CRISPR-Cas9 helps solve a piece of the puzzle of the biosynthesis of salicinoids and suggests a role in the growth-defense trade-off in poplar

Willow tree bark has been in use as a reliever of pain and fever for around 3,500 years (Desborough and Keeling, 2017). Unknown to our ancient ancestors, willow bark contains salicin, a salicinoid compound that is the original source of aspirin. Salicinoids are phenolic glycosides found only in willows and poplars. They are plant defense compounds and help willow and poplar trees to deter herbivores. Despite their medicinal value and importance in plant defense, salicinoid biosynthesis remains puzzling. Recent studies have provided a clue that a UDP-glycosyltransferase (UGT71L1) is essential for salicinoid biosynthesis in poplar (Fellenberg et al., 2020; Kulasekaran et al., 2021). In this issue, Harley Gordon and coauthors (Gordon et al., 2022) generated CRISPR-Cas9-mediated UGT71L1 knock-out mutants of poplar and revealed a central role of UGT71L1 in salicinoid biosynthesis and herbivore defense (see Figure).

Figure.

Schematic representation of the function of UGT71L1 in salicinoid biosynthesis, herbivore defense, growth, and metabolism. Photographs of plants (lower left) and graph of increased herbivore vulnerability (lower right) adapted from Gordon et al. (2022), Figures 5 and 3C, respectively.

Gordon et al. used both targeted and non-targeted metabolomic analysis of the knock-out poplars to study the impact of the loss of UGT71L1 on the concentration of the major salicinoids in leaves. Compared with control plants, mutants showed >90% reductions in tremulacin and salicortin concentrations, while salicin exhibited an 80% reduction. These data suggest a strong role for UGT71L1 in salicinoid biosynthesis. However, concentrations of other salicinoids such as salicin-7-sulfate, salireposide, and trichocarpin were not affected by the loss of UGT71L1 function. This finding, together with the existence of low concentrations of tremulacin, salicortin, and salicin in mutants, indicates the existence of parallel pathways and involvement of additional UDP-glycosyltransferases (UGTs). Recombinant UGT71L1 was shown to glucosylate potentially salicinoid biosynthetic intermediates such as salicyl salicylate, benzyl salicylate, and salicyl benzoate. Gordon et al. propose that UGT71L1 glucosylates salicyl salicylate to produce salicortin.

Strikingly, the UGT71L1 knock-out plants displayed severely reduced leaf and plant size. To test whether the dwarf phenotype is due to UGT71L1 disruption alone, the authors carried out a rescue experiment by retransforming the mutant with a functional copy of UGT71L1. Rescued plants were physically identical to the wild-type plants and showed full restoration of salicin, salicortin, and tremulacin biosynthesis. This result showed that the growth phenotype was due to the loss of function of UGT71L1 and not due to possible off-target effects of the CRISPR-Cas construct. Intriguingly, analyses of plant hormones and growth regulators revealed that salicylic acid (SA) and SA-glucoside were 10-fold higher in UGT71L1 knock-out poplars than in wild-type plants. If we assume that the disruption of UGT71L1 prevents the conversion of salicyl salicylate to salicortin, the accumulation of SA in the mutants might be explained as excess salicyl salicylate readily hydrolyzing to salicyl alcohol and free SA. Similarly, jasmonic acid (JA) and JA-isoleucine were strongly elevated in knock-out plants. Transcriptome data also showed upregulation of JA biosynthesis and metabolism genes in mutants. Since both SA and JA are known to inhibit growth as part of a defense-growth trade-off, Gordon et al. hypothesize that the combined effect of SA and JA overaccumulation on growth is most likely the principal cause of reduced leaf size and plant height of UGT71L1 knock-out plants. This was further supported by the observation of the growth inhibiting effect of exogenous application of the JA analog coronatine. Since SA and JA often show antagonism in other plants, the parallel increase in SA and JA in UGT71L1-knockout poplars is worth further investigation.

Gordon et al. further showed that herbivore defense was compromised in UGT71L1 knock-out lines. White tussock moth (Orygia leucostigma) larvae, a tree feeding lepidopteran, consumed significantly more leaves from knock-out lines than wild-type leaves. Since salicinoids provide defense against both mammalian and insect herbivores, it is assumed that they are under selection pressure by herbivores. However, the current findings of severe growth impact due to salicinoid pathway disruption could imply that salicinoids are under very strong selection pressure, beyond herbivore pressure, for maintenance through evolutionary time.

Hence, Gordon and coworkers generated valuable information for several ecological and evolutionary theories of plant resistance and growth. The study also indicated a tight connection of salicinoid biosynthesis (secondary metabolic pathways) to primary metabolism and growth. In this context, it is interesting to note that misregulation of the salicinoid pathway can give rise to a plant hormone (SA). Although this study provides evidence of the function of UGT71L1 in the synthesis of salicortin through glucosylation, how the proposed intermediate salicyl salicylate glucoside is transformed into salicortin and other salicinoids remains elusive.