WO2017190128A1 - Procédés de modulation du transport des sucres dans les plantes au niveau du phloème - Google Patents

Procédés de modulation du transport des sucres dans les plantes au niveau du phloème Download PDF

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WO2017190128A1
WO2017190128A1 PCT/US2017/030370 US2017030370W WO2017190128A1 WO 2017190128 A1 WO2017190128 A1 WO 2017190128A1 US 2017030370 W US2017030370 W US 2017030370W WO 2017190128 A1 WO2017190128 A1 WO 2017190128A1
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promoter
plant
nucleic acid
plant cell
sugar
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Wolf B. Frommer
Peter WITTICH
Davide SOSSO
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Carnegie Institution Of Washington
Syngenta Participations Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • Cereal seeds are the fundamental sources of food, feed, and fuel.
  • the large cereal seeds are composed mainly of sugars that accumulate in the endosperm as starch, in the embryo and that form the cell walls in all tissues of the seed.
  • the imported sugars serve as a source of energy for the developing seeds, which cannot sustain itself by photosynthesis.
  • Many other products of the seed also rely on the energy and carbon skeletons provided by the imported carbohydrates.
  • Crop yield depends critically on the delivery of photoassimilates from leaves.
  • Transporters for sucrose translocation have been identified, namely SUT1, which loads the sieve element companion cell complex with sucrose and SWEETs, which export sucrose from phloem parenchyma to provide sucrose for phloem loading by SUT 1 (Riesmeier, J.W., Willmitzer, L., and Frommer, W. B. (1992). The EMBO Journal 11, 4705--4713). More recently, a second class of transporters, the SWEETs, were discovered (Chen et al., 2010 Nature 468, 527-532), and the evidence for phloem loading roles was obtained in Arabidopsis (Chen et al., 2012 Science Jan 13;335(6065):207-11).
  • the transporters are key targets of regulation and the access to the respective genes provides a handle to get an understanding of the regulation and possibly a tool for increasing crop yield.
  • the work was predominantly done in dicots, although maize SUT1 (a homolog of the dicot SUT2) was recently shown to be essential for plant growth and likely responsible for one step in phloem loading in maize (Braun a nd Slewinski, 2009 Pla nt
  • compositions and methods are provided for increasing the levels of at least one sugar in a plant cell or plant part.
  • Plants are provided having a first heterologous nucleic acid sequence encoding a SWEET13 protein operably linked to a promoter active in a plant cell. Further provided are expression constructs for expression of SWEET13 in specific tissues of the plant. Based on promoter selection, SWEET13 can be used to increase transport of sugar at the source in the leaves, or at the sugar sink in the seeds.
  • SWEET13 in the leaves in order to facilitate phloem loading and concurrently in the seed in order to increase sugar unloading and seed filling, overall sugar transport can be increased in an effort to increase total yield.
  • FIG. 1 shows that SWEET13 allows HEK293T cells to accumulate sucrose, as recorded by FRET ratio changes using a FRET sucrose sensor.
  • FIGS 2 A, 2B and 2C show that SWEET13a allows HEK293T cells to accumulate sucrose, as recorded by FRET ratio changes using a FRET sucrose sensor (FUPsuc-90u-deltal V).
  • FIGS 3A, 3B and 3C show that SWEET13b allows HEK293T cells to accumulate sucrose, as recorded by FRET ratio changes using a FRET sucrose sensor (FUPsuc-90u-deltal V).
  • FIGS 4A, 4B and 4C show that SWEET13c allows HEK293T cells to accumulate sucrose, as recorded by FRET ratio changes using a FRET sucrose sensor (FUPsuc-90u-deltal V).
  • Figure 5 shows mutant corn plants deficient in SWEET13 expression demonstrate stunted growth and a dwarf phenotype.
  • Figure 6 shows an alignment of the promoter regions of ZmSWEET13a (maize).
  • ZmSWEET13b (maize), ZmSWEET13c (maize), and OsSWEET13 (rice). Highlighted regions show high identity between the sequences.
  • Figures 7A, 7B and 7C show an alignment of the promoter regions of ZmSWEET13a (maize), ZmSWEET13b (maize), ZmSWEET13c (maize), and OsSWEET13 (rice). Highlighted regions show high identity between the sequences. See also Figure 6.
  • FIGS 8A, 8B and 8C show OsSWEET13 leaf localization and cytology.
  • B GUS activity in transgenic rice flag leaf blade carrying a
  • prOsSWEET13:OsSWEET13-GUS construct Expression is delimited to minor and major veins.
  • C Cross section of a rice leaf blade carrying previous GUS construct. Leaf was stained 30min for GUS, fixed, embedded in paraffin and then sectioned. GUS presence in only detectable within phloem cells, xy, xylem; bs, bundle sheath.
  • Figures 9A, 9B, 9C, 9D and 9E show phenotypes characterization of ossweetl3 mutants.
  • Figure 9A Photograph of fully mature wild-type and same age C ISP /Cas9 osswetl3-l and -2, showing no detectable morphological differences.
  • Figure 9C Total soluble sugar measurements of wild-type and ossweetl3-l and -2 in rice flag leaf.
  • Figures 10A, 10B and IOC show SWEET13 molecular phylogenetic analysis.
  • Figure 10A Maximum Likelihood phylogenic tree of SWEET13 proteins collected from different species: A. thaliana (At), O. sativa (Os), H. vulgare (Hv), B. distachyon (Bd), S. italica (Si), S. bicolor (Sb) and Z. mays (Zm).
  • AtSWEET13 has been used as Eudicots out-group reference. Bootstrap values are out 1000 replicates.
  • Figure 10B For each protein in Figure 10A its relative gene model is presented; black boxes are exons.
  • Figure IOC Schematic representation of the potential SWEET13 duplication process in grasses. Chronogram branch divergence time-points were defined in Wu and Ge, 2012.
  • Figures 11A, 11B and 1C show ZmSWEET13s leaf localization and sucrose transport.
  • Figures 12A, 12B, 12C, 12D, 12E and 12F show morphological and physiological phenotypes of triple mutant zmsweetl3aabbcc.
  • Figure 12A Photograph of fully mature Wild-type and same age C ISP /Cas9 triple mutant.
  • Figures 12B and 12C Leaves comparison of Figure 12A plants, showing reduced growth and chlorosis in triple mutant leaves.
  • Figure 12E Relative expression (by qRT-PCR) of ZmSWEET13s in maize flag leaf from wild-type and zmsweetl3aabbcc.
  • Figures 13A, 13B, 13C and 13D show starch accumulation in wild-type
  • FIG. 12A Wild-type and triple mutant flag leaves were harvested at dawn (7:00 am) and starch was stained using a saturated IKI solution. At this time most starch has been degraded into soluble sugars in wild-type leaves. In contrast, zmsweetl3aabbcc shows higher amount of starch.
  • Figure 12B Total starch quantification of wild-type and triple mutant flag leaves harvested at 5:00 pm.
  • Figures 12C and 12D Cross sections of wild-type and triple mutants leaves harvested at dawn, fixed and embedded in LR White resin and stained with IKI for starch presence. zmsweetl3aabbcc accumulates high amounts of starch in both mesophyll and bundle sheath cells.
  • Embodiments of the present disclosure may take many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided to describe the disclosed methods and products so as to provide for making and using the disclosed methods and products.
  • compositions and methods are provided for modulating the transport of sugars in plants.
  • the compositions provided herein take advantage of the specificity of SWEET13 proteins and promoters in order to enhance the ability of sugar transporters to direct the movement of sugars away from the source of the sugar in the leaf and toward the sink of the sugar in the seed.
  • transgenic plants and plant cells are provided that express a SWEET13 protein in leaf cells so as to move sugar away from the leaf.
  • transgenic plants and plant cells are provided that express a SWEET13 protein in seed cells and tissues so as to move more sugar from the vasculature and into the seeds.
  • sugar transport can be engineered to reduce or remove bottlenecks in the sugar transport process in an effort to increase yield.
