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Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.

Summary Two putative endosperm-specific rice genes, OsPR602 and OsPR9a , were identified from database searches. The promoter regions of these genes were isolated, and transcriptional promoter: β -glucuronidase (GUS) fusion constructs were stably transformed into rice and barley. The GUS expression patterns revealed that these promoters were active in early grain development in both rice and barley, and showed strongest expression in endosperm transfer cells during the early stages of grain filling. The GUS expression was similar in both rice and barley, but, in barley, expression was exclusively in the endosperm transfer cells and differed in timing of activation relative to rice. In rice, both promoters showed activity not only in the endosperm transfer cells, but also in the transfer cells of maternal tissue and in several floral tissues shortly before pollination. The expression patterns of OsPR602 and OsPR9a in flowers differed. The similarity of expression in both rice and barley suggests that these promoters may be useful to control transgene expression in the transfer cells of cereal grains with the aim of altering nutrient uptake or enhancing the barrier against pathogens at the boundary between maternal tissue and the developing endosperm. However, the expression during floral development should be considered if the promoters are used in rice.

Cytokinins (CKs) are plant hormones that regulate a large number of processes associated with plant growth and development such as induction of stomata opening, delayed senescence, suppression of auxin-induced apical dominance, signaling of nitrogen availability, differentiation of plastids and control of sink strength. In maize, CKs are thought to play an important role in establishing seed size and increasing seed set under normal and unfavorable environmental conditions therefore influencing yield. In recent years, the discovery of isopentenyl transferase (IPT) genes in plants has shed light on the CK biosynthesis pathway in plants. In an effort to increase our understanding of the role played by CKs in maize development and sink-strength, we identified several putative IPT genes using a bioinformatics approach. We focused our attention on one gene in particular, ZmIPT2, because of its strong expression in developing kernels. The expression of the gene and its product overlays the change in CK levels in developing kernels suggesting a major role in CK biosynthesis for kernel development. We demonstrate that at 8-10 days after pollination (DAP) the endosperm and especially the basal transfer cell layer (BETL) is a major site of ZmIPT2 expression, and that this expression persists in the BETL and the developing embryo into later kernel development stages. We show that ectopic expression of ZmIPT2 in calli and in planta created phenotypes consistent with CK overproduction. We also show that ZmIPT2 preferentially uses ADP and ATP over AMP as the substrates for dimethylallyl diphosphate (DMAPP) IPT activity. The expression pattern of ZmIPT2 in the BETL, endosperm and embryo during kernel development will be discussed with an emphasis on the suggested role of CKs in determining sink-strength and grain production in crop plants.

Summary We report expression profiles of several genes of carbohydrate metabolism, cell wall invertase (CWI) in particular, to better understand sugar transport and its utilization in developing caryopses of grain sorghum [Sorghum bicolor (L.) Moench]. Gene expression analyses for CWI using RNA gel blot and real-time quantitative PCR approaches on developing caryopses, including the glumes (maternal tissue appended to the seeds), showed expression of SbIncw (ZmIncw2 ortholog) primarily in the basal sugar unloading zone of endosperm. The expression of ZmIncw1 ortholog was significantly less abundant and restricted to the glumes. The protein and enzyme activity data corroborated the temporal transcript expression profile that showed maximal CWI protein (INCW) expression preceding the starch-filling phase of endosperm development, i.e. 6–12 d-after-pollination (DAP). Protein gel blot analysis using polyclonal maize INCW1 antibodies showed a single polypeptide of 72 kDa. The highest level of enzyme activity was unique to the basal part of the endosperm, in particular the basal endosperm transfer cell (BETC) layer and the maternal pedicel region that were highly enriched for the INCW protein, as seen by immunolocalization. High hexose-to-sucrose ratio in 6–12 DAP seeds, and negligible starch deposition in glumes corroborated the CWI activity data. Additionally, we report transcription profiles of several other genes related to sugar-to-starch metabolism in developing sorghum endosperm. As in maize, the INCW-mediated apoplastic cleavage of sucrose in the BETC and pedicel during the early developmental stages of caryopses is essential for the normal development of filial tissues. The unique cell-specificity of the INCW protein to both proximal and distal ends of placental sac shown here for the first time is likely to greatly increase uptakes of both hexose sugars and water through turgor sensing into developing seed. This trait is unique to sorghum among cereals and may facilitate its survival in drought environment.

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.

To explain previously reported discrepancies between oxygen uptake rates in biological systems and the sodium sulphite test system a comparison was made between oxygen mass transfer into a sodium sulphite solution catalysed by copper(II) ions, and into a biological system, the bacterium E. coli in a mineral medium. A stirred transfer cell, with a relatively low rate of physical mass transfer per unit area, and a bubble column, with a high physical mass transfer rate per unit area, were used to make this comparison; in both cases, the areas of gas/liquid contact could be determined. In the transfer cell the gas/liquid mass transfer coefficient for the biological system was only 10–25% of that for the sulphite oxidation system (for which the absorption rate was increased by chemical reaction). In the bubble column mass transfer into both systems was controlled by physical absorption and the mean mass transfer coefficients were similar for both systems. However, the mass transfer coefficient for the biological system increased with E. coli concentration, probably due to physical effects of the small particles.

Requirement of mineral elements in different plant tissues is not often consistent with their transpiration rate; therefore, plants have developed systems for preferential distribution of mineral elements to the developing tissues with low transpiration. Here we took silicon (Si) as an example and revealed an efficient system for preferential distribution of Si in the node of rice (Oryza sativa). Rice is able to accumulate more than 10% Si of the dry weight in the husk, which is required for protecting the grains from water loss and pathogen infection. However, it has been unknown for a long time how this hyperaccumulation is achieved. We found that three transporters (Lsi2, Lsi3, and Lsi6) located at the node are involved in the intervascular transfer, which is required for the preferential distribution of Si. Lsi2 was polarly localized to the bundle sheath cell layer around the enlarged vascular bundles, which is next to the xylem transfer cell layer where Lsi6 is localized. Lsi3 was located in the parenchyma tissues between enlarged vascular bundles and diffuse vascular bundles. Similar to Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribution of Si to the panicles but increased Si to the flag leaf. Furthermore, we constructed a mathematical model for Si distribution and revealed that in addition to cooperation of three transporters, an apoplastic barrier localized at the bundle sheath cells and development of the enlarged vascular bundles in node are also required for the hyperaccumulation of Si in rice husk.

