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First published online December 23, 2005; 10.1104/pp.105.069690 Plant Physiology 140:30-48 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
A coumaroyl-ester-3-hydroxylase Insertion Mutant Reveals the Existence of Nonredundant meta-Hydroxylation Pathways and Essential Roles for Phenolic Precursors in Cell Expansion and Plant Growth1,[W],[OA]Department of Plant Metabolic Responses (N.A., J.E., C.A., S.R., P.U., D.W.-R.) and Department Cell Biology (M.E., V.S.), Institute of Plant Molecular Biology Centre National de la Recherche Scientifique-Unité Propre de Recherche 2357, Université Louis Pasteur, 67000 Strasbourg, France; Laboratoire de Chimie Biologique-Unité Mixte de Recherche 206, Institut National de la Recherche Agronomique-Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France (B.P., C.L.); Plant Cell Wall Group, Max Planck Institute for Molecular Plant Physiology, 14476 Golm, Germany (K.L., M.P.); and Unité de Recherche Génomique Végétale, 91057 Evry cedex, France (C.P., J.-P.R.)
Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.
Plants synthesize a wide variety of natural products based on the phenylpropane skeleton derived from Phe (Petersen et al., 1999  ). These compounds have an array of important functions, which are not yet fully evaluated or understood and involve a variety of ecological and physiological phenomena (Stafford, 1990; Debeaujon et al., 2000). The derivatives of hydroxycinnamic acids are essential intermediates in lignification and influence the physicochemical properties of the cell walls (Boerjan et al., 2003). Sinapoyl esters function to protect Arabidopsis (Arabidopsis thaliana) from UV irradiation (Landry et al., 1995; Booij-James et al., 2000) and caffeoyl and feruloyl esters play a significant role as antioxidants (Grace and Logan, 2000; Mathew and Abraham, 2004). Hydroxycinnamic acids are the precursors of a wide range of volatile compounds with most likely allelochemical functions (Dudareva et al., 2004). Other important groups of natural products derived from phenylpropane units are involved in plant response to environmental biotic and abiotic stimuli. For example, stilbenes and isoflavones are important phytoalexins in plants (Nicholson and Hammerschmidt, 1992).
Lignin, which originates from the oxidative polymerization of p-hydroxycinnamyl alcohols, is a major structural component of secondarily thickened cell walls of tissues with conducting and/or mechanical functions (Lewis and Yamamoto, 1990
An ethyl methanesulfonate mutant of the CYP98A3 gene has recently been described (Franke et al., 2002a , 2002b). The ref8 mutation was a single missense base change in the last one-third of the coding sequence, which was sufficient to result in a strong impact on plant development and in the inhibition of the formation of sinapoyl esters and of the G and S lignin units. Here we describe isolation of a T-DNA insertion mutant and show that complete inactivation of CYP98A3 has an even greater impact on plant development. Gene inactivation is associated with reduced cell expansion, altered sugar composition of cell walls, and decreased content in crystalline cellulose and pectins. Changes in plant and cell wall composition are associated with strong perturbations in the expression of genes involved in energetic and stress metabolism, signaling cascades, and cell wall development. In agreement with the results obtained on the ref8 mutant (Franke et al., 2002a), the lignification of the aerial part of this insertion mutant is deeply altered, with a reduced lignin content essentially made of H units and only trace amounts of G and S units. In contrast to stems, roots of the null mutant, however, display an ectopic lignification phenotype and a substantial frequency of G and S lignin units. Similar to the ref8 mutant, only trace amounts of sinapoyl malate (SM) are observed in the null mutant, which not only contains unusual p-coumaroyl derivatives, but also accumulates flavonol glycosides. To get further insight into the role of CYP98A3 and plants less severely affected in their development, we also produced cosuppressed plants. Surprisingly enough, while changes in lignification were similar to those observed in the null mutant, albeit less pronounced, our analyses revealed that the leaves of CYP98A3 cosuppressed plants overaccumulate sinapoylated anthocyanins, and that their levels of SM are similar to the control. Together, these data indicate (1) an alternative meta-hydroxylation pathway leading to G and S units in root lignin and, to a lesser extent, in the rachis; (2) cross talk between hydroxycinnamic acid/lignin and flavonoid pathways; and (3) a crucial role of phenolic precursors for cell wall expansion and homeostasis control during plant development.
