Green algae bestrophin bicarbonate transporter

Abstract

Aspects of the present disclosure relate to genetically modified plants and / or algae that have increased carbon utilization efficiency as a result of an increased ability of bicarbonate to cross membranes within plant cells. Other aspects of the present disclosure relate to methods of making such plants and / or algae, as well as methods of cultivating these genetically modified plants and / or growing these genetically modified algae for increased carbon utilization efficiency.

Description

Detailed Description of the Invention

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Application No. 62 / 769,214, filed on November 19, 2018, and U.S. Provisional Application No. 62 / 697,840, filed on July 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] [Submission of Sequence Listing by ASCII Text File] The contents of the following submission by ASCII text file are incorporated herein by reference in their entirety: the computer - readable form (CRF) of the sequence listing (file name: 794542000140SEQLIST.TXT, date of record: June 28, 2019, size: 461 KB).

[0003] [Technical Field] The present disclosure relates to genetically modified plants. In particular, the present disclosure relates to genetically modified plants containing a green algal bicarbonate transporter that preferably provides increased carbon use efficiency.

[0004] [Background] Green algae and other photosynthetic aquatic organisms are often exposed to low and fluctuating CO2 conditions in the natural environment. There are various factors that can reduce the CO2 utilization ability of these organisms, such as the slow diffusion of gases in water, the two inorganic carbon (Ci) forms, carbon dioxide (CO2) and bicarbonate (HCO3 -) includes slow interconversions and pH changes. As a result of the variable natural environment, most aquatic photosynthetic organisms have evolved a carbon dioxide concentrating mechanism (CCM) that can be induced under limited CO2 conditions. The CCM enables aquatic photosynthetic organisms such as green algae to efficiently concentrate Ci for fixation by Rubisco (Giordano et al., Ann Rev Plant Bio 56:99-131, 2005). The current CCM model for the green alga Chlamydomonas reinhardtii (Jungnick et al., Photosynth Res 121:159-173, 2014; Wang and Spalding, Plant Physiol 166:2040-2050, 2014) includes bicarbonate transporters on the plasma membrane and the chloroplast envelope as major components of the CCM that allow the movement of Ci, especially HCO3 - across the membrane. Carbonic anhydrases that interconvert CO2 and HCO3 - are additional important components of the CCM model (Mitra et al., Can J Bot 83:780-795, 2005; Moroney et al., Photosynth Res 109:133-149, 2011).

[0005] In Chlamydomonas reinhardtii, a compartment called the pyrenoid is located at the base of the chloroplast. The pyrenoid is the site where Rubisco is sequestered under limited CO2 conditions (Kuchitsu et al., Plant Cell Phys 29:1269-1278, 1988; Rawat et al., Planta 198:263-270, 1996; Borkhsenious et al., Plant Physiol 116:1585-1591, 1998). A extensive network of thylakoid tubules and minitubules is associated with the pyrenoid, presumably providing a pathway for HCO3 - to enter the pyrenoid (Engel et al., Elife 13:04889, 2015). The thylakoid carbonic anhydrase CAH3 is found in these tubules, and CAH3 catalyzes the conversion of HCO3- is hypothesized to be converted to CO2 and fixed within the lumen (Moroney and Ynalvez, Eukaryotic Cell 6:1251-1259, 2007).

[0006] Chlamydomonas reinhardtii cells grown under high CO2 conditions (5% v / v) show low affinity for Ci. When high CO2-acclimated cells are exposed to lower CO2 conditions (e.g., ambient (0.04%) to low (less than 0.01% v / v)), induction of high-affinity Ci transporters has been reported. CO2 diffuses readily across intracellular membranes (Gutknecht et al., J. Gen. Physiol 69:779-794, 1977), but since then, the need for an active transport system to facilitate the movement of Ci (especially HCO3 - ) to the sites where fixation by Rubisco can occur in cells grown under low CO2 conditions has been established by many studies (Moroney et al., Plant Physiol 83:460-463, 1987; Sultemeyer et al., Planta 176:256-260, 1988; Badger et al., Physiologia Plant 90:529-536, 1994; Ohnishi et al., Plant Cell 22:3105-3117, 2010). Furthermore, molecular and physiological studies have also confirmed the occurrence of multiple forms of Ci transporters on the plasma membrane and the chloroplast envelope of cells (Amoroso et al., Plant Physiol 116:193-201, 1998; Duanmu et al., PNAS 106:5990-5995, 2009; Atkinson et al., Plant Biotechnol J 5:12497, 2015; Gao et al., Plant 82:1-11, 2015; Yamano et al., PNAS 112:7315-7320, 2015).

[0007] In cyanobacteria inhabiting marine environments, HCO3 transport across the plasma membrane is often coupled to the high concentration of external Na ions in seawater. In the freshwater environment where Chlamydomonas reinhardtii is found, Na - transport is often coupled to the high concentration of external Na ions in seawater. In the freshwater environment where Chlamydomonas reinhardtii is found, Na + ions are often coupled to the high concentration of external Na ions in seawater. In the freshwater environment where Chlamydomonas reinhardtii is found, Na +Since the concentration is relatively low, the transport is H + is thought to be cotransported (Morth et al., Nat Rev Mol Cell Biol 12:60-70, 2011; Taylor et al., Trends Plant Sci 17:675-684, 2012). As a result, genomic studies using Chlamydomonas reinhardtii and Volvox carteri have revealed the presence of both H + and Na + cotransporters for sulfate and phosphate (Pootakham et al., Plant Physiol 153:1653-1668, 2010). Whether this type of ion coupling is also important for bicarbonate uptake is not yet clear.

[0008] To date, two high-affinity bicarbonate transport proteins and one low-affinity bicarbonate transport protein in Chlamydomonas reinhardtii have been characterized and are known to function under low CO2 conditions. The first high-affinity transporter, High Light Activated protein 3 (HLA3), is an ATP-binding cassette (ABC)-type transporter of the multidrug resistance protein family and is localized to the plasma membrane (Im and Grossman, 2002). Hla3 transcripts are induced by both high light and low CO2 conditions and are controlled by the CCM "master regulator" encoded by the Cia5 gene. Duanmu et al. (2009) showed a significant decrease in Ci affinity and Ci uptake in HLA3 RNAi knockdown mutants, with HCO3 -Supported the role of this protein in transport. The second high-affinity transporter, Low Carbon Inducible protein 1 (LCI1), is a relatively small protein. LCI1 is strongly upregulated in cells grown under low CO2 conditions and is localized to the plasma membrane (Ohnishi et al., Plant Cell 22:3105-3117, 2010). Furthermore, overexpression of the LCI1 protein in the Lcr1 (Chlamydomonas strain lacking the MYB-transcription factor) background resulted in an increase in Ci uptake. Therefore, both HLA3 and LCI1 are thought to be Ci transporters located on the plasma membrane.

[0009] The third transporter, NAR1.2 (also known as LCIA), is a chloroplast envelope protein of the formate / nitrite transporter family. The NAR1.2 protein has a lower affinity for bicarbonate (as revealed by values in the mM range), but expression of NAR1.2 in Xenopus laevis oocytes increased HCO3 1 / 2 uptake (Mariscal et al., Protist 157:421-433, 2006; Atkinson et al., Plant Biotechnol J 5:12497, 2015). NAR1.2 has been shown to be localized to the chloroplast envelope and is thought to be involved in Ci uptake, but the molecular mechanism for this remains unclear (Yamano et al., PNAS 112: 7315-7320, 2015). Experimental results indicate that the NAR1.2 and HLA3 proteins have a cooperative role within CCM (Yamano et al., PNAS 112: 7315-7320, 2015). Under extremely low CO2 conditions, NAR1.2 has been shown to interact with Low-CO2 Inducible protein B (LCIB). These results suggest that while LCIB is involved in CO2 uptake and the reuptake of CO2 that has leaked from the pyrenoid, NAR1.2 is involved in HCO3 - -A model involving the uptake and transport pathways was suggested (Wang and Spalding, Plant Physiol 166:2040-2050, 2014).

[0010] In addition to the proteins likely involved in the above CCM, the roles of multiple other proteins have been suggested. For example, two soluble proteins, LCIB and LCIC, form a complex that has been observed to associate closely with the pyrenoid when cells are acclimated to extremely low CO2 (Yamano et al., Plant Cell Physiol 51:1453-1468, 2010), but it is not yet certain what role this complex can play (Jin et al., PNAS 113:14716-14721, 2016). Another example is provided by CCP1 and CCP2, other putative Ci transporters that have been shown to localize to mitochondria (Atkinson et al., Plant Biotechnol J 5:12497, 2015). This localization suggests that mitochondria may be important in the CCM function, but what this mechanism is or what role mitochondria can play is not yet clear.

[0011] Bestrophin is Cl - shows channel activity and is a family of membrane proteins that also function as regulators of voltage-dependent Ca 2+ channels. Human and mouse bestrophin have been found to have high permeability and high conductivity to HCO3 - (Qu and Hartzell, Am J Physiol Cell Physiol 294:C1371-C1377, 2008). However, protein alignment and phylogenetic tree analysis have shown that the amino acids in photosynthetic organism bestrophin proteins exhibit high diversity compared to their counterparts in mammals. For example, in animal bestrophin, Ca 2+The residues forming the sensing mechanism are not conserved in the bestrophin-like proteins (AtBest1 and AtBest2) in Arabidopsis, suggesting that Ca 2+ is not required for AtBest channel activation in chloroplasts. Thus, during evolution, bestrophin proteins in photosynthetic organisms may have acquired electrophysiological properties different from those in mammals (Duan et al., Journal of Integrative Plant Biology 58:848-858, 2016). In Chlamydomonas reinhardtii, LCI11 and Cre16.g662600 have been suggested as putative bestrophins, and Cre16.g663400 has been proposed to be a bestrophin-like protein (Mackinder et al., Cell 171:133-147, 2017), but these have not been further characterized.

[0012] Current data only clearly support the requirement for five proteins (including HLA3, LCI1, LCIA, CAH3, and LCIB) for inorganic carbon (Ci) influx in Chlamydomonas reinhardtii (Mackinder, New Phytologist 217:54-61, 2018). As described above, these proteins are localized in the plasma membrane and the chloroplast envelope. The current model of the Chlamydomonas reinhardtii CCM suggests that at least one additional transporter or channel in the chloroplast thylakoid membrane is required to maintain the flow of HCO3 - from the stroma to CAH3 in the lumen (Mackinder New Phytologist 217:54-61, 2018). Furthermore, identifying functional bicarbonate transport proteins remains an important goal. Current studies have identified many possible bicarbonate transport proteins, but only a few of these have promising experimental results, and there is little available mechanistic data.

[0013] 〔Overview〕 To meet these needs, the present disclosure is directed to green algal bestrophin polypeptides that function as bicarbonate transporters. Certain aspects of the present disclosure relate to a genetically modified plant, or a part thereof, wherein the plant comprises one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast into the stroma. In some aspects, the present disclosure relates to a genetically modified plant, or a part thereof, wherein the plant comprises one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant chloroplast into the lumen. In some embodiments, the acquisition of the ability of bicarbonate to cross a membrane is the result of the expression of at least one green algal bestrophin polypeptide. In some embodiments, the green algal bestrophin polypeptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the increase or provision of the ability of bicarbonate to cross a membrane is: a first polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof; the result of the expression of a polypeptide selected from the group.In some embodiments, the increase or provision of the ability of bicarbonate to pass through the membrane is the result of the expression of a polypeptide selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3; or any combination thereof.In some embodiments, the increase or provision of the ability of bicarbonate to pass through the membrane is the result of the expression of a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111; preferably, it is the result of the expression of a polypeptide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, the polypeptide is localized in the chloroplast envelope or thylakoid membrane of at least one chloroplast in the plant cell. In some embodiments, the plant cell is a mesophyll cell.In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0014] In some aspects, the present disclosure relates to a plant, or a part thereof, having increased carbon use efficiency, wherein the plant comprises at least one modified nucleic acid sequence having at least one coding sequence of a green algal bestrophin polypeptide in the plant, or a part thereof, wherein the bestrophin polypeptide is expressed in the plant, or a part thereof, and wherein when the plant is grown under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, grown under ambient carbon dioxide conditions. In some embodiments, the bestrophin polypeptide is localized to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0015] In some embodiments, the present disclosure relates to a plant, or a part thereof, having increased water use efficiency, wherein the plant, or part thereof, comprises at least one modified nucleic acid sequence having at least one coding sequence of a chlorophyte bestrophin polypeptide, wherein the bestrophin polypeptide is expressed in the plant, or part thereof, and wherein, when the plant is grown under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant, or corresponding WT part, that does not overexpress the bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding wild-type (WT) plant, or corresponding WT part, that does not overexpress the bestrophin polypeptide grown under ambient carbon dioxide conditions. In some embodiments, the bestrophin polypeptide is localized in the chloroplast envelope or chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0016] In some embodiments, the present disclosure provides a plant, or a part thereof, having increased nitrogen use efficiency, wherein the plant, or the part thereof, comprises at least one modified nucleic acid sequence having at least one coding sequence of a chlorophyte bestrophin polypeptide, wherein the bestrophin polypeptide is expressed in the plant, or the part thereof, and wherein when the plant is grown under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant, or a corresponding WT part, that does not overexpress the bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding wild-type (WT) plant, or a corresponding WT part, that does not overexpress the bestrophin polypeptide grown under ambient carbon dioxide conditions. In some embodiments, the bestrophin polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0017] In some embodiments, the present disclosure relates to a plant, or a part thereof, having reduced photo-inhibition, wherein the plant comprises at least one modified nucleic acid sequence having at least one coding sequence of a green algal bestrophin polypeptide in the plant, or in a part thereof, wherein the bestrophin polypeptide is expressed in the plant, or in a part thereof, and wherein when the plant is grown under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than that of a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, or the yield, growth rate, or biomass is substantially similar to that of a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, grown under ambient carbon dioxide conditions. In some embodiments, the bestrophin polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0018] In some embodiments of any of the above embodiments, the modified nucleic acid sequence is stably integrated into the nuclear genome of the plant. In some embodiments of any of the above embodiments, the at least one modified nucleic acid sequence further comprises a second nucleic acid sequence encoding a signal peptide sequence or a targeting sequence operably linked to the at least one coding sequence of the green algal bestrophin polypeptide, and the expression of the signal peptide sequence or the targeting sequence results in the localization of the bestrophin polypeptide to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell.

[0019] In some embodiments of any of the above-described embodiments, increased carbon use efficiency, increased water use efficiency, increased nitrogen use efficiency, or decreased photoinhibition is: a first polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof; and is the result of the expression of a polypeptide selected from the group. In some embodiments of any of the above-described embodiments, increased carbon use efficiency, increased water use efficiency, increased nitrogen use efficiency, or decreased photoinhibition is: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof; and is the result of the expression of a polypeptide selected from the group.In some embodiments of any of the above-described embodiments, the increase or provision of the ability of bicarbonate to pass through the membrane is the result of the expression of a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111; preferably, it is the result of the expression of a polypeptide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63.

