Dataset Information

Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon, Cucumis melo var. makuwa Makino.

ABSTRACT: Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar "Yumeiren", encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.

SUBMITTER: Cao S

PROVIDER: S-EPMC4839669 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon, Cucumis melo var. makuwa Makino.

Cao Songxiao S Chen Hao H Zhang Chong C Tang Yufan Y Liu Jieying J Qi Hongyan H

PloS one 20160421 4

Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar "Yumeiren", encode 902 and 90 ...[more]

PMID: 27101009

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Project description:BACKGROUND:Lipoxygenases (LOXs) play significant roles in abiotic stress responses, and identification of LOX gene promoter function can make an important contribution to elucidating resistance mechanisms. Here, we cloned the CmLOX08 promoter of melon (Cucumis melo) and identified the main promoter regions regulating transcription in response to signalling molecules and abiotic stresses. RESULTS:The 2054-bp promoter region of CmLOX08 from melon leaves was cloned, and bioinformatic analysis revealed that it harbours numerous cis-regulatory elements associated with signalling molecules and abiotic stress. Five 5'-deletion fragments obtained from the CmLOX08 promoter-2054 (LP1), 1639 (LP2), 1284 (LP3), 1047 (LP4), and 418?bp (LP5)-were fused with a GUS reporter gene and used for tobacco transient assays. Deletion analysis revealed that in response to abscisic acid, salicylic acid, and hydrogen peroxide, the GUS activity of LP1 was significantly higher than that of the mock-treated control and LP2, indicating that the -?2054- to -?1639-bp region positively regulates expression induced by these signalling molecules. However, no deletion fragment GUS activity was induced by methyl jasmonate. In response to salt, drought, and wounding treatments, LP1, LP2, and LP4 promoted significantly higher GUS expression compared with the control. Among all deletion fragments, LP4 showed the highest GUS expression, indicating that -?1047 to -?1?bp is the major region regulating promoter activity and that the -?1047 to -?418-bp region positively regulates expression induced by salt, drought, and wounding, whereas the -?1284 to -?1047-bp region is a negative regulatory segment. Interestingly, although the GUS activity of LP1 and LP2 was not affected by temperature changes, that of LP3 was significantly induced by heat, indicating that the -?1284- to -?1-bp region is a core sequence responding to heat and the -?2054- to -?1284-bp region negatively regulates expression induced by heat. Similarly, the -?1047- to -?1-bp region is the main sequence responding to cold, whereas the -?2054- to -?1047-bp region negatively regulates expression induced by cold. CONCLUSIONS:We cloned the CmLOX08 promoter and demonstrated that it is a signalling molecule/stress-inducible promoter. Furthermore, we identified core and positive/negative regulatory regions responding to three signalling molecules and five abiotic stresses.

Dataset Information

Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon, Cucumis melo var. makuwa Makino.

Publications

Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon, Cucumis melo var. makuwa Makino.

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