When the 7_d old barley ( Hordeum vulgare L.) seedlings were treated with different concentrations of NaCl for 3 d, the levels of the noncovalently conjugated polyamines (PAs) in the plasma membrane and tonoplast v...When the 7_d old barley ( Hordeum vulgare L.) seedlings were treated with different concentrations of NaCl for 3 d, the levels of the noncovalently conjugated polyamines (PAs) in the plasma membrane and tonoplast vesicles and the covalently conjugated PAs in the membrane proteins were promoted by NaCl of low concentrations and suppressed by NaCl of high concentrations. Among the noncovalently conjugated PAs in the vesicles, spermidine (Spd) level was the most abundant, while putrescine (Put) content was predominant among the covalently conjugated PAs, accounted for 40%-70%, 35%-60%, respectively. In addition, the TLC (thin_layer chromatography) profiles of the benzoylated PAs presented an unknown polyamine with Rf =0.92 (X 0.92 ), which conjugated covalently and noncovalently in root tonoplast and its content changed as well as Spd with NaCl treatment. The total PA contents in the roots were higher than that in the leaves, and the types and contents of covalently and noncovalently conjugated PAs in the tonoplast were higher than those in the plasma membrane. The results showed that the above two PAs associated with the membrane might be essential in salt adaption of cells and the maintenance of membrane function.展开更多
Under NaCl stress for 2 d, H+-ATPase activity increased, and H+-PPase activity decreased in the tonoplast of salt-tolerant barley ( Hordeum vulgare L. cv. 'Tanyin 2') roots. La3+ (1 mmol/L), an inhibitor of Ca...Under NaCl stress for 2 d, H+-ATPase activity increased, and H+-PPase activity decreased in the tonoplast of salt-tolerant barley ( Hordeum vulgare L. cv. 'Tanyin 2') roots. La3+ (1 mmol/L), an inhibitor of Ca2+ channel in plasma membrane, and EGTA (5 mmol/L), a Ca2+ chelator, inhibited this NaCl-induced increase in H+-ATPase activity but stimulated the H+-PPase activity. Treatment of barley roots with CaM antagonist (trifluoperazine, TFP, 20 mumol/L) also diminished the increase of H+-ATPase activity induced by NaCl. La3+, TFP or La3+ + TFP increased Na+ uptake and decreased K+ and Ca2+ uptake in barley roots under NaCl stress. These results suggested that the activation of tonoplast H+-ATPase and the regulation of Na+ and K+ uptake under NaCl stress may be related to Ca2+-CaM system.展开更多
Effect of nitrate and ammonium on the activity of tonoplast pyrophosphatase (V-PPase) was investigated in the roots of tomato ( Lycopersicon esculentum L.). The results showed that the activity of V-PPase was increase...Effect of nitrate and ammonium on the activity of tonoplast pyrophosphatase (V-PPase) was investigated in the roots of tomato ( Lycopersicon esculentum L.). The results showed that the activity of V-PPase was increased by ammonium nutrition, compared with nitrate nutrition. The H+ transport of tonoplast vesicles by V-PPase was also stimulated by ammonium nutrition. The result of Western blot indicated that the protein amount of V-PPase was increased by ammonium nutrition.展开更多
To study the function and adaptive mechanism of tonoplast H + ATPase under salt stress, pea ( Pisum sativum L.) seedlings were treated with different concentrations of salt (100-250 mmol/L NaCl) and with 100 mmol...To study the function and adaptive mechanism of tonoplast H + ATPase under salt stress, pea ( Pisum sativum L.) seedlings were treated with different concentrations of salt (100-250 mmol/L NaCl) and with 100 mmol/L NaCl for different days (1-3 d). The ATP hydrolytic activity and the proton transport activity and the changes of the amount of tonoplast H + ATPase (subunit A) were measured. ATP hydrolytic activity of H + ATPase prepared from plants treated with 250 mmol/L NaCl was reduced by about 25% compared to that of control plants, but that of stressed plants treated with 100 mmol/L and 200 mmol/L NaCl was unchanged. The activity from plants treated with 100 mmol/L NaCl for up to 3 d was lower than that of control plants by 20%. But the proton transport activity was increased under the same salt stresses as above. These results showed that the changes of the hydrolytic activity and the proton transport activity were not in proportion and salt stress may cause the change of the coupling ratio of H + transport activity to ATP hydrolysis. The protein amount kept unchanged and reduced a little only when pea was treated with 100 mmol/L NaCl for 3 d. These results indicated that salinity stimulated the increase of the pump efficiency of the V_ATPase from pea roots, which was due to the change of the coupling ratio, but not due to the increase of ATP hydrolysis and the amount of V_ATPase.展开更多
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...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.展开更多