cyp98A3 T-DNA Insertion and Cosuppression Mutants Show Drastic Impairments in Growth and Development
A cyp98A3 insertional mutant was isolated by PCR screening of a T-DNA-mutagenized population in the accession Wassilewskija (Ws) 2 (Krysan et al., 1999
The reddish-dark bushy appearance and reduced growth of the homozygous null mutants was also shared by plants where CYP98A3 overexpression under the control of a 35S promoter led to gene cosuppression. Approximately 10% of primary transformants (ecotype Columbia [Col-0]) containing a cauliflower mosaic virus (CaMV) 35S::CYP98A3 construct showed a reduced growth phenotype (Fig. 3 ). These independently transformed plants were arrested at different stages of development. The cosuppression of CYP98A3 in each primary transformant was confirmed by RNA-blot hybridization (data not shown) and quantitative RT-PCR (see below). A gradation of phenotypes was obtained, which appears to be correlated with the onset of the cosuppression (Fig. 3). Some plants arrested earlier in development did not bolt and showed a very limited growth of the rosette (line IV). For others, bolting was delayed and growth of inflorescence stems was retarded (Fig. 3, B and D, lines V to VIII). When plants bolted, inflorescences showed purple stems and cauline leaves. If flowers developed, they were limp, in most cases male sterile, and only occasionally produced viable seeds (Fig. 3, C–E). A few seeds were recovered from primary transformants designated as lines V, VI, and VII in Figure 3, and CYP98A3 expression was monitored throughout the life cycle in T2 plants derived from each line (Fig. 3F). Cosuppression was preceded by a phase of CYP98A3 overexpression for all plants (data not shown), and was fully established only when plants were 5 to 6 weeks old. CYP98A3 transcript levels were then reduced to undetectable levels within the subsequent 3 weeks (Fig. 3F). In fully cosuppressed plants, no CYP98A3 transcripts were detectable in RNA-blot analysis, and no protein was detected based on protein-blot analyses using CYP98A3-directed polyclonal antibodies (data not shown). The timing and degree of the cosuppression observed in each cosuppressed line differed slightly and seems to be correlated with the severity of the phenotype, in particular with the onset of developmental arrest.
cyp98A3 Mutants Display Modified Lignin in Stems But Ectopic Lignin Deposition in Roots
Given the severe phenotype of the cyp98A3 insertion mutant, plants were compared to wild type both at the same age and at a comparable stature. Lignin yield and composition were determined using all aerial parts from insertion mutant plants (6 weeks old) and wild-type Ws plants (6 weeks old or same size as 6-week-old mutants). Lignin determination by the standard Klason gravimetric method could not be applied to the dwarf null plants because this method is too sample demanding. Instead, the lignin structure was analyzed by thioacidolysis, which specifically gives rise to H, G, and S monomers from H, G, or S lignin arylglycerol lignin units involved in
Phloroglucinol staining was performed in an attempt to determine the tissue localization of lignin in the insertion mutant. Cytological examination of the miniature inflorescence stems from cyp98A3 insertion plants was impossible due to the poor mechanical properties of the tissues and crushing of the sections. In contrast, the less severe phenotype of the roots permitted this cytological analysis, and phloroglucinol staining revealed an ectopic lignification phenotype. In addition to xylem cell and fibers, which stain both in wild-type and mutant plant roots, phloroglucinol staining of pith and cortex cells was observed only in the roots of cyp98A3 insertion mutants (Fig. 4, A and B ). A similar ectopic lignification phenotype in roots has been previously reported for various mutants affected in cell wall biogenesis and cell expansion (Caño-Delgado et al., 2000 ) and in plants treated with inhibitors of cellulose synthase (Caño-Delgado et al., 2003). More surprisingly, an ectopic lignification phenotype was also observed in the rachis (stem, cauline leaves, and sepals) of cosuppressed