Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Relationships between cell-wall β-1,3-endoglucanase activity and auxin-induced elongation in mung bean hypocotyl segments

Summary A β-1,3-endoglucanase (mol. wt. 43,000 and pI around 7) was immunolocalized in outer epidermal cell walls and pericycle walls in the elongation zone of mung bean hypocotyls. Hypocotyl segments infiltrated with anti-β-1,3-endoglucanase antibodies show a clear reduction in their auxin-induced elongation when the auxin treatment was maintained for a long period (15 h). These results suggest that, even in dicotyledons, rupture of some β-1,3-glycosyl linkages might be associated with the cell-wall loosening, which is responsible for auxin-induced elongation, or that some β-1,3-glucanases are indirectly involved in auxin-induced elongation through their auxin-binding properties.     

The role of the epidermis in auxin-induced and fusicoccin-induced elongation of Pisum sativum stem segments

The effects of peeling and wounding on the indole-3-acetic acid (IAA) and fusicoccin (FC) growth response of etiolated Pisum sativum L. cv. Alaska stem tissue were examined. Over a 5 h growth period, peeling was found to virtually eliminate the IAA response, but about 30% of the FC response remained. In contrast, unpeeled segments wounded with six vertical slits exhibited significant responses to both IAA and FC, indicating that peeling does not act by damaging the tissue. Microscopy showed that the epidermis was removed intact and that the underlying tissue was essentially undamaged. Neither the addition of 2% sucrose to the incubation medium nor the use of a range of IAA concentrations down to 10^-8 M restored IAA-induced growth in peeled segments, suggesting that lack of osmotic solutes and supra-optimal uptake of IAA were not important factors over this time period. It is concluded that, although the possibility remains that peeling merely allows leakage of hydrogen ions into the medium, it seems more likely that peeling off the epidermis removes the auxin responsive tissue.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.     

Cytochemical localization of NADH-ferricyanide oxido-reductase in hypocotyl segments and isolated membrane vesicles of soybean

Summary NADH-ferricyanide oxido-reductase activity was demonstrated at the inner (cytoplasmic) aspect of plasma membranes and plasma membrane vesicles from hypocotyls of etiolated soybean (Glycine max L.) seedlings by cytochemical procedures. The plasma membrane-associated activity, observed in both tissue and vesicle preparations, resisted fixation in 0.1 % glutaraldehyde, required the presence of exogenous pyridine nucleotide and was inhibited by adriamycin. With tissue, the activity could be demonstrated only with broken cells where reactants could penetrate freely. With vesicles of plasma membrane origin, activity was seen only with cytoplasmic side out vesicles (fraction E) prepared by free-flow electrophoresis. Activity was observed also on the cytoplasmic surface of the tonoplast and on putative tonoplast vesicles oriented cytoplasmic side out.Recipient of a NSF/CNRS post doctoral fellowship.     

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.     

Effect of galactose on auxin-induced cell elongation in oat coleoptile segments in mannitol solutions

Oat coleoptile segments were treated with or without 10 mM galactose in the presence or absence of 10 μM IAA and various concentrations of mannitol (pre-incubation). Auxin-induced growth was inhibited by galactose. Segments were then transferred to buffer solutions containing or not containing 10 mM galactose (post-incubation). Expansion growth due to rapid water absorption was observed. The expansion growth during the post-incubation was inhibited by galactose when galactose was applied during the post-incubation period or all through the pre- and post-incubation but was not affected by galactose when it was applied only during the pre-incubation. This result indicates that the galactose effect on the expansion growth is due to its inhibitory action during the post-incubation period. Galactose has been reported to be a specific inhibitor for cell wall synthesis. Thus, it is suggested that the expansion growth during post-incubation requires cell wall synthesis and is not just the process of passive water absorption. The primary action of auxin does not seem to require new synthesis of polysaccharides.     

Invertase and auxin-induced elongation in internodal segments of Phaseolus vulgaris

A close positive correlation was observed between segment elongation and the specific activity of soluble acid invertase in stem segments of P. vulgaris incubated for 21 hr in the presence of IAA or of several synthetic auxins and auxin analogues. Optimum concentrations for the stimulation of growth and invertase activity were similar and varied from 10^?6 M (2,4-D) through 10^?5 M (IAA, IBA, α-NAA, β-NAA) to greater than 10^?4 (IPA, PoAA, trans-cinnamic acid). The weak activity of trans-cinnamic acid, a competitive inhibitor of auxin action, may have resulted from cis-trans isomerization during incubation. The concentration of hexose sugars in the segments fell during incubation in the presence of auxin, the greatest decline in hexose concentration occurring in the presence of compounds exhibiting the greatest stimulation of growth.     