Background Transfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy. Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level. Results The simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation. Conclusions Use of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0483-8) contains supplementary material, which is available to authorized users.

In this study, we developed a device that could easily, rapidly, and completely transfer cell sheets from one material to another or transplant cell sheets onto the dorsal subcutaneous tissues of rats without leaving residual cells. Because the manipulation is as simple as pipetting, technical expertise is not required to transfer cell sheets very rapidly (the transfer time was 3.7 ± 1.6 s) using the device compared with that of a conventional method using a pipette (430 ± 180 s). After transfer by the device, C2C12 skeletal myoblast sheets showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor and stromal-derived factor-1α, indicating transfer without cell damage. Cardiac cell sheets after transfer showed spontaneous and synchronous beating, indicating intact cell-cell junctions and ion channel proteins on the cell opsurface. In addition, the device allowed us to transfer C2C12 cell sheets onto soft, rugged and curved surfaces such as human hands. Furthermore, cardiac cell sheets adhered rapidly and tightly onto the dorsal subcutaneous tissues of rats. This transfer/transplantation device may be a powerful tool in cell sheet-based tissue engineering and regenerative medicine.

Transfer cell formation in cotyledons of developing faba bean (Vicia faba L.) seeds coincides with an abrupt change in seed apoplasm composition from one dominated by hexoses to one in which sucrose is the principal sugar. On the basis of these observations, we tested the hypothesis that sugars induce and/or sustain transfer cell development. To avoid confounding effects of in planta developmental programs, we exploited the finding that adaxial epidermal cells of cotyledons, which do not become transfer cells in planta, can be induced to form functional transfer cells when cotyledons are cultured on an agar medium. Growth rates of cotyledons cultured on hexose or sucrose media were used to inform choice of sugar concentrations. The same proportion of adaxial epidermal cells of excised cotyledons were induced to form wall ingrowths independent of sugar species and concentration supplied. In all cases, induction of wall ingrowths coincided with a marked increase in the intracellular sucrose-to-hexose ratio. In contrast, further progression of wall ingrowth deposition was correlated positively with intracellular sucrose concentrations that varied depending upon external sugar species and supply. Sucrose symporter induction and subsequent maintenance behaved identically to wall ingrowth formation in response to an external supply of hexoses or sucrose. However, in contrast to wall ingrowth formation, induction of sucrose symporter activity was delayed. We discuss the possibility of intracellular sugars functioning both as signals and substrates that induce and control subsequent development of transfer cells.

Dodders are obligate parasites, requiring both water and carbohydrate to be supplied by the host plant. To achieve the successful flow of both carbohydrate and water, the searching hyphae of the dodder must penetrate the host and transform into xylic or phloic hyphae, depending on the cell type that the hyphae encounter. The phloic hyphae develop a massive handlike appendage that surrounds the host phloem, whereas the xylic hyphae are directly connected to the host. In this study, changes in wall composition and structure are monitored via structural, cytochemical, and immunocytochemical techniques that allow one to discriminate changes in the hyphae as they make the transition into functional xylem and phloem. Although the terminal structure of the phloic hyphae has been termed a transfer cell, it bears little resemblance to those cell types; few if any wall ingrowths are noted. Rather, an extensive array of smooth endoplasmic reticulum (ER) is aligned at right angles and adjacent to the host phloem cell...

To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.

The development of phloem transfer cells in expanded dark grown leaflets of pea seedlings (Pisum sativum) has been re-examined. In agreement with previous observations transfer cells in leaflets maintained in the dark did not form wall ingrowths to the same extent as those placed in the light. A previous report that exposure to light in a carbon dioxide depleted atmosphere inhibited wall ingrowth formation could not be confirmed. It was found that dark grown leaflets could be induced to form wall ingrowths without illumination by immersing them in a glucose solution, demonstrating for the first time that light is not necessary for phloem transfer cell differentiation in leaves. Attempts were made to alter the carbohydrate level in the whole seedling by removing the cotyledons, but this had no recognizable effect on wall ingrowth formation in any of the treatments. Starch grain formation in the plastids was taken as an indication of available soluble carbohydrate level in the leaflets. It is concluded that both light and the presence of soluble carbohydrate can independently induce wall ingrowth formation in phloem transfer cells of pea leaflets.

Highlight A persistent and polarized cytosolic Ca2+ signal, formed into plumes by co-operative activities of plasma membrane Ca2+ channels and Ca2+-ATPase clusters, directs papillate wall ingrowth deposition in trans-differentiating 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 Ca2+ signals generated by spatiotemporal alterations in cytosolic Ca2+ ([Ca2+]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 Ca2+ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca2+ signal intensity, by withdrawing extracellular Ca2+ or blocking Ca2+ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca2+ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca2+ by co-operative functioning of plasma membrane Ca2+-permeable channels and Ca2+-ATPases. Viewed paradermally, and proximal to the cytosol–plasma membrane interface, the Ca2+ signal was organized into discrete patches that aligned spatially with clusters of Ca2+-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca2+ were consistent with inward-directed plumes of elevated [Ca2+]cyt. Plume formation depended upon an alternating distribution of Ca2+-permeable channels and Ca2+-ATPase clusters. On further inward diffusion, the Ca2+ plumes coalesced into a uniform Ca2+ signal. Blocking or dispersing the Ca2+ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca2+ plumes define the loci at which wall ingrowth papillae are deposited.

We report here on the identification and characterization of ZmLrk-1, a member of the Lrk class of receptor-like kinases in Zea mays. This gene was found to be located at the bin21.40 region on the short arm of maize chromosome 8, closely linked to the previously reported pseudogene of the same class psiZmLrk (originally called Zm2Lrk). Transient expression experiments in onion epithelium cells, using a ZmLrk-1:GFP fusion protein, indicate that ZmLrk-1 is a membrane protein. ZmLrk-1 is ubiquitously expressed in the maize plant, including roots and aerial parts. In seeds, ZmLrk-1 transcripts can be detected by in situ hybridization exclusively at the basal endosperm transfer cell layer during the first stages of development. However, from 14 days after pollination its transcripts are preferentially detected at the upper half of the kernel, including both the aleurone and the starchy endosperm. ZmLrk-1 expression is not induced after treatment with salicylic acid, jasmonic acid or wounding, but it clearly increases after infection of germinating seeds with Fusarium oxysporum. This suggests that ZmLrk-1 could be involved in a sensing system to activate plant defence mechanisms against fungal attacks during endosperm development and seed germination.

