WO2017066200A1

WO2017066200A1 - Methods of modulating seed filling in plants

Info

Publication number: WO2017066200A1
Application number: PCT/US2016/056456
Authority: WO
Inventors: Amarjit Basra; Shib Basu; Wolf B. Frommer; Peter WITTICH
Original assignee: Syngenta Participations Ag; Carnegie Institution Of Washington
Priority date: 2015-10-12
Filing date: 2016-10-11
Publication date: 2017-04-20

Abstract

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 transfer cell-specific protein and a second heterologous nucleic acid sequence encoding a sugar transporter, such as SWEET 4c. Further provided are expression constructs for expression of the transfer cell-specific protein and sugar transporter. Methods are provided for increasing sugar content in a plant or plant cell by introducing into a plant a first heterologous nucleic acid sequence encoding a transfer cell- specific protein operably linked to a promoter functional in a plant and a second heterologous nucleic acid sequence encoding a sugar transporter operably linked to a promoter functional in a plant.

Description

METHODS OF MODULATING SEED FILLING IN PLANTS

FIELD OF THE INVENTION

The invention is drawn to methods and compositions for modulating seed filling in plants.

BACKGROUND

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. Moreover, 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. Early maize farmers subjected common agronomic traits to artificial selection in a process called domestication. In maize, several domestication loci that affect plant architecture, synchronization of growth and flowering, loss of seed dispersal and increasing fruit size have been reported. However, the genetic differences underlying the six to ten fold increase in sugar accumulation in seeds of elite maize compared to seeds of its wild ancestor teosinte (Z. mays ssp. parviglumis) have not been identified.

The seed of cereal grains consists of the embryo and the endosperm, the two products of the double fertilization event. The endosperm originates from the central cells of the embryo sac, which are fertilized by one of the two haploid male gametes, and the embryo originates from the fusion of the second gamete with the oosphere. The endosperm is a complex tissue, made from free-nuclear divisions, followed by cellularisation and the subsequent formation of a range of functional cellular domains. As a nutritive tissue for the embryo during development of the seed, the endosperm is a storage organ in maize seeds and subsequently provides nutrients to the seedling on germination.

The endosperm is commonly divided into four domains. The central area of the endosperm (first endosperm domain) consists of large cells with vacuoles, which store the reserves of starch and proteins (central starchy endosperm where genes involved in starch and in prolamin storage proteins biosynthesis are expressed), whilst the region surrounding the embryo (ESR that corresponds to the second endosperm domain) is distinguished by rather small cells, occupied for the major part by cytoplasm. The ESR may have a role in embryo nutrition or in establishing a physical barrier between the embryo and the endosperm during seed development. The aleurone, which is the outer layer of the endosperm, and accumulates proteins and oil.

Finally, the Basal Endosperm Transfer Layer (BETL that corresponds to the third endosperm domain) 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 translocate these products to the developing endosperm and embryo. These transfer cells facilitate nutrient import into the maize kernel.

SUMMARY

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 transfer cell-specific protein and a second heterologous nucleic acid sequence encoding a sugar transporter, such as SWEET 4c. Further provided are expression constructs for expression of the transfer cell-specific protein and sugar transporter. Methods are provided for increasing sugar content in a plant or plant cell by introducing into a plant a first heterologous nucleic acid sequence encoding a transfer cell-specific protein operably linked to a promoter functional in a plant and a second heterologous nucleic acid sequence encoding a sugar transporter operably linked to a promoter functional in a plant.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 presents a generic model for principal transport steps and pathways for phloem unloading in various tissues. Sucrose arriving at the phloem endings is either moving

symplasmically via plasmodesmata or unloaded (from phloem or after moving symplasmically from post-phloem cells) via a transporter (PT); it can be taken up by sink cells using either a sucrose importer (Ul), or by a hexose importer (U2) after cleavage by an extracellular invertase (Inv-CW). Imported sucrose can be catabolized to hexoses by a cytosolic invertase (Inv-N) or sucrose synthase. Subsequently sucrose or hexoses can be transferred to the vacuole by sucrose or hexose transporters on the tonoplast (VI, V2). The model represents a simplification of the phloem and postphloem unloading pathways.

Figure 2 shows that a zmsweet4c-umul mutant has reduced starch accumulation and seed weight. (A) zmsweet4c-umul homozygous seeds were excised from a segregating ear and weighted in parallel with wild-type seeds from the same ear. Teosinte seeds were collected from field-grown plants and weighted as well, showing a similar weight of the mutant, approximately 8 times smaller than wild-type seeds. (B) Starch measurement of wild-type and zmsweet4c-umul homozygous seeds at 3 developmental time points. At 10DAP the mutant has less starch content that the wild- type (-60% less), but the difference increases dramatically up to 24 DAP when the mutant starch content is as little as -10% of the wild- type.

Figure 3 reports the relative expression of ZmSWEET4c in Z. mays, Z. parviglumis, and the mutant background zmsweet4c-umul from 10 to 24 days after pollination.

Figure 4 shows the failure of the zmsweet4c-umul mutant to develop transfer cell characteristics at ~6 days after pollination.

Figure 5 reports (A) the relative expression of ZmMrpl in the zmsweet4c-umul mutant strain compared to the wild-type strain, (B) the relative expression of ZmSWEET4c induced by glucose fructose, and sucrose, and (C) the proposed model of gene activation and sugar transport in a BETL cell.

Figure 6 reports ZmSWEET4c expression in zmcwi2/mnl and Zmlncw2/Mnl expression in zmsweet4c-umul . Figure 6A shows expression levels of ZmSWEET4c in homozygous

zmincw2/mnl grains in four developmental time points. Apart from 8 DAP, ZmSWEET4c expression seems to be greatly reduced at all time points. Figure 6B shows the relative expression levels of Zmlncw2/Mnl expression during three stages of kernel development. As in the reciprocal experiment Zmlncw2/Mnl expression is strongly reduced in <?mp(empty pericarp) kernels. qRT- PCR values are means of three technical replicates normalized with the 18S gene. Error bars represent the SE. Time scale is in Days After Pollination (DAP).

Figures 7A and 7B show that SWEET4a can participate in seed filling. Three maize strains with homozygous SWEET4a gene inactivations and one heterozygous SWEET4a mutant strain demonstrated decreased kernel size (Figure 7A) and decreased kernel weight (Figure 7B) when compared to the wild-type strain.

Figures 8A and 8B demonstrate that SWEET4a can indirectly regulate SWEET4c expression. Figure 8A shows that the expression of SWEET4a is decreased in each SWEET4a mutant at both 10DAP and 20DAP. Figure 8B reports that SWEET4c expression is also decreased in the SWEET4a mutant strains at both 10DAP and 20DAP.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

/. Overview

Compositions and methods are provided for increasing the sugar content of plant cells and plant seeds. Specifically, the compositions provided herein take advantage of the specificity of transfer cell-specific proteins in order to enhance the ability of sugar transporters to direct the movement of sugars into a plant cell or seed. For example, transgenic plants and plant cells are provided that express a transfer cell-specific protein along with a sugar transporter, such as SWEET 4c. Providing a sugar transporter along with a transfer cell-specific protein can increase the sugar transport across transfer cells and increase the total amount of carbohydrate in a plant cell or seed. Along with plants and plant cells, recombinant DNA constructs are also provided for expression of the transfer cell-specific protein and sugar transporter. //. Plants and Plant Cells

Transgenic plants and plant cells are provided in which a heterologous transfer cell-specific protein is expressed along with a heterologous sugar transporter, such as SWEET 4c. 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. For example, 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. The term "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. As used herein, "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. In specific embodiments, transgenic plants or a transgenic plant cell can comprise a transgene encoding a transfer cell-specific protein and a transgene encoding a sugar transporter.

The transgenic plants and plant cells provided herein express a transfer cell-specific protein along with a sugar transporter, such as SWEET 4c, in order to increase the carbohydrate content of plant cells or seeds. "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 trans- 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 (e.g. , seeds or fruits) cells. Further, 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. By expressing a transfer cell-specific protein in a plant cell, the transport of nutrients can be increased. For example, 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). In specific embodiments, the transfer cell- specific protein is a protein expressed in the BETL. Proteins specifically expressed in the BETL include, but are not limited to, MRPl, BETL-1, BETL-2, Meg-1, and TCRR-1. MRP-1 or MRPl 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. The expression of Myb-Related Protein- 1 (MRP1) can be detected in the basal part of the endosperm as early as 3 days after pollination, when the endosperm coenocyte is still organized into nuclear- cytoplasmic domains, and continues during the development of the TCs. Further, MRP1 can regulate the expression of transfer cell-specific genes, through its interaction with a specific sequence in the corresponding promoters. Thus, in some embodiments, the transfer cell-specific protein described herein is MRP1.

In certain embodiments, the transfer cell-specific protein is an invertase. In many agriculturally important plants, 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. In the sink 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. Examples of 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: GRMZM2G095725_P01, GRMZM2G095725_P02,

GRMZM2G095725_P03, GRMZM2G463871_P01, GRMZM2G139300_P01,

GRMZM2G139300_P02, GRMZM2G018692_P01, GRMZM2G018716_P01,

GRMZM2G089836_P01, GRMZM2G089836_P02, GRMZM2G119689_P01,

GRMZM2G119689_P02, GRMZM2G119689_P03, GRMZM2G123633_P01,

GRMZM2G394450_P01, GRMZM2G174249_P01, GRMZM2G174249_P02,

GRMZM2G174249_P03, GRMZM2G174249_P04, GRMZM2G174249_P05,

GRMZM2G174249_P06, GRMZM2G119941_P01, as listed in the Maize Genomics Database. Examples of cell wall invertases include: GRMZM2G040843_P01, GRMZM2G040843_P02, GRMZM2G040843_P03, GRMZM2G022782_P01, GRMZM2G022782_P02,

GRMZM2G136139_P01, GRMZM2G136139_P02, GRMZM2G136139_P03,

GRMZM2G000829_P01, GRMZM2G007277_P01, GRMZM2G056056_P01,

GRMZM2G056056_P02, GRMZM2G084940_P01, GRMZM2G084940_P02,

GRMZM2G159896_P01, GRMZM2G477236_P03, GRMZM2G170842_P01,

GRMZM2G170842_P02, GRMZM2G118737_P01, GRMZM5G871418_P01,

GRMZM5G871418_P02, GRMZM2G084694_P01, GRMZM2G115451_P01,

GRMZM2G115451_P02, GRMZM2G115451_P03 as listed in the Maize Genomics Database. The plants and plant cells described herein can comprise a single heterologous nucleic acid encoding a transfer cell-specific protein, such as Mnl, or can comprise multiple heterologous nucleic acids encoding transfer cell specific proteins, such as Mnl and MRP1.

