Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves

Abstract

Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.

Figure 1.

Biosynthetic and proposed biosynthetic pathway for 18-carbon ketols, OPDA, and OPDA-containing MGDG. Analogous pathways are thought to convert 16:3 to dnOPDA and 16-carbon ketols, and dnOPDA to JA via two cycles of β-oxidation. The relationship of OPDA- and dnOPDA-containing MGDG, DGDG, and PG to this process is not clear, as indicated by the question marks, but two possibilities are that (1) trienoic fatty acids are released from galactolipids (or PG), converted to OPDA or dnOPDA, then reincorporated into complex lipids, or (2) conversion of trienoic fatty acids to OPDA or dnOPDA takes place on the complex lipids, and these species are later released.

Figure 2.

CID-TOF spectra of anions from lipids of unwounded (A) and wounded (B; after 5 h) Arabidopsis leaves. Unselected molecular ions in an extract were subjected to CID and analyzed with a TOF analyzer. Table I provides additional information about each peak. Note that the spectra are expanded 10× from approximately m/z 262 to 273 and 5× from 290 to 310. Insets show ranges in which there are two peaks at similar m/z.

Figure 3.

CID-TOF spectra of anions from lipids of unwounded (A) and wounded (B; after 5 h) Arabidopsis leaves after catalytic hydrogenation. Unselected molecular ions in an extract were subjected to CID and analyzed with a TOF analyzer. Table I provides additional information about each peak. Note that the spectra are expanded 20× from approximately m/z 262 to 282 and 5× from 290 to 310. Insets show areas in which there are two peaks at similar m/z.

Figure 4.

Discovery scans: precursor scanning of an unfractionated extract from wounded Arabidopsis. All scans were performed in the negative mode. The peak designations were verified by determination of the exact masses of the acyl product ions as indicated in Supplemental Table I. Most peaks indicate the [M − H]⁻ ions, while peaks indicated by labels in parentheses indicate the [M + OAc]⁻ adducts of MGDG. A, Scan for precursors of m/z 227 indicates PG species (Welti et al., 2003). B, Scan for precursors of m/z 235, a glycero-galactose minus water fragment, indicates MGDG. C, Scan for precursors of m/z 249 or the acyl anion of 16:3. D, Scan for precursors of m/z 277 or the acyl anion of 18:3. E, Scan for precursors of m/z 263 or the acyl anion of dnOPDA. F, Scan for precursors of 291 or the acyl anion of OPDA. G, Scan for precursors of m/z 309 or the acyl anion of the ketols derived from 18:3. The marked species are MGDG unless explicitly designated as PG. Note the relative scales of the sections. In addition, please note that sections A to F have breaks in the vertical scales. The same scales are used in Supplemental Figure 1.

Figure 5.

Three of the OPDA-containing lipid species from leaves of wounded Arabidopsis. Please note that the acyl position assignments and the assignment of the double bond position in the 16:1 acyl chain are not based on mass spectral data in this work. The assignments utilized in producing this figure reflect the work of others (Hisamatsu et al., 2003, 2005), knowledge of the 16- and 18-carbon acyl positional specificity in plastidically produced lipids, and knowledge of the structure of 16:1 typically found in plastidically produced PG (Miquel et al., 1998; Somerville et al., 2000).

Figure 6.

Levels of OPDA and dnOPDA-containing galactolipids in unwounded Arabidopsis. Error bars are sd, n = 5.

Figure 7.

Levels of the major oxylipin-containing MGDG and DGDG species during response to wounding in Arabidopsis leaves. Wounded leaves were collected at the time points indicated, lipids were extracted, and galactolipids containing esterified oxylipins were analyzed using multiple acyl precursor scanning. A, MGDG species. B, DGDG species. The zero time point represents unwounded plants. Error bars are sd, n = 5.

Figure 8.

Ratios of measured galactolipid species containing two oxylipins and galactolipid species containing one oxylipin. For MGDG, ([OPDA-dnOPDA MGDG] + [OPDA-OPDA MGDG])/([OPDA-16:3 MGDG] + [18:3-dnOPDA MGDG] + [OPDA-18:3 MGDG]) and for DGDG, (OPDA-OPDA DGDG)/(OPDA-18:3 DGDG) are plotted as a function of the time after wounding. The zero time point represents unwounded plants. Error bars are sd, n = 5.