OsWAK16 Regulates Seed Anti-aging Ability by Modulating Antioxidant Enzyme Activity in Rice

doi:10.11983/CBB24038

Abstract

Abstract: INTRODUCTION: The cell wall-associated kinase (WAK) family has annotated approximately 130 WAK genes in the genome of rice (Oryza sativa), which play an important role in rice growth and development and stress responses.

RATIONALE: Here, we investigated the regulation and physiological mechanism of OsWAK16, an encoding gene of the cell wall-associated kinase WAK16-RLK, on rice seed vigor and anti-aging ability.

RESULTS: The results showed that before and after artificial aging, the seed vigor of OsWAK16 knock out mutants and overexpression lines was significantly lower and higher than that of wild-type seeds, respectively, indicating that OsWAK16 positively regulates the anti-aging ability of seeds. Physiological and biochemical analyses indicated that compared with wild-type seeds before and after artificial aging treatment, malondialdehyde (MDA) content and electrical conductivity (EC) of seed soaking solution of OsWAK16 knock out mutant seeds were significantly increased, while antioxidant enzyme activity was significantly decreased. The reverse was true in overexpression seeds. In addition, the differential expression of OsWAK16 in three types of seeds, whether artificially aged or not, also caused synergistic changes in the expression of other seed vigor-related genes OsPER1A, OsbZIP23, OsPIMT1, OsSdr4, OsMSRB5 and OsHSP18.2.

CONCLUSION: Therefore, it is speculated that OsWAK16 may work synergistically with other seed vigor-related genes to clear reactive oxygen species in cells, thereby regulating seed vigor and anti-aging capacity.

Key words: OsWAK16, seed vigor, artificial aging, malondialdehyde, antioxidant enzyme

Jianhong Tian, Yan Liu, Mengqi Yin, Jing Wang, Ting Chen, Yan Wang, Xiaocheng Jiang. OsWAK16 Regulates Seed Anti-aging Ability by Modulating Antioxidant Enzyme Activity in Rice[J]. Chinese Bulletin of Botany, 2025, 60(1): 17-32.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB24038

https://www.chinbullbotany.com/EN/Y2025/V60/I1/17

Figures/Tables 8

Table 1 The primers used in the experiments

Primer name	Sequence (5′-3′)	Purpose
gRT1	CCGTTACAAGGCCCTTCTGT	CRISPR/Cas9 vector construction
OsU6aT1	ACAGAAGGGCCTTGTAACGGC
gRT2	ATTACAACTGGTATTAGTTG
OsU6bT2	CAACTAATACCAGTTGTAATC
PB-L	GCGCGCGGTCTCGCTCGACTAGTATGG
PB-R	GCGCGCGGTCTCTACCGACGCGTATCC
OsWAK16-cas9-F1	GCCAGGTACTGATACCTAC
OsWAK16-cas9-R1	CTCAAGAAAAGATCAGTCGC
OsWAK16-cas9-F2	TTTACATGCACTAGTTGCCC
OsWAK16-cas9-R2	GCGCTTTCATCTGAGATTAG
OsWAK16-pHB-F	ATGAGGTCGAGCTTTGTGGC	Overexpression vector construction
OsWAK16-pHB-R	CTAGCGTGGCAAACTGACTGAG	Overexpression vector construction
OsWAK16-1301-F	CCAATGAGTACATTTCTGCTATAGACAG	GUS vector construction
OsWAK16-1301-R	CTTTGCGCCAGCCGAGAC	GUS vector construction
OsActin1-F	CAATGTGCCAGCTATGTATGTCGCC	Quantitative internal control
OsActin1-R	TTCCCGTTCAGCAGTGGTAGTGAAG	Quantitative internal control
qRT-OsWAK16-F	GATGGCAACTTTACTACAA	OsWAK16 expression analysis
qRT-OsWAK16-R	TGTGATAATACTCTGGGTC	OsWAK16 expression analysis
OsPER1A-F	GACCCGGACGAGAAGGATTC	Gene expression analysis
OsPER1A-R	ACCACCTCATCCATGTTCCG
OsbZIP23-F	CTGGGAAATGGGCTGGTCT
OsbZIP23-R	CCATCTTGCCGAAGCCATT
OsPIMT1-F	CACCGACTGTGGTCAAGC
OsPIMT1-R	AGCACCAGGAGGCACAAA
OsSdr4-F	AAGACGGCGGAGGAGGTGGA
OsSdr4-R	CATGGACGGATGACCACTTGC
OsMSRB5-F	GCCATAAACCGAACACCG
OsMSRB5-R	GCTCATCAGTAGGCGTCTTG
OsHSP18.2-F	GCTCAAGTCCTCCGACATCAAG
OsHSP18.2-R	TCCGCAGGTACTTGCACGAC

