Transfer cells: what regulates the development of their intricate wall labyrinths?

Transfer cells (TCs) support high nutrient rates into, or at symplasmic discontinuities within, the plant body. Their transport capacity is conferred by an amplified plasma membrane surface area, enriched in nutrient transporters, supported on an intricately invaginated wall labyrinth (WL). Thus, development of the WL is at the heart of TC function. Enquiry has shifted from describing WL architecture and formation to discovering mechanisms regulating WL assembly. Experimental systems used to examine these phenomena are critiqued. Considerable progress has been made in identifying master regulators that commit stem cells to a TC fate (e.g. the maize Myeloblastosis (MYB)-related R1-type transcription factor) and signals that induce differentiated cells to undergo trans-differentiation to a TC phenotype (e.g. sugar, auxin and ethylene). In addition, signals that provide positional information for assembly of the WL include apoplasmic hydrogen peroxide and cytosolic Ca2+ plumes. The former switches on, and specifies the intracellular site for WL construction, while the latter creates subdomains to direct assembly of WL invaginations. Less is known about macromolecule species and their spatial organization essential for WL assembly. Emerging evidence points to a dependency on methyl-esterified homogalacturonan accumulation, unique patterns of cellulose and callose deposition and spatial positioning of arabinogalactan proteins.

Enzymes contributing to the hydrogen peroxide signal dynamics that regulate wall labyrinth formation in transfer cells.

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (apoROS), and cytosolic Ca2+. Apoplasmic hydrogen peroxide (apoH2O2) was confirmed as the apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H2O2 biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in apoH2O2 production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of apoH2O2. The findings were consistent with catalase activity constraining the apoH2O2 signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular apoH2O2 signatures that differed during assembly of the UWL and WI papillae.

Ethylene and hydrogen peroxide regulate formation of a sterol-enriched domain essential for wall labyrinth assembly in transfer cells.

Transfer cells (TCs) facilitate high rates of nutrient transport into, and within, the plant body. Their transport function is conferred by polarized wall ingrowth papillae, deposited upon a specialized uniform wall layer, that form a scaffold supporting an amplified area of plasma membrane enriched in nutrient transporters. We explored the question of whether lipid-enriched domains of the TC plasma membrane could serve as organizational platforms for proteins regulating the construction of the intricate TC wall labyrinth using developing Vicia faba cotyledons. When these cotyledons are placed in culture, their adaxial epidermal cells trans-differentiate to a TC phenotype regulated by auxin, ethylene, extracellular hydrogen peroxide (apoH2O2), and cytosolic Ca2+ ([Ca2+]cyt) arranged in series. Staining cultured cotyledons with the sterol-specific dye, Filipin III, detected a polarized sterol-enriched domain in the plasma membrane of their trans-differentiating epidermal transfer cells (ETCs). Ethylene activated sterol biosynthesis while extracellular apoH2O2 directed sterol-enriched vesicles to fuse with the outer periclinal region of the ETC plasma membrane. The sterol-enriched domain was essential for generating the [Ca2+]cyt signal and orchestrating construction of both the uniform wall layer and wall ingrowth papillae. A model is presented outlining how the sterol-enriched plasma membrane domain forms and functions to regulate wall labyrinth assembly.

Sucrose Transporter Localization and Function in Phloem Unloading in Developing Stems.

How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5-SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow.

Cell Wall Invertase Promotes Fruit Set under Heat Stress by Suppressing ROS-Independent Cell Death.

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.

A Ca2+-dependent remodelled actin network directs vesicle trafficking to build wall ingrowth papillae in transfer cells.

The transport function of transfer cells is conferred by an enlarged plasma membrane area, enriched in nutrient transporters, that is supported on a scaffold of wall ingrowth (WI) papillae. Polarized plumes of elevated cytosolic Ca2+ define loci at which WI papillae form in developing adaxial epidermal transfer cells of Vicia faba cotyledons that are induced to trans-differentiate when the cotyledons are placed on culture medium. We evaluated the hypothesis that vesicle trafficking along a Ca2+-regulated remodelled actin network is the mechanism that underpins this outcome. Polarized to the outer periclinal cytoplasm, a Ca2+-dependent remodelling of long actin bundles into short, thin bundles was found to be essential for assembling WI papillae but not the underlying uniform wall layer. The remodelled actin network directed polarized vesicle trafficking to sites of WI papillae construction, and a pharmacological study indicated that both exo- and endocytosis contributed to assembly of the papillae. Potential candidates responsible for the Ca2+-dependent actin remodelling, along with those underpinning polarized exo- and endocyotosis, were identified in a transcriptome RNAseq database generated from the trans-differentiating epidermal cells. Of most significance, endocytosis was controlled by up-regulated expression of a dynamin-like isoform. How a cycle of localized exo- and endocytosis, regulated by Ca2+-dependent actin remodelling, assembles WI papillae is discussed.