  • recombinant DNA constructs are also provided for expression of the SWEET13 protein in plant tissues of interest.
  • Transgenic plants and plant cells are provided in which a SWE ET13 protein is expressed in order to increase sugar transport at the sugar source in the leaves or at the sugar sink at the seeds.
  • a “transgenic plant” or “transgenic plant cell” refers to any plant in which one or more, or all, of the cells of the plant include a heterologous nucleic acid sequence.
  • a transgenic plant or transgenic plant cell may comprise a transgene integrated within a nuclear genome or organelle genome, or may comprise extra-chromosomally replicating DNA.
  • transgene refers to a nucleic acid that is partly or entirely heterologous, foreign, to a transgenic plant or plant cell into which it is introduced, or a nucleic acid that is present in the plant or plant cell in a genomic or extra-chromosomal position different from that in which the gene is found in nature.
  • heterologous in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • the transgenic plants and plant cells provided herein express a heterologous SWEET13 protein in order to increase the transfer of sugars through the vasculature of the plant.
  • vascular or “vasculature” refers to the tissue system for the transport of fluids and nutrients in plants, including the xylem and phloem.
  • sugars can be more efficiently transported to the seeds (e.g., sugar sink).
  • Leaf tissue cells included within the leaf tissue including the vascular cells are xylem, phloem, bundle sheath, mesophyll, pharenchyma (e.g., phloem pharenchyma), sieve-tube element, companion cells, plasmodesmata, and intermediary cells, among others.
  • pharenchyma e.g., phloem pharenchyma
  • sieve-tube element e.g., phloem pharenchyma
  • companion cells plasmodesmata
  • intermediary cells e.g., plasmodesmata
  • SWEET13 protein can also be used to unload sugars from the vascular tissues and into the seed. If an excess of sugar is provided from sugar source cells in the leaf into the vascular tissues such that more sugar arrives at the seed than can properly be transported into the seed, expression of SWEET13 in seed tissues can help unload more sugar from the vasculature and into the seeds.
  • Cells included within the seed are the pedicel, placenta-chalaza, BETL (Basal Endosperm Transport Layer), ESR (Embryo Surrounding Region), embryo, endosperm, pericarp, and aleurone, among others.
  • seed-specific proteins or “seed-specific” expression refers to proteins or expression that occurs specifically or preferentially in cells or tissues of the seed.
  • Transfer cell-specific proteins refers to proteins that are specifically or preferentially expressed in transfer cells or transfer cell layers. Thus the expression of transfer cell- specific proteins occurs in a greater amount in transfer cells or transfer layers when compared to other cells or layers in the plant. Transfer cells are specialized parenchyma cells that facilitate the transfer of sugars from a sugar source to a sugar sink. In some embodiments, transfer cell-specific proteins are not expressed in any cell type other than transfer cells (TCs). Transfer cells irons-differentiate from existing cell types by developing extensive wall ingrowths. The resulting increase in plasma membrane surface area enables increased densities of membrane transporters to optimize nutrient transport across apoplasmic/symplasmic boundaries at sites where TCs form.
  • Transfer cells can differentiate at many plant exchange surfaces, including phloem loading and unloading zones, such as those present in the sink organs and seeds.Thus, transfer cell-specific proteins can be expressed in transfer cells specific for transport in source (e.g., leaves) and sink
  • transfer cell specific proteins can be expressed in any area of the vascular network responsible for transport of nutrients, such as the loading areas of minor leaf veins, areas surrounding the vascular bundle at stem nodes, points of glandular secretion, and places of delivery of nutrients at sink organs, such as the base of flowers and fruits.
  • a transfer cell-specific protein in a plant cell, the transport of nutrients can be increased.
  • expressing a transfer cell-specific protein at the interface between the filial and maternal tissues of the seed beyond the expression that might exist in the wild type can facilitate nutrient uptake from the apoplasmic space in the placenta-chalaza area and increase the nutrients in the resulting seed beyond that which would be present in the seed without such heterologous expression of the transfer cell-specific protein.
  • the Basal Endosperm Transfer Layer (BETL) area is highly specialized to facilitate uptake of solutes during grain development. These transfer cells of the basal endosperm have specialized internal structures adapted to absorb solutes from the maternal pedicel tissue and apoplasmic space, and translocate these products to the developing endosperm and embryo.
  • BETL genes can be expressed between 8 to 20 days after pollination (DAP).
  • the transfer cell-specific protein is a protein expressed in the BETL. Proteins specifically expressed in the BETL include, but are not limited to, M RP1, BETL-1, BETL-2, Meg-1, and TCRR-1.
  • M RP-1 is a transfer cell-specific transcriptional activator containing a MYB-related DNA binding domain identified in several DNA binding proteins belonging to the SHAQK(Y/F)F subfamily.
  • M RP1 Myb- Related Protein-1
  • M RP1 can regulate the expression of transfer cell-specific genes, through its interaction with a specific sequence in the corresponding promoters.
  • the transfer cell-specific protein described herein is M RP1.
  • the transfer cell-specific protein is an invertase.
  • carbohydrates are distributed through the vasculature of the plant to the sink organs in the form of sucrose, the end product of photosynthesis in source organs.
  • sucrose can be cleaved, by invertase or sucrose synthase, to monomers that are used to synthesize carbohydrate polymers (e.g., starch), which are the storage forms of photosynthesis products.
  • invertases include by are not limited to Incw2 (i.e., Mnl) and Ivrl.
  • the invertases disclosed herein include both cell wall invertases and neutral invertases.
  • Examples of cell wall invertases include: GRMZM 2G095725 _P01, GRMZM 2G095725 _P02, GRMZM 2G095725 _P03, GRMZM2G463871_P01, GRMZM2G139300_P01, GRMZM2G139300_P02, GRMZM 2G018692_P01, GRMZM2G018716_P01, GRMZM2G089836_P01, GRMZM2G089836_P02, GRMZM 2G119689_P01, GRMZM 2G119689_P02, GRMZM2G119689_P03, GRMZM 2G123633_P01, GRMZM 2G394450_P01, GRMZM 2G174249.. P01, GRMZM2G1174249.. P02,
  • GRMZM 2G174249_P03 GRMZM 2G174249_P04, GRMZM2G174249_P05, GRMZM2G174249_P06, GRMZM 2G119941_P01, as listed in the Maize Genomics Database.
  • Examples of cell wall invertases include: GRMZM 2G040843_P01, GRMZM2G040843_P02, GRMZM2G040843 _P03,
  • GRMZM 2G022782_P01 GRMZM2G022782 _P02, GRMZM2G136139 _P01,
  • heterologous nucleic acid encoding a transfer cell-specific protein such as Mnl
  • SWEET13 in the source tissues of the leaf can increase transport of sugar into the vascular system, but the increased amount of sugar may be unable to be unloaded with wild- type expression of sugar transporters at the sugar sink in seed tissues.
  • a heterologous sugar transporter in seed tissues increased sugar provided by heterologous expression of SWEET13 in the leaves can ultimately be unloaded into the seed.
  • Sugar transport into the endosperm can be facilitated by expression of a heterologous sugar transporter.
  • the sugar transporter can be specific for glucose, specific for hexose, specific for fructose, or specific for sucrose.
  • the sugar transporter can be a sucrose or hexose uniporter.
  • a hexose uniporter is, as the name implies, a transporter protein that transports hexose sugars, e.g., cyclic hexoses, aldohexoses and ketohexoses.
  • sucrose or hexose uniporters that may be utilized in the methods, constructs, plants, and plant seeds described herein include but are not limited to glucose uniporters and fructose uniporters.
  • the sugar transporter is SWEET13.
  • SWEET13a At least 3 related SWEET13s were identified in maize and labeled SWEET13a, SWEET13b, and SWEET13c.
  • the maize SWE ET13s are close homologs of the two Arabidopsis genes, SWEET11 and SWEET12, which play a role in phloem loading in dicots.
  • Phylogenetic studies revealed that other grasses show different paralogy, with rice having only a single SWEET13 ortholog. On the contrary, maize and sorghum have triplicated this specific SWEET13, possibly to enhance sugar efflux from the leaves.
