Shengxiong Huang

Draft genome of the kiwifruit Actinidia chinensis

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1 Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage.

A Role of Tomato UV-Damaged DNA Binding Protein 1 (DDB1) in Organ Size Control via an Epigenetic Manner

Epigenetic modification generally refers to phenotypic changes by a mechanism other than changes in DNA sequence and plays a significant role in developmental processes. In this study, we found that overexpression of one alternatively spliced tomato DDB1 transcript, DDB1F that is prevalently present in all tested tissues, resulted in reduction of organ size. Transgenic plants constitutively expressing the DDB1F from a strong cauliflower mosaic virus (CaMV) 35S promoter displayed moderately reduced size in vegetative organs (leaves and stems) and radically decreased size in reproductive organs (flowers, seeds and fruits), in which several genes encoding negative regulators for cell division were upregulated. Significantly, reduction of organ size conferred by overexpression of DDB1F transgene appears not to segregate in the subsequent generations, suggesting the phenotypic alternations are manipulated in an epigenetic manner and can be transmitted over generations. This notion was further substantiated by analysis of DNA methylation level at the SlWEE1 gene (encoding a negative regulator of cell division), revealing a correlation between less methylation in the promoter region and elevated expression level of this gene. Thus, our results suggest DDB1 plays an important role in regulation of the epigenetic state of genes involved in organogenesis, despite the underlying mechanism remains to be elucidated.

Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis)

Genomic and transcriptomic data on kiwifruit (Actinidia chinensis) in public databases are very limited despite its nutritional and economic value. Previously, we have constructed and sequenced nine fruit RNA-Seq libraries of A. chinensis “Hongyang” at immature, mature, and postharvest ripening stages of fruit development, and generated over 66.2 million paired-end and 24.4 million single-end reads. From this dataset, here we have identified 7051 long noncoding RNAs (lncRNAs), 29,327 alternative splicing (AS) events and 2980 novel protein-coding genes that were not annotated in the draft genome of “Hongyang.” AS events were demonstrated in genes involved in the synthesis of nutritional metabolites in fruit, such as ascorbic acids, carotenoids, anthocyanins, and chlorophylls, and also in genes in the ethylene signaling pathway, which plays an indispensable role in fruit ripening. Additionally, transcriptome profiles and the contents of sugars, organic and main amino acids were compared between immature, mature, and postharvest ripening stages in kiwifruits. A total of 5931 differentially expressed genes were identified, including those associated with the metabolism of sugar, organic acid, and main amino acids. The data generated in this study provide a foundation for further studies of fruit development and ripening in kiwifruit, and identify candidate genes and regulatory elements that could serve as targets for improving important agronomic traits through marker assisted breeding and biotechnology.

PTIR: Predicted Tomato Interactome Resource

Protein-protein interactions (PPIs) are involved in almost all biological processes and form the basis of the entire interactomics systems of living organisms. Identification and characterization of these interactions are fundamental to elucidating the molecular mechanisms of signal transduction and metabolic pathways at both the cellular and systemic levels. Although a number of experimental and computational studies have been performed on model organisms, the studies exploring and investigating PPIs in tomatoes remain lacking. Here, we developed a Predicted Tomato Interactome Resource (PTIR), based on experimentally determined orthologous interactions in six model organisms. The reliability of individual PPIs was also evaluated by shared gene ontology (GO) terms, co-evolution, co-expression, co-localization and available domain-domain interactions (DDIs). Currently, the PTIR covers 357,946 non-redundant PPIs among 10,626 proteins, including 12,291 high-confidence, 226,553 medium-confidence, and 119,102 low-confidence interactions. These interactions are expected to cover 30.6% of the entire tomato proteome and possess a reasonable distribution. In addition, ten randomly selected PPIs were verified using yeast two-hybrid (Y2H) screening or a bimolecular fluorescence complementation (BiFC) assay. The PTIR was constructed and implemented as a dedicated database and is available at http://bdg.hfut.edu.cn/ptir/index.html without registration.

Tomato MBD5, a methyl CpG binding domain protein, physically interacting with UV-damaged DNA binding protein-1, functions in multiple processes.

In tomato (Solanum lycopersicum), high pigment mutations (hp-1 and hp-2) were mapped to genes encoding UV-damaged DNA binding protein 1 (DDB1) and de-etiolated-1 (DET1), respectively. Here we characterized a tomato methyl-CpG-binding domain protein SlMBD5 identified by yeast two-hybrid screening using SlDDB1 as a bait. Yeast two-hybrid assay demonstrated that the physical interaction of SlMBD5 with SlDDB1 is mediated by the C-termini of SlMBD5 and the β-propeller-C (BPC) of SlDDB1. Co-immunoprecipitation analyses revealed that SlMBD5 associates with SlDDB1-interacting partners including SlDET1, SlCUL4, SlRBX1a and SlRBX1b in vivo. SlMBD5 was shown to target to nucleus and dimerizes via its MBD motif. Electrophoresis mobility shift analysis suggested that the MBD of SlMBD5 specifically binds to methylated CpG dinucleotides but not to methylated CpHpG or CpHpH dinucleotides. SlMBD5 expressed in protoplast is capable of activating transcription of CG islands, whereas CUL4/DDB1 antagonizes this effect. Overexpressing SlMBD5 resulted in diverse developmental alterations including darker green fruits with increased plastid level and elevated pigmentation, as well as enhanced expression of SlGLK2, a key regulator of plastid biogenesis. Taken together, we hypothesize that the physical interaction of SlMBD5 with the CUL4-DDB1-DET1 complex component may affect its binding activity to methylated DNA and subsequently attenuate its transcription activation of downstream genes.

