From the Laboratory of Molecular Biology and
Biotechnology, Research Center of Medicinal Resources, Faculty of
Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku,
Chiba 263-8522, Japan and the
Institute for Fundamental
Research, Suntory Ltd., Wakayama-dai 1-1-1, Shimamoto, Misima, Osaka
618-8503, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
UDP-glucose: anthocyanin
5-O-glucosyltransferase (5-GT) is responsible for the
modification of anthocyanins to more stable molecules in complexes for
co-pigmentation, supposedly resulting in a purple hue. The cDNA
encoding 5-GT was isolated by a differential display applied to two
different forms of anthocyanin production in Perilla
frutescens var. crispa. Differential display was
carried out for mRNA from the leaves of reddish-purple and green
forms of P. frutescens, resulting in the isolation of five
cDNA clones predominantly expressed in the red form. The cDNA
encoded a polypeptide of 460 amino acids, exhibiting a low homology
with the sequences of several glucosyltransferases including
UDP-glucose: anthocyanidin 3-O-glucosyltransferase. By
using this cDNA as the probe, we also isolated a homologous
cDNA clone from a petal cDNA library of Verbena
hybrida. To identify the biochemical function of the encoded proteins, these cDNAs were expressed in Saccharomyces
cerevisiae cells. The recombinant proteins in the yeast extracts
catalyzed the conversion of anthocyanidin 3-O-glucosides
into the corresponding anthocyanidin 3,5-di-O-glucosides
using UDP-glucose as a cofactor, indicating the identity of the
cDNAs encoding 5-GT. Several biochemical properties (optimum pH,
Km values, and sensitivity to inhibitors) were
similar to those reported previously for 5-GTs. Southern blot analysis
indicated the presence of two copies of 5-GT genes in the
genome of both red and green forms of P. frutescens. The
mRNA accumulation of the 5-GT gene was detected in the
leaves of the red form but not in those of the green form and was
induced by illumination of light, as observed for other structural
genes for anthocyanin biosynthesis in P. frutescens.
Anthocyanins, representative secondary products found in most
plant species, are formed through two stages of biosynthetic reactions
(1). The early-stage reactions common to most anthocyanins are
responsible up to the formation of anthocyanidin
3-O-glucosides, which are the first stable colored
metabolites in anthocyanin biosynthesis. The late stage involves the
reactions of further modifications such as glycosylation, acylation,
and methylation. These late-stage reactions are concerned with fine
adjustment for a variety of floral color (2). All biosynthetic enzymes leading to anthocyanidin 3-O-glucoside have been
characterized with their cDNA clones, which have been
isolated by applying various cloning strategies (3-5). However,
a few genes encoding enzymes for the modification of anthocyanins, such
as UDP-glucose: anthocyanin 5-O-glucosyltransferase
(5-GT)1 and malonyl
transferase, have not yet been isolated.
Glycosylation at the 5-position is believed to allow for more stable
complexes in co-pigmentation of anthocyanins, resulting in flowers with
a bright reddish-purple color from dull violet or pure red when
5-O-glycosylation does not take place (2, 6, 7). Thus, 5-GT
is one of the most important enzymes modifying flower color,
particularly for creating a purple hue. Anthocyanins of a variety of
plants, e.g. Perilla frutescens (perilla) (8) and
Verbena hybrida (verbena) (9), are 5-glucosylated. The late-stage biosynthetic pathway involving 5-GT is postulated as depicted in Fig. 1 in P. frutescens var. crispa (10), in which malonyshisonin is
the major anthocyanin (8, 11). Cyanidin is glucosylated at the
3-position by UDP-glucose: anthocyanidin 3-O-glucosyltransferase (3-GT). Cyanidin
3-O-glucoside is then either p-coumaroylated at
the O-6'' position of glucose followed by
glucosylation at the 5-position or 5-glucosylated and then p-coumaroylated to form cyanidin
3-O-(p-coumaroyl)glucoside-5-O-glucoside (12). Finally, the malonyltransfer reaction takes place at the O-6'' position of the 5-O-glucosyl
group (12). In the literature, although 5-GT has been partially
characterized from several plant species (6, 7, 13-15), no cDNA
has been isolated, probably because of the low quantity and/or
instability of the enzyme and the subsequent difficulty of
purification.
