Transcription factors RhPIF4/8 and RhHY5 regulate autophagy-mediated petal senescence in rose ( Rosa hybrida)

Abstract

Petal senescence refers to the progressive loss of intracellular structures and functions within plant decorative organs, ultimately leading to cell death. Autophagy involves the degradation of damaged cellular components and nutrient recycling. Plant organ senescence and autophagy are highly coordinated; however, the mechanisms by which autophagy regulates petal senescence remain largely unknown. In this study, by using transmission electron microscopy, we observed that autophagic activity peaked early, at flower opening, without any senescence and other morphological symptoms in petals. We found that darkness positively regulated petal senescence and upregulated autophagy-related genes (ATGs). Dark treatment promoted the accumulation of Rosa hybrida phytochrome-interacting factor 4 ( RhPIF4) in petals. RhPIF4 silencing delayed petal senescence and repressed the expression of ATGs. In contrast, silencing of the light-responsive gene Rosa hybrida elongated hypoctyl 5 ( RhHY5) promoted petal senescence and ATG gene expression. RhPIF4/8 and RhHY5 could directly interact with RhWRKY40, and RhWRKY40 is directly bound to the promoters of RhATG7 and RhATG11. Silencing RhWRKY40 delayed petal senescence and suppressed RhATG7 and RhATG11 expression. Based on these results, we propose that RhPIF4/8 and RhHY5 transcription factors are involved in regulating petal senescence in response to dark or light conditions by modulating autophagic activity.

