Regulator Of Drought Stress Tolerance Research Articles

Integration of miRNA dynamics and drought tolerant QTLs in rice reveals the role of miR2919 in drought stress response

To combat drought stress in rice, a major threat to global food security, three major quantitative trait loci for ‘yield under drought stress’ (qDTYs) were successfully exploited in the last decade. However, their molecular basis still remains unknown. To understand the role of secondary regulation by miRNA in drought stress response and their relation, if any, with the three qDTYs, the miRNA dynamics under drought stress was studied at booting stage in two drought tolerant (Sahbaghi Dhan and Vandana) and one drought sensitive (IR 20) cultivars. In total, 53 known and 40 novel differentially expressed (DE) miRNAs were identified. The primary drought responsive miRNAs were Osa-MIR2919, Osa-MIR3979, Osa-MIR159f, Osa-MIR156k, Osa-MIR528, Osa-MIR530, Osa-MIR2091, Osa-MIR531a, Osa-MIR531b as well as three novel ones. Sixty-one target genes that corresponded to 11 known and 4 novel DE miRNAs were found to be co-localized with the three qDTYs, out of the 1746 target genes identified. We could validate miRNA-mRNA expression under drought for nine known and three novel miRNAs in eight different rice genotypes showing varying degree of tolerance. From our study, Osa-MIR2919, Osa-MIR3979, Osa-MIR528, Osa-MIR2091-5p and Chr01_11911S14Astr and their target genes LOC_Os01g72000, LOC_Os01g66890, LOC_Os01g57990, LOC_Os01g56780, LOC_Os01g72834, LOC_Os01g61880 and LOC_Os01g72780 were identified as the most promising candidates for drought tolerance at booting stage. Of these, Osa-MIR2919 with 19 target genes in the qDTYs is being reported for the first time. It acts as a negative regulator of drought stress tolerance by modulating the cytokinin and brassinosteroid signalling pathway.

CtCYP71A1 promotes drought stress tolerance and lignin accumulation in safflower and Arabidopsis

Lignin is a complex phenolic polymer that forms interwoven networks in the cell wall and is crucial to plant development and stress responses. The root tissues of safflower contain a high concentration of lignin, which may contribute to drought regulation mechanism in safflower. Cytochrome P450 is the largest oxidase family, which is often dubbed with a significant role in plant metabolism. However, little is known about the molecular mechanism of CYP450-mediated regulation of drought stress tolerance in safflower roots. In this study, a putative CtCYP71A1 gene from safflower was identified by combining genome-wide and transcriptomic analysis. In safflower, a total of 16 CtCYP71A enzyme coding genes were identified. The conserved structural topology of CtCYP71As reveals several signature motifs that are crucial to plant growth and stress response. The expression of CtCYP71A1 was shown to be diversely expressed in different tissues, however, it was significantly up-regulated in roots under normal condition. Similarly, in response to PEG stress, the expression of CtCYP71A1 in roots gradually increased, whereas it was upregulated initially and subsequently downregulated with ABA, GA3, and SA stress. In addition, the phenotypic analysis and root morphology of CtCYP71A1 overexpressed Arabidopsis lines showed enhanced drought tolerance with improved lignin content than that of wild-type and antisense plants, which was further confirmed by expression analysis, and various physiological indexes. Similarly, overexpression of CtCYP71A1 resulted in the positive regulation of several other genes involved in lignin biosynthesis compared to WT and antisense plants. Furthermore, drought sensitivity was observed in safflower roots with CtCYP71A1 virus-induced gene silencing assay. Together, these findings revealed important insights into the crucial role of CtCYP71A1 in drought tolerance via lignin accumulation in safflower.

