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Journal of Experimental Botany, Vol. 56, No. 414, pp. 1093–1103, April 2005 doi:10.1093/jxb/eri101 Advance Access publication 14 February, 2005 This paper is available online free of all access charges (see http://jxb.oupjournals.org/open_access.html for further details) RESEARCH PAPER Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves Francesco Paolocci, Tessa Bovone, Nicola Tosti, Sergio Arcioni and Francesco Damiani* Istituto di Genetica Vegetale sez. Perugia, CNR, Via Madonna Alta 130, I-06128 Perugia, Italy Received 30 August 2004; Accepted 8 December 2004 Abstract Introduction The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families. Flavonoid biosynthesis supplies plants with a large variety of secondary metabolites, such as flavonols, anthocyanins, and condensed tannins (CT). They are polyphenolic compounds, synthesized by higher plants in response to both internal metabolic cues and external signals. CT act as protectants of plants against pathogens, pests, and diseases, and they control seed permeability and dormancy (Debeaujon et al., 2000). The capacity of CT to interact strongly with amino acid residues in proteins has led to their wide industrial use. The palatability and nutritive value of forage legumes are highly influenced by the presence and structure of CT (Barry and McNabb, 1999). The presence of CT in leaves of forage plants protects ruminant animals against pasture bloat (Tanner et al., 1995). CT in fact make the conversion of plant protein into animal protein more efficient by increasing the amount of rumen bypass protein, thereby reducing the need to feed supplemental protein. However, CT only accumulate in the seed coats of the most valuable forage species such as alfalfa and clovers, and are absent from their leaves. The ability to manipulate CT synthesis and to direct biosynthesis to the leaves of forage species has been one of the most challenging goals for forage breeders over the past few decades (Damiani et al., 2000). CT are oligomers of flavan-3-ol monomers such as catechin or epicatechin and flavon 3,4-diols (leucoanthocyanidins) that form the ‘starter unit’ on which tannin condensation takes place. No information, however, is available on the nature of condensing steps, and this polymerization process is believed to occur by progressive addition of flavon 3,4-diols to the starter unit (Stafford, 1989). Some researchers have proposed a series of enzymedriven reactions; others claim that, because condensation Key words: ANS, CHS, condensed tannins, DFR, elongation factor, gene expression, light intensity, Lotus corniculatus, PAL, real-time RT-PCR. * To whom correspondence should be addressed. Fax: +39 07550 14869. E-mail: [email protected]. ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. The online version of this article has been published under an Open Access model. Users are entitled to use, reproduce, disseminate, or display the Open Access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Society for Experimental Biology are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact: [email protected] 1094 Paolocci et al. can be achieved in vitro, additional enzymes and therefore genes may not be necessary (Haslam, 1999). Until recently, the biosynthesis of anthocyanidin and CT was believed to share the same sequence of reactions, as the last common intermediate, leucocyanidin, is formed with the enzyme dihydroflavonol reductase (DFR) as the catalyst (Ray et al., 2003). However, recent findings have shown that epicatechin formation occurs via anthocyanidin synthesis and reduction, two steps catalysed by anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR), respectively (Xie et al., 2003). Anthocyanidins are consequently essential for CT biosynthesis in species that accumulate only epicatechin-based CT such as Arabidopsis thaliana (Abrahams et al., 2003). A scheme of the CT biosynthetic pathway is given in Fig. 1. The regulation of CT synthesis and its tissue accumulation is complex, involving several regulatory genes acting on different steps of the pathway. Because tannins are one of the terminal products of a highly branched pathway, which leads to the synthesis of several compounds with a broad action spectrum, several steps of activation are required. Fig. 1. Schematic representation of the CT pathway in Lotus corniculatus. Enzymatic activities are reported in italics. The question mark indicates undetermined enzymatic activities. Some of these steps control the initial phases of the synthesis, the so-called ‘early genes’ (Martin et al., 1991; Martin and Gerats, 1993; Pellettier et al., 1997); other terminal steps of the pathway (‘late genes’) lead to the synthesis of the final compounds. The characterization of mutants defective in anthocyanin accumulation in petunia flowers (an mutants) and in pigmentation in the Arabidopsis seed coat (tt mutants) has resulted in the identification of important regulatory genes. Pigment biosynthesis seems to be mainly