Advances in AP2/ERF super-family transcription factors in plant.

In the whole life process, many factors including external and internal factors affect plant growth and development. The morphogenesis, growth, and development of plants are controlled by genetic elements and are influenced by environmental stress. Transcription factors contain one or more specific DNA-binding domains, which are essential in the whole life cycle of higher plants. The AP2/ERF (APETALA2/ethylene-responsive element binding factors) transcription factors are a large group of factors that are mainly found in plants. The transcription factors of this family serve as important regulators in many biological and physiological processes, such as plant morphogenesis, responsive mechanisms to various stresses, hormone signal transduction, and metabolite regulation. In this review, we summarized the advances in identification, classification, function, regulatory mechanisms, and the evolution of AP2/ERF transcription factors in plants. AP2/ERF family factors are mainly classified into four major subfamilies: DREB (Dehydration Responsive Element-Binding), ERF (Ethylene-Responsive-Element-Binding protein), AP2 (APETALA2) and RAV (Related to ABI3/VP), and Soloists (few unclassified factors). The review summarized the reports about multiple regulatory functions of AP2/ERF transcription factors in plants. In addition to growth regulation and stress responses, the regulatory functions of AP2/ERF in plant metabolite biosynthesis have been described. We also discussed the roles of AP2/ERF transcription factors in different phytohormone-mediated signaling pathways in plants. Genomic-wide analysis indicated that AP2/ERF transcription factors were highly conserved during plant evolution. Some public databases containing the information of AP2/ERF have been introduced. The studies of AP2/ERF factors will provide important bases for plant regulatory mechanisms and molecular breeding.

The genome sequence of 'Kurodagosun', a major carrot variety in Japan and China, reveals insights into biological research and carrot breeding.

Carrot (Daucus carota L.) is one of the most economically important root vegetables in the world, providing numerous nutrients for human health. China is the largest country of carrot production in the world, and 'Kurodagosun' has been a major carrot variety in China. Carrot material used in this study was the inbred line 'DC-27', which was derived by forced selfing from 'Kurodagosun'. To understand the genetic system and plant-specific genes of 'Kurodagosun', we report the draft genome sequence of carrot 'DC-27' assembled using a combination of Roche454 and HiSeq 2000 sequencing technologies to achieve 32-fold genome coverage. A total of 31,891 predicted genes were identified. These assembled sequences provide candidate genes involved in biological processes including stress response and carotenoid biosynthesis. Genomic sequences corresponding to 371.6 Mb was less than 473 Mb, which is the estimated genome size. The availability of a draft sequence of the 'DC-27' genome advances knowledge on the biological research and breeding of carrot, as well as other Apiaceae plants. The 'DC-27' genome sequence data also provide a new resource to explore the evolution of other higher plants.

Advances in the research of celery, an important Apiaceae vegetable crop.

Celery (Apium graveolens L.), one of the most important vegetables in Apiaceae family, is cultivated worldwide and utilized in food and cosmetic industries because it is an excellent source of vitamins, phenolic compounds, volatile oils and other nutrients. Celery extracts possess various medicinal properties, such as antibacterial, anti-inflammatory and lowering blood glucose and serum lipid levels. With the rapid advancements in molecular biology and sequencing technology, studies on celery have been performed. Numerous molecular markers and regulatory genes have been discovered and applied to improve celery. Research advances, including genetic breeding, genomics research, function genes and chemical composition, regarding celery are reviewed in this paper. Further exploration and application trends are briefly described. This review provides a reference for basic and applied research on celery, an important Apiaceae vegetable crop.

Genome-wide analysis of the R2R3-MYB transcription factor genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveals their stress and hormone responsive patterns.

