Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus)

Abstract

A molecular description of the control of floral pigmentation in a multi-species group displaying various flower color patterns is of great interest for understanding the molecular bases of phenotypic diversification and pollinator-mediated speciation. Through transcriptome profiling, mutant analyses and transgenic experiments, we aim to establish a 'baseline' floral anthocyanin regulation model in Mimulus lewisii and to examine the different ways of tinkering with this model in generating the diversity of floral anthocyanin patterns in other Mimulus species. We find one WD40 and one bHLH gene controlling anthocyanin pigmentation in the entire corolla of M. lewisii and two R2R3-MYB genes, PELAN and NEGAN, controlling anthocyanin production in the petal lobe and nectar guide, respectively. The autoregulation of NEGAN might be a critical property to generate anthocyanin spots. Independent losses of PELAN expression (via different mechanisms) explain two natural yellow-flowered populations of M. cardinalis (typically red-flowered). The NEGAN ortholog is the only anthocyanin-activating MYB expressed in the M. guttatus flowers. The mutant lines and transgenic tools available for M. lewisii will enable gene-by-gene replacement experiments to dissect the genetic and developmental bases of more complex floral color patterns, and to test hypotheses on phenotypic evolution in general.

Keywords: MYB-bHLH-WD40; anthocyanin pigmentation; autoregulation; flower color; monkeyflowers (Mimulus); natural variation; phenotypic evolution.

Fig. 1

Natural flower color variation among Mimulus species. Shown on the left is a schematic illustration of the phylogenetic relationships among major Mimulus clades, based on Beardsley et al. (2004) and Grossenbacher & Whittall (2011); on the right are representative species of each clade. Images of M. norrisii, M. filicaulis, M. palmeri, M. shevockii, M. suksdorfii, M. layneae, M. mephiticus, M. angustatus, and M. pulchellus were provided by Dena Grossenbacher; images of M. aurantiacus were provided by Matt Streisfeld; the remaining images were taken by Y.W.Y.

Fig. 2

Maximum likelihood (ML) phylogenies of R2R3-MYB (subgroup 6 and related subgroups) proteins (a) and bHLH (subgroup IIIf and related subgroups) proteins (b). Subgroup classifications followed Stracke et al. (2001) and Heim et al. (2003). Trees are rooted by midpoint rooting. Bootstrap support values >50% are indicated along the branches. Mimulus lewisii sequences characterized in this study are highlighted in bold and have been deposited in GenBank (accession numbers see Table 1). All M. guttatus sequences were retrieved from Phytozome (http://www.phytozome.net), including MgMYB1-5 (MgMYB1: mgv1a023671m; MgMYB2: mgv1a024703m; MgMYB3: mgv1a024996m; MgMYB4: mgv1a025765m; MgMYB5: mgv1a019326m) and the three bHLHs (mgv1a00290, mgv1a00684, mgv1a00268). All Arabidopsis sequences were retrieved from the TAIR site (http://www.arabidopsis.org/); Other sequences were retrieved from GenBank as follows: Antirrhinum majus ROSEA1 (DQ275529); ROSEA2 (DQ275530); Venosa (DQ275531); AmMIXTA (X79108); AmDelia (AAA32663); Lilium hybrid LhMYB6 (AB534587); LhMYB12 (AB534586); Lotus japonicus TT2a (BAG12893); Malus x domestica MdMYB10a (DQ267897); Mimulus lewisii GUIDELESS (KC139356); Petunia x hybrida PhAN2 (AF146702); PhAN4 (HQ428105); PhDPL (HQ116169); PhPHZ (HQ116170); PhAN1 (AAG25928); PhJAF13 (AAC39455); Vitis vinifera VvmybA1 (BAD18977); VvmybA2 (BAD18978); VvMYBPA2 (ACK56131); VvMYC1 (EU447172); VvMYCA1 (EF193002); Zea mays ZmP (P27898); ZmB (CAA40544); ZmLc (AAA33504).

Fig. 3

Semi-quantitative RT-PCR of the structural anthocyanin biosynthetic genes in the Mimulus lewisii boo mutants. MlF3Ha, MlDFR, and MlANS are dramatically down-regulated in these mutants; MlUF3GT is also down-regulated, but to a lesser extent; MlCHSa and MlCHI are not affected. MlUBC is shown as a reference gene. PCR cycle numbers are shown after the gene names. WT, wild-type.

