| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online September 17, 2004; 10.1105/tpc.104.023705 © 2004 American Society of Plant Biologists Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism
a Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B-9052 Ghent, Belgium 1 To whom correspondence should be addressed. E-mail wout.boerjan{at}psb.ugent.be; fax 32-9-3313809.
The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.
The phenylpropanoid compounds derived from the shikimic acid pathway are precursors to diverse classes of phenolics, such as lignin, flavonoids, isoflavonoids, coumarins, and stilbenes. One of the intriguing characteristics of phenylpropanoids is the rapid modification in fluxes and rates of their synthesis and metabolism. This aspect is corroborated by the occurrence of specific phenolics in certain cell types, in response to specific stimuli (light, pathogens, and wounding) or during particular developmental stages.
The end products of the shikimate pathway, the aromatic amino acids Phe, Trp, and Tyr, are generated from the precursors phospho-enol-pyruvate and erythrose-4-phosphate. Phe ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of the phenylpropanoid pathway, removes Phe from aromatic amino acid biosynthesis to generate trans-cinnamic acid and NH3 in a nonoxidative deamination. Therefore, PAL commits the flux of primary metabolites into the phenylpropanoid pathway and becomes rate limiting when reduced below a threshold of 20 to 25% in tobacco (Nicotiana tabacum) (Bate et al., 1994
Given that alterations in the expression of monolignol biosynthesis genes can lead to differential accumulation of pathway intermediates that, in turn, can affect the transcription of other genes (Boerjan et al., 2003 Here, we show that in the absence of phenotypic alterations in the pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur at the transcriptome and metabolome levels in these mutants. Transcript-profiling approaches reveal far-reaching consequences of the disruption of the PAL function on the transcription of genes that encode enzymes of the phenylpropanoid, carbohydrate, and amino acid metabolisms, establishing a clear link between primary and secondary metabolisms. Furthermore, many of these alterations at the transcriptional level are translated into the corresponding biochemical effects as witnessed by targeted metabolite profiling. Our data indicate common as well as discrete functions of PAL1 and PAL2 and demonstrate that a single mutation can profoundly affect the transcription of a plethora of genes, also outside the phenylpropanoid pathway, and the biosynthesis of a variety of metabolites.
Morphological Characterization of the pal1, pal2, and pal1 pal2 Mutants T-DNA insertion mutants for PAL1 and PAL2 were generated by exon-trap mutagenesis in the Arabidopsis ecotype C24 (Babiychuk et al., 1997  ; see Methods). T-DNA insertions resided in the first intron of both genes (data not shown).
Although flavonoid deficiency is known to influence several aspects of plant development, such as lateral root formation, inflorescence growth, and seed viability, the pal1 and pal2 exon-trap mutants had no visible phenotypic alterations. The pal1 pal2 double mutant exhibited no gross morphological differences either, except that it had become sterile. As shown in Figure 1, the siliques remained small (3 to 4 mm versus
Although no growth differences could be seen with respect to time of bolting, height, or diameter of the inflorescence stem, slight differences in lignin accumulation were observed by lignin histochemistry in inflorescence stems of 2.5-month-old plants. As shown in Figure 1, a small reduction in lignin deposition was detected in the pal1 mutant and a more significant reduction in the pal1 pal2 double mutant (Figures 1A to 1L). Cell walls were further investigated at the ultrastructural level by transmission electron microscopy, as presented in Figure 2. The cell walls of xylem and interfascicular fibers were evenly thickened and uniformly stained in the wild type (Figures 2A and 2E). Double mutants were indistinguishable from the wild type in terms of cell size, cell number, or thickness of the cell wall. However, the cell wall of the interfascicular fibers and the large vessels appeared unevenly stained, and cell corners were often whiter (Figures 2B and 2F). In several cells, various signs of a reduced cell wall strength were observed: layers of the cell wall were loosened and had numerous little holes, or cellulose microfibrils became apparent (Figures 2C, 2D, 2G, and 2H). The latter phenomena were never observed in the wild type. At a low frequency, cell walls of the double mutant were aberrantly shaped and irregular (Figure 2B), similar to what has been observed in a more dramatic way in the lignin-deficient irregular xylem 4 Arabidopsis mutants (Jones et al., 2001 ).
In conclusion, pal1 and pal2 mutants have no obvious visible phenotype. Only the combination of both mutations in the double mutant leads to infertility and clear changes in lignin accumulation and secondary cell wall ultrastructure.
Expression of the Four Arabidopsis PAL Genes Is Altered in the Mutants
To estimate the consequences of upregulation of other isoforms at the enzyme level, PAL activity was determined in inflorescence stems of greenhouse-grown plants. As shown in Figure 4, PAL activity in the inflorescence stem was clearly reduced in the pal1 but not in the pal2 mutant. In the double mutant, the 25% residual PAL activity must be derived mainly from PAL4 transcripts (Figures 3 and 4). Additional activity measurements in siliques and leaves of these plants revealed none and a 50% reduced activity in all genotypes, respectively (data not shown). Thus, most clear-cut differences in activity were observed in the inflorescence stem, the tissue where PAL1 and PAL2 are most highly expressed. However, the lesion in PAL2 was almost completely compensated for at the enzyme level. Following the PAL activity during inflorescence growth, PAL activity was highest in 10-week-old, seed-setting but still green plants (Figure 4; data not shown). Assuming that at this age the differences in PAL activity between the wild type and mutants would also be highest, inflorescence stem material of 10-week-old plants was used to conduct all further experiments.
Expression of 10 Genes Encoding Enzymes of Monolignol Biosynthesis Is Strongly Altered in the pal Mutants Our search was extended for consequences of PAL disruption on the transcription of the remaining 30 genes encoding the nine other enzymes of monolignol biosynthesis (Raes et al., 2003 ). Their expression was assayed by RT-PCR with gene-specific primers for each of the genes. Given the rapid alterations in expression levels known to occur for many phenylpropanoid genes and the semiquantitative nature of RT-PCR, a conservative approach of fourfold change was taken to delineate those genes with clear changes in expression. As shown in Figure 5, 23 of the 30 genes were expressed in 10-week-old inflorescence stems. Among the genes that were judged important for the developmental lignification of vascular tissues based on expression, phylogenetic relationship, and occurrence of promoter elements (Raes et al., 2003), pronounced changes in expression were found for 4-coumarate:CoA ligase (4CL1), para-coumarate 3-hydroxylase (C3H1), hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD6). Interestingly, similar trends in alteration of gene expression were observed for most of them in both single pal mutants (Figure 5). Only the expression of C3H1 was higher in the mutants, whereas that of HCT, COMT, and CAD6 was lower (Figure 5). C3H1, hydroxylating the C3 position of p-coumaroyl shikimic/quinic acid, might be very prone to transcriptional regulation by the concentration of certain pathway intermediates. On the other hand, HCT activity is positively regulated by cinnamic acid (Lamb, 1977); hence, the decreased transcription probably correlated with the reduced synthesis of cinnamic acid in the pal mutants. Moreover, the HCT promoter contains both an H and a G box, known to be necessary for feed-forward induction by p-coumaric acid (Loake et al., 1992; Lindsay et al., 2002; Raes et al., 2003). Thus, our data underscore the responsiveness of the HCT gene to regulation by early pathway intermediates.