  • SWEET13a, b and c have been determined to function as sucrose transporters, just as other clade I II members from other species had been shown to predominantly function as sucrose transporters (Chen et al., 2012).
  • SWEET13 participates in sucrose efflux (unloading) from bundlesheath/mesophyll or phloem parenchyma, after which the sucrose is imported into the phloem sieve element/companion cell complex.
  • sucrose is imported into the phloem sieve element/companion cell complex by SUT1.
  • SWEETll is another clade III SWEET protein that plays a key role in the efflux of sugars from maternal tissues for seed filling. Specifically, maize SWEETll participates in kernel filling. Two paralogs of SWEETll were identified in maize and labeled as SWEETlla and SWEETllb.
  • the ScHXTl transporter can be used in the transgenic plants and expression constructs described herein.
  • the SWEET4c e.g., ZmSWEET4c
  • glucose transporter can be used in the transgenic plants, plant cells, and expression constructs described herein.
  • the SWEET proteins in general, belong to the PFAM family "MtN3_slv" (Accession No. PF03083).
  • the sugar transporter proteins utilized in the methods, plants, and plant parts disclosed herein are uniporters, which is a well-known term in the art that means a protein that facilitates transport through facilitated diffusion, i.e., the molecules being transported are being transported with the solute gradient. Uniporters do not typically utilize energy for movement of the molecules they transport, other than harnessing the solute gradient.
  • SWEET proteins are well-known in the art, and their primary amino acid structures can be found in a variety of databases including but not limited to plant membrane protein databases such as aramemnon.botanik.uni-koeln.de, C. elegans protein databases such as
  • SWEETs have a characteristic modular structure that is different from other sugar transporters.
  • SWEETs have a different three-dimensional structure from lac permease, yeast hexose transporters, human GLUTs, or human SGLTs.
  • the basic unit of a SWEET transporter is a domain composed of three transmembrane domains (TMs).
  • TMs transmembrane domains
  • proteins with 3 TMs have to form at least one dimer to create a sugar transporting pore.
  • the eukaryotic versions of the SWEET proteins contain a repeat of this subunit, which is separated by an additional TM domain.
  • This additiona l TM domain (“TM4") is not conserved a mongst fa mily mem bers, thus the specific a mino acid sequence of this domain is not critica l to proper functioning across the kingdom of SWEET proteins.
  • This additiona l TM4 domain serves as a n inversion linker that puts the two repeat units of 3 TMs into a pa ra l lel configuration, which is how the dimer is formed with the bacteria l protein.
  • This 7 TM structure is unique from a l l other known suga r tra nsporters. That the a nima l versions of these SWEET proteins as wel l as bacteria l proteins from this sa me fa mily all transport sugars is indicative that the plant version of these SWEET proteins sugar transporters.
  • SWEET transporter superfamily are defined both by conserved amino acid sequences and structural features. For example, all SWEETs are composed of 7 TM divided in two conserved MtN3/saliva motifs embedded in the tandem 3 TM repeat unit, which is connected by a central TM helix that is less conserved, indicating that this central TM serves as a linker. Structures of SWEET genes are known and have been described previously (Feng, L, and Frommer, W., (2016) Trends Biochem Sci. Feb;41(2): 118-9 and Tao Y., et al. Nature. 2015 Nov 12;527(7577):259-63). The resulting structure has been described as the 3-1-3 TM SWEET structure.
  • the first TM domain on average is predicted to be composed of 23 amino acids, but could vary between 20 and 25. Within this TM domain there are at least 4 highly conserved amino acids: G, P, T and F.
  • the second TM domain on average is predicted to be composed of 19 amino acids, but could vary between 16 and 23. Within this TM domain there are at least 3 highly conserved amino acids: P, Y and Y.
  • the third TM domain on average is predicted to be composed of 23 amino acids, but could vary between 20 and 25. Within this TM domain there are at least 3 highly conserved amino acids: T, N and G.
  • the fifth TM domain on average is predicted to be composed of 23 amino acids, but could vary between 20 and 25. Within this TM domain there are at least 3 highly conserved amino acids: G, P and L.
  • the fifth loop, linking together TM 5 and 6, has 2 highly conserved amino acids: V and T.
  • the sixth TM domain on average is predicted to be composed of 23 amino acids, but could vary between 19 and 25. Within this TM domain there are at least 7 highly conserved amino acids: S, V, M, P, L, S and Y.
  • the sixth loop, linking together TM 6 and 7, has a highly conserved amino acid: D.
  • the seventh TM domain on average is predicted to be composed of 23 amino acids, but could vary between 20 and 25. Within this TM domain there are at least 5 highly conserved amino acids: P, N, G, Q. and Y.
  • SWEETs from a particular species of plant can be categorized into clades, or groups, based on amino acid sequence similarity.
  • clades or groups, based on amino acid sequence similarity.
  • SWEET proteins based on sequence similarity within each clade.
  • Clade I in Zea mays contains SWEETS la, lb, 2, 3a and 3b;
  • Clade II contains SWEETs 4a, 4b, 4c, 6a and 6b;
  • Clade III contains SWEETs 11, 12a, 12b, 13a, 13b, 13c, 14a, 14b, 15a, and 15b;
  • Clade IV contains SWEETs 16, 17a, and 17b.
  • the number of the specific SWEET protein in maize is used to reflect the
  • SWEETs e.g., SWEET11 in maize is most closely related, by sequence comparison, to SWEET 11 in Arabidopsis, and smaller letters are used to indicate a possible gene amplification relative to Arabidopsis.
  • SWEET transporter proteins used in methods, constructs, plants, and plant parts disclosed herein are SWEET proteins from crops plants, such as a cereal crops, food crops, feed crops or biofuels crops.
  • crops plants such as a cereal crops, food crops, feed crops or biofuels crops.
  • Exemplary important crops may include corn, wheat, soybean, cotton and rice.
  • Crops also include corn, wheat, barley, triticale, soybean, cotton, millet, sorghum, sugarcane, sugar beet, potato, tomato, grapevine, citrus (orange, lemon, grapefruit, etc), lettuce, alfalfa, common bean, fava bean and strawberries, sunflowers and rapeseed, cassava, miscanthus and switchgrass.
  • plants include but are not limited to an African daisy, African violet, alfalfa, almond, anemone, apple, apricot, asparagus, avocado, azalea, banana and plantain, beet, bellflower, black walnut, bleeding heart, butterfly flower, cacao, caneberries, canola, carnation, carrot, cassava, chickpea, cineraria, citrus, coconut palm, coffee, common bean, maize, cotton, crucifers, cucurbit, cyclamen, dahlia, date palm, douglas-fir, elm, English walnut, flax, Acanthaceae, Agavaceae, Araceae, Araliaceae, Araucariacea, Asclepiadaceae, Bignoniaceae, Bromeliaceae, Cactaceae, Commelinaceae, Euphobiaceae,
  • Gentianaceae Gesneriaceae, Maranthaceae, Moraceae, Palmae, Piperaceae, Polypodiaceae, Urticaceae, Vitaceae, fuchsia, geranium, grape, hazelnut, hemp, holiday cacti, hop, hydrangea, impatiens, Jerusalem cherry, kalanchoe, lettuce, lentil, lisianthus, mango, mimulus, monkey-flower, mint, mustard, oats, papaya, pea, peach and nectarine, peanut, pear, pearl millet, pecan, pepper, Persian violet, pigeonpea, pineapple, pistachio, pocketbook plant, poinsettia, potato, primula, red clover, rhododendron, rice, rose, rye, safflower, sapphire flower, spinach, strawberry, sugarcane, sunflower, sweetgum, sweet potato, sycamore, tea, tobacco, tomato, verbena, and wild
  • the sugar transporter is a SWEET protein from Zea mays.
