Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity

Abstract

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/μ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.

Figure 1.

ALD1-mediated l-Lys conversion: formation of 2,3-DP by abstraction of the α-NH₂ group of l-Lys. The GC-MS (and GC-FTIR) analyses of plant extracts or assay samples included the methylation of carboxyl groups as a derivatization strategy (procedure A). A, Segment of overlaid ion chromatograms (m/z = 108) of GC-MS-analyzed extracts from mock- or Psm-inoculated leaves of Col-0 wild-type and ald1 mutant plants. Leaf samples were harvested 48 h post inoculation (hpi). A molecular species (1a) with m/z 108 (or m/z 126 or 141) is present exclusively in the Col-0-Psm samples. B, Segment of overlaid ion chromatograms (m/z = 108) of GC-MS-analyzed l-Lys conversion assays with ALD1 protein, l-Lys conversion assays with LysOx in the presence of catalase (Supplemental Fig. S2), and leaf extracts from Psm-inoculated Col-0 plants. Retention times of the molecular species with m/z 108 (or m/z 126 or 141) in the different samples are identical. C, At left, the mass spectra of the compound 1a derived from extracts of Psm-inoculated Col-0 plants (green), ALD1 in vitro assays (blue), and LysOx/catalase in vitro assays (red) are identical. At right, chemical structures of l-Lys and plausible structures for 1a, as deduced from the mass spectrometric information collected so far. The molecular ion (M⁺) and plausible ion fragments are indicated. The methyl group (red) is introduced by derivatization. D, The use of l-Lys-6-¹³C,ε-¹⁵N as a substrate in the ALD1 in vitro assay reveals retention of the ε-nitrogen and abstraction of the α-nitrogen in the transamination reaction leading to DP products. At left, a mass spectrum of the isotope-labeled product with shifts in fragmentation pattern by 2 mass units compared with unlabeled 1a. At right, isotope-labeled 2,3-DP-methylester is depicted as a plausible structure. The same result was observed for the LysOx/catalase assay. E, The use of l-Lys-4,4,5,5-d₄ as a substrate in the ALD1 in vitro assay excludes the formation of DP isomers with double bonds in the 3,4-, 4,5-, or 5,6-position. At left, a mass spectrum of the isotope-labeled product with shifts in fragmentation pattern by 4 mass units compared with unlabeled 1a. At right, isotope-labeled 2,3-DP-methylester is depicted as a plausible structure. The same result was observed for the LysOx/catalase assay. F, GC-FTIR analysis of 1a indicates its enaminic structure, supports its identity as methylated 2,3-DP, and excludes the ketiminic 1,2-DP derivative as a possible structure. The IR spectrum of 1a is depicted (wave numbers from 4,000 to 600 cm⁻¹). Assignments of IR absorption bands to functional groups are given in the box at right.

Figure 2.

Summarized scheme of Pip biosynthesis from l-Lys in Arabidopsis. The biochemical pathway supported by the experimental data of this study is represented by arrows with solid lines. Detected Lys-derived compounds in extracts and in vitro assays are framed. Arrows with dashed lines represent likely biochemical scenarios, supported from literature findings. Arabidopsis ALD1 first catalyzes a transamination step that transfers the α-amino group of l-Lys to an acceptor oxoacid (preferentially pyruvate). Thereby, KAC and Ala are formed. A subsequent dehydrative cyclization of KAC produces the ketimine 1,2-DP, which isomerizes to the enamine 2,3-DP. 2,3-DP is detected in plant extracts and in vitro assays as the ALD1-derived product. Arabidopsis SARD4 (alias ORNCD1) then reduces a DP isomer to Pip. Since the human SARD4 homolog CRYM has been described as a ketimine reductase, it is possible that the reduction takes place via 1,2-DP, which is supposed to be in chemical equilibrium with 2,3-DP. Alternatively, a direct reduction of the detected intermediate 2,3-DP might take place. Since the sard4-5 knockout line is still able to biosynthesize (reduced amounts of) Pip, an alternative reductive mechanism capable of generating Pip from DP intermediates supposedly exists in plants. The hypothetical pathway illustrated with dotted arrows (top), proceeding via abstraction of the ε-amino group, and the formation of AAS and 1,6-DP can be ruled out on the basis of our data.

