Carbon sequestration using transgenic plants expressing carbonic anhydrase

Abstract

Root-specific expression of carbonic anhydrase increases the plant's ability to sequester environmental carbon. Herein, plants are genetically engineered so that the plant's heritable genetic material contains a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells, such that carbonic anhydrase is transported from the plant's roots into the extracellular environment. These genetically modified plants demonstrate increased levels of carbonic anhydrase activity in the extracellular environment of plant roots, which enhances carbon sequestration 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. Genetically modified plants that express enzymes to promote CO sequestration are provided, along with enzyme sequences and their uses in various applications. The present invention further relates to such modified plant parts, such as plant cells, plant parts, plant organs, fruits, seeds, embryos, germplasm, and processed plant products. [Background technology]

[0002] Anthropogenic greenhouse gas emissions have already caused an increase in Earth's surface temperature of approximately 1°C above pre-industrial levels (Masson-Delmotte, 2018). Carbon dioxide (CO2) emissions are the primary cause of this temperature rise, and efforts to meet the Paris Agreement's requirement to stay below 2°C, and ideally below 1.5°C, to mitigate the worst impacts of climate change (Horowitz, 2016) require a combined effort to reduce global CO2 emissions and develop technologies to increase atmospheric CO2 sequestration (Masson-Delmotte, 2018). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2015 / 183935 [Patent Document 2] International Publication No. 2013 / 159228 Summary of the Invention [Means for solving the problem]

[0004] The inventors unexpectedly discovered that carbonic anhydrase genes expressed in root cells can enable CO sequestration in the extracellular environment of the root, e.g., as bicarbonate. In doing so, they provided a solution for atmospheric CO sequestration relevant to industrial biotechnology. Prior to the present invention, carbonic anhydrase genes had never been overexpressed in plant root cells, nor had carbonic anhydrase proteins been exported from plant root cells, nor had carbonic anhydrase proteins been overexpressed on the surface or in the extracellular environment of plant root cells. Thus, the approach used herein is novel. Overall, the present invention achieves enhanced carbon sequestration from the atmosphere without observable negative or adverse anatomical, physiological, biochemical, or developmental effects on the modified plant.

[0005] The present invention provides plants containing a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells, or plants in which the plant's heritable genetic material contains 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 plant's roots into the extracellular environment. Thus, the present invention provides plants whose genetic material has been modified such that the modified plant expresses carbonic anhydrase or a portion thereof in at least some of the plant's root cells. This expression in root cells is additional to any endogenous expression of carbonic anhydrase in the plant, particularly in plant roots where endogenous expression does not provide secretable / transportable carbonic anhydrase or any substantial carbonic anhydrase activity in the extracellular environment of the plant root.

[0006] The expressed carbonic anhydrase may result in an increase in the level of extracellular root carbonic anhydrase activity, or in other words, carbonic anhydrase activity in the extracellular environment of the plant roots, which may be soil or any other location in which a plant may germinate or grow or be cultivated, including, for example, aeroponic and / or hydroponic growing environments.

[0007] Also provided herein are plants in which carbonic anhydrase is overexpressed in the extracellular environment of root cells compared to plants that do not contain a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells, or plants in which the heritable genetic material of the plant does not contain a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0008] Additionally or alternatively, provided herein are plants whose heritable genetic material has been modified to contain a gene encoding a secretable / transportable carbonic anhydrase protein under the control of a promoter active in root cells. The genetic modification results in carbonic anhydrase being overexpressed in the extracellular environment of the plant root compared to a plant 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, or compared to an unaltered plant.

[0009] In some embodiments, the gene encoding the secretable / transportable carbonic anhydrase protein further comprises a signal peptide sequence to direct secretion / transport of the carbonic anhydrase into the extracellular environment of the plant root.

[0010] Also provided herein is a polynucleotide sequence comprising a sequence encoding a polypeptide sequence under the control of a suitable promoter, the polypeptide sequence comprising a carbonic anhydrase sequence and a signal peptide sequence, the promoter being active in plant roots and capable of driving expression of the carbonic anhydrase in root cells, and the signal peptide sequence being capable of directing secretion / transport of the carbonic anhydrase into the extracellular environment of the plant root. The signal peptide sequence of any polynucleotide sequence disclosed herein can be either natively encoded or translationally fused to the carbonic anhydrase sequence to ensure secretion / transport of the carbonic anhydrase into the extracellular environment of the plant root. The signal peptide sequence for directing secretion / transport can be derived from any secreted protein transported to the extracellular environment by the protein secretory pathway. The signal peptide sequence can also be derived from an extensin protein, and in some embodiments, can be derived from a carrot plant or a vacuolar sorting protein, or can be derived from a pea plant.

[0011] Carbonic anhydrase (including polynucleotides encoding it or peptides thereof) can be transported and, in some embodiments, can be secreted or secretable. Additionally, in some embodiments, carbonic anhydrase can be attached to the outside of the cell or to the plasma membrane by a transmembrane domain, or a transmembrane helix, or a membrane anchor, or a glycosylphosphatidylinositol (GPI) anchor, or any other biomolecule that keeps the carbonic anhydrase attached to the plasma membrane or cell wall after secretion from the cell.

[0012] The polynucleotide or plant promoter, signal peptide sequence, or carbonic anhydrase disclosed herein can be derived from a gene from the same plant species or variety as the plant in which it is expressed. The promoter can be root-specific, and in some embodiments, root hair and / or root epidermis-specific. The promoter's expression region can also include other cells and tissues of the plant, as long as the expression region includes the root.

[0013] Also provided herein are carbonic anhydrases that further comprise a transmembrane domain or a membrane anchor sequence. The carbonic anhydrase can be of the α, β, γ, δ, ζ, η, θ, or ι subtype. In some embodiments, it is a monomer.

[0014] Polypeptide sequences encoded by the polynucleotides disclosed herein are provided herein. Also provided are plant cells, cell lines, or progeny thereof comprising the polynucleotide sequences or polypeptide sequences disclosed herein. The progeny may be descendants of the plant cells or cell lines.

[0015] In some embodiments, the plants, polynucleotides, polypeptides, cells, cell lines, or progeny disclosed herein may be derived from a plant that is a crop. In some embodiments, the plant is a row crop or a cover crop. In some embodiments, the plant is selected from corn, soybean, pea, cotton, canola, camelina, potato, tomato, sugar beet, cassava, sweet potato, alfalfa, wheat, barley, sorghum, oat, sorghum, millet, rye, teff, rice, or clover, cress, brassica, vetch, and prairie grass. Furthermore, the polynucleotides, polypeptides, cells, cell lines, or progeny disclosed herein may be derived from a plant that is a tree. In some embodiments, the tree is poplar, spruce, pine, eucalyptus, oil palm, or rubber.

[0016] Also provided herein are plants, polypeptides, polynucleotides, cells, cell lines, or progeny disclosed herein, wherein more carbon is sequestered in the extracellular environment of the plant compared to a plant 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.

[0017] Further provided are plant parts, plant tissues, plant organs, plant cells, plant protoplasts, embryos, callus, cell cultures, pollen grains, or seeds derived from or obtained from the plants disclosed herein.

[0018] A vector comprising the polynucleotide sequence disclosed herein is provided herein. The vector is optionally a plasmid. The plasmid according to the present invention may further comprise one or more other elements selected from an enhancer, a plant selection marker, a multicloning site, or a recombination site. The construction, modification, propagation, and production of vectors or plasmids are well known to those skilled in the art.

[0019] Also provided are compositions for transforming plant cells, comprising a polynucleotide, vector, or plasmid disclosed herein, and optionally a microparticle coated with the polynucleotide or vector. The microparticle may be metallic or synthetic. In some embodiments, the metal is tungsten or gold.

[0020] Also provided is a bacterium comprising a polynucleotide, plasmid, or vector disclosed herein, optionally an E. coli or Agrobacterium species. In some embodiments, the Agrobacterium is A. tumefaciens.

[0021] Also provided herein are plants comprising a polynucleotide disclosed herein stably integrated into its genome, in some embodiments genetically integrated into its genome.

[0022] (a) providing a vector or plasmid disclosed herein; (b) activating a promoter operably linked to DNA encoding the polypeptide. Including, (c) Optionally, provided herein are methods for producing a plant or seed in which a polypeptide is overexpressed in the extracellular environment of a cell compared to a plant in which the heritable genetic material of the plant does not contain a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0023] Also provided herein is a method for increasing a plant's ability to sequester carbon in soil, comprising modifying the plant's heritable genetic material so that carbonic anhydrase protein is transported from the plant's roots into the extracellular environment. The modification of the heritable genetic material may include inserting at least one polynucleotide into the heritable genetic material of the plant cell. The modification of the heritable genetic material may further include gene editing the heritable genetic material of the plant cell.

[0024] The method according to the present invention can use classical and well-known techniques of genetic modification, including transformation methods, by which the polynucleotide of the present invention is incorporated into the plant genome. Any necessary root cell expression regulatory elements can be present. In some embodiments, this incorporation is stable and heritable, advantageously allowing the introduction of modifications into specific lines of crop plants for the purpose of crop improvement or breeding programs. Gene editing methods can be used to incorporate or generate the polynucleotide of the present invention.

[0025] Although various methods for gene editing are known, including TALE nuclease (TALEN) or zinc finger, CRISPR system can also be used.For example, the CRISPR-Cas system can be used, in which guide RNA (gRNA) is selected to target the action of CRISPR-Cas protein to desired genomic locus, and cause homologous recombination (HR) event or non-homologous end joining repair, that is, the insertion-deletion of polynucleotide into plant genome.

[0026] Thus, the polynucleotides disclosed herein can include a polynucleotide that encodes a CRISPR-Cas protein and optionally also encodes a guide RNA (gRNA), where the gRNA directs the CRISPR-Cas protein to the locus of an endogenous carbonic anhydrase coding sequence in the plant cell genome, and optionally, a regulatory element is inserted to cause expression of the carbonic anhydrase in at least some root cells of the plant.

[0027] Also provided herein is a processed plant product 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 active in at least a portion of a root cell of the plant, or (ii) a polynucleotide or at least a portion of a polynucleotide disclosed herein.

