- Split View
-
Views
-
Cite
Cite
Nobuhiro Sasaki, Katsuhiro Wada, Takatoshi Koda, Kichiji Kasahara, Taiji Adachi, Yoshihiro Ozeki, Isolation and Characterization of cDNAs Encoding an Enzyme with Glucosyltransferase Activity for cyclo-DOPA from Four O’clocks and Feather Cockscombs, Plant and Cell Physiology, Volume 46, Issue 4, April 2005, Pages 666–670, https://doi.org/10.1093/pcp/pci064
- Share Icon Share
Abstract
cDNAs encoding an enzyme with UDP-glucose:cyclo-DOPA 5-O-glucosyltransferase activity were isolated from four o’clocks and feather cockscombs. Phylogenetic analysis of the amino acid sequences deduced from the cDNAs show that they represent a single subclade distinct from those of other phenylpropanoid and flavonoid glucosyltransferases. Changes in the amount of transcripts of the cDNA in four o’clocks correlated with the accumulation of betanin during flower development. The cDNAs isolated here were candidates for the gene of the enzyme involved in another pathway of betacyanin biosynthesis via glucosylation at the cyclo-DOPA step rather than at the betanidin step.
The betalains, which color the flowers and fruits of plants in the order Caryophyllales, with the exception of Caryophyllaceae and Molluginaceae (for a review, see Strack et al. 2003), are classified into two groups: the red betacyanins and the yellow betaxanthins. Despite extensive study of the biosynthetic pathway of anthocyanins, few of the enzymes and genes involved in the biosynthesis of betalains have been identified. The presumed pathway is indicated with thin arrows in Fig. 1 (Strack et al. 2003). Only recently has one of the enzymes involved in the pathway, dihydroxyphenylalanine (DOPA) dioxygenase, been identified when its cDNA was cloned from Portulaca grandiflora (Christinet et al. 2004). The last step in the pathway is the glucosylation of betanidin using UDP-glucose to form betanidin 5-O- and 6-O-glucosides, determined in extracts prepared from suspension-cultured cells of Dorotheanthus bellidiformis (Heuer and Strack 1992, Heuer et al. 1996), and cDNAs encoding betanidin 5-O- and 6-O-glucosyltransferase have been isolated and characterized (Vogt et al. 1997, Vogt et al. 1999, Vogt 2002). However, Wyler et al. (1984) have suggested other possible steps in the glucosylation of cyclo-DOPA. They found that the content of cyclo-DOPA glucoside represented up to 46% of total betacyanins in young red beet roots and 12% in old roots, suggesting that cyclo-DOPA glucoside is involved in or related to the biosynthetic pathway of betacyanins.
Previously, we detected UDP-glucose:cyclo-DOPA 5-O-glucosyltransferase (cDOPA5GT) activity in crude extracts from four o’clocks (Mirabilis jalapa) and other betacyanin-producing plants (Sasaki et al. 2004). Increases in cDOPA5GT activity correlated with the accumulation of betanin during flower development in four o’clocks, suggesting the existence of another pathway in which betacyanins are formed in plants by glucosyltransferases acting at the cyclo-DOPA step, followed by condensation with betalamic acid (thick arrows in Fig. 1). However, our attempts to purify the enzyme were unsuccessful. Therefore, to identify cDOPA5GT, we tried cloning cDOPA5GT cDNA from four o’clocks.
