Two aquaporins, LcPIP1;4 and LcPIP1;4a, cooperatively regulate the onset of dormancy of the terminal buds in evergreen perennial litchi (Litchi chinensis Sonn.)

Abstract

Although extensively studied in various plants, the roles of aquaporin proteins in litchi remain unclear. In this study, low moisture content was observed in the dormant terminal buds of litchi. Transcriptome analysis revealed that two aquaporin genes, PLASMA MEMBRANE INTRINSIC PROTEIN 1;4 (LcPIP1;4) and LcPIP1;5, could be remarkably inhibited by exogenous ethylene (ETH), which also reduced the moisture content of litchi buds. Quantitative real-time polymerase chain reaction assays indicated that LcPIP1;4 expression was relatively elevated in the dormancy stage of litchi terminal buds. Inhibition of LcPIP1;4 in the buds of litchi during the growth stage delayed the onset of dormancy, resulting in a significantly reduced dormancy rate and increased moisture content. Further study indicated that LcPIP1;4 interacts with LcPIP1;4a, and they are capable of self-interaction. Silencing of LcPIP1;4a in litchi buds resulted in a phenotype consistent with silencing of LcPIP1;4. Additionally, simultaneous silencing of both LcPIP1;4 and LcPIP1;4a resulted in a more severe bud dormancy phenotype. Moreover, LcPIP1;4 was directly upregulated by LcRAP2.4. Silencing of LcRAP2.4 also delayed the onset of dormancy in litchi terminal buds, which is regulated by LcSVP2. ETH treatment at 1000 mg/l significantly downregulated the expression of LcPIP1;4 and LcRAP2.4, but had no significant effect on LcPIP1;4a. In contrast, abscisic acid (ABA) treatment at 200 mg/l significantly upregulated the expression of LcPIP1;4, LcPIP1;4a, and LcRAP2.4. Combined treatment with ETH and ABA exerted a stronger inhibitory effect on the bud break and upregulated LcPIP1;4 and LcRAP2.4 to lower degrees than ABA alone, suggesting that ABA reversed the inhibitory effect of ETH on the expression of LcPIP1;4 and LcRAP2.4. ABA treatment and combined treatment with ETH and ABA effectively reduced the moisture content of the terminal buds. These results demonstrate that LcRAP2.4, LcPIP1;4, and LcPIP1;4a play a vital role in dormancy onset of litchi terminal buds by regulating moisture levels.

Acknowledgements

This study was supported by the China Litchi and Longan Industry Technology Research System (project no. CARS-32), and 2023 Special Project for Key Areas of Research and Development of Guangzhou Municipality (2023B01J2002), Guangzhou Basic and Applied Basic Research Foundation (No. 2024A04J4919), Open project of Key Laboratory of Genetic Resources Evaluation and Utilization of Tropical Fruits and Vegetables (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs (ITFT2024PT0203), and Construction Plan of Guangdong Province High-level Universities and the Research Start-up Funds for the High-level Talent Introduction Project of South China Agricultural University (5300-223109).

Author contributions

The authors confirm contribution to the paper as follows. Ren-Fang Zeng and Xu-Ming Huang contribute ideas, experimental design, and data analysis. Xue Tian, Zhi-Qun Zhong, Yu Qi, Meng-Meng Ma, Fang-Yi Zhang, and Dong-Cheng Li carried out field experiment and lab analyses and provided raw data and data processing. Ming-Chao Yang and Hui-Cong Wang provided ideas and methods. Ren-Fang Zeng drafted the manuscript, and Xu-Ming Huang provided major revisions on the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Data availability

All the data used in this research can be found within the main text or the supplementary materials, and interested parties can request the raw data from the corresponding author with a reasonable justification.

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] Lang G, Early J, Darnell R. et al. Endo-, Para-, and eco-dormancy: physiological terminology and classification for dor-mancy research. HortScience. 1987; 22:371-7

[2] Rohde A, Bhalerao RP. Plant dormancy in the perennial context. Trends Plant Sci. 2007; 12:217-23

[3] Ding J, Böhlenius H, Rühl MG. et al. GIGANTEA-like genes control seasonal growth cessation in Populus. New Phytol. 2018; 218: 1491-503

[4] Ma M-M, Zhang H-F, Tian Q. et al. MIKC type MADS-box tran-scription factor LcSVP2 is involved in dormancy regulation of the terminal buds in evergreen perennial litchi (Litchi chinensis Sonn.). Hortic Res. 2024;11:uhae150

