Genome-Wide Identification and Characterization of RdHSP Genes Related to High Temperature in Rhododendron delavayi

Abstract

Heat shock proteins (HSPs) are molecular chaperones that play essential roles in plant development and in response to various environmental stresses. Understanding R. delavayi HSP genes is of great importance since R. delavayi is severely affected by heat stress. In the present study, a total of 76 RdHSP genes were identified in the R. delavayi genome, which were divided into five subfamilies based on molecular weight and domain composition. Analyses of the chromosome distribution, gene structure, and conserved motif of the RdHSP family genes were conducted using bioinformatics analysis methods. Gene duplication analysis showed that 15 and 8 RdHSP genes were obtained and retained from the WGD/segmental duplication and tandem duplication, respectively. Cis-element analysis revealed the importance of RdHSP genes in plant adaptations to the environment. Moreover, the expression patterns of RdHSP family genes were investigated in R. delavayi treated with high temperature based on our RNA-seq data, which were further verified by qRT-PCR. Further analysis revealed that nine candidate genes, including six RdHSP20 subfamily genes (RdHSP20.4, RdHSP20.8, RdHSP20.6, RdHSP20.3, RdHSP20.10, and RdHSP20.15) and three RdHSP70 subfamily genes (RdHSP70.15, RdHSP70.21, and RdHSP70.16), might be involved in enhancing the heat stress tolerance. The subcellular localization of two candidate RdHSP genes (RdHSP20.8 and RdHSP20.6) showed that two candidate RdHSPs were expressed and function in the chloroplast and nucleus, respectively. These results provide a basis for the functional characterization of HSP genes and investigations on the molecular mechanisms of heat stress response in R. delavayi.

Keywords: RNA-seq data; RdHSP gene family; expression pattern; high-temperature stress; subcellular localization.

Figure 1

Chromosomal distribution of the RdHSP family genes. The position of each RdHSP gene is marked on the right side of each chromosome (Chr). The size of the chromosome is represented by its relative length. Tandemly duplicated gene pairs are indicated with a red bar.

Figure 2

Phylogenetic analysis of HSP subfamily genes. The unrooted phylogenetic trees were constructed using the neighbor-joining method in MEGA 10.0 software with bootstrap test (replicated 1000 times). (a) An unrooted phylogenetic tree of the HSP100 subfamily genes; (b) an unrooted phylogenetic tree of the HSP90 subfamily genes; (c) an unrooted phylogenetic tree of the HSP20 subfamily genes; (d) an unrooted phylogenetic tree of the HSP60 subfamily genes; (e) an unrooted phylogenetic tree of the HSP70 subfamily genes. Different font colors represent the different Rhododendron species: R. delavayi (red), R. williamsianum (purple), R. simsii (black), R. ovatum (blue), R. henanense (green), and R. irroratum (light blue). Latin numbers (I–X) represent different groups.

Figure 3

Schematic diagram of the conserved domain, motif composition, and intron–exon distribution of RdHSP family genes in R. delavayi. (a). The conserved domain in classified members of each RdHSP subfamily protein. (b). The conserved motifs in each RdHSP protein. Schematic diagram of motif composition in R. delavayi RdHSPs was explored using MEME. The relative positions of each motif in RdHSP proteins are shown in different colors. The black lines represent non-conserved sequences. (c). Exon–intron distribution of RdHSP genes in R. delavayi. The exons are represented by orange rectangles. The black lines connecting two exons represent introns.

Figure 4

Cis-element analysis in the promoters of the RdHSP family genes. Locations of cis-elements in the 2 kb sequences upstream of RdHSP genes. Different kinds of cis-elements are represented with different colored rectangular boxes.

Figure 5

Segmentally duplicated gene pairs of RdHSP genes in the R. delavayi genome (a) and the orthologous relationships of HSP genes across six Rhododendron species (b–f). The red lines indicated segmentally duplicated gene pairs (a) and orthologous gene pairs (b–f). The chromosome number is indicated at the top of each chromosome.

Figure 6

Ka, Ks, and Ka/Ks distributions of orthologous HSP gene pairs. (a) Ka, Ks, and Ka/Ks ratio of segmental duplicated gene pairs; (b) orthologous HSP gene pairs between R. delavayi and R. henanense; (c) orthologous HSP gene pairs between R. delavayi and R. irroratum; (d) orthologous HSP gene pairs between R. delavayi and R. ovatum; (e) orthologous HSP gene pairs between R. delavayi and R. simsii; (f) orthologous HSP gene pairs between R. delavayi and R. williamsianum. The box plots are exhibiting the distributions of Ka, Ks, and Ka/Ks values among paralogs and orthologs. The small square and the line in the box represent average and median values of the Ka, Ks, and Ka/Ks values, respectively.

Figure 7

GO and KEGG enrichment analysis. (a) GO enrichment analysis of the RdHSP genes. (b) KEGG pathway enrichment analysis of the RdHSP genes. GO, Gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 8

Expression patterns of RdHSPs in R. delavayi under high-temperature treatments. The scale bars represent the log₂ transformations of the RPKM values. Light green indicates low expression and red indicates high expression.

Figure 9

Expression pattern validation of 15 RdHSPs in high-temperature treated R. delavayi leaves. The expression levels of 15 RdHSP genes relative to 18S rRNA were determined by qRT-PCR. Three technical replicates and three biological replicates were applied for each data point. Data were presented as means ± SD (N = 6). Lowercase letters above bars represent the significant differences between the high-temperature treated R. delavayi leaves and control group (p < 0.05).

Figure 10

Protein–protein interaction networks among RdHSPs. The black lines represent the interaction strength between proteins.

Figure 11

Subcellular localization of two candidate RdHSP proteins (RdHSP20.6 and RdHSP20.8). GFP, CHI, DIC, and Merge represent green fluorescence field (488 nm), chloroplast autofluorescence field, bright field, and superposition field, respectively. The proteins RdHSP20.6 and RdHSP20.8 are localized in the cytoplasm chloroplast indicated by white arrows. Bars of 20 µm.