Genome-wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.)

Abstract

Background: Heat shock factors (Hsfs) play crucial roles in plant developmental and defence processes. The production and quality of pepper (Capsicum annuum L.), an economically important vegetable crop, are severely reduced by adverse environmental stress conditions, such as heat, salt and osmotic stress. Although the pepper genome has been fully sequenced, the characterization of the Hsf gene family under abiotic stress conditions remains incomplete.

Results: A total of 25 CaHsf members were identified in the pepper genome by bioinformatics analysis and PCR assays. They were grouped into three classes, CaHsfA, B and C, based on highly conserved Hsf domains, were distributed over 11 of 12 chromosomes, with none found on chromosome 11, and all of them, except CaHsfA5, formed a protein-protein interaction network. According to the RNA-seq data of pepper cultivar CM334, most CaHsf members were expressed in at least one tissue among root, stem, leaf, pericarp and placenta. Quantitative real-time PCR assays showed that all of the CaHsfs responded to heat stress (40 °C for 2 h), except CaHsfC1 in thermotolerant line R9 leaves, and that the expression patterns were different from those in thermosensitive line B6. Many CaHsfs were also regulated by salt and osmotic stresses, as well as exogenous Ca(2+), putrescine, abscisic acid and methyl jasmonate. Additionally, CaHsfA2 was located in the nucleus and had transcriptional activity, consistent with the typical features of Hsfs. Time-course expression profiling of CaHsfA2 in response to heat stress revealed differences in its expression level and pattern between the pepper thermosensitive line B6 and thermotolerant line R9.

Conclusions: Twenty-five Hsf genes were identified in the pepper genome and most of them responded to heat, salt, osmotic stress, and exogenous substances, which provided potential clues for further analyses of CaHsfs functions in various kinds of abiotic stresses and of corresponding signal transduction pathways in pepper.

Fig. 1

Motifs identified by MEME tools in pepper Hsfs. In total, 25 motifs were identified and are indicated by increasing numbers from 1 to 25. Because motif 13 was the same as motif 8, it was labelled as motif 8. Different motifs are indicated by different borders and colours. The names of the Hsf members from pepper and their combined P-values are on the left side of the figure, and the motif sizes are indicated at the bottom of the figure. The same number in different Hsfs refers to the same motif

Fig. 2

Neighbour-Joining phylogenetic tree of Hsf proteins from pepper, tomato, Arabidopsis, rice and maize. The N-proximal regions (from the start of the DNA-binding domain to the end of the HR-A/B region) of Hsf proteins were used to construct of the phylogenetic tree with MEGA 5.10. For Arabidopsis (prefixed by AT), tomato (prefixed by Solyc), rice (prefixed by Os) and maize (prefixed by ZM) Hsf proteins, both locus ID and subclass numbers are listed. CaHsf proteins are marked in red. An unrooted Neighbour-Joining analysis was performed with pairwise deletion and Poisson correction

Fig. 3

Tissue-specific expression analysis of pepper Hsf genes. Raw data were from RNA-seq data of each tissue from CM334. The analysed tissues including root, stem, leaf, pericarp (PC) and placenta (PL) at 6, 16, 25 days post-anthesis (DPA), PC and PL at mature green (MG) and at breaker (B) stages, PC and PL at 5 and 10 days post-breaker (B5 and B10, respectively). The data from 22 pepper Hsf genes (excluding CaHsfA1e, B3b and B4 whose date are absent) were used to create a heat map using HemI. The RNA-seq data for zero is indicated as white, and the other data were normalized using log2

Fig. 4

Relative gene expression levels of CaHsfs, analysed by qRT-PCR, in response to HS treatment in B6 and R9 leaves. HS treatment: 40 °C for 2 h; B6: pepper thermosensitive line; R9: pepper thermotolerant line. qRT-PCR data were normalized using the pepper ubiquitin-conjugating protein gene (UBI-3) and are shown relative to 0 h. The relative expression levels were calculated using the -ΔΔCT method and then a heat map with HemI was created

Fig. 5

qRT-PCR analysis of relative CaHsfs transcript levels in R9 plants exposed to various abiotic stresses. qRT-PCR data of HS as seen in Fig. 4. R9: pepper thermotolerant line; L-HS: heat stress (40 °C for 2 h); R-NaCl and S-NaCl: salt stress (300 mM NaCl for 6 h) responsiveness of CaHsf genes in roots and stems, respectively; R-Mannitol and S-Mannitol: osmotic stress (5 % mannitol for 6 h) responsiveness of CaHsf genes in roots and stems, respectively. The expression levels under salt and osmotic stress treatments were relative to that of the samples treated with water. L-CaCl₂, −Put, −ABA and -MeJA: CaCl₂ (15 mM for 6 h), putrescine (Put, 1.5 mM for 6 h), abscisic acid (ABA, 100 μM for 3 h) and methyl jasmonate (MeJA, 100 μM for 6 h) responsiveness of CaHsf genes in leaves, respectively. MeJA was dissolved in 10 % ethanol and other substances were dissolved in water; therefore, control seedlings were sprayed with 10 % ethanol (for the MeJA treatment) or water (for the CaCl₂, Put and ABA treatments). The expression levels are relative to that of the control samples

Fig. 6

Transient expression of the CaHsfA2-GFP fusion protein in onion epidermal cells. (a) Schematic diagram of the construction of the recombinant CaHsfA2-GFP vector. 35S: a constitutive promoter from the cauliflower mosaic virus; GFP: green fluorescent protein; NOS: nopaline synthase terminator. (b) Transient expression of GFP and CaHsfA2-GFP in onion epidermal cells. (b 1, b 3, b 5) Onion epidermal cells transformed with 35S::GFP as control. (b 2, b 4, b 6) Onion epidermal cells transiently expressing 35S::CaHsfA2-GFP. (b 1, b 2) Merged images. (b 3, b 4) Dark field images. (b 5, b 6) Bright field images. Bars = 0.1 mm

Fig. 7

Transactivational activity of the CaHsfA2 protein in yeast. (a) Schematic diagram illustrating the CaHsfA2 cDNA fragments encoding CaHsfA2 that was fused to the DNA sequences encoding the GAL4 DNA binding domain in the yeast vector pGBKT7. (b) Transactivational analysis of CaHsfA2 in yeast. Fusion proteins of pGBKT7-CaHsfA2 and pGBKT7 were expressed in yeast strain AH109. The transformants were streaked on the SD/Trp- and SD/Trp-Ade-His- (with X-α-gal) medium. The plates were incubated at 30 °C for 3 d

Fig. 8

Expression level of CaHsfA2 in pepper in responses to HS treatment. B6: pepper thermosensitive line; R9: pepper thermotolerant line. (a) The time course of HS treatments. Triangles indicate the time points when the leaves were collected (sample a-i). (b) Expression profiles of CaHsfA2 in pepper leaves under the HS treatment at different time points. Expression data were normalized with UBI-3 as the reference gene. The expression levels are relative to that of the sample a from B6 and R9, respectively