[5] Núñez-Elisea R, Davenport TL. Effect of leaf age, duration of cool temperature treatment, and photoperiod on bud dormancy release and floral initiation in mango. Sci Hortic. 1995; 62:63-73

[6] Zeng RF, Zhou H, Fu LM. et al. Two citrus KNAT-like genes, CsKN1 and CsKN2, are involved in the regulation of spring shoot development in sweet orange. JExp Bot. 2021; 72:7002-19

[7] Zhang HF, Li H, Lai B. et al. Morphological characterization and gene expression profiling during bud development in a tropical perennial, Litchi chinensis Sonn. Front Plant Sci. 2016; 7:01517

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

[9] Böhlenius H, Huang T, Charbonnel-Campaa L. et al. CO/FT regu-latory module controls timing of flowering and seasonal growth cessation in trees. Science. 2006; 312:1040-3

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

[11] Yang Q, Yang B, Li J. et al. ABA-responsive ABRE-BINDING FAC-TOR3 activates DAM3 expression to promote bud dormancy in Asian pear. Plant Cell Environ. 2020; 43:1360-75

[12] Liu J, Sherif SM. Hormonal orchestration of bud dormancy cycle in deciduous Woody perennials. Front Plant Sci. 2019; 10:1136

[13] Pan W, Liang J, Sui J. et al. ABA and bud dormancy in peren-nials: current knowledge and future perspective. Genes (Basel). 2021; 12:1635

[14] Wang X, Wei J, Wu J. et al. Transcription factors BZR2/MYC2 modulate brassinosteroid and jasmonic acid crosstalk during pear dormancy. Plant Physiol. 2024; 194:1794-814

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

[16] Zhong W, Gao Z, Zhuang W. et al. Genome-wide expression profiles of seasonal bud dormancy at four critical stages in Japanese apricot. Plant Mol Biol. 2013; 83:247-64

[17] Götz K-P, Chmielewski F-M, Homann T. et al. Seasonal changes of physiological parameters in sweet cherry (Prunus avium L.) buds. Sci Hortic. 2014; 172:183-90

[18] Kaufmann H, Blanke M. Changes in carbohydrate levels and relative water content (RWC) to distinguish dormancy phases in sweet cherry. J Plant Physiol. 2017; 218:1-5

[19] Yooyongwech S, Sugaya S, Sekozawa Y. et al. Differential adapta-tion of high-and low-chill dormant peaches in winter through aquaporin gene expression and soluble sugar content. Plant Cell Rep. 2009; 28:1709-15

[20] Walde MG, Wenden B, Chuine I. et al. Stable water isotopes reveal the onset of bud dormancy in temperate trees, whereas water content is a better proxy for dormancy release. Tree Physiol. 2024;44:tpae028

[21] Li S, Wang L, Zhang Y. et al. Genome-wide identification and function of aquaporin genes during dormancy and sprouting periods of kernel-using apricot (Prunus armeniaca L.). Front Plant Sci. 2021; 12:690040

[22] Wang Y, Zhao Z, Liu F. et al. Versatile roles of aquaporins in plant growth and development. Int J Mol Sci. 2020; 21:9485

[23] Yooyongwech S, Horigane AK, Yoshida M. et al. Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy. Physiol Plant. 2008; 134:522-33

[24] Danielson JA, Johanson U. Unexpected complexity of the aqua-porin gene family in the moss Physcomitrella patens. BMC Plant Biol. 2008; 8:45

[25] Chen W, Yin X, Wang L. et al. Involvement of rose aquaporin RhPIP1;1 in ethylene-regulated petal expansion through inter-action with RhPIP2;1. Plant Mol Biol. 2013; 83:219-33

[26] Ma N, Xue J, Li Y. et al. Rh-PIP2;1, a rose aquaporin gene, is involved in ethylene-regulated petal expansion. Plant Physiol. 2008; 148:894-907

[27] Aharon R, Shahak Y, Wininger S. et al. Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress. Plant Cell. 2003; 15:439-47

[28] Diehn TA, Pommerrenig B, Bernhardt N. et al. Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to brassica crops and Arabidopsis. Front Plant Sci. 2015; 6:166

[29] Jaillon O, Aury JM, Noel B. et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007; 449:463-7

[30] Yaghobi M, Heidari P. Genome-wide analysis of aquaporin gene family in Triticum turgidum and its expression profile in response to salt stress. Genes. 2023; 14:202

[31] Park W, Scheffler BE, Bauer PJ. et al. Identification of the family of aquaporin genes and their expression in upland cotton (Gossyp-ium hirsutum L.). BMC Plant Biol. 2010; 10:142

