Hydroxycinnamoyl transferases in populus and their roles in vascular development




Le, Cuong Hieu

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Hydroxycinnamoyl conjugates (HCC)s are an extremely diverse class of natural products that serve a wide variety of key functions in plant physiology, for example during wood formation, and in defence. They have diverse biological properties and act as antioxidants, antimicrobials, and antivirals. The biochemical basis of HCC diversity, however, has not yet been fully elucidated. Plants in the Populus genus are known to produce a particularly diverse range of HCCs and they constitute up to 5% of the leaf dry mass in some Populus species. HCCs can be formed by hydroxycinnamoyl transferases (HCTs) and distinct HCT isoforms in Populus may have distinct biological functions related to the synthesis of specific classes of HCCs. These can be identified on the basis of their evolutionary history and I show that many of the biochemically characterised HCTs belong to the BAHD superfamily of acyltransferases. My phylogenetic reconstruction of the BAHD superfamily has also defined a subclass containing most of the already-characterised HCTs, including nine potential HCT candidates in Populus. Caffeoyl-shikimate is a central precursor in the formation of lignin, a biopolymer (along with cellulose) that imparts mechanical stability to wood. Based on the transcript abundance of two candidate genes PtHCTA1 (Potri.001G042900)) and PtHCTA2 (Potri.003G183900) were hypothesised to be responsible for caffeoyl-shikimate formation in secondary xylem (i.e., wood). As part of this project, RNAi whole-plant knock-downs were generated for the xylem-associated PtHCTA1/2. The PtHCTA1/2 RNAi knock-downs have stunted growth, reminiscent of other mutants with impaired lignin biosynthesis. Based on thioacidolysis GC-MS, I found that the mutants produced a lignin with enriched hydroxyphenyl (H) subunits, which were derived from precursors upstream of the HCT-catalysed reaction and normally do not occur in Populus lignin. Interestingly, in one of the RNAi lines, the lignin phenotype was uncoupled from the developmental dwarfing phenotype. This is of high interest from a bioethanol perspective, since wood rich in H-lignin is more easily fermented than wood that is rich in guaiacyl (G) and syringyl (S) lignin. Another candidate gene (Potri.018G109900, HCT-E2) was linked to the formation of caffeoyl-spermidine in male catkins (which function in pollen coat formation), and one candidate gene (Potri.018G104700, HCT-C2) was associated with the formation of bioactive, soluble HCCs in leaves and roots. Since RNAi-mediated down-regulation proved ineffective, CRISPR-based gene knock-out methodology was developed and utilised for the Populus hairy root system. Targeted knock-out mutants for the leaf-associated HCT-C2 were generated. HCC identity was determined by metabolite purification and subsequent MS/MS/MS from leaf extracts, and the metabolite concentrations were determined by LC-MS. A decrease in chlorogenic acid concentration was apparent in CRISPR hairy-root knockouts of HCT-C2 indicating that HCTC2 is involved in HCC biosynthesis and can directly produce chlorogenic acid. Candidates for the HCTs involved in lignin biosynthesis, soluble ester biosynthesis, and pollen coat formation were identified and plant genetics confirmed the role of the lignin and soluble ester HCT candidates.



Hydroxycinnamoyl conjugates (HCC)s, Populus, Hydroxycinnamoyl transferases (HCTs)