Graham N. Scofield

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.

Localization of sucrose synthase in developing seed and siliques of Arabidopsis thaliana reveals diverse roles for SUS during development

This study investigated the roles of sucrose synthase (SUS) in developing seeds and siliques of Arabidopsis thaliana. Enzyme activity assays showed that SUS activity was highest in developing whole siliques and young rosette leaves compared with other tissues including mature leaves, stems, and flowers. Surprisingly, quantitative PCR analyses revealed little correlation between SUS activity and transcript expression, which indicated the importance of examining the role of SUS at the protein level. Therefore, immunolocalization was performed over a developmental time course to determine the previously unreported cellular localization of SUS in Arabidopsis seed and silique tissues. At 3 d and 10 d after flowering (daf), SUS protein localized to the silique wall, seed coat, funiculus, and endosperm. By 13 daf, SUS protein was detected in the embryo and aleurone layer, but was absent from the seed coat and funiculus. Starch grains were also present in the seed coat at 3 and 10 daf, but were absent at 13 daf. Co-localization of SUS protein and starch grains in the seed coat at 3 and 10 daf indicates that SUS may be involved in temporary starch deposition during the early stages of seed development, whilst in the later stages SUS metabolizes sucrose in the embryo and cotyledon. Within the silique wall, SUS localized specifically to the companion cells, indicating that SUS activity may be required to provide energy for phloem transport activities in the silique wall. The results highlight the diverse roles that SUS may play during the development of silique and seed in Arabidopsis.

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.

Starch storage in the stems of wheat plants: localization and temporal changes

BACKGROUND AND AIMS Carbohydrate temporarily accumulates in wheat stems during the early reproductive growth phase, predominantly as water soluble carbohydrate (WSC), and is subsequently remobilized during grain filling. Starch has also been reported as a minor storage carbohydrate component in wheat stems, but the details are lacking. METHODS The accumulation and localization of starch in wheat stem and leaf sheath tissue over a developmental period from 6 d before anthesis to 35 d after anthesis was investigated. KEY RESULTS The region of the peduncle enclosed by the flag-leaf sheath, and the penultimate internode were the main tissues identified as containing starch, in which the starch grains localized to the storage parenchyma cells. In contrast, the exposed peduncle lacked starch grains. Starch grains were also found in the flag-leaf and second-leaf sheath. Plants grown in low-nitrogen conditions exhibited increased storage of both starch and WSC compared with plants grown in high-nitrogen supply. CONCLUSIONS The major accumulation and decrease of starch occurred temporally independently to that for WSC, suggesting a different functional role for starch in wheat stems. Starch reutilization concomitant with peduncle growth, and the early development of the reproductive structures, suggested a role in provision of energy and/or carbon scaffolds for these growth processes.

A xylem sap retrieval pathway in rice leaf blades: evidence of a role for endocytosis?

The structure and transport properties of pit membranes at the interface between the metaxylem and xylem parenchyma cells and the possible role of these pit membranes in solute transfer to the phloem were investigated. Electron microscopy revealed a fibrillar, almost tubular matrix within the pit membrane structure between the xylem vessels and xylem parenchyma of leaf blade bundles in rice (Oryza sativa). These pits are involved primarily with regulating water flux to the surrounding xylem parenchyma cells. Vascular parenchyma cells contain large mitochondrial populations, numerous dictyosomes, endomembrane complexes, and vesicles in close proximity to the pit membrane. Taken collectively, this suggests that endocytosis may occur at this interface. A weak solution of 5,6-carboxyfluorescein diacetate (5,6-CFDA) was applied to cut ends of leaves and, after a minimum of 30 min, the distribution of the fluorescent cleavage product, 5,6-carboxyfluorescein (5,6-CF), was observed using confocal microscopy. Cleavage of 5,6-CFDA occurred within the xylem parenchyma cells, and the non-polar 5,6-CF was then symplasmically transported to other parenchyma elements and ultimately, via numerous pore plasmodesmata, to adjacent thick-walled sieve tubes. Application of Lucifer Yellow, and, separately, Texas Red-labelled dextran (10 kDa) to the transpiration stream, confirmed that these membrane-impermeant probes could only have been offloaded from the xylem via the xylem vessel–xylem parenchyma pit membranes, suggesting endocytotic transmembrane transfer of these membrane-impermeant fluorophores. Accumulation within the thick-walled sieve tubes, but not in thin-walled sieve tubes, confirms the presence of a symplasmic phloem loading pathway, via pore plasmodesmata between xylem parenchyma and thick-walled sieve tubes, but not thin-walled sieve tubes.

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.