BACKGROUND: The MYB superfamily is one of the most abundant transcription factor (TF) families in plants. MYB proteins include highly conserved N-terminal MYB repeats (1R, R2R3, 3R, and atypical) and various C-terminal sequences that confer extensive functions. However, the functions of most MYB genes are unknown, and have been little studied in Chinese cabbage. RESULTS: Here, we analyzed 256 (55.2% of total MYBs) R2R3-MYB genes from Chinese cabbage (Brassica rapa ssp. pekinensis) and anchored them onto the 10 chromosomes and three subgenomes. The R2R3-, 3R- and atypical MYB proteins in Chinese cabbage formed 45 subgroups based on domain similarity and phylogenetic topology. Organization and syntenic analysis revealed the genomic distribution and collinear relationships of the R2R3-BrMYBs. Synonymous nucleotide substitution (Ka/Ks) analysis showed that the Chinese cabbage MYB DNA-binding domain is under strong purifying selection. Moreover, RNA-seq data revealed tissue-specific and distinct R2R3-BrMYB expression profiles, and quantitative real-time PCR (qPCR) analysis in leaves showed stress responsive expression and crosstalk with ABA-auxin signaling cascades. CONCLUSIONS: In this study, we identified the largest MYB gene family in plants to date. Our results indicate that members of this superfamily may be involved in plant development, stress responses and leaf senescence, highlighting their functional diversity.

Genome-wide analysis of the SBP-box gene family in Chinese cabbage (Brassica rapa subsp. pekinensis).

The SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box gene family contains highly conserved plant-specific transcription factors that play an important role in plant development, especially in flowering. Chinese cabbage (Brassica rapa subsp. pekinensis) is a leafy vegetable grown worldwide and is used as a model crop for research in genome duplication. The present study aimed to characterize the SBP-box transcription factor genes in Chinese cabbage. Twenty-nine SBP-box genes were identified in the Chinese cabbage genome and classified into six groups. We identified 23 orthologous and 5 co-orthologous SBP-box gene pairs between Chinese cabbage and Arabidopsis. An interaction network among these genes was constructed. Sixteen SBP-box genes were expressed more abundantly in flowers than in other tissues, suggesting their involvement in flowering. We show that the MiR156/157 family members may regulate the coding regions or 3'-UTR regions of Chinese cabbage SBP-box genes. As SBP-box genes were found to potentially participate in some plant development pathways, quantitative real-time PCR analysis was performed and showed that Chinese cabbage SBP-box genes were also sensitive to the exogenous hormones methyl jasmonic acid and salicylic acid. The SBP-box genes have undergone gene duplication and loss, evolving a more refined regulation for diverse stimulation in plant tissues. Our comprehensive genome-wide analysis provides insights into the SBP-box gene family of Chinese cabbage.

Genome-wide analysis of the GRAS gene family in Chinese cabbage (Brassica rapa ssp. pekinensis).

The GRAS gene family is one of the most important families of transcriptional regulators. In this study, 48 GRAS genes are identified from Chinese cabbage, and they are classified into eight groups according to the classification of Arabidopsis. The characterization, classification, gene structure and phylogenetic construction of GRAS proteins are performed. Distribution mapping shows that GRAS proteins are nonrandomly localized in 10 chromosomes. Fifty-five orthologous gene pairs are shared by Chinese cabbage and Arabidopsis, and interaction networks of these orthologous genes are constructed. The expansion of GRAS genes in Chinese cabbage results from genome triplication. Among the 17 species examined, 14 higher plants carry the GRAS genes, whereas two lower plants and one fungi species do not. Furthermore, the expression patterns of GRAS genes exhibit differences in three tissues based on RNA-seq data. Taken together, this comprehensive analysis will provide rich resources for studying GRAS protein functions in Chinese cabbage.

Genome-wide analysis of the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp. pekinensis).