Plasma Membrane Ca2+-Permeable Channels are Differentially Regulated by Ethylene and Hydrogen Peroxide to Generate Persistent Plumes of Elevated Cytosolic Ca2+ During Transfer Cell Trans-Differentiation.

The enhanced transport capability of transfer cells (TCs) arises from their ingrowth wall architecture comprised of a uniform wall on which wall ingrowths are deposited. The wall ingrowth papillae provide scaffolds to amplify plasma membranes that are enriched in nutrient transporters. Using Vicia faba cotyledons, whose adaxial epidermal cells spontaneously and rapidly (hours) undergo a synchronous TC trans-differentiation upon transfer to culture, has led to the discovery of a cascade of inductive signals orchestrating deposition of ingrowth wall papillae. Auxin-induced ethylene biosynthesis initiates the cascade. This in turn drives a burst in extracellular H2O2 production that triggers uniform wall deposition. Thereafter, a persistent and elevated cytosolic Ca(2+) concentration, resulting from Ca(2+) influx through plasma membrane Ca(2+)-permeable channels, generates a Ca(2+) signal that directs formation of wall ingrowth papillae to specific loci. We now report how these Ca(2+)-permeable channels are regulated using the proportionate responses in cytosolic Ca(2+) concentration as a proxy measure of their transport activity. Culturing cotyledons on various combinations of pharmacological agents allowed the regulatory influence of each upstream signal on Ca(2+) channel activity to be evaluated. The findings demonstrated that Ca(2+)-permeable channel activity was insensitive to auxin, but up-regulated by ethylene through two independent routes. In one route ethylene acts directly on Ca(2+)-permeable channel activity at the transcriptional and post-translational levels, through an ethylene receptor-dependent pathway. The other route is mediated by an ethylene-induced production of extracellular H2O2 which then acts translationally and post-translationally to up-regulate Ca(2+)-permeable channel activity. A model describing the differential regulation of Ca(2+)-permeable channel activity is presented.

Differential transcriptional networks associated with key phases of ingrowth wall construction in trans-differentiating epidermal transfer cells of Vicia faba cotyledons.

BACKGROUND: Transfer cells are characterized by intricate ingrowth walls, comprising an uniform wall upon which wall ingrowths are deposited. The ingrowth wall forms a scaffold to support an amplified plasma membrane surface area enriched in membrane transporters that collectively confers transfer cells with an enhanced capacity for membrane transport at bottlenecks for apo-/symplasmic exchange of nutrients. However, the underlying molecular mechanisms regulating polarized construction of the ingrowth wall and membrane transporter profile are poorly understood. RESULTS: An RNAseq study of an inducible epidermal transfer cell system in cultured Vicia faba cotyledons identified transfer cell specific transcriptomes associated with uniform wall and wall ingrowth deposition. All functional groups of genes examined were expressed before and following transition to a transfer cell fate. What changed were the isoform profiles of expressed genes within functional groups. Genes encoding ethylene and Ca(2+) signal generation and transduction pathways were enriched during uniform wall construction. Auxin-and reactive oxygen species-related genes dominated during wall ingrowth formation and ABA genes were evenly expressed across ingrowth wall construction. Expression of genes encoding kinesins, formins and villins was consistent with reorganization of cytoskeletal components. Uniform wall and wall ingrowth specific expression of exocyst complex components and SNAREs suggested specific patterns of exocytosis while dynamin mediated endocytotic activity was consistent with establishing wall ingrowth loci. Key regulatory genes of biosynthetic pathways for sphingolipids and sterols were expressed across ingrowth wall construction. Transfer cell specific expression of cellulose synthases was absent. Rather xyloglucan, xylan and pectin biosynthetic genes were selectively expressed during uniform wall construction. More striking was expression of genes encoding enzymes for re-modelling/degradation of cellulose, xyloglucans, pectins and callose. Extensins dominated the cohort of expressed wall structural proteins and particularly so across wall ingrowth development. Ion transporters were selectively expressed throughout ingrowth wall development along with organic nitrogen transporters and a large group of ABC transporters. Sugar transporters were less represented. CONCLUSIONS: Pathways regulating signalling and intracellular organization were fine tuned whilst cell wall construction and membrane transporter profiles were altered substantially upon transiting to a transfer cell fate. Each phase of ingrowth wall construction was linked with unique cohorts of expressed genes.

Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed.

Polarized and persistent Ca²âº plumes define loci for formation of wall ingrowth papillae in transfer cells.

Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca(2+) by co-operative functioning of plasma membrane Ca(2+)-permeable channels and Ca(2+)-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca(2+) signal was organized into discrete patches that aligned spatially with clusters of Ca(2+)-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca(2+) were consistent with inward-directed plumes of elevated [Ca(2+)]cyt. Plume formation depended upon an alternating distribution of Ca(2+)-permeable channels and Ca(2+)-ATPase clusters. On further inward diffusion, the Ca(2+) plumes coalesced into a uniform Ca(2+) signal. Blocking or dispersing the Ca(2+) plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca(2+) plumes define the loci at which wall ingrowth papillae are deposited.

Glucose and ethylene signalling pathways converge to regulate trans-differentiation of epidermal transfer cells in Vicia narbonensis cotyledons.

Transfer cells are specialized transport cells containing invaginated wall ingrowths that provide an amplified plasma membrane surface area with high densities of transporter proteins. They trans-differentiate from differentiated cells at sites where enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, the signal(s) and signalling cascades responsible for initiating their trans-differentiation are poorly understood. In culture, adaxial epidermal cells of Vicia narbonensis cotyledons were induced to trans-differentiate to a transfer cell morphology. Manipulating their intracellular glucose concentrations by transgenic knock-down of ADP-glucose pyrophosphorylase expression and/or culture on a high-glucose medium demonstrated that glucose functioned as a negative regulator of wall ingrowth induction. In contrast, glucose had no detectable effect on wall ingrowth morphology. The effect on wall ingrowth induction of culture on media containing glucose analogues suggested that glucose acts through a hexokinase-dependent signalling pathway. Elevation of an epidermal cell-specific ethylene signal alone, or in combination with glucose analogues, countered the negative effect of glucose on wall ingrowth induction. Glucose modulated the amplitude of ethylene-stimulated wall ingrowth induction by down-regulating the expression of ethylene biosynthetic genes and an ethylene insensitive 3 (EIN3)-like gene (EIL) encoding a key transcription factor in the ethylene signalling cascade. A model is presented describing the interaction between glucose and ethylene signalling pathways regulating the induction of wall ingrowth formation in adaxial epidermal cells.

Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons.

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H(2)O(2) signature. Extracellular H(2)O(2) fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H(2)O(2) flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H(2)O(2) signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H(2)O(2) production and were regulated by cross-talk with ethylene. It is concluded that H(2)O(2) functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form.

GIGANTEA is a component of a regulatory pathway determining wall ingrowth deposition in phloem parenchyma transfer cells of Arabidopsis thaliana.

Transfer cells are specialised transport cells containing invaginated wall ingrowths that generate an amplified plasma membrane surface area with high densities of transporter proteins. They trans-differentiate from differentiated cells at sites at which enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, little is known of the molecular mechanisms regulating construction of their intricate wall ingrowths. We investigated the genetic control of wall ingrowth formation in phloem parenchyma transfer cells of leaf minor veins in Arabidopsis thaliana. Wall ingrowth development in these cells is substantially enhanced upon exposing plants to high-light or cold treatments. A hierarchical bioinformatic analysis of public microarray datasets derived from the leaves of plants subjected to these treatments identified GIGANTEA (GI) as one of 46 genes that are commonly up-regulated twofold or more under both high-light and cold conditions. Histological analysis of the GI mutants gi-2 and gi-3 showed that the amount of phloem parenchyma containing wall ingrowths was reduced 15-fold compared with wild-type. Discrete papillate wall ingrowths were formed in gi-2 plants but failed to develop into branched networks. Wall ingrowth development in gi-2 was not rescued by exposing these plants to high-light or cold conditions. In contrast, over-expression of GI in the gi-2 background restored wall ingrowth deposition to wild-type levels. These results indicate that GI regulates the ongoing development of wall ingrowth networks at a point downstream of inputs from environmental signals.

An epidermal-specific ethylene signal cascade regulates trans-differentiation of transfer cells in Vicia faba cotyledons.

