Osmotic induction of fluid-phase endocytosis in onion epidermal cells

A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells.     

Root-to-shoot signalling: apoplastic alkalinization,a general stress response and defence factor in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

Summary. We used a noninvasive microprobe technique to record in substomatal cavities of barley leaves the apoplastic pH response to different stress situations. When K⁺ (or Na⁺) activity at the roots of intact plants was increased from 1 to 50 mM, the leaf apoplastic pH increased by 0.4 to 0.6 units within 8 to 12 min when stomata were open, and within 15 to 20 min when stomata were closed. This reaction was accompanied by a correlative increase in K⁺ activity. Addition of 1 μM abscisic acid caused an apoplastic alkalinization of 0.5 to 0.8 units, and low temperatures (4 °C) increased pH by 0.2 to 0.3 units. Addition of 100 mM sorbitol or pH changes in the range 4.0 to 7.9 had no effect, ruling out that osmotic potential and/or pH is the carried signal. On detached leaves, the same treatments yielded qualitatively similar results, suggesting that the xylem is the most likely signal path. Following the attack of powdery mildew, the apoplastic pH of barley leaves substantially increases. We demonstrate that in susceptible barley, pretreatment (soil drench) with the resistance-inducing chemical benzo- (1,2,3)thiadiazole-7-carbothioic acid S-methyl ester markedly enhances this pH response. This is consistent with previous finding that apoplastic alkalinization is related to the degree of resistance towards this fungus. Correspondence and reprints: Botanisches Institut I, Justus-Liebig-Universit?t, Senckenbergstra?e 17, 35390 Gie?en, Federal Republic of Germany.     

Different pH-dependences of K+ channel activity in bundle sheath and mesophyll cells of maize leaves

The isolation of bundle sheath protoplasts from leaves of Zea mays L. for patch clamp whole-cell experiments presents special problems caused by the suberin layer surrounding these cells. These problems were overcome by the isolation technique described here. Two different types of whole-cell response were found: a small response caused by MB-1 (maize bundle sheath conductance type 1) which was instantaneously activated, and another caused by MB-2 (maize bundle sheath conductance type 2) consisting of an instantaneous response (maize bundle sheath K⁺ instantaneous current type 2; MB-KI2) similar to but stronger than the current through MB-1 plus a small time-dependent outward rectifying component (maize bundle sheath activated outward rectifying current; MB-AOR) with voltage-dependent delayed activation. The occurrence of MB-AOR was often accompanied by a smaller contribution from an inward rectifying channel at negative potentials. Activation of MB-2 required ATP. It is suggested that MB-1 and MB-2 are related to bundle sheath cells with and without direct contact with the xylem vessels. In mesophyll cells, only one type of response caused by MM-2 (maize mesophyll conductance type 2) was found with an instantaneous (maize mesophyll K⁺ instantaneous current type 2, MM-KI2) and a voltage-dependent delayed component (maize mesophyll activated outward rectifying current, MM-AOR). The most striking difference between bundle sheath and mesophyll cells was the pH dependence of K⁺ uptake. At pH 7.2, uptake of K⁺ by MB-2 was identical to that by MM-2 over the whole voltage range. However, acidification stimulated K⁺ conductance in bundle sheath cells, whereas a decrease was found for MM-2. At pH 6.15, the bundle sheath channel MB-2 had more than a 10-fold higher K⁺ uptake at positive and negative potentials than MM-2. The channel MB-1, too, was stimulated by low pH. This seems to indicate a putative role for MB-1 and MB-2 in charge balance during uptake of nutrients via cotransport from the xylem into the symplasm. Received: 23 April 1999 / Accepted: 19 July 1999     

The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H₂O₂ levels in lignifying tissues, whose capacity to produce and to accumulate H₂O₂ is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H₂O₂ in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O₂ to remove ASC, it could regulate O₂ availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H₂O₂ production in lignifying tissues) at substrate level, by controlling the tension of O₂. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.     

Construction and restructuring of the cellulose-xyloglucan framework in the apoplast as mediated by the xyloglucan-related protein family—A hypothetical scheme

The cellulose-xyloglucan framework functions as the load-bearing structure of the cell wall and constrains cell shape in plants. Xyloglucan cross-links which underpin the framework structure can be modified by transferases and hydrolases encoded by xyloglucan-related protein (XRP) family genes. These enzymes are considered to play critical roles in the construction and restructuring of the three-dimensional structure of the plant cell wall. Although analyses of their protein structures and gene-expression profiles for individual members of XRPs have disclosed their potentially divergent roles in plants, the biochemical reactions catalyzed by individual XRPs and their biochemical implications remain to be clarified. This review focuses on the XRP-catalyzed chemical processes occurring in the apoplast and considers the biochemical steps involved in the construction and restructuring of the cellulose-xyloglucan framework, an ensemble of chemical reactions that are more complicated than commonly supposed.     