transformants, which developed bolting stems (Fig. 4, C–E). No phloroglucinol-reactive material was, however, detected in the rosette leaves of cosuppressed plants or in the hypocotyls and rosette leaves of the cyp98A3 insertion plants. To clarify the nature of the phloroglucinol-reactive material accumulated, we analyzed the lignin content and structure in stems of cosuppressed plants, as well as the structure of root lignin from 3-week-old cyp98A3 insertion plants. Inflorescence stems of cosuppressed plants were harvested from T2 plants derived from lines V, VI, and VII at an age of 13 weeks, when plant growth was fully arrested and CYP98A3 transcripts were undetectable (Fig. 3F). Due to the limited number of T2 seeds/plants available, material from all three lines was pooled. The extract-free stems of cosuppressed plants were characterized by a 40% reduced Klason lignin content relative to the corresponding Col-0 control (Table II). Upon thioacidolysis, the lignin from cosuppressed plants released H, G, and S monomers in much lower total yield compared to wild type (93% reduction), which is indicative of a high content in resistant interunit bonds. Similar to cyp98A3 insertion mutants, the cosuppressed plants are characterized by a strong decrease in the relative amounts of S and G units in stems and a dramatic increase in the proportion of H units, which constitute 74% of lignin thioacidolysis monomers compared to less than 1% in stems from wild-type Col-0 plants (Table II). In addition, the dithioketal derivative of p-OH coumaraldehyde was obtained in a low, but quantifiable, amount (about 1.3% of the H main monomers), which indicates that p-OH-coumaraldehyde end groups occur that could contribute to the positive phloroglucinol staining in stems of cosuppressed plants.
The analyses of wild-type Ws roots confirmed that, compared to the corresponding stem lignin, roots contain relatively less S units and correlatively more G and H units (Sibout et al., 2003 ), with H units contributing 5% to 7% to the total lignin thioacidolysis monomers, depending on the developmental stage of wild-type plants (Table I). Similar to stems, roots of the cyp98A3 insertion mutants provided a lower total thioacidolysis yield and a much higher proportion of H monomers (43%) compared to the wild-type roots. However, the frequency of G thioacidolysis monomers released by root lignin was not reduced to the same extent as in the case of lignin derived from stems, and the frequency of S monomers was comparable in wild-type and mutant roots (Table I). Indeed, G and S units in the root tissues of the cyp98A3 insertion mutant still constitute more than 56% of the thioacidolysis-derived monomers (wild-type roots contain about 94%; Table I). Together with the persistence of G and S lignin units in the mutant stem, this result further supports the occurrence of an alternative 3-hydroxylation mechanism leading to the ectopic root lignin.
To further characterize the impact of the cyp98A3 mutation on phenolic metabolism, we analyzed soluble phenolics by liquid chromatography (LC)-mass spectrometry (MS) of soluble phenolics. Given the severe phenotype of the insertion mutant, analyses were performed using 15-d-old seedlings when phenotypic differences to wild-type Ws plants are less pronounced. Despite this, the results obtained for cyp98A3 insertion plants displayed a higher variability than those for control Ws plants grown under the same conditions (Table III). The main p-hydroxycinnamic and flavonol derivatives were identified on the basis of their mass and UV spectra (see "Materials and Methods" and supplemental text for details) or, where available, from literature data (Veit and Pauli, 1999
The characterization of flavonol glycosides by negative electrospray LC-MS revealed that their level was 3-fold higher in the cyp98A3 insertion seedlings relative to the Ws control (Fig. 5; Table III). Comparison of the profiles indicates that the mutation leads to a general enrichment of all flavonol glycosides present in wild-type plants and does not seem to induce the increase of selective or specific flavonol derivatives (Table III).
