Cloning and Functional Analysis of Leaf Senescence Gene LPS1 in Oryza sativa

doi:10.16819/j.1001-7216.2021.210303

Abstract

Abstract:

【Objective】 As a good carrier, the premature aging mutant plays an important role in exploring the genetic mechanism and mechanism of action on premature aging, as well as improving grain yield and quality of rice.【Method】 In this study, a premature senescence mutant lps1 was obtained by EMS mutagenesis, and phenotypic observation, cytological and histochemical analysis, physiological and biochemical analysis, genetic analysis, gene localization and hormone treatment were performed on wild-type and mutants.【Result】 The leaves of lps1 began to turn yellow during the three-leaf stage, and the plant height, effective tiller number, seed-setting rate, and thousand-grain weight were extremely significantly reduced in the maturing stage. Electron microscopy observation found that the surface of lps1 leaves was smooth, the numbers of silicified protrusions and chloroplasts decreased, and the lamella structure was disordered. Physiological and biochemical analysis showed that a large amount of reactive oxygen species accumulated in mutant lps1, accompanied by protein degradation, cell membrane damage and large-scale cell death. Genetic analysis showed that the progeria phenotype was controlled by a single recessive nuclear gene, which encodes an ubiquitin conjugating enzyme on chromosome 5. The subcellular localization results prove that LPS1 protein is expressed in both the cytoplasm and the nucleus. Exogenous hormone treatment demonstrated that the lps1 was more sensitive to exogenous hormone treatment, and the LPS1 mutation promoted the expression of genes related to ABA synthesis.【Conclusion】 LPS1 mutation makes the rice ABA synthesis signal pathway abnormal, which in turn triggers abnormal changes in a series of aging-related physiological indicators such as H₂O₂, leading to premature senescence of lps1, and ultimately resulting in a serious decrease in rice yield.

Key words: rice, premature senescence, genetic analysis, subcellular localization, hormone

摘要：

【目的】早衰突变体是研究早衰机制的良好载体,对于探究早衰的遗传机理与作用机制及提高水稻的产量和品质具有重要作用。【方法】本研究利用EMS诱变获得了一个早衰突变体lps1,并对该突变体及其野生型进行表型观察、细胞学及组织化学分析、生理生化分析、遗传分析、基因定位和激素处理。【结果】lps1的叶片从3叶期开始发黄,成熟期株高、有效分蘖数、结实率、千粒重等极显著降低。电镜观察发现lps1叶表面光滑,硅质化突起和叶绿体数目减少、片层结构紊乱。生理生化分析表明lps1中有大量的活性氧积累,同时伴有蛋白质的降解、细胞膜的损伤以及大规模的细胞死亡。遗传分析表明该早衰表型受单隐性核基因控制,并且其在第5染色体上编码了一个泛素结合酶。亚细胞定位结果证明LPS1蛋白在细胞质与核中均有表达。外源激素处理发现,lps1对外源激素的处理更为敏感,且LPS1突变促进了ABA合成相关基因的表达。【结论】LPS1 突变使水稻ABA合成信号途径异常,进而引发H₂O₂等一系列与衰老相关生理指标的异常变动,导致lps1过早衰老,最终造成水稻产量严重降低。

关键词: 水稻, 早衰, 遗传分析, 亚细胞定位, 激素

Xiaojie CHU, Tao LU, Hanfei YE, Sheng WANG, Han LIN, Xianmei WU, Rui HE, Gang YAN, Yuexing WANG, Sanfeng LI, Mei LU, Haitao HU, Yaolong YANG, Yuchun RAO. Cloning and Functional Analysis of Leaf Senescence Gene LPS1 in Oryza sativa[J]. Chinese Journal OF Rice Science, 2021, 35(5): 427-438.

褚晓洁, 芦涛, 叶涵斐, 王盛, 林晗, 吴先美, 何瑞, 严钢, 王跃星, 李三峰, 路梅, 胡海涛, 杨窑龙, 饶玉春. 水稻叶片衰老基因LPS1的克隆与功能研究[J]. 中国水稻科学, 2021, 35(5): 427-438.

