n-Hexanal and (Z)-3-hexenal are generated from arachidonic acid and linolenic acid by a lipoxygenase in Marchantia polymorpha L.

Figures & data

Fig. 1. The amounts of n-hexanal (A) and (Z)-3-hexenal (B) formed from intact or mechanically wounded thalli, antheridiophores, and archegoniophores.

Notes: Mean ± standard error is shown (n = 3). Different letters indicate significant differences between means (p < 0.01, two-way ANOVA with Bonferroni test for n-hexanal, and one-way ANOVA with Bonferroni test for (Z)-3-hexenal).

Fig. 2. Phylogenetic analysis of MpLOXs with the LOXs from C. reinhardtii, K. flaccidum, Marchantia polymorpha, P. patens, S. moellendorffii, A. thaliana, Z. mays, P. trichocarpa, and P. abies.

Notes: All the genes that showed similarity with Arabidopsis lipoxygenase 2 are aligned, and the phylogenetic analysis is performed using the neighbor-joining method of MEGA6 software. The list of LOXs used for the phylogenetic analysis is in Supplemental Table S1. The names of LOXs mentioned in the text are shown.

Fig. 3. Amino acid sequence of MpLOX7.

Notes: Histidine, asparagine, and isoleucine residues involved in binding iron atom are indicated with stars. PLAT domain is shown in the box. Domains showing similarity among all the LOX sequences used for making the phylogenetic tree in Fig. 2 are underlined, and those conserved among non-seed plant LOX clade are double underlined. Alignment of the sequences is performed using the L-INS-i method of MAFFT version 7, and those reasonably aligned positions are chosen with Gblocks.

Table 1. Kinetic parameters and main products of recombinant MpLOX7 with fatty acid substrates.

Substrate	km (µM)	Vmax (nkat·mg^−1)	kcat (s^−1)	Main product
Linoleic acid	12.0	2270	237	13-Hydroperoxy-(9Z,11E)-octadecadienoic acid
C18:2 (n-6)
Arachidonic acid	24.5	2300	241	15-Hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid
C20:4 (n-6)
α-Linolenic acid	25.0	3000	314	13-Hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid
C18:3 (n-3)
γ-Linolenic acid	71.0	330	34.5	Not determined
C18:3 (n-6)

Fig. 4. Formation of n-hexanal from arachidonic acid (ARA) (A) and (Z)-3-hexenal from α-linolenic acid (LNA) (B) during catalysis of recombinant MpLOX7.

Notes: As a control experiment, hydroperoxide [arachidonic acid 15-hydroperoxide (HPETE) for (A) and α-linolenic acid 13-hydroperoxide (HPOT) for (B)] formed during catalysis was reacted with active or heat-inactivated MpLOX7, and n-hexanal and (Z)-3-hexenal formed were analyzed. Mean ± standard error is shown (n = 3). Different letters in (A) indicate significant differences between means (p < 0.01, one-way ANOVA with Bonferroni test).

Fig. 5. Expression level of MpLOX7 in thalli, archegoniophores before mechanical wounding and after mechanical wounding, and antheridiophore before mechanical wounding and after mechanical wounding.

Notes: MpEF1a was used as a control gene to standardize the amount of template used for PCR. Amplification with 31 or 33 cycles for MpLOX7 (137 bp) and 24 or 26 cycles for MpEF1α (448 bp) is shown in order to compare their expression levels before saturation of amplification.

Fig. 6. Proposed routes of β-scission of fatty acid hydroperoxides formed by lipoxygenases.

Notes: In route a, the non-allylic C–C bonding would be cleaved to form C5 volatiles. In route b, the allylic C–C bonding (between the carbon at the base of alkoxyl radical and that of double bond) would be cleaved to form C6 volatiles. R1 = decenoic acid or dodecadienoic acid; R2 = butyl or butenyl.