Journal of Experimental Botany, Vol. 54, No. 381, pp. 239-248,
January 2, 2003
© 2003 Oxford University Press
Sn, a maize bHLH gene, modulates anthocyanin and condensed tannin pathways in Lotus corniculatus
Received 26 June 2002; Accepted 19 August 2002
1 Cell Biology Department, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK
2 Istituto di Ricerche sul Miglioramento, Genetico della Piante Foraggere, Consiglio Nazionale della Ricerche, Perugia, Italy
3 To whom correspondence should be addressed. Fax: +44 (0)1970 823242. E-mail: mark.robbins{at}bbsrc.ac.uk
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Abstract |
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Anthocyanins and condensed tannins are major flavonoid end-products in higher plants. While the transactivation of anthocyanins by basic helix-loop-helix (bHLH) transcription factors is well documented, very little is known about the transregulation of the pathway to condensed tannins. The present study analyses the effect of over-expressing an Sn transgene in Lotus corniculatus, a model legume, with the aim of studying the regulation of anthocyanin and tannin end-products. Contrary to expectation, effects on anthocyanin accumulation were subtle and restricted to the leaf midrib, leaf base and petiole tissues. However, the accumulation of condensed tannin polymers was dramatically enhanced in the leaf blade and this increase was accompanied by a 50-fold increase in the number of tannin-containing cells in this tissue. A detailed analysis of selected lines indicated that this transactivational phenotype correlated with high steady-state transcript levels of the introduced transgene and the introduction of a single copy of the CaMV35S-Sn construct into these clonal genotypes. While the levels of condensed tannins in leaves were increased by up to 1% of the dry weight, other major secondary end-products (flavonols, lignins and inducible phytoalexins) were unaltered in transactivated lines. These results give an initial insight into the developmental and higher-order regulation of polyphenolic metabolism in Lotus and other higher plant species.
Key words: Anthocyanins, condensed tannins, Lotus, metabolic engineering, transactivation.
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Introduction |
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Secondary products perform a wide range of functions essential to the survival of higher plants (Burbulis and Winkel-Shirley, 1999). Of these secondary product pathways, those leading to flavonoid and anthocyanin end-products have been the most extensively studied due to their importance in flower pigmentation and the excellent range of genetic resources in petunia, Antirrhinum, maize, and Arabidopsis (Holton and Cornish, 1995). In maize, two major classes of transcription factors have been described. The R/B family, which shows sequence homology to the basic helix-loop-helix (bHLH) DNA binding proteins found in animal MYC protooncogenes (Ludwig et al., 1989) and the C1/P family, which encodes proteins with similarity to the DNA binding domain of the mammalian MYB oncogene products (Paz-Ares et al., 1987). By contrast, there is very little information on the molecular mechanisms that control the expression of the closely related biosynthetic pathway to condensed tannins (syn. proanthocyanidins) (Robbins and Morris, 1999). This lack of knowledge regarding condensed tannins is unfortunate as these are one of the world’s major biopolymers and they perform important functions in plant–insect interactions (Muir et al., 1999), in plant–herbivore interactions (Furstenburg and van Hoven, 1994), as well as having a range of biotechnological applications (Robbins et al., 1999). A major interest of scientists studying forage legumes is to investigate whether this pathway can be introduced into the foliar tissues of white clover and lucerne with the aim of producing bloat-safe field crops with enhanced nitrogen efficiency when grazed by ruminant livestock (Aerts et al., 1999).
Previous work has shown that the tissue-specific accumulation of anthocyanin pigments in maize and Antirrhinum is controlled by a range of anthocyanin regulatory genes that are responsible for the co-ordinate induction of structural genes in this metabolic sequence (Holton and Cornish, 1995). Also of note is the observation that the ectopic expression of bHLH transcription factors has been reported to enhance anthocyanin levels in non-tannin-containing species such as tomato, tobacco and petunia (Mooney et al., 1995; Bradley et al., 1998). In maize cell cultures, the expression of bHLH and MYB anthocyanin transactivators in combination has been reported to result in the accumulation of anthocyanin end-products contained within anthocyanoplasts (Grotewold et al., 1998). In Arabidopsis the expression of R, a maize anthocyanin MYC, increased levels of anthocyanin while the expression of R and C1 resulted in anthocyanin accumulation in novel tissue locations such as root, petal and stamen (Lloyd et al., 1992). Recently, two genes have been identified in Arabidopsis, TT8 (encoding a bHLH domain transcription factor) and TT2 (encoding a R2R3 MYB domain protein) and these have been identified as key determinants for the proanthocyanidin accumulation in developing seeds (Nesi et al., 2000, 2001).