[0020] In some embodiments of any of the above embodiments, the plant is a cowpea (i.e., black-eyed pea, Vigna unguiculata), soybean (i.e., soybean, Glycine max), cassava (i.e., manioc, Manihot esculenta), rice (i.e., Oryza sativa, Oryza glaberrima, Zizania spp), wheat (i.e., common wheat, spelt, durum, bread wheat, Triticum aestivum, Triticum spelta, Triticum durum, Triticum spp), barley (i.e., Hordeum vulgare), rye (i.e., Secale cereale), oat (i.e., Avena sativa), potato (i.e., Solanum tuberosum), tomato (i.e., Solanum lycopersicum), or another C3 crop plant. In some embodiments, the plant is tobacco (i.e., Nicotiana tabacum, Nicotiana edwardsonii, Nicotiana plumbagnifolia, Nicotiana longiflora), or Arabidopsis (i.e., rock cress, salad cress, Arabidopsis thaliana).In some embodiments of any of the above-described embodiments, the plant is not corn (i.e., maize, Zea mays), sorghum (i.e., durra, great millet, milo, Sorghum bicolor), sugarcane (i.e., sugarcane, Saccharum officinarum), millet (i.e., finger millet, common millet, pearl millet, foxtail millet, Eleusine coracana, Panicum miliaceum, Pennisetum glaucum, Setaria italica), switchgrass (i.e., tall panic grass, touchgrass, Panicum virganum), or another C4 crop plant.

[0021] In some embodiments, a plant part of any of the above-described embodiments is a leaf, stem, root, flower, seed, fruit, cell, or a part thereof. In some embodiments, the plant part is a fruit. In some embodiments, the plant part is a grain, kernel, bean, or tuber.

[0022] In some aspects, the present disclosure relates to a pollen grain or ovule of any of the above-described embodiments.

[0023] In some aspects, the present disclosure relates to a protoplast produced from any of the above-described embodiments.

[0024] In some aspects, the present disclosure relates to a tissue culture produced from a protoplast or cell of any of the above-described embodiments, where the cell or protoplast is produced from one of the plant parts in a group of leaf, anther, pistil, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, hypocotyl, embryo, or meristematic cells.

[0025] In some embodiments, the present disclosure relates to a genetically modified seed, wherein the seed comprises one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane. In some embodiments, the seed produces a plant that has the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the chloroplast of the plant to the stroma. In some embodiments, the seed produces a plant that has the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the chloroplast of the plant to the lumen. In some embodiments, the plant expresses at least one green algal bestrophin polypeptide. In some embodiments, the green algal bestrophin polypeptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or any combination thereof. In some embodiments, the plant expresses at least one polypeptide selected from the group consisting of: a first polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof. In some embodiments, the plant expresses at least one polypeptide selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.In some embodiments, the plant expresses a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111; preferably, it expresses a polypeptide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, the polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of the plant cell. In some embodiments, the plant cell is a mesophyll cell.In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant. In some embodiments, the plant is cowpea, soybean, cassava, rice, soybean, wheat, or other C3 crop plants. In some embodiments, the plant is not corn, sorghum, or other C4 crop plants.

[0026] In some embodiments of any of the above embodiments, the expression of the endogenous carbonic anhydrase is regulated. In some embodiments, the regulated expression can be increased expression, decreased expression, expression at a different location, or any combination thereof.

[0027] Certain aspects of the present disclosure relate to a method of producing a plant having increased carbon use efficiency, where the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to pass through a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma is increased or provided, or the ability of bicarbonate to pass through a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen is increased or provided, thereby increasing the carbon use efficiency of the plant.

[0028] In some aspects, the present disclosure relates to a method of producing a plant having increased water use efficiency, where the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to pass through a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma is increased or provided, or the ability of bicarbonate to pass through a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen is increased or provided, thereby increasing the water use efficiency of the plant.

[0029] In some embodiments, the present disclosure relates to a method of producing a plant having increased nitrogen use efficiency, wherein the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant chloroplasts into the lumen is increased or provided, thereby increasing the nitrogen use efficiency of the plant.

[0030] In some embodiments, the present disclosure relates to a method of producing a plant having reduced photoinhibition, wherein the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant chloroplasts into the lumen is increased or provided, thereby reducing the photoinhibition of the plant.

[0031] In some embodiments, the present disclosure relates to a method of producing a plant having increased growth or productivity, wherein the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant chloroplasts into the lumen is increased or provided, thereby increasing the growth or productivity of the plant.

[0032] In some embodiments of any of the above methods, the expression of an endogenous carbonic anhydrase is regulated. In some embodiments, the regulated expression can be increased expression, decreased expression, expression at a different location, or any combination thereof.

[0033] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased carbon use efficiency, where the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, where the seed provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast into the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast into the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases carbon use efficiency as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0034] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased water use efficiency, where the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, where the seed provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast into the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast into the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases water use efficiency as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0035] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased nitrogen use efficiency, where the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, where the seed provides a plant having the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases nitrogen use efficiency as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0036] In some embodiments, the present disclosure relates to a method of cultivating a plant having reduced photoinhibition, where the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, where the seed provides a plant having the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane decreases photoinhibition as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0037] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased growth or productivity, wherein the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, wherein the seed provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast to the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast to the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases growth or productivity as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0038] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased carbon use efficiency, wherein the steps of the method are: a) providing to the tissue culture or protoplast one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane; b) regenerating the tissue culture or protoplast into a juvenile plant; c) growing the juvenile plant into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast to the stroma, or has the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases carbon use efficiency as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0039] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased water use efficiency, where the steps of the method are: a) providing one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane to a tissue culture or protoplast; b) regenerating the tissue culture or protoplast into an undeveloped plant body; c) growing the undeveloped plant body into a plant, where the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases water use efficiency as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0040] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased nitrogen use efficiency, where the steps of the method are: a) providing one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane to a tissue culture or protoplast; b) regenerating the tissue culture or protoplast into an undeveloped plant body; c) growing the undeveloped plant body into a plant, where the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases nitrogen use efficiency as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0041] In some embodiments, the present disclosure relates to a method of cultivating a plant having reduced photo-inhibition, wherein the steps of the method are: a) providing one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane to a tissue culture or protoplast; b) regenerating the tissue culture or protoplast into an undeveloped plant body; c) growing the undeveloped plant body into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from at least a part of the plant cytoplasm of the plant chloroplast to the stroma, or the ability of bicarbonate to cross a membrane from at least a part of the stroma of the plant chloroplast to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane reduces photo-inhibition as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0042] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased growth or productivity, wherein the steps of the method are: a) providing one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane to a tissue culture or protoplast; b) regenerating the tissue culture or protoplast into an undeveloped plant body; c) growing the undeveloped plant body into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from at least a part of the plant cytoplasm of the plant chloroplast to the stroma, or the ability of bicarbonate to cross a membrane from at least a part of the stroma of the plant chloroplast to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases growth or productivity as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0043] In some embodiments of any of the above methods, the seed, tissue culture, or protoplast has one or more genetic modifications that regulate the expression of endogenous carbonic anhydrase. In some embodiments, the regulated expression can be increased expression, decreased expression, expression at a different location, or any combination thereof.

[0044] In some embodiments of any of the above methods, the increase or provision of the ability of bicarbonate to pass through the membrane is the result of the expression of at least one polypeptide selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3; or any combination thereof.In some embodiments of any of the above methods, the increase or provision of the ability of bicarbonate to pass through the membrane is: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or selected from the group of SEQ ID NO: 111; preferably, it is the result of the expression of at least one polypeptide selected from the group of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments of any of the above methods, the plant is cowpea, soybean, cassava, rice, soybean, wheat, or other C3 crop plants.In some embodiments of any of the above methods, the plant is not corn, sorghum, or other C4 crop plants.

[0045] Certain aspects of the present disclosure relate to genetically modified algae comprising one or more genetic modifications that result in an increased ability of the algae to transport bicarbonate into the lumen of the chloroplast. In some embodiments, the increased bicarbonate transport ability is the result of overexpressing at least one green algal bestrophin polypeptide from the group consisting of: a first polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof. In some embodiments, the increased bicarbonate transport ability is the result of overexpressing at least one green algal bestrophin polypeptide from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof. In some embodiments, the polypeptide is localized in the chloroplast thylakoid membrane. In some embodiments, the polypeptide is overexpressed at least when the algae are under conditions of less than 100 ppm carbon dioxide (CO2 < 0.01% [v / v] in air).

[0046] In some embodiments, the present disclosure relates to green algae, or a part thereof, having increased bicarbonate transport, comprising a modified nucleic acid sequence comprising a coding sequence of at least one green algal bestrophin polypeptide; wherein the bestrophin polypeptide is overexpressed; wherein the bestrophin polypeptide is localized in the chloroplast thylakoid membrane; wherein when the algae are cultured under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air), the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) algae or WT part thereof that does not overexpress at least one bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding WT algae or corresponding WT part thereof that does not overexpress at least one bestrophin polypeptide and is cultured under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air). In some embodiments, the at least one green algal bestrophin polypeptide is selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.

[0047] In some embodiments, the present disclosure relates to green algae, or a part thereof, having increased growth under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air), comprising a modified nucleic acid sequence encoding at least one green algal bestrophin polypeptide; wherein the bestrophin polypeptide is overexpressed; wherein the bestrophin polypeptide is localized in the chloroplast thylakoid membrane; and wherein when the algae are cultured under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air), the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) algae or WT part thereof that does not overexpress at least one bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding WT algae or corresponding WT part thereof that does not overexpress at least one bestrophin polypeptide and is cultured under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air). In some embodiments, the at least one green algal bestrophin polypeptide is selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.

[0048] In some embodiments of any of the above algae, the green algae are selected from the group consisting of: Chlamydomonas reinhardtii, Chlamydomonas eustigma, Volvox carteri f. nagariensis, Gonium pectorale.

[0049] In some embodiments, the present disclosure relates to a method of producing algae having increased carbon utilization efficiency, where the steps of the method include: a) introducing a genetic modification into the algae, including an increase in the ability to transport bicarbonate into the lumen of the chloroplast of the algae, thereby increasing the carbon utilization efficiency of the algae. In some embodiments, the acquisition of the transport ability is the result of overexpressing at least one green algal bicarbonate polypeptide selected from the group consisting of: a first polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, or at least 95% sequence identity to SEQ ID NO: 3; or any combination thereof. In some embodiments, the increase in bicarbonate transport ability is the result of overexpressing at least one green algal bicarbonate polypeptide selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.

[0050] 〔Brief Description of the Drawings〕 Figures 1A - 1D show the similarities of the Chlamydomonas reinhardtii genes (BST1, BST2, and BST3) and proteins (BST1, BST2, and BST3) to each other, as well as their similarities to other bestrophin family proteins of BST1 - 3. Figure 1A shows a schematic diagram of the BST1, BST2, and BST3 genes, where the light gray squares represent exons, the thin gray lines represent introns, the thick gray lines represent untranslated regions (UTRs), and the overlapping lines (overlapping exons and introns) indicate common regions shared by the three genes. Figure 1B shows the positions and directions of the BST1, BST2, and BST3 genes on the Chlamydomonas reinhardtii genome. Figure 1C shows the amino acid alignment of the BST1 (SEQ ID NO: 1), BST2 (SEQ ID NO: 2), and BST3 (SEQ ID NO: 3) proteins, where the asterisks in the bottom row indicate amino acids that are identical in all three proteins. Figure 1D shows a phylogenetic tree of the protein sequences of the Chlamydomonas reinhardtii BST1, BST2, and BST3 homologs in vascular plants (second group from the top), non - vascular plants (third group from the top), diatoms (fourth group from the top), and green algae (bottom group).

[0051] Figures 2A-2B show the transcriptional analysis of the Chlamydomonas reinhardtii genes BST1, BST2, and BST3. Figure 2A shows semi-quantitative RT-PCR of BST1-3 accumulation in wild-type strain D66 and cia5 cells under low CO2 (less than 0.04% CO2 in air) and high CO2 (5% (v / v) CO2 in air). Figure 2B shows the expression of BST1-3 in cDNA obtained from cells grown under high CO2 (5% (v / v) CO2 in air), and the time course of semi-quantitative RT-PCR showing the expression of BST1-3 in cDNA obtained from cells switched to low CO2 (less than 0.04% CO2 in air) for 2 hours (2h), 4 hours (4h), 6 hours (6h), or 12 hours (12h). Actin was used as a loading control in both Figure 2A and Figure 2B, and the results shown are from one of two replicates.

[0052] Figures 3A-3B show confocal microscope fluorescence protein images showing the localization of BST1-Venus, BST2-Venus, and BST3-Venus fusion proteins in Chlamydomonas reinhardtii. Figure 3A shows the location of BST1-Venus, BST2-Venus, and BST3-Venus fusion protein expression in Chlamydomonas reinhardtii ("Venus" column), the location of chloroplast thylakoids ("Chlorophyll" column), and the location of both relative to each other ("Merge" column). The scale bar is 5 μm. Figure 3B shows an enlarged image of the pyrenoid from the "BST1" row of Figure 3A. The arrow highlights the location where BST1-Venus fluorescence can be seen inside the pyrenoid in the thylakoid tubules that penetrate the pyrenoid. The scale bar is 1 μm. For both Figure 3A and Figure 3B, the images are representative images from multiple replicates.

[0053] Figure 4 shows the results of qRT-PCR analysis of BST1-3 expression in RNAi knockdown strains bsti-1 and bsti-2 compared to wild-type strain D66. Error bars indicate the standard error for three biological replicates.

[0054] Figures 5A-5C show the results of growth phenotype analysis of the RNAi knockdown strain bsti-1, as well as the pmp1 and cia3 mutant strains, compared to the wild-type strain D66 under various pH and CO2 conditions. The vertical dots represent three different cell concentrations (10,000 cells, 5,000 cells, and 2,500 cells). Figure 5A shows the results of growth phenotype analysis of the strains under extremely low CO2 (CO2 is 0.01% (v / v), in air) conditions and at pH 7 or 8.4. Figure 5B shows the results of growth phenotype analysis of the strains under low CO2 (CO2 is 0.04% (v / v), in air) and at pH 7 or 8.4. Figure 5C shows the results of growth phenotype analysis of the strains under high CO2 (CO2 is 5% (v / v), in air) conditions and at pH 7 or 8.4. The growth phenotype analysis experiments were repeated three times, and the results shown are representative.

