Genome-wide identification and functional analysis of U-box E3 ubiquitin ligases gene family related to drought stress response in Chinese white pear (Pyrus bretschneideri)

Abstract

Background: The plant U-box (PUB) proteins are a family of ubiquitin ligases (E3) enzymes that involved in diverse biological processes, as well as in responses to plant stress response. However, the characteristics and functional divergence of the PUB gene family have not yet been previously studied in the Chinese white pear (Pyrus bretschneideri).

Results: In the present study, we identified 62 PbrPUBs in Chinese white pear genome. Based on the phylogenetic relationship, 62 PUB genes were clustered into five groups. The results of conserved motif and gene structure analysis supported the classification phylogenetic tree. The PbrPUB genes were unevenly distribution on 17 pear chromosomes, chromosome 15 housed most member of PUB family, with eight PUB genes. Cis-acting element analysis indicated that PUB genes might participate in diverse biological processes, especially in the response to abiotic stresses. Based on RNA-data from 'Dangshansuli' at seven tissues, we found that PUB genes exhibited diverse of expression level in seven tissues, and qRT-PCR experiment further supported the reliable of RNA-Seq data. To identify candidate genes associated with resistance, we conducted qRT-PCR experiment the expression level of pear seed plant under four abiotic stresses, including: ABA, dehydration, salt and cold treatment. One candidate PUB gene associated with dehydration stress was selected to conduct further functional experiment. Subcellular localization revealed PbrPUB18 protein was located on cell nucleus. Furthermore, heterologous over-expression of PbrPUB18 in Arabidopsis indicated that the over-expression of PbrPUB18 could enhance resistance in drought treatment. In conclusions, we systematically identified the PUB genes in pear, and provided useful knowledge for functional identification of PUB genes in pear.

Keywords: Abiotic stresses; PUB gene family; PbrPUB18; Pyrus; Ubiquitin ligases.

Fig. 1

Phylogenetic tree analysis of PUB gene family. a A Neighbor–Joining (NJ) tree of PUB proteins from three species, including pear, tomato and Arabidopsis. The phylogenetic tree was constructed by Mega-X software with 1000 boot strap. The red star, green triangle and blue tick represents the PUB proteins in Arabidopsis, pear and tomato, respectively. All of 185 PUB proteins from three species were clustered into five subgroups, named Group I, II, III, IV and V; b Five pie plots represented the percentage of PUB genes of three species in five groups. The orange part represented the percentage of PUB genes in pear, and the blue part represented the percentage of PUB genes in tomato, and the purple part presented the percentage of PUB genes in Arabidopsis

Fig. 2

The conserved motifs and gene structure analysis of PUB gene family in pear. a A Neighbor–Joining (NJ) phylogenetic tree of 62 pear PUB genes. The phylogenetic tree was constructed by Mega-X with 1000 bootstrap. The red branches indicated group I; the blue branches indicated group II; the orange branches indicated group III; the green branches indicated group IV; the purple branches indicated group V; b The conserved motifs analysis of PbrPUB genes in pear. A total of 20 motifs were predicated by MEME tool, named Motif 1–20. The scale bar indicates 200 aa; c The gene structure analysis of PUB genes in pear, including UTR, intron and exon. The green rectangles represented UTR; the yellow rectangles represented Exon; the grey lines presented Intron. The scale bar indicates 2 kb

Fig. 3

The location distribution and synteny analysis of PUB genes in pear genome. a The distribution pattern of PbrPUBs in 17 pear chromosomes. Due to no PUB genes were mapped on Chromosomes 8, we didn’t show it in the Fig. 3; b The distribution pattern synteny analysis of PUB genes family. The red lines indicated the synteny gene pairs of PUB gene family

Fig. 4

The cis-acting elements analysis of putative promoter of 62 PbrPUB genes. a The distribution pattern of 15 cis-acting elements of putative promoter of PUB gene family in pear. The phylogenetic tree is same with the phylogenetic tree in Fig. 2a. b The number of 15 cis-acting elements of putative promoter of PbrPUB genes. The color scale at the top right indicated the number of cis-acting elements. Green color indicated the number of cis-acting elements on PUB member. 15 cis-acting elements including: (I) Abscisic acid responsiveness; (II) MeJA-responsiveness; (III) Gibberellin-responsive element; (IV) Light responsive element; (V) MYB binding site involved in drought-inducibility; (VI) Salicylic acid responsiveness; (VII) Anaerobic induction; (VIII) Auxin-responsive element; (IX) Zein metabolism regulation; (X) Defense and stress responsive element; (XI) Low-temperature responsiveness; (XII) MYB binding site involved in light responsiveness; (XIII) Endosperm expression; (XIV) Wound-responsive element; (XV) MYB binding site involved in flavonoid biosynthetic genes regulation

Fig. 5

The expression pattern analysis of PbrPUB gene family in seven different tissues. a The heatmap of expression level of PbrPUBs in seven different tissues, including stem, ovary, petal, sepal, bud, fruit and leaf. Pheatmap, an R package, were used to generate the heatmap. The color scale represented the RPKM values normalized by log₂(RPKM + 1). Red color represented high expression, while blue represented low expression; b The expression levels of 15 randomly selected PbrPUBs in seven different tissues. Seven tissues are comprised of bud, stem, ovary, petal, sepal, fruit and leaf. The x-axes represented seven different tissues; the y-axes represented the relatively expression of PUB genes

Fig. 6

The expression level of 11 randomly selected PbrPUB genes in four abiotic stresses. a For dehydration treatment, the shoots were placed on dry filter papers for 0, 1, 6, 9, 12 and 24 h; b For cold stress, the seedlings were placed in the chamber set at 4 °C for 0, 1, 6, 9, 12, 24, 48 and 96 h; c For salt stress, the seedlings were placed in solution containing 200 mM NaCl solution for 0, 2, 4, 6, 8, 12 and 36 h; d For ABA stress, The seedlings were dipped in solution containing 100 μM ABA for 0, 1, 3, 6, 9, 12 and 36 h. The x-axes represented time after treatment; the y-axes represented the relatively expression of PbrPUB genes

Fig. 7

The subcellular localization of PbrPUB18. a Tobacco leaf epidermal cells were transiently transformed with constructs containing 35S:GFP vector alone as control; b Transient expression of fusion plasmid (35S: PbrPUB18-GFP) in tobacco leaf epidermal cells. The nucleus was identified by DAPI staining. Green fluorescence images, DAPI staining mages, blight field images and the merged images are shown from left to right. Scale bars = 20 μm

Fig. 8

Drought tolerance assay of transgenic Arabidopsis plants overexpressing PbrPUB18. a Phenotypes of 20-day-old transgenic plants and WT before and after 12 days drought stress; b Images of (Fv/Fm). The false color code depicted on the right of the image ranges from 0 (black) to 1.0 (purple); Electrolyte leakage (c), MDA contents (d) in the WT, OE-4 and OE-5 after drought treatment; e Fv/Fm of WT, OE-4 and OE-5 before and after drought stress; f Histochemical staining with DAB and NBT for detection of H₂O₂ and O₂⁻, respectively, in WT, OE-4 and OE-5 after drought stress for 12 days; Levels of H₂O₂ (g) and anti-O₂⁻ (h) in Arabidopsis WT, OE-4 and OE-5 after drought treatment. Asterisks indicate that the value is significantly different from that of the WT at the same time point (**P < 0.01; ***P < 0.001)