endogenous carbonic anhydrase levels in all biological backgrounds, and these carbonic anhydrase levels will be used as baseline comparisons for plants of the present invention. The baseline level of carbonic anhydrase typically reflects the endogenous level of carbonic anhydrase, which can fluctuate with the internal and external environment of the plant. The control plant can be a wild-type plant. The control plant can be a wild-type plant of the same species as the plant of the present invention. The function of the control plant is to provide reliable reference, and the expression of the polynucleotide of the present invention or the polypeptide in the modified plant material can be compared with it. Therefore, compared with non-engineered plants, the plant of the present invention can be genetically engineered. The carbonic anhydrase activity level can be determined to define the control plant by enzyme assays known in the art and defined herein. Compared with plant of the present invention, the carbonic anhydrase activity in the control plant can be undetectable or be in very low level. Plant of the present invention shows the carbonic anhydrase activity level that increases, i.e. any amount of activity increase compared with the control plant. Usually, observe that plant of the present invention increases more than twice compared with control or unmodified plant. In some embodiments, plant of the present invention increases more than twice, three times, four times or five times compared with the control plant. In some embodiments, plant of the present invention increases carbonic anhydrase activity by more than 0.005%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to control plants. Said carbonic anhydrase activity can be measured in root cells, whole root or whole strain plant. In some embodiments, plant of the present invention increases carbonic anhydrase activity by more than two-fold, three-fold, four-fold or five-fold compared to control plants in the extracellular space.In some embodiments, carbonic anhydrase activity is quantified using the Wilbur-Anderson unit (WA) equation, or by measuring the rate at which it catalyzes the hydrolysis of p-nitrophenol acetate (pNPA) to a known concentration of p-nitrophenol (pNP) per unit time, which rate can also be expressed in units of enzyme activity such as katal (kat) or international units (IU), or by measuring the unit of change in conductivity per unit time, or by quantifying carbonic anhydrase activity using a radioactive tracer-based assay where the activity is reported as counts or decays per unit time, or by other equivalent methods well understood by those skilled in the art to quantify carbonic anhydrase activity. Optionally, the carbonic anhydrase activity of a plant of the present invention is increased two-fold, three-fold, four-fold, or five-fold in WA units compared to a control plant.

[0083] The gene encoding the transportable carbonic anhydrase protein may further comprise a signal peptide sequence for directing the carbonic anhydrase to be transported to the extracellular environment of the plant root. The term "signal peptide" may also be referred to as a signal sequence, a targeting signal, a localization signal, a localization sequence, a transit peptide, a targeting peptide, a leader sequence, or a leader peptide. The signal peptide is typically present at the N-terminal end of the polypeptide, but may also be present at the C-terminal end. In the present invention, the signal peptide is present at the N-terminal end.

[0084] Membrane-bound proteins can be of type I or type II; type I membrane-bound proteins rely on a signal peptide to direct them to the secretory pathway, which is subsequently cleaved, whereas type II proteins are targeted to the secretory pathway via their first transmembrane domain, which is not cleaved. Thus, the signal peptides of the present invention may or may not contain a transmembrane domain.

[0085] The signal peptide sequence encoded in the DNA polynucleotide sequence of the present invention may correspond to any of the polynucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence having at least 65% identity to any of said sequences; in some embodiments, a sequence having at least 70% identity to any of said sequences; and in some embodiments, a sequence having at least 80% identity to any of said sequences, or a sequence having at least 85%, at least 90%, or at least 95% identity to any of said sequences. In some embodiments, the carbonic anhydrase encoded by the above DNA sequence will have any of the percentages of homology mentioned herein and retain the enzymatic activity of carbonic anhydrase.

[0086] The polynucleotide may correspond to the full-length signal peptide or a portion thereof.

[0087] The terms "part," "variant," "homolog," or other representative terms in the art used herein with respect to a signal peptide sequence or a functional fragment thereof refer to any signal peptide sequence from any species (including plants) having a different polynucleotide or polypeptide sequence.

[0088] In terms of percent identity relative to a reference sequence (such as SEQ ID NO: 3 or 4), variants of the signal peptide sequence may have (in order of increasing preference) at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% overall sequence identity relative to the reference sequence.

[0089] The signal peptide sequence cooperates with the promoter to ensure extracellular expression and transport of the polypeptide sequence to the extracellular environment.

[0090] Also provided herein are polynucleotide sequences comprising:

[0091] A suitable promoter;

[0092] a sequence encoding a polypeptide sequence under the control of said suitable promoter; and

[0093] A sequence encoding a signal peptide sequence;

[0094] The polypeptide sequence comprises a carbonic anhydrase sequence, and the promoter is active in plant roots and capable of driving overexpression of the carbonic anhydrase, and the signal peptide sequence is capable of (before removal) directing the transport of the carbonic anhydrase to the extracellular environment of the plant root.

[0095] A suitable promoter is one that enables expression of the polypeptide sequence to which it is associated.The signal peptide sequence is sufficient to transport the carbonic anhydrase into the extracellular environment of the plant root.

[0096] The signal peptide sequence may be naturally encoded or translationally fused to the carbonic anhydrase sequence to ensure transport of the carbonic anhydrase to the extracellular environment of the plant root.

[0097] The signal peptide sequence for directing transport can be derived from a secretory protein that is transported to the extracellular environment through the protein secretion pathway (in some embodiments, through the conventional (or classical) protein secretion pathway). The signal peptide sequence can be derived from an extensin protein, and in some embodiments, it is derived from carrot plants or from a vacuolar sorting protein, or it can be derived from a pea plant.

[0098] Plants or polynucleotides according to the present invention may have promoter and / or signal peptide sequences derived from genes of the same plant species or variety as the plant. Plants or polynucleotides according to the present invention may have promoter and / or signal peptide sequences derived from genes of a different plant species, cultivar, or variety than the plant. Plant varieties are genetic variations of the same plant species that occur naturally in the environment. Cultivars are genetic variations of the same plant species that have been developed by humans through controlled breeding.

[0099] The promoter sequence, signal peptide sequence, transmembrane sequence, and any other sequences present in the polynucleotide may be derived from any species, variety, or cultivar.

[0100] Promoter of the present invention has activity in the plant tissue that brings into play root effect, and can be root hair cell and / or root epidermal cell specific in some embodiments.Root specific cell is those cells of a part for plant root system, and it can bring into play root specific function, i.e. anchoring, stability, sheltering effect, nutrition harvest and storage, water absorption, macromolecular transport.Underground stem (as other plant related organs near tuber, rhizome, bulb, corm, runner, taro and be positioned at underground root) provides the underground function similar to the root tissue of botanical definition, and can be considered as bringing into play root effect and comprise the plant tissue of root cell for the purpose of the present invention.

