Mutants impaired in normal lignin deposition provide a powerful tool to unravel the complexities underpinning the spatial and temporal control of lignin biosynthesis. The mutants pom1/elp1, eli1/cev1 and det3 all accumulate lignins in cells where they are not normally found. The three mutants arise because of mutations in three very different loci, with no obvious link between the functions of the three gene products. That all three mutations give rise to a shared phenotype is intriguing, and it may be that this occurs because the defects arising from the mutations impinge on a common regulatory pathway that governs the timing and localization of lignin deposition. Alternatively, it is possible that ectopic lignification arises in the three mutants as a result of completely nonoverlapping mechanisms. The results presented herein suggest that at least part of the ectopic lignification phenotype may be attributable to misregulation of lignin biosynthetic genes, and that this misregulation may involve both common and distinct regulators in the three different mutants.
The ectopic lignification phenotype varies between mutants
There are obvious differences in the ectopic lignification phenotypes observed in the det3 mutant relative to the pom1 and eli1 mutants that were isolated in this study. The det3 phenotype is the most striking of the three in that it stains with the greatest intensity and, on the basis of chemical analysis, has the greatest abundance of extractable lignins. The pom1 allele isolated in this study had the weakest lignification phenotype, both in terms of histochemical staining and chemical analysis. The pom1 mutant did not have significantly greater concentrations of total (acid insoluble + acid soluble) lignins compared with wild-type plants, which was a surprising finding. A decrease in the relative abundance of lignified vascular cells relative to ectopically lignifying cells could account for the apparent lack of increase in total lignins in the pom1 mutant, but there was no evidence to support this hypothesis. Alternatively, it is possible that the presence of ectopically lignifying cells without a concomitant increase in total lignin accumulation in the stem was accommodated by decreasing the amount of lignin deposition that occurred in the cell walls of the cells that are normally lignified. This hypothesis is difficult to test, but could be the subject of future investigations. Regardless of the mechanism, the pattern of lignification, and the composition of the lignins were clearly different in the pom1 mutant. The eli1 mutant was different again, with a lower S : G ratio, and an increase in extractable lignins superimposed upon the ectopic lignification phenotype
The variation in the quantity and quality of lignins was surpassed by the variation in the quality and quantity of other cell wall components. There was notable variation in cell wall carbohydrates, suggesting that there is no obvious relationship between cell wall carbohydrate composition and the manifestation of an ectopic lignification phenotype per se. Conversely, the specific changes in cell wall carbohydrates may be related to the specific changes in lignin deposition found in each mutant. Chemical analysis of cellulose synthase mutants related to eli1, such as rsw1, may determine whether there are consistent changes in cell wall carbohydrate composition that can be correlated with specific changes in lignin chemistry.
Despite the variation between the mutants in terms of lignin composition and the quantity of extractable lignins, there was one striking similarity between the mutants – the location of the ectopic lignins. While the double mutants suggested that the intensity of lignification, and the nature of the subunits in the mutants could be additive, the localization of ectopic lignin deposition, as determined by phloroglucinol staining, remained the same. Ectopic lignification, as defined by the localization of phloroglucinol staining, was only observed in the pith and occasionally the phloem fibres. Cortical cell types also stained positively with Mäule's reagent in the eli1 mutant, but this staining did not coincide with phloroglucinol staining, suggesting that whatever the Mäule's reagent was reactive with, it was not lignins. Therefore, while it is possible to deposit different kinds of lignins, it appeared that only certain cell types acquire the competency to lignify in the mutants. This suggests that the three mutants may impinge upon a common regulatory pathway to confer competency to these cells alone.
Ectopic lignification mutants provide further evidence for the central role of transcriptional regulation in the control of lignin deposition
There is a large body of evidence that suggests that lignin biosynthesis is regulated, at least in part, at the transcriptional level (Hertzberg et al., 2001; Anterola et al., 2002; Demura et al., 2002; Israelsson et al., 2003; Patzlaff et al., 2003; Raes et al., 2003). Consistent with this evidence, a high degree of accordance was found between transcript abundance and lignin accumulation in the three ectopic lignification mutants examined in this study. So strong was the correlation that it is tempting to speculate that the differences in lignin accumulation that were observed between the three mutants may be solely attributable to differences in transcriptional regulation of genes encoding lignin biosynthetic enzymes.
In examining the transcriptional regulation of lignin biosynthetic genes, it is important to examine the diurnal changes in transcript abundance. Some previous studies have hinted at the diurnal regulation of lignin biosynthetic genes (Logemann et al., 1995; Mizutani et al., 1997), but a systematic analysis of the diurnal changes in transcript abundance of lignin biosynthetic genes has not yet been reported. The data provided herein reveal that it is important to investigate the diurnal component of the regulation of lignin biosynthetic genes, otherwise important differences in transcript abundance might be overlooked. Microarray analysis of lignin biosynthetic genes revealed that they are also likely to be under circadian regulation (Harmer et al., 2000). Consequently, in dissecting the basis for differences in lignin deposition, this aspect of regulation should also be addressed.
