OsSRK1, an Atypical S-Receptor-Like Kinase Positively Regulates Leaf Width and Salt Tolerance in Rice

doi:10.1016/j.rsci.2020.01.004

Abstract

Abstract:

Receptor-like kinases (RLKs) are important for plant growth, development and defense responses. The S-receptor protein kinases (SRKs), which represent an RLK subfamily, control the self- incompatibility among Brassica species. However, little information is available regarding SRK functions in rice. We identified a gene OsSRK1 encoding an atypical SRK. The transcript of OsSRK1 was induced by abscisic acid (ABA), salt and polyethylene glycol. OsSRK1 localized to the plasma membrane and cytoplasm. Leaf width was increased in OsSRK1-overexpression (OsSRK1-OX) transgenic rice plants, likely because of an increase in cell number per leaf. Furthermore, the expression levels of OsCYCA3-1 and OsCYCD2-1, which encode positive regulators of cell division, were up-regulated in leaf primordium of OsSRK1-OX rice plants relative to those in wild type. Meanwhile, the expression level of OsKRP1, which encodes cell cycle inhibitor, was down-regulated in the OsSRK1-OX plants. Therefore, it is deduced that OsSRK1 regulates leaf width by promoting cell division in the leaf primordium. Additionally, OsSRK1-OX plants exhibited enhanced ABA sensitivity and salt tolerance compared with wild type. These results suggest that OsSRK1 plays important roles in leaf development and salt responses in rice.

Key words: rice, S-receptor-like protein kinase, OsSRK1, salt tolerance, leaf width, cell division

Jinjun Zhou, Peina Ju, Fang Zhang, Chongke Zheng, Bo Bai, Yaping Li, Haifeng Wang, Fan Chen, Xianzhi Xie. OsSRK1, an Atypical S-Receptor-Like Kinase Positively Regulates Leaf Width and Salt Tolerance in Rice[J]. Rice Science, 2020, 27(2): 133-142.

Figures/Tables 9

Supplemental Table 1. Primers used in this study.

Gene

Primer (5´ to 3´ )

For vector construction
cOsSRK F
cOsSRK R
gOsSRK F
gOsSRK R
For RT-PCR
rOsSRK F
rOsSRK R
rUBQ F
rUBQ R
For Real Time-PCR
OsMyb4 F
OsMyb4 R
OsDREB1A F
OsDREB1A R
ZOS3-22 F
ZOS3-22 R
OsWRKY8 F
OsWRKY8 R
EL5 F
EL5 R
OsKRP1F
OsKRP1 R
OsKRP4 F
OsKRP4 R
OsCDKB2-1 F
OsCDKB2-1 R
OsCYCA3-1 F
OsCYCA3-1 R
OsCYCD2-1 F
OsCYCD2-1 R
OsCYCD5-1 F
OsCYCD5-1 R
qUBQ F
qUBQ R

ATGGATCCCACCAACATGTCTG
GCACTAGTACCTGCTCTTAAC
ATTCTAGATGTCTGCTAATCCCAAC
ATGGATCCAACAATGTAATGATCCAATTG

CAACAAGCAAGCCTCCAGCAA
CGAAGCAGAAGCCGAACAGC
ATCACGCTGGAGGTGGAG
AGGCCTTCTGGTTGTAGACG

ACAACACCACGGACAGTTT
GTGCTGTCCACCGTCATT
TACCACACTCGAGCAGAGCA
GACGACTCGCCGCTCATCT
CGTTCCCGTCGTTCCAG
GCTTCTCCTGCACCTTCTT
GAACTAGGCCGAAACCCTAAA
TTCGTCTTCTTCAGCGACTG
TCGGTAGAACCGAACTGGAA
GATGCTTCTCACTGCCATCC
CAGCCGTCCACTACCTTCTG
ATCTGCACGCTCGCTTCAGC
CGATTAGCACCCCTGGATCT
TCAGTCTAGCTTGACCCATTC
CATGGACACCGACCTCAAGA
TGCAGAGCTGGTACATCAGG
TGGGCAATCCTACAACCAAG
TGAACTCCAAGGGAAGGCTA
CGAGCATCATCATAACAAGC
CATCCTCACATCTCCAATCT
ACAACAGGAGGAGCAGCAAG
CGGTGTCGGTAACATCATCA
ACCACTTCGACCGCCACTACT
ACGCCTAAGCCTGCTGGTT

Supplemental Fig. 1. The expression level of OsSRK1 in wild type (WT) and phyB1. a. The expression level of OsSRK1 in WT and phyB1 from genechip results. b. The expression level of OsSRK1 tested by qPCR. Data are means and SE of three biological replicates.

