Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H͞COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H͞COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k cat ͞K m of CAld5H for coniferyl aldehyde was Ϸ140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K i ‫؍‬ 0.59 M) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H͞ COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5-hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K m and K i values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.Regardless of its importance to tree growth, lignin is problematic to postharvest, cellulose-based wood processing for fiber, chemical, and energy production, because it must be degraded from cellulose at great expense. Certain structural constituents of lignin, such as the guaiacyl moiety, promote monomer cross-linkages that increase lignin resistance to degradation (1-3). In angiosperms, lignin is composed of a mixture of guaiacyl and syringyl monolignols, and can be degraded at considerably less energy and chemical cost than gymnosperm lignin, which consists almost entirely of guaiacyl moieties (4). As a result, there has been long standing incentive to understand the biosynthesis of syringyl monolignol in angiosperm trees in order to genetically engineer more syringyl lignin in trees to facilitate wood processing (5-8).For more than four decades, our thinking of syringyl monolignol biosynthesis in angiosperms has followed the doctrine that S-adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT) 1 -catalyzed methylation of caffeate and 5-hydroxyferulate constitutes a central pathway for the formation of syringyl monolignol via sinapate (Fig. 1A) . This methylation pathway has, however, been challenged, as the involvement in this pathway (Fig. 1A) of the ferulate 5-hydroxylation step (1...

S-adenosyl-L-methionine (SAM)-dependent