Data from this study show that the chalazal cyst is a consistent feature of the Brassicaceae that exhibits variation of potential taxonomic value. The U-shaped seeds of mustards comprise three development chambers: micropylar (MC), central (CC), and chalazal (ChC). Early in seed development the syncytial endosperm in the ChC becomes differentiated into a cyst that remains distinct from storage endosperm. In early syncytial stages, the cyst is stratified into three zones: (1) an apical zone containing nuclei, plastids and mitochondria, (2) a mid-zone rich in endomembranes but depleted of large organelles, and (3) a basal portion that is bordered by a labyrinthine wall. The basal zone has projections that range from foot-like extensions to thread-like haustoria that penetrate the underlying maternal chalazal proliferative tissues (CPT). No cell-to-cell connections occur between endosperm and maternal tissue, but the extensive labyrinthine walls closely invest cell lysate resulting from degradation of the CPT. Structural data from light, confocal and transmission electron microscopy suggest that the cyst is active in uptake and transport of metabolites into the developing seed. Four types of cyst reflecting position and size of the ChC are recognized. In general, they correlate with major tribes of Brassicaceae. Data support recent molecular studies indicating that classification of the Lepidieae is artificial and suggest that Thlaspi is a candidate for realignment. The most divergent morphology in a tribe is seen in the two species of the Hesperidae. Erysimum has a more typical chalazal chamber and cyst morphology while the tubular chalazal chamber and filiform cyst of Chorispora is of the most extreme type encountered in the survey. This suggests that the tribe is paraphyletic and should be reinvestigated to clarify relationships. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 144, 375−394.

Medicago sativa var. Gabes is a perennial glycophyte that develops new shoots even in high salinity (150 mM NaCl). In the upper exporting leaves, K(+) is high and Na(+) is low by comparison with the lower leaves, where Na(+) accumulation induces chlorosis after 4 weeks of NaCl treatment. By secondary ion mass spectroscopy, a low Na(+)/K(+) ratio was detected in the phloem complex of blade veins in these lower leaves. By transmission electron microscopy, the ultrastructural features were observed in the phloem complex. In the upper leaves of both control and NaCl-treated plants, companion cells in minor veins were found to be transfer cells. These cells may well be involved in the intravenous recycling of ions and in Na(+) flowing out of exporting leaves. Under the effect of NaCl, companion cells in the main veins develop transfer cell features, which may favor the rate of assimilate transport from exporting leaves toward meristems, allowing the positive balance necessary for the survival in salt conditions. These features no longer assist the lower leaves when transfer cells are necrotized in both minor and main veins of NaCl-treated plants. As transfer cells are the only degenerating phloem constituent, our observations emphasize their role in controlling nutrient (in particular, Na(+)) fluxes associated with the stress response.

The immunocytochemical localization of the plasma membrane H+-ATPase in epidermal cells of tomato roots was studied using a monoclonal antibody raised against purified maize P-type H+-ATPase. Plants subjected to iron starvation exhibited increased proton extrusion that was confined to the root elongation zones. Immunogold labelling of the H+-ATPase on the plasma membrane was considerably higher in rhizodermal cells within zones with intense proton extrusion than in non-acidifying areas of the roots. Transfer cells were formed in rhizodermal cells of Fe-deficient plants. Quantitative determination of immunolabelling revealed that the density of PM H+-ATPase in transfer cells was about twice that of ordinary epidermal cells. In transfer cells, H+-ATPase was most abundant on the plasma membrane lining the labyrinthine invaginations of the peripheral cell wall. While the number of immunologically detectable ATPase molecules in transfer cells was not spatially correlated with proton extrusion activity, the frequency of transfer cells was considerably higher in acidifying root areas relative to non-active segments. Split-root experiments indicated that both the steady-state level of plasma membrane H+-ATPase and proton extrusion activity are systemically regulated, indicating inter-organ regulation of rhizosphere acidification. Exogenous application of the auxin analog 2,4-dichlorophenoxyacetic acid and the ethylene precursor 1-aminocyclopropane-1-carboxlic acid caused the formation of transfer cells at a frequency similar to that observed in Fe-deficient roots. However, the number of proton pumps was not affected by the hormone treatment, suggesting that both responses are regulated independently. It is concluded that transfer cells in the rhizodermis may be important but not crucial for rhizosphere acidification.

Legume seed development is closely related to metabolism and nutrient transport. To analyse this relationship, a combination of biochemical, histological and transgenic approaches was used. Sugars within tissue sections have been quantitatively measured by metabolic imaging. During cotyledon differentiation glucose gradients emerge related to a particular cell type, with higher concentrations in non-differentiated premature regions. Sucrose in creases at the beginning of maturation in a layer underneath the outer epidermis expressing a sucrose transporter. Sucrose distribution is initially controlled by uptake activity and the permeability within the parenchyma and, later on, also by differences in growth and starch accumulation. Increased sucrose levels are accompanied by increased levels of sucrose synthase and ADP-Glc pyrophosphorylase mRNAs, but carbon flux into starch is initially still low. Rates increase at a stage when hexose concentrations become low, allowing increased flux through the sucrose synthase pathway. Transfer cell formation represents a regional specification of the cotyledonary epidermis for embryo nutrition characterized by increased transport-active cell surfaces and up-regulated expression of transport-related genes. The E2748 pea seed mutation blocks epidermal differentiation into transfer cells and leads to the loss of epidermal cell identity. Embryos with impaired epidermis cannot tolerate elevated levels of sucrose and respond with disorganized growth. The E2748 gene product is required for transfer cell formation in developing cotyledons with no other function during plant growth. Seed coat permeability provides a hypoxic environment for embryo development. However, at maturity, seed energy supply is not limited indicating fundamental developmental and metabolic adaptations. Results from transgenic seeds show that altered expression of single genes induces complex and unexpected changes. In AGP-antisense seeds the block in starch synthesis leads to pleiotropic effects of water and nitrogen content and induces temporal changes in seed development.

