Mechanisms underlying flower color variation in Asian species of Meconopsis: A preliminary phylogenetic analysis based on chloroplast DNA and anthocyanin biosynthesis genes

The Qinghai–Tibet Plateau (QTP) harbors the highest species diversity of alpine plants in the world, with a spectacular diversity of flower colors. Among these QTP plants, the genus Meconopsis comprises more than 50 species, for which flower color is a key diagnostic character. However, the mechanisms underlying flower color variation have rarely been investigated. In the present study, we used three chloroplast (cp) DNA fragments and two anthocyanin biosynthesis genes (F3H andF3′H) for phylogenetic reconstruction of Meconopsis. We revealed the presence of three well-supported clades and/or subclades in the cpDNA and nuclear gene trees; further, flower color transition occurred in each lineage. The results of selection tests and preliminary expression analyses of the anthocyanin biosynthesis genes indicate that the pigment pathway leading to cyanidin is active in blue and red flowers of Meconopsis; further, a blue–red color shift is not attributable to an on/off switching of the anthocyanin biosynthetic pathway (ABP) branches. Together with the results of previous flower pigment analyses, our findings suggest that blue–red flower color transitions in Meconopsis are attributable to modification of cyanidin. Our molecular dating results indicate that the lineage diversification inMeconopsis is closely related to the QTP uplift; thus, it is likely that environmental changes arising from the QTP uplift have played important roles in driving the diversification of flavonoids, through which species of Meconopsis have adapted physiologically to diverse habitats.     

Extensive evolutionary rate variation in floral color determining genes in the genus Ipomoea

Flower color is an important adaptive trait in many plant species because it determines reproductive success through differential attractiveness to insect pollinators. The genus Ipomoea is a pan tropically distributed plant genus characterized by showy flowers that often differ in color among closely related species. Flower color is determined primarily by products of the anthocyanin biosynthesis pathway. To determine whether flower color evolution among members of the genus is driven by adaptive molecular evolution of the anthocyanin pathway genes, we analyzed data from 10 genes sequenced from 19 species of Ipomoea . Six protein-coding genes from the anthocyanin pathway were drawn for evolutionary analysis in addition to three genes from the unrelated leucine biosynthesis pathway and one MADS box regulatory gene for comparison. The analyses provided: (i) no convincing evidence for positive selection on anthocyanin pathway structural genes, or on the other sampled genes, despite shifts in flower color among species included in the sample; (ii) pathway position correlated weakly with estimates of the intensity of evolutionary constraint on the anthocyanin pathway enzyme coding genes; and (iii) there was substantial gene-specific heterogeneity in the rates of synonymous site evolution. Synonymous rate heterogeneity does not appear to be accounted for by codon bias or local contextual or compositional sequence differences, leading us to implicate heterogeneous rates of mutation among genes as the most probable cause of synonymous rate heterogeneity.     

Bird-pollinated Macaronesian Lotus (Leguminosae) evolved within a group of entomophilous ancestors with post-anthesis flower color change

We analyzed the evolution of red/orange flowers in four putatively bird-pollinated species of Macaronesian Lotus, with the aim of investigating whether this floral trait evolved from a similar trait found in some entomophilous Lotus species, namely the ability to modify flower color to red after anthesis. First, we mapped the ability to modify flower color in this group on a well-resolved and densely sampled phylogenetic tree of the Macaronesian Lotus. Secondly, we determined differences in light reflectance and pigment composition between petals of (1) prechange and postchange flowers in bee-pollinated species and (2) between bee and putatively bird-pollinated species. Post-anthesis flower color change evolved three times within Macaronesian Lotus, and putatively bird-pollinated species evolved within a clade with this ability to change flower color to red after anthesis. The evolutionary transition to red/orange flowers in the putatively bird-pollinated species involved biochemical changes similar to those of the developmental transition to red postchange flowers. In both cases there are changes in the composition of flavonols and anthocyanidins within the same metabolic pathways, especially in the cyanidin branch of pigment production, but not the activation or inactivation of additional branches of this pathway. Post-anthesis color change in Lotus, from yellow to red, is thought to be an adaptation to reduce bee visits to already pollinated flowers. Our results are consistent with the hypothesis that constitutive red coloration for bird-pollination evolved from facultative red flower color change in Lotus. As red post-anthesis coloration is widespread in plants, this may possibly represent a widespread exaptive mechanism for the evolution of bird pollination.     

