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

Salicinoids are salicyl alcohol-containing phenolic glycosides with strong antiherbivore 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 knockout plants. Metabolomic analyses revealed substantial reductions in the major salicinoids, confirming the central role of the enzyme in salicinoid biosynthesis. Correspondingly, UGT71L1 knockouts were preferred to wild-type by white-marked tussock moth (Orgyia leucostigma) larvae in bioassays. Greenhouse-grown knockout 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 knockouts 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.

Kaempferol rhamnoside catabolism in rosette leaves of senescing Arabidopsis and postharvest stored radish.

Flavonol rhamnosides including kaempferitrin (i.e., kaempferol 3-O-α-rhamnoside-7-O-α-rhamnoside) occur throughout the plant kingdom. Mechanisms governing flavonol rhamnoside biosynthesis are established, whereas degradative processes occurring in plants are relatively unknown. Here, we investigated the catabolic events affecting kaempferitrin status in the rosette leaves of Arabidopsis thaliana L. Heynh. (Arabidopsis) and Raphanus sativus L. (radish), respectively, in response to developmental senescence and postharvest handling. On a per plant basis, losses of several kaempferol rhamnosides including kaempferitrin were apparent in senescing leaves of Arabidopsis during development and postharvest radish stored at 5 °C. Conversely, small pools of kaempferol 7-O-α-rhamnoside (K7R), kaempferol 3-O-α-rhamnoside (K3R), and kaempferol built up in senescing leaves of both species. Evidence is provided for ⍺-rhamnosidase activities targeting the 7-O-α-rhamnoside of kaempferitrin and K7R in rosette leaves of both species. An HPLC analysis of in vitro assays of clarified leaf extracts prepared from developing Arabidopsis and postharvest radish determined that these metabolic shifts were coincident with respective 237% and 645% increases in kaempferitrin 7-O-⍺-rhamnosidase activity. Lower activity rates were apparent when these ⍺-rhamnosidase assays were performed with K7R. A radish ⍺-rhamnosidase containing peak eluting from a DEAE-Sepharose Fast Flow column hydrolyzed various 7-O-rhamnosylated flavonols, as well as kaempferol 3-O-ß-glucoside. Together it is apparent that the catabolism of 7-O-α-rhamnosylated kaempferol metabolites in senescing plant leaves is associated with a flavonol 7-O-α-rhamnoside-utilizing α-rhamnosidase.

Discovery of salicyl benzoate UDP-glycosyltransferase, a central enzyme in poplar salicinoid phenolic glycoside biosynthesis.

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.

An Apoplastic ß-Glucosidase is Essential for the Degradation of Flavonol 3-O-ß-Glucoside-7-O-α-Rhamnosides in Arabidopsis.

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 ß-glucosidase (BGLU), BGLU15, hydrolyzes flavonol 3-O-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-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-ß-glucoside-7-O-α-rhamnoside or rhamnosylated flavonols, but was active with the synthetic substrate, p-nitrophenyl-ß-d-glucoside. In addition, shoots of both bglu15 mutants contained negligible flavonol 3-O-ß-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-ß-glucoside-7-O-α-rhamnosides and quercetin 3-O-ß-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-ß-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-ß-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-ß-glucoside-7-O-α-rhamnosides and flavonol 3-O-ß-glucosides in Arabidopsis.