Sugar transport into the endosperm can be facilitated by a sugar transporter. For example, the sugar transporter can be specific for glucose, specific for hexose, specific for fructose, or specific for sucrose. Specifically, 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. Examples of 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. In some embodiments, the ScHXTl transporter can be used in the transgenic plants and expression constructs described herein. In certain embodiments the SWEET4c (e.g. , ZmSWEET4c) glucose transporter can be used in the transgenic plants, plant cells, and expression constructs described herein. Maize ZmSWEET4c, as opposed to sucrose-transporting homologs, mediates trans-epithelial hexose transport across the BETL. In certain embodiments, the SWEET13a (e.g., ZmSWEET13a) sugar transporter can be used in the transgenic plants, plant cells, and expression cassettes described herein. The SWEET proteins, in general, belong to the PFAM family "MtN3_slv" (Accession No. PF03083). See pfam.sanger.ac.uk, which is a database of protein families that are determined and represented by multiple sequence alignments and hidden Markov models (HMMs). In one embodiment, 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 www.wormbase.org, and even in human transporter databases, such as www.tcdb.org. In general SWEETs have a characteristic modular structure that is different from other sugar transporters. For example, 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). In bacteria, 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 additional TM domain ("TM4") is not conserved amongst family members, thus the specific amino acid sequence of this domain is not critical to proper functioning across the kingdom of SWEET proteins. This additional TM4 domain serves as an inversion linker that puts the two repeat units of 3 TMs into a parallel configuration, which is how the dimer is formed with the bacterial protein. This 7 TM structure is unique from all other known sugar transporters. That the animal versions of these SWEET proteins as well as bacterial proteins from this same family all transport sugars is indicative that the plant version of these SWEET proteins sugar transporters.

Members of the 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. 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.

Both sugar transport and the seven TM three-dimensional structure are the two key features for this superfamily of proteins. Despite the great variability in size or sequence, and despite the broad number of organisms from which they can be isolated, all SWEETs tested using different heterologous systems have shown sugar transport function.

In general, SWEETs from a particular species of plant can be categorized into clades, or groups, based on amino acid sequence similarity. In maize, for example there are four clades of SWEET proteins based on sequence similarity within each clade. For example, 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 phylogenetic relationship to Arabidopsis 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.

In specific embodiments, the 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. 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. Other examples of 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, diseases, 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, mustar, 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 rice.

In specific embodiments, the sugar transporter is a SWEET protein from Zea mays.

Examples of nucleic acid sequences and/or amino acid sequences of the SWEET proteins include but are not limited to ZmSweetla-GRMZM2G039365, ZmSweetlb-GRMZM2G153358,

ZmSweet2-GRMZM2G324903, ZmSweet3a-GRMZM2G179679, ZmSweetSb- GRMZM2G060974, ZmSweet4a-GRMZM2G000812, ZmSweet4b-GRMZM2G144581,

ZmSweet4c-GRMZM2G137954, ZmSweet6a-GRMZM2G157675, ZmSweet6b- GRMZM2G416965 , ZmS weetll-GRMZM2G368827 , ZmS weetl2a-GRMZM2G 133322,

ZmSweetl2b-GRMZM2G099609, ZmSweetl3a-GRMZM2G173669, ZmSweetBb- GRMZM2G021706, ZmSweetl3c-GRMZM2G179349, ZmSweetl4a-GRMZM2G094955, ZmSweetl4b-GRMZM2G015976, ZmSweetl5a-GRMZM2G168365, ZmSweetl5b- GRMZM5G872392, ZmSweetl6-GRMZM2G107597, ZmSweetl7a-GRMZM2G106462,

ZmSweetl7b-GRMZM2Gl 11926. Accession numbers following the gene name, e.g. ,

"GRMZM2G039365," refer accession numbers from the Maize Genetics and Genomics database at www.maizegdb.org.

In specific embodiments, the hexose uniporter, SWEET4c from Zea mays (i.e.,

ZmSWEET4c), is used as the sugar transporter along with a transfer cell-specific protein in the plants, plant parts, constructs, and methods disclosed herein. ZmSWEET4c was previously classified as ZmSWEET4d in the art. See, for example, WO2014/149845, herein incorporated by reference in the entirety. In specific embodiments, the polynucleotide encoding SWEET4c comprises at least one intron, or portion thereof, of SWEET4c (e.g. , ZmSWEET4c). For example, the polynucleotide encoding 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. As used herein, "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. In some embodiments, 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. For example, 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. Accordingly, chimeric sugar transporter proteins comprising 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 encoding a transfer cell- specific protein and sugar transporter 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 miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrijolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oil palm (Elaeis guineensis), poplar (Populus spp.), eucalyptus (Eucalyptus spp.), oats (Avena sativa), barley (Hordeum vulgare), vegetables, ornamentals, and conifers. In specific embodiments, the transgenic plants and plants parts disclosed herein are cereal crops, such as maize (corn), rice (paddy), wheat, barley, and sorghum.

As used herein, the term 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 transfer cell-specific 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 transfer cell-specific protein and sugar transporter are not expressed. Expression Constructs

Compositions further include recombinant DNA constructs for expression of the heterologous nucleic acids (e.g. , the heterologous nucleic acid sequences) disclosed herein.

Expression constructs or expression cassettes can express a single heterologous nucleic acid or multiple nucleic acids. Likewise, 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 transfer cell-specific protein. Likewise, the expression constructs disclosed herein can comprise a promoter operably linked to a nucleic acid sequence encoding a sugar transporter, such as ZmSWEET4c. In specific embodiments an expression construct is provided comprising a promoter operably linked to a nucleic acid sequence encoding a transfer cell-specific protein and a promoter operably linked to a nucleic acid sequence encoding a sugar transporter. Expression constructs can also comprise a promoter operably linked to a nucleic acid sequence encoding a transfer cell-specific protein, a promoter operably linked to a nucleic acid sequence encoding a sugar transporter, and third promoter operably linked to a third nucleic acid. The third nucleic acid can encode a transfer cell-specific protein, a sugar transporter such as SWEET4c, SWEET4a, SWEET4b, or ScHXTl, an invertase, or any other protein disclosed herein.

In some embodiments, the promoter is heterologous to the operably linked polynucleotide. For example, 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

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 or expression construct 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) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al, (1991) Genes Dev. 5:141-149; Mogen, et al, (1990) Plant Cell 2:1261-1272; Munroe, et al, (1990) Gene 91:151-158; Ballas, et al, (1989) Nucleic Acids Res. 17:7891-7903; and Joshi, et al, (1987) Nucleic Acid Res. 15:9627- 9639, herein incorporated by reference in their entirety.

Compositions further include recombinant DNA constructs or expression constructs that encode a transfer cell- specific 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 transfer cell-specific protein and nucleic acid sequences encoding ZmSWEET4c. As used herein, "encodes" or "encoding" refers to a DNA sequence which can be processed to generate an RNA and/or polypeptide.

The terms "polynucleotide," "polynucleotide sequence," "nucleic acid sequence," and "nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. 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. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleo tides. Such 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. In specific embodiments, 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. Alternatively, 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. In specific embodiments, the recombinant polynucleotide can comprise a recombinant DNA sequence or a recombinant RNA 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.

In one embodiment, a recombinant DNA construct comprises a first heterologous nucleic acid sequence encoding a transfer cell-specific 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, operably linked to a heterologous promoter functional in a plant cell. In specific embodiments, a

recombinant DNA construct is provided having a first heterologous nucleic acid sequence encoding a transfer cell- specific protein operably linked to a first promoter functional in a plant cell and 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. In a specific embodiment, the first and second heterologous nucleic acid sequences are operably linked to the same promoter. In a specific embodiment, a recombinant DNA construct is provided having a first heterologous nucleic acid sequence encoding a first transfer cell-specific 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 second transfer cell- specific protein operably linked to a promoter functional in a plant cell.

A number of 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 transfer cell-specific protein or sugar transporter (e.g. , SWEET4c). 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.

In some embodiments, the expression constructs provided herein can be combined with constitutive, tissue-preferred, developmentally-preferred or other promoters for expression in plants. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the Γ- 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 GRP1-8 promoter and other transcription initiation regions from various plant genes known to those of skill. If low level expression is desired, 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;

5,399,680; 5,268,463; and 5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated by reference.

Examples of inducible promoters are 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 In2-2 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 US01/22169).

Examples of 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. In some embodiments, the expression comprise a tissue-preferred promoter. A "tissue preferred" promoter is a promoter that initiates transcription mostly, but not necessarily entirely or solely in certain tissues.

In some embodiments, 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.

The expression constructs described herein can also comprise seed-preferred promoters. In some embodiments, the seed-preferred promoters have expression in embryo sac, early embryo, early endosperm, aleurone, and/or basal endosperm transfer cell layer (BETL). In specific embodiments, the promoter operably linked to the polynucleotide encoding a transfer cell-specific protein is the BETL1 promoter, BETL2 promoter, MN1 promoter, ZmSWEET4c promoter,

ZmSWEET13a promoter, any BETL-specific or BETL-pref erred promoter, a drought inducible promoter.

In certain embodiments, the expression constructs (i.e., recombinant DNA constructs) comprise a ZmSWEET4c promoter operably linked to a polynucleotide encoding a MRP1 polypeptide. The expression constructs disclosed herein may comprise a BETL2 promoter operably linked to a polynucleotide encoding a MRP1 polypeptide. Further, the expression constructs disclosed herein may comprise a BETL2 promoter operably linked to a polynucleotide encoding Mnl or ScHXTl. The expression constructs described herein may comprise a BETL2 promoter operably linked to a polynucleotide encoding a SWEET4c (e.g. , ZmSWEET4c) polypeptide or a MN1 promoter operably linked to a polynucleotide encoding a SWEET4c (e.g. , ZmSWEET4c) polypeptide.