*Transfer cells (TCs) trans-differentiate by developing extensive wall ingrowths that facilitate enhanced plasma membrane transport of nutrients. Signal(s) and signalling cascades responsible for initiating this trans-differentiation event are poorly understood. We tested the hypothesis that ethylene functions as a key inductive signal for wall ingrowth formation in epidermal cells of Vicia faba cotyledons. *Scanning electron microscopy of epidermal cells monitored their propensity for wall ingrowth formation. Spatial and temporal expression profiles of ethylene biosynthetic enzymes and key elements of ethylene signalling cascades (ethylene insensitive 3 (EIN3) and ethylene response factors (ERFs)) were determined. *Wall-ingrowth formation responded positively to manipulation of ethylene biosynthesis and perception. It was preceded by a cell-specific burst in ethylene biosynthesis accompanied by a co-localized post-translational up-regulation of VfEIN3-1 and differential expression of three VfERF genes. Blocking ethylene production arrested ongoing wall ingrowth development. Wound-induced ethylene in pod walls and seed coats caused an in planta activation of ethylene biosynthetic genes in adaxial epidermal cells that coincidentally formed wall ingrowths. *A cell-specific burst of ethylene biosynthesis functions as an inductive signal initiating and sustaining trans-differentiation to a TC morphology in vitro. These events are reproduced for developing V. faba seeds in planta.

Early gene expression programs accompanying trans-differentiation of epidermal cells of Vicia faba cotyledons into transfer cells.

Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons.

Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H(+) symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level.

Transfer cells: cells specialized for a special purpose.

Transfer cells are plant cells with secondary wall ingrowths. These cells are ubiquitous, occurring in all plant taxonomic groups and in algae and fungi. Transfer cells form from differentiated cells across developmental windows and in response to stress. They are considered to play a central role in nutrient distribution by facilitating high rates of transport at bottlenecks for apo-/symplasmic solute exchange. These properties are conferred by their unique structural features--an invaginated secondary wall ensheathed by an amplified area of plasma membrane enriched in a suite of solute transporters. Recent development of transfer cell experimental systems, combined with technologies to image the three-dimensional structure of wall ingrowths, is allowing identification of inductive and regulatory signals, discovery of sequential processes involved in their differentiation, and a search for transfer cell identity genes. A model of key events in differentiation of a transfer cell is presented to highlight areas for future investigation.

Pteris umbrosa R. Br. as an arsenic hyperaccumulator: accumulation, partitioning and comparison with the established As hyperaccumulator Pteris vittata.

The capacity of the Australian native fern Pteris umbrosa to function as an arsenic (As) hyperaccumulator (shoot:soil As concentration >1) was examined by growing plants under glasshouse conditions in an inert medium supplemented with As. Arsenic preferentially accumulated in the fronds, a trait of a hyperaccumulator. The As concentration of fronds decreased with age, being particularly high in the croziers and low in the senesced fronds. Below ground, rhizomes accumulated more As than adventitious roots. Uptake from a range of solution concentrations followed Michaelis Menten kinetics up to a soil solution As concentration of 400mgl(-1). The K(m) for As uptake by roots suggested the operation of a low-affinity carrier. The predicted Nernst membrane potential indicated that uptake was against the electrochemical gradient of As. At 600mgl(-1), the rate of As uptake increased and phytotoxic effects were indicated by a significant decline in biomass. Arsenic uptake and translocation in P. umbrosa and Pteris vittata were similar at low exposure to As. At higher exposure, As uptake and translocation by P. vittata increased more than in P. umbrosa. The growth rate of both ferns was similar, whereas the biomass distribution was not, with P. vittata having a much larger root mass. This suggests that As uptake by P. umbrosa roots was very efficient and may be improved by stimulating root growth to enhance its potential.

A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells.

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.

Contribution of sucrose transporters to phloem unloading within Sorghum bicolor stem internodes.

Sucrose produced in source leaves is loaded into collection phloem, transported to sinks and unloaded for utilization or storage. In the context of long distance transport, sucrose transporters (SUTs) can function to load sucrose into collection phloem, retrieve leaked sucrose during long distance transport, and load sucrose into sink cells. SUTs have also been proposed to efflux sucrose under conditions of low proton motive force and low extracellular sucrose. The involvement of sucrose transporters in phloem unloading in a representative monocot stem, Sorghum bicolor, was evaluated during different stages of internode development. Transcript levels and functional properties of selected key transporters were measured, with both cellular and subcellular localization determined.