Growth,photosynthesis and water relations as affected by different drought regimes and subsequent recovery in <Emphasis Type="Italic">Medicago laciniata</Emphasis> (L.) populations

The aims of this study were to investigate the effects of water deficit and recovery on growth, photosynthesis and water relations in four Medicago laciniata populations from saharian (Ml-90), inferior arid (Ml-204), superior arid (Ml-306) and semi-arid (Ml-376) Tunisian regions. After 28 d of sowing with ample irrigation, the plants were subjected to 4 water regimes: optimal irrigation (100% of field capacity, FC), moderate drought (75% FC), severe drought (35% FC) and rewatering (plants submitted to 35% FC during 7 d, afterwards the plants were rewatered to 100% FC). Harvest was carried out after 28 d of treatments. The drought tolerance in M. laciniata populations was found to be increased particularly with increasing temperatures of collection site of the population. The Ml-204 and Ml-90 populations used mainly physiological strategies for survival under moderate water shortage. Higher severe drought tolerance in both signaled populations would be related to their lower photosynthesis metabolic impairment, relatively higher leaf RWC and greater osmotic potential decrease. The results suggest that plants with low values of leaf features are likely to maintain higher leaf RWC under sever drought. The largest decrease of osmotic potential was found associated with the solute accumulations such as proline and K⁺.     

Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress

Proline accumulates in a variety of plant species in response to stresses such as drought, salinity and extreme temperatures. Although its role in plant osmotolerance remains controversial, proline is thought to contribute to osmotic adjustment, detoxification of reactive oxygen species and protection of membrane integrity. In the present study, we evaluated the effects of stress-inducible proline production on osmotic adjustment, chlorophyll fluorescence and oxidative stress protection in transgenic sugarcane transformed with a heterologous P5CS gene. In well-watered conditions, free proline, malondialdehyde (MDA) levels, Fv/Fm ratios and chlorophyll contents (Chls) in transgenic sugarcane were not statistically different from non-transformed control plants. After 9 days without irrigation, proline content in transgenic events was on the average 2.5-fold higher than in controls. However, no osmotic adjustment was observed in plants overproducing proline during the water-deficit period. The photochemical efficiency of PSII observed was higher (65%) in the transgenic events at the end of the water-deficit experiment. The effects of proline on lipid peroxidation as MDA levels and on the decline of Chl in paraquat-treated leaf discs along the drought period suggest that proline protected the plants against the oxidative stress caused by the water deficit. The overall capacity of transgenic plants to tolerate water-deficit stress could be assessed by the significantly higher biomass yields 12 days after withholding water. These results suggest that stress-inducible proline accumulation in transgenic sugarcane plants under water-deficit stress acts as a component of antioxidative defense system rather than as an osmotic adjustment mediator.     

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2, by constitutive and inducible expression of tobacco <Emphasis Type="Italic">osmotin</Emphasis>

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.     

Disruption of the cellulose synthase gene, AtCesA8/IRX1, enhances drought and osmotic stress tolerance in Arabidopsis

Two allelic Arabidopsis mutants, leaf wilting 2-1 and leaf wilting 2-2 (lew2-1 and lew2-2 ), were isolated in a screen for plants with altered drought stress responses. The mutants were more tolerant to drought stress as well as to NaCl, mannitol and other osmotic stresses. lew2 mutant plants accumulated more abscisic acid (ABA), proline and soluble sugars than the wild type. The expression of a stress-inducible marker gene RD29A, a proline synthesis-related gene P5CS (pyrroline-5-carboxylate synthase) and an ABA synthesis-related gene SDR1 (alcohol dehydrogenase/reductase) was higher in lew2 than in the wild type. Map-based cloning revealed that the lew2 mutants are new alleles of the AtCesA8/IRX1 gene which encodes a subunit of a cellulose synthesis complex. Our results suggest that cellulose synthesis is important for drought and osmotic stress responses including drought induction of gene expression.     

Physiological Evaluation of Drought Stress Tolerance and Recovery in Cauliflower (Brassica oleracea L.) Seedlings Treated with Methyl Jasmonate and Coronatine

Coronatine (COR) is a chlorosis-inducing phytotoxin that mimics some biological activities of methyl jasmonate (MeJA). Although MeJA has been reported to alleviate drought stress, it is unclear if COR has the same ability. Our objective was to determine the influence of exogenously applied MeJA and COR on the growth and metabolism of cauliflower seedlings under drought stress and recovery. Both MeJA and COR enhanced the growth and accumulation of dry matter in cauliflower seedlings during drought-stressed and rewatering conditions. Treatment with MeJA or COR enhanced tolerance of drought stress through increased accumulation of chlorophyll and net photosynthetic rate. Enzymatic (superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and glutathione reductase) and nonenzymatic antioxidant (proline and soluble sugar) systems were activated, and lipid peroxidant (malondialdehyde and hydrogen peroxide) was suppressed by MeJA and COR under drought stress. MeJA and COR also increased leaf relative water content and endogenous abscisic acid level under drought-stressed conditions. After rewatering, the contents of leaf water, chlorophyll, abscisic acid, and photosynthetic characteristics as well as enzymatic and nonenzymatic antioxidant systems showed nearly complete recovery. Both MeJA and COR can alleviate the adverse effects of drought stress and enhance the ability for water stress resistance through promotion of defense-related metabolism in cauliflower seedlings.     