Figure 1 Bioinformatics analysis of OsWAK16 (A) OsWAK16 gene structure; (B) OsWAK16 protein structure; (C) Phylogenetic tree of OsWAK16 and its homologous genes from Oryza sativa (OsWAK16 is highlighted in blue).

Figure 2 Expression patterns of OsWAK16 in different tissues and seeds of rice with different vigor (A) Expression level of OsWAK16 in different tissues of Kasalath (WT); (B) Analysis of OsWAK16 promoter-driven GUS expression (a: Seedling; b: Root; c: Leaf; d: Spikelet; e: Seed); (C) Germination rate of WT seeds at different time of artificial aging; (D) Expression of OsWAK16 in WT seeds at different time of artificial aging. (B) Bars in a-d=1 cm; bar in e=0.1 cm. Data represent means±SD (n=3). Significant differences between root and other tissues (A), and significant differences between unaged seeds and seeds artificially aged for different days (C, D) were determined using Student’s t-test (* P<0.05, ** P<0.01).

Figure 3 Construction of transgenic rice lines of OsWAK16 (A) Target sites (target site 1 and target site 2) selected by CRISPR/Cas9 and changes of the nucleotide sequences as well as the corresponding amino acid sequences of mutants oswak16-1 and oswak16-2; (B) Relative expression levels of OsWAK16 in leaves of wild type (WT) and overexpression lines OE-1, OE-2, and OE-3, data represent means±SD (n=3), significant differences between WT and overexpression lines were determined using Student’s t-test (** P<0.01).

Figure 4 Seed germination phenotypes and the related indexes of rice Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines (A) Germination phenotypes of unaged seeds and seeds with 12 days of artificial aging (bar=1 cm); (B) Seedling shoot length; (C) Seedling root length; (D) Germination rate; (E) Germination potential; (F) Germination index; (G) The relative expression level of OsWAK16. Data represent means±SD (n=3), significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

Figure 5 Comparison of malondialdehyde (MDA) content (A) and relative conductivity (EC) (B) of seeds of Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines before and after artificial aging Data represent means±SD (n=3); significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

Figure 6 Comparison of antioxidase activities and H2O2 and O2-. contents in seeds before and after artificial aging of Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines (A) Superoxide dismutase (SOD); (B) Peroxidase (POD); (C) Catalase (CAT); (D) O2-. ; (E) H2O2. Data represent means±SD (n=3); significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

Figure 7 Analysis of seed vigor-related genes expression in seeds before and after artificial aging of Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines (A) OsPER1A; (B) OsbZIP23; (C) OsPIMT1; (D) OsSdr4; (E) OsMSRB5; (F) OsHSP18.2. Data represent means±SD (n=3), significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

