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.

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OsRac1, one of the Rac͞Rop family of small GTPases, plays important roles in defense responses, including a role in the production of reactive oxygen species mediated by NADPH oxidase. We have identified an effector of OsRac1, namely rice (Oryza sativa) cinnamoyl-CoA reductase 1 (OsCCR1), an enzyme involved in lignin biosynthesis. Lignin, which is polymerized through peroxidase activity by using H 2O2 in the cell wall, is an important factor in plant defense responses, because it presents an undegradable mechanical barrier to most pathogens. Expression of OsCCR1 was induced by a sphingolipid elicitor, suggesting that OsCCR1 participates in defense signaling. In in vitro interaction and two-hybrid experiments, OsRac1 was shown to bind OsCCR1 in a GTP-dependent manner. Moreover, the interaction of OsCCR1 with OsRac1 led to the enzymatic activation of OsCCR1 in vitro. Transgenic cell cultures expressing the constitutively active OsRac1 accumulated lignin through enhanced CCR activity and increased reactive oxygen species production. Thus, it is likely that OsRac1 controls lignin synthesis through regulation of both NADPH oxidase and OsCCR1 activities during defense responses in rice.defense response ͉ G protein ͉ monolignol

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4DD-induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.