| Delaying aging is an important task for safe storage of paddy rice. Since lipid peroxidation is a causitive reason for the rice aging and the lipoxygenase is a key enzyme in lipid peroxidation pathway, it is of great significance to do research work about the rice embryotic lipoxygenase. While rice seed lipoxygenase-3 (OsLOX3) is considered to be associated with storability of rice and rice lipoxygenase-1 (OsLOX1) is relevant to seedling resistance to insects, the physiological function of rice lipoxygenase-2 (OsLOX2) remains unknown. The aim of this research is to elucidate the role of OsLOX2 in seed aging and germination, which would provide a theoretical basis for the development of new rice variteties with good storability.At 7-d after flowering,OsLOX2 was expressed in all the tissues, highest in roots and stems, lower in panicles and hardly in leaves. OsLOX2 transcripts were detected in embryos through all developing stages (6-21 days after flowering), with the highest levels on the 18th day after flowering. During the process of germinating (0-12 days after imbibition), the gene was strongly induced on the second day after imbibition in the embryos, and it reached the highest level in the roots on the fourth day after imbibition and dropped to the level in mature plant thereafter, as the same trend as in leaves. Collectively, it was suggested that OsLOX2 might play a pivtal role in the seed development and germination process.OsLOX2 full-length cDNA was cloned from the Nipponbare germinating embryos. By transient expression of 35S::GFP:OsLOX2 in rice protoplast, OsLOX2 protein was found to perform in cytoplasm. In vitro, recombinant OsLOX2 (use T7 pro::His:OsLOX2:His vector by prokaryotic expression) showed a substrate preference to linoleic acid and a wide range of temperature adaptation (20℃-50℃). In order to study the relationship of OsLOX2 gene expression and its corresponding germination and aging, both OsLOX2 gene over-expression and RNA interference homozygous transgenic lines were obtained by Agrobacterium-mediated transformation. A knockout mutant of OsLOX2 due to Tos17 insertion (CU313865) was purchased from CIRAD mutant library. OsLOX2 over-expression lines can speed up germination but also aggravate the aging which was established by the declined germination rate after accelerated aging treatment. In contrast, RNAi and Tos17 knockout method down-regulated the gene expression level in different degree, they all showed lower germination speed than control. But interestingly, after accelerated aging treatment, those with mild repression showed an increased germination rate as we expected, however, those repressed severely was incapable to germinate anymore. Overall, OsLOX2 is essential for seed germination and will accelerate aging under a storage condition. Moreover, appropriate inhibition of OsLOX2 gene contributes to enhanced tolerance of a long-term storage without obstacle of germination, which makes good sense in seed production and storage. |
