Regulatory role of Prunus mume DAM6 on lipid body accumulation and phytohormone metabolism in the dormant vegetative meristem

Abstract

Bud dormancy is a crucial process in the annual growth cycle of woody perennials. In Rosaceae fruit tree species, DORMANCY-ASSOCIATED MADS-box (DAM) transcription factor genes regulating bud dormancy have been identified, but their molecular roles in meristematic tissues have not been thoroughly characterized. In this study, molecular and physiological analyses of transgenic apple plants overexpressing the Japanese apricot DAM6 gene (PmDAM6) and Japanese apricot cultivars and F₁ individuals with contrasting dormancy characteristics revealed the metabolic pathways controlled by PmDAM6. Our transcriptome analysis and transmission electron microscopy examination demonstrated that PmDAM6 promotes the accumulation of lipid bodies and inhibits cell division in the dormant vegetative meristem by down-regulating the expression of lipid catabolism genes (GDSL ESTERASE/LIPASE and OIL BODY LIPASE) and CYCLIN genes, respectively. Our findings also indicate PmDAM6 promotes abscisic acid (ABA) accumulation and decreases cytokinin (CTK) accumulation in vegetative buds by up-regulating the expression of the ABA biosynthesis gene ARABIDOPSIS ALDEHYDE OXIDASE and the CTK catabolism gene CYTOKININ DEHYDROGENASE, while also down-regulating the expression of the CTK biosynthesis genes ISOPENTENYL TRANSFERASE (IPT) and CYP735A. Additionally, PmDAM6 modulates gibberellin (GA) metabolism by up-regulating GA2-OXIDASE expression and down-regulating GA3-OXIDASE expression. Furthermore, PmDAM6 may also indirectly promote lipid accumulation and restrict cell division by limiting the accumulation of CTK and GA in buds. In conclusion, using our valuable genetic platform, we clarified how PmDAM6 modifies diverse cellular processes, including lipid catabolism, phytohormone (ABA, CTK, and GA) biosynthesis and catabolism, and cell division, in the dormant vegetative meristem.

Acknowledgements

This research was supported by the Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science, Japan (Grant-in-Aid KAKENHI Nos. 18H02198 and 21H02186) to HY. The authors thank Keiko Okamoto-Furuta and Haruyasu Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University) for technical assistance in electron microscopy. We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Author Contributions

H.Y. participated in the conceptualization. T.F.H. and M.G.T. participated in the analysis and investigation. H.Y. and R.T. participated in the resources. T.F.H. and H.Y. participated in the writing—original draft. T.F.H. participated in the visualization. R.T. participated in the writing—review and editing.

Data availability

All relevant data are provided in the manuscript and its supporting materials. The RNA-seq data for the apple transformants and Japanese apricot generated in this study were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) (accession numbers PRJNA960952 and PRJNA958110).

Conflict of interest statement

The authors have no conflicts of interest to declare.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] 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

[2] Perry TO. Dormancy of trees in winter. Science. 1971; 171:29-36

[3] Lang GA, Early JD, Martin GC. et al. Endo-, para-, and ecodormancy: physiological terminology and classification for dormancy research. HortScience. 1987; 22:371-7

[4] Faust M, Erez A, Rowland LJ. et al. Bud dormancy in perennial fruit trees: physiological basis for dormancy induction, maintenance, and release. HortScience. 1997; 32:623-9

[5] Horvath DP, Anderson JV, Chao WS. et al. Knowing when to grow: signals regulating bud dormancy. Trends Plant Sci. 2003; 8:534-40

[6] Falavigna V d S, Guitton B, Costes E. et al. I want to (bud) break free: the potential role of DAM and SVP-like genes in regulating dormancy cycle in temperate fruit trees. Front Plant Sci. 2019; 9:1-17

[7] Hsiang T-F, Chen W, Yamane H. The MADS-box gene family involved in the regulatory mechanism of dormancy and flowering in Rosaceae fruit trees. Ann Plant Rev. 2021; 4:649-86

[8] Yang Q, Gao Y, Wu X. et al. Bud endodormancy in deciduous fruit trees: advances and prospects. Hortic Res. 2021; 8:139

[9] Goralogia GS, Howe GT, Brunner AM. et al. Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees. Hortic Res. 2021; 8:167