References 64

[1]	An JY, Liu YH, Han JJ, He C, Chen M, Zhu XB, Hu WM, Song WJ, Hu J, Guan YJ (2022). Transcriptional multiomics reveals the mechanism of seed deterioration in Nicotiana tabacum L. and Oryza sativa L. J Adv Res 42, 163-176.
[2]	Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G (2010). A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci USA 107, 9452-9457. DOI PMID
[3]	Chang HW, Zhang FL, Yang ZR, Kong DJ, Zheng QL, Hao LZ (2015). Physiological and biochemical responses of allium mongolicum seeds to storage aging. Plant Physiol J 51, 1075-1081. (in Chinese)
	常海文, 张凤兰, 杨忠仁, 孔德娟, 郑清岭, 郝丽珍 (2015). 沙葱种子贮藏陈化过程中的生理生化应答反应. 植物生理学报 51, 1075-1081.
[4]	Chen BX, Fu H, Gao JD, Zhang YX, Huang WJ, Chen ZJ, Zhang Q, Yan SJ, Liu J (2022). Identification of metabolomic biomarkers of seed vigor and aging in hybrid rice. Rice 15, 7.
[5]	Chu Z, Mao GF, Wu M, Wu HK (2023). Relationship between electrical conductivity of seed soaking solution and seed vigor in rice (Oryza sativa L.). J Agric Sci Technol 25, 35-41. (in Chinese)
	曲宗普尺, 毛光锋, 吴敏, 吴洪恺 (2023). 水稻种子浸泡液电导率与种子活力的关系. 中国农业科技导报 25, 35-41. DOI
[6]	Christoff AP, de Lima JC, de Ross BCF, Sachetto-Martins G, Margis-Pinheiro M, Margis R (2014). The wall-associated kinase gene family in rice ge- nomes. Plant Sci 229, 181-192.
[7]	Delteil A, Gobbato E, Cayrol B, Estevan J, Michel-Romiti C, Dievart A, Kroj T, Morel JB (2016). Several wall-associated kinases participate positively and negatively in basal defense against rice blast fungus. BMC Plant Biol 16, 17. DOI PMID
[8]	Gao JD, Fu H, Zhou XQ, Chen ZJ, Luo Y, Cui BY, Chen GH, Liu J (2016). Comparative proteomic analysis of seed embryo proteins associated with seed storability in rice (Oryza sativa L) during natural aging. Plant Physiol Biochem 103, 31-44.
[9]	Gao QM, Lu XX, Zhu LY, Xin X, Jiang XC (2019). Correlation studies on MDA and 4-HNE contents in soybean seed aging. Seed 38(4), 1-9. (in Chinese)
	高琴梅, 卢新雄, 朱凌燕, 辛霞, 姜孝成 (2019). 大豆种子老化MDA和4-HNE的含量变化相关性研究. 种子 38(4), 1-9.
[10]	Ghassemi-Golezani K, Khomari S, Valizadeh M (2009). Effects of seed and seedling vigor on antioxidative isozyme activity and cold acclimation capability of winter oilseed rape. J Food Agric Environ 7, 452-456.
[11]	Goel A, Sheoran IS (2003). Lipid peroxidation and peroxide-scavenging enzymes in cotton seeds under natural ageing. Biol Plant 46, 429-434.
[12]	Han GH, Huang RN, Hong LH, Xu JX, Hong YG, Wu YH, Chen WW (2023). The transcription factor NAC102 confers cadmium tolerance by regulating WAKL11 expression and cell wall pectin metabolism in Arabidopsis. J Integr Plant Biol 65, 2262-2278.
[13]	Harkenrider M, Sharma R, De Vleesschauwer D, Tsao L, Zhang XT, Chern M, Canlas P, Zuo SM, Ronald PC (2016). Overexpression of rice wall-associated kinase 25 (OsWAK25) alters resistance to bacterial and fungal pathogens. PLoS One 11, e0147310.
[14]	Hazra A, Varshney V, Verma P, Kamble NU, Ghosh S, Achary RK, Gautam S, Majee M (2022). Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa). New Phytol 236, 1042-1060.
[15]	He YQ, Cheng JP, He Y, Yang B, Cheng YH, Yang C, Wang ZS (2019). Influence of isopropylmalate synthase OsIPMS1 on seed vigour associated with amino acid and energy metabolism in rice. Plant Biotechnol J 17, 322-337.
[16]	He ZH, Fujiki M, Kohorn BD (1996). A cell wall-associated receptor-like protein kinase. J Biol Chem 271, 19789-19793. DOI PMID
