Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa)
- First Online:
- 1.5k Downloads
The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.
KeywordsR2R3-MYB transcription factor Repressor Arabidopsis Lactuca sativa
Reverse transcription-polymerase chain reaction
Rapid amplification of cDNA ends-polymerase chain reaction
Cauliflower mosaic virus
UDP-glucose:flavonoid 3-O-glucosyl transferase
Flavonoid metabolism is unique to plants and produces a great number and variety of compounds including flavones, flavonols, anthocyanins, and proanthocyanins (Winkel-Shirley 2001; Park et al. 2004). These molecules play important roles in many fundamental processes in plants, such as the pigmentation of flowers and fruits, UV resistance, pollen fertility, responses to wounding and fungal attacks, and environmental stress responses (Shirley et al. 1995; Weisshaar and Jenkins 1998; Harborne and Williams 2000; Dixon and Piava 1995; Moyano et al. 1996; Pietta 2000; Cominelli et al. 2005). The regulation of the anthocyanin pigments in plants is particularly important in metabolic engineering applications because these compounds act as visual signals that attract the insects and animals required for pollination and seed dispersal (Holton and Cornish 1995).
The metabolic pathways involved in anthocyanin biosynthesis are well established, and the central pathways in this process are highly conserved in plants (Shih et al. 2006). Moreover, studies in model plants, such as Arabidopsis and maize, have facilitated a sound understanding of the genes involved and the assembly of the relevant enzyme complexes (Winkel-Shirley 2001). Two groups of genes are required for anthocyanin biosynthesis: structural genes that encode the enzymes that directly participate in the biosynthetic reactions, and genes for transcription factors that regulate the expression of these structural genes and the accumulation of flavonoid metabolites. Transcription factors can act as activators or repressors of gene expression, and mediate either increases or decreases in transcription through sequence-specific DNA binding and protein-protein interactions (Broun 2004).
MYB proteins have been identified in a large number of eukaryotes, including fungi, plants and vertebrates (Ohi et al. 1994; Martin and Paz-Ares 1997; Slamon et al. 1986), and have roles in a wide array of cellular processes. These include the regulation of secondary metabolism, signal transduction, cell division, and responses to plant diseases and various forms of stress (UV-B light, cold and drought) (Larkin et al. 1994; Borevitz et al. 2000; Stracke et al. 2001; Vailleau et al. 2002; Cominelli et al. 2005). The R2R3-MYB-related proteins activate the transcription of structural genes that function in different branches of phenylpropanoid metabolism (Martin and Paz-Ares 1997). The promoters of these structural genes each contain potential MYB protein recognition motifs and also bind with the basic helix-loop-helix (bHLH) domain and conserved WD40 repeat proteins (Park et al. 2007a, b; Martin and Paz-Ares 1997; Winkel-Shirley 2001).
The classification of MYB transcription factors is based on the strong conservation of imperfect repeats in the MYB DNA-binding domain of the member proteins (Martin and Paz-Ares 1997). In contrast to animals (R1R2R3), the MYB domain transcription factors found in plants are characterized by the R2R3-type MYB domain, comprising a DNA-binding domain and an activation/repression domain. Moreover, the balance between activators and repressors in this transcription factor family in plants may provide extra flexibility in terms of transcriptional control (Jin et al. 2000). There have been 126 R2R3 MYB genes identified in Arabidopsis thaliana, all of which exhibit strong homology within their N-terminal MYB DNA-binding domains and can be divided into 24 subgroups on the basis of their sequences (Stracke et al. 2001; Riechmann and Ratcliffe 2000; Kranz et al. 1998). To determine the biological roles of the MYB-related transcription factors in the production of phenylpropanoids, we isolated six R2R3-MYB proteins from Arabidopsis based on available information indicating that these genes are induced by environmental stress (UV-B irradiation) (Kranz et al. 1998). However, they do not have an informative phenotype, because of the structural and functional redundancy among these factors (Bouche and Bouchez 2006). Furthermore, although there have been other studies of anthocyanin accumulation in lettuce, one of the most popular and commercially important vegetables in the world, this process has not been characterized before at the level of transcriptional regulation. The regulation of anthocyanin accumulation in plants is also a key issue in our understanding of the regulation of leaf color. The identification of the factors that exert this control will provide valuable tools to moderate the extent and distribution of anthocyanin-derived pigmentation in plant tissues.