To analyze the role of CYP98A3 in older tissues, pooled cauline leaves from cosuppressed T2 plants derived from lines V, VI, and VII (Fig. 3), which were arrested at later stages of development, were used. Cauline leaves from wild-type Col-0 were analyzed in parallel by negative electrospray LC-MS. In agreement with literature data (Graham, 1988
The deep purple coloration of leaves from cosuppressed plants prompted us also to examine their anthocyanin content by positive electrospray LC-MS. In addition to the major anthocyanin of Arabidopsis, cyanidin 3-O-[2''-O-(6'''-O-(sinapoyl) xylosyl) 6''-O-(p-O-(glucosyl)-p-coumaroyl) glucoside] 5-O-(6''''-O-malonyl) glucoside (Bloor and Abrahams, 2002
In summary, the aerial parts of the cyp98A3 insertion mutant display drastically reduced, although not negligible, levels of sinapoyl esters and S and G lignin units. It is accompanied by a strong increase in p-coumaroyl esters, H lignin units, and flavonoids. This confirms the central role of CYP98A3 as the C3'H in the general phenylpropanoid pathway (Schoch et al., 2001
In an attempt to understand the observed inhibition of plant growth, cell size and cell wall modifications were investigated. A statistical analysis of the size of epidermal cells in different leaves and of different leaf areas indicated a significant 6-fold decrease of the cell size in the cyp98A3 insertion mutant (Fig. 6A ). This reduced cell size is not accompanied by modification of the cell shape, and an estimation of the total number of cells per leaf based on leaf and cell size (Fig. 6A) revealed no difference to wild-type plants. A substantial, but less dramatic, reduction in cell size was also observed in cosuppressed plants (data not shown).
In addition to decreased cell size, an alteration in the cell wall polysaccharide composition was observed (Fig. 6B). Polysaccharide analysis of 5-week-old plants revealed a significant increase in Glc released by trifluoroacetic acid (TFA) hydrolysis representing amorphous glucans (mainly amorphous cellulose). A concomitant decrease in the level of TFA-released uronic acids (UA; reflecting a decrease of pectic polysaccharides) compared to wild-type plants of the same age was observed (Fig. 6B). In addition, the amount of Glc released by Seaman hydrolysis, reflecting crystalline cellulose, was 50% lower in the cyp98A3 insertion mutant compared to mature Ws plants. It is interesting to note that the decrease in crystalline cellulose is accompanied by an increase in amorphous cellulose in cyp98A3, suggesting that the overall amount of cellulose is not altered in cell walls of the mutant, but rather its state of crystallinity, and hence possibly the architecture of the wall. However, at 5 weeks, wild-type plants are significantly larger than cyp98A3 homozygous plants. To test whether the differences in the composition of the cell wall polysaccharides did not merely reflect delayed development, wild-type plants were grown for 2 weeks until they reached a size comparable to the size of the 5-week-old null cyp98A3. Certain changes in polysaccharide composition occur during development of wild-type Ws plants. In mature plants, an increase in UAs was observed, accompanied by a slight increase in all neutral sugars analyzed with the exception of TFA-released Glc. In addition, a strong increase of crystalline cellulose was observed (Fig. 6B), which is likely due to the higher proportion of secondary walls present in the older plants. When comparing cyp98A3 insertion mutants to both developmental stages of wild-type plants, a reduction of UAs, an increase in amorphous cellulose, and a significant reduction in Ara, reflecting lower levels of arabinogalactan proteins and/or pectin-derived arabinans, was apparent (Fig. 6B).
Cytoskeleton and, in particular, cortical microtubules, are known to be intimately associated with the biogenesis of the cell wall and cellulose microfibril elongation, as well as secondary cell wall depositions and signaling of morphogenetic processes (Baskin, 2001
The strong alterations in cell wall composition, cell size, and in overall plant development observed in the cyp98A3 insertion mutant go beyond expectations and cannot be explained by modifications in phenylpropanoid metabolism alone. Gene expression-profiling analyses were thus performed to pinpoint the underlying developmental defects. Given the severe phenotype of the mutant, 15-d-old seedlings grown in vitro were used to minimize the impact of differences in plant development on gene expression and to catch a picture of the phenomenon at its onset. Although even at this early stage pleiotropic and other indirect effects cannot be excluded, they were expected to be less pronounced.
