euAP2a, a key gene that regulates flowering time in peach (Prunus persica) by modulating thermo-responsive transcription programming

Abstract

Frequent spring frost damage threatens temperate fruit production, and breeding of late-flowering cultivars is an effective strategy for preventing such damage. However, this effort is often hampered by the lack of specific genes and markers and a lack of understanding of the mechanisms. We examined a Late-Flowering Peach (LFP) germplasm and found that its floral buds require a longer chilling period to release from their dormancy and a longer warming period to bloom than the control cultivar, two key characteristics associated with flowering time. We discovered that a 983-bp deletion in euAP2a, an APETALA2 (AP2)-related gene with known roles in regulating floral organ identity and flowering time, was primarily responsible for late flowering in LFP. This deletion disrupts an miR172 binding site, resulting in a gain-of-function mutation in euAP2a. Transcriptomic analyses revealed that at different stages of floral development, two chilling-responsive modules and four warm-responsive modules, comprising approximately 600 genes, were sequentially activated, forming a unique transcription programming. Furthermore, we found that euAP2a was transiently downregulated during the activation of these thermal-responsive modules at various stages. However, the loss of such transient, stage-specific downregulation of euAP2a caused by the deletion of miR172 binding sites resulted in the deactivation or delay of these modules in the LFP flower buds, suggesting that euAP2a acts as a transcription repressor to control floral developmental pace in peaches by modulating the thermo-responsive transcription programming. The findings shed light on the mechanisms behind late flowering in deciduous fruit trees, which is instrumental for breeding frost-tolerant cultivars.

Acknowledgements

This study was supported by USDA-ARS in-house funding under CRIS 8080-21000-029-00D and 8080-21000-033-00D.

Author Contributions

Z.L. and C.D. conceived and designed experiments. J.L., D.D., M.D., T.A. E.B. and Z.L. conducted experiments. J.L., C.D., E.B. and Z.L. performed data analyses and data interpretation. Z.L., wrote the manuscript, and J.L., C.D., and T.A. assisted the manuscript preparation.

Data availability

The raw and processed data from this study have been submitted to the NCBI BioProject and GEO databases, and accession number is GSE244570.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Liu J, Sherif SM. Combating spring frost with ethylene. Front Plant Sci. 2019; 10:1408

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

[3] Irish VF. The flowering of Arabidopsis flower development. Plant J. 2010; 61:1014-28

[4] 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

[5] Arora R, Rowland LJ, Tanino K. Induction and release of bud dormancy in woody perennials: a science comes of age. HortScience. 2003; 38:911-21

[6] Atkinson CJ, Brennan RM, Jones HG. Declining chilling and its impact on temperate perennial crops. Environ Exp Bot. 2013; 91:48-62

[7] Fan S, Bielenberg DG, Zhebentyayeva TN. et al. Mapping quantitative trait loci associated with chilling requirement, heat requirement and bloom date in peach (Prunus persica). New Phytol. 2010; 185:917-30

[8] Erez A, Couvillon A, Hendershott CH. Quantitative chilling enhancement and negation in peach buds by high temperatures in a daily cycle. J Am Soc Hortic Sci. 1979; 104:536-40

[9] Liu Z, Zhu H, Abbott A. Dormancy behaviors and underlying regulatory mechanisms:from perspective of pathways to epigenetic regulation. In: Anderson J,ed. Advances in Plant Dormancy. Spring Science, 2015,75-106

[10] Wang L, Zhang L, Ma C. et al. Impact of chilling accumulation and hydrogen cyanamide on floral organ development of sweet cherry in a warm region. J Integr Agric. 2016; 15:2529-38

[11] Julian C, Rodrigo J, Herrero M. Stamen development and winter dormancy in apricot (Prunus armeniaca). Ann Bot. 2011; 108:617-25

[12] Luna V, Lorenzo E, Reinoso H. et al. Dormancy in peach (Prunus persica L.) flower buds. I. Floral morphogenesis and endogenous gibberellins at the end of the dormancy period. Plant Physiol. 1990; 93:20-5

[13] Luna V, Reinoso H, Lorenzo E. et al. Dormancy in peach (Prunus persica L.) flower buds. II. Comparative morphology and phenology in floral and vegetative buds, and the effect of chilling and gibberellin A3. Trees. 1991; 5:244-6