[0101] In some embodiments, carbonic anhydrase can further comprise a membrane spaning domain and / or a membrane anchoring sequence. The membrane spaning domain sequence can be a full-length membrane spaning domain sequence or its portion. The membrane spaning sequence can encode a membrane spaning domain, and the membrane spaning domain is partially present in the plasma membrane once it is transported, or it can span the entire plasma membrane. The membrane spaning sequence can comprise post-translational elements, such as glycoprotein and polysaccharide structure. In some embodiments, carbonic anhydrase can be attached to cell exterior or plasma membrane by a membrane spaning domain, a transmembrane helix, a membrane anchoring sequence or a glycosylphosphatidylinositol (GPI) anchor or any other biomolecule, and the biomolecule makes carbonic anhydrase keep being attached to plasma membrane or cell wall after secretion from the cell.

[0102] In some embodiments, the transmembrane domain is derived from pea (Pisum sativum) BP80. The transmembrane domain sequence encoded in the DNA polynucleotide sequence of the present invention can correspond to the polynucleotide sequence SEQ ID NO: 5, or a sequence having at least 65% identity to any of the sequences; in some embodiments, a sequence having at least 70% identity to any of the sequences; and in some embodiments, a sequence having at least 80% identity to any of the sequences, or a sequence having at least 85%, at least 90%, or at least 95% identity to any of the sequences. In some embodiments, the carbonic anhydrase will have any of the percentages of homology mentioned herein and retain the enzymatic activity of the carbonic anhydrase.

[0103] The polynucleotide may correspond to the full-length transmembrane sequence or a portion thereof. Additionally, the polynucleotide may comprise a sequence capable of directing the formation of a glycosylphosphatidylinositol (GPI) anchor or any other biomolecule that allows carbonic anhydrase to remain attached to the plasma membrane or cell wall after secretion from the cell.

[0104] The terms "part," "variant," "homolog," or other representative terms in the art used herein with respect to a transmembrane sequence or a functional fragment thereof refer to any transmembrane sequence ortholog having a different polynucleotide or polypeptide sequence from any species (including plants).

[0105] In terms of percent identity relative to a reference sequence (e.g., SEQ ID NO: 5), variants of the transmembrane sequence may have (in order of increasing preference) at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% overall sequence identity relative to the reference sequence.

[0106] In some embodiments, the membrane anchor sequence corresponds to an entire glycosylphosphatidylinositol anchor sequence or a portion thereof.

[0107] Also provided herein are isolated polynucleotide sequences of the invention, which may be fused to another polynucleotide sequence or a targeting peptide sequence or a transmembrane domain sequence.The polynucleotides may also be described as engineered or non-naturally occurring.

[0108] Also provided herein are isolated polypeptides of the present invention, which may be fused to another polypeptide or a targeting peptide or a transmembrane domain. An isolated polynucleotide or polypeptide is a DNA or protein construct that exists by itself, isolated from the internal environment of a cell. These may contain DNA or protein-related elements. These may be recombinant. The polypeptides of the present invention may temporarily contain a signal peptide, which is subsequently cleaved after entering the secretory pathway. The polypeptides of the present invention may also contain a signal peptide representing the first transmembrane domain, which acts as a signal peptide and is not cleaved after entering the secretory pathway.

[0109] Also provided herein are plant cells, cell lines, or progeny thereof comprising the polynucleotide sequences or polypeptide sequences disclosed herein. The term "plant" encompasses whole plants, ancestors and progeny of plants, and plant parts, including seeds, fruits, branches, stems, leaves, roots (including tubers), flowers, and tissues and organs, each of which comprises a polynucleotide of the present invention. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, each of which comprises a polynucleotide of the present invention.

[0110] In some embodiments, the plant is a crop plant, and in some embodiments, a row crop or a cover crop. In some embodiments, the plant is selected from corn, soybean, fava bean, mung bean, red bean, broad bean, pea, cowpea, cotton, rapeseed, camelina, potato, tomato, sugar beet, cassava, yam, sweet potato, alfalfa, wheat, barley, sorghum, oats, sorghum, millet, rye, teff, triticale, miscanthus, sorghum, rice, or clover, cress, Brassica (such as Brassica oleracea), vetch, and prairie grasses (such as switchgrass).

[0111] The plant can be a tree. In some embodiments, the tree is a poplar, spruce, pine, eucalyptus, oil palm, or rubber tree, particularly those commonly used in forestry and land management, food production, carbon capture, and biofuel or biomass production.

[0112] The plant can be an aquatic or semi-aquatic plant. In some embodiments, the plant can be duckweed, mangrove, Nymphaeales or rice.

[0113] Plants, polypeptides, polynucleotides, cells, cell lines or progeny of the present invention can sequester more carbon into the extracellular environment of the plant compared to plants whose heritable genetic material does not contain a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells. Carbon sequestration occurs by converting CO2 into bicarbonate and hydrogen ions.

[0114] Also provided herein are plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains, or seeds derived from or obtained from a plant of the invention.

[0115] Also provided herein are vectors comprising the polynucleotide sequences of the present invention. In some embodiments, the vector is a plasmid. The plasmid may comprise an origin of replication, a T-DNA right border repeat sequence of a Ti or Ri plasmid, a left border repeat sequence of a Ti or Ri plasmid, and at least one bacterial selectable marker. The plasmid may also comprise an enhancer, a plant selectable marker, a multiple cloning site, and / or a recombination site.

[0116] Also provided herein is a composition for transforming plant cells, comprising a polynucleotide of the present invention or a vector comprising a polynucleotide of the present invention. The vector may comprise a microparticle coated with the polynucleotide or the vector.

[0117] Plant transformation is a conventional technique in many species. Any of several transformation methods can be used to introduce a gene of interest into a suitable ancestral cell. According to various aspects of the present invention, the polynucleotides of the present invention are introduced into plants and expressed as transgenes by transformation. The terms "introduction" or "transformation" mentioned herein encompass the transfer of exogenous polynucleotides into host cells, regardless of the method used for transfer. The methods described for transforming and regenerating plants by plant tissue or plant cells can be used for transient transformation or stable transformation. Transformation methods include using liposomes, electroporation, chemicals that increase free DNA uptake, injecting DNA directly into plants, flower dips, biolistics, using viruses or pollen transformation, and microinjection. Methods can be selected from electroporation, microinjection, RNA-coated particle bombardment, or viral transfection, but are not limited to those methods listed herein. Transformation methods generally depend on the species selected. Polynucleotides can be introduced into host cells transiently or stably and can remain non-integrated, for example, as plasmids. Alternatively, they can be integrated into the host genome. Then, the resulting transformed plant cells can be used for regenerating transformed plants. In order to select transformed plants, the plant material obtained in the transformation is usually placed under selective conditions so that the transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above manner can be planted and, after an initial growth period, suitable selection and screening are carried out.

