Effects of low temperature, high salinity and exogenous ABA on the synthesis of two chloroplastic drought-induced proteins in Solanum tuberosum

Two chloroplastic proteins of 32 and 34 kDa were previously shown to be substantially synthesized in response to a progressive water deficit in whole Solanum tuberosum plants (G. Pruvot, S. Cuiné, N. Gault, G. Peltier and P. Rey, unpublished data; G. Pruvot, S. Cuiné, G. Peltier and P. Rey. 1996. Planta 198: 471–479). These chloroplastic drought-induced stress proteins, named CDSP 32 and CDSP 34, accumulated in the stroma and in the thylakoids, respectively. In this study, we investigated the effects of low temperature and high salinity on the synthesis of the CDSP proteins. Whereas the CDSP 32 synthesis was not modified in response to a cold treatment, an increased synthesis of CDSP 32 was observed in salt-stressed plants, resulting in accumulation of the protein. The thylakoid CDSP 34 protein exhibited enhanced synthesis and substantial accumulation in response to cold and high salinity. A significant increase in the leaf abscisic acid content (at least 2.5-fold) was measured in plants subjected to water deficit, high salinity or low temperature. The contribution of ABA to the synthesis of the two proteins was investigated by spraying well-watered plants with a 100 μ M / ABA solution for 15 days. This treatment resulted in a 15-fold increase in the leaf ABA content. Whereas synthesis of the CDSP 32 protein was not affected by exogenous ABA, synthesis of the CDSP 34 protein was substantially enhanced. Based on these results, we conclude that ABA likely mediates the increased synthesis of CDSP 34 upon drought, low temperature and high salinity and suggest that another signal, likely related to high osmolarity, is involved in the induction of CDSP 32 synthesis.     

Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants

Two chloroplastic proteins, named CDSP 32 and CDSP 34 for chloroplastic drought-induced stress protein of 32 and 34 kDa, were previously shown to be substantially synthesized in Solanum tuberosum plants subjected to water deficit. We investigated the localization of CDSPs in leaf chloroplasts from control and wilted potato plants using immunocytochemistry. Observation of electron micrographs did not reveal any important change in plastid structures of drought-stressed plants, except an increased number and a larger size of plastoglobuli. In well-watered plants, very little labeling corresponding to CDSP 32 was detected. Consecutively to water stress, a higher abundance of CDSP 32 was revealed, the protein being exclusively localized in the stroma. Immunocytochemical data indicated the presence of some CDSP 34 protein in well-watered plants and confirmed its accumulation upon water deficit. CDSP 34 was found to be preferentially associated with stromal lamellae thylakoids, but some protein was revealed in the stroma. No association of CDSP 34 with grana and plastoglobuli was noticed in chloroplasts from control and stressed plants.     

Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis

Cytokinins (CKs) may be involved in the regulation of plant adaptation to drought stress. The objectives of the study were to identify proteomic changes in leaves and roots in relation to improved drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera) containing a senescence-activated promoter (SAG12) and the isopentyl transferase (ipt) transgene that increases endogenous CK content. Leaves of SAG12-ipt bentgrass exhibited less severe senescence under water stress, as demonstrated by maintaining lower electrolyte leakage and lipid peroxidation, and higher photochemical efficiency (F(v)/F(m)), compared with the null transformant (NT) plants. SAG12-ipt plants had higher root/shoot ratios and lower lipid peroxidation in leaves under water stress than the NT plants. The suppression of drought-induced leaf senescence and root dieback in the transgenic plants was associated with the maintenance of greater antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase). The SAG12-ipt and NT plants exhibited differential protein expression patterns under well-watered and drought conditions in both leaves and roots. Under equivalent leaf water deficit (47% relative water content), SAG12-ipt plants maintained higher abundance of proteins involved in (i) energy production within both photosynthesis and respiration [ribulose 1,5-bisphosphate carboxylase (RuBisCO) and glyceraldehyde phosphate dehydrogenase (GAPDH)]; (ii) amino acid synthesis (methionine and glutamine); (iii) protein synthesis and destination [chloroplastic elongation factor (EF-Tu) and protein disulphide isomerases (PDIs)]; and (iv) antioxidant defence system (catalase and peroxidase) than the NT plants. These results suggest that increased endogenous CKs under drought stress may directly or indirectly regulate protein abundance and enzymatic activities involved in the above-mentioned metabolic processes, thereby enhancing plant drought tolerance.     