In the ectopically lignifying aerial tissues of all three mutants grown to growth stage 1.03, there did not appear to be a significant disruption in diurnal or circadian trends in transcript abundance; however, all three mutants exhibited marked increases in transcript abundance for many of the key genes encoding enzymes in the monolignol biosynthetic pathway, the genes that define the ‘lignin toolbox’ (Raes et al., 2003). The greatest changes in transcript abundance were observed for det3, which also accumulated the greatest amounts of lignins. Similarly, pom1, which had the lowest amount of lignins of all three mutants, also had the lowest transcript abundance. Under diurnal conditions, the genes encoding PAL1, C4H1, CCoAOMT1 and F5H1 had significant increases in transcript abundance in all three mutants relative to wild-type plants. In keeping with the relationship between lignin content and transcript abundance, in the eli1 and det3 mutants, but not pom1, CCR1 and COMT also exhibited significant increases in transcript abundance relative to wild type, and in the det3 mutant, but not the other two mutants, PAL2, 4CL1 and CAD6 had increased transcript abundance relative to wild type. These findings suggest that PAL1, C4H1, CCoAOMT and F5H1 may be under a common regulatory control in all three mutants; whereas, CCR1 and COMT are only affected in the mutants with the greatest accumulation of lignins. This could reflect either the expression of different regulators in the different mutants, or a threshold mechanism that results in the expression of different suites of genes depending on the strength of some common stimulus.
It is important to note that the differences in transcript abundance did not completely explain differences in lignin subunit composition in the three mutants. There are several reasons why this may be the case. In the first instance, while the Northern blot analysis was relatively comprehensive in capturing multiple time points in the expression of genes encoding lignin biosynthetic enzymes, it still only represented 2 d during the lifetime of the plants. It may be that the genes modifying lignin subunit composition are expressed at later points in development that were missed by the analyses described herein. In addition, the differences in transcript abundance documented in the aerial tissues of growth stage 1.03 plants undoubtedly do not reflect those that take place in the inflorescence stem for which the lignin composition was determined. Finally, transcript abundance only represents a portion of the picture in terms of how a biosynthetic pathway may be regulated, and it may be that translational or post-translational mechanisms operate to modify lignin subunit composition. Future studies should aim to investigate these possibilities.
Under circadian conditions, significant increases in relative transcript abundance were documented for only C3H1 and CAD6 in all three mutants relative to wild-type plants, while CCoAOMT, CCR1 and COMT had elevated transcript abundance in only eli1 and det3. These findings emphasize the correlation between the extent of transcript accumulation across the pathway and lignin accumulation. Moreover, these results suggest that diurnal and circadian stimuli have effects on overall transcript abundance that vary between the different members of the lignin biosynthetic toolbox, and that this variance is genotype dependent. Nevertheless, while there was an increase in overall transcript abundance in the mutants, the pattern of diurnal and circadian changes was not appreciably different from wild-type plants. This suggests that the mutants do not accumulate lignins by altering the regulatory mechanisms that control diurnal or circadian fluctuations in lignin biosynthesis, but rather simply alter the overall accumulation of transcripts.
A role for sugar in lignin deposition
As the patterns of diurnal and circadian changes in transcript abundance were not significantly altered in the mutants relative to wild-type plants, an alternative hypothesis was considered that may account for the ectopic lignin production in the mutants. As importance of sugar signalling in the deposition of lignins in wild-type A. thaliana plants has been shown (Rogers et al., 2005), the possibility that this may be altered in the mutants was examined. As has previously been observed (Rogers et al., 2005), on the basis of phloroglucinol staining, the ability to detect lignins in dark-grown, wild-type xylem vessels was contingent on the presence of exogenous sucrose. That is, exogenous sugar was necessary for visible phloroglucinol staining in dark-grown, wild-type seedlings. This was not the case for the ectopic lignification mutants, which accumulated phloroglucinol-positive material in the dark even in the absence of sucrose. Consistent with this finding, many of the lignin biosynthetic genes that had increased transcript abundance in wild-type plants grown in the dark in the presence of sucrose (Rogers et al., 2005) also had increased transcript abundance in the mutants. This suggests that the regulatory factors that are deployed to give rise to sugar-responsive lignification in dark-grown, wild-type plants may also be deployed in the mutants even when sugar is not present.