In specific embodiments, the expression cassette comprises a maize BET2 operably linked to a polynucleotide encoding a maize MRP1. The expression cassettes disclosed herein may comprise the native promoter of ZmSWEET4c operably linked to a polynucleotide encoding ZmSWEET4c. The expression cassettes disclosed herein may comprise the native promoter of ZmSWEET4c operably linked to maize cell wall invertase 2 (Incw2). In particular embodiments, the promoter derived from the maize NADP-malic enzyme is operably linked to sugar efflux transporter SWEET13a. The expression cassettes disclosed herein may comprise the native promoter of ZmSWEET4c operably linked to a polynucleotide encoding ZmMRPl.

Examples of 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, Zea mays early endosperm 1 gene, Zea mays early endosperm 2 gene, GRMZM2G 124663,

GRMZM2G006585, GRMZM2G120008, GRMZM2G157806, GRMZM2G176390,

GRMZM2G472234, GRMZM2G138727, Zea mays CLAVATA1, Zea mays MRP1, Oryza sativa PR602, Oryza sativa PR9a, Zea mays BET1, Zea mays BETL-2, Zea mays BETL-3, Zea mays

BETL-4, Zea mays BETL-9, Zea mays BETL- 10, Zea mays MEG1, Zea mays TCCR1, Zea mays ASP1, Oryza sativa ASP1, Triticum durum PR60, Triticum durum PR91, Triticum durum GL7, AT3G10590, AT4G18870, AT4G21080, AT5G23650, AT3G05860, AT5G42910, AT2G26320, AT3G03260, AT5G26630, AtIPT4, AtIPT8, AtLEC2, LFAH12. Additional such promoters are described in U.S. Patent Nos. 7803990, 8049000, 7745697, 7119251, 7964770, 7847160, 7700836, U.S. Patent Application Publication Nos. 20100313301, 20090049571, 20090089897,

20100281569, 20100281570, 20120066795, 20040003427; PCT Publication Nos.

WO/1999/050427, WO/2010/129999, WO/2009/094704, WO/2010/019996 and WO/2010/147825, each of which is herein incorporated by reference in its entirety for all purposes. Functional variants or functional fragments of the promoters described herein can also be operably linked to the nucleic acids disclosed herein.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, 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 In2-2 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, and the 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 et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421- 10425 and McNellis et 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 et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.

(1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.

112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Set USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

Leaf -preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Set USA 90(20):9586- 9590. In addition, the promoters of cab and rubisco can also be used. See, for example, Simpson et al. (1958) EMBO J 4:2723-2729 and Timko et al. (1988) Nature 318:57-58.

Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633- 641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen- fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus

corniculatus , and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed roIC and roID root- inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(l):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J.

8(2):343-350). The TR gene, fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and roIB promoter (Capana et al. (1994) Plant Mol. 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 mRNA structures.

Expression constructs disclosed herein can further include a promoter that terminates expression of a gene of interest. In specific embodiments, the terminators used in the expression constructs and plants described herein can be a BET2 (e.g., maize BETL2) terminator, a

Cauliflower Mosaic virus 35S terminator, a SWEET4c (e.g., maize SWEET 4c) terminator, or a NADP-malic (e.g., maize NADP-malic) enzyme terminator.

Expression constructs (i.e., expression cassettes) can further comprise nucleotide sequences that increase the expression of at least one transfer cell-specific protein or at least one sugar transporter. In some embodiments, the expression constructs described herein comprise a nucleotide sequence encoding a SWEET transporter that increases the expression of a sugar transporter. For example, expression constructs can encode a nucleotide sequence encoding SWEET4a or SWEET4b, wherein expression of SWEET4a or SWEET4b in a plant cell increases endogenous expression of SWEET4c. Similarly, expression constructs can comprise a nucleotide sequence encoding SWEET4c as the sugar transporter and can further comprise a nucleotide sequence encoding SWEET4a or SWEET4b, wherein expression of SWEET4a or SWEET4b in a plant cell increases heterologous expression of SWEET4c. In other embodiments, expression of SWEET4a can be used to increase endogenous expression of a sugar transporter disclosed herein or used to increase heterologous expression of a sugar transporter disclosed herein. In specific embodiments, an expression construct as disclosed herein can comprise a nucleic acid sequence encoding SWEET4c operably linked to a promoter specific for the BETL and a nucleic acid sequence encoding SWEET4a operably linked to a promoter active in a plant cell. Expression cassettes disclosed herein can also comprise a nucleic acid sequence encoding MRP1 or MN1 operably linked to a promoter and a nucleic acid sequence encoding SWEET4a operably linked to a promoter.

The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include, without limitation: picornavirus 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); MDMV leader (Maize Dwarf Mosaic Virus); human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al. , (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al. , (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al. , (1989) Molecular Biology of RNA, pages 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al. , (1991) Virology 81:382-385), herein incorporated by reference in their entirety. See, also, Della-Cioppa, et al. , (1987) Plant Physiology 84:965-968, herein incorporated by reference in its entirety. Methods known to enhance mRNA stability can also be utilized, for example, 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) Maydica 35:353-357) and the like, herein incorporated by reference in their entirety.

In preparing the expression cassette, 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. Toward this end, 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. For this purpose, 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. Examples of 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. Examples of 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 Mol. Biol. 16:807-820); hygromycin (Waldron, et al, (1985) Plant Mol. Biol. 5:103-108 and Zhijian, et al, (1995) Plant Science 108:219-227);

streptomycin (Jones, et al, (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al, (1996) Transgenic Res. 5:131-137); bleomycin (Hille, et al, (1990) Plant Mol. Biol. 7:171- 176); sulfonamide (Guerineau, et al, (1990) Plant Mol. Biol. 15:127-36); bromoxynil (Stalker, et al , (1988) Science 242:419-423); glyphosate (Shaw, et al, (1986) Science 233:478-481 and US Patent Application Serial Numbers 10/004,357 and 10/ '427 ',692); phosphinothricin (DeBlock, et al, (1987) EMBO J. 6:2513-2518), herein incorporated by reference in their entirety.

Other 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 Mol. Biol. Rep. 5:387), GFP (green fluorescence protein; Chalfie, et al , (1994) Science 263:802), luciferase (Riggs, et al, (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen, et al, (1992) Methods Enzymol. 216:397-414) and the maize genes encoding for anthocyanin production

(Ludwig, et al, (1990) Science 247:449), herein incorporated by reference in their entirety.

In still other embodiments, 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 developmental gene, a regulatory gene, a DNA repair gene, a transcriptional regulatory gene or any other polynucleotide and/or polypeptide of interest. In some embodiments, the expression cassette can include additional polynucleotides that downregulate the expression of genes responsible for agronomically important traits. For example, in some embodiments, the expression cassettes disclosed herein can comprise a nucleic acid sequence that downregulates expression of GW2 (grain weight 2). 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 al. (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. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. Application Serial No. 08/740,682, filed November 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Illinois), pp. 497-502; herein incorporated by reference); corn (Pedersen et al. (1986) /. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71 :359; both of which are herein incorporated by reference); and rice (Musumura et al. (1989) Plant Mol. Biol. 12: 123, herein incorporated by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

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; 5,593,881 ; and Geiser et al. (1986) Gene 48: 109); and the like.

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); glyphosate (e.g. , the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03/092360); or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and 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.

The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

Commercial traits can also be encoded on a gene or genes of the expression cassette that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Patent No. 5,602,321. Genes such as β-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) /. Bacteriol. 170:5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).

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. See, for example, W099/25821, W099/25854, WO99/25840, W099/25855, and W099/25853, all of which are herein incorporated by reference. Other 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 7 : 176-187. Additional sequence of interest that can be employed, include but are not limited to ZnFingers, meganucleases, and, TAL nucleases. See, for example, WO2010079430, WO2011072246, and US20110201118, each of which is herein incorporated by reference in their entirety.

As used herein, "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.

In some embodiments, a vector comprises the expression constructs (e.g., expression cassettes) disclosed herein. In specific embodiments a vector can comprise a single expression cassette or multiple expression cassettes. For example, a vector can comprise 1, 2, 3, 4, 5, 6 or more expression cassettes as disclosed herein. In particular embodiments, binary vector are encompassed by the disclosure herein and can comprise 1, 2, 3, 4, 5, 6 or more expression cassettes as disclosed herein. A "binary vector," as used herein refers to a vector that can replicate and express protein in multiple hosts (e.g., (E. coli and Agrobacterium tumefaciens).

IV. Methods of Increasing Sugar Content

Methods are provided for increasing the level of at least one sugar in a plant or plant part by introducing a transfer cell-specific protein and sugar transporter. Any of the various transfer cell-specific proteins or sugar transporters disclosed herein can be used to increase the level of at least one sugar. The terms "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. Specifically, 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. , raffinose, tetrasaccharides, e.g. , stachyose or 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. Indeed, 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. Once inside the seed coat or developing seed coat, however, the seed may utilize these increased hexoses and/or disaccharides to then form more complex sugars. These more complex sugars that may be contained (increased) in the seed or developing seed include but are not limited to disaccharides, trisaccharides, e.g. , raffinose, tetrasaccharides, e.g. , stachyose or polysaccharides, e.g. , amylose, amylopectin.

Thus, 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. Similarly, an "increase in fructose," for example, 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. Similarly, an "increase in sucrose," for example, 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. Given that 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, 77:667-674 (1996), Buckeridge, M.S., Plant Physiol., 154(3): 1017-1023 (2010), all of which are incorporated by reference, one of skill in the art can readily ascertain if there is an increase in the level of sugar in a seed or developing seed compared to control seeds or control developing seeds. In select embodiments, 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.