In this paper, the effect of the over-expression of Sn, a maize anthocyanin bHLH gene, has been analysed in Lotus corniculatus which is a metabolic model for condensed tannin biosynthesis. This model legume accumulates a wide range of flavonoid end-products as outlined in Fig. 1. Most notably, this species biosynthesizes condensed tannins and clonal transformable genotypes have been produced which permit the analysis of transgenic interventions in a uniform genetic background (Robbins et al., 1999). Previous work has shown that this pathway is readily manipulable using structural genes of the pathway (Carron et al., 1994; Colliver et al., 1997) and that the over-expression of pathway genes can enhance condensed tannin content and also modify tannin polymer structure (Bavage et al., 1997). In this paper, it is demonstrated that Sn, which encodes a heterologous bHLH involved in the control of anthocyanin biosynthesis, can transactivate both anthocyanin and condensed tannin pathways in Lotus. Increases in end-products occur in a tissue-specific manner and strongly suggest that there are shared regulatory mechanisms, which control the biosynthesis of anthocyanin and condensed tannin end-products.
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Materials and methods |
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Production and growth of experimental material
Hairy root transformation was performed in three well characterized genotypes (S33, S41, S50) belonging to the L. corniculatus cultivar Leo (Carron et al., 1994). The plasmid 121.Sn (Damiani et al., 1998), derived from pBI121.1 where the gus gene was replaced with the full length cDNA of Sn (Tonelli et al., 1991), was introduced through triparental mating into the wild-type A. rhizogenes strain 1855. Axenic plants were inoculated in the stem by puncturing with a needle previously dipped in the bacterial culture. Independently derived hairy roots were detached and cultured in the dark for 2 weeks on a hormone-free medium consisting of Gamborg’s B5 salts (Sigma) supplemented with sucrose (2%), agar (1.5%), kanamycin (50 mg l–1) and carbenicillin (1 g l–1) (Gamborg et al., 1968). Shoot formation from each hairy root started when plates were transferred to light (50 µM m–2 s–1 light intensity). Following 2 months of in vitro culture in the light, plants were moved to soil in a mist chamber to prevent plant desiccation and then grown on for further analysis. Typically, experimental plant material comprised mature Lotus plants grown in triplicate at 18/15 °C day/night with a day length of 14 h and a light level of 750 µmol m–2 s–1.
Screening for condensed tannin phenotype
Detached leaves from mature plants were stained for condensed tannins using 4-dimethylaminocinnamaldehyde (DMACA) according to the method of Li et al. (1996). This reagent produces a blue coloration on reaction with condensed tannins under acidified conditions and from the intensity of staining each plant was scored on a 0–6 scale.
DNA analysis and measurements of transcript levels
For Southern blotting DNA was extracted according to Cluster et al. (1996), restricted, electrophoresed and blotted to Amersham Hybond N+ filter according to standard procedures. Filters were then probed with the 32P labelled XbaI fragment of the Sn cDNA.
For RNA analysis, total RNA was isolated from leaves of transgenic lines 50/11, 50/10, 50/9, and 50/6 and from control plant S50 according to Chang et al. (1993). One µg of PolyA+ RNA, isolated from total RNA using the ‘Dynabeads mRNA purification Kit’ (Dynal), was hybridized with Sn and Rubisco SSU probes as described by Damiani et al. (1999). The DFR probe was a 610 bp PCR fragment amplified from the plasmid pGMcDFR (GenBank Accession No. AY117027) containing a full length DFR cDNA of L. corniculatus, with the primers DFRFW1/DFRGSP2 (DFRFW1: 5'- CTAACATGAAGAAGGTGAAG-3'; DFRGSP2: 5'-TGGCATTG TCGGCATTAGAAAGG-3').
