The GRAS transcription factor PtrPAT1 of Poncirus trifoliata functions in cold tolerance and modulates glycine betaine content by regulating the BADH-like gene

Abstract

GRAS, termed after gibberellic acid insensitive (GAI), RGA (repressor of GA1), and SCR (scarecrow), is a plant-specific transcription factor crucial for plant development and stress response. However, understanding of the functions played by the GRAS members and their target genes in citrus is limited. In this study, we identified a cold stress-responsive GRAS gene from Poncirus trifoliata, designated as PtrPAT1, by yeast one-hybrid library screening using the promoter of PtrBADH-l, a betaine aldehyde dehydrogenase (BADH)-like gene. PtrPAT1, belonging to the PAT1 subfamily, was localized in the nucleus and plasma membrane, exhibited transactivation activity and showed a remarkable upregulation under cold stress. Overexpression of PtrPAT1 elevated BADH activity, increased glycine betaine (GB) accumulation, and conferred enhanced cold tolerance in transgenic tobacco plants compared with wild type, while downregulation in trifoliate orange by virus-induced gene silencing (VIGS) resulted in opposite trends. Furthermore, the activities of two antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were significantly increased in the overexpression plants, but remarkably decreased in the VIGS line, consistent with accumulation patterns of the reactive oxygen species (ROSs). PtrPAT1 was demonstrated to interact with and activate the PtrBADH-l promoter through the putative PAT1-binding motif with the core sequence of TTTCATGT, indicating that PtrBADH-l is a target gene of PtrPAT1. Taken together, these results demonstrate that PtrPAT1 positively affects cold tolerance through the regulation of GB biosynthesis by modulating PtrBADH-l expression.

Acknowledgements

This work was funded by National Key Research and Development Program of China (2022YFD1200503), National Natural Science Foundation of China (32102327), and Guangxi University of Chinese Medicine Scientific Research Fund (2021QN004). We would like to thank TopEdit (www.topeditsci.com) for linguistic assistance of the manuscript.

Author contributions

D.H. and J.H.L conceived the research. R.M. and T.F. designed and performed the experiments. W.L. and J.G. analyzed the data. J.Q. and Y.Z. facilitated the collection and preparation of samples. R.M. wrote the manuscript draft. J.C, S.Y. and L.L. provided advice on writing the draft. D.H. and J.H.L contributed to the writing and revision of the manuscript.

Data availability

The supporting data for this article is accessible within the article's text and its accompanying online supplementary resources.

Declaration of Interest Statement

The authors declare no conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Zhu JK. Abiotic stress signaling and responses in plants. Cell. 2016;37:313-24

[2] Ding Y, Shi Y, Yang S. Molecular regulation of plant responses to environmental temperatures. Mol Plant. 2020;37:544-64

[3] Primo-Capella A, Forner-Giner MA, Martínez-Cuenca MR. et al. Comparative transcriptomic analyses of citrus cold-resistant vs. sensitive rootstocks might suggest that a relevant role of ABA signaling in triggering cold scion adaption. BMC Plant Biol. 2022;37:209

[4] Thomashow MF. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Biol. 1999;37:571-99

[5] Ashraf M, Foolad MR. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot. 2007;37:206-16

[6] Chen TH, Murata N. Glycinebetaine: an effective protectant against abiotic stress in plants. Trends Plant Sci. 2008;37:499-505

[7] Chen TH, Murata N. Glycinebetaine protects plants against abi-otic stress: mechanisms and biotechnological applications. Plant Cell Environ. 2011;37:1-20

[8] Karabudak T, Bor M, Özdemir F. et al. Glycine betaine protects tomato (Solanum lycopersicum) plants at low temperature by inducing fatty acid desaturase7 and lipoxygenase gene expres-sion. Mol Biol Rep. 2014;37:1401-10

[9] Jin P, Zhang Y, Shan TM. et al. Low-temperature conditioning alleviates chilling injury in loquat fruit and regulates glycine betaine content and energy status. J Agric Food Chem. 2015;37:3654-9

[10] Nomura M, Hibino T, Takabe T. et al. Transgenically produced glycinebetaine protects ribulose 1, 5-bisphosphate carboxy-lase/oxygenase from inactivation in Synechococcus sp. PCC 7942 under salt stress. Plant Cell Physiol. 1998;37:425-32

