A NAC family gene PmNAC32 associated with photoperiod promotes flower induction in Prunus mume

Abstract

The photoperiod is essential to flower induction, and the exact timing of the process can be precisely regulated based on the relative duration of light and darkness. However, the mechanisms linking photoperiod and flower induction in woody plants remain largely unexplored. Using RNA-seq, we identified a photoperiod response factor PmNAC32, which is predominantly expressed in early-flowering varieties. Overexpression of PmNAC32 in Arabidopsis thaliana, tobacco, and Prunus mume calli resulted in accelerated flowering. Binding and activation analyses revealed that PmNAC32 can be directly suppressed by REVEILLE 1 (RVE1) and REVEILLE 3 (RVE3), implying that PmNAC32 plays a role in the photoperiodic signaling pathway. Further studies established that PmNAC32 functions as a positive regulator of CONSTANS-LIKE 5 (COL5) and a negative regulator of CONSTANS-LIKE 4 (COL4). Interestingly, we identified two homologs of PmNAC32, namely PmNAC29 and PmNAC47. These three proteins can interact with each other and enhance the regulation of PmCOL4 and PmCOL5. Although PmNAC29 and PmNAC47 can promote flower induction respectively, neither of them responded to the photoperiod. Thus, our results reveal a novel mechanism by which PmNAC32 regulates flower induction in Prunus mume.

Acknowledgements

We are grateful to Jiafu Jiang from Nanjing Agricultural University for his suggestion and technical support. This work was funded through the National Natural Science Foundation of China (32372670, 31971703), the ‘JBGS’ Project of Seed Industry Revitalization in Jiangsu Province (JBGS〔2021〕019), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Postgraduate Research and Practice Innovation Program of Jiangsu Province, China (KYCX23_0798).

Author contributions

C.D.M. and Z.H.G. designed the research framework and experimental procedures. C.D.M. and P.Y.Z. conducted the experimental work. C.D.M., Y.F.M., W.T., X.H., Z.J.N., and T.S. contributed to the reagents, materials, and analysis tools. C.D.M. wrote the article, S.I. and S.S. polished the language.

Data availability

The data presented in this study are included in the manuscript or supplementary materials. The processed RNA-seq datasets have been deposited in the NCBI Sequence Read Archive (SRA; PRJNA1060359).

Conflict of interests

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Bäurle I, Dean C. The timing of developmental transitions in plants. Cell. 2006; 125:655-64

[2] Putterill J, Laurie R, Macknight R. It’s time to flower: the genetic control of flowering time. BioEssays. 2004; 26:363-73

[3] Srikanth A, Schmid M. Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci. 2011; 68:2013-37

[4] Tan F-C, Swain SM. Genetics of flower initiation and develop-ment in annual and perennial plants. Physiol Plant. 2006; 128: 8-17

[5] Kaur A, Maness N, Ferguson L. et al. Role of plant hormones in flowering and exogenous hormone application in fruit/nut trees: a review of pecans. Fruit Res. 2021; 1:1-9

[6] Sun L, Nie T, Chen Y. et al. From floral induction to blooming: the molecular mysteries of flowering in woody plants. Int J Mol Sci. 2022; 23:10959

[7] Koutinas N, Pepelyankov G, Lichev V. Flower induction and flower bud development in apple and sweet cherry. Biotechnol Biotechnol Equip. 2010; 24:1549-58

[8] Yamane H, Ooka T, Jotatsu H. et al. Expression analysis of PpDAM5 and PpDAM6 during flower bud development in peach (Prunus persica). Sci Hortic. 2011; 129:844-8

[9] Gao Z, Shi T. Botanical description of Prunus mume. In: Gao Z,ed. The Prunus Mume Genome. Cham: Springer International Publishing, 2019,9-24

[10] Simpson GG, Dean C. Arabidopsis, the rosetta stone of flowering time? Science. 2002; 296:285-9

[11] Weng X, Lovell JT, Schwartz SL. et al. Complex interactions between day length and diurnal patterns of gene expression drive photoperiodic responses in a perennial C4 grass. Plant Cell Environ. 2019; 42:2165-82

[12] Quail PH. Phytochrome photosensory signalling networks. Nat Rev Mol Cell Biol. 2002; 3:85-93

[13] Shim JS, Kubota A, Imaizumi T. Circadian clock and photope-riodic flowering in Arabidopsis: CONSTANS is a hub for signal integration. Plant Physiol. 2017; 173:5-15