Caffeoyl-CoA 3-O-methyltransferases CCoAOMT5, CCoAOMT6, and CCoAOMT7, CAD2, and cinnamoyl-CoA reductase (CCR2) with an altered expression in pal mutants had not been selected as candidates for a role in developmental lignification (Figure 5; Raes et al., 2003 ). CCR2, assigned a primary function in pathogen response (Lauvergeat et al., 2001), was also induced in the cellulose-deficient mutants de-etiolated 3, radially swollen 1, and ectopic lignification 1-1 (Caño-Delgado et al., 2003). Thus, CCR2 as well as the others might become subject to transcriptional regulation upon alterations in the concentration of pathway intermediates.
pal Mutants Are Affected in Transcription of Genes Encoding Enzymes of Amino Acid, Phenylpropanoid, and Carbohydrate Metabolisms
The transcriptomes of pal1 and pal2 mutants from the same biological material were also compared with the wild type with a 6K-cDNA microarray. As shown in Table 2, expression significantly changed (twofold at P = 0.001) for 36 genes, of which only three differential genes were common in both mutants. Convincingly, half of these genes (or 61% of the genes with a known function) fell into the same three functional categories (amino acid, phenylpropanoid, and carbohydrates) as in the cDNA-AFLP experiment. Only two genes were found with both methods: a homolog of Asn synthase (ASN3, At3g15450) and a putative protein (At1g73330). The fact that 27 of the 63 tags found in the cDNA-AFLP experiment are present on the microarray suggests that many of these tags were not detected because of cross-hybridization with transcripts of other gene family members or transcript abundance. Clearly, cDNA-AFLP detected more differentially expressed genes involved in signal transduction/gene regulation and unknowns than the microarray (15 versus 3 and 14 versus 5, respectively). This observation underscores the complementarity of both approaches and the higher sensitivity of the cDNA-AFLP method for low-abundant messengers and members of gene families.
Figure 6 shows an interesting trend by clustering all 97 tags according to their expression in the mutants: the largest group of genes linked specifically to the pal1 mutation identifies genes of the phenylpropanoid pathway. Remarkably,  50% of the differentials found in pal1 and pal2 mutants were common to pal1 and pal2, and 27 of 32 of these common differentials were also found in the double mutant (Figure 6). Only one gene (At3g12300, unknown protein; Table 1) required both mutations for a change in expression, altogether corroborating that the differentially expressed genes were linked to the defect in PAL1 and PAL2, and not to stochastic gene expression.
As summarized in Figure 7, 43 and 61% of the differentially expressed genes with a known function identified through cDNA-AFLP and microarray, respectively, were related to amino acid, phenylpropanoid, and carbohydrate metabolisms. With respect to amino acid metabolism, three tags recognized enzymes of the aromatic amino acid biosynthesis and metabolism: chorismate mutase (CM), the first committed step in Phe and Tyr biosynthesis; Trp synthase (TSB), performing the final step in biosynthesis of Trp; and Tyr transaminase (TAT), degrading Phe and Tyr (Tables 1 and 2, Figure 7). Two genes that are homologs of ASN3 are upregulated in pal2 mutants (Tables 1 and 2). Four other tags correspond to genes encoding components of the Gly cleavage complex, primarily involved in photorespiration: two different homologs of Gly dehydrogenase and two DNA binding proteins (At1g69570 and At3g25990) that recognize motifs present in the H protein promoter with the H protein itself being part of the Gly decarboxylase enzyme complex (Tables 1 and 2). In addition, Met synthase, upregulated in the pal2 mutant, represents an interesting tag because it catalyzes the methylation of homocysteine with 5-methyl-tetrahydrofolate as methyl donor. This reaction does not only occur during de novo synthesis but also during recycling of S-adenosyl-L-homocysteine after S-adenosyl-Met (SAM) has donated its methyl group. Besides the incorporation of Met into proteins, 80% of Met is converted into SAM for various transmethylations that are also very frequent in the phenylpropanoid pathway (Figure 7).
Among the phenylpropanoid pathway-related transcripts, PAL1, PAL2, and 4CL1 were expressed in a manner consistent with the results from the RT-PCR experiment (Tables 1 and 2, Figures 3 and 5). The NADPH ferrihemoprotein reductase ATR3, found particularly induced in the pal1 mutant, is probably part of a microsomal P450 system that associates multiple P450 enzymes and NADPH:cytochrome P450 reductases. ATR homologs from poplar (Populus trichocarpa x P. deltoides) and Arabidopsis were shown to efficiently support the activity of the P450 enzyme cinnamate 4-hydroxylase (C4H), when coexpressed in yeast (Urban et al., 1997 ; Mizutani and Ohta, 1998; Ro et al., 2002). Differential tags from a carboxymethylenebutenolidase, dihydroflavonol 4-reductase (DFR), sinapoylglucose:malate sinapoyltransferase (SNG1), a UDP-glucosyl transferase, a glutathione S-transferase (GST), and a glutathione S-conjugate pump (GS-X pump) correspond to enzymes active toward the endpoints of biosynthetic pathways originating from the phenylpropanoid pathway (Figure 7). The UDP-glucosyl transferase belongs to the glycosyltransferase family 1; characterized members use phenylpropanoids, such as flavonol and sinapate, as substrates (http://afmb.cnrs-mrs.fr/CAZY/). The GSTs of the  and  subgroups, to which the downregulated GST also belongs (GSTF12; Dixon et al., 2002), have been implicated in cytoplasmic stabilization of flavonoids and flavonoid conjugation before uptake into the vacuole (Mueller et al., 2000; Dixon et al., 2002). The ABC transporter (AtMRP2) identified here encodes a functional GS-X pump of the plant vacuolar membrane with a high capacity for glutathionated anthocyanins (Lu et al., 1998; Liu et al., 2001; Sánchez-Fernández et al., 2001).
