Mobilization of storage lipids is crucial for the development of oil seedlings though it provides energy and carbon supply until autotrophic metabolism is established . Therefore, the degradation of the lipid body phospholipid monolayer by a patatin-type lipase may allow access for different enzymes to the TAG-fraction . The classical pathway of storage lipid mobilization may then be initiated by the TAG lipase-dependent release of free fatty acids like LA from the lipid bodies and subsequent transfer to glyoxysomes where activation to the respective acyl-CoA and β-oxidation takes place [1, 6]. Though LA contains two cis-double bonds and the canonical β-oxidation reaction cascade only deals with trans-double bonds, two additional enzymes (Δ3,Δ2-enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase) are necessary for its degradation .
Besides this classical TAG lipase-initiated pathway, an additional LOX-initiated pathway seems to exist . A lipid body specific 13-LOX oxidizes esterified LA in the TAG-fraction and 13-HPOD is subsequently released from the lipid bodies, reduced to 13-HOD and transported to the glyoxysomes . While the 13-LOX-mediated mobilization and release of 13-HOD during germination has been described for sunflower, linseed and cucumber , the knowledge on the degradation of this oxylipin remained incomplete. In a recent in vitro study it was shown, that 13-HOD can efficiently be activated to 13-HOD-CoA by glyoxysomes isolated from sunflower seedlings. However, the absence of small chain acyl-CoAs and the apparent stable accumulation of two intermediates suggested a stop of degradation after two rounds of β-oxidation and prompted the authors to the assumption, that the hydroxy-diene system might constitute a general barrier for the β-oxidation machinery .
In the present study the protocol for the separation of β-oxidation intermediates was optimized and a methodology for the identification of these compounds has been developed. As reported by Gerhardt and coworkers before , the accumulation of two prominent intermediates with apparently higher polarity than 13-HOD-CoA was observed (Figures 4 and 5, retention times ~ 96 and 92 min). Using mass spectrometry the before made assumption that these intermediates are metabolites after one and two cycles of β-oxidation of 13-HOD-CoA was confirmed. Interestingly, these species didn’t constitute the end products of 13-HOD degradation in our experiments. They disappeared with prolonged reaction times and three main classes of other intermediates were detected. Strikingly in both systems, glyoxysomes from cucumber and sunflower seedlings, the formation of short chain acyl-CoAs (saturated acyl-CoAs with chain length between 4 and 12) lacking the hydroxy-diene system were detected. Most importantly, their formation was only detected upon addition of exogenous 13-HOD. This finding clearly demonstrates the general ability of glyoxysomes from these plants for the complete degradation of 13-HOD-CoA.
Moreover, these identified intermediates during 13-HOD degradation allow now to suggest a modified degradation path of this oxylipin (Figure 2, pathway in the middle). Three rounds of classical β-oxidation reactions yield 7-hydroxy-dodecadienoyl-CoA as the last intermediate which still contains the initial hydroxy-diene system. Beside intermediates after the complete first (11-hydroxy-hexadecadienoyl-CoA), second (9-hydroxy-tetradecadienoyl-CoA) and third cycle (7-hydroxy-dodecadienoyl-CoA) also masses that correspond to the respective dihydroxy-intermediates have been detected. They most likely correspond to the 3-hydroxy β-oxidation intermediates of this oxylipin (i.e. third compound from above in Figure 2 and compound with a retention time of ~ 96 min in Figure 3). For the further conversion three pathways are theoretically possible. The first depends on known activities, which successively degrade the hydroxy-diene system in rounds 4 to 6 of β-oxidation (Figure 2, left path) as has been suggested before [13, 18, 19]. The conjugated hydroxy-diene system of 7-hydroxy-dodecadienoyl-CoA is converted by cis-Δ3-enoyl-CoA isomerase and Δ3,Δ2-enoyl-CoA isomerase into classical β-oxidation intermediates. This pathway can be now excluded as it cannot explain the presence of the hydroxy-dodecadienoyl-CoA intermediate and the completely reduced intermediates dodecanoyl-CoA, decanoyl-CoA and octanoyl-CoA that were newly detected in the presented experiments (Figures 4 and 5).
