The OsAGO2–OsNAC300–OsNAP module regulates leaf senescence in rice

Abstract

Leaves play a crucial role in the growth and development of rice (Oryza sativa) as sites for the production of photosynthesis. Early leaf senescence leads to substantial drops in rice yields. Whether and how DNA methylation regulates gene expression and affects leaf senescence remains elusive. Here, we demonstrate that mutations in rice ARGONAUTE 2 (OsAGO2) lead to premature leaf senescence, with chloroplasts in Osago2 having lower chlorophyll content and an abnormal thylakoid structure compared with those from wild-type plants. We show that OsAGO2 associates with a 24-nt microRNA and binds to the promoter region of OsNAC300, which causes DNA methylation and suppressed expression of OsNAC300. Overexpressing OsNAC300 causes the similar premature leaf senescence as Osago2 mutants and knocking out OsNAC300 in the Osago2 mutant background suppresses the early senescence of Osago2 mutants. Based on yeast one-hybrid, dual-luciferase, and electrophoresis mobility shift assays, we propose that OsNAC300 directly regulates transcription of the key rice aging gene NAC-like, activated by APETALA3/PISTILLATA (OsNAP) to control leaf senescence. Our results unravel a previously unknown epigenetic regulatory mechanism underlying leaf senescence in which OsAGO2–OsNAC300–OsNAP acts as a key regulatory module of leaf senescence to maintain leaf function.

Keywords

DNA methylation / leaf senescence / OsAGO2 / OsNAC300 / OsNAP

References

[1] Ay, N.,Janack, B., and Humbeck, K. (2014). Epigenetic control of plant senescence and linked processes. J. Exp. Bot. 65:3875–3887.

[2] Apel, K., and Hirt, H. (2004). Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55:373–399.

[3] Balazadeh, S.,Riaño-Pachón, D.M., and Mueller-Roeber, B. (2008). Transcription factors regulating leaf senescence in Arabidopsis thaliana. Plant Biol. 10:63–75.

[4] Buchanan-Wollaston, V.,Page, T.,Harrison, E.,Breeze, E.,Lim, P.O.,Nam, H.G.,Lin, J.F.,Wu, S.H.,Swidzinski, J.,Ishizaki, K., et al. (2005). Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J. 42:567–585.

[5] Bi, Z.,Zhang, Y.,Wu, W.,Zhan, X.,Yu, N.,Xu, T.,Liu, Q.,Li, Z.,Shen, X.,Chen, D., et al. (2017). ES7, encoding a ferredoxin-dependent glutamate synthase, functions in nitrogen metabolism and impacts leaf senescence in rice. Plant Sci. 259:24–34.

[6] Brabbs, T.R.,He, Z.,Hogg, K.,Kamenski, A.,Li, Y.,Paszkiewicz, K.H.,Moore, K.A.,O’Toole, P.,Graham, I.A., and Jones, L. (2013). The stochastic silencing phenotype of Arabidopsis morc6 mutants reveals a role in efficient RNA-directed DNA methylation. Plant J. 75:836–846.

[7] Christiansen, M.W., and Gregersen, P.L. (2014). Members of the barley NAC transcription factor gene family show differential co-regulation with senescence-associated genes during senescence of flag leaves. J. Exp. Bot. 65:4009–4022.

[8] Chen, L.J.,Wuriyanghan, H.,Zhang, Y.Q.,Duan, K.X.,Chen, H.W.,Li, Q.T.,Lu, X.,He, S.J.,Ma, B.,Zhang, W.K., et al. (2013). An S-domain receptor-like kinase, OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice. Plant Physiol. 163:1752–1765.

[9] Chun, I.,Kim, H.J.,Hong, S.,Kim, Y.G., and Kim, M.S. (2023). Structural basis of DNA binding by the NAC transcription factor ORE1, a master regulator of plant senescence. Plant Commun. 4:100510.

[10] Cao, J.,Liu, H.,Tan, S., and Li, Z. (2023). Transcription factors-regulated leaf senescence: Current knowledge, challenges and approaches. Int. J. Mol. Sci. 24:9245.

[11] Chen, Y.,Shen, J.,Zhang, L.,Qi, H.,Yang, L.,Wang, H.,Wang, J.,Wang, Y.,Du, H.,Tao, Z., et al. (2021). Nuclear translocation of OsMFT1 that is impeded by OsFTIP1 promotes drought tolerance in rice. Mol. Plant 14:1297–1311.

[12] Carbonell, A. (2017). Immunoprecipitation and high-throughput sequencing of ARGONAUTE-bound target RNAs from plants. Methods Mol. Biol. 1640:93–112.

