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Pollen fertility is critical for successful fertilization and, accordingly, for crop yield. While sugar unloading affects growth and development of all types of sink organs, the molecular nature for sugar import to tomato pollen is poorly understood. However, SWEET transporters have been proposed to function in pollen development. Here, qRT-PCR revealed that SlSWEET5b was markedly expressed in flowers when compared to the remaining tomato SlSWEETs; particularly, in the stamens of maturing flower buds undergoing mitosis. Distinct accumulation of SlSWEET5b-GUS fusion proteins was present in mature flower buds, especially in anther vascular and inner cells, symplasmic isolated pollen cells and styles. The demonstration that GFP fusion proteins located to the plasma membrane support the idea that the SlSWEET5b carrier functions in apoplasmic sugar translocation during pollen maturation. Such function is in line with data from yeast complementation experiments and radiotracer uptakes, showing that SlSWEET5b operates as a low affinity hexose-specific passive facilitator, with a KM of ~36 mM. Most importantly, RNAi-mediated suppression of SlSWEET5b expression resulted in shrunken nucleus-less pollen cells, impaired germination and low seed yield. Interestingly, stamens from SlSWEET5b-silenced tomato mutants contained significantly lower amounts of sucrose and increased invertase activity, pointing to reduced carbon supply and perturbed sucrose homeostasis in this tissue. Taken together, our findings reveal an essential role of SlSWEET5b in mediating apoplasmic hexose import into phloem unloading cells and into developing pollen cells to support pollen mitosis and maturation in tomato flowers.One-sentence SummaryPlasma-membrane-localized SlSWEET5b facilitates a sequential hexose flux, from phloem to anther cells and from anther locule to pollen, to support pollen maturation and fertility in tomato flowers.

Ovule formation is essential for realizing crop yield because it determines seed number. The underlying molecular mechanism, however, remains elusive. Here, we show that cell wall invertase (CWIN) functions as a positive regulator of ovule initiation in Arabidopsis (Arabidopsis thaliana). In situ hybridization revealed that CWIN2 and CWIN4 were expressed at the placenta region where ovule primordia initiated. Specific silencing of CWIN2 and CWIN4 using targeted artificial microRNA driven by an ovule-specific SEEDSTICK promoter (pSTK) resulted in a substantial reduction of CWIN transcript and activity, which blocked ovule initiation and aggravated ovule abortion. There was no induction of carbon (C) starvation genes in the transgenic lines, and supplementing newly forming floral buds with extra C failed to recover the ovule phenotype. This indicates that suppression of CWIN did not lead to C starvation. A group of hexose transporters was downregulated in the transgenic plants. Among them, two representative ones were spatially coexpressed with CWIN2 and CWIN4, suggesting a coupling between CWIN and hexose transporters for ovule initiation. RNA-sequencing analysis identified differentially expressed genes encoding putative extracellular receptor-like kinases, MADS-box transcription factors, including STK, and early auxin response genes in response to CWIN-silencing. Our data demonstrate the essential role of CWIN in ovule initiation, which is most likely to occur through sugar signaling instead of C nutrient contribution. We propose that CWIN-mediated sugar signaling may be perceived by, and transmitted through, hexose transporters or receptor-like kinases to regulate ovule formation by modulating downstream auxin signaling and MADS-box transcription factors.

Soybean (Glycine max) seed is primarily composed of a mature embryo that provides a major source of protein and oil for humans and other animals. Early in development, the tiny embryos grow rapidly and acquire large quantities of sugars from the liquid endosperm of developing seeds. An insufficient supply of nutrients from the endosperm to the embryo results in severe seed abortion and yield reduction. Hence, an understanding of the molecular basis and regulation of assimilate partitioning involved in early embryo development is important for improving soybean seed yield and quality. Here, we used expression profiling analysis to show that two paralogous sugar transporter genes from the SWEET (Sugars Will Eventually be Exported Transporter) family, GmSWEET15a and GmSWEET15b, were highly expressed in developing soybean seeds. In situ hybridization and quantitative real-time PCR showed that both genes were mainly expressed in the endosperm at the cotyledon stage. GmSWEET15b showed both efflux and influx activities for sucrose in Xenopus oocytes. In Arabidopsis (Arabidopsis thaliana), knockout of three AtSWEET alleles is required to see a defective, but not lethal, embryo phenotype, whereas knockout of both GmSWEET15 genes in soybean caused retarded embryo development and endosperm persistence, resulting in severe seed abortion. In addition, the embryo sugar content of the soybean knockout mutants was greatly reduced. These results demonstrate that the plasma membrane sugar transporter, GmSWEET15, is essential for embryo development in soybean by mediating Suc export from the endosperm to the embryo early in seed development.

