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.)

Shanyue Liao,Yaoguang Liu,Neng Zhao,Xuemei Qin,Babar Usman,Rongbai Li,Yue Han,Gul Nawaz,Xin Wang,Liang Luo

doi:10.3390/agronomy9110728

Shanyue Liao, Yaoguang Liu + Show 8 more

Open Access

https://doi.org/10.3390/agronomy9110728

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Abstract

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’ (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.

Highlights

The leaf is a primary photosynthetic organ in plants having a decisive role in plant growth, development, and survival
We generated CRISPR mutants with several morphological imperfections, and our results revealed that the Semi-rolled leaf 1 (SRL1) and SRL2 loss-of-function mutations led to a rolled leaf phenotype that showed drought tolerance
Results showed that the expression of SRL1 and SRL2 was significantly suppressed in mutant lines (Figure 11A)

Summary

Introduction

The leaf is a primary photosynthetic organ in plants having a decisive role in plant growth, development, and survival. Drought is a prolonged dry period in the natural climate cycle that impairs rice production and challenging tasks due to its unpredictable and complex nature. Plants undergo a range of biochemical and physiological pathways and excessive accumulations of ROS (reactive oxygen species). Antioxidant enzymes, such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), play a vital role in dealing with ROS accumulation. Abscisic acid (ABA) plays the main role in regulating stomatal closure, while malondialdehyde (MDA) production indicates cell membrane damage from water-deficit stress. Leaf rolling decreases stomatal conductance and reduced water loss under severe water-deficit conditions. The development of leaf rolling genotypes may provide a source to enhance rice yield under drought conditions. In China, breeding for rolled leaf genotypes is preferred for high-yielding rice varieties, including super hybrid rice [2,7,8]

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