The monolignol pathway [i. e., fromPhe (6) formation to themonolignols 1-5 and ultimately lignin] has received recent growing interest as regards the extent of gene families/metabolic networks associated with lignification and other monolignol 1-5 related metabolism. This could thus now be fully investigated given that the Arabidopsis genome was sequenced in its entirety in 2000 (79). Furthermore, since several of the enzymatic steps [e. g., to cinnamic acid (8) (via PAL)] can also involve other unrelated phenylpropanoid-forming processes (such as to flavonoids, suberins, and other non-monolignol metabolites), it was therefore useful to determine to what extent there were functionally redundant metabolic networks and/or nodes within same that were monolignol/lignin-specific. This was nec­essary to eventually understand how these processes were (constitutively) occurring in a coordinated manner with other biochemical systems (e. g., to the cell wall biopolymers, cellulose, hemicelluloses, etc.), including identification of the various transcription factors involved.

In this regard, although possible gene families have been provisionally annotated in the TAIR database (e. g., as COMTs, CADs, etc.) (93), it became clear early on in our studies (56, 94, 122) that unambiguous biochemical/physiological functional determinations for each candidate gene (mutant) were urgently needed. Moreover, given, for example, the substrate versatility of various dehydrogenases, comparative kinetic data was also required. Thus, each of the biochemical steps discussed below had to be reconsidered in terms of the above, with particular attention being given to database (mis)annotations.

In apparent agreement with previously deduced regulatory roles for carbon allocation to the monolignol-forming pathway for C4H and pC3H (together with their associated reductases and HCT) described earlier, each exists in Arabidopsis as single gene. Moreover, while as discussed earlier, F5H does not appear to affect overall carbon allocation (77), it nevertheless serves as a “switch” between G and S lignin formation, and it is also part of a very small gene family (~2). Interestingly, until recently, enzymes such as C4H were considered to be of very limited substrate versatility. On the other hand, a recent study using recombinant C4H protein from Arabidopsis described a broader substrate versatility than previously recognized — albeit with unnatural substrates (146).

By contrast, the two preceding biosynthetic steps — leading to arogenate (37) and Phe (6) formation, are encoded by multi-gene families (e. g., 6 genes for the ADT and 4 for the PAL families in Arabidopsis). Furthermore, at least for the PAL gene family, the correspond­ing genes have quite complex patterns of expression [as demonstrated by GUS-reporter promoter fusion analyses (Kim etal., manuscript in preparation)]. That is, the level of co­expression in various tissues and organs is indicative of a partially overlapping, functionally redundant, metabolic network. This is anticipated to be the case for the ADT gene family as well. Both enzymatic steps also have quite strict substrate specificities, at least for the known/tested substrates employed to date.

The remaining downstream enzymatic steps (i. e., 4CLs, CCOMTs, COMTs, CCRs, and CADs) in the monolignol/lignin-forming pathway are — as originally/currently annotated in the TAIR database — members of rather large gene families, i. e., original annotations were 4CL (14 genes), CCR (12), CAD (17), CCOMT (5), and COMT (17), respectively (93). However, this is, in fact, a considerable overestimation as discussed below.

As indicated earlier, 4CL corresponds (at most) to a four-member gene family (122), and as for PAL, the GUS-promoter fusion studies for each display a complex, partially overlapping, metabolic network (Kim et al., manuscript in preparation), i. e., in agreement with the different phenylpropanoid pathway products (from flavonoids to lignins) being formed. Additionally, there appear to be only 2-3 CCRs (130, 131) (Cochrane et al., unpublished results) and predominantly 2 CADs (56,57,71) in Arabidopsis — at least as far aslignificationis concerned. Interestingly, just one of the 17 putative COMTs has the requisite catalytic activity in vitro (Zhang et al., manuscript in preparation) for the monolignol-forming pathway, and the CCOMTs also appear to only have at most 2 genes associated with monolignol/lignin deposition (Takahashi et al., manuscript in preparation). As for PAL and other genes, the constitutive patterns of gene expression have also been established, thus providing the basis for probing/investigating the nature of the metabolic networks operative at all stages of plant growth and development.

Taken together, the entire suite of genes excluding transcription factors possibly involved in monolignol/lignin deposition — at least to the monolignols — accounts for <25 genes from prephenic acid (36) onwards, rather than the more than 10-fold higher number originally annotated.

The opportunities and challenges that now remain in further understanding monolig — nol/lignin pathway metabolism are to complete the full biochemical/physiological charac­terization of these different gene family members and to establish the “rules” for overall pathway induction/deployment — particularly since the pathway genes are “scattered” over different chromosomes. This potentially appears to be a very straightforward task, given that each of the members has now been identified — at least through their in vitro biochem­ical conversions. The data also underscore the need for circumspection when considering database annotations: while useful repositories, these cannot be considered definitive in any way until the physiological and biochemical functions are unambiguously determined.