INTRODUCTION
Top
Abstract
Introduction
References
View larger version (17K):
[in a new window]
Fig. 1.
The postulated late-stage pathway of
anthocyanin biosynthesis in P. frutescens. The numbers
in parentheses indicate the relative activities of recombinant
5-GT (this study) and acyltransferase in crude extracts of perilla red
leaves reported by Matsune et al. (12).
Recently, we have isolated and characterized a series of genes encoding enzymes involved in anthocyanin biosynthesis from P. frutescens (10). These results indicated that the expression of these structural genes was coordinately regulated in an anthocyanin form-specific manner: the expression was observed only in a reddish-purple form but not in a green form (10). These results prompted us to examine the possibility of isolating new genes, which are expressed in a form-specific manner, by applying a differential screening system. The mRNA differential display is a powerful technique for isolating cDNAs specifically expressed in particular types of cells (16). Even cDNAs with a low level of expression such as transcriptional factors could be cloned by this technique (17).
In the present investigation, we have applied the differential display
technique to isolate genes that are expressed in an anthocyanin
form-specific manner in P. frutescens. The cDNA encoding 5-GT was successfully isolated for the first time, and its molecular and biochemical properties were characterized.
![]() |
EXPERIMENTAL PROCEDURES |
---|
Plant Materials-- The seeds of a reddish-purple form (cv. Chirimenjiso Shikun) and a green form (cv. Chirimen-aojiso Seikun) of P. frutescens var. crispa were germinated at 25 °C under illumination with a 16-h light period in a greenhouse. After 5 weeks, when the leaves grew to a length of ~3 cm, the younger leaves were used for the extraction of RNA and DNA. V. hybrida cv. Hanatemari violet (Suntory Ltd., Osaka, Japan) plants were grown in a standard greenhouse.
Differential Display of mRNA and cDNA Cloning--
0.9
µg of poly(A)+ RNA isolated from the top young leaves of
P. frutescens was reverse transcribed in a reaction mixture
of 33 µl with avian myeloblastosis virus reverse transcriptase with subsets of specific 1-base anchored oligo(dT) primers (H-T11G, H-T11A,
or H-T11C; GenHunter) that recognize different fractions of the total
poly(A)+ RNA population. The resulting cDNA was
amplified with a combination of the same anchored oligo(dT) primer used
in the reverse transcription and a 13-base pair (bp) primer of an
arbitrary sequence (one of H-AP1-H-AP8, GenHunter) by a polymerase
chain reaction (PCR) with Taq DNA polymerase (Toyobo, Osaka,
Japan). PCR was performed in a 20-µl reaction mixture that contained
2 µl of cDNA solution, 0.2 µM anchored oligo(dT)
primer, 0.2 µM arbitrary primer, 0.12 µM
deoxynucleotide triphosphates, 370 kBq [32P]dCTP, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.01% Triton
X-100, 1.25 mM MgCl2, and 1 unit of
Taq DNA polymerase. PCR conditions were as follows: a 20-s
initial heating at 72 °C, followed by 40 three-step cycles of a 30-s
denaturation at 94 °C, a 2-min annealing at 40 °C, and a 30-s
elongation at 72 °C, and a final 5-min elongation at 72 °C in a
thermal cycler. Amplified DNA was fractionated by electrophoresis in a
sequence gel (18). After drying the gel and exposing it to x-ray film,
the differentially appearing bands were cut out of the gel, and
amplified DNA was eluted with 100 µl of H2O by heating at
100 °C and then purified by ethanol precipitation. Reamplification
was carried out using eluted DNA as a template under the same PCR
conditions. The resulting reamplified DNAs were purified by agarose gel
electrophoresis, cloned into pBluescript II SK, and used
as probes for initial RNA blot analysis and cDNA screening.
The gt10 cDNA library constructed from the mRNA of leaves of
the red form of P. frutescens (10) was screened with
32P-labeled probes obtained by differential display. The
ZAP cDNA library constructed from the mRNA of colored buds
of V. hybrida cv. Hanatemari violet was screened using the
perilla 5-GT cDNA (p3R4) as a probe under a low stringency
condition (19).
