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.     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Engineering of flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway

Flower color was modified in forsythia (Forsythia x intermedia cv Spring Glory) by inducing anthocyanin synthesis in petals through sequential Agrobacterium-mediated transformation with dihydroflavonol 4-reductase from Antirrhinum majus (AmDFR) and anthocyanidin synthase from Matthiola incana (MiANS) genes. This is the second report of flower color modification of an ornamental shrub after rose, and the first time an ANS gene is used for this purpose. Double transformants (AmDFR+MiANS) displayed a novel bronze-orange petal color, caused by the de novo accumulation of cyanidin-derived anthocyanins over the carotenoid yellow background of wild type (wt), and intense pigmentation of vegetative organs. Transformation with single genes (either AmDFR or MiANS) produced no change in flower color, showing a multistep control of late anthocyanin pathway in petals of forsythia. Analysis of relevant late flavonoid pathway genes – an endogenous flavonoid glycosyltransferase (FiFGT) and transformed DFR and ANS genes – showed appropriate expression in flower organs. Functional characterization of FiFGT expressed in E. coli revealed its ability to metabolize both flavonols and anthocyanidin substrates, a prerequisite for effective anthocyanin accumulation in petals of plants transformed with constructs leading to anthocyanidin synthesis. Biochemical analyses of flavonoid compounds in petals and leaves showed that, besides anthocyanin induction in petals of double transformants, the accumulation pattern of flavan-3-ols was quantitatively and qualitatively modified in petals and leaves of transformants, in agreement with the most recent model proposed for flavan-3-ol synthesis. On the other hand, phenylpropanoid, flavone and flavonol pools were not quantitatively affected, indicating a tight regulation of early flavonoid pathway.     

Chalcone synthase activity and polyphenolic compounds of shoot tissues from adult and rejuvenated walnut trees

Changes in the metabolism of naphthoquinone and flavonoids during the growth of half-sib adult and rejuvenated walnut shoots (Juglans nigra × Juglans regia L.) were studied at the tissue level for two years after pruning. Moreover, the role of chalcone synthase (CHS; EC 2.3.1.74) in the regulation of flavonoid biosynthesis was investigated at the level of enzyme activity. The end products of walnut flavonoid biosynthesis, myricitrin and quercitrin, which accumulated in the bark and phloem at the end of growth, did not inhibit the biosynthetic process at concentrations of up to 100 μM each. There was no evidence of CHS regulation by feedback or similar mechanisms which might modulate enzyme activity. Mathematical correlation of CHS activity and flavonoid accumulation during shoot growth, however, indicated that CHS is the rate-limiting enzyme of the pathway in bark and phloem and that flavonoids seem to be transported from phloem to bark where they accumulated mainly during growth. In defoliated shoots, naphthoquinone metabolism appeared to be a marker of the walnut rejuvenation stage in the medulla, phloem and buds immediately after cutting and thereafter mainly in buds one year after cutting. Chalcone synthase and flavonoid contents appeared to be markers of the adult stage in the phloem. Received: 30 September 1996 / Accepted: 17 March 1997     

Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene

Plants can survive a limiting nitrogen (N) supply by developing a set of N limitation adaptive responses. However, the Arabidopsis nla (nitrogen limitation adaptation) mutant fails to produce such responses, and cannot adapt to N limitation. In this study, the nla mutant was utilized to understand further the effect of NLA on Arabidopsis adaptation to N limitation. Grown with limiting N, the nla mutant could not accumulate anthocyanins and instead produced an N limitation-induced early senescence phenotype. In contrast, when supplied with limiting N and limiting phosphorus (Pi), the nla mutants accumulated abundant anthocyanins and did not show the N limitation-induced early senescence phenotype. These results support the hypothesis that Arabidopsis has a specific pathway to control N limitation-induced anthocyanin synthesis, and the nla mutation disrupts this pathway. However, the nla mutation does not affect the Pi limitation-induced anthocyanin synthesis pathway. Therefore, Pi limitation induced the nla mutant to accumulate anthocyanins under N limitation and allowed this mutant to adapt to N limitation. Under N limitation, the nla mutant had a significantly down-regulated expression of many genes functioning in anthocyanin synthesis, and an enhanced expression of genes involved in lignin production. Correspondingly, the nla mutant grown with limiting N showed a significantly lower production of anthocyanins (particularly cyanidins) and an increase in lignin contents compared with wild-type plants. These data suggest that NLA controls Arabidopsis adaptability to N limitation by channelling the phenylpropanoid metabolic flux to the induced anthocyanin synthesis, which is important for Arabidopsis to adapt to N limitation.     

Cloning of FaPAL6 gene from strawberry fruit and characterization of its expression and enzymatic activity in two cultivars with different anthocyanin accumulation

The accumulation of anthocyanin pigments is one of the most important traits that turn strawberry fruit attractive to consumers. During ripening, strawberry fruit color development is associated to anthocyanin synthesis through the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) is a key enzyme in this pathway, having a determining role in strawberry fruit quality. In this work, we studied the level of anthocyanins during fruit ripening of two cultivars that differ in color development (Camarosa and Toyonoka). Toyonoka showed a lower anthocyanin accumulation that was limited to external fruit tissue, while Camarosa accumulated higher amount of anthocyanins in both internal and external sections. In addition, we cloned a full-length gene (FaPAL6) and analyzed its expression in different strawberry plant tissues. The expression of this gene is fruit specific, and increases during fruit ripening in both cultivars along with anthocyanin accumulation. The mRNA level of FaPAL6 was higher in Camarosa. PAL enzyme activity increased at similar rates in both cultivars at early ripening stages, but at the end of ripening PAL activity diminished in Toyonoka while it rose markedly in Camarosa. PAL activity was higher in internal fruit tissue, showing no correlation with anthocyanin level of the same section in both cultivars. The higher FaPAL6 expression and activity detected in Camarosa could be associated to the enhanced anthocyanin accumulation found in this cultivar.     

Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference

Suppression of biosynthetic genes involved in flower color formation is an important approach for obtaining target flower colors. Here we report that flower color of the garden plant Torenia hybrida was successfully modulated by RNA interference (RNAi) against a gene of chalcone synthase (CHS), a key enzyme for anthocyanin and flavonoid biosynthesis. By using each of the coding region and the 3'-untranslated region of the CHS mRNA as an RNAi target, exhaustive and gene-specific gene silencing were successfully induced, and the original blue flower color was modulated to white and pale colors, respectively. Our results indicate that RNAi is quite useful for modulations of flower colors of commercially important garden plants.     

Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway

Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s) of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP), suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS) at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments.     

Population genetic structure and adaptive differentiation of iron walnut Juglans regia subsp. sigillata in southwestern China

Southwestern (SW) China is an area of active tectonism and erosion, yielding a dynamic, deeply eroded landscape that influences the genetic structure of the resident populations of plants and animals. Iron walnut (Juglans regia subsp. sigillata) is a deciduous tree species endemic to this region of China and cultivated there for its edible nuts. We sampled 36 iron walnut populations from locations throughout the species' range in SW China and genotyped a total of 765 individuals at five chloroplast DNA regions and 22 nuclear microsatellite loci. Species distribution models were produced to predict the evolution and historical biogeography of iron walnut and to estimate the impacts of climate oscillations and orographic environments on the species' demography. Our results indicated that J. regia subsp. sigillata had relatively low genetic diversity, high interpopulation genetic differentiation, and asymmetric interpopulation gene flow. Based on DIYABC analysis, we identified two lineages of J. sigillata in southwestern China. The lineages (subpopulations) diverge during the last glacial period (~1.34 Ma). Southwestern China was a glacial refuge during the last glacial period, but increasingly colder and arid climates might have fostered the fragmentation of J. regia subsp. sigillata within this refugium. Finally, we found that recent habitat fragmentation has led to a reduction in population connectivity and increased genetic differentiation by genetic drift in isolated populations. Our results support a conclusion that geological and climatic factors since the Miocene triggered the differentiation, evolutionary origin, and range shifts of J. sigillata in the studied region.     

Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth

In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.     

Application of Pseudomonas fluorescens to Blackberry under Field Conditions Improves Fruit Quality by Modifying Flavonoid Metabolism

Application of a plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens N21.4, to roots of blackberries (Rubus sp.) is part of an optimised cultivation practice to improve yields and quality of fruit throughout the year in this important fruit crop. Blackberries are especially rich in flavonoids and therefore offer potential benefits for human health in prevention or amelioration of chronic diseases. However, the phenylpropanoid pathway and its regulation during ripening have not been studied in detail, in this species. PGPR may trigger flavonoid biosynthesis as part of an induced systemic response (ISR) given the important role of this pathway in plant defence, to cause increased levels of flavonoids in the fruit. We have identified structural genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways catalysing the conversion of phenylalanine to the final products including flavonols, anthocyanins and catechins from blackberry, and regulatory genes likely involved in controlling the activity of pathway branches. We have also measured the major flavonols, anthocyanins and catechins at three stages during ripening. Our results demonstrate the coordinated expression of flavonoid biosynthetic genes with the accumulation of anthocyanins, catechins, and flavonols in developing fruits of blackberry. Elicitation of blackberry plants by treatment of roots with P.fluorescens N21.4, caused increased expression of some flavonoid biosynthetic genes and an accompanying increase in the concentration of selected flavonoids in fruits. Our data demonstrate the physiological mechanisms involved in the improvement of fruit quality by PGPR under field conditions, and highlight some of the genetic targets of elicitation by beneficial bacteria.     

Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants

Initially, we isolated the caffeic acid O-methyltransferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, we produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidases expression, which are related to the phenylpropanoid pathway and antioxidant activity. The MsCOMT-down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT-down-regulated transgenic plants. We also observed a morphological difference, with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway.     

基于整合方法分析茶树响应病原真菌胁迫的共有模式

为探讨茶树(Camellia sinensis)对病菌胁迫的共有响应模式和抗病机制,运用生物信息学方法对多组RNA-seq数据进行提取、整合及功能富集,结合多种工具和数据库资源对主要调控分子及蛋白互作模块加以分析。结果表明,病原真菌胁迫下,茶树有较多细胞色素P450家族成员表达显著上调;类固醇和激素的代谢过程、苯丙烷合成途径被激活,有丝分裂细胞周期调控、DNA甲基化等生物过程及光合作用途径受到抑制;主要调控分子如转录因子WRKY和NAC、激酶RLK-Pelle和CAMK等以上调为主。差异表达的蛋白互作模块分析表明,有丝分裂周期调控、基于微管运动、淀粉和蔗糖代谢、细胞壁多糖合成、光合作用、类黄酮代谢模块明显下调,木质素合成和萜类生物合成模块上调;且模块之间可能存在互作。病菌胁迫激活的木质素和萜类合成途径的关键基因包括阿魏酸-5-羟基化酶基因F5H、过氧化物酶基因POD和萜类合成酶基因HMGR等。细胞色素P450基因可能在病菌胁迫中起关键作用,增强木质素和萜类物质的合成、削弱光合作用可能是茶树响应真菌胁迫的核心模式。