[10] Rodriguez AJ, Sherman WB, Scorza R. et al. ‘Evergreen’ peach, its inheritance and dormant behavior. J Am Soc Hortic Sci. 1994; 119:789-92

[11] Bielenberg DG, Wang Y, Li Z. et al. Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation. Tree Genet Genomes. 2008; 4:495-507

[12] Jiménez S, Reighard GL, Bielenberg DG. Gene expression of DAM5 and DAM6 is suppressed by chilling temperatures and inversely correlated with bud break rate. Plant Mol Biol. 2010; 73:157-67

[13] Wu R, Cooney J, Tomes S. et al. RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees. Tree Physiol. 2021; 41:1510-23

[14] Lloret A, Quesada-Traver C, Conejero A. et al. Regulatory circuits involving bud dormancy factor PpeDAM6. Hortic Res. 2021; 8:261

[15] Tuan PA, Bai S, Saito T. et al. Dormancy-associated MADS-box (DAM) and the abscisic acid pathway regulate pear endodormancy through a feedback mechanism. Plant Cell Physiol. 2017; 58:1378-90

[16] Falavigna V d S, Severing E, Lai X. et al. Unraveling the role of MADS transcription factor complexes in apple tree dormancy. New Phytol. 2021; 232:2071-88

[17] Sasaki R, Yamane H, Ooka T. et al. Functional and expressional analyses of PmDAM genes associated with endodormancy in Japanese apricot. Plant Physiol. 2011; 157:485-97

[18] Yamane H, Wada M, Honda C. et al. Overexpression of Prunus DAM6 inhibits growth, represses bud break competency of dormant buds and delays bud outgrowth in apple plants. PLoS One. 2019; 14:1-24

[19] Weinberger JH. Chilling requirements of peach varieties. Proc Am Soc Hortic Sci. 1950; 56:122-8

[20] Veerabagu M, Paul LK, Rinne PLH. et al. Plant lipid bodies traffic on actin to plasmodesmata motorized by myosin XIs. Int J Mol Sci. 2020; 21:7-9

[21] Chen M, Du X, Zhu Y. et al. Seed fatty acid reducer acts downstream of gibberellin signalling pathway to lower seed fatty acid storage in Arabidopsis. Plant Cell Environ. 2012; 35:2155-69

[22] Müller AO, Ischebeck T. Characterization of the enzymatic activity and physiological function of the lipid droplet-associated triacylglycerol lipase AtOBL1. New Phytol. 2018; 217:1062-76

[23] Pyc M, Gidda SK, Seay D. et al. LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis. Plant Cell. 2021; 33:3076-103

[24] Gidda SK, Park S, Pyc M. et al. Lipid droplet-associated proteins (LDAPs) are required for the dynamic regulation of neutral lipid compartmentation in plant cells. Plant Physiol. 2016; 170:2052-71

[25] Shao Q, Liu X, Su T. et al. New insights into the role of seed oil body proteins in metabolism and plant development. Front Plant Sci. 2019; 10:1568

[26] Takeuchi T, Matsushita MC, Nishiyama S. et al. RNA-sequencing analysis identifies genes associated with chilling-mediated endodormancy release in apple. J Am Soc Hortic Sci. 2018; 143:194-206

[27] Tylewicz S, Petterle A, Marttila S. et al. Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication. Science. 2018; 360:212-5

[28] Wu R, Wang T, Richardson AC. et al. Histone modification and activation by SOC1-like and drought stress-related transcription factors may regulate AcSVP2 expression during kiwifruit winter dormancy. Plant Sci. 2019; 281:242-50

[29] Takei K, Yamaya T, Sakakibara H. Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydroxylases that catalyse the biosynthesis of trans-Zeatin. J Biol Chem. 2004; 279:41866-72

[30] Redig P, Shaul O, Inzé D. et al. Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells. FEBS Lett. 1996; 391:175-80

[31] Lv L, Huo X, Wen L. et al. Isolation and role of PmRGL2 in GA-mediated floral bud dormancy release in Japanese apricot (Prunus mume Siebold et Zucc.). Front Plant Sci. 2018; 9:1-13

[32] Zheng C, Acheampong AK, Shi Z. et al. Abscisic acid catabolism enhances dormancy release of grapevine buds. Plant Cell Environ. 2018; 41:2490-503

[33] Xu H, Cao D, Chen Y. et al. Gene expression and proteomic analysis of shoot apical meristem transition from dormancy to activation in Cunninghamia lanceolata (lamb.) hook. Sci Rep. 2016; 6:1-12