As used herein, 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, as compared to the level of the same sugar in the control seed or control developing seed, even if levels of another sugar in the seed or developing seed may decrease or remain static. Of course, more than one specific, measured sugar may be increased as compared to control seed or control developing seed. In specific embodiments, 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. In other specific embodiments, 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. In other specific embodiments, 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 levels of sugar in both control and transgenic seeds can be assessed in a seed or developing seed. In one embodiment, 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. For example, in one embodiment, 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. As understood herein, 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 methods provided herein comprise introducing into a plant cell, plant, or seed a first heterologous nucleic acid sequence encoding a transfer cell-specific protein and a second heterologous nucleic acid sequence encoding a sugar transporter (e.g. , SWEET4c). In some embodiments, the first and second heterologous nucleic acid sequences are introduced to the plant cell on the same polynucleotide construct. Alternatively, 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 (i.e. plants) are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

The terms "introducing" and "introduced" are intended to mean providing a nucleic acid (e.g. , a recombinant expression construct) or protein into a cell. Introduced includes reference to the incorporation of a 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

transformation methods, as well as sexually crossing. Thus, "introduced" 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 incorporated into the genome of the cell (e.g. , chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

"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. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend et al. , U.S. Patent No. 5,563,055; Zhao et al. , U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al. , U.S. Patent No. 4,945,050; Tomes et al. , U.S. Patent No. 5,879,918; Tomes et al. , U.S. Patent No. 5,886,244; Bidney et al. , U.S. Patent No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer- Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.

87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et al. , U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via

Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer- Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al. , U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197- 209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

In specific embodiments, the recombinant expression constructs disclosed herein can be provided to a plant using a variety of transient transformation methods. In some embodiments, the recombinant expression constructs disclosed herein are provided to a plant on a vector such as a binary vector. Such 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 Set 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Set 91 : 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotides can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, 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. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).

In other embodiments, recombinant expression constructs disclosed herein may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, 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. In one embodiment, 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, WO99/25840, W099/25855, and W099/25853, all of which are herein incorporated by reference. Briefly, 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. For example, precise genome-editing technologies can be used to introduce the expression cassettes disclosed herein into the plant genome. In this manner, 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. Cell Research 23: 1229-1232, 2013, Podevin, et al. Trends Biotechnology 31: 375-383, 2013, Wei et al. 2013 / Gen Genomics 40 : 281-289, Zhang et al 2013, WO 2013/026740); Cre-lox site-specific recombination (Dale et al. (1995) Plant J 7:649-659; Lyznik, et al. (2007) Transgenic Plant J 1: 1-9; FLP-FRT recombination (Li et al. (2009) Plant Physiol 151 : 1087-1095); Bxbl-mediated integration (Yau et al. Plant J (2011) 701 : 147-166); zinc- finger mediated integration (Wright et al. (2005) Plant J 44:693-705); Cai et al. (2009) Plant Mol Biol 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy (2011) Methods Mol Biol 701: 51-65); Puchta, H. (2002) Plant Mol Biol 48: 173-182).

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. Preferably, 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. In this manner, 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.

Non-limiting embodiments include:

1. A transgenic plant cell comprising at least a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence, wherein the first heterologous nucleic acid sequence encodes the amino acid sequence of a transfer cell-specific protein, and the second heterologous nucleic acid sequence encodes the amino acid sequence of a SWEET4c, wherein the first heterologous nucleic acid is operably linked to a first promoter functional in the plant cell and the second heterologous nucleic acid is operably linked to a second promoter functional in the plant cell.

2. The transgenic plant cell of embodiment 1, wherein the transfer cell-specific protein is Myb Related Protein 1 (MRP1).

3. The transgenic plant cell of embodiment 1, wherein the transfer cell-specific protein is an invertase.

4. The transgenic plant cell of embodiment 3, wherein the invertase is MN1 (invertase cell wall 2).

5. The transgenic plant cell of embodiment 1, wherein the plant cell further comprises a third heterologous nucleic acid sequence operably linked to a third promoter functional in the plant cell. 6. The transgenic plant cell of embodiment 5, wherein the third heterologous nucleic acid encodes the amino acid sequence of a MNl (invertase cell wall 2).

7. The transgenic plant cell of embodiment 5, wherein the third heterologous nucleic acid encodes the amino acid sequence of a sugar transporter.

8. The transgenic plant cell of embodiment 7, wherein the sugar transporter is a

SWEET protein.

9. The transgenic plant cell of embodiment 8, wherein the SWEET protein is

SWEET13a.

10. The transgenic plant cell of any one of embodiments 1-9, wherein the first, the second, and/or the third promoter is selected from a SWEET4c promoter, a MRPl promoter, a

MNl promoter, a Basal Endosperm Transfer Layer (BETL) promoter, or a SWEET13a promoter.

11. The transgenic plant cell of any one of embodiments 1-10, wherein the second promoter is a BETL2 promoter.

12. The transgenic plant cell of any one of embodiments 5-9, wherein the third promoter is a BETL2 promoter.

13. The transgenic plant cell of embodiment 2, wherein the first promoter comprises a MRPl promoter and the second promoter comprises a SWEET4c promoter.

14. The transgenic plant cell of embodiment 2, wherein both the first and the second promoter comprise a SWEET4c promoter.

15. The transgenic plant cell of embodiment 2, wherein the first promoter comprises a

BETL2 promoter and the second promoter comprises a SWEET4c promoter.

16. The transgenic plant cell of embodiment 6, wherein the first promoter comprises a

MRPl promoter, the second promoter comprises a SWEET4c promoter, and the third promoter comprises a MNl promoter.

17. The transgenic plant cell of embodiment 9, wherein the first promoter comprises a

MRPl promoter, the second promoter comprises a SWEET4c promoter, and the third promoter comprises a SWEET13a promoter.

18. The transgenic plant cell of any one of embodiments 1-17, wherein the plant is a cereal grain plant.

19. The transgenic plant cell of embodiment 18, wherein the cereal grain plant comprises maize, rice, wheat, rye, oats, sorghum, millet, or barley.

20. The transgenic plant cell of embodiment 18, wherein the cereal grain plant is maize.

21. A plant comprising the plant cell of any one of embodiments 1-20. 22. The plant of claim 21, wherein said plant has an increased yield as compared to a control plant that does not contain the first, the second or the third heterologous nucleic acid sequence.

23. A transgenic seed comprising the plant cell of any one of embodiments 1-20. 24. A recombinant DNA construct comprising a polynucleotide encoding a MRP1 polypeptide operably linked to a heterologous promoter.

25. The recombinant DNA construct of embodiment 24, wherein the promoter comprises a SWEET4c promoter.

26. The recombinant DNA construct of embodiment 24, wherein the promoter comprises a BETL2 promoter.

27. A recombinant DNA construct comprising a polynucleotide encoding a SWEET4c polypeptide operably linked to a heterologous promoter.

28. The recombinant DNA construct of embodiment 27, wherein the promoter comprises a BETL2 promoter.

29. The recombinant DNA construct of embodiment 25, wherein the promoter comprises a MN1 promoter.

30. The recombinant DNA construct of any one of embodiments 27-29, wherein the polynucleotide further comprises a portion of at least one intron from SWEET4c.

31. The recombinant DNA construct of any one of embodiments 27-29, wherein the polynucleotide further comprises at least one whole intron from SWEET4c.

32. The recombinant DNA construct of any one of embodiments 30-31, wherein the intron comprises the first intron of SWEET4c.

33. A method of increasing the levels of at least one sugar in developing seeds in a plant, the method comprising introducing into a plant cell at least a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence, wherein the first heterologous nucleic acid sequence encodes the amino acid sequence of a transfer cell-specific protein, and the second heterologous nucleic acid sequence encodes the amino acid sequence of a SWEET4c;

wherein the first heterologous nucleic acid is operably linked to a first promoter functional in the plant cell and the second heterologous nucleic acid is operably linked to a second promoter functional in the plant cell; and

wherein the levels of at least one sugar are increased in the developing seed in the plant as compared to a control plant that does not contain the first or the second heterologous nucleic acid sequence. 34. The method of embodiment 33, wherein the transfer cell-specific protein is Myb Related Protein 1 (MRP1).

35. The method of embodiment 33, wherein the transfer cell-specific protein is an invertase.

36. The transgenic plant cell of embodiment 35, wherein the invertase is MN1 (invertase cell wall 2).

37. The method of embodiment 33, further comprising introducing into said plant cell a third heterologous nucleic acid sequence operably linked to a third promoter functional in the plant cell.

38. The method of embodiment 37, wherein the third heterologous nucleic acid sequence encodes the amino acid sequence of MN1.

39. The transgenic plant cell of embodiment 37, wherein the third heterologous nucleic acid encodes the amino acid sequence of a sugar transporter.

40. The method of embodiment 39, wherein the sugar transporter is a SWEET protein. 41. The method of embodiment 39, wherein the sugar transporter is SWEET13a.

42. The method of any one of embodiments 33-41, wherein the first, the second, and/or the third promoter is selected from a SWEET4c promoter, a MRP1 promoter, a MN1 promoter, a Basal Endosperm Transfer Layer 1 (BETL1) promoter, a BETL2 promoter, or a SWEET13a promoter.

43. The method of embodiment 34, wherein the first promoter comprises a MRP1 promoter and the second promoter comprises a SWEET4c promoter.

44. The method of embodiment 34, wherein both the first and the second promoter comprise a SWEET4c promoter.

45. The method of embodiment 34, wherein the first and/or second promoter comprises a BETL2 promoter.

46. The method of embodiment 34, wherein the first promoter comprises a BETL2 promoter and the second promoter comprises a SWEET4c promoter.

47. The method of embodiment 38, wherein the first promoter comprises a MRP1 promoter, the second promoter comprises a SWEET4c promoter and the third promoter comprises a MRP1 promoter.

48. The method of embodiment 41, wherein the first promoter comprises a MRP1 promoter, the second promoter comprises a SWEET4c promoter, and the third promoter comprises a SWEET13a promoter. 49. The method of any one of embodiments 33-48, wherein the plant is a cereal grain plant.

50. The method of embodiment 49, wherein the cereal grain plant comprises maize, rice, wheat, rye, oats, sorghum, millet, or barley.