The effects of NaCl stress on the H +-ATPase, H +-PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars(Glycine max L.) differ...The effects of NaCl stress on the H +-ATPase, H +-PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars(Glycine max L.) differing in salt tolerance(Wenfeng7, salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3%(W/V) NaCl for 3 d, the H +-ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H +-PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios of phospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.展开更多
We have mapped the expression of the tonoplast intrinsic protein （TIP） gene family members in Arabidopsis seeds by fluorescent protein tagging of their genomic sequences and confocal microscopy. Three isoforms （TIP...We have mapped the expression of the tonoplast intrinsic protein （TIP） gene family members in Arabidopsis seeds by fluorescent protein tagging of their genomic sequences and confocal microscopy. Three isoforms （TIP1;1, TIP2;1, and TIP2;2） have distinct patterns of expression in maternal tissues （outer integument and placento-chalazal region）. Two isoforms, TIP3;1 and the previously uncharacterized TIP3;2, are the only detectable TIPs in embryos during seed maturation and the early stages of seed germination. Throughout these developmental stages, both isoforms co-locate to the tono- plast of the protein storage vacuoles, but also appear to label the plasma membrane. Plasma membrane labeling is specific to TIP3;1 and TIP3;2, is independent of the position of the fluorescent protein tag, and appears to be specific to early seed maturation and early germination stages. We discuss these results in the context of the predicted distribution of aquaporins in Arabidopsis seeds.展开更多
Both enhanced were H^+ transport activities of tonoplast vesicles isolated from roots of the salt-tolerant mutant and wild type of wheat with treatment of NaCI, but the activity of the mutant was significantly higher ...Both enhanced were H^+ transport activities of tonoplast vesicles isolated from roots of the salt-tolerant mutant and wild type of wheat with treatment of NaCI, but the activity of the mutant was significantly higher than that of wild type. H^+ transport activity was indicated as the stable value of fluorescence quenching per mg membrane proteins. The H^+ transport activities dependent on ATP of the mutant and wild type were 1099 and 558 respectively and their activities dependent on PPi were 358 and 228 separately.展开更多
Summary Vacuolar trafficking routes and their regu- lators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a dir...Summary Vacuolar trafficking routes and their regu- lators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER- to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V- ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells.展开更多
Subject Code:C02With the support by the National Natural Science Foundation of China,the research team led by Prof.Lin Jinxing(林金星)at the College of Biological Sciences&Biotechnology,Beijing Forestry University...Subject Code:C02With the support by the National Natural Science Foundation of China,the research team led by Prof.Lin Jinxing(林金星)at the College of Biological Sciences&Biotechnology,Beijing Forestry University,overcame the limitations of existing techniques and expanded the study of protein characteristics from the plasma membrane to the vacuole membranes.This study was published in Molecular Plant(2016,DOI:展开更多
Aquaporins are implicated in a wide variety of plant physiological processes, although the mechanisms involved in their regulation are not fully understood. To gain further insight into the regulatory factors involved...Aquaporins are implicated in a wide variety of plant physiological processes, although the mechanisms involved in their regulation are not fully understood. To gain further insight into the regulatory factors involved in this process, we used a yeast two-hybrid system to screen for potential binding partners to the Arabidopsis tonoplast intrinsic protein (TIP) AtTIP1;1. This was the first protein identified to be associated with high water permeability in vacuolar membranes from Arabidopsis thaliana. Using AtTIP1;1 as bait, a novel binding protein was identified in both yeast and plant cells. This prey protein, named AtSM34, was a 309 aa polypeptide with a predicted molecular mass of 34 kD and contained a single MYB/SANT-like domain. AtSM34 promoter:: GUS histochemical staining analysis detected AtSM34 expression in flowers, stems and leaves, particularly in the vascular tissues, in response to osmotic stress. AtSM34 expression was localized in the endoplasmic reticulum membrane, and sequence deletion analysis revealed that the N-terminal coding region (amino acids 1-83) was critical for this localization. Overexpression of AtSM34 resulted in hypersensitivity to exogenous mannitol, sorbitol and abscisic acid, and caused a significant delay in germination. AtSM34 interacted with AtTIP1;2 and AtTIP2;1, which are essential proteins for modulation of tonoplast permeability and highly expressed in germinating seedlings. These data indicate AtSM34 is a novel TIPs binding protein involved in the osmotic stress response of seedlings at an early stage of development.展开更多
