Influence of K + on the Coupling Between ATP Hydrolysis and Proton Transport by the Plasma Membrane H +_ATPase from Soybean Hypocotyls

The plasma membrane vesicles were purified from soybean (Glycine max L.) hypocotyls by two_phase partitioning methods. The stimulatory effects of K + on the coupling between ATP hydrolysis and proton transport by the plasma membrane H +_ATPase were studied. The results showed that the proton transport activity was increased by 850% in the presence of 100 mmol/L KCl, while ATP hydrolytic activity was only increased by 28.2%. Kinetic studies showed that K m of ATP hydrolysis decreased from 1.14 to 0.7 mmol/L, while V max of ATP hydrolysis increased from 285.7 to 344.8 nmol Pi·mg -1 protein·min -1 in the presence of KCl. Experiments showed that the optimum pH was 6.5 and 6.0 in the presence and absence of KCl, respectively. Further studies revealed that K + could promote the inhibitory effects of hydroxylamines and vanadates on the ATP hydrolytic activity. The above results suggested that K + could regulate the coupling between ATP hydrolysis and proton transport of the plasma membrane H +_ATPase through modulating the structure and function of the kinase and phosphatase domains of the plasma membrane H +_ATPase.

Relationship Between H^+-ATPase Activity and Fluidity of Tonoplast in Barley Roots Under NaCl Stress

H +_ATPase activity of tonoplast in roots of Hordeum vulgare L. cv. 'Tanyin 2' (salt_tolerant cultivar) increased when the roots were exposed to 50-200 mmol/L NaCl for 2 d, and decreased when NaCl concentration was increased to 600 mmol/L. In 'Kepin 7' (salt_sensitive cultivar), tonoplast H +_ATPase activity in roots also increased at lower levels of NaCl (50-100 mmol/L), but decreased at higher levels of NaCl (200-600 mmol/L). Tonoplast fluidity in roots of 'Tanyin 2' decreased at 50-200 mmol/L NaCl, and increased significantly at 600 mmol/L NaCl. Under salt stress, the change of tonoplast fluidity was identical with that of the ratio of unsaturated fatty acids to saturated fatty acids in tonoplast lipid of barley roots. It is proposed that the increase of tonoplast fluidity due to increased degree of unsaturation of fatty acids is one of the reasons leading to the decrease of H +_ATPase activity under higher level of NaCl stress.

Ca^(2+)-homeostasis Differs Between Plant Species with Different Cold-tolerance at 4 ℃ Chilling

A comparative study was carried out on the EM_cytochemical localization of calcium and Ca 2+ _ATPase activity in the suspension_cultured cells between the chilling_sensitive maize ( Zea mays L. cv. Black Mexican Sweet) and chilling_insensitive Trititrigia ( Triticum sect. Trititrigia mackey) at 4 ℃ chilling. When maize and Tyititrigia cells were cultured at 26 ℃, electron microscopic observations revealed that the electron_dense calcium antimonate deposits, an indication of the calcium localization, were localized mainly in the vacuoles, and few was found in the cytosol and nuclei. The electron_dense cerium phosphate deposits, an indication of Ca 2+ _ATPase activity, were abundantly distributed on the plasma membrane (PM). When the cells from both species were cultured at 4 ℃ for 1 and 3 h, an elevation of Ca 2+ level in the cytosol and nuclei was observed, whereas the cerium phosphate deposits on the PM showed no quantitative difference from those of the 26 ℃_cultured cells, indicating that the enzymatic activities were not altered during these chilling periods. However, there was a distinct difference in the dynamics of the Ca 2+ distribution and the PM Ca 2+ _ATPase activity between maize and Trititrigia when chilled at 4 ℃ for 12, 24 and 72 h. In maize cells, a large number of Ca 2+ deposits still existed in the cytosol and nuclei, and the PM Ca 2+ _ATPase became less and less active, and even inactive at all. In Trititrigia cells, the increased cytosolic and nuclear Ca 2+ ions decreased after 12 h chilling. By chilling up to 24 and 72 h, the intracellular Ca 2+ concentration had been restored to a similar low level as those of the warm temperature_cultured cells, while the activity of the PM Ca 2+ _ATPase maintained high. The transient cytosolic and nuclear Ca 2+ increase and the activities of PM Ca 2+ _ATPase during chilling are discussed in relation to plant cold hardiness.

