硕士研究生读书报告
姓 名： 郜志栋
学科、专业： 研究方向： 指导教师：
20xx年12月12日
Advances in the transcriptional regulation of the flavonoid biosynthetic pathway Abstract: Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix–loop–helix (bHLH), and WD40 classes. This MYB–bHLH–WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins.
Key words: bHLH, flavonoids, MYB, transcription factors, WD40.
Introduction
Flavonoid compounds are secondary metabolite s widely accumulated in vascular plants and to a lesser extent in mosses. They accumulate in all organs and tissues, at different stage s of development, and depending on the environmental conditions. Beside their multiple roles in plant development and adaptation to the environment, these molecules are of major interest for human nutrition and health . Indeed, they contribute to the organoleptic quality of plant-derived products (colour, taste, flavour, etc.), and, in addition, they have been shown to be beneficial to human health and in prevention of cell ageing. In grape (Vitis vinifera L.) berries for instance, the flavonoid composition is essential for wine quality and conservation. Moreover, the regular consumption of red wine is thought to explain the ‘French pa radox’, whereby the French population suffers a relatively low incidence of coronary heart disease in spite of a diet rich in saturated fat . The mechanisms involved have long been related to the presence of flavonoids and stilbenes in red wine.
Work achieved on model plants pinpointed the tight regulation of the flavonoid biosynthetic pathway during plant development. It is now established that the transcriptional regulation of the structural gene s is controlled by MYB and basic helix–loop–helix (bHLH ) transcription factors, together with WD40 proteins. Special attention has hitherto been devoted to MYB, as demonstrated by the reported publications. Herein, the recent advances in the knowledge of the transcriptional regulation of the flavonoid pathway are discussed, with a particular focus on bHLH transcription factors.
The MYB transcription factors
The first MYB transcription factors regulating the flavonoid pathway were identified in 1987
in maize, and comprised C1 and Pl1 (Purple leaf 1), in addition to P1. At that time, identification of C1 indicated that plant transcription factors were closely related to those of mammals, constituting a milestone in plant molecular biology. Indeed, C1 showed a significant homology with the vertebrate c-MYB proto-oncogene, derived from avian myeloblastosis virus and known to control cell proliferation and differentiation. MYB transcription factors are characterized by the so-called N-terminal MYB domain, consisting of 1 to 3 imperfect repeats of almost 52 amino acids (R1, R2, and R3). While the MYB domain is involved in DNA binding and dimerization, the C-terminal region regulates target gene expression (i.e. activation or repression). Plant MYB transcription factors bind different cis-elements, called MYB-binding sites (MBSs), and some MYB transcription factors show a certain flexibility of recognition. However, MYB transcription factors belonging to different species and regulating the same pathway, such as PA biosynthesis for instance, seem to bind the same motif. MYB transcription factors regulating the flavonoid pathway have been widely investigated and identified in crop, ornamental, and model plants (Table 1). Most of them present two R repeats (R2R3 MYB proteins), and belong to subgroups 1-7 of the classification of Stracke et al. (2001). Regulators of the PA and anthocyanin pathways display the [D/E]Lx2[R/K]x3Lx6Lx3R motif necessary for interaction with bHLH transcription factors in their R3 repeat, while MYB transcription factors governing flavonoidl biosynthesis exhibit the SG7 [K/R][R/x][R/K]xGRT[S/x][R/ G]xx[M/x]K and the SG7-2 ([W/x][L/x]LS) motifs in their C-terminal end. Nevertheless all regulators of the flavonoid pathway do not fit this classification perfectly. In potato, a single domain MYB protein, similar to soybean MYB73, is 44 times more expressed in purple flesh compared with white flesh, suggesting a role in the control of anthocyanin biosynthesis.
Most of the MYB transcription factors characterized to date control only one branch of the flavonoid pathway. Specific regulators of the anthocyanin pathway have been identified in petunia, Arabidopsis, strawberry, grapevine, tomato, potato, tobacco, and pear, to name a few. Among them, the R3 AtMYBL2 is an anthocyanin repressor, and the R2R3 AtMYB60 inhibits anthocyanin synthesis in lettuce. Extensive protein sequence alignments of 134 MYB transcription factors regulating the anthocyanin pathway revealed conserved residues in the R3 repeat (arginine, valine, and alanine) of dicots, as well as a short conserved motif ANDV. In addition, the
[R/K]Px[P/A/R]xx[F/Y] motif has been identified in the C-terminal region of these anthocyanin-regulating MYBs.