The growth of barley (Hordeum vulgare L.) seedling is inhibited by 300 mmol/L NaCl. When 20 mg/L GSH is present in the hydroponic culture solution with NaCl, root length, root and shoot dry weigh, chlorophyll as well ...The growth of barley (Hordeum vulgare L.) seedling is inhibited by 300 mmol/L NaCl. When 20 mg/L GSH is present in the hydroponic culture solution with NaCl, root length, root and shoot dry weigh, chlorophyll as well as K+ contents are increased, Na+ content is decreased. At the same time the activities of H+-ATPase and H+-PPase associated with tonoplast vesicles isolated from leaves are stimulated, and electrolytic leakage are diminished by exogenous GSH.展开更多
文摘When the 7_d old barley ( Hordeum vulgare L.) seedlings were treated with different concentrations of NaCl for 3 d, the levels of the noncovalently conjugated polyamines (PAs) in the plasma membrane and tonoplast vesicles and the covalently conjugated PAs in the membrane proteins were promoted by NaCl of low concentrations and suppressed by NaCl of high concentrations. Among the noncovalently conjugated PAs in the vesicles, spermidine (Spd) level was the most abundant, while putrescine (Put) content was predominant among the covalently conjugated PAs, accounted for 40%-70%, 35%-60%, respectively. In addition, the TLC (thin_layer chromatography) profiles of the benzoylated PAs presented an unknown polyamine with Rf =0.92 (X 0.92 ), which conjugated covalently and noncovalently in root tonoplast and its content changed as well as Spd with NaCl treatment. The total PA contents in the roots were higher than that in the leaves, and the types and contents of covalently and noncovalently conjugated PAs in the tonoplast were higher than those in the plasma membrane. The results showed that the above two PAs associated with the membrane might be essential in salt adaption of cells and the maintenance of membrane function.
文摘Under NaCl stress for 2 d, H+-ATPase activity increased, and H+-PPase activity decreased in the tonoplast of salt-tolerant barley ( Hordeum vulgare L. cv. 'Tanyin 2') roots. La3+ (1 mmol/L), an inhibitor of Ca2+ channel in plasma membrane, and EGTA (5 mmol/L), a Ca2+ chelator, inhibited this NaCl-induced increase in H+-ATPase activity but stimulated the H+-PPase activity. Treatment of barley roots with CaM antagonist (trifluoperazine, TFP, 20 mumol/L) also diminished the increase of H+-ATPase activity induced by NaCl. La3+, TFP or La3+ + TFP increased Na+ uptake and decreased K+ and Ca2+ uptake in barley roots under NaCl stress. These results suggested that the activation of tonoplast H+-ATPase and the regulation of Na+ and K+ uptake under NaCl stress may be related to Ca2+-CaM system.
文摘Effect of nitrate and ammonium on the activity of tonoplast pyrophosphatase (V-PPase) was investigated in the roots of tomato ( Lycopersicon esculentum L.). The results showed that the activity of V-PPase was increased by ammonium nutrition, compared with nitrate nutrition. The H+ transport of tonoplast vesicles by V-PPase was also stimulated by ammonium nutrition. The result of Western blot indicated that the protein amount of V-PPase was increased by ammonium nutrition.
文摘To study the function and adaptive mechanism of tonoplast H + ATPase under salt stress, pea ( Pisum sativum L.) seedlings were treated with different concentrations of salt (100-250 mmol/L NaCl) and with 100 mmol/L NaCl for different days (1-3 d). The ATP hydrolytic activity and the proton transport activity and the changes of the amount of tonoplast H + ATPase (subunit A) were measured. ATP hydrolytic activity of H + ATPase prepared from plants treated with 250 mmol/L NaCl was reduced by about 25% compared to that of control plants, but that of stressed plants treated with 100 mmol/L and 200 mmol/L NaCl was unchanged. The activity from plants treated with 100 mmol/L NaCl for up to 3 d was lower than that of control plants by 20%. But the proton transport activity was increased under the same salt stresses as above. These results showed that the changes of the hydrolytic activity and the proton transport activity were not in proportion and salt stress may cause the change of the coupling ratio of H + transport activity to ATP hydrolysis. The protein amount kept unchanged and reduced a little only when pea was treated with 100 mmol/L NaCl for 3 d. These results indicated that salinity stimulated the increase of the pump efficiency of the V_ATPase from pea roots, which was due to the change of the coupling ratio, but not due to the increase of ATP hydrolysis and the amount of V_ATPase.
