Function And Regulation Of Lipoxygenase Gene Family Members In Kiwifruit

Studies of LOX gene family members function and regulation were carried out by using postharvest kiwifruit (Actinidia deliciosa) as plant materials. The main results are as followings.From the HortResearch Actinidia EST database, we identified and cloned six LOX genes, i.e. AdLox1-6. AdLox1, AdLox2, AdLox3 and AdLox4 contained full-length cDNAs with 2739, 2595, 2739 and 2709 bp respectively, AdLox5 and AdLox6 were cDNA fragments of 1359 and 1557 bp. Among the six kiwifruit LOX genes, AdLox5 and AdLox6 had the highest amino acid dequence identity, which was 74%, while AdLox3 and AdLox5 produced the lowest amino acid sequence identity, which was 49%. Phylogenetic analysis showed that AdLox2, AdLox5 clustered as 9-LOX, while AdLox1, AdLox3, AdLox4 and AdLox6 clustered as 13-LOX. There is a signal peptide sequence of 60 amino acids in all members of the 13-LOX group, but no similar sequence is found in the 9-LOX genes. AdLox1 was most similar to TomLoxD at 80% identity at the amino acid level, AdLox2 and AdLox5 matched to TomLoxA with 68 and 75% identity respectively, and AdLox4 and AdLox6 showed 62 and 63% sequence identity to TomLoxC, however, AdLox3 had less than 55% identity to LOX genes from tomato.Closely related genes that were very similar at the sequence level, such as AdLox2 and AdLox5 had 74% identity at amino acid level, and therefore, it may be difficult to determine the RNA level of a specific LOX gene member. This problem is resolved by the high specificity of real-time quantitative PCR (QPCR) using of specific oligonucleotide primers. The primers were designed at the 3'UTR, which was divergent among the LOX gene family members. The primers showed no cross-amplification to other members of the family. End-point semi-quantitative PCR (SQPCR) was used to examine the expression pattern of the six LOX genes in root, stem, leaf and petal tissues, and during fruit development. AdLox1 had similar transcript abundances in root, stem and petal; AdLox2 expressed mainly in kiwifruit root, stem and leaf; AdLox3, AdLox4 and AdLox5 had similar distributions, and expressed mainly in stem and leaf; transcripts of AdLox5 was barely detectable in the various tissues. During fruit development, young fruit at 20 days after anthesis (daa) had relatively high expression levels of LOX genes, similar to those observed in stem tissues. AdLox1, AdLox2, AdLox3 and AdLox6 exhibited similar expression patterns throughout fruit development, with transcript abundance decreasing from 20 to 80 daa and increasing again at about 100 daa. AdLox4 showed a relatively constant expression level during fruit development, and produced the strongest bands among the LOX gene family. AdLox5 transcript was almost undetectable during fruit development.For fruit stored at 20℃without ethylene treatment, transcripts of AdLox1 and AdLox5 increased with kiwifruit ripening. In contrast, expression of AdLox2, AdLox3 AdLox4 and AdLox6 decreased when kiwifruit ripening progressed to the climacteric stage. During ethylene treatment (100μl/l, 24 h, 20℃), there was an increase in total LOX enzyme activity. After the ethylene treatment, LOX activity returned to harvest levels within 24 h and subsequently increased again as fruit ripened further. MDA accumulated gradually with fruit ripening. The relative QPCR results showed that expression of AdLox1 and AdLox5 was stimulated by ethylene treatment within 24 h, however, transcript abundance of AdLox2, AdLox3, AdLox4 and AdLox6 decreased about 5-, 20-, 10- and 2-fold, respectively, within 24 h of exposure to ethylene. Over-expression of AdLox1 in tobacco leaves significantly accelerated tissue chlorophyll degradation (P<0.05) and decreased chlorophyll fluorescence (P<0.05). However, over-expression of AdLox2 had no effect on senescence as measured by these two properties.Kiwifruit held at 20℃, there was an increase in LOX enzyme activity and peaked at about 180 h after harvest, following a significant decrease (P<0.05) during fruit ripening and senescence. Fatty acid-derived C6 aldehydes, such as hexanal and (2s)-2-hexenal, which are derived from a LOX-mediated pathway, give the kiwifruit a green aroma character. Amount of the two compounds trended to decrease during kiwifruit ripening, especially for (E)-2-hexenal, which showed 7 fold declines from 180 to 276 h after harvest. Along with the time when there was a decrease in C6 aldehydes, expression levels of AdLox2, AdLox3, AdLox4 and AdLox6 showed a significant reduction. Treating kiwifruit flesh discs with LA and LeA resulted in a significant increase in LOX activity (P<0.05), and marked transcripts accumulation in AdLox4 and AdLox6. Transcripts of AdLox6 were induced by 12- and 6-fold in response to LA and LeA, respectively. In contrast, AdLox2 and AdLox3 maintained constant expression level in treated tissues.Fruit held at low temperature (0℃, 168 h) maintained firmness, and did not significantly soften after transfer to 20℃for 72 h. Transcripts of AdLox1 were strongly induced by the low temperature treatment, peaking at about 72 h after harvest, and this expression declined during shelf life. AdLox5 and AdLox6 had similar expression patterns to those of AdLox1 during the low temperature treatment. The expression profiles of the above three LOX genes matched changes in LOX enzyme activity in response to low temperature. However, AdLox2, AdLox3 and AdLox4 showed no significant changes in transcript levels during low temperature treatment.These results showed that the LOX gene family members were differentially regulated during kiwifruit ripening and senescence, and they showed different response to temperature and ethylene treatment. The possible roles of individual LOX isoforms in kiwifruit were discussed.