Biosynthesis of wall-bound phenolic esters, and of lignin, in phenylalanine ammonia-lyase-inhibited radish seedlings

Young radish (Raphanus sativus) cotyledons contain high levels of seed-derived soluble sinapoyl esters which decrease significantly during seedling development. This work tests the hypothesis that they may be recycled into wall-bound esters and lignin. When Phenylalanine ammonia-lyase (PAL) was inhibited by 100 muM 2-aminoindan-2-phosphonic acid (AIP), wall-bound p-coumaric and ferulic acids were strongly inhibited but levels of wall-bound sinapic acid were similar to that of controls, evidence that de novo hydroxycinnamic acid synthesis was blocked and seed-derived soluble sinapoyl esters were converted into wall-bound esters.; Similarly, the syringyl to guaiacyl (S/G) ratio in lignin, determined by benzaldehydes formed by alkaline cupric oxidation and Fourier-Transform infrared (FT-IR) spectra of lignothioglycolic acid (LTGA), was much increased in lignin from cotyledons grown on AIP, evidence that seed-derived sinapoyl esters were incorporated into lignin. However, hypocotyls plus roots of AIP-grown plants contained only traces of wall-bound sinapic acid, suggesting no appreciable transport of soluble sinapoyl esters from the cotyledons.; FT-IR spectra of LTGA from extractive-free cell walls conventionally prepared with 50% aqueous MeOH showed significantly protein contamination. This can cause gross overestimation of lignin quantitated by absorbance of LTGA at 280 nm. An improved technique was developed involving extraction with KPO4 buffer, 1M NaCl, and cellulytic enzymes which yielded radish lignin whose FT-IR spectrum was comparable to that of kraft hardwood LTGA.; When grown for 30 days with 10 muM AIP, soluble and wall-bound p-coumaric and ferulic acids were inhibited about 90% but this treatment had little effect on soluble and wall-bound sinapic acid and the S/G ratio in lignin increased with increasing levels of AIP. Plants grown with 10 muM AIP appeared normal if protected from UV by growth under Mylar. Without Mylar they were stunted, presumably due to UV damage of the photosynthetic apparatus. These. results suggest that research to create transgenic plants with reduced lignin content for pulping may be possible, but only if other aspects of defensive phenolic metabolisms remain functional.