Engineering gt2 type of membrane proteins for targeting to the tonoplast, and engineering sorghum to synthesize and deposit mixed-linkage glucan (MLG) in the vacuole

Abstract

Described herein are expression cassettes, plant cells, plant seeds, plants, and methods useful for improving the glucan content and growth of plants.

Description

ENGINEERING GT2 TYPE OF MEMBRANE PROTEINS FOR TARGETING TO THE TONOPLAST, AND ENGINEERING SORGHUM TO SYNTHESIZE AND DEPOSIT MIXED-LINKAGE GLUCAN (MLG) IN THE VACUOLE PRIORITY

[0001] This application claims the benefit of priority of US provisional patent application No.63 / 500,086, filed on May 4, 2023, the benefit of priority of which is claimed hereby, and which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers DE-FC02-07ER64494 and DE-SC0018409 awarded by the Department of Energy Great Lakes Bioenergy Research Center. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on May 4, 2024, is name 3000217WO1.xml and is 47,285 bytes in size. BACKGROUND

[0004] Mixed linkage (1,3;1,4)-β-D-glucan (MLG) are abundant matrix polysaccharides that can occupy up to approximately 40% of the total cell wall in grasses. For example, Brachypodium endosperm can have up to 40% mixed- linkage glucans (Guillon et al. J Exp Bot 62(3):1001-15 (2011)). MLG are pmixedolymers containing β-glucosyl residues with both (1,3)- and (1,4)- linkages. Diverse roles have been suggested for mixed-linkage glucans including regulation of cell growth, cell wall structure and energy storage. The MLG content of grains varies amongst the cereals, with barley, oats and rye having the highest amounts and wheat, maize and rice having relatively low levels. SUMMARY

[0005] Described herein are plants, plant cells, and plant seeds that provide improved glucan content, as well as methods for making and using such plants, plant cells, and plant seeds. The nucleic acids, expression cassettes, plants, seedsand methods described herein can be used to improve the quality and quantity of plant materials for biofuel production and other uses. Methods of cultivating such plant seeds and plants are also described herein that include, for example, harvesting the plants, seeds, or the tissues of the plants. Such methods can also include isolating glucans, polysaccharides, starch, and / or sugars from the plants, seeds, or tissues of the plants.

[0006] For example, plant cells, plant seeds, and plants are described herein that include an expression system comprising at least one expression vector comprising a first nucleic acid segment encoding a vacuolar targeting polypeptide linked in-frame with at least one second nucleic acid segment encoding a cellulose synthase-like Csl protein to form a fusion protein, wherein the vacuolar targeting polypeptide has an activity of localizing the fusion protein to a tonoplast without affecting the protein topology.

[0007] In addition, methods are described herein that include comprising growing a plant seed or plant comprising an expression system comprising a heterologous promoter operably linked to a nucleic acid segment encoding a vacuolar targeting polypeptide linked in-frame with a nucleic acid segment encoding a Csl protein to form a fusion protein, to thereby produce a mature plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS.1A-B are graphs depicting the regulation of MLG in elongating leaf in plants. Fig. 1A shows there a higher amount of MLG in leaf base versus tip. Fig.1B shows the similar diurnal variation of MLG in the elongation zone of wild type and the opposite pattern of expression of lichenase responsible for MLG degradation. The graphs of Figs. 1A-B illustrate that the development and light cycle regulate MLG abundance.

[0009] FIG. 2 is an image showing overexpression of CslF6 in Brachypodium results in plants that set seeds and have a shorter stature than wild-type plants.

[0010] FIG. 3 is a graph illustrating the amounts of MLG in leaves of Brachypodium that overexpress CslF6 (CslF6OX) (y-axis is leaf MLG μg / mg of AIR). Completion of MLG degradation in leaf was observed at 8 weeks. Fig. 3 shows that Brachypodium tissues that overexpress CslF6 (CslF6OX) have higher mixed-linkage glucan content than wild plant tissues.

[0011] FIG. 4 is an image of a Brachypodium plant that expresses CslF6 (CslF6OX), shown at 5 weeks, 6 weeks, and 8 weeks.

[0012] FIGS.5A-B are graphs illustrating that overexpression of Brachypodium CslF6 (BdCSLF6) increases the level of MLG in Brachypodium. Fig. 5A is a graph showing an increase of leaf MLG in Brachypodium isolates overexpressing CslF6 compared to wild-type Brachypodium. Fig. 5B is a graph showing an increase of stem MLG in Brachypodium isolates overexpressing CslF6 compared to wild-type Brachypodium.

[0013] FIG.6 is a graph illustrating the levels of MLG in the transgenic sorghum overexpressing CslF6 genes from Brachypodium (Bd) or Sorghum bicolor (Sb). MLG levels are increased in some transgenic lines compared to wild type plants, with isolates of the transgenic sorghum line overexpressing sorghum CslF6 showing marked increases in MLG production.

[0014] FIG. 7 is a graph showing the levels of biomass in transgenic Sorghum bicolor overexpressing BdCslF6 or SbCslF6. Transgenic Sorghum bicolor overproducing MLG grows normally in greenhouse conditions compared to a vector control.

[0015] FIG. 8 is a graph showing the stem height and stem biomass of the transgenic sorghum overexpressing CslF6 genes from Brachypodium (Bd) or Sorghum bicolor (Sb). As shown, transgenic sorghum overproducing MLG grows normally in field conditions.

[0016] FIG.9 is a graph showing the levels of MLG in portions of the transgenic sorghum overexpressing CslF6 genes from Brachypodium (Bd) or Sorghum bicolor (Sb) at a boot stage of growth in field conditions during the daytime at 2- 4 pm. An increased level of MLG is shown in transgenic lines at the boot stage in daylight.

[0017] FIG. 10 is a graph showing the levels of MLG in portions of the transgenic sorghum overexpressing CslF6 genes from Brachypodium (Bd) or Sorghum bicolor (Sb) at a boot stage of growth in field conditions during the nighttime at 5-6 am. An increased level of MLG is shown in each transgenic line at the boot stage at nighttime.

[0018] FIGS. 11A-C show the characterization of three sorghum MLG endoglucanases (also called lichenases). Fig. 11A is an illustration of the lichenase-mediated hydrolysis of MLG in plants, causing MLG degradation. Fig.11B is a graph showing the enzymatic activity of lichenase enzymes in Sorghum. The two Sorghum lichenases have similar enzymatic activity at their optimal condition (near pH 5 and temperature between 25˚C and 45˚C), but one lichenase (SbLCH2) has a lower activity than the other two lichenases (SbLCH1 and SbLCH3). Fig.11C is an image showing that the three Sorghum enzymes localize in the apoplast, where lichenase activity is expected to take place.

[0019] FIG.12 shows the lichenases’ expression pattern during development and light conditions and the lichenase that is likely responsible for MLG degradation in Sorghum.

[0020] FIG. 13 is a graph depicting MLG regulation in Sorghum young leaves. High MLG and low starch synthesis were observed in the actively elongating region of the leaves. Fully elongated cells start starch synthesis during the day. MLG degradation is shown not to substantially occur at night and starch is shown to be degraded at night.

[0021] FIG. 14 shows images of fluorescence microscopy of Sorghum protoplasts expressing TonoCSLF6 fusion protein. The images show localization of CSLF6 in the tonoplast.

[0022] FIG. 15 is a graph showing an increase in MLG in top and bottom portions of Sorghum leaf tops expressing TonoCSLF6 fusion protein. DETAILED DESCRIPTION

[0001] Described herein are plants, plant cells, and plant seeds that provide improved glucan content, as well as methods for making and using such plants, plant cells, and plant seeds. The nucleic acids, expression cassettes, plants, seeds and methods described herein can be used to improve the quality and quantity of plant materials for biofuel production and other uses. Methods of cultivating such plant seeds and plants are also described herein that include, for example, harvesting the plants, seeds, or the tissues of the plants. Such methods can also include isolating glucans, polysaccharides, starch, and / or sugars from the plants, seeds, or tissues of the plants.

[0002] The diets of humans and livestock rely heavily on cereals storage proteins and carbohydrates, including the simple, yet, important, glucose polymer, mixed- linkage glucan (MLG). Storage proteins and the proteins responsible for the production of MLG are synthesized by the endomembrane system.

[0003] CslF6

[0004] MLG is a significant cell wall carbohydrate in grasses and an important dietary fiber source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of members of membrane- spanning glycosyltransferase (GT) 2 family of uridine diphosphate glucose (UDP- Glc) utilizing enzymes. The genes responsible for MLG synthesis in grasses share sequence homology to the cellulose synthases (CesAs) and belong to the cellulose synthase-like (Csl) F, H, and J sub-families. CslF6 is the major isoform contributing to total MLG abundance in both vegetative and floral tissues in barley, rice, wheat, and Brachypodium distachyon.

[0005] A variety of CslF6 proteins and CslF6 nucleic acids can be used to increase plant glucan content. For example, one sequence of a CslF6 protein from Brachypodium distachyon (Bradi3g16307.1) is shown below as SEQ ID NO:1. 1 MAPAVAGGSS RGAGCKCGFQ VCVCSGSAAV ASAGSSLEVE 41 RAMAVTPVEG QAAPVDGESW VGVELGPDGV ETDESGAGVD 81 DRPVFKTEKI KGVLLHPYRV LIFVRLIAFT LFVIWRISHK 121 NPDTMWLWVT SICGEFWFGF SWLLDQLPKL NPINRIPDLA 161 VLRQRFDRAD GTSTLPGLDI FVTTADPIKE PILSTANSVL 201 SILAADYPVD RNTCYISDDS GMLMTYEAMA ESAKFATLWV 241 PFCRKHGIEP RGPESYFELK SHPYMGRAHD EFVNDRRRVR 281 KEYDDFKAKI NSLETDIQQR NDLHNAAVPQ NGDGIPRPTW 321 MADGVQWQGT WVEPSANHRK GDHAGIVLVL IDHPSHDRLP 361 GAPASADNAL DFSGVDTRLP MLVYMSREKR PGHNHQKKAG 401 AMNALTRASA LLSNAPFILN LDCDHYINNS QALRAGICFM 441 VGRDSDTVAF VQFPQRFEGV DPTDLYANHN RIFFDGTLRA 481 LDGMQGPIYV GTGCLFRRIT VYGFDPPRIN VGGPCFPALG 521 GLFAKTKYEK PSMEMTMARA NQAVVPAMAK GKHGFLPLPK 561 KTYGKSDKFV DTIPRASHPS PYAAEGIRVV DSGAETLAEA 601 VKVTGSAFEQ KTGWGSELGW VYDTVTEDVV TGYRMHIKGW 641 RSRYCSIYPH AFIGTAPINL TERLFQVLRW STGSLEIFFS 681 KNNPLFGSTY LHPLQRVAYI NITTYPFTAI FLIFYTTVPA 721 LSFVTGHFIV QRPTTMFYVY LGIVLATLLI IAVLEVKWAG 761 VTVFEWFRNG QFWMTASCSA YLAAVCQVLT KVIFRRDISF 801 KLTSKLPAGD EKKDPYADLY VVRWTPLMIT PIIIIFVNII 841 GSAVAFAKVL DGEWTHWLKV AGGVFFNFWV LFHLYPFAKG 881 LLGKHGKTPV VVLVWWAFTF VITAVLYINI PHIHGGGGKH 921 SVGHGMHHGK KFDGYYLWP