In this study, we overexpressed the AtMYB60 gene in lettuce plants (“Jinjachuckmyun” cultivar) that are highly pigmented with a red color throughout their leaf tissues. Because the loss of these pigments is easily detected, the activity and repression of the MYB-type transcription factors that affect these pathways can be investigated. In the leaves of lettuce, coordinated transcriptional regulation controls virtually each step of the anthocyanin biosynthetic pathway (Park et al. 2007a). We compared the expression of the major anthocyanin biosynthetic genes and the AtMYB60 gene by RT-PCR. We show that this transcription factor is functionally active in repressing anthocyanin accumulation and can thus generate green lettuce leaves. We also show, by functionally repressing the accumulation of this compound, that AtMYB60 plays a significant role in controlling anthocyanin biosynthesis via the inhibition of a key target gene encoding dihydroflavonol reductase (DFR). Thus, we report here for the first time that anthocyanin biosynthesis and the transcription of the DFR gene are repressed through the overexpression of Arabidopsis R2R3-MYB transcription factors in transgenic lettuce plants. We also discuss the function of transcriptional regulators in the control of the expression of the structural protein that are required for anthocyanin biosynthesis in lettuce.
Materials and methods
Plant materials and growth condition
To investigate the effects of exogenous AtMYB proteins on anthocyanin biosynthesis in lettuce leaves at the molecular level, Jinjachuckmyun plants, supplied by Syngenta (Seoul, Korea), were used. The plants were grown at the National Institute of Agricultural Biotechnology in Suwon, South Korea. Fresh lettuce leaves were plucked from the plants, frozen immediately in liquid nitrogen and stored at −80°C until required.
Isolation of AtMYB cDNAs
To isolate the AtMYB genes, cDNAs were isolated from UV-B-irradiated Arabidopsis plants using the BD SMART RACE cDNA Amplification Kit (Clontech, USA) and then used as templates for PCR cloning. The following gene-specific primers were used for RACE-PCR: AtMYB4 (forward: 5′-GTATGGGAAGGTCACCGTGCTGTGAGAAA-3′; reverse: 5′-TATTATTTCATCTCCAAGCTTCGAAAGCC-3′), AtMYB29 (forward: 5′-AAATGTCAAGAAAGCCATGTTGTGTGGGA-3′; reverse: 5′-GATCATATGAAGTTCTTGTCGTCATAATC-3′), AtMYB30 (forward: 5′-TAATGGTGAGGCCTCCTTGTTGTGACAAA-3′; reverse: 5′-CTTCAGAAGAAATTAGTGTTTTCATCCAA-3′), AtMYB34 (forward: 5′-AGATGGTGAGGACACCATGTTGCAAAGAA-3′; reverse: 5′-CGTCAGACAAAGACTCCAACCATATTGTC-3′), AtMYB51 (forward: 5′-GAATGGTGCGGACACCGTGTTGCAAAGCC-3′; reverse: 5′-ACTCATCCAAAATAGTTATCAATTTCGTC-3′) and AtMYB60 (forward: 5′-AGATGGGTAGGCCTCCATGCTGTGACAAG-3′; reverse: 5′-AATTAAAGCATATTAGAGAGCTCCATCAA-3′). The resulting PCR products were then cloned into the pGEM-T Easy vector (Promega, USA) and sequenced on both strands.