[14] Luna V, Soriano MD, Bottini R. et al. Dormancy in peach (Prunus persica L.) flower buds. III. Levels of endogenous gibberellins, abscisic acid, indole-3- acetic acid, and naringenin during dormancy of peach flower buds. Acta Hortic. 1993; 329:264-7

[15] Reinoso H, Luna V, Daurfa C. et al. Dormancy in peach (Prunus persica) flower buds. VI. Effects of gibberellins and an acyl-cyclohexanedione (trinexapac-ethyl) on bud morphogenesis in field experiments with orchard trees and on cuttings. Can J Bot. 2002a; 80:664-74

[16] Reinoso H, Luna V, Pharis RP. et al. Dormancy in peach (Prunus persica) flower buds. V. Anatomy of bud development in relation to phenological stage. Can J Bot. 2002b; 80:656-63

[17] Wang SP, Yuan CJ, Dai YT. et al. Development of flower organs in sweet cherry in Shanghai area. Acta Hortic Sin. 2004; 31:357-9

[18] Yamane H, Tao R, Ooka T. et al. Comparative analyses of dormancy-associated MADS-box genes, PpDAM5 and PpDAM6, in low- and high-chill peaches (Prunus persica L.). J Jpn Soc Hortic Sci. 2011; 80:276-83

[19] Ghrab M, Ben Mimoun M, Masmoudi MM. et al. Chilling trends in a warm production area and their impact on flowering and fruiting of peach trees. Sci Hortic. 2014; 178:87-94

[20] Pawasut A, Fujishige N, Yamane K. et al. Relationships between chilling and heat requirement for flowering in ornamental peaches. J Jpn Soc Hortic Sci. 2004; 73:519-23

[21] Ruíz D, Campoy JA, Egea J. Chilling and heat requirements of apricot cultivars for flowering. Environ Exp Bot. 2007; 61:254-63

[22] Ruíz D, Egea J, Salazar JA. et al. Chilling and heat requirements of Japanese plum cultivars for flowering. Sci Hortic. 2018; 242:164-9

[23] Alvarez-Buylla ER, Benítez M, Corvera-Poiré A. et al. Flower development. Arabidopsis Book. 2010; 8:e0127

[24] Anderson JL, Richardson EA, Kesner CD. Validation of chill unit and flower bud phenology models for ‘Montmorency’ sour cherry. Acta Hortic. 1986; 184:71-8

[25] Atagul O, Calle A, Demirel G. et al. Estimating heat requirement for flowering in peach germplasm. Agronomy. 2022; 12:1002

[26] Luedeling E, Zhang M, Girvetz EH.Climatic changes lead to declining winter chill for fruit and nut trees in California during1950-2099. PLoS One. 2009; 4:e6166

[27] Alonso JM, Ansón JM, Espiau MT. et al. Stability of the almond blooming date in a changing climate. Acta Hortic. 2011; 912:337-42

[28] Citadin I, Raseira MCB, Herter FG. et al. Heat requirement for blooming and leafing in peach. HortScience. 2001; 36:305-7

[29] Scorza R, Okie WR. Peaches (Prunus). In: Moore JM, Ballington JR,eds. Genetic Resources of Temperate Fruit and Nut Crops. Vol. 290. 1st ed. Acta Horticulturae: Leuven, Belgium, 1991,177-234

[30] Jenkitkonchai J, Marriott P, Yang W. et al. Exploring PIF4’s contribution to early flowering in plants under daily variable temperature and its tissue-specific flowering gene network. Plant Direct. 2021; 5:e339

[31] Kumar SV, Lucyshyn D, Jaeger KE. et al. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature. 2012; 484:242-5

[32] Xu Y, Zhu Z. PIF4 and PIF4-Interacting proteins: at the nexus of plant light, temperature and hormone signal integrations. Int J Mol Sci. 2021; 22:10304

[33] Box MS, Huang BE, Domijan M. et al. ELF3 controls thermoresponsive growth in Arabidopsis. Curr Biol. 2015; 25:194-9

[34] Mizuno T, Nomoto Y, Oka H. et al. Ambient temperature signal feeds into the circadian clock transcriptional circuitry through the EC night-time repressor in Arabidopsis thaliana. Plant Cell Physiol. 2014; 55:958-76