Figures/Tables 12

Table 1 Primers and sequences used in this study.

引物名称 Primer name	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）	用途 Use
M1	CACACATTCTAAATTTGGAAAAAGG	TACGTGGCTACTTGGCGTTC	精细定位Fine mapping
M2	GCTGAAGAGCCTCCTCGAA	GGTATCATGAGAGCGAGTCTGA	精细定位Fine mapping
M3	ACCCATGACCATGAGACGAT	GGGTACTAGCCGTGCTTATCC	精细定位Fine mapping
M4	AGCCATTAGGGGCTTAGGAA	CCCCTGAGTGATATGCTTGG	精细定位Fine mapping
M5	TAAATTACATCGGCCGGAGA	CCCACCAAAGAAATCTCCAA	精细定位Fine mapping
M6	ATTTGGGGGAAAGTTTGCTT	AATAGTATGCGTGCGCTGTG	精细定位Fine mapping
LPS1	TCCTAAGAAGGGCTCGGAAA	CTGCCTCAACCACACTTACA	载体构建Vector construction
LPS1-GFP	GCCCAGATCAACTAGTATGGAT CTATATGCAATTGAC	TCGAGACGTCTCTAGACGGGCTGC AGGGGATGCCGG	载体构建Vector construction
Q-YGL1	AACCTTACCGTCCTATTCCTT	CCATACATCTAACAGAGCACC	qRT-PCR
Q-CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC	qRT-PCR
Q-NYC3	TCTATCTAGGTGCCAAAGGC	ATTCTGGCACCTGCTGTTTC	qRT-PCR
Q-DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTC	qRT-PCR
Q-RLS1	CTTGGGCTGTTGATGCAGC	CTTCAACACCCGCCTCGC	qRT-PCR
Q-OsABA1	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsABA2	AGCAAACCTGAAAGGTGTGGA	AAAGCCACCATCCACCATGA	qRT-PCR
Q-OsABA3	GGGCAAGATTTTGTTCGGCA	AAGGGTACACTTGTTGCCCC	qRT-PCR
Q-OsNCED1	ACCATGAAGTCCATGAGGCT	TCTCGTAGTCTTGGTCTTGG	qRT-PCR
Q-OsNCED2	ATGGAAACGAGGATAGTGGT	CTTATTGTTGTGCGAGAAGT	qRT-PCR
Q-OsNCED3	CTCCCAAACCATCCAAACCG	TGAGCATATCCTGGCGTCGT	qRT-PCR
Q-OsNCED5	TCCGAGCTCCTCGTCGTGAA	AGGTGTTTTGGAATGAACCA	qRT-PCR
Q-OsZEP	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsZDS	CACGTGTTCTTCGGGTGTTA	ATGTAACGGAGCTCCCACAG	qRT-PCR
Q-OsABA80x1	AAGCTGGCAAAACCAACATC	CCGTGCTAATACGGAATCCA	qRT-PCR
Q-OsABA80x2	CTACTGCTGATGGTGGCTGA	CCCATGGCCTTTGCTTTAT	qRT-PCR
Q-OsABA80x3	AGTACAGCCCATTCCCTGTG	ACGCCTAATCAAACCATTGC	qRT-PCR
Actin	CAGGCCGTCCTCTCTCTGTA	AAGGATAGCATGGGGGAGAG	qRT-PCR

Table 1 Primers and sequences used in this study.

