CRISPR/Cas9 edited <i>HSFA6a and HSFA6b</i> of <i>Arabidopsis thaliana</i> offers ABA and osmotic stress insensitivity by modulation of ROS homeostasis

Abstract

The role of Heat Shock Transcription Factor 6 (<i>HSFA6a</i> & <i>HSFA6b</i>) in response to abiotic stresses such as ABA, drought, salinity, drought, and osmotic stress is individually well established. Unfortunately, the functional redundancy between the <i>HSFA6a</i> and <i>HSFA6b</i> as well as the consequences of simultaneous editing of both in response to aforementioned stresses remains elusive. Therefore, this study was designed with the aim of addressing whether there is any functional redundancy between <i>HSFA6a</i> and <i>HSFA6b</i> as well as to decipher their role in abiotic stresses tolerance in <i>Arabidopsis thaliana</i>, by using the CRISPR-Cas9. We have generated the single (<i>hsfa6a and hsfa6b</i>) as well as double mutants (<i>hsfa6a/hsfa6b-1</i> and <i>hsfa6a/hsfa6b-2</i>) of <i>HSFA6a</i> and <i>HSFA6b</i> with higher frequencies of deletion, insertion, and substitution. The phenotypic characterization of generated double and single mutants under abiotic stresses such as ABA, mannitol, and NaCl identified double mutants more tolerant to subjected abiotic stresses than those of their single mutants. It warrants mentioning that we have identified that <i>HSFA6a</i> and <i>HSFA6b</i> also involved in other major ABA responses, including ABA-inhibited seed germination, stomatal movement, and water loss. In addition to the above, the simultaneous editing of <i>HSFA6a</i> and <i>HSFA6b</i> lead to a reduced ROS accumulation, accompanied by increased expression of much abiotic stress and ABA-responsive genes, including involved in regulation of ROS level. In conclusion, these results suggest that <i>HSFA6a</i> and <i>HSFA6b</i> may offer abiotic stress tolerance by regulating the ROS homeostasis in plants.