GhTZF1 regulates drought stress responses and delays leaf senescence by inhibiting reactive oxygen species accumulation in transgenic Arabidopsis

Redox homeostasis is important for plants to be able to maintain cellular metabolism, and disrupting cellular redox homeostasis will cause oxidative damage to cells and adversely affect plant growth. In this study, a cotton CCCH-type tandem zinc finger gene defined as GhTZF1, which was isolated from a cotton cell wall regeneration SSH library in our previous research, was characterized. GhTZF1 was predominantly expressed during early cell wall regeneration, and it was expressed in various vegetative and reproductive tissues. The expression of GhTZF1 was substantially up-regulated by a variety of abiotic stresses, such as PEG and salt. GhTZF1 also responds to methyl jasmonate (MeJA) and H₂O₂ treatment. Overexpression of GhTZF1 enhanced drought tolerance and delayed drought-induced leaf senescence in transgenic Arabidopsis. Subsequent experiments indicated that dark- and MeJA-induced leaf senescence was also attenuated in transgenic plants. The amount of H₂O₂ in transgenic plants was attenuated under both drought conditions and with MeJA-treatment. The activity of superoxide dismutase and peroxidase was higher in transgenic plants than in wild type plants under drought conditions. Quantitative real-time PCR analysis revealed that overexpression of GhTZF1 reduced the expression of oxidative-related senescence-associated genes (SAGs) under drought conditions. Overexpression of GhTZF1 also enhanced oxidative stress tolerance, which was determined by measuring the expression of a set of antioxidant genes and SAGs that were altered in transgenic plants during H₂O₂ treatment. Hence, we conclude that GhTZF1 may serve as a regulator in mediating drought stress tolerance and subsequent leaf senescence by modulating the reactive oxygen species homeostasis.     

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.     

Redox regulation of water stress responses in field-grown plants. Role of hydrogen peroxide and ascorbate

Abiotic stresses, such as drought, can increase the production of reactive oxygen species (ROS) in plants. An increase in ROS levels can provoke a partial or severe oxidation of cellular components inducing redox status changes, so continuous control of ROS and therefore of their metabolism is decisive under stress conditions. The present work focuses on the contribution of one pro-oxidant, hydrogen peroxide (H₂O₂) and one antioxidant, ascorbate (AA) and its redox status, in the control of plant responses to drought-oxidative stress in resistant plants growing in field conditions. After a general introduction to the concept of drought and oxidative stress and its relationship, we describe the role of H₂O₂ in drought stress responses, emphasizing the importance of studies in H₂O₂ subcellular localization, needed for a better understanding of its role in plant responses to stress. Although more studies are needed in the study of changes of redox status in plants subjected to stress, the AA pools and its redox status can be indicative of its involvement as a part of cellular mechanisms by which the plant respond to drought-induced oxidative stress. The mechanism of resistance and/or tolerance to drought-oxidative stress is complex, especially when studies are carried out in plants growing in field conditions, where an interaction of stresses occurs. This study sheds light on the mechanisms of plant responses to water-oxidative stress in plants growing in the field.     

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.     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.     

Carbon/Nitrogen Imbalance Associated with Drought-Induced Leaf Senescence in Sorghum bicolor

Drought stress triggers mature leaf senescence, which supports plant survival and remobilization of nutrients; yet leaf senescence also critically decreases post-drought crop yield. Drought generally results in carbon/nitrogen imbalance, which is reflected in the increased carbon:nitrogen (C:N) ratio in mature leaves, and which has been shown to be involved in inducing leaf senescence under normal growth conditions. Yet the involvement of the carbon/nitrogen balance in regulation of drought-induced leaf senescence is unclear. To investigate the role of carbon/nitrogen balance in drought-induced senescence, sorghum seedlings were subjected to a gradual soil drought treatment. Leaf senescence symptoms and the C:N ratio, which was indicated by the ratio of non-structural carbohydrate to total N content, were monitored during drought progression. In this study, leaf senescence developed about 12 days after the start of drought treatment, as indicated by various senescence symptoms including decreasing photosynthesis, photosystem II photochemistry efficiency (Fv/Fm) and chlorophyll content, and by the differential expression of senescence marker genes. The C:N ratio was significantly enhanced 10 to 12 days into drought treatment. Leaf senescence occurred in the older (lower) leaves, which had higher C:N ratios, but not in the younger (upper) leaves, which had lower C:N ratios. In addition, a detached leaf assay was conducted to investigate the effect of carbon/nitrogen availability on drought-induced senescence. Exogenous application of excess sugar combined with limited nitrogen promoted drought-induced leaf senescence. Thus our results suggest that the carbon/nitrogen balance may be involved in the regulation of drought-induced leaf senescence.     