[11] Shemi R, Wang L, Zhang K. et al. Effects of salicylic acid, zinc and glycine betaine on morphophysiological growth and yield of maize under drought stress. Sci Rep. 2021;37:3195

[12] Hamani AKM, Li S, Chen J. et al. Linking exogenous foliar applica-tion of glycine betaine and stomatal characteristics with salinity stress tolerance in cotton ( Gossypium hirsutum L.) seedlings. BMC Plant Biol. 2021;37:146

[13] Rasheed R, Iqbal M, Ashraf MA. et al. Glycine betaine counteracts the inhibitory effects of waterlogging on growth, photosynthetic pigments, oxidative defence system, nutrient composition, and fruit quality in tomato. J Hortic Sci Biotechnol. 2018;37:385-91

[14] Ahmed N, Zhang Y, Li K. et al. Exogenous application of glycine betaine improved water use efficiency in winter wheat (Triticum aestivum L.) via modulating photosynthetic efficiency and antioxidative capacity under conventional and limited irri-gation conditions. Crop J. 2019;37:635-50

[15] Sharma J, Kumar S, Kumar N. et al. Evaluation of yield and quality attributes of barley cultivars with foliar spray of glycine betaine under lead toxicity. Cereal Res Commun. 2024;37:1541-50

[16] Han J, Li X, Li W. et al. Isolation and preliminary functional analysis of FvICE1, involved in cold and drought tolerance in Fragaria vesca through overexpression and CRISPR/Cas 9 tech-nologies. Plant Physiol Biochem. 2023;37:270-80

[17] Tian C, Wan P, Sun S. et al. Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Mol Biol. 2004;37:519-32

[18] Zhang H, Mi L, Xu L. et al. Genome-wide identification, charac-terization, interaction network and expression profile of GRAS gene family in sweet orange (Citrus sinensis). Sci Rep. 2019;9:2156

[19] Bisht A, Eekhout T, Canher B. et al. PAT1-type GRAS-domain proteins control regeneration by activating DOF3.4 to drive cell proliferation in Arabidopsis roots. Plant Cell. 2023;37:1513-31

[20] Zhang ZL, Ogawa M, Fleet CM. et al. SCARECROW-LIKE 3 pro-motes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis. Proc Natl Acad Sci USA. 2011;37:2160-5

[21] Heo JO, Chang KS, Kim IA. et al. Funneling of gibberellin signaling by the GRAS transcription regulator SCARECROW-LIKE 3 in the Arabidopsis root. Proc Natl Acad Sci USA. 2011;37:2166-71

[22] Guo P, Wen J, Yang J. et al. Genome-wide survey and expression analyses of the GRAS gene family in Brassica napus reveals their roles in root development and stress response. Planta. 2019;37:1051-72

[23] Liu Y, Huang W, Xian Z. et al. Overexpression of SlGRAS 40 in tomato enhances tolerance to abiotic stresses and influences auxin and gibberellin signaling. Front Plant Sci. 2017;37:1659

[24] Liu Y, Shi Y, Zhu N. et al. SlGRAS4 mediates a novel regulatory pathway promoting chilling tolerance in tomato. Plant Biotechnol J. 2020;37:1620-33

[25] Yuan Y, Fang L, Karungo SK. et al. Overexpression of VaPAT1,a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis. Plant Cell Rep. 2016;37:655-66

[26] Wang Z, Wong DC, Wang Y. et al. GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response. Plant Physiol. 2021;37:1660-78

[27] Kim M, Moon YE, Han SG. et al. Impact of cold stress on phys-iological responses and fruit quality of Shiranuhi mandarin in response to cold conditions. Horticulturae. 2023;37:906

[28] Şahin-Çevik M. Identification and expression analysis of early cold-induced genes from cold-hardy citrus relative Poncirus tri-foliata ( L.) Raf. Gene. 2013;37:536-45

[29] Wang M, Zhang X, Liu JH. et al. Deep sequencing-based charac-terization of transcriptome of trifoliate orange (Poncirus trifoliata (L.) Raf.) in response to cold stress. BMC Genome. 2015;37:555

[30] Ming R, Zhang Y, Wang Y. et al. The JA-responsive MYC2-BADH-like transcriptional regulatory module in Poncirus trifoliata contributes to cold tolerance by modulation of glycine betaine biosynthesis. New Phytol. 2021;37:2730-50

