Weibing Zhuang

High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot

BackgroundMicroRNAs (miRNAs) are a class of endogenous, small, non-coding RNAs that regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in high plants. However, the diversity of miRNAs and their roles in floral development in Japanese apricot (Prunus mume Sieb. et Zucc) remains largely unexplored. Imperfect flowers with pistil abortion seriously decrease production yields. To understand the role of miRNAs in pistil development, pistil development-related miRNAs were identified by Solexa sequencing in Japanese apricot.ResultsSolexa sequencing was used to identify and quantitatively profile small RNAs from perfect and imperfect flower buds of Japanese apricot. A total of 22,561,972 and 24,952,690 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. 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.ConclusionsThis 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 characterisation of miRNAs associated with pistil development in plants.

Comparative proteomic and transcriptomic approaches to address the active role of GA4 in Japanese apricot flower bud dormancy release

Hormones are closely associated with dormancy in deciduous fruit trees, and gibberellins (GAs) are known to be particularly important. In this study, we observed that GA4 treatment led to earlier bud break in Japanese apricot. To understand better the promoting effect of GA4 on the dormancy release of Japanese apricot flower buds, proteomic and transcriptomic approaches were used to analyse the mechanisms of dormancy release following GA4 treatment, based on two-dimensional gel electrophoresis (2-DE) and digital gene expression (DGE) profiling, respectively. More than 600 highly reproducible protein spots (P<0.05) were detected and, following GA4 treatment, 38 protein spots showed more than a 2-fold difference in expression, and 32 protein spots were confidently identified according to the databases. Compared with water treatment, many proteins that were associated with energy metabolism and oxidation–reduction showed significant changes after GA4 treatment, which might promote dormancy release. We observed that genes at the mRNA level associated with energy metabolism and oxidation–reduction also played an important role in this process. Analysis of the functions of the identified proteins and genes and the related metabolic pathways would provide a comprehensive proteomic and transcriptomic view of the coordination of dormancy release after GA4 treatment in Japanese apricot flower buds.

Genome-wide expression profiles of seasonal bud dormancy at four critical stages in Japanese apricot.

Dormancy is one of the most important adaptive mechanisms developed by perennial plants. To reveal the comprehensive mechanism of seasonal bud dormancy at four critical stages in Japanese apricot (Prunus persica), we applied Illumina sequencing to study differentially expressed genes (DEGs) at the transcriptional level. As a result, 19,759, 16,375, 19,749 and 20,800 tag-mapped genes were sequenced from libraries of paradormancy (R1), endodormancy (R2), ecodormancy (R3) and dormancy release (R4) stages based on the P. persica genome. Moreover, 6,199, 5,539, and 5,317 genes were differentially expressed in R1 versus R2, R2 versus R3, and R3 versus R4, respectively. Gene Ontology analysis of dormancy-related genes showed that these were mainly related to the cytoplasm, cytoplasmic part metabolism, intracellular metabolism and membrane-bound organelle metabolism. Pathway-enrichment annotation revealed that highly ranked genes were involved in ribosome pathways and protein processing in the endoplasmic reticulum. The results demonstrated that hormone response genes such as auxin, abscisic acid, ethylene and jasmonic acid, as well as zinc finger family protein genes are possibly involved in seasonal bud dormancy in Japanese apricot. The expression patterns of DEGs were verified using real-time quantitative RT-PCR. These results contribute to further understanding of the mechanism of bud dormancy in Japanese apricot.

Differential expression of proteins associated with seasonal bud dormancy at four critical stages in Japanese apricot.

