Studies On Lipoxygenase And Its Associated Genes Related To Persimmon Fruit Softening And Ripening

Lipoxygenase(LOX, EC1.13.11.12) is widely distributed in the plant kingdom. LOX initiates the oxygenation of polyunsaturated fatty acids(PUFAs) to form a large class of biologically active compounds, and plays important functions in plant developmental processes, fruit ripening and resistance to defence and biotic or abiotic stress. Persimmon(Diospyros kaki L.) is an important horticultural commodity with delicious taste and excellent nutritional properties in northwest China. However, the persimmon fruit softens and decays quickly, negatively affecting its transport and marketability. In this study, different persimmon cultivars differing in ripening rates were used as the materials to study the relationships between 9-LOX, fruit ripening and resistance to abiotic stress, and explore the roles of 13-LOX in persimmon fruit ripening. The main results are described as followings.1. Two full-length LOX genes, DkLOX3(KF035131) and DkLOX4(KF035132) were isolated from ‘Ganmaokui' by RACE PCR. Phylogenetic analysis indicated both DkLOX3/4 belong to 9-LOX. The persimmon DkLOX3/4 genes was expressed in Escherichia coli, the authenticity of recombinant proteins was addressed by Western blotting and further purified. In vitro enzymatic reaction assay were conducted, and the results showed that both DkLOX3 and DkLOX4 possessed the expected LOX activity.2. Transcriptional profiles of DkLOX3/4 genes in three different persimmon cultivars(‘Fupingjianshi' with worst storability, ‘Huoshi' with better storability and ‘Ganmaokui' with the best storability) were investigated using qRT-PCR. We also studied their expression in response to propylene and 1-methylcyclopropene(1-MCP) to explore the relationships between 9-LOX and ethylene in ‘Fupingjianshi' and ‘Ganmaokui'. qRT-PCR analysis showed that DkLOX3/4 genes were abundantly expressed in persimmon fruit during ripening, but distinctly different expression patterns were evident. The expression level of DkLOX3 was significantly higher than that of DkLOX4. And compared to DkLOX4, expression of DkLOX3 was particularly up-regulated by propylene treatment, down-regulated by 1-MCP treatment.3. The structural change in fruit peel of ‘Fupingjianshi' and ‘Ganmaokui' was investigated using scanning electron microscopy. The observations of fruit surface showed there was no lenticel visible in the fruit peel. In the present study, 12 days of storage resulted in increased the numbers and depths of microcracks in both persimmon varieties, which did not exhibit any evident cuticle microcracks at harvest. The microcrack depth in ‘Fupingjianshi' fruit peel was greater than in ‘Ganmaokui'.4. The relationship between DkLOX3/4 and ultrastructural changes was studied. Results from the subcellular-localization study revealed that during storage, the immuno-labelled gold particles of DkLOX3 accumulated much higher in the cell plasmalemma of ‘Fupingjianshi' than that in ‘Ganmaokui'. However, few immuno-labelled gold particles of DkLOX4 accumulation were observed in both persimmon cultivars. The results suggested that DkLOX3 plays an important role in ultrastructural changes promoting persimmon fruit ripening.5. The transcriptional profiles of Dk LOX3/4 were analysed in response to physical and hormonal treatments in ‘Fupingjianshi' persimmon fruit by qRT-PCR. qRT-PCR analysis showed that expression patterns of DkLOX3 generally paralleled the ethylene production under various treatments. The expression of DkLOX3 was positively up-regulated by mechanical damage and ABA, and was suppressed by low temperature, SA and GA. In particular, the expression of DkLOX3 differed from the ethylene trajectory after MeJA treatment. The transcriptional profile of DkLOX4 differed from DkLOX3 under various treatments in persimmon fruit. The expression of DkLOX4 was up-regulated by mechanical damage, low temperature, MeJA, SA and GA, and was suppressed by ABA.6. The promoters of DkLOX3/4 genes were cloned by Genome walking method. They were named pDkLOX3 and pDkLOX4, with Genbank accession numbers KX779272 and KX792109 respectively. An transient expression system in tobacco leaves verified that the DkLOX3/4 promoters possessed transcriptional activation. In addition, to elucidate whether the differential gene expression patterns of DkLOX3/4 in abiotic stress response are correlated with the regulation of elements in its promoter, we prepared a series of DkLOX3/4 promoters deletions. In GUS activity analysis, the promoter activities of DkLOX3/4 genes were significantly induced through various physical and hormonal treatments. According to the results of 5'deletion derivates of DkLOX3/4 promoters, we deduced that TGACG motif is important for the expression of DkLOX3 gene in persimmon under MeJA treatment. TCA element may play an important role in regulating DkLOX3/4 promoter activities after application of SA. TATC-box, P-box and GARE motif are important for regulating DkLOX4 promoter activity after application of GA.7. Transcriptome analysis of ‘Fupingjianshi' persimmon fruit under propylene and 1-MCP treatments by RNA-seq. Among the comparisons between different treatments, 41,301 differentially expressed genes were obtained from transcriptome data. A total of 1868 differentially expressed genes, log2>2, were identified. Of these 1868 differentially expressed genes, 1246 were significantly up-regulated by propylene and 622 were significantly down-regulated by propylene. In the KEGG database, we identified the LOX pathways related key genes.8. The roles of 13-LOX pathway in abiotic stress response were explored in persimmon fruit. We identified the 13-LOX pathway related key genes including DkLOX5/6, DkHPL and DkAOS from transcriptome data. Phylogenetic analysis indicated DkLOX5/6 belong to the 13-LOX family, DkHPL belong to 13-HPL and DkAOS belong to 13-AOS. qRT-PCR analysis showed that the expression of DkLOX5/6, DkHPL and DkAOS was positively up-regulated by mechanical damage and MeJA. The expression of DkLOX5/6 was suppressed by low temperature, while the expression of DkAOS, DkHPL and Dk ERF22 was up-regulated by low temperature. In addition, the expression of DkLOX5/6, DkAOS, DkHPL and DkERF26 was up-regulated after application of ABA.