Photosynthesis and antioxidative defense mechanisms in deciphering drought stress tolerance of crop plants

Crop plants are regularly exposed to an array of abiotic and biotic stresses, among them drought stress is a major environmental factor that shows adverse effects on plant growth and productivity. Because of this these factors are considered as hazardous for crop production. Drought stress elicits a plethora of responses in plants resulting in strict amendments in physiological, biochemical, and molecular processes. Photosynthesis is the most fundamental physiological process affected by drought due to a reduction in the CO₂ assimilation rate and disruption of primary photosynthetic reactions and pigments. Drought also expedites the generation of reactive oxygen species (ROS), triggering a cascade of antioxidative defense mechanisms, and affects many other metabolic processes as well as affecting gene expression. Details of the drought stress-induced changes, particularly in crop plants, are discussed in this review, with the major points: 1) leaf water potentials and water use efficiency in plants under drought stress; 2) increased production of ROS under drought leading to oxidative stress in plants and the role of ROS as signaling molecules; 3) molecular responses that lead to the enhanced expression of stress-inducible genes; 4) the decrease in photosynthesis leading to the decreased amount of assimilates, growth, and yield; 5) the antioxidant defense mechanisms comprising of enzymatic and non-enzymatic antioxidants and the other protective mechanisms; 6) progress made in identifying the drought stress tolerance mechanisms; 7) the production of transgenic crop plants with enhanced tolerance to drought stress.     

Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity

Drought and high-salinity are the important constraints that severely affect plant development and crop yield worldwide. It has been established that ethylene response factor (ERF) proteins play important regulatory roles in plant response to abiotic and biotic stresses. Our previous researches have revealed that transgenic tobacco over-expressing TERF1 (encoding a tomato ERF protein) showed enhanced tolerance to abiotic stress. Here, we further investigate the function of TERF1 in transgenic rice. Compared with the wild-type plants, overexpression of TERF1 resulted in an increased tolerance to drought and high-salt in transgenic rice. And the enhanced tolerance may be associated with the accumulation of proline and the decrease of water loss. Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and OsCDPK19 under normal growth conditions. Our analyses of cis-acting elements show that there exist DRE/CRT and/or GCC-box existing in TERF1 targeted gene promoters. Our results revealed that ectopic expression of TERF1 in rice caused a series of molecular and physiological alterations and resulted in the transgenic rice with enhanced tolerance to abiotic stress, indicating that TERF1 might have similar regulatory roles in response to abiotic stress in tobacco and rice. Shumei Gao, Haiwen Zhang and Yun Tian contributed equally to this work.     

Expression of apoplastically secreted tobacco osmotin in cotton confers drought tolerance

Osmotin or osmotin-like proteins have been shown to be induced in several plant species in response to various types of biotic and abiotic challenges. The protein is generally believed to be involved in protecting the plant against these stresses. Although some understanding of the possible mechanism underlying the defense function of osmotin against biotic stresses is beginning to emerge, its role in abiotic stress response is far from clear. We have transformed cotton plants with a tobacco-osmotin gene, lacking the sequence encoding its 20 amino acid-long, C-terminal vacuolar-sorting motif, under the control of CaMV 35S promoter. Apoplastic secretion of the recombinant protein was confirmed and the plants were evaluated for their ability to tolerate drought conditions. Under polyethylene glycol-mediated water stress, the osmotin-expressing seedlings showed better growth performance. The transformants showed a slower rate of wilting during drought and faster recovery following the termination of dry conditions in a greenhouse setting. During drought, the leaves from transgenic plants had higher relative water content and proline levels, while showing reduced H₂O₂ levels, lipid peroxidation and electrolyte leakage. Importantly, following a series of dry periods, the osmotin transformants performed better in terms of most growth and developmental parameters tested. Most relevant, the fiber yield of transgenic plants did not suffer as much as that of their non-transgenic counterparts under drought conditions. The results provide direct support for a protective role of osmotin in cotton plants experiencing water stress and suggest a possible way to achieve tolerance to drought conditions by means of genetic engineering. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression profiling of rice cultivars differing in their tolerance to long-term drought stress

