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Studies in cell wall bound invertase mutants indicate that the promoter of the transfer cell-specific transcription factor, ZmMRP - 1 , is modulated by the carbohydrate balance. Transfer cells are highly specialized plant cells located at the surfaces that need to support an intensive exchange of nutrients, such as the entrance of fruits, seeds and nodules or the young branching points along the stem. ZmMRP-1 is a one-domain MYB-related transcription factor specifically expressed at the transfer cell layer of the maize endosperm. Previous studies demonstrated that this factor regulates the expression of a large number of transfer cell-specific genes, and suggested that ZmMRP-1 is a key regulator of the differentiation of this tissue. The expression of this gene is largely dominated by positional cues, but within the ZmMRP-1 expressing cells the promoter appears to be modulated by sugars. Here we have investigated in vivo this modulation. Using maize and Arabidopsis mutants for cell wall invertase genes, we found that the absence of cell wall invertase activity is a major inductive signal of the ZmMRP-1 expression.

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.

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.

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.

Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a K-m of 45 and 54 mu M Fe-III-EDTA and a V-max of 42 and 33 nmol Fe2+.(g FW)(-1).min(-1) for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex.

Two experimental systems were developed to study the uptake of sucrose by the dermal transfer cells of developing cotyledons of Vicia faba L. First, the in-vivo state was approximated by short-term (10 min) incubation of whole cotyledons in [ 14 C]sucrose solutions. Under these conditions, a minimum of 67% of the 14 C label entered the dermal transfer cell complex. Of this, at least 40% crossed the plasma membranes of the epidermal transfer cells. Second, a protocol was developed to enzymatically isolate and purify dermal transfer cell protoplasts. The yields of the transfer cell protoplasts were relatively low and their preparation incurred a significant loss of plasma membrane. However, the protoplasts remained viable up to 24 h following purification and proved to be a suitable system to verify transport properties observed with whole cotyledons. Using these two experimental systems, it was established that [ 14 C]sucrose uptake by the dermal transfer cells exhibited features consistent with mediated energy-dependent transport. This included saturation kinetics, competition for uptake between structurally similar molecules, and inhibition of uptake by p-chloromercuribenzenesulfonic acid and several other metabolic inhibitors. For comparative purposes, sugar uptake by the storage parenchyma of the Vicia cotyledons was also examined. In contrast to the dermal transfer cell complex, sucrose uptake by the storage parenchyma displayed characteristics consistent with simple diffusion.

The subcellular distribution of membrane-bound ATPases was compared among terrestrial plants, seagrasses and marine algae by cytochemical techniques. High ATPase activity was detected in the copiously invaginated plsma membrane that was characteristic of transfer cells but not in the tonoplast of epidermal cells in mature leaves of seagrasses. Magnesium- or Ca(2+)-dependent ATPase activity was induced together with the characteristics of transfer cells during the development of leaf tissues able to resist seawater. Northern hybridization revealed the effective induction of the synthesis of mRNA for plasma-membrane H(+)-ATPase during the development of leaves. Such high ATPase activity was not detected in the smooth plasma membranes of marine macro-algae but was found in the membranes of some cytoplasmic vesicles or microvacuoles, providing evidence of the excretion of salts by exocytosis. It appears, therefore, that two essentially different methods for excreting excess salts have developed separately in these two classes of marine plants. The evolution of mechanisms of salt tolerance in the plant kingdom is discussed in terms of the differential subcellular distribution of ATPase activity.

