Salicylic acid improves chilling tolerance via CsNPR1-CsICE1 interaction in grafted cucumbers

Abstract

Salicylic acid (SA) plays a role in the regulation of grafting-induced cold tolerance. However, the molecular mechanism behind it is still unknown. Here, we established that the phenylalanine ammonia-lyase (PAL) pathway-dependent elevate in SA content in grafted cucumber leaves was not only synthesized in the leaves but also transported from the roots under chilling stress. RNAi-CsPAL with low SA content as rootstock reduced SA accumulation in grafted seedling leaves while decreasing rootstock-induced cold tolerance, as evidenced by higher electrolyte leakage (EL), hydrogen peroxide (H₂O₂), and superoxide anion (O₂·−) contents and lower expression of cold-responsive genes (CsICE1, CsDREB1A, CsDREB1B, and CsCOR47), whereas OE-CsPAL with high SA content as rootstock improved the cold tolerance of grafted plants in comparison with the wild type (WT). In addition, CsNPR1 was significantly upregulated in grafted cucumber under chilling stress, with exogenous and endogenous overexpressed SA inducing its transcriptional expression and protein stability, which exhibited higher expression in grafted plants than in self-root plants. While CsNPR1-overexpression (OE-CsNPR1) seedlings as scions were more tolerant to chilling stress than WT seedlings, CsNPR1-suppression (Anti-CsNPR1) seedlings as scions were more vulnerable to chilling stress. Notably, CsNPR1-CsICE1 interactions alleviated ROS accumulation and activated the expression of CsDREB1A, CsDREB1B, CsCOR47, CsCOR15, CsCOR413, and CsKIN1 to enhance SA-mediated chilling tolerance in grafted cucumber. Overall, our findings reveal that SA enhances chilling tolerance in grafted cucumbers via the model of the CsNPR1-CsICE1 transcriptional regulatory cascade.

Acknowledgements

This work is funded by The National Science Foundation of China (31872155, 32372685), The National Key Research and Development Program of China (2019YFD1000300), The Special Fund of Vegetable Industrial Technology System of Shandong Province in China (SDAIT-05-10), and The Funds of Shandong ‘Double Tops’ Program (SYL2017YSTD06).

Author contributions

X.F conducted the majority of the experiment, analysed the data, and finished the initial draft. X. A. and H.B. were responsible for the research design and paper editing. Y.F. and Y.Z. collaborated with X.F. to carry out the experiment.

Data availability

All relevant data can be found within the manuscript and its supporting materials.The transcriptome datasets have been uploaded to the Gene Expression Omnibus of NCBI and can be accessed through SRA accession: PRJNA701131230.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Shang XH, Zhou SM, Guo YY. et al. Advances in stress resistant changes in hetero-grafting cucumber (Cucumis sativus L.) plant. Chin. J Cell Biol. 2017; 39:364-72

[2] Davoudi M, Song M, Zhang M. et al. Long-distance control of pumpkin rootstock over cucumber scion under drought stress as revealed by transcriptome sequencing and mobile mRNAs identifications. Hortic Res. 2022; 9:uhab033

[3] Li H, Guo Y, Lan Z. et al. Methyl jasmonate mediates melatonin-induced cold tolerance of grafted watermelon plants. Hortic Res. 2021; 8:57

[4] Li H, Liu SS, Yi CY. et al. Hydrogen peroxide mediates abscisic acid-induced HSP70 accumulation and heat tolerance in grafted cucumber plants. Plant Cell Environ. 2014; 37:2768-80

[5] Niu M, Huang Y, Sun S. et al. Root respiratory burst oxidase homologue-dependent H₂O₂ production confers salt tolerance on a grafted cucumber by controlling Na⁺ exclusion and stomatal closure. J Exp Bot. 2018; 69:3465-76

[6] Lu X, Liu W, Wang T. et al. Systemic long-distance signaling and communication between rootstock and scion in grafted vegetables. Front Plant Sci. 2020; 11:460

[7] Lv C, Li F, Ai X. et al. H₂O₂ participates in ABA regulation of grafting-induced chilling tolerance in cucumber. Plant Cell Rep. 2022; 41:1115-30

[8] Ding Y, Shi Y, Yang S. Advances and challenges in uncovering cold tolerance regulatory mechanisms in plants. New Phytol. 2019; 222:1690-704

[9] Ding Y, Shi Y, Yang S. Molecular regulation of plant responses to environmental temperatures. Mol Plant. 2020; 13:544-64

[10] Shi Y, Ding Y, Yang S. Cold signal transduction and its interplay with phytohormones during cold acclimation. Plant Cell Physiol. 2015; 56:7-15

[11] Zhao C, Zhang Z, Xie S. et al. Mutational evidence for the critical role of CBF transcription factors in cold acclimation in Arabidopsis. Plant Physiol. 2016; 171:2744-59

[12] Medina J, Catalá R, Salinas J. The CBFs: three Arabidopsis transcription factors to cold acclimate. Plant Sci. 2011; 180:3-11