Perturbations in the PAL function seem to lead to adjustments in carbon fixation and carbohydrate metabolism (Tables 1 and 2, Figure 7). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is upregulated in pal1 and pal2 mutants, and Rubisco activase is upregulated in the pal1 mutant. Both are central parts of CO2 fixation into organic carbon (Table 2, Figure 7). Enzymes, such as phosphoglycerate kinase and triose phosphate isomerase, that metabolize the phosphoglycerate generated by Rubisco, are also upregulated in pal2 mutants (Tables 1 and 2, Figure 7). SNF4 (Akin
Two smaller groups of tags relate to light signal transduction and stress responses, two processes in which the importance of flavonoids is well documented. Light-related transcripts are found preferentially in the pal2 mutant. SPA1 and FAR are both specific signaling intermediates of phytochrome A signal transduction (Hoecker et al., 1999
pal1 pal2 Double Mutants Lack Three Major Flavonol Glycosides
When the soluble phenolic compounds in the inflorescence stem of the three mutants and the wild type were compared by reverse-phase HPLC, significant changes in the profile were noticed. As shown in the maxplots presented in Figure 8, reduced PAL activity led to a decreased accumulation of several phenolics. Out of 89 peaks in the analyzed chromatograms, eight peaks differed significantly in abundance between the wild type and pal mutants. As shown in Table 3, total phenolics were significantly diminished to approximately one-third of the wild-type amount in the pal1 pal2 double mutants, slightly reduced to 75% in pal1 mutants, and not affected in the pal2 mutant. This decrease in total phenolics correlates with the levels of PAL activity (Figure 4) and is mainly attributable to the depletion of the three major peaks denoted as A, B, and C in Figure 8. As shown in Figure 9A, these peaks correspond, on the basis of their retention time (RT), photodiode array, and mass spectrometry (MS) spectra, to previously identified kaempferol glycosides (Veit and Pauli, 1999
Feruloyl Malate Esters Coupled to Coniferyl Alcohol Are Less Abundant in pal1 and pal1 pal2 Double Mutants Besides the three major UV light–absorbing kaempferol glycosides, five other peaks were significantly altered in the mutants, one of which occurred only in the double mutant (RT 8.350 min; not identified, Table 3). The compound at RT 6.033 min (Figure 8) was identified as glycosylated vanillic acid by RT, photodiode array spectrum, and comparison with an authentic standard (Table 3).
The remaining three peaks were significantly reduced in pal1 and the double mutant, but not in the pal2 mutant (RT 7.331 min, 11.178 min, and 15.119 min; Table 3). MS analysis of the compound eluting at 7.331 min indicated that this compound is scopolin or isoscopolin (Figure 9B). The peak was identified as scopolin because its aglycon released after hydrolysis consisted exclusively of scopoletin (RT 11.37 min) and not of isoscopoletin (RT 10.78 min). Scopolin had been detected previously in tobacco leaf extracts (Baumert et al., 2001
The compound eluting at 11.178 min has a UV/VIS spectrum characterized by absorption maxima at 235 and 329 nm. MS analysis suggested that this peak corresponds to the 4–O–8-cross-coupling product of feruloyl malate and coniferyl alcohol, FM(4–O–8)G, or 3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-1-hydroxymethyl-ethoxy]-3-methoxy-phenyl}-acrylic acid (Figure 9C). The core structure FA(4–O–8)G (FA, ferulic acid) was authenticated using previously isolated FA(4–O–8)G from ryegrass (Lolium perenne) (Ralph et al., 1992 In summary, only downregulation of both PAL1 and PAL2 leads to significant changes in the accumulation of the major soluble phenolic compounds, the kaempferol glycosides (Table 3). On the other hand, the reduced accumulation of compounds produced by other side branches of phenylpropanoid metabolism, such as scopolin (hydroxycoumarin), and the feruloyl malates coupled to coniferyl alcohol were clearly linked with the mutation in PAL1 (Table 3, Figure 9). Sinapate esters were notably unaffected in all mutants, indicating that either this pathway is essential or that the enzymes producing sinapate esters are in metabolic complexes with PAL isoforms other than PAL1 and PAL2. This observation suggests that PAL1 and PAL2 are either associated with different cell types or with different metabolic complexes within the cell or that PAL1 has a higher activity than PAL2.
pal1 pal2 Mutants Overaccumulate Phe and Are Perturbed in Other Nonaromatic Amino Acids
Another affected amino acid was Met that was significantly increased in the pal2 mutant but decreased to 6% of the wild-type level in the double mutant (Table 4). A transcript corresponding to Met synthase was exclusively upregulated in the pal2 mutant (Table 1). Finally, and noteworthy, Gly was only detected in the wild type (Table 4). Four transcripts related to Gly degradation were exclusively expressed or upregulated in the pal mutants (Tables 1 and 2). By contrast, Asn was only detected in the double mutant (Table 4). Correspondingly transcripts encoding putative proteins with strong similarity to ASN were upregulated in pal2 and pal1 pal2 mutants (Tables 1 and 2).
In conclusion, blocking PAL led to a strong overaccumulation of Phe and imbalanced the abundance of many other amino acids. Quite surprisingly, 15 out of 20 amino acids changed significantly in at least one genotype (Table 4). The fact that the concentration of these amino acids increases, except for Met in the double mutant and Cys in pal1, suggests an overarching mechanism for homeostasis of amino acid levels that acts across individual biosynthetic pathways. Similarly, plants overexpressing a Trp or Tyr decarboxylase, thus artificially creating a metabolic sink, were altered in the aromatic and three nonaromatic amino acids (Yao et al., 1995
pal1 pal2 Mutants Have Less Lignin with a Higher Syringyl/Guaiacyl Ratio
The determination of lignin content by acetyl bromide leads to an underestimation of the amount of monomers linked by ester bonds. Therefore, isolated cell wall material was saponified and analyzed by HPLC. The HPLC profiles of the wild type and double mutant were virtually identical (data not shown), indicating that the amount of esters incorporated into the cell wall was not significantly changed.