The presence of hydroxy-dodecanoyl-CoA may theoretically be explained, if the conjugated double bond is completely reduced at the C12-stage without conversion of the hydroxy-group at C7 (Figure 2, right path). The resulting 7-hydroxy-dodecadienoyl-CoA could then in principle be further degraded using classical β-oxidation enzymes. However, this alternative route also implies the presence of hydroxylated intermediates with a chain length shorter than C12 and again cannot explain the presence of dodecanoyl-CoA, decanoyl-CoA and octanoyl-CoA. These intermediates can only be explained if the hydroxy-diene system is completely degraded at the C12-stage (Figure 2, middle path). The resulting dodecanoyl-CoA is then completely degraded using normal β-oxidation reactions and is the only path that is supported by our data.
Besides the similar formation of saturated short chain intermediates, the glyoxysomes from cucumber and sunflower differ in the formation of some intermediates. A huge quantity of oxidized free fatty acid intermediates were found during turnover of 13-HOD by glyoxysomes from cucumber indicating an exit from β-oxidation during the first two rounds. Contrary, the transformation of the hydroxy-diene system into a keto-diene system was the major observation when organelles from sunflower were used. Both observations can explain the insufficient formation of only three to four (instead of eight theoretical possible, Figure 1) molecules of NADH from each molecule 13-HOD in the optical assay. The 13-KOD derivatives may be in this way of special interest, as they bear a reactive α,β-unsaturated carbonyl group. Such compounds are termed reactive electrophilic species and this group includes not only fatty acid oxygenation products, but also secondary metabolites, products of hem metabolism, and many others . Reactive electrophilic species like the oxylipins 12-oxo phytodienoic acid and hexenal stimulate the expression of survival genes that are commonly up-regulated during environmental stress and pathogenesis . Formation of keto-dienes has been reported as response to pathogen infection of Arabidopsis leaves, where these compounds may act as phytoalexins . High concentrations of these metabolites may have toxic effects though they inactivate enzymes or change the redox state of the cell due to the reaction with nucleophilic compounds like thiols . Therefore, it is tempting to assume that this inactivation may be the reason for the weak ability of sunflower and cucumber glyoxysomes to degrade 13-KOD directly. This finding also suggests, that 13-KOD is not a necessary on-pathway intermediate for 13-HOD degradation. However, whether formation of reactive electrophilic species from 13-HOD is of relevance in vivo and supports seedling viability, or whether it is just artificially enhanced under the applied in vitro conditions, remains speculative.
Another striking difference between the cucumber and sunflower system was found for the turnover of LA. Glyoxysomes from cucumber possess a higher preference for the degradation of LA than 13-HOD in the optical assay, whereas the sunflower organelles only show very weak formation of NADH from LA. This finding and the observation, that LA is rapidly converted to 13-HOD and activated to 13-HOD-CoA in absence of NAD, prompt us to assume, that in sunflower LA degradation may be generally initiated by the oxidation to 13-HOD. However, the weak ability of sunflower glyoxysomes to create NADH from LA is somehow enigmatic as the initially formed 13-HOD should be an efficient substrate, but may be restricted to the used sunflower variety. One could even expect an initial burst in NADH-formation, when LA-derived 13-HOD is oxidized to 13-KOD in a NAD-dependent manner. That this conversion indeed depends on NAD can be derived from the observation, that huge amounts of 13-KOD are produced from 13-HOD in a complete β-oxidation assay (Figure 5), but only traces of 13-KOD are formed, when NAD is absent (Figure 3). However, the strong oxidative capacity of the sunflower organelles might explain the low amount of NADH generated during LA turnover, if enzyme inactivation proceeds faster than substrate consumption.