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First published online November 10, 2006; 10.1104/pp.106.090886 Plant Physiology 143:504-516 (2007) © 2007 American Society of Plant Biologists Ectopic Expression of a Basic Helix-Loop-Helix Gene Transactivates Parallel Pathways of Proanthocyanidin Biosynthesis. Structure, Expression Analysis, and Genetic Control of Leucoanthocyanidin 4-Reductase and Anthocyanidin Reductase Genes in Lotus corniculatus1,[W]National Research Council, Plant Genetics Institute, 130–06128 Perugia, Italy (F.P., L.M., S.A., F.D.); Institute of Grassland and Environmental Research, Aberystwyth, Ceredigion SY23 3EB, United Kingdom (M.P.R.); and Philipps Universität Marburg, Institut für Pharmazeutische Biologie, 35037 Marburg/Lahn, Germany (S.M.)
Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.
Flavonoids represent one of the oldest, largest, and most diverse families of plant secondary metabolites. These compounds fulfill a multitude of functions during plant development (Winkel-Shirley, 2001  ) and are also of significant interest as antioxidant and anticancer agents in the human diet (Dixon et al., 2005; Tohge et al., 2005). Proanthocyanidins (PAs), also known as condensed tannins, are oligomeric and polymeric end products of the flavonoid biosynthetic pathway. They are widespread in nature, occurring in numerous plant species, including many important plant-derived food materials (Tanner, 2004).
The beneficial effects of PAs on human health and the significant pharmacological activities of PAs have been recently reviewed (Marles et al., 2003
PAs vary widely in composition, depending upon the nature (stereochemistry and hydroxylation pattern) of the flavan 3-ol starter and extension units, the position and stereochemistry of the linkage to the lower unit, the degree of polymerization, and modifications of the 3-hydroxyl group (for recent and extensive reviews, see Tanner, 2004
Although the PA biosynthetic pathway has been best characterized in Arabidopsis (Arabidopsis thaliana), this species accumulates only epicatechin-based starter units and lacks any obvious LAR ortholog (Abrahams et al., 2003
The analysis of Arabidopsis seed coat mutants has yielded critical information regarding the transcriptional control of genes involved in PA biosynthesis (Lepiniec et al., 2006 ). A ternary transcriptional complex comprising TTG1 (encoding a WD-repeat protein), TT2 (encoding a R2R3 MYB-class protein), and TT8 (encoding a basic helix-loop-helix [bHLH] class protein) has been shown to regulate tissue-specific accumulation of PAs. Mutations in any of these three genes result in a transparent testa phenotype with either a reduced or complete absence of PA polymers. The MYB, bHLH, and WD complex also regulates anthocyanin biosynthesis, trichome initiation, and non-root hair cell specification in Arabidopsis. In these cases, the WD-repeat protein (TTG1) is the same in all of the different functional complexes, whereas the bHLH and MYB proteins are flexible in their participation. Therefore, mutations in TTG1 affect the functioning of all the pathways, whereas mutations in the genes encoding the bHLH proteins affect overlapping subsets of the pathways and the specificity in regulating the different pathways in different tissues is conferred by different MYB proteins (Zhang et al., 2003). In this context, mutations in TTG1 and TT8 also affect anthocyanin accumulation in seedlings, consistent with the expression of TT8 and TTG1 in both seed and seedlings. Conversely, TT2 is specifically expressed in the innermost cell layer of the inner seed tegument where it controls PA accumulation by regulating the expression of DFR, LDOX, and BAN. TT8 is similarly important for the expression of DFR and BAN in siliques and TTG1 controls BAN expression mainly by affecting TT8 function (Baudry et al., 2004).