Regulators of PA biosynthesis have been identified in Arabidopsis, grapevine, leguminous plants, persimmon, and poplar. More recently, MYBs regulating the flavonol branch have also been identified in Arabidopsis and grapevine. As already mentioned above, MYBs generally regulate only one branch of the flavonoid pathway. In grapevine for instance, overexpression of VlMYBA1-2 in hairy roots induced only expression of structural genes related to anthocyanin biosynthesis and transport. Likewise, ectopic expression of VvMYBPA1 and VvMYBPA2 in grapevine hairy roots exclusively activated genes encoding enzymes of the PA pathway such as anthocyanidin reductase and leucoanthocyanidin reductase. Despite this highly specific function, some MYB transcription factors may play different roles. Over-expression of VvMYB5b in tomato affected both phenylpropanoid and carotenoid metabolism. The single R3 repeat CAPRICE (CPC) is known to regulate epidermal cell fates such as trichome and root hair formation in Arabidopsis. Furthermore, CPC inhibits anthocyanin accumulation in homologous and heterologous hosts, by competing with R2R3 MYB transcription factors regulating the
flavonoid pathway. Since CPC does not bind to DNA, it is likely that this transcription factor interferes by interacting with bHLH partners, as demonstrated by yeast two-hybrid assays.
In summary, many recent studies, together with the analysis of new plant genomes, suggest that primary protein structures and biological functions are correlated within MYB subgroups that are conserved between divergent species. This is especially true for MYB transcription factors regulating the flavonoid pathway, where specific motifs and conserved residues have been identified in anthocyanin (Lin-Wang et al., 2010) and flavonoid regulators. However, the biological functions of the consensus motifs present in the C-terminus of the proteins are just beginning to be investigated. It would be of great interest to determine if these specific motifs can provide the specificity for a MYB transcription factor to regulate a given branch of the flavonoid pathway, by modulating interactions with DNA and/or with protein partners such as bHLH and/or WD40 proteins.
The WD40 proteins
WD40 or WDR (WD repeat) proteins are involved in many eukaryotic cellular processes including cell division, vesicle formation and trafficking, signal transduction, RNA processing, and regulation of transcription. They notably participate in chromatin remodelling, through modifications of the histone proteins, and can thus influence transcription.
WD40 proteins are characterized by a peptide motif of 44-60 amino acids, typically delimited by the GH dipeptide on the N-terminal side (11-24 residues from the N-terminus) and the WD dipeptide on the C-terminus). This motif can be tandemly repeated 4-16 times within a protein, with a large majority of Arabidopsis WD40 proteins exhibiting 4 or more WD repeats. WD40 proteins are not thought to have any catalytic activity (DNA binding or regulation of expression of a target gene), but rather seem to be a docking platform, as they can interact with several proteins simultaneously. Only Arabidopsis TTG1 (Transparent Testa Glabra 1) was clearly demonstrated, using chromatin immunoprecipitation, to bind the promoter of AtTTG2, a gene encoding a WRKY transcription factor mainly involved in trichome patterning.A small number of WD40 proteins involved in the regulation of the flavonoid pathway have been identified so far (Table 1), and include petunia AN11, Arabidopsis TTG1, perilla PFWD, maize ZmPAC1, Medicago trunculata MtWD40-1, and grapevine WDR1 and WDR2. These WD40 proteins appear to be highly conserved among species. Indeed, PFWD and PhAN11 show 81.3% identity, whereas PFWD and AtTTG1 share 77.8% identity. The WD40 protein family seems to be less expanded than the the MYB or bHLH families, since MtWD40-1, AN11, and PAC1, are single-copy genes.
WD40 proteins, regulating the flavonoid pathway, such as TTG1, can control many other physiological processes, such as trichome and root hair determination and seed mucilage production, and are accordingly expressed in tissues both accumulating and not accumulating flavonoids. In petunia, an11 mutants show a reduced anthocyanin content in the corolla. Disturbance of petal coloration is attributed both to a reduction in the expression of flavonoid structural genes and to a modifica?tion of the vacuolar pH, indicating that AN11 is involved at least in the regulation of these two metabolic events. In Medicago truncatula, MtWD40-1 mutants are deficient in accumulation of mucilage, and the synthesis of PAs, flavonols, anthocyanins, and benzoic acid in seeds, but only in PA synthesis in flowers, and finally they show no modification of epidermal cell fate. MtWD40-1 mutants show a strong reduction of the expression of flavonoid structural genes, whereas overexpression of MtWD40-1 in M. truncatula hairy root does not
induce PA accumulation.
Altogether, these data clearly indicate that WD40 proteins can be involved in various physiological and metabolic events, but also point to the fact that the underlying regulatory mechanisms of these events require the presence of additional partners.
