RNA<i>i</i>Modulation of Chlorogenic Acid and Lignin Deposition in<i>Nicotiana tabacum</i>and Insufficient Compensatory Metabolic Cross-Talk

Abstract

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities. <i>HQT</i> and <i>HCT</i> RNA<i>i</i> silencing of a commercial tobacco (<i>Nicotiana tabacum</i>) K326 line was examined herein. <i>NtHQT</i> gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNA<i>i NtHCT</i> silencing had markedly adverse phenotypes (e.g., stunted, multiple stems, delayed flowering, with senescence delayed by several months). Lignin contents were partially lowered, with a small increase in cleavable <i>p-</i>hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. <i>In vitro</i> NtHCT kinetic parameters revealed preferential <i>p</i>-coumaroyl CoA and shikimate esterification, as compared to other structurally related potential acyl group donors and acceptors. In the presence of coenzyme A, NtHCT catalyzed the reverse reaction. Site-directed mutagenesis of NtHCT (His153Ala) abolished enzymatic activity. NtHQT, by comparison, catalyzed preferential conversion of <i>p</i>-coumaroyl CoA and quinic acid to form <i>p</i>-coumaroyl quinate, the presumed CGA precursor. In sum, metabolic pathways to CGA and lignins appear to be fully independent, and previous conflicting reports of substrate versatilities and metabolic cross-talk are resolved.