Changwei Zhang

Genome-wide identification of turnip mosaic virus-responsive microRNAs in non-heading Chinese cabbage by high-throughput sequencing.

Turnip mosaic virus (TuMV) is the most prevalent viral pathogen infecting most cruciferous plants. MicroRNAs (miRNAs) are around 22 nucleotides long non-protein-coding RNAs that play key regulatory roles in plants. Recent research findings show that miRNAs are involved in plant-virus interaction. However we know little about plant defense and viral offense system networks throughout microRNA regulation pathway. In this study, two small RNA libraries were constructed based on non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino, NHCC) leaves infected by TuMV and healthy leaves, and sequenced using the Illumina-Solexa high-throughput sequencing technology. A total of 86 conserved miRNAs belonging to 25 known miRNA families and 45 novel ones were identified. Among them, twelve conserved and ten new miRNAs were validated by real-time fluorescence quantitative PCR (qPCR). Differential expression analysis showed that 42 miRNAs were down-regulated and 27 miRNAs were up-regulated in response to TuMV stress. A total of 271 target genes were predicted using a bioinformatics approach, these genes are mainly involved in growth and resistance to various stresses. We further selected 13 miRNAs and their corresponding target genes to explore their expression pattern under TuMV and/or cold (4°C) stresses, and the results indicated that some of the identified miRNAs could link TuMV response with cold response of NHCC. The characterization of these miRNAs could contribute to a better understanding of plant-virus interaction throughout microRNA regulation pathway. This can lead to finding new approach to defend virus infection using miRNA in Chinese cabbage.

Overexpression of FLOWERING LOCUS C , Isolated from Non-Heading Chinese Cabbage ( Brassica campestris ssp. chinensis Makino), Influences Fertility in Arabidopsis

Flowering, transition from vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. FLOWERING LOCUSC (FLC) in Arabidopsis encodes a dosage-dependent repressor of flowering. We have isolated a FLC-related sequence from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and named it as BcFLC1. In this study, we found that fertility was affected by the overexpression of BcFLC1 in Arabidopsis. The floral morphology of shortening anther filaments and the phenotype of reduced or small siliques were observed in BcFLC1 overexpression plants. RT-PCR showed that the reduced fertility in Arabidopsis by the overexpression of FLC was related with the enhanced expression of anther filaments suppressor, such as RGA and RGL genes, and declined expression of SEPALLATA3 (SEP3) gene, encoding a MADS-box transcription factor involved in flower and ovule development.

Over-expression of BcFLC1 from non-heading Chinese cabbage enhances cold tolerance in Arabidopsis

A gene (named BcFLC1) homologous to the AtFLC gene, which encodes a floral repressor, was isolated from the nonheading Chinese cabbage (Brassica campestris L. ssp. chinensis) cv. NJ074. The gene showed high similarity to AtFLC. For studying the gene function, we designed to introduce the BcFLC1 gene into Arabidopsis thaliana. The results showed that BcFLC1 had effects on flowering time similar to AtFLC. We also found that Arabidopsis cold-tolerance was enhanced by BcFLC1 overexpression. Under low temperature stress, the BcFLC1 transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the BcFLC1 over-expressing plants was also confirmed by the changes of electrolyte leakage and malonyldialdehyde and proline content.

Chloroplast elongation factor BcEF-Tu responds to turnip mosaic virus infection and heat stress in non-heading Chinese cabbage

Eukaryotic elongation factor Tu has been implicated in responses to heat stress and viral infection. In this study, the turnip mosaic virus (TuMV)-response gene BcLRK01, which encodes a leucine-rich repeat receptor-like kinase, was probed using the cDNA library of TuMV-infected leaves of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The BcEF-Tu gene, which encodes chloroplast elongation factor Tu, was obtained and verified by a yeast two-hybrid system to interact with the BcLRK01 gene. TuMV infection depressed the expression of this gene, whereas a heat stress induced its expression. Overexpression of BcEF-Tu enhanced the viability of Escherichia coli transformants under the heat stress. These results demonstrate that elongation factor BcEF-Tu responded to the TuMV infection and heat stress. This is the first report on chloroplast EF-Tu in non-heading Chinese cabbage which provides a theoretical basis for the functional research of EF-Tu.

Basic helix–loop–helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage

Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix–loop–helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.

Sufficient sulfur supply promotes seedling growth, alleviates oxidation stress, and regulates iron uptake and translocation in rice

We investigated the effect of sulfur (S) supply on growth, oxidative stress, and iron uptake and transport in rice (Oryza sativa L.) seedlings using a hydroponic culture with four S concentrations (0, 1.75, 3.5, and 7.0 mM). The length and fresh mass of seedlings were enhanced with the increased S concentration. In addition, the content of thiobarbituric acid reactive substances (TBARS) in rice leaves was the highest when no S was added to the nutrition solution and gradually declined with the increasing S supply. The higher S nutrition reduced the amount of Fe plaque on rice roots and increased Fe content in roots and leaves. The content of nicotianamine was significantly higher in rice roots under the S deficiency, whereas the reverse trend was observed in rice shoots. Taken together, the sufficient S nutrition promoted growth of rice, reduced oxidative stress, and ensured normal Fe uptake and distribution.