Figure 3.

ALD1-mediated l-Lys conversion: formation of 2,3-DP as determined by GC-MS analysis using propyl chloroformate derivatization (procedure B). A, Segment of overlaid ion chromatograms (m/z = 255) of GC-MS-analyzed l-Lys conversion assays with ALD1 protein showing the propyl chloroformate-derivatized transamination product 1b. Red, ALD1 enzyme assay with l-Lys as an amino acid substrate and pyruvate as an acceptor oxoacid; green, control assay lacking ALD1 protein; blue, control assay lacking l-Lys as a substrate. B, Segment of overlaid ion chromatograms (m/z = 255) from extract samples of mock- or Psm-inoculated Col-0 wild-type and ald1 mutant leaves. Leaf samples were harvested 48 hpi. Substance 1b accumulates in the Col-0-Psm samples only. C, Mass spectrum and molecular structure of substance 1b derived from ALD1-mediated l-Lys conversion assays. The mass spectral information is consistent with a 2,3-DP derivative that is propyl carbamylated at its amino group and propyl esterified at its carboxyl group. The groups introduced by propyl chloroformate derivatization are labeled in red. The molecular ion (M⁺) and plausible ion fragments are indicated. D, The use of l-Lys-6-¹³C,ε-¹⁵N as a substrate in ALD1 in vitro assays results in the formation of an isotope-labeled variant of 1b with shifts in the mass spectral fragmentation pattern by 2 mass units compared with unlabeled 1b. E, The use of l-Lys-4,4,5,5-d₄ as a substrate in ALD1 assays results in the formation of an isotope-labeled variant of 1b with shifts in the fragmentation pattern by 4 mass units compared with unlabeled 1b. F, Quantification of 2,3-DP in leaf extracts from Col-0 and ald1 plants. Leaves were mock (MgCl₂) infiltrated or Psm inoculated and harvested at 48 hpi. Bars represent means ± sd of three biological replicate samples, each consisting of six leaves. FW, Fresh weight. A correction factor for the absolute quantification of 2,3-DP was estimated as described in “Materials and Methods.” Different letters above the bars denote statistically significant differences (P < 0.001, ANOVA and posthoc Tukey’s honestly significant difference [HSD] test).

Figure 4.

ALD1 transfers the α-NH₂ group from l-Lys to acceptor oxoacids. A, ALD1 in vitro assay with l-Lys as the substrate amino acid and pyruvate as the acceptor oxoacid results in the formation of Ala as the product amino acid. The mass spectrum of Ala after propyl chloroformate derivatization and the molecular structure of the product are shown. The M⁺ ion and the main fragment ion are indicated (dark red). The groups introduced by propyl chloroformate derivatization are labeled in blue. B, ALD1 assay with isotope-labeled l-Lys-α-¹⁵N as the amino acid substrate and pyruvate as the acceptor oxoacid results in the formation of α-¹⁵N-labeled Ala. For details, see A. C, ALD1 assay with l-Lys as the substrate amino acid and α-ketoglutarate as the acceptor oxoacid results in the formation of Glu as the product amino acid. For details, see A. D, ALD1 assay with isotope-labeled l-Lys-α-¹⁵N as the amino acid substrate and α-ketoglutarate as the acceptor oxoacid results in the formation of α-¹⁵N-labeled Glu. For details, see A. E, Relative activities of ALD1 toward l-Lys and glyoxylate (glyo), pyruvate (pyr), oxaloacetate (oxalo), or α-ketoglutarate (α-KG) as the acceptor oxoacid. The formation of the corresponding product amino acid (Gly for glyoxylate, Ala for pyruvate, Asp for oxaloacetate, and Glu for α-ketoglutarate) was determined after 30 min of incubation time for activity assessments.