[0006] A nucleotide sequence that encodes the CslF6 protein from Brachypodium distachyon with SEQ ID NO:1 is shown below as SEQ ID NO:2. 1 ATGGCGCCAG CGGTGGCCGG CGGGAGCAGC CGGGGTGCAG 41 GGTGTAAGTG CGGGTTCCAG GTGTGCGTGT GCTCTGGGTC81 GGCGGCGGTG GCGTCGGCGG GTTCGTCGCT GGAGGTGGAG 121 AGAGCCATGG CGGTGACGCC GGTGGAAGGG CAGGCGGCGC 161 CGGTGGACGG CGAGAGCTGG GTCGGCGTCG AGCTCGGCCC 201 CGACGGCGTG GAGACGGACG AGAGCGGCGC CGGCGTCGAC 241 GACCGCCCCG TCTTCAAGAC CGAGAAGATC AAGGGCGTCC 281 TCCTCCACCC CTACAGGGTG CTGATCTTTG TTCGTCTGAT 321 AGCGTTCACC CTGTTCGTGA TCTGGCGTAT CTCGCACAAG 361 AACCCGGACA CGATGTGGCT GTGGGTGACC TCCATCTGCG 401 GCGAGTTCTG GTTCGGCTTC TCCTGGCTGC TGGACCAGCT 441 TCCAAAGCTC AACCCGATCA ACCGGATCCC GGACCTCGCC 481 GTGCTCCGGC AACGCTTCGA CCGCGCCGAC GGGACATCCA 521 CATTGCCGGG CCTCGACATC TTCGTCACCA CGGCCGACCC 561 CATCAAGGAA CCCATCCTGT CGACGGCCAA CTCCGTGCTC 601 TCCATCCTGG CCGCCGACTA CCCGGTGGAC CGCAACACCT 641 GCTACATCTC CGACGACAGC GGCATGCTCA TGACCTACGA 681 GGCCATGGCG GAGTCGGCCA AGTTCGCCAC CCTCTGGGTG 721 CCATTCTGCC GCAAGCACGG CATCGAACCA CGCGGGCCGG 761 AGAGCTACTT CGAGCTCAAG TCGCACCCGT ACATGGGGAG 801 AGCGCACGAC GAGTTCGTCA ATGACCGCCG CCGGGTGCGC 841 AAGGAGTATG ATGACTTCAA GGCCAAGATT AACTCTCTGG 881 AGACTGATAT CCAGCAGAGG AATGATCTGC ATAACGCTGC 921 CGTGCCGCAG AATGGGGATG GGATCCCCAG GCCTACCTGG 961 ATGGCTGATG GAGTCCAGTG GCAGGGGACT TGGGTCGAGC 1001 CGTCCGCTAA TCACCGCAAG GGAGACCACG CCGGCATCGT 1041 CCTGGTTCTG ATTGACCACC CGAGCCACGA CCGCCTTCCC 1081 GGCGCGCCGG CGAGCGCCGA CAACGCGCTG GACTTCAGCG 1121 GCGTGGACAC CCGCCTCCCG ATGCTCGTCT ACATGTCCCG 1161 CGAGAAGCGC CCAGGCCACA ACCACCAGAA GAAGGCCGGC 1201 GCCATGAACG CGCTCACCAG GGCTTCCGCG CTGCTCTCCA 1241 ACGCGCCCTT CATCCTCAAC CTCGACTGCG ACCACTACAT 1281 CAACAACTCC CAGGCCCTCC GCGCCGGGAT CTGCTTCATG 1321 GTCGGCCGGG ACAGCGACAC CGTCGCCTTC GTGCAGTTCC 1361 CGCAGCGGTT CGAGGGCGTC GACCCCACGG ACCTCTACGC 1401 CAACCACAAC CGCATCTTCT TCGACGGCAC CCTCAGGGCG 1441 CTCGACGGAA TGCAAGGCCC GATCTATGTC GGCACGGGAT 1481 GCCTCTTCCG GCGCATCACC GTCTACGGCT TCGACCCGCC 1521 CAGGATCAAC GTCGGCGGGC CATGCTTCCC TGCTCTCGGT 1561 GGCCTGTTCG CCAAGACCAA GTATGAGAAG CCCAGCATGG 1601 AGATGACCAT GGCGAGAGCC AACCAGGCCG TGGTGCCGGC 1641 CATGGCCAAG GGGAAGCACG GCTTCCTGCC GCTCCCCAAG 1681 AAGACGTACG GGAAGTCCGA CAAGTTCGTG GACACCATCC 1721 CGCGCGCGTC CCACCCGTCG CCGTACGCGG CGGAGGGGAT 1761 CCGCGTGGTG GACTCCGGCG CGGAGACTCT GGCTGAGGCC 1801 GTCAAGGTGA CCGGATCGGC ATTCGAGCAG AAGACCGGAT 1841 GGGGCAGCGA GCTCGGCTGG GTCTACGACA CTGTCACAGA 1881 GGACGTGGTG ACTGGCTACA GGATGCACAT CAAGGGCTGG 1921 AGGTCCCGCT ACTGCTCCAT CTACCCGCAC GCCTTCATCG 1961 GCACCGCCCC GATCAACCTC ACGGAGCGGC TCTTCCAGGT 2001 GCTCCGCTGG TCCACCGGCT CCCTCGAGAT CTTCTTCTCC 2041 AAGAACAACC CGCTCTTCGG CAGCACCTAC CTGCACCCGC 2081 TCCAGCGCGT CGCCTACATC AACATCACCA CATACCCGTT2121 CACCGCCATC TTCCTCATCT TCTACACCAC CGTGCCGGCG 2161 CTCTCCTTCG TCACCGGCCA CTTCATCGTG CAGCGCCCGA 2201 CGACCATGTT CTACGTCTAC CTGGGGATCG TGCTGGCGAC 2241 GCTGCTCATC ATCGCTGTTC TTGAGGTCAA GTGGGCTGGA 2281 GTGACAGTGT TCGAGTGGTT CAGGAACGGG CAGTTCTGGA 2321 TGACGGCTAG CTGCTCCGCC TACCTTGCTG CTGTGTGCCA 2361 GGTGCTCACC AAGGTGATCT TCAGGAGGGA CATCTCATTC 2401 AAGCTCACTT CCAAGCTGCC TGCTGGGGAC GAGAAGAAGG 2441 ACCCCTATGC CGATCTGTAC GTGGTGCGTT GGACTCCACT 2481 CATGATCACT CCAATCATCA TCATCTTCGT CAACATCATC 2521 GGCTCGGCGG TGGCCTTCGC CAAGGTGCTG GACGGCGAGT 2561 GGACGCACTG GCTCAAGGTG GCGGGAGGAG TCTTCTTCAA 2601 CTTCTGGGTG CTGTTCCACC TCTACCCGTT CGCCAAGGGT 2641 CTCCTGGGGA AGCATGGCAA GACCCCCGTC GTCGTGCTCG 2681 TCTGGTGGGC ATTCACCTTC GTCATCACCG CCGTCCTCTA 2721 CATCAACATC CCGCACATCC ATGGAGGAGG AGGCAAGCAC 2761 AGCGTGGGGC ATGGGATGCA CCATGGCAAG AAGTTCGACG 2801 GCTACTACCT CTGGCCGTGA

