Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses

Abstract

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis "Xuegan" plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.

Keywords: 14-3-3s; CitGF14s; Citrus sinensis; abiotic stress; citrus canker; sweet orange.

Figure 1

Phylogenetic trees of 14-3-3 family genes in Citrus sinensis (CitGF14s, indicated by the star symbol), Arabidopsis (AtGRF), Populus trichocarpa (PtGRF), and Oryza sativa (OsGRF) constructed using the MEGA6.0 program with neighbor-joining method within 1000 bootstrap replicates. The green and blue shade separates the ε and non-ε groups.

Figure 2

Genomic distribution of 14-3-3 (CitGF14s) genes across nine Citrus sinensis chromosomes. The chromosome number is indicated at the top of each chromosome. The scale is in megabases (Mb). Chromosomal locations of CitGF14s were indicated based on the physical position of each gene.

Figure 3

Structure analysis of CitGF14s genes in Citrus sinensis. The graphic representation is displayed using GSDS (http://gsds.cbi.pku.edu.cn/). The unrooted phylogenetic tree was constructed using the amino sequences of CitGF14s genes by the Neighbor-Joining method with 1000 bootstrap replicates. Exons consisted of CDS shown as orange boxes, introns are shown as thin lines, and UTRs are shown as blue boxes.

Figure 4

Expression analysis of CitGF14s in different tissue and organs of Citrus sinensis plants. (A) Expression profiles of CitGF14s derived from RNA-Seq data where RPKM expression values were log-transformed for normalization. Log2 based RPKM values were used for creating the heat map with clustering by HemI. The scale represents the relative intensity of RPKM values. (B) RT-qPCR analysis of the expression patterns of CitGF14s in different organs of C. sinensis. The relative expression was normalized using the ACTIN and GAPDH genes as references. Each bar represented the mean of four biological replications with standard error. Different letters on the top of bars indicate significant differences analyzed by Tukey’s HSD test at p < 0.05 level.

Figure 5

Expression profiles of CitGF14s in response to different stresses. (A) The microarray data was downloaded from NCBI database. Heatmap shows hierarchical clustering of CitGF14 expression after plants were inoculated with citrus canker pathogens: Xanthomonas axonopodis pv. citri (Xaa) and Xanthomonas axonopodis pv. Aurantifolii (Xac) and citrus greening pathogen: Candidatus Liberibacter asiaticus (Ca. Las). Gene expression values were calculated based on the ratios between the infection and the mock (control). Heatmap was generated based on log2 (infection expression/mock expression) values by using HemI. The color scale represents the relative intensity level of transcript abundance. (B) RT-qPCR analysis of CitGF14s expression after C. sinensis “Xuegan” was inoculated with Xac. The relative expression was normalized using the ACTIN and GAPDH genes as references using 2^−ΔΔCt method. The values were based on the means of four biological replications.

Figure 6

RT-qPCR analysis of CitGF14 expressions after C. sinensis “Xuegan” plants were sprayed with jasmonate (JA), abscisic acid (ABA), ethephon (ETH), and salicylic acid (SA) (A), exposed to low (4 °C) and high (42 °C) temperatures as well as wounding (B), and treated with NaCl (200 mM) and polyethylene glycol (PEG) (20% PEG6000), a simulated drought stress (C). The relative expression was normalized using the ACTIN and F-box gene as references by 2^−ΔΔCt method. Heatmaps were generated based on log2 (treatment expression/control expression) with HemI. The values were based on the means of four biological replications.

Figure 7

Yeast two-hybrid (Y2H) assay to test interactions among nine CitGF14 (14-3-3) proteins of Citrus sinensis. The coding sequences of the CitGF14s genes were cloned into the Y2H vectors pGADT7 (AD) and pGBKT7 (BD), and introduced into yeast cells Y2H gold. Transformants were assayed for growth on SD/-Trp-Leu (A) and SD/-Trp-Leu-His-Ade-x-α-gal (B) nutritional selection media. EV was empty vector pGADT7 used as a negative control.

Figure 8

Protein–protein interaction network of CitGF14s and target proteins. The colored nodes represent proteins of miscellaneous functions in this network (see picture detail). Solid line or dotted line represents the predicted interaction based on ortholog or domain.