In maize, the endosperm region next to the placento-chalaza is characterised by the presence of cells with extensive cell wall ingrowths (transfer cells). Specific genes, collectively named as BETL (for Basal Endosperm Transfer Layer) genes, are expressed in these cells in a way that coincides with the temporal development of the transfer cell layer. The BETL-1 gene encodes a polypeptide of 7 kDa, exclusively expressed in the transfer cell region and possibly involved in the construction of the cell wall. However, the sequence (defensin-like) is also consistent with an antipathogenic role. We found that BETL-1 is strongly associated with the cell wall ingrowths of the endosperm transfer layer. On the other hand, BETL-2 (an antifungal protein) is not bound to the cell wall of transfer cells. The two BETL-2 forms (the 8 kDa propeptide and the final 4.5 kDa peptide) are detected in different subcellular compartments. The propeptide is mostly associated with the membrane fraction of endosperm, while the low molecular weight form is soluble. Vesicles containing the 8-kDa BETL-2 propeptide are located in association with microtubules present in the transfer cell region. Together with previous evidence on the differential distribution of the BETL-2 forms, these data outline the processing pathway of BETL-2, from synthesis in the transfer cells to the site of utilisation in the placento-chalaza region. BETL-4 (which is weakly related to a family of protease inhibitors) is mainly found in the transfer cell layer, and is absent from adjacent regions. Unlike BETL-1, BETL-4 is not a cell wall protein, even though it is associated with the ingrowths of the transfer cell walls. Therefore, BETL proteins show distinct properties of localisation and distribution, which are related to their differential functions in the transfer cells.

During legume seed development the epidermis of the embryos differentiates into a transfer cell layer which mediates nutrient uptake during the storage phase. This specific function of the epidermal cells is acquired at the onset of embryo maturation. We investigated this process in the pea seed mutant E2748. The epidermal cells of the mutant embryo, instead of turning into transfer cells, enlarge considerably and become vacuolated and tightly associated with adjacent seed tissues. Expression of a sucrose transporter gene that is upregulated in wild-type transfer cells decreases in the mutant and changes its spatial pattern. This indicates that the outermost cell layer of mutant cotyledons cannot acquire transfer cell morphology but loses epidermal cell identity and does not function as a sucrose uptake system. Seed coat growth as well as composition, concentration and dynamics of sugars within the endospermal vacuole are unchanged. The loss of epidermal identity has severe consequences for further embryo development and is followed by disruption of the symplast within the parenchyma, the breach of the developmental gradient, lower sucrose and starch levels and initiation of callus-like growth. It is concluded that the E2748 gene controls differentiation of the cotyledonary epidermis into transfer cells and thus is required for the regional specialisation with a function in embryo nutrition.

Developing seeds consist of genetically distinct maternal and filial tissues, whose interactions during development are largely unknown. To better understand the molecular physiology of developing seed tissues in barley, we created a high-density cDNA macroarray bearing 711 cDNA fragments from 691 clones representing at least 620 unique genes mainly derived from a cDNA library constructed with mRNA from the early stages of caryopsis development. This array has been used to compare gene expression patterns in maternal pericarp and filial embryo sac tissues of caryopses sampled 1-7 days after flowering (DAF). The profiles obtained for both tissues revealed that at least 26 genes in pericarp and 12 genes in embryo sac tissues were up-regulated by more than a factor of two during this period. RNAs expressed at high levels in the pericarp mainly encode enzymes involved in carbohydrate and lipid metabolism, but also include mRNA for a transcription factor related to FILAMENTOUS FLOWER (FIL). Genes preferentially expressed in the embryo sac are mainly related to degradation and/or processing of proteins or are involved in the process of starch accumulation, which begins in the seed at this time. Some of the most conspicuously regulated genes were studied in more detail by Northern analysis and in situ hybridization. The mRNA with the highest apparent signal intensity encodes a methionine synthase (MSY). MSY is highly expressed throughout the pericarp and to a lower extent in the transfer cell layer of the endosperm. Of special interest is a gene of unknown function because its high-level expression is restricted to the nucellar projection, the maternal transfer tissue of the caryopsis. This gene, represented by clone HY09L21, may play a central role in transport processes and thus in embryo growth.

Patterning of epidermal cells is subject to genetic regulation but also influenced by environmental stimuli. To adapt to unfavorable environmental conditions plants have developed various mechanisms to increase the plasma membrane's surface area of epidermal root cells, for example through the formation of root hairs and differentiation of rhizodermal transfer cells. Mechanisms controlling cell fate speciation in the rhizodermis were investigated by application of hormones and hormone antagonists. In addition, the effect of Fe deficiency on root epidermal patterning and Fe(III)-reduction activity was examined. In the iron-hyperaccumulating pea mutants dgl and brz and in the Arabidopsis mutant man1 Fe(III)-reduction activity was found to be up-regulated under both high and low iron supply. In contrast, morphological responses such as the development of transfer cells and extranumerary root hairs was repressed by a high iron concentration in the external medium. All morphological responses can be mimicked by exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D). Conversely, Fe(III)-reduction rates were not influenced or only slightly affected by the hormone treatment. Application of inhibitors of ethylene synthesis, ethylene action or auxin transport was effective only in inhibiting the formation of extra root hairs, indicating that these hormones are not required for transfer cell formation or expression of Fe(III) reduction. These data suggest that the Fe reductase induced by iron stress does not depend on the formation of transfer cells and further imply separate regulatory pathways for the two responses. The data are compatible with a model in which root reduction activity is modulated by a shoot-borne signal coordinating iron uptake with the shoot demand, while the epidermal phenotype is primarily dependent on the intracellular iron concentration of root cells.