RT-PCR amplification from DNA-free total RNA was carried out, run on agarose gel, blotted and hybridized basically as described by Damiani et al. (1999). As control for the presence of DNA contaminating the RNA preparations, RNA samples were processed both in the presence and/or in the absence of MMLV reverse transcriptase (Gibco BRL) and submitted to PCR amplification. The primer pairs used both for RT-PCR analysis and probe preparation for Southern of the RT-PCR blots were: Sn1/Sn2 (Sn1: 5'-TCT GGCTGTGCAACGCGCACC-3' and Sn2: 5'- CTTCTCTCGTCG CTTTCGCTC-3') to amplify a 810 bp Sn fragmentand EF1F/EF1B (EF1F: 5'-ATTGTGGTCATTGGCCACGT-3'; EF1B: 5'- CCAA TCTTGTACACATCCTG-3') to amplify a 710 bp fragment of EF-1
gene. The primer pair used for RT-PCR analysis of DFR mRNA levels was DFRFW1/DFRGSP2, which can amplify all known L. corniculatus DFR cDNA sequences.
Cellular location of anthocyanin-containing and tannin-containing cells in transgenics and cell counts
Cell counts were performed on trifoliate leaves selected at random. Anthocyanin-containing cells were red in colour and visible with the naked eye. Cells containing condensed tannins were visible after staining with 4-dimethylaminocinnamaldehyde (DMACA). Shoot tissues were harvested and decolourized in ethanol overnight. After discarding the ethanol, samples were stained using 0.3% (w/v) DMACA in 6 M HCl for 1 h. After four changes of distilled water, samples were then analysed and blue colour indicated the presence of condensed tannin polymers.
For quantification, mature leaves were mounted on a slide and viewed using an Olympus BH light microscope (10x magnification).
Quantification of phenolic and flavonoid end-products
Condensed tannin levels were determined in freeze-dried samples as described by Terrill et al. (1992). Flavonols were quantified essentially as described by Robbins et al. (1998) by summing peaks after performing diode array high performance liquid chromatography using a Waters 996 machine. Thioglycolic acid (TGA) lignin was determined using the method of Whitmore (1978). Inducible phytoalexins were analysed after elicitation with 10mM glutathione as described by Robbins et al. (1995).
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Results |
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Initial screening of Lotus corniculatus for condensed tannin phenotype
Standard transformation procedures were employed to introduce a CaMV35S-Sn gene construct into Lotus corniculatus. Clonal genotypes were used in order to assess effects in a uniform genetic background. Regenerated plants were analysed and scored for condensed tannin content as shown in Fig. 2. In genotype S50 lines were noted with reduced condensed tannin content and these appear to be similar to lines described by Damiani et al. (1999) where the introduction of Sn apparently reduced end-product accumulation in leaves. More notable in this study were a number of co-transformed lines in the S50 background with markedly higher levels of condensed tannin than found in control lines (Fig. 2a). Similar effects were noted in the S33 background with a number of lines showing enhanced tannin content relative to controls (Fig. 2b). Interestingly when a high tannin genotype (S41) was transformed, no lines were noted with levels of tannins higher than controls (Fig. 2c).
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A limited number of lines were subjected to RT-PCR analysis in order to determine whether there was any obvious correlation between transgene expression and derived chemical phenotype. In the S33 background high levels of steady-state transcript were noted for lines 33/19 and 33/1. Curiously one other enhanced line, 33/6, showed no obvious expression of transgene, however, evidence suggests that this was due to silencing of transgene between initial screening for condensed tannin phenotype and sampling for molecular analysis by RT-PCR (data not shown). In the S50 transgenics Sn expression was clearly detected in lines with high tannin scores, 50/23, 50/11, 50/10; but not in lines with control or reduced levels, 50/14, 50/13, 50/9, 50/8, 50/6, and 50/5. Line 50/1 was noted to silence between initial analysis and RT-PCR analysis (data not shown). Only two S41 lines were analysed for transgene expression, 41/6 and 41/17 and neither showed detectable transgene expression. However, analysis of CaMV-gus lines in this genotype confirmed expression of the CaMV promotor in the S41 genetic background.
