An OsKala3, R2R3 MYB TF, Is a Common Key Player for Black Rice Pericarp as Main Partner of an OsKala4, bHLH TF

Abstract

Rice (Oryza sativa) pericarp exhibits various colors due to the accumulation of anthocyanins and/or proanthocyanidins. Previous work revealed that the two basic helix-loop-helix (bHLH) transcription factors OsKala4 and OsRc are key regulators for the black and red pericarp traits, respectively, and their inactivation results in rice with white pericarp. However, their pericarp-specific R2R3 MYB partner remained unknown. Here, we characterized the role of the R2R3 MYB gene OsKala3 in rice pericarp pigmentation through genetic and molecular approaches. A rice protoplast transfection assay showed that OsKala3 is a nuclear-localized protein. Furthermore, OsKala3 physically interacted with OsKala4 in a yeast two-hybrid analysis. Co-transfection assays in rice protoplasts revealed that OsKala3 and OsKala4 mediate the activation of anthocyanin biosynthetic genes. Notably, the OsKala3 promoter region exhibited an insertion polymorphism specifically in rice cultivars with black pericarp, creating two tandem repeats while red and white varieties harbor only one. The number of repeats within the OsKala3 promoter correlated with increased transactivation by OsKala3, thus providing a rationale for the black pericarp characteristic of cultivars with two repeats. These results thus provide evidence for the molecular basis of anthocyanin biosynthesis in rice pericarp and may facilitate the introduction of this beneficial trait to other rice cultivars through marker-assisted breeding.

Keywords: OsKala3; OsKala4; anthocyanin; regulation system; rice; tandem repeats in promoters.

FIGURE 1

Anthocyanin contents and expression analysis of OsKala3 and other putative anthocyanin regulatory genes from developing rice seeds and young seedling stage. (A) Mean anthocyanin accumulation in developing rice seeds from non-pigmented (IM), black (HN and HJJ), and red (JJJ) rice varieties during seed maturation (top). Relative transcript levels for regulators of anthocyanin biosynthesis (bottom). DAP, days after pollination. (B) Anthocyanin contents (top) and transcript level (bottom) of anthocyanin regulator genes in various tissues of the young seedlings stage (14 days after sowing). L, leaves; R, roots; Sd, seedlings. Results represent mean values ± SD from three independent biological replicates. Different letters above the bars indicate significantly different values (p < 0.05) calculated using two-way ANOVA followed by Duncan’s multiple range tests.

FIGURE 2

Phylogenetic relationships between anthocyanin biosynthetic regulators in rice and other species. (A) Phylogenetic tree of rice OsKala3 and R2R3 MYB proteins from other plants. The phylogenetic tree was constructed using the neighbor-joining method with MEGA6 software. GenBank database accession numbers are Allium cepa AcMYB1 (KX785130); Antirrhinum majus AmRos1 (ABB83826); Arabidopsis thaliana AtPAP1 (AAG42001), AtTT2 (CAC40021); Dahlia pinnata (syn. variabilis) DvMYB1 (AB601003); Gerbera hybrida GhMYB10 (CAD87010); Ipomoea nil InMYB1 (BAE94391); Lilium hybrida LhMYB6 (BAJ05399), LhMYB12 (BAJ05398); Lotus japonicas LjTT2a (AB300033); Malus × domestica MdMYB9 (ABB84757), MdMYB10 (ACQ45201); Medicago truncatula MtLAP1 (ACN79541); Oncidium Gower Ramsey OgMYB1 (ABS58501); Oryza sativa OsC1 (BAD04024), OsKala3 (BAA23339); Petunia × hybrida PhAN2 (AAF66727); Phalaenopsis schilleriana PsUMyb6 (FJ039860); Picea mariana PmMBF1 (U39448); Trifolium arvense TaMYB14 (AFJ53053); Triticum aestivum TaPpm1 (MG066451); Vanda hybrida VhMYB6 (ADQ57817); Vitis vinifera VvMYBA (BAD18977), VvMYBPA1 (AM259485); Zea mays ZmC1 (AAA33482). (B) Multiple sequence alignment of part of the C-terminal region of the R2R3-MYB sequences, showing the SG5 and SG6 motifs. Amino acids matching either the SG6 motif or the SG5 motif are indicated in red. The starting amino acid position of the sequences is given in the second column. (C) Multiple sequence alignment of the R2 and R3 domains across R2R3 MYB proteins shown in (A). The conserved residues of DNEI and ANDV are represented using the red and blue boxes, respectively. Inverted blue triangles indicate the conserved residues forming the inner hydrophobic core of the R2 and R3 domains.