Minor-vein anatomy, sugar content, sugar synthesis, and translocation were studied in mature leaves of nine members of the Scrophulariaceae to determine if there is a correlation between companion-cell type and class of sugar translocated. Three types of companion cell were found: intermediary cells with extensive plasmodesmatal connections to the bundle sheath; transfer cells with wall ingrowths and few plasmodesmata; and ‘ordinary’ companion cells with few plasmodesmata and no wall ingrowths. Alonsoa warscewiczii Regal., Verbascum chaixi Vill., and Mimulus cardinalis Dougl. ex. Benth. have intermediary cells and ordinary companion cells in the minor veins. These plants synthesize large amounts of raffinose and stachyose as well as sucrose. Nemesia strumosa Benth., and Rhodochiton atrosanguineum Zucc. have both intermediary cells and transfer cells and make proportionately less raffinose oligosaccharide than the species above. In N. strumosa, a single sieve element may abut both an intermediary cell and a transfer cell. The minor veins of Asarina scandens (Cav.) Penn. have transfer cells and what appear to be modified intermediary cells that have fewer plasmodesmata than other species, and occasional wall ingrowths. Asarina scandens synthesizes little raffinose or stachyose. Cymbalaria muralis P. Gaertn et al. and Linaria maroccana Hook.f. have only transfer cells and Digitalis grandiflora Mill. has only ordinary companion cells; these species make a trace of galactinol and raffinose, but no stachyose. Translocation experiments indicate that there is long-distance movement of raffinose oligosaccharide in these plants, even when it is synthesized in very small quantities in the leaves. We conclude that intermediary cells are as distinct a cell type as the transfer cell. In contrast to transfer cells, which are specialized for uptake of solute from the apoplast, intermediary cells are specialized for symplastic transfer of photoassimilate from the mesophyll and for synthesis of raffinose oligosaccharide. This supports our contention that raffinose oligosaccharide synthesis and symplastic phloem loading are mechanistically linked (Turgeon and Gowan 1990, Plant Physiol. 94, 1244–1249). Minor-vein anatomy and sugar synthesis may be useful characters in determining the phylogenetic relationships of plants in this family.

Tubular evaginations of the plasmalemma of the scutellar epithelial cells of barley are described. The evaginations are similar to those present at other sites where solute flux occurs for a limited period only and wall development of the transfer-cell form has not occured. Differential uptake of the fluorescent dyes fluorescein, which moves into the symplast, and 8-anilino-1-naphthalene sulphonic acid, which remains in the apoplast only, indicates that the scutellar epithelial cells contain the boundary between the apoplast and symplast. We suggest that i) the plasmalemma evaginations, which have a specific form and localisation, may be referred to as plasmatubules rather than by the general term plasmalemmasome, and that ii) the plasmatubules may act in membrane amplification in a short-term structural modification which is an alternative to transfer cells.

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461-465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.

Abscisic acid (ABA) treatment of secondary protonema of Physcomitrium pyriforme Brid in the presence of sucrose does not prevent cell division but results in shorter cells with vesicular cytoplasm and an accumulation of lipid. When transferred to sucrose medium without ABA and with low irradiance isodiametric intercalary cells are cut off which give rise to apogamous sporophytes either directly or after the formation of a small amount of callus. The organization of the cells leading up to the apogamous sporophyte is described. The cells initiating the sporophyte develop dense cytoplasm and the walls become labyrinthine and callosed, but they do not form any recognizable placenta. It is proposed that labyrinthine walls are a consequence of a perturbation of cell wall metabolism as growth changes from gametophytic to sporophytic. The use of the term “transfer cell” for this kind of cell is questioned and the need for a causal approach to the investigation of labyrinthine walls is stressed.

Helianthus annuus L. responds to iron deficiency by forming a thickened cortex and abundant root hairs in a zone near the root apex that corresponds to the primary developmental stage. Cytological investigations revealed that within 24 to 48 h of iron deficiency most of the peripheral cells differentiate into transfer cells. The wall labyrinth is always situated on the peripheral walls that face the external medium. The cytoplasm of these cells is characterized by numerous mitochondria, extensive rough endoplasmic reticulum, and large leucoplasts containing protein bodies. These observations are discussed in relation to the fact that Helianthus, as an "iron efficient" plant, responds physiologically to iron deficiency by extrusion of H(+), production of reducing substances, and a steep increase in the uptake efficiency of Fe.

The development of the placental complex including the gametophyte vaginula and the bulbous foot of the sporophyte in the liverwort Targionia hypophylla L. (Marchantiales) was studied by transmission electron microscopy. The vaginula and foot are separated by an intervening space and each consist of parenchymatous cells without intercellular spaces. Transfer cells begin to differentiate at the gametophyte-sporophyte interface just prior the onset of meiosis. While a single epidermal transfer-cell layer has developed in the foot by the end of meiosis, a multilayered pattern of transfer cells is formed in the vaginula. Gametophyte transfer cells have wall labyrinths which decrease in complexity with distance from the foot, lack plasmodesmata, and show signs of degeneration in the proximity of the foot. During meiosis, amyloplasts of both vaginula and foot lack starch and develop some thylakoid grana. In the subsequent stage of spore maturation, obliteration of the wall labyrinth occurs in both gametophyte and sporophyte transfer cells. The developmental pattern of the placental complex in Targionia is discussed in relation to that of mosses.