Identification and Expression Profiling of the Auxin Response Factors in Dendrobium officinale under Abiotic Stresses

Abstract

Auxin response factor (ARF) proteins play roles in plant responses to diverse environmental stresses by binding specifically to the auxin response element in the promoters of target genes. Using our latest public Dendrobium transcriptomes, a comprehensive characterization and analysis of 14 DnARF genes were performed. Three selected DnARFs, including DnARF1, DnARF4, and DnARF6, were confirmed to be nuclear proteins according to their transient expression in epidermal cells of Nicotiana benthamiana leaves. Furthermore, the transcription activation abilities of DnARF1, DnARF4, and DnARF6 were tested in a yeast system. Our data showed that DnARF6 is a transcriptional activator in Dendrobium officinale. To uncover the basic information of DnARF gene responses to abiotic stresses, we analyzed their expression patterns under various hormones and abiotic treatments. Based on our data, several hormones and significant stress responsive DnARF genes have been identified. Since auxin and ARF genes have been identified in many plant species, our data is imperative to reveal the function of ARF mediated auxin signaling in the adaptation to the challenging Dendrobium environment.

Keywords: ARF; Dendrobium officinale; abiotic stress; auxin; transcriptional activator.

Figure 1

Phylogenetic relationships of Arabidopsis and D. officinale ARF proteins. An unrooted phylogenetic tree was constructed using MEGA 5.1 (The Biodesign Institute, Tempe, AZ, USA) by N–J method. Bootstrap values are presented for all branches. All ARF family genes were grouped into six subgroups named from I to VI. Different subfamilies were showed by different colorized shading.

Figure 2

Protein sequences alignment and domain analysis of DnARF family proteins. Alignment of DnARF proteins obtained with the ClustalW program under default parameters. Multiple alignments of the domains DBD, ARF, MR, and CTD of the DnARF proteins were showed by different color lines. DBD: B3 DNA-binding domain; ARF: AUX_RESP domain; MR: middle region; CTD: C-terminal dimerization domain; NLS: nuclear localization signals. Colorized shading indicates identical and conversed amino acid residues, respectively. Two NLSs were marked by black asterisks.

Figure 3

Analysis of motif distribution in DnARF proteins. Analysis of motif distribution in DnARF proteins. (a) Ten classical motifs in DnARF proteins were analyzed by MEME (Multiple Em for Motif Elicitation) online tool. The width of each motif ranged from six to 50 amino acids. Different color blocks represent different motifs. (b) Analysis of specific amino acid conservation in each motif. The height of each character represents different conservative degrees.

Figure 4

Analysis and classification of ARF family in D. officinale. (a) Amino acid compositions of the MR domains in various DnARF proteins. Different colors indicated different types of amino acids. (b) Classification of DnARF proteins based on their amino acid preferences and domain structures. DBD: B3 DNA-binding domain; MR: middle region; CTD: C-terminal dimerization domain; Q: glutamine; S: serine; L: leucine; G: glycine; P: proline; AD: activation domain; RD: repression domain.

Figure 5

Subcellular localization and transcriptional activation of three selected DnARFs. (a) DnARF gene-GFP fusion constructs transiently expressed in tobacco epidermis cells. Localization of DnARF6, DnARF1, and DnARF4 fusion protein. Left to right: green fluorescence, red fluorescence, bright-field and merged. (b) Transcriptional activities of DnARF6, DnARF1, and DnARF4 were tested by the yeast system. The growth of transformed yeast strain AH109 with constructs under SD/-Trp and SD/-Trp-His-A nutrition-deficient medium. BD refers to the pGBKT7 vector, which serves as the negative control.

Figure 6

Organ-specific expression of DnARF family genes. Expression patterns of DnARF genes in four organs, including root, flower, stem, and leaf. The highest expression accumulation in organs was marked by dash line circles.

Figure 7

Expression analysis of DnARF genes under various hormone treatments. Total RNA was extracted from the seedlings of D. officinale for basal expression. The relative expression levels of 14 DnARF genes under (a) IAA, (b) ABA, (c) GA, and (d) 6-BA treatments. Significant differences in expression of DnARF genes between control and hormone treatments were indicated by ‘*’.

Figure 8

Expression analysis of DnARF genes under various abiotic treatments. Total RNA was extracted from the seedlings of D. officinale for basal expression. The relative expression levels of 14 DnARF genes under (a) NaCl, (b) PEG, (c) 4 °C, and (d) 30 °C treatments. Significant differences in expression of DnARF genes between control and abiotic treatments were indicated by “*”.