Basic helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotic organisms and are thought to be one of the largest families of regulatory proteins. This important family of transcriptional regulators plays crucial roles in plant development. However, a systematic analysis of the bHLH transcription factor family has not been reported in Chinese cabbage. In this study, 230 bHLH transcription factors were identified from the whole Chinese cabbage genome and compared with proteins from other representative plants, fungi and metazoans. The Chinese cabbage bHLH (BrabHLH) gene family could be classified into 24 subfamilies. Phylogenetic analysis of BrabHLHs along with bHLHs from Arabidopsis and rice indicated 26 subfamilies. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns and interaction networks of BrabHLHs were analyzed. Distribution mapping showed that BrabHLHs were non-randomly located on the ten Chinese cabbage chromosomes. One hundred and twenty-four orthologous bHLH genes were identified between Chinese cabbage and Arabidopsis, and the interaction networks of the orthologous genes were constructed in Chinese cabbage. Quantitative RT-PCR analysis showed that expressions of BrabHLH genes varied widely under different abiotic stress treatments for different times. Thus, this comprehensive analysis of BrabHLHs represents a rich resource, aiding the elucidation of the roles of bHLH family members in plant growth and development. Furthermore, the comparative genomics analysis deepened our understanding of the evolution of this gene family after a polyploidy event.

Genetic diversity and evolutionary characterization of Chinese porcine reproductive and respiratory syndrome viruses based on NSP2 and ORF5.

To more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the Nsp2 and ORF5 gene sequences of 35 representative PRRSV isolates from 2008 to 2012. Sequence analysis revealed that the Nsp2 and ORF5 genes have undergone genetic variation. Furthermore, the isolate FJLYDX04 contains five insertions at positions 599 to 603 and is the first isolate from China reported to have an insertion in Nsp2. Our results suggest that the highly pathogenic PRRSV has become the dominant strain in China and that Chinese PRRSV has undergone rapid evolution and can circumvent immune responses induced by currently used vaccines.

Low light intensity elongates period and defers peak time of photosynthesis: a computational approach to circadian-clock-controlled photosynthesis in tomato.

Photosynthesis is involved in the essential process of transforming light energy into chemical energy. Although the interaction between photosynthesis and the circadian clock has been confirmed, the mechanism of how light intensity affects photosynthesis through the circadian clock remains unclear. Here, we propose a first computational model for circadian-clock-controlled photosynthesis, which consists of the light-sensitive protein P, the core oscillator, photosynthetic genes, and parameters involved in the process of photosynthesis. The model parameters were determined by minimizing the cost function ( [Formula: see text]), which is defined by the errors of expression levels, periods, and phases of the clock genes (CCA1, PRR9, TOC1, ELF4, GI, and RVE8). The model recapitulates the expression pattern of the core oscillator under moderate light intensity (100 µmol m -2 s-1). Further simulation validated the dynamic behaviors of the circadian clock and photosynthetic outputs under low (62.5 µmol m-2 s-1) and normal (187.5 µmol m-2 s-1) intensities. When exposed to low light intensity, the peak times of clock and photosynthetic genes were shifted backward by 1-2 hours, the period was elongated by approximately the same length, and the photosynthetic parameters attained low values and showed delayed peak times, which confirmed our model predictions. Our study reveals a potential mechanism underlying the circadian regulation of photosynthesis by the clock under different light intensities in tomato.

AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis.

The calcium-dependent protein kinase (CDPK) family is needed in plant signaling during various physiological pathways. The Arabidopsis AtCPK6 gene belongs to the subclass of stress-inducible CDPKs, which is stimulated by salt and osmotic stress. To elucidate the physiological function of AtCPK6, transgenic Arabidopsis plants under the control of double CaMV 35S promoter were obtained. AtCPK6 over-expressing plants showed enhanced tolerance to salt/drought stresses. The elevated tolerance of the AtCPK6 over-expressing plants was confirmed by the change of proline and malondialdehyde (MDA). Real-time PCR analyses revealed that the expression levels of several stress-regulated genes were altered in AtCPK6 over-expressing plants. However, cpk6 mutant displayed no obvious difference with control. These results are likely to indicate that AtCPK6 is functionally redundant and a positive regulator involved in the tolerance to salt/drought stress in Arabidopsis.

Cyanobacteria MT gene SmtA enhance zinc tolerance in Arabidopsis.

