The Molecular Mechanism Of Pistil Abortion Based On ’Omics’ In Japanese Apricot

Japanese apricot (Prunus mume Sieb. et Zucc) originated in China and is an important economical fruit crops in China and Japan. Owing to its charming flowers and flavorsome fruit, it has been widely planted as an ornamental plant and an economic fruit tree. The fruit of Japanese apricot has consistently been a valuable processing material used in the food and vintage industry and is believed to contain many physiochemical substances, which are beneficial to human health. However, the phenomenon of imperfect flowers widely occurs and has seriously affected the production yield. Two Japanese apricot cultivars’Longyan’ and’Daqiandi’were taken as the materials to study the pistil differentiation. The quality of flower organs, the progress of pistil differentiation and the related physiological indexes were investigated. To understand the role of miRNAs and genes in pistil development, we used high-throughput sequencing to identify pistil development-related miRNAs and transcrpts in Japanese apricot. Meanwhile, we used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) approaches to identify the differentially expressed proteome between perfect and imperfect flower buds in Japanese apricot. The main results were as follows:1. We have investigated biological traits of flowers and pollen germination among 49 Japanese apricot cultivars, including aroma, the rate of incomplete flower and pollen quantity and measured the rate of pollen germination. Subsequently, we have got some results as follows through the correlative statistics analysis:Chinese cultivars’ aroma is more fragrant than Japanese cultivars’; the rate of incomplete flower covers 1.17～74.53%; there is an obvious diversity among the pollen quantity of different cultivars, and the coverage is from 0 to 1265.63; there is an obvious diversity among the rate of pollen germination of different cultivars, the highest rate is 85.26% and the lowest rate is 0.2. Two Japanese apricot cultivars ’Longyan’ and ’Daqiandi’ were taken as the materials to study the pistil differentiation. The quality of flower organs, the progress of pistil differentiation and the related physiological indexes were investigated. The results showed that the imperfect flower rate of ’Longyan’ was 5%, and ’Daqiandi’ was 76.3% at the stage of full bloom. The pistil differentiation of ’Longyan’ was progressed smoothly and formed perfect flower finally. The pistil of’Daqiandi’did not continue to develop at about December 10th, which suggested that it was the key stage of the pistil abortion in ’Daqiandi’. As a whole, the contents of soluble sugars and protein of ’Longyan’ were higher than that of ’Daqiandi’, but the content of starch was lower than that of ’Daqiandi’. The content of soluble sugars and protein of the perfect flowers were higher than that of imperfect flower, but the content of starch was lower than that of imperfect flowers in ’Daqiandi’ cultivar.3. To understand the role of miRNAs in pistil development, we used high-throughput sequencing to identify pistil development-related miRNAs in Japanese apricot. Solexa sequencing was used to identify and quantitatively profile small RNAs from perfect and imperfect flower buds of Japanese apricot. A total of 22561972 and 24952690 reads were sequenced from two small RNA libraries constructed from perfect and imperfect flower buds, respectively. Sixty-one known miRNAs, belonging to 24 families, were identified. Comparative profiling revealed that seven known miRNAs exhibited significant differential expression between perfect and imperfect flower buds. A total of 61 potentially novel miRNAs/new members of known miRNA families were also identified by the presence of mature miRNAs and corresponding miRNA s in the sRNA libraries. Comparative analysis showed that six potentially novel miRNAs were differentially expressed between perfect and imperfect flower buds. Target predictions of the 13 differentially expressed miRNAs resulted in 212 target genes. The majority of genes encoding transcription factors or F-box proteins have a significant role in the plant development. In the present study, it was found that the predicted targets of miR319, miR319a, miR319e, miR160, miR393, miR394, miR6274, miR6295 and miR171d were either transcription factors or F-box proteins. In addition, it is predicted that miR319/miR319a/miR319e target ARF2 genes and that miR160a targets ARF16/17. Auxin regulates a variety of physiological and developmental processes in plants. ARF has been reported to regulate flower and leaf development. Gene ontology (GO) annotation revealed that high-ranking miRNA target genes are those implicated in the developmental process, the regulation of transcription and response to stress. This study represents the first comparative identification of miRNAomes between perfect and imperfect Japanese apricot flowers. Seven known miRNAs and six potentially novel miRNAs associated with pistil development were identified, using high-throughput sequencing of small RNAs. The findings, both computationally and experimentally, provide valuable information for further functional characterization of miRNAs associated with pistil development in plants.4. To investigate genes related to the pistil development of Japanese apricot, high-throughput sequencing technology (Illumina) was employed to survey gene expression profiles from perfect and imperfect Japanese apricot flower buds.3476249 and 3580677 tags were sequenced from two libraries constructed from perfect and imperfect flower buds of Japanese apricot, respectively. There were 689 significant differentially expressed genes between the two libraries. GO annotation revealed that highly ranked genes were those implicated in small molecule metabolism, cellular component organisation or biogenesis at the cellular level and fatty acid metabolism. The expression patterns of 36 differentially expressed genes were confirmed by real-time PCR. According to the results, we assumed that LEA gene, coding a late embryogenesis abundant protein, DICER-LIKE 3 (DCL3) gene, XTH2 gene, coding a xyloglucan endotransglucosylase/hydrolase, PPME1 gene, coding a protein with pectin methylesterase activity, LTP3 gene, coding a PR (pathogenesis-related) protein belonged to a lipid transfer protein, FATTY ACID BIOSYNTHESIS I (FAB1) gene and FAD5 gene, coding a fatty acid desaturase, all might have relationships with the pistil abortion in Japanese apricot. This is the first report of the Illumina RNA-seq technique being used for the analysis of differentially-expressed gene profiles related to pistil abortion that both computationally and experimentally provides valuable information for the further functional characterisation of genes associated with pistil development in woody plants.5. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) approaches to identify the differentially expressed proteome between perfect and imperfect flower buds in Japanese apricot. More than 400 highly reproducible protein spots (P<0.05) were detected and 27 protein spots showed a greater than two-fold difference in their expression values. The proteins identified were classified into eight functional classifications and ten process categories, according to the Gene Ontology (GO). Acetyl-CoA produced by ATP citrate lyase (ACL) as a structural substance during formation of the cell wall could regulate pistil abortion in Japanese apricot. S-adenosylmethionine (SAM), xyloglucan endotransglucosylase/hydrolases (XTHs) and caffeoyl-CoA-O-methyl transferase (CCoAOMT) could promote cell wall formation in perfect flower buds of Japanese apricot, greatly contributing to pistil development. Spermidine hydroxycinnamoyl transferase (SHT) may be involved in the O-methylation of spermidine conjugates and could contribute to abnormal floral development. The identification of such differentially expressed proteins provides new targets for future studies that will assess their physiological roles and significance in pistil abortion.