引物名称 Primer name	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）	用途 Use
M1	CACACATTCTAAATTTGGAAAAAGG	TACGTGGCTACTTGGCGTTC	精细定位Fine mapping
M2	GCTGAAGAGCCTCCTCGAA	GGTATCATGAGAGCGAGTCTGA	精细定位Fine mapping
M3	ACCCATGACCATGAGACGAT	GGGTACTAGCCGTGCTTATCC	精细定位Fine mapping
M4	AGCCATTAGGGGCTTAGGAA	CCCCTGAGTGATATGCTTGG	精细定位Fine mapping
M5	TAAATTACATCGGCCGGAGA	CCCACCAAAGAAATCTCCAA	精细定位Fine mapping
M6	ATTTGGGGGAAAGTTTGCTT	AATAGTATGCGTGCGCTGTG	精细定位Fine mapping
LPS1	TCCTAAGAAGGGCTCGGAAA	CTGCCTCAACCACACTTACA	载体构建Vector construction
LPS1-GFP	GCCCAGATCAACTAGTATGGAT CTATATGCAATTGAC	TCGAGACGTCTCTAGACGGGCTGC AGGGGATGCCGG	载体构建Vector construction
Q-YGL1	AACCTTACCGTCCTATTCCTT	CCATACATCTAACAGAGCACC	qRT-PCR
Q-CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC	qRT-PCR
Q-NYC3	TCTATCTAGGTGCCAAAGGC	ATTCTGGCACCTGCTGTTTC	qRT-PCR
Q-DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTC	qRT-PCR
Q-RLS1	CTTGGGCTGTTGATGCAGC	CTTCAACACCCGCCTCGC	qRT-PCR
Q-OsABA1	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsABA2	AGCAAACCTGAAAGGTGTGGA	AAAGCCACCATCCACCATGA	qRT-PCR
Q-OsABA3	GGGCAAGATTTTGTTCGGCA	AAGGGTACACTTGTTGCCCC	qRT-PCR
Q-OsNCED1	ACCATGAAGTCCATGAGGCT	TCTCGTAGTCTTGGTCTTGG	qRT-PCR
Q-OsNCED2	ATGGAAACGAGGATAGTGGT	CTTATTGTTGTGCGAGAAGT	qRT-PCR
Q-OsNCED3	CTCCCAAACCATCCAAACCG	TGAGCATATCCTGGCGTCGT	qRT-PCR
Q-OsNCED5	TCCGAGCTCCTCGTCGTGAA	AGGTGTTTTGGAATGAACCA	qRT-PCR
Q-OsZEP	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsZDS	CACGTGTTCTTCGGGTGTTA	ATGTAACGGAGCTCCCACAG	qRT-PCR
Q-OsABA80x1	AAGCTGGCAAAACCAACATC	CCGTGCTAATACGGAATCCA	qRT-PCR
Q-OsABA80x2	CTACTGCTGATGGTGGCTGA	CCCATGGCCTTTGCTTTAT	qRT-PCR
Q-OsABA80x3	AGTACAGCCCATTCCCTGTG	ACGCCTAATCAAACCATTGC	qRT-PCR
Actin	CAGGCCGTCCTCTCTCTGTA	AAGGATAGCATGGGGGAGAG	qRT-PCR

Fig. 1. Phenotype and agronomic traits of the wild type and lps1. A, Plant phenotype(Bar=3 cm); B, Leaf phenotype at the three-leaf stage(Bar=1 cm); C, Tillering stage(Bar=6 cm); D, Maturity stage(Bar=8 cm); E, Seed phenotype of wild type and lps1(Bar=10 cm); F-I, Agronomic traits of wild type and lps1; J-L, Statistics of seed thickness, length and width of the wild type and lps1. * and ** indicate that the wild-type and mutant lps1 are significantly different at the levels of 0.05 and 0.01, respectively.

Fig. 2. Chlorophyll contents and related gene expression analysis at the tillering stage.A, Leaf phenotype of the wild type and lps1 in the tillering stage, bar=2 cm; B-D, Chlorophyll a, chlorophyll b and carotenoid contents of the wild type and lps1; E, Chlorophyll synthesis and metabolism-related gene expression level. L1, First leaf from the top; L2, Second leaf from the top; L3, Third leaf from the top. * and ** indicate that the wild type and lps1 are significantly different at the levels of 0.05 and 0.01, respectively.

Fig. 3. Scanning electron microscope (SEM) observations of the epidermis of the wild type (A-C) and lps1(D-F).