[0007] A nucleotide sequence that encodes the CslF6 protein from Brachypodium distachyon with SEQ ID NO:1 and that has been codon-optimized for expression in Brachypodium distachyon is shown below as SEQ ID NO:3. 1 ATGGCTCCAG CTGTTGCTGG CGGCTCCTCT AGGGGCGCTG 41 GCTGCAAGTG CGGCTTCCAG GTGTGCGTGT GCTCCGGCTC 81 TGCCGCCGTG GCCTCCGCCG GCTCATCCCT CGAGGTCGAG 121 AGGGCCATGG CTGTTACCCC AGTTGAGGGC CAGGCCGCTC 161 CAGTGGACGG CGAGTCCTGG GTGGGCGTTG AGCTTGGCCC 201 AGACGGCGTC GAGACCGACG AGTCCGGCGC TGGCGTGGAC 241 GACAGGCCAG TGTTCAAGAC CGAGAAGATC AAGGGCGTGC 281 TCCTCCACCC ATACAGGGTG CTCATCTTCG TGAGGCTGAT 321 CGCCTTCACC CTCTTCGTGA TCTGGCGCAT CTCCCACAAG 361 AACCCGGACA CCATGTGGCT CTGGGTGACC TCTATTTGCG 401 GCGAGTTCTG GTTCGGCTTC TCCTGGCTCC TCGACCAGCT 441 CCCAAAGCTC AACCCGATCA ACCGCATCCC AGATCTCGCC 481 GTTCTCAGGC AGAGGTTCGA TAGGGCCGAC GGCACCTCCA 521 CCCTCCCAGG CCTTGATATT TTCGTGACCA CCGCCGACCC 561 CATCAAGGAG CCAATTCTCT CAACCGCCAA CTCCGTGCTC 601 TCTATCCTCG CCGCCGATTA CCCGGTGGAT AGGAACACGT 641 GCTACATCTC CGACGACAGC GGCATGCTCA TGACCTACGA 681 GGCTATGGCC GAGTCCGCCA AGTTCGCTAC CCTCTGGGTG 721 CCATTCTGCC GCAAGCACGG CATCGAGCCA AGGGGCCCAG 761 AGTCCTACTT CGAGCTTAAG TCCCACCCGT ACATGGGCAG 801 GGCCCATGAC GAGTTCGTGA ACGATAGGCG CAGGGTGAGG 841 AAGGAGTACG ACGACTTCAA GGCCAAGATC AACTCCCTCG 881 AGACGGACAT CCAGCAGAGG AACGACCTCC ATAACGCCGC 921 CGTGCCACAG AACGGGGACG GCATCCCAAG GCCAACCTGG 961 ATGGCCGATG GCGTGCAGTG GCAGGGCACC TGGGTTGAGC 1001 CATCTGCCAA CCATAGGAAG GGCGATCACG CCGGCATTGT 1041 GCTCGTGCTC ATCGACCATC CATCCCACGA CAGGCTCCCA1081 GGCGCCCCAG CCTCTGCCGA CAACGCCCTC GACTTCTCCG 1121 GCGTGGACAC CAGGCTTCCA ATGCTCGTTT ACATGTCCCG 1161 CGAGAAGAGG CCAGGCCACA ACCACCAGAA GAAGGCTGGC 1201 GCTATGAACG CCCTTACCAG GGCTTCTGCT CTCCTCTCCA 1241 ACGCCCCGTT CATCCTCAAC CTCGACTGCG ACCACTACAT 1281 CAACAACAGC CAGGCTCTCA GGGCCGGCAT CTGCTTCATG 1321 GTGGGCAGGG ATTCTGACAC CGTGGCCTTC GTTCAGTTCC 1361 CGCAGCGCTT CGAGGGGGTT GACCCAACCG ATCTCTACGC 1401 CAACCACAAC AGGATTTTCT TCGATGGCAC CCTCAGGGCC 1441 CTCGATGGCA TGCAGGGCCC TATCTACGTG GGCACCGGCT 1481 GCCTCTTCAG GCGCATCACC GTGTACGGCT TCGACCCGCC 1521 AAGGATTAAC GTTGGCGGCC CATGCTTCCC AGCTCTCGGC 1561 GGCCTCTTCG CTAAGACCAA GTACGAGAAG CCCAGCATGG 1601 AGATGACCAT GGCCAGGGCC AACCAGGCCG TTGTTCCAGC 1641 TATGGCTAAG GGGAAGCACG GCTTCCTGCC ACTCCCGAAG 1681 AAGACCTACG GCAAGAGCGA CAAGTTCGTC GACACCATTC 1721 CAAGGGCCTC CCACCCATCT CCATACGCTG CCGAGGGCAT 1761 TAGGGTTGTG GACTCTGGCG CCGAGACCCT CGCCGAGGCC 1801 GTGAAGGTGA CCGGCTCCGC CTTCGAGCAG AAGACCGGCT 1841 GGGGCTCCGA GCTTGGCTGG GTTTACGACA CCGTGACCGA 1881 GGATGTGGTC ACCGGCTACA GGATGCACAT TAAGGGCTGG 1921 CGCAGCAGGT ACTGCTCCAT CTACCCACAT GCCTTCATCG 1961 GCACCGCCCC CATTAACCTC ACCGAGAGGC TTTTCCAGGT 2001 GCTCAGGTGG TCTACCGGCA GCCTCGAGAT CTTCTTCAGC 2041 AAGAACAACC CGCTGTTCGG CTCCACCTAC CTGCATCCAC 2081 TCCAGAGGGT GGCCTACATT AACATCACCA CCTACCCGTT 2121 CACCGCCATC TTCCTCATCT TCTACACGAC CGTGCCCGCC 2161 CTCTCATTCG TGACCGGCCA TTTCATTGTG CAGAGGCCGA 2201 CCACCATGTT CTACGTGTAC CTCGGGATCG TGCTCGCCAC 2241 CCTCCTCATT ATTGCCGTGC TCGAGGTTAA GTGGGCTGGC 2281 GTGACCGTGT TCGAGTGGTT CCGCAACGGC CAGTTCTGGA 2321 TGACCGCCTC TTGCTCTGCT TACCTCGCCG CTGTTTGCCA 2361 GGTCCTCACC AAGGTTATCT TCCGCAGGGA CATCTCCTTC 2401 AAGCTCACCT CCAAGCTCCC AGCCGGCGAC GAGAAGAAGG 2441 ACCCATACGC CGATCTGTAC GTGGTGAGGT GGACCCCGCT 2481 CATGATCACC CCGATCATCA TCATTTTCGT CAACATCATC 2521 GGCTCCGCGG TCGCCTTCGC CAAGGTGCTC GATGGCGAGT 2561 GGACCCATTG GCTTAAGGTC GCCGGCGGCG TGTTCTTCAA 2601 CTTCTGGGTT CTCTTCCACC TCTACCCTTT CGCGAAGGGC 2641 CTTCTTGGCA AGCACGGCAA GACCCCAGTG GTGGTTCTTG 2681 TCTGGTGGGC CTTCACCTTC GTCATCACCG CCGTGCTGTA 2721 CATCAACATC CCGCACATCC ATGGCGGCGG CGGCAAGCAC 2761 TCCGTGGGCC ACGGCATGCA CCATGGCAAG AAGTTCGACG 2801 GCTACTACCT CTGGCCGTGA

[0008] CslF6 proteins and nucleic acids from a variety of species can be used in the plants, seeds, plant cells and methods described herein. For example, a CslF6 amino acid sequence from wheat (Triticum aestivum) can be used that has about86% sequence identity with the CslF6 from Brachypodium distachyon that has SEQ ID NO:1. This wheat CslF6 sequence is shown below with SEQ ID NO:5. 1 MAPAVAGGGR VRSNEPAAAA TAPASGKPCV CGFQVCACTG 41 SAAVASAASS LDMDIVAMGQ IGAVNDESWV GVELGEDGET 81 DESGAAVDDR PVFRTEKIKG VLLHPYRVLI FVRLIAFTLF 121 VIWRISHKNP DAMWLWVTSI CGEFWFGFSW LLDQLPKLNP 161 INRVPDLAVL RQRFDRPDGT STLPGLDIFV TTADPIKEPI 201 LSTANSVLSI LAADYPVDRN TCYVSDDSGM LLTYEALAES 241 SKFATLWVPF CRKHGIEPRG PESYFELKSH PYMGRAQDEF 281 VNDRRRVRKE YDEFKARINS LEHDIKQRND GYNAANAHRE 321 GEPRPTWMAD GTQWEGTWVD ASENHRRGDH AGIVLVLLNH 361 PSHRRQTGPP ASADNPLDFS GVDVRLPMLV YMSREKRPGH 401 DHQKKAGAMN ALTRASALLS NSPFILNLDC NHYINNSQAL 441 RAGICFMVGR DSDTVAFVQF PQRFEGVDPT DLYANHNRIF 481 FDGTLRALDG MQGPIYVGTG CLFRRITVYG FDPPRINVGG 521 PCFPRLAGLF AKTKYEKPGL EMTMAKAKAA PVPAKGKHGF 561 LPLPKKTYGK SDAFVDSIPR ASHPSPYAAA AEGIVADEAT 601 IVEAVNVTAA AFEKKTGWGK EIGWVYDTVT EDVVTGYRMH 641 IKGWRSRYCS IYPHAFIGTA PINLTERLFQ VLRWSTGSLE 681 IFFSKNNPLF GSTYLHPLQR VAYINITTYP FTAIFLIFYT 721 TVPALSFVTG HFIVQRPTTM FYVYLGIVLS TLLVIAVLEV 761 KWAGVTVFEW FRNGQFWMTA SCSAYLAAVC QVLTKVIFRR 801 DISFKLTSKL PSGDEKKDPY ADLYVVRWTP LMITPIIIIF 841 VNIIGSAVAF AKVLDGEWTH WLKVAGGVFF NFWVLFHLYP 881 FAKGILGKHG KTPVVVLVWW AFTFVITAVF YINIPHMHSS 921 GGKHTTVHGH HGKKFVDAGY YNWP