[34] van der Schoot C, Paul LK, Paul SB. et al. Plant lipid bodies and cell-cell signaling a new role for an old organelle? Plant Signal Behav. 2011; 6:1732-8

[35] Saito T, Wang S, Ohkawa K. et al. Lipid droplet-associated gene expression and chromatin remodelling in LIPASE 5′-upstream region from beginning- to mid-endodormant bud in ‘Fuji’ apple. Plant Mol Biol. 2017; 95:441-9

[36] Kuroda H, Sagisaka S. Ultrastructural changes in cortical cells of apple (Malus pumila mill.) associated with cold hardiness. Plant Cell Physiol. 1993; 34:357-65

[37] Lv J, Dai CB, Wang WF. et al. Genome-wide identification of the tobacco GDSL family and apical meristem-specific expression conferred by the GDSL promoter. BMC Plant Biol. 2021; 21:1-16

[38] Ma R, Yuan H, An J. et al. A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis. PLoS One. 2018; 13:1-18

[39] Ding LN, Guo XJ, Li M. et al. Improving seed germination and oil contents by regulating the GDSL transcriptional level in Brassica napus. Plant Cell Rep. 2019; 38:243-53

[40] Huang LM, Lai CP, Chen LFO. et al. Arabidopsis SFAR4 is a novel GDSL-type esterase involved in fatty acid degradation and glucose tolerance. Bot Stud. 2015; 56:1-12

[41] Seo M, Aoki H, Koiwai H. et al. Comparative studies on the Arabidopsis aldehyde oxidase (AAO) gene family revealed a major role of AAO 3 in ABA biosynthesis in seeds. Plant Cell Physiol. 2004; 45:1694-703

[42] Aksenova NP, Sergeeva LI, Konstantinova TN. et al. Regulation of potato tuber dormancy and sprouting. Russ J Plant Physiol. 2013; 60:301-12

[43] Fabian T, Lorbiecke R, Umeda M. et al. The cell cycle genes cycA1;1 and cdc2Os-3 are coordinately regulated by gibberellin in planta. Planta. 2000; 211:376-83

[44] Achard P, Gusti A, Cheminant S. et al. Gibberellin signaling controls cell proliferation rate in Arabidopsis. Curr Biol. 2009; 19:1188-93

[45] Riou-Khamlichi C, Huntley R, Jacqmard A. et al. Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science. 1999; 283:1541-4

[46] Wang H, Zhou Y, Bird DA. et al. Functions, regulation and cellular localization of plant cyclin-dependent kinase inhibitors. J Microsc. 2008; 231:234-46

[47] Hartmann A, Senning M, Hedden P. et al. Reactivation of meristem activity and sprout growth in potato tubers require both cytokinin and gibberellin. Plant Physiol. 2011; 155:776-96

[48] Hsiang TF, Lin YJ, Yamane H. et al. Characterization of Japanese apricot (Prunus mume) floral bud development using a modified BBCH scale and analysis of the relationship between BBCH stages and floral primordium development and the dormancy phase transition. Horticulturae. 2021; 7:142

[49] Japelaghi RH, Haddad R, Garoosi GA. Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Mol Biotechnol. 2011; 49:129-37

[50] Yamane H, Kashiwa Y, Ooka T. et al. Suppression subtractive hybridization and differential screening reveals endodormancy-associated expression of an SVP/AGL24-type MADS-box gene in lateral vegetative buds of Japanese apricot. J Am Soc Hortic Sci. 2008; 133:708-16

[51] Chen S, Zhou Y, Chen Y. et al. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34:i884-90

[52] Daccord N, Celton JM, Linsmith G. et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet. 2017; 49:1099-106

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

[54] Dobin A, Davis CA, Schlesinger F. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29:15-21

[55] Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014; 30:923-30

[56] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019; 20:1-14

[57] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:1-21

[58] Yu G, Wang LG, Han Y. et al. ClusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16:284-7

[59] Velasco R, Zharkikh A, Affourtit J. et al. The genome of the domesticated apple (Malus × domestica Borkh.). Nat Genet. 2010; 42:833-9

[60] Kuroda H, Saonosuke S. Ultrastructural changes in apical meristem cells of apple flower buds associated with dormancy and cold tolerance. Chem Pharm Bull. 2002; 70:553-60