Reduced cell wall invertase (CWIN) activity has been shown to be associated with poor seed and fruit set under abiotic stress. Here, we examined whether genetically increasing native CWIN activity would sustain fruit set under long-term moderate heat stress (LMHS), an important factor limiting crop production, by using transgenic tomato (Solanum lycopersicum) with its CWIN inhibitor gene silenced and focusing on ovaries and fruits at 2 d before and after pollination, respectively. We found that the increase of CWIN activity suppressed LMHS-induced programmed cell death in fruits. Surprisingly, measurement of the contents of H2O2 and malondialdehyde and the activities of a cohort of antioxidant enzymes revealed that the CWIN-mediated inhibition on programmed cell death is exerted in a reactive oxygen species-independent manner. Elevation of CWIN activity sustained Suc import into fruits and increased activities of hexokinase and fructokinase in the ovaries in response to LMHS Compared to the wild type, the CWIN-elevated transgenic plants exhibited higher transcript levels of heat shock protein genes Hsp90 and Hsp100 in ovaries and HspII17.6 in fruits under LMHS, which corresponded to a lower transcript level of a negative auxin responsive factor IAA9 but a higher expression of the auxin biosynthesis gene ToFZY6 in fruits at 2 d after pollination. Collectively, the data indicate that CWIN enhances fruit set under LMHS through suppression of programmed cell death in a reactive oxygen species-independent manner that could involve enhanced Suc import and catabolism, HSP expression, and auxin response and biosynthesis.

Seed number and quality are key traits determining plant fitness and crop yield and rely on combined competence in male and female fertilities. Sucrose metabolism is central to reproductive success. It remains elusive, though, how individual sucrose metabolic enzymes may regulate the complex reproductive processes. Here, by silencing vacuolar invertase (VIN) genes in cotton (Gossypium hirsutum) reproductive organs, we revealed diverse roles that VIN plays in multiple reproductive processes. A set of phenotypic and genetic studies showed significant reductions of viable seeds in GhVIN1-RNAi plants, attributed to pollination failure and impaired male and female fertilities. The former was largely owing to the spatial mismatch between style and stamen and delayed pollen release from the anthers, whereas male defects came from poor pollen viability. The transgenic stamen exhibited altered expression of the genes responsible for starch metabolism and auxin and jasmonic acid signaling. Further analyses identified the reduction of GhVIN expression in the seed coat as the major cause for the reduced female fertility, which appeared to disrupt the expression of some key genes involved in trehalose and auxin metabolism and signaling, leading to programmed cell death or growth repression in the filial tissues. Together, the data provide an unprecedented example of how VIN is required to synchronize style and stamen development and the formation of male and female fertilities for seed development in a crop species, cotton.