[0118] Also provided herein are bacteria comprising polynucleotides of the present invention or bacteria comprising vectors containing polynucleotides of the present invention. In some embodiments, the bacteria are Escherichia coli or Agrobacterium, and in some embodiments are Agrobacterium tumefaciens. Any suitable cloning system can be used. Genetically modified plants (including crops) are produced in some embodiments by transformation mediated by Agrobacterium tumefaciens. Such conventional methods are also used to introduce gene editing proteins, such as CRISPR-Cas nucleases and base editors, which can be used to edit natural carbonic anhydrase sequences to make them transportable and have increased expression in root cells.

[0119] Also provided herein are plants comprising a polynucleotide of the present invention stably integrated into their genome; and in some embodiments, heritably integrated into their genome.

[0120] Also provided is a method for producing the plant or seed of the present invention, comprising the steps of:

[0121] (a) providing a vector comprising a polynucleotide of the present invention;

[0122] (b) activating a promoter operably linked to the polypeptide-encoding DNA;

[0123] (c) Optionally, wherein the polypeptide is overexpressed in the extracellular environment of the cell compared to plants whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0124] Also provide the method that increases plant carbon sequestration ability in soil, described method comprises the heritable genetic material that changes plant so that carbonic anhydrase protein is transported to the extracellular environment from the root of plant.The normal ability of plant carbon sequestration in soil is indicated by the plant that is not genetically modified in the same manner as plant of the present invention.This ability can reflect the upper limit of the speed that CO2 is converted into bicarbonate and hydrogen ion in the extracellular environment of the plant that is not genetically modified in the same manner as plant of the present invention.This speed realizing in modified plant depends in essence on the transshipment of carbonic anhydrase from plant root to the extracellular environment.Changing heritable genetic material can comprise using transgenic manipulation method well known in the art that at least one polynucleotide is inserted in the heritable genetic material of vegetable cell.

[0125] Changing the heritable genetic material can also include using gene modification methods known in the art (such as CRISPR, TALENs and meganucleases (Meganucleases)) to carry out gene editing to the heritable genetic material of plant cells. Base editing can also be used, wherein a single nucleotide can be manipulated in the target gene. Guide editing (Primeediting) can also be used, which is a search and replace genome editing method for inserting accurate small insertions and deletions (indel), including single base or multi-base replacement, conversion and transversion, without the need for double-strand breaks and donor repair templates. Any regulatory sequence can also be manipulated using these technologies to cause the expression of carbonic anhydrase to increase, such as by using CRISPR-Cas systems to introduce random insertions and deletions into natural regulatory sequences.

[0126] Any of the polynucleotides disclosed herein may further comprise a polynucleotide sequence encoding a CRISPR-Cas protein, optionally comprising a guide RNA (gRNA), wherein the gRNA directs the CRISPR-Cas protein to the locus of at least one copy of an endogenous carbonic anhydrase gene in the genome of a plant cell, thereby inserting a root-specific promoter such that one or more copies of carbonic anhydrase are expressed in at least some root cells of the plant.

[0127] Gene editing tools can also be used to manipulate native carbonic anhydrase to introduce a signal peptide into the native carbonic anhydrase gene using targeted nucleases and homology-directed repair or non-homologous end joining repair mechanisms and a template sequence comprising a signal peptide. Such gene knock-in techniques are well known in the art.

[0128] Also provided are processed plant products obtained from plants or plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus cultures, pollen grains, or seeds disclosed herein. The processed plant products may comprise a detectable nucleic acid sequence comprising: (i) a transportable carbonic anhydrase downstream of a gene expression regulatory element active in at least some root cells of the plant, or (ii) a polynucleotide of the invention, or at least a portion thereof, present in the product. Processing may involve chemical or physical manipulation of the product to form a product having desired properties suitable for a specific function (i.e., granulation).

[0129] Example

[0130] Example 1

[0131] To demonstrate that plant roots can be engineered to produce active carbonic anhydrase protein, the soybean (Glycine max) carbonic anhydrase gene (Accession No. Glyma.19G135900 [SEQ ID NO: 1 and SEQ ID NO: 8]) was overexpressed. The protein sequence of this carbonic anhydrase contains a domain annotated as "eukaryotic carbonic anhydrase" (Pfam #PF00194.24).

[0132] The carbonic anhydrase gene (SEQ ID NO: 1 and SEQ ID NO: 8) was expressed under the control of the 35S promoter and cloned into the pCAMBIA-1300 plant expression vector. The vector contains a herbicide resistance gene (kanamycin) and the expression cassette is flanked by Agrobacterium LB and RB t-DNA sequences. The vector was transformed into Agrobacterium rhizogenes strain K599 using electroporation and used to transform soybean roots using the hairy root transformation method according to the protocol in Chen et al., 2018.

[0133] After transformation, positively transformed root tissue was visually identified by visualizing reporter gene expression. Root tissue was isolated from the transformed roots, and an equivalent sample of untransformed root tissue was sampled as a control. Root tissue was homogenized in 700 μl of extraction buffer in an ice-cold pestle and mortar. The homogenized roots were transferred to a 2 ml tube and centrifuged at 5000 rpm for 15 minutes at 4°C. All assays were performed using the supernatant following this centrifugation step.

[0134] The carbonic anhydrase assay was performed to measure carbonic anhydrase activity. This assay measures the rate of pH change of CO2-saturated water after addition of root homogenates of transformed and untransformed (also known as wild-type controls) root tissue. The pH change is caused by the protons (H + ) release. The pH change is proportional to the amount of bicarbonate produced by carbonic anhydrase. Such assays are known to those skilled in the art.

[0135] For each carbonic anhydrase assay, 100 μl of supernatant from homogenized root samples was used. For the no-root tissue control, 100 μl of extraction buffer without root tissue was used. The sample was added to a 2 ml tube containing 1100 μl of 20 mM Tris-HCl solution (containing 0.003% bromothymol blue) at pH 8.3. Afterwards, 800 μl of ice-cold CO 2 saturated water was added. The time at which the color changed from blue (pH 8.3) to yellow (pH 5.3) was recorded. All reactions were carried out on ice.

[0136] Activity was calculated using the Wilbur-Anderson Unit (WA) equation:

[0137] WA unit = (t0-ts) / ts

[0138] where t0 is the time to color change in the no-root control reaction and ts is the time to color change in the sample containing supernatant from homogenized roots.