Transfer cell (TCs) develop unique wall ingrowth networks which amplify plasma membrane surface area and thus maximize nutrient transporter density at key anatomic sites for nutrient exchange within plants and their external environment. These sites fall into 4 main groups corresponding to 4 categories of trans-membrane flux: absorption/secretion of solutes from or to the external environment, and absorption/secretion of solutes from or to internal, extra-cytoplasmic compartments. Research on TC biology over recent decades has demonstrated correlations between wall ingrowth deposition in TCs and enhanced transport capacity in many major agricultural species such as pea, fava bean, cotton and maize. Consequently, there is general consensus that the existence of wall ingrowth morphology implies an augmentation in membrane transport capacity. However, this may not be entirely applicable for phloem parenchyma (PP) TCs in Arabidopsis. Our recent survey of PP TC abundance and distribution in Arabidopsis veins indicated that PP TC development reflects heteroblastic status. A consequence of this observation is the suggestion that PP TCs, or at least wall ingrowth deposition in these cells, potentially act as a physical barrier to defend access of invading pathogens to sugar-rich sieve elements rather than solely in facilitating the export of photoassimilate from collection phloem in leaves.

Transfer cells are ubiquitous plant cells that play an important role in plant development as well as in responses to biotic and abiotic stresses. They are highly specialized and differentiated cells playing a central role in the acquisition, distribution and exchange of nutrients. Their unique structural traits are characterized by augmented ingrowths of invaginated secondary wall material, unsheathed by an amplified area of plasma membrane enriched in a suite of solute transporters. Similar morphological features can be perceived in vascular root feeding cells induced by sedentary plant-parasitic nematodes, such as root-knot and cyst nematodes, in a wide range of plant hosts. Despite their close phylogenetic relationship, these obligatory biotrophic plant pathogens engage different approaches when reprogramming root cells into giant cells or syncytia, respectively. Both nematode feeding-cells types will serve as the main source of nutrients until the end of the nematode life cycle. In both cases, these nematodes are able to remarkably maneuver and reprogram plant host cells. In this review we will discuss the structural, functional and morphogenetic characteristics function and formation of these specialized multinucleate cells that act as nutrient transfer cells to accumulate and synthesize components needed for survival and successful offspring of plant-parasitic nematodes. Plant cells with transfer-like functions are also a renowned subject of interest involving still poorly understood molecular and cellular transport processes.

In the course of a project aimed to isolate transfer cells-specific genes in maize endosperm we have identified the BETL9 gene. BETL9 encodes for a small protein very similar in sequence to the product of the barley transfer cell-specific gene END-1. Both BETL9 and END-1 proteins are lipid transfer proteins, but their function is currently unknown. In situ hybridization analysis confirms that the BETL9 gene is exclusively transcribed in the basal endosperm transfer cell layer during seed development since 10 days after pollination. However, immunolocalization data indicates that the BETL9 protein accumulates in the maternal placento-chalaza cells located just beside the transfer cell layer. This suggests that the BETL9 protein should be transported to the maternal side to exert its, still unknown, function. In addition, we have identified a second maize gene very similar in sequence to BETL9 and we have named it BETL9like. In situ hybridization shows that BETL9like is also specifically transcribed in the developing maize endosperm within the same time frame that BETL9, but in this case it is exclusively expressed in the aleurone cell layer. Consequently, the BETL9 and BETL9like genes are transcribed in a non-overlapping pattern on the outer surface of the maize endosperm. The BETL9 and BETL9like promoter sequences, fused to the GUS reporter gene, accurately reflected the expression pattern observed for the genes in maize. Finally, we have identified in the Arabidopsis genome a set of four genes orthologous to BETL9 and BETL9like and analyzed the activity of their promoters in Arabidopsis transgenic plants carrying fusions of their promoter sequences to the GUS reporter. As in the case of the maize genes, the Arabidopsis orthologs showed highly complementary expression patterns.

Phyto-endoparasitic nematodes induce specialized feeding cells (NFCs) in their hosts, termed syncytia and giant cells (GCs) for cyst and root-knot nematodes, respectively. They differ in their ontogeny and global transcriptional signatures, but both develop cell wall ingrowths to facilitate high rates of apoplastic/symplastic solute exchange showing transfer cell (TC) characteristics. Regulatory signals for TC differentiation are not still well known. The two-component signalling system (2CS) and reactive oxygen species are proposed as inductors of TC identity, while, 2CSs-related genes are not major contributors to differential gene expression in early developing NFCs. Additionally, transcriptomic and functional studies have assigned a major role to auxin and ethylene as regulatory signals on early developing TCs. Genes encoding proteins with similar functions expressed in both early developing NFCs and typical TCs are putatively involved in upstream or downstream responses mediated by auxin and ethylene. Yet, no function directly associated to the TCs identity of NFCs, such as the formation of cell wall ingrowths is described for most of them. Thus we reviewed similarities between transcriptional changes observed during the early stages of NFCs formation and those described during differentiation of TCs to hypothesize about putative signals leading to TC-like differentiation of NFCs with particular emphasis on auxin an ethylene.

Tocopherols (vitamin E) are lipid-soluble antioxidants produced by all plants and algae, and many cyanobacteria, yet their functions in these photosynthetic organisms are still not fully understood. We have previously reported that the vitamin E deficient 2 (vte2) mutant of Arabidopsis thaliana is sensitive to low temperature (LT) due to impaired transfer cell wall (TCW) development and photoassimilate export associated with massive callose deposition in transfer cells of the phloem. To further understand the roles of tocopherols in LT induced TCW development we compared the global transcript profiles of vte2 and wild-type leaves during LT treatment. Tocopherol deficiency had no significant impact on global gene expression in permissive conditions, but significantly affected expression of 77 genes after 48 h of LT treatment. In vte2 relative to wild type, genes associated with solute transport were repressed, while those involved in various pathogen responses and cell wall modifications, including two members of callose synthase gene family, GLUCAN SYNTHASE LIKE 4 (GSL4) and GSL11, were induced. However, introduction of gsl4 or gsl11 mutations individually into the vte2 background did not suppress callose deposition or the overall LT-induced phenotypes of vte2. Intriguingly, introduction of a mutation disrupting GSL5, the major GSL responsible for pathogen-induced callose deposition, into vte2 substantially reduced vascular callose deposition at LT, but again had no effect on the photoassimilate export phenotype of LT-treated vte2. These results suggest that GSL5 plays a major role in TCW callose deposition in LT-treated vte2 but that this GSL5-dependent callose deposition is not the primary cause of the impaired photoassimilate export phenotype.