[13] Gregersen, P.L., and Holm, P.B. (2007). Transcriptome analysis of senescence in the flag leaf of wheat Triticum aestivum (L.). Plant Biotechnol. J. 5:192–206.

[14] Gallego-Bartolomé J. (2020). DNA methylation in plants: Mechanisms and tools for targeted manipulation. New Phytol. 227:38–44.

[15] Guo, Y., and Gan, S. (2006). AtNAP, a NAC family transcription factor, has an important role in leaf senescence. Plant J. 46:601–612.

[16] He, L.,Wu, W.,Zinta, G.,Yang, L.,Wang, D.,Liu, R.,Zhang, H.,Zheng, Z.,Huang, H.,Zhang, Q., et al. (2018). A naturally occurring epiallele associates with leaf senescence and local climate adaptation in Arabidopsis accessions. Nat. Commun. 9:460.

[17] Jiang, H.,Chen, Y.,Li, M.,Xu, X., and Wu, G. (2011). Overexpression of SGR results in oxidative stress and lesion-mimic cell death in rice seedlings. J. Integr. Plant Biol. 53:375–387.

[18] Kong, Z.,Li, M.,Yang, W.,Xu, W., and Xue, Y. (2006). A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. Plant Physiol. 141:1376–1388.

[19] Kinoshita, Y.,Saze, H.,Kinoshita, T.,Miura, A.,Soppe, W.J.,Koornneef, M., and Kakutani, T. (2007). Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats. Plant J. 49:38–45.

[20] Kim, J.H.,Woo, H.R.,Kim, J.,Lim, P.O.,Lee, I.C.,Choi, S.H.,Hwang, D., and Nam, H.G. (2009). Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science 323:1053–1057.

[21] Kim, H.J.,Park, J.H.,Kim, J.,Kim, J.J.,Hong, S.,Kim, J.,Kim, J.H.,Woo, H.R.,Hyeon, C.,Lim, P.O., et al. (2018). Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 115: E4930–E4939.

[22] Kim, H.J.,Hong, S.H.,Kim, Y.W.,Lee, I.H.,Jun, J.H.,Phee, B.K.,Rupak, T.,Jeong, H.,Lee, Y.,Hong, B.S., et al. (2014). Gene regulatory cascade of senescence-associated NAC transcription factors activated by Ethylene-insensitive2-mediated leaf senescence signalling in Arabidopsis. J. Exp. Bot. 65:4023–4036.

[23] Kim, H.J.,Nam, H.G., and Lim, P.O. (2016). Regulatory network of NAC transcription factors in leaf senescence. Curr. Opin. Plant Biol. 33:48–56.

[24] Lim, P.O.,Kim, H.J., and Nam, H.G. (2007). Leaf senescence. Annu. Rev. Plant Biol. 58:115–136.

[25] Liang, C.,Wang, Y.,Zhu, Y.,Tang, J.,Hu, B.,Liu, L.,Ou, S.,Wu, H.,Sun, X.,Chu, J., et al. (2014). OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice. Proc. Natl. Acad. Sci. U.S.A. 111:10013–10018.

[26] Lee, S.,Seo, P.J.,Lee, H.J., and Park, C.M. (2012). A NAC transcription factor NTL4 promotes reactive oxygen species production during drought-induced leaf senescence in Arabidopsis. Plant J. 70:831–844.

[27] Lang, Z.,Wang, Y.,Tang, K.,Tang, D.,Datsenka, T.,Cheng, J.,Zhang, Y.,Handa, A.K., and Zhu, J.K. (2017). Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit. Proc. Natl. Acad. Sci. U.S.A. 114: E4511–E4519.

[28] Lu, J.,Jiang, Z.,Chen, J.,Xie, M.,Huang, W.,Li, J.,Zhuang, C.,Liu, Z., and Zheng, S. (2024). SET DOMAIN GROUP 711-mediated H3K27me3 methylation of cytokinin metabolism genes regulates organ size in rice. Plant Physiol. 194:2069–2085.

[29] Liu, L., and Chen, X. (2016). RNA quality control as a key to suppressing RNA silencing of endogenous genes in plants. Mol. Plant 6:826–836.

[30] Mao, C.,Lu, S.,Lv, B.,Zhang, B.,Shen, J.,He, J.,Luo, L.,Xi, D.,Chen, X., and Ming, F. (2017). A rice NAC transcription factor promotes leaf senescence via ABA biosynthesis. Plant Physiol. 174:1747–1763.

[31] Mahmood, K.,El-Kereamy, A.,Kim, S.H.,Nambara, E., and Rothstein, S.J. (2016). ANAC032 positively regulates age-dependent and stress-induced senescence in Arabidopsis thaliana. Plant Cell Physiol. 57:2029–2046.

