Transcriptomic And Proteomic Analysis Of The Effects Of Gibberellin On Increased Fruit Sink Strength In Pear

Gibberellin(GA) regulates numerous developmental processes in Asian pear(Pyrus pyrifolia Nakai) such as fruit set, stem and leaf elongation and the development of fruit and seed. GA is commonly used to improve fruit yield and quality in tree fruit production. To better understand the mechanism by which GA increases fruit size at the transcriptome and proteome level, fruits from 6-year-old Asian pear cv. ‘Cuiguan’ plants were treated with GA(30 mg. L-1) on 30 days after anthesis(DAA). Fruits were sampled at the early and middle stages of fruit expansion and the maturation stage for comparative transcriptomic and proteomic analysis.1. This study aims to investigate the Asian pear transcriptome by the use of RNA-Seq normalized fruits cDNA library to create a genomic resource for Unigenes and marker discovery. 37649 contigs were generated using a de novo assembly approach. Alternative splicing was found within 4 121 contigs. In addition, we obtained a total of 30 560 single-nucleotide polymorphisms and 7 443 simple sequence repeats. After searching the reference sequences using BLAST2 GO against Nr, GO, COG and KEGG, we found 28,590 Unigenes providing significant matches results, including 28550, 17766, 10451 and 3682 Unigenes with Nr, GO, KEGG and COG annotation, respectively. The majority of the genes that are associated with gibberellin metabolism and signal transduction were identified. The targets for signal transduction of other plants hormones(117 Unigenes) and carbohydrate transmembrane transporter(132 Unigenes) were also enumerated.2. The objective of this study is to construct the Digital Gene Expression(DGE) tag profiling and find out the potential genes related to the effects of gibberellin on increased fruit sink strength in Asian pear. Taking early maturing variety cultivar from Asian pear named Cuiguan as tested materials and 12 DEGs of different expansion stages by Illumina sequencing technique were constructed and the enrichment analysis of GO and Pathway function was conducted based on the data of transcriptome. The results showed that there were 36953467, 29792878 and 31149878 tags respectively which obtained from DGE during the expansion stage and maturing phase. Pair-wise comparison of GA-treated and control samples detected 678, 32, 13 genes in mesocarp, 56, 21, 235 genes in core with an over two-fold change in expression 14, 28, and 90 days after GA treatment, respectively. Significantly modulated expression included genes encoding major carbohydrate metabolism, hormone metabolism, transport process, signal transduction and regulation. Bioinformatics analysis revealed that, cell wall synthesis genes and genes involved in sugar transport, sucrose-starch synthesis, and cell wall precursor were enhanced under GA treatment. The quantitative real time-PCR analysis supports the RNA sequencing results. The temporal gene-expression patterns of cytoskeleton and carbohydrate transmembrane transporter, regulation of hormone levels and cell division process revealed a pivotal role in GA-induced pear fruit expansion. Altogether, these data showed that GA might have acted as an important sink enhancer for reserve mobilization contributing to the pear fruit expansion. Indicated that GA-induced pear fruit expansion and maturation involved in source-sink mechanism.3. Our results revealed that 115 protein spots showed significant differences in abundance between GA-treated and untreated fruits. Among these spots, 64 proteins were successfully identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS). Increases were observed for proteins related to primary metabolism, transport, ion channels and stress responses. During the fruit expansion process, decreases in abundance were observed for proteins related to photorespiration, epigenetic, and glycolysis. The greatest protein abundance changes occurred at the early stage of fruit expansion. Seventeen highly abundant proteins that accumulated after GA application and were related to increases in sink strength were further validated by quantitative real time PCR. Increased levels of H2O2, which is associated with advanced cell wall loosening and fruit maturation, were observed after GA application. Microscopic observations of the mesocarp cell wall supported the finding that GA has promoting effects on pear fruit expansion. Increases in the activities of ATPase, RubisCO, serine hydroxymethyltransferases and NAD-dependent formate dehydrogenase were significantly correlated with sugar accumulation. In addition, increased activities of vacuole-associated annexin, the proteasome and adenylate kinase were closely involved in the cell expansion process. Our proteomic results and subsequent validation describes a dynamic protein network supporting the hypothesis that GA application during rapid fruit growth increases both sink size and sink activity, resulting in larger fruit in P. pyrifolia.4. Fruit weight 2.2(fw2.2) is an important quantitative trait locus(QTL) gene in controlling the fruit weight of plants. Based on the database of Asian pear transcriptome, an Asian pear homolog of the tomato fw2.2 gene, here named Ppfw2.2(P. Pyrifolia fw2.2-like protein), were obtained by the homologous cloning and RACE technology. Bioinformatics analyses showed that the complete cDNA sequences of Ppfw2.2 were 812 bp, and it included a 5′-untranslated region(UTR) of 144 bp, a 3′-UTR of 41 bp and an open reading frame that encoded a protein of 208 amino acid residues with a predicted molecular mass of 23.08 kD. Both the nucleotides and amino acids sequences were high homologous with those of the known fw2.2 genes in other species. Ppfw2.2 had no signal peptide, locating in the plasma membrane with transmembrane structure and the typical cysteine-rich protein domain. Phylogenetic tree analyses indicated that Ppfw2.2 belonged to the same branch with a close genetic relationship with apple. Expression difference was detected between GA treatment and its control by q RT-PCR. The results of qRT-PCR and DGEseq demonstrated that the Ppfw2.2 was negatively regulated by GA.5. To clarify the mechanism of fruit sink strength increased by GA, we investigated the contents of fructose, glucose, sucrose, sorbitol, total soluble sugar and the key genes related to sorbitol and sucrose metabolism by real-time PCR. Our results showed that sorbitol-6-phosphate dehydrogenase(S6PDH) genes were mainly expressed in fruits and up-regulated by GA treatment, indicating that sorbitol could also be resynthesized from monosaccharides, especially during later stages of fruit development. Gene expression of NAD-sorbitol dehydrogenase(NAD-SDH) was depressed by GA at the early stage of fruit expansion. GA significantly up-regulated the gene expression of soluble acid invertase(SI) and sucrose phosphate synthase(SPS). SPS gene expression increased along with fruit maturation and mainly located in the core. GA upregulated SPS during the middle and late stages of fruit expansion. A high gene expression level of sucrose synthase(SS) was measured at the early and middle stages of fruit expansion, and GA treatment depressed SS gene expression. The content of total soluble sugar in the mesocarp and core increased after GA treatment and the expression of SS, SI, SPS, NAD-SDH and S6 PDH genes exhibits temporal and special differences between the core and the mesocarp in the fruit. Scanning electron microscopy was performed to study the cell wall morphology of mesocarp, and surprisingly, we found that the secondary cell wall was significantly thicker after GA application.