[0055] Figures 6A-6F show the photosynthetic oxygen evolution activities of BST-RNAi lines 1 and 2, namely bsti-1 and bsti-2, as well as D66. Figure 6A shows the K 0.5 (C i ) value (the C i concentration required for half of the maximum oxygen evolution), which was calculated from the O2 evolution for the C i curve for bsti-1 and D66 acclimated to low CO2 (less than 0.04% CO2) for 12 hours at pH 8.4. Figure 6B shows the oxygen evolution activities measured at different C i amounts and different pH values, and is plotted as a curve for bsti-1 and D66 acclimated to low CO2 (less than 0.04% CO2) for 12 hours at pH 8.4. Figure 6C shows the K 0.5 (C i ) value (the C i concentration required for half of the maximum oxygen evolution), which was calculated from the O2 evolution for the C i curve for bsti-1, bsti-2, and D66 acclimated to low CO2 (less than 0.04% CO2) for 12 hours at pH 7.8. The symbol "*" indicates that the difference in K 0.5 (C i ) was significant (P < 0.05 by Student's t-test). Figure 6D shows different Ci The amount and oxygen evolution activity measured at different pH values ​​are shown and plotted as curves for bsti-1, bsti-2, and D66 acclimated to low CO2 (CO2 < 0.04%) at pH 7.8 for 12 hours. 0.5 (C i ) value (C required for half the maximum oxygen generation i The concentration of C is shown for bsti-1 and D66 acclimated to high CO2 (CO2 > 5%) at pH 7.8 for 12 hours. i The O2 evolution was calculated from the curve. Figure 6F shows the results of the different C i The amount and oxygen evolution activity measured at different pH values ​​are shown and plotted as curves for bsti-1 and D66 acclimated to high CO2 (CO2 > 5%) at pH 7.8 for 12 h. i Concentrations were run in triplicate and error bars represent three biological replicates and are based on standard deviation. Vmax for all strains was set to 100% oxygen evolving activity.

[0056] Figures 7A-7D show inorganic carbon uptake in bsti-1 and D66. i Figure 7B shows the time course of CO2(C i ) fixation time course. Figure 7C shows the intracellular C at pH 8.4. i Figure 7D shows the time course of CO2 fixation at pH 8.4. Cells were grown in elevated CO2 (5% CO2 in air) and then acclimated to low CO2 (CO2 < 0.04%) for 12 hours before analysis. Cells were harvested and endogenous C i After depletion of IgG, assays were performed and triplicate samples were run for each time point. Error bars in Figures 7A-7D represent three biological replicates.

[0057] Figures 8A - 8C show that BST3 is knocked out in the mutant bst3 derived from the Chlamydomonas Library Project (CLiP). Figure 8A shows a schematic diagram of the BST3 gene, where exons are depicted as gray squares, introns as black lines, and untranslated regions as dark gray squares. The position of the insert is shown as a light gray triangle, and the primers for detecting the insert are shown as small black arrows. Figure 8B shows the results of a PCR reaction using the primers shown in Figure 8A to confirm the position of the insert in bst3. Lane 1, BST3F and BST3R primers using WT strain D66 DNA as a template; Lane 2, CIB1F and BST3F primers using bst3 DNA as a template; Lane 3, CIB1R and BST3R primers using bst3 DNA as a template; Lane 4, BST3F and BST3R primers using bst3 DNA as a template. The size difference between Lane 1 and Lane 4 indicates the presence of an 1800 bp cassette. Figure 8C shows semi - quantitative RT - PCR indicating BST1 - 3 accumulation in D66 and bst3 cells under low CO2 (CO2 < 0.04% in air) and high CO2 (CO2 = 5% (v / v) in air). As indicated by the red square, BST3 is not expressed in bst3 cells. Actin was used as a loading control.

[0058] Figures 9A - 9D show the measurement of growth and inorganic carbon affinity of the knockout strain bst3 compared to the WT strain D66. Figure 9A shows the growth of bst3 and WT at low CO2 (CO2 < 0.04%) and pH 8.6 over 6 days, measured using OD 730 . Figure 9B shows the growth of bst3 and WT at low CO2 (CO2 < 0.04%) and pH 8.6 over 6 days, which was measured using chlorophyll estimation at wavelengths 645 and 663. Figure 9C shows the oxygen evolution activity measured at pH 7.4, plotted as curves for bst3 and D66. Each C iThree repetitions were performed at the concentration. Figure 9D shows the K i calculated from O2 generation for bst3 and D66 at pH 7.4 0.5 (C i ) value (C required for the half-value of the maximum oxygen generation i concentration).

[0059] Figures 10A - 10D show protein structure models for Chlamydomonas reinhardtii BST1, BST2, and BST3. Figure 10A shows the structural models for Chlamydomonas reinhardtii BST1 - 3 and Klebsiella pneumonia bestrophin (Kpbest) as monomers with conserved residues arranged as protruding shapes and labeled selective pores. Figure 10B shows the secondary structure of the BST1 homotetramer model shaded using the local QMEAN score (structural quality assessment scoring function). Figure 10C shows the contour of the channel cavity depicted on the BST1 homotetramer model in black. The conserved residues from the BST1 model in Figure 10A are depicted as protruding shapes extending into the channel cavity. Figure 10D shows the electrostatic potential on the calculated BST1 homotetramer model. The electrostatic potential is displayed on a potential scale of -4kT / e (negative) to +4kT / e (positive).

[0060] Figure 11 shows the current Chlamydomonas reinhardtii CCM model for inorganic carbon (C i ) transport. BST1, BST2, and BST3 are shown as bicarbonate transport channels (light gray, gray, and dark gray rectangles) localized in the chloroplast thylakoid membrane.

[0061] 〔Detailed Description〕 The following description describes exemplary methods, parameters, etc. However, such description is not intended as a limitation on the scope of the present disclosure, but rather should be recognized as provided as an explanation of exemplary embodiments.

[0062] [Genetically Modified Plants and Seeds] Certain aspects of the present disclosure relate to a genetically modified plant, or a part thereof, wherein the plant comprises one or more genetic modifications that increase or provide the ability of bicarbonate to pass through a membrane from the plant cytosol of at least a portion of the plant chloroplasts into the stroma. In some aspects, the present disclosure relates to a genetically modified plant, or a part thereof, wherein the plant comprises one or more genetic modifications that increase or provide the ability of bicarbonate to pass through a membrane from the stroma of at least a portion of the plant chloroplasts into the lumen. In some embodiments, the acquisition of the ability of bicarbonate to pass through a membrane is the result of the expression of at least one green algal bestrophin polypeptide. In some embodiments, the acquisition of the ability of bicarbonate to pass through a membrane is the result of the expression of two or three green algal bestrophin polypeptides. In some embodiments, the acquisition of the ability of bicarbonate to pass through a membrane is the result of the expression of four or more (e.g., 5, 6, 7, 8, 9, 10) green algal bestrophin polypeptides. In some embodiments, the green algal bestrophin polypeptide is SEQ ID NO: 1 (i.e., BESTROPHIN1, BST1, Cre16.g662600.t1.2), SEQ ID NO: 2 (i.e., BESTROPHIN2, BST2, Cre16.g663400.t2.1), or SEQ ID NO: 3 (i.e., BESTROPHIN3, BST3, Cre16.g663450.t1.2).In some embodiments, the increase or provision of the ability of bicarbonate to cross the membrane is the result of the expression of at least one polypeptide selected from the following group: a first polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof. In some embodiments, the increase or provision of the ability of bicarbonate to cross the membrane is: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof; the result of the expression of a polypeptide selected from the group.

[0063] As used herein, the term "BST1", as well as its capitalized and italicized forms, refers to green algal genes and proteins. As used herein, in some embodiments, this term may refer to the Chlamydomonas reinhardtii gene and protein. In other embodiments, this term may refer to one or more homologs or orthologs of the genes and proteins of any green algal species that also transports bicarbonate. In some embodiments, this term may refer to one or more paralogs of the genes and proteins of any green algal species. In some embodiments, the green algal species is Chlamydomonas reinhardtii (C. reinhardtii), Chlamydomonas eustigma, Volvox carteri f. nagariensis, or Gonium pectorale. SEQ ID NO: 1 provides the Chlamydomonas reinhardtii BST1 protein. When shown in all lowercase italicized form, a mutant (e.g., knockout) form of the gene / protein is intended. In Chlamydomonas reinhardtii, the mutant form may be a single gene / protein. In other green algal species, the mutant form may be one, some, or all of the homologs, orthologs, and / or paralogs of the gene / protein.

[0064] As described herein, the term "BST2", as well as its capitalized and italicized form, refers to green algal genes and proteins. As described herein, in some embodiments, this term may refer to the Chlamydomonas reinhardtii gene and protein. In other embodiments, this term may refer to one or more homologs or orthologs of the genes and proteins of any green algal species that also transports bicarbonate. In some embodiments, this term may refer to one or more paralogs of the genes and proteins of any green algal species. In some embodiments, the green algal species is Chlamydomonas reinhardtii (C. reinhardtii), Chlamydomonas eustigma, Volvox carteri f. nagariensis, or Gonium pectorale. SEQ ID NO: 2 provides the Chlamydomonas reinhardtii BST2 protein. When shown in all lowercase italicized form, a mutant (e.g., knockout) form of the gene / protein is intended. In Chlamydomonas reinhardtii, the mutant form may be a single gene / protein. In other green algal species, the mutant form may be one, several, or all of the homologs, orthologs, and / or paralogs of the gene / protein.

[0065] As described herein, the term "BST3", as well as its capitalized and italicized form, refers to green algal genes and proteins. As described herein, in some embodiments, this term may refer to Chlamydomonas reinhardtii genes and proteins. In other embodiments, this term may refer to one or more homologs or orthologs of genes and proteins of any green algal species that also transports bicarbonate. In some embodiments, this term may refer to one or more paralogs of genes and proteins of any green algal species. In some embodiments, the green algal species is Chlamydomonas reinhardtii (C. reinhardtii), Chlamydomonas eustigma, Volvox carteri f. nagariensis, or Gonium pectorale. SEQ ID NO: 3 provides the Chlamydomonas reinhardtii BST3 protein. When shown in all lower case and italicized, a mutant (e.g., knockout) form of the gene / protein is intended. In Chlamydomonas reinhardtii, the mutant form can be a single gene / protein. In other green algal species, the mutant form can be one, several, or all of the homologs, orthologs, and / or paralogs of the gene / protein.

[0066] In some embodiments, the increase or provision of the ability of bicarbonate to pass through the membrane is: a polypeptide from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111, or a polypeptide having a high percentage of identity with one of the group; preferably, it is the result of the expression of a polypeptide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63.In the context of two polynucleotides or polypeptides, the phrases "high percentage identical", "high percentage of identity", or "high sequence identity" and their grammatical variants refer to two or more sequences or subsequences that have at least about 80%, identity, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acid identity when compared and aligned for maximum correspondence using an alignment algorithm or by visual inspection. In an exemplary embodiment, a high percentage of identity is present over a region of a sequence that is at least about 16 nucleotides or amino acids in length. In another exemplary embodiment, a high percentage of identity is present over a region of a sequence that is at least about 50 nucleotides or amino acids in length. In yet another exemplary embodiment, a high percentage of identity is present over a region of a sequence that is at least about 100 nucleotides or amino acids in length or more. In one exemplary embodiment, the sequences are highly identical over the entire length of the polynucleotide or polypeptide sequence.

[0067] In some embodiments, the polypeptide is localized in the chloroplast envelope or thylakoid membrane of at least one chloroplast within a plant cell. In some embodiments, the polypeptide is capable of transporting bicarbonate (HCO3 - ) across the membrane. In some embodiments, the polypeptide is capable of transporting chloride anions (Cl - ) across the membrane. In some embodiments, the polypeptide is capable of transporting HCO3 - and Cl -Both can be moved. In some embodiments, the polypeptide can move negatively charged ions across the membrane. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant. In some embodiments, the polypeptide oligomerizes to form a pentamer. In some embodiments, the polypeptide oligomerizes to form a homopentamer. In some embodiments, the pentamer or homopentamer has an entry pocket with a predominantly negative hydrostatic potential and a selective pore with a neutral / positive charge. In some embodiments, the homopentamer transports negatively charged ions.

[0068] In some embodiments, the present disclosure relates to a plant, or a part thereof, having increased carbon use efficiency, wherein the plant, or part thereof, comprises at least one modified nucleic acid sequence having at least one coding sequence of a bestrophin polypeptide, wherein the bestrophin polypeptide is expressed in the plant, or part thereof, and wherein, when the plant is cultivated under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant, or corresponding WT part, that does not overexpress the bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding wild-type (WT) plant, or corresponding WT part, that does not overexpress the bestrophin polypeptide cultivated under ambient carbon dioxide conditions. As used herein, the term "ambient carbon dioxide" refers to the carbon dioxide content in air that is not added to or removed from CO2. In some embodiments, the ambient carbon dioxide conditions are carbon dioxide conditions of 400-500 ppm, 400-550 ppm, 400-600 ppm, 400-650 ppm, 400-700 ppm, 450-500 ppm, 450-550 ppm, 450-600 ppm, 450-650 ppm, 450-700 ppm, 500-550 ppm, 500-600 ppm, 500-650 ppm, 500-700 ppm, 550-600 ppm, 550-650 ppm, 550-700 ppm, 600-650 ppm, 600-700 ppm, or 650-700 ppm. As used herein, the term "carbon use efficiency" may refer to the proportion of carbon obtained from the environment that is incorporated into the biomass of the plant. Carbon use efficiency can be measured by any method known in the art (e.g., subtracting the amount of carbon lost by plant respiration from the total amount of carbon taken up by the plant and then dividing this value by the total amount of carbon taken up by the plant, etc.). In some embodiments, the bestrophin polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of the plant cell.In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0069] In some aspects, the present disclosure provides a plant, or a part thereof, having increased water use efficiency, wherein the plant comprises at least one modified nucleic acid sequence having at least one coding sequence of a green algal bestrophin polypeptide in the plant, or a part thereof, wherein the bestrophin polypeptide is expressed in the plant, or a part thereof, and wherein when the plant is grown under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, grown under ambient carbon dioxide conditions. As used herein, the term "water use efficiency" refers to the ratio of carbon assimilation to water consumption in a plant. Measures of water use efficiency include, but are not limited to, intrinsic water use efficiency and instantaneous water use efficiency. Instantaneous water use efficiency can be calculated by determining the ratio between plant carbon assimilation and plant transpiration. Intrinsic water use efficiency can be calculated by determining the ratio between plant carbon assimilation and plant stomatal conductance. Measures of carbon assimilation can include, but are not limited to, plant photosynthesis rate, yield, and biomass. In some embodiments, the bestrophin polypeptide is localized to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of the plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0070] In some embodiments, the present disclosure relates to a plant, or a part thereof, having increased nitrogen use efficiency, wherein the plant comprises at least one modified nucleic acid sequence having at least one coding sequence of a green algal bestrophin polypeptide in the plant, or a part thereof, wherein the bestrophin polypeptide is expressed in the plant, or a part thereof, and wherein when the plant is cultivated under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than, or the yield, growth rate, or biomass is substantially similar to, the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof. As used herein, the term "nitrogen use efficiency" refers to the ratio of nitrogen used by a plant for metabolism to the total nitrogen supplied to the plant. Nitrogen use efficiency can be measured by any method known in the art (e.g., 15 N isotope labeling, agronomic efficiency, apparent nitrogen recovery). Nitrogen sources supplied to the plant include, but are not limited to, nitrogen contained in soil, nitrogen supplied by nitrogen-fixing bacteria, and nitrogen contained in fertilizers. In some embodiments, the bestrophin polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0071] In some embodiments, the present disclosure relates to a plant, or a part thereof, having reduced photo-inhibition, wherein the plant comprises at least one modified nucleic acid sequence having at least one coding sequence of a green algal bestrophin polypeptide in the plant, or a part thereof, wherein the bestrophin polypeptide is expressed in the plant, or a part thereof, and wherein when the plant is cultivated under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, cultivated under ambient carbon dioxide conditions. As used herein, the term "photo-inhibition" refers to the light-induced decrease in photosynthetic production in a plant. Photo-inhibition can be measured by any method known in the art (e.g., the rate of light-saturated oxygen evolution, the ratio of variable levels of chlorophyll a fluorescence to maximum levels). In some embodiments, the bestrophin polypeptide is localized to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

[0072] In some embodiments of any of the above embodiments, the modified nucleic acid sequence is stably integrated into the nuclear genome of the plant. In some embodiments of any of the above embodiments, at least one modified nucleic acid sequence further comprises a signal peptide sequence or a second nucleic acid sequence encoding a target sequence operably linked to at least one coding sequence of the green algal bestrophin polypeptide, and the expression of the signal peptide sequence or the target sequence results in the localization of the bestrophin polypeptide to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of the plant cell. In some embodiments, the signal peptide sequence or the target sequence is a leader sequence. In some embodiments, the signal peptide sequence or the target sequence is any sequence known in the art to result in polypeptide expression in the chloroplast envelope or the chloroplast thylakoid membrane.