The genus Lotus offers a wide range of options for studying PAs and includes species that accumulate different quantities of condensed tannins; some species accumulate these end products in the majority of plant tissues, whereas others (Lotus japonicus, Lotus tenuis) do not produce condensed tannin polymers in leaf tissues (Harborne, 1988 Here, we report on the cloning and characterization of genes encoding ANR and LAR from L. corniculatus. Expression analysis of these genes in wild-type and transgenic lines showing different levels of leaf PAs and the analysis of cell types in leaf tissues, which accumulate detectable levels of PAs, have developed our understanding of the genetic control of PA biosynthesis in this forage legume. Our findings may also be of importance in understanding the genetic mechanisms controlling PA accumulation in other tanniferous plants, where both branches of the PA pathway are operating.
Cloning of ANR cDNAs By aligning the ANR cDNA sequences from Medicago truncatula (AY184243) and Phaseolus coccineus (BN000164), degenerate primers were designed and used to amplify an orthologous leaf cDNA fragment from L. corniculatus S41, which was then extended to the 5' and 3' ends by RACE. The ANR amplicons resulting from end-to-end PCR on leaf cDNA were cloned in a plasmid vector and six clones were selected. Four clones harbored a fragment of about 1.2 kb, whereas the other two harbored fragments of about 1,100 bp and 500 bp, respectively. Sequence analysis showed that length differences among clones resided basically in the coding sequences. The 5'- and 3'-untranslated regions (UTR) ranged from 73 to 79 bp and from 104 to 109 bp; the longest clones, named LcANR1-1 to LcANR1-4, encoded a 338-amino acid protein and the other two, named LcANR2 and LcANR3, encoded proteins of 302 and 107 amino acids, respectively (Supplemental Fig. S1). The four LcANR1 clones differed slightly from each other, with a total of six conservative amino acid substitutions, and all showed a high (82%) sequence identity at the amino acid level to the ANR cDNAs from M. truncatula and P. coccineus. The identity of LcANR2 and LcANR3 to the above-mentioned cDNAs was still high. However, LcANR2 differed from LcANR1 and ANR cDNAs from other species, such as M. truncatula, P.coccineus, and grapevine (Vitis vinifera), because it lacked most of the coding sequence that showed homology to the NAD-dependent epimerase/dehydratase domain as per the National Center for Biotechnology Information (NCBI) conserved domain search. The same search did not show any putative conserved domain in LcANR3.
A BLASTn similarity search was carried out to identify putative homologous sequences to LAR cDNA from D. uncinatum (AJ550154), the only LAR gene present in public databases when this research was initiated. This search revealed that at least five regions in the M. truncatula bacterial artificial chromosome clone mth2-103j7 (AC146570.4) displayed high nucleotide similarity with the query sequence (data not shown). From these highly homologous regions, degenerate primer pairs were designed to amplify leaf cDNA from the S41 genotype. The cDNA amplicons resulting from amplification with the primer pairs 2LarF/3larR (about 800 bp long) and 5LarF/6larR (about 500 bp long) were then used as targets for 5'- and 3'-RACE PCR amplification. Primers were designed for each set of both 5' and 3' ends at the extremity of the longest amplicons and used to amplify the respective full-length cDNAs. Using the different forward/reverse primer combinations, amplicons of two lengths were recovered, the longest, generated by 5Lar1cl/3larbis primers, was named LcLAR1, the other, named LcLAR2, was generated by the 14fwUTR/x3Lar primer combination.