[0028] The invention will now be further described by reference to the examples and accompanying drawings. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 shows an exemplary diagram of the genetic vector used to transform soybean to express a secreted version of soybean carbonic anhydrase in roots. [Figure 2]Figure 2 demonstrates that carbonic anhydrase activity in the extracellular environment around the roots is enhanced in genetically engineered plants compared to wild-type (control) plants. A) Carbonic anhydrase activity in the supernatant after enzyme release from root tissue of control and engineered soybean hairy roots. Standard error bars are shown. B) Carbonic anhydrase activity in the hydroponic solution in which control and engineered plants were grown. Standard error bars are shown. C) The genetically engineered plants produce a measurable decrease in soil pH compared to both the soil-only control pots and the control plants. This demonstrates that carbonic anhydrase catalyzed the conversion of atmospheric CO2 to bicarbonate and protons in the soil. Standard error bars are shown. D) Engineered plant roots sequester CO2 from the atmosphere, whereas control plant roots cannot. CO2 sequestration is shown for pots containing soil and plant roots (aerial tissue excised) over a 30-minute measurement period. Standard error bars are shown. E) Carbonic anhydrase activity in the extracellular environment of wild-type and transgenic plant roots, indicating that the transgenic plants have higher carbonic anhydrase activity. F) No discernible differences in growth phenotype exist between control and engineered Arabidopsis plants that secrete carbonic anhydrase from their roots. Photographs taken of a 5-week-old control plant (left) and an engineered plant of the invention (right). Scale bar indicates 5 cm. [Figure 3] Figure 3 shows a general schematic diagram of the DNA sequence of a fusion carbonic anhydrase protein. Such a construct is characterized by including a gene sequence encoding a protein with carbonic anhydrase activity, a promoter ensuring expression of this sequence in, for example, plant root tissue, a terminator, and a peptide sequence specifying cellular export, such as an N-terminal signal peptide. [Figure 4]Figure 4 demonstrates that carbonic anhydrase activity in the extracellular environment around the roots is enhanced in genetically engineered plants compared to wild-type (control) plants. Figure A) shows that carbonic anhydrase activity in the hydroponic solution of genetically engineered soybean plants that transport carbonic anhydrase from the plant roots into the extracellular environment is higher compared to control plants. Standard error bars are shown. Figure B) shows that genetically engineered soybean plants that transport carbonic anhydrase from the plant roots into the extracellular environment produce a measurable decrease in soil pH compared to control plants, demonstrating that carbonic anhydrase catalyzed the conversion of atmospheric CO2 to bicarbonate and protons in the soil. Standard error bars are shown. Figure C) shows that genetically engineered soybean plants with carbonic anhydrase embedded on the root surface produce a measurable decrease in soil pH compared to control plants, demonstrating that carbonic anhydrase catalyzed the conversion of atmospheric CO2 to bicarbonate and protons in the soil. Standard error bars are shown. D) Shows an increase in carbonic anhydrase activity in the hydroponic solution in which engineered soybean plants with alternative carbonic anhydrases were grown compared to the hydroponic solution in which control plants were grown. Standard error bars are shown. E) Demonstrates an increase in carbonic anhydrase activity from the roots of engineered soybean plants compared to the control plants. Standard error bars are shown. F) Shows that there are no discernible differences in growth phenotype between the control and engineered soybean plants that secrete carbonic anhydrase from their roots. Photographs taken of a 5-week-old control plant (left) and an engineered plant of the invention (right). Scale bar indicates 5 cm. [Figure 5]Figure 5A+B shows confocal images of soybean protoplasts expressing fluorescent protein-targeted carbonic anhydrase proteins. A) Conticribra weissflogii carbonic anhydrase fused to a fluorescent protein. B) Conticribra weissflogii carbonic anhydrase fused to a fluorescent protein with a PsBP80 signal peptide at the N-terminus of the fusion protein and a PsBP80 transmembrane domain at the C-terminus of the fusion protein. C+D: Light microscope 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 indicate the following: A) 10 μm, B) 20 μm, C) 50 μm, D) 50 μm. The arrows in A) highlight cytoplasmic GFP expression, and in B) highlight fluorescent protein expression localized to the plasma membrane of soybean protoplasts. The arrows in C) left and right images and D) left image highlight the area of ​​GUS-stained roots. [Figure 6] Figure 6 demonstrates that carbonic anhydrase activity in the extracellular environment around the roots of genetically engineered plants enhanced inorganic carbon sequestration in the soil compared to wild-type (control) plants. A) Analysis of soil inorganic carbon content demonstrating that soils growing engineered soybean plants of the present invention accumulated more inorganic carbon than soils growing control soybean plants and soils without plants. B) Shows that soils growing engineered Arabidopsis thaliana plants of the present invention accumulated more soil inorganic carbon than soils growing control Arabidopsis thaliana plants after treatment with hydrated lime, Ca(OH). C) Shows that the results shown in B are consistent when soil inorganic carbon content is normalized by soil organic carbon content. D) Shows that soils growing engineered soybean plants of the present invention accumulated more soil inorganic carbon than soils growing control soybean plants. E) Shows that the results shown in D are consistent when soil inorganic carbon content is normalized by soil organic carbon content. DETAILED DESCRIPTION OF THE INVENTION

[0030] Weathering-mediated CO2 sequestration One way that CO2 is naturally sequestered from the atmosphere is through the process of chemical 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, it breaks down the rocks, releasing bicarbonate ions (HCO3 - ) and calcium ions (Ca 2+ Chemical reactions occur that release ions (e.g., ions from rocks). These ions are transported by surface waters to the oceans, where they precipitate and fall to the ocean floor, resulting in 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 the proposal of methods to accelerate chemical rock weathering. These methods, known as enhanced weathering, involve substantially increasing the surface area of ​​rock exposed to rain (containing dissolved CO2) by mechanically grinding it into a powder and distributing it over terrestrial ecosystems. This has been proposed to greatly increase the rate at which rocks are weathered and, therefore, the rate at which CO2 is sequestered from the atmosphere (Strefler et al., 2018).

[0031] Carbonic anhydrase Carbonic anhydrase converts gaseous carbon dioxide (CO2) and water (H2O) into bicarbonate ions (HCO3 - ) and hydrogen ions (H + ) to [CO2 + H2O ⇔ HCO3 - +H +Carbonic anhydrase is a group of naturally occurring enzymes that catalyze the removal of CO2 from industrial flue gases. Carbonic anhydrase is a widely distributed enzyme, with members of this enzyme family found in organisms spanning the tree of life, from bacteria and archaea to plants and animals. Given the enzyme's activity, there has been interest in using carbonic anhydrase in industrial settings to capture CO2 from industrial flue gases (as discussed in Boone et al., 2013 and Steger et al., 2022). WO 2015 / 183935 also describes the use of modified carbonic anhydrase to catalyze the removal of CO2 from industrial flue gases. The use of modified carbonic anhydrase bound to a matrix and cross-linked with inorganic ions, such that bicarbonate precipitates, thereby removing CO2, is also disclosed. This process is used in desalination and extracorporeal CO2 removal. Similarly, WO 2013 / 159228 describes an industrial process in which CO2 is contacted with a carbonic anhydrase solution containing additional absorbing compounds that together sequester the CO2.

[0032] Carbonic anhydrase (CA) requires a metal cofactor (typically Zn 2+ However, in some cases, Cd 2+ , Co 2+ , Fe 2+ and Mn 2+However, one newly described class of carbonic anhydrases is the metalloenzyme, which has been shown to be active under metal-free conditions (Hirakawa et al., 2021). To date, eight classes of carbonic anhydrases (α, β, γ, δ, ζ, η, θ, and ι) have been identified, with varying distribution across three domains of life. α, β, and γ carbonic anhydrases are present in eukaryotes and prokaryotes, while other classes are found in some species of microalgae and protists. The recently described ι-CA was 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 their diversity in form, carbonic anhydrases have diverse functions both between and within organisms. For example, in plants, carbonic anhydrases are present in several different plant tissues, and their subcellular localization varies (DiMario et al., 2017). All carbonic anhydrases, regardless of their class or the organism in which they are found, perform the same biochemical reaction: the conversion of gaseous CO2 and water (H2O) to bicarbonate ions (HCO3), as described above. - ) and hydrogen ions (H + ) to catalyze the interconversion of

[0033] Recently, Matt et al. (2022) described the expression of a glycosylphosphatidylinositol-anchored membrane-bound extracellular carbonic anhydrase in the calcifying primary mesenchyme cells of sea urchin larvae. These findings demonstrated that extracellular CO2 hydration suggests that the resulting bicarbonate is taken up by cells for the construction of carbonate structures that form the skeletal tissue of this organism.

[0034] Plants offer an intriguing opportunity to utilize carbonic anhydrase for CO2 sequestration as bicarbonate in the soil and for long-term sequestration of that bicarbonate via groundwater in a manner similar to chemical weathering processes. Plant roots are in direct contact with the soil and with gaseous CO2 in the soil, which is itself in equilibrium with atmospheric CO2.

[0035] 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 phytase from the fungus Aspergillus ficuum fused at its N-terminus to a signal peptide sequence from the carrot extensin gene. This recombinant gene was then fused to a promoter from the Arabidopsis Pky10 gene, conferring gene expression in the roots of transgenic soybean plants.

[0036] Herein, we propose engineering plants to express in their root cells (specifically, the outer cell layer, i.e., epidermal cells) an engineered form of carbonic anhydrase that is secreted from the cells into the extracellular environment. In particular, targeting plants that cover large surface areas of the Earth (e.g., crop plant species) provides a means by which atmospheric CO2 can be sequestered on a large scale as bicarbonate ions in soil. After transport by surface waters, some of this bicarbonate is expected to be transported to and buried in the oceans.

[0037] Bioremediation refers to any process that uses biological systems (e.g., plants or bacteria) to remove environmental pollutants, such as CO2, from natural or industrial environments. Bioremediation methods, such as those presented by the present invention, have certain advantages. Bioremediation often requires fewer resources and less energy than industrial, artificial techniques and can be specifically modified through genetic engineering to address the specific needs of particular environments. Furthermore, these biological methods do not produce hazardous waste as by-products and therefore do not require expensive, specialized waste disposal.