Figure 5.

ALD1 catalyzes the conversion of DAP to 6-carboxy-2,3-DP in vitro, but the conversion does not occur to detectable levels in planta. A, Reaction scheme of the in vitro conversion of l,l-DAP or meso-DAP to 6-carboxy-2,3-DP (2) by ALD1. B, In vitro ALD1 activity assays with l-Lys as the amino acid substrate (red), l,l-DAP as the amino acid substrate (black), or no amino acid substrate (blue). Pyruvate served as the acceptor oxoacid in all cases. Overlaid ion chromatograms (m/z 108) are depicted after applying workup procedure A. The m/z 108 ion occurs both in the l-Lys-derived 2,3-DP product (1a) and in the l,l-DAP-derived 6-carboxy-2,3-DP product (2a). C, Mass spectrum and chemical structure of 2a obtained from 6-carboxy-2,3-DP (2) by procedure A. The two methyl groups (blue) are introduced by derivatization. The molecular ion (M⁺), plausible ion fragments, and fragment losses are indicated. D, Mass spectrum and chemical structure of 2b obtained from 6-carboxy-2,3-DP (2) by procedure B. The propyl and propyl carbamate groups introduced by derivatization are depicted in blue. The molecular ion (M⁺), plausible ion fragments, and fragment losses are indicated. E, Segments of overlaid ion chromatograms from extract samples of Psm-inoculated Col-0 wild-type leaves, as analyzed by GC-MS procedure A. Leaf samples were harvested 48 hpi. Whereas the presence of l-Lys-derived 2,3-DP (1a) in the extracts evokes characteristic ion chromatograms (m/z 141, 126, and 108) with the expected ratios of abundance (Fig. 1C) at a retention time (RT) of 12 min (left), 6-carboxy-2,3-DP (2a; m/z 199, 140, and 108; Fig. 5C) is not detected at the supposed retention time of 17.65 min (right).

Figure 6.

The ald1 mutant is able to convert exogenously supplied 2,3-DP to Pip in response to P. syringae inoculation. A, Segment of overlaid ion chromatograms (m/z = 170) from leaf extract samples of differently treated ald1 mutant plants. Leaves were coinfiltrated with Psm and 2,3-DP obtained from ALD1 in vitro assays (black; Fig. 3C). The assays containing 20 mm l-Lys as a substrate were run to completion (greater than 99% of Lys used), enzyme inactivated, and diluted 6-fold with 10 mm MgCl₂. The mixture was then supplied with the bacterial suspension to a final OD₆₀₀ of 0.005 and infiltrated into three leaves of 5-week-old ald1 plants. At 48 h later, the infiltrated leaves were harvested and processed by analytical procedure B. Coinfiltration of the assay mixture not containing ALD1 enzyme (green), MgCl₂ (mock) infiltration alone (blue), and Psm treatment alone (red) served as the control treatments. The peak at 11.8 min is observed only in the Psm/2,3-DP sample (black) and corresponds to derivatized Pip. Similar results were obtained when 2,3-DP generated by the LysOx/catalase assay was used. B, Mass spectrum of propyl chloroformate-derivatized Pip (compare Návarová et al., 2012) derived from 2,3-DP-supplemented and Psm-inoculated ald1 leaves (peak at 11.8 min in A). The groups introduced by derivatization are labeled in red. C, Segment of overlaid ion chromatograms (m/z = 174) from leaf extract samples of differently treated ald1 mutant plants. Leaves were coinfiltrated with Psm and d₄-labeled 2,3-DP obtained from ALD1 in vitro assays (black; Fig. 3E), coinfiltrated with Psm and the assay mixture not containing 2,3-DP (green), infiltrated with mock (10 mm MgCl₂) solution alone (blue), or infiltrated with Psm alone. Further experimental details are identical to those described in A. The peak at 11.76 min observed only in the Psm/d₄-2,3-DP sample (black) corresponds to derivatized d₄-Pip. D, Mass spectrum of propyl chloroformate-derivatized d₄-Pip derived from d₄-2,3-DP-supplemented and Psm-inoculated ald1 leaves (peak at 11.76 min in C). E, Segment of overlaid ion chromatograms (m/z = 174) from leaf extract samples of differently treated Col-0 wild-type plants. Plants were coinfiltrated in leaves with 5 mm l-Lys-4,4,5,5-d₄ (d₄-Lys) and Psm (OD₆₀₀ = 0.005; black), coinfiltrated with 5 mm d₄-Lys and mock (10 mm MgCl₂) solution (green), infiltrated with mock solution alone (blue), or infiltrated with Psm alone. The peak at 11.76 min accumulating in the Psm/d₄-Lys sample (black) corresponds to derivatized d₄-Pip. It is noteworthy that, in addition to labeled Pip, Psm/d₄-Lys-treated Col-0 plants also accumulate endogenous, unlabeled Pip. F, Mass spectrum of propyl chloroformate-derivatized d₄-Pip derived from d₄-Lys-supplemented and Psm-inoculated Col-0 leaves (peak at 11.76 min in E).

