Characterisation of recombinant Hevea brasiliensis allene oxide synthase: Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity

Gareth Norton, Arokiaraj Pappusamy, Faridah Yusof, Valerie Pujade-Renaud, Mark Perkins, David Griffiths, Heddwyn Jones

Research output: Contribution to journalArticle

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Abstract

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a Km value of 4.02 ± 0.64 μM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.
Original languageEnglish
Pages (from-to)129-138
Number of pages10
JournalPlant Physiology and Biochemistry
Volume45
Issue number2
Early online date13 Jan 2007
DOIs
Publication statusPublished - Feb 2007

Fingerprint

Hevea
Lipoxygenase Inhibitors
salicylates
Hevea brasiliensis
Salicylates
lipoxygenase
jasmonic acid
Salicylic Acid
enzyme activity
salicylic acid
Enzymes
Complementary DNA
enzymes
Molecular Cloning
acid treatment
gene transfer
cytochrome P-450
Cytochrome P-450 Enzyme System
abiotic stress
hydroperoxide dehydratase

Keywords

  • allene oxide synthase
  • Hevea brasiliensis
  • jasmonic acid
  • laticifers

Cite this

Characterisation of recombinant Hevea brasiliensis allene oxide synthase : Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity. / Norton, Gareth; Pappusamy, Arokiaraj; Yusof, Faridah; Pujade-Renaud, Valerie; Perkins, Mark; Griffiths, David ; Jones, Heddwyn.

In: Plant Physiology and Biochemistry, Vol. 45, No. 2, 02.2007, p. 129-138.

Research output: Contribution to journalArticle

Norton, Gareth ; Pappusamy, Arokiaraj ; Yusof, Faridah ; Pujade-Renaud, Valerie ; Perkins, Mark ; Griffiths, David ; Jones, Heddwyn. / Characterisation of recombinant Hevea brasiliensis allene oxide synthase : Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity. In: Plant Physiology and Biochemistry. 2007 ; Vol. 45, No. 2. pp. 129-138.
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abstract = "Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a Km value of 4.02 ± 0.64 μM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.",
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T1 - Characterisation of recombinant Hevea brasiliensis allene oxide synthase

T2 - Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity

AU - Norton, Gareth

AU - Pappusamy, Arokiaraj

AU - Yusof, Faridah

AU - Pujade-Renaud, Valerie

AU - Perkins, Mark

AU - Griffiths, David

AU - Jones, Heddwyn

N1 - Acknowledgements The authors would like to thank Dr Graeme Nicol for his assistance in the phylogenetic analysis.

PY - 2007/2

Y1 - 2007/2

N2 - Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a Km value of 4.02 ± 0.64 μM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.

AB - Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a Km value of 4.02 ± 0.64 μM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.

KW - allene oxide synthase

KW - Hevea brasiliensis

KW - jasmonic acid

KW - laticifers

U2 - 10.1016/j.plaphy.2007.01.003

DO - 10.1016/j.plaphy.2007.01.003

M3 - Article

VL - 45

SP - 129

EP - 138

JO - Plant Physiology and Biochemistry

JF - Plant Physiology and Biochemistry

SN - 0981-9428

IS - 2

ER -