Transcriptional modulation of AREB-1 by CRISPRa improves plant physiological performance under severe water deficit.

Plants are sessile organisms, which are vulnerable to environmental stresses. As such, plants have developed multiple molecular, physiological, and cellular mechanisms to cope with natural stressors. However, these environmental adversities, including drought, are sources of the main agribusiness problems since they interfere with plant growth and productivity. Particularly under water deprivation conditions, the abscisic acid-responsive element-binding protein AREB1/ABF2 plays an important role in drought stress response and physiological adaptation. In this investigation, we provide substantial confirmation for the role of AREB1/ABF2 in plant survival under severe water deficit using the CRISPR activation (CRISPRa) technique to enhance the AREB1 gene expression. In our strategy, the inactive nuclease dCas9 was fused with an Arabidopsis histone acetyltransferase 1, which improves gene expression by remodeling chromatin. The AREB1 overexpression promotes an improvement in the physiological performance of the transgenic homozygous plants under drought, which was associated with an increase in chlorophyll content, antioxidant enzyme activity, and soluble sugar accumulation, leading to lower reactive oxygen species accumulation. Finally, we found that the CRISPR-mediated up-regulation of AREB1 changes the abundance of several downstream ABA-inducible genes, allowing us to report that CRISPRa dCas9-HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

Implications of ethylene biosynthesis and signaling in soybean drought stress tolerance.

BACKGROUND: Ethylene is a phytohormone known for inducing a triple response in seedlings, leaf abscission and other responses to various stresses. Several studies in model plants have evaluated the importance of this hormone in crosstalk signaling with different metabolic pathways, in addition to responses to biotic stresses. However, the mechanism of action in plants of agricultural interest, such as soybean, and its participation in abiotic stresses remain unclear. RESULTS: The studies presented in this work allowed for the identification of 176 soybean genes described elsewhere for ethylene biosynthesis (108 genes) and signal transduction (68 genes). A model to predict these routes in soybean was proposed, and it had great representability compared to those described for Arabidopsis thaliana and Oryza sativa. Furthermore, analysis of putative gene promoters from soybean gene orthologs permitted the identification of 29 families of cis-acting elements. These elements are essential for ethylene-mediated regulation and its possible crosstalk with other signaling pathways mediated by other plant hormones. From genes that are differentially expressed in the transcriptome database, we analyzed the relative expression of some selected genes in resistant and tolerant soybean plants subjected to water deficit. The differential expression of a set of five soybean ethylene-related genes (MAT, ACS, ACO, ETR and CTR) was validated with RT-qPCR experiments, which confirmed variations in the expression of these soybean target genes, as identified in the transcriptome database. In particular, two families of ethylene biosynthesis genes (ACS and ACO) were upregulated under these experimental conditions, whereas CTR (involved in ethylene signal transduction) was downregulated. In the same samples, high levels of ethylene production were detected and were directly correlated with the free fraction levels of ethylene's precursor. Thus, the combination of these data indicated the involvement of ethylene biosynthesis and signaling in soybean responses to water stress. CONCLUSIONS: The in silico analysis, combined with the quantification of ethylene production (and its precursor) and RT-qPCR experiments, allowed for a better understanding of the importance of ethylene at a molecular level in this crop as well as its role in the response to abiotic stresses. In summary, all of the data presented here suggested that soybean responses to water stress could be regulated by a crosstalk network among different signaling pathways, which might involve various phytohormones, such as auxins, ABA and jasmonic acid. The integration of in silico and physiological data could also contribute to the application of biotechnological strategies to the development of improved cultivars with regard to different stresses, such as the isolation of stress-specific plant promoters.