MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening.

Fruit ripening is a complex developmental process, which is modulated by both transcriptional and post-translational events. Control of fruit ripening is important in maintaining moderate quality traits and minimizing postharvest deterioration. In this study, we discovered that the transcription factor MaMYB4 acts as a negative regulator of fruit ripening in banana. The protein levels of MaMYB4 decreased gradually with banana fruit ripening, paralleling ethylene production, and decline in firmness. DNA affinity purification sequencing combined with RNA-sequencing analyses showed that MaMYB4 preferentially binds to the promoters of various ripening-associated genes including ethylene biosynthetic and cell wall modifying genes. Furthermore, ectopic expression of MaMYB4 in tomato delayed tomato fruit ripening, which was accompanied by downregulation of ethylene biosynthetic and cell wall modifying genes. Importantly, two RING finger E3 ligases MaBRG2/3, whose protein accumulation increased progressively with fruit ripening, were found to interact with and ubiquitinate MaMYB4, contributing to decreased accumulation of MaMYB4 during fruit ripening. Transient overexpression of MaMYB4 and MaBRG2/3 in banana fruit ripening delayed or promoted fruit ripening by inhibiting or stimulating ethylene biosynthesis, respectively. Taken together, we demonstrate that MaMYB4 negatively modulates banana fruit ripening, and that MaMYB4 abundance could be regulated by protein ubiquitination, thus providing insights into the role of MaMYB4 in controlling fruit ripening at both transcriptional and post-translational levels.

MaXB3 Modulates MaNAC2, MaACS1, and MaACO1 Stability to Repress Ethylene Biosynthesis during Banana Fruit Ripening.

Ethylene plays a critical regulatory role in climacteric fruit ripening, and its biosynthesis is fine-tuned at the transcriptional and posttranslational levels. Nevertheless, the mechanistic link between transcriptional and posttranslational regulation of ethylene biosynthesis during fruit ripening is largely unknown. This study uncovers a coordinated transcriptional and posttranslational mechanism of controlling ethylene biosynthesis during banana (Musa acuminata) fruit ripening. NAC (NAM, ATAF, and CUC) proteins MaNAC1 and MaNAC2 repress the expression of MaERF11, a protein previously known to negatively regulate ethylene biosynthesis genes MaACS1 and MaACO1 A RING E3 ligase MaXB3 interacts with MaNAC2 to promote its ubiquitination and degradation, leading to the inhibition of MaNAC2-mediated transcriptional repression. In addition, MaXB3 also targets MaACS1 and MaACO1 for proteasome degradation. Further evidence supporting the role of MaXB3 is provided by its transient and ectopic overexpression in banana fruit and tomato (Solanum lycopersicum), respectively, which delays fruit ripening via repressing ethylene biosynthesis and thus ethylene response. Strikingly, MaNAC1 and MaNAC2 directly repress MaXB3 expression, suggesting a feedback regulatory mechanism that maintains a balance of MaNAC2, MaACS1, and MaACO1 levels. Collectively, our findings establish a multilayered regulatory cascade involving MaXB3, MaNACs, MaERF11, and MaACS1/MaACO1 that controls ethylene biosynthesis during climacteric ripening.

Integrative comparative analyses of metabolite and transcript profiles uncovers complex regulatory network in tomato (Solanum lycopersicum L.) fruit undergoing chilling injury.

Tomato fruit are especially susceptible to chilling injury (CI) when continuously exposed to temperatures below 12 °C. In this study, integrative comparative analyses of transcriptomics and metabolomics data were performed to uncover the regulatory network in CI tomato fruit. Metabolite profiling analysis found that 7 amino acids, 27 organic acids, 16 of sugars and 22 other compounds had a significantly different content while transcriptomics data showed 1735 differentially expressed genes (DEGs) were down-regulated and 1369 were up-regulated in cold-stored fruit. We found that the contents of citrate, cis-aconitate and succinate were increased, which were consistent with the expression of ATP-citrate synthase (ACS) and isocitrate dehydrogenase (IDH) genes in cold-treated tomato fruit. Cold stress promotes the expression of ACS and IDH which may increase the synthesis of citrate, cis-aconitate and succinate. Alanine and leucine had increased contents, which may result from alanine aminotransferase (ALT) and branched-chain amino acid aminotransferase (BcAT)'s high expression levels, respectively. Overall the transcriptomics and metabolomics data in our study explain the molecular mechanisms of the chilling injury and expands our understanding of the complex regulatory mechanisms of a metabolic network in response to chilling injury in tomato fruit.

Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes.

Genetic Divergence between Camellia sinensis and Its Wild Relatives Revealed via Genome-Wide SNPs from RAD Sequencing.

Tea is one of the most popular beverages across the world and is made exclusively from cultivars of Camellia sinensis. Many wild relatives of the genus Camellia that are closely related to C. sinensis are native to Southwest China. In this study, we first identified the distinct genetic divergence between C. sinensis and its wild relatives and provided a glimpse into the artificial selection of tea plants at a genome-wide level by analyzing 15,444 genomic SNPs that were identified from 18 cultivated and wild tea accessions using a high-throughput genome-wide restriction site-associated DNA sequencing (RAD-Seq) approach. Six distinct clusters were detected by phylogeny inferrence and principal component and genetic structural analyses, and these clusters corresponded to six Camellia species/varieties. Genetic divergence apparently indicated that C. taliensis var. bangwei is a semi-wild or transient landrace occupying a phylogenetic position between those wild and cultivated tea plants. Cultivated accessions exhibited greater heterozygosity than wild accessions, with the exception of C. taliensis var. bangwei. Thirteen genes with non-synonymous SNPs exhibited strong selective signals that were suggestive of putative artificial selective footprints for tea plants during domestication. The genome-wide SNPs provide a fundamental data resource for assessing genetic relationships, characterizing complex traits, comparing heterozygosity and analyzing putatitve artificial selection in tea plants.

[Rice area change in Northeast China and its correlation with climate change.] / 气候变化背景下东北水稻的时空分布特征.

Based on the time-series map of rice area, a spatial production allocation model (SPAM) which has been applied for mapping the global level crop allocation datasets was deve-loped to simulate the spatio-temporal dynamics of rice area in Northeast China during 1980-2010 within 5'×5' grid cells. The spatio-temporal variations of rice area with temperature and precipita-tion during past 30 years were explored. The results indicated that the rice area expanded significantly northwards to46° N before 2000. After that, the increased sown area mainly occurred in the northern parts of Northeast China. Meanwhile, rice area also expanded eastwards to 131° E and toward the higher elevation regions (above 200 m). Due to a northward movement of accumulated temperature belts, the new rice area mainly appeared in the regions with an annual accumulated temperature (AAT) between 2800 and 3400 ℃·d. The trend of precipitation during the study period increased before 2000 and decreased afterwards. The increased rice area was found mainly in the regions with precipitation range from 300 mm to 600 mm.

Model-based online learning with kernels.

New optimization models and algorithms for online learning with Kernels (OLK) in classification, regression, and novelty detection are proposed in a reproducing Kernel Hilbert space. Unlike the stochastic gradient descent algorithm, called the naive online Reg minimization algorithm (NORMA), OLK algorithms are obtained by solving a constrained optimization problem based on the proposed models. By exploiting the techniques of the Lagrange dual problem like Vapnik's support vector machine (SVM), the solution of the optimization problem can be obtained iteratively and the iteration process is similar to that of the NORMA. This further strengthens the foundation of OLK and enriches the research area of SVM. We also apply the obtained OLK algorithms to problems in classification, regression, and novelty detection, including real time background substraction, to show their effectiveness. It is illustrated that, based on the experimental results of both classification and regression, the accuracy of OLK algorithms is comparable with traditional SVM-based algorithms, such as SVM and least square SVM (LS-SVM), and with the state-of-the-art algorithms, such as Kernel recursive least square (KRLS) method and projectron method, while it is slightly higher than that of NORMA. On the other hand, the computational cost of the OLK algorithm is comparable with or slightly lower than existing online methods, such as above mentioned NORMA, KRLS, and projectron methods, but much lower than that of SVM-based algorithms. In addition, different from SVM and LS-SVM, it is possible for OLK algorithms to be applied to non-stationary problems. Also, the applicability of OLK in novelty detection is illustrated by simulation results.

Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to 'Candidatus Liberibacter asiaticus' infection.

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by 'Candidatus Liberibacter asiaticus'. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to 'Ca. L. asiaticus' infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to 'Ca. L. asiaticus'. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.

