Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale

Abstract

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

Keywords: AP2 transcription factor; Dendrobium officinale; development; dual-luciferase reporter gene system; gene expression.

Figure 1

Phylogenetic analysis and conserved domain of the AP2 proteins from D. officinale, O. sativa and A. thaliana. (A) Phylogenetic tree of DoAP2 proteins. A total of 14 AP2 proteins from D. officinale, 16 from O. sativa and 18 from A. thaliana were aligned using ClustalX to generate a FASTA alignment file. The phylogenetic tree was constructed using the MEGA 7.0 program and the neighbor-joining (NJ) method with 1000 bootstrap replications based on the alignment file; (B) Conserved domain of DoAP2 proteins. The location and size of AP2 domains are shown by different colors.

Figure 2

Protein–protein interaction network analysis of DoAP2 proteins using STRING 11. Cyan line represents data from curated databases, green lines indicate gene neighborhoods, and black lines indicate co-expression. Red text = DoAP2s; black text = the proteins in A. thaliana.

Figure 3

Prediction of cis-responsive elements in the 2-kbp upstream region of the initiation codon of 14 DoAP2 genes. Different colored boxes indicated different cis-responsive elements. MeJA, methyl jasmonate.

Figure 4

Expression analysis of DoAP2 genes during plant regeneration from PLBs by qRT-PCR. PLB, protocorm-like bodies; MS, multiple shoots. Each data bar represents the mean ± standard deviation (SD) of three biological replicates (n = 3). ND, not detected.

Figure 5

Expression analysis of DoAP2 genes during flower development by qRT-PCR. FB1, small flower buds (about 5 mm long); FB2, medium flower buds (about 10 mm long); FBF, fully bloomed flowers. Each data bar represents the mean ± standard deviation (SD) of three biological replicates (n = 3). ND, not detected.

Figure 6

Expression analysis of DoAP2 genes in response to abiotic stresses (cold, PEG and NaCl) by qRT-PCR. PEG treatment, 15% polyethylene glycol (PEG)-6000; NaCl treatment, 250 mM NaCl; cold treatment, 4 °C. Each data bar represents the mean ± standard deviation (SD) of three biological replicates (n = 3).

Figure 7

Subcellular localization of four DoAP2 proteins (DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11) in A. thaliana protoplasts. Bars = 5 μm.

Figure 8

Transcriptional activity assay of DoAP2 genes in tobacco leaves. (A) Schematic presentation of the reporter and effector vectors; (B) Transcriptional repression ability of DoAP2 proteins in tobacco leaves. Double asterisks (**) indicate significant differences in two-treatment comparisons (p < 0.01) using Dunnett’s test, compared with the negative control (pBD). Each value represents the means of six biological replicates.