Both LAR1 and LAR2 fragments were cloned and two clones for each gene, named LcLAR1-1, LcLAR1-2 and LcLAR2-1, LcLAR2-2, were sequenced. The 1,418-bp-long LcLAR1-1 cDNA contained 99 nucleotides upstream of the first in-frame ATG, with an open reading frame (ORF) of 1,047 bp (349 amino acids) and a 3' UTR of 272 bp, the poly(A+) tail not included. Both LAR1-1 and LAR1-2 showed the microsatellite-like motif (TG)4(TGAG)4 in the 5' UTR. LAR1-2 differed from LAR1-1 by two amino acid substitutions (T-S and N-K at positions 192 and 333, respectively). As regards the LcLAR2 amplification products, LAR2-1 cDNA was 1,114 bp long and contained an ORF of 339 amino acids with a 14-bp 5' UTR and 79 nucleotides after the stop codon and before the poly(A) tail. LAR2-2 cDNA was 1,120 bp long and contained an ORF of 339 amino acids with a 19-bp 5' UTR and 81 nucleotides after the stop codon and before the poly(A) tail. At a protein level, three amino acid substitutions were detected between the LAR2-1 and LAR2-2 cDNA clones (Q-L, I-V, and P-S at positions 37, 79, and 229, respectively). The LAR gene cloned from Desmodium was most closely related to isoflavone reductase-like proteins (Tanner et al., 2003 The cloned LAR1 and LAR2 sequences showed 71% sequence identity at the amino acid level when compared with each other. A BLASTP search showed that both LAR1 genes had the highest sequence identity at the amino acid level to a LAR gene cloned from Lotus uliginosus (96%) and, to a lesser extent, to the LAR genes from M. truncatula and P. coccineus (66%). LAR2-1 cDNA, by contrast, displayed a higher sequence identity to LAR genes cloned from M. truncatula and P. coccineus (78% and 76%, respectively). A phylogenetic tree showing the relation of LcLAR1 and LcLAR2 with LAR sequences from other legume species is shown in Figure 2 .
Organization of ANR and LAR Genes The primer pairs designed to amplify the full-length cDNA of ANR and of the two LAR genes were also used to amplify and clone the corresponding genomic regions from L. corniculatus to gain insight into their organization and structure within the genome. Amplification from the S41 genome using end-to-end PCR with ANR-specific primers produced two products. Alignment of the 2,860-bp-long genomic sequence, named LcANR1, with the longest cDNAs, showed the ANR coding region is organized into six exons and that the ORF of LcANR2 encodes a 302-amino acid polypeptide that lacks the N-terminal part of the second exon with respect to the longest ANR cDNAs (LcANR1). The short ANR genomic fragment, LcANR2, perfectly matched the short ANR cDNA fragment (LcANR3) and was about 540 bp long. In addition to extensive deletion on the 3' end of the first exon, LcANR2 lacked the exons 2, 3, and 4, and the 5' end of the fifth exon, while retaining the sixth exon in its entirety with respect to LcANR1 (Fig. 3A ).
The LAR1-specific primer pair gave rise to amplicons highly divergent in length. Their sequencing and alignment with the respective LcLAR1 cDNA showed that two genomic fragments coded for LAR1, although with differences in terms of both intron structure and length. The shortest gene (about 2,300 bp), named LcLAR1-1, exhibited a single intron. By contrast, in the second LAR1 gene, the coding sequence was split into five exons (Fig. 3B). The microsatellite-like motif in the 5' UTR of both genomic fragments was (TG)4(TGAG)5. A similar approach was employed to isolate and characterize the S41 genomic fragments corresponding to LAR2. The two LAR2 genomic fragments cloned had a quite similar length (about 2,400 bp) and were organized into five exons (Fig. 3C).
To determine the gene copy numbers of LAR1, LAR2, and ANR in this tetraploid species, gene-specific probes were used to hybridize against genomic DNA restricted with XbaI and SacI restriction sites, which our sequence analysis showed were absent from the genes. To discriminate between LAR1 and LAR2 genes, the respective probes were PCR amplified from regions of intron sequences that did not show appreciable sequence homology between these two genes. Southern analysis showed the presence of multiple hybridizing bands for all probes (Supplemental Fig. S2). However, due to the high molecular weight of the hybridizing fragments, it was difficult to discriminate single from comigrant bands and determine the exact number of copies.
Northern analyses were first performed to assess steady-state expression levels for LAR and ANR in different S41 plant organs harvested at late-flowering stages. Given the sequence similarity between the LAR1 and LAR2 genes, probes for differentially labeling the respective mRNAs in northern analysis were sought at the 5' end of both cDNAs, where nucleotide similarities between the two coding sequences were only about 55%. The hybridizing signals from northern blots were faint, especially when the filters were hybridized with the LAR2 probe. A clearer picture emerged when they were probed with ANR, where a transcript of approximately 1,200 bp was detected in all tissues analyzed. Importantly, this analysis demonstrated that the shortest ANR mRNAs, identified through the initial end-to-end reverse transcription (RT)-PCR analyses, were only poorly expressed (data not shown).