[0038] There is a need in the art to find methods for accelerating long-term CO2 sequestration by utilizing bioremediation methods. Furthermore, there is a need to achieve this via scalable methods that can be implemented rapidly and globally to urgently address the problems of anthropogenic CO2 emissions and elevated atmospheric CO2 concentrations. The present invention addresses this need by demonstrating that expression of carbonic anhydrase in plant roots, which results in increased levels of extracellular carbonic anhydrase activity, results in enhanced CO2 sequestration via the conversion of CO2 to bicarbonate. The present invention also demonstrates that multiple different native or engineered carbonic anhydrase genes from different species can provide this function in plants. Unexpectedly, the inventors have found that this function can be provided when the carbonic anhydrase protein is secreted from the plant and / or when the carbonic anhydrase protein is anchored to the external periphery of the cell, for example, via either a transmembrane domain or an anchor. Optionally, the N- or C-terminus can be fused to a transmembrane domain, a transmembrane helix, or a glycosylphosphatidylinositol (GPI) anchor, or other biomolecule that keeps the carbonic anhydrase bound to the plasma membrane or cell wall after secretion from the cell. The present invention also demonstrates that the various compositional forms of carbonic anhydrase proteins described above can be expressed in plants using various promoters to achieve the desired expression in root tissue.

[0039] The following detailed description conveys exemplary embodiments of the invention in sufficient detail to enable those skilled in the art to practice the invention. Features or limitations of the various described embodiments do not necessarily limit other embodiments of the invention, or the invention as a whole. Accordingly, the following detailed description does not limit the scope of the invention, which is defined solely by the claims.

[0040] Conventional techniques in botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, and recombinant DNA technology, and bioinformatics, for use in practicing the present invention, are all readily known and available to those skilled in the art, and specific techniques are explained fully in the literature.

[0041] The term "genetically engineered" is used herein, and as is well known to those skilled in the art, this term may encompass the terms "genetically modified" and "gene editing," each of which is understood in the art as having different meanings and underlying technical methodologies. However, the term "modified" in its open-ended sense herein may more simply be understood to mean "altered," "changed," or "different." The terms "altered," "altered," and "modified" may be used interchangeably herein. The terms "increase," "improve," or "enhance" are used interchangeably herein.

[0042] The present invention provides a plant in which the heritable genetic material of the plant contains 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 plant roots into the extracellular environment.

[0043] Plants of the present invention may be genetically modified plants, genetically enhanced plants, non-naturally occurring plants, and / or genetically engineered plants. In some examples, the plants may be transgenic plants.

[0044] Plants according to the present invention can be monocotyledonous or dicotyledonous; in some embodiments, they are crop plants, such as fruits, vegetables, grains, oilseeds, and legumes, which can be commonly 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. The plants can also be trees, particularly those common in forestry and land management, carbon capture, and biofuel or biomass production. The plants can be horticultural plants. In some embodiments, the plants are cover crops, which are plants used to cover exposed soil between harvests. Cover crops are used to mitigate soil erosion, increase soil fertility and quality, conserve water, manage weeds / pests and diseases, and maintain biodiversity and wildlife in agricultural ecosystems. Cover crops can be used during fallow periods in arable agriculture. During fallow periods, arable land is not planted with harvestable seeds for at least one or more vegetative cycles. This restores arable land. Cover crops can be planted during the fallow period to help restore the land.

[0045] Heritable genetic material refers to any polynucleotide sequence and any associated genetic elements in any cell or organelle that can be passed from one generation to subsequent generations via sexual or asexual reproduction. Sexual plant breeding methods can include propagation. Asexual reproduction, also known as clonal propagation, by grafting plant cuttings also counts as incorporating heritable genetic material into the plant's genome. Thus, heritable genetic material can only be present in plant parts.

[0046] The term "gene" refers to a polynucleotide sequence that encodes a polypeptide, including introns and exons, and all or part of an open reading frame (ORF) to enable transcription. The term code refers to the ability of a nucleotide sequence to act as a template for translation, whereby the arrangement of nucleotides indicates codons that further indicate amino acids. A gene may further include additional genetic elements, such as promoters, repressors, terminators, enhancers, and / or transcription factor binding elements. A gene may be inserted into the genome of an organism using genetic engineering methods known in the art, such as transformation. A gene may be exogenous (i.e., non-native, inserted into an existing genome) or endogenous (i.e., present in the genome as a native gene).

[0047] A polypeptide of the invention may be a fusion protein, which is a polypeptide derived from a single nucleotide sequence containing two or more genes or portions of genes that encode separate polypeptides that are not natively transcribed and translated together.

[0048] Typically, a polynucleotide of the present invention comprises 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., anchors it in the plasma membrane, a stop codon, and a terminator, as represented in Figure 3.

[0049] The coding sequence of the carbonic anhydrase encoded by the DNA polynucleotide sequence of the present invention may correspond to any of the polynucleotide sequences of SEQ ID NO:1 or SEQ ID NO:2, or a sequence at least 65% identical to any of said sequences; or a sequence at least 70% identical to any of said sequences; or a sequence at least 80% identical to any of said sequences, or a sequence at least 85%, at least 90%, or at least 95% identical to any of said sequences. In some embodiments, the carbonic anhydrase has any of the percent homologies referred to herein and retains carbonic anhydrase activity.

[0050] The amino acid sequence of the carbonic anhydrase encoded by the polynucleotide sequences of the invention may correspond to any of the amino acid sequences of SEQ ID NO:8 or SEQ ID NO:9, or a sequence at least 65% identical to any of said sequences; or a sequence at least 70% identical to any of said sequences; or a sequence at least 80% identical to any of said sequences, or a sequence at least 85%, at least 90%, or at least 95% identical to any of said sequences. In some embodiments, the carbonic anhydrase has any of the percent identities referred to herein, and preferably retains carbonic anhydrase activity in the extracellular root environment.

[0051] The polynucleotide may correspond to a full-length carbonic anhydrase or a portion thereof. As used herein, the art-recognized terms "portion," "variant," "homologue," or other representative language used in reference to a carbonic anhydrase sequence or functional fragment thereof refers to any carbonic anhydrase ortholog of a different polynucleotide or polypeptide sequence from any species, including plants.

[0052] In terms of percentage identity to a reference sequence, for example SEQ ID NO: 1, 2, 8 or 9, a variant of carbonic anhydrase 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 to the reference sequence.

[0053] Carbonic anhydrase converts water (H2O) and carbon dioxide (CO2) into bicarbonate (HCO3 - ) and hydrogen (H + ) to: H2O+CO2⇔HCO3 - +H +

[0054] In the absence of carbonic anhydrase activity, the pH of the aqueous environment determines whether the forward or reverse reaction is favored. Carbonic anhydrase is widely distributed in all living organisms. Carbonic anhydrase determines the rate at which this reaction occurs and the ratio of CO2 to HCO3. - Carbonic anhydrases from different species may have minimal sequence similarity but may adopt similar tertiary structures that catalyze this reaction in very different contexts.

[0055] In some embodiments, the carbonic anhydrase can be an α, β, γ, δ, ζ, η, θ, or ι subtype of carbonic anhydrase, and in some embodiments, is monomeric. Three classes of carbonic anhydrases exist natively in plants: α, β, and γ subtypes. The β subtype is better characterized and includes isoforms with high efficiency for CO hydration; for example, the β subtype functions as part of C4 photosynthesis in C4 plants. The β subtype is dimeric, while the α subtype is monomeric. Monomeric subtypes have the advantage that there is no requirement for multimer formation at the surface of the cell.

[0056] The carbonic anhydrase sequences used in the present invention may be homologs, analogs, orthologs, or paralogs of any subtype of carbonic anhydrase isolated from any species. It is expected by those skilled in the art that any carbonic anhydrase will function equivalently in the present invention, so long as it is suitable for expression by the plants of the present invention (e.g., it is composed of amino acids naturally occurring in protein form). The examples of the present application demonstrate that sequence identity or similarity between carbonic anhydrases is not required for the plants of the present invention to exhibit their surprising ability to sequester CO2. As noted above, carbonic anhydrases from different species can have similar tertiary structures regardless of sequence similarity. Thus, those skilled in the art will understand that plants of the present invention overexpressing any type of carbonic anhydrase in the extracellular environment of root cells will function to sequester CO2. Those skilled in the art will understand that carbonic anhydrases are defined by their function. Thus, the conversion of gaseous CO2 and water (HO) to bicarbonate ions (HCO3) is essential. - ) and hydrogen ions (H + ) is contemplated as a carbonic anhydrase protein as part of the present invention.

[0057] In some embodiments, carbonic anhydrases are selected that have high catalytic efficiency in the direction of bicarbonate formation so that the reverse reaction is not favored. Carbonic anhydrases can be selected based on efficacy, e.g., determined via enzyme activity assays. Additionally, codon usage can be altered to enhance expression for specific plant species.

[0058] In some embodiments, in any aspect of the 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 promoter active in root cells is a promoter that currently enables expression of a polynucleotide sequence in root cells, i.e., it is sufficient for protein production in root cells. The promoter may be active in other cells of the plant, and in some embodiments, is relatively high in root cells. The activity of the promoter may be enhanced when compared to a wild-type promoter. The activity of the promoter may be enhanced conditionally. The promoter may be an inducible promoter. The activity of the promoter may vary, for example, seasonally, daily, or in response to external signals, i.e., soil temperature, pH, nutrients. The promoter may be constitutively active.

[0059] The promoter may be a synthetic promoter composed of various selected elements. For example, such a synthetic promoter may include a root cell-specific transcription factor binding element upstream of the promoter element. Two or more transcription factor binding elements, which may be the same or different, may be present. Multiple such transcription factor binding elements may function to enhance the activity and / or specificity of the promoter in root cells. The promoter may be derived from a non-plant organism, for example, a 35S promoter.

[0060] In another aspect, 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 in other plant cell types at endogenous levels. The promoter can be selected based on its predicted function in a particular species. Those skilled in the art are familiar with many root cell-specific promoters. A root cell-specific promoter can be highly expressed in roots, although it may be active in some or all other plant cells at some expression level.

[0061] The root-specific promoter encoded by the DNA polynucleotide sequence of the present invention may correspond to any of the polynucleotide sequences of SEQ ID NO:6 or SEQ ID NO:7, or a sequence at least 65% identical to any of said sequences; in some embodiments, a sequence at least 70% identical to any of said sequences; in some embodiments, a sequence at least 80% identical to any of said sequences, or a sequence at least 85%, at least 90%, or at least 95% identical to any of said sequences. In some embodiments, the encoded carbonic anhydrase has any of the percent identities referred to herein and retains carbonic anhydrase activity.

