UGT76B1, a promiscuous hub of small molecule-based immune signaling, glucosylates N-hydroxypipecolic acid, and balances plant immunity

Abstract

Glucosylation modulates the biological activity of small molecules and frequently leads to their inactivation. The Arabidopsis thaliana glucosyltransferase UGT76B1 is involved in conjugating the stress hormone salicylic acid (SA) as well as isoleucic acid (ILA). Here, we show that UGT76B1 also glucosylates N-hydroxypipecolic acid (NHP), which is synthesized by FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) and activates systemic acquired resistance (SAR). Upon pathogen attack, Arabidopsis leaves generate two distinct NHP hexose conjugates, NHP-O-β-glucoside and NHP glucose ester, whereupon only NHP-O-β-glucoside formation requires a functional SA pathway. The ugt76b1 mutants specifically fail to generate the NHP-O-β-glucoside, and recombinant UGT76B1 synthesizes NHP-O-β-glucoside in vitro in competition with SA and ILA. The loss of UGT76B1 elevates the endogenous levels of NHP, SA, and ILA and establishes a constitutive SAR-like immune status. Introgression of the fmo1 mutant lacking NHP biosynthesis into the ugt76b1 background abolishes this SAR-like resistance. Moreover, overexpression of UGT76B1 in Arabidopsis shifts the NHP and SA pools toward O-β-glucoside formation and abrogates pathogen-induced SAR. Our results further indicate that NHP-triggered immunity is SA-dependent and relies on UGT76B1 as a common metabolic hub. Thereby, UGT76B1-mediated glucosylation controls the levels of active NHP, SA, and ILA in concert to balance the plant immune status.

Figure 1

Accumulation of an NHP-hexoside following treatment with resistance enhancers is dependent on UGT76B1. A–C, Pip, NHP, and m/z 308.1346 content in rosette leaves of 5-week-old Wt, ugt76b1, and a UGT76B1-OE line 48 h after watering with control solution or 1 mM dl-Pip. Rosette leaves were harvested, extracted, and analyzed by LC–MS. Bars are means ± sd, n = 4; significant differences are indicated by letters according to one-way ANOVA (P.adj. < 0.05). D–F, Detection of Pip, NHP, and m/z 308.1346 in leaf extracts of 3-week-old plants sprayed with a control solution or 1 mM BTH. Leaves were harvested 48 h after the treatment and analyzed by LC–MS. Bars are means ± sd, n = 4; significant differences are indicated by letters according to one-way ANOVA (P.adj. < 0.05). G, MS/MS fragmentation of m/z 308.1346 ± 0.005 identified in plant extracts. H, Detection of m/z 308.1346 in extracts of Wt Col and of ugt76b1, fmo1, and fmo1 ugt76b1 mutant lines using LC–MS.

Figure 2

Inoculation with Psm induces accumulation of two distinct NHP-H derivatives dependent on a functional NHP biosynthetic pathway. A, GC–MS analysis reveals the accumulation of two distinct NHP-hexose conjugates (NHP-H1 and NHP-H2) in leaves of Psm-inoculated Col-0 plants. Overlaid ion chromatograms (m/z 172) are shown (blue: Wt, red: ugt76b1). NHP-H1 accumulates independently of UGT76B1 and represents the NHP-hexose-conjugate previously described by Hartmann and Zeier (2018). The accumulation of NHP-H2 is absent in ugt76b1 mutants. Moreover, NHP-H2 is synthesized in vitro by recombinant UGT76B1 (green: full enzyme assay with recombinant UGT76B1, black control assay without enzyme). Sample derivatization by trimethylsilylation was performed prior to GC–MS analyses. B, Mass spectrum of the penta-trimethylsilylated NHP-hexoside NHP-H2 (molecular weight: 667 g mol⁻¹) from plant extracts. The M⁺-CH₃ ion at m/z 652, which produces an m/z 562 ion by loss of Si(CH₃)₃OH, is clearly discernible. The structure of the trimethylsilyl-N-hydroxypiperidine fragment at m/z 172 is indicated. Fragment ions characteristic for per-trimethlysilylated hexose conjugates are m/z 450, m/z 361, m/z 271, m/z 217, and m/z 204 (Ehmann, 1974). Note that the mass spectrum of penta-trimethylsilylated NHP-H1 exhibits similar fragmentation patterns, including a more prominent m/z 172 (Hartmann and Zeier, 2018). C, Both NHP-H2 and NHP-H1 are biosynthetically derived from NHP. Feeding of deuterated D₉-NHP to Psm-inoculated fmo1 plants results, in contrast to feeding with D₉-Pip, in the formation of D₉-labeled NHP-H2 and D₉-labeled NHP-H1. Ion chromatograms of m/z 181 are depicted. The m/z 181 ion corresponds to a D₉-trimethlysilyl-hydroxypiperidine fragment. GC–MS analyses as described in (A) and (B). D, Relative levels of NHP-H2 and NHP-H1 in Psm-inoculated or Mock-treated (MgCl₂-infiltrated) leaves of Wt and ugt76b1 plants at 48 hpi. Data represent the mean ± sd of three biological replicates. Mean levels in Psm-inoculated Col-0 leaves are set to 1. Different letters denote significant differences (P < 0.05, ANOVA and post hoc Tukey HSD test). E and F, Relative levels of NHP-H1 (E) and NHP-H2 (F) in Psm-inoculated or Mock-treated leaves of Wt plants, NHP biosynthesis-defective mutants (ald1, sard4, and fmo1), SA pathway mutants (sid2 and npr1), as well as eds1 and pad4 mutants at 24 hpi. Other details as described in (D).