Detailed molecular analysis of enhanced and suppressed lines in S50 background
In view of phenotypes noted in the S50 genotype, a subset of enhanced and suppressed lines was selected for a more focused analysis. In particular, two enhanced lines (50/10 and 50/11) were selected and compared with two suppressed lines (50/6 and 50/9) together with a control line. Southern blot analysis (Fig. 3a) showed that lines contained between one and six transgene copies and this is typical for co-transformation experiments in Lotus (Carron et al., 1994). One aspect of interest is that, while suppressed lines contained multiple copies of CaMV-Sn, the two enhanced lines had single transgene copies within their genomic DNA complement.
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In order to clarify the molecular basis of the phenotype, Northern analysis was performed (data not shown) and Sn transcript was clearly detectable in PolyA+ RNA extracted from leaf tissues of 50/10 and 50/11. Probing of the same blots with a probe encoding dihydroflavonol reductase (DFR), a gene common to anthocyanin and condensed tannin pathways, showed enhanced steady-state levels of this gene which would be consistent with up-regulation conferred by the introduced transcriptional activator. RT-PCR analysis was also carried out on control and selected lines for Sn, DFR and a housekeeping gene (EF-1
), for normalization, and confirmed the results from Northern analysis, i.e. expression of the Sn transgene in 50/10 and 50/11 combined with enhanced levels of DFR mRNA in these two lines when compared with control and suppressed lines (Fig. 3b).
Analysis of anthocyanin and condensed tannin cell types in S50 background
Careful observation of lines grown under control conditions confirmed a subtle anthocyanin phenotype in Sn-expressing lines in the S50 genetic background, 50/10 and 50/11. Similar general phenotypes were also noted in the S33 background. When grown in tissue culture under high light conditions, control Lotus plants did not accumulate visible quantities of anthocyanin pigment (Fig. 4b). However, lines which had been identified with enhanced levels of condensed tannin exhibited a characteristic anthocyanin pigmentation which was particularly marked in juvenile leaf and stem tissues (Fig. 4a and insert). Close examination of mature control plants showed some anthocyanin-containing cells in leaf petiole tissues adjacent to leaf base tissues (Fig. 4d). When line 50/10 was analysed, intense pigmentation was noted in leaf bases, at the end of the petiole and along the midrib of the leaf; no alteration in anthocyanin accumulation was noted in other parts of the plant (Fig. 4c). By contrast, line 50/9 which had been identified with reduced tannin content showed a marked reduction in anthocyanin cells in the petiole (Fig. 4f) relative to a control line (Fig. 4e). Anthocyanins were induced in a tissue-specific manner and cross-sections of petiole (Fig. 4i), leaf (Fig. 4g) and leaf base (Fig. 4h) showed that anthocyanin-containing cells were restricted to the subepidermal cell layer.
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In order to examine the effect of Sn upon the distribution of condensed tannin cells in leaves, one Sn-suppressed line (50/9) was compared with an Sn-enhanced line (50/10) and also an S50 control. The presence of cells containing condensed tannins was determined using DMACA, a reagent which specifically stains cell types that accumulate condensed tannin polymers (Li et al., 1996). Under normal conditions Lotus leaves can contain tannin cells in three positional locations: adjacent to vascular tissues, distributed through the palisade mesophyll and in a matrix formation in the spongy mesophyll (Robbins and Morris, 1999). These observations were confirmed in this study. Leaves from a control plant contained condensed tannin cells in leaf mesophyll tissues (Fig. 5b, e) and analysis of cell distributions at the base of the trifoliate leaf indicated that tannin-containing cells were restricted to the vascular mesophyll (Fig. 5h, k). By contrast, control leaves contained very few tannin-containing cells at the leaf tip (Fig. 5n, q) while petioles had tannin-containing cells adjacent to central vascular tissue (Fig. 5t).