[32] Kong W, Yang S, Wang Y. et al. Genome-wide identification and characterization of aquaporin gene family in Beta vulgaris. PeerJ. 2017; 5:e3747

[33] Reuscher S, Akiyama M, Mori C. et al. Genome-wide identifica-tion and expression analysis of aquaporins in tomato. PLoS One. 2013; 8:e79052

[34] WenB, DengC, TianQ. et al. Application of ethephon manually or via drone enforces bud dormancy and enhances flowering response to chilling in litchi (Litchi chinensis Sonn.). Horticulturae. 2024; 10:1109

[35] Bienert MD, Diehn TA, Richet N. et al. Heterotetramerization of plant PIP1 and PIP2 Aquaporins is an evolutionary ancient fea-ture to guide PIP1 plasma membrane localization and function. Front Plant Sci. 2018; 9:382

[36] Fetter K, Van Wilder V, Moshelion M. et al. Interactions between plasma membrane aquaporins modulate their water channel activity. Plant Cell. 2004; 16:215-28

[37] Shibasaka M, Horie T, Katsuhara M. Mechanisms activating latent functions of PIP aquaporin water channels via the inter-action between PIP1 and PIP2 proteins. Plant Cell Physiol. 2021; 62: 92-9

[38] Guo H, Ecker JR. The ethylene signaling pathway: new insights. Curr Opin Plant Biol. 2004; 7:40-9

[39] Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol. 2000; 3:217-23

[40] Cronje RB, Hajari E, Jonker A. et al. Foliar application of ethep-hon induces bud dormancy and affects gene expression of dormancy-and flowering-related genes in ’Mauritius’ litchi (Litchi chinensis Sonn.). J Plant Physiol. 2022; 276:153768

[41] Yu XS, Wang HR, Lei FF. et al. Structure and functional divergence of PIP peptide family revealed by functional studies on PIP1 and PIP2 in Arabidopsis thaliana. Front Plant Sci. 2023; 14:1208549

[42] Hussain S, Wang W, Ahmed S. et al. PIP2, an auxin induced plant peptide hormone regulates root and hypocotyl elongation in Arabidopsis. Front Plant Sci. 2021; 12:646736

[43] Tian S, Wang X, Li P. et al. Plant aquaporin AtPIP1;4 links apoplas-tic H2O2 induction to disease immunity pathways. Plant Physiol. 2016; 171:1635-50

[44] Yao X, Mu Y, Zhang L. et al. AtPIP1;4 and AtPIP2; 4 cooperatively mediate H(2)O(2) transport to regulate plant growth and disease resistance. Plan Theory. 2024; 13:1018

[45] Alexandersson E, Danielson JA, Råde J. et al. Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress. Plant J. 2010; 61:650-60

[46] Jang JY, Kim DG, Kim YO. et al. An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Mol Biol. 2004; 54: 713-25

[47] Coulon D, Faure L, Grison M. et al. LPIAT, a lyso-phosphatidylinositol acyltransferase, modulates seed germination in Arabidopsis thaliana through PIP signalling pathways and is involved in hyperosmotic response. Int J Mol Sci. 2020; 21:1654

[48] Footitt S, Clewes R, Feeney M. et al. Aquaporins influence seed dormancy and germination in response to stress. Plant Cell Envi-ron. 2019; 42:2325-39

[49] Saito T, Tuan PA, Katsumi-Horigane A. et al. Development of flower buds in the Japanese pear (Pyrus pyrifolia) from late autumn to early spring. Tree Physiol. 2015; 35: 653-62

[50] Yue C, Cao H, Wang L. et al. Molecular cloning and expression analysis of tea plant aquaporin (AQP) gene family. Plant Physiol Biochem. 2014; 83:65-76

[51] Boursiac Y, Chen S, Luu D-T. et al. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. Plant Physiol. 2005; 139:790-805

[52] Li L, Wang H, Gago J. et al. Harpin Hpa1 interacts with aquaporin PIP1;4 to promote the substrate transport and photosynthesis in Arabidopsis. Sci Rep. 2015; 5:17207

[53] Feng K, Hou XL, Xing GM. et al. Advances in AP2/ERF super-family transcription factors in plant. Crit Rev Biotechnol. 2020; 40:750-76

[54] Iwase A, Mitsuda N, Koyama T. et al. The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis. Curr Biol. 2011; 21:508-14

[55] Lin RC, Park HJ, Wang HY. Role of Arabidopsis RAP2.4 in regulat-ing light-and ethylene-mediated developmental processes and drought stress tolerance. Mol Plant. 2008; 1:42-57