[0073] In some embodiments of any of the above-described embodiments, increased carbon use efficiency, increased water use efficiency, increased nitrogen use efficiency, or decreased photo-inhibition is: a first polypeptide having at least 70% sequence identity, at least 71%, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof; as a result of the expression. In some embodiments of any of the above-described embodiments, increased carbon use efficiency, increased water use efficiency, increased nitrogen use efficiency, or decreased photo-inhibition is: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or; any combination thereof; as a result of the expression.

[0074] In some embodiments of any of the above embodiments, the increase or provision of the ability of bicarbonate to pass through the membrane is: a polypeptide from the group of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111; preferably, it is the result of the expression of a polypeptide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63.

[0075] In some embodiments of any of the above-described embodiments, the plant is a cowpea (i.e., black-eyed pea, Vigna unguiculata), soybean (i.e., soybean, Glycine max), cassava (i.e., manioc, Manihot esculenta), rice (i.e., Oryza sativa, Oryza glaberrima, Zizania spp), wheat (i.e., common wheat, spelt, durum, bread wheat, Triticum aestivum, Triticum spelta, Triticum durum, Triticum spp), barley (i.e., Hordeum vulgare), rye (i.e., Secale cereale), oat (i.e., Avena sativa), potato (i.e., Solanum tuberosum), tomato (i.e., Solanum lycopersicum), or another C3 crop plant. In some embodiments, the plant is tobacco (i.e., Nicotiana tabacum, Nicotiana edwardsonii, Nicotiana plumbagnifolia, Nicotiana longiflora), or Arabidopsis (i.e., rockcress, salad cress, Arabidopsis thaliana).In some embodiments of any of the above embodiments, the plant is not corn (i.e., maize, Zea mays), sorghum (i.e., durra, great millet, milo, Sorghum bicolor), sugarcane (i.e., sugarcane, Saccharum officinarum), millet (i.e., finger millet, common millet, pearl millet, foxtail millet, Eleusine coracana, Panicum miliaceum, Pennisetum glaucum, Setaria italica), switchgrass (i.e., tall panic grass, touchgrass, Panicum virganum), or another C4 crop plant.

[0076] In some embodiments, the plant part of any of the above embodiments is a leaf, stem, root, flower, seed, fruit, cell, or a part thereof. In some embodiments, the plant part is a fruit. In some embodiments, the plant part is a grain, kernel, bean, or tuber.

[0077] In some aspects, the present disclosure relates to pollen grains or ovules of any of the above embodiments.

[0078] In some aspects, the present disclosure relates to protoplasts produced from any of the above embodiments.

[0079] In some aspects, the present disclosure relates to a tissue culture produced from protoplasts or cells of any of the above embodiments, where the cells or protoplasts are produced from one of the plant parts in a group of leaf, anther, pistil, stem, petiole, root, root tip, fruit, seed, flower, cotyledon, hypocotyl, embryo, or meristematic cells.

[0080] In some embodiments, the present disclosure relates to a genetically modified seed, wherein the seed comprises one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane. In some embodiments, the seed produces a plant that has the ability for bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the chloroplast of the plant into the stroma. In some embodiments, the seed produces a plant that has the ability for bicarbonate to cross a membrane from the stroma of at least a portion of the chloroplast of the plant into the lumen. In some embodiments, the plant expresses at least one green algal bestrophin polypeptide. In some embodiments, the green algal bestrophin polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or any combination thereof. In some embodiments, the plant expresses: a first polypeptide having at least 70% sequence identity to SEQ ID NO: 1, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity; a second polypeptide having at least 70% sequence identity to SEQ ID NO: 2, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity; a third polypeptide having at least 70% sequence identity to SEQ ID NO: 3, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity, or any combination thereof.In some embodiments, the plant expresses a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3; or any combination thereof.In some embodiments, the plant expresses a polypeptide from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111; preferably, the plant expresses a polypeptide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63. In some embodiments, the polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of the plant cell. In some embodiments, the plant cell is a mesophyll cell.In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of the plant. In some embodiments of any of the above embodiments, the plant is cowpea (i.e., black-eyed pea, Vigna unguiculata), soybean (i.e., soybean, Glycine max), cassava (i.e., manioc, Manihot esculenta), rice (i.e., Oryza sativa, Oryza glaberrima, Zizania spp), wheat (i.e., common wheat, spelt, durum, bread wheat, Triticum aestivum, Triticum spelta, Triticum durum, Triticum spp), barley (i.e., Hordeum vulgare), rye (i.e., Secale cereale), oat (i.e., Avena sativa), potato (i.e., Solanum tuberosum), tomato (i.e., Solanum lycopersicum), or another C3 crop plant. In some embodiments, the plant is tobacco (i.e., Nicotiana tabacum, Nicotiana edwardsonii, Nicotiana plumbagnifolia, Nicotiana longiflora), or Arabidopsis (i.e., rock cress, salad cress, Arabidopsis thaliana).In some embodiments of any of the above embodiments, the plant is not corn (i.e., maize, Zea mays), sorghum (i.e., durra, great millet, milo, Sorghum bicolor), sugarcane (i.e., sugarcane, Saccharum officinarum), millet (i.e., finger millet, common millet, pearl millet, foxtail millet, Eleusine coracana, Panicum miliaceum, Pennisetum glaucum, Setaria italica), switchgrass (i.e., tall panic grass, touchgrass, Panicum virganum), or another C4 crop plant.

[0081] In some embodiments of any of the above embodiments, the expression of the endogenous carbonic anhydrase is regulated. In some embodiments, the regulated expression can be increased expression, decreased expression, expression at a different location, or any combination thereof.

[0082] [Method for producing and cultivating genetically modified plants] Certain aspects of the present disclosure relate to a method for producing a plant having increased carbon use efficiency, where the steps of the method are: a) introducing a genetic modification into a plant, such that the ability of bicarbonate to pass through a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts to the stroma is increased or provided, or the ability of bicarbonate to pass through a membrane from the stroma of at least a portion of the plant's chloroplasts to the lumen is increased or provided, thereby increasing the carbon use efficiency of the plant.

[0083] In some embodiments, the present disclosure relates to a method of producing a plant having increased water use efficiency, where the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts into the lumen is increased or provided, thereby increasing the water use efficiency of the plant.

[0084] In some embodiments, the present disclosure relates to a method of producing a plant having increased nitrogen use efficiency, where the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts into the lumen is increased or provided, thereby increasing the nitrogen use efficiency of the plant.

[0085] In some embodiments, the present disclosure relates to a method of producing a plant having reduced photoinhibition, where the steps of the method are: a) introducing a genetic modification into a plant, such that as a result, the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts into the lumen is increased or provided, thereby reducing the photoinhibition of the plant.

[0086] In some embodiments, the present disclosure relates to a method of producing a plant having increased growth or productivity, where the steps of the method are: a) introducing a genetic modification into a plant, such that the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts into the stroma is increased or provided, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts into the lumen is increased or provided, thereby increasing the growth or productivity of the plant. Indicators of increased growth can include, but are not limited to: faster growth rate, larger plant, increased biomass, increased dry mass, increased shoot mass, and increased root mass. Indicators of increased productivity can include, but are not limited to: higher crop yield, greater number of leaves, and fewer days to crop maturity.

[0087] In some embodiments of any of the above methods, the expression of an endogenous carbonic anhydrase is regulated. In some embodiments, the regulated expression can be increased expression, decreased expression, expression at a different location, or any combination thereof.

[0088] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased carbon use efficiency, where the steps of the method are: a) providing to a seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, where the seed provides a plant having the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant's chloroplasts into the stroma, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant's chloroplasts into the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases carbon use efficiency, compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0089] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased water use efficiency, wherein the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, wherein the seed provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast into the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast into the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases water use efficiency as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0090] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased nitrogen use efficiency, wherein the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, wherein the seed provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast into the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast into the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases nitrogen use efficiency as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0091] In some embodiments, the present disclosure relates to a method of cultivating a plant having reduced photoinhibition, wherein the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, wherein the seed provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the plant cytoplasm of the plant chloroplast into the stroma, or provides a plant having the ability of bicarbonate to cross a membrane from at least a portion of the stroma of the plant chloroplast into the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane reduces photoinhibition as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0092] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased growth or productivity, wherein the steps of the method are: a) providing to the seed one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane, wherein the seed provides a plant having the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts to the stroma, or a plant having the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant chloroplasts to the lumen; b) cultivating the plant under conditions where the ability of bicarbonate to cross a membrane increases growth or productivity as compared to one or more plants lacking the genetic modification cultivated under the same conditions.

[0093] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased carbon use efficiency, wherein the steps of the method are: a) providing to the tissue culture or protoplast one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane; b) regenerating the tissue culture or protoplast into a juvenile plant; c) growing the juvenile plant into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts to the stroma, or the ability of bicarbonate to cross a membrane from the stroma of at least a portion of the plant chloroplasts to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases carbon use efficiency as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0094] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased water use efficiency, wherein the steps of the method are: a) providing one or more genetic modifications to a tissue culture or protoplast that increase or provide the ability of bicarbonate to cross a membrane; b) regenerating the tissue culture or protoplast into an undeveloped plant; c) growing the undeveloped plant into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts to the stroma or from the stroma of at least a portion of the plant chloroplasts to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases water use efficiency as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0095] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased nitrogen use efficiency, wherein the steps of the method are: a) providing one or more genetic modifications to a tissue culture or protoplast that increase or provide the ability of bicarbonate to cross a membrane; b) regenerating the tissue culture or protoplast into an undeveloped plant; c) growing the undeveloped plant into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a portion of the plant chloroplasts to the stroma or from the stroma of at least a portion of the plant chloroplasts to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases nitrogen use efficiency as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0096] In some embodiments, the present disclosure relates to a method of cultivating a plant having reduced photoinhibition, wherein the steps of the method are: a) providing one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane to a tissue culture or a protoplast; b) regenerating the tissue culture or protoplast into a juvenile plant; c) growing the juvenile plant into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a part of the chloroplast of the plant to the stroma, or the ability of bicarbonate to cross a membrane from the stroma of at least a part of the chloroplast of the plant to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane reduces photoinhibition as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0097] In some embodiments, the present disclosure relates to a method of cultivating a plant having increased growth or productivity, wherein the steps of the method are: a) providing one or more genetic modifications that increase or provide the ability of bicarbonate to cross a membrane to a tissue culture or a protoplast; b) regenerating the tissue culture or protoplast into a juvenile plant; c) growing the juvenile plant into a plant, wherein the plant has the ability of bicarbonate to cross a membrane from the plant cytoplasm of at least a part of the chloroplast of the plant to the stroma, or the ability of bicarbonate to cross a membrane from the stroma of at least a part of the chloroplast of the plant to the lumen; d) transplanting the plant into conditions where the ability of bicarbonate to cross a membrane increases growth or productivity as compared to one or more plants lacking the genetic modification grown under the same conditions.

[0098] In some embodiments of any of the above methods, the seed, tissue culture, or protoplast has one or more genetic modifications that regulate the expression of endogenous carbonic anhydrase. In some embodiments, the regulated expression can be increased expression, decreased expression, expression at a different location, or any combination thereof.

[0099] In some embodiments of any of the above methods, the increase or provision of the ability of bicarbonate to pass through the membrane is the result of the expression of at least one polypeptide selected from the group consisting of a first polypeptide having at least 95% sequence identity with SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity with SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity with SEQ ID NO: 3, or any combination thereof.The increase or provision of the ability of bicarbonate to pass through a membrane, which is any of the above methods, is the result of the expression of at least one polypeptide selected from the following group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111; preferably, it is the result of the expression of at least one polypeptide selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 42, SEQ ID NO: 62, or SEQ ID NO: 63.In some embodiments, the polypeptide is localized in the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell. In some embodiments, the plant cell is a mesophyll cell. In some embodiments, the polypeptide is expressed in at least 70% of the mesophyll cells of a plant. In some embodiments of any of the above embodiments, the plant is cowpea (i.e., black-eyed pea, Vigna unguiculata), soybean (i.e., soybean, Glycine max), cassava (i.e., manioc, Manihot esculenta), rice (i.e., Oryza sativa, Oryza glaberrima, Zizania spp), wheat (i.e., common wheat, spelt, durum, bread wheat, Triticum aestivum, Triticum spelta, Triticum durum, Triticum spp), barley (i.e., Hordeum vulgare), rye (i.e., Secale cereale), oat (i.e., Avena sativa), potato (i.e., Solanum tuberosum), tomato (i.e., Solanum lycopersicum), or another C3 crop plant. In some embodiments, the plant is tobacco (i.e., Nicotiana tabacum, Nicotiana edwardsonii, Nicotiana plumbagnifolia, Nicotiana longiflora), or Arabidopsis (i.e., rock cress, salad cress, Arabidopsis thaliana).In some embodiments of any of the above-described embodiments, the plant is not corn (i.e., maize, Zea mays), sorghum (i.e., durra, great millet, milo, Sorghum bicolor), sugarcane (i.e., sugarcane, Saccharum officinarum), millet (i.e., finger millet, common millet, pearl millet, foxtail millet, Eleusine coracana, Panicum miliaceum, Pennisetum glaucum, Setaria italica), switchgrass (i.e., tall panic grass, touchgrass, Panicum virganum), or another C4 crop plant.