Due to the very low expression levels of both LAR1 and LAR2 genes and, to a lesser extent, ANR, we took advantage of sensitivity of the real-time RT-PCR analysis to monitor their expression profiles in the different tissues of S41 plants obtained after botanical fractionation. The LAR1- and LAR2-specific primer pairs were therefore designed against the respective 5' gene ends and the primer specificity was tested against the cloned LAR1 and LAR2 cDNAs. To test for ANR expression by real-time RT-PCR, specific primers were designed against the 5' gene end common to all ANR cDNAs cloned from L. corniculatus. Figure 4
shows the expression profiles of LAR1, LAR2, and ANR within each plant organ using elongation factor-1
Expression Levels of LAR and ANR in Leaves of Wild-Type and Sn Transgenic Lines
Real-time RT-PCR analyses were performed to test the steady-state levels of ANR, LAR1, and LAR2 genes in leaves of either naturally or ectopically induced PA polymorphic genotypes. Wild-type S50 and S41 plants displayed a different pattern of PA accumulation in leaves; the S50 genotype produces much lower leaf PA levels than S41, the clonal genotype of the L. corniculatus line that constitutively accumulates the highest amount of these compounds in leaves (Robbins et al., 2003
Functional Analysis of ANR, LAR1, and LAR2 Genes
The catalytic function of the proteins encoded by LcANR1, LcLAR1-1, LcLAR1-2, LcLAR2-1, and LcLAR2-2 was determined by heterologous expression of the genes in Escherichia coli. Recombinant proteins were incubated with suitable 14C-labeled or unlabeled precursors, here anthocyanindins and leucoanthocyanidins, in the presence of NADPH as an absolutely required cofactor. Incubation of LcANR1 protein with cyanidin (Cy) and NADPH resulted in the formation of a novel product identified as epicatechin by cochromatography with an authentic standard and specific derivatization with dimethylcinnamaldehyde (DMACA) reagent as described in Pfeiffer et al. (2006
Quantification and Localization of PA-Containing Cells
The number and distribution of PA-containing cells was evaluated in leaves of a number of clonal and Sn transgenic lines polymorphic for the level of leaf PAs to assess whether these differences in levels of PA were consistent with a difference in the distribution of tanniferous cells. As shown in Figure 7
, the genetic transformation with Sn resulted in an altered number and distribution of PA-containing cells with respect to the control, untransformed plants. In spongy mesophyll layers, the number of cells accumulating PAs was dramatically increased or decreased and correlated with expression or suppression of the introduced transgene as measured by Robbins et al. (2003)
According to the most recent models, ANR and LAR participate in two separate pathways to PA biosynthesis in most species (Dixon et al., 2005 ). However, the specific contribution of each branch of this pathway to PA biosynthesis in different plant tissues and species is not clear. The isolation and characterization of ANS/ANR and LAR cDNAs from the same species has been reported in grapevine (Bogs et al., 2005) and apple (Malus domestica; Takos et al., 2006); however, whether the two branches of the PA pathway are controlled by the same or independent regulators has not yet been addressed. Here, we report the isolation and characterization of ANR and LAR cDNAs and corresponding genomic fragments from L. corniculatus, a legume species that accumulates epicatechin- and catechin-based PA in both reproductive and vegetative tissues. In addition, by means of real-time RT-PCR analyses on a range of plant lines polymorphic for leaf PA accumulation, we provide clear evidence that ANR and one LAR gene family are subject to coordinate transcriptional regulation mediated by a bHLH transcription factor and that their steady-state levels correlate with levels of leaf PAs.