[17]	He ZH, He DZ, Kohorn BD (1998). Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response. Plant J 14, 55-63. DOI PMID
[18]	Hu W, Lv YY, Lei WR, Li X, Chen YH, Zheng LQ, Xia Y, Shen ZG (2014). Cloning and characterization of the Oryza sativa wall-associated kinase gene OsWAK11 and its transcriptional response to abiotic stresses. Plant Soil 384, 335-346.
[19]	Huang JX, Cai MH, Long QZ, Liu LL, Lin QY, Jiang L, Chen SH, Wan JM (2014). OsLOX2, a rice type I lipoxygenase, confers opposite effects on seed germination and longevity. Transgenic Res 23, 643-655. DOI PMID
[20]	Kanneganti V, Gupta AK (2011). RNAi mediated silencing of a wall associated kinase, OsiWAK1 in Oryza sativa results in impaired root development and sterility due to anther indehiscence: wall associated kinases from Oryza sativa. Physiol Mol Biol Plants 17, 65-77.
[21]	Kaur H, Petla B, Kamble NU, Singh A, Rao V, Salvi P, Ghosh S, Majee M (2015). Differentially expressed seed aging responsive heat shock protein OsHSP18.2 implicates in seed vigor, longevity and improves germination and seedling establishment under abiotic stress. Front Plant Sci 6, 713. DOI PMID
[22]	Kim DH, Han SH (2018). Seed coat and aging conditions affect germination and physiological changes of aging Korean pine seeds. J For Res 23, 372-379.
[23]	Kohorn BD, Kobayashi M, Johansen S, Friedman HP, Fischer A, Byers N (2006). Wall-associated kinase 1 (WAK1) is crosslinked in endomembranes, and transport to the cell surface requires correct cell-wall synthesis. J Cell Sci 119, 2282-2290. PMID
[24]	Kohorn BD, Kohorn SL (2012). The cell wall-associated kinases, WAKs, as pectin receptors. Front Plant Sci 3, 88. DOI PMID
[25]	Koornneef M, Bentsink L, Hilhorst H (2002). Seed dormancy and germination. Curr Opin Plant Biol 5, 33-36. DOI PMID
[26]	Kumari S, Joshi R, Singh K, Roy S, Tripathi AK, Singh P, Singla-Pareek SL, Pareek A (2015). Expression of a cyclophilin OsCyp2-P isolated from a salt-tolerant landrace of rice in tobacco alleviates stress via ion homeostasis and limiting ROS accumulation. Funct Integr Genomics 15, 395-412.
[27]	Lally D, Ingmire P, Tong HY, He ZH (2001). Antisense expression of a cell wall-associated protein kinase, WAK4, inhibits cell elongation and alters morphology. Plant Cell 13, 1317-1331. DOI PMID
[28]	Li H, Zhou SY, Zhao WS, Su SC, Peng YL (2009). A novel wall-associated receptor-like protein kinase gene, OsWAK1, plays important roles in rice blast disease resistance. Plant Mol Biol 69, 337-346.
[29]	Li SM, Dong LP, Sun JY, Ma J (2012). Effect of artificial accelerated aging of 2 wheat cultivars on seed germination and physiological and biochemical characteristics. J Jilin Agricul Sci 37(5), 18-20. (in Chinese)
	李淑梅, 董丽平, 孙君艳, 马俊 (2012). 人工加速老化对2个小麦品种发芽和种子生理生化特性的影响. 吉林农业科学 37(5), 18-20.
[30]	Li XF, Zhou XX, Liu ZM (2005). On physiological and biochemical changes of artificially aged pepper seeds. J Hunan Agricul Univ Nat Sci 31, 265-268. (in Chinese)
	李雪峰, 邹学校, 刘志敏 (2005). 辣椒种子人工老化及劣变的生理生化变化. 湖南农业大学学报(自然科学版) 31, 265-268.
[31]	Lim S, Park J, Lee N, Jeong J, Toh S, Watanabe A, Kim J, Kang H, Kim DH, Kawakami N, Choi G (2013). ABA-INSENSITIVE3, ABA-INSENSITIVE5, and DELLAs interact to activate the expression of SOMNUS and other high-temperature-inducible genes in imbibed seeds in Arabidopsis. Plant Cell 25, 4863-4878.
