Effect of NdCl_3 on the Injury of Membrane and ABA Content under Osmotic Stress

ＥｆｆｅｃｔｏｆＮｄＣｌ_３ｏｎｔｈｅＩｎｊｕｒｙｏｆＭｅｍｂｒａｎｅａｎｄＡＢＡＣｏｎｔｅｎｔｕｎｄｅｒＯｓｍｏｔｉｃＳｔｒｅｓｓ￥ＡｎＪｉａｎｐｉｎｇ（安建平）（ＢｉｏｌｏｇｙＤｅｐａｒｔｍｅｎｔ，ＴｉａｎｓｈｕｉＮｏｒｍａｌＣｏｌｌｅｇｅ，Ｔｉ?..     

Characterization of an Arabidopsis thaliana mutant that has a defect in ABA accumulation: ABA-dependent and ABA-independent accumulation of free amino acids during dehydration

In an attempt to elucidate the physiological role of ABA in seed dormancy and the adaptive response to dehydration, we isolated an ABA-deficient mutant of Arabidopsis thaliana (L.) Heynh. which germinated in the presence of a gibberellin biosynthetic inhibitor. Genetic analysis showed this mutation is a new allele of a recently reported locus aba2, and therefore has been designated aba2-2. The levels of endogenous ABA in fresh and dehydrated tissues of the aba2-2 mutant were highly reduced compared to those of wild-type plants. As a consequence, aba2-2 plants wilt and produce seeds with reduced dormancy. Dark germinated seedlings of the aba2-2 mutant showed true leaves, which were not observed in those of the wild type, indicating that aba2-2 embryos grew precociously during seed maturation. In the dehydrated tissues of the wild-type plants, the levels of free proline, isoleucine and leucine were elevated to a content approximately 100-fold higher than those in fresh tissues. In contrast to the wild-type plants, dehydration-induced accumulation of proline was highly suppressed in the aba2-2 mutant plants while that of leucine and isoleucine accumulated. Furthermore, exogenous application of ABA to wild-type plants promoted accumulation of free proline, but not leucine nor isoleucine. These results suggest that dehydration-induced accumulation of free leucine and isoleucine is achieved independent of ABA.     

Effect of yttrium on photosynthesis and water relations in young maize plants

Despite an increase in spectrum of industrial applications of yttrium(Y) and the fact that it is widely present in the soils and plants,some of which are agronomically important crops,its effects on plant growth and metabolism are still obscure.Therefore,the aim of this work was to examine the effect of different concentrations of Y on its accumulation and distribution,photosynthetic responses,water relations,free proline concentration and growth of young maize plants.The experiment was done with maize(Zea mays L.,hybrid NS-640),in water cultures,under semi-controlled conditions of a greenhouse.Plants were supplied with half-strength complete Hoagland nutrient solution,to which was added either 0(control),10–5,10–4 or 10–3 mol/L Y,in the form of Y(NO)3·5H2O.Each variant was set in thirteen replications,with six plants in each replication.Plants were grown for 21 d and they were at the stage of 3 and 4 leaves when they were analyzed.The presence of Y reduced maize growth and photosynthetic performance.Dimensions of stomata significantly decreased while their density significantly increased on both adaxial and abaxial epidermis.Plant height,root length,total leaf area and dry mass also declined.Concentration of photosynthetic pigments(chl a and b and carotenoids) and free proline decreased.Photosynthesis and transpiration were impaired in the presence of Y – their intensities were also reduced,and the same stands for stomatal conductance of water vapor,photosynthetic water use efficiency(WUE) and water content.Although the highest concentration of Y was found in maize roots in each treatment,Y concentration in the second leaf and shoot also significantly increased with an increase in Y concentration in the nutrient solution.Albeit Y concentration was much higher in roots than in shoots,shoot metabolism and growth were much more disrupted.The results demonstrated that young maize plants accumulated significant amount of Y and that this element,when present in higher concentrations,had unfavorable effect on physiological processes and therefore plant growth.     

Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss

Desiccation of plants during drought can be detrimental to agricultural production. The phytohormone abscisic acid (ABA) reduces water loss by triggering stomatal pore closure in leaves, a process requiring ion-channel modulation by cytoplasmic proteins. Deletion of the Arabidopsis farnesyltransferase gene ERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing. ERA1 was expressed in guard cells. Double-mutant analyses of era1 with the ABA-insensitive mutants abi1 and abi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment.     

Reversible and Irreversible Drought-Induced Changes in the Anther Proteome of Rice(Oryza sativa L.)Genotypes IR64 and Moroberekan

Crop yield is most sensitive to water deficit during the reproductive stage.For rice,the most sensitive yield component is spikelet fertility and the most sensitive stage is immediately before heading.Here,we examined the effect of drought on the anther proteome of two rice genotypes:Moroberekan and IR64.Water was withheld for 3 d before heading(3DBH)in well watered controls for 5 d until the flag leaf relative water content(RWC)had declined to 45-50%.Plants were then re-watered and heading occurred 2-3 d later,representing a delay of 4-5 d relative to controls.The anther proteins were separated at 3 DBH,at the end of the stress period,and at heading in stressed/re-watered plants and controls by two-dimensional(2-D)gel electrophoresis,and 93 protein spots were affected reproducibly in abundance by drought during the experiment across two rice genotypes.After drought stress,upon re-watering,expressions of 24 protein spots were irreversible in both genotypes,60 protein spots were irreversible in IR64 but reversible in Moroberekan,only nine protein spots were irreversible in Moroberekan while reversible in IR64.Among them,there were 14 newly drought-induced protein spots in IR64;none of them was reversible on re-watering.However,there were 13 newly drought-induced protein spots in Moroberekan,10 of them were reversible on re-watering,including six drought-induced protein spots that were not reversed in IR64.Taken together,our proteomics data reveal that drought-tolerant genotype Moroberekan possessed better recovery capability following drought and re-watering at the anther proteome level than the drought-sensitive genotype IR64.The disruptions of drought to rice anther development and pollen cell functions are also discussed in the paper.     

