Induction of Glucoraphasatin Biosynthesis Genes by MYB29 in Radish (Raphanus sativus L.) Roots.

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish (Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor (RSG00789) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.

Identification of source-sink tissues in the leaf of Chinese cabbage (Brassica rapa ssp. pekinensis) by carbohydrate content and transcriptomic analysis.

BACKGROUND: A leaf of Chinese cabbage (Brassica rapa ssp. pekinensis) is composed of a photosynthetic blade and a non-photosynthetic large midrib; thus each leaf contains both source and sink tissues. This structure suggests that, unlike in other plants, source-sink metabolism is present in a single leaf of Chinese cabbage. OBJECTIVE: This study was designed to identify the transport route of photosynthetic carbon and to determine whether both source and sink tissues were present in a leaf. METHODS: Plant samples were collected diurnally. Their carbohydrate contents were measured, and a genome-wide transcriptome analysis was performed using the Br300K microarray. Expression profiles of selected genes were validated using qRT-PCR analysis. RESULTS: The presence of two contrasting tissues (blade as source and midrib as sink) in a leaf was demonstrated by (1) diurnal distribution patterns of starch and sucrose content; (2) Gene Ontology (GO) enrichment analysis of microarray data; (3) expression profiles of photosynthetic and sucrose biosynthetic genes; and (4) expression patterns of a variety of sugar transporter genes. CONCLUSION: Source and sink tissues were both present in Chinese cabbage leaves, but the midrib functioned as a sink tissue as well as a site exporting to roots and other sink tissues. Function of most genes discriminating between source and sink tissue appeared to be regulated largely at the post-transcriptional level, not at the transcriptional level.

Carotenoid Biosynthesis in Oriental Melon (Cucumis melo L. var. makuwa).

Full-length cDNAs encoding ξ-carotene desaturase (CmZDS), lycopene ε-cyclase (CmLCYE), ß-ring carotene hydroxylase (CmCHXB), and zeaxanthin epoxidase (CmZEP), and partial-length cDNA encoding ε-ring carotene hydroxylase (CmCHXE) were isolated in Chamoe (Cucumis melo L. var. makuwa), an important commercial fruit. Sequence analyses revealed that these proteins share high identity and common features with other orthologous genes. Expression levels of entire genes involved in the carotenoid biosynthetic pathway were investigated in the peel, pulp, and stalk of chamoe cultivars Ohbokggul and Gotgam. Most of the carotenoid biosynthetic genes were expressed at their highest levels in the stalk, whereas carotenoids were highly distributed in the peel. The expression levels of all carotenoid biosynthetic genes in fruits of the native cultivar Gotgam chamoe were higher than those in the cultivar Ohbokggul chamoe, consistent with the abundant carotenoid accumulation in Gotgam chamoe fruits and trace carotenoid content of Ohbokggul chamoe fruit. Lutein and ß-carotene were the dominant carotenoids; high levels (278.05 µg g-1 and 112.02 µg g-1 dry weight, respectively) were found in the peel of Gotgam chamoe. Our findings may provide a foundation for elucidating the carotenoid biosynthetic mechanism in C. melo and inform strategies for developing new chamoe cultivars with improved characteristics.

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.

Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton.

Sophorae Radix (Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.

Brassinosteroids regulate glucosinolate biosynthesis in Arabidopsis thaliana.

Plants must constantly adjust their growth and defense responses to deal with the wide variety of stresses they encounter in their environment. Among phytohormones, brassinosteroids (BRs) are an important group of plant steroid hormones involved in numerous aspects of the plant lifecycle including growth, development and responses to various stresses including insect attacks. Here, we show that BRs regulate glucosinolate (GS) biosynthesis and function in insect herbivory. Preference tests and larval feeding experiments using the generalist herbivore, diamondback moth (Plutella xylostella), revealed that the larvae prefer to feed on Arabidopsis thaliana brassinosteroid insensitive 1 (bri1-5) plants over wild-type Ws-2 or BRI1-Flag (bri1-5 background) transgenic plants, which results in an increase in larval weight. Analysis of GS contents showed that 3-(methylsulfinyl) propyl GS (C3) levels were higher in bri1-5 than in Ws2 and BRI1-Flag transgenic plants, whereas sinigrin (2-propenylglucosinolate), glucoerucin (4-methylthiobutylglucosinolate) and glucobrassicin (indol-3-ylmethylglucosinolate) levels were lower in this mutant. We investigated the effect of brassinolide (BL) on GS biosynthesis in Arabidopsis and radish (Raphanus sativus L.) by monitoring the expression levels of GS biosynthetic genes, including MAM1, MAM3, BCAT4 and AOP2, which increased in a BL-dependent manner. These results suggest that BRs regulate GS profiles in higher plants, which function in defense responses against insects.