AtMYB coding regions were cloned into an expression vector containing the CaMV 35S promoter and the nopaline synthase 3′ terminator. The resulting constructs were then introduced into the binary vector 22103 (a derivative of pCAMBIA3301; Cambia, Australia). The complete coding sequence of each AtMYB cDNA was amplified with a specific forward primer designed to introduce an MluI restriction site and a reverse primer designed to introduce an XbaI restriction site to allow subcloning, as follows: AtMYB4 (forward: 5′-ATACGCGTATGGGAAGGTCACCGTGCTGTG-3′; reverse: 5′-TATCT AGATTATTTCATCTCCAAGCTTCGA-3′), AtMYB29 (forward: 5′-ATACGCGTATGTCAAGAAAGCCATGTTGTG-3′; reverse: 5′-TATCTAGATCATATGAAGTTCTTGTCGTCA-3′), AtMYB30 (forward: 5′-ATACGCGTATGGTGAGGCCTCCTTGTTGTG-3′; reverse: 5′-TATCTAGATCAGAAGAAATTAGTGTTTTCA-3′), AtMYB34 (forward: 5′- ATACGCGTATGGTGAGGACACCATGTTGCA-3′; reverse: 5′-TATCTAGATCAGACAAAGACTCCAACCATA-3′), AtMYB51 (forward: 5′-ATACGCGTATGGTGCGGACACCGTGTTGCA-3′; reverse:5′-TATCTAGATCATCCAAAATAGTTATCAATT-3′), AtMYB60 (forward: 5′-ATACGCGTATGGGTAGGCCTCCATGCTGTG-3′; reverse: 5′-TATCTAGATTAAAGCATATTAGAGAGCTCC-3′). After PCR and digestion, the MluI/XbaI fragments were cloned into the binary vector 22103 between CaMV 35S promoter and the nopaline synthase poly(A) addition site, creating the binary vector 22103-AtMYB plasmids (Fig. 2). These constructs were then introduced into the Agrobacteriumtumefaciens EHA105 strain using the freeze–thaw method.
Generation of AtMYB transgenic lettuce
Transgenic lettuce plants were generated using the Agrobacterium-mediated transformation of cotyledons. Lettuce seeds of the Jinjachuckmyun cultivar were supplied by Syngenta (Korea) and were surface sterilized in 25% (v/v) bleach for 7 min, washed (three changes) in sterile water, and placed on phytogel-solidified (0.2%, w/v) MS medium at pH 5.8. The seeds were germinated and maintained at 25°C (16 h photoperiod, 200 μmol m−2 s−1, daylight fluorescent tubes). The cotyledons were excised from the 4-day-old lettuce seedling and inoculated with freshly grown A. tumefaciens. The inoculated cotyledons were then co-cultivated on MS salt medium solidified with 0.2% purified phytogel (Sigma, USA), for 2 days at 24°C in the dark. The cotyledon explants were then transferred to shoot initiation medium supplemented with 400 mg l−1 carbenicillin (Duchefa, the Netherlands). After a washing step, these explants were cultured (ten per plate) on selection medium (MS salt 4.43 g l−1, sucrose 30 g l−1, phytogel 2 g l−1, kinetin 0.5 mg l−1, carbenicillin 400 mg l−1, phosphinothricin 1.0 mg l−1 and cysteine 10 mg l−1) and subcultured every 2 weeks on the same medium. Shoots that regenerated from explants on medium containing phosphinothricin were rooted in rooting medium (MS salt 4.43 g l−1, sucrose 30 g l−1, phytogel 2 g l−1 and phosphinothricin 1.0 mg l−1), before transfer to the greenhouse, where they were allowed to self-pollinate and to set seed. The seeds were harvested and stored at 4°C.
Anthocyanin extraction and HPLC analysis
Anthocyanin in the lettuce plants was quantified by HPLC at a detection wavelength of 510 nm, comparing the sample retention times and peaks with those of known standards. The relative quantification of each phenolic compound samples was expressed as the equivalent quantity of purified standards for each treatment.
About 0.2 g of ground and freeze-dried leaves was accurately weighed and extracted with 1 ml of a solution of methanol:water (80:20, v/v) at 80°C for 1 h, then sonicated for 20 min. The extracted solution was filtered into a 50-ml volumetric flask, and the flask and filter were rinsed with a solution of methanol:water (80:20, v/v). The filtrate was then made up to the required volume with the same solvent. Approximately 1 mL of the sample solution was passed through a 0.4 μm filter before analysis by HPLC. A Shimadzu HPLC system with 10AD dual pumps was used, with an RP18 (LiChrospher, 250 mm × 4 mm × 5 μm) column. The HPLC parameters were as follows: column temperature 30°C, solvent A = 0.1% trifluoroacetic acid in water, solvent B = 98% acetonitrile with 0.1% trifluoroacetic acid, solvent gradient, 0 min = 0% B, 3 min = 6% B 12 min = 18% B, 25 min = 25% B, 35 min = 100% B, 40 min = 100% B. The flow rate was 0.35 mL min−1. The peaks were classified as either cyanidin or delphinidin derivatives by UV spectral analysis.