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.     

Proline accumulation and nitrate reductase activity in contrasting sorghum lines during mid-season drought stress

Six lines of sorghum ( Sorghum bicolor L. Moench) with differing drought resistance (IS 22380, ICSV 213, IS 13441 and SPH 263, resistant and IS 12739 and IS 12744, susceptible) were grown under field conditions in the semi-arid tropics and analysed for proline and nitrate reductase activity (NRA; EC 1.6.6.1) during a mid-season drought. The resistant lines accumulated high levels of proline, while the susceptible lines showed no significant proline accumulation. Most of the proline was accumulated after growth of the plants had ceased. In a separate greenhouse experiment, most of the proline was found in the green rather than the fired portions of leaves. The levels returned to that of irrigated controls within 5 days of rewatering. Proline levels increased as leaf water potential and relative water content fell, and there was no apparent difference among the different sorghum lines with change in plant water status. Susceptible lines accumulated less proline than resistant lines as leaf death occurred at higher water potentials. Proline accumulation may, however, contribute to the immediate recovery of plants from drought. Leaf NRA reached high levels at about 35 days after sowing in both the stressed and irrigated plants, after which it declined. The decline in NRA was more pronounced in the stressed than in the irrigated plants and closely followed changes in the growth rate. Upon rewatering, NRA increased several-fold in all the lines and, in contrast to proline accumulation, genotypic differences in NRA were small, both during stress and upon rewatering. The high sensitivity of NRA to mild drought stress was reflected in the rapid decline of activity with small changes in leaf water potential and relative water content. The results are discussed in the light of a possible role for proline during recovery from drought, and the maintenance of NRA during stress and its recovery upon rewatering.     

Response of proteome and morphological structure to short‐term drought and subsequent recovery in Cucumis sativus leaves

Drought is the primary limitation to plant growth and yield in agricultural systems. Cucumber (Cucumis sativus) is one of the most important vegetables worldwide and has little tolerance for water deficit. To understand the drought stress response strategy of this plant, the responses of cucumber to short‐term drought and rewatering were determined in this study by morphological structure and proteomic analyses. The leaf relative water content was significantly decreased under drought, and the cell structure was altered, while rewatering obviously alleviated the symptoms of water shortage and cell damage. A total of 320 and 246 proteins exhibiting significant abundance changes in response to drought and recovery, respectively, were identified. Our proteome analysis showed that 63 co‐regulated proteins were shared between drought and rewatering, whereas most of the responsive proteins were unique. The proteome is adjusted through a sequence of regulatory processes including the biosynthesis of secondary metabolites and the glutathione metabolism pathway, which showed a high correlation between protein abundance profile and corresponding enzyme activity. Drought and recovery regulated different types of proteins, allowing plants to adapt to environmental stress or restore growth, respectively, which suggests that short‐term drought and recovery are almost fully uncoupled processes. As an important component of the antioxidant system in plants, glutathione metabolism may be one of the main strategies for regulating antioxidant capacity during drought recovery. Our results provide useful information for further analyses of drought adaptability in cucumber plants.     

Cloning of Gossypium hirsutum Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (GhSnRK2) and Its Overexpression in Transgenic Arabidopsis Escalates Drought and Low Temperature Tolerance

The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that GhSnRK2 participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.     

香蕉MaASR1基因的抗干旱作用

ASR(ABA, stress, ripening induced protein)是一类响应植物干旱胁迫的关键转录因子, 在许多植物中已有报道, 然而尚未见香蕉(Musa acuminata)中ASR与抗旱作用的相关研究。该实验从香蕉果实cDNA文库中筛选出1个ASR基因, 即MaASR1(登录号为AY628102)。干旱胁迫下, 该基因在叶片中的表达量高于根部。将MaASR1转入拟南芥(Arabidopsis thaliana), Southern检测确定了两株独立表达的转基因株系(命名为L14和L38)。表型观察发现, 此两转基因株系的叶片变小且变厚; Northern和Western检测结果表明, MaASR1在L14和L38中表达。控水处理后, L14和L38的存活率及脯氨酸含量均高于野生型。经干旱胁迫和外源ABA处理后, 对MaASR1转基因株系中ABA/胁迫响应基因的表达分析, 发现MaASR1可增强转基因株系对ABA信号的敏感度, 但不能增强植株依赖于ABA途径的抗旱性。     

Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress

The biochemical basis of heat/drought tolerance was investigated by comparing the response of antisense and sense transgenic soybean plants (containing the L-delta1-pyrroline-5-carboxylate reductase gene) with non-transgenic wild-type plants. The plants were subjected to a simultaneous drought and heat stress of 2 days, whereafter they were rewatered at 25 degrees C. During this time the sense plants only showed mild symptoms of stress compared to the antisense plants which were severely stressed. Upon stress, nicotinamide adenine dinucleotide phosphate (NADP+) levels decreased in antisense while it increased in sense plants. Recovery with respect to NADP+ levels was best in sense plants. Sense plants had the highest ability to accumulate proline during stress and to metabolise proline after rewatering. Analyses of the fast phase chlorophyll-a fluorescence transients showed dissociation of the oxygen-evolving complex (OEC) upon stress in all plants tested. In the sense plants, which best resisted the stress, OEC dissociation was bypassed by proline feeding electrons into photosystem 2 (PSII), maintaining an acceptable nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) level, preventing further damage. Upon recovery, NADPH is consumed during oxidation of accumulated proline providing high Levels of NADP+ to act as electron acceptor to PSII, which indirectly may ameliorate the inhibition and/or the effect of uncoupling of the OEC.     

MusaDHN-1, a novel multiple stress-inducible SK3-type dehydrin gene, contributes affirmatively to drought- and salt-stress tolerance in banana

Dehydrins are highly hydrophilic proteins involved in playing key adaptive roles in response to abiotic stress conditions having dehydration as a common component. In the present study, a novel banana SK(3)-type dehydrin, MusaDHN-1, was identified and later characterized using transgenic banana plants to investigate its functions in abiotic stress tolerance. Expression profiling in native banana plants demonstrated that MusaDHN-1 was induced in leaves by drought, salinity, cold, oxidative and heavy metal stress as well as by treatment with signalling molecules like abscisic acid, ethylene and methyl jasmonate. Promoter analysis carried out by making a MusaDHN-1 promoter: β-glucuronidase fusion construct reconfirmed the abiotic stress inducibility of MusaDHN-1. Transgenic banana plants constitutively overexpressing MusaDHN-1 were phenotypically normal and displayed improved tolerance to drought and salt-stress treatments in both in vitro and ex vitro assays. Enhanced accumulation of proline and reduced malondialdehyde levels in drought and salt-stressed MusaDHN-1 overexpressing plants further established their superior performance in stressed conditions. This study is the first to report generation of transgenic banana plants engineered for improved drought and salt-stress tolerance.     

Effect of Putrescine, 4-PU-30, and Abscisic Acid on Maize Plants Grown under Normal, Drought, and Rewatering Conditions

The experiments were carried out with maize (Zea mays L.) seedlings, hybrid Kneja 530, grown hydroponically in a growth chamber. Twelve-day-old plants were foliar treated with putrescine, N¹-(2-chloro-4-pyridyl)-N²-phenylurea (4-PU-30), and abscisic acid (ABA) at concentrations of 10⁻⁵ m. Twenty-four hours later the plants were subjected to a water deficit program, induced by 15% polyethylene glycol (PEG; molecular weight, 6,000). Three days after drought stress half of the plants were transferred to nutrient solution for the next 3 days. The effects of the water shortage, rewatering, and plant growth regulator (PGR) treatment on the fresh and dry weights, leaf pigment content, proline level, relative water content (RWC), transpiration rate, activities of catalase and guaiacol peroxidase, hydrogen peroxide content, and level of the products of lipid peroxidation were studied. It was established that the application of PGRs alleviated to some extent the plant damage provoked by PEG stress. At the end of the water shortage program the plants treated with these PGRs possessed higher fresh weight than drought-subjected control seedlings. It was found also that putrescine increased the dry weight of plants. Under drought, the RWC and transpiration rate of seedlings declined, but PGR treatment reduced these effects. The accumulation of free proline, malondialdehyde, and hydrogen peroxide was prevented in PGR-treated plants compared with the water stress control. The results provided further information about the influence of putrescine, 4-PU-30, and ABA on maize plants grown under normal, drought, and rewatering conditions. Received September 25, 1997; accepted August 10, 1998     

棒叶落地生根对干旱与复水的生理响应

为探讨棒叶落地生根(Kalanchoe tubiflora)耐旱的机制,在干旱与复水条件下,对其叶片的一些生理生化指标进行了测定。结果表明,随干旱时间延长,棒叶落地生根叶片中O2-·生成速率增大,H2O2含量升高,导致脂质过氧化产物MDA含量增高;同时SOD活性升高,CAT活性降低;可溶性糖与脯氨酸含量增加,但复水后这些指标均恢复到干旱前的水平。这说明棒叶落地生根能够耐受干旱环境是通过积累渗透调节物质,提高活性氧的清除能力,从而减少氧化胁迫造成的伤害。