  • SWEET proteins include but are not limited to ZmSweetla-GRMZM2G039365, ZmSweetlb- GRMZM2G 153358, ZmSweetZ-
  • GRMZM2G324903 ZmSweet3a-GRMZM2G179679, ZmSweet3b-GRMZM2G060974, ZmSweet4a-
  • GRMZM2G157675 ZmSweet6b-GRMZM2G416965, ZmSweetll-GRMZM2G368827, ZmSweetl2a-
  • GRMZM2G133322 ZmSweetl2b-GRMZM2G099609, ZmSweetl3a-GRMZM2G173669,
  • GRMZM2G094955 ZmSweetl4b-GRMZM2G015976, ZmSweetl5a-GRMZM2G168365, ZmSweetl5b-GRMZM5G872392, ZmSweetl6-GRMZM2G107597, ZmSweetl7a-GRMZM2G106462, ZmSweetl7b-GRMZM2G 111926. Accession numbers following the gene name, e.g.,
  • GRMZM2G039365 refer accession numbers from the Maize Genetics and Genomics database at www.maizegdb.org.
  • Sucrose transporters are polynucleotides that encode a class of sucrose/H+ symporters that facilitate transport of sucrose across plant membranes in various plant tissues.
  • Sucrose transporters are present in many plants including, but not limited to, maize, spinach, potato, tomato, pea, Arabidopsis, celery, grape, tobacco, Lotus, broad bean, and rice (for a review see Kuhn, C. (2003) Plant biol. 5:215-232; Allen et al. U.S. Pat. No. 7,288,645).
  • the terms "sucrose transporter” and SUT are used interchangeably herein. Three subfamilies of SUTs are known in plants.
  • the SUT1 subfamily is defined as high affinity, low capacity transporters; the SUT2 subfamily is defined as low affinity or very low affinity, high capacity transporters; and, the SUT4 subfamily is defined as medium or low affinity, high capacity transporters (Rosche et al. (2002) The Plant Journal 30(2): 165-175; Kuhn, C. (2003) Plant biol. 5:215-232; Sauer, N. (2007) FEBS Letters 581:2309-2317; Lalonde et al. (2004) Ann. Rev. Plant Biol. 55:341-372).
  • the SUT1 subfamily of high affinity transporters is not relevant to this invention.
  • AtSUT2 is a member of the SUT2 group (Schulze et al.
  • the SUT4 group of which AtSUT4 is a member, are low affinity transporters which are expressed in sink tissues and may function in phloem loading within source tissues (Weise et al. (2000) The Plant Cell 12:1345-1355). Active variants and fragments of sucrose transporters will retain the ability to transport sucrose.
  • the hexose uniporter, SWEET4c from Zea mays i.e., ZmSWEET4c
  • ZmSWEET4c was previously classified as ZmSWEET4d in the art. See, for example, W02014/149845, herein incorporated by reference in the entirety.
  • the polynucleotide encoding SWEET4c comprises at least one intron, or portion thereof, of SWEET4c (e.g., ZmSWEET4c).
  • SWEET4c can comprise the first intron, of SWEET4c.
  • the polynucleotide encoding SWEET4c can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 introns or any portion of introns present in the polynucleotide.
  • intron is any nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA product of a gene is being generated. The term refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts.
  • the sugar transporter can be synthetically constructed using portions of other known sugar transporters in order to optimize the transport of sugar in a plant cell.
  • portions of two or more SWEET proteins can be combined in order to alter the activity of the sugar transporter to be specific for the individual plant, cell, or substrate being used.
  • chimeric sugar transporter proteins comprising of two or more different sugar transporters can be used in the methods and compositions disclosed herein.
  • Any plant species can comprise the heterologous nucleic acids SWEET13, or a homolog or paralog thereof as described herein, including, but not limited to, monocots and dicots.
  • Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miiiaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Heiianthus annuus), safflower (Carthamus tinctorius), wheat ( Triticum aestivum), soybean (Glycine max), tobacco (Nicotian a tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Mani
  • pineapple Ananas comosus
  • citrus trees citrus trees
  • cocoa Theobroma cacao
  • tea Anamellia sinensis
  • banana Ananas comosus
  • banana Ananas comosus
  • avocado Persea americana
  • fig Ficus casica
  • guava Psidium guajava
  • mango Manifera indica
  • olive Olea europaea
  • papaya Carica papaya
  • cashew Anacardium occidentale
  • macadamia Macadamia integrifolia
  • almond Panus amygdalus
  • sugar beets Beta vulgaris
  • sugarcane Sane
  • poplar Populus spp.
  • eucalyptus Eucalyptus spp.
  • oats Avena sativa
  • barley Haordeum vulgare
  • plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the heterologous nucleic acids disclosed herein.
  • a “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of the subject plant or plant cell.
  • a control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which does not express the SWEET13 protein and sugar transporter as described herein); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; or (d) the subject plant or plant cell itself, under conditions in which heterologous nucleic acids encoding a SWEET13 are not expressed.
  • compositions further include recombinant DNA constructs for expression of the
  • heterologous nucleic acids e.g., the heterologous nucleic acid sequences or heterologous nucleic acid molecules
  • Expression constructs can express a single heterologous nucleic acid or multiple nucleic acids.
  • plants disclosed herein can comprise a single expression construct for expression of a single nucleic acid, a single expression construct for expression of multiple nucleic acids, multiple expression constructs each expressing a single nucleic acid, or a combination of expression constructs expressing a single nucleic acid and multiple nucleic acids ⁇ e.g., two, three, four, five, or more nucleic acids).
  • the expression constructs disclosed herein can comprise a promoter operably linked to a nucleic acid sequence encoding a SWEET13 protein.
  • the expression constructs disclosed herein can comprise a promoter operably linked to a nucleic acid sequence encoding a sugar transporter, such as ZmSWEET4c or SUT1.
  • expression constructs can comprise a promoter operably linked to a gene of the C4 assimilation pathway to increase sucrose supply at the source, such as SWEET13.
  • Expression constructs can also comprise a promoter operably linked to nucleic acid sequences encoding proteins that enhance sink strength, such as those that enhance kernel size, kernel number per ear, or sugar transport/starch synthesis.
  • genes for increase in sink strength include, but are not limited to, invertase, genes for gluconeogenesis, and starch biosynthesis, and those genes related to vacuolar accumulation of sugars.
  • an expression construct comprising a promoter operably linked to a nucleic acid sequence encoding a SWEET13 protein and a promoter operably linked to a nucleic acid sequence encoding a sugar transporter or transfer cell-specific protein.
  • Expression constructs can also comprise a promoter operably linked to a nucleic acid sequence encoding a SWEET13 protein, a promoter operably linked to a nucleic acid sequence encoding a sugar transporter, and third promoter operably linked to a third nucleic acid sequence.
  • the third nucleic acid can encode a transfer cell-specific protein, a sugar transporter such as SWEET4c or ScHXTI, an invertase, or any other protein disclosed herein.
  • the promoter is heterologous to the operably linked polynucleotide.
  • a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the
  • the same/analogous species one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
  • the expression cassette can include, in the 5'-3' direction of transcription, a transcriptional initiation region (i.e., a promoter), a translational initiation region, a heterologous nucleic acid sequence, a translational termination region and optionally, a transcriptional termination region functional in the host plant.
  • the regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotide of the embodiments may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the polynucleotide of the embodiments may be heterologous to the host cell or to each other.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence being expressed, the plant host, or any combination thereof).
  • Convenient termination regions are available from the Ti-plasmid of A tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mai. Gen. Genet. 262: 141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev.
  • Compositions further include recombinant DNA constructs or expression constructs that encode a SWEET13 protein and a sugar transporter each operably linked to a promoter functional in a plant cell.
  • Exemplary components of the expression constructs include, for example, nucleic acid sequences encoding a SWEET13 and nucleic acid sequences encoding ZmSWEET4c or SWEET11.
  • encodes or “encoding” refers to a DNA sequence which can be processed to generate an RNA and/or polypeptide.
  • polynucleotide polynucleotide sequence
  • nucleic acid sequence nucleic acid fragment
  • a polynucleotide may be a polymer of RNA or DNA that is single-or double- stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • the use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA.
  • polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides.
  • deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • the polynucleotides provided herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
  • a “recombinant polynucleotide” or “recombinant DNA construct” comprises a combination of two or more chemically linked nucleic acid segments which are not found directly joined in nature. By “directly joined” is intended the two nucleic acid segments are immediately adjacent and joined to one another by a chemical linkage.
  • the recombinant polynucleotide comprises a polynucleotide of interest or active variant or fragment thereof such that an additional chemically linked nucleic acid segment is located either 5', 3' or internal to the polynucleotide of interest.
  • the chemically-linked nucleic acid segment of the recombinant DNA construct can be formed by deletion of a sequence.
  • the additional chemically linked nucleic acid segment or the sequence deleted to join the linked nucleic acid segments can be of any length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater nucleotides.
  • Various methods for making such recombinant polynucleotides are disclosed herein, including, for example, by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques.
  • the recombinant polynucleotide can comprise a recombinant DNA sequence or a recombinant NA sequence.
  • a "fragment of a recombinant polynucleotide" comprises at least one of a combination of two or more chemically linked amino acid segments which are not found directly joined in nature.
  • a recombinant DNA construct comprises a first heterologous nucleic acid sequence encoding a SWEET13 protein operably linked to a first promoter functional in a plant cell.
  • a recombinant DNA construct as disclosed herein can further comprise a heterologous nucleic acid sequence encoding a sugar transporter, such as SWEET4c or SWEET11 operably linked to a heterologous promoter functional in a plant cell.
  • a recombinant DNA construct having a first heterologous nucleic acid sequence encoding a SWEET11 protein operably linked to a first promoter functional in a plant cell and a second heterologous nucleic acid sequence encoding a transfer cell-specific protein, such as M RP1 or Incw2, operably linked to a second heterologous promoter functional in a plant cell.
  • the first and second heterologous nucleic acid sequences are operably linked to the same promoter.
  • a recombinant DNA construct having a first heterologous nucleic acid sequence encoding a SWEET13 protein operably linked to a first promoter functional in a plant cell, a second heterologous nucleic acid sequence encoding a sugar transporter, such as SWEET4c, operably linked to a second heterologous promoter functional in a plant cell, and a third heterologous nucleic acid sequence encoding a transfer cell-specific protein operably linked to a promoter functional in a plant cell.
  • promoters can be used in the various expression constructs provided herein.
  • the promoters can be selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of different promoters in the recombinant expression constructs to modulate the timing, location and/or level of expression of the SWEET13 protein.
  • Such recombinant expression constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • the expression constructs provided herein can be combined with constitutive, tissue-preferred, developmentally-preferred or other promoters for expression in plants.
  • constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'-promoter derived from T- DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRPI-8 promoter and other transcription initiation regions from various plant genes known to those of skill.
  • CaMV cauliflower mosaic virus
  • 1'- or 2'-promoter derived from T- DNA of Agrobacterium tumefaciens
  • the ubiquitin 1 promoter the Smas promoter
  • the cinnamyl alcohol dehydrogenase promoter U.S
  • weak promoter(s) may be used.
  • Weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like.
  • Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608, 149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;
  • inducible promoters examples include the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the ln2-2 promoter which is safener induced (U.S. Pat. No. 5,364,780), the ERE promoter which is estrogen induced, and the Axigl promoter which is auxin induced and tapetum specific but also active in callus (PCT/USOl/22169).
  • promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
  • a "tissue specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, promoters from homologous or closely related plant species can be preferable to use to achieve efficient and reliable expression of transgenes in particular tissues.
  • the expression comprises a tissue-preferred promoter.
  • tissue preferred is a promoter that initiates transcription mostly, but not necessarily entirely or solely in certain tissues.
  • the expression construct comprises a cell type specific promoter.
  • a "cell type specific” promoter is a promoter that primarily drives expression in certain cell types in one or more organs, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells.
  • the expression construct can also include cell type preferred promoters.
  • a "cell type preferred” promoter is a promoter that primarily drives expression mostly, but not necessarily entirely or solely in certain cell types in one or more organs, for example, BETL cells, vascular cells in roots, leaves, stalk cells, and stem cells.
  • leaf preferred and leaf specific promoters include the PepC, ZmNADPME, ZmSWEET13a, ZmSWEET13b, and ZmSWEET13c promoter.
  • the expression constructs described herein can also comprise seed-preferred or seed- specific promoters.
  • the seed-preferred or seed-specific promoters have expression in embryo sac, early embryo, early endosperm, aleurone, pedicel, placenta-chalaza, and/or basal endosperm transfer cell layer (BETL).
  • the promoter operably linked to the polynucleotide encoding a transfer cell-specific protein is the BETL1 promoter, BETL2 promoter.. M Nl promoter, ZmSWEET4c promoter, any BETL-specific or BETL-preferred promoter, a drought inducible promoter.
  • Promoters can be used from any of the members of the MADS gene family, as described in W02005102034.
  • the OsMADS6 promoter or OsMADS13 promoter can be used to direct expression of SWEET13, or any other protein, to the kernel.
  • the expression constructs (i.e., recombinant DNA constructs) comprise a ZmSWEET13 promoter operably linked to a polynucleotide encoding a SWEET13 polypeptide.
  • the expression constructs disclosed herein may comprise an OsMADS6 promoter operably linked to a polynucleotide encoding a SWEET13 polypeptide, such as SWEET13a or SWEET13c. Introduction of this construct into a plant should increase sugar unloading and transport to the kernel.
  • an expression cassette comprising the OsMADS6 promoter operably linked to SWEET13a is introduced into a plant cell along with an expression cassette comprising an OsMADS6 promoter operably linked to ZmSWEET14b for maintaining sugar transport into the kernel, even during drought stress.
  • the expression constructs disclosed herein may comprise a ZmM RPl promoter operably linked to a polynucleotide encoding a MRP1 polypeptide, a construct comprising a ZmNADPE promoter operably linked to a polynucleotide encoding ZmSWEET13a and a ZmSWEET4c promoter operably linked to a polypeptide encoding Zmlncw2.
  • the expression constructs described herein may comprise a OsMADS6 promoter operably linked to SWEET13c for introduction into a plant to facilitate unloading of sugar at the kernel.
  • seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A. et al. (1986) Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21):6426; and Kloesgen, R. B. et al. (1986) Mol. Gen. Genet. 203:237-244.
  • Promoters that express in the embryo, pericarp, and endosperm are disclosed in U.S. Pat. No. 6,225,529 and PCT publication WO 00/12733. The disclosures for each of these are incorporated herein by reference in their entirety.
  • Promoters that can drive gene expression in a plant seed- preferred manner with expression in the embryo sac, early embryo, early endosperm, aleurone and/or basal endosperm transfer cell layer (BETL) can be used in the compositions and methods disclosed herein.
  • Such promoters include, but are not limited to, promoters that are naturally linked to Zea mays early endosperm 5 gene, lea mays early endosperm 1 gene, Zea mays early endosperm 2 gene, GRMZM2G124663, GRMZM2G006585, GRMZM2G120008, GRMZM 2G157806, GRMZM2G176390, GRMZM2G472234, GRMZM2G138727, Zea mays CLAVATA1, Zea mays MRP1, Oryza sativa PR602, Oryza sativa PR9a, Zea mays BETL-1, Zea mays BETL-2, Zea mays BETL-3, Zea mays BETL
  • Promoters specific for expression in the stem parenchyma can be used to direct sugars for vacuolar storage in the stem. Accordingly, plants such as sugarcane and sweet sorghum could benefit from such a targeted expression of SWEET13 to the stem provided by stem-specific or stem-preferred promoters.
  • stem-specific and stem-preferred promoters include, for example, the SHOM2 promoter (US20130247252) or any other stem-specific or stem-preferred promoter known in the art. Many promoters have been described that are phloem-specific to a greater or lesser degree. Among these, several have been reasonably well-characterized.
