SlUPA-like, a bHLH Transcription Factor in Tomato (Solanum lycopersicum), Serves as the Crosstalk of GA, JA and BR

Abstract

The bHLH (basic Helix-Loop-Helix) transcription factor serves as pivotal controller in plant growth and development. In a previous study, the overexpression of SlUPA-like in Solanum lycopersicum L. Ailsa Craig (AC⁺⁺) altered the JA (Jasmonic acid) response and endogenous GA (Gibberellic acid) content. However, the detailed regulation mechanism was not fully explored. In the present research, we found that the overexpression of SlUPA-like influenced the accumulation of GA, JA and BR (Brassinolide). RNA-Seq data illustrated that the expression levels of genes related to these plant hormones were significantly affected. Additionally, the interaction of SlUPA-like with SlMYB21, SlMYC2 and SlDELLA was characterized by employing Y2H (Yeast Two-Hybrid) and BiFC (Bimolecular Fluorescence Complementation) assay. Furthermore, Dual-LUC (Dual-Luciferase) assay and EMSA (Electrophoretic Mobility Shift Assay) identified that SlUPA-like directly targeted the E-box motif in the promoter of SlGID2 and activated the transcription of SlGID2. These results shed light on the potential role of SlUPA-like in mediating crosstalk among multiple plant hormones and established a robust theoretical framework for further unraveling the functions of SlUPA-like transcription factors in the context of plant growth and hormone signal transduction.

Keywords: SlGID2; SlUPA-like; plant hormone; tomato (Solanum lycopersicum).

Figure 1

(A) The protein multiple sequence alignment of SlUPA-like and homologous genes from various plant species, including At (Arabidopsis thaliana, NP_568745), Os (Oryza sativa, NP_001409737), Md (Malus domestica, XP_028959633), Vv (Vitis vinifera, XP_002284464), Nt (Nicotiana tabacum, NP_001312696), Gm (Glycine max, XP_006578721), Mt (Medicago truncatula, XP_003630566), Sb (Sorghum bicolor, XP_002462650). (B) The prediction of cis-acting element based on the 3 kb sequence upstream of SlUPA-like in the SGN database, using online database PlantCare.

Figure 2

(A) The phenotype of leaves collected in the present study. (B–H) The measurement of plant hormone in wild-type and transgenic lines, (B) brassinolide; (C) 6-deoxocastasterone; (D) CS; (E) GA₇; (F) JA; (G) OPDA; (H) JA-ILE. (I–K) The detection of transcript level of genes involved in GA ((I), CPS, KAO, GA20ox1, GA20ox2, GA2ox2, GA2ox4, GAST1, GID2 and GAI), JA ((J), OPR3, LOXD, JA1, COI1 and MYC2) and BR ((K), BRI1, BZR1, DWERF, IBH1 and CYP734A7). Error bars represent the standard error of the mean (n = 3). (**) p < 0.01 and (*) p < 0.05 between the AC⁺⁺ and transgenic plants by the t test.

Figure 3

RNA-seq analyses of the mature leaves in both AC⁺⁺ and 35S:SlUPA-like lines. (A) A Venn diagram illustrating the overlap and unique expressed genes identified in the samples. (B) A volcano plot depicting the differentially expressed genes (DEGs) between the AC⁺⁺ and 35S:SlUPA-like lines, with 3404 upregulated and 2131 downregulated DEGs. (C) Gene Ontology (GO) analyses were performed on the DEGs to determine their functional categories. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted to identify enriched pathways among the major upregulated and downregulated DEGs. The “Rich factor” indicates the ratio of DEGs associated with a specific KEGG pathway to the total number of DEGs.

Figure 4

(A–C) Metabolic and signaling transduction network diagram of GA, JA and BR. (D–F) The visualization of DEGs involved in GA, JA and BR. The heatmap of DEGs based on RNA-Seq involved in JA (A), GA (B) and BR (C).

Figure 5

The protein-protein identification between SlUPA-like and MYB21, MYC2 and GAI through Y2H (A) and BiFC assay (B). Bar means 25 μm. In Y2H assay, pGADT7-T and pGBKT7-53 were set as the positive control, while pGADT7-T and pGBKT7-Lam were set as the negative control. BD refers to the DNA-binding domain, and AD represents the activation domain. SlUPA-like was cloned into pGADT7, and others were cloned into pGBKT7. The protein–protein interaction was identified using the synthetic defined double dropout (SD DDO) medium (left) and SD quadruple dropout (SD QDO) medium with X-α-gal(5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside) (right). In BiFC assay, the HY5-RFP was set as nuclear localization signal. SlUPA-like was cloned into GFP^N, while GAI, MYB21 and MYC2 were cloned into GFP^C. The first column is the field of view under the green fluorescence signal, the second column is the bright field of view, the third column is the field of view under the red fluorescence signal, and the fourth column is the merged image of the three fields of view. The bar means 25 μm.

Figure 6

The regulation of SlUPA-like on SlGID2. (A) Schematic diagram of Dual-LUC assay. The empty pGreen-62SK was set as the control, and the pGreen-62SK-SlUPA-like was set as the effector. The 2 kb promoter sequence before ATG of SlGID2 was cloned into pGreen 0800LUC to drive the LUC and 35S promoter drives REN as internal control. (B) The results of Dual-LUC showed that SlUPA-like dramatically activated the transcript of SlGID2. Error bars represent the standard error of the mean (n = 3). * means p < 0.05. (C) There were two E-box motifs in the 2 kb promoter sequence before ATG. (D,E) EMSA assay identified that SlUPA-like regulated SlGID2 by binding to motif 2 in its promoter. The underlined sequences represent E-box elements, and the bold and underlined sequences represent mutated E-box elements. Bio-P and Bio-mP mean probe and mutant probe labeled by biotin, respectively. Cold-P and Cold-mP mean cold probe and mutant cold probe without biotin label, respectively. The image was acquired using the ChemiDocTM MP Image system from BIO-RAD, Image Lab Touch Software (version 3.0.1.14).