CRISPR/Cas9 disruption of UGT71L1 in poplar connects salicinoid and salicylic acid metabolism and alters growth and morphology

Abstract

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong anti-herbivore effects found only in poplars and willows. Their biosynthesis is poorly understood but recently a UDP-dependent glycosyltransferase, UGT71L1, was shown to be required for salicinoid biosynthesis in poplar tissue cultures. UGT71L1 specifically glycosylates salicyl benzoate, a proposed salicinoid intermediate. Here we analyzed transgenic CRISPR/Cas9-generated UGT71L1 knock-out plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knock-outs were preferred to wild-type by white tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knock-out plants showed substantial growth alterations, with decreased internode length and smaller serrated leaves. Reinserting a functional UGT71L1 gene in a transgenic rescue experiment demonstrated that these effects were due only to the loss of UGT71L1. The knock-outs contained elevated salicylate (SA) and jasmonate (JA) concentrations, and also had enhanced expression of SA- and JA-related genes. SA is predicted to be released by UGT71L1 disruption, if salicyl salicylate is a pathway intermediate and UGT71L1 substrate. This idea was supported by showing that salicyl salicylate can be glucosylated by recombinant UGT71L1, providing a potential link of salicinoid metabolism to SA and growth impacts. Connecting this pathway with growth could imply that salicinoids are under additional evolutionary constraints beyond selective pressure by herbivores.

Anti-Inflammatory Salicin Derivatives from the Barks of Salix tetrasperma

“…This information coupled with the comparable ECD curve with those of 1 – 3 determined the absolute configuration of 6 as 1′ S , 2′ R , 3′ S , 4′ S , 5′ R , 1″ S , which was corroborated further by TDDFT-ECD analysis (Figure ). The absolute configurations of compounds 4 and 5 were assigned as the same as that of 6 , as deduced from their consistent 1D NMR data of fragment B and similar tendencies of the ECD curves, and supported by TDDFT-ECD analysis of 4 (Figure ), as well as their shared biosynthesis from salicin derivatives. , …”

“…The absolute configurations of compounds 4 and 5 were assigned as the same as that of 6, as deduced from their consistent 1D NMR data of fragment B and similar tendencies of the ECD curves, and supported by TDDFT-ECD analysis of 4 (Figure 3), as well as their shared biosynthesis from salicin derivatives. 49,50 Compounds 7 (salixteroside G) and 8 (salixteroside H) were initially obtained as pale-yellow gum. Their molecular formulas were determined as C 23 H 30 O 12 based on the HRESIMS and 13 C NMR data, implying nine IHDs.…”

Genomic and transcriptomic analyses reveal polygenic architecture for ecologically important traits in aspen (Populus tremuloides Michx.)

“…The genes underlying the salicinoid phenolic glycoside biosynthesis pathway have been largely elusive (Fellenberg et al, 2020 ). Recent studies that have identified and validated candidate genes underlying salicinoid phenolic glycosides have used methods that can better account for a polygenic architecture (Fellenberg et al, 2020 ; Gordon et al, 2022 ). Taken together, our work and these studies underscore the importance of accounting for the polygenic nature of most ecologically important traits in characterizing their genetic architectures accurately.…”

CRISPR/Cas9-based gene activation and base editing in Populus

“…The most widely used programmable RNA-guided DNA endonuclease for genome editing is the type II SpCas9 system derived from Streptococcus pyogenes , which has been applied for targeted mutagenesis in different plant species [ 18 , 20 – 22 ]. CRISPR/Cas9-mediated mutagenesis has also been widely applied in poplars, mainly in the hybrid poplar clone “717-1B4” due to its transformation capacity [ 23 , 24 ]. In addition, knockout mutants can be generated effectively in poplar in the primary transformants [ 25 ].…”