In plants, the carotenoid biosynthesis pathway (CBP) is essential for the production of photosynthetic and protective pigments, plant hormones, and visual/olfactory attractants for animal pollinators and seed dispersers. The regulation of carotenoid biosynthesis at the transcriptional level is vitally important for all of these functions and has been the subject of intensive research. Many putative transcriptional regulators, both direct and indirect, have been identified through conventional mutant analysis, transcriptome profiling, yeast one-hybrid screening, and candidate gene approaches. Despite this progress, our understanding of the transcriptional regulation of carotenoid biosynthesis remains fragmented and incomplete. Frequently, a stimulus or regulator is known, but the mechanism by which it affects transcription has not been elucidated. In other cases, mechanisms have been proposed (such as direct binding of a CBP gene promoter by a transcription factor), but function was tested only in vitro or in heterologous systems, making it unclear whether these proteins actually play a role in carotenoid regulation in their endogenous environments. Even in cases where the mechanism is relatively well understood, regulators are often studied in isolation, either in a single plant species or outside the context of other known regulators. This presents a conundrum: why so many candidate regulators but so little consensus? Here we summarize current knowledge on transcriptional regulation of the CBP, lay out the challenges contributing to this conundrum, identify remaining knowledge gaps, and suggest future research directions to address these challenges and knowledge gaps.

SummaryCarotenoids are yellow, orange, and red pigments that contribute to the beautiful colors and nutritive value of many flowers and fruits. The structural genes in the highly conserved carotenoid biosynthetic pathway have been well characterized in multiple plant systems, but little is known about the transcription factors that control the expression of these structural genes.By analyzing a chemically induced mutant of Mimulus lewisii through bulk segregant analysis and transgenic experiments, we have identified an R2R3-MYB, Reduced Carotenoid Pigmentation 1 (RCP1), as the first transcription factor that positively regulates carotenoid biosynthesis during flower development.Loss-of-function mutations in RCP1 lead to down-regulation of all carotenoid biosynthetic genes and reduced carotenoid content in M. lewisii flowers, a phenotype recapitulated by RNA interference in the wild-type background. Overexpression of this gene in the rcp1 mutant background restores carotenoid production and, unexpectedly, results in simultaneous decrease of anthocyanin production in some transgenic lines by down-regulating the expression of an activator of anthocyanin biosynthesis.Identification of transcriptional regulators of carotenoid biosynthesis provides the 'toolbox' genes for understanding the molecular basis of flower color diversification in nature and for potential enhancement of carotenoid production in crop plants via genetic engineering.

Many organisms exhibit visually striking spotted or striped pigmentation patterns. Turing's reaction-diffusion model postulates that such periodic pigmentation patterns form when a local autocatalytic feedback loop and a long-range inhibitory feedback loop interact. At its simplest, this network only requires one self-activating activator that also activates a repressor, which inhibits the activator and diffuses to neighboring cells. However, the molecular activators and repressors fully fitting this versatile model remain elusive. Here, we characterize an R2R3-MYB activator and an R3-MYB repressor in monkeyflowers that correspond to Turing's model and explain how periodic anthocyanin spots form. Notably, disrupting this pattern impacts pollinator visitation. Thus, subtle changes in simple reaction-diffusion networks are likely essential contributors to the evolution of the remarkable diversity of periodic pigmentation patterns in flowers. MaintextPeriodic pigmentation patterns like the stripes of zebras, the spiral pigmentation of seashells, and the petal spots of many flowers have fascinated biologists and mathematicians for centuries. One proposed developmental explanation for how such periodic patterns form is Turing's reactiondiffusion model (1), in which dynamic and autonomous patterns are generated simply owing to the interaction of an activator and a repressor. The activator self-activates and activates the repressor, which then diffuses and inhibits the activator along the diffusion path. This mechanism amplifies initial cellular fluctuations into tissue-level spatial patterns (2-4). Computer simulations suggest that by tinkering with the diffusion constants and the kinetics of the activator-repressor interaction, this simple circuit can recapitulate the immense diversity of pigmentation patterns observed in nature (3). However, the molecular identities and dynamics of actual activator-repressor pairs that fulfill the classic Turing model for pigment patterning have remained elusive. Anthocyanin spots in flower petals provide an excellent empirical system to reveal the molecular basis for the formation and evolution of periodic pigmentation patterns. These patterns, which are highly diverse in the angiosperms even among different varieties of the same species (5,6), are known to serve as critical cues in plant-pollinator interactions (7-9); and the genetic network controlling anthocyanin pigment production is otherwise well described (10, 11).

Flower color patterns have long served as a model for developmental genetics because pigment phenotypes are visually striking, yet generally not required for plant viability, facilitating the genetic analysis of color and pattern mutants. The evolution of novel flower colors and patterns has played a key role in the adaptive radiation of flowering plants via their specialized interactions with different pollinator guilds (e.g., bees, butterflies, birds), motivating the search for allelic differences affecting flower color pattern in closely related plant species with different pollinators. We have identified LIGHT AREAS1 (LAR1), encoding an R2R3-MYB transcription factor, as the causal gene underlying the spatial pattern variation of floral anthocyanin pigmentation between two sister species of monkeyflower: the bumblebee-pollinated Mimulus lewisii and the hummingbirdpollinated Mimulus cardinalis. We demonstrated that LAR1 positively regulates FLAVONOL SYNTHASE (FLS), essentially eliminating anthocyanin biosynthesis in the white region (i.e., light areas) around the corolla throat of M. lewisii flowers by diverting dihydroflavonol into flavonol biosynthesis from the anthocyanin pigment pathway. FLS is preferentially expressed in the light areas of the M. lewisii flower, thus prepatterning the corolla. LAR1 expression in M. cardinalis flowers is much lower than in M. lewisii, explaining the unpatterned phenotype and recessive inheritance of the M. cardinalis allele. Furthermore, our gene-expression analysis and genetic mapping results suggest that cis-regulatory change at the LAR1 gene played a critical role in the evolution of different pigmentation patterns between the two species.flower color pattern | Mimulus | anthocyanins | flavonols | R2R3-MYB M any flowers display interesting color patterns (e.g., spots, stripes, picotees, bull's-eyes) that are precisely programmed during development. Numerous studies have shown that these color patterns are critically important for plant-pollinator interactions (1-8). Among the most captivating examples are deceptive orchids that display floral pigment patterns remarkably similar to female bees or wasps to lure male counterparts for pseudocopulation, thereby achieving pollination (9-11). Despite the obvious aesthetic and ecological significance of these flower color patterns, the molecular mechanisms of pigment pattern formation is not well understood, nor is the genetic basis underlying pattern variation between related species in nature.From a genetic and developmental viewpoint, the most extensively studied flower color pattern is venation. Studies in snapdragon (Antirrhinum majus) and petunia (Petunia hybrida) have revealed a conserved mechanism for the formation of vein-associated anthocyanin pigmentation pattern in petal epidermis. Pigments are only produced in the overlapping expression domains of the R2R3-MYB and bHLH coregulators of anthocyanin biosynthetic genes; the bHLH expression is confined to the petal epidermis and the R2R3-MYB expression is specific to ce...

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