Keywords

Rose / Flower senescence / Autophagy / Darkness / ATG

References

[1]	Al-Sady B, Ni W, Kircher S, Sch?fer E, Quail PH. Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell. 2006;23:439–46. https://doi.org/10.1016/j.molcel.2006.06.011.
[2]	Avila-Ospina L, Moison M, Yoshimoto K, Masclaux-Daubresse C. Autophagy, plant senescence, and nutrient recycling. J Exp Bot. 2014;65:3799–811. https://doi.org/10.1093/jxb/eru039.
[3]	Broda M, Millar AH, Van Aken O. Mitophagy: a mechanism for plant growth and survival. Trends Plant Sci. 2018;23:434–50. https://doi.org/10.1016/j.tplants.2018.02.010.
[4]	Burko Y, Seluzicki A, Zander M, Pedmale UV, Ecker JR, Chory J. Chimeric activators and repressors define HY5 activity and reveal a light-regulated feedback mechanism. Plant Cell. 2020;32:967–83 https://doi.org/10.1105/tpc.19.00772.
[5]	Castillon A, Shen H, Huq E. Phytochrome interacting factors: central players in phytochrome-mediated light signaling networks. Trends Plant Sci. 2007;12:514–21. https://doi.org/10.1016/j.tplants.2007.10.001.
[6]	Chen C, Ma Y, Zuo L, Xiao Y, Jiang Y, Gao J. The calcineurin B-like 4/CBL-interacting protein 3 module degrades repressor JAZ5 during rose petal senescence. Plant Physiol. 2023;193:1605–20. https://doi.org/10.1093/plphys/kiad365.
[7]	Cheng C, Yu Q, Wang Y, Wang H, Dong Y, Ji Y, et al. Ethylene-regulated asymmetric growth of the petal base promotes flower opening in rose (Rosa hybrida). Plant Cell. 2021;33:1229–51. https://doi.org/10.1093/plcell/koab031.
[8]	Chi C, Li X, Fang P, Xia X, Shi K, Zhou Y, et al. Brassinosteroids act as a positive regulator of NBR1-dependent selective autophagy in response to chilling stress in tomato. J Exp Bot. 2020;71:1092–106. https://doi.org/10.1093/jxb/erz466.
[9]	Chung T, Phillips AR, Vierstra RD. ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A and ATG12B loci. Plant J. 2010;62:483–93. https://doi.org/10.1111/j.1365-313X.2010.04166.x.
[10]	Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000;5:199–206. https://doi.org/10.1016/s1360-1385(00)01600-9.
[11]	Feng Q, De Rycke R, Dagdas Y, Nowack MK. Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap. Curr Biol. 2022;32:2110–19.e3. https://doi.org/10.1016/j.cub.2022.03.053.
[12]	Gangappa SN, Kumar SV. DET1 and HY5 control PIF4-mediated thermosensory elongation growth through distinct mechanisms. Cell Rep. 2017;18:344–51. https://doi.org/10.1016/j.celrep.2016.12.046.
[13]	Guan B, Lin Z, Liu D, Li C, Zhou Z, Mei F, et al. Effect of waterlogging-induced autophagy on programmed cell death in Arabidopsis roots. Front Plant Sci. 2019;10:468. https://doi.org/10.3389/fpls.2019.00468.
[14]	Guo Y, Ren G, Zhang K, Li Z, Miao Y, Guo H. Leaf senescence: progression, regulation, and application. Mol Hortic. 2021;1:5. https://doi.org/10.1186/s43897-021-00006-9.
[15]	Hoeberichts FA, de Jong AJ, Woltering EJ. Apoptotic-like cell death marks the early stages of gypsophila (Gypsophila paniculata) petal senescence. Postharvest Biol Technol. 2005;35:229–36. https://doi.org/10.1016/j.postharvbio.2004.10.005.
[16]	Jiang C, Liang Y, Deng S, Liu Y, Zhao H, Li S, et al. The RhLOL1–RhILR3 module mediates cytokinin-induced petal abscission in rose. New Phytol. 2022;237:483–96. https://doi.org/10.1111/nph.18556.
[17]	Kurusu T, Koyano T, Hanamata S, Kubo T, Noguchi Y, Yagi C, et al. OsATG7 is required for autophagy-dependent lipid metabolism in rice postmeiotic anther development. Autophagy. 2014;10:878–88. https://doi.org/10.4161/auto.28279.
[18]	Kwon SI, Cho HJ, Jung JH, Yoshimoto K, Shirasu K, Park OK. The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis Plant J. 2010;64:151–64. https://doi.org/10.1111/j.1365-313X.2010.04315.x.
[19]	Li F, Chung T, Pennington JG, Federico ML, Kaeppler HF, Kaeppler SM, et al. Autophagic recycling plays a central role in maize nitrogen remobilization. Plant Cell. 2015;27:1389–408. https://doi.org/10.1105/tpc.15.00158.
[20]	Ma N, Cai L, Lu W, Tan H, Gao J. Exogenous ethylene influences flower opening of cut roses (Rosa hybrida) by regulating the genes encoding ethylene biosynthesis enzymes. Sci China Life Sci. 2005;48:434–44. https://doi.org/10.1360/062004-37.