Fourteen different cDNA fragments with similarity to the known nucleotide sequences of the gene for flavonoid glycosyltransferase (hereafter designated simply as ‘GT’) were isolated by polymerase chain reaction (PCR) using degenerate primers and cDNA derived from the petals of four o’clocks as a template. The 5′-terminal nucleotide sequences of 13 of these cDNAs were then isolated; following that, primers were designed to include the first ATG and used to PCR-amplify full-length coding regions for each of the 13 cDNAs. These were cloned into pDEST14 and transformed into Escherichia coli. One of the 13 extracts prepared from recombinant E. coli showed a weak but remarkable cDOPA5GT activity instead of the crude extract prepared from the petals of four o’clocks under the same assay conditions previously reported (Sasaki et al. 2004). To confirm this result, the cDNA was cloned into a yeast expression vector pYES-DEST52 and transformed into yeast. The reaction mixture consisted of cyclo-DOPA, UDP-glucose and a crude extract from the yeast, and was incubated for 8 and 30 min at 30°C. The enzymatic reaction was stopped by the addition of phosphoric acid, and the products reacted chemically with betalamic acid for 2 h at 25°C (Fig. 2A). Reaction mixtures without either UDP-glucose or the crude extract became red after 8 min incubation and subsequent chemical reaction with betalamic acid by the formation of betanidin and its C-15 epimer, isobetanidin (Fig. 2B, b), because the synthetic betalamic acid prepared here consisted of both the (2S)- and (2R)-isoforms, which produced betanindin and isobetanidin, respectively. However, a longer reaction for 30 min at 30°C produced negligible red color without either UDP-glucose or the crude extract (30 min in Fig. 2A), because most of the cyclo-DOPA had been oxidized and disappeared during the longer incubation in the reaction mixture at neutral pH (Wyler and Chiovini 1968). This was supported by high-performance liquid chromatography (HPLC) analysis in that the high peaks corresponding to betanidin and isobetanidin after the 8 min reaction were reduced after 30 min incubation in the absence of UDP-glucose (blue charts in Fig. 2B). When the crude extract from the transformed yeast was added and incubated for 30 min, red products were clearly observed (Fig. 2A). The reaction mixture was subjected to HPLC analysis and the formation of betanin and isobetanin was confirmed (Fig. 2B). These results indicate that this cDNA isolated from four o’clocks encodes a protein with cDOPA5GT activity, and we here designate the gene MjcDOPA5GT. We also isolated a cDNA encoding a protein with cDOPA5GT activity from feather cockscombs (Celosia cristata), using the same approach as described above, from six cDNA fragments encoding flavonoid GT homologs. The feather cockscombs gene was designated CccDOPA5GT.
Although extracts of yeasts carrying both MjcDOPA5GT and CccDOPA5GT cDNAs in an expression vector showed cDOPA5GT activity, the deduced amino acid sequence of MjcDOPA5GT is only 62% similar to that of CccDOPA5GT. To determine any similarities in their enzymatic properties, the substrate specificities for sugar acceptors of the crude extracts prepared from recombinant yeasts were studied. The crude extract prepared from yeast carrying MjcDOPA5GT cDNA in an expression vector did not contain any glucosylated products of tyrosine, DOPA, dopamine, quercetin, cyanidin or betanidin (data not shown). However, although extract from yeast carrying CccDOPA5GT cDNA also contained no tyrosine, DOPA or dopamine glucosylation products, significant glucosylated products of quercetin and cyanidin were observed, together with low but detectable levels of glucosylated betanidin, when the reaction mixture contained 10 mM ascorbic acid (data not shown). Although CccDOPA5GT might show broader substrate specificity than MjcDOPA5GT in vitro, both proteins represent a new GT subclade distinct from those of the other GTs (Fig. 3), despite being isolated from different plant families within the order Centrospermae (four o’clocks are in the family Nyctaginaceae and feather cockscombs in the family Amaranthaceae).
We analyzed the expression of MjcDOPA5GT in four o’clock petals at each stage of flower development, and in leaves, stems and roots (Fig. 4). In petals, MjcDOPA5GT transcripts were detectable at stage 2 of flower development, when betanin synthesis begins. Transcript levels increased in parallel with the progression of flower development and the accumulation of betanin in the petals (see Fig. 3 in Sasaki et al. 2004). MjcDOPA5GT transcripts were most abundant at stage 5 of flower development (fully opened flowers, Fig. 4A). These results accord well with the betanin content and cDOPA5GT activity previously observed in the petals at various stages of flower development (Sasaki et al. 2004). Analysis of the tissue specificity of MjcDOPA5GT expression in four o’clocks showed that low but distinct expression was observed in leaves and stems, compared with high expression in petals. The plants bearing red flowers used here and in our previous study (Sasaki et al. 2004) have stems with a faint but distinct red color, although their leaves show no visible red color. Because the amount of betanin in both the leaves and stems was very low relative to that in the petals, their amounts were below the level of detection under our analytical conditions, whereas cDOPA5GT activity was detectable in both stems and leaves (Sasaki et al. 2004). The low level expression of MjcDOPA5GT in stems and leaves is in accordance with the level of cDOPA5GT activity in each tissue.
