Genome-wide analysis of basic helix-loop-helix transcription factors in papaya ( Carica papaya L.)

Abstract

The basic helix-loop-helix (bHLH) transcription factors (TFs) have been identified and functionally characterized in many plants. However, no comprehensive analysis of the bHLH family in papaya (Carica papaya L.) has been reported previously. Here, a total of 73 CpbHLHs were identified in papaya, and these genes were classified into 18 subfamilies based on phylogenetic analysis. Almost all of the CpbHLHs in the same subfamily shared similar gene structures and protein motifs according to analysis of exon/intron organizations and motif compositions. The number of exons in CpbHLHs varied from one to 10 with an average of five. The amino acid sequences of the bHLH domains were quite conservative, especially Leu-27 and Leu-63. Promoter cis-element analysis revealed that most of the CpbHLHs contained cis-elements that can respond to various biotic/abiotic stress-related events. Gene ontology (GO) analysis revealed that CpbHLHs mainly functions in protein dimerization activity and DNA-binding, and most CpbHLHs were predicted to localize in the nucleus. Abiotic stress treatment and quantitative real-time PCR (qRT-PCR) revealed some important candidate CpbHLHs that might be responsible for abiotic stress responses in papaya. These findings would lay a foundation for further investigate of the molecular functions of CpbHLHs.

Keywords: Abiotic stress; Expression analysis; Genome-wide analysis; bHLH transcription factors; Papaya.

Figure 1. Phylogenic and family members analysis of bHLHs from papaya, rice and Arabidopsis.

(A) The 73 CpbHLHs are clustered into 18 subfamilies. Phylogenetic tree was constructed based on the neighbor-joining method. Bootstrapping with 1,000 replicates was used to assess the statistical reliability of nodes in the tree. (B) Comparison of bHLH family members from papaya, rice and Arabidopsis. Different colors represent the different plants. Black: OsbHLHs, red: AtbHLHs, purple: CpbHLHs.

Figure 2. Gene structure and motif distribution of the papaya bHLH family.

(A) Exon-intron organization of CpbHLH genes. Exons and introns are presented as filled orange lines and thin black single lines, respectively. The brackets and Roman numerals separate each subfamily and clearly present the member conservation of each subfamily. (B) Arrangements of conserved motifs in 73 CpbHLH proteins. Three predicted motifs are represented by different colored boxes, motif 1 (red block), motif 2 (black block) and motif 3 (yellow block).

Figure 3. Multiple sequence alignment of the bHLH domains in papaya.

Amino acids with more than 50% identity are labeled with colored boxes.

Figure 4. Motif composition and logos of papaya bHLH proteins.

(A) The logos of motif 1 and 2, which together constitute the bHLH domain in papaya. The overall height of the character represents the conservation of an amino acid at the specific position. Each color of the English letters represents a type of amino acid residue. (B) The logo of motif 3, which is another conserved motif.

Figure 5. Cis-acting element analysis of the promoter of bHLH genes in papaya.

(A) Percentage of total cis-acting elements in the promoter region of CpbHLH genes. (B) The percentage of each cis-acting element in the abiotic and biotic stresses categories. Abscisic acid (29.15%), methyl jasmonate (23.62%), salicylic acid (9.78%), auxin (7.20%), gibberellin (8.86%), drought (8.67%), wound (1.11%), defense and stress (6.27%), low temperature (5.35%).

Figure 6. Gene ontology (GO) annotation of Cp bHLH proteins.

The annotation was performed on three categories: (A) molecular function, (B) biological processes and (C) cellular components.

Figure 7. Quantitative RT-PCR analysis of 22 selected CpbHLH genes under cold stress condition (4 °C).

The data are expressed as means ± SD of three independent biological determinations. Untreated seedlings were used as the control groups. ^∗P < 0.05 and ^∗∗P < 0.01 (Student’s t test) indicate significant differences between treated seedlings and control groups.