[56] Iwase A, Harashima H, Ikeuchi M. et al. WIND1 promotes shoot regeneration through transcriptional activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis. Plant Cell. 2016; 29: 54-69

[57] Li Z, Sheerin DJ, von Roepenack-Lahaye E. et al. The phytochrome interacting proteins ERF55 and ERF58 repress light-induced seed germination in Arabidopsis thaliana. Nat Commun. 2022; 13:1656

[58] Anh Tuan P, Bai S, Saito T. et al. Involvement of EARLY BUD-BREAK, an AP2/ERF transcription factor gene, in bud break in Japanese pear (Pyrus pyrifolia Nakai) lateral flower buds: expres-sion, histone modifications and possible target genes. Plant Cell Physiol. 2016; 57:1038-47

[59] Azeez A, Zhao YC, Singh RK. et al. EARLY BUD-BREAK 1 and EARLY BUD-BREAK 3 control resumption of poplar growth after winter dormancy. Nat Commun. 2021; 12:1123

[60] Yordanov YS, Ma C, Strauss SH. et al. EARLY BUD-BREAK 1 (EBB1) is a regulator of release from seasonal dormancy in poplar trees. Proc Natl Acad Sci U S A. 2014; 111:10001-6

[61] Busov V, Carneros E, Yakovlev I. EARLY BUD-BREAK1 (EBB1) defines a conserved mechanism for control of BUD-BREAK in woody perennials. Plant Signal Behav. 2016; 11:e1073873

[62] Vélez ML, Costamagna E, Kimura ET. et al. Bacterial lipopolysac-charide stimulates the thyrotropin-dependent thyroglobulin gene expression at the transcriptional level by involving the transcription factors thyroid transcription factor-1 and paired box domain transcription factor 8. Endocrinology. 2006; 147: 3260-75

[63] Liu M, Wang C, Ji Z. et al. Regulation of drought tolerance in Ara-bidopsis involves the PLATZ4-mediated transcriptional repres-sion of plasma membrane aquaporin PIP2;8. Plant J. 2023; 115: 434-51

[64] Ding L, Uehlein N, Kaldenhoff R. et al. Aquaporin PIP2;1 affects water transport and root growth in rice (Oryza sativa L.). Plant Physiol Biochem. 2019; 139:152-60

[65] Li DD, Ruan XM, Zhang J. et al. Cotton plasma membrane intrin-sic protein 2s (PIP2s) selectively interact to regulate their water channel activities and are required for fibre development. New Phytol. 2013; 199:695-707

[66] Lian HL, Yu X, Lane D. et al. Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment. Cell Res. 2006; 16:651-60

[67] Liu S, Fukumoto T, Gena P. et al. Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake. Plant J. 2020; 102:779-96

[68] Vasiljevic T, Toebes A, Huppertz T. Moisture sorption by dairy powders studied by low-field NMR. Int Dairy J. 2021; 119: 105062

[69] Chen T, Zhang W, Liu Y. et al. Water status and predictive models of moisture content during drying of soybean dregs based on LF-NMR. Molecules. 2022; 27:4421

[70] Ezeanaka MC, Nsor-Atindana J, Zhang M. Online low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) for food quality optimization in food processing. Food Bioproc Tech. 2019; 12:1435-51

[71] Sun Y, Zhang M, Mujumdar AS. et al. Pulse-spouted microwave freeze drying of raspberry: control of moisture using ANN model aided by LF-NMR. J Food Eng. 2021; 292:110354

[72] Mao J, Li J, Wang Y. et al. Selection and validation of reference genes for qRT-PCR in cultivated octoploid strawberry. Fruit Res. 2024; 4:0

[73] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. MolBiolEvol. 1987; 4:406-25

[74] Thompson JD, Gibson TJ, Higgins DG. Multiple sequence align-ment using ClustalW and ClustalX. Curr Protoc Bioinformatics. 2002;Chapter 2:Unit 2.3

[75] Wise AA, Liu Z, Binns AN. Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol. 2006; 343:43-53

[76] Cheng C, Yu Q, Wang Y. et al. Ethylene-regulated asymmetric growth of the petal base promotes flower opening in rose (Rosa hybrida). Plant Cell. 2021; 33:1229-51

[77] Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989; 340:245-6

[78] Stagljar I, Korostensky C, Johnsson N. et al. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A. 1998; 95: 5187-92

[79] Wang F, Wang X, Zhang Y. et al. SlFHY3 and SlHY5 act com-pliantly to enhance cold tolerance through the integration of myo-inositol and light signaling in tomato. New Phytol. 2022; 233: 2127-43