While superficially simple, endosperm development is a complex, dynamic process. Cereal endosperms contain three major cell types: starchy endosperm, transfer cells and aleurone. The localized accumulation of the END1 transcript in the syncitial endosperm suggests that signals from the maternal placental tissue specify transfer cell type early. Aleurone fate is plastic and requires the continual input of positional cues to maintain cell identity. Starchy endosperm appears to be the default cell type. Mutant patterns suggest that a regulatory hierarchy integrates endosperm development. Requirements for gametic imprinting, maternal : paternal genome ratios and putative chromatin modeling factors indicate the importance of genomic control.

Root hair formation and the development of transfer cells in the rhizodermis was investigated in various existing auxin-related mutants of Arabidopsis thaliana and in the tomato mutant diageotropica. Wild-type Arabidopsis plants showed increased formation of root hairs when the seedlings were cultivated in Fe- or P-free medium. These extranumerary hairs were located in normal positions and in positions normally occupied by nonhair cells, e.g., over periclinal walls of underlying cortical cells. Defects in auxin transport or reduced auxin sensitivity inhibited the formation of root hairs in response to Fe deficiency completely but did only partly affect initiation and elongation of hairs in P-deficient roots. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or the auxin analog 2,4-dichlorophenoxyacetic acid did not rescue the phenotype of the auxin-resistant axr2 mutant under control and Fe-deficient conditions, indicating that functional AXR2 product is required for translating the Fe deficiency signal into the formation of extra hairs. The development of extra hairs in axr2 roots under P-replete conditions was not affected by auxin antagonists, suggesting that this process is independent of auxin signaling. In roots of tomato, growth under Fe-deficient conditions induced the formation of transfer cells in the root epidermis. Transfer cell frequency was enhanced by application of 2,4-dichlorophenoxyacetic acid but was not inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid. In the diageotropica mutant, which displays reduced sensitivity to auxin, transfer cells appeared to develop in both Fe-sufficient and Fe-deficient roots. Similar to the wild type, no reduction in transfer cell frequency was observed after application of the above auxin transport inhibitor. These data suggest that auxin has no primary function in inducing transfer cell development; the formation of transfer cells, however, appears to be affected by the hormonal balance of the plants.

Endosperm transfer cells (ETCs) are positioned at the intersection of maternal and filial tissues in seeds of cereals and represent a bottleneck for apoplasmic transport of assimilates into the endosperm. Endosperm cellularization starts at the maternal-filial boundary and generates the highly specialized ETCs. During differentiation barley ETCs develop characteristic flange-like wall ingrowths to facilitate effective nutrient transfer. A comprehensive morphological analysis depicted distinct developmental time points in establishment of transfer cell (TC) morphology and revealed intracellular changes possibly associated with cell wall metabolism. Embedded inside the grain, ETCs are barely accessible by manual preparation. To get tissue-specific information about ETC specification and differentiation, laser microdissection (LM)-based methods were used for transcript and metabolite profiling. Transcriptome analysis of ETCs at different developmental stages by microarrays indicated activated gene expression programs related to control of cell proliferation and cell shape, cell wall and carbohydrate metabolism reflecting the morphological changes during early ETC development. Transporter genes reveal distinct expression patterns suggesting a switch from active to passive modes of nutrient uptake with the onset of grain filling. Tissue-specific RNA-seq of the differentiating ETC region from the syncytial stage until functionality in nutrient transfer identified a high number of novel transcripts putatively involved in ETC differentiation. An essential role for two-component signaling (TCS) pathways in ETC development of barley emerged from this analysis. Correlative data provide evidence for abscisic acid and ethylene influences on ETC differentiation and hint at a crosstalk between hormone signal transduction and TCS phosphorelays. Collectively, the data expose a comprehensive view on ETC development, associated pathways and identified candidate genes for ETC specification.

Endosperm transfer cells (ETC) are one of four main types of cells in endosperm. A characteristic feature of ETC is the presence of cell wall in-growths that create an enlarged plasma membrane surface area. This specialised cell structure is important for the specific function of ETC, which is to transfer nutrients from maternal vascular tissue to endosperm. ETC-specific genes are of particular interest to plant biotechnologists, who use genetic engineering to improve grain quality and yield characteristics of important field crops. The success of molecular biology-based approaches to manipulating ETC function is dependent on a thorough understanding of the functions of ETC-specific genes and ETC-specific promoters. The aim of this review is to summarise the existing data on structure and function of ETC-specific genes and their products. Potential applications of ETC-specific genes, and in particular their promoters for biotechnology will be discussed.

Transfer-cell-like wall ingrowth deposition is induced in adaxial epidermal cells ofVicia faba L. cotyledons grown in vitro in the presence of high hexose concentrations. We have further characterised this putative transfer cell induction system by examining initiation of secondary-wall ingrowth deposition and expression of sucrose transport-related genes. Wall ingrowth deposition, as wall thickening and scattered papillate ingrowths, was visualised on the outer periclinal walls of adaxial epidermal cells of cotyledons after 1 day in culture. Over the next 2 days, wall deposition increased significantly to form a distinct band of discrete and coalescing ingrowths. Thereafter, further wall deposition was arrested. Densities of Golgi, endoplasmic reticulum, and mitochondria increased concurrently with wall ingrowth deposition. Transcripts of a H+/sucrose symporter (SUT) and a sucrose-binding protein (SBP) were detected by in situ hybridisation in differentiating transfer cells. Antibodies raised against an H+-ATPase immunolocalised evenly around the perimeter of the adaxial epidermal cells in day 1 cotyledons. Thereafter, labelling became increasingly localised to the developing wall ingrowth regions. In contrast, SBP antibodies immunolocalised exclusively to wall ingrowth regions. However, SBP exhibited a transient pattern of expression, being detected only in 2-day-cultured cotyledons. A proton gradient, sufficient to facilitate sulphorhodamine G accumulation, was established by adaxial epidermal cells after 1 day in culture. [14C]sucrose uptake by cotyledons became sensitive to an inhibitor of carrier-mediated transport of sucrose,para-chloromercuribenzene sulfonic acid after 2 days. The initial 37% inhibition of sucrose transport by this compound declined to 5% for cotyledons cultured for 3 days. Collectively, these results suggest that differentiation of the key functional characteristics of transfer cells can be induced in vitro, providing an exciting tool for further exploration of transfer cell development.