[14] Robson F, Costa MMR, Hepworth SR. et al. Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants. Plant J. 2001; 28:619-31

[15] Imaizumi T, Schultz TF, Harmon FG. et al. FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science. 2005; 309:293-7

[16] Sawa M, Nusinow DA, Kay SA. et al. FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidop-sis. Science. 2007; 318:261-5

[17] Fornara F, Panigrahi KCS, Gissot L. et al. Arabidopsis DOF tran-scription factors act redundantly to reduce CONSTANS expres-sion and are essential for a photoperiodic flowering response. Dev Cell. 2009; 17:75-86

[18] Goralogia GS, Liu TK, Zhao L. et al. CYCLING DOF FACTOR 1 represses transcription through the TOPLESS co-repressor to control photoperiodic flowering in Arabidopsis. Plant J. 2017; 92: 244-62

[19] Laubinger S, Marchal V, Gentilhomme J´. et al. Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability. Development. 2006; 133:3213-22

[20] Zuo Z, Liu H, Liu B. et al. Blue light-dependent interaction of CRY 2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis. Curr Biol. 2011; 21:841-7

[21] Liu B, Zuo Z, Liu H. et al. Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev. 2011; 25:1029-34

[22] Sheerin DJ, et al.Menon C, zur Oven-Krockhaus S. Light-activated phytochrome A and B interact with members of the SPA family to promote photomorphogenesis in Arabidopsis by reorganizing the COP1/SPA complex. Plant Cell. 2015; 27:189-201

[23] Takagi H, Hempton AK, Imaizumi T. Photoperiodic flowering in Arabidopsis: multilayered regulatory mechanisms of CON-STANS and the florigen FLOWERING LOCUS T. Plant Commun. 2023; 4:100552

[24] Tripathi P, Carvallo M, Hamilton EE. et al. Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering. Proc Natl Acad Sci. 2017; 114:172-7

[25] Wang L, Sun J, Ren L. et al. CmBBX8 accelerates flowering by targeting CmFTL1 directly in summer chrysanthemum. Plant Biotechnol J. 2020; 18:1562-72

[26] Zhai Y, Zhu Y, Wang Q. et al. BBX7 interacts with BBX8 to accelerate flowering in chrysanthemum. Mol Hortic. 2023; 3:7

[27] Zhang X, An L, Nguyen TH. et al. The cloning and func-tional characterization of peach CONSTANS and FLOWERING LOCUS T homologous genes PpCO and PpFT. PLoS One. 2015; 10: e0124108

[28] ShiG, AiK, YanX. et al. Genome-wide analysis of the BBX genes in Platanus × acerifolia and their relationship with flowering and/or dormancy. Int J Mol Sci. 2023; 24:8576

[29] Hassidim M, Dakhiya Y, Turjeman A. et al. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and the circadian control of stomatal aperture. Plant Physiol. 2017; 175:1864-77

[30] Sun C, Zhang K, Zhou Y. et al. Dual function of clock component OsLHY sets critical day length for photoperiodic flowering in rice. Plant Biotechnol J. 2021; 19:1644-57

[31] Rawat R, Schwartz J, Jones MA. et al. REVEILLE1, a Myb-like transcription factor, integrates the circadian clock and auxin pathways. Proc Natl Acad Sci. 2009; 106:16883-8

[32] Farinas B, Mas P. Functional implication of the MYB transcrip-tion factor RVE8/LCL5 in the circadian control of histone acety-lation. Plant J. 2011; 66:318-29

[33] Sorkin ML, Tzeng SC, King S. et al. COLD REGULATED GENE 27 and 28 antagonize the transcriptional activity of the RVE8/LNK1/LNK2 circadian complex. Plant Physiol. 2023; 192: 2436-56

[34] Rawat R, Takahashi N, Hsu PY. et al. REVEILLE8 and PSEUDO-REPONSE REGULATOR5 form a negative feedback loop within the Arabidopsis circadian clock. PLoS Genet. 2011; 7:e1001350

[35] Hsu PY, Devisetty UK, Harmer SL. Accurate timekeeping is controlled by a cycling activator in Arabidopsis. eLife. 2013; 2: e00473

[36] Hughes CL, Harmer SL. Myb-like transcription factors have epistatic effects on circadian clock function but additive effects on plant growth. Plant Direct. 2023; 7:e533