Figure 3

UGT76B1 glucosylates NHP in vitro. A, LC–MS separation of the in vitro reaction of UGT76B1 (blue curve; control in stippled red) with NHP and UDP glucose producing an NHP glucoside with m/z 308.1346, which shows the correct MS/MS fragmentation pattern (Supplemental Figure S1C). B, NHP inhibits the UGT76B1-dependent SA glucosylation. Means ± sd, n = 3. Additional mutual inhibitions of the UGT76B1 substrates SA, NHP, and ILA are shown in Supplemental Figure S5.

Figure 4

Effects of esterase and β-glucosidase treatments on NHP-H1, NHP-H2, SGE, and SAG. An extract of Arabidopsis leaves inoculated with Psm that accumulated NHP- and SA-conjugates was buffered to pH 6.0 with 0.1 mM sodium phosphate, and aliquots thereof incubated for 15 h with 10 U mL⁻¹ esterase (+), 10 U mL⁻¹ β-glucosidase (+), or with buffer only (−). Samples were analyzed by GC–MS (Figure 2), and amounts of analytes were related to ribitol as internal standard. All four hexose conjugates were stably detectable by GC–MS when incubated overnight at pH 6.0. Values are expressed relative to the means of the buffer only condition (n = 3). Asterisks indicate significant differences between buffer only (−) and enzyme-treatments (^**P < 0.001 and ^*P < 0.01 (two-tailed Student’s t test).

Figure 5

Exogenous ILA enhances the accumulation of SA, Pip, and NHP, and induces expression of FMO1. A and B, Pip and NHP levels of leaves of 12-day-old Wt seedlings 24 and 48 h after incubation in half MS medium without (gray bar) and with 500 µM ILA (black bars). Bars represent means ± sd; n = 3–4. Differences between treated or untreated plants were analyzed by Welch two-sample t test; *P < 0.05. C, Transcript abundance of FMO1 of leaves of 14-day-old plantlets grown in liquid culture was measured by RT-qPCR 48 h after the application of 500 µM ILA. Gene expression was normalized to S16 and UBQ5; bars are means ± sd; n = 4. Differences between treated or untreated plants were analyzed by Welch two-sample t test; ^*P < 0.05. D, SA levels of leaves of 12-day-old Wt seedlings as described for (A and B).

Figure 6

Enhanced accumulation of defense-related transcripts and metabolites by the ugt76b1 mutant is dependent on FMO1. A, RT-qPCR analysis of FMO1 transcript abundance of leaves of 4-week-old Wt and ugt76b1 plants grown under short-day conditions; the normalized relative quantity was determined based on the UBQ5 and S16 internal standards; bars show means ± sd, n = 4; differences between genotypes were analyzed by Welch two-sample t test; *P < 0.05. B–F, Pip- and SA-related metabolites were determined using leaf extracts of 4-week-old Wt, ugt76b1, fmo1, and fmo1 ugt76b1 plants grown under short-day conditions using LC–MS. Bars show means ± sd, n = 4; significant differences between genotypes were analyzed by Welch two-sample t test; ^*P < 0.05 (C) and by one-way ANOVA with post hoc Lincon test as indicated by letters (P.adj. < 0.05) (B and D–F).