Understanding the molecular basis of plant performance under water-limiting conditions will help to breed crop plants with a lower water demand. We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4) and drought-sensitive (Nipponbare and Taipei 309) rice (Oryza sativa L.) cultivars to 18 days of drought stress in climate chamber experiments. Drought stressed plants grew significantly slower than the controls. Gene expression profiles were measured in leaf samples with the 20 K NSF oligonucleotide microarray. A linear model was fitted to the data to identify genes that were significantly regulated under drought stress. In all drought stressed cultivars, 245 genes were significantly repressed and 413 genes induced. Genes differing in their expression pattern under drought stress between tolerant and sensitive cultivars were identified by the genotype x environment (G x E) interaction term. More genes were significantly drought regulated in the sensitive than in the tolerant cultivars. Localizing all expressed genes on the rice genome map, we checked which genes with a significant G x E interaction co-localized with published quantitative trait loci regions for drought tolerance. These genes are more likely to be important for drought tolerance in an agricultural environment. To identify the metabolic processes with a significant G x E effect, we adapted the analysis software MapMan for rice. We found a drought stress induced shift toward senescence related degradation processes that was more pronounced in the sensitive than in the tolerant cultivars. In spite of higher growth rates and water use, more photosynthesis related genes were down-regulated in the tolerant than in the sensitive cultivars.     

Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana

Tocopherol cyclase (VTE1, encoded by VTE1 gene) catalyzes the penultimate step of tocopherol synthesis. Transgenic tobacco plants overexpressing VTE1 from Arabidopsis were exposed to drought conditions during which transgenic lines had decreased lipid peroxidation, electrolyte leakage and H(2)O(2) content, but had increased chlorophyll compared with the wild type. Thus VTE1 can be used to increase vitamin E content of plants and also to enhance tolerance to environmental stresses.     

Expression of ethylene response factor JERF1 in rice improves tolerance to drought

Ethylene response factor (ERF) proteins regulate a variety of stress responses in plant. JERF1, a tomato ERF protein, can be induced by abscisic acid (ABA). Overexpression of JERF1 enhanced the tolerance of transgenic tobacco to high salt concentration, osmotic stress, and low temperature by regulating the expression of stress-responsive genes by binding to DRE/CRT and GCC-box cis-elements. In this research, we further report that overexpression of JERF1 significantly enhanced drought tolerance of transgenic rice. The overexpression activated the expression of stress-responsive genes and increased the synthesis of the osmolyte proline by regulating the expression of OsP5CS, encoding the proline biosynthesis key enzyme deltal-pyrroline-5-carboxylate synthetase. JERF1 also activated the expression of two ABA biosynthesis key enzyme genes, OsABA2 and Os03g0810800, and increased the synthesis of ABA in rice. Analysis of cis-elements of JERF1-targeted genes pointed to the existence of DRE/CRT and/or GCC box in their promoters, indicating that JERF1 could activate the expression of related genes in rice by binding to these cis-elements. Unlike some other ERF proteins, constructive overexpression of JERF1 did not change the growth and development of transgenic rice, which makes JEFR1 a potentially useful source in breeding for greater tolerance to abiotic stress.     

Salinity and drought tolerance of mannitol-accumulating transgenic tobacco

Tobacco plants (Nicotiana tabacum L.) were transformed with a mannitol-1-phosphate dehydrogenase gene resulting in mannitol accumulation. Experiments were conducted to determine whether mannitol provides salt and/or drought stress protection through osmotic adjustment. Non-stressed transgenic plants were 20–25% smaller than non-stressed, non-transformed (wild-type) plants in both salinity and drought experiments. However, salt stress reduced dry weight in wild-type plants by 44%, but did not reduce the dry weight of transgenic plants. Transgenic plants adjusted osmotically by 0.57 MPa, whereas wild-type plants did not adjust osmotically in response to salt stress. Calculations of solute contribution to osmotic adjustment showed that mannitol contributed only 0-003-0-004 MPa to the 0.2 MPa difference in full turgor osmotic potential (π_o) between salt-stressed transgenic and wild-type plants. Assuming a cytoplasmic location for mannitol and that the cytoplasm constituted 5% of the total water volume, mannitol accounted for only 30–40% of the change in π_o of the cytoplasm. Inositol, a naturally occurring polyol in tobacco, accumulated in response to salt stress in both transgenic and wild-type plants, and was 3-fold more abundant than mannitol in transgenic plants. Drought stress reduced the leaf relative water content, leaf expansion, and dry weight of transgenic and wild-type plants. However, π_o was not significantly reduced by drought stress in transgenic or wild-type plants, despite an increase in non-structural carbohydrates and mannitol in droughted plants. We conclude that (1) mannitol was a relatively minor osmolyte in transgenic tobacco, but may have indirectly enhanced osmotic adjustment and salt tolerance; (2) inositol cannot substitute for mannitol in this role; (3) slower growth of the transgenic plants, and not the presence of mannitol per se, may have been the cause of greater salt tolerance, and (4) mannitol accumulation was enhanced by drought stress but did not affect π_o or drought tolerance.     

