| Autor: |
Cardenas CL; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States., Costa MA; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States., Laskar DD; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States., Moinuddin SGA; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States., Lee C; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States., Davin LB; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States., Lewis NG; Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, United States. |
| Abstrakt: |
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. HQT and HCT RNA i silencing of a commercial tobacco ( Nicotiana tabacum ) K326 line was examined herein. NtHQT gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNA i NtHCT 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 p- hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. In vitro NtHCT kinetic parameters revealed preferential p -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 p -coumaroyl CoA and quinic acid to form p -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. |