regulated at the transcription level since most of the regulatory genes identified encode transcription factors. The majority of them belong to the R2R3-MYB and bHLH families, with help from TTG1, a WD40 protein that may recruit these and other transcription factors to the promoters of target genes (Walker et al., 1999). Factors belonging to the homeodomain WRKY and WIP families have also been shown to be involved in regulating certain aspects of flavonoid accumulation in Arabidopsis (Kubo et al., 1999; Eulgem et al., 2000; Sagasser et al., 2002). The activation of flavonoid biosynthesis results from a complex network into which multiple signals input to regulate gene expression. There is a large body of factors determining how external stimuli affect flavonoid synthesis. High light and UV, pathogen attack, nitrogen, phosphorus, and iron deficiencies, low temperature, and wounding, all affect the synthesis of different compounds derived from the flavonoid pathway (Dixon and Paiva, 1995), but in no case was CT accumulation affected by such environmental conditions. Carter et al. (1999) showed that CT levels in Lotus corniculatus plants were positively affected by CO2 concentration and negatively influenced by drought and temperature. However, a significant influence of tissue type and genotype, as well as an interaction between the different treatments, was observed. Preliminary observations also showed that light intensity can modify CT accumulation in a genotype-dependent manner in L. corniculatus leaves (Paolocci et al., 1999). Lotus corniculatus transgenic lines expressing Sn, a gene encoding a bHLH protein from maize that promotes anthocyanin accumulation, had been produced previously (Paolocci et al., 1999). Lotus plants expressing Sn showed either an enhanced or a decreased accumulation of CT in their leaves, depending on whether the transgene generated Sn protein or whether it was involved in gene silencing (Robbins et al., 2003). These lines may help to scale-down the order of complexity of the regulatory machine of flavonoid biosynthesis. The aim of the present study was to investigate the role of genetic and environmental factors on condensed tannin biosynthesis. To achieve this, the effects of Sn and light on the production of condensed tannins, and on the steady-state transcript levels of four key genes in the pathway, were estimated in Lotus corniculatus leaves. Gene expression and the synthesis of condensed tannins Materials and methods Plant material Lotus corniculatus L. genotypes S50 and S41, isolated from the cultivar ‘Leo’, and characterized for their different levels of CT accumulation in leaves were kindly provided by Dr Mark Robbins (IGER, Aberystwyth, UK). S50 plants transformed with the maize bHLH regulatory gene Sn, showing either an enhanced (plants S5010 and S50-11) or a reduced (named S50-6, S50-9, S50-20) level of CT in leaves, were obtained as described previously (Paolocci et al., 1999; Robbins et al., 2003). All the plants were repetitively assessed for their accumulation of CT by DMACA staining, as reported by Li et al. (1996), and for anthocyanin-containing cells by monitoring the leaves with the naked eye and under a stereomicroscope after an overnight ethanol treatment to decolorize chlorophyll. Light treatment For each of the seven plants under investigation (S50, S41, S50-10, S50-11, S50-6, S50-9, S50-20) cuttings were produced from stem segments rooted in sand in a humid chamber. Once roots were developed, the plantlets were transferred to pots. After 4 weeks in a humid chamber (95% humidity, 23 8C), the plants were moved to a growth chamber (12 h of light at 650 lmol photons mÿ2 sÿ1, 22 8C) and 6 weeks later were split into three groups. All the groups were maintained in the same growth chamber but they were differentially shadowed to irradiate plants with 100, 400, or 1000 lmol photons mÿ2 sÿ1 of light. Each genotype was represented by four cuttings per light treatment. Leaves were collected on all plants after 4 weeks of light treatment and 4 h after the light was switched on. Leaves from each plant were partly stained for CT, and partly freeze-dried in liquid nitrogen and stored at ÿ80 8C for molecular analysis. Quantification of CT was performed on five randomly selected young leaves per each treated plant. After DMACA staining, the leaves were washed four times with distilled water and observed under a stereomicroscope (36 magnification) and the number of CTcontaining cells counted as a proportion of the total number of cells in five optical fields per leaf. RNA isolation and analysis Total RNA from four young leaves was isolated using the Nucleo Spin RNA Plant isolation kit (Macherey Nagel) according to the supplier’s instructions, to which a further DNase treatment was added. Spectrophotometric RNA determination was carried out three times for each sample. Poly (A)+ RNA was recovered from total RNA by using the poly (A)+ tract system II (Promega) and resuspended in 20 ll of RNAsi-free water. Five microlitres of mRNA were reverse transcribed at 37 8C for 60 min in the presence of 200 units of SuperScript II H- Reverse Transcriptase (Invitrogen) and 100 pmol of random hexamers (Pharmacia Biotech) according to the enzyme’s supplier. 