Figure 7

Local and systemic immunity is enhanced by ugt76b1 loss-of-function, whereas overexpression of UGT76B1 compromises SAR. A, Susceptibility of ugt76b1 introgressed into fmo1 and NahG sid2 toward Pst DC3000. Four-week-old Wt, ugt76b1, fmo1, fmo1 ugt76b1, NahG sid2, and NahG sid2 ugt76b1 plants were infiltrated with 5 × 10⁴ cfu (OD₆₀₀ = 0.0001) of Pst DC3000. Bacterial growth was monitored after 72 h. Bars are means ± sd of 15 replicates from three independent experiments, each experiment consisting of five biological replicates. The presented values are log₁₀-transformed. Different letters denote significant differences (P < 0.01, ANOVA and post hoc Tukey HSD test). The smaller growth of ugt76b1 in comparison to Wt is ameliorated by the introgression of fmo1 or NahG sid2 (Supplemental Figure S10). B, The ugt76b1 mutant is able to induce SAR, whereas the UGT76B1-OE line is deficient in SAR. To assess SAR, three 1° leaves of a plant were mock-treated or Psm-inoculated (OD₆₀₀ = 0.005). Two days later, three 2° leaves were challenge-infected with a bioluminescent Psm strain (Psm lux; OD₆₀₀ = 0.001), and growth of Psm lux was assessed after 2.5 days by luminescence measurements. Bacterial numbers were determined as rlus per leaf area (rlu cm⁻²). The presented values are log₁₀-transformed. Bars are the mean ± sd of 12 or more replicate leaf samples from 6 to 7 different plants. Different letters denote significant differences (P < 0.01, ANOVA and post hoc Tukey HSD test). Independent experiments yielded similar results with some variability of the SAR phenotype of ugt76b1 (Supplemental Figure S11).

Figure 8

The Psm-induced local and systemic accumulation of NHP, SA, and the respective glucose conjugates is deranged in ugt76b1 knockout and UGT76B1 OE lines. A, Levels of NHP, NHP-H2, NHP-H1, SA, SAG, and SGE in Psm-inoculated or Mock-treated leaves of the indicated Arabidopsis lines at 24 hpi. Bars represent means ± sd of four biological replicates Different letters denote significant differences (P < 0.05, Kruskal–Wallis H test). B, Levels of defense metabolites in 2°-distant leaves upon Psm- or Mock-inoculation of 1° leaves at 48 hpi. Details as described in (A). The accumulation of Pip in these scenarios is depicted in Supplemental Figure S12.

Figure 9

Biochemistry and modulatory action of UGT76B1 in plant basal immunity and SAR. A, In addition to ILA, UGT76B1 glycosylates the two SAR-regulatory metabolites NHP and SA in parallel to form the respective O-β-glucosides NHP-H2 and SAG. The glucosyltransferases UGT74F1 and UGT74F2 also contribute to SA glucosylation. The enzyme catalyzing esterification of NHP to NHP-H1 is not yet characterized. B–E, Model for the function of UGT76B1 as a central hub in the regulation of plant basal immunity and SAR. The model illustrates the four major defense scenarios addressed in this study. Relative metabolite levels are depicted by the size of the letters of aglyca and glucosides, which also subsumes the enhanced or repressed biosynthetic activities. Increasing darkness of the central disc, sizes of letters, and widths of the dark red arrows symbolize the activity of UGT76B1 for each scenario. The strength of immune/SAR signaling is indicated by the sizes of purple arrows. B, Naïve Wt is in a state of contained basal defense. UGT76B1 provides a metabolic hub controlling the levels of the unconjugated, immune-stimulating NHP and SA. Both substrates can be alternatively glucosylated by UGT76B1 to the putatively inactive SAG and NHP-H2. Thereby, the mutual amplification loops of NHP and SA (+) and, consequently, basal defense are contained. ILA is an additional, competing substrate that inhibits SAG and NHP-H2 formation. C, The loss of UGT76B1 glucosylation releases this control. NHP and SA mutually enforce each other and promote a SAR-like, NHP- and SA-dependent enhanced basal immune status. The loss of UGT76B1 abolishes the formation of ILA glucoside and NHP-H2, whereas SAG and SGE are still produced due to the presence of other Arabidopsis SA glucosyltransferases. In addition, the elevated NHP level is accompanied by the enhanced formation of the UGT76B1-independent NHP-H1. D, The pathogen-induced activation of FMO1-mediated NHP biosynthesis and ICS1-regulated SA biosynthesis triggers SAR. In the Wt, UGT76B1 is transcriptionally induced as well. This inducible UGT76B1 expression modulates the levels of free NHP and SA and thereby dampens immune signaling once SAR is activated. E, In UGT76B1-OE plants, a constitutively de-regulated UGT76B1 expression shifts the basal und pathogen-inducible NHP and SA metabolic pools toward O-β-glucoside (NHP-H2, SAG) formation. This compromises NHP and SA accumulation, reduces basal immunity, and abrogates SAR.