Fig. 4. Transmission electron microscopy (TEM) observation of the wild type (A-C) and lps1(D-F) leaves. N, Nucleus; C, Chloroplast; Thy, Thylakoid; S, Starch granules; Og, Osmophilic granules.

Fig. 5. Physiological and biochemical detection of the wild type and lps1.A, DAB staining; bar=2 cm; B, NBT staining, bar=2 cm; C, Determination of aging-related physiological indicators (H2O2, MDA, SP, CAT, POD, SOD). * and ** indicate that the wild-type and lps1 are significantly different at the levels of 0.05 and 0.01, respectively.

Fig. 6. TUNEL treatment results of the wild type (A and B) and lps1 (C and D) leaves. A-D, bar=100 μm. Blue fluorescence represents normal cells, green fluorescence represents apoptotic cells.

Table 2 Statistical results of F2 segregating population.

杂交组合 Combination	F₁表型 Phenotype of F₁	F₂表型 Phenotype of F₂			χ²_(3:1)
杂交组合 Combination	F₁表型 Phenotype of F₁	正常表型 Normal	突变表型 Mutant	总株数 Total	χ²_(3:1)
lps1/浙辐802 lps1/Zhefu 802	正常表型 Normal	253	82	335	0.049
浙辐802/lps1 Zhefu 802/lps1	正常表型 Normal	233	76	309	0.027

Fig. 7. Map-based cloning of LPS1 gene.

Fig. 8. Subcellular localization of LPS1 protein.

Fig. 9. Effect of exogenous hormone treatment on the wild type and lps1 seedlings. A, Effect of exogenous hormone treatment on the phenotype of the wild type(left) and lps1(right), bar=3 cm; B, Length of the aerial part after hormone treatment; C, Length of the underground part after hormone treatment. * and ** indicate significant difference between the wild type and lps1 at 0.05 and 0.01 level, respectively.

Fig. 10. ABA synthesis and degradation related gene expression levels. A, ABA-related gene expression levels in the wild type and lps1 before treatment; B, ABA-related gene expression levels in the wild type and lps1 after treatment; * and ** indicate significant difference between the wild type and lps1 at 0.05 and 0.01 level, respectively.