The MYB-bHLH-WD40 (MBW) complex
Although flavonoid subgroups are derived from the same biosynthetic pathway, they accumulate differentially in plant organs and tissues, depending on the developmental stage and the environmental conditions, since they fulfil different biological functions. Thus, their distribution implies an accurate spatial and temporal regulation of the flavonoid biosynthetic pathway, requiring a specific combination of transcription factors. The involvement of a ternary complex formed by proteins from the bHLH, MYB, and WD40 families, the MBW complex, has been clearly demonstrated in Arabidopsis and petunia.
The MBW complex is highly organized, and each subunit fulfils a specific function such as binding to DNA, activation of expression of a target gene, or stabilization of the transcription factor complex. The interaction between members of the MBW complex may determine the sub- cellular localization of the complex itself. For instance, bHLH-WD40 interaction seems to be necessary for trans-location of the WD40 proteins into the nucleus. Indeed, the PFWD-green fluorescent protein (GFP) fusion protein is localized in the cytosol when expressed alone, and co?expression of PFWD and MYC-RP in onion cells allows PFWD transport to the nucleus (Payne et al., 2000; Sompornpailin et al., 2002). Similar results have been described in petunia, where AN11 has also been localized in the cytosol. Likewise, in tobacco leaves infiltrated with the grapevine WDR1, the encoded protein is localized either in the cytosol or in the cytosol and nucleus depending on the observed cell, while VvMYCA1 is localized in both cellular compartments. Moreover, within the nucleus, members of the MBW complex can influence each other’s accumulation. In Arabidopsis ttg1 and gl1 mutants, GL3-yellow fluorescent protein (YFP) is partitioned to the nucleus, but is unevenly distributed into speckles, indicating that TTG1 and GL1 transcription factors are required for the proper subnuclear distribution of GL3
Using knockout mutants and overexpression experiments, two MBW complexes have been clearly identified so far and described in Arabidopsis and petunia, namely TT2/ TT8/TTG1 (Transparent Testa 2/Transparent Testa 8/ Transparent Testa Glabra 1) and AN2/AN1/AN11 (Antho?cyanin 2/1/11), respectively.
In Arabidopsis, the MBW complex TT2/TT8/TTG1 regulates PA accumulation in the seed coat, whereas the GL1/GL3- EGL3-TT8/TTG1 (Glabrous 1/Glabra 3-Enhancer of Glabra 3-Transparent Testa 8/Transparent Testa Glabra 1) complex controls trichome initiation and formation. A physical interaction between TT8 and TT2, as well as between TT8 and TTG1, has been demonstrated using yeast two-hybrid experiments. In addition, TTG1 can also directly interact with TT2 or the trichome regulator GL1, but without showing any obvious catalytic activity. Thus, it has been proposed that TTG1 may act as a bridge to stabilize the MBW complex. As described above, the ttg1 mutant phenotype indicates that TTG1 is involved in several physiological responses. bHLH proteins TT8, GL3, and EGL3 also show partially overlapping functions (Zhang et al., 2003). Consequently, the target gene specificity of the MBW complex seems to be conferred by the MYB protein. Indeed, PAP1/PAP2 (Production of Anthocyanin Pigment 1/2), TT2, GL1, WER (WEREWOLF), and AtMYB61 regulate anthocyanin
accumulation in seedlings, PA biosynthesis in seed teguments, trichome formation, root hair initiation, and mucilage production in seed teguments, respectively. Except for TT2, none of its closest homologues (PAP1, PAP2, WER, and AtMYB111) could activate the AtBAN pro?moter (BAN encodes an anthocyanidin reductase). In contrast, TT2 could interact either with TT8, EGL3, or GL3 to increase BAN activity significantly.
Rather than participating in the specific recognition of a target gene promoter, WD40 proteins are more likely to enhance gene activation. Dissection of the AtBAN promoter revealed that a fragment of 86 bp, including an MBS and a G-box at a distance of 36 bp, is sufficient to drive expression of the uidA reporter gene specifically in PA- accumulating cells. If the TT2-TT8 dimer can bind to the BAN promoter in yeast and activate it in planta, co-expression of TT2, TT8, and TTG1 in Arabidopsis protoplasts activates the BAN promoter almost four times more than the TT2-TT8 double transformation.
In petunia, the AN2/AN1/AN11 complex controls anthocyanin biosynthesis in the corolla, mainly by regulating DFR and CHSJ expression. Similarly to AN1, AN11 is involved in the regulation of anthocyanin biosynthesis in the corolla, but also regulates the vacuolar pH in petal limb cells and the morphology of the seed epidermal cells. However, AN2 does not affect these traits and exclusively regulates anthocyanin biosynthesis, while a second MYB transcription factor, PH4, controls the vacuolar pH. Again, these results are consistent with the specificity of MYB transcription factors. Removal of the AN1 C-terminal end only affects vacuolar pH and morphology of the seed coat cells, indicating that this domain is a domain which interacts with different MYB partners.