Apple WRKY transcription factor MdWRKY56 positively modulates drought stress tolerance

Drought seriously threatens the growth, development, and yield of crops. WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, the study of WRKY TFs in woody plant apple (Malus domestica) in terms of drought tolerance regulation is limited. This study aimed to functionally characterize an apple group IIc WRKY gene MdWRKY56. The transcription abundance of MdWRKY56 was significantly upregulated under drought stress. The subcellular localization analysis revealed that the MdWRKY56 protein localized to the nucleus. Transcriptional activation experiments proved that MdWRKY56 had transcriptional activity in yeast, and its N-terminal played a decisive role in this activity. The functional analysis using MdWRKY56 transgenic Arabidopsis, apple calli, and apple plants indicated that the MdWRKY56 TF functioned positively in the modulation of osmotic and drought stress tolerance, with the MdWRKY56-overexpressed transgenic materials showing lower electrolyte leakage, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation, and higher fresh weight, proline content, and antioxidant enzyme activities. In conclusion, our findings indicated that MdWRKY56 served as an important positive regulator of drought stress tolerance in apple.

Effects of fulvic acid on photosynthetic characteristics, yield and quality of cucumber under drought stress

Fulvic acid (FA) participates in the regulation of drought stress tolerance in plants, but the underlying mechanisms remain unclear. We carried out an experiment with cucumber cultivar 'Jinyou 35' as the test material and the polyethylene glycol (PEG-6000) being used to simulate drought stress. The concentration effect of FA on drought alleviation of cucumber as well as the effect of FA on photosynthetic enzymes activities, chloroplast ultrastructure, fluorescence parameters, water use efficiency, yield and quality of cucumber plants were studied through spraying FA with different concentrations (0, 100, 300, 500, 700 and 900 mg·L-1). The results showed that FA with different concentrations significantly promoted the relative water content and leaf area and decreased drought injure index as well as the electrolyte leakage and malondialdehyde (MDA) content, compared with the control (0 mg·L-1) under drought stress. The mitigative effect of FA increased first and then decreased with the increases of FA concentration, with 700 mg·L-1 FA showing the best effect. FA significantly enhanced the chlorophyll content, Rubiasco and Rubisco activase (RCA) activities and gene expression, net photosynthesis (Pn), maximal photochemical efficiency of PSⅡin darkness, actual photochemical efficiency, absorbed light energy per unit area, captured light energy per unit area, quantum yield of electron transport and PSⅠ activity, decreased the increase of K point and maintained chloroplast ultrastructure. The experiment in solar-greenhouse showed that FA obviously increased water use efficiency, promoted dry matter accumulation as well as the contents of Vc, soluble sugar, soluble protein and free amino acid, and decreased tannin content. Our results suggested that FA could improve the yield and quality of cucumber in solar greenhouse under drought stress.

Molecular Characterization of U-box E3 Ubiquitin Ligases (TaPUB2 and TaPUB3) Involved in the Positive Regulation of Drought Stress Response in Arabidopsis.

Plant U-box E3 ubiquitin ligase (PUB) is involved in various environmental stress conditions. However, the molecular mechanism of U-box proteins in response to abiotic stress in wheat remains unknown. In this study, two U-box E3 ligase genes (TaPUB2 and TaPUB3), which are highly expressed in response to adverse abiotic stresses, were isolated from common wheat, and their cellular functions were characterized under drought stress. Transient expression assay revealed that TaPUB2 was localized in the cytoplasm and Golgi apparatus, whereas TaPUB3 was expressed only in the Golgi apparatus in wheat protoplasts. Additionally, TaPUB2 and TaPUB3 underwent self-ubiquitination. Moreover, TaPUB2/TaPUB3 heterodimer was identified in yeast and the cytoplasm of wheat protoplasts using a pull-down assay and bimolecular fluorescence complementation analysis. Heterogeneous overexpression of TaPUB2 and TaPUB3 conferred tolerance to drought stress. Taken together, these results implied that the heterodimeric form of U-box E3 ubiquitin ligases (TaPUB2/TaPUB3) responded to abiotic stress and roles as a positive regulator of drought stress tolerance.

Genome-Wide Analysis of MADS-Box Genes in Foxtail Millet (Setaria italica L.) and Functional Assessment of the Role of SiMADS51 in the Drought Stress Response.

