Christina E. Offler

Post-sieve element transport of photoassimilates in sink regions

Photoassimilate transport from the sieve elements to the recipient sink cells, principally in the form of sucrose, provides a link between sink metabolism and compartmentation with phloem import. Phloem unloading has focused attention on photoassimilate transport across the sieve element boundary. However, post-sieve element transport can be of equal or greater significance. Three cellular pathways of sieve element unloading and post-sieve element transport are identified. These are apoplastic, symplastic and symplastic interrupted by an apoplastic step. The symplastic path is considered to be the common path, while the remaining pathways serve specialized functions. In particular, the apoplastic step isolates the sieve element transport function from the effects of solute concentration or osmotic changes in the sink cells. Switching between apo- and symplastic routes within a given sink has been found to be linked with such changes. Plasmodesmatal transport undoubtedly involves a diffusive component, but whether bulk flow contributes to the symplastic flux of photoassimilate from the sieve elements to the recipient sink cells is yet to be established unequivocally. Efflux across the plasma membranes of the sieve element-companion cell (se-cc) complexes and other vascular cells occurs by passive diffusion. Along the axial route, retrieval from the phloem apoplast is mediated by sucrose/proton symport. However, this mechanism is absent in terminal sinks. Non-vascular efflux from the maternal tissues of developing seed is passive in cereals and energy-coupled in certain grain legumes. Accumulation of sugars from the apoplast of all sinks with an apoplastic step universally occurs by a plasma membrane-bound sugar/proton symport mechanism. Regulation of symplastic transport could be mediated by a combination of sink metabolism and compartmentation coupled with changes in the transport properties of the interconnecting plasmodesmata.

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

SummaryIn developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H+/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H+-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H+-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.

Role of membrane transport in phloem translocation of assimilates and water

Most growth and storage organs (sinks) of higher plants import assimilates in solution by bulk flow through the phloem, driven by differences in hydrostatic pressure. These differences in pressure, located between the ends of the interconnecting phloem path, are generated by osmotic water movement, driven in turn by membrane transport of solutes. Sucrose, amino-nitrogen compounds and potassium represent the osmotically important solutes found in phloem contents of most species. Phloem loading and unloading events of these assimilate species play central roles in determining phloem translocation rates and partitioning of assimilates and water. Depending on plant species, leaf vein order and sink type, phloem loading and unloading may follow apoplasmic or symplasmic routes. Irrespective of the cellular pathway followed, assimilates are transported across plasma and organellar membranes. Aquaporins, amino-nitrogen transporters, sucrose transporters and potassium channels have been detected in key sites along the source-phloem-sink transport pathway. Reverse genetics has demonstrated that sucrose/proton symporters are important in transport events necessary for phloem loading in Solanaceous plant species. Drawing on circumstantial evidence, we review possible functions the remaining membrane transporters and channels may serve in driving phloem translocation of assimilates and water from source to sink.

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.

Transfer cell induction in cotyledons ofVicia faba L.

SummaryImmediately prior to seed fill, a dermal transfer cell complex, comprised of epidermal and subepidermal cells, differentiates on the abaxial surface of the cotyledons in seed ofVicia faba. Over the period of differentiation of this complex in vivo, the principal sugars of the seed apoplasmic sap change from hexoses, glucose and fructose, to sucrose. Cotyledons were removed from seeds before differentiation of the transfer cell complex and cultured for 6 days on an agar-based medium in the dark with their abaxial surface in contact with a medium containing either 100 mM hexoses (glucose and fructose in equimolar concentrations) or 100 mM sucrose. On both media, cotyledon growth rate was maintained throughout the culture period at, or above, that of in vivo grown cotyledons of equivalent developmental age. When cotyledons were cultured on a medium containing glucose and fructose, epidermal cells of both the ab- and adaxial surfaces developed wall ingrowths on their outer periclinal walls and their cytoplasm became dense, vesicular, and rich in mitochondria. Extensive ingrowth deposition also occurred on walls of the subepidermal cells and several rows of underlying storage cells where they abutted intercellular spaces. This latter ingrowth development was apparent on both cotyledon surfaces, but extended into more of the underlying cell layers on the abaxial surface at the funicular end of the cotyledon. In in vivo grown cotyledons, such ingrowth development is restricted to the subepidermal cells of the abaxial surface. Ingrowth morphology was commensurate with that of transfer cells of in vivo grown cotyledons. In contrast to the observed induction on a medium containing glucose and fructose, cotyledons cultured with sucrose as the sole sugar source exhibited no ingrowth deposition or small wall ingrowths in some abaxial epidermal cells. While the potential sugar signalling mechanism is unknown, this culture system offers an exciting opportunity to explore the molecular biology of transfer cell development.

