A R1-type MYB CmREVEILLE2 regulates light-mediated chlorophyll biosynthesis and green color formation in chrysanthemum flowers

Abstract

Chrysanthemum (Chrysanthemum ×  morifolium Ramat.) is a widely cultivated ornamental species, among which green-flowered cultivar representing rarity and distinctiveness. However, the underlying mechanisms governing the formation and regulation of green color remain poorly understood. In this study, exposure to high-intensity light resulted in the loss of green coloration in green chrysanthemum, while low-intensity light preserved it during flower opening in chrysanthemum ‘Lv Dingdang’. A R1-type MYB transcription factor, CmREVEILLE2 (CmRVE2), was identified through transcriptomic analysis of flowers subjected to different light intensities (3,000 Lux for low light, LL; 6,000 Lux for moderate light, ML; and 12,000 Lux for high light, HL). CmRVE2 expression was significantly upregulated under HL conditions, while its expression was lower under LL compared to ML. Additionally, CmRVE2 was upregulated by abscisic acid (ABA) and downregulated by gibberellin (GA). Virus-induced gene silencing (VIGS) of CmRVE2 led to a marked increase in chlorophyll content and the upregulation of chlorophyll biosynthesis-related genes, while CmRVE2 overexpression resulted in the opposite trend. Yeast one-hybrid, electrophoretic mobility shift assays (EMSA), and transient activation assays demonstrated that CmRVE2 directly bound to the promoter of CHLI1, a gene encodes magnesium chelatase I subunit in chlorophyll biosynthesis. Furthermore, CmRVE2 repressed the expression of MAGNESIUM-PROTOPORPHYRIN IX MONOMETHYL ESTER OXIDATIVE CYCLASE1 (CRD1) and PROTOCHLOROPHYLLIDE OXIDOREDUCTASE1 (PORA1), thereby inhibiting chlorophyll synthesis. These findings provide new insights into how CmRVE2 mediates light signaling to negatively regulate chlorophyll biosynthesis in chrysanthemum flowers.

Keywords

Chrysanthemum / Light / Flower color / Chlorophyll / CmREVEILL2 / Biological Sciences / Genetics

References

[1] AnJ, WeiX, HuoH. Transcriptome analysis reveals the accelerated expression of genes related to photosynthesis and chlorophyll biosynthesis contribution to shade-tolerant in Phoebe bournei. BMC Plant Biol, 2022, 22: 268.

[2] Bonjoch NP , Tamayo PR. Protein content quantification by bradford method. In: Reigosa Roger MJ, editor. Handbook of plant ecophysiology techniques. Dordrecht: Springer;2001.p.283–95. https://doi.org/10.1007/0-306-48057-3_19.

[3] ChenJ, WuS, DongF, LiJ, ZengL, TangJ, et al. . Mechanism underlying the shading-induced chlorophyll accumulation in tea leaves. Front Plant Sci, 2021, 12. 779819

[4] Chen S, Wang H, Li R, Wang Z, Luo J, Wang C. Characterization of CmMYC2 in formation of green color in ray florets of Chrysanthemum. Acta Horticulturae Sinica. 2022;49:2377–87. https://doi.org/10.16420/j.issn.0513-353x.2021-0524.

[5] DongW, LiM, LiZ, LiS, ZhuY, WangZ. Transcriptome analysis of the molecular mechanism of Chrysanthemum flower color change under short-day photoperiods. Plant Physiol Biochem, 2020, 146: 315-328.

[6] DubosC, StrackeR, GrotewoldE, WeisshaarB, MartinC, LepiniecL. MYB transcription factors in Arabidopsis. Trends Plant Sci, 2010, 15: 573-581.

[7] FankhauserC, ChristieJM. Plant phototropic growth. Curr Biol, 2015, 25: R384-R389.

[8] FuH, ZengT, ZhaoY, LuoT, DengH, MengC, et al. . Identification of chlorophyll metabolism and photosynthesis-related genes regulating green flower color in chrysanthemum by integrative transcriptome and weighted correlation network analyses. Genes (Basel), 2021, 12: 449.

[9] GaoS, GaoJ, ZhuX, SongY, LiZ, RenG, et al. . ABF2, ABF3, and ABF4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis. Mol Plant, 2016, 9: 1272-1285.

[10] GrabherrMG, HaasBJ, YassourM, LevinJZ, ThompsonDA, AmitI, et al. . Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat Biotechnol, 2011, 29: 644-652.

[11] GuoY, RenG, ZhangK, LiZ, MiaoY, GuoH. Leaf senescence: progression, regulation, and application. Mol Horticulture, 2021, 1: 5.

[12] HanX, ShenY, WangY, ShenJ, WangH, DingS, et al. . Transcriptome revealed the effect of shading on the photosynthetic pigment and photosynthesis of overwintering tea leaves. Agronomy, 2023, 13: 1701.