[0062] The polynucleotide may correspond to a full-length root-specific promoter or a portion thereof. As noted above, it is expected by those skilled in the art that any promoter active in plant roots will function equivalently in the present invention.

[0063] As used herein, the art-recognized terms "portion," "variant," "homologue," or other representative terminology used in connection with a root-specific promoter sequence or functional fragment thereof refers to any root-specific promoter ortholog of a different polynucleotide or polypeptide sequence from any species, including plants.

[0064] In terms of percentage identity to a reference sequence, e.g., SEQ ID NO: 6 or 7, a variant of a 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 to the reference sequence.

[0065] In one embodiment, a plant is provided in which the heritable genetic material of the plant 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 having at least 65% identity to SEQ ID NO:1 or SEQ ID NO:2, and optionally the promoter comprises SEQ ID NO:6 or SEQ ID NO:7 or a sequence having at least 65% identity to SEQ ID NO:6 or SEQ ID NO:7.

[0066] In one embodiment, a plant is provided in which the heritable genetic material of the plant 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 having at least 65% identity to SEQ ID NO:1 or SEQ ID NO:2, and optionally the promoter comprises SEQ ID NO:6 or SEQ ID NO:7 or a sequence having at least 65% identity to SEQ ID NO:6 or SEQ ID NO:7; and the signal peptide sequence comprises SEQ ID NO:3 or SEQ ID NO:4 or a sequence having at least 65% identity to SEQ ID NO:3 or SEQ ID NO:4.

[0067] A root cell-specific promoter can be ubiquitously active at endogenous levels in all plant cells. Endogenous levels of carbonic anhydrase mRNA and full-length polypeptide are lower in non-root cells. Carbonic anhydrase expression levels can exceed endogenous levels in any biological context in which carbonic anhydrase is expressed. The biological context can be in response to changes in the environment.

[0068] The term secreted is used to describe the transport of a protein from inside to outside a cell. It is used interchangeably with transported or exported. Thus, a secretable / transportable carbonic anhydrase is capable of being secreted / transported to the extracellular environment. In some embodiments, the carbonic anhydrase is transported across the plasma membrane via a secretory pathway. The secretory pathway can be conventional or non-conventional. In some embodiments, the carbonic anhydrase is transported via the conventional secretory pathway, resulting in transport of the carbonic anhydrase across the plasma membrane at the periplasmic periphery. Non-conventional transport of proteins, such as carbonic anhydrase, can occur via one of four pathways: direct protein translocation across the plasma membrane via membrane transport proteins, lysosomal secretion, exosome release, and blebbing. Transport of the carbonic anhydrase can be active or passive. In some embodiments, a secreted protein, such as a carbonic anhydrase of the present invention, is located in the plasma membrane (also known as the extracellular membrane) in a cell. Carbonic anhydrase may contact the outer cell membrane via an extracellular matrix protein, e.g., a glycoprotein, e.g., an extensin, or via a polysaccharide. Suitable glycoproteins and polysaccharide anchors are well known in the art. Carbonic anhydrase may contact the plasma membrane via a molecular anchor, e.g., a transmembrane domain, a transmembrane helix, or a glycosylphosphatidylinositol (GPI) anchor, or other biomolecules that keep the carbonic anhydrase bound to the plasma membrane or cell wall after secretion from the cell. Carbonic anhydrase may contact the plasma membrane via a portion of the carbonic anhydrase polypeptide of the present invention, where this portion of the polypeptide represents a transmembrane protein. One or more transmembrane domains may be present. Carbonic anhydrase may not contact the plasma membrane. Secreted / transported carbonic anhydrase may be adjacent to the plasma membrane or may be in the extracellular environment in close proximity to the plasma membrane, e.g., in the cell wall region. The carbonic anhydrase may remain in close proximity to the plasma membrane but may extend beyond the cell wall region, hi some embodiments, the carbonic anhydrase is secreted into the environment surrounding the root, e.g., soil or alternative bedding medium if the plant is present in such a medium.In some embodiments, carbonic anhydrase is not secreted into plant root nodules. Carbonic anhydrase may continue to be secreted / transported ex vivo / ex planta when the plant is separated from other parts of itself, i.e., in the case of cuttings. Once secreted, secreted / transported carbonic anhydrase may diffuse into the extracellular environment or may remain on the root surface. Carbonic anhydrase secreted from root cells may diffuse to the extracellular environment of other tissues or cells, for example, adjacent to the root cells. This may include any cells in close proximity to the root or rhizosphere.

[0069] Carbonic anhydrase can be released from cells via non-secretory pathways, such as apoptosis, or any physical disruption of the cell membrane that exposes intracellular carbonic anhydrase to the extracellular environment.

[0070] Carbonic anhydrase can be overexpressed in the extracellular environment of root cells 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.

[0071] The term "overexpressed," in relation to carbonic anhydrase, refers to the expression level of the produced carbonic anhydrase protein compared to endogenous baseline expression. The term "produced" is understood to refer to the production of carbonic anhydrase protein and can be used interchangeably with the term expressed. 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 exhibit endogenous levels of carbonic anhydrase in all biological contexts, and these levels of carbonic anhydrase are used as baseline comparison controls for plants of the present invention. The baseline level of carbonic anhydrase typically reflects the endogenous level of carbonic anhydrase, which may vary depending on the internal and external environment of the plant. An increase in the level of carbonic anhydrase, i.e., any amount above the baseline, is considered to indicate overexpression of carbonic anhydrase protein. In some embodiments, the plants of the present invention express 0.005%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 25%, 50%, 75%, 100%, or 200% more carbonic anhydrase protein compared to endogenous baseline expression. Endogenous baseline expression can be established in a control plant, which can be a wild-type plant. Cells of the same type and morphology in the same location of the plant, i.e., root cells, should be compared to each other.

[0072] A plant 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 is a plant lacking a gene in its genome encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells. This plant has not been modified in the same manner as a plant of the present invention. This plant may be known as an untransformed plant compared to a transformed plant of the present invention. The plant may contain other genetically modified elements related or unrelated to carbonic anhydrase, separate from those of the present invention. A plant 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 may be called or used as a "control" plant. Thus, the control plant has not been genetically modified to alter the expression of the polynucleotides and / or polypeptides disclosed herein. This plant exhibits endogenous levels of carbonic anhydrase in all biological contexts, and these levels of carbonic anhydrase are used as a baseline comparison for the plants of the present invention. The baseline level of carbonic anhydrase typically reflects the endogenous level of carbonic anhydrase, which may vary depending on the plant's internal and external environment. 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 a reliable reference against which the expression of the polynucleotide or polypeptide of the present invention in modified plant material can be compared. Thus, the plant of the present invention can be genetically engineered compared to an unengineered plant. The control plant can be defined by determining the level of carbonic anhydrase activity by enzyme assays known in the art and defined herein. Carbonic anhydrase activity can be undetectable or at very low levels in the control plant compared to the plant of the present invention. The plant of the present invention exhibits an increased level of carbonic anhydrase activity compared to the control plant, i.e., any amount of increased activity described above. Typically, a greater than two-fold increase in carbonic anhydrase activity is observed in the plant of the present invention compared to a control or unmodified plant.In some embodiments, plants of the present invention have a greater than 2-fold, 3-fold, 4-fold, or 5-fold increase in carbonic anhydrase activity compared to control plants. In some embodiments, plants of the present invention have a greater than 0.005%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase in carbonic anhydrase activity compared to control plants. Carbonic anhydrase activity can be measured in root cells, whole roots, or whole plants. In some embodiments, plants of the present invention have a greater than 2-fold, 3-fold, 4-fold, or 5-fold increase in carbonic anhydrase activity in the extracellular space compared to control plants. In some embodiments, carbonic anhydrase activity is quantified using the Wilbur-Anderson units (WA) equation, or carbonic anhydrase esterase activity is measured by the rate of catalyzing the hydrolysis of a known concentration of p-nitrophenol acetate (pNPA) to p-nitrophenol (pNP) per unit time, which may alternatively be expressed in enzyme activity units, e.g., katals (kat) or international units (IU), or carbonic anhydrase activity is measured in units of conductivity changed per unit time, or carbonic anhydrase activity is quantified using a radiotracer-based assay in which activity is reported in counts or decays per unit time, or other equivalent methods for quantifying carbonic anhydrase activity well understood by those of skill in the art. Optionally, plants of the invention have a 2-fold, 3-fold, 4-fold, or 5-fold increase in carbonic anhydrase activity in WA units compared to control plants.

[0073] The gene encoding the transportable carbonic anhydrase protein may further comprise a signal peptide sequence to direct the transport of the carbonic anhydrase into the extracellular environment of the plant root. The term "signal peptide" may also be known as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, target peptide, leader sequence, or leader peptide. A signal peptide is usually located at the N-terminus of a polypeptide, but may also be located at the C-terminus. In the present invention, the signal peptide is located at the N-terminus.

[0074] Membrane-bound proteins can be type I or type II; type I membrane-bound proteins rely on a signal peptide that is subsequently cleaved to direct them towards the secretory pathway, while 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.

[0075] The signal peptide sequence encoded by 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 at least 65% identical to any of said sequences; in some embodiments, a sequence at least 70% identical to any of said sequences; in some embodiments, a sequence at least 80% identical to any of said sequences, or a sequence at least 85%, at least 90%, or at least 95% identical to any of said sequences. In some embodiments, the carbonic anhydrase encoded from the above DNA sequences has any of the percent identities referred to herein and retains carbonic anhydrase activity.

[0076] The polynucleotide may correspond to the full-length signal peptide or a portion thereof.

[0077] As used herein, the art-recognized terms "portion," "variant," "homologue," or other representative terminology used in reference to a signal peptide sequence or functional fragment thereof refers to any signal peptide sequence of a different polynucleotide or polypeptide sequence from any species, including plants.

[0078] In terms of percentage identity to a reference sequence, e.g., SEQ ID NO: 3 or 4, variants of a 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 to the reference sequence.

[0079] The signal peptide sequence cooperates with the promoter to ensure extracellular expression and transport of the polypeptide sequence into the extracellular environment.

[0080] · Suitable promoters; a sequence encoding a polypeptide sequence under the control of said suitable promoter; and A sequence encoding a signal peptide sequence wherein the polypeptide sequence comprises a carbonic anhydrase sequence, the promoter is active in plant roots and capable of driving overexpression of the carbonic anhydrase, and the signal peptide sequence is (prior to removal) capable of directing transport of the carbonic anhydrase into the extracellular environment of the plant root.

