Identification and functional characterization of JrbZIP40 in walnut reveals its role in salt and drought stress tolerance in transgenic Arabidopsis seedlings

Abstract

Background: Salt and drought are the primary environmental stress factors that severely threaten plant growth, development, and yield. bZIP transcription factors reportedly play crucial roles in plant responses to both biotic and abiotic stressors. However, the biological function of bZIP transcription factors in oil crops, particularly walnuts, under salt and drought stress remains unclear.

Results: In this study, members of the walnut bZIP gene family were identified based on the walnut genome Chandler 2.0. Transcriptome data and RT-qPCR results were used to analyze the expression patterns of various JrbZIP genes under biotic and abiotic stress, revealing that JrbZIP40 was strongly induced by both drought and salt stress. Subcellular localization and transcriptional activation assays demonstrated that JrbZIP40 localized to the nucleus and exhibited transcriptional activation activity. Overexpression of JrbZIP40 in transgenic Arabidopsis seedlings significantly enhanced resistance to salt and drought stress. DAP-seq and Dual-luciferase results indicated that JrbZIP40 may bind to the JrHB7 and JrATG8G promoters and activate their expression, contributing to stress resistance.

Conclusions: Overall, this study elucidates the regulatory network and biological functions of JrbZIP40 in drought and salt tolerance, providing a theoretical foundation and candidate gene resources for the future use of genetic engineering to improve walnut stress resistance.

Keywords: Juglans regia; Drought stress; Functional characterization; Salt stress; bZIP transcription factor.

Fig. 1

Expression heat map of JrbZIP genes in response to Colletotrichum gloeosporioides infection. The expression data for JrbZIP genes in response to C. gloeosporioides infection were obtained from the supplementary data of a previously published study [27]. After re-analysis of the RNA-Seq data, relative expression levels were calculated with respect to control samples (i.e., 0 hpi). Detailed values are listed in Table S3

Fig. 2

Expression profiles of JrbZIP genes under abiotic stress. A Expression heat map of JrbZIP genes in response to drought and salt stresses. After re-analysis of RT-qPCR data, relative expression levels were calculated with respect to control samples (i.e., 0 d for drought; 0 h for salt). B Expression heat map of JrbZIP genes in response to heat and cold stresses. After re-analysis of RT-qPCR data, relative expression levels were calculated with respect to control samples (i.e., 0 h for heat and cold). Heat maps were generated using TIGR MeV v4.8.1 software. Detailed primers and values are listed in Table S1 and Table S4, respectively

Fig. 3

Amino acid sequence, subcellular localization, and transcriptional activation analysis of JrbZIP40. A Amino acid sequence analysis of the JrbZIP40 protein. The basic region is indicated by the blue area, and the leucine zipper is indicated by the gray area. Structural features of JrbZIP40 were established based on the description by Jakoby et al. [4]. B The subcellular localization of JrbZIP40 was observed by confocal laser scanning microscopy in Nicotiana benthamiana. Green fluorescence represents JrbZIP40-GFP, and blue fluorescence corresponds to the nucleus stained with fluorescent probe (DAPI). bars = 20 μm. C Transcriptional activation of JrbZIP40 in yeast cells. The indicated pGBKT7-JrbZIP40 fusion bait vectors were transformed into yeast cells. Yeast synthetic dropout medium lacking Trp (SD/-Trp) served as the transformation control, while medium lacking Trp, Ade, and His (SD/-Trp-His-Ade) was used for screening. The pGBKT7-MdWRKY115 bait vector served as a positive control, and the empty pGBKT7 vector was used as a negative control

Fig. 4

Phenotypic analysis of transgenic JrbZIP40-overexpressing Arabidopsis seedlings under NaCl and mannitol treatments. A Representative images of five-day-old ‘Col’ and transgenic seedlings grown for 11 days on MS medium supplemented with 0 mM, 100 mM NaCl, or 300 mM mannitol. Bars = 1 cm. B Primary root lengths; C fresh weights; D MDA contents; and E electrolyte leakage of five-day-old ‘Col’ and transgenic seedlings measured after 11 days of exposure to NaCl and osmotic stresses. Error bars indicate the standard deviation of three biological replicates. Different letters in each panel indicate significant differences at P < 0.05, based on one-way ANOVA and Duncan’s test

Fig. 5

Genome-wide analysis of JrbZIP40-binding sites. A Venn diagrams of bZIP40-1 and bZIP40-2 analysis of JrbZIP40. bZIP40-1 and bZIP40-2 represent two technical replicates. B Distribution of JrbZIP40 peaks on different chromosomes. C Analysis of JrbZIP40-enriched regions in the DAP-seq assay. D GO analysis of the candidate target genes bound by JrbZIP40. E KEGG analysis of the candidate target genes bound by JrbZIP40

Fig. 6

Identification of potential target genes of the JrbZIP40 protein by DAP-seq. A Identification of the DNA-binding motif of the JrbZIP40 protein. Recombinant JrbZIP40 proteins were used for DAP-seq analysis. B Schematic representation of the JrbZIP40 binding site in the JrHB7 (Jr07_09110) promoter, identified through DAP-seq. The black circle represents the length of the DNA fragment, and the black arrow indicates the 5′ to 3′ direction. The dotted line indicates the position of the JrbZIP40 binding motif. The figure was created using IGV software (version 2.17.1) with a track height of 100. C Phylogenetic tree of JrHB7, rice and Arabidopsis homeodomain-leucine zipper proteins. The tree was constructed using MEGA software (version 6.06) with the neighbor-joining method and a bootstrap test of 1000 replicates. JrHB7 is marked with an asterisk. D Schematic representation of the JrbZIP40 binding site in the JrATG8G (Jr05_03470) promoter, identified through DAP-seq. E The phylogenetic tree of JrATG8G, rice and Arabidopsis autophagy 8 proteins. F Relative expression levels of JrHB7 and JrATG8G under drought and salt stress. Error bars indicate the standard deviation of three biological replicates. Different letters in each panel indicate significant differences at P < 0.05, based on one-way ANOVA and Duncan’s test. G Schematic diagram of the constructed reporter and effector vectors. H, I Fluorescence observations (H) and relative LUC/REN activity measurements (I) in Dual-LUC assays. Error bars indicate the standard deviation of three biological replicates. Different letters in each panel indicate significant differences at P < 0.05, based on one-way ANOVA and Duncan’s test