[32] Nuruzzaman, M.,Manimekalai, R.,Sharoni, A.M.,Satoh, K.,Kondoh, H.,Ooka, H., and Kikuchi, S. (2010). Genome-wide analysis of NAC transcription factor family in rice. Gene 465:30–44.

[33] Nakashima, K.,Tran, L.S.,Van Nguyen, D.,Fujita, M.,Maruyama, K.,Todaka, D.,Ito, Y.,Hayashi, N.,Shinozaki, K., and Yamaguchi-Shinozaki, K. (2007). Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 51:617–630.

[34] Ogneva, Z.V.,Dubrovina, A.S., and Kiselev, K.V. (2016). Age-associated alterations in DNA methylation and expression of methyltransferase and demethylase genes in Arabidopsis thaliana. Biol. Plant. 60:628–634.

[35] Oh, S.A.,Park, J.H.,Lee, G.I.,Paek, K.H.,Park, S.K., and Nam, H.G. (1997). Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. Plant J. 12:527–535.

[36] Ostrowska-Mazurek, A.,Kasprzak, P.,Kubala, S.,Zaborowska, M., and Sobieszczuk-Nowicka, E. (2020). Epigenetic landmarks of leaf senescence and crop improvement. Int. J. Mol. Sci. 21:5125.

[37] Oda-Yamamizo, C.,Mitsuda, N.,Sakamoto, S.,Ogawa, D.,Ohme-Takagi, M., and Ohmiya, A. (2016). The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves. Sci. Rep. 6:23609.

[38] Pandey, R.,Müller, A.,Napoli, C.A.,Selinger, D.A.,Pikaard, C.S.,Richards, E.J.,Bender, J.,Mount, D.W., and Jorgensen, R.A. (2002). Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes. Nucleic Acids Res. 30:5036–5055.

[39] Podzimska-Sroka, D.,O’Shea, C.,Gregersen, P.L., and Skriver, K. (2015). NAC transcription factors in senescence: From molecular structure to function in crops. Plants 4:412–448.

[40] Qi, Y.,He, X.,Wang, X.J.,Kohany, O.,Jurka, J., and Hannon, G.J. (2006). Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443:1008–1012.

[41] Rong, H.,Tang, Y.,Zhang, H.,Wu, P.,Chen, Y.,Li, M.,Wu, G., and Jiang, H. (2013). The stay-green rice like (SGRL) gene regulates chlorophyll degradation in rice. J. Plant Physiol. 170:1367–1373.

[42] Ren, Y.,Li, M.,Wang, W.,Lan, W.,Schenke, D.,Cai, D., and Miao, Y. (2022). MicroRNA840 (MIR840) accelerates leaf senescence by targeting the overlapping 3′UTRs of PPR and WHIRLY3 in Arabidopsis thaliana. Plant J. 109:126–143.

[43] Schippers, J.H. (2015). Transcriptional networks in leaf senescence. Curr. Opin. Plant Biol. 27:77–83.

[44] Satyaki, P.R., and Gehring, M. (2017). DNA methylation and imprinting in plants: Machinery and mechanisms. Crit. Rev. Biochem. Mol. Biol. 52:163–175.

[45] Sato, Y.,Morita, R.,Katsuma, S.,Nishimura, M.,Tanaka, A., and Kusaba, M. (2009). Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J. 57:120–131.

[46] Sakuraba, Y.,Piao, W.,Lim, J.H.,Han, S.H.,Kim, Y.S.,An, G., and Paek, N.C. (2015). Rice ONAC106 inhibits leaf senescence and increases salt tolerance and tiller angle. Plant Cell Physiol. 56:2325–2339.

[47] Sperotto, R.A.,Ricachenevsky, F.K.,Duarte, G.L.,Boff, T.,Lopes, K.L.,Sperb, E.R.,Grusak, M.A., and Fett, J.P. (2009). Identification of up-regulated genes in flag leaves during rice grain filling and characterization of OsNAC5, a new ABA-dependent transcription factor. Planta 230:985–1002.

[48] Tang, Y.,Li, M.,Chen, Y.,Wu, P.,Wu, G., and Jiang, H. (2011). Knockdown of OsPAO and OsRCCR1 cause different plant death phenotypes in rice. J. Plant Physiol. 168:1952–1959.

[49] Tolia, N.H., and Joshua-Tor, L. (2007). Slicer and the argonautes. Nat. Chem. Biol. 3:36–43.

[50] Takasaki, H.,Maruyama, K.,Takahashi, F.,Fujita, M.,Yoshida, T.,Nakashima, K.,Myouga, F.,Toyooka, K.,Yamaguchi-Shinozaki, K., and Shinozaki, K. (2015). SNAC-As, stress-responsive NAC transcription factors, mediate ABA-inducible leaf senescence. Plant J. 84:1114–1123.