51. The method of embodiment 50, wherein the cereal grain plant is maize.

52. The method of any one of embodiments 33-51, wherein the at least one sugar comprises a hexose sugar.

53. The method of embodiment 52, wherein the hexose sugar comprises glucose and/or fructose.

54. The method of any one of embodiments 33-53, wherein said plant has an increased yield as compared to a control plant that does not contain the first, the second or the third heterologous nucleic acid sequence.

55. A method of producing a transgenic plant that produces seeds having

increased levels of at least one sugar, the method comprising

(a) transforming a plant cell with at least a first heterologous nucleic acid sequence encoding the amino acid sequence of a transfer cell-specific protein, and a second heterologous nucleic acid sequence encoding the amino acid sequence of a SWEET4c; and

(b) regenerating a transgenic plant from the transformed plant

cell, wherein said plant comprises in its genome the at least first heterologous nucleic acid sequence and second heterologous nucleic acid sequence.

56. The method of embodiment 55, wherein the transfer cell-specific protein is Myb Related Protein 1 (MRP1).

57. The method of embodiment 55, wherein the transfer cell-specific protein is an invertase.

58. The method of embodiment 57, wherein the invertase is MN1 (invertase cell wall 2).

59. The method of embodiment 55, further comprising transforming the plant or plant cell with a third heterologous nucleic acid sequence

60. The method of embodiment 59, wherein the third heterologous nucleic acid sequence encodes the amino acid sequence of MN1.

61. The method of embodiment 59, wherein the third heterologous nucleic acid encodes the amino acid sequence of a sugar transporter.

62. The method of embodiment 61, wherein the sugar transporter is a SWEET protein.

63. The method of embodiment 62, wherein the sugar transporter is SWEET13a. 64. The method of any one of embodiments 55-63, wherein the at least one sugar comprises a hexose sugar.

65. The method of embodiment 64, wherein the hexose sugar comprises glucose and/or fructose.

66. A transgenic plant cell comprising at least a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence, wherein the first heterologous nucleic acid sequence encodes the amino acid sequence of a transfer cell-specific protein, and the second heterologous nucleic acid sequence encodes the amino acid sequence of a sugar transporter, wherein the first heterologous nucleic acid is operably linked to a first promoter functional in the plant cell and the second heterologous nucleic acid is operably linked to a second promoter functional in the plant cell.

67. The transgenic plant cell of embodiment 66, wherein said sugar transporter comprises an amino acid sequence derived from at least 2 different sugar transporters.

68. The transgenic plant cell of embodiment 67, wherein said sugar transporter comprising an amino acid sequence derived from at least 2 different sugar transporters comprises an amino acid sequence derived from at least 2 different SWEET proteins.

69. The transgenic plant cell of embodiment 5, wherein the third heterologous nucleic acid sequence encodes a SWEET transporter that increases expression of a sugar transporter.

70. The transgenic plant cell of embodiment 69, wherein the third heterologous nucleic acid sequence encodes SWEET4a or SWEET4b, and wherein expression of said SWEET4a or

SWEET4b in the plant cell increases expression of SWEET4c.

71. The recombinant DNA construct of any one of embodiments 24-32, further comprising a polynucleotide encoding a SWEET transporter operably linked to a promoter active in a plant cell.

72. The recombinant DNA construct of embodiment 71, wherein expression of said

SWEET transporter in the plant cell increases expression of SWEET4c.

73. The recombinant DNA construct of any one of embodiments 71 or 72, wherein said polynucleotide encoding a SWEET transporter encodes SWEET4a or SWEET4b. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Example 1. Clade III SWEETs in seed filling

Based on the finding that SWEETs act as cellular efflux transporters in phloem parenchyma and nectaries, we hypothesized that they may also contribute to assimilate transfer from maternal seed coat to developing embryo. GFP fusions show that AtSWEETll, 12 and 15 are expressed in seed coat and endosperm. Analysis of the triple knockout mutant showed a severe delay in embryo development and a wrinkled seed phenotype at maturity due to lower starch and lipid content and a smaller embryo (Figure 2C). These findings are exciting because the identification of SWEETs involved in seed coat efflux opens up the possibility of identifying the mechanisms that coordinate the delivery of sucrose from leaves with the needs of the developing seed. Extensive studies of the regulation of post-phloem unloading into seeds by osmotic gradients and sucrose provide guidance for further analysis. A generalized model of phloem unloading is presented in Figure 1. Example 2. Analysis of SWEET4c role in sugar transport

Maize plant material and growth conditions.

The zmsweet4c-umul and zmsweet4c-umu2 alleles were obtained from the UniformMu transposon population: zmsweet4c-umul (UFMu-07993) from the Maize Genetics Cooperation Stock Center, and zmsweet4c-umu2 by Mu-seq profiling of seed mutants from the population. Allelism of zmsweet4c-umul and zmsweet4c-umu2 was confirmed by genetic complementation tests with reciprocal crosses between heterozygous plants. The zmsweet4c-umul mutation was propagated as a heterozygote by transmission through the female and selection for emp kernels. Homozygous seeds were germinated on sterile filter paper with sterile water, 3 days in dark, 30C. Wild-type controls were grown side by side for each experiment either in summer field conditions (Stanford Campus, CA) or in greenhouses under long-day conditions (16 hr day:8 hr night). To further characterize emp mutants, sagittal sections of 15 DAP seed were cut with an utility knife. Seed weight was assessed on seed isolated form segregating ears grown in the summer field. 10 seeds were weighted together on a lab scale (Sartorius Extend), 3 pulls of 10 seeds per ear, for a total of 10 ears.

Genotyping and transcript analysis.

Genomic DNA was extracted from maize seedling leaves using Biosprint96 (Qiagen), accordingly with supplier's directions. Primer specific to ZmSWEET4c sequence flanking the Mutator element was SWT4c-5R while as transposon specific primer we used MuINT19. PCR were performed accordingly with Terra™ PCR Direct Red Dye Premix Protocol (Clontech Laboratories, Inc.) with a Tm of 60C. Total RNA was extracted from wild-type maize, teosinte, and zmsweet4c-umul seeds between 4pm and 6pm using Spectrum™ Plant Total RNA Kit (Sigma). First strand cDNA was synthesized using Quantitech Reverse Transcription Kit following the instruction of the supplier (Qiagen). Primers specific for ZmSWEET4c last exon and 3'UTR (ZmSWT4cqF and ZmSWT4cqR) were used for qRT-PCR to determine expression levels using LightCycler 480 System (Roche). Zml8s and ZmLUG (Zml8sF, Zml8sR and ZmLUGqF, ZmLUGqR) served as reference genes. The 2-AACt method was used for relative quantification.

In Situ Hybridization

The ZmSWEET4c gene was selected for in situ hybridization analysis. The gene was cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The probe made using ^g total DNA (M13F+R PCR product) with SP6 polymerase for antisense and T7 polymerase for sense. The Roche DIG RNA Labeling Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used for in vitro transcription and labeling of antisense and sense RNA probes. In situ hybridizations were done essentially as described (Wang C, et al. (2010) Plant Mol Biol Report 28:438-449).

Caryopses of maize 12 DAP inbred W22 were harvested, fixed, dehydrated, and embedded in Paraplast Plus paraffin (Sherwood Medical Co., St. Louis, MO). A rotary microtome (Microm 325; Carl Zeiss) was used for sectioning paraffin-embedded caryopses.

FRET sucrose and glucose sensors analysis in HEK293T cells

ZmSWEET4c coding sequence of was cloned into the Gateway entry vector pDONR221fl and mixed with destination vectors pcDNA3.2V5 for constructs expressing in HEK293T cells by LR reactions. The analysis was performed as described using both FRET sucrose and glucose sensor instead of a FRET glucose sensor (14-16). Briefly, HEK293T cells were co-transfected with a plasmid carrying ZmSWEET4c CDS and the sucrose sensor FLIPsuc9(^Al V or the glucose sensor FLIPglu60(^D13V (100 ng), using Lipofectamine2000 (Invitrogen) in 6-well plates. For FRET imaging, HEK293T/FLIPsuc90 μ Δ1 V cells were perfused with HBSS medium followed by a pulse of 10 mM sucrose, whereas HEK293T/FLIPglu600μD13V cells were perfused with medium followed by of pulse of 2.5-5-20 mM glucose. A Leica inverted fluorescence microscope DM IRE2 with Quant EM camera was used for imaging with SlideBook 4.2 (Intelligent Imaging Innovations) with 200 msec exposure and time interval 10 sec. FRET analyses were performed as described (16). AtSWEET12 and AtSWEETl were used as positive controls for sucrose and glucose respectively, while empty vector was transfected serving as negative control.

EBY4000 growth complementation assay

ZmSWEET4c coding sequence of was cloned into the Gateway entry vector pDONR221fl and mixed with destination vectors pDRfl-GW for constructs expressing

in yeast by LR reactions. Transformants of the yeast hexose transporter mutant EBY4000 with pDRfl-GW-ZmSWEET4c were first grown on selective synthetic complete (SC) (-Ura) medium containing 2% (vol/vol) maltose (Sigma) as the sole carbon source. Drop tests were used to assess the transformed yeast growth on glucose or fructose 2% solid SD media at 5 concentrations

(OD500 = 1, 10-1, 10-2, 10-3, 10-4). Yeast HXT5 served as positive control, while empty vector was used as negative grown at 30°C for 4d. Maltose medium was used as positive growth medium, while W/O sugars medium was used as negative growth medium. Growth was recorded by scanning the plates on a flatbed scanner.