Transfer cell wall architecture: a contribution towards understanding localized wall deposition

Summary. A survey is presented of the architecture of secondary wall ingrowths in transfer cells from various taxa based on scanning electron microscopy. Wall ingrowths are a distinguishing feature of transfer cells and serve to amplify the plasma membrane surface area available for solute transport. Morphologically, two categories of ingrowths are recognized: reticulate and flange. Reticulate-type wall ingrowths are characterized by the deposition of small papillae that emerge from the underlying wall at discrete but apparently random loci, then branch and interconnect to form a complex labyrinth of variable morphology. In comparison, flange-type ingrowths are deposited as curvilinear ribs of wall material that remain in contact with the underlying wall along their length and become variously elaborate in different transfer cell types. This paper discusses the morphology of different types of wall ingrowths in relation to existing models for deposition of other secondary cell walls.

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.

Wall ingrowth formation in transfer cells: novel examples of localized wall deposition in plant cells

The formation of wall ingrowths increases plasma membrane surface areas of transfer cells involved in membrane transport of nutrients in plants. Construction of these ingrowths provides intriguing and diverse examples of localized wall deposition. Flange wall ingrowths resemble secondary wall thickenings of tracheary elements in morphology and probable mechanisms of deposition. By contrast, reticulate wall ingrowths, deposited as discrete papillate projections, branch and fuse to create a fenestrated wall labyrinth representing a novel form of localized wall deposition. Papillate wall ingrowths are initiated as patches of disorganized cellulosic material and are compositionally similar to primary walls, except for a surrounding layer of callose and enhanced levels of arabinogalactan proteins at the ingrowth/membrane interface. How this unusual form of localized wall deposition is constructed is unknown but may involve constraining cellulose-synthesizing rosette complexes at their growing tips.

Nucellar projection transfer cells in the developing wheat grain

SummaryTransfer cells in the nucellar projection of wheat grains at 25 ±3 days after anthesis have been examined using light and electron microscopy. Within the nucellar tissue, a sequential increase in non-polarized wall ingrowth differentiation and cytoplasmic density was evident. Cells located near the pigment strand were the least differentiated. The degree of differentiation increased progressively in cells further removed from the pigment strand and the cells bordering the endosperm cavity had degenerated. Four stages of transfer cell development were identified at the light microscope level. Wall ingrowth differentiation followed a sequence from a papillate form through increased branching (antler-shaped ingrowths) which ultimately anastomosed to form a complex labyrinth. The final stage of wall ingrowth differentiation was compression which resulted in massive ingrowths. In parallel with wall ingrowth deposition cytoplasmic density increased. During wall deposition, paramural and multivesicular bodies were prominent and were in close association with the wall ingrowths. The degeneration phase involved infilling of cytoplasmic islets within the wall ingrowths. This was accompanied by complete loss of the protoplast. The significance of this transfer cell development for sucrose efflux to the endosperm cavity was assessed by computing potential sucrose fluxes across the plasma membrane surface areas of the nucellar projection cells. Transfer cell development amplified the total plasma membrane surface area by 22 fold. The potential sucrose flux, when compared with maximal rates of facilitated membrane transport of sugars, indicated spare capacity for sucrose efflux to the endosperm cavity. Indeed, when the total flux was partitioned between the nucellar projection cells at the three stages of transfer cell development, the fully differentiated stage III cells located proximally to the endosperm cavity alone exhibited spare transport capacity. Stage II cells could accommodate the total rate of sucrose transfer, but stage I cells could not. It is concluded that the nucellar projection tissue of wheat provides a unique opportunity to study transfer cell development and the functional role of these cells in supporting sucrose transport.