Change in XET activities,cell wall extensibility and hypocotyl elongation of soybean seedlings at low water potential

In dark-grown soybean (Glycine max [L.] Merr.) seedlings, exposing the roots to water-deficient vermiculite (_w=–0.36 MPa) inhibited hypocotyl (stem) elongation. The inhibition was associated with decreased extensibility of the cell walls in the elongation zone. A detailed spatial analysis showed xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity on the basis of unit cell wall dry weight was decreased in the elongation region after seedlings were transplanted to low _w. The decrease in XET activity was at least partially due to an accumulation of cell wall mass. Since cell number was only slightly altered, wall mass had increased per cell and probably led to increased wall thickness and decreased cell wall extensibility. Alternatively, an increase in cell wall mass may represent a mechanism for regulating enzyme activity in cell walls, XET in this case, and therefore cell wall extensibility. Hypocotyl elongation was partially recovered after seedlings were grown in low-_w vermiculate for about 80 h. The partial recovery of hypocotyl elongation was associated with a partial recovery of cell wall extensibility and an enhancement of XET activity in the hypocotyl elongation zone. Our results indicate XTH proteins may play an important role in regulating cell wall extensibility and thus cell elongation in soybean hypocotyls. Our results also showed an imperfect correlation of spatial elongation and XET activity along the hypocotyls. Other potential functions of XTH and their regulation in soybean hypocotyl growth are discussed.     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

Dictyosome vesicle production and plasma membrane turnover in auxin-stimulated outer epidermal cells of coleoptile segments fromAvena sativa (L.)

Summary Dark grown coleoptile segments were floated on solutions of IAA alone and of IAA and the secretion inhibitors cytochalasin and monensin. The secretion inhibitors prevented normal elongation of the tissue segments, the monensin inhibition being virtually complete while cytochalasin gave a 40% reduction over the first six hours with little further further elongation in the following 18 hours. Vesicle production was assessed in outer epidermal cells after 6 hours of IAA-stimulated elongation using the vesicle accumulation method following a cytochalasin-block of vesicle transport. The results were compared with the area of plasma membrane required to enable cell elongation to proceed at the observed rate. The area of vesicle membrane delivered to the cell surface exceeded this requirement to such an extent that at least 65% of the delivered membrane must be recycled back into the cytoplasm. Expressed in terms of the whole cell, the plasma membrane turnover rate was found to be once every 200 minutes. It is concluded that limitation of elongation by secretion inhibitors is more likely to reflect a requirement for the vesicle contents than the vesicle membrane. These results are compared with those obtained from other secretory systems using a similar approach.Abbreviations IAA indole acetic acid - DMSO dimethyl sulphoxide - D dictyosome - ER endoplasmic reticulum - V vesicle     

An atypical soybean leucine-rich repeat receptor-like kinase, GmLRK1, may be involved in the regulation of cell elongation

A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro. However, the phosphorylation of GmRLK1 could be induced after incubation with plant protein extracts, suggesting that some plant proteins may interact with GmLRK1 and phosphorylate the protein in vivo. Analyses of the expression profiles of GmLRK1 and its Arabidopsis ortholog At2g36570 revealed that they may be involved in regulation of more fundamental metabolic and/or developmental pathways, rather than a specialized developmental program such as seed development. Our results further indicate that the GmLRK1 and At2g36570 may play a role in the regulation of certain cellular processes that lead to cell elongation and expansion. S. Kim and S.-J. Kim have contributed equally to this work.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures

The time-dependent accumulation of phosphatidyldimethylethanolamine in formaldehyde-induced vesicles obtained from a somatic cell hybrid line was investigated. From a number of considerations including a two-fold enrichment of cholesterol and sphingomyelin it was concluded that these vesicles were derived from the cell plasma membrane.A progressive depletion of phosphatidylcholine, the major vesicle phospholipid, was observed in cells supplemented for various time periods with dimethylethanolamine. This depletion was accompanied by a concomitant increase in the amount of lipid analog.The time-dependent alteration of the phospholipid polar head group in intact cells was almost identical to that observed in isolated plasma membrane vesicles, suggesting a rapid equilibration of the de novo synthesized phospholipid with the cell surface compartment. From the initial velocity rate, the time required for the phosphatidylcholine pool to double was about 12 h.Agarose-linked phospholipase A₂ was used to measure the relative composition of choline- and dimethylethanolamine-phosphoglycerides in the outer surface of vesicles prepared from cells with different degrees of polar head group substitution. The gradual appearance of lysodimethylethanolamine lipid analog in vesicles treated with phospholipase A₂ suggested an asymmetric distribution of the phospholipid between the interior and the exterior part of the vesicle. This asymmetry was maximal up to about 4 h following the addition of dimethylethanolamine to the culture medium and was of a transient nature as the lipid analog accumulated on both sides of the plasma membrane. Based on these measurements a fast followed by a slow translocation component could be distinguished with apparent doubling times of 7 and 43 h for the lipid analog, respectively. As the analog becomes the predominant cellular phospholipid a significant increase in the vesicle lipid fluidity was measured.     

Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments

Branca, C, De Lorenzo, G. and Cervone, F. 1988. Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments. - Physiol. Plant. 72: 499–504.
α-D-galacturonide oligomers (OG) were prepared by partial hydrolysis of sodium polypectate with an homogeneous Aspergillus niger endopolygalacturonase (EC 3.2.1.15). OG, obtained after digestion for 10, 20, 30, 60, 120 min and 24 h, were assayed for their ability to interfere with the IAA-induced elongation of pea ( Pisum sativum L. cv. Alaska) stems. Maximum inhibiting activity was exhibited by oligomers with an approximate degree of polymerization higher than 8. Inhibition by longer OG was much lower, and the products of the 24 h digestion and the unhydrolysed polypectate were ineffective. The addition of OG to pea stems caused a parallel shift to the right of the IAA dose-effect curve. The shift depended on the amount of OG used, showing that oligogalacturonides behave as competitive antagonists of IAA. The presence of OG caused the disappearance of the second maximum of the elongation rate and reduced the first maximum. OG were also tested for their ability to inhibit IAA-induced ethylene evolution of pea stem segments. Maximal inhibition was obtained with OG of the same size as those that interfered with IAA-induced elongation. Inhibition of the auxin action seemed to be specific as OG did not interfere with the activity of gibberellic acid (GA₃) or kinetin. It was concluded that oligogalacturonides strongly interfere with the activity of IAA, although they are by themselves incapable to influence the elongation of pea stem segments directly.     

The effect of calcium antagonists and inhibitors of secretory processes on auxin-induced elongation and fine structure ofPisum sativum stem segments

Summary The treatment of dark grown pea stem segments with chelators of divalent cations (EGTA, EDTA, CTC), various Ca²⁺ antagonists (LaCl₃, A-23187, verapamil) and inhibitors of secretory processes (monensin, CB) reduced elongation in the presence of indole-3-acetic acid (IAA). Generally the inhibition increased with increasing concentrations of the substances. The timing of the responses can be correlated with maximum auxin-stimulated secretion of cell wall material. Examination of cell ultrastructure showed that changes in dictyosome activity could explain a reduced deposition of cell wall material and so cause inhibition of elongation.The inhibitors affected the morphology and vesiculation of the dictyosomes, and the appearance of the plasma membrane, ER and mitochondria in different ways. The most pronounced effects on ultrastructure resulted from monensin and LaCl₃ treatments with the dictyosomes being most affected; large vesicles appeared in the cytoplasm. Less pronounced effects on cell structure were seen in EGTA, A-23187 and verapamil treated tissue. The effects on the dictyosomes are considered to be due to disturbances of Ca²⁺ and other ionic levels within the cells.Abbreviations ATPase adenosine triphosphatase - CB cytochalasin B - CTC chlorotetracycline - DMSO dimethyl sulphoxide - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(B-amino ethyl ether)N,N¹-tetraacetic acid - ER endoplasmic reticulum - ver verapamil     

Calcium and gibberellin-induced elongation of lettuce hypocotyl sections

The relationship between calcium ions and gibberellic acid (GA₃)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood (non-flow-past) or by switching solution flowing past the base of hypocotyl (flow-past), resulting in differences in growth behavior. Drops of CaCl₂ added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA₃. A continuous H₂O flow resulted in growth rates comparable to those in response to GA₃. Addition of CaCl₂ to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl₂ concentration. The growth rate was found to be a function of CaCl₂ concentration. When a constant CaCl₂ concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl₂, causing an increase in growth rate similar to that found previously when GA₃ was added to hypocotyls grown in H₂O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca²⁺ by the hypocotyl cells.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid - GA gibberellin - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Stimulation of onion root elongation by ascorbate and ascorbate free radical inAllium cepa L.

Summary We report that ascorbate free radical stimulates onion root growth at 15 °C and 25 °C. The fully reduced form, ascorbate, also stimulates root elongation if culture conditions allow its oxidation. When ascorbate oxidation was inhibited, no stimulation of root growth was found. The effect of the fully oxidized form, dehydroascorbate, was inhibitory. We show also that ascorbate free radical generated by ascorbate oxidation, is reduced back probably by a transplasmalemma reductase. These results are discussed on the basis of an activation of a transplasma membrane redox system likely involved in processes related to cell growth.Abbreviations AFR ascorbate free radical - ASC ascorbate - DHA dehydroascorbate