CRISPR‐Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba

Abstract

Summary Lignins are cell wall‐located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR‐Cas9‐generated cse1 and cse2 single mutants had a wild‐type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced‐lignin content translated into a fourfold increase in cellulose‐to‐glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p‐coumaroyl, 5‐hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl‐shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar

“…Unlike the report of de Vries et al (2021) [44], our CSE1-sg2 and CSE2-sg3 poplars had significantly reduced lignin levels (up to 29.1%) and thus showed a dramatic increase in saccharification efficiency (Figure 6a). It is not clear why the results are different at this point, but perhaps the different species and the different target sites of CRISPR might also be the reason.…”

“…Reducing CSE function has been proven to produce better biomass feedstock by reducing the recalcitrance of Arabidopsis and hybrid poplar to high saccharification [15,22]. Very recently, de Vries et al (2021) [44] reported CRISPR-Cas9 editing of CSE in Populus tremula × P. alba, an approach very similar to that used in this study. However, in their study, CRISPR-Cas9-generated cse1 and cse2 single mutants had no significant phenotype and a wild-type lignin level; only cse1 cse2 double mutants showed a reduction in lignin (35%) with a severe growth penalty.…”

“…However, in their study, CRISPR-Cas9-generated cse1 and cse2 single mutants had no significant phenotype and a wild-type lignin level; only cse1 cse2 double mutants showed a reduction in lignin (35%) with a severe growth penalty. The cse1 cse2 double mutants had a four-fold increase in cellulose-to-glucose conversion upon limited saccharification [44].…”

Phylogenetic Occurrence of the Phenylpropanoid Pathway and Lignin Biosynthesis in Plants

“…In addition to causing a 35% reduction in lignin content, the cse1/cse2 double mutant significantly improved celluloseto-glucose transformation efficiency. As such, CSEs in Populus could be promising target genes in biorefinery although their growth penalty should be managed to avoid (de Vries et al, 2021). Noticeably, CSE has also been shown to be functional in gymnosperms, such as Larix kaempferi (Wang et al, 2019).…”

“…Recently, a ccr2 mutant was generated by the CRISPR/Cas9 approach that contain a null and haplo-insufficient allele in Populus. This mutant line does not have growth penalty, but still has low lignin content and improved saccharification efficiency (De Meester et al, 2020). Therefore, CCR2 gene could be a useful target that can be deployed in genetic engineering of bioenergy woody crops.…”