[0100] [Genetically Modified Algae] Certain aspects of the present disclosure relate to genetically modified algae comprising one or more genetic modifications that result in an increased ability of the algae to transport bicarbonate into the lumen of the chloroplast. In some embodiments, the increased bicarbonate transport ability is the result of overexpressing at least one green algal bestrophin polypeptide selected from the group consisting of: a first polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 3; or any combination thereof. In some embodiments, the increased bicarbonate transport ability is the result of overexpressing at least one green algal bestrophin polypeptide selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof. In some embodiments, the polypeptide is localized to the chloroplast thylakoid membrane. In some embodiments, the polypeptide is overexpressed when the algae are under conditions of less than 100 ppm carbon dioxide (CO2 < 0.01% [v / v] in air).

[0101] In some embodiments, the present disclosure relates to a green alga having increased bicarbonate transport, or a part thereof, comprising a modified nucleic acid sequence comprising a coding sequence of at least one green algal bestrophin polypeptide; wherein the bestrophin polypeptide is overexpressed; wherein the bestrophin polypeptide is localized in the chloroplast thylakoid membrane; wherein when the alga is cultured under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air), the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) alga or its WT part that does not overexpress at least one bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from a corresponding WT alga or its corresponding WT part that does not overexpress at least one bestrophin polypeptide and is cultured under conditions of less than 100 ppm carbon dioxide (CO2 less than 0.01% [v / v] in air). In some embodiments, the at least one green algal bestrophin polypeptide is selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.

[0102] In some embodiments, the present disclosure relates to green algae, or a part thereof, having increased growth under conditions of less than 100 ppm carbon dioxide (CO2 < 0.01% [v / v] in air), comprising a modified nucleic acid sequence comprising a coding sequence for at least one green algal bestrophin polypeptide; wherein the bestrophin polypeptide is overexpressed; wherein the bestrophin polypeptide is localized in the chloroplast thylakoid membrane; and wherein when the algae are cultured under conditions of less than 100 ppm carbon dioxide (CO2 < 0.01% [v / v] in air), the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from the corresponding wild-type (WT) algae or WT part thereof that do not overexpress at least one bestrophin polypeptide, or the yield, growth rate, or biomass is substantially similar to the yield, growth rate, or biomass from the corresponding WT algae or corresponding WT part thereof that do not overexpress at least one bestrophin polypeptide and are cultured under conditions of less than 100 ppm carbon dioxide (CO2 < 0.01% [v / v] in air). In some embodiments, the at least one green algal bestrophin polypeptide is selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.

[0103] In some embodiments of any of the above algae, the green algae are selected from the group consisting of: Chlamydomonas reinhardtii, Chlamydomonas eustigma, Volvox carteri f. nagariensis, Gonium pectorale.

[0104] [Method for producing genetically modified algae] In some embodiments, the present disclosure relates to a method of producing algae having increased carbon utilization efficiency, where the steps of the method include: a) introducing a genetic modification into the algae, including an increase in the ability to transport bicarbonate into the lumen of the chloroplast of the algae, thereby increasing the carbon utilization efficiency of the algae. In some embodiments, the acquisition of the transport ability is the result of overexpressing at least one green algal bicarbonate polypeptide selected from the group consisting of: a first polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 1; a second polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 2; a third polypeptide having at least 70% sequence identity, at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or at least 95% sequence identity to SEQ ID NO: 3; or any combination thereof. In some embodiments, the increase in bicarbonate transport ability is the result of overexpressing at least one green algal bicarbonate polypeptide selected from the group consisting of: a first polypeptide having at least 95% sequence identity to SEQ ID NO: 1, a second polypeptide having at least 95% sequence identity to SEQ ID NO: 2, a third polypeptide having at least 95% sequence identity to SEQ ID NO: 3, or any combination thereof.

[0105] ​One embodiment of the present invention provides a plant or plant cell comprising one or more modified plant genes and / or introduced genes. For example, the present disclosure provides a transgenic plant having an increased or provided ability for bicarbonate to pass through a membrane from at least a portion of the plant cell cytoplasm to the stroma of the plant chloroplast. Further, the present disclosure provides a transgenic plant having an increased or provided ability for bicarbonate to pass through a membrane from the stroma of at least a portion of the plant chloroplast to the lumen. Further, the present disclosure provides a transgenic plant having at least one modified nucleic acid sequence comprising at least one coding sequence of a green algal bestrophin polypeptide. The regulated expression of other genetic elements (e.g., endogenous carbonic anhydrase) is also contemplated and described herein.

[0106] The transformation and production of genetically modified monocotyledonous and dicotyledonous plant cells are known in the art. See, for example, Weising et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Patent No. 5,679,558; Agrobacterium Protocols, edited by Gartland, Humana Press Inc (1995); and Wang et al., Acta Hort. 461:401-408 (1998). The choice of method varies with the type of plant to be transformed, the particular application and / or desired result. Suitable transformation techniques are readily selected by those skilled in the art.

[0107] Any methodology known in the art for deleting, inserting or otherwise modifying cellular DNA (e.g., genomic DNA and organellar DNA) can be used in practicing the invention described herein. For example, an unarmed Ti plasmid containing a gene construct for deletion or insertion of a target gene in Agrobacterium tumefaciens can be used to transform plant cells, and the transformed plants can then be regenerated from the transformed plant cells using procedures described in the art, for example, as follows: EP0116718, EP0270822, PCT publication WO84 / 02913, and published European patent application ("EP") 0242246. Each of the Ti plasmid vectors contains a gene located between the border sequences of the T-DNA of the Ti plasmid, or at least a gene located to the left of the right border sequence. Of course, other types of vectors can be used to transform plant cells using procedures such as: direct gene transfer (e.g., as described in EP0233247), pollen-mediated transformation (e.g., as described in EP0270356, PCT publication WO85 / 01856, and U.S. Patent No. 4,684,611), plant RNA virus-mediated transformation (e.g., as described in EP0067553 and U.S. Patent No. 4,407,956), liposome-mediated transformation (e.g., as described in U.S. Patent No. 4,536,475), and methods for transforming specific lines of maize (e.g., U.S. Patent No. 6,140,553; Fromm et al., Bio / Technology (1990) 8, 833-839); Gordon-Kamm et al., The Plant Cell (1990) 2, 603-618), and methods for transforming specific lines of rice (Shimamoto et al., Nature (1989) 338, 274-276; Datta et al., Bio / Technology, (1990) 8, 736-740), and methods for generally transforming monocotyledonous plants (PCT publication WO92 / 09696), and other methods.For the transformation of cotton, the method described in PCT Patent Publication WO00 / 71733 can be used. For the transformation of soybean, methods known in the art (e.g., Hinchee et al. (Bio / Technology, (1988) 6, 915) and Christou et al. (Trends Biotech, (1990) 8, 145) or the method of WO00 / 42207) are referred to.

[0108] The transgenic plants of the present invention can be used in conventional plant breeding schemes to produce more transgenic plants having the same characteristics or to introduce genetic modifications in other varieties of the same or related plant species. Seeds obtained from the transformed plants preferably contain genetic modifications such as stable inserts in chromosomal DNA or organellar DNA. Plants containing the genetic modification according to the present invention include plants containing roots and rhizomes of plants containing the genetic modification of the present invention, such as fruit trees or ornamental plants, or plants derived therefrom. Accordingly, any non-transgenic grafted plant parts inserted into the transformed plants or plant parts are included in the present invention.

[0109] The introduced genetic element, whether it is an expression vector or an expression cassette that results in the expression of the introduced gene, will typically utilize a plant-expressible promoter. As used herein, "plant-expressible promoter" refers to a promoter that ensures the expression of the genetic modification of the present invention in plant cells.Examples of promoters that drive constitutive expression in plants are known in the art and include the following: for example, the strong constitutive 35S promoter of cauliflower mosaic virus (CaMV) (“35S promoter”) (e.g., isolated CM1841 (Gardner et al., Nucleic Acids Res, (1981) 9, 2871 - 2887), CabbB S (Franck et al., Cell (1980) 21, 285 - 294), and CabbB JI (Hull and Howell, Virology, (1987) 86, 482 - 483)); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., Plant Mol Biol, (1992) 18, 675 - 689), the gos2 promoter (de Pater et al., The Plant J (1992) 2, 834 - 844), the emu promoter (Last et al., Theor Appl Genet, (1990) 81, 581 - 588), the promoters described by An et al. (The Plant J (1996) 10, 107), actin promoters such as the rice actin promoter described by Zhang et al. (Plant Cell, (1991) 3, 1155 - 1165); the promoter of cassava vein mosaic virus (WO97 / 48819, Verdaguer et al. (Plant Mol Biol, (1998) 37, 1055 - 1067), the pPLEX series of promoters from subterranean clover stunt virus (WO96 / 06932, particularly the S4 or S7 promoter), the alcohol dehydrogenase promoter, e.g., pAdh1S (GenBank accession numbers X04049, X00581), and the TR1' promoter and TR2' promoter that drive the expression of the 1' and 2' genes of T - DNA, respectively (each “TR1' promoter” and “TR2' promoter”) (Velten et al., EMBO J, (1984) 3, 2723 - 2730).

[0110] Alternatively, a plant-expressible promoter can be a tissue-specific promoter, i.e., a promoter that directs a higher level of expression in some cells or tissues of a plant (e.g., green tissue), such as the promoter of PEP carboxylase. The plant PEP carboxylase promoter (Pathirana et al., Plant J, (1997) 12:293-304) has been described as a strong promoter for expression in vascular tissue and is useful in one embodiment of the present invention. Alternatively, a plant-expressible promoter can also be a wound-inducible promoter, such as the promoter of the pea cell wall invertase gene (Zhang et al., Plant Physiol, (1996) 112:1111-1117). As used herein, a "wound-inducible" promoter means that upon injury to the plant, either by mechanical means or insect feeding, the expression of the coding sequence under the control of the promoter is significantly increased in such plants. These plant-expressible promoters can be combined with enhancer elements, and they can be combined with minimal promoter elements or can contain repeat elements to ensure the desired expression profile.

[0111] In some embodiments, genetic elements can be utilized to increase expression in plant cells. For example, an intron at the 5' or 3' end of the introduced gene, or the coding sequence of the introduced gene, such as the hsp70 intron. Other such genetic elements can include, but are not limited to: promoter enhancer elements; double or triple promoter regions; a 5' leader sequence that is different from another introduced gene or from the endogenous (plant host) gene leader sequence; a 3' trailer sequence that is different from another introduced gene used in the same plant or from the endogenous (plant host) trailer sequence.

[0112] The introduced gene of the present invention can be inserted into the host cell DNA, and the inserted gene portion is upstream (i.e., 5') of an appropriate 3' terminal transcriptional regulatory signal (i.e., a transcriptional product formation signal and a polyadenylation signal). This is preferably achieved by inserting the gene into the plant cell genome (nucleus or chloroplast). Preferred polyadenylation signals and transcriptional product formation signals include polyadenylation signals and transcriptional product formation signals such as those that function as 3' untranslated DNA sequences in transformed plant cells: nopaline synthase gene (Depicker et al., J. Molec Appl Gen, (1982) 1, 561-573), octopine synthase gene (Gielen et al., EMBO J, (1984) 3:835 845), SCSV or Malic enzyme terminator (Schunmann et al., Plant Funct Biol, (2003) 30:453-460), and T DNA gene 7 (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981 6998). In some embodiments, one or more introduced genes are stably integrated into the nuclear genome. Stable integration exists when the nucleic acid sequence remains integrated into the nuclear genome and continues to be expressed through subsequent plant generations (i.e., detectable mRNA transcripts or proteins are produced). Stable integration into the nuclear genome can be achieved by any method known in the art (e.g., particle bombardment, Agrobacterium-mediated transformation, CRISPR / Cas9, electroporation of protoplasts, microinjection, etc.).

[0113] The term recombinant nucleic acid or modified nucleic acid refers to a polynucleotide made by the combination of two sequence segments that are separated by other means, achieved by the manipulation of isolated segments of polynucleotides by genetic engineering techniques or chemical synthesis. In so doing, polynucleotide segments of desired functions can be ligated together to generate a combination of desired functions.

[0114] As used herein, the terms "overexpression" and "upregulation" refer to increased expression (e.g., of mRNA, polypeptide, etc.) as a result of genetic modification, compared to expression in a wild-type organism (e.g., a plant, an alga). In some embodiments, the increase in expression is a slight increase, about 10% more than the expression in the wild type. In some embodiments, the increase in expression is an increase of 50% or more (e.g., 60%, 70%, 80%, 100%, etc.) compared to the expression in the wild type. In some embodiments, an endogenous gene is overexpressed. In some embodiments, an exogenous gene is overexpressed by being expressed. Overexpression of a gene in a plant or alga can be achieved by any method known in the art, including but not limited to the following uses: a constitutive promoter, an inducible promoter, a high-expression promoter (e.g., the PsaD promoter), an enhancer, a transcriptional regulatory sequence and / or a translational regulatory sequence, codon optimization, a modified transcription factor, and / or a mutant gene or a modified gene that controls the expression of the overexpressed gene.

[0115] When a recombinant nucleic acid is intended for the expression, cloning, or replication of a particular sequence, the DNA construct prepared for introduction into a host cell typically includes a replication system (i.e., a vector) recognized by the host, which includes a DNA fragment intended to encode the desired polypeptide, and may also include a transcriptional regulatory sequence and a translation initiation regulatory sequence operably linked to the segment encoding the polypeptide. Further, such a construct may include a cell localization signal (e.g., a chloroplast localization signal). In a preferred embodiment, such a DNA construct is introduced into the genomic DNA, chloroplast DNA, or mitochondrial DNA of the host cell.

[0116] In some embodiments, a non-integrating expression system can be used to induce the expression of one or more introduced genes. The expression system (expression vector) can include, for example: an origin of replication or an autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer, and necessary processing information sites (such as ribosome binding sites, RNA splice sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilization sequences). A signal peptide can also, where appropriate, be included from a suitable secreted polypeptide of the same or a related species that enables the protein to pass through and / or remain in the cell membrane, cell wall, and / or be secreted from the cell.

[0117] Selectable markers useful in practicing the methodology of the invention disclosed herein can be positive selectable markers. Typically, positive selection refers to the case where genetically modified cells can survive in the presence of a toxic substance only when the recombinant polynucleotide of interest is present within the cell. Negative selectable markers and screening markers are also known in the art and are contemplated by the present invention. One of ordinary skill in the art will recognize that any relevant marker available can be utilized in the practice of the invention disclosed herein.

[0118] Screening and molecular analysis of the recombinant strains of the present invention can be performed using nucleic acid hybridization techniques. Hybridization procedures, such as those modified using the techniques described herein, are useful for identifying polynucleotides and have sufficient homology with target regulatory sequences to be useful as taught herein. Specific hybridization techniques are not essential to the present invention. As improvements are made to hybridization techniques, they can be readily applied by those skilled in the art. Hybridization probes can be labeled using any suitable label known to those skilled in the art. Hybridization conditions and washing conditions (e.g., temperature and salt concentration) can be varied to change the stringency of the detection threshold. For further guidance regarding hybridization conditions, see, for example, Sambrook et al. (1989) (see below), or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley and Sons, NY, NY.Y.