In a preliminary experiment, the expression of selected genes upstream in the phenylpropanoid pathway was compared using quantitative real-time RT-PCR analysis. Given that genes encoding phenylpropanoid enzymes display diurnal changes in their expression (Rogers et al., 2005
Microarray analysis of gene expression was thus performed using 15-d-old homozygous cyp98A3 insertion plants compared to wild-type Ws. Two biological replicates were analyzed, with two technical replicates (dye swaps) each for a total of four microarrays. The CATMA Arabidopsis near-full genome array was employed, which contains 24,576 gene-specific elements representing 21,371 annotated genes. Statistical analysis (see "Materials and Methods" for details) identified 1,944 elements representing 1,889 annotated genes to be differentially expressed (adjusted p [t test] < 0.05). Among these, 1,067 elements (1,038 genes) changed more than 2-fold, and 362 elements representing 352 annotated genes changed more than 3-fold (94 genes overexpressed and 260 genes repressed in the mutant).
Supplemental Table I lists normalized expression ratios and annotation details for all differentially expressed genes; the complete dataset was deposited into the ArrayExpress database (accession E-MEXP-346). Functional grouping of differentially expressed genes using the Functional Category Database at MatDB (Schoof et al., 2002
The higher expression of genes related to stress response, in particular mediated by jasmonate, prompted us to dissect the potential involvement of this plant hormone and to compare it to the impact of other hormones. Therefore, we retrieved from The Arabidopsis Information Resource (TAIR; Garcia-Hernandez et al., 2002 ) curator-annotated lists of genes that are involved in the "biosynthesis of," in the "signal transduction mediated by," and in the "response to" the plant hormones auxin, abscisic acid, brassinosteroid, cytokinin, ethylene, gibberellic acid, jasmonic acid, and salicylic acid. Within each of these groups of genes, the frequency of differentially expressed genes was determined and compared to the frequency of differentially expressed genes among all genes represented on the array. Genes expressed to higher levels in the cyp98A3 mutant were clearly over-represented in the functional group related to jasmonate signaling (Fig. 8B). Indeed, many genes involved in the biosynthesis of jasmonate are significantly higher expressed in the mutant compared to wild type (Ws); These include LOX2 (see above), LOX1, allene oxide synthase (CYP74A, At5g42650), and allene oxide cyclase 4 (At1g13280).
In addition, genes related to the abscisic acid cascade are over-represented in the group of genes expressed to higher levels in cyp98A3 insertion mutants (Fig. 8B). These include the gene responsive to dehydration 22 (RD22I, At5g25610), which is strongly induced by abscisic acid and drought (Yamaguchi-Shinozaki and Shinozaki, 1993
None of the other plant hormones analyzed appears to have a similar relevance for the group of genes expressed to higher levels in the cyp98A3 insertion mutant. To a lesser extent, genes related to gibberellic acid and salicylic acid are more frequently found in the group of genes expressed to lower levels in cyp98A3 insertion mutants than expected by chance (Fig. 8B). Among genes related to salicylic acid signaling and expressed to lower levels in cyp98A3 insertion mutants, defective in induced resistance 1 (At5g48485) encodes a lipid transfer protein that, when mutated, results in specific loss of systemic acquired resistance (Maldonado et al., 2002
Finally, in an attempt to explain the dramatic decrease in cell growth and plant development observed in the mutant, we further explored the expression of genes involved in cell expansion and cell division. This analysis revealed that no gene annotated in TAIR gene ontology (GO) term "cell division" is differentially expressed more than 2-fold. In addition, none of the genes involved in the core cell cycle machinery (Vandepoele et al., 2002
To ensure that the observed phenotype, in particular the drastic growth and developmental inhibition of the null mutant, was indeed the result of the CYP98A3 gene inactivation, a genetic complementation was performed by transformation of heterozygous plants with a construct containing the CYP98A3 coding region under control of the CaMV 35S promoter. Transformants were identified based on the BASTA resistance marker present in the construct, and homozygous cyp98A3 plants in the progeny of BASTA-resistant plants were identified using PCR (Fig. 9A ). Homozygous cyp98A3 insertion plants containing the 35S::CYP98A3 constructs were morphologically indistinguishable from wild-type plants (Fig. 9B). They displayed the same phenotype, including growth and development, as wild-type and heterozygous plants. Based on phloroglucinol staining, no signs of ectopic lignification were detected in roots of complemented plants (data not shown).