References 52

[1]	Cao J, Jiang F, Sodmergen, Cui K M. Time-course of programmed cell death during leaf senescence in Eucommia ulmoides[J]. Journal of Plant Research, 2003, 116(1): 7-12.
[2]	Vicky B W. The molecular biology of leaf senescence[J]. Journal of Experimental Botany, 1997, 48(2): 181-199.
[3]	Lim P O, Kim H J, Nam H G. Leaf senescence[J]. Annual Review of Plant Biology, 2007, 58(1): 115-136.
[4]	Zhang H S, Zhou C J. Signal transduction in leaf senescence[J]. Plant Molecular Biology, 2013, 82(6): 539-545.
[5]	Panda D, Sarkar R K. Natural leaf senescence: Probed by chlorophyll fluorescence, CO2 photosynthetic rate and antioxidant enzyme activities during grain filling in different rice cultivars[J]. Physiology and Molecular Biology of Plants, 2013, 19(3): 43-51.
[6]	Li Z H, Zhang Y, Zou D, Zhao Y, Wang H L, Zhang Y, Xia X L, Luo J C, Zhang Z. LSD 3.0: A comprehensive resource for the leaf senescence research community[J]. Nucleic Acids Research, 2020, 48(D1): D1069-D1075.
[7]	Mao C J, Lu S C, Lü B, Zhang B, Shen J B, He J M, Luo L Q, Xi D D, Chen X, Ming F. A rice NAC transcription factor promotes leaf senescence via ABA biosynthesis[J]. Plant Physiology, 2017, 174(3): 1747-1763.
[8]	Liang C Z, Wang Y Q, Zhu Y N, Tang J Y, Hu B, Liu L C, Ou S J, Sun X H, Chu J F, Chu C C. OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(27): 10013-10018.
[9]	Han M, Kim C Y, Lee J, Lee S K, Jeon J S. OsWRKY42 represses OsMT1d and induces reactive oxygen species and leaf senescence in rice[J]. Molecular Cells, 2014, 37(7): 532-539.
[10]	Uji Y, Akimitsu K, Gomi K. Identification of OsMYC2-regulated senescence-associated genes in rice[J]. Planta, 2017, 245(6): 1241-1246.
[11]	Lu G W, Casaretto J A, Ying S, Mahmood K, Liu F, Bi Y M, Rothstein S J. Overexpression of OsGATA12 regulates chlorophyll content, delays plant senescence and improves rice yield under high density planting[J]. Plant Molecular Biology, 2017, 94(1): 215-227.
[12]	Liu W Z, Fu Y P, Hu G C, Si H M, Wu C, Sun Z X. Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (Oryza sativa L.)[J]. Planta, 2007, 226(3): 785-795.
[13]	Liu C H, Zhu H T, Xing Y, Tan J J, Chen X H, Zhang J J, Peng H F, Xie Q J, Zhang Z M. Albino Leaf 2 is involved in the splicing of chloroplast group I and II introns in rice[J]. Journal of Experimental Botany, 2016, 67(18): 5339-5347.
[14]	Kusaba M, Ito H, Morita R, Iida S, Sato Y, Fujimoto M, Kawasaki S, Tanaka R, Hirochika H, Nishimura M, Tanaka A. Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence[J]. Plant Cell, 2007, 19(4): 1362-1375.
[15]	Rong H, Tang Y Y, Zhang H, Wu P Z, Chen Y P, Li M R, Wu G J, Jiang H W. The Stay-Green Rice like (SGRL) gene regulates chlorophyll degradation in rice[J]. Journal of Plant Physiology, 2013, 170(15): 1367-1373.
[16]	Fang C Y, Zhang H, Wan J, Wu Y Y, Chen W, Wang S C, Wang W S, Zhang H W, Zhang F, Qu L H, Liu X Q, Zhou D X, Luo J. Control of leaf senescence by an MeOH-jasmonates cascade that is epigenetically regulated by OsSRT1 in rice[J]. Molecular Plant, 2016, 9(10): 1366-1378.
[17]	Xu W Y, Kong Z S, Li M N, Yang W Q, Xue Y B. A novel nuclear-localized CCCH-type ainc finger protein, OsDOS, is involved in delaying leaf senescence in rice[J]. Plant Physiology, 2006, 141(4): 1376-1388.
[18]	Chen Y, Xu Y Y, Luo W, Li W X, Chen N, Zhang D J, Chong K. The F-Box protein OsFBK12 targets OsSAMS1 for degradation and affects pleiotropic phenotypes, including leaf senescence, in rice[J]. Plant Physiology, 2013, 163(4): 1673-1685.