[0139] Carbonic anhydrase activity in transgenic root samples was significantly increased by 2-fold compared with wild-type root samples (P = 0.022669, one-tailed Student's t-test, unequal variance) ( Figure 2 A). Thus, soybean root cells can be engineered to have increased carbonic anhydrase activity by overexpressing a gene encoding a carbonic anhydrase protein, and the protein is catalytically active when released from the root cells into the extracellular environment.

[0140] Example 2

[0141] In order for carbonic anhydrase to promote CO2 in the plant extracellular environment to the conversion of HCO3, they need to be secreted / transported into the extracellular environment from the plant cell. Cells have several mechanisms by which proteins are targeted to the secretion / transport from the cell to the extracellular environment. Two main ways to achieve this are conventional secretion pathways and unconventional secretion pathways. Conventional secretion pathways are achieved by using an N-terminal signal peptide to secrete / transport proteins.

[0142] To demonstrate that carbonic anhydrase proteins can be engineered for secretion / translocation from plant cells, we translated the signal peptide of the experimentally characterized carrot (Daucus carota) expansin gene (G. Li et al., 2009) [SEQ ID NO: 3] into a soybean carbonic anhydrase [SEQ ID NO: 1 and SEQ ID NO: 8] provided in Example 1. This fusion protein was placed under the control of the root-specific promoter [SEQ ID NO: 6] of the soybean expansin gene (Glyma.17G133100) and cloned into a plant transformation vector that also contained a herbicide resistance gene (spectinomycin) and was flanked by Agrobacterium t-DNA sequences ( Figure 1 The vector was transformed into Agrobacterium tumefasciens (strain LBA4404) using electroporation, and positive colonies were selected and cultured using standard techniques. To demonstrate the broad applicability of this technique across all plant species, the vector was transformed into Arabidopsis thaliana using the floral dip method (Clough & Bent, 1998), and stable transgenic lines were generated.

[0143] It should be noted that although the soybean expansin gene promoter was used in this example, other root promoters (e.g., Y. Li et al., 2019; Marquès-Bueno et al., 2016) are also known in the art. One of ordinary skill in the art would expect that any root promoter selected from any species would function equally well to drive expression in roots. It should also be noted that if the carbonic anhydrase protein is expressed throughout the plant or in other plant tissues, as long as the expression domain includes expression in the roots, the technology of the present invention is expected to function.

[0144] Experiment is carried out to prove that the plant that can secrete / transport carbonic anhydrase in root cell causes the carbonic anhydrase activity in the extracellular environment of root.At this, transformed plant and wild type control plant are grown for six weeks under hydroponic condition.In the time of six weeks, individual plant is transferred in the fresh hydroponic culture medium of 15ml and allowed to grow two days.After the time period of these two days, hydroponic solution is sampled and used for carbonic anhydrase activity assay, to measure the carbonic anhydrase activity amount that has been secreted / transported to in the hydroponic culture medium from root.

[0145] Here, 100 μl of the hydroponic solution was added to a 2 ml snap-cap microcentrifuge tube containing 1050 μl of 20 mM Tris-HCl solution (pH 8.3) containing 0.003% bromothymol blue and 100 μl of extraction buffer (200 mM Tris HCl pH 8.3, 1 mM EDTA, 20 mM MgCl2, 50 mM NaCl, 100 mM Na2SO4). Subsequently, 750 μl of ice-cold CO2-saturated water (generated by a soda water machine) was added to start the reaction. The time for the color to change from blue (pH 8.3) to yellow (pH 5.3) was recorded and used to calculate the enzyme activity as described in Example 2. The reaction was carried out on ice.

[0146] The carbonic anhydrase activity in the hydroponic culture medium samples of the transgenic plants was significantly increased by 11 times compared with that in the hydroponic culture medium samples of the wild-type control plants (P = 0.029404, one-tailed Student's t-test, unequal variance) ( Figure 2 B) Thus, plants transformed with the present invention successfully expressed and secreted active carbonic anhydrase protein into the extracellular environment of the roots, which in turn catalyzed the conversion of CO2 into bicarbonate.

[0147] Example 3

[0148] To demonstrate that this technology can also be used to sequester CO2 in soil, separate experiments were conducted on soil-grown plants. Given that carbonic anhydrase catalyzes the conversion of CO2 and water into bicarbonate and protons, the sequestration of atmospheric CO2 in soil is directly proportional to the pH change of the soil. Here, transgenic plants and wild-type plants (as described in Example 2) were grown in 6.5 cm pots containing 110 g of Westland John Innes No. 3 mature plant compost for six weeks. Soil control pots without any plants were also constructed. For each pot, HANNA TM Soil pH was measured 2 cm below the surface using a soil pH meter (HI-981030) at three random locations. Figure 2 As shown in Figure C, the soil pH in pots containing transgenic plants of the present invention was significantly lower than that in pots without plants ("soil control") (P = 0.000092, one-tailed Student's t-test, unequal variance) or pots containing wild-type plants (P = 0.000449, one-tailed Student's t-test, unequal variance). Therefore, pots containing plants of the present invention sequestered more CO2 in the soil than pots containing control plants or pots without plants.

[0149] The plants were also photographed as they grew. There were no obvious phenotypic differences between the transgenic and wild-type plants in terms of plant development, growth rate, flowering, or fruiting ( Figure 2 F) Therefore, secretion of carbonic anhydrase from roots and sequestration of CO2 as bicarbonate in the soil have no detrimental effects on plant growth.

[0150] Example 4

[0151] A CO2 soil flux experiment was conducted to further demonstrate that the transgenic plants of the present invention increase the rate at which CO2 diffuses from the air into the soil and is converted into bicarbonate.

[0152] Here, transgenic plants and wild-type plants (as described in Example 2) were grown in 6.5 cm pots containing 110 g of Westland John Innes No. 3 mature plant compost for 6 weeks. After 6 weeks, all above-ground biomass (i.e. leaves, stems, etc.) was removed and the pots containing all underground biomass in the soil were placed in a LI-COR 6800 small plant chamber in complete darkness (achieved by covering the chamber with tin foil). The CO2 absorbed or released by the soil in the pots was then measured using the LI-COR 6800. To do this, the chamber was equilibrated in CO2-free air for 1 hour. After 1 hour, the CO2 level rose to 1000 ppm (~2.5 times the atmospheric CO2 concentration). After acclimation for 1 minute, the soil CO2 sequestration rate was measured every second over a 30-minute recording window. Net carbon sequestration was calculated by summing the carbon sequestration over the recording window. Figure 2As shown in Figure 3D, this indicates that the pots containing roots of the control plants did not sequester CO2 from the airflow, but instead produced CO2 through soil respiration. In contrast, the pots containing roots of the transgenic plants of the present invention consumed CO2 from the airflow. Thus, the plants of the present invention promote CO2 sequestration into the soil. Moreover, the magnitude of this sequestration is significantly greater than the CO2 released through soil respiration.