Plastic embedding and sectioning

Arabidopsis freshly collected seeds were placed into a fixative solution of 0.1M cacodylate buffer with 2% paraformaldehyde and 2% glutaraldehyde. Dehydration followed the fixation step with an increasing ethanol gradient (10-30-50-75-95%). Plastic embedding was performed accordingly with the LR White embedding kit protocol (Electron Microscopy Science). Semi-thin cross sections (1 μιη) were cut by Ultracut (Reichert) and stained with 0.1% (w/v) Safranin O for 30sec, then washed twice with distilled water. All sections were mounted with CytoSeal 60 (EM Science). Endosperm in vitro culture and growth conditions 5 DAP ears were sterilized with a 95% alcohol followed by 5min immersion in 0.6% bleach under the laminar flow hood. A scalpel was used to remove and longitudinally dissect each grain into two halves. Using a small lab scoop the endosperm was removed from the two halves and placed on plastic petri dishes with solid medium. The preparation of media was essentially as described by Cheng (1999)(18): media were supplemented with 1 mg/1 of 2,4-dichlorophenoxyacetic acid (2,4-D). All media were adjusted to pH 5.8 prior to the addition of agar (2.5 g/1) and phytagel (2 g/1). After autoclaving, streptomycin sulfate (10 mg/1) was filter-sterilized into the medium and 3% glucose, 3% fructose, 3% sucrose, 1.5% glucose / 1.5% fructose or no sugars were added separately. After 4 days in the dark at 30 °C endosperms from same treatments were pulled together and RNA was extracted from these samples as described above.

Rice plant material, TALEN nucleases mutations and expression profile.

The japonica rice variety Kitaake was used for TALEN (TAL effector nuclease) mediated mutagenesis in OsSWEET4 (Os02gl9820). The methods of TALEN gene synthesis and rice transgenic for production of ossweet4-l and -2 were as described (Li T., et al. (2014) Methods, 69:9-19.). Genes encoding for TALENs recognizing two opposing sub-sites in exon 3 of

OsSWEET4 were expressed under the CaMV 35S promoter and maize Ubiquitinl promoter respectively in rice embryogenic callus cells. Primary transgenic plants with site-specific mutations (4-bp and 7-bp deletions) were self-pollinated and progeny for homozygous mutations were selected. Plants were grown in either growth chamber or green house under conditions of the temperature 28-30 °C, relative humidity 50-75% and day/night 12/12 hours. Analysis of

OsSWEET4 expression using real time RT-PCR was performed as described (Li T., et al. (2012) Nature Biotechnology 30:390-392).

Starch quantification

Segregating wild type and zmsweet4c-umul seeds were harvested at 10, 17, and 24 DAP.

Seeds were ground to a fine powder with mortar and pestle in liquid nitrogen, and 50 mg of tissue was incubated with 1 ml 70% [v/v] ethanol for 1 h on ice, with frequent vigorous vortexing.

Subsequently, the samples were spun 5 min at 4°C, 13Ό00 g, and the supernatant was removed.

This extraction was repeated twice. The pellet was

subsequently dried in a vacuum concentrator, and resuspended in water. Starch was

quantified accordingly with Starch Assay kit protocol - UV method (Roche). Example 3. Seed filling in domesticated maize depends on SWEET-mediated trans -epithelial hexose transport.

Changes in gene expression are prevalent among several domestication genes. Thus, we compared ZmSWEET4c expression profiles in developing maize and teosinte grains. In maize, ZmSWEET4c expression increases by a factor of 3.4 from 10 to 17 days after pollination (DAP), coincident with the onset of massive sugar import into wild-type seed. In contrast, expression in teosinte averages 75% less than in maize at 17 DAP. In maize, two different regulatory regions, i.e. promoter and first intron of ZmSWEET4c, were altered through selection, possibly leading to changes in expression.

In fact, seeds having zmsweet4c insertional mutations show extreme grain defects, in particular an almost complete loss of the endosperm. The zmsweet4c-umul mutation is caused by a Mutator transposon in the last exon of ZmSWEET4c and behaves as a monogenic recessive trait classified as empty pericarp (emp). This emp phenotype, apparent at ~8 DAP, manifests as collapsed grains at -15 DAP. Similar observations were made for zmsweet4c-umu2, which carries an insertion in exon 3. Mature zmsweet4c-umul grains contained ~10x less starch and weighed ~8x less than wild-type (Fig. 2A and 2B). ZmSWEET4c transcript abundance was greatly reduced in mutant seeds at all measured time -points (Fig. 3). The zmsweet4c-umul mutation also affected embryo size, however less dramatically than the endosperm, suggesting an alternate sugar translocation path. Homozygous mutant embryos were able to germinate and develop into normal- appearing, fertile plants, implying that ZmSWEET4c is specific and essential to seed filling.

Filial tissues, i.e. embryo and endosperm, are symplasmically isolated from the maternal seed coat, thus requiring a set of at least two transporters: one that secretes sugars from the maternal layers and one that takes up sugars into the filial tissues. Recent work has identified several SWEETs as being involved in the efflux of sucrose from maternal tissues (L.Q. Chen et al. , The Plant Cell (2015)). ZmSWEET4c could therefore be responsible for sucrose efflux along the phloem-unloading path, such as release of sucrose at the phloem termini or the placentochalaza. There are additional sites at which sugar transport activity is required. Further, mutation of a cell wall invertase, Zmlncw2/Mnl, also dramatically reduces endosperm size (S. Bihmidine, et al , Front Plant Sci 4, 177 (2013), K. E. Koch, Current Opinion in Plant Biology 7, 235-246 (2004)). Therefore, ZmSWEET4c could function in the transfer of sugars between cells within the endosperm. Analysis of in situ hybridization and RNAseq data indicated that ZmSWEET4c expression was abundant in the basal endosperm transfer layer (BETL) rather than maternal tissues such as phloem termini or placentochalaza. The function of ZmSWEET4c could thus be delineated to three alternative roles: sucrose transfer across the BETL, hexose transfer across the BETL, or transient vacuolar storage within the BETL. Subcellular localization of ZmSWEET4c-eGFP fusions to the plasma membrane supports a role in mono- or disaccharide transport into the BETL cells. The function of ZmSWEET4c could thus be one of three alternative roles: sucrose transfer across the BETL, hexose transfer across the BETL, or transient vacuolar storage within the BETL.

Knowledge of the actual substrate of the transporter should allow us to further specify the role of ZmSWEET4c. Transport specificity was tested by co-expressing ZmSWEET4c with sucrose or glucose FRET sensors in human embryonic kidney HEK293T cell cultures (L.-Q. Chen et al, Science 335, 207-211 (2012)). ZmSWEET4c failed to show detectable sucrose transport activity, but enabled HEK293T the cells to accumulate glucose. The emp phenotype seen in mutants of the BETL-expressed Zmlncw2/Mnl invertase gene (K. E. Koch, Current Opinion in Plant Biology 7, 235-246 (2004)), implicating the presence of unknown glucose and fructose transporters. We demonstrated that ZmSWEET4c is likely responsible for the uptake of glucose into the BETL, leaving the remaining question of how equimolar amounts of fructose are imported into the BETL. Taken together, our findings intimate that ZmSWEET4c does not translocate sucrose but rather transfers cell wall invertase-derived hexoses across the BETL as a necessary prerequisite step for seed filling.

Thus, ZmSWEET4c in maize seeds can be analogous to the glucose uniporter HsGLUT2 in the human intestine, which, at high external glucose and fructose supply, mediates trans-epithelial hexose transport across the intestinal epithelial cell. This work places Zmlncw2/Mnl and

ZmSWEET4c into a linear pathway for sugar cleavage/import/export and implies the existence of additional sucrose efflux transporters (possibly SWEET homologs) at the phloem termini and placentochalaza. Sucrose efflux may be potentially mediated by sucrose transporting SWEET homologs, as has been observed in Arabidopsis (L.-Q. Chen et al, The Plant Cell (2015)).

The amplification of cell surface area in the BETL is likely necessary for sustaining the large flux necessary for seed filling. Interestingly, transfer cell characteristics are regulated by sugars, in particular glucose in legume seeds, and link membrane surface area and flux directly to sugar transport. We also observe a coincidence of transfer cells, sugar transporters and sugar supply. In maize, the BETL characteristics, i.e. vertical cell elongation and cell wall ingrowth, appear at ~6 DAP. Strikingly, at this stage both zmsweet4c-umul (Fig. 4) and zmincw2/mnl mutants fail to develop transfer cell characteristics (W. H. Cheng, et al, The Plant Cell 8, 971-983 (1996)). In maize, BETL cell fate is established early during seed development (~5 DAP) via the transcriptional master regulator ZmMrpl. ZmMrpl expression, and therefore BETL cell fate, is controlled by glucose (G. Hueros et al , Planta 229, 235-247 (2008)). In wild-type seeds, expression of Zmlncw2/Mnl and ZmSWEET4c appears to precede that of ZmMrpl suggesting that ZmSWEET4c-mediated import of glucose (derived from invertase activity) signals activation of the ZmMrpl promoter. Consistent with this model, ZmMrpl expression is strongly reduced in zmsweet4c-umul, a probable cause of failure to establish the BETL (Fig. 5 A). Furthermore, both Zmlncw2/Mnl and ZmSWEET4c may also be regulated by sugar accumulation in the basal endosperm, as indicated by the reduced Zmlncw2/Mnl gene expression in zmsweet4c-umul and the reduced ZmSWEET4c gene expression in zmincw2/mnl (Fig. 6 A and 6B). Indeed,

ZmSWEET4c expression is glucose-inducible during in vitro culture of endosperm (Fig. 5B). Based on these data, we propose two hypotheses for modulation of the BETL precursor cells: (i) glucose induces ZmMrpl, Zmlncw2/Mnl and ZmSWEET4c in parallel, or (ii) glucose import by basal levels of Zmlncw2/Mnl and ZmSWEET4c in turn induces ZmMrpl and further activates the transfer cell machinery, including Zmlncw2/Mnl and ZmSWEET4c. Both models involve a 'feedforward mechanism' in which rising levels of glucose are coupled to enhanced membrane surface area as well as increased capacity to hydrolyze sucrose and import the resultant hexoses into the BETL. At present, zmmrpl mutants are not available and zmsweet4c-umul is difficult to transform, obstructing direct testing of this model. (Fig. 5C)

Another way to test this model is to characterize its conservation across species. Maize has three closely related SWEET4 paralogs, but the striking phenotype of zmsweet4c-umul and the low expression levels of ZmSWEET4a and 4b in the grain suggest a dominant role for

ZmSWEET4c in endosperm sugar import. Phylogenetic comparison shows that both Sorghum bicolor and S. italica have three SWEET4 genes, while Brachy podium distachyon has only two. Rice (O. sativa) has only a single homolog, OsSWEET4, suggesting that all three genes evolved from a single gene in the common ancestor of rice and maize.

