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 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.

Figure 1

Proposed biosynthetic pathway for salicinoids based on prior work in various Populus or Salix species and the current experiments. Alternate entry points starting with BEBT and SABT are shown. Solid black arrows and blue font indicate established enzymatic reactions (Chedgy et al., 2015; Fellenberg et al., 2020; Kulasekaran et al., 2021); dashed black arrows are proposed biosynthetic steps based on published in vivo labeling experiments (Zenk, 1967; Babst et al., 2010); dashed gray arrows represent known nonenzymatic or enzymatic decomposition steps (Ruuhola et al., 2003; Julkunen-Tiitto and Sorsa, 2001); gray arrows indicate hypothesized biosynthetic reactions. Compounds within the shaded background and purple arrows indicate proposed biosynthetic steps and products based on the current work. Red arrows in the shaded area represent additional reactions deduced to be enhanced in UGT71L1-KO plants based on metabolomic analysis in this study.

Figure 2

Detection of biallelic mutations and indels four independent UGT71L1-knockout lines. Upper panel shows Sanger sequencing traces of the UGT71L1 gRNA target site. The gRNA is underlined, and the magenta dashed line marks the Protospacer Adjacent Motif (PAM) necessary for Cas9 double-strand cleavage. N indicates ambiguous bases in the sequence readout. The lower panels show read-outs of the TIDE (shinyapps.datacurators.nl/tide/) webtool used to detect mutations in the target sequence. The vertical blue dashed line (left panel) indicates the position of the gRNA PAM site, while the vertical green traces indicate variant sequences in chromatograms indicating a mutation in the downstream sequence. Lower right panel shows the number of nucleotides inserted or deleted along the horizontal axis for each mutated line. Lines UGT71-1 to UGT71-3 are biallelic knockouts with frameshifts in both alleles. Line UGT71-4 has mutations in both alleles but only one results in a frameshift. This line is designated as partial-KO.

Figure 3

Nontargeted metabolomic analysis of UGT71L1-KO plants. A, Principal component score plots of non-targeted metabolomic analysis of three independent UGT71L1-KO lines (five biological replicates each), one partial-KO line (four biological replicates), and controls consisting of three empty vector lines and a WT (four or five biological replicates each). B, Unweighted pair group method with arithmetic mean hierarchical cluster analysis of nontargeted metabolomic samples. Multivariate analysis was conducted using Metaboanalyst 5.0 (Chong et al., 2019). Blue color (left-most cluster in A, lowest branch in B) indicates UGT71L1-KO lines, green (middle cluster in A, central branch in B) indicates partial-KO lines, and magenta (right cluster in A, top branch in B) indicates empty vector or WT control lines. C, Orgyia leucostigma feeding preference (third and fourth instar larvae) expressed as percent leaf mass consumed. Leaf disks from two independent UGT71L1-KO lines were pooled for choice bioassays with disks from two independent empty vector lines. The entire choice test was conducted twice for a total of 25 arena assays (error bars indicate �se). *Indicates significant difference (two-tailed t test, P < 0.005).

Figure 4

Phenotypic effects of UGT71L1 disruption in transgenic poplar. A, Fully expanded leaves from 3-month-old greenhouse grown UGT71L1-KO plants (left) and empty vector control plants (right). Scale bar represents 10 cm. B, Abaxial view of fully expanded 3-month-old greenhouse grown UGT71L1-KO leaf. C, Abaxial view of corresponding control leaf. D, Whole plant image of UGT71L1-KO (left) and control (right) plants after 3 months of greenhouse growth. E, Change in plant height for UGT71L1-KO, partial-KO line, and control plants grown in greenhouse for 9 weeks. Data for seven to eight biological replicates each of three independent UGT71L1-KO lines, nine replicates of the partial-KO line, and pooled control samples from three independent empty vector lines and a WT plant (four to five replicates per line) are shown. Data are shown as mean � se.

Figure 5

Rescue of UGT71L1-KO plant phenotype by retransformation with a synthetic UGT71L1 coding sequence. Upper left (top) shows the gRNA target sequence for native UGT71L1 and the corresponding sequence modified to be resistant to the CRISPR/Cas9 construct. Degenerate codons were modified as indicated in gray highlights in the lower nucleotide sequence. Lower left images show two independently transformed 2-month-old rescue plant lines, together with a wild-type and a UGT71L1-KO line. Right show concentrations of major salicinoids and salicylic acid glucoside in expanded leaves of 2-month-old greenhouse-grown poplars. Values shown are mean � se. WT, wild-type with five biological replicates; KO, UGT71L1-KO with four biological replicates. Rescue-1, -2, and -3 represent independently transformed rescue lines, with four to five biological replicates per line. Statistical significance was determined using a t test. *P < 0.05 indicates significant differences from WT plants; **P < 0.01 indicates.

Figure 6

Gene ontology (GO) terms significantly enriched in UGT71L1-KO transcriptome compared to WT analyzed by the PoplarGene webtool. Green bars indicate on left significant enrichment of GO terms in UGT71L1-KO lines (log₂FC >2); magenta bars on right show significant enrichment of GO terms in WT plants (log₂FC >2).

Figure 7

Enzymatic activity of recombinant UGT71L1 and UGT78M1 with potential salicinoid biosynthetic intermediates. Bars show means (n = 3) of relative product formation relative to UGT78M1 glucosylation of salicyl benzoate under standard conditions. Empty vector controls were carried out in duplicate and consisted of extracts from bacterial host strains without the recombinant enzyme. Each assay contained 5-�g protein and 250-�M substrate and was conducted as described in “Materials and methods”. Product formation was quantified by UPLC-UV and absorbance at 280 nm. Data shown are mean � se.

Figure 8

Proposed metabolic and hormonal interactions leading to growth and metabolic phenotypes in UGT71L1-KO plants. Flat magenta arrows represent inhibited responses, and pointed green arrows represent stimulated processes. The interruption of salicinoid biosynthesis by CRISPR/Cas9 is shown with a magenta X, suggested to cause the hyperaccumulation of SA in these plants. The dashed arrow represents a hypothesized but not yet demonstrated stimulation of jasmonate by SA in poplar. Stimulation of biotic stress responses and inhibition of growth reflect the GO categories of enhanced and repressed genes observed in RNA-seq data (Figure�5), as well as the direct effects of coronatine on poplar (Figure�4). Feedback inhibition of the shikimate pathway is hypothesized based on RNA-seq and metabolomics data (Table�5;Supplementary Tables S3 and S4), including the accumulation of possible allosteric regulator caffeic acid.