[0009] A nucleotide sequence that encodes the CslF6 protein from Triticum aestivum with SEQ ID NO:5 is shown below as SEQ ID NO:6. 1 ATGGCGCCAG CGGTGGCCGG AGGAGGCCGC GTGCGGAGCA ACGAGCCGGC TGCTGCCGCT 61 ACCGCGCCCG CCAGCGGGAA GCCCTGCGTC TGCGGCTTCC AGGTGTGCGC CTGCACGGGA 121 TCGGCCGCGG TGGCGTCTGC CGCCTCCTCG CTCGACATGG ACATCGTGGC CATGGGGCAG 181 ATAGGCGCCG TCAACGACGA GAGCTGGGTG GGCGTGGAGC TCGGCGAGGA CGGCGAGACC 241 GACGAAAGCG GTGCCGCCGT TGACGACCGC CCCGTCTTCC GCACCGAGAA AATCAAGGGT 301 GTCCTCCTCC ACCCCTACCG GGTGCTGATC TTCGTCCGTC TCATCGCCTT CACCCTGTTC 361 GTGATCTGGC GTATTTCCCA CAAGAACCCT GACGCCATGT GGCTGTGGGT GACATCCATC 421 TGCGGCGAGT TCTGGTTCGG TTTCTCCTGG CTGCTGGATC AGCTCCCCAA GCTGAACCCC481 ATCAACCGCG TGCCGGACCT GGCCGTGCTG CGGCAGCGCT TCGACCGCCC CGACGGCACC 541 TCCACCCTCC CGGGGCTGGA CATCTTCGTC ACCACGGCCG ACCCCATCAA GGAGCCCATC 601 CTCTCCACTG CCAACTCGGT GCTCTCCATC CTCGCCGCCG ACTACCCCGT CGACCGCAAC 661 ACATGCTACG TTTCTGACGA CAGTGGCATG CTGCTCACCT ACGAGGCCCT GGCTGAGTCC 721 TCCAAGTTCG CCACCCTCTG GGTGCCCTTC TGCCGCAAGC ACGGGATCGA GCCCAGGGGC 781 CCGGAGAGCT ACTTTGAGCT CAAGTCCCAC CCCTACATGG GGAGAGCCCA GGACGAGTTC 841 GTGAACGACC GTCGCCGCGT CCGCAAGGAG TACGACGAGT TCAAGGCCAG GATCAACAGC 901 CTGGAGCATG ACATCAAGCA GCGCAACGAC GGGTACAACG CCGCCAACGC CCACCGGGAA 961 GGCGAGCCCC GACCCACCTG GATGGCCGAC GGCACCCAGT GGGAGGGCAC CTGGGTCGAC 1021 GCCTCCGAGA ACCACCGCAG GGGCGACCAC GCCGGCATCG TCCTGGTGCT GCTGAACCAC 1081 CCGAGCCACC GCCGGCAGAC GGGCCCGCCG GCGAGTGCTG ACAACCCACT GGACTTCAGC 1141 GGCGTGGATG TGCGTCTCCC CATGCTGGTG TACGTGTCCC GTGAGAAGCG CCCCGGACAC 1201 GACCACCAGA AGAAGGCCGG CGCCATGAAC GCGCTCACCC GCGCCTCCGC GCTGCTCTCC 1261 AACTCCCCCT TCATCCTCAA CCTCGACTGC GACCACTACA TCAACAACTC CCAGGCCCTC 1321 CGCGCCGGCA TCTGCTTCAT GGTGGGACGC GACAGCGACA CCGTCGCCTT CGTCCAGTTC 1381 CCGCAGCGCT TCGAGGGCGT CGACCCCACC GACCTCTACG CCAACCACAA CCGCATCTTC 1441 TTCGACGGCA CCCTCCGTGC CCTCGACGGC ATGCAGGGCC CCATCTACGT CGGCACCGGC 1501 TGTCTCTTCC GCCGCATCAC CGTCTACGGC TTCGACCCGC CCAGGATCAA CGTCGGCGGG 1561 CCCTGCTTCC CCAGGCTCGC CGGGCTCTTC GCCAAGACCA AGTACGAGAA GCCTGGGCTC 1621 GAGATGACCA TGGCCAAGGC CAAGGCTGCG CCGGTGCCCG CCAAGGGCAA GCACGGCTTC 1681 CTGCCTCTGC CCAAGAAGAC GTACGGCAAG TCGGACGCCT TCGTGGACAG CATCCCGCGC1741 GCGTCGCACC CGTCGCCTTA CGCCGCGGCG GCTGAGGGCA TCGTGGCCGA CGAGGCGACC 1801 ATCGTGGAGG CGGTGAACGT GACGGCGGCG GCGTTCGAGA AGAAGACCGG GTGGGGCAAA 1861 GAGATCGGCT GGGTGTACGA CACCGTGACG GAGGACGTGG TGACCGGGTA CCGGATGCAT 1921 ATCAAGGGGT GGCGGTCACG CTACTGCTCC ATCTACCCAC ACGCCTTCAT CGGCACCGCA 1981 CCCATCAACC TCACGGAGAG GCTCTTCCAG GTGCTTCGCT GGTCCACGGG CTCCCTCGAG 2041 ATCTTCTTCT CCAAGAACAA CCCGCTCTTC GGCAGCACCT ACCTCCACCC GCTGCAGCGC 2101 GTCGCCTACA TCAACATCAC CACCTACCCG TTCACCGCCA TCTTCCTCAT CTTCTACACC 2161 ACCGTGCCGG CGCTCTCCTT CGTCACTGGC CACTTCATCG TGCAGCGCCC CACCACCATG 2221 TTCTACGTCT ACCTGGGCAT CGTGCTCTCC ACGCTGCTCG TCATCGCCGT GCTGGAGGTC 2281 AAGTGGGCCG GGGTCACCGT CTTCGAGTGG TTCAGGAACG GCCAGTTCTG GATGACGGCA 2341 AGTTGCTCCG CCTACCTCGC CGCCGTGTGC CAGGTGCTCA CCAAGGTGAT ATTCCGGCGT 2401 GACATCTCCT TCAAGCTCAC ATCCAAGCTA CCGTCGGGAG ACGAGAAGAA GGACCCCTAC 2461 GCCGACCTCT ACGTGGTGCG CTGGACGCCG CTCATGATCA CACCCATCAT CATCATTTTC 2521 GTCAACATCA TCGGGTCGGC GGTGGCCTTC GCCAAGGTGC TCGACGGCGA GTGGACGCAC 2581 TGGCTCAAGG TCGCCGGCGG CGTCTTCTTC AACTTCTGGG TGCTCTTCCA CCTCTACCCG 2641 TTCGCCAAGG GCATCCTGGG GAAGCACGGC AAGACGCCAG TCGTGGTGCT CGTCTGGTGG 2701 GCATTCACCT TCGTCATCAC CGCCGTGCTC TACATCAACA TCCCCCACAT GCATAGCTCG 2761 GGAGGCAAGC ACACAACGGT GCATGGTCAC CATGGCAAGA AGTTCGTCGA CGCAGGGTAC 2821 TACAACTGGC CGTGAGGTCC CTGTTGACGA CTTTGCCGCC GCGCATCCGG ACAAGACAAC 2881 GACGACCTGC GACAAGAAAC AACAAGTCAT CCACTGAACA GTGCA

[0010] A CslF6 amino acid sequence from barley (Hordeum vulgare) has about 86% sequence identity with the CslF6 from Brachypodium distachyon that has SEQ ID NO:1. This barley CslF6 sequence is shown below with SEQ ID NO:7.1 MAPAVAGGGR VRSNEPVAAA AAAPAASGKP CVCGFQVCAC 41 TGSAAVASAA SSLDMDIVAM GQIGAVNDES WVGVELGEDG 81 ETDESGAAVD DRPVFRTEKI KGVLLHPYRV LIFVRLIAFT 121 LFVIWRISHK NPDAMWLWVT SICGEFWFGF SWLLDQLPKL 161 NPINRVPDLA VLRQRFDRPD GTSTLPGLDI FVTTADPIKE 201 PILSTANSVL SILAADYPVD RNTCYVSDDS GMLLTYEALA 241 ESSKFATLWV PFCRKHGIEP RGPESYFELK SHPYMGRAQD 281 EFVNDRRRVR KEYDEFKARI NSLEHDIKQR NDGYNAAIAH 321 SQGVPRPTWM ADGTQWEGTW VDASENHRRG DHAGIVLVLL 361 NHPSHRRQTG PPASADNPLD LSGVDVRLPM LVYVSREKRP 401 GHDHQKKAGA MNALTRASAL LSNSPFILNL DCDHYINNSQ 441 ALRAGICFMV GRDSDTVAFV QFPQRFEGVD PTDLYANHNR 481 IFFDGTLRAL DGMQGPIYVG TGCLFRRITV YGFDPPRINV 521 GGPCFPRLAG LFAKTKYEKP GLEMTTAKAK AAPVPAKGKH 561 GFLPLPKKTY GKSDAFVDTI PRASHPSPYA AAAEGIVADE 601 ATIVEAVNVT AAAFEKKTGW GKEIGWVYDT VTEDVVTGYR 641 MHIKGWRSRY CSIYPHAFIG TAPINLTERL FQVLRWSTGS 681 LEIFFSKNNP LFGSTYLHPL QRVAYINITT YPFTAIFLIF 721 YTTVPALSFV TGHFIVQRPT TMFYVYLGIV LSTLLVIAVL 761 EVKWAGVTVF EWFRNGQFWM TASCSAYLAA VCQVLTKVIF 801 RRDISFKLTS KLPSGDEKKD PYADLYVVRW TPLMITPIII 841 IFVNIIGSAV AFAKVLDGEW THWLKVAGGV FFNFWVLFHL 881 YPFAKGILGK HGKTPVVVLV WWAFTFVITA VLYINIPHMH 921 TSGGKHTTVH GHHGKKLVDT GLYGWLH

[0011] A nucleotide sequence that encodes the CslF6 protein from Hordeum vulgare with SEQ ID NO:7 is shown below as SEQ ID NO:8. 1 cgtaaaggag agtgagtgcg tgcattgagg acgacggcca tggcgccagc ggtggccgga 61 gggggccgcg tgcggagcaa tgagccggtt gctgctgctg ccgccgcgcc ggcggccagc 121 ggcaagccct gcgtgtgcgg cttccaggtt tgcgcctgca cggggtcggc cgcggtggcc 181 tccgccgcct cgtcgctgga catggacatc gtggccatgg ggcagatcgg cgccgtcaac 241 gacgagagct gggtgggcgt ggagctcggc gaagatggcg agaccgacga aagcggtgcc 301 gccgttgacg accgccccgt attccgcacc gagaagatca agggtgtcct cctccacccc 361 taccgggtgc tgattttcgt tcgtctgatc gccttcacgc tgttcgtgat ctggcgtatc 421 tcccacaaga acccagacgc gatgtggctg tgggtgacat ccatctgcgg cgagttctgg 481 ttcggtttct cgtggctgct agatcagctg cccaagctga accccatcaa ccgcgtgccg 541 gacctggcgg tgctgcggca gcgcttcgac cgccccgacg gcacctccac gctcccgggg 601 ctggacatct tcgtcaccac ggccgacccc atcaaggagc ccatcctctc caccgccaac 661 tcggtgctct ccatcctggc cgccgactac cccgtggacc gcaacacatg ctacgtctcc 721 gacgacagtg gcatgctgct cacctacgag gccctggcag agtcctccaa gttcgccacg781 ctctgggtgc ccttctgccg caagcacggg atcgagccca ggggtccgga gagctacttc 841 gagctcaagt cacaccctta catggggaga gcccaggacg agttcgtcaa2641 ggcggcgtct tcttcaactt ctgggtgctc ttccacctct accccttcgc caagggcatc 2701 ctggggaagc acggaaagac gccagtcgtg gtgctcgtct ggtgggcatt