OsPFA-DSP1, a rice protein tyrosine phosphatase, negatively regulates drought stress responses in transgenic tobacco and rice plants

Dephosphorylation plays a pivotal role in regulating plant growth, development and abiotic/biotic stress responses. Here, we characterized a plant and fungi atypical dual-specificity phosphatase (PFA-DSP) subfamily member, OsPFA-DSP1, from rice. OsPFA-DSP1 was determined to be a functional protein tyrosine phosphatase (PTP) in vitro using phosphatase activity assays. Quantitative real-time PCR and GENEVESTIGATOR analysis showed that OsPFA-DSP1 mRNA was induced by drought stress. Transfection of rice protoplasts showed that OsPFA-DSP1 accumulated in both the cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP1 in tobacco increased sensitivity to drought stress and insensitivity to ABA-induced stomatal closure and inhibition of stomatal opening. Furthermore, overexpression of OsPFA-DSP1 in rice also increased sensitivity to drought stress. These results indicated that OsPFA-DSP1 is a functional PTP and may act as a negative regulator in drought stress responses.     

Differential antioxidative responses of indica rice cultivars to drought stress

The present study investigated the linkages between drought stress, oxidative damages and variations in antioxidants in the three rice varieties IR-29 (salt-sensitive), Pokkali (salt-tolerant) and aromatic Pusa Basmati (PB), to elucidate the antioxidative protective mechanism governing differential drought tolerance. Water deficit, induced by 20% (w/v) polyethylene glycol (PEG-6000), provoked severe damages in IR-29 and PB in the form of huge chlorophyll degradation and elevated H₂O₂, malondialdehyde and lipoxygenase (LOX, EC 1.13.11.12) levels as compared to Pokkali. The protein oxidation was more conspicuous in IR-29. Increment in antioxidants, particularly flavonoids and phenolics was several folds higher over control in Pokkali, while much lesser in IR-29 and PB. The activity of catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) were decreased in IR-29 and PB, but unaltered in Pokkali. However, marked drought-induced increase in guaiacol peroxidase (GPX, EC 1.11.1.7) activity was noted in both IR-29 and PB. Induction in radical scavenging activity, being the maximum in IR-29, and increased reducing power ability in all the cultivars, accompanied with drought stress, were observed as a defense mechanism. The novelty of our work is that it showed the aromatic rice PB behaving more closely to IR-29 in greater susceptibility to dehydration stress, while the salt-tolerant Pokkali also showed effective drought tolerance properties.     

A cotton group C MAP kinase gene, GhMPK2, positively regulates salt and drought tolerance in tobacco