[0153] Example 5

[0154] To demonstrate that other carbonic anhydrase genes from other species can be used in various embodiments of the present invention, we expressed the carbonic anhydrase from the diatom Thalassiosira weissflogii [SEQ ID NO: 2]. As previously described, this carbonic anhydrase was translationally fused to the signal peptide of the experimentally characterized carrot (Daucus carota) extensin gene (G. Li et al., 2009) [SEQ ID NO: 3]. This fusion protein was placed under the control of the root-specific promoter of the soybean expansin gene (Glyma.17G133100) [SEQ ID NO: 6] and cloned into a plant transformation vector that also contained a herbicide resistance gene (spectinomycin) and was flanked by Agrobacterium tumefaciens LB and RB T-DNA sequences. The vector was transformed into Agrobacterium tumefaciens (strain LBA4404) by electroporation, and positive colonies were selected and cultured using standard techniques. This vector was transformed into Arabidopsis thaliana to generate stable transgenic lines.

[0155] Seeds of transgenic plants expressing the secretable / transportable carbonic anhydrase of Thalassiosira wissenii (SEQ ID NOs 2 and 9) and seeds of wild-type plants were germinated on 1 / 2 Murashige and Skoog + 1% sucrose + 2.5 mM MES agar plates. After 7 days, the seedlings were transferred to 96-well plates with only the roots entering each well. Each well of the 96-well plate contained 350 μl of 1 / 2 Murashige and Skoog + 1% sucrose solution and 30 mg / L bromocresol purple. After 4 hours, the seedlings were removed and the absorbance at 429 nm was measured using a plate reader. Carbonic anhydrase activity was estimated by the rate of pH change over a 4-hour period. Carbonic anhydrase activity was significantly higher in the root environment of the transgenic plants than in the root environment of the wild-type control plants (P = 0.017435, one-tailed Student's t-test, unequal variance) ( Figure 2 E). Thus, plants transformed with the secretable / transportable carbonic anhydrase of Thalassiosira wieldingii successfully expressed and secreted active carbonic anhydrase protein into the extracellular environment of the root, catalyzing the conversion of CO2 to bicarbonate. This indicates that carbonic anhydrase proteins from any species and any carbonic anhydrase isoform are expected to function equally well.

[0156] Example 6

[0157] To demonstrate that soybean plants can be engineered to secrete active carbonic anhydrase proteins from their roots into the extracellular environment, we translationally fused the signal peptide of the carrot (Daucus carota) extensin gene (SEQ ID NO: 3), experimentally characterized in Example 2, to the soybean carbonic anhydrase (SEQ ID NO: 1 and SEQ ID NO: 8) provided in Example 1. This fusion protein was placed under the control of the root-specific promoter (SEQ ID NO: 6) of the soybean extensin gene (Glyma.17G133100) and cloned into a plant transformation vector that also contained a herbicide resistance gene (spectinomycin) and was flanked by Agrobacterium t-DNA sequences (LB and RB). Figure 1 ). The vector was transformed into Agrobacterium tumefaciens using standard techniques and cultured. The vector was transformed into soybean to generate stable engineered strains. Engineered plants and azygous control plants were grown in Levington advance F2S Seed & Modular & Sand Compost for 3 weeks, after which the roots were washed and transplanted into 4.5 L hydroponic boxes. Plants were grown in hydroponic medium for two weeks as previously described by Hata and Futamura (2020). After two weeks, the hydroponic medium was replenished with fresh medium based on the rate at which the plants consumed the hydroponic medium and the plants were grown for 5 days. Samples of the hydroponic medium from the hydroponic boxes containing control or engineered plants were filtered through a Falcon 40 μm cell strainer to remove plant debris. The proteins present in the filtered samples were then separated and concentrated using a Pierce 10 kD MWCO protein concentration column. The total protein concentration of each isolated protein sample was calculated using the Bradford protein assay, and the total protein concentration of all samples was adjusted to be equal. To measure carbonic anhydrase activity, a modified version of the protocol described by Ozdemir (2009) was used. Here, 160 μl of concentrated hydroponic culture medium sample was added to 120 μl of 50 mM Tris-HCl (pH 7.5) in the wells of a 96-well microplate. Subsequently, 30 μl of 10 mM p-nitrophenyl acetate in acetonitrile was added to each well to initiate the colorimetric assay. The absorbance at 400 nm of each well was measured at 25°C over a period of 16 hours using a spectrophotometric microplate reader. Carbonic anhydrase activity is expressed as μM p-nitrophenol synthesized per second. The average carbonic anhydrase activity was calculated from two independent non-integrated lines and three independent engineered lines ( Figure 4A) Protein samples isolated from hydroponic cultures containing non-integrated plants exhibited baseline p-nitrobenzene hydrolysis activity. Protein samples isolated from hydroponic cultures containing engineered plants exhibited significantly higher p-nitrobenzene hydrolysis activity. This is consistent with the presence of secreted carbonic anhydrase in the hydroponic cultures containing engineered plants.

[0158] Plants grown in soil were also photographed. There were no significant phenotypic differences between the engineered soybean plants and control plants in terms of plant development, growth rate, flowering, or fruiting ( Figure 4 F) Therefore, secretion of carbonic anhydrase from roots and sequestration of CO2 as bicarbonate in the soil have no detrimental effects on plant growth.

[0159] Example 7

[0160] In order to prove that the soybean plant that has been engineered to secrete active carbonic anhydrase protein to the extracellular environment can sequester CO2 in soil, carried out the following experiment.To express in root hair cells can secrete carbonic anhydrase engineered plant (as described in Example 6) and wild type control plant and be planted in the basin of 13cm, contain the LevingtonM3 compost, Melcourt topsoil, Melcourt coarse sand and perlite of 1:1:2:2 in the basin, be mixed with 5ml Miracle-Gro All Purpose Continuous Release Plant Food (Miracle-Gro All Purpose Continuous Release Plant Food).When plant growth is to 17 weeks old, use the soil sampling probe of diameter 2.1cm to collect soil core (soilcore) from the random position near each basin root zone, be transferred in the falcon pipe.Add ddH2O isopyknic with soil weight, mix 30 minutes and leave standstill 1 hour.Use METLER pH probe to measure the pH of soil slurry. The pH of the soil in the pots containing the engineered plants of the present invention was significantly lower than that in the pots containing wild-type plants (P=0.00798, one-tailed Student's t-test, unequal variances) ( Figure 4 B) This decrease in soil pH is consistent with the presence of active carbonic anhydrase protein and the resulting increase in bicarbonate concentration in the soil of the pots containing the engineered plants of the present invention. Therefore, the soil growing the plants of the present invention sequestered more CO2 than the soil growing the control plants.