1095 Gene cloning Extension of the partial DFR sequences of Lotus already published (Paolocci et al., 1999) was performed using the 59-RACE and 39RACE System for Rapid Amplification cDNA Ends Version 2.0 (GIBCO-BRL) according to the supplier’s instructions, by using the adaptor primers provided with the kits in combination with the DFR gene-specific primers (59-TGGCATTGTCGGCATTAGAAAGG-39) and (59-AGAAGGCCAAAACTGTACGGAGG-39) for the 59- and 39-RACE, respectively. Primers were then designed on the extreme 59 (59-CAAGCCAACCCAAACATAG-39) and 39 (59-ACAATGCAAACACAG-39) sequence ends, to amplify the full-length cDNA as well as the DFR gene from L. corniculatus. The full-length cDNA sequence and a genomic DFR sequence have been registered in the NCBI’s sequence database under the accession numbers AF117263 and AY633707, respectively. The amplification of elongation factor 1-a (EF) cDNA was achieved using the primer pairs reported previously (Robbins et al., 2003). The resulting partial cDNA fragment has been deposited in NCBI’s sequence database under the accession number AY633710. By aligning cDNA sequences encoding PAL and b-tubulin (Tub) from plant species closely related to the Lotus genus, the primer pairs PalFw (59-GGRCAATGYGGYAACCARATCGG-39) and PalRev (59-GCATCYTGGTAYTGYTGGTAYTC-39), and TubFw (59AARCAYCAYCCNGGICARATIGARGC-39) and Tubrev (59TTNACRTCYTGRTTRTGYTGYTGYTCIGC-39) were designed and used in a 50 ll volume of PCR reaction on cDNA from poly (A)+ RNA (see above) in the presence of 10 pmol of each primer. The resulting partial cDNA fragments encoding PAL and Tub were deposited in Genbank under the accession numbers AY633709 and AY633708, respectively. Sequence analysis Double-strand sequence analysis was carried out, either directly on PCR products or on PCR fragments previously cloned in the pGEM-T Easy Vector System I (Promega), using the Big Dye Terminator Cycle Sequencing Kit and an ABI Prism 310 Sequence Analyser (Applied Biosystems) according to the supplier’s instructions. The sequencing primers were the Sp6 and T7 vector primers for cloned PCR fragments and gene-specific primers for direct sequencing reactions. Primer selection for real-time PCR analysis Primers for quantitative RT-PCR analysis were designed using OligoExpress Software (Applied Biosystems) on the PAL, Tub, DFR, and EF cDNA sequences obtained from L. corniculatus accession S50 and from the ANS and CHS gene sequences from L. corniculatus deposited in the databases (accession numbers AY028931 and AF308143, respectively). All primers used in realtime RT-PCR experiments are listed in Table 1. Real-time RT-PCR analysis For real-time RT- PCR analysis 18 preparations of first-strand cDNA were made from total RNA from six different genotypes Table 1. List of primer pairs used for real-time RT-PCR analysis Target gene Forward primer 59–39 Reverse primer 59–39 EF1-a Tub DFR Sn CHS PAL ANS AAGGAGGCCGCTGAGATGA GTGGTAACCAGATCGGTTCGA CAATCATTCCACCCCTTGTTG ACGGGAGCAGCACAGGAA TTGATGGCCACCTTTGTGAA CTCAGTGGCTTGGACCTCAGA TCCACCTTATCTTTCCAGAAGACAA CGCTCAGCCTTCAATTTGTCA CTCCCCTGCGGATCTAAGC GATCGAATAATGGGCCTCGTT GTCGCTTTCGCTCCGACAT TGAAACAATCCCAGGAACATCTT CGATCATCTTGCTTGCATGC TCGCTGGTAACTTCCGTATAATATGA 1096 Paolocci et al. (S50, S50-10; S50-11, S50-6, S50-9, S50-20) grown under three different light conditions. cDNAs were diluted to 100 ll, and 4 ll used for each real-time PCR experiment. The PCR reaction was made up using the SyBrGreen PCR core mix (Applied Biosystems) according to the supplier’s instructions in 25 ll of final volume in the presence of 2.5 pmol of each primer. Cycling parameters were: two initial steps of 50 8C for 2 min and 95 8C for 10 min for UNG and AmpliTaq Gold activation, respectively, a two-step cycle of 95 8C for 30 s and 60 8C for 1 min repeated 50 times, and a final step of 10 min at 60 8C. Afterwards, the dissociation protocol was performed. Amplifications were performed on ABI PRISM 5700 SDS apparatus (Applied Biosystems). Four replicates for each RNA sample per target gene were amplified and, for each transcript, the average Ct (threshold cycle) was determined. Ct is defined as the point at which fluorescence rises appreciably above the background. Standard curves for target and housekeeping genes were obtained by the amplification of a serially diluted mixture of cDNA samples, with six dilution points, each one replicated four times. Different strategies were investigated to search for the most reliable reference for the data sets. Firstly, spectrophotometric quantification of RNAs was used. However, accurate measurement of total RNA levels is not a particularly reliable standard for analyses based on PCR. Consequently, a gene quantification method based on the relative expression of the target gene versus a reference gene, that is supposed to be stably expressed, was