[19]	Zhao Y, Chan Z L, Gao J H, Xing L, Cao M J, Yu C M, Hu Y L, You J, Shi H T, Zhu Y F, Gong Y H, Mu Z X, Wang H Q, Deng X, Wang P C, Bressan R A, Zhu J K. ABA receptor PYL9 promotes drought resistance and leaf senescence[J]. Proceedings of the National Academy of Sciences of The United States of America, 2016, 113(7): 1949-1954.
[20]	Yang X, Gong P, Li K Y, Huang F D, Cheng F M, Pan G. A single cytosine deletion in the OsPLS1 gene encoding vacuolar-type H+-ATPase subunit A1 leads to premature leaf senescence and seed dormancy in rice[J]. Journal of Experimental Botany, 2016, 67(9): 2761-2776.
[21]	Zhou Y, Liu L, Huang W F, Yuan M, Zhou F, Li X H, Lin Y J. Overexpression of OsSWEET5 in rice causes growth retardation and precocious senescence[J]. PloS One, 2014, 9(4): e94210.
[22]	Singh S, Singh A, Nandi A K. The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death[J]. Journal of Biosciences, 2016, 41(3): 445-453.
[23]	Wu L W, Ren D Y, Hu S K, Li G M, Dong G J, Jiang L, Hu X M, Ye W J, Cui Y T, Zhu J, Zhang G H, Gao Z Y, Zeng D L, Qian Q, Guo L B. Down-regulation of a nicotinate phosphoribosyltransferase gene OsNaPRT1 leads to withered leaf tips[J]. Plant Physiology, 2016, 171(2): 1085-1098.
[24]	Lee R H, Hsu J H, Huang H J, Lo S F, Grace S C. Alkaline α-galactosidase degrades thylakoid membranes in the chloroplast during leaf senescence in rice[J]. New Phytology, 2009, 184(3): 596-606.
[25]	Kang K, Kim Y S, Park S, Back K. Senescence-induced serotonin biosynthesis and its role in delaying senescence in rice leaves[J]. Plant Physiology, 2009, 150(3): 1380-1393.
[26]	Kudo T, Makita N, Kojima M, Tokunaga H, Sakakibara H. Cytokinin activity of cis-zeatin and phenotypic alterations induced by overexpression of putative cis-zeatin glucosyltransferase in rice[J]. Plant Physiology, 2012, 160(1): 319-331.
[27]	Wiseman B R. Plant-resistance to insects in integrated pest-management[J]. Plant Disease, 1994, 78(9): 927-932.
[28]	Liang C Z, Chu C C. Towards understanding abscisic acid-mediated leaf senescence[J]. Science China: Life Sciences, 2015, 58(5): 506-508.
[29]	Pan H R, Liu S M, Tang D Z. HPR1, a component of the THO/TREX complex, plays an important role in disease resistance and senescence in Arabidopsis[J]. Plant Journal, 2012, 69(5): 831-843.
[30]	Hu Y N, Jiang Y J, Han X, Wang H P, Pan J J, Yu D Q. Jasmonate regulates leaf senescence and tolerance to cold stress: Crosstalk with other phytohormones[J]. Journal of Experimental Botany, 2017, 68(6): 1361-1369.
[31]	Dani K G S, Fineschi S, Michelozzi M, Loreto F. Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence[J]. Plant Cell Environment, 2016, 39(5): 1103-1111.
[32]	Kim J I, Murphy A S, Baek D, Lee S W, Yun D J, Bressan R A, Narasimhan M L. YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana[J]. Journal of Experimental Botany, 2011, 62(11): 3981-3992.
[33]	Chen L G, Xiang S Y, Chen Y L, Li D B, Yu D Q. Arabidopsis WRKY45 interacts with the DELLA protein RGL1 to positively regulate age-triggered leaf senescence[J]. Molecular Plant, 2017, 10, 1174-1189.
[34]	Riefler M, Novak O, Strnad M, Schmülling T. Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism[J]. Plant Cell, 2006, 18(1): 40-54.
[35]	Wang T, Li C X, Wu Z H, Jia Y C, Wang H, Sun S Y, Mao C Z, Wang X L. Abscisic acid regulates auxin homeostasis in rice root tips to promote root hair elongation[J]. Frontier in Plant Science, 2017, 8: 1121-1138.
[36]	Xu N, Chu Y L, Chen H L, Li X X, Wu Q, Jin L, Wang G X, Huang J L. Rice transcription factor OsMADS25 modulates root growth and confers salinity tolerance via the ABA-mediated regulatory pathway and ROS scavenging[J]. PLoS Genet, 2018, 14(10): e1007662.