Dormancy is of great significance in the growth and development of deciduous fruit trees. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS) to identify the differentially expressed proteome of Japanese apricot flower buds at four critical stages, from paradormancy before leaf fall to dormancy release. More than 400 highly reproducible protein spots (P < 0.05) were detected: 34 protein spots showed a greater than twofold difference in expression values, of which 32 protein spots were confidently identified from databases. Identified proteins were classified into six functional categories: stress response and defence (11), energy metabolism (ten), protein metabolism (five), cell structure (three), transcription (one) and unclassified (two). The glyoxalase I homologue could help Japanese apricot survival under various abiotic and biotic stresses, greatly contributing to its dormancy. Enolase, thioredoxin family proteins and triose phosphate isomerase provide adequate energy to complete consecutive dormancy release and bud break in Japanese apricot. Cinnamyl alcohol dehydrogenase 9 and arginase enhance the resilience of plants, enabling them to complete dormancy safely. Analysis of functions of identified proteins and related metabolic pathways will increase our knowledge of dormancy in woody plants.

Identification of Differentially-Expressed Genes Associated with Pistil Abortion in Japanese Apricot by Genome-Wide Transcriptional Analysis

The phenomenon of pistil abortion widely occurs in Japanese apricot, and imperfect flowers with pistil abortion seriously decrease the yield in production. Although transcriptome analyses have been extensively studied in the past, a systematic study of differential gene expression has not been performed in Japanese apricot. 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. 3,476,249 and 3,580,677 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. According to the results, we assumed that late embryogenesis abundant protein (LEA), Dicer-like 3 (DCL3) Xyloglucan endotransglucosylase/hydrolase 2 (XTH2), Pectin lyase-like superfamily protein (PPME1), Lipid transfer protein 3 (LTP3), Fatty acid biosynthesis 1 (FAB1) and Fatty acid desaturase 5 (FAD5) might have relationships with the pistil abortion in Japanese apricot. The expression patterns of 36 differentially expressed genes were confirmed by real-time (RT)-PCR. 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.

Evaluation of different kinds of organic acids and their antibacterial activity in Japanese Apricot fruits

The fruit of Japanese apricot is rich in organic acids, which have strong antibacterial activities. The types and contents of organic acids in six different cultivars of Japanese apricot fruit were evaluated by reverse-phase high performance liquid chromatography (HPLC). The antibacterial activity against Escherichia coli, Bacillus subtilis and Streptococcus suis was also determined. The results revealed that there are nine types of organic acids in the presence of the extracts of Japanese apricot fruit, including oxalic, tartaric, malic, ascorbic, acetic, citric, maleic, fumaric and succinic acids. The total organic acid content of ‘Zhonghong’ was the highest among the studied cultivars, and the main organic acids present were citric and malic acids. The antibacterial activity on the growth of E. coli and B. subtilis was higher than on the growth of S. suis. The antibacterial effect of acetic acid against bacteria was the best, and the minimum antibacterial concentration (MIC) against E. coli and B. subtilis was 0.417 mg/mL. The citric and maleic acids were also against these three strains of bacteria. The results suggest that the antibacterial activity is related to the organic acid composition and content of Japanese apricot fruit.

Comparative proteomic analysis of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low temperature.

Low-temperature stress is one of the major abiotic stresses in plants worldwide, and the dehydration responsive element binding protein (DREB) transcription factor induces expression of genes involved in environmental stress tolerance in plants. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) and subsequent mass spectrometric identification was used to study the changes in the leaf proteome profiles of rd29A:RdreB1BI transgenic and non-transgenic strawberries exposed to low-temperature conditions. By comparing the proteomic profiles, we located 21 protein spots that were reproducibly up- or down-regulated by more than twofold between transgenic and non-transgenic strawberries. Eight identified proteins function in energy and metabolism, four in biosynthetic processes, four were stress and defense related, three spots were identified as cold-stress related expressed sequence tags (ESTs), and two were unknown proteins. The change patterns of low-temperature tolerance proteins, including photosynthetic proteins (RuBisCO large subunit and RuBisCO activase), cytoplasmic Cu/Zn-superoxide dismutase (Cu/Zn-SOD), late embryogenesis abundant protein 14-A (Lea14-A), eukaryotic translation initiation factor 5A (eIF5A), and cold-stress related ESTs, were differentially regulated between non-transgenic and rd29A:RdreB1BI transgenic strawberries. They are likely important gene products in the regulatory network of the RdreB1BI gene. Consequently, this study provides the first characterization of the transgenic strawberry proteome and the predicted target proteins of the RdreB1BI gene by using proteomic approaches.