The unusual thick-walled cells in contact with host and parasite vessels, first noted by Calvin 1967 in sinkers (structures composed of tracheary elements and parenchyma that originate from parasite bark strands that grow centripetally to the host vascular cambium and become embedded by successive development of xylem) of the mistletoePhoradendron macrophyllum (Englem.) Cockerell, have been investigated by modern methods of microscopy. The wall is thickest in cells abutting large-diameter host vessels, less so against smaller host vessels and those abutting sinker vessels. Transmission electron microscopy reveals the wall to be complex, consisting of a basement primary wall, upon which two developments of secondary-wall material occur. These are represented by lignified thickenings, in the form of flanges, and a labyrinth of wall ingrowths characteristic of a transfer cell. The wall ingrowths occur mostly in the primary-wall regions between the flanges, but when in contact with a large host vessel the ingrowths also differentiate on top of the flanges. Cells with such a transfer cell labyrinth have not been previously reported in the endophytic system of a mistletoe. The cells are confined to the xylary portion of the primary haustorium and sinkers. InP. macrophyllum, however, the cells differ from ordinary transfer cells in that they have differentiated as part of a flange parenchyma cell. This arrangement represents a novel anatomical situation. The name flange-walled transfer cell is used for these cells. The xylem of primary haustorium and sinkers also contain numerous ordinary flange cells. In both flange-walled transfer cells and ordinary flange cells the flanges are lignified and form a reticulate pattern of thickenings, separated by rounded areas of primary pit fields. The extent of development of the flange wall can vary in different parts of a sinker. At the host interface, the existence of a flange-walled transfer cell in direct contact with a vessel reflects a site associated with high loading into the parasite. Similarly, a labyrinth against a sinker vessel indicates a site of unloading from surrounding sinker tissue into the vessel for subsequent longdistance transport within the parasite.

Abnormally thick cell walls of a clonal maize cell line with the labyrinth wall morphology found in transfer cells were analyzed and compared to the relatively thin and even archetypical walls of a sister cell line. Despite a drastic difference in wall morphology between the transfer and archetypical cell walls, the chemical composition of the walls was essentially the same. There were no major differences in the glycosyl residue composition, in the amount of total lipid, and in the amount of total protein. The amounts of wall material released by chemical extraction of cellulosic, hemicellulosic, and pectic fractions were the same for the two types of walls. There were some differences in the protein profile and in the inorganic ion content between the transfer and archetypical walls. These results indicate that profound changes in wall morphology can be brought about in the absence of gross changes in wall composition and suggest that major changes in time- or place-dependent deposition and/or subtle changes in arrangement of rare wall constituents may be responsible.

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (< 100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.

The early interaction of lily roots with the cortical rot pathogen Fusarium oxysporum f.sp. lilii was studied using roots of lily bulblets grown in Hoagland's solution, inoculated with the pathogen, and sampled up to 48 h later. Conidia produced germ tubes within 6 h, which extended towards and into the mucilage covering the root elongation zone, and along and into the anticlinal grooves and middle lamellae of epidermal cells. By 24–48 h, infecting hyphae had reached the periclinal walls and intercellular spaces between the epidermis and the outermost cells of the cortex. Penetration of intercellularly growing hyphae directly across host cell walls was not observed; invasion of the cell lumen only occurred by gradual infringing of hyphae upon successive primary wall layers. Non-cellulosic wall appositions rich in vesicles and covered by a cellulosic protective-like layer were formed in response to approaching hyphae in resistant cv.Connecticut King, but rarely in susceptible cv. Esther which seemed more susceptible to plasmolysis and rot. Finger-like projections of the appositions into the host cell cytoplasm likely represent early stages of transfer cell formation.

The most basal endosperm cells of maize (Zea mays L.) began differentiating into transfer cells in 10 days after pollination (DAP). The thickening and ingrowths forming in the transfer cell wall were slow during 10 and 15 DAP. There were many vesicles, silky and string ball objects in cytoplasm, and the number of mitochondria and rough endoplasm reticulum increased. After 15 DAP, the wall thickening and ingrowths forming in the transfer cells sped up. By 20 DAP, the transfer cell zone had developed, there appeared 65 - 70 rows of cells in width and 3 - 4 layers of cell in depth, the obvious cell wall ingrowths presented strong positive reaction with periodic acid Schiff's reagent. After 20 DAP, no significant change appeared in the shape and structure of the transfer cells, and the transfer cells entered function stage. In the mature kernels (53 DAP), the most basal transfer cells were filled with ingrowths, however, dense cytoplasm was also found in these cells. The nuclei had quite irregular shapes in these cells. Some transfer cells contained black grains and crystals. A black layer formed in the pericarp tissue adjacent to the transfer cell zone. Full development of endosperm transfer cells was important for reduction of kernel abortion and increase of kernel mass.

Chicory root, stem, and leaf tissues have the propensity to regenerate organogenetic nodules following a wounding treatment. Using histological methods, we described this phenomenon on leaves ofCichorium intybus L. cv. ‘Witloof’ cultivatedin vitro. Nodule morphogenesis and bud regeneration from nodules were characterized with the assistance of light and transmission microscopy techniques. Semithin and ultrathin sections of leaves and nodules were prepared from nodule induction to bud regeneration phases. Starch and inulin in differentiating tissues were histochemically detected in semithin and handmade sections. Our study demonstrates that nodules are formed in direct contact with vascular bundles of the leaves. The nodules consist of organized structures: a vascular center is surrounded by a large parenchyma, which is delimited by a periderm of multiple cork cell layers. Inside the vascular center near phloem tissues, a secretory network of articulated pseudolaticifers develops. From parenchymatous cells, in the vicinity of vascular centers, endogenous buds can be induced. In this region, several cords of vessel initials formed the vascular connection between neoformed buds and vascular centers. Simultaneously, transfer cell specialization occurs near the neoformed vascular strands. Vascular centers play a major role in this kind of organogenesis. Sugar metabolism appears to be closely related to nodule morphogenetic events.

