Tongkun Liu

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.

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 MADS-box gene family in Brassica rapa (Chinese cabbage)

The MADS-box gene family is an ancient and well-studied transcription factor family that functions in almost every developmental process in plants. There are a number of reports about the MADS-box family in different plant species, but systematic analysis of the MADS-box transcription factor family in Brassica rapa (Chinese cabbage) is still lacking. In this study, 160 MADS-box transcription factors were identified from the entire Chinese cabbage genome and compared with the MADS-box factors from 21 other representative plant species. A detailed list of MADS proteins from these 22 species was sorted. Phylogenetic analysis of the BrMADS genes, together with their Arabidopsis and rice counterparts, showed that the BrMADS genes were categorised into type I (Mα, Mβ, Mγ) and type II (MIKCC, MIKC*) groups, and the MIKCC proteins were further divided into 13 subfamilies. The Chinese cabbage type II group has 95 members, which is twice as much as the Arabidopsis type II group, indicating that the Chinese cabbage type II genes have been retained more frequently than the type I genes. Finally, RNA-seq transcriptome data and quantitative real-time PCR analysis revealed that BrMADS genes are expressed in a tissue-specific manner similar to Arabidopsis. Interestingly, a number of BrMIKC genes showed responses to different abiotic stress treatments, suggesting a function for some of the genes in these processes as well. Taken together, the characterization of the B. rapa MADS-box family presented here, will certainly help in the selection of appropriate candidate genes and further facilitate functional studies in Chinese cabbage.

Genome-wide identification, classification and expression analysis of the heat shock transcription factor family in Chinese cabbage

The Hsf gene family, one of the most important transcription factor families, plays crucial roles in regulating heat resistance. However, a systematic and comprehensive analysis of this gene family has not been reported in Chinese cabbage. Therefore, systematic analysis of the Hsf gene family in Chinese cabbage has profound significance. In this study, 35 BrHsf genes were identified from Chinese cabbage, which could be classified into three groups according to their structural characteristics and phylogenetic comparisons with Arabidopsis and rice. Thirty-three BrHsf genes mapped on chromosomes were further assigned to three subgenomes and eight ancestral karyotypes. Distribution mapping showed that BrHsf genes were non-randomly localized on chromosomes. Chinese cabbage and Arabidopsis shared 22 orthologous gene pairs. The expansion of BrHsf genes mainly resulted from genome triplication. Comparative analysis showed that the most Hsf genes were in Chinese cabbage among the five species analyzed. Interestingly, the number of Hsf genes of heat-resistant plants (Theobroma cacao and Musaacuminata) was fewer than that in Chinese cabbage. The expression patterns of BrHsf genes were different in six tissues, based on RNA-seq. Quantitative real-time-PCR analysis showed that the expression level of BrHsf genes varied under various abiotic stresses. In conclusion, this comprehensive analysis of BrHsf genes will provide rich resources, aiding the determination of Hsfs functions in plant heat resistance. Furthermore, the comparative genomics analysis deepened our understanding of Hsf genes’ evolution accompanied by the polyploidy event of Chinese cabbage.

Identification of differentially accumulating pistil proteins associated with self-incompatibility of non-heading Chinese cabbage

Non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), an important vegetable crop in China, exhibits a typical sporophytic self-incompatibility (SI) system. To better understand the mechanism of SI response and identify potential candidate proteins involved in the SI system of this vegetable crop, the proteomic approach was taken to identify differential accumulating pistil proteins. Pistils were collected at 0 h and 2 h after self-pollination at anthesis in self-incompatible and compatible lines of non-heading Chinese cabbage, and total proteins were extracted and separated by two-dimensional gel electrophoresis (2-DE). A total of 25 protein spots that displayed differential abundance were identified by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF/TOF MS) and peptide mass fingerprinting (PMF). Among them, 22 protein spots were confidently established. The mRNA levels of the corresponding genes were detected by quantitative RT-PCR. The 22 identified protein spots are involved in energy metabolism (four), protein biosynthesis (three), photosynthesis (six), stress response and defence (five), and protein degradation (four). Among these potential candidate proteins, UDP-sugar pyrophosphorylase could be involved in sucrose degradation to influence pollen germination and growth. Glutathione S-transferases could be involved in pollen maturation, and affect pollen fertility. Senescence-associated cysteine protease, which is related to programmed cell death, could be mainly related to self pollen recognition of non-heading Chinese cabbage. The study will contribute to further investigations of molecular mechanism of sporophytic SI in Brassicaceae.

Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of CO-like genes in plant kingdom.

In plants, flowering is the most important transition from vegetative to reproductive growth. The flowering patterns of monocots and eudicots are distinctly different, but few studies have described the evolutionary patterns of the flowering genes in them. In this study, we analysed the evolutionary pattern, duplication and expression level of these genes. The main results were as follows: (i) characterization of flowering genes in monocots and eudicots, including the identification of family-specific, orthologous and collinear genes; (ii) full characterization of CONSTANS-like genes in Brassica rapa (BraCOL genes), the key flowering genes; (iii) exploration of the evolution of COL genes in plant kingdom and construction of the evolutionary pattern of COL genes; (iv) comparative analysis of CO and FT genes between Brassicaceae and Grass, which identified several family-specific amino acids, and revealed that CO and FT protein structures were similar in B. rapa and Arabidopsis but different in rice; and (v) expression analysis of photoperiod pathway-related genes in B. rapa under different photoperiod treatments by RT-qPCR. This analysis will provide resources for understanding the flowering mechanisms and evolutionary pattern of COL genes. In addition, this genome-wide comparative study of COL genes may also provide clues for evolution of other flowering genes.