Metabolic changes upon flower bud break in Japanese apricot are enhanced by exogenous GA4.

Gibberellin (GA4) has a significant effect on promoting dormancy release in flower buds of Japanese apricot (Prunus mume Sieb. et Zucc). The transcriptomic and proteomic changes that occur after GA4 treatment have been reported previously; however, the metabolic changes brought about by GA4 remain unknown. The present study was undertaken to assess changes in metabolites in response to GA4 treatment, as determined using gas chromatography–mass spectrometry and principal component analysis. Fifty-five metabolites that exhibited more than two-fold differences in abundance (P < 0.05) between samples collected over time after a given treatment or between samples exposed to different treatments were studied further. These metabolites were categorized into six main groups: amino acids and their isoforms (10), amino acid derivatives (7), sugars and polyols (14), organic acids (12), fatty acids (4), and others (8). All of these groups are involved in various metabolic pathways, in particular galactose metabolism, glyoxylate and dicarboxylate metabolism, and starch and sucrose metabolism. These results suggested that energy metabolism is important at the metabolic level in dormancy release following GA4 treatment. We also found that more than 10-fold differences in abundance were observed for many metabolites, including sucrose, proline, linoleic acid, and linolenic acid, which might play important roles during the dormancy process. The current research extends our understanding of the mechanisms involved in budburst and dormancy release in response to GA4 and provides a theoretical basis for applying GA4 to release dormancy.

Sequence Analysis of New S-RNase and SFB alleles in Japanese Apricot (Prunus mume)

As with other self-incompatible Prunus species, cultivars of Japanese apricot (Prunus mume Sieb. et Zucc.) display the S-RNase-based gametophytic self-incompatibility system. In this study, S-genotypes of ten Japanese apricot cultivars native to China were subjected to polyacrylamide gel electrophoresis (PAGE) analysis using an efficient Prunus S-RNase primer pair, Pru-C2 and PCE-R. In addition, three new S-RNase genes (S34, S35 and S36-RNase) and six new SFB genes (PmSFB14, PmSFB18, PmSFB22, PmSFB24, PmSFB31 and PmSFB34) were identified and their sequences were characterized and deposited in the GenBank database.

Expression analysis of ABA- and GA-related genes during four stages of bud dormancy in Japanese apricot (Prunus mume Sieb. et Zucc)

ABSTRACT Seasonal dormancy in deciduous fruit trees is a complex physiological state, which is regulated by many plant hormones and genes. Abscisic acid (ABA) and gibberellic acid (GA3) are known to be particularly important in controlling dormancy. We measured the concentrations of these two hormones in flower buds (collected during para-, endo-, and eco-dormancy, and at the dormancy-release stage) and vegetative buds (collected monthly from 20 November 2012 to 20 February 2013) of two cultivars of Japanese apricot (Prunus mume Sieb. et Zucc), ‘Bungo’ and ‘Taoxingmei’, using liquid chromatography and tandem mass spectrometry (LC–MS/MS). Levels of expression of two zinc-finger family genes CCCH and C2H2 and the GA20ox (gibberellin 20 oxidase) gene (GenBank Accession No. Pm013607), as candidates for dormancy-controlling genes, were analysed at each sampling time using real-time RT-quantitative PCR (RT-qPCR). Expression analyses indicated that both zinc-finger family genes exhibited high transcript levels in flower buds of both cultivars during para-dormancy, concomitant with high ABA concentrations. The zinc-finger family genes were relatively highly expressed in vegetative buds of both cultivars on 20 November 2012 (autumn). In addition, high concentration of GA3 in flower buds at the dormancy-release stage suggested that GA3 plays an important role in controlling the release of bud dormancy. High levels of expression of the GA20ox gene during eco-dormancy and at the dormancy-release stage might have a prominent effect on dormancy-release by regulating gibberellin (GA) signalling in Japanese apricot. These results will contribute to increasing our understanding of ABA- and GA-mediated seasonal dormancy mechanisms in Japanese apricot.