Detail-enhanced exposure fusion.

In a typical processing chain of image enhancement, an exposure fusion scheme can be used to synthesize a more detailed low dynamic range (LDR) image directly from a set of differently exposed LDR images, without generation of an intermediate high dynamic range image. In this brief, we introduce a new quadratic optimization-based method to extract fine details from a vector field. The new method extracts fine details from a set of differently exposed LDR images simultaneously. The extracted fine details are then added to an intermediate LDR image which is fused by simply using an existing exposure fusion scheme. With this, the proposed scheme can enhance fine details to produce sharper images.

Comparative iTRAQ proteome and transcriptome analyses of sweet orange infected by "Candidatus Liberibacter asiaticus".

Citrus Huanglongbing (HLB) has been threatening citrus production worldwide. In this study, a comparative proteomic approach was applied to understand the pathogenic process of HLB in affected sweet orange leaves. Using the isobaric tags for relative and absolute quantification (iTRAQ) technique, we identified 686 unique proteins in the mature leaves of both mock-inoculated and diseased 'Madam Vinous' sweet orange plants. Of the identified proteins, 20 and 10 were differentially expressed in leaves with and without symptoms of HLB (fold change > 2.5), respectively, compared with mock-inoculated controls. Most significantly, upregulated proteins were involved in stress/defense response, such as four miraculin-like proteins, chitinase, Cu/Zn superoxide dismutase and lipoxygenase. Microarray analysis also showed that stress-related genes were significantly upregulated at the transcriptional level. For example, remarkable upregulations of miraculin-like proteins and Cu/Zn superoxide dismutase transcripts were observed. Moreover, the transcriptional patterns of miraculin-like protein 1 and Cu/Zn superoxide dismutase were examined at different stages of HLB disease development. Combined with the transcriptomic data, the proteomic data can provide an enhanced understanding of citrus stress/defense responses to HLB.

Characterization of three terpenoid glycosyltransferase genes in 'Valencia' sweet orange (Citrus sinensis L. Osbeck).

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in 'Valencia' sweet orange (Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730-874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

Isolation and characterization of a novel lignocellulose decomposing fungal strain.

A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70 degrees C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28 degrees C (pH 6.0) in flasks stirred at 150 x g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization.

Isolation of single chain variable fragments against six esters of pyrethrins by subtractive phage display.

Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger(4)Ser)(3) and short-linker (Ger(4)Ser) were established to contain 1.04 x 10(7) or 6.07 x 10(6) transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.

[Construction and preliminary analysis of subtractive library for peel pitting of 'Fengjie' navel orange fruit].

In this study, the pitted peel and non-pitted peel of 'Fengjie' navel orange fruits were used as experimental materials to construct and screen the peel pitting related genes by suppression subtractive hybridization (SSH). The results showed that suppression subtractive hybridization was very effective. A cDNA library of differentially expressed genes was constructed. The library included about 200 clones with an average insert size of around 300 bp. Part of the positive clones were picked up randomly and sequenced. Six of the 50 clones had no homologous sequences being found and three had unknown functions in GenBank. According to the analysis of the homology, four homologous (Ca2+ binding protein, cysteine proteinase, NAC-domain protein and expansin) genes were chosen to examine their expressions through semi-quantitative RT-PCR analysis in pitted and non-pitted navel orange fruits. The expression of four genes were all higher in pitted peel than that in non-pitted peel. It suggests that these genes in the SSH cDNA library may be involved with peel pitting and can be subject of future investigation to explore the molecular biological mechanism of the pitting of citrus fruit.

[The relationship between activity and gene expression of phenylalanine ammonia-lyase and peel pitting in 'Fengjie' navel orange fruits].