[13] He C, Liu X, Teixeira da Silva JA, Liu N, Zhang M, Duan J. Transcriptome sequencing and metabolite profiling analyses provide comprehensive insight into molecular mechanisms of flower development in Dendrobium officinale (Orchidaceae). Plant Mol Biol. 2020;104:529–48. https://doi.org/10.1007/s11103-020-01058-z.

[14] HongY, BaiX, SunW, JiaF, DaiS. The numerical classification of chrysanthemum flower color phenotype. Acta Horticulturae Sinica, 2012, 39: 1330-1340

[15] JeongYS, ChoiH, KimJK, BaekSA, YouMK, LeeD, et al. . Overexpression of OsMYBR22/ OsRVE1 transcription factor simultaneously enhances chloroplast-dependent metabolites in rice grains. Metab Eng, 2022, 70: 89-101.

[16] JiaT, ItoH, HuX, TanakaA. Accumulation of the NON-YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana. Plant J, 2015, 81: 586-596.

[17] JiaoY, LauOS, DengX. Light-regulated transcriptional networks inhigher plants. Nat Rev Genet, 2007, 8: 217-230.

[18] JobN, DattaS. PIF3/HY5 module regulates BBX11 to suppress protochlorophyllide levels in dark and promote photomorphogenesis in light. New Phytol, 2021, 230: 190-204.

[19] LescotM, DéhaisP, ThijsG, MarchalK, MoreauY, Van de PeerY, et al. . PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 2002, 30: 325-327.

[20] LiS, GaoJ, YaoL, RenG, ZhuX, GaoS, et al. . The role of ANAC072 in the regulation of chlorophyll degradation during age-and dark-induced leaf senescence. Plant Cell Rep, 2016, 35: 1729-1741.

[21] LiuX, LiY, ZhongS. Interplay between light and plant hormones in the control of Arabidopsis seedling chlorophyll biosynthesis. Front Plant Sci, 2017, 8: 1433.

[22] LiuL, LinN, LiuX, YangS, WangW, WanX. From chloroplast biogenesis to chlorophyll accumulation: the interplay of light and hormones on gene expression in Camellia sinensis cv. Shuchazao Leaves Front Plant Sci, 2020, 11: 256.

[23] LiuW, LiuS, ZhangK, XieM, SunH, HuangX, et al. . Chlorophyllase is transcriptionally regulated by CsMYB308/CsDOF3 in young leaves of tea plant. Hortic Plant J, 2023, 9: 1162-1176.

[24] Liu S, Cheng Y, Zhao X, Wang E, Liu T, Zhang H, et al. The transcription factor StMYB113 regulates light-induced greening by modulating steroidal glycoalkaloid biosynthesis in potatoes (Solanum tuberosum L.). Hortic Adv. 2024;2:7. https://doi.org/10.1007/s44281-023-00025-0.

[25] LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^{− ΔΔCT} method. Methods, 2001, 25: 402-408.

[26] LoveMI, HuberW, AndersS. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 2014, 15: 1-21.

[27] LuoJ, WangH, ChenS, RenS, FuH, LiR, et al. . CmNAC73 mediates the formation of green color in chrysanthemum flowers by directly activating the expression of chlorophyll biosynthesis genes HEMA1 and CRD1. Genes (Basel), 2021, 12: 704.

[28] MathurS, JainL, JajooA. Photosynthetic efficiency in sun and shade plants. Photosynthetica, 2018, 56: 354-365.

[29] MekapoguM, KwonOK, SongHY, JungJA. Towards the improvement of ornamental attributes in chrysanthemum: recent progress in biotechnological advances. Int J Mol Sci, 2022, 23: 12284.

[30] MishaninVI, TrubitsinBV, PatsaevaSV, PtushenkoVV, SolovchenkoAE, TikhonovAN. Acclimation of shade-tolerant and light-resistant Tradescantia species to growth light: chlorophyll a fluorescence, electron transport, and xanthophyll content. Photosynth Res, 2017, 133: 87-102.

[31] NakanoM, HirakawaH, FukaiE, ToyodaA, KajitaniR, MinakuchiY, et al. . A chromosome-level genome sequence of Chrysanthemum seticuspe, a model species for hexaploid cultivated chrysanthemum. Commun Biol, 2021, 4: 1167.

[32] NarbonaE, del ValleJC, WhittallJB. Painting the green canvas: how pigments produce flower colours. Biochemist, 2021, 43: 6-12.

[33] OhmiyaA, Oda-YamamizoC, KishimotoS. Overexpression of CONSTANS-like 16 enhances chlorophyll accumulation in petunia corollas. Plant Sci, 2019, 280: 90-96.

[34] Potosí-Calvache DC, Vanegas-Mahecha P, Martínez-Correa HA. Convective drying of squash (Cucurbita moschata): Influence of temperature and air velocity on effective moisture diffusivity, carotenoid content and total phenols. Dyna 2017;84:112–9. https://doi.org/10.15446/dyna.v84n202.63904.