Compartmentation of Assimilate Fluxes in Leaves

Abstract: Sugar levels in the apoplast of assimilate exporting leaves were studied in two groups of plant species with contrasting structures of companion cells in minor veins. These species are termed either "symplastic" (with intermediary cells) or "apoplastic" (with transfer or ordinary cells). Sugars were measured in intercellular washing fluid after extracting the apoplast by an infiltration-centrifugation technique. During the course of a day, sugar contents in the apoplast were, in general, lower in species with intermediary cells than in species with transfer or ordinary cells. In "symplastic" species, apoplastic sucrose concentrations were between 0.3 and 1 mM. In "apoplastic" species with transfer cells, they ranged between 2 and 6 mM. Apoplastic hexose contents were between 0.3 and 1 mM irrespective of presumed transport mode. "Symplastic" and "apoplastic" plants differed markedly in their response to a'translocation block. In "symplastic" plants, inhibition of assimilate export left apoplastic concentrations of sucrose and hexoses unchanged, whereas in "apoplastic" plants sugar levels increased, the maximal increase being observed with sucrose. In these plants, concentrations of sucrose were two to six times higher in the apoplast under export inhibition than in control leaves. The data suggest a different role of the leaf apoplast in the compartmentation and export of assimilates in the two plant groups under study.     

Removal of root apices enables study of direct toxic effects of aluminum on rice (Oryza sativa L.) leaf cells

Root growth inhibition is a well-known symptom of aluminum (Al) toxicity in intact plants, mainly because the mechanisms of Al exclusion or resistance that operate outside the root endodermis prevent the ascent of this metal from roots into shoots. This work presents a new method to better understand the direct effects of this metal on rice leaves. For this, Al-sensitive and tolerant rice genotypes, having had their root apices removed, were incubated in AlCl₃ solutions to evaluate unblocked metal ascension toward the leaf cells. To avoid regrowth and closing of roots, apices were removed daily and also verified for a lack of tyloses production and consequent obstruction in tracheal elements. Thus, seedlings of both cultivars, which were root apex-free, accumulated differentially high amounts of Al in the leaves, highlighting the importance of mechanisms of Al exclusion or resistance in roots of intact plants. Also, Al moved freely toward leaf cells, clearly inducing necrosis-like mesophyll alterations in both genotypes. Added ultrastructural analyses revealed significant cytoplasmatic damage, mainly in chloroplasts. These results suggest that differential responses to Al sensitivity/tolerance preserved in roots between the genotypes studied are also expressed in leaves. Therefore, this method allowed for development of a possible biological model suitable for investigating the direct effect of Al on cells and, alternatively, other compounds in plant leaf cell physiology.     

Leaf structure in relation to solute transport and phloem loading in Zea mays L.

Small and intermediate (longitudinal) vascular bundles of the Zea mays leaf are surrounded by chlorenchymatous bundle sheaths and consist of one or two vessels, variable numbers of vascular parenchyma cells, and two or more sieve tubes some of which are associated with companion cells. Sieve tubes not associated with companion cells have relatively thick walls and commonly are in direct contact with the vessels. The thick-walled sieve tubes have abundant cytoplasmic connections with contiguous vascular parenchyma cells; in contrast, connections between vascular parenchyma cells and thin-walled sieve tubes are rare. Connections are abundant, however, between the thin-walled sieve tubes and their companion cells; the latter have few connections with the vascular parenchyma cells. Plasmolytic studies on leaves of plants taken directly from lighted growth chambers gave osmotic potential values of about-18 bars for the companion cells and thin-walled sieve tubes (the companion cell-sieve tube complexes) and about-11 bars for the vascular parenchyma cells. Judging from the distribution of connections between various cell types of the vascular bundles and from the osmotic potential values of those cell types, it appears that sugar is actively accumulated from the apoplast by the companion cell-sieve tube complex, probably across the plasmalemma of the companion cell. The thick-walled sieve tubes, with their close spatial association with the vessels and possession of plasmalemma tubules, may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream. The transverse veins have chlorenchymatous bundle sheaths and commonly contain a single vessel and sieve tube. Parenchymatic elements may or may not be present. Like the thick-walled sieve tubes of the longitudinal bundles, the sieve tubes of the transverse veins have plasmalemma tubules, indicating that they too may play a role in retrieval of solutes entering the leaf apoplast in the transpiration stream.     

Effects of fusicoccin and indole-3-acetic acid on the levels of ascorbic acid and dehydroascorbic acid in the apoplast during elongation of epicotyl segments of Vigna angularis

The levels of ascorbic acid (AA) and dehydroascorbic acid (DHA) in the apoplast of epicotyl segments from Vigna angularis L. cv. Erimoshouzu decreased to nearly zero and about 35%, respectively, of their initial levels, 3 h after the preparation of the epicotyl segments. The decreased level was kept nearly constant between 3 and 7 h. Fusicoccin (FC) and indole-3-acetic acid (IAA) slightly amplified the initial decrease in the level of AA, but suppressed the initial decrease in the level of DHA while enhancing elongation growth. During incubation for 3 and 7 h, FC then increased the levels of both AA and DHA, whereas IAA did so only with DHA. By the addition of FC 4 h after the start of incubation, the levels of both AA and DHA were also increased. The uncoupler carbonylcyanide m -chlorophenyl hydrazone increased the levels of both AA and DHA in the apoplast inhibiting elongation growth. These results suggest that the electrochemical proton gradient across the plasma membrane is one of the factors that control the apoplastic levels of AA and DHA.