GA signaling protein LsRGL1 interacts with the abscisic acid signaling-related gene LsWRKY70 to affect the bolting of leaf lettuce

Abstract

A variety of endogenous hormone signals, developmental cues, and environmental stressors can trigger and promote leaf lettuce bolting. One such factor is gibberellin (GA), which has been linked to bolting. However, the signaling pathways and the mechanisms that regulate the process have not been discussed in full detail. To clarify the potential role of GAs in leaf lettuce, significant enrichment of GA pathway genes was found by RNA-seq, among which the LsRGL1 gene was considered significant. Upon overexpression of LsRGL1, a noticeable inhibition of leaf lettuce bolting was observed, whereas its knockdown by RNA interference led to an increase in bolting. In situ hybridization analysis indicated a significant accumulation of LsRGL1 in the stem tip cells of overexpressing plants. Leaf lettuce plants stably expressing LsRGL1 were examined concerning differentially expressed genes through RNA-seq analysis, and the data indicated enhanced enrichment of these genes in the ‘plant hormone signal transduction’ and ‘phenylpropanoid biosynthesis’ pathways. Additionally, significant changes in LsWRKY70 gene expression were identified in COG (Clusters of Orthologous Groups) functional classification. The results of yeast one-hybrid, β-glucuronidase (GUS), and biolayer interferometry (BLI) experiments showed that LsRGL1 proteins directly bind to the LsWRKY70 promoter. Silencing LsWRKY70 by virus-induced gene silencing (VIGS) can delay bolting, regulate the expression of endogenous hormones, abscisic acid (ABA)-linked genes, and flowering genes, and improve the nutritional quality of leaf lettuce. These results strongly associate the positive regulation of bolting with LsWRKY70 by identifying its vital functions in the GA-mediated signaling pathway. The data obtained in this research are invaluable for further experiments concerning the development and growth of leaf lettuce.

Acknowledgements

We appreciate Cathay Green Seeds (Beijing) Co., Ltd for supplying testing materials. For his wise counsel, Dr Ji Tian (Beijing University of Agriculture, China) is gratefully acknowledged. The Beijing Joint Research Program for Germplasm Innovation and New Variety Breeding (G20220628003), The Beijing Natural Science Foundation Program (22JF0014), and The National Natural Science Foundation of China (3177110780) all contributed financially to the present project.

Author contributions

L.C.: software, verification, methodology, and writing original draft. C.L.: resources, software, supervision, and project management. J.H.: methodology and validation. S.F.: supervision, resources, and seeking funding. Y.H.: conceptualization, writing review and editing, and project administration.

Data availability

The datasets generated for this study can be found in the NCBI SRA database: PRJNA880996 and SRP076512.

Conflict of interest

The authors declare that they have no conflict of interest.

References

[1] Assefa AD, Choi S, Lee JE et al. Identification and quantification of selected metabolites in differently pigmented leaves of lettuce (Lactuca sativa L.) cultivars harvested at mature and bolting stages . BMC Chem. 2019; 13: 56.

[2] Chen Z, Zhao W, Ge D et al. LCM-seq reveals the crucial role of LsSOC1 in heat-promoted bolting of lettuce (Lactuca sativa L.). Plant J. 2018; 95: 516-28.

[3] Lee ON, Fukushima K, Han YP et al. QTL analysis of stem elongation and flowering time in lettuce using genotyping-by-sequencing. Genes. 2021; 12: 947.

[4] Hao JH, Zhang LL, Li PP et al. Quantitative proteomics analysis of lettuce (Lactuca sativa L.) reveals molecular basis-associated auxin and photosynthesis with bolting induced by high temperature . Int J Mol Sci. 2018; 19: 2967.

[5] Chen L, Xu M, Liu C et al. LsMYB15 regulates bolting in leaf lettuce (Lactuca sativa L.) under high-temperature stress . Front Plant Sci. 2022; 13: 921021.

[6] Liu R, Su Z, Zhou H et al. LsHSP70 is induced by high temperature to interact with calmodulin, leading to higher bolting resistance in lettuce. Sci Rep. 2020; 10: 15155.

[7] Li W, Wang H, Yu D . Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions . Mol Plant. 2016; 9: 1492-503.

[8] Zou L, Pan C, Wang MX et al. Progress on the mechanism of hormones regulating plant flower formation. Hereditas (Beijing). 2020; 42: 739-51.

[9] Fang J, Chai Z, Yao W et al. Interactions between ScNAC23 and ScGAI regulate GA-mediated flowering and senescence in sugarcane. Plant Sci. 2021; 304: 110806.