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

Response to mild water stress in transgenic Pssu-ipt tobacco

The response of antioxidant enzymes to cyclic drought was studied in control non-transformed tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) and two types of transgenic Pssu-ipt tobacco (grafted on wild rootstock and poorly rooted progeny of F1 generation) grown under different conditions of irradiation (greenhouse, referred as high light, versus growth chamber, referred as low light). Water stress cycles started with plants at two contrasting developmental stages, i.e., at the stage of vegetative growth (young) and at the onset of flowering (old). Drought reduced the growth of SR1 plants compared with transgenic ones, particularly, when treatment started in earlier stage of plant development. Relative leaf water content was significantly lower (below 70%) in all transgenic grafts and plants compared with the wild type, irrespective of age, drought, and growth conditions. The response of antioxidant enzymes was significantly dependent on plant type and plant age; nevertheless, growth conditions and water stress also affected enzyme activities. Contrary to non-transgenic tobacco, where about half of glutathione reductase activity was found in older plants, both transgenic types exhibited unchanged activities throughout plant development and stress treatment. No differences were found in catalase activity, although the growth in the greenhouse caused a moderate increase in all older plants. In contrast to non-transgenic and Pssu-ipt rooted plants, peroxidase activities (ascorbate, guaiacol, and syringaldazine peroxidase) in older Pssu-ipt grafts were up to four times higher, irrespective of growth and stress, nevertheless, the effect seemed to be age-dependent. Superoxide dismutase (SOD) activity was affected particularly by plant age but also by growth conditions. Unlike in older plants, water stress caused an increase of SOD activities in all younger plants. The differences observed in activities of enzymes of intermediary metabolism (i.e., malic enzyme and glucose-6-phosphate dehydrogenase) revealed that transgenic grafts probably compensated differently for a decrease of ATP and NADPH than control and transgenic rooted plants under stress.     

An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

The HUB2 gene encoding histone H2B monoubiquitination E3 ligase is involved in seed dormancy, flowering timing, defence response and salt stress regulation in Arabidopsis thaliana. In this study, we used the cauliflower mosaic virus (CaMV) 35S promoter to drive AtHUB2 overexpression in cotton and found that it can significantly improve the agricultural traits of transgenic cotton plants under drought stress conditions, including increasing the fruit branch number, boll number, and boll‐setting rate and decreasing the boll abscission rate. In addition, survival and soluble sugar, proline and leaf relative water contents were increased in transgenic cotton plants after drought stress treatment. In contrast, RNAi knockdown of GhHUB2 genes reduced the drought resistance of transgenic cotton plants. AtHUB2 overexpression increased the global H2B monoubiquitination (H2Bub1) level through a direct interaction with GhH2B1 and up‐regulated the expression of drought‐related genes in transgenic cotton plants. Furthermore, we found a significant increase in H3K4me3 at the DREB locus in transgenic cotton, although no change in H3K4me3 was identified at the global level. These results demonstrated that AtHUB2 overexpression changed H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus, leading the GhDREB gene to respond quickly to drought stress to improve transgenic cotton drought resistance, but had no influence on transgenic cotton development under normal growth conditions. Our findings also provide a useful route for breeding drought‐resistant transgenic plants.     

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.