It could not be confirmed yet whether the cDNAs identified here absolutely correspond to the cDOPA5GT gene or not, especially because CccDOPA5GT showed broad substrate specificity. The data currently available indicate that several GTs exhibit broader substrate specificity in vitro than in vivo (Paquette et al. 2003). According to the suggestion by Bowles’ group that the substrate specificities of the GTs are conserved within each individual subclade on the GT phylogenetic tree (Jackson et al. 2001, Li et al. 2001, Lim et al. 2001, Ross et al. 2001, Lim et al. 2002, Lim et al. 2003), it was possible that both cDNAs isolated here were candidates for the gene of cDOPA5GT involved in another pathway of betacyanin biosynthesis via glucosylation at the cyclo-DOPA step rather than at the betanidin step.
Materials and Methods
The petals and other tissues of four o’clocks were collected in the garden of Tokyo University of Agriculture and Technology (Koganei, Tokyo, Japan), and stored at –80°C after freezing in liquid nitrogen. Feather cockscombs were purchased from a flower shop.
Total RNAs from the red petals of four o’clocks and the purple flowers of feather cockscombs were prepared using a modified guanidinium thiocyanate–CsCl ultracentrifugation method (Chirgwin et al. 1979). Poly(A)+ RNA was purified from total RNA using Oligotex-dT30<Super> (TaKaRa Bio, Shiga, Japan), according to the manufacturer’s manual. Full-length cDNAs were synthesized using the GeneRacer™ Kit (Invitrogen, Carlsbad, CA, USA). Three reverse degenerate primers were designed based on the conserved regions of the amino acid sequences in various GTs previously deposited in the databases. These primers were as follows: 5′-NARDATNARNACYTGNGGNGCCCA-3′, designated GT-1Rv, which corresponds to the amino acid sequence WAPQVLIL; 5′-NSWRTTCCANCCRCARTGNGT-3′, designated GT-2Rv, which corresponds to THCGWNS; and 5′-NGGCCANGTNACCATNGGNACNCC-3′, designated GT-3Rv, which corresponds to GVPMVTWP. The order of the primers from 5′ to 3′ was GT-1Rv, GT-2Rv, and GT-3Rv. The forward primer, 5′-TAGCGGCCGCTGACATGGACTGAAGGAGTA-3′, designated Not-GR, was designed to contain the recognition site for NotI, and a partial GeneRacer™ 5′ nested primer sequence. The first PCR (30 cycles of denaturion at 92°C for 30 s, annealing at 60°C for 40 s, and extension at 72°C for 1 min) was performed using Not-GR as the forward primer and GT-1Rv, GT-2Rv or GT-3Rv as the reverse primer. For the second round of PCR (under the same conditions as above except for an annealing temperature of 58°C), the Not-GR primer was used for the 5′ end, and the 3′ end primer was either the same degenerate primer used in the first round of PCR, or a nested primer corresponding to a sequence located inside the end of the first round PCR products. PCR products were cloned into EcoRV/NotI-digested pBluescript+ plasmids, and their sequences determined using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). A set of gene-specific GT forward primers corresponding to the nucleotide sequence of the putative 5′-untranslated region of each of the candidate GT cDNAs were synthesized, and full-length four o’clock and feather cockscomb GT cDNAs were amplified by PCR using the specific primer and the 3′ Gene Racer™ primer. The full-length cDNAs were subjected to PCR using appropriate primers adding SD sequence, attB1 and attB2 sequences to the cDNA sequences, and they were introduced into pDONR221 (Invitrogen) using BP clonase (Invitrogen). The GT amino acid sequences were aligned using the multiple alignment program CLUSTAL W (Thompson et al. 1994) on the server at DDBJ. Based on this alignment, phylogenetic analysis was performed by the neighbor-joining method (Saitou and Nei 1987), and a phylogenetic tree was drawn using the Treeview software (Page 1996).