[0012] 82% sequence identity with the CslF6 from Brachypodium distachyon that has SEQ ID NO:1. This corn CslF6 sequence is shown below with SEQ ID NO:9. MAAVASAASSADMDRVAVAATEGQIGAVNDESWIAVDLSDDGLSADGADPGVALEDRPVF RTEKIKGVLLHPYRVLIFVRLIAFTLFVIWRISHRNPDALWLWVTSIAGEFWFGFSWLLD QLPKLNPINRVPDLAALRQRFDRAGGGAGGGTSLLPGLDVFVTTADPFKEPILSTANSVL SILAADYPVERNTCYLSDDSGMLLTYEAMAEAAKFATVWVPFCRKHGIEPRGPESYFDLK SHPYMGRSQEDFVNDRRRVRKDYDEFKARINGLDHDIKQRSDAYNAARGLKDGEPRATWM ADGTQWEGTWVEPSENHRKGDHAGIVLVLLNHPSHSRQLGPPASADNPLDLSMVDVRLPM LVYVSREKRPGHNHQKKAGAMNALTRCSAVLSNSPFILNLDCDHYINNSQALRAGICFML GRDSDTVAFVQFPQRFEGVDPTDLYANHNRIFFDGTLRALDGMQGPIYVGTGCLFRRITL YGFDPPRINVGGPCFPALGGMFAKAKYEKPGLELTTKAAVAKGKHGFLPMPKKSYGKSDA FADTIPMASHPSPFAAAAAAVVVADEATIAEAVAVCAAAYEKKTGWGSDIGWVYGTVTED VVTGYRMHIKGWRSRYCSIYPHAFIGTAPINLTERLFQVLRWSTGSLEIFFSRNNPLFGS TFLHPLQRVAYINITTYPFTAIFLIFYTTVPALSFVTGHFIVQRPTTMFYVYLAIVLGTL LILAVLEVKWAGVTVFEWFRNGQFWMTASCSAYLAAVCQVLVKVVFRRDISFKLTSKQPA GDEKKDPYADLYVVRWTWLMVTPIIIILVNIIGSAVAFAKVLDGEWTHWLKVAGGVFFNF WVLFHLYPFAKGILGRHGKTPVVVLVWWAFTFVITAVLYINIPHIHGPGGKHGGAIGRHG GDAHHHGKKFDGYYLWP

[0013] A nucleotide sequence that encodes the CslF6 protein from Zea mays with SEQ ID NO:9 is shown below as SEQ ID NO:10. 1 cttccgccag gcaggcagag agggagggcg cgcccggcat tatggcggcg gtggcgtccg 61 ccgcgtcctc cgccgacatg gaccgcgtgg ccgtggccgc caccgagggc cagatcggcg 121 ccgtcaacga cgagagctgg atcgccgtcg acctcagcga cgacggcctg tccgccgacg 181 gcgccgaccc gggcgtcgcg ctcgaggacc gccccgtctt ccgcaccgag aagatcaagg 241 gcgtcctcct ccacccctac agggtgctga tcttcgtccg gctgatcgcg ttcacgctgt301 tcgtgatctg gcgcatctcg caccgcaacc cggacgcgct gtggctgtgg gtgacgtcga 361 tcgcgggcga gttctggttc ggcttctcgt ggctgctgga ccagctgccg2161 ccatgttcta cgtgtacctg gccatcgtgc tgggcacgct gctgatcctg gccgtcctgg 2221 aggtgaagtg ggcgggcgtg acggtgttcg agtggttcag gaacgggcag

[0014] has about 82% sequence identity with the CslF6 from Brachypodium distachyon that has SEQ ID NO:1. This Sorghum CslF6 sequence is shown below with SEQ ID NO:11. 1 MAPGGGDGRR NGEGQQQANG NNNNNNSNAK AKHGCVCGFP 41 VCACAGAAAV ASAASSADMD RVAAAQTEGQ IGAVNDESWI 81 AVDLSDDLSG DGGGADPGVA IEDRPVFRTE KIKGILLHPY 121 RVLIFVRLIA FTLFVIWRIS HRNPDAMWLW VTSIAGEFWF 161 GFSWLLDQLP KLNPINRVPD LAVLRQRFDR ADGTSRLPGL 201 DIFVTTADPF KEPILSTANS ILSILAADYP VERNTCYLSD 241 DSGMLLTYEA MAEAAKFATV WVPFCRKHGI EPRGPESYFE 281 LKSHPYMGRS QEDFVNDRRR VRKEYDEFKA RINGLEHDIK 321 QRSDAFNAAR GLKDGEPRAT WMADGNQWEG TWVEPSENHR 361 KGDHAGIVYV LLNHPSHSRQ LGPPASADNP LDFSMVDVRL 401 PMLVYVSREK RPGFNHEKKA GAMNALTRCS AVISNSPFIL 441 NLDCDHYINN SQALRAGICF MLGRDSDTVA FVQFPQRFEG 481 VDPTDLYANH NRIFFDGTLR ALDGMQGPIY VGTGCMFRRI 521 TLYGFDPPRI NVGGPCFPSL GGMFAKTKYE KPGLELTTKA 561 AVAKGKHGFL PLPKKSYGKS DAFVDTIPRA SHPSPFLSAD 601 EAAAIVADEA MITEAVEVCT AAYEKKTGWG SDIGWVYGTV 641 TEDVVTGYRM HIKGWRSRYC SIYPHAFIGT APINLTERLY 681 QVLRWSTGSL EIFFSRNNPL FGSTFLHPLQ RVAYINITTY 721 PFTALFLIFY TTVPALSFVT GHFIVQRPTT MFYVYLAIVL 761 GTLLILAVLE VKWAGVTVFE WFRNGQFWMT ASCSAYLAAV 801 CQVLVKVVFR RDISFKLTSK QPAGDEKKDP YADLYVVRWT 841 WLMVTPIIII LVNIIGSAVA FAKVLDGEWT HWLKVAGGVF 881 FNFWVLFHLY PFAKGLLGRH GKTPVVVLVW WAFTFVITAV 921 LYINIPHIHG PGGKHGGAIG KHGAAHHGKK FDLDNLSYNW 961 P

[0015] A nucleotide sequence that encodes the CslF6 protein from Sorghum bicolor with SEQ ID NO:11 is shown below as SEQ ID NO:12. 1 gagggcgcag ccggcattat ggcgccgggc ggcggagacg gccggcgcaa cggcgaggga 61 cagcagcagg cgaacggcaa caacaacaac aacaacagca acgctaaggc taagcacggg 121 tgcgtgtgcg ggttccccgt gtgcgcgtgc gccggcgcgg ccgcggtggc gtccgcggcc 181 tcctccgccg acatggaccg cgtggccgcc gcgcagaccg agggccagat cggcgccgtc 241 aacgacgaga gctggatcgc cgtcgacctg agcgacgacc tctccggcga cggcggcggc 301 gccgaccccg gcgtcgcgat cgaggaccgc cccgtcttcc gcaccgagaa gatcaagggc 361 atcctcctcc acccctacag ggtgctcatc ttcgtgcgcc tgatcgcgtt cacgctgttc 421 gtcatctggc gtatctcgca ccgcaacccg gacgcgatgt ggctgtgggt gacgtcgatc 481 gcgggcgagt tctggttcgg cttctcctgg ctgctggacc agctccccaa gctgaacccg 541 atcaaccgcg tcccggacct cgcggtgctc cggcagcggt tcgaccgcgc cgacggcacg 601 tcccgcctcc cgggcctgga catcttcgtc accacggcgg acccgttcaa ggagcccatc 661 ctgagcacgg ccaactccat cctctccatc cttgccgccg actaccccgt ggagcgcaac 721 acgtgctacc tctccgacga ctccgggatg ctgctcacct acgaggccat ggcggaggcc 781 gccaagttcg ccaccgtctg ggtgcccttc tgccggaagc acggcatcga gcctcgtggc 841 cccgagagct acttcgagct caagtcgcac ccctacatgg ggaggtcgca ggaggacttc 901 gtcaacgacc gccgccgtgt ccgcaaggag tacgacgagt tcaaggcccg gatcaatggc 961 ctcgagcatg atatcaagca gaggtccgac gcgtttaacg ccgctagggg gcttaaggac 1021 ggcgagccca gagctacgtg gatggccgac gggaaccagt gggagggcac atgggttgag 1081 ccatcggaga accaccgcaa gggtgaccac gccggcatcg tctatgtgct tctgaaccac 1141 ccgagccaca gccgtcagct cggcccgccg gcgagcgcgg acaacccgct ggacttcagc 1201 atggtggacg ttcgcctccc catgctggtg tacgtctccc gtgagaagcg gcccgggttc 1261 aaccacgaga agaaggccgg cgccatgaac gcgctgaccc gctgctccgc cgtgatctcc 1321 aactcgccct tcatcctcaa cctggactgc gaccactaca tcaacaactc gcaggcgctt 1381 cgcgccggca tctgcttcat gctcggccgg gacagcgaca cggtggcgtt cgtgcagttc 1441 ccgcagcggt tcgagggcgt ggaccccacg gacctgtacg ccaaccacaa ccgcatcttc 1501 ttcgacggca cgctccgggc gctggacggc atgcagggcc ccatctacgt cggcaccggc 1561 tgcatgttcc gccgcatcac gctctacggc ttcgacccgc cgaggatcaa cgtcggcggg 1621 ccgtgcttcc cgtcgctcgg cggcatgttc gccaagacca agtacgagaa gcccgggctg1681 gagctcacca ccaaggccgc cgtcgccaag ggcaagcacg gcttcctccc gttgcccaag 1741 aagtcgtacg gcaagtcgga cgcgttcgtc gacaccatcc cgagggcgtc tcacccgtcg 1801 ccgttcctga gcgccgacga ggccgccgcc atcgtcgccg acgaggccat gatcaccgag 1861 gccgtggagg tgtgcacggc ggcgtacgag aagaagaccg gctggggcag cgacatcggc 1921 tgggtgtacg gcaccgtcac cgaggacgtg gtgacggggt accggatgca catcaagggg 1981 tggcggtctc gctactgctc catctacccg cacgccttca tcggcaccgc cccgatcaac 2041 ctgacggagc ggctgtacca ggtgctccgc tggtccacgg ggtcgctgga gatcttcttc 2101 tcccggaaca acccgctgtt cggcagcacg ttcctgcacc cgctgcagcg cgtggcgtac 2161 atcaacatca ccacctaccc gttcacggcg ctgttcctca tcttctacac caccgtgccg 2221 gcgctgtcgt tcgtgacggg gcacttcatc gtgcagcggc cgaccaccat gttctacgtg 2281 tacctggcca tcgtgctggg gacgctgctc atcctggccg tcctggaggt gaaatgggcg 2341 ggcgtcaccg tcttcgagtg gttcaggaac gggcagttct ggatgacggc cagctgctcc 2401 gcgtacctgg ccgccgtgtg ccaggtgctg gtgaaagtgg tgttccggcg agacatctcc 2461 ttcaagctca catccaagca gcccgccggc gacgagaaga aggaccccta cgccgacctg 2521 tacgtggtgc gctggacctg gctcatggtg acccccatca tcatcatcct cgtcaacatc 2581 atcggatccg ccgtggcgtt cgccaaggtg ctggacggcg agtggacgca ctggctcaag 2641 gtggccggcg gcgtcttctt caacttctgg gtgctgttcc acctctaccc gttcgccaag 2701 ggcctcctcg ggaggcacgg caagaccccc gtggtggtgc tcgtctggtg ggcattcacc 2761 ttcgtcatca ccgccgtgct ctacatcaac atcccccaca tccatggccc cggcggcaag 2821 cacggcggcg cgatcggaaa gcacggcgcc gcccaccacg gcaagaagtt cgacctcgac 2881 aacctctcct acaactggcc gtgaag

[0016] Overexpression of CslF6

[0017] To increase the levels of MLG in Sorghum bicolor, methods of increasing MLG synthesis and reducing or preventing MLG degradation in the plant are described. In plants, MLG abundance is controlled by MLG synthase, CslF6 and MLG hydrolase, lichenase through developmental program (Figs. 1A-B). Overproduction of MLG hampers growth in some species, such as Brachypodium (Fig. 2), rice and barley. Nevertheless, transgenic Sorghum was produced to overexpress an MLG synthase (CslF6) from either Brachypodium or Sorghum (constructs named BdCslF6 and SbCslF6, respectively), which generate adifferent frequency of β1- 3 linkages in MLG, resulting in different types of the fine structure of this polymer.

