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.

Purple Brassica oleracea var. capitata F. rubra is due to the loss of BoMYBL2-1 expression.

BACKGROUND: Water-soluble anthocyanin pigments are important ingredients in health-improving supplements and valuable for the food industry. Although great attention has been paid to the breeding and production of crops containing high levels of anthocyanin, genetic variation in red or purple cabbages (Brassica oleracea var. capitata F. rubra) has not yet been characterized at the molecular level. In this study, we identified the mechanism responsible for the establishment of purple color in cabbages. RESULTS: BoMYBL2-1 is one of the regulatory genes in the anthocyanin biosynthesis pathway in cabbages. It is a repressor whose expression is inversely correlated to anthocyanin synthesis and is not detectable in purple cabbages. Sequence analysis of purple cabbages revealed that most lacked BoMYBL2-1 coding sequences, although a few had a substitution in the region of the promoter 347 bp upstream of the gene that was associated with an absence of BoMYBL2-1 expression. Lack of transcriptional activity of the substitution-containing promoter was confirmed using transgenic Arabidopsis plants transformed with promoter::GUS fusion constructs. The finding that the defect in BoMYBL2-1 expression was solely responsible for purple coloration in cabbages was further demonstrated using genomic PCR and RT-PCR analyses of many other structural and regulatory genes in anthocyanin biosynthesis. Molecular markers for purple cabbages were developed and validated using 69 cabbage lines. CONCLUSION: Expression of BoMYBL2-1 was inversely correlated to anthocyanin content, and purple color in cabbages resulted from a loss of BoMYBL2-1 expression, caused by either the promoter substitution or deletion of the gene. This is the first report of molecular markers that distinguish purple cabbages. Such markers will be useful for the production of intraspecific and interspecific hybrids for functional foods, and for industrial purposes requiring high anthocyanin content.

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.

Molecular characterization of the Brassica rapa auxin-repressed, superfamily genes, BrARP1 and BrDRM1.

Two auxin-repressed superfamily genes, auxin-repressed protein 1 (ARP1) and dormancy-associated protein 1 (DRM1), are highly expressed in both the dormant buds and non-growing tissues of several plant species. To further identify the function of these proteins in Chinese cabbage (Brassica rapa L. ssp. pekinensis), we examined comprehensive expression patterns of BrARP1 and BrDRM1 under various developmental and stress conditions. We also examined these same genes in transgenic Arabidopsis plants. Both genes were expressed in all tissues tested, but their levels were highest in mature tissues accompanied by low levels of the growth-associated marker, B. rapa ribosomal protein 27. Expression of both genes was induced by abiotic stresses, such as chilling, heat shock, and salt treatment. Overexpression of either BrARP1 or BrDRM1 in Arabidopsis causes a reduction in vegetative growth and seed productivity, without affecting morphology. The lengths of petioles and siliques were greatly reduced. Simultaneous expression of both genes showed an additive effect on the growth suppression, resulting in significant reduction in plant size. Knock-out of Arabidopsis ARP1, DRM1, or both, neither affected growth rate nor final size. Results suggest BrARP1 and BrDRM1 are either involved in growth arrest, or stop growth, possibly from inhibition of either cell elongation or cell expansion, thereby creating a "growth brake".

Overexpression of BrMORN, a novel 'membrane occupation and recognition nexus' motif protein gene from Chinese cabbage, promotes vegetative growth and seed production in Arabidopsis.

Proteins that contain membrane occupation and recognition nexus (MORN) motifs regulate various aspects of cellular metabolism by localizing proteins in different cellular organelles. The full-length Brassica rapa MORN motif protein (BrMORN) cDNA consists of 1,510 bp encoding 502 deduced amino acids with a predicted molecular mass of 55.8 kDa and an isoelectric point of 9.72. BrMORN is a novel protein composed of two N-terminal transmembrane helices and seven C-terminal MORN motifs and it appears to be localized on the plastid envelope. BrMORN expression was relatively high in actively-growing tissues, but low in mature tissues and under some abiotic stresses. Arabidopsis thaliana plants overexpressing BrMORN showed an enhanced rate of growth, hypocotyl elongation, and increases in the size of vegetative organs and seed productivity under normal growth conditions. In addition, cell size in Arabidopsis plants overexpressing BrMORN was 24% larger than that of wild-type plants, implying that the increase in the size of vegetative organs is due to cell enlargement. The increased size of the vegetative organs also led to increased seed production. Our data suggest that the MORN motif of BrMORN may act at the plastid envelope and facilitate plant growth via cell enlargement.

Developmental expression patterns of alpha1H T-type Ca2+ channels during spermatogenesis and organogenesis in mice.

The objectives of the present study were to investigate the expression patterns of T-type Ca2+ channel mRNA during spermatogenesis and organogenesis in mice. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify the subtypes of calcium channels present in the round spermatids isolated from mouse testes by flow cytometry. Transcripts of L-type (alpha1D), non-L-type (alpha1E) and T-type Ca2+ channels were detected in round spermatids. Analysis of PCR products of T-type Ca2+ channels indicated that only alpha1H subunits were detected in round spermatids. The appearance and differential distribution of alpha1H T-type Ca2+ channel mRNA during mouse spermatogenesis and postimplantation embryogenesis (embryonic (E) days E9, E12, E15) were investigated by in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. In testes from adult and immature mice (postnatal 2 and 3 weeks), alpha1H T-type Ca2+ channel mRNA was expressed in all developing germ cells and sertoli cells. On E9 and E12, tissues of the central nervous system, such as the telencephalon, expressed alpha1H T-type Ca2+ channel mRNA. On E15, signals were detected throughout all organs of the embryo. These findings indicate that the expression of alpha1H T-type Ca2+ channels is spatio-temporally regulated during spermatogenesis and organogenesis.