Zinc is essential but toxic in excess. A bacterial metallothionein, SmtA from Synechococcus PCC 7942, has high affinity for Zn2+ and the intracellular exclusively handling of Zn2+. In this study, we report a functional analysis of SmtA in Arabidopsis thaliana and its response to zinc stress. After high zinc stress, the transgenic plants over-expressing SmtA showed higher survival rate than the wild type. We also found that over-expression of SmtA in Arabidopsis increased the activities of SOD and POD, and enhanced the tolerance to zinc stress. Together, our results indicate that SmtA may play an important role in the response to zinc stress in Arabidopsis.

A chromosome-level reference genome of non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis].

Non-heading Chinese cabbage (NHCC) is an important leafy vegetable cultivated worldwide. Here, we report the first high-quality, chromosome-level genome of NHCC001 based on PacBio, Hi-C, and Illumina sequencing data. The assembled NHCC001 genome is 405.33 Mb in size with a contig N50 of 2.83 Mb and a scaffold N50 of 38.13 Mb. Approximately 53% of the assembled genome is composed of repetitive sequences, among which long terminal repeats (LTRs, 20.42% of the genome) are the most abundant. Using Hi-C data, 97.9% (396.83 Mb) of the sequences were assigned to 10 pseudochromosomes. Genome assessment showed that this B. rapa NHCC001 genome assembly is of better quality than other currently available B. rapa assemblies and that it contains 48,158 protein-coding genes, 99.56% of which are annotated in at least one functional database. Comparative genomic analysis confirmed that B. rapa NHCC001 underwent a whole-genome triplication (WGT) event shared with other Brassica species that occurred after the WGD events shared with Arabidopsis. Genes related to ascorbic acid metabolism showed little variation among the three B. rapa subspecies. The numbers of genes involved in glucosinolate biosynthesis and catabolism were higher in NHCC001 than in Chiifu and Z1, due primarily to tandem duplication. The newly assembled genome will provide an important resource for research on B. rapa, especially B. rapa ssp. chinensis.

The genome sequence of celery (Apium graveolens L.), an important leaf vegetable crop rich in apigenin in the Apiaceae family.

Celery (Apium graveolens L.) is a vegetable crop in the Apiaceae family that is widely cultivated and consumed because it contains necessary nutrients and multiple biologically active ingredients, such as apigenin and terpenoids. Here, we report the genome sequence of celery based on the use of HiSeq 2000 sequencing technology to obtain 600.8 Gb of data, achieving ~189-fold genome coverage, from 68 sequencing libraries with different insert sizes ranging from 180 bp to 10 kb in length. The assembled genome has a total sequence length of 2.21 Gb and consists of 34,277 predicted genes. Repetitive DNA sequences represent 68.88% of the genome sequences, and LTR retrotransposons are the main components of the repetitive sequences. Evolutionary analysis showed that a recent whole-genome duplication event may have occurred in celery, which could have contributed to its large genome size. The genome sequence of celery allowed us to identify agronomically important genes involved in disease resistance, flavonoid biosynthesis, terpenoid metabolism, and other important cellular processes. The comparative analysis of apigenin biosynthesis genes among species might explain the high apigenin content of celery. The whole-genome sequences of celery have been deposited at CeleryDB (http://apiaceae.njau.edu.cn/celerydb). The availability of the celery genome data advances our knowledge of the genetic evolution of celery and will contribute to further biological research and breeding in celery as well as other Apiaceae plants.

Induction of immune responses in mice after oral immunization with recombinant Lactobacillus casei strains expressing enterotoxigenic Escherichia coli F41 fimbrial protein.

