Xiangshu Dong

Genome-Wide Transcriptome Analysis of Two Contrasting Brassica rapa Doubled Haploid Lines under Cold-Stresses Using Br135K Oligomeric Chip

Genome wide transcription analysis in response to stresses is important to provide a basis of effective engineering strategies to improve stress tolerance in crop plants. We assembled a Brassica rapa oligomeric microarray (Br135K microarray) using sequence information from 41,173 unigenes and analyzed the transcription profiles of two contrasting doubled haploid (DH) lines, Chiifu and Kenshin, under cold-treatments. The two DH lines showed great differences in electrolyte leakage below −4°C, but similar patterns from 4°C to −2°C. Cold-treatments induced 885 and 858 genes in Chiifu and Kenshin, respectively. Overall, 134, and 56 genes showed an intrinsic difference in expression in Chiifu and Kenshin, respectively. Among 5,349 genes that showed no hit found (NHF) in public databases, 61 and 24 were specifically expressed in Chiifu and Kenshin, respectively. Many transcription factor genes (TFs) also showed various characteristics of expression. BrMYB12, BrMYBL2, BrbHLHs, BrbHLH038, a C2H2, a WRKY, BrDREB19 and a integrase-type TF were induced in a Chiifu-specific fashion, while a bHLH (Bra001826/AT3G21330), bHLH, cycling Dof factor and two Dof type TFs were Kenshin specific. Similar to previous studies, a large number of genes were differently induced or regulated among the two genotypes, but many genes, including NHFs, were specifically or intrinsically expressed with genotype specificity. Expression patterns of known-cold responsive genes in plants resulted in discrepancy to membrane leakage in the two DH lines, indicating that timing of gene expression is more important to conferring freezing tolerance rather than expression levels. Otherwise, the tolerance will be related to the levels of transcripts before cold-treatment or regulated by other mechanisms. Overall, these results indicate common signaling pathways and various transcriptional regulatory mechanisms are working together during cold-treatment of B. rapa. Our newly developed Br135K oligomeric microarray will be useful for transcriptome profiling, and will deliver valuable insight into cold stresses in B. rapa.

Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes

We investigated the mechanism regulating cytoplasmic male sterility (CMS) in Brassica rapa ssp. pekinensis using floral bud transcriptome analyses of Ogura-CMS Chinese cabbage and its maintainer line in B. rapa 300-K oligomeric probe (Br300K) microarrays. Ogura-CMS Chinese cabbage produced few and infertile pollen grains on indehiscent anthers. Compared to the maintainer line, CMS plants had shorter filaments and plant growth, and delayed flowering and pollen development. In microarray analysis, 4646 genes showed different expression, depending on floral bud size, between Ogura-CMS and its maintainer line. We found 108 and 62 genes specifically expressed in Ogura-CMS and its maintainer line, respectively. Ogura-CMS line-specific genes included stress-related, redox-related, and B. rapa novel genes. In the maintainer line, genes related to pollen coat and germination were specifically expressed in floral buds longer than 3mm, suggesting insufficient expression of these genes in Ogura-CMS is directly related to dysfunctional pollen. In addition, many nuclear genes associated with auxin response, ATP synthesis, pollen development and stress response had delayed expression in Ogura-CMS plants compared to the maintainer line, which is consistent with the delay in growth and development of Ogura-CMS plants. Delayed expression may reduce pollen grain production and/or cause sterility, implying that mitochondrial, retrograde signaling delays nuclear gene expression.

GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

Global Gene-Expression Analysis to Identify Differentially Expressed Genes Critical for the Heat Stress Response in Brassica rapa.