[0119] Furthermore, screening and molecular analysis of genetically modified strains, as well as the production of desired isolated nucleic acids, can be carried out using the polymerase chain reaction (PCR). PCR is an iterative, enzymatic, preparative synthesis of nucleic acid sequences. This procedure is known and commonly used by those skilled in the art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159; Saiki et al., (1985) Science 230:1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest, flanked by two oligonucleotide primers that hybridize to the reverse strands of the target sequence. The primers are oriented such that their 3' ends face each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers using DNA polymerase result in the amplification of the segment defined by the 5' ends of the PCR primers. Since the extension products of each primer can serve as templates for the other primer, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in an exponential accumulation of a specific target fragment up to millions of times within a few hours. By using a thermostable DNA polymerase such as Taq polymerase isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be fully automated. Other enzymes that can be used are known to those skilled in the art.

[0120] The nucleic acids and proteins of the present invention may also include homologs of the specifically disclosed sequences. The homology (e.g., sequence identity) can be from 50% to 100%. In some cases, such homology exceeds 80%, exceeds 85%, exceeds 90%, or exceeds 95%. The degree of homology or identity required for any intended use of the sequence is readily determined by one of ordinary skill in the art. As used herein, the percentage of sequence identity between two nucleic acids is determined using algorithms known in the art as disclosed by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 and modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. The NBLAST nucleotide search is performed using the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences having the desired percentage of sequence identity. To obtain gap alignments for comparison purposes, gap BLAST is used as described in Altschul et al. (1997) Nucl. Acids Res. 25:3389-3402. When utilizing the BLAST and gap BLAST programs, the default parameters of each program (NBLAST and XBLAST) are used. See www.ncbi.nih.gov.

[0121] Preferred host cells are plant cells or algal cells. A recombinant host cell, in the context of this text, contains an isolated nucleic acid molecule, contains one or more deletions or other non-functional genes that are normally present and functional in the host cell, or is genetically modified to contain one or more genes for producing at least one recombinant protein. The nucleic acid encoding the protein of the present invention can be introduced by any means known in the art that is appropriate for a particular type of cell, including but not limited to transformation, lipofection, electroporation, or any other methodology known to those skilled in the art.

[0122] While the invention has been generally described, it will be better understood by reference to specific, non-limiting examples included herein and not intended to limit the scope of the invention as defined by the claims.

[0123] 〔Examples〕 The present disclosure is described in more detail in the following examples, which are in no way intended to limit the scope of the disclosure any more than the claims do. The accompanying drawings are intended to be considered an integral part of the specification and description of the present disclosure. The following examples are provided for illustration purposes only and do not limit the claimed disclosure.

[0124] 〔Example 1: General materials and methods used for cell culture and growth〕 The following example describes the cell culture conditions and growth conditions used in all of the following examples. Chlamydomonas reinhardtii cells were maintained on either Tris-acetate-phosphate (TAP) medium or yeast-acetate (YA) medium on Petri plates. Prior to the experiment, the cells were inoculated into minimal (i.e., carbon source-free) medium and grown at high CO2 (CO2 higher than 5% in air) at 2 - 3×10 6 cells·mL -1They were grown to the concentration of. Subsequently, these cells were diluted in minimal medium and grown at the indicated pH and CO2 concentrations.

[0125] The Chlamydomonas reinhardtii culture conditions were the same as those described in Ma et al., Plant Physiol 156:884 - 896, 2011. The D66 strain (nit2 - , cw15, mt + ) was obtained from Dr. Rogene Schnell (University of Arkansas, Little Rock). CMJ030 (CC - 4533; cw15, mt - ) and bst3 (BST3 knockout LMJ.RY0402.089365) were obtained from the CLiP collection of the Chlamydomonas culture collection (Zhang et al., Plant Cell 26(4):1398 - 1409, 2014). Tris - acetate - phosphate (TAP) medium, yeast - acetate (YA) medium, and minimal (MIN; without acetate, i.e., without carbon source) were prepared according to Sueoka, PNAS 46:83 - 91, 1960. Both TAP and YA petri plates were prepared by adding 1.2% (w / v) agar, and Chlamydomonas reinhardtii cells were maintained on either type of petri plate. Cell cultures were initiated for mixotrophic growth by inoculating colonies from TAP plates into 100 mL of TAP liquid medium in a Erlenmeyer flask. The cultures were grown to the early logarithmic phase under continuous illumination (100 μmol·m -2 ·s -1 ) and shaking for 48 hours. Early - logarithmic - phase TAP growth cultures were harvested, washed with MIN medium, then resuspended in MIN medium, bubbled with high CO2 (CO2 was 5% [v / v] in air), and grown to an OD 6 of 0.2 - 0.3 (~2 - 3×10 -1 cells·mL 730 . For CCM induction, the cells were transferred to low CO2 (CO2 less than 0.01% [v / v] in air) bubbling for 12 hours or to ambient CO2 (CO2 was 0.04% - 0.045% [v / v] in air).

[0126] 〔Example 2: Phylogenetic tree of bestrophin family genes〕 The following example describes the construction of a phylogenetic tree depicting the evolutionary relationships of bestrophin family genes in various photosynthetic organisms. In mammals, bestrophin proteins are known to conduct chloride and bicarbonate. In plants, most bestrophin proteins have not yet been characterized.

[0127] 〔Materials and methods〕 Three Chlamydomonas reinhardtii genes, BESTROPHIN1 (BST1), BESTROPHIN2 (BST2), and BESTROPHIN3 (BST3), were selected for use in this phylogenetic tree for several reasons. One is that the BST1, BST2, and BST3 genes share a common region (see Figures 1A - 1B). Another is that the amino acid sequences of the BST1 (SEQ ID NO: 1), BST2 (SEQ ID NO: 2), and BST3 (SEQ ID NO: 3) proteins are more than 80% identical to each other (see Figure 1C). Chlamydomonas reinhardtii contains seven other predicted bestrophins, but none of these other bestrophins have more than 45% identity with these three proteins. Finally, all three selected bestrophins were predicted to be targeted to the chloroplast.

[0128] The amino acid sequences for System Trees: BST1 (Cre16.g662600.t1.2), BST2 (Cre16.g663400.t2.1), and BST3 (Cre16.g663450.t1.2) were BLASTED against the following: NCBI Genbank (Benson et al., Nucleic Acids Res, 21(13):2963-2965, 1993), and Phytozome v12.1 (https: / / Phytozome.jgi.doe.gov / pz / portal.html; Goodstein et al., Nucleic Acids Res 40(Database issue):D1178-1186, 2012). The top hits identified using these databases were downloaded. Additionally, the amino acid sequences encoding Homo sapiens BEST1 (SJM31533.1) and Klebsiella pneumoniae bestrophin (pdb_4WD8_A) were downloaded from NCBI Genbank and included as outgroups in the phylogenetic analysis. A total of 63 initial sequences were aligned in Geneious 11.1.4 (Kearse et al., Bioinformatics 28(12):1647-1649, 2012) using the ClustalW (Thompson et al., Nucleic Acids Res 22(22):4673-4680, 1994) algorithm with the amino acid substitution matrix BLOSUM62 (Henikoff and Henikoff, PNAS 89(22):10915-10919, 1992). Duplicate sequences from the two databases (NCBI and Phytozome), as well as sequences with less than 70% pairwise percentage positive identity (BLOSUM62), were excluded. The final alignment included 30 sequences and was manually trimmed to remove variable-length termini from the sequences. Phylogenetic analysis was completed in MEGA X (Kumar et al., Comput Appl Biosci 10(2):189-191, 1994).The best maximum likelihood (ML) model for phylogenetic analysis of the alignment was calculated using the model selection function in MEGA X. The ML tree was constructed using the LG substitution model (Le and Gascuel, Mol Biol Evol 25(7):1307-1320, 2008) with a gamma distribution (5 discrete categories) and 500 bootstrap replicates (see Figure 1D). The tree was formatted and shaded using Adobe Illustrator CC. A phylogenetic tree of the protein sequences of Chlamydomonas reinhardtii BST1, BST2, and BST3 homologs was constructed using maximum likelihood (ML).

[0129] 〔Results〕 Figure 1A shows a schematic diagram of the BST1, BST2, and BST3 genes. The light gray squares represent exons, the thin gray lines represent introns, the thick gray lines represent untranslated regions (UTRs), and the overlapping lines (overlapping exons and introns) indicate the common regions shared by the three genes. Additionally, the length of each of the three bestrophin genes is shown, with BST1 being the shortest, less than 3000 kb, and BST2 being the longest, exceeding 5000 kb. In Figure 1B, the positions and orientations of the BST1, BST2, and BST3 genes on the Chlamydomonas reinhardtii genome are shown. BST1 (Cre16.g662600), BST2 (Cre16.g663400), and BST3 (Cre16.g663450) are paralogous genes located within a 130 kbp region on chromosome 16 of Chlamydomonas reinhardtii. Furthermore, this image shows the relative positions of BST1, BST2, and BST3 to each other. Figure 1C shows the amino acid alignment of the BST1, BST2, and BST3 proteins, where the asterisks in the bottom row indicate the amino acids that are identical in all three proteins. The BST1, BST2, and BST3 proteins are more than 80% identical to each other.

[0130] Figure 1D shows a phylogenetic tree representing the relationship between the protein sequences of Chlamydomonas reinhardtii BST1, BST2, and BST3 homologs in various photosynthetic organisms. These organisms include vascular plants (second group from the top), non-vascular plants (third group from the top), diatoms (fourth group from the top), and green algae (bottom group). The Chlamydomonas reinhardtii vestrophin proteins are shown in bold within the green algae group. An outgroup consisting of proteins from the non-photosynthetic organisms human (Homo sapiens) and bacterium (Klebsiella pneumoniae) is also shown (top group). Sequence alignment of Chlamydomonas reinhardtii BST1, BST2, and BST3 with human bestrophin 1 (BEST1) showed low sequence identity (21 - 23%) between BEST1 and BST1 - 3. The Chlamydomonas reinhardtii vestrophin proteins show considerable sequence similarity to each other and to other green algae vestrophin proteins, as indicated by their proximity within the tree and the shallower branching pattern connecting these proteins. In particular, Volvox carteri and Chlamydomonas eustigma are very closely related to Chlamydomonas reinhardtii, while Dunaliella salina is more distantly related to Chlamydomonas reinhardtii. The diatom species (highlighted in blue-green) are very distantly related to Chlamydomonas reinhardtii and actually arose from different endosymbiotic events. This distant relationship is clearly depicted by the deeper branching pattern connecting these proteins. The vascular plant and non-vascular plant proteins shown in Figure 1D have only low sequence similarity of 30 - 35% to the Chlamydomonas reinhardtii vestrophin proteins, as depicted by their more distant position and the deeper branching pattern connecting these proteins. For example, the Arabidopsis thylakoid-localized VCCN1 protein has approximately 30% sequence identity with BST1 - 3. The other proteins used to construct this phylogenetic tree are also bestrophin family genes, but they are slightly more distantly related to the Chlamydomonas reinhardtii vestrophins.At the amino acid level, Chlamydomonas reinhardtii bestrophin has less than 50% identity with bestrophin proteins found in higher plants or mammals. Furthermore, human bestrophin proteins are sufficiently different from Chlamydomonas reinhardtii bestrophin and can be used as an outgroup to root the phylogenetic tree, along with the bacterial Klebsiella pneumoniae bestrophin protein.

[0131] 〔Example 3: Bestrophins are upregulated under low CO2 and their expression is controlled by CIA5〕 CIA5 is a transcription factor that controls many CCM genes. In particular, CIA5 controls all known CCM transporters. The following examples describe the analysis of bestrophin expression in wild-type (WT) D66 Chlamydomonas reinhardtii strains and cia5 mutant Chlamydomonas reinhardtii strains grown under either high CO2 or low CO2 conditions.

[0132] 〔Materials and methods〕 The cell culture conditions and growth conditions for Chlamydomonas reinhardtii cells were as described in Example 1. For the experiments, WT D66 Chlamydomonas reinhardtii strains and cia5 mutant Chlamydomonas reinhardtii strains were cultured under either high CO2 (CO2 at 5% [v / v] in air) or low CO2 conditions (CO2 less than 0.04% [v / v] in air, i.e., ambient CO2 conditions).

[0133] RNA samples were obtained from both high-CO2 and low-CO2 acclimated cultures of Chlamydomonas reinhardtii strains. RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer's instructions. 1 μg of RNA per sample was used as a template for cDNA, which was prepared using ProtoScript® First Strand cDNA Synthesis Kit (NEB) according to the manufacturer's instructions. 100 ng of RNA per sample was used, and qRTPCR was performed using QuantStudio 6 and Luna® Universal One-Step RT-qPCR Kit from NEB according to the manufacturer's instructions. Semi-quantitative RT-PCR was performed using primers specific for BST1, BST2, or BST3, with actin primers as a control (listed in Table 1). PCR products were analyzed using gel electrophoresis. The experiments were repeated twice.

[0134]

Table 1

[0135] Analysis of the time-course changes in BST1, BST2, and BST3 expression was also performed. For this analysis, cells grown under high CO2 were transferred to low CO2 for 2 - 12 hours, then RNA was extracted and cDNA was prepared as described above. This analysis was repeated twice.

[0136] 〔Results〕 As shown in Fig. 2A, in D66(WT), the expression levels of all three bestrophin genes were up-regulated under low CO2 conditions compared to high CO2 conditions. In particular, BST3 expression was very low in cells grown under high CO2 conditions and much higher in cells grown under low CO2 conditions. The increased band intensity from D66 cells grown under low CO2 conditions compared to D66 cells grown under high CO2 conditions clearly shows that all three genes are up-regulated under low CO2 conditions. In contrast to D66, low CO2 conditions did not induce increased expression of BST1, BST2, or BST3 in the cia5 mutant. Furthermore, BST1 and BST3 were not expressed under any conditions in the cia5 mutant, which is the same transcriptional pattern as other CCM genes in the cia5 mutant (Xiang et al., PNAS 98(9):5341-5346, 2001; Fukuzawa et al., PNAS 98(9):5347-5352, 2001; Moroney et al., Plant Physiol 89(3):897-903, 1989). The BST2 transcript level in cia5 cells was the same under both high CO2 and low CO2 conditions and did not show significant induction under low CO2. This is in contrast to D66 cells in which BST2 transcript levels increase under low CO2 conditions.

[0137] Fig. 2B shows the expression of the three BST genes in D66 at different time points after transfer from high CO2 to low CO2 conditions. All three genes had increased transcript levels within 2 hours after switching to low CO2, and these expression levels were maintained for at least 12 hours after induction. These results demonstrate that all three BST genes were up-regulated under low CO2 growth conditions known to induce CCM. Furthermore, this up-regulation was not seen in the cia5 mutant, suggesting that the expression of the bestrophin genes is regulated by CIA5. Collectively, these results indicate the potential role of the bestrophin genes in CCM.