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In Sn-suppressed leaves numbers of tannin-containing cells in leaves were reduced (Fig. 5c, f, i, l). No tannin-containing cells were noted at the leaf tip and some reduction in the numbers of tannin-containing cells was evident in the petiole (Fig. 5u). By contrast, Sn-enhanced lines had higher numbers of tannin cells than suppressed or control lines and had a modified developmental expression profile for these cell types. Increases in numbers of tannin cells were clear after initial staining of trifoliate leaves (Fig. 5a, d) and more detailed analysis indicated that expression of Sn had induced the production of tannin-containing cells both in palisade and spongy mesophyll cell layers (Fig. 5g, j). Sn-enhanced lines also contained tannins at the leaf tip (Fig. 5m), in contrast to control lines and this was accompanied by the appearance of tannin-containing cells, which were predominantly located in the spongy mesophyll (Fig. 5p).
Analysis of the tissue-specific modulation of anthocyanin and tannin-containing cells in Sn transgenics
In view of changes in anthocyanin and tannin cell numbers, cell counts were performed in a range of tissue locations within the trifoliate leaf and these data are presented in Fig. 6. In the enhanced line selected for study, 50/10, increases in anthocyanin cell counts were recorded in petiole and leaf base and anthocyanin cells were noted in layers directly above leaf midrib and this confirms initial observations (Fig. 4d). However, no anthocyanin containing cells were found in the leaf blade, i.e. in areas outside the leaf midrib. By contrast, tannin-containing cells were increased in number relative to controls in the petiole, leaf base and in tannin-containing cells directly adjacent to vascular tissues. More notably, however, tannin-containing cells were present in the lamina of the leaf and a dramatic increase in the numbers of these cells was noted in 50/10. By contrast, lower numbers of tannin-containing cells were found in 50/9, the Sn-suppressed line included in this analysis.
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Effects of Sn transgene upon other flavonoid and phenolic end-products in Lotus leaves
This study has shown that the introduction of Sn modifies the accumulation of condensed tannins and anthocyanin end-products. In order to analyse the effects on other secondary products, both phenolic and flavonoid levels were determined in leaves from three control lines, a phenotype negative line (50/13) together with 50/10 and 50/9. These data are displayed in Fig. 7. In control lines, the major flavonoid end-products were condensed tannins (0.98 mg g–1 DW) and flavonols, primarily kaempferol glycosides (82±3 mg g–1 DW). No isoflavonoid end-products were detectable in leaf tissues, but after elicitation with glutathione measurable levels of vestitol could be determined (515±178 µg g–1 FW) together with trace amounts of sativan (typically 20±16 µg g–1 FW). Free and wall-bound phenolics were present at levels below detectability, but levels of thioglycolic acid (TGA) lignin were measured at 5.5±0.5 mg g–1 DW in control lines.
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Line 50/13 had similar levels of flavonols, lignins and inducible isoflavans to control lines. 50/10 contained 10.3±1.4 mg g–1 DW condensed tannin which corresponds to an increase of nearly 1% of the dry weight of this particular end-product. In other experiments under environmental conditions where plants accumulate higher levels of tannins, leaves from 50/10 accumulated over 2% DW more condensed tannin than corresponding controls. In this up-regulated line, levels of lignin and inducible phytoalexins were unaltered (data not shown). Mean flavonol levels in 50/10 were reduced relative to controls, 72±9 mg g–1 DW as compared with 82±3 mg g–1 DW. However, this difference was not significant and there is no other evidence that increases in condensed tannins are matched by a corresponding decrease in flavonols in other Sn up-regulated lines (data not shown).
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Discussion |
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Previous studies have implicated bHLH transcription factors as anthocyanin pathway regulators in a number of higher plant systems. The maize R gene family (R, B, Lc, and Sn) has been extensively studied and ectopic expression of Lc results in enhanced pigmentation in tomato and petunia (Goldsbrough et al., 1996; Bradley et al., 1998), while expression of R increases anthocyanin production in tobacco and Arabidopsis (Lloyd et al., 1992). By contrast, expression of Lc resulted in no visible phenotypic alteration in vegetative or floral pigmentation in pelargonium or lisianthus (Bradley et al., 1999). Expression of B-Peru in white clover has been reported to induce a novel pattern of anthocyanin accumulation in leaf tissues (de Majnik et al., 2000). Sn hairy roots produced from a range of dicotyledonous plant species showed patterns of red pigmentation dependent upon species, genotype and transformation event (Damiani et al., 1998). The phenotypes resulting from the expression of Sn in Lotus as outlined in this paper are in general agreement with this class of effects upon anthocyanin pathways, however, in this system, alterations in pigmentation were restricted to petiole, leaf base and, in some cases, the leaf midrib.
