#2

Genes encoding cinnamate-4-hydroxylase (C4H, EC 1.14.13.11), p-coumarate-3-hydroxylase (C3H,) and ferulate-5-hydroxylase (F5H)

The introduction of hydroxyl groups onto the aromatic ring of various phenylpropanoid pathway intermediates, i.e. at C–4 (C4H), C–3 (C3H) and C–5 (F5H), respectively, are catalyzed by specific NADPH-dependent cytochrome P-450’s. The initial hydroxylation step (C4H) affords entry into all of the phenylpropanoid pathway derived metabolites (i.e. lignins, suberins, sporopollenins, hydroxycinnamic acids, etc.), whereas C3H and F5H are primarily involved in coniferyl and sinapyl alcohol formation. These in turn are the two major precursors of the Arabidopsis lignins (i.e. the so-called guaiacyl and syringyl lignins).

In Arabidopsis, each of these steps is represented by either a single gene (C4H and C3H) or by two genes (F5H), and in related work, we established that carbon allocation to the phenylpropanoid pathway was determined (i.e. regulated) by Phe availability, as well as by the activities of both C4H and C3H, respectively (3-5). On the other hand, the third hydroxylation step, catalyzed by F5H, has no rate-limiting role in carbon allocation to the pathway.

Because there are only one or at most two genes involved in each aromatic ring hydroxylation step, this places these particular genes in a rather unique metabolic situation when compared with the large multigene families associated with other phenylpropanoid pathway steps. Of these, C4H has already been well-studied, and its patterns of gene expression examined in detail. Available EST database data (1) also reveals that both C4H and C3H are expressed in all tissue/organ types examined, this being in accordance with their central role in formation of (essentially) all phenylpropanoid derivatives. On the other hand, the EST databases for F5H, which permits entry to the sinapate esters and sinapyl alcohol, is clearly incomplete.

Much to our surprise, some researchers have emphatically stated that neither the composition nor content of lignin is particularly important, and that the plant simply only needs a polymer derived from one of the monolignols, or if they cannot be formed, some surrogate thereof (6-8). There are many flaws in this argument, and much if not all of the previous data interpretation and claims have been in error. These assertions have been roundly criticized (4,5,9), and need not be repeated here.

On the other hand, manipulation of the NADPH-dependent cytochrome P-450’s, encoding C3H and F5H, provide an excellent opportunity to study how monolignol targeting and lignin assembly is achieved in vivo. In this way, it is possible to obtain Arabidopsis transformants with H (so-called ref8), G (so-called fah 1-2) and S (so-called C4H-F5H) enriched lignin skeleta (10-12).

With these transformants in hand, we first needed to develop methodologies to study lignin deposition in distinct cell types: in this regard, Arabidopsis stems have two distinct cell types that undergo lignification, namely xylem and interfascicular fiber cells.

The analysis of the different Arabidopsis cell lines to study lignin assembly was thus carried out as follows: (i) characterization of gross growth parameters and lignin deposition in Arabidopsis C3H and C4H-F5H transformants relative to their wild type counterparts; and (ii) patterns of lignin deposition in Arabidopsis H (ref8), G (fah 1-2), S (C4H-F5H) and wild type plants: confocal microscopy laser cell microdissection excision and pyrolysis GC-MS analysis.