Genome-wide analyses of genes encoding FK506-binding proteins reveal their involvement in abiotic stress responses in apple

Abstract

Background: The FK506-binding proteins (FKBPs) play diverse roles in numerous critical processes for plant growth, development, and abiotic stress responses. However, the FKBP gene family in the important fruit crop apple (Malus × domestica Borkh.) has not been studied as thoroughly as in other species. Our research objective was to investigate the mechanisms by which apple FKBPs enable apple plants to tolerate the effects of abiotic stresses.

Results: Using bioinformatics-based methods, RT-PCR, and qRT-PCR technologies, we identified 38 FKBP genes and cloned 16 of them in the apple genome. The phylogenetic analysis revealed three major groups within that family. The results from sequence alignments, 3-D structures, phylogenetics, and analyses of conserved domains indicated that apple FKBPs are highly and structurally conserved. Furthermore, genomics structure analysis showed that those genes are also highly and structurally conserved in several other species. Comprehensive qRT-PCR analysis found various expression patterns for MdFKBPs in different tissues and in plant responses to water-deficit and salt stresses. Based on the results from interaction network and co-expression analyses, we determined that the pairing in the MdFKBP62a/MdFKBP65a/b-mediated network is involved in water-deficit and salt-stress signaling, both of which are uniformly up-regulated through interactions with heat shock proteins in apple.

Conclusions: These results provide new insight for further study of FKBP genes and their functions in abiotic stress response and multiple metabolic and physiological processes in apple.

Keywords: Apple; Drought stress; Expression analysis; FKBP gene family; Genome-wide; Salt stress.

Fig. 1

Multiple alignments of FK506-binding domain encoded by 36 apple FKBP genes

Fig. 2

The secondary and three-dimensional structure of FKBP genes. Sequence logos of FK506-binding domains (a) and TPR domains (b) in 25 FKBP12 and 25 FKBP42 genes, respectively. Heights of symbols within each stack indicate relative frequency of each amino acid at that position. Logos were obtained through multiple alignments of 25 FKBP12 and 25 FKBP42 protein sequences. The a-helix and b-sheet appear at top of corresponding amino acid sequences. Predicted three-dimensional tertiary structural modes of MdFKBP12 (c), MdFKBP42 (d), MdFKBP62 (e), and MdFKBP72 (f) proteins (PDB IDs: 5HWB.1.A, 2IF4.1.A, 1KT1.1.A, and 3JYM.1.A, respectively). Yellow, red, and green-blue indicate β-Sheets, α-helices, and strands, respectively. RasTop software was used to generate 3-D representation

Fig. 3

Phylogenetic tree of amino acid sequences from 36 MdFKBPs, ZmFKBP12, OsFKBP12, VvFKBP12, PpFKBP12, and AtFKBP12. Neighbor-Joining phylogenetic tree was constructed with MEGA 5 software, using 41 full-length amino acid sequences from 6 species. Three protein groups on tree are represented by Roman numerals

Fig. 4

Distribution of functional domains in 38 apple FKBPs. Three conserved domains shown in colored boxes were identified in apple FKBP protein sequences using NCBI batch web Conserved Domains Search tool. Blue box, FKBP_C domain; red box, TPR domain; and purple box, Tigger_C domain. Order in which motifs appears corresponds with their positions in individual protein sequences

Fig. 5

Chromosomal locations of apple FKBP genes. Scale is in megabases (Mb). Two genes could not be localized to specific chromosome. Red font, tandem duplications; blue font, segmental duplications; dark-grey area, genome-wide duplications

Fig. 6

Exon length distribution analysis of FKBP12, FKBP42, FKBP62, and FKBP72 in various plant species. Analysis of exon length distributions for FKBP12 (a), FKBP42 (b), FKBP62 (c), and FKBP72 (d), based on Boxplot depictions in SigmaPlot 12.0 program. Each box represents exon size range in which 50% of values for a particular exon are grouped. Mean value is indicated by long red line

Fig. 7

Expression heat map of MdFKBPs in various tissues. Expression values are log2-transformed

Fig. 8

Expression heat map of MdFKBPs under water deficit stress. After qRT-PCR data were re-analyzed, relative expression was calculated with respect to control samples (i.e., Day 0). Heat maps were generated using TIGR MeV v4.8.1 software. Bar at bottom of each heat map presents relative expression values: 0, down-regulated; 1.0, expression unaltered; or 1.4, up-regulated

Fig. 9

Expression heat map of MdFKBPs under NaCl stress. After qRT-PCR data were re-analyzed, relative expression was calculated with respect to control samples (i.e., Day 0). Heat maps were generated using TIGR MeV v4.8.1 software. Bar at bottom of each heat map presents relative expression values: 0, down-regulated; 1.0, expression unaltered; or 1.4, up-regulated

Fig. 10

Interaction network and co-expression analyses of FKBP62 and FKBP65 in Arabidopsis and apple