Molecular Phenotyping Is Informative Genome-wide studies of the transcriptome allow explaining a mutant phenotype at the molecular level at a depth that was not possible before the development of genomic tools. The extension of the phenotypic description to the molecular level increases the resolution so that functional redundancy of closely related gene family members can be separated. Thus, these tools are excellent to provide an insight into the functional significance of individual gene family members. Furthermore, our study underscores that the disruption of a single gene can have wide-ranging consequences on the transcription of other genes, revealing the intricacy of interactions among metabolic pathways, even in the absence of a visible phenotype (Tables 1 and 2). Elements of most of the pathways, altered in pal mutants, have been previously described for their responsiveness to pathogen attack or elicitation (Figure 7; Somssich and Hahlbrock, 1998 ). However, in contrast with transcriptome profiling after challenging the plant, typically designed to reconstruct a pathway response to a particular agent or environmental stress, our aim was to describe the adaptation of the transcriptome to genetic differences at a particular locus. In a similar study on the brevipedicellus mutant that is disrupted in a KNOX gene and displays premature lignin deposition, expression differed in 12 genes related to lignification or cell wall biosynthesis (Mele et al., 2003). Among these are 4CL1, PAL1, TAT, and cellulose synthase, genes that are also differentially expressed in the pal mutants (Tables 1 and 2). Moreover, ASN, differentially expressed in the brevipedicellus and pal2 mutant, is known to respond dramatically at the transcriptional level to metabolites, light, and development (Lam et al., 1998). Thus, our study might have identified genes that are under metabolic regulation, as opposed to genes involved in a pathway's response to environmental or developmental signals. Altogether, the pal1 and pal2 mutations cause 58 and 72 genes, respectively, to change in expression within our experiment and degree of transcriptome resolution (20% with the microarray and 60% with the cDNA-AFLP; Figure 6). In the pal mutants, the expression of several differentially expressed genes depends probably on the level of one or more biochemical compounds generated or consumed by PAL, such as Phe and cinnamic acid or their upstream and downstream derivatives. However, among the differentially expressed genes, there will also be genes that are expressed as a result of secondary effects of altered cellular processes.
A Reduced Carbon Flux into the Phenylpropanoid Pathway Affects the Homeostasis of Amino Acid and Carbohydrate Metabolism Assuming that most transcriptomic changes concern the same few cell types, the three major downstream branches of the phenylpropanoid pathway (flavonoids, lignins, and sinapate esters) undergo a different adaptation to the reduced flux into the pathway in the mutants: the production of flavonoids and lignin is clearly reduced. Additionally, hydroxycoumarins, glycosylated vanillic acid, and FM(4–O–8)G and FM(5–8)G are reduced in abundance (Table 3), whereas sinapate esters remain unchanged. Because all flavonoids, sinapate esters, and hydroxycoumarins have UV light-protecting capacities, the sinapate esters remain probably produced because they are most effective in UV light shielding. Alternatively, sinapate esters could be derived from a metabolic complex involving a PAL isoform other than PAL1 or PAL2.
As summarized in Figure 7, signs of molecular adjustments are not only found in the phenylpropanoid pathway but also in the shikimate pathway, in the related cell wall biosynthesis, in components of central metabolism such as glycolysis, and even in distant stress-related pathways. In the transcriptome data (Figures 3 and 5, Tables 1 and 2), the complete sequence of the early phenylpropanoid biosynthesis is reconstructed, except for C4H (Figure 7). Similarly, in PAL-suppressed tobacco plants, C4H expression remains unchanged (Blount et al., 2000
With respect to the shikimate pathway generating the aromatic amino acids, transcripts for the enzyme at the branch point (CM) and two enzymes at the respective end points (TSB and TAT) were differentially expressed (Figure 7). All three hold a great potential for exerting control over the flux in this pathway. Phe, accumulating to 100-fold in the double mutant, and Trp, accumulating to fourfold in the double mutant, exert negative and positive feedback control on CM, respectively, and Trp also negatively regulates anthranilate synthase in its own biosynthesis (Bentley, 1990
Clearly, many changes also occur in the primary metabolism of the pal mutants (Figure 7). Several studies reveal the reverse, namely, consequences of downregulation or disruption of enzyme functions in primary metabolism for secondary metabolism. For example, downregulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (shikimate pathway) or plastid transketolase (TK, Figure 7) leads to a decrease in aromatic amino acids, phenylpropanoids, and lignin (Jones et al., 1995
Reduction in Lignin Is Not Compensated for by Cellulose in pal Mutants
A Role in Cross-Linking for the Novel Coupling Products of Feruloyl Malate and Coniferyl Alcohol?
The Molecular Phenotypes of pal1 and pal2 Mutants Reveal Distinct Roles for PAL1 and PAL2 in Phenylpropanoid Production pal1 and pal2 mutants are indistinguishable at the phenotypic level, and only the double mutant has slight phenotypic changes together with a clear reduction in kaempferol glycosides and lignin (Figures 1 and 2, Tables 3 and 5). Considering the 34 genes that code for the enzymes of monolignol biosynthesis, similar trends in alteration of gene expression were observed for most of them in both single pal mutants (e.g., HCT, CCoAOMT5, COMT, and CAD6, Figure 5). Although this observation argues for a functional overlap of PAL1 and PAL2, the fact that expression levels of these four genes were closer to the wild-type level in the double mutant than in the single mutants corroborates a more complex metabolic interaction (Figure 5). On the other hand, expression of several genes was only significantly increased in the double mutant (4CL1 and CCoAOMT6; Figure 5), arguing for a synergistic action of PAL1 and PAL2 in the generation of particular phenylpropanoids. Still, the differential expression of several genes is correlated with only one of the two mutations: PAL3, C3H1, and CCoAOMT7 with pal2, and CCR2 with pal1 (Figures 3 and 5). Although it is too early to speculate on how this transcriptional regulation corresponds to the differential accumulation of a particular metabolite or with particular metabolic complexes, these transcriptional differences clearly indicate directions toward the specific roles of these two PAL isoforms. When the altered transcripts of other pathways in either pal1 or pal2 (and the double mutant) are compared, more tags relate to light, UV light protection, and stress in pal2 than in pal1 mutants (Figures 5, 6, and 7). On the other hand, pal1 mutants are more altered in phenylpropanoid-related transcripts (PAL1, PAL2, ATR3, 4CL1, DFR, CHS, GST, GS-X pump, and SNG1, Figure 7). In accordance with these transcriptomic changes, the pal1, but not the pal2, mutant has reduced total extractable phenolics because of a decrease in sugar-conjugated kaempferols, scopolin, FM(4–O–8)G, and FM(5–8)G (Table 3). The difference in aromatic carbon flux seems to translate in different ways to carbon flux in both mutants. The lack of C6-C3 carbon probably signals in both mutants a demand for more carbon; transcripts correlated with photosynthetic carbon fixation and with sucrose degradation are upregulated in both mutants (e.g., Rubisco and invertase; Figure 7). However, based on the transcriptomic changes that exclusively occur in pal1 mutants, the excess carbon is stored away as starch in pal1 mutants (starch synthase: up, starch phosphorylase; involved in starch degradation: down; Figure 7). On the other hand, in pal2 mutants, carbon is rather redirected toward other processes; transcripts related to glycolysis and gluconeogenesis (e.g., trehalose 6P-synthase, triose-P isomerase, phosphoglycerate kinase: up; Figure 7). Possibly part of this carbon is used to generate more amino acids because the levels of many amino acids are higher in pal2 than in pal1 (Table 4). The understanding of the transcriptome and biochemistry provide novel insight into the functional differences of the two PAL isoforms. On the basis of these observations, a PAL1 knock down has more severe consequences for the phenylpropanoid pathway than a PAL2 knock down. Whereas both enzymes contribute to the production of lignin precursors, PAL1 is of higher importance for the generation of flavonoids in the inflorescence stem. To elaborate on the specific functions of the PAL isoforms in Arabidopsis, future experiments need to address the function of the two other PALs and the potentially different localization of all PALs at the cellular and tissue levels.