  • sucrose transporters that drive expression of sucrose transporters are highly active in source leaf phloem since these proteins are involved in phloem loading (Stadler et al., "Phloem Loading by the PmSUC2 Sucrose Carrier from Plantago major Occurs into Companion Cells," Plant Cell 7:1545- 1554 (1995).
  • sucrose symport activity is widespread, perhaps ubiquitous, in plant tissues; as a result, these promoters are active in the phloem of several tissue types.
  • the SUC2 promoter directs beta.
  • sucrose,/H ⁇ sym porter, as well as one HVATPase promoter region indicate expression in major (large) veins and sink tissue as well as in minor veins.
  • Yield for a Plasma Membrane Proton Pump in Phloem Cells of Higher Plants Plant 1. 1:121-128 (1991)
  • Kuhn et a I. " Macromolecular Trafficking Indicated by Localization and Turnover of Sucrose Transporters in Enucleate Sieve Elements” Science 275: 1298-1300 (1997).
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize
  • GST promoter which is activated by hydrophobic electrophilic compounds that are used as pre- emergent herbicides
  • tobacco PR-la promoter which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena ef al. (1991) Proc. Natl. Acad. Sci. USA 88:10421- 10425 and McNellis ef al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and
  • tetracycline-repressible promoters see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229- 237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
  • Tissue-preferred promoters can be utilized to target enhanced expression of an expression construct within a particular plant tissue.
  • Tissue-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata ef al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen ef al. (1997) Mai. Gen Genet. 254(3):337-343; Russell ef al. (1997)
  • Leaf-preferred promoters are known in the art. See, for example, Yamamoto ef al. (1997) Plant J. 12(2):255-265; Kwon ef al. (1994) Plant Physiol. 105:357-67; Yamamoto ef al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor ef al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant M al. Biol.
  • Root-preferred promoters are known and can be selected from the many available from the literature or isolated de nova from various compatible species. See, for example, Hire ef al. (1992)
  • Plant Mai. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and
  • VfENOD-GRP3 gene promoter Kuster et al. (1995) Plant M ol. Biol. 29(4):759-772
  • rolB promoter Capana et al. (1994) PLant Mo!. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
  • the phaseolin gene (Murai et al. (1983) Science 23:476-482 and Sengopta-Gopalen et al. (1988) PNAS 82:3320-3324.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats and other such well-characterized sequences that may be deleterious to gene expression.
  • the G-C content of the heterologous nucleotide sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary m RNA structures.
  • the expression cassettes may additionally contain 5' leader sequences.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include, without limitation: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison, et al., (1986) Virology 154:9-20);
  • M DMV leader (Maize Dwarf Mosaic Virus); human immunoglobulin heavy-chain binding protein
  • introns such as the maize Ubiquitin intron (Christensen and Quail, (1996) Transgenic Res. 5:213-218; Christensen, et al., (1992) Plant Molecular Biology 18:675-689) or the maize Adhl intron (Kyozuka, et al., (1991) Mol. Gen. Genet. 228:40-48; Kyozuka, et al., (1990) M aydica 35:353-357) and the like, herein incorporated by reference in their entirety.
  • introns such as the maize Ubiquitin intron (Christensen and Quail, (1996) Transgenic Res. 5:213-218; Christensen, et al., (1992) Plant Molecular Biology 18:675-689) or the maize Adhl intron (Kyozuka, et al., (1991) Mol. Gen. Genet. 228:40-48; Kyozuka, et al.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, for example, transitions and transversions may be involved.
  • Reporter genes or selectable marker genes may also be included in the expression cassettes of the present invention.
  • suitable reporter genes known in the art can be found in, for example, Jefferson, et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al., (Kluwer Academic Publishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO J. 9:2517-2522; Kain, et al., (1995) Bio Techniques 19:650-655 and Chiu, et al., (1996) Current Biology 6:325-330, herein incorporated by reference in their entirety.
  • Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance or resistance to herbicides.
  • suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213; Meijer, et al., (1991) Plant Mai. Biol. 16:807-820); hygromycin (Waldron, et al., (1985) Plant M ai. Biol. 5: 103-108 and Zhijian, et al., (1995) Plant Science 108:219-227);
  • streptomycin Jones, et al., (1987) Mai. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131-137); bleomycin (Hille, et al., (1990) Plant Mai. Biol. 7: 171- 176); sulfonamide (Guerineau, et al., (1990) Plant Mai. Biol.
  • polynucleotides that could be employed on the expression cassettes disclosed herein include, but are not limited to, examples such as GUS (beta-glucuronidase; Jefferson, (1987) Plant
  • the expression cassette can include an additional polynucleotide encoding an agronomically important trait, such as a plant hormone, plant defense protein, a nutrient transport protein, a biotic association protein, a desirable input trait, a desirable output trait, a stress resistance gene, a disease/pathogen resistance gene, a male sterility, a
  • Agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Patent Nos. 5,703,049, 5.885,801, 5.885.802, and 5,990.389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Patent No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson et ai. (1987) Eur. J. Biochem. 165:99-106, the disclosures of which are herein incorporated by reference.
  • Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide.
  • the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S.
  • Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like.
  • Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;
  • Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Patent No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78: 1089); and the like.
  • Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene);
  • ALS acetolactate synthase
  • glyphosate e.g. the EPSPS gene and the GAT gene; see., for example, U.S. Publication No.
  • the bar gene encodes resistance to the herbicide basta
  • the nptll gene encodes resistance to the antibiotics kanamycin and geneticin
  • the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.
  • Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Patent No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
  • Exogenous products include plant enzymes and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like.
  • the level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
  • Additional polypeptides that can be encoded by a polynucleotide on the expression cassettes disclosed herein can include, for example, polypeptides such as various site specific recombinases and systems employing the same.
  • polynucleotides can include various meganucleases to target polynucleotides are set forth in WO 2009/114321 (herein incorporated by reference), which describes "custom" meganucleases. See, also, Gao et al. (2010) Plant Journal 1: 176-187. Additional sequence of interest that can be employed, include but are not limited to ZnFingers, meganucleases, and, TAL nucleases. See, for example, W02010079430, W02011072246, and US20110201118, each of which is herein incorporated by reference in their entirety.
  • vector refers to a DNA molecule such as a plasmid, cosmid or bacterial phage for introducing a nucleotide construct, for example, an expression cassette, into a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.
  • SWEET13s can be expressed at the source of sugar production in the leaves in order to increase sugar transport from the source in to the vascular tissues (e.g., phloem) for transport to the seed.
  • the SWEET13s can be expressed with other sugar transporters, such as SUT1 to enhance source sugar transport, or can be expressed with C4 assimilation pathway genes to increase sucrose supply.
  • SWEET13s can also be expressed in the seeds in order to increase the sink strength to increase kernel size, increase kernel number per ear, or to increase starch synthesis.
  • SWEET13 can be expressed in vascular tissues in the stem (e.g., phloem tissue) in order to increase sugar transport and storage in vacuoles of stem parenchyma cells.
  • SWEET13s operably linked to a leaf-specific or leaf-preferred promoter to enhance source strength can be stacked with constructs that enhance sink strength.
  • sugar is well known in the art and is used to mean a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide or polysaccharide.
  • the sugar or sugars measured may or may not be modified, such as being acetylated.
  • the sugars that are increased are selected from the groups consisting of sucrose, fructose, glucose, mannose and galactose.
  • the sugars that are increased may or may not be part of more complex compounds, such as trisaccharides, e.g., raff i nose, tetrasaccharides, e.g., stachyose or polysaccharides, e.g., amylose, amylopectin.
  • trisaccharides e.g., raff i nose
  • tetrasaccharides e.g., stachyose
  • polysaccharides e.g., amylose, amylopectin.
  • the compositions and methods disclosed herein are not limited to the identity of the specific sugars that are increased in the seeds and plants of the present invention.
  • the sugar transporters of the present invention predominantly transport hexoses, such as but not limited to, glucose, mannose, fructose and galactose, as well as disaccharides, such as but not limited to, sucrose, lactose, maltose, trehalose, cellobiose into the developing seed.