[0081] A suitable promoter is one that allows for the expression of the polypeptide sequence with which it is associated. The signal peptide sequence is sufficient to allow the transport of carbonic anhydrase into the extracellular environment of the plant root.

[0082] The signal peptide sequence can be either natively encoded or translationally fused to the carbonic anhydrase sequence to ensure transport of the carbonic anhydrase to the extracellular environment of the plant root.

[0083] The signal peptide sequence for directing transport may be derived from a protein secretory pathway, and in some embodiments, from a secreted protein that is transported to the extracellular environment by the conventional (or canonical) protein secretory pathway. The signal peptide sequence may be derived from an extensin protein, and in some embodiments, from a carrot plant or from a vacuolar sorting protein, or from a pea plant.

[0084] A plant or polynucleotide according to the invention may have a promoter and / or signal peptide sequence derived from a gene from the same plant species or variety as the plant. A plant or polynucleotide according to the invention may have a promoter and / or signal peptide sequence derived from a gene from a different plant species, cultivar, or variety than the plant. Plant varieties are genetic variations of plants of the same species that occur naturally in the environment. Cultivars are genetic variations of plants of the same species that have been developed by humans through controlled breeding.

[0085] 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.

[0086] The promoters of the present invention are active in plant tissues that function as roots, and in some embodiments, may be specific to root hair cells and / or root epidermal cells. Root-specific cells are cellular portions of a plant's root system that can function in root-specific capacities, i.e., anchorage, stability, shelter, nutrient harvesting and storage, water absorption, and macromolecule transport. Rhizomes (e.g., tubers, rhizomes, bulbs, corms, stolon, taro, and other plant-related organs located underground near the roots) provide similar underground functions to botanically defined root tissue and, for purposes of the present invention, can be considered plant tissues that function as roots and contain root cells.

[0087] The carbonic anhydrase may further comprise a transmembrane domain and / or a membrane anchor sequence. The transmembrane domain sequence may be the full-length transmembrane domain sequence or a portion thereof. The transmembrane sequence may encode a transmembrane domain that, once transported, resides partially in the plasma membrane or may span the entire plasma membrane. The transmembrane sequence may include post-translational elements, such as glycoprotein and polysaccharide structures. In some embodiments, the carbonic anhydrase may be bound to the outside of the cell or to the plasma membrane by a transmembrane domain, or a transmembrane helix, or a membrane anchor, or a glycosylphosphatidylinositol (GPI) anchor, or any other biomolecule that keeps the carbonic anhydrase bound to the plasma membrane or cell wall after secretion from the cell.

[0088] In some embodiments, the transmembrane domain is derived from Pisum sativum BP80. The transmembrane domain sequence encoded by the DNA polynucleotide sequences of the present invention may correspond to the polynucleotide sequence of SEQ ID NO: 5, or a sequence at least 65% identical to any of the foregoing sequences; in some embodiments, a sequence at least 70% identical to any of the foregoing sequences; in some embodiments, a sequence at least 80% identical to any of the foregoing sequences, or a sequence at least 85%, at least 90%, or at least 95% identical to any of the foregoing sequences. In some embodiments, the carbonic anhydrase has any of the percent identities referred to herein and retains carbonic anhydrase activity.

[0089] The polynucleotide may correspond to the full-length transmembrane sequence or a portion thereof. Additionally, the polynucleotide may include a sequence capable of directing the formation of a glycosylphosphatidylinositol (GPI) anchor or any other biomolecule that keeps the carbonic anhydrase bound to the plasma membrane or cell wall after secretion from the cell.

[0090] As used herein, the art-recognized terms "portion," "variant," "homologue," or other representative language used in connection with a transmembrane sequence or functional fragment thereof refers to any transmembrane sequence ortholog of a different polynucleotide or polypeptide sequence from any species, including plants.

[0091] In terms of percentage identity to a reference sequence, e.g., SEQ ID NO: 5, variants of a 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 to the reference sequence.

[0092] In some embodiments, the membrane anchor sequence corresponds to an entire glycophosphatidylinositol anchor sequence or a portion thereof.

[0093] Also provided herein are isolated polynucleotide sequences of the invention that may be fused to another polynucleotide sequence or a targeting peptide sequence or a transmembrane domain sequence. Polynucleotides may also be described as engineered or non-naturally occurring.

[0094] Also provided herein are isolated polypeptides of the present invention that can be fused to another polypeptide, a targeting peptide, or a transmembrane domain. Isolated polynucleotides or polypeptides are DNA or protein constructs that exist independently, away from the internal environment of a cell. They may contain DNA or protein-related elements. They may be recombinant. Polypeptides of the present invention may temporarily contain a signal peptide that is subsequently cleaved upon entry into the secretory pathway. Polypeptides of the present invention may also contain a signal peptide that is not cleaved upon entry into the secretory pathway and that indicates a first transmembrane domain that acts as a signal peptide.

[0095] 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, plant ancestors and progeny, as well as plant parts, including seeds, fruits, buds, stems / stem, leaves, roots (including tubers), and flowers, as well as tissues and organs, each of which contains a polynucleotide of the invention. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, each of which contains a polynucleotide of the invention.

[0096] In some embodiments, the plant is a crop, 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, adzuki bean, broad bean, pea, cowpea, cotton, canola, camelina, potato, tomato, sugar beet, cassava, yam, sweet potato, alfalfa, wheat, barley, sorghum, oat, sorghum, millet, rye, teff, triticale, Miscanthus, sorghum rice, or clover, cress, brassica (e.g., Brassica oleracea), vetch, and prairie grass (e.g., switchgrass).

[0097] The plant may be a tree, hi some embodiments, the tree is a poplar, spruce, pine, eucalyptus, oil palm, or rubber, among others, common in forestry and land management, food production, carbon capture, and biofuel or biomass production.

[0098] The plant may be an aquatic or semi-aquatic plant, hi some embodiments, the plant may be a duckweed, mangrove, Nymphalis, or rice.

[0099] The plants, polypeptides, polynucleotides, cells, cell lines, or progeny of the present invention may sequester more carbon in the extracellular environment of the plant compared to plants in which the plant's 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 via the conversion of CO2 to bicarbonate and hydrogen ions.

[0100] 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 the plants of the present invention.

[0101] 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 of a Ti or Ri plasmid, a left border repeat of a Ti or Ri plasmid, and at least one bacterial selection marker. The plasmid may also comprise an enhancer, a plant selection marker, a multiple cloning site, and / or a recombination site.

[0102] Also provided herein is a composition for transforming plant cells, comprising a polynucleotide of the invention or a vector comprising a polynucleotide of the invention. The vector may comprise a microparticle coated with the polynucleotide or vector.

[0103] Plant transformation is a common technique in many species. Any of several transformation methods can be used to introduce a gene of interest into a suitable ancestor cell. According to various aspects of the present invention, a polynucleotide of the present invention is introduced and expressed in a plant as a transgene via transformation. The terms "introduction" or "transformation," as used herein, encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. The described methods for plant transformation and regeneration from plant tissues or cells can be utilized for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, direct injection of DNA into plants, floral dipping, biolistics, virus or pollen transformation, and microinjection. Methods can be selected from, but are not limited to, electroporation, microinjection, RNA-coated particle bombardment, or viral transfection. The transformation method typically depends on the selected species. The polynucleotide can be transiently or stably introduced into the host cell and can be maintained non-integratively, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants. To select transformed plants, the plant material obtained in the transformation is usually subjected to selection conditions so that transformed plants can be distinguished from non-transformed plants. For example, seeds obtained in the above manner can be planted and, after an initial growth period, subjected to appropriate selection and screening.

[0104] Also provided herein are bacteria comprising a polynucleotide of the invention or a vector comprising a polynucleotide of the invention. In some embodiments, the bacterium is Escherichia coli or an Agrobacterium species, and in some embodiments, Agrobacterium tumefaciens. Any suitable cloning system may be used. Genetically modified plants, including crop plants, are produced in some embodiments via Agrobacterium tumefaciens-mediated transformation. 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 native carbonic anhydrase sequences to make them transportable and increase expression in root cells.

[0105] Also provided herein are plants comprising a polynucleotide of the invention genetically integrated into their genome, in some embodiments, whereby the polynucleotide is stably integrated into their genome.

[0106] (a) providing a vector comprising a polynucleotide of the present invention; (b) activating a promoter operably linked to DNA encoding the polypeptide. Including, (c) Optionally, methods for producing a plant or seed of the invention are also provided, wherein the polypeptide is overexpressed in the extracellular environment of the cell compared to a plant in which the heritable genetic material of the plant does not contain a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

[0107] Also provided is a method for increasing a plant's ability to sequester carbon in soil, comprising modifying the plant's heritable genetic material so that carbonic anhydrase protein is transported from the plant's roots into the extracellular environment. The normal ability of a plant to sequester carbon in soil is exhibited by plants that are not genetically modified in the same manner as the plants of the present invention. This ability may reflect an upper limit to the rate of CO2 conversion to bicarbonate ions and hydrogen ions in the extracellular environment of plants that are not genetically modified in the same manner as the plants of the present invention. This rate achieved in modified plants essentially depends on the transport of carbonic anhydrase from the plant's roots into the extracellular environment. Modifying the heritable genetic material may include inserting at least one polynucleotide into the heritable genetic material of a plant cell using transgenic engineering methods well known in the art.

[0108] Altering heritable genetic material can also include gene editing of the heritable genetic material of plant cells using genetic modification methods well known in the art, such as CRISPR, TALEN, and meganucleases. Base editing, whereby a single nucleotide can be engineered in a targeted gene, can also be used. Prime editing, a search-and-replace genome editing method used to insert precise small indels, including single or multiple base substitutions, transitions, and transversions, without the need for double-strand breaks and donor repair templates, can also be used. Any regulatory sequence can also be engineered using these techniques to result in increased expression of carbonic anhydrase, for example, by introducing random indels into the native regulatory sequence using the CRISPR-Cas system.

[0109] Any polynucleotide disclosed herein can further comprise a polynucleotide sequence encoding a CRISPR-Cas protein, and optionally a guide RNA (gRNA), where the gRNA directs the CRISPR-Cas protein to the locus of at least one copy of its endogenous carbonic anhydrase gene in the plant cell genome, whereby the root-specific promoter is inserted to cause expression of one or more copies of carbonic anhydrase in at least some root cells of the plant.