In summary, maize SWEET4 genes encode hexose transporters likely acting downstream of a cell wall invertase that hydrolyzes phloem-derived sucrose. ZmSWEET4c is responsible for transferring hexoses across the BETL to sustain development of the large starch-storing endosperm of the maize grain and contributes to its sink strength. Sequence variation at ZmSWEET4c intimates selection during domestication. Alteration by molecular breeding of SWEET hexose transporters might be particularly useful for generating high-yield maize varieties. Based on regulatory studies, we propose a model for a glucose-mediated feed-forward cycle that couples plasma membrane amplification to invertase and SWEET activity. Our work highlights analogous processes relating to trans-epithelial sugar import across cells with amplified membrane surface, mediated by SWEET4 in plants and GLUT2 in humans.

Reduced sequence variation at ZmSWEET4c intimates that both loci were targets of selection during domestication. Engineering of SWEET hexose transporters may help generating new high- yield maize and rice varieties. We propose a model for glucose-mediated feed-forward regulation that couples plasma membrane amplification to invertase and SWEET activity. The instant disclosure highlights processes analogous to irans-epithelial sugar import across cells with amplified membrane surface, mediated by SWEET4 in plants and GLUT2 in humans.

Example 4. SWEET4a can participate in seed filling

Generation of mutant for ZmSWEET4a

The ZmSWEET4a knockouts were obtained by genome editing using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system. The stable expression of endonuclease Cas9 generates double strand breaks in the target genomic DNA defined by a RNA guide specific of the target. Two guide RNA sequences were designed to target specifically a conserved region for all ZmSWEET4a, ZmSWEET4b, ZmSWEET4c Transformed plants were propagated and the presence of CRISPR/Cas9 was confirmed by PCR using 3 couples of primers. The target region were amplified by PCR with corresponding primers and sequenced. In T2 generation, mutants for ZmSWEET4a gene only were isolated, the samples showing single mutation and multi peaks in the target sites were marked as homozygotes and heterozygous, respectively.

Sequences

RNA guide 1 GCTGCTGAACTGCATGATGTGG (SEQ ID NO: 25)

RNA guide

RNA guide 2 GGTGCACCCGCACAGCATGCTGG (SEQ ID NO: 26)

Cas9-1F CATCGGTTGGAAGAATCGTT (SEQ ID NO: 27)

Cas9-1R CGCATTGATGGGATTCTCTT (SEQ ID NO: 28)

Cas9-2F CCAGCCTGGGTACGTATCAT (SEQ ID NO: 29)

Cas9 PCR

Cas9-2R CGCTCCCTGCTATTTTTCTG (SEQ ID NO: 30)

Cas9-3F GTTGGGGATCACGATTATGG (SEQ ID NO: 31)

Cas9-3R AACTCGCTGATTTGCTCGAT (SEQ ID NO: 32)

ZmSWT4a-F2 TCATAGTACTAGCTAGTACGTG (SEQ ID NO: 33)

ZmSWT4a-R2 ATACGAACGACTCTCGCCCTC (SEQ ID NO: 34) Target site

ZmSWET4b-Fl TGTTGCCACAAAGGAAAGGACG (SEQ ID NO: 35) PCR and

ZmSWET4b-Rl CTTTGTTCCTACATGTACGCCG (SEQ ID NO: 36) sequencing

ZmSWT4c-F2 GCGCAAACAAACAAACAAAC (SEQ ID NO: 37) ZmSWT4c-R2 CACAGCAGATCAGAGACACCA (SEQ ID NO: 38)

ZmSweet4a-qF Actgaccgacgatcaagtgacgat (SEQ ID NO: 39)

ZmSweet4a-qR Acggacaagcagcgtgcatataga (SEQ ID NO: 40)

Swt4c_qF3 Aggaaacaatacccctcatctct (SEQ ID NO: 41)

qRT-PCR

Swt4c_3utr_qR3 Tgctaactggaaatagcatttttg (SEQ ID NO: 42)

Actin-F TACCCGATTGAGCATGGCA (SEQ ID NO: 43)

Actin-R TCTTCAGGCGAAACACGGA (SEQ ID NO: 44)

Kernel measurements

ZmSWEET4a knockout plants and wild-type were grown in the same greenhouses (16 hours day 35°C, 8 hours night 20°C). Kernel weight and area have been measured for the same set of mature kernels. 48 individual kernels have been measured from T2 generation of 2 homozygous ears and 2 segregating ears generated by crosses as reported in the table Fig7A. For the wild-type, the ears of 3 different plants have been used. Error bars correspond to the standard deviation calculated from all measurements and p- values correspond to Student tests to determine if the measures of kernel from mutant ears were significantly different from the wild-type.

As described in Figure 7, seed size (Figure 7A) and weight (Figure 7B) can be decreased in maize plants deficient in SWEET4a expression. 48 kernels were measured per cob, and p- values assigned using the Student T-test.

Example 5.

Analysis of expression were conducted with pools of five kernels provided from wild-type and mutants ears at 10 and 20 days after pollination and harvested around 6pm. Total RNA was extracted with the TRIzol extraction reagent (Thermo fisher), DNase treated and reverse transcribed with Quantitech Reverse Transcription Kit (Quiagen) following supplier instructions. Quantitative PCR was carried out with the FastStart SYBR Green Master mix (Roche) on a LightCycler 480 System (Roche), according to the manufacturer's protocol. Expression levels were calculated using Actin as reference gene and the 2-AACT method (Schmittgen and Livak 2008, doi:

10.1038/nprot.2008.73).

As presented in Figure 8, mutant maize strains deficient in SWEET4a expression also demonstrated decreased expression of SWEET4c, particularly at 20DAP.

Example 6. Transgenic Maize Expressing MRP1, ZmSWEET4c, and Incw2

A total of 6 binary constructs were constructed. Standard cloning techniques such as PCR, restriction enzyme digestion, gel electrophoresis and subsequence fragment purification, DNA ligation, bacterial cell transformation and selection, and the like were used to generate the vectors (see Sambrook 1985 for standard methods). Some of the components of the expression vectors described were synthesized by a commercial DNA synthesis lab (Gene Art, Germany).

The binary vector 23321 contains an expression cassette with the following components operatively linked together in this order: the promoter of the maize Basel Endosperm Transfer

Layer (BETL) 2 gene promoter (prZmBETL2-02) (SEQ ID NO: 1); maize MYB-Related Protein-1 CDS (MRP1) (SEQ ID NO: 3); and the maize Basal Endosperm Transfer Layer 2 terminator (SEQ ID NO: 4).

The binary vector 22837 contains an expression cassette with the following components operatively linked together in this order: Maize native promoter of ZmSWEET4c (SEQ ID NO: 5); coding sequence of maize seven trans -membrane sugar transporter ZmSWEET4c (SEQ ID NO: 6); and the maize native terminator of ZmSWEET4c (SEQ ID NO: 7).

The binary vector 23341 contains two expression cassettes. Expression cassette one has the following components operatively linked together in this order: Maize native promoter of

ZmSWEET4c (SEQ ID NO: 5); coding sequence of maize seven trans-membrane sugar transporter ZmSWEET4c (SEQ ID NO: 6); and the maize native terminator of ZmSWEET4c (SEQ ID NO:

7) ; the second expression cassette has the following components operatively linked together in this order: Maize native promoter of ZmMRPl (SEQ ID NO: 2); ZmMRPl (Zea mays MYB-Related Protein-1) CDS (SEQ ID NO: 3); and the Cauliflower Mosaic virus 35S terminator (SEQ ID NO: 8).

The binary vector 23422 contains three expression cassettes. Expression cassette one has the following components operatively linked together in this order: Maize native promoter of ZmSWEET4c (SEQ ID NO: 5); coding sequence of maize seven trans-membrane sugar transporter ZmSWEET4c (SEQ ID NO: 6); and the maize native terminator of ZmSWEET4c (SEQ ID NO: 7); the second expression cassette has the following components operatively linked together in this order: Maize native promoter of ZmMRPl (SEQ ID NO: 2); ZmMRPl (Zea mays MYB-Related Protein-1) CDS (SEQ ID NO: 3); and the Cauliflower Mosaic virus 35S terminator (SEQ ID NO:

8) . The third expression cassette contains Maize native promoter of ZmSWEET4c (SEQ ID NO: 5), maize cell wall invertase 2 (Incw2) CDS (SEQ ID NO: 9), and the maize native terminator of ZmSWEET4c (SEQ ID NO: 7).

The binary vector 23379 contains three expression cassettes. Expression cassette one has the following components operatively linked together in this order: Maize native promoter of ZmMRPl (SEQ ID NO: 2); ZmMRPl (Zea mays MYB-Related Protein-1) CDS (SEQ ID NO: 3); and the Cauliflower Mosaic virus 35S terminator (SEQ ID NO: 8). The second expression cassette has the following components operatively linked together in this order: the promoter derived from the maize NADP-malic enzyme (SEQ ID NO: 10); the maize putative seven transmembrane sugar efflux transporter SWEET13a gene CDS (SEQ ID NO: 11); and the transcriptional terminator derived from the maize NADP-malic (SEQ ID NO: 12). The third expression cassette contains

Maize native promoter of ZmSWEET4c (SEQ ID NO: 5), maize cell wall invertase 2 (Incw2) CDS (SEQ ID NO: 9), and the maize native terminator of ZmSWEET4c (SEQ ID NO: 7).

The binary vector 23409 contains two expression cassettes. Expression cassette one has the following components operatively linked together in this order: Maize native promoter of

ZmSWEET4c (SEQ ID NO: 5); ZmMRPl (Zea mays MYB-Related Protein- 1) CDS (SEQ ID NO: 3); and the maize native terminator of ZmSWEET4c (SEQ ID NO: 7); the second expression cassette has the following components operatively linked together in this order: Maize native promoter of ZmSWEET4c (SEQ ID NO: 5); coding sequence of maize seven trans-membrane sugar transporter ZmSWEET4c (SEQ ID NO: 6); and the maize native terminator of ZmSWEET4c (SEQ ID NO: 7).