The body of knowledge on the genetic regulation of late flavonoid biosynthetic genes has been gained mainly from studies on anthocyanin-defective mutants in several species, with the control of flavonoid biosynthesis being regulated principally at the transcriptional level. The model described for MYB-bHLH interaction in the regulation of anthocyanin biosynthesis in maize (Goff et al., 1992
We have previously shown that the ectopic expression of Sn, a maize bHLH gene, increases the steady-state levels of expression of DFR and ANS genes, two gene families committed also to the accumulation of anthocyanins (Paolocci et al., 2005
Whereas Sn is not an ortholog of TT8, overexpression of this EGL3 (group IIIf) ortholog nevertheless appears to enhance the level of leaf PAs as well as anthocyanins in a limited number of tissues (Robbins et al., 2003
The LcANR1 gene, selected as a representative of the ANR gene class most highly expressed in Lotus leaves, encodes a protein that will convert Cy, its expected major substrate, to epicatechin, confirming its biochemical activity as an ANR (Fig. 6). We also show that in L. corniculatus there are two LAR gene families that encode proteins that share 71% identity at the amino acid level and that, within LAR1 and ANR gene families, members are differently arranged in terms of the number and position of their introns, most likely as result of gene duplication events (Fig. 3). The phylogenetic analyses of LAR proteins from different legume species show that LcLAR1 is most similar to the LAR proteins from species accumulating PAs in their leaves, such as L. uliginosus and D. uncinatum. In contrast, the LAR2 sequences cluster with proteins cloned from species that lack appreciable PA biosynthesis in stem, leaf, and root tissues (i.e. M. truncatula and P. coccineus; Fig. 2). Although LcLAR1 and LcLAR2 retain the diagnostic amino acid stretches specific to LAR proteins (Bogs et al., 2005
Whether the LAR and ANR pathways are coordinately regulated by the same class of transcription factors is of importance in understanding the synthesis of PAs. To address these points, we first analyzed the expression profiles of LAR1, LAR2, and ANR genes in different organs of L. corniculatus and then we monitored their expression levels in leaves producing different levels of PAs. Real-time RT-PCR analyses allowed us to detect the transcripts of our target genes in all organs analyzed. High ANR expression levels combined with the observation that L. corniculatus mainly accumulates epicatechin extension units (Foo et al., 1996
Given that all three gene families (i.e. ANR, LAR1, and LAR2) are transcribed in Lotus leaves, we sought to gain information on their genetic control by comparing their relative expression levels in leaves of natural and ectopically induced PA polymorphic L. corniculatus lines. The overexpression and silencing of the maize bHLH gene Sn in the S50 background results in a significant up- and down-regulation, respectively, of leaf PAs (Paolocci et al., 1999
To understand whether the ranges of PA-specific mRNA transcripts and total amounts of leaf PAs correlated with the presence of detectable tannin-accumulating cells, we recorded the number and distribution of PA-containing cells in S41, S50, S50/10 (PA-enhanced), and S50/9 (PA-suppressed) lines. The increase of steady-state levels of ANR and LAR1 mRNAs in S41 and S50/10 was paralleled by an increase in the number of PA cells in palisade and spongy mesophyll tissues when compared with the S50 control line (Fig. 6). Similarly, there were less PA cells in palisade and, most significantly, in spongy mesophyll of the S50/9 line than in S50. In stark contrast, the number of PA cells adjacent to veins remained unaltered in all genotypes. Thus, it is tempting to speculate that Sn activates the expression of both ANR and LAR1 in palisade and spongy mesophyll and that the high levels of PAs in the S41 genotype are due to constitutive expression of a gene encoding a protein functionally equivalent to Sn throughout the leaf blade. The significant reduction of levels of DFR, ANS, ANR, and LAR1 transcripts and PAs in leaves of Sn-suppressed lines (Paolocci et al., 2005 ; this study) suggests that, in these lines, the transgene has caused the silencing of an endogenous bHLH regulator. However, unlike Sn, the bHLH protein endogenous to Lotus may also participate in controlling LAR2 expression in leaves because this target gene was significantly down-regulated in S50 Sn-suppressed lines with respect to the control S50 (Fig. 5). Therefore, these studies on L. corniculatus indicate that it is likely that the accumulation of PAs in leaves is limited by the expression of a bHLH gene.