Protective Effects of Glycinebetaine on Brassica chinensis Under Salt Stress

Brassica chinensis L. were foliarly applied with glycinebetaine (GB), as this species is unable to synthesis GB and sensitive to osmotic stress such as salt. The exogenous GB was easily absorbed and transported by the leaf of B. chinensis . Its application (0-20 mmol/L) enhanced the plant tolerance to salt stress. The treatment of 15 mmol/L GB significantly decreased the Na + accumulation in leaf and root under NaCl stress. This difference in accumulating Na + and K + is caused by higher selectivity of root absorption. Furthermore, GB increased H +_ATPase activity of root plasma membrane evidently. This result strongly suggested that in root the decreased Na + accumulation was caused by the GB accumulation that enhanced the extrusion of Na + from the cell in some way through plasma membrane transporter, e.g. Na +/H + antiport driven by H +_ATPase. The GB application was also found to stabilize the plasma membrane, to decrease the loss of chlorophyll, and to stimulate the osmosis induced proline response under salt stress.

Cloning, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat

Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .

Existence and Characteristics of Tonoplast-bound Protein Kinase in the Tip Cell of Maize Root

For understanding the function of tonoplast protein in plant cell signal pathway, we have identified an integral protein kinase activity from the highly purified tonoplast isolated from maize ( Zea mays L.) root by a new nonradioactive method in which a color labeled peptide was used as substrate. The protein kinase was Ca 2+ _dependent and CaM and phosphatidylserine_independent, like the calmodulin_like domain protein kinase (CDPK) in many plants. The optimal pH value and Ca 2+ concentration were 6.5 and 10 μmol/L, respectively. According to the optimal pH value and the effect of detergent, it could be inferred that the active site of this protein kinase is oriented toward the cytoplasm. Zn 2+ had no obvious effect on its activity, indicating that this protein kinase has no zinc_finger domain that exists in some mammalian protein kinases. At the same time, when tonoplast proteins were prephosphorylated in the presence of Ca 2+ and ATP, both the ATP_hydrolysis and the proton_transport activity of vacuolar H +_ATPase were stimulated. This stimulation could be reversed by an alkaline_phosphatase. These results indicate that a Ca 2+ _dependent protein kinase was located in the tonoplast, and a Ca 2+ _dependent phosphorylation, probably caused by this kinase, activated the vacuolar H +_ATPase activity. These results are helpful for further research on the function of CDPK in the course of signal transduction in plants.

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H^+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso_PC) on ATP and ρ_nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H +_ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso_PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso_PC was within 0-0.03%, and increased slightly when lyso_PC was higher than 0.03%. At the concentration of 0.03%, lyso_PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46 μmol P i·mg -1 protein·min -1 to 0.87 μmol P i·mg -1 protein·min -1 while K m increased from 0.88 mmol/L to 1.15 mmol/L under lyso_PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0 . Moreover, it was found lyso_PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso_PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso_PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C_terminal autoinhibitory domain in the plant plasma membrane H +_ATPase.

低温胁迫下北海道黄杨叶肉细胞Ca^(2+)和Ca^(2+)-ATPase的变化

利用焦锑酸钙沉淀和硝酸铅沉淀的电镜细胞化学方法,以室温生长的北海道黄杨植株为对照,研究了人工4℃低温胁迫过程中北海道黄杨(Euonymusjaponicus‘Cuzhi’)叶肉细胞Ca2+和Ca2+-ATPase的动态变化。在4℃低温胁迫的初期(3~12h),北海道黄杨叶肉细胞间隙和液泡内的Ca2+沉淀颗粒减少,而细胞质和细胞核内的Ca2+水平升高,但Ca2+-ATPase在细胞的分布几乎没有变化,主要分布在质膜和液泡膜上,有较高的活性;低温胁迫24h,细胞质和细胞核内增加的Ca2+开始回到细胞间隙和液泡中,Ca2+-ATPase在质膜和液泡膜上活性增强;在低温胁迫48~96h,细胞内的Ca2+又回到低温胁迫前的低水平,但Ca2+-ATPase在质膜和液泡膜上仍有很高的活性。叶肉细胞内Ca2+稳态平衡和Ca2+-ATPase的活性变化与植物的抗寒性存在一定的相关性。