The observed growth inhibition in cyp98A3 insertion plants may result either from a depletion of 3-hydroxylated products or from the accumulation of potentially toxic or bioactive upstream or side products (e.g. flavonoids that were reported to modulate auxin transport [Peer et al., 2004 ]). In an attempt to dissect these hypotheses, chemical complementation tests were performed. CYP98A3 metabolizes with high efficiency both p-coumaroyl shikimate and p-coumaroyl quinate, both of which can be generated from p-coumaric acid by HCT. HCT is also expected to catalyze the conversion of the CYP98A3 product, caffeoyl shikimate/quinate, to caffeoyl-CoA (Hoffmann et al., 2003; Niggeweg et al., 2004). Complementation was therefore first attempted using the commercially available caffeic acid and caffeoyl quinate (chlorogenic acid). cyp98A3 insertion plants were grown on media containing these compounds, but no stimulation of plant growth was observed (data not shown). Given that caffeoyl shikimate is likely the major CYP98A3 product in vivo based on catalytic parameters (Schoch et al., 2001; M. Morant, G. Schoch, P. Ullmann, T. Ertunç, D. Little, C.E. Olsen, M. Petersen, J. Negrel, D. Werck-Reichhart, unpublished data), we synthesized this compound enzymatically from p-coumaroyl shikimate using recombinant CYP98A3 and purified the product caffeoyl shikimate in sufficient amounts to attempt complementation. Exogenously applied caffeoyl shikimate at least partially restored plant growth (Fig. 9C). Growth inhibition is thus more likely to result from the depletion of an essential 3-hydroxylated product rather than from the accumulation of a toxic precursor. In the absence of a commercially available source of radiolabeled compounds, we cannot exclude a differential uptake of caffeic acid, caffeoyl quinate, and caffeoyl shikimate, but it seems likely that chlorogenic acid or caffeic acid cannot be converted into this essential growth-promoting compound in cyp98A3 insertion mutants.
Analysis of cyp98A3 Mutants Reveals the Existence of an Alternative meta-Hydroxylation Pathway of Phenolic Compounds
The genes belonging to the CYP98 family recently emerged as the cytochrome P450 monooxygenases catalyzing the meta-hydroxylation of phenolic compounds in several plant species. These enzymes catalyze the meta-hydroxylation of p-coumaric derivatives esterified to shikimate, quinate, or phenyllactate (Schoch et al., 2001
This analysis of Arabidopsis cyp98A3 insertion and cosuppressed lines confirms the essential role of CYP98A3 in the synthesis of G and S lignin monomers in aerial parts of the plant and, to some extent, also in the roots. It also confirms that, under normal growth and homeostasis conditions, CYP98A3 is the major contributor to the biosynthesis of soluble sinapoyl derivatives as previously reported by Franke et al. (2002a)
It is interesting to note that the alternative meta-hydroxylation pathway observed in cyp98A3 mutants appears to be activated when the prevalent pathway is not functional. However, this alternative pathway cannot complement the defects observed in cyp98A3 mutants (e.g. reduced growth, reduced lignin in stems, limp stems, and male sterility). It does not ensure normal growth and appears to be active in tissues that do not normally lignify, as judged from the ectopic phloroglucinol staining observed in cyp98A3 insertion and cosuppressed plants. This alternative pathway seems to be ectopically activated under conditions of perturbed metabolism, but the nature and normal physiological relevance, in specific tissues or at specific stages of plant development, remains to be elucidated. Interestingly enough, the ectopic CYP98A3 overexpression for complementation of the null mutant does not promote any ectopic lignification. It is worth mentioning that the analysis of precursor conversion by peltate glands isolated from two sweet basil (Ocimum basilicum) lines that differ in their ability to produce eugenol also led Gang et al. (2002)
The ectopic activation of an alternative meta-hydroxylation pathway in cyp98A3 mutants is paralleled with strong morphological changes and with an accumulation of flavonoids. The accumulation of flavonoids (flavonol glycosides and anthocyanins) in null and cosuppressed cyp98A3 mutants is not surprising, since creating a bottleneck at the level of CYP98A3 leads to an accumulation of p-coumaroyl-CoA precursors that may subsequently simply overflow into the flavonoid pathway (Fig. 1).
