Analysis Of Rice Lipoxygenase Isozymes, Cloning Of OsLOX3 And Structural Analysis Of A Gene Cluster Encoding Lipoxygenase Related To Seed Storability

Food security is a global strategy issue and food storage is one of the key rings in this chain. Under normal storage condition, rice grain deterioration and development of a stale flavor often appear in the second year. According to statistics from Ministry of Agriculture, the loss during storage of rough rice for food and seeds accounts for about 3% of the total production per year in China. In recent years, many investigators have demonstrated that the degradation of lipids was mainly responsible for reducing rice quality and producing stale flavor during storage, and lipoxygenase (LOX, EC 1.13.11.12) was the key enzyme for lipid degradation in above processes. Therefore, the absence of LOX enzymes in crops might decrease lipid peroxidation, alleviate the accumulation stale flavor, and thus increase storability. This assumption has been preliminarily clarified in the relationship between LOX-3-less rice and storability. However, the simple method of screening rice embryos for LOX-3-null has not established. Analysis of LOX isozymes in developing rice seeds, cloning of OsLOX3 gene and its relationship with LOX-3-null have not carried out either.In this paper, based on co-oxidation secondary reaction of LOX-3 and the oxidative ability of 9-hydroperoxide, the catalytic product of LOX-3, two reliable, rapid, simple and inexpensive spectrophotometic methods, named as carotene bleaching and iodide-starch methods, were developed to screen rice embryos for LOX-3-null in comparison with the monoclonal antibodies technique. Thus, above two methods could be used in breeding for storable rice cultivars.There are several LOX isozymes in maturing rice seeds, including some isozymes with molecular weight less than 68 kDa. According to different biochemical separating methods, there exist at least 4-9 isozyme bands. For example, in comparison with rice cultivar (Koshihikari, Japanese rice cultivar) with normal LOX-3 activity, the LOX-3-null cultivar Daw Dam reported by Suzuki et al (1996a, 2000) lack of the first and third types of LOX isozymes by native gradient PAGE (5-20%), also at least four bands disappeared. Furthermore, by using IEF methods (pH3.5-10), LOX isozyme (type II, pI4.56) could not be easily detected in Daw Dam. Meanwhile, two types of LOX isozymes disappeared confirmed by using western blotting method. In view of the fact that the lack of LOX isozymes in Daw Dam was the type of LOX isozyme with lowest pI rather than a single isozyme, we suggested that the LOX isozymes during rice maturation process was classified as three type, named as type I, II and III.In maturing rice seeds, the majority of LOX protein was present in protein storage vacuole (PSV) rather than oil body of both wild-type and LOX-3-null seeds, suggesting that LOX is separated from its substrate. However, physical damage during storage caused direct contact of the lipase with triacylglycerols, contributing the release of free fatty acids, especially the increase of the polyunsaturated fatty acid. Furthermore, LOX could catalyze the oxidation of polyunsaturated fatty acids, including linoleic and linolenic acids, into conjugated hydroperoxy fatty acids. Hydroperoxides were further transformed by autooxidation and enzymatic degradation, including hydroperoxide lyases (HPLs) and hydroperoxide isomerases (HPIs), into various volatile carbonyl compounds, such as hexanal, pentanal and pentanol, adding off-flavor which decreased the quality of strored rice and seriously influenced its storability. Additionally, the possibility that rice embryo LOXs may function as seed storage proteins or be involved in the synthesis signal molecule responsible for abiotic and biotic stresses could not be easily ruled out.Furthermore, cloning of a novel LOX gene OsLOX3 from developing rice seeds, which was located in Chromosome 3, also formed LOX gene cluster with OsLOX1/2. In comparison with the expression products of OsLOX1/2, OsLOX3 exhibited the lowest pI. The location of OsLOX3 in chromosome 3 was also consistent with QTL position related to rice storability reported by our research groups. The expression, purification and characterize of Mini0sL0X3 were also carried out. The 1 bp deletion of adenine residue, which causes a frame-shift, was at the 5' region of the fourth exon, leads to the product of MiniOsLOX3 with molecular weight being 28 kDa. Meanwhile, lower LOX activity was also detected. Finally, the bioinformatical analysis of LOX gene cluster located in chromosome 3 also illusted three-dimensional structures, domain distribution, phylogenetic analysis and the expressions profiles of the deduced OsLOX1/2/3. Additionally, we deduced that there exists the possibility of alternative splicing.