FIGURE 3

OsKala3, OsKala4, and OsTTG1 form a ternary complex. (A) Interactions between OsKala3, OsKala4, and OsTTG1 proteins, as revealed by yeast two-hybrid (Y2H) analysis. (B) Schematic representation of constructs used to delineate the interaction interface between OsKala3, OsKala4, and OsTTG1. Amino acid positions are indicated in the diagrams. (C) Interactions between truncated OsKala3, OsKala4, and full-length OsTTG1. AD, activation domain; BD, binding domain; SD, synthetic defined medium; 3AT, 3-amino-1,2,4-triazole; SD/–TL, minimal medium lacking Trp and Leu; SD/–TLH + 3AT, synthetic defined medium lacking Trp, Leu, His and containing 10 mM 3AT.

FIGURE 4

OsKala3, OsKala4, and OsTTG1 cooperate to induce the expression of anthocyanin biosynthetic genes. (A) Schematic representation of effector and reporter constructs used in the transcriptional activation assay. Effector constructs consist of the OsKala3, OsKala4, and OsTTG1 open reading frames (ORFs) driven by the CaMV 35S promoter. In the reporter constructs, the firefly luciferase (fLUC) reporter gene is driven by the OsCHS, OsCHI, OsF3H, OsF3’H, OsDFR, or OsANS promoters. In the internal control constructs, the Renilla luciferase (rLUC) reporter gene is driven by the ubiquitin promoter. (B) Transactivation reporter assays showing the effects of OsKala3, OsKala4, and OsTTG1 on the transcription of anthocyanin biosynthetic genes. Results represent mean values ± SD from three independent biological replicates. Different letters above the bars indicate significantly different values (p < 0.0001) calculated using one-way ANOVA followed by a Duncan’s multiple-range test.

FIGURE 5

Validation of OsKala3 promoter variation. (A) Diagram of the genomic structure of OsKala3 from black, red, and white pericarp cultivars. The light gray box and dark gray box indicate repeat unit 1 (RU1) and RU2, respectively, in the OsKala3 promoter region. (B) The amplicons’ representative gel image corresponds to promoter region (left) and ORF region (right). The primers used in this study are shown as arrows and listed in Supplementary Table 1.

FIGURE 6

The number of repeat units in the OsKala3 promoter region affects its transactivation rate. (A) Schematic representation of the promoter constructs used in the assay, bearing one, two, four, or eight repeat units (RU). The IM cultivar carries one RU copy in the OsKala3 promoter, while the cultivar HJJ carries two copies of the element. RUs are indicated in light and dark gray. (B) Increasing the number of RU copies raises the activation of OsKala3 transcription when co-transformed with 35S:OsKala3, 35S:OsKala4, and 35S:OsTTG1. Results represent mean values ± SD from three independent biological replicates. The fLUC/rLUC ratio in the absence of effectors was set to 1.

FIGURE 7

Variation at OsKala3, OsKala4, OsRc, and OsDFR predicts pericarp color across 40 rice cultivars. (A) Schematic representation of OsKala3 promoter region. The light gray box and dark gray box indicate RU1and RU2, respectively, in the OsKala3 promoter region. The position of the RU is indicated relative to the ATG translation start site. (B) Representative photographs of rice seeds showing pericarp colors for the indicated cultivars (top). Genotyping results of the 40 rice cultivars with the and InDel markers designed against OsKala3, OsKala4, and OsRc and CAPS marker designed against OsDFR (bottom).

FIGURE 8

Proposed model of anthocyanin biosynthesis of seed and vegetative tissue in rice. (A) Heat map diagram of expression level for anthocyanin regulators consisting of MYB (M), bHLH (B), and WD40 (W) from rice pericarp and leaf, respectively. Color scale indicates fold changes in gene expression. (B) Working models of anthocyanin coloration in different tissues of rice. Both OsKala3 and OsKala4 had extremely high expression levels in rice pericarp, while OsC1 and OsRb were more strongly expressed in respective rice leaf. When all of OsDFR was functional, it forms the functional MBW complex consisting of OsKala3-OsKala4-OsTTG1 and OsC1-OsRb-OsTTG1 activating anthocyanin biosynthesis in rice.