PARALLEL EVOLUTION AT MULTIPLE LEVELS IN THE ORIGIN OF HUMMINGBIRD POLLINATED FLOWERS IN IPOMOEA

A transition in flower color accompanying a shift in pollinator guilds is a prominent and repeated adaptation in angiosperms. In many cases, shifts to similar pollinators are associated with similar flower‐color transitions. The extent to which this parallelism at the phenotypic level results from parallel changes at the biochemical, developmental, and genetic levels, however, remains an open question. There have been few attempts to determine whether parallelism at these lower levels results from mutation bias or fixation bias of different classes of mutation. We address these issues by examining the biochemical, developmental, and genetic changes that have occurred in red‐flowering species of the Mina lineage of morning glories (Ipomoea) and compare these to the changes reported for I. horsfalliae, which has independently evolved red flowers. Using transgenic techniques, we demonstrate that the transition from blue to red flowers in Mina species is due primarily to down‐regulation of the enzyme flavonol‐3′‐hydroxylase (F3′H) in flowers but not in vegetative tissues, and that this down‐regulation is at least partly due to cis‐regulatory change in the gene for F3′H. These changes are similar to those exhibited by I. horsfalliae, indicating parallelism at the biochemical and developmental levels, and possibly at the genetic level.     

PREDICTABILITY AND IRREVERSIBILITY OF GENETIC CHANGES ASSOCIATED WITH FLOWER COLOR EVOLUTION IN PENSTEMON BARBATUS

Two outstanding questions in evolutionary biology are whether, and how often, the genetic basis of phenotypic evolution is predictable; and whether genetic change constrains evolutionary reversibility. We address these questions by studying the genetic basis of red flower color in Penstemon barbatus. The production of red flowers often involves the inactivation of one or both of two anthocyanin pathway genes, Flavonoid 3′,5′‐hydroxylase (F3′5′h) and Flavonoid 3′‐hydroxylase (F3′h). We used gene expression and enzyme function assays to determine that redundant inactivating mutations to F3′5′h underlie the evolution of red flowers in P. barbatus. Comparison of our results to previously characterized shifts from blue to red flowers suggests that the genetic change associated with the evolution of red flowers is predictable: when it involves elimination of F3′5′H activity, functional inactivation or deletion of this gene tends to occur; however, when it involves elimination of F3′H activity, tissue‐specific regulatory substitutions occur and the gene is not functionally inactivated. This pattern is consistent with emerging data from physiological experiments indicating that F3′h may have pleiotropic effects and is thus subject to purifying selection. The multiple, redundant inactivating mutations to F3′5′h suggest that reversal to blue‐purple flowers in this group would be unlikely.     

The Genetic Basis of a Rare Flower Color Polymorphism in Mimulus lewisii Provides Insight into the Repeatability of Evolution

A long-standing question in evolutionary biology asks whether the genetic changes contributing to phenotypic evolution are predictable. Here, we identify a genetic change associated with segregating variation in flower color within a population of Mimulus lewisii. To determine whether these types of changes are predictable, we combined this information with data from other species to investigate whether the spectrum of mutations affecting flower color transitions differs based on the evolutionary time-scale since divergence. We used classic genetic techniques, along with gene expression and population genetic approaches, to identify the putative, loss-of-function mutation that generates rare, white flowers instead of the common, pink color in M. lewisii. We found that a frameshift mutation in an anthocyanin pathway gene is responsible for the white-flowered polymorphism found in this population of M. lewisii. Comparison of our results with data from other species reveals a broader spectrum of flower color mutations segregating within populations relative to those that fix between populations. These results suggest that the genetic basis of fixed differences in flower color may be predictable, but that for segregating variation is not.     

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.     

Patterns of evolutionary rate variation among genes of the anthocyanin biosynthetic pathway

The anthocyanin biosynthetic pathway is responsible for the production of anthocyanin pigments in plant tissues and shares a number of enzymes with other biochemical pathways. The six core structural genes of this pathway have been cloned and characterized in two taxonomically diverse plant species (maize and snapdragon). We have recently cloned these genes for a third species, the common morning glory, Ipomoea purpurea. This additional information provides an opportunity to examine patterns of evolution among genes within a single biochemical pathway. We report here that upstream genes in the anthocyanin pathway have evolved substantially more slowly than downstream genes and suggest that this difference in evolutionary rates may be explained by upstream genes being more constrained because they participate in several different biochemical pathways. In addition, regulatory genes associated with the anthocyanin pathway tend to evolve more rapidly than the structural genes they regulate, suggesting that adaptive evolution of flower color may be mediated more by regulatory than by structural genes. Finally, for individual anthocyanin genes, we found an absence of rate heterogeneity among three major angiosperm lineages. This rate constancy contrasts with an accelerated rate of evolution of three CHS-like genes in the Ipomoea lineage, indicating that these three genes have diverged without coordinated adjustment by other pathway genes.     