Heterologous Expression in Yeast-- The cDNA inserts of p3R4, p3R6, and pSHGT8 were cloned into a yeast expression vector to create pY3R4, pY3R6, and pYHGT8, respectively, in which the expression of cDNAs was controlled by a strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from yeast. Transformation and culture of yeast were carried out as described previously (20). The harvested cells were washed, resuspended in a buffer (100 mM phosphate buffer, pH 8.5, 14 mM 2-mercaptoethanol, 10 µM (p-aminophenyl)methanesulfonyl fluoride, and 100 µM UDP-glucose), and disrupted with glass beads (Sigma). After centrifugation at 15,000 rpm for 20 min, the supernatant was used as a crude enzyme extract.
Enzymatic Activity Assay of 5-GT-- For an in vitro assay of 5-GT, 100 µl of standard reaction mixture containing 100 mM phosphate buffer, pH 8.5, 200-250 µM anthocyan substrates, 1 mM UDP-glucose, and 100 µg of protein from crude enzyme extract from transformed yeast were incubated at 30 °C for 5 min. For the determination of Km values, the concentrations of cyanidin 3-O-glucoside and UDP-glucose were varied; Km values were obtained by double-reciprocal plots. The reaction was stopped by adding Folch solution (CH3OH-CHCl3-HCl, 1:2:0.015). After centrifugation at 15,000 rpm for 5 min, the aqueous supernatant was filtrated through a membrane filter and analyzed by high performance liquid chromatography (HPLC).
HPLC analysis of anthocyanins was carried out on a reverse phase column
Asahipak ODA-50 (4.6 mm × 250 mm; Showa-Denko) with a gradient
elution of 10-50% acetonitrile with 0.5% trifluoroacetic acid for 20 min, followed by an isocratic elution of 50% acetonitrile with 0.5%
trifluoroacetic acid at a flow rate of 0.6 ml/min at 40 °C with a
monitoring absorbance of 520 nm. Anthocyanins were identified by
comparing the retention times and spectral patterns (250-600 nm) of
standard compounds.
Miscellaneous Techniques--
DNA sequencing and nucleic acid
hybridization analysis were carried out as described previously (10).
Protein concentration was determined by the method of Bradford (21)
using bovine serum albumin as a standard.
![]() |
RESULTS |
---|
Differential Display of mRNA between Red and Green Forms-- The mRNAs from the reddish-purple and green forms were reverse transcribed into cDNAs, and then PCR amplification was carried out with various combinations of an oligo(dT) primer and a 13-bp primer of an arbitrary sequence. Using three oligo(dT) primers with A, G, or C at the 3'-position and eight different arbitrary primers, 36 fragments (of ~2,600 amplified fragments in total) were differentially displayed as specifically expressed cDNAs in the red forma leaves but not in the green forma leaves. The 33 differentially expressed fragments were isolated and reamplified using the same primer combinations as in the first PCR and subsequently cloned into the plasmid vector. RNA blot analysis for mRNA from leaves from the red and green forms was performed using these cloned fragments as probes. Among these fragments, five were selected as red form specifically expressed cDNA fragments. The cDNA library of red form leaves was screened using these five fragments to isolate the full-length cDNA clones.
Table I summarizes the cDNAs that were isolated from the red form of P. frutescens by the differential display strategy and whose functions were identified. The p3R4 cDNA was eventually confirmed to encode 5-GT. The insert of p3R6 was highly homologous with that of p3R4; however, it presumably encoded a nonfunctional form of 5-GT (see below). The cDNAs carried on p8R5 and p2R3 were identified as those encoding anthocyanidin synthase (leucoanthocyanidin dioxygenase) and a homologue of Myb transcriptional factor, respectively, by sequence comparison with corresponding genes reported previously. However, we have also isolated a few additional cDNAs whose functions are unknown.