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) F41 infections, we have developed a surface antigen display system using poly-gamma-glutamate synthetase A (PgsA) as an anchoring matrix. The recombinant fusion proteins comprised of PgsA and fimbrial protein of F41 were stably expressed in Lactobacillus casei 525. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy, and flow cytometry. Oral inoculation of recombinant L. casei 525 into specific-pathogen-free BALB/c mice resulted in significant mucosal immunoglobulin A (IgA) titers that remained elevated for >16 weeks. High levels of IgG responses in sera specific for F41 fimbriae were also induced, with prominent IgG1 titers as well as IgG2a and IgG2b titers. The helper T-cell (Th) response was Th2-cell dominant, as evidenced by increased mucosal and systemic interleukin-4-producing T cells and a concomitant elevation of serum IgG1 antibody responses. More than 80% of the mice were protected against challenge with a 2 x 10(4)-fold 50% lethal dose of standard-type F41 (C83919). The induced antibodies were important for eliciting a protective immune response against F41 infection. These results indicated that the use of recombinant L. casei 525 could be a valuable strategy for future vaccine development for ETEC.

Genetic dissection of flowering time in Brassica rapa responses to temperature and photoperiod.

The Brassica rapa (B. rapa) species displays enormous phenotypic diversity, with leafy vegetables, storage root vegetables and oil crops. These different crops all have different flowering time, which determine their growing season and cultivation area. Little is known about the effects of diverse temperature and day-lengths on flowering time QTL associated with FLC paralogues. We phenotyped the flowering time of a doubled haploid population, established from a cross between Yellow sarson and Pak choi under diverse environmental conditions. We identified flowering-time QTL (fQTL) in different photoperiod and temperature regimes in the greenhouse, and studied their colocation with known flowering time genes. As several fQTL colocalized with FLC paralogues, we studied the expression patterns of four FLC paralogues during the course of vernalization in parental lines. Under all environmental conditions tested the major fQTL that mapped to the BrFLC2_A02 locus was detected, however its effect decreased when plants were grown at low temperatures. Another fQTL that mapped to the FLC paralogue, BrFLC5_A03 was also identified under all tested environments, while no fQTL colocated with BrFLC1_A10 or BrFLC3_A03. Furthermore, the vernalization treatment decreased expression of all BrFLC paralogues in the parental lines, and showed the lowest transcript level after 28 days of vernalization. Transcript abundance stayed low after returning the plants for seven days to normal growth temperature. Interestingly, transcript abundance of BrFLC3_A03 and BrFLC5_A03 was repressed much stronger and already reached lowest levels after 14d in the early-flowering type YS-143. This study improves understanding of the effects of daylength and vernalization on flowering time in B. rapa and the role of the different BrFLC paralogues therein.

Advances in research on the carrot, an important root vegetable in the Apiaceae family.

Carrots (Daucus carota L.), among the most important root vegetables in the Apiaceae family, are cultivated worldwide. The storage root is widely utilized due to its richness in carotenoids, anthocyanins, dietary fiber, vitamins and other nutrients. Carrot extracts, which serve as sources of antioxidants, have important functions in preventing many diseases. The biosynthesis, metabolism, and medicinal properties of carotenoids in carrots have been widely studied. Research on hormone regulation in the growth and development of carrots has also been widely performed. Recently, with the development of high-throughput sequencing technology, many efficient tools have been adopted in carrot research. A large amount of sequence data has been produced and applied to improve carrot breeding. A genome editing system based on CRISPR/Cas9 was also constructed for carrot research. In this review, we will briefly summarize the origins, genetic breeding, resistance breeding, genome editing, omics research, hormone regulation, and nutritional composition of carrots. Perspectives about future research work on carrots are also briefly provided.

CeleryDB: a genomic database for celery.

Celery (Apium graveolens L.) is a plant belonging to the Apiaceae family, and a popular vegetable worldwide because of its abundant nutrients and various medical functions. Although extensive genetic and molecular biological studies have been conducted on celery, its genomic data remain unclear. Given the significance of celery and the growing demand for its genomic data, the whole genome of 'Q2-JN11' celery (a highly inbred line obtained by artificial selfing of 'Jinnan Shiqin') was sequenced using HiSeq 2000 sequencing technology. For the convenience of researchers to study celery, an online database of the whole-genome sequences of celery, CeleryDB, was constructed. The sequences of the whole genome, nucleotide sequences of the predicted genes and amino acid sequences of the predicted proteins are available online on CeleryDB. Home, BLAST, Genome Browser, Transcription Factor and Download interfaces composed of the organizational structure of CeleryDB. Users can search the celery genomic data by using two user-friendly query tools: basic local alignment search tool and Genome Browser. In the future, CeleryDB will be constantly updated to satisfy the needs of celery researchers worldwide.Database URL: http://apiaceae.njau.edu.cn/celerydb.

