Molecular Mechanism Of Stony Hard Flesh And Screening Of Ripening Related MicroRNAs In Peaches

The stony hard flesh peaches possess greater market competence for the special fruit texture. As for research, this phenotype is an excellent materials for the investigation mechanisms of peach fruit ripening and softening. So far, research on the molecular mechanisms of peach ripening and texture regulation are far from knowing enough, further investigations to understand the regulatory mechanism of peach fruit maturation and softening is needed. This study was presented as three parts: based on the nectarine ‘CN13’(melting type) and ‘CN16’(stony hard type) cultivars, the accumulation patterns of ACS genes family in fruit development and ripening were examined; the expression profiles of IAA homeostasis related genes between the stony hard and the ordinary peaches were identified by digital gene expression profiling analysis, genes involved in stony hard flesh regulaition were isolated, and then the possible role of a candidate gene was for stony hard flesh was elucidated; the expression profiles of micro RNAs during peach ripening were examined in stony hard and ordinary peaches. The main results were as follows:1 Characterization of 1-aminocyclopropane-1-carboxylic acid synthase genes during fruit development and ripening(1) Six ACS sequences were isolated from nectarine genome, and named as PpACS1~6. Expression of ACS genes during stony hard nectarine cv. ‘CN16’ and melting nectarine ‘CN13’ fruit development and ripening were determined. In nectarine ‘CN13’, PpACS1, PpACS4, and PpACS5 transcripts accumulated as fruit development and ripening progressed, while the Pp ACS2, Pp ACS3, and Pp ACS6 genes exhibited minimal expression. Expression of both Pp ACS1 and Pp ACS4 increased with ripening. Gene expression in ‘CN13’ differed markedly from that in ‘CN16’ for a number of genes. PpACS1 was expressed at substantially lower levels in ‘CN16’ than in ‘CN13’ during fruit development and ripening. Interestingly, peak expression of three genes, Pp ACS2, Pp ACS3, and Pp ACS6, was detected at S4-1 in ‘CN16’. No expression differences were noted between the two cultivars for Pp ACS4 and Pp ACS5.(2) The Pp ACS genes was affected by postharvest treatment(auxin, ethylene, and LT). Transcription of Pp ACS1, Pp ACS4, and Pp ACS5 increased in fruits treated with auxin, of these, Pp ACS1 was induced most strongly. By contrast, expression of Pp ACS1, Pp ACS4, and Pp ACS5 was repressed by ethylene treatment. Expression of Pp ACS1 and Pp ACS5 was upregulated under LT treatment, but Pp ACS4 expression was unaffected.(3) The probable model in nectarine fruit. In immature nectarine fruit, system I ethylene relies on the expression of Pp ACS5. In mature nectarine fruit, the transition from system I to system II is induced throughout the induction and/or repression of unknown ethylene-independent factors so that the fruit gains the competence to ripen. These factors could be the increase of auxin and other development factors. PpACS1 and Pp ACS4 mRNAs are not expressed during this transition period in an ethylene-dependent manner, but rather in an auxin- or development factor-dependent manner.2 Comprehensive analysis of auxin-homeostasis-related genes between stony hard peach cv. ‘Yumyeong’ and melting peach ‘Goldhoney 3’ during fruit ripening(1) IAA concentration and digital gene expression(DGE) analysis of auxin-homeostasis-related genes. IAA levels significantly increased in a melting flesh cultivar ‘Goldhoney 3’ at the climacteric stage, although the IAA concentration in the mesocarp tissue of a stony hard peach cultivar ‘Yumyeong’ were low and did not increase. Interestingly, six auxin-homeostasis-related genes were found to cluster closely with Pp ACS1. Taking the gene functions into account, the YUC gene(ppa008176m, Pp YUC11) may regulate IAA concentration during the fruit ripening stage of the melting flesh peach.(2) The expression profiles of the nine YUC-like genes were analysed in selected tissues of the two types cultivars. Both Pp YUC10(ppa007054m) and Pp YUC11(ppa008176m) are enriched in the fruit ripening stage of ‘Goldhoney 3’, but Pp YUC11 cannot be detected in ‘Yumyeong’ fruit during the same period. Pp YUC11 was expressed at various stages in the seeds of ‘Goldhoney 3’ and ‘Yumyeong’. However, only in the melting flesh fruit ‘Goldhoney 3’ did Pp YUC11 show obvious tissue-specific expression in S4 fruits.(3) The distribution of all Pp YUC11 variations can be found in both flesh types, with the exception of the TC dinucleotide microsatellite. We found at least twelve polymorphic loci in the Pp YUC11 gene using a combination of cloning and direct Sanger sequencing of PCR products, the distribution of all Pp YUC11 variations can be found in both flesh types, with the exception of the TC dinucleotide microsatellite.(TC)n microsatellites were found in at least four different lengths(the repeat number ‘n’ of Hd1, Hd2, Hd3, and hd are 29, 26, 24, and 20, respectively) in these peach cultivars. The diversity of microsatellite length variants exhibited dramatic differences between the 36 normal and the seven stony hard flesh peach cultivars. All seven stony hard peaches were homozygous for hd. By contrast, all of the normal peach cultivars had at least one Hd allele(Hd1, Hd2 or Hd3).(4) Co-ordinated variation of Pp YUC11 expression, IAA concentration, PpACS1 expression, ethylene production, and fruit firmness in ripening peaches. A total of 12 peach cultivars with different genotypes in the Pp YUC11 SSR loci were selected, overall, Pp YUC11 and PpACS1 transcripts were highly expressed in the mesocarp of normal fleshed S4 fruits, but were either low or undetectable in the mesocarp of stony hard or S3 fruits. These expression profiles were accompanied by similar IAA content profiles or ethylene production in the mesocarp of these peach cultivars. Flesh firmness decreased towards harvest time along with ethylene emissions.Taken together, the expression pattern, predicted function, and tissue distribution make Pp YUC11 an excellent candidate gene for stony hard type peaches.3 Comparative profiling of miRNAs related to fruit ripening between SH and melting flesh peaches. To understand further the relationship between miRNAs and their target genes during fruit ripening in peach, small RNA library construction and Solexa sequencing were performed at stages during fruit ripening in stony hard nectarine cv. ‘CN16’ and melting nectarine ‘CN13’. We identified 142 conserved miRNAs and 239 novel miRNA candidates in peach by deep sequencing. Some mi RNAs that showed different expression patterns during ripening stage between the SH and M phenotype, 24 miRNAs with different expression pattern were selected out, Interestingly, the expression levels of miR171b/z significantly increased in a melting flesh cultivar ‘CN13’ at the climacteric stage, although the its concentration in the mesocarp tissue of a stony hard peach cultivar ‘CN16’ were low and did not increase. The results of this study lay a foundation for unraveling key miRNA regulators of peach fruit ripening on post-transcriptional level.