Genome-wide characterisation of HD-Zip transcription factors and functional analysis of PbHB24 during stone cell formation in Chinese white pear (Pyrus bretschneideri)

Abstract

Background: The homodomain-leucine zipper (HD-Zip) is a conserved transcription factor family unique to plants that regulate multiple developmental processes including lignificaion. Stone cell content is a key determinant negatively affecting pear fruit quality, which causes a grainy texture of fruit flesh, because of the lignified cell walls.

Results: In this study, a comprehensive bioinformatics analysis of HD-Zip genes in Chinese white pear (Pyrus bretschneideri) (PbHBs) was performed. Genome-wide identification of the PbHB gene family revealed 67 genes encoding PbHB proteins, which could be divided into four subgroups (I, II, III, and IV). For some members, similar intron/exon structural patterns support close evolutionary relationships within the same subgroup. The functions of each subgroup of the PbHB family were predicted through comparative analysis with the HB genes in Arabidopsis and other plants. Cis-element analysis indicated that PbHB genes might be involved in plant hormone signalling and external environmental responses, such as light, stress, and temperature. Furthermore, RNA-sequencing data and quantitative real-time PCR (RT-qPCR) verification revealed the regulatory roles of PbHB genes in pear stone cell formation. Further, co-expression network analysis revealed that the eight PbHB genes could be classified into different clusters of co-expression with lignin-related genes. Besides, the biological function of PbHB24 in promoting stone cell formation has been demonstrated by overexpression in fruitlets.

Conclusions: This study provided the comprehensive analysis of PbHBs and highlighted the importance of PbHB24 during stone cell development in pear fruits.

Keywords: Genome-wide analysis; HD-Zip; Lignin; Pear; Stone cell; Transcription factor.

Fig. 1

Genome distribution and collinearity of the pear HD-Zip family. HD-Zip genes are mapped to pear chromosomes by the TBtools. Pear chromosomes arranged in a circle. Red lines mark the position of genes on chromosomes. Blue lines indicate a collinear relationship among genes

Fig. 2

HD- and LZ domains are highly conserved across all HD-Zip proteins in the pear genome. Sequence logos of the HD- and LZ-domain are based on the sequence alignments of 56 PbHB proteins. Bit score indicates the information content for each position in the sequence. Multiple alignment analysis of 56 PbHB proteins was performed with ClustalW (see Supplementary Fig. S1)

Fig. 3

Phylogenetic relationships and gene structures of PbHBs. (A) Phylogenetic tree of 56 PbHB proteins. Phylogenetic tree was created in IQ-TREE v.1.6 using the Maximum Likelihood model with 1,000 bootstrap replicates. (B) Gene structure of PbHB family TFs in Chinese white pear. Abscissa represents the length of the nucleic acid sequence (bp)

Fig. 4

Phylogenetic analysis and motif compositions of HD-Zips from pear and Arabidopsis. (A) Phylogenetic tree of HD-Zip proteins from pear and Arabidopsis was constructed with IQ-TREE v.1.6 software. PbHBs were clustered into 4 distinct clades, marked by curves of different colours. (B) Schematic representation of the conserved motifs of the PbHBs identified by MEME. Each motif is indicated by a coloured box numbered at the bottom. Abscissa represents the amino acid length (C) Schematic representation of the distinctive domains exhibited by each subfamily of HD-Zip proteins. Abbreviations: MEKHLA domain, named after the highly conserved amino acids Met, Glu, Lys, His, Leu, Ala; N-term, N-terminus consensus; SAD, START adjacent domain; START, steroidogenic acute regulatory protein-related lipid transfer domain

Fig. 5

Cis-acting elements analysis of the promoter of PbHB genes. (A) Distribution of 15 cis-acting elements in the promoters of 56 PbHB genes. Abscissa represents the promoter sequence length. Scale bar indicates 300 bp. Different coloured boxes indicate different types of cis-acting elements. (B) Number of 15 cis-acting elements in promoters of 56 PbHB genes. The colour of the box indicates the number of cis-acting elements

Fig. 6

Heatmap indicates the expression pattern of HD-Zip genes in pear fruits. Gene expression of 40 PbHB genes was scaled according to the bar in colour that measures Z-scores of mean FPKM values. Five genes indicated in red means HD-Zip genes of subfamily III. Detailed information has listed in Table S5. Red represents up-regulated and green represents down-regulated. DAF, days after flowering

Fig. 7

Co-expression network of PbHBs with lignin-related genes. RNA-seq data were used to measure the expression similarity between gene pairs as PCC values. Detailed information is listed in Table S6. Size and colour depth of each object represent degree values measured by CytoNCA [84]. The values were then filtered with Excel software (with the parameter set as > 0.6). Data were visualised using Cytoscape software

Fig. 8

Tissue-specific expression of pear HD-Zip III genes. Primer sequences are listed in Table S7. Bar values represent the mean ± SD of three biological replicates. Different coloured bars represent different genes

Fig. 9

Transient overexpression of PbHB24 in pear fruitlets. (A) Phloroglucinol staining of the pulp of ‘Dangshansuli’ fruitlets after Agrobacterium-mediated overexpression. 35 S::EV, fruitlets injected by empty vector as a control; 35 S::PbHB24, PbHB24 overexpression vector mediated by 35 S strong promoter. (B) Relative expression of PbHB24 7 days after injection. The error bars are the means ± SD (n = 3). (C) Stone cell content of pear fruits 7 days after injection. The error bars are the means ± SD (n = 12). (D) Acetyl bromide lignin content of pear fruits 7 days after injection. The error bars are the means ± SD (n = 6). The vertical bars are the means ± SD of biological replicates and asterisks indicate significant differences by two-tailed Student’s t-test (*P < 0.05, **P < 0.01)