[0110] Native carbonic anhydrases can also be engineered using targeted nucleases and homology-directed repair or non-homologous end joining repair mechanisms, and gene editing tools to introduce a signal peptide into the native carbonic anhydrase gene using a template sequence containing the signaling peptide. Such gene knock-in techniques are well known in the art.

[0111] Also provided herein are processed plant products obtained from the 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 contain 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 the plant's root cells, or (ii) a polynucleotide or at least a portion of a polynucleotide of the invention present in the product. Processing may include chemical or physical manipulation of the product, i.e., pelleting, to form a product with desired properties suitable for a particular function. [Example]

[0112] Example 1 To demonstrate that plant roots can be engineered to produce active carbonic anhydrase protein, the soybean (Glycine max) carbonic anhydrase gene was selected for overexpression (accession number Glyma.19G135900 [SEQ ID NOs: 1 and 8]). This carbonic anhydrase contains an annotated "eukaryotic-type carbonic anhydrase" domain (Pfam #PF00194.24) in its protein sequence.

[0113] Expression of the carbonic anhydrase gene (SEQ ID NO:1 and SEQ ID NO:8) was placed under the control of a 35S promoter and cloned into the pCAMBIA-1300 plant expression vector. This vector contained a herbicide resistance gene (kanamycin), and the expression cassette was flanked by Agrobacterium LB and RB t-DNA sequences. This 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).

[0114] After transformation, reliably transformed root tissue was visually identified by expression of the visual reporter. Root tissue was isolated from the transformed roots, and equivalent untransformed root tissue was sampled as a control. Root tissue was homogenized in 700 μl of extraction buffer in a chilled 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 on the supernatant after this spin step.

[0115] Carbonic anhydrase assays were performed to measure carbonic anhydrase activity. This assay measures the rate of change in pH of CO2-saturated water after addition of root homogenates from transformed and untransformed (also known as wild-type control) root tissue. The pH change is determined by the release of protons (H) by carbonic anhydrase. +) The change in pH is directly proportional to the amount of bicarbonate produced by carbonic anhydrase. Such assays are known to those skilled in the art.

[0116] For each carbonic anhydrase assay, 100 μl of supernatant from homogenized root samples was used. For the non-root tissue control, 100 μl of extraction buffer without root tissue was used. Samples were added to 2 ml tubes containing 1100 μl of 20 mM Tris-HCl solution, pH 8.3, containing 0.003% bromothymol blue. This was followed by the addition of 800 μl of ice-cold CO2-saturated water. The time taken for the color to change from blue (pH 8.3) to yellow (pH 5.3) was recorded. All reactions were performed on ice.

[0117] Activity was calculated using the Wilbur-Anderson unit (WA) equation: WA unit = (t0-ts) / ts where t0 is the time it takes for a color change to occur in the no-root control reaction and ts is the time it takes for a color change to occur in the sample containing the supernatant from the homogenized roots.

[0118] Carbonic anhydrase activity was significantly increased by approximately 2× in transgenic root samples compared to wild-type root samples (P = 0.022669, one-tailed Student's t-test, unequal variance) (Figure 2A). Thus, soybean root cells can be engineered to have increased carbonic anhydrase activity through overexpression of a gene encoding a carbonic anhydrase protein, which is catalytically active when released from root cells into the extracellular environment.

[0119] Example 2 For carbonic anhydrases to facilitate the conversion of CO2 to HCO3 in the extracellular environment of plants, they must be secreted / transported from plant cells into the extracellular environment. There are several mechanisms by which cells target proteins for secretion / transport from cells into the extracellular environment. The two main ways this is achieved are the conventional secretory pathway and the non-conventional secretory pathway. The conventional secretory pathway achieves protein secretion / transport through the use of an N-terminal signal peptide.

[0120] To demonstrate that carbonic anhydrase proteins can be engineered for secretion / export from plant cells, we translationally fused the experimentally characterized signal peptide from the carrot (Daucus carota) extensin gene (G. Li et al., 2009) [SEQ ID NO: 3] to the soybean carbonic anhydrase gene provided in Example 1 [SEQ ID NO: 1 and SEQ ID NO: 8]. This fusion protein was placed under the control of a root-specific promoter [SEQ ID NO: 6] from the soybean expansin gene (Glyma.17G133100) and cloned into a plant transformation vector (Figure 1) that also contains a herbicide resistance gene (spectinomycin) and is flanked by Agrobacterium LB and RB t-DNA sequences. This vector was transformed into Agrobacterium tumefasciens (strain LBA4404) by electroporation, and positive colonies were selected and cultured using standard techniques. To demonstrate the broad utility of this technology across all plant species, this vector was transformed into Arabidopsis thaliana using the floral dip method (Clough and Bent, 1998) to generate stable transgenic lines.

[0121] It should be noted that while the soybean expansin gene promoter was used in this example, other root promoters are known in the art (e.g., (Y. Li et al., 2019; Marques-Bueno et al., 2016)). It would be expected by one of skill in the art that any root promoter selected from any species would be expected to function equivalently to drive expression in roots. It should also be noted that the technology of the present invention is expected to work when the carbonic anhydrase protein is expressed throughout the plant or in other plant tissues, so long as the expression region encompasses expression in the roots.

[0122] Experiments were conducted to demonstrate that plants expressing secretable / transportable carbonic anhydrase in root cells produced carbonic anhydrase activity in the extracellular environment of the root. Here, transformed plants and wild-type control plants were grown in hydroponic conditions for 6 weeks. At the 6-week point, individual plants were transferred to 15 ml of fresh hydroponic medium and allowed to grow for 2 days. After this 2-day period, the hydroponic solution was sampled and used in a carbonic anhydrase activity assay to measure the amount of carbonic anhydrase activity secreted / transported from the roots into the hydroponic medium.

[0123] Here, 100 μl of the hydroponic solution was added to a 2 ml snap-lid microcentrifuge tube containing 1050 μl of 20 mM Tris-HCl solution (pH 8.3) with 0.003% bromothymol blue and 100 μl of extraction buffer (200 mM Tris HCl pH 8.3, 1 mM EDTA, 20 mM MgCl, 50 mM NaCl, 100 mM NaSO). Subsequently, 750 μl of ice-cold CO2-saturated water (generated by a soda stream) was added to initiate the reaction. The time taken for the color change from blue (pH 8.3) to yellow (pH 5.3) was recorded and used to calculate enzyme activity as in Example 2. The reaction was performed on ice.

[0124] Carbonic anhydrase activity was significantly increased at 11× height in hydroponic medium samples from transgenic plants compared to wild-type control plants (P = 0.029404, one-tailed Student's t-test, unequal variance) (Figure 2B). Thus, plants transformed with this invention successfully expressed and secreted active carbonic anhydrase protein into the extracellular environment of the root, which then catalyzed the conversion of CO2 to bicarbonate.

[0125] Example 3 To demonstrate that this technology also works to sequester CO2 in soil, a separate experiment was conducted on soil-grown plants. Given that carbonic anhydrase catalyzes the conversion of CO2 and water to bicarbonate and protons, atmospheric CO2 sequestration in soil is directly proportional to soil pH changes. Here, transgenic and wild-type plants (described in Example 2) were grown for 6 weeks in 6.5 cm pots containing 110 g of Westland John Innes No. 3 Mature Plant Compost. Soil control pots containing no plants were also constructed. For each pot, the pH of the soil 2 cm below the surface was measured in three random locations using a HANNA™ soil pH tester (HI-981030). As shown in Figure 2C, soil pH in pots containing transgenic plants of the invention was significantly lower than either pots containing no 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). Thus, pots containing plants of the invention sequestered more CO in the soil than pots containing control plants or pots containing no plants.

[0126] Once the plants were grown, they were imaged. There were no obvious phenotypic differences between the transgenic and wild-type plants in terms of plant development, growth rate, flowering, or fruiting (Figure 2F). Therefore, secreting carbonic anhydrase from roots and sequestering CO2 as bicarbonate in the soil is not detrimental to plant growth.

[0127] Example 4 CO2 soil flux experiments were performed to further demonstrate that the transgenic plants of the present invention increased the rate at which CO2 diffused from the air into the soil and was converted to bicarbonate.

[0128] Here, transgenic and wild-type plants (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 aboveground biomass (i.e., leaves, stems / stems, etc.) was removed, and the pots containing all of the belowground biomass in the soil were placed in a LI-COR 6800 plantlet chamber in complete darkness (achieved by covering the chamber with aluminum foil). The CO absorbed or released by the soil in the pot 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 was raised to 1000 ppm (approximately 2.5 times atmospheric CO2 concentration). After 1 minute of acclimation, soil CO2 sequestration rates were measured every second for a 30-minute recording window. Net carbon sequestration was calculated by summing the carbon sequestration over the recording window. As shown in Figure 2D, this revealed that pots containing control plant roots did not sequester CO from the airstream, but instead produced CO via soil respiration. In contrast, pots containing roots of transgenic plants of the present invention depleted CO from the airstream. Thus, plants of the present invention promoted CO sequestration into the soil. Furthermore, the magnitude of this sequestration was substantially greater than the CO released via soil respiration.

[0129] Example 5 To demonstrate that other carbonic anhydrase genes from other species can be used in different embodiments of the present invention, we expressed the carbonic anhydrase (SEQ ID NO: 2) from the diatom Conticlibula weissflogii (also known as Thalassiosira weissflogii). As described above, this carbonic anhydrase was translationally fused to the experimentally characterized signal peptide (G. Li et al., 2009) (SEQ ID NO: 3) from the carrot (Daucus carota) extensin gene. This fusion protein was placed under the control of a root-specific promoter (SEQ ID NO: 6) from the soybean expansin gene (Glyma.17G133100) and cloned into a plant transformation vector flanked by Agrobacterium LB and RB t-DNA sequences, which also contains a herbicide resistance gene (spectinomycin). This vector was transformed into Agrobacterium tumefasciens (strain LBA4404) by electroporation, and positive colonies were selected and cultured using standard techniques. This vector was then transformed into Arabidopsis thaliana to generate stable transgenic lines.