A protein extract from the recombinant E. coli (strain BL21-AI, Invitrogen) was prepared by sonication in extraction buffer (100 mM potassium phosphate, pH 7.5, 7 mM 2-mercaptoethanol) and that from the yeasts (strain INVSc1, Invitrogen) was done using a FastPrep Instrument (FP120, Funakoshi, Tokyo, Japan) with Lysing Matrix C (Funakoshi) according to the supplier’s manual. After centrifugation of the lysate, the supernatant was used as a crude extract for enzyme assay. Preparation of substrates, the assay condition for cDOPA5GT activity and the product analysis by HPLC were essentially the same as those mentioned in our previous paper (Sasaki et al. 2004).
For Northern blot analysis, total RNA or poly(A)+ RNA was isolated as described above. Total RNA (25 µg) prepared from the petals of four o’clock flowers at each developmental stage and poly(A)+ RNA (1 µg) prepared from the roots, stems, leaves and stage 4 petals were separated by 1.2% agarose denaturing gel electrophoresis, and blotted onto Nytran Plus membranes (Schleicher and Schuell, Keene, NH, U.S.A.). The membranes were hybridized with 32P-labeled MjcDOPA5GT cDNA or carnation actin cDNA (the latter as a loading control) for 16 h at 65°C, and then washed twice for 15 min with 2× SSC, 0.5% SDS at room temperature, followed by two washes for 30 min with 0.1× SSC, 0.1% SDS at 65°C for MjcDOPA5GT cDNA or 1× SSC, 0.1% SDS at 55°C for actin cDNA. The membranes were exposed to X-ray film.
Acknowledgments
We are sincerely grateful to San-Ei Gen F.F.I., Inc. for supporting our experiments and providing materials. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by the National Institute of Floricultural Science (NIFS), the National Agricultural Research Organization (NARO), and the Japan Food Chemical Research Foundation.
The nucleotide sequences reported in this paper have been submitted to the DDBJ under accession numbers MjcDOPA5GT cDNA, AB182643; CccDOPA5GT cDNA, AB182644.
Abbreviations
- cyclo-DOPA
5,6-dihydroxyindoline-2-carboxylic acid
- cDOPA5GT
UDP-glucose:cyclo-DOPA 5-O-glucosyltransferase
- DOPA
dihydroxyphenylalanine
References
Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (
Christinet, L., Burdet, F.X., Zaiko, M., Hinz, U. and Zryd, J.P. (
Heuer, S. and Strack, D. (
Heuer, S., Vogt, T., Boehm, H. and Strack, D. (
Jackson, R.G., Lim, E.K., Li, Y., Kowalczyk, M., Sandberg, G., Hoggett, J., Ashford, D.A. and Bowles, D.J. (
Li, Y., Baldauf, S., Lim, E.K. and Bowles, D.J. (
Lim, E.K., Li, Y., Parr, A., Jackson, R., Ashford, D.A. and Bowles, D.J. (
Lim, E.K., Doucet, C.J., Li, Y., Elias, L., Worrall, D., Spencer, S.P., Ross, J. and Bowles, D.J. (
Lim, E.K., Baldauf, S., Li, Y., Elias, L., Worrall, D., Spencer, S.P., Jackson, R.G., Taguchi, G., Ross, J. and Bowles, D.J. (
Page, R.D. (
Paquette, S., Moller, B.L. and Bak, S. (
Ross, J., Li, Y., Lim, E. and Bowles, D.J. (
Saitou, N. and Nei, M. (
Sasaki, N., Adachi, T., Koda, T. and Ozeki, Y. (
Strack, D., Vogt, T. and Schliemann, W. (
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (
Vogt, T. (
Vogt, T., Grimm, R. and Strack, D. (
Vogt, T., Zimmermann, E., Grimm, R., Meyer, M. and Strack, D. (
Wyler, H. and Chiovini, J. (