[13] Gilmour SJ, Zarka DG, Stockinger EJ. et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J. 1998; 16:433-42

[14] Jia Y, Ding Y, Shi Y. et al. The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis. New Phytol. 2016; 212:345-53

[15] Liu Q, Kasuga M, Sakuma Y. et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell. 1998; 10:1391-406

[16] Chinnusamy V, Ohta M, Kanrar S. et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 2003; 17:1043-54

[17] Thomashow MF. PLANT COLD ACCLIMATION: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol. 1999; 50:571-99

[18] Ding Y, Li H, Zhang X. et al. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis. Dev Cell. 2015; 32:278-89

[19] Dong CH, Agarwal M, Zhang Y. et al. The negative regulator of plant cold responses, HOS1, is a RING E 3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci USA. 2006; 103:8281-6

[20] Agarwal M, Hao Y, Kapoor A. et al. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J Biol Chem. 2006; 281:37636-45

[21] An JP, Xu RR, Liu X. et al. Abscisic acid insensitive 4 interacts with ICE1 and JAZ proteins to regulate ABA signaling-mediated cold tolerance in apple. J Exp Bot. 2022; 73:980-97

[22] Hu Y, Jiang L, Wang F. et al. Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor 1 cascade and freezing tolerance in Arabidopsis. Plant Cell. 2013; 25:2907-24

[23] Klessig DF, Malamy J. The salicylic acid signal in plants. Plant Mol Biol. 1994; 26:1439-58

[24] Kumar D. Salicylic acid signaling in disease resistance. Plant Sci. 2014; 228:127-34

[25] Pál M, Szalai G, Kovács V. et al. Salicylic Acid-Mediated Abiotic Stress Tolerance. In: Hayat S, Ahmad A, Alyemeni MN,eds. SALICYLIC ACID: Plant Growth and Development. Springer Netherlands: Dordrecht, 2013,183-247

[26] Vlot AC, Dempsey DA, Klessig DF. Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol. 2009; 47:177-206

[27] Dong CJ, Li L, Shang QM. et al. Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings. Planta. 2014; 240:687-700

[28] Pan DY, Fu X, Zhang XW. et al. Hydrogen sulfide is required for salicylic acid-induced chilling tolerance of cucumber seedlings. Protoplasma. 2020; 257:1543-57

[29] Shang QM, Li L, Dong CJ. Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L. Planta. 2012; 236:1093-105

[30] Khalvandi M, Siosemardeh A, Roohi E. et al. Salicylic acid alleviated the effect of drought stress on photosynthetic characteristics and leaf protein pattern in winter wheat. Heliyon. 2021; 7:e05908

[31] Khan MI, Fatma M, Per TS. et al. Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front. Plant Sci. 2015; 6:462

[32] Miura K, Tada Y. Regulation of water, salinity, and cold stress responses by salicylic acid. Front Plant Sci. 2014; 5:4

[33] Szepesi A, Csiszár J, Gémes K. et al. Salicylic acid improves acclimation to salt stress by stimulating abscisic aldehyde oxidase activity and abscisic acid accumulation, and increases Na⁺ content in leaves without toxicity symptoms in Solanum lycopersicum L. J Plant Physiol. 2009; 166:914-25

[34] Fu X, Feng YQ, Zhang XW. et al. Salicylic acid is involved in rootstock-scion communication in improving the chilling tolerance of grafted cucumber. Front. Plant Sci. 2021; 12:693344

[35] Wu Y, Zhang D, Chu JY. et al. The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Rep. 2012; 1:639-47

[36] Després C, DeLong C, Glaze S. et al. The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell. 2000; 12:279-90

[37] Olate E, Jiménez-Gómez JM, Holuigue L. et al. NPR1 mediates a novel regulatory pathway in cold acclimation by interacting with HSFA1 factors. Nat Plants. 2018; 4:811-23

[38] Li S, He L, Yang Y. et al. INDUCER OF CBF EXPRESSION 1 promotes cold-enhanced immunity by directly activating salicylic acid signaling. Plant Cell. 2024; 36:2587-606

[39] Zhang Y, Fu X, Feng Y. et al. Abscisic acid mediates salicylic acid induced chilling tolerance of grafted cucumber by activating H₂O₂ biosynthesis and accumulation. Int J Mol Sci. 2022; 23:16057

[40] Peng Y, Yang J, Li X. et al. Salicylic acid: biosynthesis and signaling. Annu Rev Plant Biol. 2021; 72:761-91

[41] Ding Y, Sun T, Ao K. et al. Opposite roles of salicylic acid receptors NPR1 and NPR3/NPR4 in transcriptional regulation of plant immunity. Cell. 2018; 173:1454-1467.e15

[42] Wang D, Amornsiripanitch N, Dong X. A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog. 2006; 2:e123

[43] Fan W, Dong X. In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis. Plant Cell. 2002; 14:1377-89

[44] Cheng F, Gao M, Lu J. et al. Spatial-temporal response of reactive oxygen species and salicylic acid suggest their interaction in pumpkin rootstock-induced chilling tolerance in watermelon plants. Antioxidants (Basel). 2021; 10:2024

