Genome-Wide Identification and Characterization of Vacuolar Processing Enzyme Gene Family and Diverse Expression Under Stress in Apple (Malus × Domestic)

Abstract

Vacuolar processing enzymes (VPEs) play an important role in stress resistance and development of plants. Despite their diverse roles, little information is available in apple (Malus × domestic). This study firstly presents the genome-wide identification of VPE family genes in apple, resulting in 20 family members those are unevenly distributed across six out of the 17 chromosomes. Phylogenetic analysis assigned these genes into four groups. Analysis of exon-intron junctions and motifs of each candidate gene revealed high levels of conservation within and between phylogenetic groups. Cis-element including w box, ABRE, LTR, and TC-rich repeats were found in promoters of MdVPEs. NCBI-GEO database shown that the expression of MdVPEs exhibited diverse patterns in different tissues as well as the infection of Pythium ultimum and Apple Stem Grooving Virus. Furthermore, qRT-PCR showed that MdVPE genes were responsive to salt, cadmium, low-temperature, and drought. Overexpression of MDP0000172014, which was strongly induced by salt and drought stress, significantly decreased Arabidopsis tolerance to salt stress. The genome-wide identification and characterization of MdVPEs in apple provided basic information for the potential utilization of MdVPEs in stress resistance.

Keywords: Genome-wide analysis; VPE gene family; apple (Malus × domestic); diverse expression; salt stress.

FIGURE 1

Phylogenetic analysis of apple and Arabidopsis VPE proteins. A total of 20 VPE proteins from apple and four from Arabidopsis were aligned using Clustal W. The phylogenetic tree was constructed using the MEGA 7.0 program by the neighbor-joining (NJ) method with bootstrap values 1,000 using protein sequences. According to Arabidopsis VPE protein sequcences, MdVPE proteins were devided into four groups. Different colors represent different groups.

FIGURE 2

Phylogenetic relationship, gene structure, conserved motif and proteins structure analysis of MdVPE genes. (A) Phylogenetic tree of 20 MdVPE proteins. The unrooted neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using full-length amino acid sequences of 20 MdVPE proteins, and the bootstrap test replicate was set as 1,000 times. (B) The exon-intron structure of MdVPE genes. Black boxes represent exons and black lines represent introns. (C) Distributions of conserved motifs in MdVPE genes. Ten putative motifs are indicated in different colored boxes. For details of motifs refer to Table 2.

FIGURE 3

The distribution of MdVPE genes in apple chromosomes. (A) The number of MdVPE genes in each chromosome. (B) MdVPEs were mapped onto apple chromosomes. The black boxes represented clusters of genes.

FIGURE 4

Predicted stress-related cis-elements in MdVPE promoters. Promoter sequences (–2,000 bp) of MdVPEs are analyzed by Plant CARE. Different color boxes represent different cis-element. According to the bottom scale, the upstream length to the translation start site can be inferred.

FIGURE 5

Expression profiles of MdVPE genes in different tissues of apple. NCBI-GEO data (GSE42873) were downloaded for the expression profile analysis. GSM1052622 flower-M67 and GSM1052625 flower-M74 represent the whole flower of M67 and the whole flower of M74, respectively. GSM1052624 fruit-M74-100aa, GSM1052627 fruit-M20-100aa, and GSM1052631 fruit-M20-harvest represent the 100 days after anthesis-fruit of M74, the 100 days after anthesis-fruit of M20 and fruit flesh at harvest of M20, respectively. GSM1052629 leaf-M14 and GSM1052634 leaf-M49 represents the whole leaf of M14, the whole leaf of M49, respectively. GSM1052637 Root-GD and GSM1052641 Root-X8877 represents the in vitro root of Golden Delicious, the in vitro root of X8877, respectively. GSM1052639 stem-X8877 and GSM1052643 stem-GD represent the fully developed-stem of X8877, the fully developed-stem of Golden Delicious, respectively. GSM1052646 seeding-GD and GSM1052649 seeding-X41002 represents the 10 days old-seedling of Golden Delicious, the 10 days old-seedling of X41002, respectively. GSM1052648 seed-X4442 × X2596 represents the dormant seed from cross X4442 and X2596. M67, M74, M20, M14, M49, X8877, Golden Delicious, X41002, X4442, and X2596 represent apple cultivars. RPKM values of MdVPE genes were transformed by log2 and the heatmap was constructed by TBtools.

FIGURE 6

Expression profile of MdVPEs under P. ultimum and Apple Stem Grooving Virus (ASGV) stress. (A) Expression profile of MdVPEs under Pythium ultimum stress. NCBI-GEO data (GSE62103) were downloaded for the expression profile analysis. GSM1519658 represents the apple root organization at 0 h, GSM1519662, GSM1519663, GSM1519664, and GSM1519665 represents the simulated inoculation mold 24, 48, 72, and 96 h, and the apple root organization, GSM1519669, GSM1519670, GSM1519671, and GSM1519672 represents apple root tissue when inoculated with humus 24, 48, 72, and 96 h, respectively. (B) Expression profile of MdVPEs under ASGV stress. NCBI-GEO data (GSE53825) were downloaded for the expression profile analysis. AP-Vinfect represents ASGV-infected asymptomatic apple plantlets and AP-Vfree represents virus-free apple plantlets. RPKM values of MdVPE genes were transformed by log2 and the heatmap was constructed by TBtools.

FIGURE 7

Expression profiles of MdVPE genes under salt, cadmium, cold, and drought stresses. Quantitative RT-PCR was used to investigate the expression levels of each MdVPE genes in apple roots. The data are presented as means ± standard deviations (SD) of three independent measurements from three individual plants. Means with different small letters in the column are significantly different at P < 0.05.

FIGURE 8

Overexpression of MDP0000172014 decreased the tolerance of Arabidopsis to salt stress. (A) Phenotype comparisons (B) and root length between the wild type (WT) and overexpressed MDP0000172014 transgenic Arabidopsis seedlings (T3-1, T3-2, and T3-3) after 100 mM NaCl treatment, and the length of the black line represents 1 cm. (C) The MDA, (D) H₂O₂, (E) rate of cell death, and (F) VPE activity of WT and transgenic Arabidopsis after 200 mM NaCl treatment. The data are presented as means ± standard deviations (SD) of three independent measurements from three individual plants. Means with different small letters in the column are significantly different at P < 0.05.