MADS-box transcription factors play vital roles in multiple biological processes in plants. At present, a comprehensive investigation into the genome-wide identification and classification of MADS-box genes in foxtail millet (Setaria italica L.) has not been reported. In this study, we identified 72 MADS-box genes in the foxtail millet genome and give an overview of the phylogeny, chromosomal location, gene structures, and potential functions of the proteins encoded by these genes. We also found that the expression of 10 MIKC-type MADS-box genes was induced by abiotic stresses (PEG-6000 and NaCl) and exogenous hormones (ABA and GA), which suggests that these genes may play important regulatory roles in response to different stresses. Further studies showed that transgenic Arabidopsis and rice (Oryza sativa L.) plants overexpressing SiMADS51 had reduced drought stress tolerance as revealed by lower survival rates and poorer growth performance under drought stress conditions, which demonstrated that SiMADS51 is a negative regulator of drought stress tolerance in plants. Moreover, expression of some stress-related genes were down-regulated in the SiMADS51-overexpressing plants. The results of our study provide an overall picture of the MADS-box gene family in foxtail millet and establish a foundation for further research on the mechanisms of action of MADS-box proteins with respect to abiotic stresses.

The durum wheat NAC transcription factor TtNAC2A enhances drought stress tolerance in Arabidopsis

Plant specific NAC transcription factors play diverse roles in plant development and stress responses. However, their biological roles in crop plants such as wheat remain poorly described. Here, we report the isolation and functional characterization of novel durum wheat TtNAC2A gene which acts as a transcription activator in yeast. Expression profiling analyses revealed that TtNAC2A gene is expressed in several wheat tissues and at different stages of seed development. Moreover, TtNAC2A was up-regulated in leaves and roots after exposure to salt, PEG and exogenous abscisic acid treatments, and this up-regulation was confirmed by functional promoter analysis in Arabidopsis. Furthermore, transgenic Arabidopsis plants overexpressing TtNAC2A showed enhanced drought tolerance and hypersensitivity to ABA. This phentotype was accompanied by a greater water retention capacity, attenuated oxidative stress, lower electrolyte leakage and higher proline contents, compared to wild type plants. The TtNAC2A-mediated drought tolerance also corroborated with an activation of a subset of stress-responsive genes, including AtRD22, AtRD29A and AtRD29B. Thus, TtNAC2A functions as a positive regulator of drought stress tolerance and constitutes an attractive candidate for stress breading in wheat crops.

Expression of the Kale WRKY Gene BoWRKY10 in Transgenic Tobacco Confers Drought Stress Tolerance

WRKYs play important roles in plant growth, defense regulation, and the stress response. However, the mechanisms through which WRKYs are involved in drought tolerance have been rarely characterized in kale (Brassica oleracea var. acephala DC). In this study, we cloned the BoWRKY10 gene from kale and its expression was induced with PEG4000, NaCl, gibberellic acid, cold, H2O2, and abscisic acid (ABA). Bo-WRKY10 was localized in the nucleus. The protein had a WRKY domain and a C2H2 zinc finger structure and belonged to subgroup II. The analysis of yeast transcriptional activity showed that BoWRKY10 may have transcriptional activation activity, which was mainly determined by the carboxyl terminal sequence. BoW-RKY10-overexpressing tobacco (Nicotiana tabacum L.) showed enhanced drought tolerance. After the drought treatment, relative water content and proline contents as well as superoxide dismutase activity were higher in transgenic plants, while malondialdehyde and H2O2 contents were lower. In addition, several genes related to the ABA signaling pathway, sucrose, and the reactive oxygen species scavenging system, were significantly upregulated in the transgenic lines. These results demonstrate that BoWRKY10 confers drought tolerance in tobacco. These results provide clues regarding the mechanism by which BoWRKY10 contributes to the regulation of drought stress tolerance.