One interpretation of the data in this paper is that Lotus contains an orthologue to maize R transcription factors and that the ectopic expression of Sn can functionally complement the expression of this Lotus orthologue. An attempt to clone an endogenous Lotus bHLH gene was made, but so far the only one cloned was found to be homologous to PG1, a ubiquitous bHLH protein not functionally related to anthocyanin regulation (Kawagoe and Murai, 1996). However, preliminary quantitative analyses have shown that the cloned bHLH gene is down-regulated in suppressed lines (F Paolocci, unpublished results). In the absence of cloned Sn orthologues, and appropriate mutants for complementation studies, no direct conclusions regarding the effects of Sn upon anthocyanin biosynthesis in Lotus can be drawn.
Evidence has also been provided that both the introduction of Sn and expression of Sn can modulate condensed tannin biosynthesis. In two of the genotypes under study, lines were noted with enhanced levels of condensed tannin. Analysis of two lines, 50/10 and 50/11, indicated that these were single copy transformation events and that leaves from these two lines contained detectable levels of the Sn transgene. Similarly, the analysis of two lines, 50/6 and 50/9, that are suppressed for condensed tannin accumulation, showed that there had been complex transformational events, which are associated with transgene suppression. Levels of DFR mRNA in leaf tissues were enhanced in these two lines and in view of the limited anthocyanin phenotype in leaves, it can be deduced that a major proportion of this Sn-mediated induction of DFR transcript is related to the condensed tannin pathway. Increases in tannin content were accompanied by increases in tannin cell number and these effects were particularly dramatic in the leaf lamina, a tissue that does not biosynthesize anthocyanin end-products. Increases in cell numbers occurred in a cell lineage, which normally contains tannin cells, i.e. the vascular mesophyll. However, tannin cells were also found in cell layers which had no tannin cells in control lines, i.e. in the leaf spongy and palisade mesophyll. It is noted, however, that glasshouse-grown Lotus plants of the high tannin S41 genotype contain tannin cells in all three mesophyll layers, i.e. vascular, palisade and spongy (data not shown). Therefore, in S50 and S33, cells in spongy and palisade mesophyll may be competent for the biosynthesis of condensed tannins, but have no expression of an Sn orthologue or functionally equivalent genes in these cell layers.
Finally, taking the anthocyanin and tannin data together, there is evidence that, in these experiments, Sn has modulated the anthocyanin and condensed tannin pathways. Anthocyanin cell numbers are enhanced in the subepidermal cell layer while alterations in tannin-containing cells occur in leaf mesophyll layers. This co-ordinate induction of independent biosynthetic pathways by a bHLH class gene is surprising, but, nevertheless, gives an interesting insight into the higher order regulation of condensed tannin biosynthesis. Until recently, most anthocyanin bHLH genes have been assumed to have fairly well described biochemical functions. However, it was noted that the TT8 locus in Arabidopsis encodes a bHLH domain protein, which has been reported to regulate the biosynthesis of both anthocyanins and proanthocyanidins (syn. condensed tannins) in siliques (Nesi et al., 2000). This dual metabolic function of TT8 is similar to the effects reported in this paper where Sn can up-regulate tannin and anthocyanins but does not appear to alter lignin, isoflavonoid or flavonoid pathways.
In conclusion, the use of Sn and related anthocyanin bHLH transcription factors may give rise to useful approaches for the modification of levels of condensed tannins in crop species. Additionally, these transgenics may be a valuable resource for cloning the terminal steps of the condensed tannin pathway and also for cloning genes involved in the design of cells that biosynthesize anthocyanin and condensed tannin end-products.
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Acknowledgements |
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IGER is grant-funded by BBSRC, JWH was funded by a BBSRC-RASP studentship ref 4648. We would like to thank other members of the laboratory for helpful assistance; Rolando Barahona, Gordon Allison, Teri Davies, and others. Thanks also to Dr Helen Ougham and Dr Joe Gallagher for constructive comments on this manuscript. IRMGPF is funded by Consiglio Nazionale della Richerche.
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