Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

Abstract

The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the "Beta type" promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening.

Fig 1. Distribution of SNPs and In-dels in CYC-B gene.

Variants identified using Eco-TILLING and Sanger sequencing, and from tomato structural variant browser are represented with a modified PARSESNP output diagram. Black and purple triangles indicate the positions of nonsynonymous and synonymous nucleotide substitutions respectively. Red triangle represents the position of the nonsense substitution G570A in tomato accession EC20636. The red squares represent frameshift changes.

Fig 2. Evolutionary relationships of CYC-B gene sequences from 58 tomato accessions.

The evolutionary history was inferred for 1098 bp region of the gene using MEGA6. The optimal tree with the sum of branch length = 0.11 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. Number of variants present in CYC-B gene for each accession is represented with bars (right).

Fig 3. Evolutionary relationships of CYC-B promoter sequences from 49 tomato accessions.

All sequences corresponding to –1 to –876 bp region of the reference variety (S. lycopersicum cv. Arka Vikas) was used for constructing the tree using MEGA6. The optimal tree with the sum of branch length = 0.38 is shown. The number of RIN binding sites in the CYC-B promoter sequence of the accessions are indicated with coloured boxes (right): CARGATCONSENSUS (green), CARGCW8GAT (red) and CARGNCAT (yellow).

Fig 4. Frequency of transition/transversion ratios in CYC-B gene and promoter.

Green fruited accessions have more transitions, whereas, coloured fruited accessions carry more transversions.

Fig 5. Comparison of three different CYC-B promoter sequences.

CYC-B promoter fragments amplified from WT (Arka Vikas), Beta (B) mutant (LA3000) and B^og mutant (LA0348) used for transient expression (A). Modified diagram generated using PlantPAN shows the alignment of the promoter sequences and 256 bp insertion in B^og (LA0348) (B). Relative expression of FLUC under different promoters in pGreenII 0800-LUC constructs (C).

Fig 6. Variability of RIN TF binding sites in CYC-B promoters.

The nucleotide sequences upstream of CYC-B gene in WT, Beta (B) type and LA0348 type are diagrammatically represented with green bars. The red coloured portion in LA0348 type represents the probable NUPT fragment. The RIN binding sites are indicated with arrows, and the altered sites are represented by triangles above the bars. The exact position of the first nucleotide in each case is mentioned below the bars. Different RIN binding sequences predicted by PLACE are indicated with different colours. The element CCTATAAAGG is CARGATCONSENSUS. The elements CAAAATATTG, CAAAAAAAAG, CATATTTTAG, CAATATAAAG and CAAATAATAG are CARGCW8GAT type and the element CCAATATAAAGG is CARGNCAT type.

Fig 7. Phylogenic tree showing the similarities between amino acid sequences of CYC-B and its homologues.

The analysis involved 32 amino acid sequences. Evolutionary analyses were conducted in MEGA6.