Identification of a new R3 MYB type repressor and functional characterization of the members of the MBW transcriptional complex involved in anthocyanin biosynthesis in eggplant (S. melongena L.)

Abstract

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.

Fig 1. Phylogenetic tree of bHLH transcription factors in S. melongena genome.

The optimal NJ tree with the sum of branch length = 31.39844401 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. This analysis involved 97 amino acid sequences, 84 from Eggplant genome [12] and 13 from known anthocyanin related bHLH in other species. The AN1 and the JAF13 clade described in the text are marked with orange and purple triangle, respectively.

Fig 2. Phylogenetic tree of MYB transcription factors in S. melongena genome.

The optimal NJ tree with the sum of branch length = 45.96037176 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. This analysis involved 154 amino acid sequences, 129 from Eggplant and 25 from known anthocyanin related MYBs in other plant species. Clades containing MYB proteins involved in positive and negative anthocyanin regulation are marked with purple and blue circles, respectively.

Fig 3

(A) Phylogenetic tree of MYB transcription factors related to flavonoid synthesis. The optimal NJ tree with the sum of branch length = 8,61587540 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The analysis included 39 amino acid sequences. Positive and negative candidate MYBs analysed in this work are marked with pink and purple square, respectively. (B) Exon/intron structure of S. melongena MYBL1 gene. The exons and introns are represented by purple boxes and black lines, respectively. (C) Domain structure of MYBL1 type repressors.

Fig 4

qRT-PCR based transcription profiling of eggplant MYBL1, AN2, ANT1, AN1, JAF13, DFR, AN11 in two stages of fruit ripening (stage A and B) and in flower organs. Expression levels, measured by qPCR, are shown as relative units using SmelGAPDH as reference gene. Data are means of three biological replicates ± SD.

Fig 5. Y2H assay.

ANT1, AN2 and MYBL1 were cloned in the prey plasmid pDEST22 and transformed with the bait plasmid pDEST32 (containing JAF13 and AN1). pDEST22 and pDEST32 were used as a negative control. Yeast cells were grown for three days on (A) synthetic complete medium lacking tryphtophan and leucine (-W/-L), (B) on selective medium lacking tryptophan, leucine and uracil (-W/-L/-U) and (C) on Whatman 541 filter papers, saturated 2% X-gal solution.

Fig 6. Bimolecular fluorescent complementation assay.

SmelAN1-nYFP fusion proteins was co-expressed transiently with SmelAN2-cYFP, SmelMYBL1-cYFP or SmelANT1-cYFP fusion proteins in freshly isolated Arabidopsis mesophyll protoplasts. GUS protein fused to both nYFP or cYFP was used as negative control. The cellular localization of interactions was investigated through DAPI staining.

Fig 7. The effects of over-expression of ANT1, AN2 and MYBL1 in Nicotiana benthamiana.

Leaves of N. benthamiana after agroinfiltration with ANT1, AN2 and MYBL1 and a combination of MYBL1 with ANT1 or AN2. Anthocyanin accumulation is indicated by dotted white circles.

Fig 8. The effects of over-expression of ANT1, AN2 and MYBL1 in Nicotiana benthamiana.

Concentration of delphinidin 3-O-rutinoside in tissue extracts of N. benthamiana control (CTR) and transiently transformed leaves (ANT1, AN2, AN1, JAF13, MYBL1 and a combination of MYBL1 with ANT1 or AN2). Error bars represent SD (n = 3). Asterisk indicates significance based on Tukey’s test (P≤0.05).