Expression and molecular cloning of drought-induced genes in the wild tomato Lycopersicon chilense.

Protein synthesis and translatable mRNA population changes induced during water stress were studied in leaves of a drought-resistant wild relative of tomato, Lycopersicon chilense, using one- and two-dimensional polyacrylamide gel electrophoresis. Under our experimental conditions, water deficit did not significantly affect total protein synthesis capacity. However, it induced biphasic synthesis of a new set of proteins. These newly synthesized proteins resumed to control levels upon rehydration of the plants. Certain drought-induced proteins also accumulated in leaves subjected to heat shock (39 degrees C) or exogenous abscisic acid (ABA, 1 mM) treatments. A cDNA library was constructed using poly(A)+ RNA from leaves of plants exposed to drought stress for 4 days. Differential screening of the library identified three groups of clones corresponding to drought- and ABA-induced mRNAs. Northern blot analysis showed that the genes of selected clones respond differently to the different environmental stresses. Our data clearly demonstrate that water stress alters gene expression in L. chilense plants resulting in the synthesis of new proteins, of which several respond to high temperature stress and others to an osmotic effect. These responses are in part modulated by ABA.     

Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants

In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.     

Potato plants lacking the CDSP32 plastidic thioredoxin exhibit overoxidation of the BAS1 2-cysteine peroxiredoxin and increased lipid Peroxidation in thylakoids under photooxidative stress

The CDSP32 protein (chloroplastic drought-induced stress protein of 32 kD) is a thioredoxin participating in the defense against oxidative damage. We recently have identified in vitro the BAS1 2-Cys peroxiredoxin, a peroxide-detoxifying enzyme, as a target for CDSP32. Here, we report the characterization under stress conditions of transgenic potato (Solanum tuberosum) plants lacking CDSP32 with regard to the BAS1 redox state and the level of lipid peroxidation. Under control conditions, BAS1 is present at similar levels both in wild-type (WT) and transgenic plants. Under drought and methyl viologen treatment, CDSP32-lacking plants display, compared with WT, an increased proportion of BAS1 monomer corresponding to an overoxidized form of the protein. Leaf discs from transgenic plants treated with methyl viologen exhibit earlier degradation of BAS1 than WT plants do. Using several approaches, i.e. a probe emitting fluorescence when reacting with peroxides, high-performance liquid chromatography determination of lipid hydroxy fatty acid content, and measurement of chlorophyll thermoluminescence, we show a higher lipid peroxidation level under methyl viologen treatment in thylakoids from CDSP32-lacking plants compared with WT. These data show that CDSP32 is a critical component in the defense system against lipid peroxidation in photosynthetic membranes, likely as a physiological electron donor to the BAS1 peroxiredoxin.     

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.     

Fine structural quantification of drought-stressed Picea abies (L.) organelles based on 3D reconstructions

