Establishment of a transgenic hairy root system in wild and domesticated watermelon (Citrullus lanatus) for studying root vigor under drought

Root vigor is an important trait for the growth of terrestrial plants, especially in water-deficit environments. Although deserts plants are known for their highly developed root architecture, the molecular mechanism responsible for this trait has not been determined. Here we established an efficient protocol for the genetic manipulation of two varieties of watermelon plants: a desert-grown wild watermelon that shows vigorous root growth under drought, and a domesticated cultivar showing retardation of root growth under drought stress. Agrobacterium rhizogenes-mediated transgenic hairy roots were efficiently induced and selected from the hypocotyls of these plants. Transgenic GUS expression was detected in the roots by RT-PCR and histochemical GUS staining. Moreover, a liquid culture system for evaluating their root growth was also established. Interestingly, growth of the hairy roots derived from domesticated variety of watermelon strongly inhibited under high osmotic condition, whereas the hairy roots derived from wild variety of watermelon retained substantial growth rates under the stress condition. The new protocol presented here offers a powerful tool for the comparative study of the molecular mechanism underlying drought-induced root growth in desert plants.     

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.     

Proteome analysis of soybean roots subjected to short-term drought stress

Drought is one of the most important constraints on the growth and productivity of many crops, including soybeans. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we carried out a proteome analysis in combination with physiological analyses of soybean roots subjected to severe but recoverable drought stress at the seedling stage. Drought stress resulted in the increased accumulation of reactive oxygen species and subsequent lipid peroxidation. The proline content increased in drought-stressed plants and then decreased during the period of recovery. The high-resolution proteome map demonstrated significant variations in about 45 protein spots detected on Comassie briliant blue-stained 2-DE gels. Of these, 28 proteins were identified by mass spectrometry; the levels of 5 protein spots were increased, 21 were decreased and 2 spots were newly detected under drought condition. When the stress was terminated by watering the plants for 4 days, in most cases, the protein levels tended towards the control level. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and nitrogen metabolism, cell wall modification, signal transduction, cell defense and programmed cell death, and they contribute to the molecular mechanism of drought tolerance in soybean plants. Analysis of protein expression patterns revealed that proteins associated with osmotic adjustment, defense signaling and programmed cell death play important roles for soybean plant drought adaptation. The identification of these proteins provides new insight that may lead to a better understanding of the molecular basis of the drought stress responses.     

高粱ATP合成酶E亚基基因(SbATPase-E)的克隆及其抗逆功能研究

高粱是一种抗旱性较强的禾谷类作物。本研究在高粱中克隆到一个全长为693 bp的编码ATP合成酶E亚基的基因(SbATPase-E)。在高粱幼苗期,SbATPase-E基因受Na Cl和脱落酸(ABA)处理诱导上调表达。该基因在拟南芥中过量表达可提高转基因植株的耐旱性和耐盐性,在逆境胁迫条件下转基因拟南芥植株较野生型植株根系发达,可能是转基因植株耐旱性和耐盐性提高的主要原因。在干旱胁迫条件下,转基因植株中DREB2A、P5CS1、RD29A、RAB18和ABI1基因的表达量相对于野生型植株中的表达量提高更为显著;在高盐处理条件下,转基因植株中SOS1和SOS2基因的表达量也较野生型植株中的表达量明显提高。这些抗逆相关基因的上调表达可能是转基因植株抗逆性提高的主要分子机制。     

Influence of Drought on Graminicide Phytotoxicity in Wild Oat (Avena fatua) Grown under Different Temperature and Humidity Conditions

Controlled environmental experiments were carried out to determine the phytotoxicity of several graminicides on wild oat (Avena futua L.) as influenced by combination of drought and temperature stress or drought and low relative humidity. Compared with unstressed conditions (20/15°C plus adequate soil moisture), imazamethabenz phytotoxicity to wild oat was reduced significantly when plants were exposed to a combination of drought and high temperature (30/20°C) stress. Imazamethabenz phytotoxicity was reduced almost as much by high temperature stress alone as by a combined temperature and drought stress. When herbicides were applied to wild oat plants subjected to drought alone or to drought plus high temperature, the observed reduction in phytotoxicity from greatest to least was: fenoxaprop = diclofop > flamprop > imazamethabenz. Fenoxaprop performance was most inhibited by the combination of drought plus high temperature, although drought alone and to a lesser degree, high temperature alone, inhibited fenoxaprop action. High temperature had an adverse effect on the efficacy of fenoxaprop at lower application rates. Raising fenoxaprop application rates to 400 g ha⁻¹ overcame the inhibition caused by high temperature alone but only partially alleviated the effect of drought combined with high temperature. When plants were grown under a low temperature regimen the imposition of drought stress had little effect on imazamethabenz phytotoxicity but did reduce fenoxaprop phytotoxicity. At 25/15°C drought reduced the phytotoxicity of fenoxaprop and diclofop greatly but had no significant impact on the performance of any of the herbicides examined, regardless of soil moisture regimen. Received April 14, 1997; accepted September 22, 1997     