Roles of UV-damaged DNA binding protein 1 (DDB1) in epigenetically modifying multiple traits of agronomic importance in tomato

Epigenetic regulation participates broadly in many fundamentally cellular and physiological processes. In this study, we found that DDB1, a protein originally identified as a factor involved in DNA repairing, plays important roles in regulating organ size, growth habit and photosynthesis in tomato via an epigenetic manner. We generated transgenic tomato plants overexpressing an alternatively spliced DDB1 transcript (DDB1F, prevalently present in tomato tissues) and found the primary transformants displayed small-fruited “cherry tomato” in companion with strikingly enhanced shoot branching and biomass, dark-green leaves with elevated chlorophyll accumulation, and increased soluble solids in fruits. Significantly, these phenotypic alterations did not segregate with the DDB1F transgene in subsequent generations, suggesting that the effect of DDB1F on multiple agronomic traits is implemented via an epigenetic manner and is inheritable over generations. We speculate that DDB1, as a core subunit in the recently identified CUL4-based E3 ligase complex, mediates the 26S proteasome-dependent degradation of a large number of proteins, some of which might be required for perpetuating epigenetic marks on chromatins.

Determination of garlic phenolic compounds using supercritical fluid extraction coupled to supercritical fluid chromatography/tandem mass spectrometry

Graphical abstract Figure. No caption available. HighlightsAn off‐line SFE‐SFC‐MS/MS method was established for determination of nine phenolic compounds in garlic.A response surface methodology (RSM) was used for optimization of SFE parameters.0.1 mM oxalic acid and 1 mM ammonium formate in methanol were chosen as the most suitable modifiers in SFC‐MS/MS.The method achieved its successful application on garlic samples from four origins. ABSTRACT A rapid, simple and environmentally friendly supercritical fluid extraction and supercritical fluid chromatography coupled to mass spectrometry (SFE‐SFC‐MS/MS) method has been developed for the analysis of nine phenolic compounds in garlic. Through the optimization of the SFE parameters using response surface methodology (RSM), 15 phenolic compounds were successfully extracted at 50 °C in 9 min with the addition of 30% methanol using a method that was automatic, efficient, green and prevented oxidation. Moreover, the operating conditions of the SFC‐MS/MS were also optimized for the analysis of the SFE extracts. 15 phenolic compounds in the garlic showed good separation performance on a Shim‐pack UC‐X Diol column, and 0.1 mM oxalic acid and 1 mM ammonium formate in methanol were chosen as the most suitable mobile phase components. Furthermore, a calibration curve was developed using a matrix‐matched standard, and 9 phenolic compounds showed good linearity (R2 > 0.99). The limits of detection of the 9 phenolic compounds varied from 0.6 to 12 ng/g, and the quantifications ranged from 2 to 40 ng/g. The validation data also showed acceptable recoveries for 9 phenolic compounds, and the relative standard deviations (RSDs) for the overall precision were lower than 10%. The developed method has been utilized to analyze the phenolic compounds in garlic from different geographical areas of China (Cangshan, Jinxiang, Pizhou, Dali), which suggests that this off‐line SFE‐SFC‐MS/MS method could be adapted to the quantitative analysis of phenolic compounds in garlic.

The transcriptome analyses of Tagetes erecta provides novel insights into secondary metabolite biosynthesis during flower development

Genomic and transcriptomic sequences of Tagetes erecta are very limited in public databases, despite its nutritional and economical value. In this study, nine cDNA libraries were constructed from leaves, immature and mature flowers and sequenced using Illumina Hiseq 2000. Over 45.66 Gb nucleotides were generated and a total of 72,499 unigenes were assembled, 34,892 (48.13%) of which were annotated in NR, Swiss-prot, COG, GO, KOG, KEGG and Pfam. 11,721 differentially expressed genes were identified in leaves, immature flowers and mature flowers. The differentially expressed genes between immature flowers and mature flowers were mainly involved in photosynthesis and secondary metabolite biosynthesis. Additionally, the catalysis-related unigenes and their expression involved in carotenoids biosynthesis were determined. Using our newly identified reference genes as internal control, the expression profiles of carotenoids biosynthetic genes were verified by real-time qRT-PCR, and four of the unigenes were cloned for full length sequences confirmation. Furthermore, gene expansions occurred among the catalytic gene families in carotenoids biosynthesis pathway, which might explain the high pigment content in T. erecta.