[0130] Seeds from transgenic plants expressing secretable / transportable carbonic anhydrase from Conticlibra weissfrogii (SEQ ID NOs: 2 and 9) and wild-type plants were germinated on 1 / 2 Murashige's Koog + 1% sucrose + 2.5 mM MES agar plates. After 7 days, seedlings were transferred to 96-well plates, with only roots in each well. Each well in the 96-well plate contained 350 μl of 1 / 2 Murashige's Koog + 1% sucrose solution and 30 mg / L bromocresol purple. After 4 hours, seedlings were removed, and absorbance at 429 nm was measured using a plate reader. Carbonic anhydrase activity was estimated from the rate of change in pH over a 4-hour period. Carbonic anhydrase activity was significantly higher in the root environment of transgenic plants than in the root environment of wild-type control plants (P = 0.017435, one-tailed Student's t-test, unequal variances) (Figure 2E). Thus, plants transformed with a secretable / transportable carbonic anhydrase from Conticribra weissflogi successfully expressed and secreted active carbonic anhydrase protein into the extracellular environment of the root, which then catalyzed the conversion of CO2 to bicarbonate. This demonstrates that carbonic anhydrase proteins from any species and any carbonic anhydrase subtype are predicted to function equivalently.

[0131] Example 6 To demonstrate that soybean plants can be engineered so that their roots secrete active carbonic anhydrase protein into the extracellular environment, we translationally fused the experimentally characterized signal peptide [SEQ ID NO: 3] from the carrot (Daucus carota) extensin gene provided 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 a root-specific promoter [SEQ ID NO: 6] from the soybean expansin gene (Glyma.17G133100) and cloned into a plant transformation vector (Figure 1) that also contains a herbicide resistance gene (spectinomycin) and is flanked by Agrobacterium LB and RB t-DNA sequences. This vector was transformed into cultured Agrobacterium tumefasciens using standard techniques. This vector was transformed into Glycine max to generate stable engineered lines. Manipulated and unmanipulated (azygous) control plants were grown on 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 on hydroponic medium for 2 weeks as previously described by Hata and Futamura (2020). After 2 weeks, the hydroponic medium was replenished with fresh medium, and plants were grown for 5 days, depending on the rate at which the plants consumed the hydroponic medium. Samples of hydroponic medium from hydroponic boxes containing either control or manipulated plants were filtered through a Falcon 40 μm cell strainer to remove plant debris. Proteins present in the filtered samples were then isolated and concentrated using Pierce 10 kD MWCO protein concentration columns. The total protein concentration of each isolated protein sample was calculated using the Bradford protein assay, and the total protein concentration was adjusted to be equal across all samples. To measure carbonic anhydrase activity, an adapted version of the protocol described by Ozdemir (2009) was used.Here, 160 μl of concentrated hydroponic medium sample was added to 120 μl of 50 mM Tris-HCl pH 7.5 in wells of a 96-well microplate. Then, 30 μl of 10 mM p-nitrophenyl acetate dissolved in acetonitrile was added to each well to initiate the colorimetric assay. A spectrophotometer plate reader was used to measure absorbance at 400 nm in each well over a 16-hour period at 25°C. Carbonic anhydrase activity was reported as μM of p-nitrophenol synthesized per second. The average carbonic anhydrase activity was calculated from two independent unmanipulated lines and three independent engineered lines (Figure 4A). Protein samples isolated from hydroponic medium containing unmanipulated plants exhibited baseline p-nitrophenyl hydrolysis activity. Protein samples isolated from hydroponic medium containing engineered plants exhibited significantly higher p-nitrophenyl hydrolysis activity. This is consistent with the presence of secreted carbonic anhydrase in the hydroponic medium containing the engineered plants.

[0132] Once the plants were grown on soil, they were also imaged. There were no obvious phenotypic differences between the engineered soybean plants and the control plants across plant development, growth rate, flowering, or fruit set (Figure 4F). Thus, secreting carbonic anhydrase from roots and sequestering CO2 as bicarbonate in the soil was not detrimental to plant growth.

[0133] Example 7 To demonstrate that soybean plants engineered to secrete active carbonic anhydrase protein into the extracellular environment are capable of sequestrating CO2 in soil, the following experiment was performed. Engineered plants expressing secretable carbonic anhydrase in root hair cells (described in Example 6) and wild-type control plants were grown in 13 cm pots containing a 1:1:2:2 mixture of Levington M3 compost, Melcourt topsoil, Melcourt sharp sand, and perlite mixed with 5 ml Miracle-Gro All Purpose Continuous Release Plant Food. When the plants were 17 weeks old, soil cores were collected from each pot at random locations near the root zone using a 2.1 cm diameter soil sampler probe and transferred to falcon tubes. A volume of ddH2O equal to the soil mass was added, mixed for 30 minutes, and allowed to settle for 1 hour. The pH of the soil slurry was measured using a Metler pH probe. The pH of the soil from pots containing engineered plants of the present invention was significantly lower than that of pots containing wild-type plants (P = 0.00798, one-tailed Student's t-test, unequal variance) (Figure 4B). This reduction in soil pH is consistent with the presence of active carbonic anhydrase protein in the soil of pots containing engineered plants of the present invention, and therefore an increase in bicarbonate concentration. Thus, soil in which plants of the present invention were grown sequestered more CO than soil in which control plants were grown.

[0134] Example 8 To demonstrate that a carbonic anhydrase gene from any species can be used in the engineered plants of the present invention, the soybean carbonic anhydrase gene used in Examples 1 and 2 above was replaced with a seaweed-derived carbonic anhydrase gene [SEQ ID NO: 2]. All other components of the vector for plant transformation were maintained the same. To demonstrate that the expressed carbonic anhydrase protein is secreted from soybean roots, the active engineered plants of the present invention and unengineered control plants were grown on Levington Advance F2S Seed & Modular & Sand Compost for three weeks, after which the roots were washed and transplanted into 4.5 L hydroponic boxes. The plants were cultivated on hydroponic medium as previously described (Hata and Futamura 2020). The plants were grown hydroponically for one week. The hydroponic medium was then replenished with fresh medium, and the plants were grown for an additional seven days. Samples of medium from the hydroponic boxes containing only control plants or engineered plants were isolated and filtered through a Falcon 40 μm cell strainer to remove plant debris. Proteins present in the filtered samples were then isolated and concentrated using Pierce 10 kD MWCO protein concentration columns. The total protein concentration of each sample was calculated using the Bradford protein assay, and enzyme activity was normalized to total protein content. To measure carbonic anhydrase activity, an adapted version of the protocol described by Ozdemir (2009) was used. Here, 160 μl of concentrated hydroponic medium sample was added to 120 μl of 50 mM Tris-HCl pH 7.5 in a well of a 96-well microplate. After this, 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 in each well was measured over an 11-hour period at 25°C using a spectrophotometer plate reader.

[0135] As shown in Figure 4C, protein samples isolated from hydroponic media grown with unengineered plants exhibited baseline p-nitrophenyl hydrolysis activity. Protein samples isolated from hydroponic media grown with engineered plants of the present invention exhibited significantly higher p-nitrophenyl hydrolysis activity, consistent with the presence of secreted carbonic anhydrase in the hydroponic media containing the engineered plants.

[0136] Example 9 To demonstrate that soybean plants engineered to express active carbonic anhydrase protein on their root surfaces enable CO2 sequestration in soil, the following experiment was performed. Engineered soybean plants, as described above, were translationally fused with soybean carbonic anhydrase (SEQ ID NO: 3) to an experimentally characterized signal peptide and transmembrane domain (SEQ ID NO: 4 and SEQ ID NO: 5) from pea (Pisum sativum). This fusion protein was placed under the control of a root-specific promoter (SEQ ID NO: 6) from the soybean expansin gene (Glyma.17G133100) and cloned into a plant transformation vector (Figure 1) that also contains a herbicide resistance gene (spectinomycin) and is flanked by Agrobacterium LB and RB t-DNA sequences. This vector was transformed into Agrobacterium tumefasciens cultured using standard techniques. This vector was transformed into Glycine max to generate stable engineered lines. Engineered plants and unengineered control plants were grown in 13 cm pots containing John Innes Cereal Mix. When the plants were 13 weeks old, a 2.1 cm diameter soil sampler probe was used to collect soil core samples from each pot at random locations near the root zone and transferred to a falcon tube. A volume of ddH2O equal to the soil mass was added, mixed for 30 minutes, and allowed to settle for 1 hour. The pH of the soil slurry was measured using a Metler pH probe. As shown in Figure 4D, the soil pH in the pots growing engineered plants of the present invention was significantly lower than that in the pots containing wild-type plants. This reduction in soil pH is consistent with the presence of active carbonic anhydrase protein in the soil of pots containing engineered plants of the present invention, and therefore an increase in bicarbonate concentration. Therefore, pots containing engineered plants of the present invention sequestered more CO2 in the soil than pots containing control plants or pots without plants.

[0137] Example 10 To demonstrate that other carbonic anhydrase genes from other species can be engineered and localized extracellularly on soybean root hairs, the engineered plants used in Figure 4C and their respective unengineered controls were grown on Levington Advance F2S Seed & Modular & Sand Compost for 3 weeks and then transplanted into 4.5 L hydroponic boxes. Plants were maintained on hydroponic medium as previously described by Hata and Futamura (2020). Plants were grown hydroponically for 2 weeks, refreshing the medium weekly. To measure carbonic anhydrase activity on the root surface, an adapted carbonic anhydrase p-nitrophenyl hydrolysis activity protocol from Ozdemir (2009) was used. 500 mg of fresh soybean roots were removed, briefly rinsed with sterile water to remove excess hydroponic 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 at 400 nm of a 300 μl sample was recorded at 1 and 33 minutes using a spectrophotometer plate reader. Carbonic anhydrase activity was reported as μM p-nitrophenol synthesized per second.

[0138] The engineered plant roots demonstrated significantly higher (P = 0.030458, one-tailed Student's t-test, unequal variance) p-nitrophenyl hydrolysis activity compared to control plant roots (Figure 4E), consistent with the detection of carbonic anhydrase activity extracellularly from the engineered plant roots.