GmNAC8 acts as a positive regulator in soybean drought stress

NAC proteins represent one of the largest transcription factor (TF) families involved in the regulation of plant development and the response to abiotic stress. In the present study, we elucidated the detailed role of GmNAC8 in the regulation of drought stress tolerance in soybean. The GmNAC8 protein was localized in the nucleus, and expression of the GmNAC8 gene was significantly induced in response to drought, abscisic acid (ABA), ethylene (ETH) and salicylic acid (SA) treatments. Thus, we generated GmNAC8 overexpression (OE1 and OE2) and GmNAC8 knockout (KO1 and KO2) lines to determine the role of GmNAC8 in drought stress tolerance. Our results revealed that, compared with the wild type (WT) plant, GmNAC8 overexpression and GmNAC8 knockout lines exhibited significantly higher and lower drought tolerance, respectively. Furthermore, the SOD activity and proline content were significantly higher in the GmNAC8 overexpression lines and significantly lower in the GmNAC8 knockout lines than in the WT plants under drought stress. In addition, GmNAC8 protein was found to physically interact with the drought-induced protein GmDi19-3 in the nucleus. Moreover, the GmDi19-3 expression pattern showed the same trend as the GmNAC8 gene did under drought and hormone (ABA, ETH and SA) treatments, and GmDi19-3 overexpression lines (GmDi19-3-OE9, GmDi19-3-OE10 and GmDi19-3-OE31) showed enhanced drought tolerance compared to that of the WT plants. Hence, the above results indicated that GmNAC8 acts as a positive regulator of drought tolerance in soybean and inferred that GmNAC8 probably functions by interacting with another positive regulatory protein, GmDi19-3.

Characterization and functional analysis of miR166f in drought stress tolerance in mulberry (Morus multicaulis)

MicroRNAs (miRNAs) regulate gene expression accurately and effectively at the posttranscriptional level by repressing translation or directly degrading target mRNAs. Over 40 plant miRNA family genes have been associated with abiotic stress; however, few reports have identified miRNAs and their molecular functions under abiotic stress in woody plants. Mulberry trees are ecologically and economically important perennial woody plants, but few studies have examined the stress physiology, biochemistry, and molecular biology of mulberry miRNAs. Previously, we identified miR166f in Morus alba and found that its target genes encode homeobox-leucine zipper (HD-Zip) transcription factors and histone arginine demethylase, which are involved in responses to heat, cold, drought, and salt stresses. In this study, we further characterized miR166f from mulberry, and found that it targets HD-Zip induced by drought stress. We isolated, cloned, and identified pre-miR166f, the precursor of miR166f, from the leaves of mulberry (Morus multicaulis); pre-miR166f was 91 bp in length with a minimum folding free energy of − 51.70 kcal/mol. Bioinformatics analysis showed that the 3000-bp 5′ upstream region of miR166f contained not only TATA and CAAT boxes that functioned in transcription initiation, but also had many cis-acting elements for stress responses, such as MBS, HSE, ARE, TC-rich repeats, and several hormones response elements, such as AuxRR-core, CGTCA-motif, and P-box. This analysis suggested that miR166f might participate in abiotic and biotic stress responses by regulating the expression of stress-related genes in mulberry. We constructed a miR166f binary overexpression vector, pCAMBIA-35S-GUS-miR166f, and established a transient transformation system in mulberry. Histochemical β-glucuronidase (GUS) staining of miR166f-overexpression in transient transgenic mulberry leaves showed the best effect and highest GUS gene expression compared to the wild-type control plants at day four posttransformation using an optimized concentration of transformation liquid (OD600 = 0.7). The target genes of miR166f—two HD-Zips and one histone arginine demethylase gene (JMJD6)—showed markedly lower expression levels in miR166f-overexpression transient transgenic mulberry leaves. Further investigation showed that transient transgenic mulberry trees had higher relative water content, free proline content, soluble protein content, and superoxide dismutase and peroxidase activities, and lower malondialdehyde content compared to the wild-type control plants, which suggested that overexpression of miR166f in mulberry could enhance tolerance to drought stresses in transient transgenic mulberry. The results suggested that miR166f might function as a positive regulator of drought stress tolerance in mulberry.