[45] Ntatsi G, Savvas D, Druege U. et al. Contribution of phytohormones in alleviating the impact of sub-optimal temperature stress on grafted tomato. Sci Hortic. 2013; 149:28-38

[46] Wu J, Zhu W, Zhao Q. Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis. J Integr Plant Biol. 2023; 65:881-7

[47] Scott IM, Clarke SM, Wood JE. et al. Salicylate accumulation inhibits growth at chilling temperature in Arabidopsis. Plant Physiol. 2004; 135:1040-9

[48] Cheng F, Lu J, Gao M. et al. Redox signaling and CBF-responsive pathway are involved in salicylic acid-improved photosynthesis and growth under chilling stress in watermelon. Front. Plant Sci. 2016; 7:1519

[49] Huang P, Dong Z, Guo P. et al. Salicylic acid suppresses apical hook formation via NPR1-mediated repression of EIN3 and EIL 1 in Arabidopsis. Plant Cell. 2020; 32:612-29

[50] Kilian J, Whitehead D, Horak J. et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 2007; 50:347-63

[51] Glazebrook J, Rogers EE, Ausubel FM. Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening. Genetics. 1996; 143:973-82

[52] Liu W, Wang Q, Zhang R. et al. Rootstock-scion exchanging mRNAs participate in the pathways of amino acids and fatty acid metabolism in cucumber under early chilling stress. Hortic Res. 2022; 9:uhac031

[53] Wang L, Wu B, Chen G. et al. The essential role of jasmonate signaling in Solanum habrochaites rootstock-mediated cold tolerance in tomato grafts. Hortic Res. 2023; 10:uhac227

[54] Backer R, Naidoo S, van den Berg N. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and related family: mechanistic insights in plant disease resistance. Front Plant Sci. 2019; 10:102

[55] Rochon A, Boyle P, Wignes T. et al. The coactivator function of Arabidopsis NPR1 requires the core of its BTB/POZ domain and the oxidation of C-terminal cysteines. Plant Cell. 2006; 18:3670-85

[56] Khan IU, Ali A, Zareen S. et al. Non-expresser of PR-genes 1 positively regulates Abscisic acid signaling in Arabidopsis thaliana. Plants (Basel). 2022; 11:815

[57] Kim YS, Lee M, Lee JH. et al. The unified ICE-CBF pathway provides a transcriptional feedback control of freezing tolerance during cold acclimation in Arabidopsis. Plant Mol Biol. 2015; 89:187-201

[58] Steponkus PL, Uemura M, Joseph RA. et al. Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc Natl Acad Sci USA.. 1998; 95:14570-5

[59] Zuo ZF, Kang HG, Park MY. et al. Zoysia japonica MYC type transcription factor ZjICE1 regulates cold tolerance in transgenic Arabidopsis. Plant Sci. 2019; 289:110254

[60] Semeniuk P, Moline HE, Abbott JA. A comparison of the effects of ABA and an Antitranspirant on chilling injury of coleus, cucumbers, and dieffenbachia. J Am Soc Hortic Sci. 1986; 111:866-8

[61] Campos PS, Quartin V, Ramalho JC. et al. Electrolyte leakage and lipid degradation account for cold sensitivity in leaves of Coffea sp. plants. J Plant Physiol. 2003; 160:283-92

[62] Zhao SJ, Cang J. Plant Physiology Experiment Instruction. Beijing: China Agricultural Press; 2015:

[63] Li Z, Gong M. Improvement of measurement method for superoxide anion radical in plant. Acta Bot Yunnanica. 2005; 27:211-6

[64] Thordal-Christensen H, Zhang Z, Wei Y. et al. Subcellular localization of H₂O₂ in plants. H₂O₂ accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J. 1997; 11:1187-94

[65] Jabs T, Dietrich RA, Dangl JL. Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science. 1996; 273:1853-6

[66] Zhang ZX, Li X, Ma S. et al. A protocol for agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) from cotyledon explants. Protocol. Exchange. 2017

[67] Chen J, Mohan R, Zhang Y. et al. NPR1 promotes its own and target gene expression in plant defense by recruiting CDK8. Plant Physiol. 2019; 181:289-304

[68] Liu Y, Huang W, Wang LJ. Response of 14C-salicylic acid to heat stress after being fed to leaves of grape plants. Agric Sci China. 2005; 4:106-12

[69] Zhang Y, Tessaro MJ, Lassner M. et al. Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA 6 reveals their redundant and essential roles in systemic acquired resistance. Plant Cell. 2003; 15:2647-53

[70] Kim Y, Park S, Gilmour SJ. et al. Roles of CAMTA transcription factors and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis. Plant J. 2013; 75:364-76

[71] Cao H, Bowling SA, Gordon AS. et al. Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance. Plant Cell. 1994; 6:1583-92

[72] Cao H, Glazebrook J, Clarke JD. et al. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell. 1997; 88:57-63