[0139] Example 11 To demonstrate that carbonic anhydrase can be engineered to localize extracellularly on the plasma membrane of cells, a reporter-gene fusion was created and tested in protoplasts. Here, Conticlibula weissfrogii carbonic anhydrase [SEQ ID NO: 2] was translationally fused to the coding sequence of a fluorescent protein to produce 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 from the pea (Pisum sativum) gene PsBP80 [SEQ ID NO: 4 and SEQ ID NO: 5], respectively. This final fusion protein was placed under the control of the cassava vein mosaic virus promoter (Verdaguer et al., 1996) and cloned into a plant transformation vector. To provide a cytoplasmic localization control, Conticlibula weissfrogii carbonic anhydrase translationally fused to the coding sequence of a fluorescent protein, without the PsPB80 signal peptide and transmembrane domain, was also placed under the control of the Cassava Vein Mosaic Virus promoter (Verdaguer et al., 1996) and cloned into a plant transformation vector.

[0140] Soybean protoplasts were transfected with these two expression vectors according to the protocol described by Sultana et al. (2019). After 2 days, transfected protoplasts were imaged using a Leica TCS SP5 confocal microscope. Protoplasts expressing Conticlibra weissfroggi carbonic anhydrase fused to the coding sequence of a fluorescent protein without the PsBP80 signal peptide and transmembrane domain showed clear cytosolic localization (Figure 5A). In contrast, protoplasts expressing Conticlibra weissfroggi carbonic anhydrase fused to the coding sequence of a fluorescent protein with the N-terminal PsBP80 signal peptide and C-terminal PsBP80 transmembrane domain localized to the plasma membrane (Figure 5B).

[0141] Example 12 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 used independently to drive expression of GUS(Cat1+) (the beta-glucuronidase gene containing the first intron Cat1 of castor bean catalase). These promoter-GUS fusions were separately 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 tumefasciens (strain LBA4404) by electroporation, and positive colonies were selected and cultured using standard techniques. These Agrobacterium strains were transformed into Arabidopsis thaliana using the floral-dip method (Clough and Bent, 1998). The engineered plants were germinated on 1 / 2 Murashige-Skoog + 1% sucrose + 2.5 mM MES agar plates. After 10 days, the seedlings were transferred to 6-well plates for GUS histochemical staining. Five ml of GUS staining buffer (0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6 3H2O, 50 mM sodium phosphate buffer, 10 mM Na2EDTA HO, 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 and cleared overnight in 70% ethanol. The stained, chlorophyll-cleared seedling roots were imaged using an Optika B-383FL microscope in conjunction with a OneView camera and imaging software.

[0142] GUS expression driven by the pGmPIN2b promoter (SEQ ID NO: 6) was localized to the root tip epidermis, with some expression extending upward to the primary root (Figure 5C). GUS expression driven by the pGmEXPA7 promoter was localized to root hair cells (Figure 5D).

[0143] Example 13 To demonstrate that the engineered plants of the present invention are capable of increasing soil inorganic carbon accumulation, the following experiment was conducted. Engineered plants described in Example 6 expressing secreted soybean carbonic anhydrase [SEQ ID NO: 6] from root hair cells and unengineered control plants were grown until the end of their life cycle in 13 cm pots containing a 1:1:2:2 mixture of Levington M3 compost, Melcourt topsoil, Melcourt sharp sand, and perlite soil mixed with 5 ml Miracle-Gro All Purpose Continuous Release Plant Food. After seed harvest and soil drying, the dried 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 checks to analyze soil inorganic carbon (SIC) and soil organic carbon (SOC) content.

[0144] The average inorganic carbon content of soil from pots growing manipulated plants was higher than that of soil from pots growing unmanipulated control plants or no plants (Figure 6A).

[0145] Example 14 To demonstrate that exogenous soil treatments can be used in combination with engineered plants of the present invention to enhance CO2 sequestration in soil, the following experiment was performed. Here, engineered Arabidopsis thaliana plants described in Example 2 expressing secretable carbonic anhydrase in root hair cells, and wild-type control plants, were grown in 6.5 cm pots containing 160 g of Levington advance F2S Seed & Modular & Sand Compost mixed with 4.16 g L-1 Ca(OH)2. The soil pH was adjusted to pH 8 to allow for the release of CO3, which can combine with Ca from the Ca(OH)2 to then form CaCO3. - to HCO3 - promoted enhanced conversion of

[0146] The plants were grown for 10 weeks. At that time, all aboveground biomass was removed, and the soil was sent to Eurofins AgroTesting in the UK for a soil carbon check to analyze soil inorganic carbon (SIC) and soil organic carbon (SOC) content. Soil grown with engineered plants of the present invention exhibited increased soil inorganic carbon (Figure 6B) and significantly higher soil inorganic carbon normalized to soil organic carbon (P = 0.002078, one-tailed Student's t-test, unequal variance) (Figure 6C). This is consistent with the presence of carbonic anhydrase activity in soil grown with engineered plants of the present invention, and therefore enhanced conversion of CO to bicarbonate. Thus, soil treatments can be used in conjunction with engineered plants of the present invention to enhance CO sequestration. It would be expected by one skilled in the art that extension of this technology to any soil treatment rich in positively charged cations, such as magnesium oxide, or cations resulting from crushed rock soil treatments or fertilization treatments, or soils that are naturally rich in cations, would be expected to function equivalently when used in conjunction with the present invention to increase inorganic soil carbon.

[0147] Example 15 To demonstrate that the engineered plants of the present invention are capable of increasing soil inorganic carbon accumulation, the following experiment was conducted. Engineered soybean plants described in Example 8 expressing soybean carbonic anhydrase anchored to the plasma membrane of root hair cells, and their respective unengineered control plants, were grown until the end of their life cycle in 13 cm pots containing a 1:1:2:2 mixture of Levington M3 compost, Melcourt topsoil, Melcourt sharp sand, and perlite soil mixed with 5 ml Miracle-Gro All Purpose Continuous Release Plant Food. After seed harvesting and soil drying, the dried 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, where soil inorganic carbon (SIC) and soil organic carbon (SOC) content was analyzed.

[0148] Soils grown with engineered plants of the present invention exhibited significantly increased soil inorganic carbon (P = 0.032395, one-tailed Student's t-test, unequal variances) (Figure 6D) and significantly higher soil inorganic carbon normalized to soil organic carbon (P = 0.048304, one-tailed Student's t-test, unequal variances) (Figure 6E) than soils grown with control plants.

[0149] This is consistent with the presence of increased carbonic anhydrase activity in the soil in which the plants of the invention were grown, and therefore the enhanced conversion of CO to bicarbonate to produce inorganic carbon, thus demonstrating that the engineered plants of the invention enhance carbon sequestration in soil.

[0150] References

[0151] [Table 1]

[0152] [Table 2]

Claims

1. A plant in which the heritable genetic material of the plant contains 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 plant roots into the extracellular environment.

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

3. 3. The plant of claim 1, wherein the gene encoding the transportable carbonic anhydrase protein further comprises a signal peptide sequence for directing transport of the carbonic anhydrase into 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 capable of driving overexpression of the carbonic anhydrase, and the signal peptide sequence is capable of directing the transport of the carbonic anhydrase into the extracellular environment of the plant root.

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

7. 7. The plant of claim 3, 5 or 6, or the polynucleotide sequence of claim 4, 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. 8. The plant or polynucleotide of claim 1, wherein the promoter and / or the signal peptide sequence, if present, is derived from a gene from the same plant species or variety as the plant.

9. 9. The plant or polynucleotide according to claim 1, wherein the promoter is root-specific, preferably root hair- and / or root epidermis-specific.

10. 10. The plant or polynucleotide of claim 1, wherein the carbonic anhydrase further comprises a transmembrane domain or a membrane anchor sequence.

11. 11. The plant or polynucleotide according to any one of claims 1 to 10, wherein the carbonic anhydrase is a carbonic anhydrase of the α, β, γ, δ, ζ, η, θ or ι subtype, preferably a monomeric one.

12. 4. A plant according to 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 having at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2, and optionally the promoter comprises SEQ ID NO: 6 or SEQ ID NO: 7 or a sequence having at least 65% identity to SEQ ID NO: 6 or SEQ ID NO:

7.

13. 4. 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 having at least 65% identity to SEQ ID NO: 1 or SEQ ID NO: 2, and optionally the promoter comprises SEQ ID NO: 6 or SEQ ID NO: 7 or a sequence having at least 65% identity to SEQ ID NO: 6 or SEQ ID NO: 7; and the signal peptide sequence comprises SEQ ID NO: 3 or SEQ ID NO: 4 or a sequence having at least 65% identity to SEQ ID NO: 3 or SEQ ID NO:

4.

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

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

16. 16. The plant, polynucleotide, polypeptide, cell, cell line or progeny of any one of claims 1 to 15, wherein the plant is a crop, preferably a row crop or cover crop, preferably selected from corn, soybean, pea, cotton, canola, camelina, potato, tomato, sugar beet, cassava, sweet potato, alfalfa, wheat, barley, sorghum, oat, sorghum, millet, rye, teff, rice plants, or clover, cress, brassica, vetch and prairie grass.

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

18. 18. The plant, polypeptide, polynucleotide, cell, cell line or progeny of any one of claims 1 to 17, wherein more carbon is sequestered in the extracellular environment of the plant compared to a plant 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.

19. 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 a plant according to any one of claims 15 to 18.

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

21. 21. A composition for the transformation of plant cells comprising a polynucleotide according to any one of claims 4 to 11 or 16 to 18 or a vector according to claim 20, and optionally a microparticle coated with said polynucleotide or said vector.

22. 21. A bacterium comprising a polynucleotide according to any one of claims 4 to 11 or 16 to 18 or a vector according to claim 20, optionally being Escherichia coli or Agrobacterium species, more preferably Agrobacterium tumefaciens.

23. 19. A plant comprising a polynucleotide according to any one of claims 4 to 11 or 16 to 18 stably integrated into its genome, preferably genetically integrated into its genome.

24. (a) providing the vector of claim 20; (b) activating a promoter operably linked to DNA encoding the polypeptide. Including, (c) Optionally, a method for producing a plant or seed, wherein said polypeptide is overexpressed in the extracellular environment of said cell compared to a plant in which the heritable genetic material of the plant does not contain a gene encoding a transportable carbonic anhydrase protein under the control of a promoter active in root cells.

25. A method for increasing the ability of a plant to sequester carbon in soil, comprising modifying the heritable genetic material of the plant so that carbonic anhydrase protein is transported from the plant's roots into the extracellular environment.

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

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

28. 26. 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 control element active in at least some of the root cells of the 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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