The application of a similar molecular phenotyping strategy to other mutants in the 34 genes that encode the 10 known enzymes in the monolignol pathway (Raes et al., 2003
Plant Growth Conditions For germination, seeds were surface sterilized and placed on MS medium (Duchefa, Haarlem, The Netherlands) supplemented with 10 g L–1 sucrose (and if required with 50 mg L–1 kanamycin). After the seeds had undergone a cold treatment for homogenous germination (overnight at 4°C), they were exposed to 20°C, 50 µmol m–2 s–1 light intensity, and 70% humidity under a 16-h-light/8-h-dark cycle. Plants were transferred to a greenhouse after 14 d. Conditions were as follows: 50 µmol m–2 s–1 light intensity at plant level (MBFR/U 400-W incandescent lamps; Philips, Eindhoven, The Netherlands), a 16-h-light/8-h-dark cycle, 40% relative humidity, 23°C, and without shielding from incident daylight. Plant material was always harvested between 11 AM and 1 PM.
Generation of the pal Mutants and Double Mutant and Identification of the Genotypes The pal1 pal2 double mutant was generated by crossing pal1 and pal2, using either as female and male parent. In the F2 progeny of both crosses, sterile double mutants were identified in the expected Mendelian ratios. Therefore, the double mutant had to be identified for each experiment in the progeny of plants homozygous for one but heterozygous for the other mutation. To identify the wild-type and mutated PAL1 and PAL2 genes, the following primers were used as sense and antisense primers, respectively: for pal1, 5'-ATGGAGATTAACGGGGCACAC-3' and 5'-GCATCAGAGCAGCCGATTGTCTGTT-3'; for PAL1, 5'-ATGGAGATTAACGGGGCACAC-3' and 5'-CATGGCGGCTCTTGTGGCGG-3'; for pal2, 5'-ATGGATCAAATCGAAGCAATG-3' and 5'-GCATCAGAGCAGCCGATTGTCTGTT-3'; for PAL2, 5'-ATGGATCAAATCGAAGCAATG-3' and 5'-CATGGCGGCTCTTGTGGCGG-3'. PCR reactions (reaction buffer as supplied with the Taq polymerase, deoxynucleotide triphosphate at 200 pmol, primers at 100 ng) were performed as follows: 30 cycles at 94°C for 30 s, at 50°C for 30 s, at 72°C for 2 min, and a final extension step at 72°C for 10 min. PCR products were separated in 1% agarose gels.
DNA and RNA Extractions
RT-PCRs In RT-PCR experiments, reactions in 25 µL (reaction buffer as supplied with the Taq polymerase, 50 ng of each primer) contained a modified nucleotide mix: dCTP, dTTP, and dGTP were at 200 pmol, whereas dATP was reduced to 20 pmol. To each reaction, 0.1 µL of 33P-labeled dATP (10 mCi mL–1, 2500 Ci mmol–1) was added, resulting in a hot-to-cold dATP ratio of 1:2500. Products were separated on 4.5% polyacrylamide or 1% agarose gels and visualized on dried gels through autoradiography. To assure at least semiquantitative assays, the linear range of the PCR reaction was determined for each gene by testing at least two template concentrations (1 µL 1:10 diluted cDNA and 1 µL undiluted cDNA) and three different PCR reaction cycles (21, 24, and 30).
Transcript Profiling and Handling of Differentially Expressed cDNA Fragments
Microarray Hybridization
Microarray Data Analysis
Determination of PAL Activity
Light Microscopy and Lignin Histochemistry
Transmission Electron Microscopy
Analysis of Soluble Phenolics and Amino Acids Liquid chromatography–mass spectrometry analysis of the water phase was performed using a gradient separation from 83% solvent A (aqueous, 1% acetic acid) to 77% solvent B (acetonitrile, 1% acetic acid) in 21 min on a reverse-phase Luna C18 column (150 x 2.1 mm, 3 µm; Phenomenex, Torrance, CA) at a flow of 0.30 mL min–1 (SpectraSystem P1000XR HPLC pump; Thermo Separation Products, Riviera Beach, FL) and a column temperature of 40°C (SpectraSystem AS1000 autosampler; Thermo Separation Products). UV/VIS absorption spectra were taken between 200 and 450 nm on a SpectraSystem UV6000LP detector (Thermo Separation Products). Full MS spectra were obtained in the positive or negative ionization mode on a LCQ Classic MS instrument (ThermoQuest, San Jose, CA), using atmospheric pressure chemical ionization as ion source. Vaporizer temperature, capillary temperature, sheath gas, aux gas, and the source current were set at 450°C, 150°C, 64, 3, and 5 µA, or at 400°C, 150°C, 23, 6, and 5 µA in the positive or negative ionization mode, respectively. MSn spectra were obtained by single reaction monitoring using a collision energy of 35%. The methanol extracts were also used for amino acid analysis. Of the extracts, 5 µL was directly applied to an amino acid analyzer (420A derivatizer with an online 130A separation system; Applied Biosystems, Foster City, CA). Amino acids were identified by cochromatography with authentic standards. Peak height/dry weight were analyzed with one-way ANOVA, with P = 0.05 for the F test and the Dunnett's post-hoc test.
The lyophilized scopolin peak and synthetic scopolin were subjected to acid hydrolysis (1 N HCl, 3 h at 90°C). The reaction products were resolved on HPLC, as described above, and authenticated with standards of scopoletin and isoscopoletin. Synthetic compounds for authentication were vanillate (Acros, Geel, Belgium), scopoletin (Sigma-Aldrich, St. Louis, MO), isoscopoletin (Fluka, Buchs, Switzerland), and FA(4-O-8)G (Ralph et al., 1992
Substrate Feeding Assays and Thin-Layer Chromatography of Sinapate Esters
Acetyl Bromide Soluble Lignin Determination and DFRC Analysis of Lignin
Cell Wall Isolation and Saponification
Carbohydrate Analysis
Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers
We thank Paul Van Hummelen for microarray hybridization, Hoon Kim (U.S. Dairy Forage Research Center) for synthesizing scopolin and FA(8–5)G, Debbie Bishop and Fachuang Lu (U.S. Dairy Forage Research Center) for the ABSL and DFRC analyses, and Martine De Cock for help in preparing the manuscript. This research was funded in part by the European Commission program EDEN (QLK5-CT-2001-00443) and the Department of Energy Biosciences program (DE-AI02-00ER15067 to J.R.). A.R. is a postdoctoral fellow of the Fund for Scientific Research, Flanders.