[35] RoguzK, GallagherMK, SendenE, Bar-LevY, LebelM, HeliczerR, et al. . All the colors of the rainbow: diversification of flower color and intraspecific color variation in the genus Iris. Front Plant Sci, 2020, 11. 569811

[36] SchelbertS, AubryS, BurlaB, AgneB, KesslerF, KrupinskaK, et al. . Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell, 2009, 21: 767-785.

[37] SinghT, BalaM. Effect of foliar spray of benzyl adenine, gibberellic acid and putrescine on post-harvest keeping quality of Chrysanthemum. Agricultural Research Journal, 2018, 55: 386-388.

[38] SongC, LiuY, SongA, DongG, ZhaoH, SunW, et al. . The Chrysanthemum nankingense genome provides insights into the evolution and diversification of Chrysanthemum flowers and medicinal traits. Mol Plant, 2018, 11: 1482-1491.

[39] StolárikT, HedtkeB, ŠantrůčekJ, IlíkP, GrimmB, PavlovičA. Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce. Photosynth Res, 2017, 132: 165-179.

[40] TanXL, FanZQ, KuangJF, LuWJ, ReiterRJ, LakshmananP, et al. . Melatonin delays leaf senescence of Chinese flowering cabbage by suppressing ABFs-mediated abscisic acid biosynthesis and chlorophyll degradation. J Pineal Res, 2019, 67. e12570

[41] TanakaA, ItoH. Chlorophyll degradation and its physiological function. Plant Cell Physiol, 2025, 66: 139-152.

[42] TanakaA, TanakaR. Chlorophyll metabolism. Curr Opin Plant Biol, 2006, 9: 248-255.

[43] WangP, GrimmB. Connecting chlorophyll metabolism with accumulation of the photosynthetic apparatus. Trends Plant Sci, 2021, 26: 484-495.

[44] WangT, WeiQ, WangZ, LiuW, ZhaoX, MaC, et al. . CmNF-YB8 affects drought resistance in Chrysanthemum by altering stomatal status and leaf cuticle thickness. J Integr Plant Biol, 2022, 64: 741-755.

[45] WangY, JingT, LiuX, AiX, BiH. SlTDC modulates photosynthesis of senescent leaves in tomato. Hortic Adv, 2024, 2: 16.

[46] Xu L, Xu H, Cao Y, Yang P, Feng Y, Tang Y, et al. Validation of reference genes for quantitative real-time PCR during bicolor tepal development in asiatic hybrid lilies (Lilium spp.). Front Plant Sci. 2017;8:669. https://doi.org/10.3389/fpls.2017.00669.

[47] YamataniH, ItoT, NishimuraK, YamadaT, SakamotoW, KusabaM. Genetic analysis of chlorophyll synthesis and degradation regulated by balance of chlorophyll metabolism. Plant Physiol, 2022, 189: 419-432.

[48] Yang T, Ali M, Lin L, Li P, He H, Zhu Q, et al. Recoloring tomato fruit by CRISPR/Cas9-mediated multiplex gene editing. Hortic Res. 2023;10:uhac214. https://doi.org/10.1093/hr/uhac214.

[49] YongS, ChenQ, XuF, FuH, LiangG, GuoQ. Exploring the interplay between angiosperm chlorophyll metabolism and environmental factors. Planta, 2024, 260: 25.

[50] YueC, WangZ, YangP. The effect of light on the key pigment compounds of photosensitive etiolated tea plant. Bot Stud, 2021, 62: 21.

[51] Zeng T, Li JW, Xu ZZ, Zhou L, Li JJ, Yu Q, et al. TcMYC2 regulates Pyrethrin biosynthesis in Tanacetum cinerariifolium. Hortic Res. 2022;9:uhac178. https://doi.org/10.1093/hr/uhac178.

[52] ZhangX, LiY, YanH, CaiK, LiH, WuZ, et al. . Integrated metabolomic and transcriptomic analyses reveal different metabolite biosynthesis profiles of Juglans mandshurica in shade. Front Plant Sci, 2022, 13. 991874

[53] ZhaoX, JiaT, HuX. HCAR is a limitation factor for chlorophyll cycle and chlorophyll b degradation in chlorophyll-b-overproducing plants. Biomolecules, 2020, 10: 1639.

[54] ZhengX, LanJ, YuH, ZhangJ, ZhangY, QinY, et al. . Arabidopsis transcription factor TCP4 represses chlorophyll biosynthesis to prevent petal greening. Plant Commun, 2022, 3. 100309

[55] ZhuX, ChenJ, QiuK, KuaiB. Phytohormone and light regulation of chlorophyll degradation. Front Plant Sci, 2017, 8: 1911.

Funding

the Expert Workstation in Wuhan Institute of Landscape Architecture(0220210393)

Key Technologies Research and Development Program(2019YFD1001500)

Cooperation Development of Chrysanthemum Industry Technology Innovation Demonstration Base in Macheng(0220220063)

RIGHTS & PERMISSIONS

The Author(s)