Temporal and spatial control of expression of anthocyanin biosynthetic genes in developing flowers of Antirrhinum majus

The co-ordination of expression of anthocyanin biosynthetic genes was studied in developing flowers. Four genes encoding enzymes operating late in the anthocyanin biosynthetic pathway are induced together during flower development but the early steps appear to be induced more rapidly. Co-ordination of expression could imply a common regulatory mechanism controlling the expression of metabolically related genes. The data presented here show that while four genes may share such a mechanism for the control of their expression during flower development, different control processes regulate the early steps of the pathway. Spatially, gene expression is patterned across the flower and appears to be very similar for all the biosynthetic genes. However, the observed influence of the regulatory gene Delila shows that the spatial co-ordination of gene expression must involve more than one regulatory system. Delila itself appears to have a dual function, being required for activation of expression of the later genes in the flower tube but repressing chalcone synthase gene expression in the mesophyll of the corolla lobes. It is postulated that common signals induce the expression of genes in the pathway during flower development. The data presented here suggest that the same regulatory mechanism interprets these signals for four of the genes encoding the later biosynthetic enzymes, but that different or modified mechanisms interpret the signals to control expression of chalcone synthase and chalcone isomerase genes in Antirrhinum flowers.     

花色改造基因工程

自1987年世界首例成功运用转基因技术改造矮牵牛花色以来,花色改造基因工程技术不断展现它在培育新花色品系上的无穷魅力。介绍了近年来运用基因工程技术成功改造花色的3种主要策略:(1)采用反义RNA及共抑制的方法来改变花颜色的深浅;(2)通过导入新基因产生新奇花色;(3)利用转座子构建特殊表达载体,随机激活花色合成的基因来产生嵌合花色。此外,还对转基因株花色不稳定原因进行了讨论。     

Genes that determine flower color: the role of regulatory changes in the evolution of phenotypic adaptations

A central goal of evolutionary genetics is to trace the causal pathway between mutations at particular genes and adaptation at the phenotypic level. The proximate objective is to identify adaptations through the analysis of molecular sequence data from specific candidate genes or their regulatory elements. In this paper, we consider the molecular evolution of floral color in the morning glory genus (Ipomoea) as a model for relating molecular and phenotypic evolution. To begin, flower color variation usually conforms to simple Mendelian transmission, thus facilitating genetic and molecular analyses. Population genetic studies of flower color polymorphisms in the common morning glory (Ipomoea purpurea) have shown that some morphs are subject to complex patterns of selection. Striking differences in floral color and morphology are also associated with speciation in the genus Ipomoea. The molecular bases for these adaptive shifts can be dissected because the biosynthetic pathways that determine floral pigmentation are well understood and many of the genes of flavonoid biosynthesis have been isolated and extensively studied. We present a comparative analysis of the level of gene expression in Ipomoea for several key genes in flavonoid biosynthesis. Specifically we ask: how frequently are adaptive shifts in flower color phenotypes associated with changes in regulation of gene expression versus mutations in structural genes? The results of this study show that most species differences in this crucial phenotype are associated with changes in the regulation of gene expression.     

环境因子调控植物花青素苷合成及呈色的机理

花青素苷（anthocyanin）是决定被子植物花、果实和种皮等颜色的重要色素之一。花青素苷的合成与积累过程往往与植物发育过程密切相关,由内外因子共同控制。环境因子通过诱导植物体内花青素苷合成途径相关基因的表达来调控花青素苷的呈色反应。该文追踪了国内外相关研究,认为光是影响花青素苷呈色的主要环境因子之一,光质和光强均能在一定程度上影响花青素苷的合成,其中光质起着更为关键的作用;低温能诱导花青素苷的积累,高温则会加速花青素苷的降解;不同的糖类物质均能影响花青素苷的合成,大部分结构基因和调节基因的表达均受糖调控。关于花发育与花青素苷呈色的关系、观赏植物花色对环境因子的响应以及花青素苷抵御逆境的机理尚待深入研究。因此,综合考察花发育与植物花青素苷合成及其呈色之间的关系,特别是光周期对花发育的影响导致花青素苷合成及呈色的机理是花色研究的一个重要课题。利用环境因子调控花色将会极大地提高花卉的观赏价值。     