adopted according to the ABI PRISM 5700 User Bulletin 2 (Applied Biosystems). Relative quantification of gene expression was achieved using both EF and Tub as reference genes. The amount of each endogenous reference per sample was determined from the appropriate standard curve, then for each RNA sample the amount of target transcript was divided by the amount of the endogenous reference to obtain a normalized value of the relative expression levels. The final number was then treated as a quantitative trait and statistically analysed. Two-way analysis of variance (Model II, all effects random) and the Duncan test for mean comparison were performed using the GLM Procedure of the SAS program (SAS Institute). Results Effects of light intensity on levels of CT It has been shown previously that in the Lotus lines under investigation the increment/decrement of leaf CT quantity is reflected by the increment/decrement of the number of CT-containing leaf cells (Robbins et al., 2003) as determined by DMACA staining, which specifically binds to CT-containing cells. Under natural outdoor growing conditions, S50 and S41 genotypes showed different levels of CT in their leaves. The first genotype accumulated very few leaf tannins, the second many more (Carron et al., 1994), to levels similar to those of Lotus uliginosus Schk., the Lotus species accumulating the greatest amounts of CT in leaves. Interestingly, the S41 genotype accumulates CT in all three mesophyll layers – vascular, palisade, and spongy (data not shown) – unlike S50 which has detectable CT only in vascular tissues. These two genotypes also behaved very differently following transformation with Sn (Paolocci et al., 1999; Robbins et al., 2003). Nevertheless, both S50 and S41 showed a progressive increase in CT levels at increasing levels of irradiation, although, irrespective of the light treatment, the S41 accession always showed a larger number of CT-containing cells than S50 (Fig. 2). S50-10 and S50-11 transgenic lines, hereinafter termed ‘E-plants’ (enhanced) because of the expression of the maize gene Sn which encodes a bHLH regulatory protein, had CT levels intermediate between those of S50 and S41. They had also accumulated CT in spongy and palisade mesophyll cells, regardless of the light treatment, in contrast to control S50 (data not shown). Conversely, the plants S50-6, S50-9, and S50-20, hereinafter called ‘S-plants’ (suppressed), due to the Sn silencing effect (Paolocci et al., 1999; Robbins et al., 2003), showed very low CT accumulation and were very depleted in CT compared with control plants (S50-C) at medium and high light intensities. Light positively increased CT levels in all the accessions, but its effect was greatest in S50-C and E plants due to increases in the numbers of CT-containing cells (Fig. 2). Conversely, within each plant class the number of anthocyanin-containing cells was unaffected by the light treatment. Selection of target genes and gene expression quantification procedures To investigate by real-time RT-PCR analysis the expression profiles of the genes involved in CT biosynthesis on S50-C, E, and S lines grown in the three light regimes, four genes in the phenylpropanoid pathway were selected: PAL, CHS, DFR, and ANS. While the first two structural genes code for enzymes in the early steps of the phenylpropanoid pathway, DFR and ANS encode for later steps and are the most downstream genes that, although only partially, have been cloned so far from Lotus. To quantify and compare the expression of target genes or, better, the steady-state levels of their mRNA under specific treatments, normalization for the amount of RNA used in the different samples was needed. This is generally achieved by comparing the level of the target gene with Fig. 2. CT levels in leaves of C, S, E, and S41 plants grown at low (L), medium (M), and high (H) light conditions, estimated as a percentage of CT-containing cells. Values are averages of three S plants, two E plants, and one control replicated four times. Gene expression and the synthesis of condensed tannins those of a reference that is constitutively expressed. However, it is conceivable that reference genes are also affected by the specific environmental conditions being examined, by the possible effects resulting from the activity of the exogenous transcription factor (Sn), or by the rol genes delivered to the host genome during transformation using Agrobacterium rhizogenes. There is increasing evidence that generally used housekeeping genes (such as GAPDH, actins, tubulins, etc.) may vary significantly under different experimental conditions (Pfaffl et al., 2001). The three methods used for RNA standardization (total RNA, steady-state of EF, and Tub mRNA) did not in fact yield identical results. To determine which was the most accountable reference the correlation among all the references was calculated (data not shown). A positive and significant correlation was observed between EF and RNA, while the Tub transcript was totally unrelated to the amount