III. Results
3. Lipoxygenase pathway of selected cyanobacterial strains
3.3 Characterisation of a peroxidase-LOX fusion protein from Nostoc sp
3.3.5 Comparison of endogenous oxylipins in different cyanobacterial strains
So far, the in vitro characterisation of NspFP isolated from the N. sp. strain SAG 25.82, which is a duplicate strain to N. sp PCC7120 as kept in the Pasteur Culture Collection, (PCC, Paris, France) was described. Analysis of the isolated sequences showed, that SAG 25.82 and PCC7120 both harbour an insertion of a transposon in the NspFP sequence, as described above. In order to compare the dependency of cyanobacterial oxylipin formation on the NspFP protein, two additional N. sp. strains, PCC7120 A and P, were obtained (a gift from Prof. Peter Wolk, Michigan State University, East Lansing, MI, USA and Prof. James Golden, Texas A&M University, College Station, TX, USA, respectively), which do not harbour a disruptive insertion in the NspFP-gene. These two strains were ordered from the PCC in Paris years ago, but cannot be regarded as specific
102 mutants in the NspFP gene since these strains may slowly drift genetically as well and thus may harbour mutations in different genes.
The insertion likely results in the formation of an inactive enzyme with a deduced amino acid sequence and this may influence the oxylipin metabolism of the cyanobacteria. The identification of these two different alleles of the Nostoc gene encoding NspFP, raised interest in comparing the "wildtype” (wt) strains PCC 7120-A and -P with the “mutant”
(mt) strain SAG 25.82 (8.5). In addition, it was aimed to compare endogenous products of the LOX pathway of wounded and non-wounded cyanobacteria as it is known from the literature that the LOX pathway is induced upon wounding in higher plants (Schilmiller and Howe 2005a). Therefore cyanobacterial cultures were additionally subjected to wounding by ultrasonification (10.11), and the effects on oxylipin metabolism were monitored, as described previously (Lang and Feussner 2007). In all samples free and esterified hydro(pero)xy dienoic FAs were measured.
In both non-wounded and wounded Nostoc sp. PCC7120 strains only C18-PUFA-derived hydroxides were detected, and no C16 or C20 derived oxylipins were present. In addition, traces of FA hydroperoxides were detected. The analysis of free oxylipins in the wt strains showed, that the most abundant hydroxy FAs were derived from 9-LOX activity (0.3-1.0 nmol/g f.w, Fig. 33A and B). The amount of the hydroxy FAs increased upon wounding, in particular that of 9-hydroxy FAs, which showed an almost tenfold increase with wounding (9.1-11.7 nmol/g f.w., Fig. 33A and B). The amount of the other hydroxy FAs (13-HODE, 13HOTE, 12-HOTE and 16-HOTE) increased only slightly to about 0.8 nmol/g f.w.. In comparison, the mt strain contained similar amounts of hydroxy FAs of about 0.7 nmol/g f.w., and there was no preference for either one of the hydroxy FA isomers (Fig. 33C). In addition, only a weak induction of oxylipin formation was observed upon wounding. The difference between wt and mt strains was even more remarkable when the stereochemistry of the different hydroxy FAs was analysed (Fig. 33D). The oxylipins HODE and HOTE of the wt strains were in R-configuration, whereas 9-HODE and 9-HOTE of the mt strain were racemic. These data indicated an induction of (9R)-LOX activity in the wt strains upon wounding and a loss of LOX activity in the mt strain, most likely due to the insertion of a disruptive transposase insert into the NspFP-gene. Interestingly, neither in wt nor in mt strains any dihydroxylated FAs products of NspFP were detected, contrasting to the results from the in vitro tests with the recombinant enzyme.
103 0
5 10 15 20
13-HODE
13-HOTE
16-HOTE
12-HOTE
9-HODE
9-HOTE
(nmol/g f.w.)
A
0 2 4 6 8 10
13 -H OD E
13 -H OT E
16 -H OT E
12 -H OT E
9- H OD E
9- H OT E
(nmol/g f.w.)
B
0 5 10 15 20
13-HODE
13-HOTE
16-HOTE
12-HOTE
9-HODE
9-HOTE
(nmol/g f.w.)
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0 2 4 6 8 10
13 -H OD E
13 -H OT E
16 -H OT E
12 -H OT E
9- H OD E
9- H OT E
(nmol/g f.w.)
B
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Figure 33 Free oxylipin profiles of different cyanobacterial strains. The oxylipin levels were determined in control cells as well as in wounded cells. A, Nostoc. sp. PCC7120P B, PCC7120A and C, SAG 25.82. The bars represent the amounts of detected hydroxy FAs from non-wounded (grey) or wounded (black) cyanobacteria D, relative proportion of the R-(black bars) and S-(grey bars) enantiomers. The data represent mean values of three independent measurements. Error bars represent standard deviation.
.
105
106
Figure 34. Esterified oxylipin profiles of different cyanobacterial strains. The oxylipin levels were determined in control cells as well as in wounded cells. The bars represent the amounts of detected hydroxy FAs from non-wounded (grey) or wounded (black) cyanobacteria. A, N. sp. PCC7120P; B, PCC7120A and C, SAG 25.82. The data represent mean values of three independent measurements. Error bars indicate standard deviations.
The analysis of oxylipins in the fraction of esterified FAs revealed that the two wt strains showed significant differences in the levels of esterified oxylipins and only for PCC7120A a slight increase of oxylipins upon wounding was detectable (Fig. 34A and B).
Furthermore there was no preference for a certain hydroxy FA product like (9R)-HODE and (9R)-HOTE, indicating that changes in oxylipins upon wounding are restricted to the free oxylipin fraction (Fig. 33A and B). The amount of esterified oxylipins in wt and mt strains showed no remarkable differences. In the case of mt strain SAG 25.85 the oxylipin level was higher in non-wounded than in wounded samples (Fig. 34C). Moreover the amount of esterified oxylipins varied between three independent measurements, resulting in high standard deviations, which are too high for any conclusions.
CP-analysis yielded in 9-hydroxy FAs in R-configuration for the wt strains, which is an indication for LOX activity, whereas the 9-hydroxy FAs of the mt strain were racemic and therefore products of autoxidation.
107 Since the detection of any dihydroxy FAs that may derive from the activity of NspFP failed, the extractable enzyme activity that may derive from NspFP was determined.
Therefore cell extracts obtained from wounded and non-wounded mt and wt strains were analysed either after incubation with LA or [1-14C]-labelled LA. The samples were extracted and analyzed for products by reversed phase HPLC (RP-HPLC). In the untreated samples, formation of neither monohydroxylated nor dihydroxylated FAs was detected.
Comparison of non-wounded and wounded samples however, revealed wound-induced formation of (9R)-H(P)ODE in wt strains. Again, neither formation of 9,14-diHODE nor that of any other dihydroxy FA was detected. The results for the two wt strains were similar. For the mutant strain SAG 25.82 no oxylipins were found.
108