[51] Ueda, M., and Seki, M. (2020). Histone modifications form epigenetic regulatory networks to regulate abiotic stress response. Plant Physiol. 182:15–26.

[52] Woo, H.R.,Kim, H.J.,Lim, P.O., and Nam, H.G. (2019). Leaf senescence: Systems and dynamics aspects. Annu. Rev. Plant Biol. 70:347–376.

[53] Woo, H.R.,Kim, J.H.,Nam, H.G., and Lim, P.O. (2004). The delayed leaf senescence mutants of Arabidopsis,ore1,ore3, and ore9 are tolerant to oxidative stress. Plant Cell Physiol. 45:923–932.

[54] Wu, L.,Zhou, H.,Zhang, Q.,Zhang, J.,Ni, F.,Liu, C., and Qi, Y. (2010). DNA methylation mediated by a microRNA pathway. Mol. Cell 38:465–475.

[55] Wu, L.,Zhang, Q.,Zhou, H.,Ni, F.,Wu, X., and Qi, Y. (2009). Rice microRNA effector complexes and targets. Plant Cell 21:3421–3435.

[56] Yuan, L.,Wang, D.,Cao, L.,Yu, N.,Liu, K.,Guo, Y.,Gan, S., and Chen, L. (2020). Regulation of leaf longevity by DML3-mediated DNA demethylation. Mol. Plant 13:1149–1161.

[57] Yamatani, H.,Sato, Y.,Masuda, Y.,Kato, Y.,Morita, R.,Fukunaga, K.,Nagamura, Y.,Nishimura, M.,Sakamoto, W.,Tanaka, A., et al. (2013). NYC4, the rice ortholog of Arabidopsis THF1, is involved in the degradation of chlorophyll-protein complexes during leaf senescence. Plant J. 74:652–662.

[58] Yang, Y.,Xu, J.,Huang, L.,Leng, Y.,Dai, L.,Rao, Y.,Chen, L.,Wang, Y.,Tu, Z.,Hu, J., et al. (2016a). PGL, encoding chlorophyllide a oxygenase 1, impacts leaf senescence and indirectly affects grain yield and quality in rice. J. Exp. Bot. 67:1297–1310.

[59] Yang, X.,Nian, J.,Xie, Q.,Feng, J.,Zhang, F.,Jing, H.,Zhang, J.,Dong, G.,Liang, Y.,Peng, J., et al. (2016b). Rice ferredoxin-dependent glutamate synthase regulates nitrogen-carbon metabolomes and is genetically differentiated between japonica and indica subspecies. Mol. Plant 9:1520–1534.

[60] Zhang, X.M.,Zhao, H.W.,Gao, S.,Wang, W.C.,Katiyar-Agarwal, S.,Huang, H.D.,Raikhel, N., and Jin, H.L. (2011). Arabidopsis Argonaute 2 regulates innate immunity via miRNA393*-mediated silencing of a Golgi-localized SNARE gene, MEMB12. Mol. Cell 42:356–366.

[61] Zicola, J.,Liu, L.,Tänzler, P., and Turck, F. (2019). Targeted DNA methylation represses two enhancers of FLOWERING LOCUS T in Arabidopsis thaliana. Nat. Plants 5:300–307.

[62] Zhang, K., and Gan, S.S. (2012). An abscisic acid-AtNAP transcription factor-SAG113 protein phosphatase 2C regulatory chain for controlling dehydration in senescing Arabidopsis leaves. Plant Physiol. 158:961–969.

[63] Zheng, S.,Li, J.,Ma, L.,Wang, H.,Zhou, H.,Ni, E.,Jiang, D.,Liu, Z., and Zhuang, C. (2019). OsAGO2 controls ROS production and the initiation of tapetal PCD by epigenetically regulating OsHXK1 expression in rice anthers. Proc. Natl. Acad. Sci. U.S.A. 116:7549–7558.

[64] Zheng, S.,Ye, C.,Lu, J.,Liufu, J.,Lin, L.,Dong, Z.,Li, J., and Zhuang, C. (2021). Improving the rice photosynthetic efficiency and yield by editing OsHXK1 via CRISPR/Cas9 system. Int. J. Mol. Sci. 22:9554.

[65] Zheng, S.,Dong, J.,Lu, J.,Li, J.,Jiang, D.,Yu, H.,Ye, S.,Bu, W.,Liu, Z.,Zhou, H., et al. (2022). A cytosolic pentatricopeptide repeat protein is essential for tapetal plastid development by regulating OsGLK1 transcript levels in rice. New Phytol. 234:1678–1695.

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2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.