Some of the genes show very consistent trends in transcript accumulation between the plants grown in the light to growth stage 1.03 for Northern blot analysis and those grown in the dark for microarray analysis, including PAL1 and F5H. By contrast, other genes, such as PAL2 and 4CL1, had increased transcript abundance in only a limited subset of mutants under one of the conditions and had increased transcript abundance in all three mutants under the other conditions. This observation underlines the fact that different conditions have varying effects on overall transcript abundance of different members of the lignin biosynthetic toolbox, and that this variance in overall abundance is genotype dependent. It suggests that different genes, including some of those genes that are ‘like’ those in the lignin biosynthetic toolbox, respond to the mutations in a manner that depends on growth stage or the environmental conditions. Importantly, it suggests that different suites of genes, including those not normally thought to be in the lignin biosynthetic toolbox, may be involved in the deposition of lignins, depending on developmental stage or growth conditions, and suggests that multiple stimuli must feed into the regulation of lignin biosynthesis. It is striking that these stimuli all impinge upon transcript abundance of genes involved in lignin deposition and suggests that transcription factors play a key role in regulating the process.
Relationship between transcriptome activity in dark-grown wild-type plants grown in the presence of sucrose vs dark-grown ectopic lignification mutants grown without sucrose
Comparative transcriptome analysis of 14-d-old seedlings that had been germinated and grown in the dark was undertaken, as these growth conditions accentuated difference in the ectopic lignification phenotype of the mutants relative to wild-type plants. Moreover, analysis of plants grown under these conditions simplified the dissection of regulatory mechanisms that may be involved in giving rise to lignin accumulation by eliminating diurnal and circadian inputs. Importantly, these analyses revealed regulatory factors that may be involved in the control of lignin deposition in wild-type plants in response to sucrose, which are constitutively active in the ectopic lignification mutants.
Cluster analysis of microarray data identified a set of 31 genes with pronounced increases in transcript abundance in dark-grown, wild-type plants grown in the presence of sucrose, and in the three ectopic lignification mutants. Two genes of the lignin biosynthetic toolbox are included in this group of genes, PAL1 and 4CL1 (Raes et al., 2003), in keeping with the lignin accumulation that occurs in wild-type plants grown in the presence of sugar, and in the three mutants grown without sugar. Under these simple growth conditions, it suggests that the mechanism that controls lignin accumulation in wild-type plants in response to sugar, does so by altering the expression of these two genes, and that this mechanism is constitutively active in the three mutants. Of the genes that show this pattern of regulation, 12 are involved in chloroplast function, which is curious, given that the plants were germinated and grown in the dark. Exogenous sucrose can function as a proxy for light signalling in dark-grown plant tissues (Cheng et al., 1992), and, in these experiments, it may be that the exogenously supplied sucrose ‘misinforms’ wild-type plants, indicating that they are undergoing photosynthesis, and activating gene expression related to chloroplast function. That these same genes are also activated in the three ectopic lignification mutants, in the absence of sucrose, suggests that this sugar-signalling pathway is constitutively active in the mutants. These findings are in keeping with the fact that all three mutants are also dark photomorphogenic (Cano-Delgado et al., 2000, 2003; Zhong et al., 2002; Mouille et al., 2003).
One of the genes contained within the 31 gene cluster, which has increased transcript abundance under conditions that result in lignin accumulation, encodes a cytochrome P450 that is involved in auxin biosynthesis, CYP79B2 (Zhao et al., 2002). CYP79B2 is a critical component of the auxin biosynthetic machinery in A. thaliana (Zhao et al., 2002). Auxin is well-known to be important in the differentiation of lignifying cells, such as xylem, and auxin can induce lignification (Demaggio, 1972; Sugiyama & Komamine, 1990; Sieburth, 1999; Sachs, 2000; Mellerowicz et al., 2001). It may be that the elevated level of CYP79B2 gene expression results in increased auxin biosynthesis in dark-grown wild-type plants growing in the presence of sucrose, and in the three ectopic lignification mutants, even in the absence of sucrose. Increased auxin levels, in turn, may induce the lignin biosynthesis observed in these plants.