[37] Hughes CL, An Y, Maloof JN. et al. Light quality-dependent roles of REVEILLE proteins in the circadian system. Plant Direct. 2024; 8:e573

[38] Han K, Zhao Y, Sun Y. et al. NACs, generalist in plant life. Plant Biotechnol J. 2023; 21:2433-57

[39] Ning Y-Q, Ma ZY, Huang HW. et al. Two novel NAC transcription factors regulate gene expression and flowering time by asso-ciating with the histone demethylase JMJ14. Nucleic Acids Res. 2015; 43:1469-84

[40] Xu B, Sathitsuksanoh N, Tang Y. et al. Overexpression of AtLOV1 in switchgrass alters plant architecture, lignin content, and flowering time. PLoS One. 2012; 7:e47399

[41] Shahnejat-Bushehri S, Tarkowska D, Sakuraba Y. et al. Arabidop-sis NAC transcription factor JUB1 regulates GA/BR metabolism and signalling. Nat Plants. 2016; 2:16013

[42] Chen X, Lu S, Wang Y. et al. OsNAC2 encoding a NAC tran-scription factor that affects plant height through mediating the gibberellic acid pathway in rice. Plant J. 2015; 82:302-14

[43] MaoC, LuS, LvB. et al. A rice NAC transcription factor promotes leaf senescence via ABA biosynthesis. Plant Physiol. 2017; 174: 1747-63

[44] Chong L, Xu R, Huang P. et al. The tomato OST1-VOZ1 mod-ule regulates drought-mediated flowering. Plant Cell. 2022; 34: 2001-18

[45] Huang X, Ni Z, Shi T. et al. Novel insights into the dissemination route of Japanese apricot (Prunus mume Sieb. et Zucc.) based on genomics. Plant J. 2022; 110:1182-97

[46] Zheng T, Li P, Zhuo X. et al. The chromosome-level genome provides insight into the molecular mechanism underlying the tortuous-branch phenotype of Prunus mume. New Phytol. 2022; 235:141-56

[47] Shi T, Bai Y, Wu X. et al. PmAGAMOUS recruits polycomb protein PmLHP1 to regulate single-pistil morphogenesis in Japanese apricot. Plant Physiol. 2023; 193:466-82

[48] Yao D, Ni X, Shi T. et al. Identification and tissue expression analysis of NAC gene family in Prunus mume. J Nucl Agric Sci. 2019; 33:226-39

[49] Sibéril Y, Doireau P, Gantet P. Plant bZIP G-box binding factors. Modular structure and activation mechanisms. Eur J Biochem. 2001; 268:5655-66

[50] Fu J, Wang L, Wang Y. et al. Photoperiodic control of FT-like gene ClFT initiates flowering in Chrysanthemum lavandulifolium. Plant Physiol Biochem. 2014; 74:230-8

[51] Harmer SL, Hogenesch JB, Straume M. et al. Orchestrated tran-scription of key pathways in Arabidopsis by the circadian clock. Science. 2000; 290:2110-3

[52] Alabadı´ D, Oyama T, Yanovsky MJ. et al. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 2001; 293:880-3

[53] Wang ZY, Kenigsbuch D, Sun L. et al. A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidop-sis Lhcb gene. Plant Cell. 1997; 9:491-507

[54] Carre IA. MYB transcription factors in the Arabidopsis circadian clock. JExp Bot. 2002; 53:1551-7

[55] Farré EM, Harmer SL, Harmon FG. et al. Overlapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circadian clock. Curr Biol. 2005; 15:47-54

[56] Hassidim M, Harir Y, Yakir E. et al. Over-expression of CONSTANS-LIKE 5 can induce flowering in short-day grown Arabidopsis. Planta. 2009; 230:481-91

[57] Steinbach Y. The Arabidopsis thaliana CONSTANS-LIKE 4 (COL4)-a modulator of flowering time. Front Plant Sci. 2019; 10:651

[58] Kim HJ, Park JH, Kim J. et al. Time-evolving genetic networks reveal a NAC troika that negatively regulates leaf senescence in Arabidopsis. Proc Natl Acad Sci. 2018; 115:4930-9

[59] Singh RK, Svystun T, AlDahmash B. et al. Photoperiod-and temperature-mediated control of phenology in trees - a molec-ular perspective. New Phytol. 2017; 213:511-24

[60] Meng J, Li Z, Wang H. et al. Haplotype-resolved genome assembly provides new insights into the genomic origin of purple colour in Prunus mume. Plant Biotechnol J. 2025; 23:1416-36