Synthesis and radiopharmacology of O-(2-[18F]fluoroethyl)-L-tyrosine for tumor imaging

The impact of simultaneous environmental stresses on plants and how they respond to combined stresses compared with single stresses is largely unclear. By using a transgene (RD29A-LUC) consisting of the firefly luciferase coding sequence (LUC) driven by the stress-responsive RD29A promoter, we investigated the interactive effects of temperature, osmotic stress, and the phytohormone abscisic acid (ABA) in the regulation of gene expression in Arabidopsis seedlings. Results indicated that both positive and negative interactions exist among the studied stress factors in regulating gene expression. At a normal growth temperature (22 degrees C), osmotic stress and ABA act synergistically to induce the transgene expression. Low temperature inhibits the response to osmotic stress or to combined treatment of osmotic stress and ABA, whereas low temperature and ABA treatments are additive in inducing transgene expression. Although high temperature alone does not activate the transgene, it significantly amplifies the effects of ABA and osmotic stress. The effect of multiple stresses in the regulation of RD29A-LUC expression in signal transduction mutants was also studied. The results are discussed in the context of cold and osmotic stress signal transduction pathways.     

Cerium alleviates drought-induced stress in Phaseolus vulgaris

Extreme drought events are expected to be one of the main challenges for tropical agriculture and,consequently,for global food security.For this reason,it is urgent to develop strategies to increase water use efficiency,as well as to increase crops' ability to cope with drought.Because rare earth elements(REEs) are known to alleviate damages in plants under abiotic stresses,we hypothesized that addition of Ce~(3+)to nutrient solution would promote the growth of common bean,especially under simulated drought stress.To test this hypothesis,we conducted an experiment evaluating the effect of six Ce~(3+)concentrations on the development and physiological traits of common bean grown in nutrient solution under normal condition and under induced drought stress,caused by the addition of polyethylene glycol6000 to the nutrient solution.Our results show that Ce~(3+)alleviates water stress in common bean plants,increasing their survival rate and growth.In addition,Ce~(3+)application increases photosynthesis rate,chlorophyll content and water use efficiency under water stress.However,we observe no significant effect of Ce~(3+)on plants growing under normal condition.Therefore,Ce~(3+)seems to be a promising attenuator of drought stress for common bean.     

Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst

In order to understand the molecular mechanisms which are responsible for desiccation tolerance in the resurrection plant Craterostigma plantagineum Hochst. a thorough analysis of the CDeT11-24 gene family was performed. CDeT11-24 comprises a small gene family whose genes are expressed in response to dehydration, salt stress and abscisic acid (ABA) treatment in leaves. The gene products are constitutively expressed in roots and disappear only when the plants are transferred to water. It is therefore suggested that the proteins are involved in sensing water status. The predicted proteins are very hydrophilic; they share some features with late-embryogenesis-abundant proteins, and sequence similarities were found with two ABA- and drought-regulated Arabidopsis genes. The analysis of beta-glucuronidase reporter genes driven by the CDeT11-24 promoter showed high activity in mature seeds in both transgenic Arabidopsis and tobacco. In vegetative tissues the promoter activity in response to ABA was restricted to young Arabidosis seedlings. The responsiveness to ABA during later developmental stages was regained in the presence of the Arabidopsis gene product ABI3. Dehydration-induced promoter activity was only observed in Arabidopsis leaves at a particular developmental stage. This analysis indicates that some components in the signal transduction pathway of the resurrection plant are not active in tobacco or Arabidopsis.     

Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5'-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ >/= 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126-2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.     

Characterization of the gene for delta1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L

A cDNA for delta1-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.     

Betaine aldehyde dehydrogenase in sorghum

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance

In common with other zwitterionic quarternary ammonium compounds (QACs), glycine betaine acts as an osmoprotectant in plants, bacteria, and animals, with its accumulation in the cytoplasm reducing adverse effects of salinity and drought. For this reason, the glycine betaine biosynthesis pathway has become a target for genetic engineering of stress tolerance in crop plants. Besides glycine betaine, several other QAC osmoprotectants have been reported to accumulate among flowering plants, although little is known about their distribution, evolution, or adaptive value. We show here that various taxa of the highly stress-tolerant family Plumbaginaceae have evolved four QACs, which supplement or replace glycine betaine-namely, choline O-sulfate and the betaines of beta-alanine, proline, and hydroxyproline. Evidence from bacterial bioassays demonstrates that these QACs function no better than glycine betaine as osmoprotectants. However, the distribution of QACs among diverse members of the Plumbaginaceae adapted to different types of habitat indicates that different QACs could have selective advantages in particular stress environments. Specifically, choline O-sulfate can function in sulfate detoxification as well as in osmoprotection, beta-alanine betaine may be superior to glycine betaine in hypoxic saline conditions, and proline-derived betaines may be beneficial in chronically dry environments. We conclude that the evolution of osmoprotectant diversity within the Plumbaginaceae suggests additional possibilities to explore in the metabolic engineering of stress tolerance in crops.     

Role of water in protein kinase C catalysis and its binding to membranes

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.     

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.