Total RNA from the transgenic and wild-type lettuce leaves was extracted using RNeasy Plant Mini Kit (Qiagen, USA), according to the manufacturer’s instructions. To estimate the transcript levels of the exogenous AtMYB4 and AtMYB60 genes in lettuce, we used the primers 5′-ATGGGAAGATCGCCTTGTTGTGAA-3′ (F) and 5′-TCATTTCATCTCTAAGCTTCTGTAGTCCAAAA-3′ (R) for AtMYB4 and 5′-ATGGGGAGGCCTCCTTGTTGTG-3′ (F) and 5′-TCAATTATCGAAAAAATTAGGGTTTTCATCA-3′ (R) for AtMYB60. The mRNA expression patterns of the CHS, F3H, DFR and UFGT genes were analyzed by RT-PCR with the All-in-one RT/PCR Premix (SuperBio P7003, Suwon, Korea). The amplification of rRNA was used as the internal control. Total RNA (100 ng) in a volume of 20 μL containing 20 mM Tris–HCl (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, M-MLV reverse transcriptase (RNaseH Minus), Super Taq Plus DNA polymerase and 10 pmol of each gene-specific amplification primer was used for RT-PCR, according to the manufacturer’s instructions. Gene-specific primers for CHS (forward: 5′-GGTTTGCTCTGAGATTACAGCGGTTACC-3′, reverse: 5′-TCCTTGAGACCAAGCTTGAGCTCCACCT-3′), F3H (forward: 5′-GAGATCTTATCAGAGGCAATGGGCCTTG-3′, reverse: 5′-ACAACGGCCCGATGGTCTGCGTTCTTAA-3′), DFR (forward: 5′-GGTCTATGACGAGTCTCATTGGAGCGAT-3′, reverse: 5′-CTATCAATTGCTCCTTTGAACATCTCCT-3′), UFGT (forward: 5′-TGGAGAAGCGGGTTAGACAGTTGATGGA-3′, reverse: 5′-TATAGCTACCATGATTCAACCAACTTCG-3′), rRNA (forward: 5′-TACGGCACTGAAGGTGCCAAGCTCGTG-3′, reverse: 5′-CATCCTCTTGGCAGTCTTGGCGTAGGG-3′) and bar (forward 5′-GCCGCAGGAACCGCAGGAGT-3′, reverse: 5′-AGCCCGATGACAGCGACCAC-3′) were used to confirm the expression levels of these genes in the lettuce plants. Reverse transcription of the total RNA was carried out at 50°C for 30 min, and the reaction was inactivated at 96°C for 3 min. The amplification conditions comprised 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. PCR was performed using a GeneAmp PCR system 9700 Cycler (Perkin-Elmer, MA, USA). The resultant RT-PCR products were resolved on a 1% agarose gel, stained with ethidium bromide, and photographed. The amplified products of the CHS, F3H, DFR, UFGT, rRNA and bar transcripts were 398, 319, 534, 153, 386 and 267 bp, respectively.
Cloning of MYB-type transcription factor genes from Arabidopsis
Expression of AtMYB genes in lettuce plants
Plant morphology in AtMYB-transformed lettuce
In our previous study, we demonstrated that the wild-type lettuce leaf accumulates anthocyanin, with cyanidin and delphinidin representing the major and minor components, respectively (Park et al. 2007a). To further characterize the functions of the AtMYB genes in anthocyanin biosynthesis, we compared the morphologies of transgenic lettuce plants overexpressing these genes with that of wild-type lettuce.
