A Genome-Wide Analysis and Expression Profile of Heat Shock Transcription Factor (Hsf) Gene Family in Rhododendron simsii

Abstract

Heat shock transcription factors are key players in a number of transcriptional regulatory pathways that function during plant growth and development. However, their mode of action in Rhododendron simsii is still unclear. In this study, 22 RsHsf genes were identified from genomic data of R. simsii. The 22 genes were randomly distributed on 12 chromosomes, and were divided into three major groups according to their phylogenetic relationships. The structures and conserved motifs were predicted for the 22 genes. Analysis of cis-acting elements revealed stress-responsive and phytohormone-responsive elements in the gene promoter regions, but the types and number varied among the different groups of genes. Transcriptional profile analyses revealed that RsHsfs were expressed in a tissue-specific manner, with particularly high transcript levels in the roots. The transcriptional profiles under abiotic stress were detected by qRT-PCR, and the results further validated the critical function of RsHsfs. This study provides basic information about RsHsf family in R. simsii, and paves the way for further research to clarify their precise roles and to breed new stress-tolerant varieties.

Keywords: Rhododendron; RsHsf family; abiotic stress; gene expression.

Figure 1

Chromosomal locations of RsHsfs in R. simsii. The scale represents megabases (Mb) and the sizes of the chromosomes can be determined using the scale given at the left.

Figure 2

Phylogenetic analysis among the identified Hsf-conserved proteins in A. thaliana, C. sinensis and R. Simsii. The 21 A. thaliana, 25 C. sinensis and 22 R. simsii Hsf sequences were aligned using Muscle. The phylogenetic tree was constructed by MEGA11.0 with the maximum likelihood method, and the bootstrap value was set at 1000 repetitions. Different families and subclasses are indicated by different colors.

Figure 3

Phylogenetic tree, gene structure, and distribution of conserved motifs. (A) Phylogenetic tree constructed using MEGA 11.0 software. (B) Schematic of gene structure constructed using tools at gene structure display server. Coding sequences, untranslated regions and introns were represented by yellow boxes, purple boxes and black lines, respectively. The relative position was proportionally displayed based on the kilobase scale at the bottom of the figure. (C) Conserved motifs of RsHsf proteins. Each colored box represented a motif in each of the RsHsf proteins, with the motif’ s number represented. The sizes of the gene can be determined using the scale given at the bottom.

Figure 4

Cis-acting element analysis of promoter regions of RsHsfs. (A) Phylogenetic tree constructed using MEGA 11.0 software. (B) Cis-acting element analysis of RsHsfs. Different colored boxes were represented by different cis-acting elements. The coordinates at the bottom of the figure indicated the length of the gene promoter, which was defined as 2 kb before the start codon.

Figure 5

Transcriptional profiles of RsHsfs in different tissues. B, buds; TL, tender leaves; ML, mature leaves; TS, tender stems; MS, mature stems; F, flowers; R, roots. Gene expression in buds was regarded as control. Data are mean ± standard deviation (SD), calculated from three biological replicates. Vertical lines represent standard deviation. * and ** indicate significant difference at p < 0.05 and p < 0.01, respectively.

Figure 6

Expression profiles of RsHsfs under drought treatment. The detection of transcript levels was performed in leaves. Gene expression at 0 h was normalized to “1”. Data are mean ± standard deviation (SD), calculated from three biological replicates. Vertical lines represent standard deviation. * and ** indicate significant difference at p < 0.05 and p < 0.01, respectively.

Figure 7

Heat map of the expression profiles of RsHsf genes under heat and melatonin treatment. The detection of expressions was performed in leaves. CK, 25 °C melatonin−free plants; 35 °C, 35 °C, melatonin−free plants; 40 °C, 40 °C, melatonin−free plants; 35 °C/MT, 35 °C, melatonin−treated plants; 40 °C/MT, 40 °C, melatonin−treated plants. Log2 transformed FPKM values were used to create heat map. Yellow or blue indicate higher or lower relative abundance or each transcript in each sample.

Figure 8

Subcellular localization of RsHsfs. (A) 35sGFP. (B) RsHsf15-GFP fusion proteins. (C) RsHsf16-GFP fusion proteins. (D) RsHsf19-GFP fusion proteins. For (A–D), 35sGFP or RsHsfs-GFP fusion proteins were transiently expressed in tobacco. Left to right: green fluorescence, bright-field and merged microscope images. Scale bars: 100 µm.