A coumaroyl-ester-3-hydroxylase Insertion Mutant Reveals the Existence of Nonredundant meta-Hydroxylation Pathways and Essential Roles for Phenolic Precursors in Cell Expansion and Plant Growth

Abstract

Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.

Flavonoid Accumulation in Arabidopsis Repressed in Lignin Synthesis Affects Auxin Transport and Plant Growth

“…Interestingly, these alterations in lignin structure are much more important than those observed in HCT-silenced N. benthamiana plants submitted to virus-induced gene silencing at later stages of development (Hoffmann et al, 2004). In agreement with the results obtained with a C3H mutant (Abdulrazzak et al, 2005), G and S lignin units could be unequivocally evidenced in HCT-silenced stems, from the specific G and S thioacidolysis monomers. The remanence of some G and S lignin units may be due to the low level of HCT activity measured in HCT-silenced stems (Figure 2).…”

“…First of all, sinapoylmalate levels did not change significantly in HCT-deficient plants that only synthesized trace amounts of S lignin unit. Similar observations have been made recently in the study of C3H-deficient plants (Abdulrazzak et al, 2005). These data suggest that sinapoylmalate and S lignin unit are produced through distinct biosynthetic routes, a hypothesis strengthened by the unusual accumulation of sinapoylated cyanidin derivatives in the leaves of HCT-deficient plants (Table 1, C2 and C5).…”

“…It is noteworthy that, in addition to HCT itself, genes directly downstream of HCT, namely C3H, CCR, and CCoAOMT, are those whose repression has been shown to have the strongest impact on plant growth (Piquemal et al, 1998;Jones et al, 2001;Pinç on et al, 2001aPinç on et al, , 2001bFranke et al, 2002;Goujon et al, 2003;Abdulrazzak et al, 2005;Patten et al, 2005). HCT repression had a particularly strong impact on growth, likely due to its double role in lignin biosynthesis upstream and downstream of the C3H step (see Figure 1).…”

“…The changes affecting growth and development of HCT-silenced plants that accumulated flavonoids are reminiscent of those Structure of flavonol glycosides and cyanidin derivatives were inferred from their UV-visible and mass spectral properties (Table 1) and from available data (Graham, 1998;Veit and Pauli, 1999;Bloor and Abrahams, 2002;Jones et al, 2003;Abdulrazzak et al, 2005;Tohge et al, 2005).…”

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

“…The 3 0 -hydroxylation of the phenylpropanoid ring is essential not only for the synthesis of lignin (Franke et al 2002;Abdulrazzak et al 2006;Chen and Dixon 2007), but is also an important step in the synthesis of chlorogenic acid (Schoch et al 2006;Mahesh et al 2007). The CYP98 family of plant cytochromes P450 has been designated as the family of enzyme that performs the meta-hydroxylation step in the phenylpropanoid pathway ( Fig.…”