High-performance liquid chromatography (HPLC) analysis of AtMYB60 transgenic lettuce shows that accumulation of anthocyanin compounds is inhibited
RNA expression profiles of anthocyanin biosynthetic enzymes suggest their coordinated regulation in AtMYB60 transgenic lettuce plants
Total RNA extracts were prepared from the leaves of six-week-old lettuce plants of wild-type, AtMYB4-101 and AtMYB60-117 lines and were analyzed by RT-PCR for the expression of the previously determined structural genes that are involved in the major biochemical pathways of anthocyanin biosynthesis (Fig. 6b). No reduction in the expression of any of these genes was observed in wild-type or AtMYB4-101 lines, both of which exhibited red coloration in their leaves. Moreover, the mRNA levels of the CHS, F3H and UFGT genes, which catalyze the synthesis of chalcones, flavanones and anthocyanin, respectively, were unchanged in any of the plants. In contrast, the mRNA levels of DFR, the production of which catalyzes the conversion of dihydroquercetin and dihydrokaempferol to leucocyanidin and leucopelargonidin (of the leucoanthocyanidins), respectively, was inhibited in the AtMYB60-117 line, which exhibits green-colored leaves with no red pigmentation.
To isolate the MYB-type transcription factor genes involved in phenylpropanoid metabolism, the transgenic expression of six AtMYB genes was analyzed in lettuce. To date, a large number of transcription factors containing DNA-binding domains that are similar to those of the MYB proteins have been identified across the eukaryotic kingdom (Kranz et al. 1998). Several MYB transcription factors are known to regulate phenylpropanoid metabolism, and the quantities of the various end-products of the associated pathways that accumulate in specific cells, tissues and organs are thought to respresent the balanced expression of several interacting MYB proteins (Tamagnone et al. 1998; Espley et al. 2007). Recently, biochemical studies have also suggested that the MYB-type transcription factors are involved in regulating the branches of the phenylpropanoid metabolic networks in higher plants. Multiple MYBs are also thought to regulate the expression of the structural proteins that drive several downstream pathways of flavonoid metabolism (Moyano et al. 1996). A previous study has also shown that MYB proteins can regulate flavonoid biosynthesis via the transcriptional regulation of the gene encoding CHS, an enzyme that catalyzes the first committed step in flavonoid biosynthesis (Feldbrugge et al. 1997).
The lettuce cultivar “Jinjachuckmyun”, which we used in our current transformation experiments, shows anthocyanin pigment accumulation in its leaves during development. We have shown in a recent study that environmental stresses upregulate the expression of the genes that encode anthocyanin biosynthetic enzymes in this same lettuce variety, as demonstrated by their responsiveness to UV-B irradiation (Park et al. 2007a).
The lettuce has merit as a model plant system to further characterize the functions of those gene products in anthocyanin metabolism, because of its leaf color is red under field conditions and green under chamber condition when exposed to UV-B irradiation. The findings described in this study further validate the use of lettuce as a model plant because it offers experimental versatility in terms of its genetic regulation of the inhibition of leaf pigmentation. The function of the MYB proteins as activators (green to red in a growth chamber) or repressors (red to green in the field) of anthocyanin biosynthesis can be characterized directly from a phenotypic analysis of the lettuce leaf color. This system has potential applications as a research tool in a number of areas of plant molecular biology.
We show in our present experiments that the anthocyanin accumulation in leaves is strongly inhibited in AtMYB60 transgenic plants compared with that in wild-type plants (Fig. 4a, b). These results suggest that AtMYB60 functions as a repressor of anthocyanin biosynthesis. Based upon our current findings of the inhibition of anthocyanin biosynthesis and our metabolite analysis of AtMYB60-overexpressing lettuce plants compared with the wild-type, we speculate that the synthesis of anthocyanin is principally controlled by the AtMYB60 transcription factor. The production of anthocyanin is inhibited in the AtMYB60-117 and AtMYB60-112 lines, and this is likely to underlie the green leaf phenotype of these plants. Hence, this characterization of the function of AtMYB60 represents a crucial step in furthering our understanding of the molecular regulation of anthocyanin biosynthesis and the transcription of related genes in lettuce and other plants. It is noteworthy in this context that MYB-related proteins generally act as transcriptional activators (Foos et al. 1994).