Flavonoid biosynthesis, at least in Arabidopsis, appears to be regulated only by MYB transcription factors that do not exhibit a motif for interaction with bHLH proteins in their R3 repeat. Indeed, AtMYB11, AtMYB12, and AtMYB111 activate on their own the CHS, CHI, F3H, and FLS promoters, but neither DFR nor UFGT. In grapevine, VvMYBF1 regulates VvFLS1 (Flavonoid Synthase 1) expression without the need for a bHLH partner, and can complement Arabidopsis myb12 mutants. Surprisingly, co-expression of ZmC1 and ZmLc driven by the fruit-specific E8 promoter in tomato led to a 60-fold increase in the flavonoid kaempferol level in the flesh, while plants transformed with each transcription factor independently showed no significant accumulation of flavonoids compared with wild-type plants. In maize, ZmFLS1 expression is controlled by the anthocyanin promoting the MYB-bHLH dimer C1/PL1 + R/B or by the phlobaphene promoting MYB P1. These results indicate that, depending on the plant species, regulation of the flavonoid pathway may differ, and involves either a MYB transcription factor alone or a MYB-bHLH dimer.
Transcriptional regulation of the regulators
Besides governing the expression of flavonoid structural genes, the members of the MBW complex also regulate their own expression in a complex circuit. TT8, for instance, interacts with TTG1 and MYB transcription factors such as TT2 or PAP1 to regulate its own transcription. Other MYB-bHLH dimers, such as PAP1/GL3, can regulate TT8 expression, as shown by yeast one-hybrid experiments and confirmed in planta. In petunia, the MYB proteins AN2 and AN4 specifically regulate AN1 expression, without influencing JAF13. In grapevine, VvMYC1 regulates its own expression by interacting with the MYB PA regulator VvMYBPA1. In gentian flower petals, GtMYB3 may control GtbHLH1 expression as well. In addition to bHLH, MYB
proteins can also control their own expression. In red-fleshed apples, MYB10 binds to and transactivates its own promoter. Indeed, in these red varieties, a minisatellite located in the promoter region of MdMYB10 constitutes an autoregulatory element, comprising five direct tandem repeats of a 23 bp motif, each one predicted to contain an MBS.
In these intricate loops, it can also be noted that ttg1 mutants can be complemented with varying degrees of efficiency by MYB transcription factors such as GL1, or bHLH proteins such as ZmR or GL3, which allow restoration of trichome formation. These results indicate that WD40 proteins act upstream of MYB and bHLH, and are also observed in Japanese morning glory (Ipomoea nil) flowers, where InbHLH2 expression is reduced in InWDR1 mutants. In an11 mutants, DFR activity is restored only by AN2 and not AN1 overexpression, indicating that AN11 may act upstream of AN2. However, the AN2 transcript level is identical in wild- type and an11 plants, indicating that AN11 could be involved in the post-translational control of AN2.
The complexity of the regulation of the MYB/bHLH network is also revealed by the transcriptomic analyses of plants from various species overexpressing a MYB transcription factor controlling the flavonoid pathway. In Gerbera callus and stamens overexpressing GMYB10 and strongly pigmented, a MYB transcription factor exhibiting a repressive motif similar to that of the V. vinifera C2 MYB protein is in turn overexpressed. Expression of this C2 repressor is also induced in grape roots overexpressing the specific anthocyanin regulator VlMYBA1, as well as in roots of grapes overexpressing the specific PA regulator VvMYBPA2. It is interesting to note that, in both cases, no significant change of bHLH or WD40 gene expression levels has been observed.
To conclude, a tight autoregulation of the MBW network does not appear systematic. In maize, PAC1 (WD40), R (bHLH), and C1 (MYB) seem to be independently regulated. In apple as well, MdMYB10 does not seem to regulate MdbHLH3 and MdbHLH33 expression.
prospect
Given the particular attention devoted to health and disease prevention through a balanced diet including natural products, flavonoids appear as possible nutraceuticals widely distributed in vegetables and fruits. In this context, an important research effort is currently underway to understand the biosynthetic pathway and the regulatory mechanisms of flavonoid biosynthesis in various plant species. If the pathway itself is now quite well understood, its regulation appears to be under a hierarchy of complex events, which are slowly being deciphered. The identification of new transcription factors involved in flavonoid biosynthesis should be conducted together with the investigation of the parameters controlling their expression. Modulating the expression of target transcription factors through cultural practices or adequate environmental conditions in order to modify flavonoid contents in plants may provide a good opportunity to avoid genetic engineering. Likewise, determining the endogenous factors which trigger the expression of the regulatory genes can be another path to follow. Finally, investigating the allelic variability between cultivars of the same plant species is likely to allow the use of these transcription factors as molecular markers of the fruit/vegetable quality.
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