Two other genes in the cluster of 31 genes may be linked to the ectopic lignification phenotype. The first of these genes encodes glutamine synthetase (GS). Like glycine decarboxylase, which is also encoded by a gene in the cluster of 31 genes, GS is implicated in photorespiration (Lam et al., 1996). It also plays a central role in nitrogen recycling in plant tissues (Lam et al., 1996). The requirement for nitrogen recycling is particularly high in lignifying cells, as the first enzyme in the lignin toolbox, PAL, catalyses the deamination of phenylalanine and thereby liberates large quantities of nitrogen. This nitrogen would be lost to the plant if there was not an efficient recycling mechanism. Glutamine synthetase is hypothesized to play a role in recovering the nitrogen liberated during lignification (Razal et al., 1996). Recent evidence suggests that the regulation of the transcription of genes encoding lignin biosynthetic enzymes and GS may be linked in some plants (Gomez-Maldonado et al., 2004), consistent with what has been observed here. The second of the two genes encodes S-adenosylmethionine synthetase (SAM synthetase). This is involved in one-carbon metabolism, and plays a central role in generating the SAM that is used as a methyl donor in methylation of the 3- and 5-hydroxyl groups of the phenyl ring during the elaboration of monolignols (Whetten & Sederoff, 1995). The level of SAM synthetase is important in the maintenance of lignin biogenesis (Shen et al., 2002), and therefore the elevated transcript abundance observed for the gene encoding this protein in the ectopic lignification mutants, and in wild-type plants grown in the presence of sucrose, may reflect an increased requirement for this activity for lignin biosynthesis.
Candidate regulators of lignin deposition
When the 5′ noncoding regions of the 31 genes in the cluster described above were subjected to a Gibbs sampling-based analysis to identify statistically over-represented sequence motifs (Thijs et al., 2002), two motifs emerged that had previously known regulatory function. One of these motifs was similar to known R2R3-MYB binding sites, and the other is a known binding site for members of the Dof family of transcription factors. R2R3-MYB binding sites, also known as AC elements, have previously been shown to be present in the putative regulatory regions of all but one of the genes that define the lignin toolbox in A. thaliana (Raes et al., 2003), and have been shown to be important in the regulation of lignin biosynthetic gene expression in a number of different plant species (Hauffe et al., 1993; Hatton et al., 1995; Lacombe et al., 2000). Consistent with this, R2R3-MYB family members that can bind to these elements have been shown to have the capacity to modulate lignin deposition (Tamagnone et al., 1998; Patzlaff et al., 2003; Feng et al., 2004). To date, Dof binding sites have not been implicated in the regulation of lignin biosynthetic genes.
Cluster analysis was used to identify specific R2R3-MYB or Dof family members that might be involved in the regulation of the genes in the lignin toolbox under conditions that increase lignin deposition. Genes encoding two other classes of transcription factors postulated to be involved in lignin deposition, KNOX family members related to KNAT1/BREVIPEDICELLUS (Mele et al., 2003) and A. thaliana homologues of Nicotiana tobaccum LIM1 (Kawaoka et al., 2000), were also included in this analysis. As might have been predicted, all of the genes in the lignin toolbox had increased transcript abundance relative to the control in all three ectopic lignification mutants and in dark-grown wild-type plants in the presence of sugar. The genes in the lignin toolbox formed a cluster at one end of the Eisengram. Embedded in this cluster were genes encoding several R2R3-MYB family members (AtMYB84, At3g49690; AtMYB61, At1g09540; AtMYB92, At5g10280; AtMYB42, At4g12350) and one Dof family member (AtDof2.1, At2g34140). It might be predicted that the gene products encoded by these genes play a role in increasing the transcript abundance of the genes of the lignin toolbox. Consistent with this hypothesis, AtMYB61 has been shown to contribute to the ectopic lignin deposition observed in the det3 mutant (Newman et al., 2004). Clearly, AtMYB61 is not involved in the manifestation of the eli1 phenotype, as AtMYB61 expression is lower in this mutant relative to the other lignin accumulation conditions, and therefore, other regulators must be involved in giving rise to the eli1 phenotype.
While it is possible that lignin deposition is controlled solely by positive regulators of gene expression, it may also be that the downregulation of transcriptional repressors of lignin biosynthetic genes contributes to increased lignin deposition. Several genes, encoding either R2R3-MYB, Dof, or NtLIM-like transcription factors, all had decreased transcript abundance relative to the control in all three ectopic lignification mutants and in dark-grown wild-type plants in the presence of sugar. These genes may encode repressors of lignin deposition that, when they themselves are repressed, allow the expression of lignin biosynthetic genes to proceed apace. Consistent with this hypothesis, AtMYB4 (At1g22640), a known repressor of some of the genes involved in lignin biosynthesis (Jin et al., 2000), has decreased transcript abundance under those conditions that result in increased lignin deposition. It may be that the other transcription factors in this category function in the same manner.
The approach described herein has identified several putative regulators of the lignin biosynthetic pathway. Thorough gain-of-function/loss-of-function analyses has already shown that one of these regulators, AtMYB61, is both necessary and sufficient to explain the ectopic lignin deposition phenotype in the det3 mutant (Newman et al., 2004). Future studies will clarify the roles of the other candidate regulators in the control of lignin deposition.