The R2R3-MYB-related proteins have so far been shown to activate the transcription of structural proteins that act in different branches of the phenylpropanoid metabolism machinery (Martin and Paz-Ares 1997) or that play roles in the plant response to stress signals (Cominelli et al. 2005). Anthocyanin expression in maize is dependent on the presence of both the MYB and MYC types of regulatory factors. It is of some interest, therefore, that in this study, the expression of AtMYB60 alone, under the control of the CaMV 35S promoter, was sufficient to repress anthocyanin accumulation in a red lettuce variety.
It is interesting to note that the transcriptional response to UV-B irradiation that operates through AtMYB60 focuses on the regulation of DFR expression and anthocyanin accumulation. Recently there has been some debate about the identity of the phenylpropanoids involved in the response to UV-B, which may provide the most effective components of sunscreen formulations. MYB transcription factors that are known to regulate the transcription of genes in the phenylpropanoid biosynthetic pathway have been studied previously (Jin and Martin 1999). The overexpression of PAP1 in Arabidopsis results in the up-regulation of the genes encoding phenylalanine ammonia-lyase (PAL), CHS and DFR (Borevitz et al. 2000; Tohge et al. 2005). Moreover, mutations in the CHS, DFR and LDOX genes reduce the pigmentation of plants (Shirley et al. 1995; Abrahams et al. 2003), suggesting that these proteins are required for the synthesis of both anthocyanidin and proanthocyanidin. Because MYB-binding sites are present in the promoter regions of these flavonoid biosynthetic genes (Nesi et al. 2001; Debeaujon et al. 2003), it is possible that the AtMYB60 transcription factor might also specifically and directly target them. It is also possible that additional target genes that are related to phenylpropanoid metabolism can be negatively regulated by AtMYB60 when it is overexpressed in lettuce.
The effects of AtMYB60 suggest that this MYB protein acts as a direct transcriptional regulator by binding to the MYB motifs common to the promoters of many structural genes in the same metabolic pathway, thus resulting in the downregulation of this pathway. The precise determination of this regulatory system at the molecular level remains to be achieved. Recently, Cominelli et al. (2005) reported that AtMYB60 is involved in the plant response to stress and its expression is negatively modulated during conditions of drought (Cominelli et al. 2005). A null mutation in AtMYB60 results in a constitutive reduction in stomatal openings and in decreased wilting under water-stress conditions. Therefore, we hypothesize that AtMYB60 has multiple functions in the regulation of anthocyanin biosynthesis and in the plant responses to different environmental conditions, including drought and exposure to UV-B light.
AtMYB60 is the first MYB protein identified that functions as a transcriptional repressor of the lettuce DFR gene in anthocyanin biosynthesis, and thus extends the known roles of the R2R3-MYB proteins in secondary metabolism. Evidence from our transgenic lettuce experiments suggests that the direct repression exerted by AtMYB60 protein produces phenotypic effects in transgenic plants. The engineering of the anthocyanin biosynthetic pathway is potentially of great commercial significance. Because the AtMYB60 transcription factor should act effectively in most plant species, it can potentially be used to control anthocyanin biosynthesis in agriculturally and industrially important plants. This method of modifying the anthocyanin content in plant tissues also opens up new avenues to engineering improvements in commercial crops by altering metabolic pathways via the regulation of different structural genes in these pathways.
This research was supported by a grant from a basic research fund (06–011) of the National Institute of Agricultural Biotechnology, the Cooperative Research Project for Bioenergy Crop Development (20070201036015) and BioGreen21 Program (20050501034844) of the Rural Development Administration (RDA), South Korea.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Meissner RC, Jin H, Cominelli E, Denekamp M, Fuertes A, Greco R, Kranz HD, Penfield S, Petroni K, Urzainqui A (1999) Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes. Plant Cell 11:1827–1840PubMedCrossRefGoogle Scholar
- Park K-I, Ishikawa N, Morita Y, Choi J-D, Hoshino A, Lida S (2007b) A bHLH regulatory gene in the common morning glory, Ipomoea purpurea, controls anthocyanin biosynthesis in flowers, proanthocyanidin and phytomelanin pigmentation in seeds, and seed trichome formation. Plant J 49:641–654PubMedCrossRefGoogle Scholar