Fig. 4

Phenotypes of the wild-type Mimulus lewisii LF10 and the boo3, boo5, and boo14 mutants. (a) Petal lobe color. (b) Anthocyanin spots in the nectar guide. (c) Color of the stem base. (d) Color of the seed coat.

Fig. 5

Mutation characterization of the Mimulus lewisii boo mutants. (a) boo3 has a premature stop codon in the PELAN R2R3-MYB gene. (b) boo1 and boo5 have a non-synonymous mutation (D172N) and a premature stop codon, respectively, in the MlWD40a gene. (c) boo14 has a mutation in an intron/exon junction in MlANbHLH1, leading to non-splicing of the last intron. (d) Expression pattern of the PELAN MYB and the NEGAN MYB in the wild-type petal lobe (PL) and nectar guide (NG).

Fig. 6

Phenotypic and molecular characterization of the RNAi transgenic lines of PELAN (a), NEGAN (b), MlANbHLH1 (c), and MlWD40a (d) in the Mimulus lewisii LF10 background. The proportion of transgenic lines with strong or intermediate phenotypes is indicated by the numbers in parentheses below the flower images. The strong RNAi lines of PELAN, MlANbHLH1, and MlWD40a accurately reproduce the phenotypes of boo3, boo14, and boo5, respectively, including the stem base color and seed coat color (not shown). Note that no EMS-induced mutants are available for NEGAN. (e) Quantitative RT-PCR of the four genes at 15-mm corolla stage. MlUBC was used as the reference gene. All four genes show substantial knock-down in their corresponding RNAi lines. Asterisks highlight the complete absence of NEGAN transcripts in the NEGAN and MlWD40a RNAi lines after 40 cycles of PCR. WT, wild-type. Bars, ±1 SD from three biological replicates.

Fig. 7

A regulatory network model of anthocyanin pigmentation in Mimulus lewisii LF10. The bottom plant images (from left to right) show the color of nectar guide spots, petal lobes, stem base, and seed coat, respectively. The dashed lines indicate putative protein–protein interactions; the arrow indicates self-activation.

Fig. 8

NEGAN transgene activates the endogenous NEGAN gene expression in Mimulus lewisii over-expression lines. (a) A representative 35S:NEGAN transgenic line showing the whole-plant phenotype. (b) The entire leaf can be changed to dark purple in strong 35S:NEGAN lines. (c) RT-PCR showing activation of the endogenous NEGAN gene, which can be distinguished from the transgene by the presence of 3′UTR, in three independent 35S:NEGAN lines. WT, wild-type.

Fig. 9

Molecular characterization of the two natural yellow-flowered Mimulus cardinalis populations. (a) M. cardinalis inbred line CE10 showing the typical red flower. (b) M. cardinalis (SM). (c) M. cardinalis (CI). The dissected nectar guide on the upper-right corner of the flower images show the anthocyanin spots in the nectar guide. (d) Structure of the PELAN gene and the positions of PCR primers used in RT-PCR and genomic PCR. (e) RT-PCR (primer pair SP3F4 & SP3R2) shows no expression of PELAN in CI or SM. (f) Genomic PCR by multiple primer pairs suggest that PELAN has probably been deleted from the SM genome. Primer sequences are listed in Supporting Information Table S4 (also see Fig. S7). GenBank accession numbers for the M. cardinalis CE10 and CI PELAN gene sequences are KJ595587- KJ595588.

Fig. 10

Anthocyanin pigmentation in Mimulus guttatus inbred line IM767. (a) Flower images showing the anthocyanin spots in the nectar guide. (b) The NEGAN ortholog is the only anthocyanin-activating MYB expressed in the corolla of IM767, indicated by the red arrow. Genomic DNA was used as control to test primer quality. The M. guttatus UBC ortholog (MgUBC) was used as a reference gene. The larger size of the MgUBC genomic amplicon is due to the presence of an intron in the amplified fragment. Primer sequences are listed in Supporting Information Table S1.