Figure 7.

Coupled assays with ALD1 and the human reductase CRYM or its Arabidopsis ortholog SARD4 (ORNCD1) yield Pip in vitro with l-Lys as a substrate. Reactions were carried out in an assay mix (200 µL) containing purified recombinant enzymes (ALD1, CRYM, and SARD4) at a final concentration of 100 µg mL⁻¹ with 20 mm l-Lys, 20 mm pyruvate, 5 mm MgCl₂, 100 µm PLP, and 200 µm NADH in 20 mm Tris buffer, pH 8. All reactions contained 5% (v/v) glycerol for additional enzyme stability and were incubated at 37°C for 16 h. At the end of the incubation period, the reaction was stopped by inactivating the enzymes at 85°C for 10 min. The formation of 2,3-DP and Pip was monitored using GC-MS after derivatization of the assays with trimethylsilyl diazomethane (procedure A) or propyl chloroformate (procedure B), respectively. From left to right are no enzyme (control), ALD1 single-enzyme assay, CRYM single-enzyme assay, ALD1 and CRYM coincubation, SARD4 single-enzyme assay, and ALD1 and SARD4 coincubation. The amounts of generated 2,3-DP and Pip products are given in nmol. Bars represent means ± sd of three replicate incubations (n.d., not detected).

Figure 8.

Transcript levels of SARD4 and metabolite levels of 2,3-DP and Pip in inoculated (1°) and distal (2°) leaves of wild-type Col-0, sard4-5, and sard4-6 mutant plants in response to P. syringae and mock treatments. A, SARD4 transcript levels in 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated with Psm (OD₆₀₀ = 0.005) or with 10 mm MgCl₂ (mock treatment) at 24 h after treatment. Transcript values are given in µg µL⁻¹ and represent means ± sd of at least three biological replicate samples from different plants, each replicate consisting of six leaves. Values for biological replicates were calculated as means of two technical replicates. Different letters above the bars denote statistically significant differences (P < 0.002, ANOVA and posthoc Tukey’s HSD test); n.d., no transcripts detected. The results were confirmed in two independent experiments. B, SARD4 transcript levels in untreated distal (2°) leaves of Col-0, sard4-5, and sard4-6 plants infiltrated in 1° leaves with Psm (OD₆₀₀ = 0.005) or 10 mm MgCl₂ (mock treatment) at 48 h after treatment. Other experimental details were as described for A. Different letters above the bars denote statistically significant differences (P < 0.002, ANOVA and posthoc Tukey’s HSD test); n.d., no transcripts detected. The results were confirmed in two independent experiments. C, Levels of Pip in treated 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated with Psm (OD₆₀₀ = 0.005) or 10 mm MgCl₂ (mock treatment) at 24 h (left graph) and 48 h (middle graph) after treatment and in untreated distal (2°) leaves at 48 h after treatment of 1° leaves (right graph). Data represent means ± sd of at least three biological replicates from different plants, each replicate consisting of six leaves from two plants. Different letters above the bars denote statistically significant differences (P < 0.05, ANOVA and posthoc Tukey’s HSD test). The results were confirmed in two independent experiments. FW, Fresh weight. D, Levels of 2,3-DP in inoculated (1°) and distal (2°) leaves of Col-0, sard4-5, and sard4-6 plants. Experimental details were as described for C. The results were confirmed in two independent experiments. E, Levels of Pip in untreated 2° leaves of Col-0, sard4-5 (s4-5), and sard4-6 (s4-6) plants infiltrated with Psm (OD₆₀₀ = 0.005) or 10 mm MgCl₂ at 24, 48, 72, and 96 h after treatment. Data represent means ± sd of three biological replicates. Asterisks denote statistically significant differences between Psm- and mock-treated samples of a given plant genotype and time point (P < 0.01, two-tailed Student’s t test).