Ultrastructural investigations of cells and organelles by transmission electron microscopy (TEM) usually lead to two-dimensional information of cell structures without supplying exact quantitative data due to the limited number of investigated ultrathin sections. This can lead to misinterpretation of observed structures especially in context of their three-dimensional (3D) assembly. 3D investigations and quantitative morphometric analysis are therefore essential to get detailed information about the arrangement and the amount of subcellular structures inside a cell or organelle, respectively, especially when the plant sample was exposed to environmental stress. In the present research, serial sectioned chloroplasts, mitochondria, and peroxisomes from first year spruce needles (Picea abies (L.) Karst.) were 3D reconstructed and digitally measured using a computer-supported image analysis system in order to obtain a detailed quantitative characterization of complete cell organelles including precise morphological data of drought-induced fine structural changes. In control plants, chloroplast volume was composed of 56% stroma, 15% starch, 27% thylakoids, and 2% plastoglobules. In drought-stressed chloroplasts, the relative volume of both the thylakoids and the plastoglobules significantly increased to 37% and 12%, respectively. Chloroplasts of stressed plants differed from control plants not only in the mean thylakoid and plastoglobules content but also in the complete lack of starch grains. Mitochondria occurred in variable forms in both control and stressed samples. In stressed plants, mitochondria showed a significant smaller mean volume which was only 81% when compared with the control organelles. Peroxisomes were inconspicuous in both samples and their volume did not differ between control and drought-stressed samples. The present study shows that specific subcellular structures are subject to significant quantitative changes during drought stress of spruce needles giving a detailed insight in adaptation processes of the investigated cell organelles.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

Drought-induced proline accumulation is uninvolved with increased nitric oxide,which alleviates drought stress by decreasing transpiration in rice

Accumulation of proline is trusted to be an adaptive response of plants against drought stress, and exogenous application of nitric oxide (NO) enhances proline accumulation in Cu-treated algae. In order to investigate whether NO works as a necessary signaling molecule in drought-induced proline accumulation in rice leaves, effects of drought stress on endogenous NO content and proline accumulation were studied in rice leaves, using sodium nitroprusside (SNP, a NO donor) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a NO scavenger). The results showed that drought treatment increased both endogenous NO and proline contents in rice leaves, while foliar spray of various concentrations of SNP failed to induce proline accumulation in the leaves of well-watered rice and foliar spray of cPTIO failed to inhibit proline accumulation in the leaves of drought-stressed rice. These results indicate that increase of endogenous NO is dispensable for proline accumulation in the leaves of rice under drought stress. Further studies indicate that exogenous application of NO alleviates drought-induced water loss and ion leakage by decreasing transpiration rate of rice leaves.     

Genetic, proteomic and metabolic analysis of the regulation of energy storage in rice seedlings in response to drought

We used proteomic analysis to determine the response of rice plant seedlings to drought-induced stress. The expression of 71 protein spots was significantly altered, and 60 spots were successfully identified. The greatest down-regulated protein functional category was translation. Up-regulated proteins were mainly related to protein folding and assembly. Additionally, many proteins involved in metabolism (e.g. carbohydrate metabolism) also showed differences in expression. cDNA microarray and GC-MS analysis showed 4756 differentially expressed mRNAs and 37 differentially expressed metabolites. Once these data were integrated with the proteomic analysis, we were able to elucidate the metabolic pathways affected by drought-induced stress. These results suggest that increased energy consumption from storage substances occurred during drought. In addition, increased expression of the enzymes involved in anabolic pathways corresponded with an increase in the content of six amino acids. We speculated that energy conversion from carbohydrates and/or fatty acids to amino acids was increased. Analysis of basic metabolism networks allowed us to understand how rice plants adjust to drought conditions.     

干旱胁迫下杨树无性系苗期光合与气孔形态变异研究

为了解不同干旱胁迫处理下,杨树叶片光合特性和气孔形态的变化规律,本研究以4个杨树无性系为材料,对其干旱胁迫下光合指标与气孔形态指标进行测定分析,方差分析结果表明：除气孔器长在处理间差异不显著外,其他性状在无性系间、处理间和无性系与处理交互作用间均达极显著差异水平。随着土壤相对含水量的逐渐降低,无性系净光合速率、蒸腾速率和气孔导度都呈现出不同程度的下降;而胞间二氧化碳浓度呈现为先降后升趋势,表明当土壤相对含水量大于40%时,光合作用的主要限制因子是气孔因素,当土壤相对含水量低于40%时,光合作用的主要限制因子转换为非气孔因素。气孔形态研究结果表明,各指标均随土壤相对含水量的持续降低呈现出持续下降趋势。利用隶属函数法对不同干旱胁迫下4个杨树无性系的抗旱性进行综合评价,结果表明：白城小青黑杨的抗旱性优于其他三个无性系。     

