Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

doi:10.1016/j.rsci.2023.10.001

Abstract

Abstract:

The morphological development of rice (Oryza sativa L.) leaves is closely related to plant architecture, physiological activities, and resistance. However, it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment. In this study, a drought-sensitive, roll-enhanced, and narrow-leaf mutant (renl1) was induced from a semi-rolled leaf mutant (srl1) by ethyl methane sulfonate (EMS), which was obtained from Nipponbare (NPB) through EMS. Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase, allelic to NRL1/OsCLSD4. The RENL1 mutation resulted in reduced vascular bundles, vesicular cells, cellulose, and hemicellulose contents in cell walls, diminishing the water-holding capacity of leaves. In addition, the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species (ROS) was decreased, leading to an increase in ROS after drought stress. Meanwhile, genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components. Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance, and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance, thereby realizing simultaneous improvement of multiple traits in rice.

Key words: cellulose, cell wall, drought tolerance, leaf morphology, rice

Liu Dan, Zhao Huibo, Wang Zi’an, Xu Jing, Liu Yiting, Wang Jiajia, Chen Minmin, Liu Xiong, Zhang Zhihai, Cen Jiangsu, Zhu Li, Hu Jiang, Ren Deyong, Gao Zhenyu, Dong Guojun, Zhang Qiang, Shen Lan, Li Qing, Qian Qian, Hu Songping, Zhang Guangheng. Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance[J]. Rice Science, 2024, 31(1): 103-117.

Figures/Tables 8

Fig. 1. Phenotypic characterization of Nipponbare (NPB) and mutants (srl1 and renl1). A-D, Phenotypes of tillering plant (A, scale bars are 10 cm), local flag leaf (B, scale bar is 1 cm), blade cross section (C, scale bar is 1 cm), and flag leaf morphology (D, scale bar is 10 cm). E-I, Comparisons of plant height (E), flag leaf length (F), flag leaf width (G), flag leaf crimp (H), and chlorophyll content (I) between wild type (NPB) and mutants (srl1 and renl1). Data are Mean ± SD (n = 3), and the lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

Fig. 2. Leaf cytological morphology analysis. A and B, Frozen sections of main veins (A, scale bars are 50 μm), and large and small veins of Nipponbare (NPB), srl1, and renl1 (B, scale bars are 100 μm). BC, Bulliform form. C-E, Numbers of large (C) and small (D) vascular bundles, and bulliform cells (E). Data are Mean ± SD (n = 3). The lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

Fig. 3. RENL1 affects rice root development. A, Phenotypes of seedling root. Scale bar is 2 cm. B-F, Length of main root (B), the number of roots per plant (C), diameter of main root (D), total root length per plant (E), and total root surface area per plant (F) in Nipponbare (NPB) and mutants (srl1 and renl1). Data are Mean ± SD (n = 3). The lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

Fig. 4. Map-based cloning of RENL1. A, RENL1 gene was mapped to an interval between molecular markers B-15 and B-16 on chromosome (Chr.) 12 and further delimited to a 12.7 kb genomic region between markers C-2 and C-7. The numbers below the markers indicate the number of recombinants (Rec). B, Structure of RENL1 gene, a G→A base substitution occurred at position 1 775 of the open reading fragment. C, Gene knockout site detection. ‘T’ in red represents the site knocked by CRISPR/Cas9. D, Phenotypes of Nipponbare (NPB), renl1, and NRL1-cas9 at the tillering stage. Scale bar is 10 cm. E, Flag leaf type of NPB, renl1, and NRL1-cas9. Scale bar is 1 cm. F, Cross section of flag leaves of NPB, renl1, and NRL1-cas9. Scale bar is 1 cm.

Fig. 5. Morphology, physiology, and scanning electron microscope observation of rice leaves in wild type (Nipponbare, NPB) and mutants (srl1, nrl1, and renl1). A and B, Flag leaf fluorescence section under blue fluorescence with a light-emitting diode (LED) wavelength of 470 nm (A) and under green fluorescence with an LED wavelength of 530 nm (B). Scale bars are 100 μm. C-E, Scanning electron microscope observation of abaxial epidermis. Cork-silica cell pairs are represented by red boxes (C), papillae and nodular papillae are indicated with arrows (D), and stomata on the abaxial epidermis (E). Bars in C, D, and E are 20 μm, 10 μm, and 2 μm, respectively. F-M, Blade stomatal width (F), blade stomatal length (G), leaf epidermal stomatal density (H), net photosynthetic rate (I), stomatal conductance (J), transpiration rate (K), intercellular CO2 concentration (L), and proportion of mesophyll cells to leaf cells (M) in wild type and mutants. Data are Mean ± SD (n = 3). The lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

Fig. 6. Measurements of physiological indices and relative expression levels of genes in rice leaves. A and B, Cellulose (A) and hemicellulose (B) contents of rice leaves in wild type (Nipponbare, NPB) and mutants (srl1, nrl1, and renl1). C and D, qRT-PCR analysis of cell wall cellulose-related synthetic genes (C for OsCSLD family and D for OsCESA family). E and F, Analysis of the expression levels of NRL1 and SRL1 in mature leaves (E) and in NPB and NRL1-overexpressing lines (F). G-I, Leaf water content (G), relative water content (H), and water loss rate in leaf (I) in NPB, srl1, nrl1, and renl1. Data are Mean ± SD (n = 3). The lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

Fig. 7. RENL1 and SRL1 affect drought tolerance of rice. A, Drought tolerance of wild type (Nipponbare, NPB) and mutants (srl1, nrl1, and renl1) under 18% polyethylene glycol (PEG) stress treatment. Scale bars are 10 cm. B, Survival rates of NPB, srl1, nrl1, and renl1 after rewatering. C and D, Relative expression levels of drought tolerant genes in NPB, srl1, nrl1, and renl1 before (C) and after (D) 18% PEG stress for uninterrupted 48 h. E, Relative expression of SRL1 and NRL1 genes in NPB, srl1, nrl1, and renl1 at the seedling stage. F, Relative expression of drought-induced genes after 18% PEG treatment for different times. In B to F, data are Mean ± SD (n = 3). The lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

Fig. 8. Drought stress on antioxidant enzyme activities of rice leaves in wild type (Nipponbare, NPB) and mutants (srl1, nrl1, and renl1). A-E, Changes of H2O2 content (A), malonaldehyde (MDA) content (B), superoxide (SOD) activity (C), peroxidase (POD) activity (D), and catalase (CAT) activity (E) in NPB, srl1, nrl1, and renl1 under drought stress. Data are Mean ± SD (n = 3). The lowercase letters above the bars represent the significant differences at the 0.05 level by one-way analysis of variance.

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