Supplemental Fig. 2. Identity between OsSRK1 and the four reported SRK proteins based on amino acid sequence of serine/threonine-protein kinase domain.

Fig. 1. Subcellular localization and gene expression pattern analysis. A, Schematic representation of OsSRK1. EX, External; TM, Transmembrane. B, Subcellular localization of OsSRK1 protein. Bars = 10 µm. C, Relative expression levels of OsSRK1 in rice leaves treated with 15% polyethylene glycol 4000 (PEG), 150 mmol/L NaCl and 100 μmol/L abscisic acid (ABA). D, Tissue expression pattern of OsSRK1. Data are Mean ± SE (n = 3).

Fig. 2. Overexpression of OsSRK1 affected leaf development. A, Schematic diagrams of the construct used to generate transgenic OsSRK1-overexpression (OsSRK1-OX) rice lines. The expression of OsSRK1 was driven by the ubiquitin promoter. B, Analysis of OsSRK1 expression in transgenic and wild type (WT) plants. #1-1, #1-2 and #1-3 represent three individual plants of OsSRK1-OX line #1; #2-1 and #2-2 represent two individual plants of OsSRK1-OX line #2; #8-1 and #8-2 represent two individual plants of OsSRK1-OX line #8. C, Phenotypes of transgenic seedlings at the 4-leaf stage. Bar = 1 cm. D, Leaf angle of WT and OsSRK1-OX plants. E, Phenotypes of the second leaves. Bar = 1 cm. F, Phenotypes of the third leaves. Bar = 1 cm. G, Leaf length of the first, second, third and fourth leaves. H, Leaf width of the first, second, third and fourth leaves. I, Flag leaf length. J, Flag leaf width. #1, #2 and #8 represent three independent transgenic OsSRK1-OX lines. Data are Mean ± SE (n = 12). Significant differences were determined with the Student’s t-test (*, P < 0.05; **, P < 0.01).

Fig. 3. Overexpression of OsSRK1 affected cell division in leaf. A, Number of non-stomata epidermal cells per mm2 in the fourth leaves of WT and OsSRK1-OX lines. Values are Mean ± SE (n = 5). B, Transverse sections of leaves in WT and OsSRK1-OX lines. MV, Middle vascular bundle; LV, Large vascular bundle; SV, Small vascular bundle. Insets are enlargements showing image of MV and SV. C, Number of small vascular bundles in WT and OsSRK1-OX lines. Values are Mean ± SE (n = 15 to 20). D, Transcript levels of cell division-related genes in the leaf primordium of OsSRK1-OX lines and WT. Values are Mean ± SE (n = 3).WT, Wild type. #1, #2 and #8 represent three independent transgenic OsSRK1-over-expression lines. Significant differences were determined with the Student’s t-test (*, P < 0.05; **, P < 0.01).

Fig. 4. Overexpression of OsSRK1 enhanced abscisic acid (ABA) sensitivity and salt tolerance. A, Plant heights of OsSRK1-OX (#1, #2 and #3) and wild type (WT) plants treated with exogenous ABA. Values are Mean ± SE (n = 30). B, Tolerance assays of OsSRK1-OX lines to NaCl. Bar = 1 cm. C, Survival rates of WT and the OsSRK1-OX lines treated with 150 mmol/L NaCl for 4 d. Values are Mean ± SE (n = 15 to 20). D, Dead leaf rate of WT and the OsSRK1-OX lines treated with 150 mmol/L NaCl for 4 d. Values are Mean ± SE (n = 15 to 20). Significant differences were determined with the Student’s t-test (*, P < 0.05; **, P < 0.01).

Supplemental Table 2. Down-regulated or up-regulated twofold genes identified by Gene-chip.

Fig. 5. Transcript levels of genes upregulated in the OsSRK1- overexpressing lines. Three OsSRK1-OX lines (#1, #2 and #8) and wild type (WT) plants were cultured for 14 d in a growth chamber under 14 h light (28 °C)/10 h dark (25 °C). Aboveground parts were harvested to analyze the transcript levels of genes up-regulated by OsSRK1 in the microarray results. Values are Mean ± SE (n = 3). Significant differences were determined with Student’s t-test (*, P < 0.05; **, P < 0.01).

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