Mitogen-activated protein kinase (MAPK) cascades play important roles in mediating biotic and abiotic stress responses. In plants, MAPKs are classified into four major groups (A-D) according to their sequence homology and conserved phosphorylation motifs. Compared with well-studied MAPKs in groups A and B, little is known about group C. In this study, we functionally characterised a stress-responsive group C MAPK gene (GhMPK2) from cotton (Gossypium hirsutum). Northern blot analysis indicated that GhMPK2 was induced by abscisic acid (ABA) and abiotic stresses, such as NaCl, PEG, and dehydration. Subcellular localization analysis suggested that GhMPK2 may activate its specific targets in the nucleus. Constitutive overexpression of GhMPK2 in tobacco (Nicotiana tabacum) conferred reduced sensitivity to ABA during both seed germination and vegetative growth. Interestingly, transgenic plants had a decreased rate of water loss and exhibited enhanced drought and salt tolerance. Additionally, transgenic plants showed improved osmotic adjustment capacity, elevated proline accumulation and up-regulated expression of several stress-related genes, including DIN1, Osmotin and NtLEA5. β-glucuronidase (GUS) expression driven by the GhMPK2 promoter was clearly enhanced by treatment with NaCl, PEG, and ABA. These results strongly suggest that GhMPK2 positively regulates salt and drought tolerance in transgenic plants.     

In vitro screening for increased drought tolerance in rice

Summary In vitro screening at the cellular level was performed with mature seed-derived callus from five rice varieties, viz. IR 18351-229-3, IR 3185-6-3-3-2, SR 26-B, Nona Bokra, and C 14-8 of diverse geographical origin and with differential drought resistance at the in planta level. Callus was induced from mature seeds on Murashige and Skoog medium supplemented with 2.0 mgl⁻¹ (9 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0, 10.0, and 15.0 gl⁻¹ of high molecular weight polyethylene glycol (PEG, 6000) as stressing agent to create chemical drought. Simultaneous efforts were also made to assess the effects of chemical drought in altering morphogenetic response in different varieties under in vitro culture. Seed germination was almost unaffected in SR 26-B and C 14-8, unlike in other varieties where germination was seriously affected. In general, seed germination was found to be decreased in three genotypes, viz. IR 18351-229-3, IR 3185-6-3-3-2, and Nona Bokra, with increased PEG concentrations. All genotypes displayed callus induction percentage in decreasing order with increased PEG concentrations supplemented in the callus induction medium (CIM), except SR 26-B and Nona Bokra. Callus induction was found to be more on CIM fortified with 5.0 gl⁻¹ PEG. In general, embryogenic callus induction and plantlet regeneration was found to be indirectly proportional to increased PEG concentrations used in CIM. Considering all characters, C 14-8 was found to be most appropriate in developing drought-tolerant lines under in vitro culture conditions followed by SR 26-B and Nona Bokra. A number of putative drought-tolerant plants were developed in C 14-8, SR 26-B, Nona Bokra, and IR 18351-29-3, and forwarded for field evaluation. In the majority of the progenies, a monogenic inheritance pattern for the drought tolerance character was observed.     

ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.     

Identification of functional candidate genes for drought tolerance in rice

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Bin-Ying Fu and Jian-Hua Xiong are contributed to this work equally.     

Evolution of drought tolerance and defense: dependence of tradeoffs on mechanism, environment and defense switching

Plants evolve defenses against herbivores and pathogens in stressful environments; however, plants that evolve tolerances to other environmental stressors may have compromised defenses. Such tradeoffs involving defenses may depend on limited resources or otherwise stressful environments; however, the effect of stressful environments on defense expression might be different for different genotypes (G×E). To test these predictions, we studied genetic variation and co‐variation of drought stress tolerance and defenses at two levels of genetic variation: between and within closely related species. We did this across an experimental drought stress gradient in a growth room for species for which genetic variation in drought tolerance was likely. In apparent contrast to predictions, the species Boechera holboellii (Brassicaceae) from lower and dryer elevations had slower inherent growth rates and correspondingly higher total defensive glucosinolate concentrations than the closely related species B. stricta from higher elevations. Thus, B. holboellii was both drought tolerant and defended; however, optimality theory does predict tradeoffs between defense and growth. Differences between species in the direct effect of water deficiency on glucosinolate production did not obscure the grow‐or‐defend tradeoff. B. holboellii may also have been more resistant to the specialist herbivore Plutella xylostella; a trend that was less clear because it depended on plant development and water deficient conditions. At finer scales of genetic variation, there was significant variation among families and naturally occurring inbred lines of B. stricta in drought tolerance measured as inherent growth, the reaction norm of growth across drought treatments, shoot water potential, and transpiration rates. Evidence for tradeoffs was also found within B. stricta in genetic correlations between resistance and transpiration rates, or glucosinolates and growth rates. No G×E was detected at these finer scales of genetic variation, although sometimes the tradeoff was dependent on drought conditions. Direct effects of drought stress resulted in an apparent plastic switch between resistance and tolerance to damage, which might be a cost avoidance mechanism because tradeoffs never involved tolerance to damage. Thus, when drought tolerance is manifest as slow inherent growth rates, plants may also have relatively high defense levels, especially in stressful environments. Otherwise, defenses may be compromised by drought‐coping mechanisms, although plastic switches to less costly defenses may alleviate constraints in stressful environments.     

