Effect of HCO - 3 concentration in the absorption solution on the energetic coupling of H+-cotransports in roots of Zea mays L.

The effect of HCO ₃ ^- on ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO₂ partial pressure in air bubbled through the solution. The surface pH shift in the vicinity of the outer surface of the plasmalemma induced by active H⁺ excretion was estimated using the initial uptake rate of acetic acid as a pH probe (Sentenac and Grignon (1987) Plant Physiol. 84, 1367). Acetic acid and orthophosphate uptake rates and NO ₃ ^- accumulation were slowed down, while ⁸⁶Rb⁺ uptake and K⁺ accumulation rates were increased by HCO ₃ ^- . These effects were similar to those induced by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid/2-amino-2-(hydroxymethyl)-1,3-propanediol (Hepes-Tris). They were more pronounced when the H⁺ excretion was strong, were rapidly reversible and were not additive to those of Hepes-Tris. The hypothesis is advanced that the buffering system CO₂/H₂CO₃/HCO ₃ ^- accelerated the diffusion of equivalent H⁺ inside the cell wall towards the medium. This attenuated the surface pH shift in the vicinity the plasma membrane and affected the coupling between the proton pump and cotransport systems.Abbreviations FW fresh weight - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - J_aa acetic acid influx - J_K ⁺ K⁺ influx - J_Pi orthophosphate influx - Mes 2-(N-morpholino)ethanesulfonic acid - pCO₂ CO₂ partial pressure - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Potassium stimulation of corn root plasmalemma ATPase : I. Hydrolytic activity of native vesicles and purified enzyme

Potassium stimulation of the plasmalemma (Zea mays L. var Mona) was studied by using a constant ionic strength to prevent indirect stimulation by the electrostatic effect of K⁺ salts. The transmembrane electrochemical H⁺ gradient was eliminated by using gramicidin. In these conditions, K⁺ stimulation was attributable to a direct effect of the cation on plasmalemma proteins. We used both native vesicles isolated on a sucrose cushion, and solubilized and purified ATPase from phase-partitioned plasmalemma, according to the method of T. Nagao, W. Sasakawa, and T. Sugiyama ([1987] Plant Cell Physiol 28: 1181-1186). The purified enzyme had a high specific activity (15 micromoles per minute per milligram protein), but was only about 20% stimulated by K⁺. In both preparations, potassium (in the range around 1 millimolar) specifically decreased two-fold the vanadate inhibition constant, and increased the maximum rate of ATP hydrolysis. In plasmalemma vesicles, the Eadie-Scatchard graph of the K⁺-dependent ATPase activity as a function of K⁺ concentration was linear only at constant ionic strength. The purified ATPase preparation appeared as two closely spaced bands in the 100 kilodalton region with isoelectric point about 6.5. Nevertheless, this biochemical heterogeneity seems unlikely to be related to K⁺ stimulation, since K⁺ modified neither the pH optimum of the activity (pH 6.5) nor the monophasic kinetics of the vanadate inhibition, in both native plasmalemma and purified enzyme preparation.     

Sulphate/proton cotransport in plasma-membrane vesicles isolated from roots of Brassica napus L.: increased transport in membranes isolated from sulphur-starved plants

The characteristics of sulphate uptake into right-side-out plasma-membrane vesicles isolated from roots of Brassica napus L., Metzger, cv. Drakkar, and purified by aqueous polymer two-phase partitioning, were investigated. Sulphate uptake into the vesicles was driven by an artificially imposed pH gradient (acid outside), and could be observed for 5–10 min before a plateau was reached and no further net uptake occurred. The uptake was partially inhibited in the presence of depolarizing agents and little uptake was observed in the absence of an imposed pH gradient. Uptake was strongly pH-dependent, being greatest at more acidic pH. After imposition of a pH gradient, the capacity for uptake decreased slowly (t1/2>10 min). The uptake had a high-affinity component which was strongly dependent on the external proton concentration (K _m=10μM at pH 5.0, 64 μM at pH 6.5). The K _m for protons varied from 0.4–1.9 μM as the sulphate concentration was reduced from 33 to 1 μM. A low-affinity component was observed which could be resolved at low temperatures (0 °C). Microsomal membranes that partitioned into the lower phase of the two-phase system gave no indication of high-affinity sulphate transport. Sulphate uptake into plasma-membrane vesicles isolated from sulphur-starved plant material was approximately twofold greater than that observed in those isolated from sulphate-fed plant material. Isolated vesicles therefore mirror the well-known in-vivo response of roots, indicating an increase in the number of transporters to be, at least in part, the underlying cause of derepression.     