[61] Zdepski A, Wang W, Priest HD. et al. Conserved daily tran-scriptional programs in Carica papaya. Trop Plant Biol. 2008; 1: 236-45

[62] Esumi T, Hagihara C, Kitamura Y. et al. Identification of an FT ortholog in Japanese apricot (Prunus mume Sieb. et Zucc.). JHortic Sci Biotechnol. 2009; 84:149-54

[63] Esumi T, Kitamura Y, Hagihara C. et al. Identification of a TFL1 ortholog in Japanese apricot (Prunus mume Sieb. et Zucc.). Sci Hortic. 2010; 125:608-16

[64] Li Y, Xu Z, Yang W. et al. Isolation and functional characteri-zation of SOC1-like genes in Prunus mume. J Am Soc Hortic Sci. 2016; 141:315-26

[65] Ahmad S, Li Y, Yang Y. et al. Isolation, functional characteriza-tion and evolutionary study of LFY1 gene in Prunus mume. Plant Cell Tissue Organ Cult. 2019; 136:523-36

[66] Gao F, Segbo S, Huang X. et al. PmRGL2/PmFRL3-PmSVP module regulates flowering time in Japanese apricot (Prunus mume Sieb. et Zucc.). Plant Cell Environ. 2025; 48:3415-30

[67] Shan W, Kuang JF, Chen L. et al. Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening. JExp Bot. 2012; 63:5171-87

[68] Fang J, Chai Z, Yao W. et al. Interactions between ScNAC23 and ScGAI regulate GA-mediated flowering and senescence in sugarcane. Plant Sci. 2021; 304:110806

[69] Zhu Y, Narsai R, He C. et al. Coordinated regulation of the mitochondrial retrograde response by circadian clock regulators and ANAC017. Plant Commun. 2023; 4:100501

[70] Zhong R, Lee C, Ye Z-H. Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Mol Plant. 2010; 3:1087-103

[71] Lindemose S, Jensen MK, de Velde JV. et al. A DNA-binding-site landscape and regulatory network analysis for NAC transcrip-tion factors in Arabidopsis thaliana. Nucleic Acids Res. 2014; 42: 7681-93

[72] Ma M, Hao T, Ren X. et al. NAC family gene CmNAC34 positively regulates fruit ripening through interacting with CmNAC-NOR in Cucumis melo. J Integr Agric. 2024; 24:2601-18

[73] Wang J, Zhang H, Wang Y. et al. Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis. Plant Physiol. 2024; 195:1365-81

[74] Song S, Willems LAJ, Jiao A. et al. The membrane associated NAC transcription factors ANAC060 and ANAC040 are functionally redundant in the inhibition of seed dormancy in Arabidopsis thaliana. JExp Bot. 2022; 73:5514-28

[75] Wu A, Allu AD, Garapati P. et al. JUNGBRUNNEN1,a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant Cell. 2012; 24:482-506

[76] Sakuraba Y, Bülbül S, Piao W. et al. Arabidopsis EARLY FLOWERING 3 increases salt tolerance by suppressing salt stress response pathways. Plant J. 2017; 92:1106-20

[77] Shahnejat-Bushehri S, Allu AD, Mehterov N. et al. Arabidop-sis NAC transcription factor JUNGBRUNNEN1 exerts conserved control over gibberellin and brassinosteroid metabolism and signaling genes in tomato. Front Plant Sci. 2017; 8:214

[78] Si J, Xiao XM, Xu YM. et al. BrJUB1, a NAC family transcription factor, regulates postharvest leaf senescence of Chinese flower-ing cabbage through the transcriptional activation of BrCCGs. N Z J Crop Hortic Sci. 2021; 49:92-105

[79] Zhang Q, Chen W, Sun L. et al. The genome of Prunus mume. Nat Commun. 2012; 3:1318

[80] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12:357-60

[81] Jiang S, Ji Y, Wang M. et al. Establishing a high-efficiency in vitro regeneration system for Agrobacterium-mediated transfor-mation in Fragaria nilgerrensis. Fruit Res. 2023; 3:0

[82] Liu W, Zheng T, Qiu L. et al. A 49-bp deletion of PmAP2L results in a double flower phenotype in Prunus mume. Hortic Res. 2024;11:uhad278

[83] Kost B, Schnorf M, Potrykus I. et al. Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay. Plant J. 1995; 8:155-66