Introduction of the rd29A:AtDREB2A CA gene into soybean (Glycine max L. Merril) and its molecular characterization in leaves and roots during dehydration

The loss of soybean yield to Brazilian producers because of a water deficit in the 2011–2012 season was 12.9%. To reduce such losses, molecular biology techniques, including plant transformation, can be used to insert genes of interest into conventional soybean cultivars to produce lines that are more tolerant to drought. The abscisic acid (ABA)-independent Dehydration Responsive Element Binding (DREB) gene family has been used to obtain plants with increased tolerance to abiotic stresses. In the present study, the rd29A:AtDREB2A CA gene from Arabidopsis thaliana was inserted into soybean using biolistics. Seventy-eight genetically modified (GM) soybean lines containing 2–17 copies of the AtDREB2A CA gene were produced. Two GM soybean lines (P1397 and P2193) were analyzed to assess the differential expression of the AtDREB2A CA transgene in leaves and roots submitted to various dehydration treatments. Both GM lines exhibited high expression of the transgene, with the roots of P2193 showing the highest expression levels during water deficit. Physiological parameters examined during water deficit confirmed the induction of stress. This analysis of AtDREB2A CA expression in GM soybean indicated that line P2193 had the greatest stability and highest expression in roots during water deficit-induced stress.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Expression of Arabidopsis DREB1C improves survival, growth, and yield of upland New Rice for Africa (NERICA) under drought

Dehydration-responsive-element-binding protein 1 genes have important roles in response to stress. To improve the drought tolerance of an upland rice cultivar NERICA1, we introduced Arabidopsis AtDREB1C or rice OsDREB1B driven by a stress-inducible rice lip9 promoter. Plants of some transgenic lines survived better than non-transgenic plants under severe drought. AtDREB1C transgenic plants had higher dry weights than non-transgenic plants when grown under moderate drought until the late vegetative growth stage. On the other hand, OsDREB1B transgenic plants had lower dry weights than non-transgenic plants under the same condition. Similar results were obtained under osmotic stress. The AtDREB1C transgenic plants headed earlier, had a larger sink capacity, and had more filled grains than non-transgenic plants. These results suggest that AtDREB1C expressed in NERICA1 improves not only survival under severe drought, but also growth and yield under moderate drought.     

Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.     

Chimeric promoter mediates guard cell-specific gene expression in tobacco under water deficit

The engineering of stomatal activity under water deficit through guard cell-specific gene regulation is an effective approach to improve drought tolerance of crops but it requires an appropriate promoter(s) inducible by water deficit in guard cells. We report that a chimeric promoter can induce guard cell-specific gene expression under water deficit. A chimeric promoter, p4xKST82-rd29B, was constructed using a tetramer of the 82 bp guard cell-specific regulatory region of potato KST1 promoter (4xKST82) and Arabidopsis dehydration-responsive rd29B promoter. Transgenic tobacco plants carrying p4xKST82-rd29B:mGFP-GUS exhibited GUS expression in response to water deficit. GUS enzyme activity of p4xKST82-rd29B:mGFP-GUS transgenic plants increased ~300 % by polyethylene glycol treatment compared to that of control plant but not by abscisic acid (ABA), indicating that the p4xKST82-rd29B chimeric promoter can be used to induce the guard cell-specific expression of genes of interest in response to water deficit in an ABA-independent manner.     

Molecular tailoring of farnesylation for plant drought tolerance and yield protection

Protecting crop yield under drought stress is a major challenge for modern agriculture. One biotechnological target for improving plant drought tolerance is the genetic manipulation of the stress response to the hormone abscisic acid (ABA). Previous genetic studies have implicated the involvement of the beta-subunit of Arabidopsis farnesyltransferase (ERA1) in the regulation of ABA sensing and drought tolerance. Here we show that molecular manipulation of protein farnesylation in Arabidopsis, through downregulation of either the alpha- or beta-subunit of farnesyltransferase enhances the plant's response to ABA and drought tolerance. To test the effectiveness of tailoring farnesylation in a crop plant, transgenic Brassica napus carrying an ERA1 antisense construct driven by a drought-inducible rd29A promoter was examined. In comparison with the non-transgenic control, transgenic canola showed enhanced ABA sensitivity, as well as significant reduction in stomatal conductance and water transpiration under drought stress conditions. The antisense downregulation of canola farnesyltransferase for drought tolerance is a conditional and reversible process, which depends on the amount of available water in the soil. Furthermore, transgenic plants were more resistant to water deficit-induced seed abortion during flowering. Results from three consecutive years of field trial studies suggest that with adequate water, transgenic canola plants produced the same amount of seed as the parental control. However, under moderate drought stress conditions at flowering, the seed yields of transgenic canola were significantly higher than the control. Using protein farnesyltransferase as an effective target, these results represent a successful demonstration of engineered drought tolerance and yield protection in a crop plant under laboratory and field conditions.     

RD20, a stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana

Plants overcome water deficit conditions by combining molecular, biochemical and morphological changes. At the molecular level, many stress-responsive genes have been isolated, but knowledge of their physiological functions remains fragmentary. Here, we report data for RD20, a stress-inducible Arabidopsis gene that belongs to the caleosin family. As for other caleosins, we showed that RD20 localized to oil bodies. Although caleosins are thought to play a role in the degradation of lipids during seed germination, induction of RD20 by dehydration, salt stress and ABA suggests that RD20 might be involved in processes other than germination. Using plants carrying the promoter RD20::uidA construct, we show that RD20 is expressed in leaves, guard cells and flowers, but not in root or in mature seeds. Water deficit triggers a transient increase in RD20 expression in leaves that appeared predominantly dependent on ABA signaling. To assess the biological significance of these data, a functional analysis using rd20 knock-out and overexpressing complemented lines cultivated either in standard or in water deficit conditions was performed. The rd20 knock-out plants present a higher transpiration rate that correlates with enhanced stomatal opening and a reduced tolerance to drought as compared with the wild type. These results support a role for RD20 in drought tolerance through stomatal control under water deficit conditions.     

Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.