Genome-wide dissection of the heat stress response (HSR) is necessary to overcome problems in crop production caused by global warming. To identify HSR genes, we profiled gene expression in two Chinese cabbage inbred lines with different thermotolerances, Chiifu and Kenshin. Many genes exhibited >2-fold changes in expression upon exposure to 0.5– 4 h at 45°C (high temperature, HT): 5.2% (2,142 genes) in Chiifu and 3.7% (1,535 genes) in Kenshin. The most enriched GO (Gene Ontology) items included ‘response to heat’, ‘response to reactive oxygen species (ROS)’, ‘response to temperature stimulus’, ‘response to abiotic stimulus’, and ‘MAPKKK cascade’. In both lines, the genes most highly induced by HT encoded small heat shock proteins (Hsps) and heat shock factor (Hsf)-like proteins such as HsfB2A (Bra029292), whereas high-molecular weight Hsps were constitutively expressed. Other upstream HSR components were also up-regulated: ROS-scavenging genes like glutathione peroxidase 2 (BrGPX2, Bra022853), protein kinases, and phosphatases. Among heat stress (HS) marker genes in Arabidopsis, only exportin 1A (XPO1A) (Bra008580, Bra006382) can be applied to B. rapa for basal thermotolerance (BT) and short-term acquired thermotolerance (SAT) gene. CYP707A3 (Bra025083, Bra021965), which is involved in the dehydration response in Arabidopsis, was associated with membrane leakage in both lines following HS. Although many transcription factors (TF) genes, including DREB2A (Bra005852), were involved in HS tolerance in both lines, Bra024224 (MYB41) and Bra021735 (a bZIP/AIR1 [Anthocyanin-Impaired-Response-1]) were specific to Kenshin. Several candidate TFs involved in thermotolerance were confirmed as HSR genes by real-time PCR, and these assignments were further supported by promoter analysis. Although some of our findings are similar to those obtained using other plant species, clear differences in Brassica rapa reveal a distinct HSR in this species. Our data could also provide a springboard for developing molecular markers of HS and for engineering HS tolerant B. rapa.

Genome-wide analysis of genes associated with bolting in heading type chinese cabbage

Bolting or flowering time affects productivity of the leafy vegetable Chinese cabbage (Brassica rapa ssp. pekinensis) and also the time required for its breeding programs. Understanding the bolting process at the molecular level and identifying the genes involved will provide valuable tools for genetic engineering and development of functional markers. To achieve these goals, microarray analyses using either outer leaves or core tissues of completely headed Chinese cabbage were performed at three developmental stages, and the findings were confirmed using RT-PCR. A large number of genes were specifically or preferentially expressed in each tissue: photosynthesis and abiotic signal responding genes were expressed in outer leaves, while genes involved in hormone responses, flower development, and histone modification were expressed in core tissues. Genes promoting bolting, such as BrPIF4, BrPIF5, and BrCOLs, were highly expressed in outer leaves, a signal-perceiving tissue, whereas floral repressors, such as BrFLCs, BrFRL, and BrMAF1s, were predominantly expressed in core tissues, with levels of expression decreasing in later stages. Although the B. rapa genome contains three BrFT paralogs, only BrFT1 was expressed. BrFD and BrFE, genes related to the transport of BrFTs, showed consistent expression. The late bolting phenotype of Huissen, the F1 variety examined in this study, may result from expression of repressors of flowering, such as BrGASA5, BrTFL1, BrMAF1, BrFLCs, and BrKNAT1, in core tissues. Coordinated regulation of several floral promoters and repressors appears to be necessary for control of the bolting process or flowering time in B. rapa.

Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa

Flowering time is a very important agronomic trait in Brassica crops and regulation of the time is one of major factor in the breeding program. To understand the control of flowering time in Brassica rapa, we have carried out Br300K microarray with two contrasting Brassica inbred lines, Rapid Cycling B. rapa (RCBr) as rapid cycling type and B. rapa ssp. pekinensis inbred line Chiifu as slow flowering phenotype. Reproductive process-related genes were specifically expressed in RCBr, whereas environmental stimuli-responsive genes in Chiifu. Flowering stimulating genes, such as BrFT and BrSOC1, were preferentially expressed in RCBr, while flowering repressing genes, such as BrFLC and BrMAF4, expressed in Chiifu. Several paralogues present in B. rapa, BrFLCs and BrCOLs, were expressed with paralog-specific pattern depending on flowering phenotypes: i.e., BrFLC1 and BrFLC2, major floral repressors, were expressed in Chiifu, BrFLCL/BrFLC5 in RCBr and BrFLC3 in both plants. The expression of several flowering repressing genes was gradually decreased in RCBr growth, but increased in Chiifu growth. However, the expression of genes involved in photoperiodic flowering was no difference between these two plants under LD and SD conditions, indicating photoperiodic pathway is not major factor to distinguish fast vs. slow flowering in B. rapa. The mechanism underlined in the rapid or fast flowering of RCBr would be further elucidated in association with the controlling mechanism of its short life span.