[35] Silva CS, Nayak A, Lai X. et al. Molecular mechanisms of evening complex activity in Arabidopsis. Proc Natl Acad Sci USA. 2020; 117:6901-9

[36] Jung JH, Domijan M, Klose C. et al. Phytochromes function as thermosensors in Arabidopsis. Science. 2016; 354:886-9

[37] Zhu H, Chen P-Y, Zhong S. et al. Thermal-responsive genetic and epigenetic regulation of DAM cluster controlling dormancy and chilling requirement in peach floral buds. Hortic Res. 2020; 7:114

[38] Cutler S, Ghassemian M, Bonetta D. et al. SA protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science. 1996; 273:1239-41

[39] Running MP, Lavy M, Sternberg H. et al. Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases. Proc Natl Acad Sci USA. 2004; 101:7815-20

[40] Gattolin S, Cirilli M, Pacheco I. et al. Deletion of the miR172 target site in a TOE-type gene is a strong candidate variant for dominant double-flower trait in Rosaceae. Plant J. 2018; 96:358-71

[41] Cirilli M, Rossini L, Chiozzotto R. et al. Less is more: natural variation disrupting a miR172 gene at the di locus underlies the recessive double-flower trait in peach (P. persica L. Batsch). BMC Plant Biol. 2022; 22:318

[42] Zhu H, Xia R, Zhao B. et al. ( 2012). Unique expression, processing regulation, and regulatory network of peach (Prunus persica) miRNAs. BMC Plant Biol, 12.

[43] Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9:559

[44] Maere S, Heymans K, Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005; 21:3448-9

[45] Alvarez JM, Schinke AL, Brooks MD. et al. Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade. Nat Commun. 2020; 11:1157

[46] Swift J, Coruzzi GM. A matter of time - how transient transcription factor interactions create dynamic gene regulatory networks. Biochim Biophys Acta Gene Regul Mech. 2017; 1860:75-83

[47] Wollmann H, Mica E, Todesco M. et al. On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development. Development. 2010; 137:3633-42

[48] Mlotshwa S, Yang Z, Kim Y. et al. Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana. Plant Mol Biol. 2006; 61:781-93

[49] Yant L, Mathieu J, Dinh TT. et al. Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2. Plant Cell. 2010; 22:2156-70

[50] Castillejo C, Pelaz S. The balance between CONSTANS and TEMPRANILLO activities determines FT expression to trigger flowering. Curr Biol. 2008; 18:1338-43

[51] Aukerman MJ, Sakai H. Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell. 2003; 15:2730-41

[52] Kim S, Soltis PS, Wall K. et al. Phylogeny and domain evolution in the APETALA2-like gene family. Mol Biol Evol. 2006; 23:107-20

[53] Park W, Li J, Song R. et al. CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol. 2002; 12:1484-95

[54] Jung JH, Seo YH, Seo PJ. et al. The GIGANTEA-regulated microRNA172 mediates photoperiodic flowering independent of CONSTANS in Arabidopsis. Plant Cell. 2007; 19:2736-48

[55] Schmid M, Uhlenhaut NH, Godard F. et al. Dissection of floral induction pathways using global expression analysis. Development. 2003; 130:6001-12

[56] Chen X. (2004). A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development. Science, 303, 2022-2025.

[57] Mathieu J, Yant LJ, Mürdter F. et al. ( 2009). Repression of Flowering by the miR172 target SMZ. PLoS Biol, 7, e1000148.

[58] Cirilli M, Micali S, Aranzana MJ. et al. The multisite PeachRefPop collection: a true cultural heritage and international scientific tool for fruit trees. Plant Physiol. 2020; 184:632-46

[59] Song YH, Niu L, Liu S. et al. Inheritance tendency of several characters of ornamental peach. Acta Agric Sin. 2010; 25:78-83

[60] Dobin A., Davis CA, Schlesinger F. et al. ( 2012). STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29, 15-21.

[61] Robinson MD, McCarthy DJ, Smyth GK. et al. ( 2009). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26, 139-40.

[62] Shannon P, Markiel A, Ozier O. et al. ( 2023). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 13, 2498-2504.

[63] Wang Y, Georgi LL, Reighard GL. et al. Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch]. J Hered. 2002; 93:352-8