[21]	Ma N, Ma C, Liu Y, Shahid MO, Wang C, Gao J. Petal senescence: a hormone view. J Exp Bot. 2018;69:719–32. https://doi.org/10.1093/jxb/ery009.
[22]	Nozue K, Covington MF, Duek PD, Lorrain S, Fankhauser C, Harmer SL, et al. Rhythmic growth explained by coincidence between internal and external cues. Nature. 2007;448:358–61. https://doi.org/10.1038/nature05946.
[23]	Osterlund MT, Hardtke CS, Wei N, Deng XW. Targeted destabilization of HY5 during light-regulated development of Arabidopsis Nature. 2000;405:462–6. https://doi.org/10.1038/35013076.
[24]	Otegui MS. Vacuolar degradation of chloroplast components: autophagy and beyond. J Exp Bot. 2018;69:741–50. https://doi.org/10.1093/jxb/erx234.
[25]	Pérez-Pérez ME, Lemaire SD, Crespo JL. Reactive oxygen species and autophagy in plants and algae. Plant Physiol. 2012;160:156–64. https://doi.org/10.1104/pp.112.199992.
[26]	Rubinsztein DC, Cuervo AM, Ravikumar B, Sarkar S, Korolchuk V, Kaushik S, et al. In search of an "autophagomometer" Autophagy. 2009;5:585–9. https://doi.org/10.4161/auto.5.5.8823.
[27]	Saijo Y, Sullivan JA, Wang H, Yang J, Shen Y, Rubio V, et al. The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes Dev. 2003;17:2642–7. https://doi.org/10.1101/gad.1122903.
[28]	Sánchez-Sevilla JF, Botella MA, Valpuesta V, Sanchez-Vera V. Autophagy is required for strawberry fruit ripening. Front Plant Sci. 2021;12:688481. https://doi.org/10.3389/fpls.2021.688481.
[29]	Scherz-Shouval R, Elazar Z. ROS, mitochondria and the regulation of autophagy. Trends Cell Biol. 2007;17:422–7. https://doi.org/10.1016/j.tcb.2007.07.009.
[30]	Shen Y, Khanna R, Carle CM, Quail PH. Phytochrome induces rapid PIF5 phosphorylation and degradation in response to red-light activation. Plant Physiol. 2007;145:1043–51. https://doi.org/10.1104/pp.107.105601.
[31]	Shen H, Zhu L, Castillon A, Majee M, Downie B, Huq E. Light-induced phosphorylation and degradation of the negative regulator phytochrome-interacting facotr1 from Arabidopsis depend upon its direct physical interactions with photoactivated phytochromes. Plant Cell. 2008;20:1586–602. https://doi.org/10.1105/tpc.108.060020.
[32]	Shibutani ST, Yoshimori T. A current perspective of autophagosome biogenesis. Cell Res. 2014;24:58–68. https://doi.org/10.1038/cr.2013.159.
[33]	Shibuya K, Yamada T, Suzuki T, Shimizu K, Ichimura K. InPSR26, a putative membrane protein, regulates programmed cell death during petal senescence in Japanese morning glory. Plant Physiol. 2009;149:816–24. https://doi.org/10.1104/pp.108.127415.
[34]	Shibuya K, Niki T, Ichimura K. Pollination induces autophagy in petunia petals via ethylene. J Exp Bot. 2013;64:1111–20. https://doi.org/10.1093/jxb/ers395.
[35]	Shibuya K, Shimizu K, Niki T, Ichimura K. Identification of a NAC transcription factor, EPHEMERAL1, that controls petal senescence in Japanese morning glory. Plant J. 2014;79:1044–51. https://doi.org/10.1111/tpj.12605.
[36]	Shimizu S, Arakawa S, Nishida Y. Autophagy takes an alternative pathway. Autophagy. 2010;6:290–1. https://doi.org/10.4161/auto.6.2.11127.
[37]	Smith MT, Sarks Y, van Staden J. Ultrastructural changes in the petals of senescing flowers of Dianthus caryophyllus L. Ann Bot. 1992;69:277–85. https://doi.org/10.1093/oxfordjournals.aob.a088341.
[38]	Song Y, Yang C, Gao S, Zhang W, Li L, Kuai B. Age-triggered and dark-induced leaf senescence require the bHLH transcription factors PIF3, 4, and 5. Mol Plant. 2014;7:1776–87. https://doi.org/10.1093/mp/ssu109.
[39]	Su T, Li X, Yang M, Shao Q, Zhao Y, Ma C, et al. Autophagy: an intracellular degradation pathway regulating plant survival and stress response. Front Plant Sci. 2020;11:164. https://doi.org/10.3389/fpls.2020.00164.
[40]	Teper-Bamnolker P, Danieli R, Peled-Zehavi H, Belausov E, Abu-Abied M, Avin-Wittenberg T, et al. Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death. Autophagy. 2021;17:3109–23. https://doi.org/10.1080/15548627.2020.1856492.