Stable Plant Transformation

In order to compare transient expression to stable transgenic plants, each binary construct as described in Example 1 was stably transformed into maize. Maize transformation was carried out as reported (Negrotto et al., 2000; Li et al., 2003; Ishida, 1996). The transgenic events were sent to greenhouse for various analyses, such as DNA, RNA and Protein expression, and to collect seeds from primary transformants. All 6 constructs generated were used for stable agrobacterium transformation using a well-established embryo transformation protocol. TO plants were generated and seeds harvested. For two constructs we had excess TO plants (single cassette insert) and they were selected for sampling 14 DAP kernels. Wild type 14 DAP kernels were collected as a control.

QRT PCR

The TO plants were sampled for qRT-PCR to verify expression cassette function. The data are summarized in Table 1.

EHA101 Agro

only 0 0 32.39 19.48 41.73 51.7 0 0

23321 7567.29 2545.6 74.18 20.75 81.32 79.24 13325.5 5730.94

22837 0 0 8777.75 6136.92 103.09 96.09 8686.14 3813.73

23341 2459.41 1308.3 8383.45 3711.13 105.07 81.02 9253.91 4772.68

23422 164.36 76.31 638.14 172.82 9949.02 3268.84 746.94 429.89

23379 748.28 510.73 160.19 147.11 17372.01 8934.44 1093.18 623.81

23409 743.36 478.02 563.09 310.57 47.3 31.8 812.25 460.03

Medium only 0 0 85.44 43.95 77.8 49.54 80.35 113.63

EHA101 Agro

only 0 0 47.6 19.02 84.53 44.91 0 0

Table 2 describes the primers and probes used for qRT-PCR.

Claims

That which is claimed is:

1. A transgenic plant cell comprising at least a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence, wherein the first heterologous nucleic acid sequence encodes the amino acid sequence of a transfer cell- specific protein, and the second heterologous nucleic acid sequence encodes the amino acid sequence of a SWEET4c, wherein the first heterologous nucleic acid is operably linked to a first promoter functional in the plant cell and the second heterologous nucleic acid is operably linked to a second promoter functional in the plant cell.

2. The transgenic plant cell of claim 1, wherein the transfer cell-specific protein is Myb Related Protein 1 (MRP1).

3. The transgenic plant cell of claim 1, wherein the transfer cell-specific protein is an invertase.

4. The transgenic plant cell of claim 3, wherein the invertase is MN1 (invertase cell wall 2).

5. The transgenic plant cell of claim 1, wherein the plant cell further comprises a third heterologous nucleic acid sequence operably linked to a third promoter functional in the plant cell.

6. The transgenic plant cell of claim 5, wherein the third heterologous nucleic acid encodes the amino acid sequence of a MN1 (invertase cell wall 2).

7. The transgenic plant cell of claim 5, wherein the third heterologous nucleic acid encodes the amino acid sequence of a sugar transporter.

8. The transgenic plant cell of claim 7, wherein the sugar transporter is a SWEET protein.

9. The transgenic plant cell of claim 8, wherein the SWEET protein is SWEET13a.

10. The transgenic plant cell of any one of claims 1-9, wherein the first, the second, and/or the third promoter is selected from a SWEET4c promoter, a MRPl promoter, a MNl promoter, a Basal Endosperm Transfer Layer (BETL) promoter, or a SWEET 13a promoter.

11. The transgenic plant cell of any one of claims 1-10, wherein the second promoter is a BETL2 promoter.

12. The transgenic plant cell of any one of claims 5-9, wherein the third promoter is a BETL2 promoter.

13. The transgenic plant cell of claim 2, wherein the first promoter comprises a MRPl promoter and the second promoter comprises a SWEET4c promoter.

14. The transgenic plant cell of claim 2, wherein both the first and the second promoter comprise a SWEET4c promoter.

15. The transgenic plant cell of claim 2, wherein the first promoter comprises a BETL2 promoter and the second promoter comprises a SWEET4c promoter.

16. The transgenic plant cell of claim 6, wherein the first promoter comprises a MRPl promoter, the second promoter comprises a SWEET4c promoter, and the third promoter comprises a MNl promoter.

17. The transgenic plant cell of claim 9, wherein the first promoter comprises a MRPl promoter, the second promoter comprises a SWEET4c promoter, and the third promoter comprises a SWEET 13a promoter.

18. The transgenic plant cell of any one of claims 1-17, wherein the plant is a cereal grain plant.

19. The transgenic plant cell of claim 18, wherein the cereal grain plant comprises maize, rice, wheat, rye, oats, sorghum, millet, or barley.

20. The transgenic plant cell of claim 18, wherein the cereal grain plant is maize.

21. A plant comprising the plant cell of any one of claims 1-20.

22. The plant of claim 21, wherein said plant has an increased yield as compared to a control plant that does not contain the first, the second or the third heterologous nucleic acid sequence.

23. A transgenic seed comprising the plant cell of any one of claims 1-20.

24. A recombinant DNA construct comprising a polynucleotide encoding a MRP1 polypeptide operably linked to a heterologous promoter.

25. The recombinant DNA construct of claim 24, wherein the promoter comprises a SWEET4c promoter.

26. The recombinant DNA construct of claim 24, wherein the promoter comprises a BETL2 promoter.

28. The recombinant DNA construct of claim 27, wherein the promoter comprises a BETL2 promoter.

29. The recombinant DNA construct of claim 25, wherein the promoter comprises a MN1 promoter.

30. The recombinant DNA construct of any one of claims 27-29, wherein the polynucleotide further comprises a portion of at least one intron from SWEET4c.

31. The recombinant DNA construct of any one of claims 27-29, wherein the polynucleotide further comprises at least one whole intron from SWEET4c.

32. The recombinant DNA construct of any one of claims 30-31, wherein the intron comprises the first intron of SWEET4c.

33. A method of increasing the levels of at least one sugar in developing seeds in a plant, the method comprising introducing into a plant cell at least a first heterologous nucleic acid sequence and a second heterologous nucleic acid sequence, wherein the first heterologous nucleic acid sequence encodes the amino acid sequence of a transfer cell- specific protein, and the second heterologous nucleic acid sequence encodes the amino acid sequence of a SWEET4c;

wherein the levels of at least one sugar are increased in the developing seed in the plant as compared to a control plant that does not contain the first or the second heterologous nucleic acid sequence.

34. The method of claim 33, wherein the transfer cell-specific protein is Myb Related Protein 1 (MRP1).

The method of claim 33, wherein the transfer cell-specific protein is an invertase.

36. The transgenic plant cell of claim 35, wherein the invertase is MNl (invertase cell wall 2).

37. The method of claim 33, further comprising introducing into said plant cell a third heterologous nucleic acid sequence operably linked to a third promoter functional in the plant cell.

38. The method of claim 37, wherein the third heterologous nucleic acid sequence encodes the amino acid sequence of MNl.

39. The transgenic plant cell of claim 37, wherein the third heterologous nucleic acid encodes the amino acid sequence of a sugar transporter.

40. The method of claim 39, wherein the sugar transporter is a SWEET protein.

41. The method of claim 39, wherein the sugar transporter is SWEET13a.

42. The method of any one of claims 33-41, wherein the first, the second, and/or the third promoter is selected from a SWEET4c promoter, a MRPl promoter, a MNl promoter, a Basal Endosperm Transfer Layer 1 (BETLl) promoter, a BETL2 promoter, or a SWEET 13a promoter.

43. The method of claim 34, wherein the first promoter comprises a MRPl promoter and the second promoter comprises a SWEET4c promoter.

44. The method of claim 34, wherein both the first and the second promoter comprise a SWEET4c promoter.

45. The method of claim 34, wherein the first and/or second promoter comprises a BETL2 promoter.

46. The method of claim 34, wherein the first promoter comprises a BETL2 promoter and the second promoter comprises a SWEET4c promoter.

47. The method of claim 38, wherein the first promoter comprises a MRP1 promoter, the second promoter comprises a SWEET4c promoter and the third promoter comprises a MRP1 promoter.

48. The method of claim 41, wherein the first promoter comprises a MRP1 promoter, the second promoter comprises a SWEET4c promoter, and the third promoter comprises a SWEET13a promoter.

49. The method of any one of claims 33-48, wherein the plant is a cereal grain plant.

50. The method of claim 49, wherein the cereal grain plant comprises maize, rice, wheat, rye, oats, sorghum, millet, or barley.

51. The method of claim 50, wherein the cereal grain plant is maize.

52. The method of any one of claims 33-51, wherein the at least one sugar comprises a hexose sugar.

53. The method of claim 52, wherein the hexose sugar comprises glucose and/or fructose.

54. The method of any one of claims 33-53, wherein said plant has an increased yield as compared to a control plant that does not contain the first, the second or the third heterologous nucleic acid sequence.

55. A method of producing a transgenic plant that produces seeds having increased levels of at least one sugar, the method comprising

(c) transforming a plant cell with at least a first heterologous nucleic acid sequence encoding the amino acid sequence of a transfer cell-specific protein, and a second heterologous nucleic acid sequence encoding the amino acid sequence of a SWEET4c; and

(d) regenerating a transgenic plant from the transformed plant

56. The method of claim 55, wherein the transfer cell-specific protein is Myb Related Protein 1 (MRP1).

57. The method of claim 55, wherein the transfer cell-specific protein is an invertase.

58. The method of claim 57, wherein the invertase is MN1 (invertase cell wall

2).

59. The method of claim 55, further comprising transforming the plant or plant cell with a third heterologous nucleic acid sequence

60. The method of claim 59, wherein the third heterologous nucleic acid sequence encodes the amino acid sequence of MN1.

61. The method of claim 59, wherein the third heterologous nucleic acid encodes the amino acid sequence of a sugar transporter.

62. The method of claim 61, wherein the sugar transporter is a SWEET protein.

63. The method of claim 62, wherein the sugar transporter is SWEET13a.

64. The method of any one of claims 55-63, wherein the at least one sugar comprises a hexose sugar.

65. The method of claim 64, wherein the hexose sugar comprises glucose and/or fructose.