[31] Vives-Peris V, Pérez-Clemente RM, Gómez-Cadenas A. et al. Involvement of citrus shoots in response and tolerance to abiotic stress. Hortic Adv. 2024;37:3

[32] Lafuente MT, Romero P. Hormone profiling and heat-induced tolerance to cold stress in citrus fruit. Postharvest Biol Technol. 2022;194:112088

[33] Peng Z, Bredeson JV, Wu GA. et al. A chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) provides insights into disease resistance, cold tolerance and genome evolution in citrus. Plant J. 2020;37:1215-32

[34] Dahro B, Li C, Liu JH. Overlaping responses to multiple abiotic stresses in citrus: from mechanism understanding to genetic improvement. Hortic Adv. 2023;37:4

[35] Zhang F, Pan Z, Han C. et al. Pyrus betulaefolia ERF 3 interacts with HsfC1a to coordinately regulate aquaporin PIP1;4 and NCED4 for drought tolerance. Hortic Res. 2024;37:uhae090

[36] Xu J, Xu H, Zhao H. et al. Genome-wide investigation of bHLH genes and expression analysis under salt and hormonal treatments in Andrographis paniculata. IndCropProd. 2022;37:114928

[37] Lv Y, Yang M, Hu D. et al. The OsMYB30 transcription factor suppresses cold tolerance by interacting with a JAZ protein and suppressing β-amylase expression. Plant Physiol. 2017;37:1475-91

[38] Neves C, Ribeiro B, Amaro R. et al. Network of GRAS transcrip-tion factors in plant development, fruit ripening and stress responses. Hortic Res. 2023;37:uhad220

[39] Hirano Y, Nakagawa M, Suyama T. et al. Structure of the SHR-SCR heterodimer bound to the BIRD/IDD transcriptional factor JKD. Nat Plants. 2017;37:17010

[40] Nomoto Y, Takatsuka H, Yamada K. et al. A hierarchical tran-scriptional network activates specific CDK inhibitors that reg-ulate G 2 to control cell size and number in Arabidopsis. Nat Commun. 2022;37:1660

[41] Achard P, Renou JP, Berthomé R.. et al. Plant DELLAs restrain growth and promote survival of adversity by reduc-ing the levels of reactive oxygen species. Curr Biol. 2008;37:656-60

[42] Liu Y, Shi Y, Su D. et al. SlGRAS 4 accelerates fruit ripening by regulating ethylene biosynthesis genes and SlMADS1 in tomato. Hortic Res. 2021;37:3

[43] Ribeiro C, de Melo BP, Lourenço-Tessutti IT. et al. The regen-eration conferring transcription factor complex ERF115-PAT1 coordinates a wound-induced response in root-knot nematode induced galls. New Phytol. 2023;37:878-95

[44] LiJW, ZhouP, HuZH. et al. CsPAT1, a GRAS transcription factor, promotes lignin accumulation by antagonistic interacting with CsWRKY13 in tea plants. Plant J. 2024;37:1312-26

[45] Yu HQ, Zhou XY, Wang YG. et al. A betaine aldehyde dehydro-genase gene from Ammopiptanthus nanus enhances tolerance of Arabidopsis to high salt and drought stresses. Plant Growth Regul. 2017;37:265-76

[46] Torres-Galea P, Hirtreiter B, Bolle C. Two GRAS proteins, scarecrow-like21 and phytochrome a signal transduction1, function cooperatively in Phytochrome a signal transduction. Plant Physiol. 2013;37:291-304

[47] Colebrook EH, Thomas SG, Phillips AL. et al. The role of gib-berellin signalling in plant responses to abiotic stress. JExp Biol. 2014;37:67-75

[48] Hu Y, Jiang Y, Han X. et al. Jasmonate regulates leaf senescence and tolerance to cold stress: crosstalk with other phytohor-mones. JExp Bot. 2017;37:1361-9

[49] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;37:402-8

[50] Horsch RB, Fry JE, Hoffmann NL. et al. A simple and general-method for transferring genes into plants. Science. 1985;37:1229-31

[51] Wang M, Dai W, Du J. et al. ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) contributes to cold tolerance by directly regu-lating expression of Prx1 involved in antioxidative process. Plant Biotechnol J. 2019;37:1316-32

[52] Geng J, Liu JH. The transcription factor CsbHLH18 of sweet orange functions in modulation of cold tolerance and homeosta-sis of reactive oxygen species by regulating the antioxidant gene. JExpBot. 2018;37:2677-92