[0018] The transgenic Sorghum grown in a greenhouse showed higher MLG levels compared to the vector control without any noticeable growth defects (Figs. 6 and 7). Field trials in East Lansing, MI were conducted with the T3 generation of CslF6-sorghum transgenics and found no defects in growth (Fig. 8). MLG analyses from the field-grown Sorghum were performed during development within single stem internodes. The transgenic lines had higher levels of MLG compared to wild-type and vector controls, even in the fully elongated region of the internode where MLG degradation normally occurs (Figs. 9-10). MLG abundance was determined in Sorghum young leaves (Fig. 13). High MLG and low starch synthesis was found in the actively elongating region of leaves. On the contrary, unchanged low level of MLG and dynamic starch metabolism were observed in the mature region of leaves.

[0019] Furthermore, to modulate and reduce MLG degradation in Sorghum and increase levels of MLG, three Sorghum MLG endoglucanases (also called lichenases) were identified and characterized (Figs. 11A-C). The three lichenases have similar enzymatic activity, with an optimal pH near 5, between 25˚C and 45˚C and digest both synthetic substrate and MLG in flours (Fig.11B). The three enzymes were localized in the apoplast, where lichenase activity is expected to take place (Fig.11C). The lichenases’ expression patterns were determined during development and light conditions, which led us to identify the lichenase that is responsible for MLG degradation in sorghum (Fig.12).

[0020] Vacuolar Targeting Polypeptide

[0021] A variety of vacuolar targeting polypeptides can be linked to a Csl protein, such as CslF6, to form the fusion protein that can be targeted to the tonoplast (Fig. 14). Expression of the fusion protein can result in higher levels of MLG production in plants such as Sorghum compared to wild type plants not expressing the fusion protein (Fig. 15). Vacuoles perform a multitude of functions in plant cells, including the storage of amino acids and sugars. Vacuoles are the largest plant organelles, constituting up to 90% of the cell volume. They are indispensable for multiple cellular functions, including the maintenance of turgor to shape the individual cell and the entire plant. Two types of vacuoles have been described in plants, a lytic vacuole and a protein storage vacuole. The lytic vacuole has acidicpH, is abundant in vegetative tissues, and is homologous to the animal lysosome. The protein storage vacuole (PSV) has a neutral pH and is the main protein storage compartment in developing seeds.

[0022] Tonoplast-localized transporters catalyze the import and release of these molecules. For example, the vacuolar targeting polypeptide can be a beta fructosidase 4 (βFruct4) polypeptide vacuole targeting domain. Fruct4 genes encode vacuolar forms of invertase. Fruct4 proteins comprise an N-terminal cytosolic domain that can transport the protein from the ER to the central vacuole (the “vacuole targeting domain”).

[0023] Fruct4 protein has a short cytosolic region of 42 amino acids at the N- terminus (“signal”) followed by a single transmembrane domain (“TMD”) and a luminal C-terminal region, which contains a functional fructosidase domain. The AtFruct4 the N-terminal region contains the sequence information for trafficking to the tonoplast (the “vacuole targeting domain”). Jung, et. al., Identification of Sorting Motifs of AtβFruct4 for Trafficking from the ER to the Vacuole Through the Golgi and PVC, Traffic (2011) 12(12): 1774-1792.

[0024] An amino acid sequence for the Fruct4 vacuole targeting domain from Arabidopsis thaliana is shown below with SEQ ID NO: 13. MASSDALLPI SAREEEPLCP YTRLPMADPN QETHGPRRRR PFKG

[0025] A nucleotide sequence that encodes the Fruct4 vacuole targeting domain from Arabidopsis thaliana with SEQ ID NO:13 is shown below as SEQ ID NO:14. 1 cgatgctctc ttgccaatct ccgccagaga agaagaacca ttatgtcctt acacgagatt 61 accaatggcc gacccgaatc aagaaaccca tggcccccgg agaagaagac cctttaaagg 121 tctcctcgcc gtctcatttg gt

[0026] In some cases, another vacuole targeting domain can include a tonoplast intrinsic protein (TIP), including TIP1-1 or TIP3-1. TIPs facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane. In barley, pea and Arabidopsis root tips, the lytic vacuole is marked by the presence of TIP1-1. TIP3-1 localizes to the protein storage vacuole. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent.

[0027] The TIP1-1 and TIP3-1 whole protein can be fused to a Csl protein, such as Csl6, to form a fusion protein that is targeted to the tonoplast (Fig. 14). An amino acid sequence for TIP1-1 from Arabidopsis thaliana is shown below with SEQ ID NO: 15. MPIRNIAIGR PDEATRPDAL KAALAEFIST LIFVVAGSGS GMAFNKLTEN GATTPSGLVA AAVAHAFGLF VAVSVGANIS GGHVNPAVTF GAFIGGNITL LRGILYWIAQ LLGSVVACLI LKFATGGLAV PAFGLSAGVG VLNAFVFEIV MTFGLVYTVY ATAIDPKNGS LGTIAPIAIG FIVGANILAG GAFSGASMNP AVAFGPAVVS WTWTNHWVYW AGPLVGGGIA GLIYEVFFIN TTHEQLPTTD Y

[0028] A nucleotide sequence that encodes the TIP1-1 from Arabidopsis thaliana with SEQ ID NO:15 is shown below as SEQ ID NO:16. 1 gctcttctag catcttgaag cttaaatctc agccgtccga tcatgccgat cagaaacatc 61 gccattggcc gtccagatga agccacccgt cccgatgcct taaaggcggc gttggctgag 121 ttcatttcaa ctttgatctt tgtcgtcgcc ggttcaggct ctggcatggc tttcaacaag 181 ctcactgaaa acggagccac cactccttct ggtctcgtag ctgctgcagt ggctcatgcc 241 tttggactct tcgtcgctgt ctcagttggt gccaacatct ctggtggaca cgttaaccct 301 gccgtcactt tcggtgcttt cattggtggt aacatcactc tcctccgtgg tatcctctac 361 tggattgctc agcttctcgg ctccgtcgtc gcttgcctca tccttaaatt cgccaccgga 421 ggcttggctg tgcccccttt tggtctctct gctggagtag gagtgttgaa cgctttcgtt 481 ttcgagatcg tgatgacatt cgggcttgtt tacaccgtct acgctacagc cattgacccc 541 aaaaacggga gtcttggaac aattgctccc atcgcaatcg gtttcattgt tggagccaac 601 atcttagctg gaggagcttt ctctggagcc tccatgaatc ccgccgtggc tttcggacca 661 gcggtggtga gctggacatg gaccaaccac tgggtctact gggccggacc tctcgtcggc 721 ggtggaatcg ctggactcat ctacgaagtt ttcttcatca acaccacaca cacgagcagc 781 tccaaccaca gactactgaa ttaatctctc tttttctctc ttgtgtaatt ttattcgact 841 ttgaatttga attttaatgt ctttttaatt tccgtttgtg taatttcgaa tcatcaaggg 901 gtttatgatc gttcgatctt ttagatgaat ccttgtccgt tggtttcaag agtggccatt 961 gcaatttcga ctatataatg tgaattatgt atc

[0029] An amino acid sequence for TIP3-1 from Arabidopsis thaliana is shown below with SEQ ID NO: 17. MATSARRAYG FGRADEATHP DSIRATLAEF LSTFVFVFAA EGSILSLDKL YWEHAAHAGT NTPGGLILVA LAHAFALFAA VSAAINVSGG HVNPAVTFGALVGGRVTAIR AIYYWIAQLL GAILACLLLR LTTNGMRPVG FRLASGVGAV NGLVLEIILT FGLVYVVYST LIDPKRGSLG IIAPLAIGLI VGANILVGGP FSGASMNPAR AFGPALVGWR WHDHWIYWVG PFIGSALAAL IYEYMVIPTE PPTHHAHGVH QPLAPEDY

[0030] A nucleotide sequence that encodes the TIP3-1 from Arabidopsis thaliana with SEQ ID NO:17 is shown below as SEQ ID NO:18. 1 acactttgtt gttaatttca agtgtttgat cataatggca acatcagctc gtagagcata 61 cggtttcggt agagccgatg aggctacaca ccctgactcc attagagcaa ctttagctga 121 gtttctctcc acttttgtct tcgtctttgc agctgaaggc tctatcctct ctctcgataa 181 gttgtattgg gaacatgcgg ctcatgcggg gacaaacaca ccaggagggc tgattttagt 241 agcgttggct catgcgtttg ctctgtttgc tgctgtttca gcagccatta atgtctccgg 301 cggacacgtt aacccggcag tcacttttgg tgctcttgtt ggaggcagag ttacagcgat 361 ccgcgccatc tactactgga tcgctcagct tcttggagcc atcctcgctt gtctcttgtt 421 aaggctcaca acaaacggca tgagaccagt tggtttccgt ctagcatcag gtgttggagc 481 ggttaatgga cttgtattag agatcatttt aacatttggc ttagtctacg tagtgtattc 541 cactttgatt gatccaaaac gtggaagcct cgggatcata gcaccgcttg caatcggact 601 catagttggg gcaaacatct tagtaggtgg accattttct ggtgcttcga tgaatccagc 661 tagagctttt ggtccagcgt tggtgggatg gagatggcat gaccactgga tctattgggt 721 cggaccattc atcggtagtg ctttagccgc ccttatatat gagtacatgg tcatacccac 781 cgaaccacct acccaccacg cacatggtgt acaccagccc ttggcccctg aagattacta 841 gatggaactt tcctcatgtc accactgctt ttgttcgttg ttcaagctct ctttgtctgt 901 atgatgagat cacttgcata gatacttttt ttcttatgct actcttatgt tgtaataata 961 aaaaataaag aacgatttcg ctaaaaaaaa aaaaaaaa