[0161] Example 8

[0162] To prove that carbonic anhydrase genes from any species can be used for the engineered plants of the present invention, the soybean carbonic anhydrase gene used in Examples 1 and 2 above was replaced with a carbonic anhydrase gene [SEQ ID NO: 2] from marine algae. All other components of the vector used for plant transformation remained unchanged. In order to prove that the expressed carbonic anhydrase protein was secreted from soybean roots and was active, the engineered plants of the present invention and the control plants without integration were grown for 3 weeks in Levington advance F2S seeds and modular sandy compost (Levington advance F2S Seed & Modular & Sand Compost), then the roots were cleaned and transplanted into a 4.5L hydroponic box. As previously mentioned by Hata and Futamura (2020), plants were supported for growth in hydroponic culture medium. Plants were grown hydroponically for 1 week. The hydroponic culture medium was supplemented with fresh culture medium and the plants were allowed to continue growing for 7 days. The culture medium samples in the hydroponic boxes containing only control plants or engineered plants were separated and filtered through a Falcon 40 μm cell strainer to remove plant debris. Proteins present in the filtered samples were then separated and concentrated using a Pierce 10kD MWCO protein concentration column. The total protein concentration of each sample was calculated using the Bradford protein assay, and enzyme activity was normalized to the total protein content. To measure carbonic anhydrase activity, a modified version of the protocol described by Ozdemir (2009) was used. Here, 160 μl of concentrated hydroponic culture medium sample was added to 120 μl of 50 mM Tris-HCl (pH 7.5) in a well of a 96-well microplate. Subsequently, 30 μl of 10 mM p-nitrophenyl acetate dissolved in acetonitrile was added to each well to initiate the colorimetric assay. The absorbance at 400 nm for each well was measured using a spectrophotometric microplate reader at 25°C over an 11-hour period.

[0163] like Figure 4 As shown in Figure C, protein samples isolated from hydroponic culture medium grown without the integration plant exhibited baseline p-nitrobenzene hydrolysis activity. Protein samples isolated from hydroponic culture medium grown with the engineered plants of the present invention exhibited significantly higher p-nitrobenzene hydrolysis activity. This is consistent with the presence of secreted carbonic anhydrase in the hydroponic culture medium containing the engineered plants.

[0164] Example 9

[0165] To demonstrate that soybean plants engineered to express an active carbonic anhydrase protein on their root surfaces were capable of sequestering CO2 in the soil, the following experiments were performed. As described above, soybean carbonic anhydrase [SEQ ID NO:3] from the engineered soybean plants was translationally fused to the experimentally characterized signal peptide and transmembrane domain of pea (Pisum sativum) [SEQ ID NO:4 and SEQ ID NO:5]. This fusion protein was placed under the control of the root-specific promoter [SEQ ID NO:6] of the soybean expansin gene (Glyma.17G133100) and cloned into a plant transformation vector containing a herbicide resistance gene (spectinomycin) and flanked by Agrobacterium t-DNA sequences (LB and RB). Figure 1 ). The vector was transformed into Agrobacterium tumefaciens using standard techniques and cultured. The vector was transformed into soybean to generate a stable engineered strain. The engineered plants and non-integrated control plants were planted in 13 cm pots containing John Innes CerealMix. When the plants were 13 weeks old, soil core samples were collected from random locations near the root zone of each pot using a 2.1 cm diameter soil sampling probe and transferred to a falcon tube. Add ddH2O in an amount equal to the weight of the soil, mix for 30 minutes and let it stand for 1 hour. The pH of the soil slurry was measured using a METLER pH probe. Figure 4 As shown in Figure 3D, the pH of the soil in pots containing the engineered plants of the present invention was significantly lower than the pH of the soil in pots containing wild-type plants. This decrease in soil pH is consistent with the presence of active carbonic anhydrase protein and the resulting increase in bicarbonate concentration in the soil of pots containing the engineered plants of the present invention. Therefore, pots containing the engineered plants of the present invention sequestered more CO2 in the soil compared to pots containing control plants or pots without plants.

[0166] Example 10

[0167] To demonstrate that other carbonic anhydrase genes from other species can be engineered and localized extracellularly in soybean root hairs, Figure 4The engineered plants used in C and their respective non-integrated control plants were grown for 3 weeks on Levington Advanced F2S seeds and modular sandy compost and transplanted into 4.5 L hydroponic boxes. Plants were supported in hydroponic culture medium as previously described by Hata and Futamura (2020). Plants were grown hydroponically for 2 weeks, with weekly medium changes. To measure carbonic anhydrase activity on the root surface, a modified protocol for carbonic anhydrase p-nitrophenyl hydrolysis by Ozdemir (2009) was used. 500 mg of fresh soybean roots were briefly washed with sterile water to remove excess hydroponic culture medium and then placed in a 30 mL beaker containing 18 mL of 50 mM Tris-HCl, pH 7.5. The reaction was initiated with 2 mL of 10 mM p-nitrophenyl acetate dissolved in acetonitrile. The absorbance of 300 μL of the sample at 400 nm was recorded at 1 and 33 minutes using a spectrophotometric microplate reader. Carbonic anhydrase activity was expressed as μM p-nitrophenol synthesized per second.

[0168] Compared with control plant roots, engineered plant roots showed significantly higher p-nitrobenzene hydrolysis activity (P = 0.030458, one-tailed Student's t-test, unequal variance) ( Figure 4 E). This is consistent with the carbonic anhydrase activity detected outside the cells of the engineered plant roots.

[0169] Example 11

[0170] To demonstrate that carbonic anhydrase can be engineered to localize extracellularly to the cytoplasmic membrane, a reporter gene-gene fusion was constructed and tested in protoplasts. Here, the Thalassiosira welchii carbonic anhydrase [SEQ ID NO:2] was translationally fused to the coding sequence for a fluorescent protein to generate a gene-reporter fusion. This gene-reporter fusion was then translationally fused at the N- and C-termini to the signal peptide and transmembrane domain of the pea (Pisum sativum) gene PsBP80 [SEQ ID NO:4 and SEQ ID NO:5]. This final fusion protein was placed under the control of the cassava stem mosaic virus promoter (Verdaguer et al., 1996) and cloned into a plant transformation vector. To provide a control for cytoplasmic localization, the Thalassiosira welchii carbonic anhydrase, translationally fused to the coding sequence for a fluorescent protein (without the PsPB80 signal peptide and transmembrane domain), was also placed under the control of the cassava stem mosaic virus promoter (Verdaguer et al., 1996) and cloned into a plant transformation vector.