|
Nucleotide and Deduced Amino Acid Sequences of 5-GT cDNA-- The nucleotide sequences of cDNA inserts carried on p3R4 and p3R6 were 1,507 and 1,474 bp long, respectively, and contained open reading frames of 1,380 and 1,329 bp encoding 460 amino acids forming a 50,973-Da polypeptide and 443 amino acids forming a 49,109-Da polypeptide, respectively (Fig. 2). Using the insert of p3R4 as a probe, we isolated homologous cDNAs from a library constructed from pigmented petals of V. hybrida. One of the cDNA clones, pSHGT8, contained an insert of 2,062 bp exhibiting an open reading frame of 1,383 bp that encoded 461 amino acids forming a 51,347-Da protein.
|
The deduced amino acid sequence of p3R4 exhibited a 92% and 69% identity with those of p3R6 and pSHGT8, respectively; the identity between the amino acid sequences of p3R6 and pSHGT8 was 65%. As shown in Fig. 2, the amino acid sequences of 5-GTs share low but significant homologies with those of 3-GT. Identities and similarities between the sequences of 5-GTs and 3-GTs were 21-26% and 32-39%, respectively. In the deduced amino acid sequence of p3R6, 18 amino acids were different from that of p3R4, and 19 amino acids at the C terminus that are present in the sequences of p3R4 and pSHGT8 were deleted. The 44-amino acid region, which exhibits the signature motif commonly found in UDP-glycosyltransferases, is conserved in all three predicted polypeptides (underlined in Fig. 2), suggesting that the predicted proteins are members of glycosyltransferase family.
Heterologous Expression in Yeast and Detection of 5-GT Activity-- The cDNAs of p3R4, p3R6, and pSHGT8 were subcloned into a yeast expression vector, resulting in the construction of pY3R4, pY3R6, and pYHGT8, respectively, in which the expression of cDNAs was regulated by a strong constitutive promoter. The crude protein extracts of yeast transformed with these expression vectors were assayed for anthocyanin glucosyltransferase in vitro. As shown in Fig. 3, cyanidin 3-O-glucoside was converted to cyanidin 3,5-diglucoside in the presence of UDP-glucose and the extract of yeast transformed with pY3R4. The identity of cyanidin 3,5-diglucoside formed by the in vitro reaction was confirmed by co-chromatography on HPLC with the authentic cyanidin 3,5-diglucoside and by its UV-visible absorption spectrum. The extract of yeast transformed with the empty vector could not convert cyanidin 3-O-glucoside to the product. The protein extracts of yeast transformed with pY3R6 exhibited no activity, suggesting coding for a nonfunctional form of 5-GT. The extract of transformant carrying pYHGT8 encoding V. hybrida 5-GT also exhibited 5-GT activity for cyanidin 3-O-glucoside (data not shown). These results confirmed that the cDNAs of p3R4 from P. frutescens and pSHGT8 from V. hybrida encode a novel glucosyltransferase that catalyzes the glucosylation at the O-5 position of anthocyanidin 3-glucosides.
|
Biochemical Properties of the Recombinant 5-GT-- As shown in Table II, both recombinant enzymes of P. frutescens (pY3R4) and V. hybrida (pYHGT8) exhibited 5-GT activities transferring the glucose moiety from UDP-glucose to the 5-position of various anthocyanins in which the 3-position is glycosylated or acylglycosylated. No activities toward cyanidin and cyanidin 3,5-diglucoside were detected. For both enzymes, cyanidin 3-O-glucoside is the best substrate among those examined, and anthocyanidin 3-O-glucosides are more preferable substrates than anthocyanidin 3-O-(6''-O-acyl)glucosides. Several biochemical properties of recombinant 5-GT from P. frutescens are shown in Table III. The optimum pH is in the 8.0-8.5 range, similar to those reported previously for crude 5-GT preparations from petunia (6) and Matthiola incana (7). The reaction catalyzed by recombinant 5-GT obeys the simple Michaelis-Menten equation. The Km values for cyanidin 3-O-glucoside and UDP-glucose are comparable with those for petunia 5-GT (6), although the petunia 5-GT was only active with anthocyanidin 3-(p-coumaroyl)-rutinosides. The activity was inhibited by p-chloromercuribenzoate and, to a lesser extent, by divalent metal cations, as reported with 5-GT from M. incana (7). These experimentally obtained biochemical characteristics and the calculated molecular mass of ~51 kDa for both enzymes from P. frutescens and V. hybrida coincide well with those reported for 5-GT in the crude enzyme preparations from different plant species.