[37]	Chang Y, Nguyen B H, Xie Y J, Xiao B Z, Tang N, Zhu W L, Mou T M, Xiong L Z. Co-overexpression of the constitutively active form of OsbZIP46 and ABA-activated protein kinase SAPK6 improves drought and temperature stress resistance in rice[J]. Frontier in Plant Science, 2017, 8: 1102-1117.
[38]	Promchuea S, Zhu Y J, Chen Z Z, Zhang J, Gong Z Z. ARF2 coordinates with PLETHORAs and PINs to orchestrate ABA-mediated root meristem activity in Arabidopsis[J]. Journal of Integrative Plant Biology, 2017, 59(1): 30-43.
[39]	Sun L R, Wang Y B, He S B, Hao F S. Mechanisms for abscisic acid inhibition of primary root growth[J]. Plant Signaling & Behavior, 2018, 13(9): e1500069.
[40]	Hung K T, Yi T H, Kao C H. Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves[J]. Physiology Plantarum, 2006, 127(2): 293-303.
[41]	李兆伟. 水稻叶片早衰突变体的糖代谢基因表达与抗氧化生理调控[D]. 杭州: 浙江大学, 2014.
	Li Z W. The expression alteration of various genes related to sugar metabolism in senescing leaves and its antioxidation modulation for esl mutant[D]. Hangzhou: Zhejiang University, 2014.
[42]	Lichtenthaler H K. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes[J]. Methods in Enzymology, 1987, 148: 350-382.
[43]	游均, 方玉洁, 熊立仲. 活性氧检测[J/OL].Bio-101, 2018: e1010170.
	You J, Fang Y J, Xiong L Z. Reactive oxygen detection [J/OL]. Bio-101, 2018: e1010170. (in Chinese)
[44]	Rao Y C, Jiao R, Wang S, Wu X M, Ye H F, Pan C Y, Li S F, Xin D D, Zhou W Y, Dai G X, Hu J, Ren D Y, Wang Y X. SPL36 encodes a receptor-like protein kinase that regulates programmed cell death and defense responses in rice[J]. Rice, 2021, 14(1): 34-47.
[45]	沈恒胜, 陈君琛, 黄进华, 汤葆莎. 水稻叶表皮硅体显微结构及其分布[J]. 福建农业大学学报, 2005, 34(2): 137-140.
	Shen H S, Chen J C, Huang J H, Tang B S. Microtructure and distribution of silica bodies rice epidermis[J]. Fujian Academy of Agricultural Sciences, 2005, 34(2): 137-140. (in Chinese with English abstract)
[46]	杨秉耀, 陈新芳, 刘向东, 郭海滨. 水稻不同品种叶表面硅质细胞的扫描电镜观察[J]. 电子显微学报, 2006, 25(2): 68-72.
	Yang B Y, Chen X F, Liu X D, Guo H B. Scanning electron microscopic observation of silicon cells on the leaf surface of different rice varieties[J]. Journal of Chinese Electron Microscopy Society, 2005, 25(2): 68-72.
[47]	Christopher E. A possible mechanism of biological silicification in plants[J]. Frontier in Plant Science, 2015, 6: 853-859.
[48]	Yang L, Han Y Q, Li P, Li F, Ali S, Hou M L. Silicon amendment is involved in the induction of plant defense responses to a phloem feeder[J]. Scientific Report, 2017, 7(1): 4232-4240.
[49]	Hideg E, Kálai T, Kós P B, Asada K, Hideg K. Singletoxygen in plants: Its significance and possible detection with double (fluorescent and spin) indicator reagents[J]. Photochemistry and Photobiology, 2006, 82(5): 1211-1218.
[50]	华春, 王仁雷. 杂交稻及其三系叶片衰老过程中SOD、CAT活性和MDA含量的变化[J]. 西北植物学报, 2003, 23(3): 406-409.
	Hua C, Wang R L. Changes of SOD and CAT activities and MDA content during senescence of hybrid rice and three lines leaves[J]. Acta Botanica Boreali- Occidentalia Sinica, 2003, 23(3): 406-409.
[51]	Hu B, Zhu C G, Li F, Tang J Y, Wang Y Q, Liu L C, Che R H, Chu C C. LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice[J]. Plant Physiology, 2011, 156(3): 1101-1115.
[52]	Lee I C, Hong S W, Whang S S, Lim P O, Nam H G, Koo J C. Age-dependent action of an ABA-inducible receptor kinase, RPK1, as a positive regulator of senescence in Arabidopsis leaves[J]. Plant and Cell Physiology, 2011, 52(4): 651-662.