The authors 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.plantcell.org) are: Antje Rohde (antje.rohde{at}psb.ugent.be) and Wout Boerjan (wout.boerjan{at}psb.ugent.be). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.023705. Received April 28, 2004; accepted July 14, 2004.
Allwood, E.G., Davies, D.R., Gerrish, C., Ellis, B.E., and Bolwell, G.P. (1999). Phosphorylation of phenylalanine ammonia-lyase: Evidence for a novel protein kinase and identification of the phosphorylated residue. FEBS Lett. 457, 47–52.[CrossRef][Web of Science][Medline]
Anterola, A.M., Jeon, J.-H., Davin, L.B., and Lewis, N.G. (2002). Transcriptional control of monolignol biosynthesis in Pinus taeda: Factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism. J. Biol. Chem. 277, 18272–18280.
Babiychuk, E., Fuangthong, M., Van Montagu, M., Inzé, D., and Kushnir, S. (1997). Efficient gene tagging in Arabidopsis thaliana using a gene trap approach. Proc. Natl. Acad. Sci. USA 94, 12722–12727.
Bate, N.J., Orr, J., Ni, W., Meromi, A., Nadler-Hassar, T., Doerner, P.W., Dixon, R.A., Lamb, C.J., and Elkind, Y. (1994). Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis. Proc. Natl. Acad. Sci. USA 91, 7608–7612. Baumert, A., Mock, H.-P., Schmidt, J., Herbers, K., Sonnewald, U., and Strack, D. (2001). Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase. Phytochemistry 56, 535–541.[CrossRef][Web of Science][Medline] Bentley, R. (1990). The shikimate pathway–A metabolic tree with many branches. Crit. Rev. Biochem. Mol. Biol. 25, 307–384.[Web of Science][Medline]
Blount, J.W., Korth, K.L., Masoud, S.A., Rasmussen, S., Lamb, C., and Dixon, R.A. (2000). Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant Physiol. 122, 107–116. Boerjan, W., Ralph, J., and Baucher, M. (2003). Lignin biosynthesis. Annu. Rev. Plant Biol. 54, 519–546.[CrossRef][Medline] Bolwell, G.P., Cramer, C.L., Lamb, C.J., Schuch, W., and Dixon, R.A. (1986). L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Modulation of the levels of active enzyme by trans-cinnamic acid. Planta 169, 97–107.
Bouly, J.-P., Gissot, L., Lessard, P., Kreis, M., and Thomas, M. (1999). Arabidopsis thaliana proteins related to the yeast SIP and SNF4 interact with AKIN
Breyne, P., Dreesen, R., Vandepoele, K., De Veylder, L., Van Breusegem, F., Callewaert, L., Rombauts, S., Raes, J., Cannoot, B., Engler, G., Inzé, D., and Zabeau, M. (2002). Transcriptome analysis during cell division in plants. Proc. Natl. Acad. Sci. USA 99, 14825–14830. Bunzel, M., Ralph, J., Kim, H., Hatfield, R.D., and Steinhart, H. (2004). Are cereal grains lignified? J. Agric. Food Chem., in press. Caño-Delgado, A., Penfield, S., Smith, C., Catley, M., and Bevan, M. (2003). Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana. Plant J. 34, 351–362.[CrossRef][Web of Science][Medline] Caño-Delgado, A.I., Metzlaff, K., and Bevan, M.W. (2000). The eli1 mutation reveals a link between cell expansion and secondary cell wall formation in Arabidopsis thaliana. Development 127, 3395–3405.[Abstract] Chambat, G., Cartier, N., Lefèbvre, A., Marais, M.-F., and Joseleau, J.-P. (1997). Changes in cell wall extracellular polysaccharides during the culture cycle of Rubus fruticosus cells in suspension culture. Plant Physiol. Biochem. 35, 655–664. Chaudhury, D.N., Holland, R.A., and Robertson, A. (1948). The synthesis of glycosides. Part XII. Fabiatrin. J. Chem. Soc. 1948, 1671–1672. Cheng, S.-H., Sheen, J., Gerrish, C., and Bowlell, G.P. (2001). Molecular identification of phenylalanine ammonia-lyase as a substrate of a specific constitutively active Arabidopsis CDPK expressed in maize protoplasts. FEBS Lett. 503, 185–188.[CrossRef][Web of Science][Medline] Dixon, D.P., Lapthorn, A., and Edwards, R. (2002). Plant glutathione transferases. Genome Biol. 3, 2004.1–2004.10. Eastmond, P.J., and Graham, I.A. (2003). Trehalose metabolism: A regulatory role for trehalose-6-phosphate? Curr. Opin. Plant Biol. 6, 231–235.[CrossRef][Web of Science][Medline]
Edwards, K., Johnstone, C., and Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19, 1349.
Elkind, Y., Edwards, R., Mavandad, M., Hedrick, S.A., Ribak, O., Dixon, R.A., and Lamb, C.J. (1990). Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene. Proc. Natl. Acad. Sci. USA 87, 9057–9061.
Ellis, C., Karafyllidis, I., Wasternack, C., and Turner, J.G. (2002). The Arabidopsis mutant cev1 links well wall signaling to jasmonate and ethylene responses. Plant Cell 14, 1557–1566.
Frishman, D., et al. (2003). The PEDANT genome database. Nucleic Acids Res. 31, 207–211. Fry, S.C. (2004). Primary cell wall metabolism: Tracking the careers of wall polymers in living plant cells. New Phytol. 161, 641–675.[CrossRef][Web of Science] Fukushima, R.S., and Hatfield, R.D. (2001). Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method. J. Agric. Food Chem. 49, 3133–3139.[CrossRef][Web of Science][Medline] Grabber, J.H., Ralph, J., and Hatfield, R.D. (2002). Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: Implications for lignification in grasses. J. Agric. Food Chem. 50, 6008–6016.[CrossRef][Web of Science][Medline] Graham, T.L. (1998). Flavonoid and flavonol glycoside metabolism in Arabidopsis. Plant Physiol. Biochem. 36, 135–144.
