Xin-Ding Wang

Pathway of Sugar Transport in Germinating Wheat Seeds

Three homeologous genes encoding a sucrose (Suc) transporter (SUT) in hexaploid wheat (Triticum aestivum), TaSUT1A, 1B, and 1D, were expressed in germinating seeds, where their function is unknown. All three TaSUT1 proteins were confirmed to be capable of transporting both Suc and maltose by complementation tests with the SUSY7/ura3 yeast (Saccharomyces cerevisiae) mutant strain. The role of Suc transporters in germinating grain was examined by combining in situ hybridization, immunolocalization, fluorescent dye tracer movement, and metabolite assays. TaSUT1 transcript and SUT protein were detected in cells of the aleurone layer, scutellar epidermis, scutellar ground cells, and sieve element-companion cell complexes located in the scutellum, shoot, and root. Ester loading of the membrane-impermeable fluorescent dye carboxyfluorescein into the scutellum epidermal cells of germinating seeds showed that a symplasmic pathway connects the scutellum to the shoot and root via the phloem. However, the scutellar epidermis provides an apoplasmic barrier to solute movement from endosperm tissue. Measurements of sugars in the root, shoot, endosperm, and scutellum suggest that, following degradation of endosperm starch, the resulting hexoses are converted to Suc in the scutellum. Suc was found to be the major sugar present in the endosperm early in germination, whereas maltose and glucose predominate during the later stage. It is proposed that loading the scutellar phloem in germinating wheat seeds can proceed by symplasmic and apoplasmic pathways, the latter facilitated by SUT activity. In addition, SUTs may function to transport Suc into the scutellum from the endosperm early in germination and later transport maltose.

Expression and localisation analysis of the wheat sucrose transporter TaSUT1 in vegetative tissues

Previously we reported the isolation of three sucrose transporter genes, TaSUT1A, 1B and 1D, all expressed at high levels in the developing grains of hexaploid wheat (Triticum aestivum L.), but also in a variety of other tissues [N. Aoki et al. (2002) Plant Mol Biol 50:453–462]. In order to further characterise the expression of the TaSUT1 genes in wheat plants, we have analysed TaSUT1 expression in their vegetative tissues using semi-quantitative reverse transcription–polymerase chain reaction, in situ hybridisation and immunolocalisation. The three TaSUT1 genes, which encode 98% identical SUT proteins, all appeared to be expressed at the same level in leaf blades, leaf sheaths and internodes, as well as developing grains, of hexaploid wheat. In mature leaf blades, TaSUT1 protein localised to the plasma membrane of phloem sieve elements in all classes of veins. In contrast, TaSUT1 mRNA was found to be localised to phloem companion cells. A similar localisation pattern for TaSUT1 protein was observed in veins of leaf sheaths and internodes. These results suggest that the wheat SUT1 has a transport function in enucleate sieve elements, in both veins responsible for loading photoassimilates, and in veins for axial transport. Furthermore, transport of the fluorescent dye carboxyfluorescein was used to investigate symplasmic connectivity between sieve element–companion cell complexes and non-phloem cells. Observations in source leaves indicated that sieve element–companion cell complexes of minor veins were symplasmically restricted, suggesting a role of TaSUT1 in apoplasmic phloem loading. In contrast, the dye was able to move symplasmically out of the phloem in internodes. In these circumstances TaSUT1 may also have a role in retrieving sucrose leaked to the phloem apoplasm.

Spatial and temporal expression of sucrose transport-related genes in developing cotyledons ofVicia faba L.

SummaryDry-matter accumulation by developing cotyledons of grain legumes includes a mandatory influx of photoassimilates, largely in the form of sucrose, from the seed apoplasm across the plasma membranes of the cotyledon cells. This study examined the temporal and spatial expression of an H+/sucrose symporter, a P-type H+-ATPase, and a sucrose-binding protein (SBP) in cotyledons ofVicia faba L. throughout their development. The flux of dry matter and sucrose symporter activity exhibited identical temporal trends. These were a marked increase during cotyledon expansion to a plateau maintained until cotyledon maturity. Thereafter both parameters declined precipitously. The temporal changes in sucrose symporter activity were accounted for by shifts in its Vmax. Transcript levels of the H+/sucrose symporter followed a similar temporal pattern to the sucrose symporter activity suggesting regulation by gene expression. Equivalent conclusions were drawn for SBP and the H+-ATPase expression during cotyledon expansion. Thereafter, during seed filling, the transcript levels of SBP and H+-ATPase did not closely follow that found for the sucrose symporter. A progressive wave of gene expression in the abaxial epidermal cells spread from the cotyledon region juxtaposed to the non-vascular region of the seed coat at the pole distal from the funicle. The pattern of expression progressed most rapidly along the median longitudinal plane of the cotyledons and more slowly outward to their margins. The densities of SBP and H+-ATPase, inserted into the plasma membranes of the abaxial epidermal cells, increased throughout cotyledon expansion. Gene expression (sucrose symporter) and membrane insertion of the gene products (SBP, H+-ATPase) were closely associated with the initiation and development of wall ingrowths in the abaxial epidermal cells.