  • hexoses such as but not limited to, glucose, mannose, fructose and galactose
  • disaccharides such as but not limited to, sucrose, lactose, maltose, trehalose, cellobiose into the developing seed.
  • the seed may utilize these increased hexoses and/or disaccharides to then form more complex sugars.
  • sugars that may be contained (increased) in the seed or developing seed include, but are not limited to, disaccharides, trisaccharides, e.g., raff i nose, tetrasaccharides, e.g., stachyose or polysaccharides, e.g., amylose, amylopectin.
  • an "increase" in the level of a sugar can be any statistically significant increase, such as at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 80%.. 100%, 150%, 200%, 250%, 300%, 500% or more increase in the level of sugar when compared to an appropriate control.
  • an "increase in glucose,” for example, is used herein to mean that the levels of glucose are increased over controls, regardless of whether the glucose is free glucose, i.e., occurs as a monosaccharide, or if the glucose subunit is part of a more complex compound, such as but not limited to disaccharides, trisaccharides, tetrasaccharides, or even polysaccharides.
  • an "increase in glucose” is used herein to mean that the levels of glucose are increased over controls, regardless of whether the glucose is free glucose, i.e., occurs as a monosaccharide, or if the glucose subunit is part of a more complex compound, such as but not limited to disaccharides, trisaccharides, tetrasaccharides, or even polysaccharides.
  • fructose is used herein to mean that the levels of fructose are increased over controls, regardless of whether the fructose is free fructose, i.e., occurs as a monosaccharide, or if the fructose subunit is part of a more complex compound, such as but not limited to disaccharides, trisaccharides, tetrasaccharides, or even polysaccharides.
  • an "increase in sucrose,” for exam ple, is used herein to mean that the levels of sucrose are increased over controls, regardless of whether the sucrose is free sucrose, i.e., occurs as a disaccharide, or if the fructose is part of a more complex compound, such as but not limited to trisaccharides, tetrasaccharides, or even polysaccharides.
  • the building blocks of di-, tri-, tetra- and polysaccharides are well known, and that methods are well established for analyzing sugar content in seeds, e.g., Hirst, E. L, et al., Biochem. J., 95:453-458 (1965), Steadman, K., et al., Ann. Botany,
  • methods of assessing or measuring levels of sugar and/or starch content in seeds include but are not limited to HPLC, NM and mass spectroscopy.
  • the phase "increase in the levels at least one sugar,” or “increase at least one sugar,” or some derivation thereof, means an increase in the levels of at least one specific, measured sugar in the seed or developing seed, the phloem, any vascular cell or tissue, or the leaf, as compared to the level of the same sugar in the control cell or tissue, even if levels of another sugar in the cell or tissue may decrease or remain static.
  • more than one specific, measured sugar may be increased as compared to control cell or tissue.
  • the phrase "increase in the levels of at least one sugar” refers to an increase in at least one of at least, glucose, mannose, fructose, galactose, sucrose, lactose, maltose, trehalose or cellobiose into the seed or developing seed.
  • the phrase "increase in the levels of at least one sugar” means an increase in at least two of. glucose, mannose, fructose, galactose, sucrose, lactose, maltose, trehalose or cellobiose into the seed or developing seed, the phloem, any vascular cell or tissue, or the leaf.
  • the phrase "increase in the levels of at least one sugar” means an increase in at least three of, glucose, mannose, fructose, galactose, sucrose, lactose, maltose, trehalose or cellobiose into the seed or developing seed the phloem, any vascular cell or tissue, or the leaf.
  • at least one sugar is increased in cells of the stem by expression of SWEET13, such as in sweet sorghum or sugarcane. Increases in sugar of this nature can increase yield in plants harvested for sugar stored in the stems.
  • the levels of sugar in both control and transgenic seeds can be assessed in a seed or developing seed.
  • the levels of sugar are measured in seeds or developing seeds from transgenic and non-transgenic plants when the seeds or developing seeds are at roughly the same stage of development.
  • the levels of sugar are measured in seeds or developing seeds from transgenic and non-transgenic plants at the zygote stage of seed development, the pre-globular stage of seed development, the globular stage of seed development, the transition stage of seed development, the heart stage of seed development, the torpedo stage of seed development, the linear cotyledon stage of seed development, the bending cotyledon stage of seed development, or the maturation green stage of seed development.
  • levels of sugar in seeds from transgenic plants are considered as "increased" over levels of sugar in seeds from non-transgenic plants if levels are higher in at least one of these stages of seed development.
  • the method and compositions disclosed herein can be used to increase phloem loading and transport of sugar through the phloem.
  • introduction of SWEET13 operably linked to a leaf-preferred or leaf-specific promoter can increase the amount of sugar transported through the phloem or accumulating in the leaf.
  • SWEET13 can be expressed in the seed (i.e., kernel) in order to increase transport of sugar from the phloem into the seed. Methods for measuring phloem transport and unloading are known in the art.
  • a kinetic model of cellular carbohydrate metabolism has been described that could be applied to growing maize endosperm or embryo cells (Alonso, A.P., Val, D. L, and Shachar-Hill, Y. (2011. Metabolic Engineering 13: 96-107).
  • the model includes sugar uptake, hydrolysis by invertase and sucrose synthase, utilization of resulting hexoses for sucrose re-synthesis, starch and cell wall synthesis, and catabolism.
  • the enzyme parameters obtained give insights into regulation by metabolic control analysis, and the model predicts the effects of changes in transport and enzymatic rates. Contributions of central metabolism were obtained from 13C-steady state flux analysis, allowing the dynamic model to focus on flux of sugars and their derivatives. Transport and metabolic fluxes into and within kernels will be analyzed by measuring transfer rates from the cob, phenotyping flux differences in mutants and transgenics and by spatio-temporal modeling of transport and metabolism.
  • the methods provided herein comprise introducing into a plant cell, plant, or seed a first heterologous nucleic acid sequence encoding a SWEET13 protein and, optionally, a second heterologous nucleic acid sequence encoding another sugar transporter (e.g., SWEET4c).
  • the first and second heterologous nucleic acid sequences are introduced to the plant cell on the same polynucleotide construct.
  • the first and second heterologous nucleic acid sequences are introduced into the plant cell on different polynucleotide constructs.
  • the methods provided herein do not depend on a particular method for introducing a sequence into the host cell , only that the polynucleotide gains access to the interior of a least one cell of the host.
  • Methods for introducing polynucleotides into host cells are known in the art and include, but are not limited to, stable transformation methods, transient
  • introducing and “introduced” are intended to mean providing a nucleic acid
  • nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient
  • nucleic acid e.g., a recombinant expression construct
  • introduction in the context of inserting a nucleic acid (e.g., a recombinant expression construct) into a cell , means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be
  • “Stable transformation” is intended to mean that the nucleotide construct introduced into a host (i.e., a plant) integrates into the genome of the plant and is capable of being inherited by the progeny thereof.
  • “Transient transformation” is intended to mean that a polynucleotide is introduced into the host (i.e., a plant) and expressed temporally.
  • Transformation protocols as well as protocols for introducing polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al.
  • the recombinant expression constructs disclosed herein can be provided to a plant using a variety of transient transformation methods.
  • transient transformation methods include, but are not limited to, the introduction of the recombinant expression constructs directly into the plant.
  • Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference.
  • the polynucleotides can be transiently transformed into the plant using techniques known in the art.
  • techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA.
  • the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced.
  • methods include the use of particles coated with polyethylimine (PEI; Sigma #P3 143).
  • recombinant expression constructs disclosed herein may be introduced into plants by contacting plants with a virus or viral nucleic acids.
  • such methods involve incorporating a nucleotide construct provided herein within a viral DNA or RNA molecule.
  • Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.
  • Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome.
  • the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, W099/25821, W099/25854, W099/25840, W099./25855, and W099./25853, all of which are herein incorporated by reference.
  • the recombinant expression constructs can be contained in a transfer cassette flanked by two non-identical recombination sites.
  • the transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette.