[32]	Liu J, Gui J, Gao W, Ma JF, Wang QZ (2016). Review of the physiological and biochemical reactions and molecular mechanisms of seed aging. Acta Ecol Sin 36, 4997-5006. (in Chinese)
	刘娟, 归静, 高伟, 马俊峰, 王佺珍 (2016). 种子老化的生理生化与分子机理研究进展. 生态学报 36, 4997-5006.
[33]	Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods 25, 402-408. DOI PMID
[34]	Ma XL, Zhang QY, Zhu QL, Liu W, Chen Y, Qiu R, Wang B, Yang ZF, Li HY, Lin YR, Xie YY, Shen RX, Chen SF, Wang Z, Chen YL, Guo JX, Chen LT, Zhao XC, Dong ZC, Liu YG (2015). A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8, 1274-1284. DOI PMID
[35]	Ma YX, Wang ZH, Humphries J, Ratcliffe J, Bacic A, Johnson KL, Qu GQ (2024). WALL-ASSOCIATED KINASE Like 14 regulates vascular tissue development in Arabidopsis and tomato. Plant Sci 341, 112013.
[36]	McDonald MB (1999). Seed deterioration: physiology, repair and assessment. Seed Sci Technol 27, 177-237.
[37]	Osakabe Y, Maruyama K, Seki M, Satou M, Shinozaki K, Yamaguchi-Shinozaki K (2005). Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis. Plant Cell 17 1105-1119. PMID
[38]	Pérez-Rodríguez JL, Ramos Aquino RG, Lorente González GY, González-Olmedo JL, Martínez Montero ME (2023). ROS production and antioxidant enzyme activity in relation to germination and vigor during tobacco seed development. Vegetos 36, 506-515.
[39]	Petla BP, Kamble NU, Kumar M, Verma P, Ghosh S, Singh A, Rao V, Salvi P, Kaur H, Saxena SC, Majee M (2016). Rice PROTEIN L-ISOASPARTYL METHYLTRANS- FERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity. New Phytol 211, 627-645.
[40]	Qun S, Wang JH, Sun BQ (2007). Advances on seed vigor physiological and genetic mechanisms. Agric Sci China 6, 1060-1066.
[41]	Sattler SE, Gilliland LU, Magallanes-Lundback M, Pollard M, DellaPenna D (2004). Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16, 1419-1432. DOI PMID
[42]	Shiu SH, Bleecker AB (2001). Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci USA 98, 10763-10768. DOI PMID
[43]	Sun Q, Wang JH, Sun BQ (2007). Advances on seed vigor physiological and genetic mechanisms. Agric Sci China 6, 1060-1066.
[44]	Tamzil MS, Alfiko Y, Mubarok AF, Purwantomo S, Suwanto A, Budiarti S (2021). Development of auxotrophic Agrobacterium tumefaciens AGL1 by Tn5 transposon for rice (Oryza sativa L.) transformation. Biotechnol Bioproc Eng 26, 641-649.
[45]	Tripathi RK, Aguirre JA, Singh J (2021). Genome-wide analysis of wall associated kinase (WAK) gene family in barley. Genomics 113, 523-530. DOI PMID
[46]	Wagner TA, Kohorn BD (2001). Wall-associated kinases are expressed throughout plant development and are required for cell expansion. Plant Cell 13, 303-318. DOI PMID
[47]	Wang WQ, Xu DY, Sui YP, Ding XH, Song XJ (2022). A multiomic study uncovers a bZIP23-PER1A-mediated deto- xification pathway to enhance seed vigor in rice. Proc Natl Acad Sci USA 119, e2026355119.
[48]	Wang YC, Wang Y, Lu M, Wu HK, Cao DD (2018). Effects of artificial aging on physiological characteristics of rice seeds in different dormancy properties. Seed 37(6), 15-19. (in Chinese)
	王仪春, 王洋, 陆敏, 吴洪恺, 曹栋栋 (2018). 人工老化处理对不同休眠特性水稻种子生理特性的影响. 种子 37(6), 15-19.
[49]	Wang YH, Xie HG, Chen FH, Lin Q, Cui LL, Wu FX, Wei YD, Luo X, Chen LP, Cai QH, Xie HA, Zhang JF (2023). Analysis of the high seed storability trait in indica rice “Fuxiangzhan”. Chin Sci Bull 68, 3857-3868. (in Chinese)
