Page 7 - V. Proteomics

Identification of rice caleosin gene OsPXG9 with peroxygenase activity
involved in oxylipin pathways

Duc Tran Anh , Kyoungwon Cho , Oksoo Han*
*
*
*Department of Molecular Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National
University, Gwangju, South Korea
BACKGROUND

Caleosin is group of genes that are specialized by several structural features, including Calcium binding EF hand motif, Proline-rich knot region and heme-binding residues. Caleosins are
generally involved in variety of biological functions. Rice, among cereals, is a key crop plant that plays significant roles among human population. Oxylipin pathways in rice has been researched
for a long time, especially octadecanoid pathways that is responsible for producing jasmonic acid under various stresses (1). However, there are many other oxylipin pathways are remain
enigmatical. In some plants, caleosins that are reported to have peroxygenase activity are generally play roles in responding to either biotic or abiotic stresses by involving in oxylipin pathways.
In this study, we investigated to study about one protein in rice, OsPXG9 (Accession number AK104598.1), turned out to be a caleosin with peroxygenase activity and create several enzymatic
products which are related to plant defense.
RESULTS

Figure 1. OsPXG9 structurally belongs to caleosin family. Figure 2. OsPXG9 possesses peroxygenase activity,
and is inhibited by peroxygenase specific inhibitors.
A B
A D

C D

B C
E
E 280 nm
407 nm

(A) OsPXG9’s UV spectrum absorbance reduction at 407 nm by addition of cumene
(A) Multiple alignment of OsPXG9 sequence with A. thaliana AtPXG1, AtPXG2, AtPXG3, AtPXG4, AtPXG7, S. lycopersicum SlPXG, A. Sativa AsPXG1. The black
and red boxed areas correspond to calcium binding and the proline knot domain, respectively. Triangles indicate positions of conservative Histidine which are hydroperoxide 1 mM after 2, 5, 8, and 10 minutes. OsPXG9 activity decrease with time after
critical for heme binding. Stars indicate position of conservative Proline that are responsible for transmembrane region. (B) Phylogentic tree of OsPXG9 with addition of cumene hydroperoxide in (B) 9-HPOD and (C) 9-HPOT used as substrate. (D)
corresponding orthologs in A. thaliana, S. lycopersicum , A. Sativa. (C) Predicted 3D structure of OsPXG9 is shown from N to C terminus by rainbow colours. (D) Relative activity remained of OsPXG9 under the addition of terbufos and β-mecarptoethanol
Predicted transmembrane structure. Sequence alignment and phylogenetic tree are generated online by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/); with 9 HPOD and 9 HPOT used as substrates. (E) OsPXG9 is able to oxidize Aniline with
3D structure and transmembrane structure are generated by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2). (E) Light absorption spectra analysis of OsPXG9,
OsPXG9 shows a high peak at 407 nm, feature of hemoproteins (2). CuOOH used as the oxygen donor.
Figure 3. OsPXG9 kinetic activity Table 1. OsPXG9 enzymatic product analysis
Substrate Product name Chemical formula Molecular
weight
9,12,13-trihydroxy-11-octadecenoic acid C 18 H 34 O 5 330.24
LA + HOOH
9,10,13-trihydroxy-11-octadecenoic acid C 18 H 34 O 5 330.24
9,12,13-trihydroxy-11-octadecenoic acid C 18 H 34 O 5 330.24
LA + CuOOH
9,10,13-trihydroxy-11-octadecenoic acid C 18 H 34 O 5 330.24
LnA + CuOOH None None None
LnA + HOOH None None None
9,12,13-trihydroxy-11-octadecenoic acid (major) C 18 H 34 O 5 330.24
9-HPOD 11-hydroxyl-13-oxo-9-octadecenoic acid (minor) C 18 H 32 O 4 312.23
9-hydroxy-13-oxo-10-octadecenoic acid (minor) C 18 H 32 O 4 312.23
9,12,13-trihydroxy-10,15-octadecadienoic acid (major) C 18 H 32 O 5 328.22
9 HPOT
OsPXG9 kinetic activity toward 9-HPOD and 9-HPOT. Data is calculated from
substrate consumed against time. Experiment is triplicated. ANOVA test was 13-oxo-9,11-octadecadienoic acid (minor) C 18 H 30 O 3 294.21
performed, P<0.05 gives no significant difference among triplicated data.
CONCLUSION REFERENCES
Our study demonstrates that OsPXG9 is structurally a caleosin 1. Agrawal, G.K., S. Tamogami, O. Han, H. Iwahashi, and R. Rakwal. 2004. Rice
which possesses peroxygenase acitivity. OsPXG9 shows ability to octadecanoid pathway. Biochemical and biophysical research
communications. 317:1-15.
consume 9-HPOD and 9-HPOT, well-known LOX products which 2. Hanano, A., M. Burcklen, M. Flenet, A. Ivancich, M. Louwagie, J. Garin, and E.
are critical in Jasmonic acid pathways, suggesting that OsPXG9 Blée. 2006. Plant seed peroxygenase is an original heme-oxygenase with an
might contribute function as a oxilipin derivatives synthesis protein. EF-hand calcium binding motif. Journal of Biological Chemistry. 281:33140-
33151.
That OsPXG9 kinetic activity is higher toward 9-HPOT than 9- 3. Martin-Arjol, I., M. Bassas-Galia, E. Bermudo, F. Garcia, and A. Manresa.
HPOD might be explained as Linolenic acid (precursor of 9-HPOT) 2010. Identification of oxylipins with antifungal activity by LC–MS/MS from the
is the natural substrate in plant. OsPXG9’s enzymatic products are supernatant of Pseudomonas 42A2. Chemistry and physics of lipids. 163:341-
346.
mainly trihydroxyl fatty acid, which are determined to contain
antifungal activity (3).