Citrus fruit is prone to develop peel pitting during development and storage, which greatly decreases its fresh market value because of the deterioration of the peel. In the present study, we have examined the effect of different temperatures (15 degrees C and 4 degrees C), waxing and mechanical damage on the changes in the activity of phenylalanine ammonia-lyase (PAL) and the incidence of peel pitting in 'Fengjie' navel orange (Citrus sinensis Osbeck) fruits. The expression levels of PAL2, PAL6 genes in the peel during the development of peel pitting have been investigated through semi-quantitative PCR method. The incidence of peel pitting was greatly enhanced by waxing and mechanical damage and was decreased in lower temperature storage (4 degrees C) (Fig.1). Waxing and mechanical damage might be the important factors inducing peel pitting and suitable low temperature could decrease the incidence of this disease. The PAL activity increased during the whole storage period in accordance with the development of this pitting (Fig.2). The expression levels of PAL2 and PAL6 genes in damaged peel were higher than those in healthy peel and the expression of PAL2 is much more higher than that of PAL6 (Figs.4 and 5). The results suggested that the enzyme activity of PAL, along with the expression of PAL2 gene is highly related to this peel pitting occurred on 'Fengjie' navel orange fruits.

Expression and purification of varicella-zoster virus glycoprotein I gene in insect cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To express the cloned gene glycoprotein I (gpI) of varicella-zoster virus (VZV), Beijing VZV 84-7 strain in insect cells and to purify its expression product.</p><p><b>METHODS</b>The gene coding for gpI of VZV was amplified from viral DNA by PCR and cloned into baculovirus transfer vector (pBacPAK9), and recombinant transfer vector plasmid pBacVZVgpI was obtained. The inserted gpI gene in the pBacVZVgpI was sequenced. Insect cells Sf 9 were co-transfected with the recombinant transfer vector plasmid pBacVZVgpI and wild type linear baculovirus BacPAK6 (digested with Bsu36I) DNA. The recombinant baculoviruses containing the VZV 84-7 gpI gene was isolated through several rounds of limited dilution. Recombinant protein gpI was expressed in insect cells Sf 9, postinfected with recombinant baculoviruses. The expressed recombinant gpI was purified by lectin affinity chromatography and its antigenicity and immunogenicity were investigated.</p><p><b>RESULTS</b>The gene coding for gpI of VZV was obtained by PCR and the gpI gene of pBacPAK9 was confirmed by DNA sequencing. The recombinant gpI was expressed in insect cells Sf 9, post-infected with recombinant baculovirus and identified by SDS-PAGE and western blotting, with its product in cell culture reaching the peak in 72 hours and with a molecular mass of 58 kd and 70 kd, the same as theoretical values. Results of immunoassay with cell lysates infected by recombinant baculoviruses indicated that recombinant protein expressed in insect cells had ability of eliciting specific antibodies against native VZV in mice and complement-dependent neutralizing antibodies. The purified recombinant gpI gave a product with a purity of more than 80%. ELISA and Western-blot analysis demonstrated that purified protein had specific VZV antibody-binding activity. This suggested that the recombinant gpI expressed in insect cells had the same biological characteristics as its native counterpart.</p><p><b>CONCLUSION</b>Baculovirus-insect cells could be used to express the gene of VZV gpI, which could provide a basis for quantitative analysis of VZV antigen, and preparation of its subunit vaccine.</p>

Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit.

Following differential screening of gene expression during tomato fruit development, we isolated developmentally regulated (DR) clones, including several putative transcription factors. Based on sequence homology, DR1, DR3, DR4 and DR8 are members of the Aux/IAA family, and DR12 belongs to the auxin response factor (ARF) family of transcription factors. Importantly, mRNA accumulation for the Aux/IAA-like genes was regulated by ethylene in tomato fruit but not in the leaves, indicating that these putative auxin response components also participate to the ethylene-dependent regulation of gene expression in a tissue-specific manner. The functional significance of DR12, the ARF-like gene, was investigated by cellular biology and reverse genetics approaches. Heterologous protein targeting studies, carried out using a DR12-GFP gene fusion construct, revealed specific nuclear localization of the DR12-encoded protein, in accordance with its putative function as a transcriptional regulator. Transgenic plants over- and under-expressing DR12 were generated in order to explore the physiological role of the gene. Both antisense and sense co-suppressed DR12-inhibited lines displayed a pleiotropic phenotype that included dark-green immature fruit, unusual cell division in the fruit pericarp, blotchy ripening, enhanced fruit firmness, upward curling leaves and increased hypocotyl and cotyledon growth. While a perturbation of the response to auxin may explain some of the phenotypes, surprisingly, the expression of members of four classes of early auxin-regulated genes was unaffected in the DR12-inhibited plants. The involvement of this ARF-like encoded protein in mediating the auxin response is discussed along with the possibility that it might affect responsiveness to other phytohormones in the tomato.