Expression of RsPORB Is Associated with Radish Root Color

Abstract

Radish (Raphanus sativus) plants exhibit varied root colors due to the accumulation of chlorophylls and anthocyanins compounds that are beneficial for both human health and visual quality. The mechanisms of chlorophyll biosynthesis have been extensively studied in foliar tissues but remain largely unknown in other tissues. In this study, we examined the role of NADPH:protochlorophyllide oxidoreductases (PORs), which are key enzymes in chlorophyll biosynthesis, in radish roots. The transcript level of RsPORB was abundantly expressed in green roots and positively correlated with chlorophyll content in radish roots. Sequences of the RsPORB coding region were identical between white (948) and green (847) radish breeding lines. Additionally, virus-induced gene silencing assay with RsPORB exhibited reduced chlorophyll contents, verifying that RsPORB is a functional enzyme for chlorophyll biosynthesis. Sequence comparison of RsPORB promoters from white and green radishes showed several insertions and deletions (InDels) and single-nucleotide polymorphisms. Promoter activation assays using radish root protoplasts verified that InDels of the RsPORB promoter contribute to its expression level. These results suggested that RsPORB is one of the key genes underlying chlorophyll biosynthesis and green coloration in non-foliar tissues, such as roots.

Keywords: RsPORB; chlorophyll biosynthesis; green radish; polymorphisms; promoter variation.

Figure 1

NADPH:protochlorophyllide oxidoreductase (POR) enzymes convert protochlorophyllide (Pchlide) into chlorophyllide (Chlide) by reducing the double bond between C17 and C18, using NADPH as the H donor. The gray box indicates the single bond resulting from the reduction.

Figure 2

Phenotypes and chlorophyll content of root skin and flesh of four different radishes. (A) Root skin (top) and root flesh (bottom) of representative samples of two white (W1, W2) and two green (G1, G2) radishes. Bar = 1 cm. (B) Chlorophyll contents in root skin and root flesh. Results represent the mean values ± SD from three independent experiments. Different letters indicate significantly different values (p < 0.05), as determined using a one-way ANOVA followed by Duncan’s multiple range tests.

Figure 3

Transcript levels of RsPORA, RsPORB, and RsPORC in root skin, root flesh, and leaf from white (W1, W2) and green (G1, G2) radishes. Results represent the mean values ± SD from three independent biological replicates. Different letters indicate significantly different values (p < 0.05), as determined using a one-way ANOVA followed by Duncan’s multiple range tests.

Figure 4

Multiple sequence alignment and RsPORB protein structure analysis. (A) Multiple sequence alignment of PORB proteins. α-Helices and β-strands are indicated by red and cyan lines, respectively, and the conserved Rossmann-fold domain, Pchlide loop, and YxxxK motif are outlined by orange boxes. The putative N-terminal cleavage sites for the plastid transit peptides are indicated by the arrow. (B) Structural modeling of RsPORB protein. The 3D protein structure modeling based on PDB ID: 7JK9 from Arabidopsis thaliana built using SWISS-MODEL. The α-helices and β-strands are shown in red and cyan, respectively. The enlarged view showed the binding of RsPORB protein to Pchlide and NADPH.

Figure 4

Multiple sequence alignment and RsPORB protein structure analysis. (A) Multiple sequence alignment of PORB proteins. α-Helices and β-strands are indicated by red and cyan lines, respectively, and the conserved Rossmann-fold domain, Pchlide loop, and YxxxK motif are outlined by orange boxes. The putative N-terminal cleavage sites for the plastid transit peptides are indicated by the arrow. (B) Structural modeling of RsPORB protein. The 3D protein structure modeling based on PDB ID: 7JK9 from Arabidopsis thaliana built using SWISS-MODEL. The α-helices and β-strands are shown in red and cyan, respectively. The enlarged view showed the binding of RsPORB protein to Pchlide and NADPH.

Figure 5

Silencing of RsPORB reduces the chlorophyll contents. (A) Representative radish plants 3 weeks after post-infiltration with the empty vector as the control (TRV2-EV), RsPORB (TRV2-PORB) and RsPOR genes (TRV2-PORs). Bar = 1 cm. (B) Chlorophyll contents in RsPORB- and RsPOR-silenced radish leaves. (C) Transcript levels of RsPORA, RsPORB, and RsPORC in leaves. Different letters indicate significantly different values (p < 0.05), as determined using a one-way ANOVA followed by Duncan’s multiple range tests.

Figure 6

Polymorphic sites in the RsPORB promoter region in G1 vs. W1 radishes. Numbers indicate positions from the ATG start codon of RsPORB in the G1 radish.

Figure 7

Promoter variation of the RsPORB gene can alter its expression level. Schematic representations of RsPORB promoter constructs from G1, W1, and constructs with deleted InDel regions of the RsPORB-W1 promoter (dP1) and dP2 (left). dP1 and dP2 were deleted from −441 to −448 and from −353 to −390 of RsPORB-W1 promoter, respectively. We used a dual-luciferase promoter plasmid encoding the firefly luciferase gene driven by the RsPORB promoters G1, W1, dP1, and dP2 as well as a Renilla luciferase (REN) gene driven by the CaMV 35S promoter to measure LUC and REN levels (right). Data denote protoplast accumulation of each fusion protein at 16 h after transfection. Different letters indicate significantly different values (p < 0.05), as determined using a one-way ANOVA followed by Duncan’s multiple range tests.