[0171] Soybean protoplasts were transfected with the two expression vectors according to the protocol of Sultana et al. (2019). Two days later, the transfected protoplasts were imaged using a Leica TCS SP5 confocal microscope. Protoplasts expressing the Thalassiosira wissenii carbonic anhydrase fused to a fluorescent protein coding sequence (without the PsPB80 signal peptide and transmembrane domain) showed clear cytoplasmic localization ( Figure 5 A). In contrast, protoplasts expressing the Thalassiosira wissenii carbonic anhydrase fused to a fluorescent protein coding sequence containing the PsBP80 signal peptide at the N-terminus and the PsBP80 transmembrane domain at the C-terminus localized to the plasma membrane ( Figure 5 B).

[0172] Example 12

[0173] To demonstrate that the promoters used to generate the engineered plants described herein drive expression in specific root tissues, GUS reporter lines were generated. Here, pGmEXPA7 (SEQ ID NO: 6) and pGmPIN2b (SEQ ID NO: 7) were each independently used to drive expression of GUS (Cat1+) (a β-glucuronidase gene with the first intron of castor bean catalase, Cat1). These promoter-GUS fusions were individually cloned into plant transformation vectors containing a herbicide resistance gene (kanamycin) and flanked by Agrobacterium LB and RB t-DNA sequences. The resulting vectors were transformed into Agrobacterium tumefaciens (strain LBA4404) by electroporation, and positive colonies were selected and cultured using standard techniques. These Agrobacterium strains were used to transform Arabidopsis thaliana using the floral dip method (Clough and Bent, 1998). Engineered plants were germinated on ½ Murashige and Skoog agar plates containing 1% sucrose and 2.5 mM MES. After 10 days, the seedlings were transferred to 6-well plates and subjected to GUS histochemical staining. 5 ml of GUS staining buffer (0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)63H2O, 50 mM sodium phosphate buffer, 10 mM Na2EDTA H2O, 1 mM X-Gluc, and 0.1% Triton-X (v:v)) was added to the seedlings and incubated at 37°C for 3 hours. The seedlings were washed with 70% ethanol and cleared overnight. Chlorophyll-stained and cleared seedling roots were imaged using an Optika B-383FL microscope in conjunction with a OneView camera and imaging software.

[0174] GUS expression driven by the pGmPIN2b promoter [SEQ ID NO: 6] was localized to the root apical epidermis, and some expression extended to the primary root ( Figure 5 C). GUS expression driven by the pGmEXPA7 promoter is localized to root hair cells ( Figure 5D).

[0175] Example 13

[0176] To demonstrate that the engineered plants of the present invention can increase the accumulation of soil inorganic carbon, the following experiments were performed. Engineered plants expressing secreted soybean carbonic anhydrase in root hair cells [SEQ ID NO: 6] and non-integrated control plants described in Example 6 were planted in 13 cm pots containing a 1:1:2:2 ratio of Levington M3 compost, Melcourt topsoil, Melcourt coarse sand, and perlite, mixed with 5 ml of Miracle-Gro general purpose sustained release plant fertilizer until the end of the life cycle. After the seeds were harvested and the soil dried, the dry biomass and roots in the soil were removed and the remaining soil was sieved through a 2 mm sieve. Soil samples were sent to Eurofins AgroTesting in the UK for soil carbon testing to analyze soil inorganic carbon (SIC) and organic carbon (SOC) content.

[0177] The average inorganic carbon content of soil in pots planted with engineered plants was higher than that in soil in pots planted with unintegrated control plants or no plants ( Figure 6 A).

[0178] Example 14

[0179] To demonstrate that exogenous soil treatments can be used in combination with the engineered plants of the present invention to enhance CO2 sequestration in soil, the following experiment was conducted. Here, engineered Arabidopsis plants expressing secretable carbonic anhydrase in root hair cells, as described in Example 2, and wild-type control plants were planted in 6.5 cm pots containing 160 g of Levington's Advanced F2S seeds and modular sandy compost mixed with 4.16 g / L of Ca(OH)2. The soil pH was adjusted to pH 8 to promote HCO3 sequestration. - To CO3 - of Ca(OH)2, which can then combine with Ca in Ca(OH)2 to form CaCO3.

[0180] Plants were grown for 10 weeks. At 10 weeks, all soil biomass was removed and the soil was sent to Eurofins AgroTesting in the UK for soil carbon testing to analyze soil inorganic carbon (SIC) and organic carbon (SOC) content. The soil planted with the engineered plants of the present invention showed increased soil inorganic carbon (SIC). Figure 6 B), and soil inorganic carbon normalized to soil organic carbon was significantly higher (P = 0.002078, one-tailed Student's t-test, unequal variances) ( Figure 6C). This is consistent with the presence of carbonic anhydrase activity in the soil in which the engineered plants of the present invention are grown, and the resulting enhanced conversion of CO2 to bicarbonate. Thus, soil treatments can be used in conjunction with the engineered plants of the present invention to enhance CO2 sequestration. One of ordinary skill in the art would anticipate that any soil treatment rich in positively charged cations (such as magnesium oxide or cations from gravel soil treatments or fertilizer treatments), or deployment of this technology in soils naturally rich in cations, when used in conjunction with the present invention, would be expected to function equally well to increase soil inorganic carbon.

[0181] Example 15

[0182] To demonstrate that the engineered plants of the present invention can increase the accumulation of soil inorganic carbon, the following experiment was performed. The engineered soybean plants expressing soybean carbonic anhydrase anchored to the plasma membrane of root hair cells described in Example 8 and their respective non-integrated control plants were planted in 13 cm pots containing a 1:1:2:2 ratio of Levington M3 compost, Melcourt topsoil, Melcourt coarse sand and perlite, mixed with 5 ml of Miracle-Gro general purpose sustained release plant fertilizer until the end of the life cycle. After the seeds were harvested and the soil dried, the dry biomass and roots in the soil were removed and the remaining soil was sieved through a 2 mm sieve. The soil samples were sent to Eurofins AgroTesting in the UK for soil carbon testing to analyze the soil inorganic carbon (SIC) and organic carbon (SOC) content.

[0183] Compared with the soil planted with the control plants, the soil planted with the engineered plants of the present invention showed a significant increase in soil inorganic carbon (P = 0.032395, one-tailed Student's t-test, unequal variance) ( Figure 6 D), and soil inorganic carbon normalized to soil organic carbon was significantly higher (P = 0.048304, one-tailed Student's t-test, unequal variances) ( Figure 6 E).