Distribution of NO3− reduction between roots and shoots of peachtree seedlings as affected by NO3− uptake rate

The distribution of NO₃^? reduction between roots and shoots was studied in hydro-ponically-grown peach-tree seedlings (Prunus persica L.) during recovery from N starvation. Uptake, translocation and reduction of NO₃^?, together with transport through xylem and phloem of the newly reduced N were estimated, using ¹⁵N labellings, in intact plants supplied for 90 h with 0.5 mM NH₄⁺ and 0.5, 1.5 or 10 mM NO₃^?. Xylem transport of NO₃^? was further investigated by xylem sap analysis in a similar experiment. The roots were the main site of NO₃^? reduction at all 3 levels of NO₃^? nutrition. However, the contribution of the shoots to the whole plant NO₃^? reduction increased with increasing external NO₃^? availability. This contribution was estimated to be 20, 23 and 42% of the total assimilation at 0.5, 1.5 and 10 mM NO₃^?, respectively. Both ¹⁵N results and xylem sap analysis confirmed that this trend was due to an enhancement of NO₃^? translocation from roots to shoots. It is proposed that the lack of NO₃^? export to the shoots at low NO₃^? uptake rate resulted from a competition between NO₃^? reduction in the root epidermis/cortex and NO₃^? diffusion to the stele. On the other hand, net xylem transport of newly reduced N was very efficient since ca 70% of the amino acids synthesized in the roots were translocated to the shoots, regardless of the level of NO₃^? nutrition. This net xylem transport by far exceeded the net downward phloem transport of the reduced N assimilated in shoots. As a consequence, the reduced N resulting from NO₃^? assimilation, principally occurring in the roots, was mainly incorporated in the shoots.     

Immunolocalization of some plasmatic proteins in basement membranes during earliest rat morphogenesis with special reference to the gonadal differentiation

Summary Rat albumin, transferrin, angiotensinogen and T kininogen were examined immunohistochemically in the epithelial basement membranes (BMs) during the earliest rat morphogenesis. As a specific marker for BMs, laminin was used. Albumin and transferrin immunostaining appeared as early as the 11th day of gestation in all epithelial BMs. In 13-day-old mesonephric-gonadal complex, just after the onset of the sexual cord differentiation, all BMs were weakly stained. One day later, a stronger immunoreactivity was distributed along the coelomic epithelium, the Wolffian duct, the mesonephric tubules, the differentiating sexual cords and the blood vessels. The epidermal BM and all epithelial BMs of differentiating organs are also immunoreactive. The accumulation of albumin and transferrin in the BMs is probably the result of a strong release of these two major liver proteins in the embryonic blood and their diffusion in extracellular spaces. At these stages, the lack of angiotensinogen and T kininogen BM labeling is consistent with their low hepatic and plasmatic concentrations. During embryogenesis, some plasma proteins are probably trapped in the epithelial BMs and not produced by local cells.     

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

Summary The purified (H⁺ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H⁺-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H⁺)ATPase molecules were closely associated with liposomes, exhibiting a high H⁺-pumping activity (150,000% quenching· min^–1·mg^–1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H⁺)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H⁺)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (inside-out orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H⁺)ATPase could also functionally insert into bilayer from PCPEPG or PCPEPI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H⁺)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H⁺)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation     

Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima.

Cakile maritima is a local oilseed halophyte exhibiting potential for secondary metabolite production. In the present study, plant growth, leaf polyphenol content and antioxidant activity were comparatively analyzed in two C. maritima Tunisian accessions (Jerba and Tabarka, respectively sampled from arid and humid bioclimatic stages) under salt constraint. Three-week-old plants were subjected to 0, 100, and 400 mM NaCl for 28 days under glasshouse conditions. A significant variability in salt response was found between both accessions: while Tabarka growth (shoot biomass, leaf expansion) was significantly restricted at 100 and 400 mM NaCl, compared to the control, Jerba growth increased at 100mM before declining at 400 mM NaCl. The better behaviour of Jerba salt-challenged plants, compared to those of Tabarka, may be related to their higher polyphenol content (1.56- and 1.3-fold the control, at 100 and 400 mM NaCl respectively) and antioxidant activity (smaller IC(50) values for both 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging), associated with lower leaf MDA accumulation (ca. -66% of the control at 100mM NaCl). Taken together, our findings suggest that halophytes may be interesting for production of antioxidant compounds, and that the accession-dependent capacity to induce antioxidative mechanisms in response to salt, may result in a corresponding variability for growth sustainability.