of total RNA; this implies that by using EF or Tub as a reference can give rise to differing results. EF is reported to be constitutively expressed and has been used widely to normalize gene expression of flavonol genes in different plant tissues and at different developmental stages (Rosati et al., 1997; Debeaujon et al., 2003). Because EF is positively correlated to total RNA levels and, importantly, because its levels also reflect variation at the RNA reverse transcription step, it was selected as the most suitable reference. Gene expression and light As expected, Sn expression was not detectable after 50 cycles of amplification in control and S lines (Robbins et al., 2003), and was low even in E lines, which, under low light conditions, showed a Ct of 34.6 against a Ct of 26.9 for DFR (which was the most highly expressed gene). Because Sn in the transgenic lines is driven by a strong promoter (CAMV 35S) it can be excluded that it is poorly transcribed; conversely, the low steady-state level of Sn mRNA can be accounted for by a high rate of transcript degradation, suggesting that transcript decay is as important as transcription rate in determining the net level of gene expression (Khodursky and Bernstein, 2003). No differences in Sn transcript levels were observed between highand low-light-treated E plants, indicating that light had no effect on the expression of this transgene or on the degradation of its transcript. From the real-time RT-PCR a quantitative analysis of the results of these expression experiments was undertaken. There were four replicates for each plant (six) under each light treatment (three), giving a total of 72 observations for each of the four genes analysed. Since the aim was to compare the effect of light on different genetic backgrounds (S, E, and C plants), data obtained from S50-10 and S50-11, as well as from S50-6, S50-20, and S50-9, were pooled. A two-way ANOVA was performed; light and plant type were 1097 the main sources of variation, and their interaction, if significant, the error. Table 2 reports the mean square values and their significance for every gene analysed. The significance of the interactions indicated that different plant types responded in a different way to light. This happened for PAL and CHS but not for DFR and ANS which behaved similarly. For such genes, E plants showed significantly higher values (1.4 for DFR, 1.2 for ANS) than the other two classes which had the same result for ANS (both 0.9). C plants showed a higher DFR level than S (1.0 and 0.8, respectively). Light had a similar effect on both genes in C and S plants; the ANS and DFR values at high light turned out to be significantly higher than values at low light, whereas their values at intermediate light were not significantly different from those under low and high light conditions. A more detailed picture of the effect of light on gene expression is given in Fig. 3, where, for all genes, the level of each transcript in each plant type under different light treatments is recorded. The high regime did not have a significant effect on PAL gene expression of S plants, although in C plants medium irradiation reduced PAL transcript levels relative to low irradiation. PAL gene expression was reduced significantly by high light compared with low light in E plants. Generally higher light decreased CHS transcript levels in the different lines as well, although high light significantly increased CHS transcript levels in E plants compared with low light conditions. DFR and ANS show a similar pattern of RNA accumulation at different light levels; the higher light level produced no effect in E and C plants, though the level of RNA in S plants grown in high light conditions was significantly different with respect to S plants at low light levels. From the quantitative analyses it is concluded that, at least for the isoforms studied, transcript levels of PAL and CHS are not correlated with CT accumulation. Conversely, higher levels of DFR and ANS transcripts are found in CT-enhanced genotypes. But, while increases in the expression of these genes could explain the higher CT accumulation of E plants relative to C/S plants, they do not explain the difference in CT accumulation shown by E plants under different light treatments (Figs 2, 3). In fact, E plants at low and high light intensity did not show significant differences in DFR and ANS transcript levels, Table 2. Scheme of ANOVA and s2 values for each source of variation d.f. PALa Plant type (P) 2 Light treatment (L) 2 Interaction (P3L) 4 Error 63 CHSa 0.350 n.s. 0.198 n.s. 0.002 n.s. 0.562 n.s. 0.061 ** 0.319 ** 0.013 0.041 DFRa ANSa 1.871 ** 0.155 ** 0.004 n.s. 0.027 0.843 ** 0.144 * 0.093 n.s. 0.043 a n.s., Not significant; **, significant at P >0.01; *, significant at P >0.05. 