Cloning and expression analysis of a CMS-related gene BcCoi1 from Brassica campestris ssp. chinensis

BcCoi1, a cytoplasmic male sterility related gene, which was isolated from flower buds of Brassicacampestris ssp. chinensis Makino using the RACE technology, was characterized and submitted to the NCBI GenBank (accession no. GU263836). The gene encodes a 67.78-kD protein containing 16 leucine-rich repeats and an N-terminal F-box motif and is extremely similar to Arabidopsis thaliana Coi1 gene. The Southern blot showed that BcCoi1 belongs to a multigene family. In A. thaliana, the Coi1 gene is involved in jasmonate signaling, and Coi1 mutant displayed male sterility. In this study, qPCR results demonstrated that BcCoi1 was accumulated in stamens and was significantly higher expressed in flower organs of the maintainer line than in the CMS one. At the microsporocyte development stage, the gene was expressed at a significantly lower extent in the CMS line than in the maintainer line. This expression profile presumes that BcCoi1 plays a role in early microspore development in non-heading Chinese cabbage.

Genome-Wide Identification, Classification, and Expression Analysis of SNARE Genes in Chinese Cabbage (Brassica rapa ssp. pekinensis) Infected by Turnip mosaic virus

Turnip mosaic virus (TuMV) is a widely distributed pathogen that seriously affects the yield and quality of brassica crops. The SNARE genes in the host encode proteins that are very important for virus replication and movement. However, systematic and comprehensive analyses of these genes have not been reported for Chinese cabbage. In the present study, 78 BrSNARE genes were identified in Chinese cabbage. We analyzed the classification of these genes, their phylogenetic relationships (including their orthologous and paralogous relationships with those in Arabidopsis and rice), conserved motifs, and distribution on the ten chromosomes of Chinese cabbage. Of the 78 BrSNAREs, 77 were unevenly distributed among ten chromosomes. In the phylogenetic analyses of SNARES from Chinese cabbage, Arabidopsis, and rice, the 78 BrSNAREs were classified into four subfamilies. Analyses of the transcript levels of BrSNAREs in six different tissues revealed tissue-specific expression of some BrSNARE genes. We detected the expression of 55 BrSNAREs and grouped them according to their change trends and subcellular location in different organelles. On the basis of our analyses, we concluded that nine BrSNAREs may be associated with cell-to-cell movement and 15 BrSNAREs may be associated with long-distance transport. These results indicate that many BrSNAREs have evolved in Chinese cabbage and that some of them are related to TuMV infection.

Comparative transcriptome discovery and elucidation of the mechanism of long noncoding RNAs during vernalization in Brassica rapa

Flowering time is an important agronomic trait in Brassica rapa (B. rapa). However, our current understanding of the role of long noncoding RNAs (lncRNAs) in flowering time responded to vernalization is limited. The rapid development of the omics sequencing technology has facilitated the identification of thousands of lncRNAs in various plant species. Here, we used comparative transcriptome analysis between control and vernalized B. rapa to identify differentially expressed genes (DEGs) and lncRNAs (DELs). A total of 300 DEGs and 254 DELs were identified. Co-localization networks consisting of 128 DEGs and 127 DELs were established, followed by analyses of hierarchical categories, functional annotations, and correlation from mRNA-to-lncRNA. We found that the BraZF-HD21 (Bra026812) gene which responds to photoperiods and vernalization is correlated with lncRNA TCONS_00035129. The correlated genes that were mapped to the plant hormone signal transduction pathway and increased gibberellin A3 (GA3) content demonstrated that vernalization influences plant hormone levels. These findings suggest that vernalization alters the process of hormone biosynthesis, which in turn regulates flowering. This study provides an approach to elucidation of the regulatory mechanism of lncRNAs during vernalization in B. rapa.

Efficient virus-induced gene silencing in Brassica rapa using a turnip yellow mosaic virus vector

Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method used for unraveling gene functions. As an attractive alternative to mutant collections or stable transgenic plants, it has been widely used in reverse-genetics studies owing to its ease use and quick turnaround time. Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana. However, the conventional vector construction is difficult and the efficiencies of the infection methods are low. Here, we improved the vector construction and viral infection methods, inserted an inverted-repeat fragment of the phytoene desaturase gene into a TYMV-derived vector by homologous recombination and transformed Brassica rapa with plasmid DNA harboring a cDNA copy of the TYMV genome through particle bombardment. An apparent photobleaching phenotype was detected and efficient VIGS was induced. An 80-bp fragment was sufficient to produce VIGS in leaves, stems, roots, flowers, siliques, and stalks of B. rapa. Because TYMV has a wide host range in Brassica, the VIGS system described here will contribute to the improvement of high-throughput technology and efficient functional research in B. rapa and other Brassicaceae crops.