Development and evaluation of enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection of porcine epidemic diarrhea (PEDV) antibodies.

An enzyme-linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of porcine epidemic diarrhea (PEDV) infection. The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein was migrated at 48 kDa and reacted with six histidine tag specific monoclonal antibody by immunoblotting. Recombinant N protein ELISA (rnELISA) demonstrated 98.7% specificities among (80) PEDV-free individuals, and 98% sensitivity ranging among (103) clinical samples with PEDV. On testing 884 field samples, an overall agreement of 88.3% was generated between the SN and rnELISA. Taken together, these results indicated that nucleocapsid protein may be a useful antigen for the sera-diagnosis of PEDV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies to PEDV.

[Genetic model analysis of leaf-weight traits in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino)].

The mixed major-gene plus polygene inheritance model was used to analyze the genetics of leafblade-weight and petiole-weight trait in SIxQiu017 of non-heading Chinese cabbage. The results showed that the leafblade-weight trait was controlled by one pair of negative complete dominant major genes plus additive-dominant polygene (D-4). The additive effect in the major gene was 1.8991, the dominant effect was -1.8991; the additive effect in the polygene was -1.2934, and the dominant effect was 1.7933; the potential ratio was -1.3865, and the dominant degree was -1.0000; the heritability of the major gene was 6.98% in B1 generation, 4.33% in B2 generation, and 36.08% in F2 generation; the heritability of the polygene was 16.03% in B1 generation, 7.39% in B2 generation, and 23.96% in F2 in the leafblade-weight trait. The peti-ole-weight trait in SIxQiu017 was controlled by one pair of additive major genes plus additive-dominant polygene (D-2). The additive effect in major gene was -1.1457, the dominant effect was 0.0000; the additive effect in polygene was 1.3472, and the dominant effect was 2.5788. The potential ratio was 1.9142, and the dominant degree was 0.0000; the heritability of the major gene was 31.72% in B1 generation, 5.27% in B2 generation, and 57.94% in F2 generation. The heritability of the polygene was 0.42% in B1 generation, 4.59% in B2 generation, and 4.80% in F2 in the petiole-weight trait. The improve-ment of leaf-weight trait of SIxQiu017 should be selected in late generation, and the improvement of petiole-weight trait of SIxQiu017 should be selected in early generation.

Genome-wide analysis of auxin transport genes identifies the hormone responsive patterns associated with leafy head formation in Chinese cabbage.

Auxin resistant 1/like aux1 (AUX/LAX), pin-formed (PIN) and ATP binding cassette subfamily B (ABCB/MDR/PGP) are three families of auxin transport genes. The development-related functions of the influx and efflux carriers have been well studied and characterized in model plants. However, there is scant information regarding the functions of auxin genes in Chinese cabbage and the responses of exogenous polar auxin transport inhibitors (PATIs). We conducted a whole-genome annotation and a bioinformatics analysis of BrAUX/LAX, BrPIN, and BrPGP genes in Chinese cabbage. By analyzing the expression patterns at several developmental stages in the formation of heading leaves, we found that most auxin-associate genes were expressed throughout the entire process of leafy head formation, suggesting that these genes played important roles in the development of heads. UPLC was used to detect the distinct and uneven distribution of auxin in various segments of the leafy head and in response to PATI treatment, indicated that the formation of the leafy head depends on polar auxin transport and the uneven distribution of auxin in leaves. This study provides new insight into auxin polar transporters and the possible roles of the BrLAX, BrPIN and BrPGP genes in leafy head formation in Chinese cabbage.