[0031] An amino acid sequence for a TIP protein from Brachypodium distachyon is shown below with SEQ ID NO: 19. MPVSRIAIGS PGELSHPDTF KAAVAEFISM LIFVFAGSGS GMAFGKLTDG GATTPSGLIA AALAHAFALF VAVSVGANIS GGHVNPAVTF GAFIGGNISL LKAIVYWVAQ LLGSTAACLL LQISTGGASV GAFSLSAGVG VWNAVVFEIV MTFGLVYTVY ATAVDPKRGD LGVIAPIAIG FIVGANILAG GAFDGASMNP AVSFGPAVVS GVWENHWVYW LGPFAGAAIA ALVYDIVFIG QRPHDQLPSA EY

[0032] An amino acid sequence for another TIP protein from Brachypodium distachyon is shown below with SEQ ID NO: 20.MPISRVAVGS HHEVYQAGAL KAAVAEFIST LIFVFAGQGS GMAFNKLNAG GAATPAGLIS AAIAHAFALF VAVSVGANIS GGHVNPAVTF GAFLGGNITL FRGLLYWVAQ LLGSTAACFL LRFSTGGLPT GTFGLTGVSV WEALVLEIVM TFGLVYTVYA TAVDPKKGSI GTIAPLAIGF IVGANILVGG AFDGASMNPA VSFGPALVSW EWGYQWVYWV GPLIGGGLAG VIYEVLFISH THEQLPTTDY

[0033] An amino acid sequence for a TIP1-1 protein from Triticum aestivum is shown below with SEQ ID NO: 21. EFISTLIFVF AGQGSGMAFS KLSPDGAATP AGLISAAIAH AFALFVAVSV GANISGGHVN PAVTFGAFVG GNITLFRGLL YWIAQLLGST AACFLLRFST GGLPTGTFGL SGIGAWEAVV LEIVMTFGLV Y

[0034] A nucleotide sequence that encodes the TIP1-1 protein from Triticum aestivum with SEQ ID NO:21 is shown below as SEQ ID NO:22. 1 acacgagccg atccatccat ccagaaaaaa aaaaaagagt gcccgtcgaa aatgccggtg 61 cgcaggatcg ccatcggcgc cccgggggag ctgtcccacc cggacacctt ccgcgccggc 121 gtcgccgagt tcatctccat gctcatcttc gtcttcgccg gctcaggctc cggcatggcc 181 ttcggcaagc tgactgacgg cggcgcgacg acgccggcgg ggcttatcgc ggcggcgctg 241 gcgcacgcgt tcgcgctctt cgtggccgtc tccgtgggcg ccaacatctc cggcgggcac 301 gtgaacccgg ccgtcacatt cggcgccttc gtgggcggca acatcagcct gctcaaggcc 361 gtggtgtact gggtggcgca gctgctcggc tccgtcgtgg cctgcctcct cctcaagatc 421 gccaccggcg gcgaggccgt gggcgccttc tcgctctccg ccggcgtcgg cgtctggaac 481 gccgtggtgt tcgagatcgt catgaccttc gggctcgtct acacggtgta cgccaccgcg 541 gtcgacccca agcgcggcga cctcggggtg atcgcgccca tcgccatcgg cttcatcgtc 601 ggcgccaaca tcctcgccgg cggcgccttc gacggcgcct ccatgaaccc cgccgtctcc 661 ttcggccccg ctgtcgtcag cggcgtctgg gagaaccact gggtctactg gctcggcccc 721 ttcgtcggcg ccgccatcgc cgccctcgtc tacgacatct gcttcatcgg ccagcgcccc 781 cacgagcagc tccccaccgc cgactactga aaacctcaat ctcatctcgt ccatccgtca 841 tcgccatgag agattgaaat cgcttcaaaa attccagcaa gtgtacgtcg tcgcgtcatc 901 atcagtaagc cagtttagtt tcattgcgct atcttgtcct tgagaaattc agggtaaacg 961 gtgtgtgcga gccagtcagt caggaataat ctcatccttc tcttatcttg gtgtatgtat 1021 gtacgtcctc aattatgaat gcttctattc catggtcaag attgatgttt gtacaaaaaa 1081 aaaaaaaaaa aa

[0035] An amino acid sequence for a TIP1-1 protein from Zea mays is shown below with SEQ ID NO: 23. MPINRIALG SHQEVYHPG ALKAAFAEF ISTLIFVFA GQGSGMAFS KLTGGGPTT PAGLIAAAV AHAFALFVA VSVGANISG GHVNPAVTF GAFVGGNIT LFRGLLYWV AQLLGSTVA CFLLRFSTG GQATGTFGL TGVSVWEAL VLEIVMTFG LVYTVYATA VDPKKGSLG TIAPIAIGF IVGANILVG GAFDGASMN PAVSFGPAL VSWEWGYQW VYWVGPLIG GGLAGVIYE LLFISHTHE QLPSTDY

[0036] The nucleic acids and polypeptides allow identification and isolation of related nucleic acids and their encoded enzymes that provide a means for production of healthy plants with increased glucan.

[0037] The related nucleic acids can be isolated and identified by mutation of the SEQ ID NOS: 2, 3, 6, 8, 10, 12, 14, 16, 18, and 22 nucleic acid sequences and / or by hybridization to DNA and / or RNA isolated from other plant species using segments of these nucleic acids as probes. The sequence of the CSLF6, Fruct4, Tip1-1, and Tip3-1 proteins (e.g., SEQ ID NOs: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, and 23) can also be examined and used a basis for designing alternative CslF6, Fruct4, Tip1-1, and / or Tip3-1 nucleic acids that encode related CslF6, Fruct4, Tip1-1, and / or Tip3-1 polypeptides.

[0038] The CslF6, Fruct4, Tip1-1, and / or Tip3-1 nucleic acids described herein can include any nucleic acid that can selectively hybridize to any of SEQ ID NOS: 2, 3, 6, 8, 10, 12, 14, 16, 18, or 22 nucleic acids. The term “selectively hybridize” includes hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence (e.g., any of the SEQ ID NOS: 2, 3, 6, 8, 10, 12, 14, 16, 18, or 22 nucleic acids) to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences. Such selective hybridization substantially excludes non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or 60-99% sequence identity, or 70- 99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 97-99% sequence identity, or 100% sequence identity (or complementarity) with each other. In some embodiments, a selectively hybridizing sequence has about at leastabout 80% sequence identity or complementarity with SEQ ID NOS: 2, 3, 6, 8, 10, 12, 14, 16, 18, or 22.

[0039] Thus, the nucleic acids of the invention include those with about 500 of the same nucleotides as SEQ ID NOS: 2, 3, 6, 8, 10, 12, 14, 16, 18, or 22, or about 600 of the same nucleotides, or about 700 of the same nucleotides, or about 800 of the same nucleotides, or about 900 of the same nucleotides, or about 1000 of the same nucleotides, or about 1100 of the same nucleotides, or about 1200 of the same nucleotides as SEQ ID NOS: 2, 3, 6, 8, 10, 12, 14, 16, 18, or 22. The identical nucleotides or amino acids can be distributed throughout the nucleic acid or the protein, and need not be contiguous.

[0040] Note that if a value of a variable that is necessarily an integer, e.g., the number of nucleotides or amino acids in a nucleic acid or protein, is described as a range, e.g., 90-99% sequence identity what is meant is that the value can be any integer between 90 and 99 inclusive, i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, or any range between 90 and 99 inclusive, e.g., 91-99%, 91-98%, 92-99%, etc.

[0041] The terms “stringent conditions” or “stringent hybridization conditions” include conditions under which a probe will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are somewhat sequence-dependent and can vary in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences can be identified with up to 100% complementarity to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of sequence similarity are detected (heterologous probing). The probe can be approximately 20-500 nucleotides in length but can vary greatly in length from about 18 nucleotides to equal to the entire length of the target sequence. In some embodiments, the probe is about 10-50 nucleotides in length, or about 18-25 nucleotides in length, or about 18-50 nucleotides in length, or about 18-100 nucleotides in length.

[0042] Typically, stringent conditions will be those where the salt concentration is less than about 1.5 M Na+ion (or other salts), typically about 0.01 to 1.0 M Na+ion concentration (or other salts), at pH 7.0 to 8.3 and the temperature is at least about 30 °C for shorter probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for longer probes (e.g., greater than 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide or Denhardt’s solution. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 °C, and a wash in 1 x SSC to 2 x SSC (where 20 x SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55 °C Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1M NaCl, 1% SDS at 37 °C, and a wash in 0.5 x SSC to 1 x SSC at 55 to 60 °C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C, and a wash in 0.1 x SSC at 60 to 65 °C. Specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution. Thus, high stringency conditions can include a wash that includes 0.1 x SSC at 60 to 65 °C.

[0043] For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem.138:267-84 (1984)): Tm = 81.5 °C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L where M is the molarity of monovalent cations; % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. The Tm is reduced by about 1 °C for each 1% of mismatching. Thus, the Tm, hybridization and / or wash conditions can be adjusted to hybridize to sequences of the desired sequence identity. For example, if sequences with greater than or equal to 90% sequence identity are sought, the Tmcan be decreased 10 °C. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can include hybridization and / or a wash at 1, 2, 3 or 4° C lower than the thermal melting point (Tm). Moderately stringent conditions can include hybridization and / or a wash at 6, 7, 8, 9 or 10 °C lower than the thermal melting point (Tm). Low stringency conditions can include hybridization and / or a wash at 11, 12, 13, 14, 15 or 20 °C lower than the thermal melting point (Tm). Using the equation, hybridization andwash compositions, and a desired Tm, those of ordinary skill can identify and isolate nucleic acids with sequences related to any of SEQ ID NO: 2, 3, 6, 8, 10, 12, 14, 16, 18, or 22.

