Genome-wide analysis of the GH3 family in apple (Malus × domestica)

Abstract

Background: Auxin plays important roles in hormone crosstalk and the plant's stress response. The auxin-responsive Gretchen Hagen3 (GH3) gene family maintains hormonal homeostasis by conjugating excess indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acids (JAs) to amino acids during hormone- and stress-related signaling pathways. With the sequencing of the apple (Malus × domestica) genome completed, it is possible to carry out genomic studies on GH3 genes to indentify candidates with roles in abiotic/biotic stress responses.

Results: Malus sieversii Roem., an apple rootstock with strong drought tolerance and the ancestral species of cultivated apple species, was used as the experimental material. Following genome-wide computational and experimental identification of MdGH3 genes, we showed that MdGH3s were differentially expressed in the leaves and roots of M. sieversii and that some of these genes were significantly induced after various phytohormone and abiotic stress treatments. Given the role of GH3 in the negative feedback regulation of free IAA concentration, we examined whether phytohormones and abiotic stresses could alter the endogenous auxin level. By analyzing the GUS activity of DR5::GUS-transformed Arabidopsis seedlings, we showed that ABA, SA, salt, and cold treatments suppressed the auxin response. These findings suggest that other phytohormones and abiotic stress factors might alter endogenous auxin levels.

Conclusion: Previous studies showed that GH3 genes regulate hormonal homeostasis. Our study indicated that some GH3 genes were significantly induced in M. sieversii after various phytohormone and abiotic stress treatments, and that ABA, SA, salt, and cold treatments reduce the endogenous level of axuin. Taken together, this study provides evidence that GH3 genes play important roles in the crosstalk between auxin, other phytohormones, and the abiotic stress response by maintaining auxin homeostasis.

Figure 1

Phylogenetic analysis and gene structure of apple GH3 members. A Phylogeny of apple GH3 proteins, generated using MEGA5 (using the neighbor-joining method and a bootstrap test with 1000 iterations). B Gene structure of the corresponding apple GH3 proteins, generated by a gene structure display server. The black boxes represent exons and lines represent introns. 0, 1, and 2 represent phase 0, 1, and 2 introns.

Figure 2

Mapping of MdGH3s and segmental duplication regions on apple chromosomes. Segmental duplication regions were determined using the SyMAP database. Genes and segmental duplication regions were mapped to the apple chromosomes via the Circos tool. The apple chromosomes were arranged in a circle. Ribbon links represent segmental duplication regions.

Figure 3

Phylogenetic tree of M. domestica and Arabidopsis GH3 proteins. The phylogeny was constructed using the neighbor-joining method and a bootstrap test with 1000 iterations, using MEGA5 software, and alignments were generated with ClustalW.

Figure 4

qRT-PCR analysis of MdGH3 genes under normal growth conditions. Seedlings were grown on Hoagland solution for Data were normalized to the expression level of the apple HistoneH3 gene. The expression level of apple HistoneH3 was assumed to be 1e⁺⁵. Mean expression values were calculated from three independent replicates. Error bars represent the standard error of the mean.

Figure 5

qRT-PCR analyses of MdGH3 genes in plants subjected to various abiotic treatments. A heat map shows the relative RNA level of MdGH3 genes in plants under IAA, ABA, SA, JA, drought, cold, and salt treatments, compared to the basal expression level of MdGH3 genes in plants under normal growth conditions. Data were normalized to apple HistoneH3 gene expression. Fold difference was designated as the log2 value. The color scale representing the relative RNA level is shown to the right of the heat map.

Figure 6

GUS activity assays of whole DR5::GUS transgenic seedlings. (A) DR5::GUS seedlings were incubated for an increasing number of hours with 10 μM IAA. (B) DR5::GUS seedlings were incubated for 3 h with 10 μM IAA and increasing concentrations of ABA. (C) DR5::GUS seedlings were incubated for 3 h with 10 μM IAA and increasing concentrations of SA. (D) DR5::GUS seedlings were incubated for 12 h with 10 μM IAA and increasing concentrations of NaCl. (E) DR5::GUS seedlings were incubated with 10 μM IAA and grown in a growth chamber set to 4°C under a 16/8 h light/dark cycle. The means and SEs of three replicates are shown.

Figure 7

The effect of IAA, ABA, SA, JA, NaCl, and cold on the expression of DR5::GUS in transgenic seedlings. GUS staining of representative root segments of DR5::GUS seedlings after treatment with (A) 10 μM IAA for an increasing number of hours, (B) 10 μM IAA and an increasing concentration of ABA for 3 h, (C) 10 μM IAA and an increasing concentration of SA for 3 h, (D) 10 μM IAA and an increasing concentration of JA for 3 h, (E) 10 μM IAA and an increasing concentration of NaCl for 12 h, and (F) 10 μM IAA and transfer to a growth chamber set at 4°C under the 16/8 h light/dark cycle for an increasing number of hours. Scale bars = 1 cm.