Plants utilize isoprene emission as a thermotolerance mechanism

Isoprene is a volatile compound emitted from leaves of many plant species in large quantities, which has an impact on atmospheric chemistry due to its massive global emission rate (5 x 10(14) carbon g year(-1)) and its high reactivity with the OH radical, resulting in an increase in the half-life of methane. Isoprene emission is strongly induced by the increase in isoprene synthase activity in plastids at high temperature in the day time, which is regulated at its gene expression level in leaves, while the physiological meaning of isoprene emission for plants has not been clearly demonstrated. In this study, we have functionally overexpressed Populus alba isoprene synthase in Arabidopsis to observe isoprene emission from transgenic plants. A striking difference was observed when both transgenic and wild-type plants were treated with heat at 60 degrees C for 2.5 h, i.e. transformants revealed clear heat tolerance compared with the wild type. High isoprene emission and a decrease in the leaf surface temperature were observed in transgenic plants under heat stress treatment. In contrast, neither strong light nor drought treatments showed an apparent difference. These data suggest that isoprene emission plays a crucial role in a heat protection mechanism in plants.     

Identification of changes in Triticum aestivum L. leaf proteome in response to drought stress by 2D-PAGE and MALDI-TOF/TOF mass spectrometry

Drought is an abiotic stress that strongly influences plant growth, development and productivity. To gain a better understanding of the drought-stress responses at physiological and molecular level in wheat plants (Triticum aestivum cv. KTC86211), we performed a comparative physiological and proteomics analysis. Eight-day-old wheat seedlings were treated with polyethylene glycol-simulated drought stress for 0, 24, 48 and 72 h. Drought treatment resulted in alterations of morphology, increased relative electrolyte leakage and reduced length and weight on leaf and root. Stress-induced proteome changes were analyzed by two-dimensional gel electrophoresis in conjunction with MALDI-TOF/TOF. Twenty-three spots differed significantly between control and treated plants following 48 h of drought stress, with 19 upregulated, and 4 downregulated, in leaf tissues. All of the differentially expressed protein spots were identified, revealing that the majority of proteins altered by drought treatment were involved in reactive oxygen species scavenging enzymes and photosynthesis. Other proteins identified were involved in protein metabolism, cytoskeleton structure, defense response, acid metabolism and signal transduction. All proteins might contribute cooperatively to reestablish cellular homeostasis under drought stress. The present study not only provides new insights into the mechanisms of acclimation and tolerance to drought stress in wheat plants, but also provides clues for improving wheat’s drought tolerance through breeding or genetic engineering.     

Beyond osmolytes and transcription factors: drought tolerance in plants <Emphasis Type="Italic">via</Emphasis> protective proteins and aquaporins

Mechanisms of drought tolerance have been studied by numerous groups, and a broad range of molecules have been identified to play important roles. A noteworthy response of stressed plants is the accumulation of novel protective proteins, including heat-shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins. Identification of gene regulatory networks of these protective proteins in plants will allow a wide application of biotechnology for enhancement of drought tolerance and adaptation. Similarly, aquaporins are involved in the regulation of water transport, particularly under abiotic stresses. The molecular and functional characterization of protective proteins and aquaporins has revealed the significance of their regulation in response to abiotic stresses. Herein, we highlight new findings regarding the action mechanisms of these proteins. Finally, this review also surveys the current advances in engineering drought tolerant plants, particularly the engineering of protective proteins (sHSPs and LEA) and aquaporins for imparting drought stress tolerance in plants.     

Recent methods of drought stress tolerance in plants

In the climate change scenario the drought has been diagnosed as major stress affecting crop productivity. This review demonstrates some recent findings on the amelioration of drought stress. Nanoparticles, synthetic growth regulators viz. Trinexapac-ethyl, and Biochar addition helps to economize the water budget of plants, enhances the bioavailability of water and nutrients as well as overcomes drought induced osmotic and oxidative stresses. Besides ABA, SA and JA are also involved in inducing tolerance to drought stress through modulation of physiological and biochemical processes in plants. Plant growth promoting rhizobacteria (PGPR) offer new opportunities in agricultural biotechnology. These beneficial microorganisms colonize the rhizosphere/endo-rhizosphere of plants and impart drought tolerance by improving root architechture, enhancing water use efficiency, producing exopolysaccharides, phytohormones viz, ABA, SA and IAA and volatile compounds. Further PGPR also play positive role in combating osmotic and oxidative stresses induced by drought stress through enhancing the accumulation of osmolytes, antioxidants and upregulation or down regulation of stress responsive genes. In transgenic plants stress inducible genes enhanced abiotic stress tolerance by encoding key enzymes regulating biosynthesis of compatible solutes. The role of genes/cDNAs encoding proteins involved in regulating other genes/proteins, signal transduction process and strategies to improve drought stress tolerance have also been discussed.     

