Transcriptional profiling reveals changes in gene regulation and signaling transduction pathways during temperature stress in wucai (Brassica campestris L.).

BACKGROUND: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a cold-tolerant plant that is vulnerable to high temperature. This study explored the response mechanism of wucai to low temperature. In this study, wucai seedlings were treated with different temperatures, including low temperature (LT), high temperature (HT), and a control. RESULTS: According to transcriptomics analysis, the number of differentially expressed genes (DEGs) in HT and LT was 10,702 and 7267, respectively, compared with the control. The key genes associated with the physiological response of wucai to the treatments were analyzed. The Kyoto Encyclopedia of Genes and Genomes and Gene Ontology annotations indicated the importance of the photosynthesis and photosynthetic-antenna protein pathways. We found that a high-temperature environment greatly inhibited the expression of important genes in the photosynthetic pathway (BrLhc superfamily members, PsaD, PsaE, PsaD, PsaD, PsbO, PsbP, PsbQ, PsbR, PsbS, PsbW, PsbY, Psb27, and Psb28), whereas low temperature resulted in the expression of certain key genes (BrLhc superfamily members, Psa F, Psa H, Psb S, Psb H, Psb 28). In addition, the wucai seedlings exhibited better photosynthetic performance under low-temperature conditions than high-temperature conditions. CONCLUSIONS: Based on the above results, we speculate that upon exposure to low temperature, the plants developed higher cold tolerance by upregulating the expression of genes related to photosynthesis. Conversely, high-temperature stress inhibited the expression of pivotal genes and weakened the self-regulating ability of the plants.

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.

CYCLING DOF FACTOR 1 represses transcription through the TOPLESS co-repressor to control photoperiodic flowering in Arabidopsis.

CYCLING DOF FACTOR 1 (CDF1) and its homologs play an important role in the floral transition by repressing the expression of floral activator genes such as CONSTANS (CO) and FLOWERING LOCUS T (FT) in Arabidopsis. The day-length-specific removal of CDF1-dependent repression is a critical mechanism in photoperiodic flowering. However, the mechanism by which CDF1 represses CO and FT transcription remained elusive. Here we demonstrate that Arabidopsis CDF proteins contain non-EAR motif-like conserved domains required for interaction with the TOPLESS (TPL) co-repressor protein. This TPL interaction confers a repressive function on CDF1, as mutations of the N-terminal TPL binding domain largely impair the ability of CDF1 protein to repress its targets. TPL proteins are present on specific regions of the CO and FT promoters where CDF1 binds during the morning. In addition, TPL binding increases when CDF1 expression is elevated, suggesting that TPL is recruited to these promoters in a time-dependent fashion by CDFs. Moreover, reduction of TPL activity induced by expressing a dominant negative version of TPL (tpl-1) in phloem companion cells results in early flowering and a decreased sensitivity to photoperiod in a manner similar to a cdf loss-of-function mutant. Our results indicate that the mechanism of CDF1 repression is through the formation of a CDF-TPL transcriptional complex, which reduces the expression levels of CO and FT during the morning for seasonal flowering.

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.

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.