[10] Rao S, Li Y, Chen J . Combined analysis of microRNAs and target genes revealed miR156-SPLs and miR172-AP2 are involved in a delayed flowering phenomenon after chromosome doubling in black goji (Lycium ruthencium). Front Genet. 2021; 12: 1192.

[11] Abolghasemi R, Haghighi M, Etemadi N et al. Transcriptome architecture reveals genetic networks of bolting regulation in spinach. BMC Plant Biol. 2021; 21: 179.

[12] Chen L, Xiang S, Chen Y et al. Arabidopsis WRKY45 interacts with the DELLA protein RGL1 to positively regulate age-triggered leaf senescence . Mol Plant. 2017; 10: 1174-89.

[13] Lei W, Li Y, Yao X et al. NAP is involved in GA-mediated chlorophyll degradation and leaf senescence by interacting with DELLAs in Arabidopsis. Plant Cell Rep. 2020; 39: 75-87.

[14] Ali MY, Chang Q, Yan Q et al. Highly efficient biosynthesis of glycyrrhetinic acid glucosides by coupling of microbial glycosyltransferase to plant sucrose synthase. Front Bioeng Biotechnol. 2021; 9: 645079.

[15] Liu X, Lv S, Liu R et al. Transcriptomic analysis reveals the roles of gibberellin-regulated genes and transcription factors in regulating bolting in lettuce (Lactuca sativa L.). PLoS One. 2018; 13: e0191518.

[16] Wen CK, Chang C . Arabidopsis RGL1 encodes a negative regulator of gibberellin responses . Plant Cell. 2002; 14: 87-100.

[17] Gao S, Chu C . Gibberellin metabolism and signaling: targets for improving agronomic performance of crops. Plant Cell Physiol. 2020; 61: 1902-11.

[18] Wu K, Xu H, Gao X et al. New insights into gibberellin signaling in regulating plant growth-metabolic coordination. Curr Opin Plant Biol. 2021; 63: 102074.

[19] Guan H, Huang X, Zhu Y et al. Identification of DELLA genes and key stage for GA sensitivity in bolting and flowering of flowering Chinese cabbage . Int J Mol Sci. 2021; 22: 12092.

[20] Wang Z, Liu L, Cheng C et al. GAI functions in the plant response to dehydration stress in Arabidopsis thaliana. Int J Mol Sci. 2020; 21: 819.

[21] Zhang L, Chen L, Yu D . Transcription factor WRKY75 interacts with DELLA proteins to affect flowering. Plant Physiol. 2018; 176: 790-803.

[22] Daviere JM, Achard P . A pivotal role of DELLAs in regulating multiple hormone signals. Mol Plant. 2016; 9: 10-20.

[23] Yang DL, Yao J, Mei CS et al. Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade. Proc Natl Acad Sci USA. 2012; 109: E1192-200.

[24] Piskurewicz U, Jikumaru Y, Kinoshita N et al. The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and AB15 activity . Plant Cell. 2008; 20: 2729-45.

[25] Hou X, Li Y, Xia K et al. DELLAs modulate jasmonate signaling via competitive binding to JAZs. Dev Cell. 2010; 19: 884-94.

[26] Cheng H, Song S, Xiao L et al. Gibberellin acts through jasmonate to control the expression of MYB21, MYB24, and MYB57 to promote stamen filament growth in Arabidopsis. PLoS Genet. 2009; 5: e1000440.

[27] Chen M, Maodzeka A, Zhou L et al. Removal of DELLA repression promotes leaf senescence in Arabidopsis. Plant Sci. 2014; 219-220: 26-34.

[28] Bai MY, Shang JX, Oh E et al. Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis. Nat Cell Biol. 2012; 14: 810-7.

[29] Han Y, Chen Z, Lv S et al. MADS-box genes and gibberellins regulate bolting in lettuce (Lactuca sativa L.). Front Plant Sci. 2016; 7: 1889.

[30] Ou X, Wang Y, Zhang J et al. Identification of BcARR genes and CTK effects on stalk development of flowering Chinese cabbage . Int J Mol Sci. 2022; 23: 7412.

[31] Yin Y, Li J, Guo B et al. Exogenous GA3 promotes flowering in Paphiopedilum callosum (Orchidaceae) through bolting and lateral flower development regulation . Hortic Res. 2022; 9: uhac091.

[32] Lange MJP, Knop N, Lange T . Stamen-derived bioactive gibberellin is essential for male flower development of Cucurbita maxima L. J Exp Bot. 2012; 63: 2681-91.

[33] Zhang L, Gu L, Ringler P et al. Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells. Plant Sci. 2015; 236: 214-22.

