CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.)

Rice leaf morphology is an essential agronomic trait to develop drought-tolerant genotypes for adequate and stable crop production in drought-prone areas. Here, rolled leaf mutant plants were acquired by CRISPR/Cas9-based mutagenesis of Semi-rolled leaf1,2 (SRL1 and SRL2) genes, and isobaric tags for relative and absolute quantification (iTRAQ) based proteomic analysis was performed to analyze the subsequent proteomic regulation events. Homozygous mutants exhibit decreased chlorophyll content, transpiration rate, stomatal conductance, vascular bundles (VB), stomatal number, and agronomic traits with increased panicle number and bulliform cells (BCs). Under drought stress, mutant plants displayed lower malondialdehyde (MDA) content while higher survival rate, abscisic acid (ABA) content, superoxide dismutase (SOD), catalase (CAT) activities, and grain filling percentage compare with their wild type (WT). Proteomic results revealed that 270 proteins were significantly downregulated, and 107 proteins were upregulated in the mutant line compared with WT. Proteins related to lateral organ boundaries&rsquo: (LOB) domain (LBD) were downregulated, whereas abiotic stress-responsive proteins were upregulated in the CRISPR mutant. LBD proteins (Q5KQR7, Q6K713, Q7XGL4, Q8LQH4), probable indole-3-acetic acid-amido synthetase (Q60EJ6), putative auxin transporter-like protein 4 (Q53JG7), Monoculm 1 (Q84MM9) and AP2 (Apetala2) domain-containing protein (Q10A97) were found to be hub-proteins. The hybrids developed from mutant restorers showed a semi-rolled leaf phenotype with increased panicle number, grain number per panicle, and yield per plant. Our findings reveal the intrinsic value of genome editing and expand the knowledge about the network of proteins for leaf rolling and drought avoidance in rice.