The epidermal transfer cells in developingVicia faba L. cotyledons are highly polarized. Extensive wall ingrowths occur on their outer periclinal walls and extend part way down both anticlinal walls. This ingrowth development serves to increase the surface area of the plasma membrane and thus maximize porter-dependent uptake of sugars from the seed apoplasm. In contrast, the inner periclinal walls of these transfer cells do not form wall ingrowths. We have commenced a study of the mechanisms responsible for establishing this polarity by first analysing the microtubule (MT) cytoskeleton in developing transfer cells. Thin sections of fixed cotyledons embedded in methacrylate resin were processed for immunofluorescence microscopy using monoclonal anti-β-tubulin and counterstained with Calcofluor White to visualize wall ingrowths. In epidermal cells of young cotyledons where wall ingrowths were yet to develop, MT labelling was detected around all cortical regions of the cell. However, in cells where wall ingrowths were clearly established, MT labelling was detected almost exclusively in cortical regions adjacent to the wall ingrowths. Little, if any, MT labelling was detected on the anticlinal or inner periclinal walls of these cells. This distribution of MTs was most prominent in cells with well developed wall ingrowths. In these cells, a subpopulation of MTs were also detected emanating from the subcortex and extending towards the wall ingrowth region. The possible role of MT distribution in establishing transfer cell polarity and wall ingrowth formation is discussed.

Temperature-responsive hydration/dehydration changes in surface-grafted poly(N-isopropylacrylamide) (PIPAAm) were utilized for hydrophilic/hydrophobic surface property alterations in cell culture. In this report, we utilized PIPAAm-grafted surfaces to recover confluently-cultured vascular endothelial cells as coherent monolayers from this cell culture substrate and to transfer to new cell culture substrates. For this purpose, we used two different methods to recover and transfer cell monolayer cultures: (1) chitin membranes used as an apical side cell support during cultured cell transfer, allowing cell basal side reattachment to new culture substrates after transfer; and (2) a cell culture insert (porous PET) used as both a support as well as new substrate, allowing basal surfaces of cultured cells to be exposed to the medium after transfer. In both cases, all cells grown on PIPAAm-grafted surfaces detach completely with maintenance of basement membrane-like structure. Recovered cells attach to the second culture surfaces, covering more than 60% of the new substrate, and retain approximately 90% viability and their original function as judged from tissue-type plasminogen activator secretion. This technique could be utilized to prepare novel bioartificial organs as well as cell co-culture systems by multi-layering different cell types to mimic tissue structures for tissue engineering.

In 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.

To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

Immediately 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.

In developing seed of Vicia faba L., solutes imported through the phloem of the coats move symplastically from the sieve elements to a specialized set of cells (the thin-walled parenchyma transfer cells) for release to the seed apoplast. Potassium (K + ) is the predominant cation released from the seed coats. To elucidate the mechanisms of K + efflux from seed coat to seed apoplast, whole-cell currents across the plasma membranes of protoplasts of thin-walled parenchyma transfer cells were measured using the whole-cell patch-clamp technique. Membrane depolarization elicited a time-dependent and an instantaneous outward current. The reversal potential (E R ) of the time-dependent outward current was close to the potassium equilibrium potential (E K ) and it shifted in the same direction as E K upon changing the external K + concentration, indicating that this current was largely carried by an efflux of K + . The activation of the time-dependent outward K + current could be well fitted by two exponential components plus a constant. The instantaneous outward current could also be carried by K + efflux as suggested by ion substitution experiments. These K + outward rectifier currents elicited by membrane depolarization are probably too small to represent the mechanism for the normal K + efflux from seed coat cells. Membrane hyperpolarization more negative than -80 mV activated a time-dependent inward current. K + influx was responsible for the inward current as the current reversed at membrane voltage close to E K and shifted in the same direction as E K when external [K + ] was varied. Activation of this K + inward rectifier current was well fitted with two exponential components plus a constant. A regulating function for this current is suggested.

In this study, we developed a device that could easily, rapidly, and completely transfer cell sheets from one material to another or transplant cell sheets onto the dorsal subcutaneous tissues of rats without leaving residual cells. Because the manipulation is as simple as pipetting, technical expertise is not required to transfer cell sheets very rapidly (the transfer time was 3.7 ± 1.6 s) using the device compared with that of a conventional method using a pipette (430 ± 180 s). After transfer by the device, C2C12 skeletal myoblast sheets showed active cell metabolism, cell viability, and very high production of vascular endothelial growth factor and stromal-derived factor-1α, indicating transfer without cell damage. Cardiac cell sheets after transfer showed spontaneous and synchronous beating, indicating intact cell-cell junctions and ion channel proteins on the cell surface. In addition, the device allowed us to transfer C2C12 cell sheets onto soft, rugged and curved surfaces such as human hands. Furthermore, cardiac cell sheets adhered rapidly and tightly onto the dorsal subcutaneous tissues of rats. This transfer/transplantation device may be a powerful tool in cell sheet-based tissue engineering and regenerative medicine.

The mechanism of carrier-mediated sucrose uptake by the dermal transfer cells of developing Vicia faba L. cotyledons was studied using excised cotyledons and isolated transfer cell protoplasts. Addition of sucrose resulted in a transitory alkalinization of the bathing solution whereas additions of glucose, fructose or raffinose had no effect. Dissipating the proton motive force by exposing cotyledons and isolated transfer cell protoplasts to an alkaline pH, carbonylcyanide m-chlorophenylhydrazone, weak acids (propionic acid and 5,5′-dimethyl-oxazolidine-2,4-dione) or tetraphenylphos-phonium ion resulted in a significant reduction of sucrose uptake. The ATPase inhibitors, erythrosin B (EB), diethylstilbestrol (DES) and N,N′-dicyclohexylcarbodiimide (DCCD) were found to abolish the sucrose-induced medium alkanization as well as reduce sucrose uptake. Cytochemical localization of the ATPase, based on lead precipitation, demonstrated that the highest activity was present in the plasma membranes located in wall ingrowth regions of the dermal transfer cells. The presence of a transplasma-membrane redox system was detected by the extracellular reduction of the electron acceptor, hexacyanoferrate III. The reduction of the ferric ion was coupled to a release of protons. The redox-induced proton extrusion was abolished by the ATPase inhibitors EB, DES and DCCD suggesting that proton extrusion was solely through the H+-ATPase. Based on these findings, it is postulated that cotyledonary dermal transfer cells take up sucrose by a proton symport mechanism with the proton motive force being generated by a H + -ATPase. Sucrose uptake by the storage parenchyma and inner epidermal cells of the cotyledons did not exhibit characteristics consistent with sucrose-proton symport.