|
|
Molecular Phylogenic Relations of 5-GT Proteins with Other Glycosyltransferases-- The molecular phylogenic tree of the amino acid sequences of glycosyltransferases from plants was constructed as shown in Fig. 4. This tree suggested that 5-GT proteins phylogenically belong to a new family of the glycosyltransferase superfamily. This glycosyltransferase superfamily of plants comprises a variety of enzymes with diverse functions. They are involved in anthocyanin metabolism (5-GT, 3-GT, and anthocyanin rhamnosyltransferase (22, 23)), auxin metabolism (indole-3-acetic acid glucosyltransferase (24)), and unknown functions induced by methyl jasmonate (25) and salicylic acid (26).
|
Genomic Complexity of the 5-GT Gene in P. frutescens-- Southern blot analysis of genomic DNA of P. frutescens indicated that at least two copies of the 5-GT gene exist in genomes of both the red and green forms (Fig. 5). The copy number of the 5-GT gene was the same as those of other structural genes for anthocyanin biosynthesis in P. frutescens (10). This genomic complexity presumably originates from the amphidiploidy of P. frutescens (27). Restriction fragment length polymorphism between the red and green forms was observed for EcoRV digestion.
|
Expression of the 5-GT Gene in P. frutescens-- The major transcript of the 5-GT gene was 1.5-kb long and was detected in the leaves of the red form but not in those of the green form (Fig. 6A). This expression pattern was quite similar to those of other genes for the enzymes involved in anthocyanin biosynthesis in P. frutescens (11). In the roots, no 5-GT mRNA accumulated, even in the red form plant. The illumination with white light remarkably induced the accumulation of the transcript of 5-GT in the leaves of red perilla (Fig. 6B). The pattern of the time course for light induction of the 5-GT gene was again identical with that of other genes involved in anthocyanin biosynthesis (11). These results suggest that the expression of all structural genes for anthocyanin biosynthesis is coordinately regulated, presumably by common regulatory gene(s).
|
![]() |
DISCUSSION |
---|
In the present study, we applied a mRNA differential display technique to isolate cDNAs expressed in an anthocyanin form-specific manner in P. frutescens and successfully cloned a novel cDNA encoding 5-GT. In addition, the molecular and biochemical properties of 5-GT were elucidated using the cDNA obtained and recombinant 5-GT protein.
Several cloning strategies were explored for the isolation of genes for anthocyanin biosynthesis (3, 4): (i) the strategy involving purifying the enzyme and a subsequent preparation of antibodies or determination of the amino acid sequence was successfully used for the initial cDNA cloning of chalcone synthase and chalcone isomerase, (ii) the transposon tagging strategy was applied for cloning the dihydroflavonol 4-reductase, 3-GT, and anthocyanindin synthase genes, (iii) a PCR strategy using degenerated primers that encode common amino acid sequences to the same protein family was applied for cDNA isolation of flavonoid 3',5'-hydroxylase and flavonol synthase, and (iv) conventional differential screening for duplicate filters with cDNAs from wild and mutant plants as probes has lead to the isolation of cDNAs encoding flavanone 3-hydroxylase and anthocyanidin-3-glucoside rhamnosyltransferase. Despite these trials for isolating new genes for the modification of anthocyanin, no cDNA encoding 5-GT has been cloned thus far. The reasons for this failure would be: (i) no available appropriate mutant line(s) lacking 5-GT activity that causes a remarkable alteration of flower color, (ii) a very low or unstable 5-GT enzyme activity and the subsequent difficulty in purifying 5-GT proteins in a sufficient amount for peptide sequencing or antibody preparation, and (iii) a low amount of mRNA encoding 5-GT sufficient for differential screening by a conventional way. In this context, mRNA differential display is quite powerful for cloning low amounts of cDNA, because a difference in expression level can be amplified by PCR even for a small quantity of cDNA. Besides 5-GT cDNA, we also isolated cDNAs encoding anthocyanidin synthase, a homologue of Myb factor, and putative proteins of unknown functions.