1098 Paolocci et al. Qualitative analysis of gene expression Fig. 3. cDNA levels of PAL, CHS, DFR, and ANS genes relative to the EF cDNA measured through real-time RT-PCR. Values are averages of three S plants, two E plants and one control replicated four times. Within each graph independently, values with the same letter do not differ significantly (P <0.05). suggesting that once DFR and/or ANS are expressed above a threshold level, other genes, whose expression is enhanced by light, need to be activated to lead to higher CT biosynthesis and accumulation. SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye. Binding of SG to double-stranded DNA is non-specific, and additional testing such as DNA melting curve analysis is required to confirm the generation of a specific amplicon. The use of melting curve analysis eliminates the necessity for agarose gel electrophoresis, providing an even more accurate and informative tool for the qualitative control of PCR reactions, allowing even single nucleotide polymorphisms (SNPs) to be revealed among the amplicons generated. A dissociation protocol was therefore performed at the end of each experiment as a quality control for the PCR, and to check for the presence of different gene members potentially co-amplified by the primer pairs designed. In fact, some of the genes under investigation are generally arranged in multi-gene families in legumes, but compared with these genes only a few or partial L. corniculatus sequences are present in public databases. While dissociation curves showed only a single peak relative to Sn, EF, Tub, PAL, and ANS in any treatment, two or multiple peaks were revealed in cDNA samples amplified with CHS and DFR primer pairs. Specifically, CHS showed a main peak at 76 8C and a less-abundant one at 73 8C. The dimension of this secondary peak was variable between samples and might explain the contrasting results between E and C plants. C and S plants showed two peaks at low light and one at high light; E plants, on the contrary, maintained the polymorphic condition, even at high light (Fig. 4). For DFR transcripts, a primer pair spanning the fourth and fifth exons was specifically designed to amplify a region of the transcript known to be polymorphic in Lotus (F Paolocci et al., unpublished data). The melting curve analysis showed the presence of a major peak, between 81 and 83 8C, and one or more secondary ones at 78 and 74 8C. Figure 5 shows the DFR dissociation curve resulting from C, E, and S plants under high and low light conditions. The products of the real-time RT-PCR were sequenced from samples producing multiple peaks. While confirming an amplicon size of 107 bp, as predicted for the amplification of DFR cDNA, sequencing revealed the presence of several differences (SNPs) between the DFR cDNAs. After cloning these cDNA fragments, three polymorphic sequences were obtained, named col2, col3, and col16 (Fig. 6). These cDNA transcripts potentially encode DFR proteins with distinctive amino acid composition and potentially different catalytic specificities. To identify which of the three distinct DFR transcripts was expressed under the condition tested the melting curve analysis was performed on the three plasmids carrying the polymorphic cDNA fragments. When amplified in purity, the three plasmids showed a Tm profile comprising a single dissociation peak of about 83, 82, and 81 8C for col16, col2, and col3, respectively, in Gene expression and the synthesis of condensed tannins 1099 Fig. 4. Dissociation pattern of CHS amplicons from cDNA of C, E, and S plants grown under high and low light conditions. accordance with the reduction in the pyrimidine number. When a mix consisting of equal amounts of col2 and col3 was amplified, a major peak at 82 8C and a secondary one at 78 8C were observed, the secondary one likely representing the dissociation of non-totally homologous sequence strands. When the relative concentration of col3 increased, the main peak shifted toward 81 8C. When only col2 and col16 were present, the dissociation pattern showed only one peak at 82 8C, regardless of their relative concentrations. When col3 and col16 were equally represented in the mix, the dissociation pattern showed three peaks; at 83 8C and 81 8C, each one specific for the pure cDNA fragments, and one at 76 8C that was attributed to the dissociation of the non-homologous annealed stretches. Since, in the leaf cDNA analysed, a single peak was never observed, then at least two different DFR forms must always be co-expressed in Lotus leaves. At high light, all plants showed two peaks at 81–82 8C and 78 8C indicating that the forms col2 and col3 were present. At low light, E plants showed a major peak at 81 8C along with a minor one at 78 8C, indicating that the form equivalent to col3 was predominant and a col2-like DFR form was also moderately expressed. Conversely, in C and S genotypes, one large peak at 82 8C and two more at 78 and 74 8C were present, indicating that col2, col3, and col16 isoforms were simultaneously transcribed and accumulated. Of course, the present analysis may not have identified all the different DFR transcripts expressed in L. corniculatus leaves. Discussion The effect of environmental conditions on CT accumulation is quite