New Plant Growth Regulators Protect Photosynthesis and Enhance Growth Under Drought of Jack Pine Seedlings

To determine whether natural plant growth regulators (PGRs) can enhance drought tolerance and the competitive ability of transplanted seedlings, 1.5-year-old jack pine (Pinus banksana Lamb.) seedlings were treated with homobrassinolide, salicylic acid, and two polyamines, spermine and spermidine, triacontanol, abscisic acid (ABA), and the synthetic antioxidant, Ambiol. PGRs were fed into the xylem for 7 days and plants were droughted by withholding water for 12 days. ABA, Ambiol, spermidine, and spermine at a concentration of 10 μg L⁻¹ stimulated elongation growth under drought, whereas ABA, Ambiol, and spermidine maintained higher photosynthetic rates, higher water use efficiency, and lower Ci/Ca ratio under drought compared with control plants. The damaging effects of drought on membrane leakage was reversed by Ambiol, ABA, triacontanol, spermidine, and spermine. Because ABA, Ambiol, and both polyamines enhanced elongation growth and also reduced membrane damage in jack pine under drought, they show promise as treatments to harden seedlings against environmental stress. The protective action of these compounds on membrane integrity was associated with an inhibition of ethylene evolution, with a reduction in transpiration rate and an enhancement of photosynthesis, which together increased water use efficiency under drought. Although most of the tested compounds acted as antitranspirants, the inhibition in membrane leakage in ABA-, Ambiol-, and polyamine-treated plants appeared more closely related to the antiethylene action. Received December 30, 1998; accepted October 14, 1999     

Physiological and morphological traits associated with adaptation of lucerne (Medicago sativa) to severely drought‐stressed and to irrigated environments

Adaptation to severe drought and to irrigated cropping can both contribute to increased water use efficiency of lucerne, but knowledge on the relevant adaptive traits is limited. Five cultivars featuring contrasting adaptive responses for 3‐year forage yield across 10 agricultural environments of the western Mediterranean basin were currently studied, to identify physiological and morphological traits associated with specific and wide‐adaptation responses. The landraces Mamuntanas, Demnat 203 and Erfoud 1, and the varieties SARDI 10 and Prosementi, were grown in replicated metal containers (55 cm long × 12 cm wide × 75 cm deep; 21 plants per container) under irrigation (weekly restoring soil field capacity) and under moderate and severe drought stress (implying decreased irrigation for 30 days followed by withheld irrigation for 33 and 58 days, respectively). Cultivar post‐stress survival reflected the known cultivar adaptation to drought‐prone agricultural environments. Demnat 203, specifically adapted to irrigated, frequently mown environments, displayed higher amounts of starch, soluble proteins and total nitrogen in the crown and the root under irrigation. This was due to outstanding organ size and, for starch, higher concentrations. Mamuntanas, specifically adapted to drought‐prone environments, exhibited high water‐soluble carbohydrate concentration in storage organs under severe stress, along with a water‐conservation strategy implying less water used in initial drought‐stress phases due to limited early root development that resulted in more water available under severe stress. Drought‐tolerant germplasm also displayed lower wilting under early stress, more plants with green tissues under severe stress, and more stems per plant in stress or favourable conditions. Multivariate patterns of cultivar variation for physiological and morphological traits were strictly associated with cultivar variation for adaptation pattern. Our results highlighted the difficulty to combine some traits of environment‐specific adaptive value into a unique widely adapted variety, supporting the selection of varieties specifically adapted to irrigated or severely drought‐prone environments.     

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     

Physiology and proteomics of drought stress acclimation in sunflower (Helianthus annuus L.)