[34] Fang X, Bo C, Wang M et al. Overexpression of the maize WRKY114 gene in transgenic rice reduce plant height by regulating the biosynthesis of GA . Plant Signal Behav. 2021; 16: 1967635.

[35] Singh D, Debnath P, Roohi SAP et al. Expression of the tomato WRKY gene, SlWRKY23, alters root sensitivity to ethylene, auxin and JA and affects aerial architecture in transgenic Arabidopsis. Physiol Mol Biol Plants. 2020; 26: 1187-99.

[36] Wang T, Yang B, Guan Q et al. Transcriptional regulation of Lonicera japonica Thunb. during flower development as revealed by comprehensive analysis of transcription factors . BMC Plant Biol. 2019; 19: 198.

[37] Shu K, Chen Q, Wu Y et al. ABSCISIC ACID-INSENSITIVE 4 negatively regulates flowering through directly promoting Arabidopsis FLOWERING LOCUS C transcription . J Exp Bot. 2016; 67: 195-205.

[38] Brocard IM, Lynch TJ, Finkelstein RR . Regulation and role of the Arabidopsis abscisic acid-insensitive 5 gene in abscisic acid, sugar, and stress response . Plant Physiol. 2002; 129: 1533-43.

[39] Yu S, Ligang C, Liping Z et al. Overexpression of OsWRKY72 gene interferes in the abscisic acid signal and auxin transport pathway of Arabidopsis. J Biosci Bioeng. 2010; 35: 459-71.

[40] Wang Y, Li B, Li Y et al. Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.). Open Life Sci. 2022; 17: 438-46.

[41] Wang YD, Huang X, Huang XM et al. BcSOC1 promotes bolting and stem elongation in flowering Chinese cabbage . Int J Mol Sci. 2022; 23: 3459.

[42] Ullah A, Sun H, Hakim et al . A novel cotton WRKY gene, GhWRKY6-like, improves salt tolerance by activating the ABA signaling pathway and scavenging of reactive oxygen species . Physiol Plant. 2018; 162: 439-54.

[43] Chapman JM, Muhlemann JK, Gayomba SR et al. RBOH-dependent ROS synthesis and ROS scavenging by plant specialized metabolites to modulate plant development and stress responses. Chem Res Toxicol. 2019; 32: 370-96.

[44] Pu Y, Liu L, Wu J et al. Transcriptome profile analysis of winter rapeseed (Brassica napus L.) in response to freezing stress, reveal potentially connected events to freezing stress . Int J Mol Sci. 2019; 20: 2771.

[45] Aziz A, Akram NA, Ashraf M . Influence of natural and synthetic vitamin C (ascorbic acid) on primary and secondary metabolites and associated metabolism in quinoa (Chenopodium quinoa Willd.) plants under water deficit regimes . Plant Physiol Biochem. 2018; 123: 192-203.

[46] Yousaf M, Bashir S, Raza H et al. Role of nitrogen and magnesium for growth, yield and nutritional quality of radish. Saudi J Biol Sci. 2021; 28: 3021-30.

[47] Yu YY, Xu JD, Huang TX et al. Combination of beneficial bacteria improves blueberry production and soil quality. Food Sci Nutr. 2020; 23: 5776-84.

[48] Wang Z, Zhu Y, Wang L et al. A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter . Planta. 2009; 230: 1155-66.

[49] Song J, Huang H, Hao Y et al. Nutritional quality, mineral and antioxidant content in lettuce affected by interaction of light intensity and nutrient solution concentration. Sci Rep. 2020; 10: 2796.

[50] Jiang D, Lu B, Liu L et al. Exogenous melatonin improves salt stress adaptation of cotton seedlings by regulating active oxygen metabolism. PeerJ. 2020; 8: e10486.

[51] Habib S, Lwin YY, Li N . Down-regulation of SlGRAS10 in tomato confers abiotic stress tolerance . Genes (Basel). 2021; 12: 623.

[52] Li M, Zhang C, Hou L et al. Multiple responses contribute to the enhanced drought tolerance of the autotetraploid Ziziphus jujuba Mill. var. spinosa. Cell Biosci. 2021; 11: 119.

[53] Li H, Han M, Yu L et al. Transcriptome analysis identifies two ethylene response factors that regulate proanthocyanidin biosynthesis during Malus crabapple fruit development . Front Plant Sci. 2020; 11: 76.

[54] Ma H, Yang T, Li Y et al. The long noncoding RNA MdLNC499 bridges MdWRKY1 and MdERF109 function to regulate early-stage light-induced anthocyanin accumulation in apple fruit. Plant Cell. 2021; 33: 3309-30.