Figure 9.

Basal resistance responses, SAR, Pip-induced resistance, and 2,3-DP-induced resistance in wild-type Col-0, sard4-5, sard4-6, and/or ald1 mutant plants. A, Basal resistance to P. syringae of Col-0, sard4-5, sard4-6, and ald1 plants. Naive plants (three leaves each) were inoculated with bioluminescent Psm lux (OD₆₀₀ = 0.001), and bacterial growth was assessed 60 h later by luminescence and expressed as relative light units (rlu) per cm² of leaf area. Data represent means ± sd of the growth values of at least 15 leaf replicates. Different letters above the bars denote statistically significant differences (P < 0.05, ANOVA and posthoc Tukey’s HSD test). The results were confirmed in three other independent experiments. B, SAR in Col-0, sard4-5, and sard4-6 plants. Three lower, 1° leaves per plant were infiltrated with either 10 mm MgCl₂ or Psm (OD₆₀₀ = 0.005), and three upper, 2° leaves were challenge infected with Psm lux (OD₆₀₀ = 0.001) 2 d later. Growth of Psm lux in 2° leaves was assessed 60 h after 2° challenge inoculation by luminescence measurements. Experimental details and statistical analyses were as described for A. The results were confirmed in two other independent experiments. C, Levels of SA in treated 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated with Psm (OD₆₀₀ = 0.005) or 10 mm MgCl₂ (mock treatment) at 24 h (left graph) and in untreated distal (2°) leaves at 48 h after treatment of 1° leaves (right graph). Data represent means ± sd of at least three biological replicates from different plants, each replicate consisting of six leaves from two plants. Different letters above the bars denote statistically significant differences (P < 0.05, ANOVA and posthoc Tukey’s HSD test). The results were confirmed in two other independent experiments. FW, Fresh weight. D, Pip-induced resistance in Col-0, sard4-5, and sard4-6 plants. Plants were each supplied with 10 mL of 1 mm Pip (dose of 10 µmol) or with 10 mL of water (control treatment) via the root system (Návarová et al., 2012), and three leaves per plant were challenge infected 1 d later with Psm lux (OD₆₀₀ = 0.001). Bacterial growth was assessed 60 hpi and analyzed as described for A. The results were confirmed in two other independent experiments. E, 2,3-DP-induced resistance in Col-0 and ald1 plants. Three leaves per plant were coinfiltrated with 2,3-DP (∼2 mm) obtained by the ALD1 in vitro assay and Psm lux (OD₆₀₀ = 0.001), and bacterial growth was assessed 60 h later. The determination of bacterial growth and statistical analysis were performed as described for A. The results were confirmed in two other independent experiments.