Guillet, G., Poupart, J., Basurco, J., and De Luca, V. (2000). Expression of tryptophan decarboxylase and tyrosine decarboxylase genes in tobacco results in altered biochemical and physiological phenotypes. Plant Physiol. 122, 933–943. Hatfield, R.D., Ralph, J., and Grabber, J.H. (1999). Cell wall cross-linking by ferulates and diferulates in grasses. J. Sci. Food Agric. 79, 403–407.[CrossRef]
Henkes, S., Sonnewald, U., Badur, R., Flachmann, R., and Stitt, M. (2001). A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell 13, 535–551. Herrmann, K.M. (1995). The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. Plant Cell 7, 907–919.[CrossRef][Web of Science][Medline]
Hoecker, U., Tepperman, J.M., and Quail, P.H. (1999). SPA1, a WD-repeat protein specific to phytochrome A signal transduction. Science 284, 496–499. Howles, P.A., Sewalt, V.J.H., Paiva, N.L., Elkind, Y., Bate, N.J., Lamb, C., and Dixon, R.A. (1996). Overexpression of L-phenylalanine ammonia-lyase in transgenic tobacco plants reveals control points for flux into phenylpropanoid biosynthesis. Plant Physiol. 112, 1617–1624.[Abstract] Hu, W.-J., Harding, S.A., Lung, J., Popko, J.L., Ralph, J., Stokke, D.D., Tsai, C.-J., and Chiang, V.L. (1999). Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat. Biotechnol. 17, 808–812.[CrossRef][Web of Science][Medline]
Hudson, M., Ringli, C., Boylan, M.T., and Quail, P.H. (1999). The FAR1 locus encodes a novel nuclear protein specific to phytochrome A signaling. Genes Dev. 13, 2017–2027. Jacquet, G., Pollet, B., Lapierre, C., Mhamdi, F., and Rolando, C. (1995). New ether-linked ferulic acid-coniferyl alcohol dimers identified in grass straws. J. Agric. Food Chem. 43, 2746–2751. Jones, J.D., Henstrand, J.M., Handa, A.K., Herrmann, K.M., and Weller, S.C. (1995). Impaired wound induction of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and altered stem development in transgenic potato plants expressing a DAHP synthase antisense construct. Plant Physiol. 108, 1413–1421.[Abstract] Jones, L., Ennos, A.R., and Turner, S.R. (2001). Cloning and characterization of irregular xylem4 (irx4): A severely lignin-deficient mutant of Arabidopsis. Plant J. 26, 205–216.[CrossRef][Web of Science][Medline]
Kao, Y.-Y., Harding, S.A., and Tsai, C.-J. (2002). Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifiying cells of quakin aspen. Plant Physiol. 130, 796–807. Korth, K.L., Blount, J.W., Chen, F., Rasmussen, S., Lamb, C., and Dixon, R.A. (2001). Changes in phenylpropanoid metabolites associated with homology-dependent silencing of phenylalanine ammonia-lyase and its somatic reversion in tobacco. Physiol. Plant. 111, 137–143.[CrossRef] Lam, H.-M., Hsieh, M.-H., and Coruzzi, G. (1998). Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana. Plant J. 16, 345–353.[CrossRef][Web of Science][Medline] Lamb, C. (1977). trans-cinnamic acid as a mediator of the light-stimulated increase in hydroxycinnamoyl:CoA-quinate hydroxycinnamoyl transferase. FEBS Lett. 75, 37–40.[Medline] Landry, L.G., Chapple, C.C.S., and Last, R.L. (1995). Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiol. 109, 1159–1166.[Abstract] Lauvergeat, V., Lacomme, C., Lacombe, E., Lasserre, E., Roby, D., and Grima-Pettenati, J. (2001). Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry 57, 1187–1195.[CrossRef][Web of Science][Medline] Legrand, M., Fritig, B., and Hirth, L. (1976). Enzymes of the phenylpropanoid pathway and the necrotic reaction of hypersensitive tobacco to tobacco mosaic virus. Phytochemistry 15, 1353–1359.[CrossRef]
Lehfeldt, C., Shirley, A.M., Meyer, K., Ruegger, M.O., Cusumano, J.C., Viitanen, P.V., Strack, D., and Chapple, C. (2000). Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12, 1295–1306. Leyva, A., Jarillo, J.A., Salinas, J., and Martinez-Zapater, J.M. (1995). Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. Plant Physiol. 108, 39–46.[Abstract]
Li, L., Zhou, Y., Cheng, X., Sun, J., Marita, J.M., Ralph, J., and Chiang, V.L. (2003). Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. USA 100, 4939–4944. Lindsay, W.P., McAlister, F.M., Zhu, Q., He, X.-Z., Dröge-Laser, W., Hedrick, S., Doerner, P., Lamb, C., and Dixon, R.A. (2002). KAP-2, a protein that binds to the H-box in a bean chalcone synthase promoter, is a novel plant transcription factor with sequence identity to the large subunit of human Ku autoantigen. Plant Mol. Biol. 49, 503–514.[CrossRef][Web of Science][Medline]
Liu, G., Sánchez-Fernández, R., Li, Z.-S., and Rea, P.A. (2001). Enhanced multispecificity of Arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2. J. Biol. Chem. 276, 8648–8656. Liu, H., Orjala, J., Sticher, O., and Rali, T. (1999). Acylated flavonol glycosides from leaves of Stenochlaena palustris. J. Nat. Prod. 62, 70–75.[Medline]
Loake, G.J., Faktor, O., Lamb, C.J., and Dixon, R.A. (1992). Combination of H-box [CCTACC(N)7CT] and G-box [CACGTG] cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid. Proc. Natl. Acad. Sci. USA 89, 9230–9234. Lu, F., and Ralph, J. (1997). Derivatization followed by reductive cleavage (DFRC method), a new method for lignin analysis: Protocol for analysis of DFRC monomers. J. Agric. Food Chem. 45, 2590–2592.[CrossRef]
Lu, Y.-P., Li, Z.-S., Drozdowicz, Y.M., Hörtensteiner, S., Martinoia, E., and Rea, P.A. (1998). AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: Functional comparisons with AtMRP1. Plant Cell 10, 267–282. Matt, P., Krapp, A., Haake, V., Mock, H.-P., and Stitt, M. (2002). Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants. Plant J. 30, 663–677.[CrossRef][Web of Science][Medline]
Mavandad, M., Edwards, R., Liang, X., Lamb, C.J., and Dixon, R.A. (1990). Effects of trans-cinnamic acid on expression of the bean phenylalanine ammonia-lyase gene family. Plant Physiol. 94, 671–680.
Mele, G., Ori, N., Sato, Y., and Hake, S. (2003). The knotted1-like homeobox gene BREVIPEDICELLUS regulates cell differentiation by modulating metabolic pathways. Genes Dev. 17, 2088–2093.
Meyermans, H., et al. (2000). Modification in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis. J. Biol. Chem. 275, 36899–36909.
Mizutani, M., and Ohta, D. (1998). Two isoforms of NADPH:cytochrome P450 reductase in Arabidopsis thaliana. Gene structure, heterologous expression in insect cells, and differential regulation. Plant Physiol. 116, 357–367.
