Genome-Wide Identification of Brassinosteroid Signaling Downstream Genes in Nine Rosaceae Species and Analyses of Their Roles in Stem Growth and Stress Response in Apple

Abstract

Brassinosteroid signaling downstream genes regulate many important agronomic traits in rice. However, information on such genes is limited in Arabidopsis and Rosaceae species. We identified these genes in Arabidopsis and nine Rosaceae species. They were, respectively, named based on chromosomal locations. Segmental duplication and whole-genome duplication under purifying selection, as determined by Ka/Ks analysis, likely contributed to Rosaceae gene expansion. Apple (Malus domestica), Arabidopsis, and rice genes were generally similar, while several Rosaceae genes differed from their rice homologs in various characteristics, such as gene length, subcellular localization, transmembrane topology, conserved domains, secondary structures, and responses to external signals. The brassinosteroid downstream genes in apple were, respectively, induced or repressed by five phytohormones. Furthermore, these apple downstream genes were differentially expressed in different apple grafting combinations ("Nagafu No. 2"/"Malling 9" and "Nagafu No. 2"/"Nagafu No. 2") and long-short shoot varieties ("Yanfu No. 6" and "Nagafu No. 2"). Responses of the MdBZR genes to diverse stress signals were examined and candidate hub genes were identified. These findings indicated that several brassinosteroid signaling downstream genes in Rosaceae functionally differed from their rice homologs, and certain apple genes may play roles in plant height and stress responses. This study provided valuable information and presented enriched biological theories on brassinosteroid signaling downstream genes in apple. Identification of such genes serve to help expand apple breeding and growth. This study provides useful information for brassinosteroid signaling downstream genes.

Keywords: Rosaceae; apple; brassinosteroid signaling downstream genes; stem growth; stress responses.

FIGURE 1

Numbers of BR downstream genes for each classification in Rosaceae species. (A) Each species; (B) instability index; (C) transmembrane structure; (D) tandem gene duplications; (E) segmental gene duplications; (F) genes without introns.

FIGURE 2

Phylogenetic analysis of rice, Arabidopsis, and Rosaceae BR downstream genes.

FIGURE 3

Expression profiles of apple-specific BR downstream genes in different tissues. Each value represents mean ± standard error of three biological replicates. Different lowercase letters indicate significant differences at 0.05 level.

FIGURE 4

Effects of BR, IAA, and GA on MdBZR gene expression. qRT-PCR data are shown relative to day 0. Bars show mean ± standard error (n = 3). Asterisks indicate significant differences at 0.05 level (*).

FIGURE 5

Effects of BR, IAA, and GA on apple BR downstream genes (except for MdBZRs). qRT-PCR data are shown relative to 0 day. Bars show mean ± standard error (n = 3). *, ** indicate significant differences at 0.05 and 0.01 level, respectively.

FIGURE 6

Expression analysis of apple BR downstream genes in YF-CF and CF/M9-CF/CF. DABB: days after bud break. Each value represents mean ± standard error of three biological replicates. Asterisks indicate significant differences at 0.05 level (*) and 0.01 level (**).

FIGURE 7

Expression levels of MdBZRs after hormone (ABA, MeJA, and SA) and stress (drought and salt) treatments. qRT-PCR data are shown relative to 0 h. Bars show mean ± standard error (n = 3). Different lowercase letters indicate significant differences at 0.05 level.

FIGURE 8

Subcellular localization analysis of MdBZR1. Confocal images of transiently transformed tobacco epidermal cells with green fluorescent protein (GFP) or MdZR1-GFP.

FIGURE 9

Venn diagram indicating various functions of apple BR downstream genes in growth and stress responses. The left side of figure shows the number of genes responding to BR, IAA, GA, CK, as well as stress, and differently expressed in YF-CF and CF/M9-CF/CF. Black columns display the details of genes that are in the above 1–7 classes.