Effects of Russian wheat aphid infestation on barley plant response to drought stress

The influence of Russian wheat aphid ( Diuraphis noxia Mordvilko) infestation on the response of barley ( Hordeum vulgare L. ev Hazen) plants to drought stress was investigated. Fourteen-day-old plants were infested with eight apterous adult aphids, which were removed 7 days later with systemic insecticide. Leaves previously infested with aphids had lower relative water content, reduced stomatal conductance, more negative water potential, lower levels of chlorophyll and higher levels of amino-N, proline and glycinebetaine than corresponding leaves from uninfested plants. When water was withheld for a period of 7 days after aphids were removed, the relative water content of previously infested plants dropped steadily from 0.89 to 0.60, while the relative water content of uninfested plants remained at about 0.94 for the first 4 days of the drought stress period followed by a steady drop to about 0.77 by the end of the drought stress period. Leaf water potentials dropped steadily during the drought stress period in both previously infested (-1.14 to -1.91 MPa) and unin-fested (-0.54 to -1.52 MPa) plants. Analysis of glycinebetaine and proline levels at the end of the drought stress period indicated that leaves of previously infested plants accumulated lower levels of these solutes than leaves from uninfested plants. Upon alleviation of drought stress, plants previously infested with aphids showed little increase in dry weight while younger leaves and tillers from uninfested plants showed large increases. It is concluded that Russian wheat aphids cause drought-stress symptoms in leaves of infested plants even in the presence of ample root moisture. The observations of low levels of glycinebetaine and proline present in leaves after water was withheld from roots and lack of leaf growth upon alleviation of drought stress in previously-infested plants, suggest that aphid infestation limits the capacity of barley plants to adjust successfully to drought stress.     

Differences in fennel seed responses to drought stress at the seed formation stage in sensitive and tolerant genotypes

To understand the effects of drought on fennel seed production and determine the underlying molecular processes, various fennel genotypes were exposed to drought stress. The yield and quality, including aromatic oil content, of fennel seeds were reduced by drought during seed development. To explore drought-induced biological processes in fennel, a label-free/gel-free proteomic analysis was performed. In Gaziantep and Tatmaj cultivars, which are sensitive and tolerant fennel genotypes, respectively, 106 and 92 drought-responsive proteins were identified. Comparison of protein-functional profiles indicated that proteins classified in stress, cell, and protein synthesis/degradation categories consisted important responsive mechanisms against drought stress. Pathway analysis visualized that the tricarboxylic acid cycle is important for both cultivars. In Tatmaj, moderate activation of proteins related to oxidative pentose phosphate pathway was detected along with an increase in photosynthesis-related proteins. Furthermore, cluster analysis of drought-responsive proteins using protein abundance at milky, dough, and mature stages identified protein homeostasis as a mechanism of drought tolerance in fennel. These results suggest that coordinated energy consumption and supply might be a drought-tolerance mechanism in fennel plants.

     