Despite substantial evidence on the essential roles of cell wall invertase (CWIN) in seed filling, it remains largely unknown how CWIN exerts its regulation early in seed development, a critical stage that sets yield potential. To fill this knowledge gap, we systematically examined the spatial and temporal expression patterns of a major CWIN gene, GhCWIN1, in cotton (Gossypium hirsutum) seeds from prefertilization to prestorage phase. GhCWIN1 messenger RNA was abundant at the innermost seed coat cell layer at 5 d after anthesis but became undetectable at 10 d after anthesis, at the onset of its differentiation into transfer cells characterized by wall ingrowths, suggesting that CWIN may negatively regulate transfer cell differentiation. Within the filial tissues, GhCWIN1 transcript was detected in endosperm cells undergoing nuclear division but not in those cells at the cellularization stage, with similar results observed in Arabidopsis (Arabidopsis thaliana) endosperm for CWIN, AtCWIN4. These findings indicate a function of CWIN in nuclear division but not cell wall biosynthesis in endosperm, contrasting to the role proposed for sucrose synthase (Sus). Further analyses revealed a preferential expression pattern of GhCWIN1 and AtCWIN4 in the provascular region of the torpedo embryos in cotton and Arabidopsis seed, respectively, indicating a role of CWIN in vascular initiation. Together, these novel findings provide insights into the roles of CWIN in regulating early seed development spatially and temporally. By comparing with previous studies on Sus expression and in conjunction with the expression of other related genes, we propose models of CWIN- and Sus-mediated regulation of early seed development.

Sucrose (Suc) synthase (Sus) is the major enzyme of Suc breakdown for cellulose biosynthesis in cotton (Gossypium hirsutum) fiber, an important source of fiber for the textile industry. This study examines the tissue-specific expression, relative abundance, and temporal expression of various Sus transcripts and proteins present in cotton. A novel isoform of Sus (SusC) is identified that is expressed at high levels during secondary cell wall synthesis in fiber and is present in the cell wall fraction. The phylogenetic relationships of the deduced amino acid sequences indicate two ancestral groups of Sus proteins predating the divergence of monocots and dicots and that SusC sequences form a distinct branch in the phylogeny within the dicot-specific clade. The subcellular location of the Sus isoforms is determined, and it is proposed that cell wall-localized SusC may provide UDP-glucose for cellulose and callose synthesis from extracellular sugars.

Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due, in part, to the complexity of multicellular plant tissues. Here, we used cotton (Gossypium spp.) fibers, fast-growing single-celled seed trichomes, to address this issue. VIN activity in elongating fibers was approximately 4-6-fold higher than that in leaves, stems, and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Furthermore, a genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than a slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1, which displayed VIN sequence features and localized to the vacuole. Once introduced to Arabidopsis (Arabidopsis thaliana), GhVIN1 complemented the short-root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber, GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNA interference or overexpression constructs reduced or enhanced fiber elongation, respectively. Together, these analyses provide evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contributions of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively.

Cotton fibers are single-celled hairs that elongate to several centimeters long from the seed coat epidermis of the tetraploid species (Gossypium hirsutum and Gossypium barbadense). Thus, cotton fiber is a unique system to study the mechanisms of rapid cell expansion. Previous work has shown a transient closure of plasmodesmata during fiber elongation (Y.-L. Ruan, D.J. Llewellyn, R.T. Furbank [2001] Plant Cell 13: 47–60). To examine the importance of this closure in fiber elongation, we compared the duration of the plasmodesmata closure among different cotton genotypes differing in fiber length. Confocal imaging of the membrane-impermeant fluorescent molecule carboxyfluorescein revealed a genotypic difference in the duration of the plasmodesmata closure that positively correlates with fiber length among three tetraploid genotypes and two diploid progenitors. In all cases, the closure occurred at the rapid phase of elongation. Aniline blue staining and immunolocalization studies showed that callose deposition and degradation at the fiber base correlates with the timing of plasmodesmata closure and reopening, respectively. Northern analyses showed that the expression of a fiber-specific β-1,3-glucanase gene, GhGluc1, was undetectable when callose was deposited at the fiber base but became evident at the time of callose degradation. Genotypically, the level of GhGluc1 expression was high in the short fiber genotype and weak in the intermediate and long fiber genotypes. The data provide genotypic and developmental evidence that (1) plasmodesmata closure appears to play an important role in elongating cotton fibers, (2) callose deposition and degradation may be involved in the plasmodesmata closure and reopening, respectively, and (3) the expression of GhGluc1 could play a role in this process by degrading callose, thus opening the plasmodesmata.

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberlessseed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of theFLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the flsmutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.