[0184] This is consistent with the increase in carbonic anhydrase activity in the soil in which the plants of the present invention are grown and the resulting enhanced conversion of CO2 to bicarbonate to generate soil inorganic carbon. Therefore, it is demonstrated that the engineered plants of the present invention enhance carbon sequestration in the soil.

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Claims

1. A plant whose heritable genetic material comprises a gene encoding a transportable carbonic anhydrase protein, said gene being under the control of a promoter active in root cells such that said carbonic anhydrase is transported from the roots of said plant into the extracellular environment.

2. The plant of claim 1 , wherein the carbonic anhydrase is overexpressed in the extracellular environment of root cells compared to plants whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

3. The plant according to claim 1 or 2, wherein the gene encoding the transportable carbonic anhydrase protein further comprises a signal peptide sequence, wherein the signal peptide sequence is used to direct the carbonic anhydrase to be transported to the extracellular environment of the plant root.

4. A polynucleotide sequence comprising a sequence encoding a polypeptide sequence under the control of a suitable promoter, wherein the polypeptide sequence comprises a carbonic anhydrase sequence and a signal peptide sequence; wherein the promoter is active in plant roots and can drive overexpression of the carbonic anhydrase, and the signal peptide sequence can direct the carbonic anhydrase to be transported to the extracellular environment of the plant roots.

5. The plant of claim 3 or the polynucleotide sequence of claim 4, wherein the signal peptide sequence is naturally encoded or translationally fused to the carbonic anhydrase sequence to ensure transport of the carbonic anhydrase to the extracellular environment of the plant root.

6. The plant according to any one of claims 1 to 3 or 5 or the polynucleotide sequence according to claim 4 or 5, wherein the signal peptide sequence for directing transport is derived from a secretory protein transported to the extracellular environment via a protein secretion pathway.

7. The plant according to any one of claims 3, 5 or 6 or the polynucleotide sequence according to any one of claims 4 to 6, wherein the signal peptide sequence is derived from an extensin protein, preferably from a carrot plant, or from a vacuolar sorting protein, preferably from a pea plant.

8. The plant or polynucleotide according to any one of the preceding claims, wherein the promoter and / or the signal peptide sequence, where present, is derived from a gene of the same plant species or variety as the plant.

9. The plant or polynucleotide according to any one of the preceding claims, wherein the promoter is root-specific, preferably root hair and / or root epidermis-specific.

10. The plant or polynucleotide of any one of the preceding claims, wherein the carbonic anhydrase further comprises a transmembrane domain or a membrane anchoring sequence.

11. The plant or polynucleotide of any preceding claim, wherein the carbonic anhydrase is an alpha, beta, gamma, delta, zeta, eta, theta or iota subtype carbonic anhydrase, preferably a monomer.

12. The plant of any one of claims 1 to 3, wherein the gene encoding the transportable carbonic anhydrase protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the promoter comprises SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence at least 65% identical to SEQ ID NO: 6 or SEQ ID NO:

7.

13. The plant of claim 3, wherein the gene encoding the transportable carbonic anhydrase protein comprises SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence at least 65% identical to SEQ ID NO: 1 or SEQ ID NO: 2, optionally wherein the promoter comprises SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence at least 65% identical to SEQ ID NO: 6 or SEQ ID NO: 7; and wherein the signal peptide sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence at least 65% identical to SEQ ID NO: 3 or SEQ ID NO:

4.

14. A polypeptide sequence encoded by the polynucleotide of any one of claims 4 to 11.

15. A plant cell, cell line or progeny thereof comprising the polynucleotide sequence or polypeptide sequence of any one of claims 4 to 11 or 14.

16. The plant, polynucleotide, polypeptide, cell, cell line or progeny of any preceding claim, wherein the plant is a crop plant, preferably a row crop or cover crop, preferably selected from corn, soybean, pea, cotton, rapeseed, camelina, potato, tomato, sugar beet, cassava, sweet potato, alfalfa, wheat, barley, sorghum, oats, sorghum, millet, rye, teff, rice plants, or clover, cress, Brassica, vetch and prairie grasses.

17. The plant, polynucleotide, polypeptide, cell, cell line or progeny of any one of claims 1 to 15, wherein the plant is a tree, preferably wherein the tree is a poplar, spruce, pine, eucalyptus, oil palm or rubber tree.

18. The plant, polypeptide, polynucleotide, cell, cell line or progeny of any preceding claim, wherein more carbon is sequestered into the extracellular environment of the plant compared to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

19. A plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus, cell culture, pollen grain or seed derived from or obtained from the plant according to any one of claims 15 to 18.

20. A vector comprising the polynucleotide sequence of any one of claims 4 to 11 or 16 to 18, wherein the vector is optionally a plasmid.

21. A composition for transforming plant cells, comprising the polynucleotide of any one of claims 4 to 11 or 16 to 18 or the vector of claim 20; optionally comprising microparticles coated with the polynucleotide or the vector.

22. A bacterium comprising the polynucleotide of any one of claims 4 to 11 or 16 to 18 or the vector of claim 20; optionally wherein the bacterium is Escherichia coli or Agrobacterium sp., more preferably Agrobacterium tumefaciens.

23. A plant comprising the polynucleotide of any one of claims 4 to 11 or 16 to 18 stably integrated into its genome; preferably, the polynucleotide is heritably integrated into the genome.

24. A method for producing a plant or seed comprising the steps of: (d) providing the vector according to claim 20; (e) activating a promoter operably linked to the polypeptide-encoding DNA; (f) Optionally, wherein the polypeptide is overexpressed in the extracellular environment of the cell compared to a plant whose heritable genetic material does not comprise a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

25. A method of increasing the carbon sequestration capacity of a plant in soil, said method comprising altering the heritable genetic material of said plant such that carbonic anhydrase protein is transported from the roots of said plant to the extracellular environment.

26. The method of claim 25, wherein altering the heritable genetic material comprises inserting at least one polynucleotide into the heritable genetic material of the plant cell.

27. The method of claim 25, wherein altering the heritable genetic material comprises gene editing the heritable genetic material of the plant cell.

28. A processed plant product obtained from the plant or plant part, plant tissue, plant organ, plant cell, plant protoplast, embryo, callus, cell culture, pollen grain or seed of any one of claims 1 to 3, 5 to 19 and / or 23; optionally, wherein the processed product comprises a detectable nucleic acid sequence of: (i) a transportable carbonic anhydrase downstream of a gene expression regulatory element active in at least a portion of a root cell of a plant, or (ii) at least a portion of a polynucleotide of any one of claims 4 to 11 or 16 to 18.

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