  • An appropriate recombinase is provided and the transfer cassette is integrated at the target site.
  • the recombinant expression construct is thereby integrated at a specific chromosomal position in the plant genome.
  • any method can be used to introduce the nucleic acids and expression cassettes disclosed herein into a plant or plant cell.
  • precise genome-editing technologies can be used to introduce the expression cassettes disclosed herein into the plant genome.
  • a nucleic acid sequence will be inserted proximal to a native plant sequence encoding the transporter protein of interest through the use of methods available in the art.
  • Such methods include, but are not limited to, meganucleases designed against the plant genomic sequence of interest (D'Halluin et al. 2013 Plant Biotechnol J 11: 933-941); CRISPR-Cas9, TALENs, and other technologies for precise editing of genomes (Feng, et al.
  • the cells that have been transformed may be grown into plants in accordance with conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, transformed seed (also referred to as "transgenic seed”) having a recombinant expression construct disclosed herein, stably incorporated into their genome is provided.
  • Plant cells that have been transformed to have a recombinant expression construct provided herein can be grown into whole plants.
  • the regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84; Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif., (1988).
  • This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated.
  • the resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
  • the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
  • the compositions presented herein provide transformed seed (also referred to as "transgenic seed") having a polynucleotide provided herein, for example, a recombinant miRNA expression construct, stably incorporated into their genome.
  • a transgenic plant cell comprising at least a first heterologous nucleic acid sequence, wherein the first heterologous nucleic acid sequence encodes the amino acid sequence of a SWEET13 protein, or a homolog or paralog thereof, and wherein the first heterologous nucleic acid sequence is operably linked to a first promoter functional in the plant cell.
  • SWEET13 protein is SWEET13a, SWEET13b, or SWEET13c or a homolog or paralog thereof.
  • transgenic plant cell of embodiment 3, wherein the first promoter is the OsMADS6 promoter or the ZmSWEET4c promoter.
  • transgenic plant cell of embodiment 5 wherein the promoter is the PepC promoter, the ZmNADPME promoter, or the ZmSWEET13a promoter.
  • the plant cell further comprises a second heterologous nucleic acid sequence operably linked to a second promoter functional in the plant cell.
  • transgenic plant cell of embodiment 8 wherein the transfer cell-specific protein is Myb Related Protein 1 (M RP1).
  • M RP1 Myb Related Protein 1
  • transgenic plant cell of embodiment 8 wherein the transfer cell-specific protein is an invertase.
  • transgenic plant cell of embodiment 18, wherein the cereal grain plant comprises maize, rice, wheat, rye, oats, sorghum, millet, or barley.
  • a plant comprising the plant cell of any one of claims 1-20.
  • a transgenic seed comprising the plant cell of any one of embodiments 1-20.
  • a recombinant DNA construct comprising a polynucleotide encoding a SWEET13 polypeptide operably linked to a heterologous promoter.
  • leaf-specific promoter is selected from the group consisting of: the PepC promoter, the Zm NADPM E promoter, or the ZmSWEET13a promoter.
  • a method of increasing the levels of at least one sugar in developing seeds in a plant comprising introducing into a plant cell at least a first heterologous nucleic acid molecule encoding the amino acid sequence of a SWEET13 protein, or a paralog or homolog thereof,
  • first heterologous nucleic acid is operably linked to a first promoter functional in the plant cell
  • the levels of at least one sugar are increased following expression of the SWEET13 protein, or paralog or homolog thereof, in the developing seed in the plant as compared to a control plant that does not contain the first heterologous nucleic acid sequence.
  • SWEET13 protein is SWEET13a, SWEET13b, or SWEET13c or a homolog or paralog thereof.
  • SWEET protein is SWEET4c or SWEET 11, or a homolog or paralog thereof.
  • a method of producing a transgenic plant that produces seeds having increased levels of at least one sugar comprising
  • SWEET13 protein is SWEET13a, SWEET13b, or SWEET13c or a homolog or paralog thereof.
  • hexose sugar comprises glucose and/or fructose.
  • SWEET13a The maize genome was searched bioinformatically for SWEET homologs and a set of three closely related SWEETs (SWEET13a, SWEET13b, and SWEET13c) were identified to be most highly expressed in leaf veins (Li-Qing Chem, et al., Nature (2010) Nov 25; 468(7323): 527-532).
  • SWEET13a Three closely related SWEETs
  • SWEET13s are close homologs of the two Arabidopsis genes SWEET11 and 12, which play a role in phloem loading in dicots. Phylogenic studies revealed that other grasses show different paralogy: rice and Brachypodium have only a single SWEET13 ortholog, while Setaria has duplicated
  • SWEET13s On the contrary, maize and sorghum have triplicated this specific SWEET13, possibly to enhance sugar efflux from the leaves. In accordance with the phylogenetic studies, it was hypothesized that SWEET13a, b and c might function as sucrose transporters as they fall into
  • SWEET clade III SWEET clade III. Clade III members from other species had been shown to predominantly function as sucrose transporters (Chen et al., 2012). Our functional studies, using a combination of mammalian cells (HEK293T) co-transfected with our sucrose FRET sensor FLIPsuc90mAlV), demonstrated that SWEET13 a, b and c can all transport sucrose (Fig. 1 and Figs. 2-5).
  • This 3rd exon is predicted, after translation, to encode for the 2nd and 3rd transmem brane domains (out of the seven that usually constitute a SWEET protein), so out-of frame mutations and even small deletions will create non-functional transporters. More than 200 TO events were obtained, regenerated from embryonic calli.. and 100 events were selected to be brought to Tl. This new generation was used to cross out the Cas9 construct, in order to get stable alleles. Among these, we grew 50 events and found two single, three double and ten triple mutants in various states of homozygocity as verified by PCR and sequencing across the target site.
  • GRMZM 2G173669 nucleic acid (SEQ ID NO:2); ZmSWEET13b-GRMZM2G021706 - amino acid
  • GRMZM 2G179349 - amino acid SEQ I D NO:5
  • CC3 ⁇ 4C CCeS3 ⁇ 4Ci3 ⁇ 4SC « ⁇ CCCSACCTGACC&CGCOTS ⁇
  • A3 ⁇ 4 3 ⁇ 4SSSC "A3 ⁇ 4OmA3 ⁇ 4SC ⁇ AS IiCCIJ iT CC ⁇
  • TTASC SGGATT&?C CC3 ⁇ 4A3 ⁇ 4 ⁇

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Abstract

La présente invention concerne des compositions et des procédés permettant d'augmenter le taux d'au moins un sucre dans une cellule végétale ou dans une partie de la plante. L'invention concerne des plantes comprenant une première séquence d'acide nucléique hétérologue codant une protéine SWEET13 liée de manière fonctionnelle à un promoteur actif dans une cellule végétale. L'invention concerne en outre des constructions d'expression pour l'expression de SWEET13 dans des tissus spécifiques de la plante. En fonction de la sélection du promoteur, le gène SWEET13 peut être utilisé pour augmenter le transport du sucre au niveau de la source dans les feuilles, ou au niveau du puisard dans les graines. De même, en exprimant le gène SWEET13 dans les feuilles afin de faciliter le chargement du phloème, et dans les graines afin d'augmenter le déchargement du sucre et le remplissage des graines, le transport global du sucre peut être augmenté dans le but d'augmenter le rendement total.
PCT/US2017/030370 2016-04-29 2017-05-01 Procédés de modulation du transport des sucres dans les plantes au niveau du phloème WO2017190128A1 (fr)

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CN113481209A (zh) * 2021-07-21 2021-10-08 吉林农业大学 一种玉米糖转运蛋白ZmSWEET15a及其应用

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CN109548652A (zh) * 2018-12-06 2019-04-02 深圳市仙湖植物园管理处（深圳市园林研究中心） 空气凤梨愈伤组织及其培养方法与应用
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CN113481209A (zh) * 2021-07-21 2021-10-08 吉林农业大学 一种玉米糖转运蛋白ZmSWEET15a及其应用

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