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Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

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Drought is among the most common abiotic constraints of crop growth, development, and productivity. Integrating different omics approaches offers a possibility for deciphering the metabolic pathways and fundamental mechanisms involved in abiotic stress tolerance. Here, we explored the transcriptional and post-transcriptional changes in drought-stressed tomato plants using transcriptomic and proteomic profiles to determine the molecular dynamics of tomato drought stress responses. We identified 22467 genes and 5507 proteins, among which the expression of 3765 genes and 294 proteins was significantly changed under drought stress. Furthermore, the differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) showed a good correlation (0.743). The results indicated that integrating different omics approaches is promising in exploring the multilayered regulatory mechanisms of plant drought resistance. Gene ontology (GO) and pathway analysis identified several GO terms and pathways related to stress resistance, including response to stress, abiotic stimulus, and oxidative stress. The plant hormone abscisic acid (ABA) plays pivotal roles in response to drought stress, ABA-response element binding factor (AREB) is a key positive regulator of ABA signaling. Moreover, our analysis indicated that drought stress increased the abscisic acid (ABA) content, which activated AREB1 expression to regulate the expression of TAS14, GSH-Px-1, and Hsp, ultimately improving tomato drought resistance. In addition, the yeast one-hybrid assay demonstrated that the AREB1 could bind the Hsp promoter to activate Hsp expression. Thus, this study involved a full-scale analysis of gene and protein expression in drought-stressed tomato, deepening the understanding of the regulatory mechanisms of the essential drought-tolerance genes in tomato.

RESUMEN

Drought stress hinders the growth and development of crop plants and ultimately its productivity. It is expected that drought stress will be frequent and intense in the future due to drastic changes in the global climate. It is necessary to make crop plants more resilient to drought stress through various techniques; drought-hardening is one of them. Defining various metabolic strategies used by tobacco plants to confer drought tolerance will be important for maintaining plant physiological functions, but studies addressing this topic are limited. This study was designed to elucidate the drought tolerance and adaptation strategies used by tobacco plants via the application of different circular drought-hardening cycles (control: no drought-hardening, T1: one cycle of drought hardening, T2: two cycles of drought-hardening, and T3: three cycles of drought-hardening) to two tobacco varieties namely Honghuadajinyuan (H) and Yun Yan-100 (Y). The results revealed that drought-hardening decreased the fresh and dry biomass of the tobacco plants. The decrease was more pronounced in the T3 treatment for both H (23 and 29%, respectively) and Y (26 and 31%, respectively) under drought stress. The MDA contents, especially in T1 and T2 in both varieties, were statistically similar compared with control under drought stress. Similarly, higher POD, APX, and GR activities were observed, especially in T3, and elevated amounts of AsA and GSH were also observed among the different circular drought-hardening treatments under drought stress. Thus circular drought-hardening mitigated the oxidative damage by increasing the antioxidant enzyme activities and elevated the content of antioxidant substances, a key metabolic strategy under drought stress. Similarly, another important plant metabolic strategy is the osmotic adjustment. Different circular drought-hardening treatments improved the accumulation of proline and soluble sugars contents which contributed to osmoregulation. Finally, at the molecular level, circular drought-hardening improved the transcript levels of antioxidant enzyme-related genes (CAT, APX1, and GR2), proline and polyamines biosynthesis-related genes (P5CS1 and ADC2), and ABA signaling (SnRK2), and transcription factors (AREB1 and WRKY6) in response to drought stress. As a result, circular drought-hardening (T2 and T3 treatments) promoted tolerance to water stress via affecting the anti-oxidative capacity, osmotic adjustment, and regulation of gene expression in tobacco.

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KEY MESSAGE: The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

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