Genome-wide identification of PbrbHLH family genes, and expression analysis in response to drought and cold stresses in pear (Pyrus bretschneideri)

Abstract

Background: The basic helix-loop-helix (bHLH) transcription factors play important roles in many processes in plant growth, metabolism and responses to abiotic stresses. Although, the sequence of Chinese white pear genome (cv. 'Dangshansuli') has already been reported, there is still a lack of clarity regarding the bHLH family genes and their evolutionary history.

Results: In this work, a genome-wide identification of the bHLH genes in Chinese white pear was performed, and we characterized the functional roles of these PbrbHLH genes in response to abiotic stresses. Based on the phylogenetic analysis and structural characteristics, 197 identified bHLH genes could be well classified into 21 groups. Expansion of PbrbHLH gene family was mainly driven by WGD and dispersed duplication with the purifying selection from the recent WGD. The functional annotation enrichment showed that the majority of PbrbHLHs were enriched in the GO terms and KEGG pathways involved in responds to stress conditions as TFs. Transcriptomic profiles and qRT-PCR revealed that PbrbHLH7, PbrbHLH8, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 were significantly up-regulated under cold and drought treatments. In addition, PbrbHLH195-silenced pear seedlings display significant reduced cold tolerance, exhibiting reduced chlorophyll content, as well as increased electrolyte leakage and concentrations of malondialdehyde and H₂O₂.

Conclusion: For the first time, a comprehensive analysis identified the bHLH genes in Chinese white pear and demonstrated that PbrbHLH195 is involved in the production of ROS in response to cold stress, suggesting that members of the PbrbHLH family play an essential role in the stress tolerance of pear.

Keywords: Chinese white pear; Cold stress tolerance; Drought stress tolerance; Evolution; Gene family; VIGS; bHLH TF.

Fig. 1

Un-rooted phylogenetic tree of PbrbHLH proteins.MEGA 7 was used to construct the phylogenetic tree based on the protein sequences. iTOL was used to annotate and review the phylogenic tree. The proteins were clustered into 21 groups. Different background colors indicate the different group of the PbrbHLH proteins

Fig. 2

Schematics of the gene structure and conserved motifs in the PbrbHLH family. a Subgroup classification. Phylogenetic tree was generated using 197 PbrbHLH genes with MEGA7. The subgroup names were labeled accordingly. b Conserved motif analysis. Twenty distinct motifs were identified with MEME suite and each motif was represented with different color. c. Gene structural analysis

Fig. 3

Distribution and collinearity of the PbrbHLHs. Red lines along the circumference of the circle mark the positions of genes on chromosomes. The lines in different colors inside the circle indicate collinearity relationships among PbrbHLH genes

Fig. 4

Expression profile of PbrbHLHs under drought and cold stresses. Expression analyses of PbrbHLHs using previous published transcriptome data under cold and drought stress conditions

Fig. 5

Expression analysis of PbrbHLHs undercold and drought stresses. a Relative expression of PbrbHLH8, PbrbHLH80, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 with cold treatment. b Relative expression of PbrbHLH7, PbrbHLH119, PbrbHLH128, PbrbHLH160, PbrbHLH161 and PbrbHLH195 with drought treatment. The pear Actin was used as internal reference for the normalization. The statistical analyses were performed using student’s t-test (* p < 0.05)

Fig. 6

Cold tolerance assay of PbrbHLH195-silenced Pyrus betulaefolia plants. a-c Phenotype of 1-month-old PbrbHLH195-silenced plants before and after cold treatment for 8 days (a). Electrolyte leakage (EL) (b) and malondialdehyde (MDA) concentrations (c) after cold treatment. d-g Chlorophyll fluorescence imaging of silenced plants and WT plants(d), Fv/Fm ratios (e), Chl content (f) and phenotype (g) of WT and pTRV-PbrbHLH195 silencing plants (pTRV-1, pTRV-2 and pTRV-3) at the end of the chilling stress. h-i In situ accumulation of H₂O₂ of WT and silencing plants, as revealed by histochemical staining with 3, 3-diaminobenzidine (DAB) (h) after cold treatment. Quantitative measurement of H₂O₂ (i) levels after cold treatment. The expression of PbrbHLH195 was detected by RT-PCR (j) and qRT-PCR (k) at 8d after cold treatment