Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant

Abstract

As the second largest transcription factor family in plant, the basic helix-loop-helix (bHLH) transcription factor family, characterized by the conserved bHLH domain, plays a central regulatory role in many biological process. However, the bHLH transcription factor family of strawberry has not been systematically identified, especially for the anthocyanin biosynthesis. Here, we identified a total of 113 bHLH transcription factors and described their chromosomal distribution and bioinformatics for the diploid woodland strawberry Fragaria vesca. In addition, transcription profiles of 113 orthologous bHLH genes from various tissues were analyzed for the cultivar 'Benihoppe', its white-flesh mutant 'Xiaobai', and the 'Snow Princess' from their fruit development to the ripening, as well as those under either the ABA or Eth treatment. Both the RT-PCR and qRT-PCR results show that seven selected FabHLH genes (FabHLH17, FabHLH25, FabHLH27, FabHLH29, FabHLH40, FabHLH80, FabHLH98) are responsive to the fruit anthocyanin biosynthesis and hormone signaling according to transcript profiles where three color modes are observed for strawberry's fruit skin and flesh. Further, prediction for the protein interaction network reveals that four bHLHs (FabHLH25, FabHLH29, FabHLH80, FabHLH98) are involved in the fruit anthocyanin biosynthesis and hormone signaling transduction. These bioinformatics and expression profiles provide a good basis for a further investigation of strawberry bHLH genes.

Figure 1

Chromosomal distributions of FvbHLH genes. The name on the side of each chromosome corresponds to the approximate location of each bHLH gene.

Figure 2

Phylogenetic tree constructed from the neighbor-joining method using the bHLH transcription factor domain for strawberry. Genes marked by the red asterisk indicates the seven candidates of FvbHLH involved in the anthocyanin biosynthesis.

Figure 3

The characterization and distribution of bHLH domains. The top: sequence logo of the FvbHLH domain by MEME. The bottom: distribution of amino acids in the bHLH consensus motif among strawberry. Asterisk symbol corresponds to each column above, which stands for the percentage of presence of amino acids at each site and the color of the asterisk symbol corresponds bHLH regions from the top insert. The analysis of the amino acids composition at each site marked by the asterisk indicates that the conservation of conserved amino acids is over 50%.

Figure 4

Materials of strawberry used in this study. (A) The fruit of ‘Benihoppe’, ‘Xiaobai’, and ‘Snow Princess’. (B) Seven fruit development and ripening stages of ‘Benihoppe’, ‘Xiaobai’, and ‘Snow Princess’. Bar = 1 cm.

Figure 5

Transcript accumulation profiles of 113 FabHLH genes from different tissues and seven fruit development and ripening stages using semi-quantitative PCR for the three cultivated strawberry varieties. (A) Lanes: R, roots; YL, young leaves; ML, mature leaves; RN, runners; RT, runner tips; RTL, runner with tips and one leaf; A, anthotaxy; F, flowers. (B) Lanes: S1, small green fruit; S2, middle green fruit; S3, large green fruit; S4, white fruit; S5, initial red; S6, partial red; S7, full red. FvActin, FvRib413 and FvGAPDH2 were used as an internal control.

Figure 7

qRT-PCR transcription analysis of seven selected FabHLH genes during fruit development and ripening stages, and them under either ABA or Eth treatment for the three strawberry varieties. FvRib413 is used as an internal control. The experiments were repeated three times and gave consistent results. The mean values and SDs were obtained from three biological and technical replicates. Different letters indicate the statistical difference among samples at P ≤ 0.01 and P ≤ 0.05 (fruit development and ripening), and P ≤ 0.01 (ABA and Eth treatments) according to Duncan’s multiple range test.

Figure 6

Transcript accumulation patterns of 113 bHLH genes for the three strawberry varieties under hormone stress (ABA and Eth). FvActin, FvRib413 and FvGAPDH2 were used as an internal control. The transcript accumulation profiles were generated by semi-quantitative PCR and were visualized as heat maps. The color scale represents the relative transcript level with increased (red) and decreased (green) transcript abundance. The FvbHLH genes marked by red asterisk indicate their candidacy in the anthocyanin biosynthesis.

Figure 8

Interaction network analysis of bHLH proteins identified for strawberry and related genes for Arabidopsis. Line thickness is related to the combined score (FvbHLH25 score >0.7, the others score >0.9). The homologous genes of strawberry are in red.