[41]	üstün S, Hafrén A, Hofius D. Autophagy as a mediator of life and death in plants. Curr Opin Plant Biol. 2017;40:122–30. https://doi.org/10.1016/j.pbi.2017.08.011.
[42]	van Doorn WG, Woltering EJ. Many ways to exit? Cell death categories in plants. Trends Plant Sci. 2005;10(3):117–22. https://doi.org/10.1016/j.tplants.2005.01.006.
[43]	van Doorn WG, Woltering EJ. Physiology and molecular biology of petal senescence. J Exp Bot. 2008;59:453–80. https://doi.org/10.1093/jxb/erm356.
[44]	van Doorn WG, Balk PA, van Houwelingen AM, Hoeberichts FA, Hall RD, Vorst O, et al. Gene expression during anthesis and senescence in Iris flowers. Plant Mol Biol. 2003;53:845–63. https://doi.org/10.1023/B:PLAN.0000023670.61059.1d.
[45]	van Doorn WG, Kirasak K, Ketsa S. Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals. J Plant Physiol. 2015;177:67–73. https://doi.org/10.1016/j.jplph.2015.01.006.
[46]	Wang Y, Cai S, Yin L, Shi K, Xia X, Zhou Y, et al. Tomato HsfA1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy. Autophagy. 2015;11:2033–47. https://doi.org/10.1080/15548627.2015.1098798.
[47]	Wang Y, Cao J, Wang K, Xia X, Shi K, Zhou YH, et al. BZR1 mediates brassinosteroid-induced autophagy and nitrogen starvation in tomato. Plant Physiol. 2019;179:671–85. https://doi.org/10.1104/pp.18.01028.
[48]	Woo HR, Kim HJ, Lim PO, Nam HG. Leaf senescence: systems and dynamics aspects. Annu Rev Plant Biol. 2019;70:347–76. https://doi.org/10.1146/annurev-arplant-050718-095859.
[49]	Xia K, Liu T, Ouyang J, Wang R, Fan T, Zhang M. Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L). DNA Res. 2011;18:363–77. https://doi.org/10.1093/dnares/dsr024.
[50]	Yang C, Luo M, Zhuang X, Li F, Gao C. Transcriptional and epigenetic regulation of autophagy in plants. Trends Genet. 2020;36:676–88. https://doi.org/10.1016/j.tig.2020.06.013.
[51]	Yang C, Shen W, Yang L, Sun Y, Li X, Lai M, et al. HY5-HDA9 module transcriptionally regulates plant autophagy in response to light-to-dark conversion and nitrogen starvation. Mol Plant. 2020;13:515–31. https://doi.org/10.1016/j.molp.2020.02.011.
[52]	Yao W, Li Y, Wu L, Wu C, Zhang Y, Liu J, et al. Atg11 is required for initiation of glucose starvation-induced autophagy. Autophagy. 2020;16:2206–18. https://doi.org/10.1080/15548627.2020.1719724.
[53]	Zhang S, Feng M, Chen W, Zhou X, Lu J, Wang Y, et al. In rose, transcription factor PTM balances growth and drought survival via PIP2;1 aquaporin. Nat Plants. 2019;5:290–9. https://doi.org/10.1038/s41477-019-0376-1.
[54]	Zhang B, Shao L, Wang J, Zhang Y, Guo X, Peng Y, et al. Phosphorylation of ATG18a by BAK1 suppresses autophagy and attenuates plant resistance against necrotrophic pathogens. Autophagy. 2020;17:2093–110. https://doi.org/10.1080/15548627.2020.1810426.
[55]	Zhang Y, Wu Z, Feng M, Chen J, Qin M, Wang W, et al. The circadian-controlled PIF8-BBX28 module regulates petal senescence in rose flowers by governing mitochondrial ROS homeostasis at night. Plant Cell. 2021;33:2716–35. https://doi.org/10.1093/plcell/koab152.
[56]	Zhang Z, Liu Y, Yang T, Wu S, Sun H, Wu J, et al. Haplotype-resolve genome assembly and resequencing provide insights into the origin and domestication of modern rose. bioRxiv. 2023:2023.06.02.543351. https://doi.org/10.1101/2023.06.02.543351.
[57]	Zhou X, Zhao P, Wang W, Zou J, Cheng TH, Peng XB, et al. A comprehensive, genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues. DNA Res. 2015;22:245–57. https://doi.org/10.1093/dnares/dsv012.
[58]	Zhou S, Hong Q, Li Y, Li Q, Wang M. Autophagy contributes to regulate the ROS levels and PCD progress in TMV-infected tomatoes. Plant Sci. 2018;269:12–9. https://doi.org/10.1016/j.plantsci.2017.11.002.
[59]	Zhu T, Zou L, Li Y, Yao X, Xu F, Deng X, et al. Mitochondrial alternative oxidase-dependent autophagy involved in ethylene-mediated drought tolerance in Solanum lycopersicum Plant Biotechnol J. 2018;16:2063–76. https://doi.org/10.1111/pbi.12939.

Funding

National Natural Science Foundation of China(32002079); Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects(CEFFPXM2019_014207_000032); 2115 Talent Development Program of China Agricultural University