Sucrose transport-related genes are expressed in both maternal and filial tissues of developing wheat grains

In developing wheat grains (Triticum turgidum var. durum cv. Fransawi), post-sieve element transport of phloem-imported photoassimilates (sucrose) includes membrane transport, to and from the grain apoplasm, between symplasmically-isolated maternal and filial tissues. The cellular location and mechanism of these membrane transport steps were explored during rapid grain fill. Genomic Southern analysis indicated the presence of a multigene family of sucrose/H + symporters (SUTs). One or more SUTs were highly expressed in developing grains, as were P-type H + /ATPase(s) and a sucrose binding protein (SBP). Transcripts of these genes were detected in vascular parenchyma, nucellar projection and aleurone cells. Antibodies, raised against a SUT, an H + /ATPase and a SBP, were selectively bound to plasma membranes of vascular parenchyma cells, nucellar projection transfer cells and modified aleurone/sub-aleurone transfer cells. The nucellar projection transfer cells and modified aleurone/sub-aleurone transfer cells exhibited strong proton pumping activity. In contrast, SUT transport function was restricted to filial tissues containing modified aleurone/sub-aleurone transfer cells. Based on these findings, we conclude that SUTs expressed in maternal tissues do not function as sucrose/H + symporters. Membrane exchange from nucellar projection transfer cells to the endosperm cavity occurs by an as yet unresolved mechanism. Sucrose uptake from the endosperm cavity into filial tissues is mediated by a SUT localised to plasma membranes of the modified aleurone/sub-aleurone transfer cells.

Cellular localisation and function of a sucrose transporter OsSUT1 in developing rice grains

We previously reported the cloning and tissue-specific expression of a gene encoding a sucrose/proton symporter in rice (Oryza sativa L.), OsSUT1 (Hirose et al. 1997, Plant Cell Physiology38, 1389–1396). This gene is expressed at high levels in the filling grain, leaf sheath and stem. Expression in these tissues occurred only after heading i.e. during the development of the reproductive structure as a major sink. In this paper, we report localisation of the transcript and protein to specific cells in the filling rice grain by in situ hybridisation with a probe from theOsSUT1 cDNA, and immunolocalisation of OsSUT1 and proton-pumping ATPase (H+-ATPase). An OsSUT1 cDNA probe recognises a transcript of approximately 2.4 kb, and the SUT1 antibody recognises a protein of approximately 55 kDa in total membrane protein extracts from the filling grain, leaf sheath and stem. In the developing grain, OsSUT1 is expressed at low levels before heading, with expression reaching a peak approximately ten days after emergence of the panicle from the sheath. Transcript is then present throughout seed development, with expression falling substantially after about 25 days post-heading. Both transcript and protein are localised to the aleurone cells of the developing grain, and are also detected in the maternal tissue, particularly the nucellus, vascular parenchyma tissue and the nucellar projection. Tissue slices from filling rice grain showed high rates of sucrose uptake that were inhibited by pCMBS. The role of OsSUT1 in sucrose transport to the filling grain endosperm is discussed.

Induction of wall ingrowths of transfer cells occurs rapidly and depends upon gene expression in cotyledons of developing Vicia faba seeds

Summary.Abaxial epidermal cells of developing faba bean (Vicia faba) cotyledons are modified to a transfer cell morphology and function. In contrast, the adaxial epidermal cells do not form transfer cells but can be induced to do so when excised cotyledons are cultured on an agar medium. The first fenestrated layer of wall ingrowths is apparent within 24 h of cotyledon exposure to culture medium. The time course of wall ingrowth formation was examined further. By 2 h following cotyledon excision, a 350 nm thick wall was deposited evenly over the outer periclinal walls of adaxial epidermal cells and densities of cytoplasmic vesicles increased. After 3 h in culture, 10% of epidermal cells contained small projections of wall material on their outer periclinal walls. Thereafter, this percentage rose sharply and reached a maximum of 90% by 15 h. Continuous culture of cotyledons on a medium containing 6-methyl purine (an inhibitor of RNA synthesis) completely blocked wall ingrowth formation. In contrast, if exposure to 6-methyl purine was delayed for 1 h at the start of the culture period, the adaxial epidermal cells were found to contain small wall ingrowths. Treating cotyledons for 1 h with 6-methyl purine at 15 h following cotyledon excision halted further wall ingrowth development. We conclude that transfer cell induction is rapid and that signalling and early events leading to wall ingrowth formation depend upon gene expression. In addition, these gene products have a high turnover rate.

Genotypic differences in seed growth rates of Phaseolus vulgaris L. II. Factors contributing to cotyledon sink activity and sink size

A previous study [Thomas et al. (2000) Aust. J. Plant Physiol. 27, 109–118] showed that genotypic dif-ferences in seed growth rates of Phaseolus vulgaris L. cultivars was accounted for by variation in dry matter flux and seed size. Bulk cotyledon saps contained identical concentrations of sucrose across cultivars suggesting that geno-typic variation in capacities for sucrose transport and metabolism are equally matched. Cotyledon sucrose transport, monitored as in vitro uptake of [14C]sucrose, exhibited genotypic variation and this was abolished by para-chloromercuribenzene- sulfonate. Eadie–Hofstee transformations of concentration-dependent [14C]sucrose uptake showed that genotypic variation in sucrose flux resulted from differences in maximal transporter activity. Maximal sucrose fluxes and levels of transcript and microsomal protein for the sucrose/H+ symporter and H+-ATPase were positively correlated. In contrast, sucrose binding protein transcript and microsomal protein levels correlated negatively with sucrose fluxes. In all cultivars, a sucrose/H+ symporter and H+-ATPase were co-localised to plasma membranes of the dermal cell complexes. Total plasma membrane surface areas of the dermal cell complexes and total volume of storage parenchyma cells correlated with cultivar variation in seed growth rates. Differences in cell number and size accounted for cultivar variation in total plasma membrane surface area of the dermal cell complexes and total storage parenchyma cell volume.