Mueller, L.A., Goodman, C.D., Silday, R.A., and Walbot, V. (2000). AN9, a petunia glutathione S-transferase required for anthocyanin sequestration, is a flavonoid-binding protein. Plant Physiol. 123, 1561–1570. Payne, R.W., and Arnold, G.M. (2002). GenStat Release 6.1 Reference Manual Part 3: Procedure Library PL14. (Hemel Hempstead, UK: VSN International).
Raes, J., Rohde, A., Christensen, J.H., Van de Peer, Y., and Boerjan, W. (2003). Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol. 133, 1051–1071. Ralph, J., Hatfield, R.D., Grabber, J.H., Jung, H.-J. G., Quideau, S., and Helm, R.F. (1999). Cell wall cross-linking in grasses by ferulates and diferulates. In Lignin and Lignan Biosynthesis, ACS Symposium Series, Vol. 697, N.G. Lewis and S. Sarkanen, eds (Washington, D.C.: American Chemical Society), pp. 209–236. Ralph, J., Helm, R.F., Quideau, S., and Hatfield, R.D. (1992). Lignin–feruloyl ester cross-links in grasses. Part 1. Incorporation of feruloyl esters into coniferyl alcohol dehydrogenation polymers. J. Chem. Soc. Perkin Trans. 1 1, 2961–2969.[CrossRef] Ralph, J., and Lu, F. (1998). The DFRC method for lignin analysis. 6. A simple modification for identifying natural acetates on lignins. J. Agric. Food Chem. 46, 4616–4619.[CrossRef] Ralph, J., Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P.F., Marita, J.M., Hatfield, R.D., Ralph, S.A., Christensen, J.H., and Boerjan, W. (2004). Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Rev. 3, in press.
Rasmussen, S., and Dixon, R.A. (1999). Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11, 1537–1551.
Ro, D.-K., Ehlting, J., and Douglas, C.J. (2002). Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductase from hybrid poplar. Plant Physiol. 130, 1837–1851.
Sánchez-Fernández, R., Davies, T.G.E., Coleman, J.O.D., and Rea, P.A. (2001). The Arabidopsis thaliana ABC protein superfamily, a complete inventory. J. Biol. Chem. 276, 30231–30244. Sarma, A.D., Sreelakshmi, Y., and Sharma, R. (1998). Differential expression and properties of phenylalanine ammonia-lyase isoforms in tomato leaves. Phytochemistry 49, 2233–2243.[Medline]
Schluepmann, H., Pellny, T., van Dijken, A., Smeekens, S., and Paul, M. (2003). Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 100, 6849–6854. Sewalt, V.J.H., Ni, W., Blount, J.W., Jung, H.G., Masoud, S.A., Howles, P.A., Lamb, C., and Dixon, R.A. (1997). Reduced lignin content and altered lignin composition in transgenic tobacco down-regulated in expression of L-phenylalanine ammonia-lyase or cinnamate 4-hydroxylase. Plant Physiol. 115, 41–50.[Abstract] Somssich, I.E., and Hahlbrock, K. (1998). Pathogen defence in plants–A paradigm of biological complexity. Trends Plant Sci. 3, 86–90.[CrossRef][Web of Science] Spearing, J.K. (1953). Simultaneous double-staining technique for histology of vascular plants. J. R. Microsc. Soc. 73, 162–163.[Medline]
Taylor, N.G., Scheible, W.-R., Cutler, S., Somerville, C.R., and Turner, S.R. (1999). The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Cell 11, 769–779.
Urban, P., Mignotte, C., Kazmaier, M., Delorme, F., and Pompon, D. (1997). Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176–19186. Veit, M., and Pauli, G.F. (1999). Major flavonoids from Arabidopsis thaliana leaves. J. Nat. Prod. 62, 1301–1303.[CrossRef][Medline] Wahl, A., Roblot, F., and Cavè, A. (1995). Isolation and structure elucidation of xylobuxin, a new neolignan from Xylopia buxifolia. J. Nat. Prod. 58, 786–789. Wanner, L.A., Li, G., Ware, D., Somssich, I.E., and Davis, K.R. (1995). The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol. Biol. 27, 327–338.[CrossRef][Medline] Wolfinger, R.D., Gibson, G., Wolfinger, E.D., Bennett, L., Hamadeh, H., Bushel, P., Afshari, C., and Paules, R.S. (2001). Assessing gene significance from cDNA microarray expression data via mixed models. J. Comput. Biol. 8, 625–637.[CrossRef][Web of Science][Medline] Yamamoto, A., Nitta, S., Miyase, T., Ueno, A., and Wu, L.-J. (1993). Phenylethanoid and lignan-iridoid complex glycosides from roots of Buddleja davidii. Phytochemistry 32, 421–425.[Medline] Yang, Y.H., Dudoit, S., Luu, P., Lin, D.M., Peng, V., Ngai, J., and Speed, T.P. (2002). Normalization for cDNA microarray data: A robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15. Yao, K., De Luca, V., and Brisson, N. (1995). Creation of a metabolic sink of tryptophan alters the phenylpropanoid pathway and the susceptibility of potato to Phytophthora infestans. Plant Cell 7, 1787–1799.[Abstract] This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | THE PLANT CELL | PLANT PHYSIOLOGY | |
|---|---|---|---|
TOP
ABSTRACT
INTRODUCTION
-3 fatty acid desaturase; JA, jasmonate. Methionine metabolism: CysS, cystathionine ß-lyase (Cys synthase); MetS, Met synthase. Shikimate pathway: DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate; Ery4P, erythrose 4-phosphate; 4-HBA, para-hydroxybenzoate; 4-HBald, para-hydroxybenzaldehyde; PEP, phospho-enol-pyruvate; UbiRed, NADH-ubiquinone oxidoreductase. Recycling of NH4+1: ASN3, Asn synthase 3. Phenylpropanoid pathway: ATR3, NADPH-ferrihemoprotein reductase 3; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; 4-HBA, para-hydroxybenzoate. Monolignol biosynthesis: G lignin, guaiacyl lignin monomers; S lignin, syringyl lignin monomers. Flavonoid biosynthesis: CHS, chalcone synthase.
), upregulated in all pal mutants, plays important regulatory roles in carbohydrate metabolism in yeast and Arabidopsis (
-L-rhamnopyranoside (Glc-Glc-Rha-kaempferol), kaempferol 3-O-ß-D-glucopyranoside-7-O-
max at 242 and 332 nm) of the third compound (RT 15.119 min) was similar to that of FM(4–O–8)G. Based on MS analysis and its partial authentication with FA(5–8)G, this compound is suggested to be the related FM(5–8)G compound 2-{3-[2-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-2,3-dihydro-benzofuran-5-yl]-acryloyloxy}-succinic acid (
R x G for each spot (Yang et al., 2002
