Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon

Abstract

Background Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called “esters” pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the “reverse” direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an “incomplete” shikimate shunt. Results The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. Conclusions Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen

“…Such downregulation should also increase metabolite flux toward the H units of lignin. Indeed, an increase in H unit levels and lignin reduction have been achieved by HCT ‐suppression in alfalfa ( Medicago sativa L.), loblolly pine ( Pinus taeda L.), Brachypodium distachyon (Serrani‐Yarce et al ., 2021) and poplar ( Populus alba × P. glandulosa ) hybrids (Shadle et al ., 2007; Wagner et al ., 2007; Zhou et al ., 2018). Moreover, fiber‐specific knockout of HCT in Arabidopsis thaliana also induces lignin reduction with elevated H unit levels (Liang et al ., 2019).…”

Phylogenetic Occurrence of the Phenylpropanoid Pathway and Lignin Biosynthesis in Plants

“…Noticeably, CSE has also been shown to be functional in gymnosperms, such as Larix kaempferi (Wang et al, 2019). These findings suggest that CSE may be evolved prior to the divergence of gymnosperms and angiosperms, but was lost in many monocots (Wang et al, 2019;Serrani-Yarce et al, 2021).…”

RNA-Seq Reveals Differentially Expressed Genes Associated with High Fiber Quality in Abaca (Musa textilis Nee)

“…Additionally, KEGG pathway analysis further showed the enrichment of genes associated with the biosynthesis of secondary metabolites, one of which is shikimate O-hydroxycinnamoyltransferase (LOCUS_014669-RA), a key enzyme in lignin biosynthesis [51]. Modification of this gene through RNA interference causes reduction of lignin levels in Brachypodium distachyon [52]. The GO term cellulose synthase activity (molecular function (MF); GO:0016759, GO:0016760) (see Figure S8 for graphical representation) was, likewise, significantly enriched.…”