Mining of Brassica-Specific Genes (BSGs) and Their Induction in Different Developmental Stages and under Plasmodiophora brassicae Stress in Brassica rapa

Orphan genes, also called lineage-specific genes (LSGs), are important for responses to biotic and abiotic stresses, and are associated with lineage-specific structures and biological functions. To date, there have been no studies investigating gene number, gene features, or gene expression patterns of orphan genes in Brassica rapa. In this study, 1540 Brassica-specific genes (BSGs) and 1824 Cruciferae-specific genes (CSGs) were identified based on the genome of Brassica rapa. The genic features analysis indicated that BSGs and CSGs possessed a lower percentage of multi-exon genes, higher GC content, and shorter gene length than evolutionary-conserved genes (ECGs). In addition, five types of BSGs were obtained and 145 out of 529 real A subgenome-specific BSGs were verified by PCR in 51 species. In silico and semi-qPCR, gene expression analysis of BSGs suggested that BSGs are expressed in various tissue and can be induced by Plasmodiophora brassicae. Moreover, an A/C subgenome-specific BSG, BSGs1, was specifically expressed during the heading stage, indicating that the gene might be associated with leafy head formation. Our results provide valuable biological information for studying the molecular function of BSGs for Brassica-specific phenotypes and biotic stress in B. rapa.

Genome-Wide Identification and Characterization of Warming-Related Genes in Brassica rapa ssp. pekinensis

For sustainable crop cultivation in the face of global warming, it is important to unravel the genetic mechanisms underlying plant adaptation to a warming climate and apply this information to breeding. Thermomorphogenesis and ambient temperature signaling pathways have been well studied in model plants, but little information is available for vegetable crops. Here, we investigated genes responsive to warming conditions from two Brassica rapa inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories “response to heat”, “heat acclimation”, “response to light intensity”, “response to oxidative stress”, and “response to temperature stimulus” were upregulated under warming treatment in both lines, but genes involved in “response to auxin stimulus” were upregulated only in Kenshin under both warming and minor-warming conditions. We identified 16 putative high temperature (HT) adaptation-related genes, including 10 heat-shock response genes, 2 transcription factor genes, 1 splicing factor gene, and 3 others. BrPIF4, BrROF2, and BrMPSR1 are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of BrHSFA2, and heat shock memory appear to be indispensable for HT adaptation in B. rapa. These results lay the foundation for molecular breeding and marker development to improve warming tolerance in B. rapa.

Suppression of ASKβ (AtSK32), a Clade III Arabidopsis GSK3, Leads to the Pollen Defect during Late Pollen Development.

Arabidopsis Shaggy-like protein kinases (ASKs) are Arabidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are comprised of 10 genes with diverse functions. To dissect the function of ASKβ (AtSK32), ASKβ antisense transgenic plants were generated, revealing the effects of ASKβ down-regulation in Arabidopsis. Suppression of ASKβ expression specifically interfered with pollen development and fertility without altering the plants’ vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of ASKβ and its co-expressed genes. In the aborted pollen of ASKβ antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate ASKβ is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that ASKβ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

Expression characteristics of LSH genes in Brassica suggest their applicability for modification of leaf morphology and the use of their promoter for transgenesis.

The functions of DUF640/ALOG (Arabidopsis LSH1 and Oryza G1) domain proteins, which are found in most land plants, have not been well characterized, but some of these proteins regulate inflorescence architecture in rice and specify organ boundaries in Arabidopsis. Arabidopsis DUF640-domain genes are initially identified as LIGHT-SENSITIVE HYPOCOTYLS (LSH) genes. Chinese cabbage leaves have large, white midribs and photosynthetic leaf blades (or lamina). A DUF640 domain gene of Brassica rapa, BrLSH2, is specifically expressed in the midrib of Chinese cabbage. Arabidopsis and rice possess ten LSH family genes, but B. rapa has 24 LSH genes, which can be categorized into two or four groups based on sequence identity. Here, we examined the expression patterns of the LSHs in various Brassica species and analyzed the promoter sequence of the BrLHS2 gene. The transcript levels of most LSH genes were very high in the midrib but low in the leaf blade. These genes were evenly expressed throughout the petiole region of Korean cabbage and highly expressed in the leaf base region near the stem and in the border area in B. oleracea. In addition, BrLSHs were expressed in both bundle and mesophyll cells of the midrib. These expression patterns suggest the possible use of these genes to generate leafy vegetables with altered leaf morphology. The BrLSH2 promoter, which contains auxin- and cytokinin-responsive elements as well as leaf development-related elements, may confer midrib-specific expression, suggesting that this promoter may be useful for the production of midrib-targeted transgenic Chinese cabbage.