Abstract

Red kiwifruit (Actinidia chinensis) is a popular fresh fruit with a high market value due to its unique color, caused by anthocyanin accumulation. The R2R3-MYB transcription factors (TFs) have important roles in plant development and anthocyanin metabolism. In this first comprehensive study of R2R3-MYBs in kiwifruit, a total of 93 R2R3-MYB genes, including five novel previously unannotated AcMYBs, were identified. Their phylogenic relationship, exon-intron structures, and conserved motifs were analyzed. Based on transcriptome data, 60 AcMYBs were expressed (FPKM > 1) across seven developmental stages of kiwifruit, revealing five expression patterns. One of the 5 newly identified R2R3 TFs, AcMYB75, showed an anthocyanin accumulation-linked expression pattern during fruit development. AcMYB75 localized to the nucleus and has an active transactivation domain, verifying it as a transcription factor. AcMYB75 protein specifically bound the promoter of the anthocyanin biosynthesis gene ANS in yeast one-hybrid system and in vivo. In 35 S:AcMYB75 Arabidopsis plants, anthocyanin significantly accumulated in leaves, and the expression of anthocyanin biosynthetic genes was greatly up-regulated. Together, these results suggest that AcMYB75 is involved in anthocyanin biosynthesis in kiwifruit. These findings will increase our understanding of AcMYBs involved in anthocyanin biosynthesis, and also benefit further functional characterization of R2R3-MYB genes in kiwifruit.

Highlights

  • The rise of colorful flowers and fruits is a crux of plant biology and evolution

  • Five R2R3-MYB genes (AcMYB20, AcMYB70, AcMYB73, AcMYB77, and AcMYB75) were identified through a Tophat-Cufflinks pipeline using our previous transcriptome data and they were unannotated in the public genome database

  • Manual checking with Integrative Genomics Viewer (IGV) further confirmed that the pair-end reads of each of these five genes were mapped on a genome region without gene annotation, respectively, and they were identified in different samples of transcriptome data (Supplementary Fig. S1), strongly supporting that these genes were novel MYBs and not previously identified

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Summary

Introduction

The rise of colorful flowers and fruits is a crux of plant biology and evolution. Fruit that is more attractive to insects and animals aids in the dissemination of genes and improves the survival rate of progeny[1]. Research has led to the understanding that fruit development and anthocyanin biosynthesis are largely controlled by specific transcription factors (TFs)[3,4], especially R2R3-MYB TFs5–7. PcMYB10 and PyMYB10 in pear[24,25], FaMYB10 in strawberry[26] and AdMYB110a in kiwifruit flower pigmentation[27]. These studies reveal potential approaches for breeding and/or biotechnological development of diverse colors in horticultural fruits. Searches of high-throughput genome sequences in different species has identified large numbers of MYB TFs based on conservation of the highly conserved DNA-binding MYB domain[7,28,29,30]. The draft kiwifruit genome sequence (http:// bioinfo.bti.cornell.edu/cgi-bin/kiwi/home.cgi)[31] and our high-quality transcriptome data from the red-fleshed Hongyang kiwifruit[32] provide an excellent opportunity for genome-wide analysis of kiwifruit genes

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