[0138] [Example 4: Localization of Bestrophin in Chloroplasts] The current CCM model indicates that bicarbonate transporters in the chloroplast thylakoid membrane are required for bicarbonate uptake, but these transporters have not yet been identified. The following examples describe the localization of chimeric Bestrophin-Venus proteins in Chlamydomonas reinhardtii cells. A fluorescence imaging approach was selected to identify specific locations within the chloroplast because computational analysis predicted chloroplast targeting based on the leader sequences of the three Bestrophin proteins rather than on specific locations within the chloroplast.

[0139] [Materials and Methods] Fluorescent Protein Tagging: For this experiment, the coding sequences of BST1, BST2, or BST3 were fused to the coding sequence of Venus to generate the fusion proteins BST1-Venus, BST2-Venus, and BST3-Venus. The BST1-3 genes were cloned and transformed into the Chlamydomonas strain CC-4533 as described by Mackinder et al., Cell 171(1):133-147 e114, 2017. Briefly, the open reading frames of the BST1-3 genes were PCR amplified from genomic DNA (primers are listed in Table 2) and cloned into pLM005 via Gibson assembly with the C-terminal Venus-3xFLAG and the PSAD promoter. Three separate constructs were generated, one for each BST. PSAD is a high-expression promoter that drives a nuclear gene encoding a abundant chloroplast protein located on the stromal side of photosystem I in Chlamydomonas reinhardtii (Fischer and Rochaix, 2001). For transformation, wild-type cultures were grown to mid-log phase and concentrated to 2×10 8 cells·mL -1 . The suspension was mixed with the constructed plasmid linearized by EcoRV before electroporation. The suspension was then supplemented with TAP paromomycin (20 μg·mL -1) plated thereon. Three separate strains expressing BST1-Venus, strains expressing BST2-Venus, and strains expressing BST3-Venus were generated. Fluorescent colonies were identified using a Typhoon 8610 scanner. The laser settings for Venus were 532 nm for excitation and 555 / 20 for emission, and chlorophyll autofluorescence was excited at 633 nm and emitted at 670 / 30.

[0140]

Table 2

[0141] Confocal microscopy: Next, to determine the localization of the bestrophin protein, fluorescent images of the three generated strains were taken using a confocal microscope. The identified fluorescent colonies were grown heterotrophically in TAP medium until they reached the mid-logarithmic phase. The cultures were then collected and resuspended in Tris minimal medium overnight before imaging. Images were taken using a Laser-scanning microscope LSM880 (Zeiss) equipped with an Airyscan module, using a 1.4 NA x63 objective lens. 514 nm and 561 nm argon lasers were used for the excitation of Venus and chlorophyll, respectively. The filters were set at 525 - 550 nm for Venus emission and 620 - 670 nm for chlorophyll emission. Chlorophyll fluorescence was used to localize the chloroplast thylakoid stacks in these strains, and the overlay of Venus and chlorophyll fluorescence was used to identify the position of the bestrophin protein relative to the chloroplast thylakoid stacks. Multiple replicates were imaged.

[0142] 〔Results〕 As shown in Fig. 3A, all three bestrophin fusion proteins showed a heterogeneous signal within the chloroplasts, as indicated by the Venus signal (shown in the left column). When the Venus signal and the chlorophyll signal (shown in the middle column) were merged, an overlap was seen between the Venus signal and the thylakoid stacks in the chloroplasts. All three BST-Venus fusion proteins were localized to the thylakoid membranes of the chloroplasts. Furthermore, the thylakoid tubules of the pyrenoid showed the same overlap between the Venus signal and the chlorophyll signal. This is clearly depicted in Fig. 3B, which shows an enlarged pyrenoid image of the BST1-Venus strain. The arrow indicates the location where BST1-Venus fluorescence is seen inside the pyrenoid in the thylakoid tubules that penetrate the pyrenoid. Both of these results confirm the computer prediction (based on the bestrophin leader sequence) that bestrophin is targeted to the chloroplasts and strongly indicate the thylakoid localization of the bestrophin protein.

[0143] 〔Example 5: Bestrophin is required for Chlamydomonas reinhardtii growth under low CO2〕 The following example describes the effect of reducing the expression of the three bestrophins BST1, BST2, and BST3 on the growth of Chlamydomonas reinhardtii. The growth phenotype of the triple bestrophin RNAi knockdown strain bsti-1 was compared with that of the WT strain D66, as well as the mutant strains cia3 and pmp1, under growth conditions of various CO2 concentrations and pH. The two mutant strains have mutations in known CCM components; the cia3 strain has a mutation in CAH3, which is a thylakoid carbonic anhydrase, while the pmp1 strain has a mutation in LCIB / LCIC, which is a stromal carbonic anhydrase.

[0144] 〔Materials and methods〕 BST1, BST2 and BST3 knockdown using RNAi: To target all three bestrophin genes simultaneously, an RNAi knockdown approach was selected. Currently, only the bst3 knockout strain is available from the Chlamydomonas Culture Collection. The bst1 and bst2 knockout strains are not available. Furthermore, as described in detail in Example 7, the bst3 knockout strain was tested under low CO2 conditions and was found to grow normally. This means both that only the bst3 knockout strain has been tested and that the material to generate a triple knockout strain is not currently available. Therefore, the RNAi knockdown approach was considered the best option.

[0145] Artificial microRNA constructs for knockdown of BST proteins were made using the protocol of Molnar et al., Plant J 58(1):165-174, 2009. Briefly, the web microRNA designer (WMD3) website (http: / / WMD3.weigelworld.org / cgi-bin / webapp.cgi) was used to design two sets of oligos complementary to various positions in the "common region" of the three BST coding sequences. Two independent constructs were designed and cloned into the pChlamyRNA3int plasmid obtained from the Chlamydomonas resource center. Oligos designed to target the three bestrophins used for RNAi knockdown are shown in Table 3. Two triple knockdown lines (BST-RNAi line 1 and 2 or bsti-1 and bsti-2) were isolated from 400 transformants. Bsti-1 was generated using oligo B1 forward and reverse, and bsti-2 was generated using oligo B2 forward and reverse.

[0146]

Table 3

[0147] The pChlamyRNA3int plasmid having the AphVIII gene conferring paromomycin resistance (para R ) was transformed into D66 by electroporation (Shimogawara et al., Genetics 148(4):1821-1828, 1998). The transformants were selected on TAP agar medium containing the antibiotic paromomycin (4 μg·mL -1 ; Invitrogen). The resistant strains were then screened for the "sick on low CO2" phenotype by replica plating onto MIN plates. Next, these were placed in a high CO2 chamber (CO2 was 5% [v / v], in air) and a low CO2 chamber (CO2 was 0.01% [v / v], in air) equipped with continuous illumination (100 μmol·m -2 ·s -1 ) for 7 days. Spot tests were performed by suspending the growing cells in liquid MIN medium to the same cell concentrations (OD 730 = 0.1, 0.05 and 0.025), and 15 μL was spotted onto MIN plates. These plates were placed in the high CO2, ambient CO2 and low CO2 chambers for 7 days. The CO2 concentration was measured using an environmental gas monitor (EGM-4, PP Systems, Massachusetts).

[0148] Quantitative RT-PCR (qRT-PCR; qPCR): RNA extraction was performed using Trizol reagent (Invitrogen) according to the manufacturer's instructions. 1 μg of RNA per sample was used as a template for cDNA, which was prepared using ProtoScript® First Strand cDNA Synthesis Kit (NEB) according to the manufacturer's instructions. 100 ng of RNA per sample was used, and qRT-PCR was performed using QuantStudio 6, using Luna® Universal One-Step RT-qPCR Kit from NEB according to the manufacturer's instructions. The primers used for qPCR are listed in Table 4; the CBLP primers were used as a control.

[0149]

Table 4

[0150] Growth phenotype examination under various pH and CO2 levels: The WT strain D66, the mutant strain cia3 (CAH3 knockout), the mutant strain pmp1 (LCIB / LCIC knockout), and bsti-1 were grown under extremely low CO2 (CO2 is 0.01% (v / v) in air), low (or ambient) CO2 (CO2 is 0.04% - 0.045% (v / v) in air), and high CO2 (CO2 is 5% (v / v) in air). Growth under each of these three CO2 conditions was tested at both pH 7 and pH 8.4. Three different cell concentrations were used to inoculate the plates; the highest concentration was 10,000 cells, the intermediate concentration was 5,000 cells, and the lowest concentration was 2,500 cells. Three spots of three different concentrations were applied to each test plate for all four strains. The growth phenotype examination experiment was repeated three times.

[0151] 〔Results〕 To investigate the role of bestrophins in the growth of Chlamydomonas reinhardtii under low CO2 conditions, an RNAi approach was adopted to reduce the expression of all three bestrophin genes simultaneously. Using RNAi targeting BST1, BST2, and BST3, two RNAi knockdown lines, bsti-1 and bsti-2, were generated. The expression levels of BST1, BST2, and BST3 in bsti-1, bsti-2, and the WT control strain D66 were measured using quantitative RT-PCR. As shown in Figure 4, the expression levels of all three bestrophin genes were lower in the bsti-1 and bsti-2 RNAi knockdown lines compared to the WT control strain D66 (the error bars shown indicate the standard error for three biological replicates). In particular, bsti-1 and bsti-2 (BST-RNAi lines 1 and 2) showed approximately 60 - 90% knockdown in the expression of BST1, BST2, and BST3 compared to D66. This result indicates that the expression of all three bestrophin genes in the bsti-1 and bsti-2 RNAi knockdown lines was effectively reduced using the RNAi approach.

[0152] Next, the bsti-1 RNAi knockdown lines, wild-type strain D66, as well as the CAH3 mutant cia3, and the LCIB / LCIC mutant pmp1 were tested for their ability to grow under their respective pH and CO2 levels. Cultures were grown at pH 7 or 8.4, and under extremely low CO2, low CO2, or high CO2 conditions. At extremely low CO2, bsti-1 showed significantly reduced growth, which was further exacerbated at high pH and resembled the growth of the CCM mutants cia3 and pmp1 (Figure 5A). At pH 7, the growth of bsti-1 was slightly better than at pH 8.4. At low CO2, bsti-1, cia3, and pmp1 all showed reduced growth compared to the wild-type D66 strain (Figure 5B). Also, all four strains showed slightly better growth at pH 7 compared to pH 8.4. However, at high CO2, the growth of bsti-1 was equivalent to that of the wild-type, cia3, and pmp1 (Figure 5C). Collectively, these results indicate that RNAi knockdown of all three bestrophins (bsti-1) reveals a pathogenic phenotype at low CO2 compared to both the WT (D66) and other mutants (cia3, pmp1) that are known to have a pathogenic phenotype at low CO2. Furthermore, CAH3 (cia3 is a CAH3 knockout) is a carbonic anhydrase found in the lumen of the thylakoid of Chlamydomonas reinhardtii and is required for CCM to function. The three bestrophin proteins may be proteins that deliver bicarbonate to CAH3 within the thylakoid. Thus, these results indicate that all three BSTs are required for wild-type-like growth of Chlamydomonas reinhardtii under low CO2 conditions.

[0153] [Example 6: The triple bestrophin RNAi knockdown strains have a reduced ability to accumulate inorganic carbon (C i )] The following example describes the inorganic carbon affinity tests of the triple bestrophin RNAi knockdown strains bsti-1 and bsti-2 (described in Example 5) compared to the WT strain D66.

[0154] [Materials and Methods] Affinity for inorganic carbon: In this analysis, the rate of photosynthesis (such as O2 generation) was measured at various inorganic carbon levels (inorganic carbon = CO2 and HCO3 - ). The affinity for external C i (K 1 / 2 [DIC]) (dissolved inorganic carbon) was estimated according to Ma et al., Plant Physiol 156:884-896, 2011. Specifically, cells equivalent to 100 μg of chlorophyll were bubbled with inert nitrogen gas, i.e., suspended in a CO2-free HEPES-NaOH buffer (pH 7.4), HEPES-NaOH buffer (pH 7.8), or 25 mM EPPS-NaOH buffer (pH 8.4). The cells were transferred to an O2 electrode chamber (Rank Brothers, Cambridge, UK) illuminated at 300 μmol·m -2 ·s -1 and left standing to deplete the DIC remaining in the buffer and intracellular space. With the depletion of endogenous CO2, no net O2 generation was observed. Known concentrations of NaHCO3 were injected into the chamber and the rate of O2 generation was measured. In this experiment, the inorganic carbon levels were varied from 25 μM to 2 mM. K 1 / 2 [DIC] was calculated as the DIC concentration required for half (50%) of the maximum rate of oxygen generation (i.e., photosynthesis) (Badger, 1985). The chlorophyll content was measured by combining chlorophyll a and chlorophyll b. Chlorophyll was extracted in 100% methanol and measured using a spectrophotometer. K 1 / 2 (CO2) is considered the CO2 concentration required to reach half of the V max of O2 generation.

[0155] C i Affinity was estimated for bsti-1 and D66 acclimated to low CO2 (CO2 less than 0.04%) for 12 hours at pH 8.4 (Figure 6A). The K 0.5 (C i ) values (C i concentration required for half of the maximum oxygen generation) shown in Figure 6A are the C iCalculated from O2 generation for the curve. Further, C i affinities were estimated for bsti-1, bsti-2, and D66 acclimated to low CO2 (less than 0.04% CO2) for 12 h at pH 7.8 (Figure 6C). The K 0.5 (C i ) values (C i concentration required for half-maximal oxygen generation) were calculated from O2 generation for the C i curve shown in Figure 6D. Further, C i affinities were estimated for bsti-1 and D66 acclimated to high CO2 (greater than 5% CO2) for 12 h at p7.8 (Figure 6E). The K 0.5 (C i ) values (C i concentration required for half-maximal oxygen generation) were calculated from O2 generation for the C i curve shown in Figure 6F. Three tests were performed at each C i concentration. The symbol “*” indicates that the difference in K 0.5 (C i ) was significant (P < 0.05 by Student's t-test). At pH 7.8 and low CO2 acclimation, the Vmax of D66 was 121 μmol O2 mg -1 Chl hr -1 , the Vmax of bsti-1 was 105 μmol O2 mg -1 Chl hr -1 , and the Vmax of bsti-2 was 95 μmol O2 mg -1 Chl hr -1 . At pH 7.8 and high CO2 acclimation, the Vmax of D66 was 121 μmol O2 mg -1 Chl hr -1 , and the Vmax of bsti-1 was 120 μmol O2 mg -1 Chl hr -1 . At pH 8.4 and low CO2 acclimation, the Vmax of D66 was 124 μmol O2 mg -1 Chl hr -1 , and the Vmax of bsti-1 was 85.5 μmol O2 mg -1 Chl hr -1It is. The Vmax of all strains was set to 100% oxygen evolution activity.