Proteomic analysis of non-heading Chinese cabbage infected with Hyaloperonospora parasitica

UNLABELLED Downy mildew is a serious fungal disease in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) that is caused by Hyaloperonospora parasitica, which infects members of the Brassicaceae family. For breeding improvement, researchers must understand the defence mechanisms employed by non-heading Chinese cabbage to combat H. parasitica infection. Using 2-DE protein analysis, we compared the proteomes from leaves of non-heading Chinese cabbage seedlings that were infected with H. parasitica or that were only treated with water at different time points post-infection. By MS analysis, 91 protein spots with significant differences in abundance (>2-fold, p<0.05) were identified in mock- and H. parasitica-inoculated leaves. Next, a resistance strategy for incompatible interactions was proposed. This network consisted of several functional components, including enhanced ethylene biosynthesis and energy supply, balanced ROS production and scavenging, accelerated protein metabolism and photorespiratory, reduced photosynthesis, and induced photosystem repair. These findings increase our knowledge of incompatible interactions between plants and pathogens and also provide new insight regarding the function of plant molecular processes, which should assist in the discovery of new strategies for pathogen control. BIOLOGICAL SIGNIFICANCE This study reported the proteomic analysis of the incompatible interactions between non-heading Chinese cabbage and downy mildew using 2-DE and MS. In total, 91 protein spots that were related to the resistance response were identified. These proteins were assigned to different functional categories, such as amino acid and carbohydrate metabolism, photosynthesis and photorespiration, protein metabolism, signal transduction, redox homeostasis, and ethylene biosynthesis. Meanwhile, several key proteins were determined to be associated with ethylene signalling, ROS scavenging and resistance-related proteins. Consistent with these results, the expression of ethylene biosynthesis genes and response genes, as well as the activity of antioxidant enzymes, increased after inoculation. These findings provide new insight for further understanding the molecular mechanisms of plant resistance.

Nitrate or NaCl regulates floral induction in Arabidopsis thaliana

Flowering, the transition from the vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. Exposure to an extended period of stress (such as low nitrate or NaCl) can also promote flowering in many species, but little is known about how these forms of stress regulate floral induction. In this study, we found that stress induced by low concentrations of nitrate or NaCl activated the biosynthesis of gibberellin (GA) as evidenced by increased expression of the GA biosynthetic enzyme GA1. Expression of CO and SOC1 were also enhanced, leading to an acceleration of flowering. The effects of nitrate and NaCl on the photoperiod pathway were distinct, however. Two genes related to the photoperiod pathway, CCA1 and LHY, were repressed only under low NaCl treatment, while expression was unaltered by nitrate. Therefore, we suggest that the biosynthesis of gibberellin (GA) may play an important role in integrating signals induced by exogenous stress to regulate flowering in Arabidopsis.

Identification and Validation of Reference Genes for RT-qPCR Analysis in Non-Heading Chinese Cabbage Flowers.

Non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino) is an important vegetable member of Brassica rapa crops. It exhibits a typical sporophytic self-incompatibility (SI) system and is an ideal model plant to explore the mechanism of SI. Gene expression research are frequently used to unravel the complex genetic mechanism and in such studies appropriate reference selection is vital. Validation of reference genes have neither been conducted in Brassica rapa flowers nor in SI trait. In this study, 13 candidate reference genes were selected and examined systematically in 96 non-heading Chinese cabbage flower samples that represent four strategic groups in compatible and self-incompatible lines of non-heading Chinese cabbage. Two RT-qPCR analysis software, geNorm and NormFinder, were used to evaluate the expression stability of these genes systematically. Results revealed that best-ranked references genes should be selected according to specific sample subsets. DNAJ, UKN1, and PP2A were identified as the most stable reference genes among all samples. Moreover, our research further revealed that the widely used reference genes, CYP and ACP, were the least suitable reference genes in most non-heading Chinese cabbage flower sample sets. To further validate the suitability of the reference genes identified in this study, the expression level of SRK and Exo70A1 genes which play important roles in regulating interaction between pollen and stigma were studied. Our study presented the first systematic study of reference gene(s) selection for SI study and provided guidelines to obtain more accurate RT-qPCR results in non-heading Chinese cabbage.

Genes associated with agronomic traits in non-heading Chinese cabbage identified by expression profiling

BackgroundThe genomes of non-heading Chinese cabbage (Brassica rapa ssp. chinensis), heading Chinese cabbage (Brassica rapa ssp. pekinensis) and their close relative Arabidopsis thaliana have provided important resources for studying the evolution and genetic improvement of cruciferous plants. Natural growing conditions present these plants with a variety of physiological challenges for which they have a repertoire of genes that ensure adaptability and normal growth. We investigated the differential expressions of genes that control adaptability and development in plants growing in the natural environment to study underlying mechanisms of their expression.ResultsUsing digital gene expression tag profiling, we constructed an expression profile to identify genes related to important agronomic traits under natural growing conditions. Among three non-heading Chinese cabbage cultivars, we found thousands of genes that exhibited significant differences in expression levels at five developmental stages. Through comparative analysis and previous reports, we identified several candidate genes associated with late flowering, cold tolerance, self-incompatibility, and leaf color. Two genes related to cold tolerance were verified using quantitative real-time PCR.ConclusionsWe identified a large number of genes associated with important agronomic traits of non-heading Chinese cabbage. This analysis will provide a wealth of resources for molecular-assisted breeding of cabbage. The raw data and detailed results of this analysis are available at the website http://nhccdata.njau.edu.cn.