	王颖姮, 谢鸿光, 陈飞鹤, 林强, 崔丽丽, 吴方喜, 魏毅东, 罗曦, 陈丽萍, 蔡秋华, 谢华安, 张建福 (2023). 籼稻福香占耐储藏性的蛋白质组学分析. 科学通报 68, 3857-3868.
[50]	Xia Y, Yin SJ, Zhang KL, Shi XT, Lian CL, Zhang HS, Hu ZB, Shen ZG (2018). OsWAK11, a rice wall-associated kinase, regulates Cu detoxification by alteration the immobilization of Cu in cell walls. Environ Exp Bot 150, 99-105.
[51]	Xu J, Yang J, Duan XG, Jiang YM, Zhang P (2014). Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz). BMC Plant Biol 14, 208. DOI PMID
[52]	Xu M, Li JL, Zhu H, Jin LL, Wang ZS (2018). Comparative study on physiological and biochemical indexes changes in the aging process of cotton seeds. Seed 37(2), 14-18. (in Chinese)
	徐敏, 李憬霖, 朱鹤, 金路路, 王子胜 (2018). 棉花种子老化过程中生理生化指标变化比较研究. 种子 37(2), 14-18.
[53]	Yadav S, Parihar S (2013). Seed germination and viability testing—principles and techniques. In: Basu S, Pariha SS, Lal SK, Arun Kumar MB, eds. Emerging Paradigms in Hybrid Seed Production, Plant Variety Protection, Value Addition and Quality Assurance for Enhancing Productivity and Sustainable Crop Production. New Delhi: Indian Agricultural Research Institute. pp. 205-217.
[54]	Yan WQ, Hu PL, Ni YX, Zhao H, Liu XT, Cao HC, Jia M, Tian BM, Miao HM, Liu HY (2023). Genome-wide characterization of the wall-associated kinase-like (WAKL) family in sesame (Sesamum indicum) identifies a SiWAKL6 gene involved in resistance to Macrophomina Phaseolina. BMC Plant Biol 23, 624.
[55]	Yang YQ, Wang XF (2004). Advances on relation-ship between biomembrane and seed vigor. Chin Bull Bot 21(6), 641-648. (in Chinese)
	杨永青, 汪晓峰 (2004). 种子活力与生物膜的研究现状. 植物学通报 21(6), 641-648.
[56]	Yin XY, Hou XW (2017). Role of OsWAK124, a rice wall-associated kinase, in response to environmental heavy metal stresses. Pak J Bot 49, 1255-1261.
[57]	Zhang CQ, Xu Y, Lu Y, Yu HX, Gu MH, Liu QQ (2011). regulates stem elongation and seed development in rice. Planta 234, 541-554.
[58]	Zhang HB, Yang GJ, Gao WD, Zhu Y, Huang F, Pei HF, Li QM (2019). Study on the seed vigor of Toona sinensis under specific storage conditions. For Res 32(2), 152-159.
	张海波, 杨桂娟, 高卫东, 祝燕, 黄放, 裴昊斐, 李庆梅 (2019). 香椿种子特定贮藏条件下活力变化的研究. 林业科学研究 32(2), 152-159.
[59]	Zhang RG, Guo XC, Zhang YL, Tian CR (2020). Influence of modified atmosphere treatment on post-harvest reactive oxygen metabolism of pomegranate peels. Nat Prod Res 34, 740-744. DOI PMID
[60]	Zhang YX, Fan F, Zhang QJ, Luo YJ, Liu QJ, Gao JD, Liu J, Chen GH, Zhang HQ (2022). Identification and functional analysis of long non-coding RNA (lncRNA) in response to seed aging in rice. Plants 11, 3223.
[61]	Zhao B, Zhang H, Chen TX, Ding L, Zhang LY, Ding XL, Zhang J, Qian Q, Xiang Y (2022). Sdr4dominates pre- harvest sprouting and facilitates adaptation to local climatic condition in Asian cultivated rice. J Integr Plant Biol 64, 1246-1263.
[62]	Zhao J, He YQ, Huang SL, Wang ZF (2021). Advances in the identification of quantitative trait loci and genes involved in seed vigor in rice. Front Plant Sci 12, 659307.
[63]	Zhao S, Zhao YL, Pan XQ, Zhang JJ, Huang DF (2019). Artificial aging of cabbage seeds and biological effects. North Hortic (24), 7-13. (in Chinese)
	赵硕, 赵颖雷, 潘学勤, 章竞瑾, 黄丹枫 (2019). 甘蓝种子的人工老化及其生物学效应. 北方园艺 (24), 7-13.
[64]	Zhou YL, Chu P, Chen HH, Li Y, Liu J, Ding Y, Tsang EWT, Jiang LW, Wu KQ, Huang SZ (2012). Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis. Planta 235, 523-537.