well documented, the effects of temperature and 1100 Paolocci et al. Fig. 5. Dissociation pattern of DFR amplicons from cDNA of C, E, and S plants grown under high and low light conditions. Fig. 6. Nucleotide and deduced amino acid sequences of three cloned DFR amplicons (col2, accession no. AY633711; col3, no. AY633712; col16, no. AY633713) resulting from real-time RT-PCR of control plants under low light condition. Boxed areas indicate polymorphisms. seasonality of plant growth on biosynthesis having been analysed (Grebrehiwot et al., 2002). However, no information about the effect of light on their accumulation has yet been reported. It is now reported that light and Sn expression induce CT accumulation synergistically in L. corniculatus leaves and that the light effect is more evident in plants with low levels of CT (S50) compared with those producing higher amounts of CT, such as S41 plants (Fig. 2). Within each plant class, light seems to induce quantitative modulation on the target genes tested within quite a narrow range (Fig. 3), but ANOVA shows a significant and positive effect of light on DFR and ANS transcript accumulation (Table 2). Conversely, no significant increases in PAL and CHS transcripts in response to light were observed, although there was a significant interaction between plant type and light. However, CHS transcript(s) decreased in control and S plants when they are moved from low to high light conditions (Fig. 3), and this trend was paralleled by a reduction in the number of CHS isoforms expressed (Fig. 4). On the contrary, E plants showed a positive response of CHS transcript levels to increased light, although isoform expression did not change with varying light conditions. The present data contrast with the observation reported by Ray et al. (2003) that Gene expression and the synthesis of condensed tannins transgenic alfalfa lines expressing Lc (a Sn paralogue) had higher levels of CHS RNA, although Lc did not detectably affect DFR transcript levels in alfalfa. The role of CHS in tannin biosynthesis is somewhat controversial. Colliver et al. (1997) obtained an increase of CHS expression and CT levels in L. corniculatus hairy root lines transformed with a heterologous CHS gene in antisense orientation. They proposed that the silencing of the gene encoding one CHS isoform might induce the overexpression of the other gene members, thereby inducing a possible re-routing of the phenylpropanoid biosynthetic pathway In pea, CHS is encoded by a multi-gene family, and the response of different members to elicitor and UV irradiation varies during development and in response to various external stimuli (Ito et al., 1997). Therefore, for a finely tuned assessment of the effect of light and Sn on CHS, the cloning and transcription analysis of each individual gene member is required. The finding that the primer pair used can amplify two distinct CHS transcripts, that have a differential expression pattern, confirms that the regulation of CHS activity is complex and multi-genic. Altogether, the present data stress the existence of a lightinsensitive phenylpropanoid pathway in Lotus leaves that may ensure CT synthesis even when environmental conditions are not optimal. Interestingly, within each gene family, the activity of one or only few gene members may be dedicated to CT synthesis. This model is confirmed by the expression of PAL. This gene was selected as a potential endogenous control of the phenylpropanoid path, since it is an early gene in this pathway and in maize is not transactivated by Sn (Tonelli et al., 1991). Unexpectedly, a down-regulation of PAL transcripts by Sn was observed, although this did not prevent the accumulation of higher levels of CT in response to Sn. For this gene, extra care is required for interpretation of the results because the pair of primers selected amplify preferentially one particular member of the PAL gene family that is highly homologous to PAL2 which, in Populus tremuloides, appeared to be less associated with CT synthesis than other PAL genes (Kao et al., 2002). Thus, the possibility cannot be excluded that light and/or Sn stimulates expression of a highly expressed PAL gene, that was not detected with the present primer pair, which may significantly affect CT levels. Expression of later steps in the pathway is more strictly correlated with CT levels. In fact, at higher levels of DFR and ANS there are correspondingly increased CT levels in S and C plants. In E plants the effect of light on DFR and ANS steady-state transcript levels is masked by Sn expression, proving that the constitutive expression of the exogenous regulator overrides the activity of the endogenous activator of these genes. However, light affects CT levels in E plants, probably through an effect on expression of endogenous regulatory genes which may promote transcription/activity of genes/enzymes downstream of DFR in the pathway. Candidate genes are BAN (Devic et al., 1999), coding for 1101 anthocyanidin reductase (Xie et al., 2003), and LAR (Tanner et al., 2003), whose cloning in Lotus is in progress in the authors’ laboratory. It is worth mentioning that, in alfalfa, the maize Lc gene does transactivate LAR transcript levels (Ray et al., 2003), while in Arabidopsis TT8, a bHLH protein, positively regulates DFR and BAN (Nesi et al., 2000). Previous observations showed that Sn silencing negatively affects LAR activity in transgenic Lotus (Damiani et al., 1999). It can thus be speculated that, if LAR/BAN expression is induced by Sn in the same way as DFR and ANS, the effect of light would probably operate through later genes in the CT pathway, such as those controlling polymerization and/or CT compartmentalization in vacuoles. The use of the SG instead of an allele-specific probe in real-time RT-PCR analysis was successful in monitoring the expression of different members of a small DFR multigene family in Lotus (Robbins et al., 1998). Sn seems not only to trigger the expression of col3-like DFR specifically, but also to prevent the expression of those DFR forms activated under low light conditions. In this scenario, the col3 DFR gene should be the one strictly committed to CT synthesis and Sn should show a differential activity on the promoter regions of the different DFR genes. Preliminary results have proved the presence of multiple DFR genes in the S50 genome. Additionally, the predicted DFR proteins encoded by col3, col2, and col16 clones showed two amino acid substitutions (Fig. 6) downstream of the region that determines the DFR substrate specificity (Johnson et al., 2001). Extension of the cDNA sequences to the 59 end of col16 and col3 did not reveal any amino acid substitutions at the key positions responsible for substrate specificity of DFR (F. Paolocci, unpublished results). Thus, it is likely that all the DFR members identified as differentially expressed in response to light treatment and transgene expression code for enzymes with similar substrate specificity; also it is likely that they may substitute functionally for each other, as is also suggested for CHS. This could explain the results obtained by Robbins et al. (1998) who increased the level of CT through DFR antisense. However, very recently Xie et al. (2004) identified two DFR genes in Medicago truncatula, with remarkably similar patterns of transcript accumulation in most tissues, coding for two different and catalytically active DFR proteins. They proposed that, beside the single amino acid change in the region identified by Johnson et al. (2001), other active site residues also have an effect on substrate preferences of the enzyme. Therefore, the possibility that the three cDNAs identified in Lotus encode for functionally distinct DFR proteins cannot be ruled out completely. The expression analysis of ANS in a species accumulating both epicatechin- and catechin-based CT such as L. corniculatus (Fig. 1) is of importance to both anthocyanin and CT synthesis. In Arabidopsis, TT8 was shown to have the dual metabolic function in boosting both CT and anthocyanin routes (Nesi et al., 2000). Foo et al. (1996) 1102 Paolocci et al. showed that the CT extender flavan units consist mostly of epicatechin in L. corniculatus. A previous observation showed that anthocyanins accumulated very poorly in L. corniculatus leaves and that Sn expression increases anthocyanin accumulation in only a few cells located in the petiole, leaf base, and next to the central leaf midrib region (Robbins et al., 2003). CT quantification carried out through cell counts and HPLC analyses is highly concordant in leaves of Sn transgenic Lotus lines (Robbins et al., 2003), while the leaf anthocyanin spectrophotometric quantitation carried out according to Ray et al. (2003) did not allow differences between leaves of Sn-induced and -suppressed plants to be appreciated under the different light conditions (data not shown). The observation that ANS is clearly transactivated by Sn, irrespective of the light treatment, and that this transactivation as well as the light effect is largely devoted to accumulation of CT rather than anthocyanins lead to the argument that both Sn and light increase the flux of flavonoids in Lotus leaves mainly towards the production of CT through the synthesis of both catechin and epicatechin. Despite the complexity of the genome, the organization of most of the phenylpropanoid genes as small multi-gene families in legumes, and the relatively few genes sequenced, an experimental procedure has been set up to assay gene modulation in L. corniculatus by real-time PCR analysis. Real-time PCR and the use of SG were demonstrated to be powerful, as well as inexpensive, methods of investigation to assess quantitative and qualitative modulation of gene expression. The design of primer pairs spanning polymorphic regions proved to be an efficient method to detect the internal and external stimuli that specifically trigger the expression/silencing of a specific gene, among different members of a gene family. 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