栓皮栎幼苗对土壤干旱胁迫的生理响应

以栓皮栎一年生盆栽苗为实验材料,采用称重控水的方法,设置不同土壤水分胁迫梯度,系统分析其幼苗在不同干旱胁迫条件下的生理生化响应特征,以探索栓皮栎耐旱特性.结果显示:(1)栓皮栎幼苗叶片中3种保护酶(SOD、POD、CAT)活性在对照(CK,土壤相对含水量19.5％～21.5％)条件下保持稳定,而中度干旱(T2,9.5％～11.5％)和重度干旱(T3,5.5％～7.5％)条件下,随着胁迫时间的延长呈先增高后降低的趋势,且变化的幅度在不同胁迫强度下存在差异.(2)在整个干旱胁迫过程中,各胁迫处理叶片丙二醛(MDA)含量均呈上升趋势,不同胁迫强度的变化幅度不同;叶片中的可溶性蛋白含量和根系活力随着干旱胁迫程度的增强呈先增高后降低的趋势.(3)栓皮栎幼苗叶片的脯氨酸含量随着干旱胁迫时间的延长表现出先增加后降低的趋势;叶片叶绿素a、叶绿素b、总叶绿素含量以及叶绿素a/b值均呈逐渐降低的趋势.研究表明,栓皮栎幼苗在短期和轻度干旱胁迫下通过提高自身的保护酶活性、增加可溶性蛋白和脯氨酸含量、提高根系活力等来抵御干旱环境的伤害,从而表现出较强的耐旱特性;而在重度干旱胁迫条件下,栓皮栎幼苗自我调节能力丧失,体内代谢紊乱,导致保护酶活性、可溶性蛋白、脯氨酸含量和根系活力等下降,从而受到干旱伤害.     

干旱胁迫对欧洲鹅耳枥幼苗生理生化特征的影响

以一年生欧洲鹅耳枥幼苗为材料,通过盆栽控水试验,设置5个土壤含水量梯度,研究不同梯度干旱胁迫对其幼苗生理生化指标的影响,以明确欧洲鹅耳枥的耐旱特性。结果显示:（1）随着干旱胁迫的加剧,欧洲鹅耳枥幼苗受到损害程度逐渐加重,形态表现出明显的缺水特征,叶片相对含水量和叶绿素总量随着干旱胁迫程度的增加而下降,且胁迫强度越强、时间越久其变化幅度越大。（2）幼苗叶片SOD活性、POD活性、可溶性糖含量和可溶性蛋白含量均随着干旱程度的增加表现出先升高后降低的趋势。（3）随着干旱胁迫程度的增加和干旱时间持续,幼苗叶片MDA含量和相对电导率总体上呈增大趋势,并在干旱末期达到最大值。研究表明,欧洲鹅耳枥幼苗在短期和轻度（田间最大持水量的60%）、中度（田间最大持水量的45%）干旱胁迫下能通过调节自身保护酶活性和渗透调节物质含量来减轻干旱造成的危害;而在重度（田间最大持水量的30%）干旱胁迫下,幼苗自我调节能力受影响,极度（田间最大持水量的15%）干旱胁迫对其造成一定的损害。研究认为,欧洲鹅耳枥具有一定的抗干旱特性,可在园林中推广运用。     

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23^fyp and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.     

水分胁迫及复水过程中小麦抗氧化酶的变化

对两个抗旱性不同的小麦品种进行水分胁迫和复水处理,研究其抗氧化酶活性的响应。在水分胁迫下,陇春-20的相对含水量高于优鉴-24,复水24h后,优鉴-24的相对含水量恢复较快且高于陇春-20。水分胁迫下,优鉴-24中H2O2含量增加迅速,而且各阶段含量均高于陇春-20,复水后两个品种的H2O2含量都下降,这表明优鉴-24在水分胁迫时受到更严重的氧化胁迫。采用温和胶电泳结合抑制剂实验发现小麦有3条Mn—SOD,一条Fe—SOD和Cu／Zn-SOD同工酶带,CAT同工酶有3条谱带。在水分胁迫和复水期间,优鉴-24的SOD和CAT活性高于陇春-20,随着水分胁迫程度的增加,两个品种的SOD和CAT活性都增强,复水后,优鉴-24的SOD活性继续增强,而陇春-20的Mn—SOD—3活性略微降低,Fe—SOD和Cu／Zn—SOD活性略微升高,陇春-20的CAT活性降低。水分胁迫诱导了Mn—SOD—1在优鉴-24及Mn—SOD-2和Fe—SOD在陇春-20中的表达。