The severe modification in plant development and the complete growth arrest observed in both cyp98A3 insertion and cosuppressed mutants, however, is more surprising. Given the reduced cell size, which is not accompanied by a reduction of the total number of cells per leaf, these morphological changes are likely due to an inhibition of cellular growth. Consistent with this hypothesis, our microarray analysis revealed no differences in transcript abundance of genes related to the cell cycle, while several genes related to cell expansion are expressed to different levels in wild-type and cyp98A3 insertion plants. Our data suggest that threshold amounts of meta-hydroxylated lignin monomers or of other caffeoyl shikimate-derived compounds are required for normal organization of the cell wall and plant growth. This conclusion is supported by the fact that external application of caffeoyl shikimate complements, at least partially, the growth phenotype of the null mutant. Such a meta-hydroxylated derivative might be needed as essential building blocks of the primary cell wall or might more indirectly promote cell wall modifications and growth (e.g. via Ca2+ chelation). Alternatively, it may directly act as a growth regulator. Phenolic compounds have been reported to play a role in cell division and expansion, either directly or as components of a cytokinin-dependent signaling cascade (Lynn et al., 1987
Another possibility is that growth arrest simply results from global perturbations of plant metabolism. A block in lignin biosynthesis suppresses a major carbon sink and induces an accumulation of flavonoids and possibly of indolic derivatives. The latter are stress-related metabolites. Many of the differences in gene expression observed in the cyp98A3 insertion mutant compared to wild type are reminiscent of a stress response. These include a general down-regulation of photosynthesis/chloroplastic genes, indicating reduced energy and carbon assimilation, and elevated expression levels of genes related to defense responses with activation of the jasmonate and abscisic acid signaling pathways. The observed ectopic lignification in roots of the cyp98A3 insertion mutant and in the rachis of cosuppressed plants can also be considered as indicative of a constitutive induction of a stress response. Ectopic lignification, resulting from mutations in genes encoding cellulose synthase, a chitinase-like enzyme, or a vacuolar ATPase (Schumacher et al., 1999
Our data indicate that the defect in the biosynthesis of an essential phenolic component leads to a similar phenotype, including elevated expression levels of genes related to jasmonate signaling, reduced growth, and activation of several stress/defense-related genes. Phenolics thus seem to be one of the elements that relate cell wall biosynthesis with growth promotion and stress signaling. No significant activation of the ethylene signaling pathway was observed in our experiments, but expression profiling indicated higher transcript levels for genes related to abscisic acid signaling in cyp98A3 mutants. Abscisic acid has been implicated as a signal transduction component in mainly abiotic stress responses, in particular responses to salt, cold, and drought stress (Pastori and Foyer, 2002
The proposed alternative meta-hydroxylation pathway is not expressed during developmental stem lignification and cannot complement developmental lignification, because mutations in CYP98A3 lead to an almost complete lack of G and S lignin units in stem (Franke et al., 2002a | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
-O-4 bonds (Lapierre et al., 1995