The involvement of 5-GT in anthocyanin formation has been proven by partial characterization of 5-GT enzyme from flowers of Silene dioica (13, 14), Petunia hybrida(6), M. incana (7), and Callistephus chinensis (15). In these plants, glycosylation at the 5-position was indicated to be responsible for the modulation of flower color from dull violet to bright violet or for partially committing a change from gray to purple (2, 6, 7). The molecular masses of 5-GT of S. dioica and P. hybrida were reported as 55,000 and 52,000 Da, respectively, in agreement with those calculated from cDNAs of P. frutescens and V. hybrida. No data regarding molecular masses are available for native 5-GTs from P. frutescens and V. hybrida. Other biochemical properties of the recombinant 5-GT (optimum pH, Km values, and sensitivity to inhibitors) were also similar to those reported previously for 5-GTs in crude enzyme preparations from petunia (6) and M. incana (7). The recombinant 5-GTs of P. frutescens and V. hybrida accept anthocyanidin 3-glucosides and anthocyanidin 3-(6''-acyl)glucosides as substrates (Table II). Anthocyanidin 3-glucosides were better substrates than anthocyanidin 3-(acyl)glucosides for both 5-GTs. Although cyanidin 3-glucoside was the best substrate for both 5-GTs, they exhibited a broad substrate specificity.
Acyltransferase catalyzing acylation of the glucose moiety of anthocyanidin 3-glucoside from perilla also uses anthocyanidin 3-glucoside as a better substrate than anthocyanidin 3,5-diglucoside (12), as shown in Fig. 1. These results indicate that the biosynthetic pathway from anthocyanidin 3-glucoside to anthocyanidin 3-(acyl)glucoside-5-glucoside forms a metabolic grid comprising two bypass routes in perilla. A similar metabolic grid was also proposed for flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase (3).
In the deduced amino acid sequences of 5-GT, the signature sequence motif common to the glycosyltransferase family was identified (Fig. 2). This motif is also present in the sequences of 3-GT (10, 19, 28) and putative glycosyltransferases whose mRNA levels were induced by methyl jasmonate (25) and salicylic acid (26). In addition to this signature motif, several conserved stretches in the deduced amino acid sequences were found in the family of these structurally related proteins. In the deduced amino acid sequence of p3R6, 19 amino acids of the C terminus that were relatively conserved in 5-GTs and 3-GTs were deleted, suggesting that these 19 amino acids may be essential for the activity of 5-GT. Molecular phylogenic study indicated that 5-GT and 3-GT could have evolved from a common ancestor protein so as to define which hydroxyl group can be glucosylated. One can also speculate that proteins induced in mRNA by jasmonate and salicylic acid may catalyze the glycosylation of the hydroxyl group of flavonoid-related or unknown metabolites.
The 5-GT gene in P. frutescens was specifically
expressed in the leaves of the red form, but not in those of the green
form, and the expression of the 5-GT gene was induced by
illumination with white light. This mode of expression is totally
similar to that of other structural genes of anthocyanin biosynthesis
as reported previously (10). These results confirmed that the
expression of all anthocyanin formation genes, from early-stage to
late-stage genes, is regulated coordinately. Consequently, we
hypothesize the presence of epistatic regulatory gene(s) that governs
the expression of structural genes in red form but does not promote it
in green form. The investigation to clarify the postulated epistatic
gene(s) is now underway in our laboratories.
![]() |
FOOTNOTES |
---|
* This research was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan, by the Research for the Future Program (96I00302) from the Japan Society for the Promotion of Science, by the Takeda Foundation, and by the Inohana Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB013596 (P. frutescens 5-GT of p3R4), AB013597 (P. frutescens p3R6), AB013598 (V. hybrida 5-GT), and AB013599 (P. frutescens Myb homologue).
§ These authors contributed equally to this study.
¶ Supported in part by a fellowship from the Uehara Memorial Foundation.
** To whom correspondence should be addressed. Tel./Fax: 81-43-290-2905; E-mail: ksaito{at}p.chiba-u.ac.jp.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: 5-GT, UDP-glucose: anthocyanin 5-O-glucosyltransferase; 3-GT, UDP-glucose: anthocyanidin 3-O-glucosyltransferase; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|