This model is in contrast to the proposal that TTG1 is the rate-limiting regulatory factor for the expression of phenylpropanoid pathways in Arabidopsis (Zimmermann et al., 2004 The successful engineering of PAs in the leaves of forage plants requires a deep understanding of the network of structural and regulatory genes involved. Here we show the presence of two LAR gene families in L. corniculatus and provide evidence that LAR1 and ANR provide two parallel and coregulated routes for PA biosynthesis in leaves and that this regulation is at least partly under the control of a bHLH transcription factor. Analysis of gene promoters and DNA-binding proteins combined with mutant analysis in the related genetic model L. japonicus will assist future studies relating to the mechanisms that control the expression of PAs in the leaves of forage legumes.
Plant Material
Lotus corniculatus genotypes S50 and S41, isolated from the cultivar Leo and characterized for their different levels of PA accumulation in leaves, have been described previously (Carron et al., 1994
For RNA isolation, the following organs were collected from S41 clonal plants at late-flowering stage: flowers, trifoliate unexpanded leaves, fully expanded leaves, stems, roots, flower pods, and flower pods deprived of the immature seeds (seedless fruits). For RNA isolation from leaves of Sn and GUS transgenic lines in both S50 and S41 genetic backgrounds and from control nontransformed S50 and S41 lines, plants were grown contemporaneously in a glasshouse under identical outdoor conditions and only healthy young (unexpanded) trifoliate leaves were harvested. RNA was isolated using the Nucleo Spin RNA plant isolation kit (Macherey-Nagel) according to the supplier's instructions, after which a further DNase treatment was added. The quality and quantity of RNA was verified by agarose gel electrophoresis and spectrophotometric analysis.
Cloning of ANR Gene Sequences from L. corniculatus
Cloning of LAR Genes from L. corniculatus The forward primers 14FxUTR (5'-agtgcaRtgctgcaaaaccatgg-3') and 5Lar1cl (5'-acYgcataccatacactcagtaa-3') and the reverse primers x3Lar (5'-catcaacatggggtccatatc-3') and x3Larbis (5'-caaccatacatgcatgtggagcatatgatcc-3') were then designed at the extreme 5' and 3' cDNA ends to amplify the full-length LAR cDNAs and the related genes (see "Results"), which then were cloned into the pGeM T-Easy vector.
Full-length LAR cDNA sequences from legume species were retrieved from public databases and two grapevine (Vitis vinifera) LAR sequences (Bogs et al., 2005
About 20 µg of total RNA from a number of different organs of the L. corniculatus S41 genotype were run and blotted on Hybond N membrane (Amersham Biosciences) according to the supplier's instructions. The ANR probe (about 1,200 bp long) was obtained by amplifying the clone containing LcANR1-1 cDNA using the 5' and 3' extreme primer pairs (see above). Similarly, both the LAR1 (about 239 bp) and LAR2 (128 bp) probes were obtained by amplifying the respective cDNAs from plasmids carrying them with the primer pairs 5Lar1cl/lar1qRn (5'-gaccctggccttagaagcagat-3') and 14FwUTR/larQ2Rn (5'-ggcggacRagcaaataggt-3') for the former and the latter gene, respectively. RNA hybridization and washes were performed as reported in Damiani et al. (1998) Final figures from this analysis were then treated in the same manner as quantitative traits and were statistically analyzed accordingly. ANOVA and Duncan tests for mean comparison were performed using the GLM procedure of the SAS program (SAS Institute). Due to the high value of the interactions, tissue x mRNA and genotype x mRNA, only the results of one-way ANOVA are reported.