[0156] Inorganic carbon uptake: Using silicone oil centrifugation, the intracellular concentration of dissolved C was measured according to Moroney et al., Plant Physiol 79(1):177-183, 1985. i Briefly, cells were centrifuged and resuspended in 25 mM EPPS-NaOH (pH 7.8 or 8.4) with 25 μg Chl mL concentration depleted of C, and incubated in the light until net O2 evolution was zero. Cells were maintained in the light until use. Then, 300 μL of C-depleted cells were centrifuged in a tube containing 25 μL of 1 M glycine (pH 10) containing 0.75% (w / v) SDS covered with 75 μL of Dow Corning AR 20 silicone oil. The assay was performed at 25 °C in the light of 200 μmol·m -1 ·s i Either 3 μL of 25 mM (added concentration 25 μm) at pH 7.8 or 3 μL of 50 mM (added concentration 50 μm) at pH 8.4 of NaH i CO3 was added, and then placed for the indicated irradiation time (15 - 120 seconds in the light of 200 μmol·m -2 ·s -1 ), and C 14 uptake was initiated. For each time point, three replicate samples were assayed. The reaction was stopped by centrifuging for 15 seconds in a microcentrifuge B (Beckman). The internal C -2 ·s -1 was calculated using the difference between the total C in the pellet and the acid-stable C i and corrected for cell volume as described by Machingura et al., J Exp Bot 68(14):3879 - 3890, 2017. i in the pellet 14 total C 14 and acid-stable C

[0157] 〔Results〕 Two characteristics of algal cells with CCM are, first, that they are inorganic carbon (C iFirst, they have a very high affinity for , and second, they have the ability to accumulate to levels higher than those obtained by diffusion. Therefore, the photosynthetic oxygen evolution activities of the RNAi knockdown lines bsti-1 and bsti-2, as well as the WT strain D66, were tested. The RNAi knockdown lines bsti-1 and bsti-2 acclimated to low CO2 showed a 3- to 10-fold lower affinity for at pH 7.8 as determined by (Figures 6A and 6C). When acclimated to high CO2, bsti-1 also showed a decreased affinity for compared to D66 (Figure 6E). This indicates that the decrease in the expression of all three BST genes caused a decrease in the cellular affinity for . At pH 8.4, the ( ) for bsti-1 is 95 μM, while in contrast, the ( ) for D66 is as low as 35 μM. At pH values higher than 8.4, the dominant species in the medium is bicarbonate, and therefore, the higher affinity of D66 (WT) cells for reflects their ability to actively take up and utilize bicarbonate. These results indicate that bsti-1 and bsti-2 have higher requirements for inorganic carbon for photosynthetic O2 evolution, providing strong evidence for the role of bestrophin in CCM. i To evaluate the importance of BST1-3 in the accumulation and fixation of , uptake activities were also measured in D66 and bsti-1. Bsti-1 acclimated to low CO2 showed significantly lower accumulation and fixation of at both pH 7.8 and pH 8.4 compared to D66 (Figures 7A-7D). At both pH 7.8 and 8.4, bsti-1 accumulated and fixed to only 20-25% of the levels seen in D66 cells. 0.5 ( i ) as shown, and showed a 3- to 10-fold lower affinity for at pH 7.8. When acclimated to high CO2, bsti-1 also showed a decreased affinity for compared to D66 (Figure 6E). This indicates that the decrease in the expression of all three BST genes caused a decrease in the cellular affinity for . At pH 8.4, the ( ) for bsti-1 is 95 μM, while in contrast, the ( ) for D66 is as low as 35 μM. At pH values higher than 8.4, the dominant species in the medium is bicarbonate, and therefore, the higher affinity of D66 (WT) cells for reflects their ability to actively take up and utilize bicarbonate. These results indicate that bsti-1 and bsti-2 have higher requirements for inorganic carbon for photosynthetic O2 evolution, providing strong evidence for the role of bestrophin in CCM. i i affinity decreased (Figure 6E). This shows that the decrease in the expression of all three BST genes caused a decrease in the cellular affinity for . At pH 8.4, the ( ) for bsti-1 is 95 μM, while in contrast, the ( ) for D66 is as low as 35 μM. At pH values higher than 8.4, the dominant species in the medium is bicarbonate, and therefore, the higher affinity of D66 (WT) cells for reflects their ability to actively take up and utilize bicarbonate. These results indicate that bsti-1 and bsti-2 have higher requirements for inorganic carbon for photosynthetic O2 evolution, providing strong evidence for the role of bestrophin in CCM. i 0.5 ( i ) is 95 μM, while in contrast, the ( ) for D66 is as low as 35 μM. At pH values higher than 8.4, the dominant species in the medium is bicarbonate, and therefore, the higher affinity of D66 (WT) cells for reflects their ability to actively take up and utilize bicarbonate. These results indicate that bsti-1 and bsti-2 have higher requirements for inorganic carbon for photosynthetic O2 evolution, providing strong evidence for the role of bestrophin in CCM. 0.5 ( i ) i species is bicarbonate, and therefore, the higher affinity of D66 (WT) cells for reflects their ability to actively take up and utilize bicarbonate. These results indicate that bsti-1 and bsti-2 have higher requirements for inorganic carbon for photosynthetic O2 evolution, providing strong evidence for the role of bestrophin in CCM. i

[0158] i To evaluate the importance of BST1-3 in the accumulation and fixation of , uptake activities were also measured in D66 and bsti-1. Bsti-1 acclimated to low CO2 showed significantly lower accumulation and fixation of at both pH 7.8 and pH 8.4 compared to D66 (Figures 7A-7D). At both pH 7.8 and 8.4, bsti-1 accumulated and fixed to only 20-25% of the levels seen in D66 cells. i 14 i accumulation and fixation of were significantly lower (Figures 7A-7D). At both pH 7.8 and 8.4, bsti-1 accumulated and fixed to only 20-25% of the levels seen in D66 cells.14 C i was not accumulated. This difference was observed at both the earliest time point (15 seconds) and the latest time point (up to 2 minutes in light) when most of the 14 C i was exhausted. These results indicate that BST1-3 play an important role in C i uptake under low CO2 conditions in Chlamydomonas reinhardtii and also provide strong evidence for the role of bestrophins in CCM.

[0159] [Example 7: The bst3 knockout strain has normal growth under low CO2 conditions and has similar inorganic carbon affinity when compared to WT] The following example describes the growth and C i affinity tests of the Chlamydomonas reinhardtii bst3 knockout strain under low CO2 conditions compared to the WT strain D66.

[0160] [Materials and Methods] Chlamydomonas reinhardtii knockout strains: The knockout strains were generated using random insertional mutagenesis and are part of the Chlamydomonas Library Project (CLiP). Strains can be ordered from the Chlamydomonas Resource Center (https: / / www.chlamycollection.org / ). Currently, only the bst3 knockout strain is available. The bst1 and bst2 knockout strains are not available. To confirm the insertion position in the bst3 knockout strain, PCR was performed using primers specific to the BST3 gene and the insert (see Table 5). Information on the insert-specific primers (CIB1F and CIB1R) and the adjacent regions was obtained from the CLiP website (https: / / www.chlamylibrary.org / allMutants).

[0161] [Table 5]

[0162] Semi - quantitative RT - PCR analysis was performed in the same manner as in Example 3.

[0163] Growth measurement: The WT strain D66 and the knockout strain bst3 were grown at pH 8.6 using the method described in the above examples. Growth was measured using OD 730 and chlorophyll estimation was performed at wavelengths 645 and 663. The measurements were carried out for 6 days under low CO2 (CO2 less than 0.04%). Cells were grown in TAP for 48 hours and then transferred to MIN at an OD of 0.01. 730

[0164] Affinity for inorganic carbon: This was carried out using the method described in Example 6. Oxygen evolution activity was measured at pH 7.4 and the K 0.5 (C i ) value (the C i concentration required for half - maximum oxygen evolution) was calculated from O2 evolution against the C i curve. Three replicate trials were carried out at each C i concentration.

[0165] [Results] The BST3 knockout (bst3) was obtained from a collection of CLiP mutants having a paromomycin insert in the first exon of the bst3 gene (Figure 8A). The position of the insert was confirmed using PCR (Figure 8B). Furthermore, BST1 - 3 expression was analyzed in the bst3 knockout strain under high CO2 and low CO2 conditions and compared with the WT strain D66. BST3 expression was not detected in bst3 under both conditions, while BST1 and BST2 expression was equivalent between the bst3 knockout strain and the WT strain D66 under both conditions (Figure 8C).

[0166] No significant growth differences were seen in the bst3 strain compared to wild - type cells under low CO2 (CO2 less than 0.04%) (Figures 9A - 9B), extremely low (CO2 is 0.02%) or high CO2 (CO2 is 5% (v / v) in air) (data not shown). The C iThere was also no significant difference in affinity (Figure 9C). In the RNAi knockdown line bsti-1, the expression of all three BST genes was reduced, and C i affinity decreased at both pH 7.8 and pH 8.4. This is in stark contrast to bst3, a knockout strain that only lacks BST3 expression and has no difference in affinity with WT (Figures 9C - 9D). These results support the hypothesis that the functions of the three BSTs overlap and that the expression of all three genes must be reduced in order to observe a physiological effect. i

[0167] [Example 8: Structural Characterization of BST1, BST2, and BST3, and the Current Chlamydomonas Reinhardtii CCM Model] The following examples describe the structural characterization of the Chlamydomonas Reinhardtii BST1, BST2, and BST3 proteins. The examples also describe the Chlamydomonas Reinhardtii CCM model of the manuscript.

[0168] [Materials and Methods] ​The peptide sequences of BST1, BST2, and BST3 were obtained from Phytozome v12.1. Homology modeling was performed using Klebsiella pneumonia bestrophin (Kpbest; PDB: 4DW8) as a template and generated using the Swiss-Model web server (Yang et al., Science 346(6216):1498-1501, 2014). Kpbest was selected because it was identified as the top template for BST1-3 by Swiss-Model. The resulting structure and the structure of Kpbest are shown as monomers with conserved residues that align the selective pores shown as protruding shapes in Figure 10A. The obtained BST1 homotetramer model was subjected to energy minimization with the gromos 43B1 force field, and the electrostatic potential was calculated using atomic partial charges using Swiss-PDBviewer (V4.01). Then, an electric field was displayed on the BST1 homotetramer model to generate Figure 10D. The electrostatic potential was displayed on a potential scale of -4kT / e (negative) to +4kT / e (positive), and the cavity was emphasized. The quality of the BST1 homotetramer structure (Figure 10B) was obtained using the QMEAN score (Benkert et al., Proteins 71(1):261-277, 2008).

[0169] [Results] To predict whether BST1-3 function to transport bicarbonate as an anion, homology modeling of BST1-3 was performed using Klebsiella pneumoniae bicarbonate. BST1-3 contain nonpolar residues along those selective pores that are conserved in bicarbonate family proteins (Figure 10A; Qu et al., J Neurosci 26(20):5411-5419, 2006). Structural studies have shown that human and Klebsiella pneumoniae bicarbonate are pentamers, and the modeling of BST1 in the pentamer assembly is of high reliability (Figure 10B). The entry pocket predominantly has a negative electrostatic potential, and the selective pore is neutrally / positively charged, supporting the hypothesis that BST1-3 transport negatively charged ions (Figures 10C-10D; Yang et al., Science 346(6216):1498-1501, 2014; Kane et al., Nature 516(7530):213-218, 2014). These results suggest the possibility that Chlamydomonas reinhardtii bicarbonate is an anion transporter.

[0170] The results described in Examples 2-8 demonstrate the important roles of the three bicarbonate proteins in the context of Chlamydomonas reinhardtii CCM. Collectively, these results indicate that the three bicarbonate proteins are thylakoid-localized bicarbonate transport channels. Figure 11 shows the integration of BST1, BST2, and BST3 into the current Chlamydomonas reinhardtii CCM model for transport. i Integration into the current Chlamydomonas reinhardtii CCM model for transport.

Brief Description of the Drawings

[0171]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2A

Figure 2B

Figure 3

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 8A

Figure 8B

Figure 8C

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 11

Claims

1. A genetically modified plant, or a part thereof, wherein the plant has a genetic modification that increases or provides the ability of bicarbonate to pass through a membrane from the plant cell cytoplasm of at least a part of the chloroplasts of the plant to the stroma, or increases or provides the ability of bicarbonate to pass through a membrane from the stroma of at least a part of the chloroplasts of the plant to the lumen, and the green algal bestrophin polypeptide is localized in the chloroplast envelope or thylakoid membrane of at least one chloroplast in the plant cell, the green algal bestrophin polypeptide functions as a bicarbonate transporter and has at least 90% identity to SEQ ID NO: 1, a plant, or a part thereof.

2. The plant cell is a mesophyll cell, the plant, or a part thereof according to claim 1.

3. The green algal bestrophin polypeptide is expressed in at least 70% of the mesophyll cells of the plant, the plant, or a part thereof according to claim 2.

4. The plant, or a part thereof according to claim 1, further comprising regulated expression of an endogenous carbonic anhydrase.

5. The regulated expression is selected from the group consisting of increased expression, decreased expression, expression at different positions, and any combination thereof, the plant, or a part thereof according to claim 4.

6. The plant is selected from the group consisting of cowpea, soybean, cassava, rice, soybean, wheat, and other C3 crop plants, the plant is not selected from the group consisting of maize, sorghum, and other C4 crop plants, the plant, or a part thereof according to claim 1.

7. A method for producing a plant or a part thereof, comprising: The method includes a step of introducing into the plant or a part thereof a nucleic acid sequence containing a coding sequence of a green algal bestrophin polypeptide, wherein the green algal bestrophin polypeptide is expressed in the plant or a part thereof, The nucleic acid sequence further includes a second nucleic acid sequence encoding a signal peptide sequence or a target sequence operably linked to the coding sequence of the green algal bestrophin polypeptide, Expression of the signal peptide sequence or the target sequence results in the localization of the green algal bestrophin polypeptide to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell, The green algal bestrophin polypeptide functions as a bicarbonate transporter and has at least 90% identity to SEQ ID NO: 1, wherein (i) when the plant is cultivated under ambient carbon dioxide conditions, the yield, growth rate, or biomass is greater than the yield, growth rate, or biomass from a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, or (ii) the green algal bestrophin polypeptide increases or provides the ability of bicarbonate to pass through a membrane from the plant cytoplasm of at least a part of the chloroplast of the plant to the stroma, or increases or provides the ability of bicarbonate to pass through a membrane from the stroma of at least a part of the chloroplast of the plant to the lumen, compared to a corresponding wild-type (WT) plant that does not overexpress the bestrophin polypeptide, or a corresponding WT part thereof, or or (iii) both (i) and (ii). A method.

8. The green algal bestrophin polypeptide is localized to the chloroplast envelope or the chloroplast thylakoid membrane of at least one chloroplast of a plant cell, The plant cell is a mesophyll cell, The method according to claim 7, wherein the polypeptide is expressed in at least 70% of the mesophyll cells of the plant.

9. The method according to claim 7, wherein the nucleic acid sequence is stably integrated into the nuclear genome of the plant.

10. The method according to claim 7, further comprising regulated expression of endogenous carbonic anhydrase in the plant or a part thereof.

11. The method according to claim 10, wherein the regulated expression is selected from the group consisting of increased expression, decreased expression, expression at a different position, and any combination thereof.

12. The plant is selected from the group consisting of cowpea, soybean, cassava, rice, soybean, wheat, and other C3 crop plants, The method according to claim 7, wherein the plant is not selected from the group consisting of maize, sorghum, and other C4 crop plants.

Images