Overexpression of the Transcription Factor NtNAC2 Confers Drought Tolerance in Tobacco

NAC proteins constitute one of the largest families of plant-specific transcription factors and play an important role in biological processes, including plant development and phytohormone homeostasis, and in responses to various environmental stresses. In this study, we isolated an NAC group A gene (named NtNAC2) from Nicotiana tabacum L. Quantitative RT-PCR (qRT-PCR) analysis indicated that NtNAC2 was significantly upregulated under drought stress, which implied NtNAC2 was important in tobacco under such conditions. Overexpression of NtNAC2 in tobacco plants exhibited enhanced drought tolerance by means of improved seedling growth. Under drought stress, organic osmoprotectants were significantly accumulated in these plants. Additionally, the activities of antioxidant defense enzymes, like superoxide dismutase (SOD) and peroxidase (POD), which could effectively scavenge accumulated reactive oxygen species (ROS), increased in NtNAC2-overexpression transgenic tobacco plants compared with wild-type plants. The net photosynthetic rate was also significantly increased in NtNAC2-overexpression transgenic lines compared with wild-type plants, and the content of malondialdehyde (MDA) and proline was lower in NtNAC2-overexpression transgenic lines than that in wild-type plants (P < 0.01). Furthermore, the expression of NtWRKY28, a drought resistance gene, was significantly increased and the δ-OAT gene was downregulated in NtNAC2-overexpression plants relative to wild-type plants. Taken together, these results indicated that NtNAC2 functions as a positive regulator of drought stress tolerance. This study provides a basis for further study of drought resistance conferred by the NtNAC2 gene.

Effect of overexpression of citrus 9-cis-epoxycarotenoid dioxygenase 3 (CsNCED3) on the physiological response to drought stress in transgenic tobacco.

9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψwleaf), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H2O2) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψwleaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H2O2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.

Tomato SR/CAMTA transcription factors SlSR1 and SlSR3L negatively regulate disease resistance response and SlSR1L positively modulates drought stress tolerance.

BackgroundThe SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress.ResultsExpression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast.ConclusionsVIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0286-3) contains supplementary material, which is available to authorized users.

High-level expression of sugar inducible gene2 (HSI2) is a negative regulator of drought stress tolerance in Arabidopsis

BackgroundHIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2 (HSI2), also known as VAL1, is a B3 domain transcriptional repressor that acts redundantly with its closest relative, HSI2-LIKE1 (HSL1), to suppress the seed maturation program following germination. Mutant hsi2 hsl1 seedlings are arrested early in development and differentially express a number of abiotic stress-related genes. To test the potential requirement for HSI2 during abiotic stress, hsi2 single mutants and plants overexpressing HSI2 were subjected to simulated drought stress by withholding watering, and characterized through physiological, metabolic and gene expression studies.ResultsThe hsi2 mutants demonstrated reduced wilting and maintained higher relative water content than wild-type after withholding watering, while the overexpressing lines displayed the opposite phenotype. The hsi2 mutant displayed lower constitutive and ABA-induced stomatal conductance than wild-type and accumulated lower levels of ABA metabolites and several osmolytes and osmoprotectants following water withdrawal. Microarray comparisons between wild-type and the hsi2 mutant revealed that steady-state levels of numerous stress-induced genes were up-regulated in the mutant in the absence of stress but down-regulated at visible wilting. Plants with altered levels of HSI2 responded to exogenous application of ABA and a long-lived ABA analog, but the hsi2 mutant did not show altered expression of several ABA-responsive or ABA signalling genes 4 hr after application.ConclusionsThese results implicate HSI2 as a negative regulator of drought stress response in Arabidopsis, acting, at least in part, by regulating transpirational water loss. Metabolic and global transcript profiling comparisons of the hsi2 mutant and wild-type plants do not support a model whereby the greater drought tolerance observed in the hsi2 mutant is conferred by the accumulation of known osmolytes and osmoprotectants. Instead, data are consistent with mutants experiencing a relatively milder dehydration stress following water withdrawal.