An easy and manageable in vitro screening system for drought tolerance of sunflower seedlings based on MS media supplemented with polyethylene glycol 6000 was evaluated. Morphological and physiological parameters were compared between control (-0.05 MPa) and drought-stressed (-0.6 MPa) seedlings of Helianthus annuus L. cv. Peredovick. There was a significant growth deficit in drought-stressed plants compared to control plants in terms of hypocotyl length, and shoot and root fresh mass. Shoot growth was more restricted than root growth, resulting in an increased root/shoot ratio of drought-stressed plants. Accumulation of osmolytes such as inositol (65-fold), glucose (58-fold), proline (55-fold), fructose (11-fold) and sucrose (eightfold), in leaves of drought-stressed plants could be demonstrated by gas-liquid chromatography. Soluble protein patterns of leaves were analysed with two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry. A set of 46 protein spots allowed identification of 19 marker proteins. Quantitative changes in protein expression of drought-stressed versus control plants were detected. In leaves of drought-stressed sunflower seedlings six proteins were significantly up-regulated more than twofold: a putative caffeoyl-CoA 3-O-methyltransferase (4.5-fold), a fructokinase 3 (3.3-fold), a vegetative storage protein (2.5-fold), a glycine-rich RNA binding protein (2.2-fold), a CuZn-superoxide dismutase (2.1-fold) and an unknown low molecular weight protein (2.3-fold). These proteins represent general stress proteins induced under drought conditions or proteins contributing to basic carbon metabolism. The up-regulated proteins are interesting candidates for further physiological and molecular investigations regarding drought tolerance in sunflower.     

Mechanisms of selection for drought stress tolerance and avoidance in Impatiens capensis (Balsaminaceae)

For longer lived annual plants, high water-use efficiency (WUE) and low stomatal conductance are hypothesized to confer a fitness advantage under drought stress. To directly test the adaptive significance of WUE and stomatal conductance under drought stress, inbred lines of Impatiens capensis were grown in two field environments (watered and not-watered), in a year of unusual early-season drought. In contrast to the results from a previous study of late-season drought in the same system, selection was detected for lower WUE, increased stomatal conductance, and early flowering time. These findings suggest that early-season drought conditions may select for drought avoidance traits such as low WUE and early reproduction, whereas later drought selects for tolerance traits such as high WUE.     

Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits

Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.     

Enhancing drought tolerance in C(4) crops

Adaptation to abiotic stresses is a quantitative trait controlled by many different genes. Enhancing the tolerance of crop plants to abiotic stresses such as drought has therefore proved to be somewhat elusive in terms of plant breeding. While many C(4) species have significant agronomic importance, most of the research effort on improving drought tolerance has focused on maize. Ideally, drought tolerance has to be achieved without penalties in yield potential. Possibilities for success in this regard are highlighted by studies on maize hybrids performed over the last 70 years that have demonstrated that yield potential and enhanced stress tolerance are associated traits. However, while our understanding of the molecular mechanisms that enable plants to tolerate drought has increased considerably in recent years, there have been relatively few applications of DNA marker technologies in practical C(4) breeding programmes for improved stress tolerance. Moreover, until recently, targeted approaches to drought tolerance have concentrated largely on shoot parameters, particularly those associated with photosynthesis and stay green phenotypes, rather than on root traits such as soil moisture capture for transpiration, root architecture, and improvement of effective use of water. These root traits are now increasingly considered as important targets for yield improvement in C(4) plants under drought stress. Similarly, the molecular mechanisms underpinning heterosis have considerable potential for exploitation in enhancing drought stress tolerance. While current evidence points to the crucial importance of root traits in drought tolerance in C(4) plants, shoot traits may also be important in maintaining high yields during drought.     

Lipidomic reprogramming associated with drought stress priming‐enhanced heat tolerance in tall fescue (Festuca arundinacea)

Stress priming by exposing plants to a mild or moderate drought could enhance plant tolerance to subsequent heat stress. Lipids play vital roles in stress adaptation, but how lipidomic profiles change, affecting the cross‐stress tolerance, is largely unknown. The objectives of this study were to perform lipidomics, to analyse the content, composition, and saturation levels of lipids in leaves of tall fescue (Festuca arundinacea) following drought priming and subsequent heat stress, and to identify major lipids and molecular species associated with priming‐enhanced heat tolerance. Plants were initially exposed to drought for 8 days by withholding irrigation and subsequently subjected to 25 days of heat stress (38/33°C day/night) in growth chambers. Drought‐primed plants maintained significantly higher leaf relative water content, chlorophyll content, photochemical efficiency, and lower electrolyte leakage than nonprimed plants under heat stress. Drought priming enhanced the accumulation of phospholipids and glycolipids involved in membrane stabilization and stress signalling (phosphatidic acid, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, and digalactosyl diacylglycerol) during subsequent exposure to heat stress. The reprogramming of lipid metabolism for membrane stabilization and signalling in response to drought priming and subsequent exposure to heat stress could contribute to drought priming‐enhanced heat tolerance in cool‐season grass species.