[0044] Those of skill in the art also understand how to vary the hybridization and / or wash solutions to isolate desirable nucleic acids. For example, if the desired degree of mismatching results in a Tmof less than 45 °C (aqueous solution) or 32 °C (formamide solution), it may be preferred to increase the SSC concentration so that a higher temperature can be used.

[0045] An extensive guide to the hybridization of nucleic acids is found in Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY – HYBRIDIZATION WITHNUCLEICACIDPROBES, part 1, chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (1993); and in CURRENTPROTOCOLS INMOLECULARBIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishing and Wiley-Interscience, New York (1995).

[0046] Unless otherwise stated, in the present application high stringency is defined as hybridization in 4 x SSC, 5 x Denhardt’s (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg / ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65 °C, and a wash in 0.1 x SSC, 0.1% SDS at 65 °C. However, because specificity is typically a function of post-hybridization washes, where the factors controlling hybridization include the ionic strength and temperature of the final wash solution, the high stringency conditions can more simply be expressed as including a wash in 0.1 x SSC at 60 to 65 °C.

[0047] The following terms are used to describe the sequence relationships between two or more nucleic acids or polypeptides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity” and (e) “substantial identity.”

[0048] As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence can be a nucleic acid sequence (e.g., any of SEQ ID SEQ ID NO:2, 3, 4, or 10) or an amino acid sequence (e.g., any of SEQ ID NO:1, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15). A reference sequence may be a subset or the entirety of a specified sequence. For example, a reference sequence may be a segment of a full-length cDNA or of a genomic DNA sequence,or the complete cDNA or complete genomic DNA sequence, or a domain of a polypeptide sequence.

[0049] As used herein, "comparison window" refers to a contiguous and specified segment of a nucleic acid or an amino acid sequence, wherein the nucleic acid / amino acid sequence can be compared to a reference sequence and wherein the portion of the nucleic acid / amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window can vary for nucleic acid and polypeptide sequences. Generally, for nucleic acids, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or more nucleotides. For amino acid sequences, the comparison window is at least about 10 amino acids, and can optionally be 15, 20, 30, 40, 50, 100 or more amino acids. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the nucleic acid or amino acid sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

[0050] Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, may permit optimal alignment of compared sequences; by the homology alignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53; by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) Nucleic Acids Res.16:10881-90; Huang, et al., (1992) Computer Applications in the Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. An example of a good program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60, whichis similar to the method described by Higgins and Sharp, (1989) CABIOS 5:151- 53 (and is hereby incorporated by reference). The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0051] GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP makes a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more.

[0052] GAP presents one member of the family of best alignments. There may be many members of this family. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the WisconsinGenetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0053] For example, sequence identity / similarity values provided herein can refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402).

[0054] As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low- complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (C1-ayerie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.

[0055] The terms “substantial identity” indicates that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, or at least 60%, or at least 70%, or at least 80%, or at least 90% or at least 95% sequence identity, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or any percentage value within the range of 55-100% sequence identity relative to the reference sequence over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra.

[0056] One indication that two CslF6-related polypeptide sequences are substantially identical is that both polypeptides have glucan synthase activity with glucose as a substrate.

[0057] The polypeptide that is substantially identical to a CslF6, Fruct4, Tip1-1, or Tip3-1 with a SEQ ID NOS: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, or 23 sequence may not have exactly the same level of activity as the CslF6, Fruct4, Tip1-1, or Tip3-1 with a SEQ ID NOS: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, or 23. Instead, the substantially identical polypeptide may exhibit greater or lesser levels of activity than the CslF6 Fruct4, Tip1-1, or Tip3-1 with SEQ ID NOS: 1, 5, 7, 9, 11,13, 15, 17, 19, 20, 21, or 23, as measured by assays available in the art or described herein (e.g., glucan synthase activity and / or protein folding activity). For example, the substantially identical polypeptide can have at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 100%, or at least about 105%, or at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 200% of the activity of the CslF6 and / or IRE1 with the SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, and 23 sequence when measured by similar assay procedures.

[0058] Alternatively, substantial identity is present when second polypeptide is immunologically reactive with antibodies raised against the first polypeptide (e.g., a polypeptide with SEQ ID NOS: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, and 23). Thus, a polypeptide is substantially identical to a first polypeptide, for example, where the two polypeptides differ only by a conservative substitution. In addition, a polypeptide can be substantially identical to a first polypeptide when they differ by a non-conservative change if the epitope that the antibody recognizes is substantially identical. Polypeptides that are "substantially similar" share sequences as noted above except that some residue positions, which are not identical, may differ by conservative amino acid changes.

[0059] The CslF6, Fruct4, Tip1-1, and / or Tip3-1 polypeptides can include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 N-terminal amino acid residues of a the SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, or 23 sequence. Alternatively, the CslF6, Fruct4, Tip1-1, and / or Tip3-1 polypeptides may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 C-terminal amino acid residues of the SEQ ID NO: 1, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, or 23 sequence.

[0060] The specific methods, devices and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0061] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

[0062] Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

[0063] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.

[0064] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description ofthe invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0065] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

What is Claimed:

1. A fusion protein comprising a vacuolar targeting polypeptide linked to a cellulose synthase like (Csl) protein, wherein the vacuolar targeting polypeptide has an activity of localizing the fusion protein to a tonoplast.

2. The fusion protein of claim 1, wherein the vacuolar targeting polypeptide is a tonoplast intrinsic protein (TIP) protein.

3. The fusion protein of claim 1, wherein the TIP protein comprises a sequence with an amino acid sequence of at least 95% sequence identity to SEQ ID NOS: 15, 17, 19, 20, 21, or 23.

4. The fusion protein of claim 1, wherein the vacuolar targeting polypeptide is a fruc4 vacuole targeting domain.

5. The fusion protein of claim 4, wherein the fruc4 protein comprises a sequence with an amino acid sequence of at least 95% sequence identity to SEQ ID NO:

13.

6. The fusion protein of claim 1, wherein the Csl protein comprises CslF6.

7. The fusion protein of claim 6, wherein the CslF6 protein comprises a sequence with an amino acid sequence of at least 95% sequence identity to SEQ ID NOS: 1, 5, 7, 9, or 11.

8. The fusion protein of claim 1, wherein the vacuolar targeting polypeptide is linked to an N-terminus of the Csl protein.

9. An expression system comprising at least one expression vector comprising a first nucleic acid segment encoding a vacuolar targeting polypeptide linked in-frame with at least one second nucleic acid segment encoding a Csl protein to form a fusion protein, wherein the vacuolar targeting polypeptide has an activity of localizing the fusion protein to a tonoplast.

10. The expression system of claim 9, wherein the vacuolar targeting polypeptide is a tonoplast intrinsic protein (TIP) protein.

11. The expression system of claim 10, wherein the TIP protein comprises a sequence with an amino acid sequence of at least 95% sequence identity to SEQ ID NOS: 15, 17, 19, 20, 21, or 23.

12. The expression system of claim 9, wherein the vacuolar targeting polypeptide is a fruc4 vacuole targeting domain.

13. The expression system of claim 9, wherein the Csl protein comprises CslF6.

14. The expression system of claim 13, wherein the CslF6 protein comprises a sequence with an amino acid sequence of at least 95% sequence identity to SEQ ID NOS: 1, 5, 7, 9, or 11.

15. The expression system of claim 9, wherein the vacuolar targeting polypeptide is linked to an N-terminus of the Csl protein.

16. The expression system of claim 9, wherein the expression vector further comprises a heterologous promotor operably linked to the first nucleic acid segment encoding the vacuolar targeting polypeptide linked in-frame with the at least one second nucleic acid segment encoding the Csl protein.

17. A plant cell, plant seed, or plant comprising the expression system of claim 9.

18. The plant cell, plant seed, or plant of claim 17, wherein the plant cell, plant seed, or plant are selected from the group comprising Brachypodium distachyon, wheat, barely, corn, rice, or sorghum.

19. A method comprising growing a plant seed or plant comprising an expression system comprising a heterologous promoter operably linked to a nucleic acid segment encoding a vacuolar targeting polypeptide linked in-frame with a nucleic acid segment encoding a Csl protein to form a fusion protein, to thereby produce a mature plant.

20. The method of claim 19, further comprising harvesting biomass from the mature plant.

21. The method of claim 20, further comprising isolating glucan, oligosaccharides, disaccharides, monosaccharides, or a combination thereof from the biomass.

22. The method of claim 19, wherein the heterologous promoter is an inducible, or tissue specific promoter.

23. The method of claim 19, wherein the heterologous promoter is selected from a CaMV 35S promoter, CaMV 19S promoter, nos promoter, Adh1 promoter, sucrose synthase promoter, α-tubulin promoter, ubiquitin promoter, actin promoter, cab promoter, PEPCase promoter, R gene complex promoter, poplar xylem-specific secondary cell wall specific cellulose synthase 8 promoter, cauliflower mosaic virus promoter, Z10 promoter from a gene encoding a 10 kDa zein protein, Z27 promoter from a gene encoding a 27 kDa zein protein, pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)) and the actin promoter from rice promoter, or phaseolin promoter.

24. The method of claim 19, wherein the plant seed or plant are selected from the group comprising Brachypodium distachyon, wheat, barely, corn, rice, or sorghum.

25. A plant cell, plant seed, or plant comprising an expression system comprising a heterologous promoter operably linked to the first nucleic acid segment encoding a Csl protein, wherein the plant cell, plant seed, or plant is Sorghum bicolor.

26. The plant cell, plant seed, or plant of claim 25, wherein the Csl protein is CslF6.

27. The plant cell, plant seed, or plant of claim 26, wherein the CslF6 protein comprises a sequence with an amino acid sequence of at least 95% sequence identity to SEQ ID NOS: 1, 5, 7, 9, or 11.

28. A method comprising growing a plant seed or plant comprising an expression system comprising a heterologous promoter operably linked to a nucleic acid segment encoding a Csl protein to thereby produce a mature plant, wherein the plant cell, plant seed, or plant is Sorghum bicolor.

29. The method of claim 28, further comprising harvesting biomass from the mature plant.

30. The method of claim 29, further comprising isolating glucan, oligosaccharides, disaccharides, monosaccharides, or a combination thereof from the biomass.

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