环境因子调控植物花青素苷合成及呈色的机理

花青素苷(anthocyanin)是决定被子植物花、果实和种皮等颜色的重要色素之一。花青素苷的合成与积累过程往往与植物发育过程密切相关, 由内外因子共同控制。环境因子通过诱导植物体内花青素苷合成途径相关基因的表达来调控花青素苷的呈色反应。该文追踪了国内外相关研究, 认为光是影响花青素苷呈色的主要环境因子之一, 光质和光强均能在一定程度上影响花青素苷的合成, 其中光质起着更为关键的作用; 低温能诱导花青素苷的积累, 高温则会加速花青素苷的降解;不同的糖类物质均能影响花青素苷的合成, 大部分结构基因和调节基因的表达均受糖调控。关于花发育与花青素苷呈色的关系、观赏植物花色对环境因子的响应以及花青素苷抵御逆境的机理尚待深入研究。因此, 综合考察花发育与植物花青素苷合成及其呈色之间的关系, 特别是光周期对花发育的影响导致花青素苷合成及呈色的机理是花色研究的一个重要课题。利用环境因子调控花色将会极大地提高花卉的观赏价值。     

Five anthocyanin polymorphisms are associated with an R2R3-MYB cluster in Mimulus guttatus (Phrymaceae)

? Premise of study: Botanists have long been interested in the reasons for genetic variation among individuals, populations, and species of plants. The anthocyanin pathway is ideal for studying the evolution of such phenotypic variation. ? Methods: We used a combination of quantitative trait loci mapping and association studies to understand the genetic basis of variation in five anthocyanin phenotypes including calyx, corolla, and leaf coloration patterns that vary within and among populations of Mimulus guttatus. We then examined what genes might be responsible for this phenotypic variation and whether one of the traits, calyx spotting, is randomly distributed across the geographic range of the species. ? Key results: All five phenotypes in M. guttatus were primarily controlled by the same major locus (PLA1), which contains a tandem array of three R2R3-MYB genes known to be involved in the evolution of flower color in a related species of Mimulus. Calyx spotting was nonrandomly distributed across the range of M. guttatus and correlated with multiple climate variables. ? Conclusions: The results of this study suggest that variation in R2R3-MYB genes is the primary cause of potentially important anthocyanin phenotypic variation within and among populations of M. guttatus, a finding consistent with recent theoretical and empirical research on flower color evolution.     

A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization,heterologous expression in transgenic plants and transactivation in yeast cells

The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s). A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P. frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon. The Myc-rp gene was expressed in leaves and roots of both red and green P. frutescens equally. Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P. frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP. The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato. However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1–115 amino acids deleted) of Myc-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed. In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P. frutescens. A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins. Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.     

Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-3′,5′-hydroxylase: specific switching of the pathway in petunia

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, which are generally required for the expression of blue or purple flower color. It has been predicted that the introduction of this enzyme into a plant species that lacks it would enable the production of blue or purple flowers by altering the anthocyanin composition. We present here the results of the genetic engineering of petunia flower color, pigmentation patterns and anthocyanin composition with sense or antisense constructs of the F3'5'H gene under the control of the CaMV 35S promoter. When sense constructs were introduced into pink flower varieties that are deficient in the enzyme, transgenic plants showed flower color changes from pink to magenta along with changes in anthocyanin composition. Some transgenic plants showed novel pigmentation patterns, e.g. a star-shaped pattern. When sense constructs were introduced into blue flower petunia varieties, the flower color of the transgenic plants changed from deep blue to pale blue or even pale pink. Pigment composition analysis of the transgenic plants suggested that the F3'5'H transgene not only created or inhibited the biosynthetic pathway to 3',5'-hydroxylated anthocyanins but switched the pathway to 3',5'-hydroxylated or 3'-hydroxylated anthocyanins.     

Evolution of Anthocyanin Biosynthesis in Maize Kernels: The Role of Regulatory and Enzymatic Loci

Understanding which genes contribute to evolutionary change and the nature of the alterations in them are fundamental challenges in evolution. We analyzed regulatory and enzymatic genes in the maize anthocyanin pathway as related to the evolution of anthocyanin-pigmented kernels in maize from colorless kernels of its progenitor, teosinte. Genetic tests indicate that teosinte possesses functional alleles at all enzymatic loci. At two regulatory loci, most teosintes possess alleles that encode functional proteins, but ones that are not expressed during kernel development and not capable of activating anthocyanin biosynthesis there. We investigated nucleotide polymorphism at one of the regulatory loci, c1. Several observations suggest that c1 has not evolved in a strictly neutral manner, including an exceptionally low level of polymorphism and a biased representation of haplotypes in maize. Curiously, sequence data show that most of our teosinte samples possess a promoter element necessary for the activation of the anthocyanin pathway during kernel development, although genetic tests indicate that teosinte c1 alleles are not active during kernel development. Our analyses suggest that the evolution of the purple kernels resulted from changes in cis regulatory elements at regulatory loci and not changes in either regulatory protein function nor the enzymatic loci.