SUMMARY The salicinoids are anti‐herbivore phenolic glycosides unique to the Salicaceae (Populus and Salix). They consist of a salicyl alcohol glucoside core, which is usually further acylated with benzoic, cinnamic or phenolic acids. While salicinoid structures are well known, their biosynthesis remains enigmatic. Recently, two enzymes from poplar, salicyl alcohol benzoyl transferase and benzyl alcohol benzoyl transferase, were shown to catalyze the production of salicyl benzoate, a predicted potential intermediate in salicinoid biosynthesis. Here, we used transcriptomics and co‐expression analysis with these two genes to identify two UDP‐glucose‐dependent glycosyltransferases (UGT71L1 and UGT78M1) as candidate enzymes in this pathway. Both recombinant enzymes accepted only salicyl benzoate, salicylaldehyde and 2‐hydroxycinnamic acid as glucose acceptors. Knocking out the UGT71L1 gene by CRISPR/Cas9 in poplar hairy root cultures led to the complete loss of salicortin, tremulacin and tremuloidin, and a partial reduction of salicin content. This demonstrated that UGT71L1 is required for synthesis of the major salicinoids, and suggested that an additional route can lead to salicin. CRISPR/Cas9 knockouts for UGT78M1 were not successful, and its in vivo role thus remains to be determined. Although it has a similar substrate preference and predicted structure as UGT71L1, it appears not to contribute to the synthesis of salicortin, tremulacin and tremuloidin, at least in roots. The demonstration of UGT71L1 as an enzyme of salicinoid biosynthesis will open up new avenues for the elucidation of this pathway.

Flavonol bisglycosides accumulate in plant vegetative tissues in response to abiotic stress, including simultaneous environmental perturbations (i.e. nitrogen deficiency and low temperature, NDLT), but disappear with recovery from NDLT. Previously, we determined that a recombinant Arabidopsis β-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-glucosides, forming flavonol 7-O-α-rhamnosides and flavonol aglycones, respectively. In this study, the transient expression of a BGLU15-Cherry fusion protein in onion epidermal cells demonstrated that BGLU15 was localized to the apoplast. Analysis of BGLU15 T-DNA insertional inactivation lines (bglu15-1 and bglu15-2) revealed negligible levels of BGLU15 transcripts, whereas its paralogs BGLU12 and BGLU16 were expressed in wild-type and bglu15 plants. The recombinant BGLU16 did not hydrolyze quercetin 3-O-β-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-β-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-β-glucoside-7-O-α-rhamnoside hydrolase activity, whereas this activity increased by 223% within 2 d of NDLT recovery in wild-type plants. The levels of flavonol 3-O-β-glucoside-7-O-α-rhamnosides and quercetin 3-O-β-glucoside were high and relatively unchanged in shoots of bglu15 mutants during recovery from NDLT, whereas rapid losses were apparent in wild-type shoots. Moreover, losses of two flavonol 3-O-β-neohesperidoside-7-O-α-rhamnosides and kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside were evident during recovery from NDLT, regardless of whether BGLU15 was present. A spike in a kaempferol 7-O-α-rhamnoside occurred with stress recovery, regardless of germplasm, suggesting a contribution from hydrolysis of kaempferol 3-O-β-neohesperidoside-7-O-α-rhamnosides and/or kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside by hitherto unknown mechanisms. Thus, BGLU15 is essential for catabolism of flavonol 3-O-β-glucoside-7-O-α-rhamnosides and flavonol 3-O-β-glucosides in Arabidopsis.

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.

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Remobilization of carbon storage compounds in trees is crucial for the resilience to disturbances, stress, and the requirements of their perennial lifestyle, all of which can impact photosynthetic carbon gain. Trees contain abundant non-structural carbohydrates (NSC) in the form of starch and sugars for long term carbon storage, yet questions remain about the ability of trees to remobilize non-conventional carbon compounds under stress. Aspens, like other members of the genus Populus, have abundant specialized metabolites called salicinoid phenolic glycosides, which contain a core glucose moiety. In this study, we hypothesized that the glucose-containing salicinoids could be remobilized as an additional carbon source during severe carbon limitation. We made use of genetically modified hybrid aspen (Populus tremula x P. alba) with minimal salicinoid content and compared these to control plants with high salicinoid content during resprouting (suckering) in dark (carbon limited) conditions. As salicinoids are abundant anti-herbivore compounds, identification of such a secondary function for salicinoids may provide insight to the evolutionary pressures that drive their accumulation. Our results show that salicinoid biosynthesis is maintained during carbon limitation and suggests that salicinoids are not remobilized as a carbon source for regenerating shoot tissue. However, we found that salicinoid-producing aspens had reduced resprouting capacity per available root biomass when compared to salicinoid-deficient aspens. Therefore, our work shows that the constitutive salicinoid production in aspens can reduce the capacity for resprouting and survival in carbon limited conditions.

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