文摘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.
文摘The effects of NaCl stress on the H +-ATPase, H +-PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars(Glycine max L.) differing in salt tolerance(Wenfeng7, salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3%(W/V) NaCl for 3 d, the H +-ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H +-PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios of phospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.
文摘We have mapped the expression of the tonoplast intrinsic protein （TIP） gene family members in Arabidopsis seeds by fluorescent protein tagging of their genomic sequences and confocal microscopy. Three isoforms （TIP1;1, TIP2;1, and TIP2;2） have distinct patterns of expression in maternal tissues （outer integument and placento-chalazal region）. Two isoforms, TIP3;1 and the previously uncharacterized TIP3;2, are the only detectable TIPs in embryos during seed maturation and the early stages of seed germination. Throughout these developmental stages, both isoforms co-locate to the tono- plast of the protein storage vacuoles, but also appear to label the plasma membrane. Plasma membrane labeling is specific to TIP3;1 and TIP3;2, is independent of the position of the fluorescent protein tag, and appears to be specific to early seed maturation and early germination stages. We discuss these results in the context of the predicted distribution of aquaporins in Arabidopsis seeds.
文摘Both enhanced were H^+ transport activities of tonoplast vesicles isolated from roots of the salt-tolerant mutant and wild type of wheat with treatment of NaCI, but the activity of the mutant was significantly higher than that of wild type. H^+ transport activity was indicated as the stable value of fluorescence quenching per mg membrane proteins. The H^+ transport activities dependent on ATP of the mutant and wild type were 1099 and 558 respectively and their activities dependent on PPi were 358 and 228 separately.
基金supported by Major Research Plan(2013CB945102) from the Ministry of Science,Technology of ChinaNational Natural Science Foundation of China(31261160490 to Y.Z.)Natural Science Foundation of Shandong Province(ZR2014CM027 to S.L.)
文摘Summary Vacuolar trafficking routes and their regu- lators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER- to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V- ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells.
文摘Subject Code:C02With the support by the National Natural Science Foundation of China,the research team led by Prof.Lin Jinxing(林金星)at the College of Biological Sciences&Biotechnology,Beijing Forestry University,overcame the limitations of existing techniques and expanded the study of protein characteristics from the plasma membrane to the vacuole membranes.This study was published in Molecular Plant(2016,DOI:
基金supported by the National Natural Science Foundation of China (30770193)the National Basic Research Program of China (2006CB100100)the "111 Project" (B06003)
文摘Aquaporins are implicated in a wide variety of plant physiological processes, although the mechanisms involved in their regulation are not fully understood. To gain further insight into the regulatory factors involved in this process, we used a yeast two-hybrid system to screen for potential binding partners to the Arabidopsis tonoplast intrinsic protein (TIP) AtTIP1;1. This was the first protein identified to be associated with high water permeability in vacuolar membranes from Arabidopsis thaliana. Using AtTIP1;1 as bait, a novel binding protein was identified in both yeast and plant cells. This prey protein, named AtSM34, was a 309 aa polypeptide with a predicted molecular mass of 34 kD and contained a single MYB/SANT-like domain. AtSM34 promoter:: GUS histochemical staining analysis detected AtSM34 expression in flowers, stems and leaves, particularly in the vascular tissues, in response to osmotic stress. AtSM34 expression was localized in the endoplasmic reticulum membrane, and sequence deletion analysis revealed that the N-terminal coding region (amino acids 1-83) was critical for this localization. Overexpression of AtSM34 resulted in hypersensitivity to exogenous mannitol, sorbitol and abscisic acid, and caused a significant delay in germination. AtSM34 interacted with AtTIP1;2 and AtTIP2;1, which are essential proteins for modulation of tonoplast permeability and highly expressed in germinating seedlings. These data indicate AtSM34 is a novel TIPs binding protein involved in the osmotic stress response of seedlings at an early stage of development.
文摘The growth of barley (Hordeum vulgare L.) seedling is inhibited by 300 mmol/L NaCl. When 20 mg/L GSH is present in the hydroponic culture solution with NaCl, root length, root and shoot dry weigh, chlorophyll as well as K+ contents are increased, Na+ content is decreased. At the same time the activities of H+-ATPase and H+-PPase associated with tonoplast vesicles isolated from leaves are stimulated, and electrolytic leakage are diminished by exogenous GSH.