Oxidative stress, protein carbonylation, proteolysis and antioxidative defense system as a model for depicting water deficit tolerance in Indica rice seedlings

Water deficit is an important constraint to rice (Oryza sativa L.) productivity. The present study was undertaken to investigate whether the level of oxidative stress, carbonylation of proteins, proteolysis and status of antioxidative defense could serve as a model to distinguish water deficit tolerant and sensitive rice cultivars. When 10-day-grown seedlings of two rice cultivars, Malviya-36 (drought-sensitive) and Brown Gora (drought-tolerant) were subjected to ?1.0 and ?2.1 MPa water deficit treatments for 24–72 h with polyethylene glycol 6000 in the medium, a greater decline in the growth of the seedlings and levels of leaf water potential, relative water content, Chl a, Chl b, carotenoids and greater increase in leaf water loss were observed in the sensitive cultivar than the tolerant. Under similar level of water deficit seedlings of sensitive cultivar showed higher level of superoxide anion generation, H₂O₂, lipid peroxidation and proteolysis in roots as well as shoots compared to the tolerant. Drought-tolerant cultivar had higher constitutive level of antioxidative enzymes superoxide dismutase and catalase and the activities of these two enzymes alongwith of guaiacol peroxidase showed greater increase in this cultivar under water deficit compared to the sensitive. A significant decline in the level of protein thiol and a higher increase in protein carbonyls content, also confirmed by protein gel blot analysis with an antibody against 2,4-dinitrophenylhydrazine was observed in the seedlings of drought sensitive cv. Malviya-36 compared to the tolerant cv. Brown Gora when subjected to similar level of water deficit. Seedlings of drought sensitive cultivar, under water deficit, showed higher proteolytic activity, higher number of in-gel activity stained proteolytic bands and higher expression of oxidized proteins in roots compared to the tolerant cultivar. Results suggest that poor capacity of antioxidative enzymes could be, at least partly, correlated with water deficit sensitivity of sensitive cultivar and that higher activity of antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, low proteolytic activity, lower level of protein carbonyls and protein thiolation could serve as a model to depict water deficit tolerance in Indica rice seedlings.     

OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice

Recent genomic and genetic analyses based on Arabidopsis suggest that ubiquitination plays crucial roles in the plant response to abiotic stress and the phytohormone abscisic acid (ABA). However, few such studies have been reported in rice as a monocotyledonous model plant. Taking advantage of strategies in biochemistry, molecular cell biology and genetics, the RING-finger containing E3 ligase OsSDIR1 (Oryza sativa SALT-AND DROUGHT-INDUCED RING FINGER 1) was found to be a candidate drought tolerance gene for engineering of crop plants. The expression of OsSDIR1 was detected in all tissues of rice and up-regulated by drought and NaCl, but not by ABA. In vitro ubiquitination assays demonstrated that OsSDIR1 is a functional E3 ubiquitin ligase and that the RING finger region is required for its activity. OsSDIR1 could complement the drought sensitive phenotype of the sdir1 mutant and overexpressing transgenic Arabidopsis were more sensitive to ABA, indicating that the OsSDIR1 gene is a functional ortholog of SDIR1. Upon drought treatment, the OsSDIR1-transgenic rice showed strong drought tolerance compared to control plants. Analysis of the stomata aperture revealed that there were more closed stomatal pores in transgenic plants than those of control plants. This result was also confirmed by the water loss assay and leaf related water content (RWC) measurements during drought treatment. Thus, we demonstrated that monocot- and dicot- SDIR1s are conserved yet have diverse functions.