Isolation, blotting, and Southern hybridization of genomic DNA were performed as in Damiani et al. (1999)
The coding regions of LcANR1-1, LcLAR1-1, LcLAR1-2, LcLANR2-1, and LcLANR2-2 were amplified from the clones harboring the corresponding full-length cDNAs with Pfx50 DNA polymerase (Invitrogen Life Technology) in the presence of the primer pairs exANRF (5'-caccatggcaagcatcaagccaatttatg)/exANRR (5'-ttaattcttcagggccccctttgtcttcaa); exLAR1F (5'-caccatggtgtcaacggcagccact)/exLAR1R (5'-tcagcccatagcagtgattggcgcta); and exLAR2F (5'-caccatggcaaccaagggcagagtcctg)/exLAR2R (5'-tcaacaggaagctgtgattggcactgctt), respectively. The resulting amplicons were cloned into the pET160/GW/D/TOPO vector (Invitrogen) and sequenced to confirm their identity. Expression and protein isolation was done according the manufacturer's instructions (Invitrogen).
Catechin, epicatechin, and Cy were obtained from Roth. [14C]DHQ was synthesized using recombinant CHS, CHI, FHT, and flavonoid 3'-hydroxylase from various plant sources as described (Martens et al., 2003
The coupled DFR/LAR assay (500 µL in total; 0.1 M Tris-HCl buffer, pH 7.5) contained either 14C-labeled (0.03 nmol) or unlabeled DHQ (1 mmol), 25 µL 20 mmol/L NADPH, and recombinant DFR and LAR (approximately 200–500 µg total protein of each determined according to Bradford, 1976
Lotus PA cell counts were carried out on leaves of S41, S50, S50/10, and S50/9 genotypes essentially as described by Robbins et al. (2003) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ349108 to DQ349112 and DQ349114 (ANR cDNAs), DQ349113 and DQ349115 (ANR genes), DQ349100 and DQ349101 (LcLAR1 cDNAs), DQ349102 and DQ349103 (LcLAR1 genes), DQ349104 and DQ349105 (LcLAR2 cDNAs), and DQ349106 and DQ349107 (LcLAR2 genes).
The following materials are available in the online version of this article.
We thank Dr. Andrea Rubini (Plant Genetics Institute) and Professor Phil Morris and colleagues in the Plant Cell Biology Team at the Institute of Grassland and Environmental Research. We also acknowledge Dr. John-Wayne Hughes for access to unpublished data and Dr. Greg Tanner and colleagues (Commonwealth Scientific and Industrial Research Organization, Canberra) and Professor Clint Chapple (Purdue University) for their assistance in the work of the Institute of Grassland and Environmental Research laboratory on PAs in legumes and Arabidopsis, respectively. Received October 6, 2006; accepted November 2, 2006; published November 10, 2006.
1 This work was supported by the Italian Ministry of University and Research (Fondo per gli Investimenti della Ricerca di Base project "Post genomica di leguminose foraggere" code n.RBAU01BKAN to the Plant Genetics Institute) and by the Biotechnology and Biological Science Research Council (to the Institute of Grassland and Environmental Research for work in the area of plant natural products). Travel funding from Consiglio Nazionale della Ricerche to the Institute of Grassland and Environmental Research was supplied by the Consiglio Nazionale delle Ricerche Short-Term Mobility Program. This is contribution number 77 from Plant Genetics Institute-Consiglio Nazionale delle Ricerche.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Francesco Paolocci (francesco.paolocci{at}igv.cnr.it).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.090886 * Corresponding author; e-mail francesco.paolocci{at}igv.cnr.it; fax 39–075–5014869.
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(EF-1
-glucuronidase (GUS), and Sn transgenic lines were grown contemporaneously in a glasshouse and RNA was isolated from young leaves. Because lines S50/9 and S50/6 are suppressed for PA accumulation and lines S50/10 and S50/11 are enhanced in PA accumulation when compared with the host nontransformed or GUS-transformed S50 genotypes (Paolocci et al., 1999