Identification and Characterization of Anthocyanin Biosynthesis-Related Genes in Kohlrabi.

Kohlrabi (Brassica oleracea var. gongylodes L.) is an important vegetable of the Brassicaceae family. The main edible part of kohlrabi is the swollen stem. The purple cultivars make anthocyanin mainly in the peel of the swollen stem, while in the leaf, it is limited to the midrib, but green cultivars do not. Anthocyanins are advantageous for both plants as well as humans. Two anthocyanin compounds were detected by high pressure liquid chromatography (HPLC) only in the peel of the purple kohlrabi cultivar. Three MYBs, three bHLHs, and one WD40 TF were identified as the candidate regulatory genes in kohlrabi. There was an abundance of transcript levels for the late biosynthetic genes more specifically for BoF3'H, BoDFR, BoLDOX, and BoGST in the purple peel while scarcely detectable in other tissues for both cultivars. The expression of BoPAP2 and BoTT8 was higher in the peel of the purple cultivar than the green cultivar. The expression of BoMYBL2.2 orthologue of Arabidopsis MYBL2, a negative regulator of anthocyanins, was dramatically decreased in the purple peel. The expression of BoACO1, a key gene for ethylene biosynthesis, and BoNCED3, an important gene of the ABA pathway, was down- and upregulated, respectively, in the peel of purple kohlrabi.

Intracellular Ca(2+) and K(+) concentration in Brassica oleracea leaf induces differential expression of transporter and stress-related genes.

BACKGROUND: One of the most important members of the genus Brassica, cabbage, requires a relatively high level of calcium for normal growth (Plant Cell Environ 7: 397-405, 1984; Plant Physiol 60: 854-856, 1977). Localized Ca(2+) deficiency in cabbage leaves causes tip-burn, bringing about serious economic losses (Euphytica 9:203-208, 1960; Ann Bot 43:363-372, 1979; Sci Hortic 14:131-138, 1981). Although it has been known that the occurrence of tip-burn is related to Ca(2+) deficiency, there is limited information on the underlying mechanisms of tip-burn or the relationship between Ca(2+) and tip-burn incidence. To obtain more information on the genetic control of tip-burn symptoms, we focused on the identification of genes differentially expressed in response to increasing intracellular Ca(2+) and K(+) concentrations in B. oleracea lines derived from tip-burn susceptible, tip-burn resistant cabbages (B. oleracea var. capitata), and kale (B. oleracea var. acephala). RESULTS: We compared the levels of major macronutrient cations, including Ca(2+) and K(+), in three leaf segments, the leaf apex (LA), middle of leaf (LM), and leaf base (LB), of tip-burn susceptible, tip-burn resistant cabbages, and kale. Ca(2+) and K(+) concentrations were highest in kale, followed by tip-burn resistant and then tip-burn susceptible cabbages. These cations generally accumulated to a greater extent in the LB than in the LA. Transcriptome analysis identified 58,096 loci as putative non-redundant genes in the three leaf segments of the three B. oleracea lines and showed significant changes in expression of 27,876 loci based on Ca(2+) and K(+) levels. Among these, 1844 loci were identified as tip-burn related phenotype-specific genes. Tip-burn resistant cabbage and kale-specific genes were largely related to stress and transport activity based on GO annotation. Tip-burn resistant cabbage and kale plants showed phenotypes clearly indicative of heat-shock, freezing, and drought stress tolerance compared to tip-burn susceptible cabbages, demonstrating a correlation between intracellular Ca(2+) and K(+) concentrations and tolerance of abiotic stress with differential gene expression. We selected 165 genes that were up- or down-regulated in response to increasing Ca(2+) and K(+) concentrations in the three leaf segments of the three plant lines. Gene ontology enrichment analysis indicated that these genes participated in regulatory metabolic processes or stress responses. CONCLUSIONS: Our results indicate that the genes involved in regulatory metabolic processes or stress responses were differentially expressed in response to increasing Ca(2+) and K(+) concentrations in the B. oleracea leaf. Our transcriptome data and the genes identified may serve as a starting point for understanding the mechanisms underlying essential macronutrient deficiencies in plants, as well as the features of tip-burn in cabbage and other Brassica species.

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.

Characterization of the wzc gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii.

To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test. The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.

Functional innovations of three chronological mesohexaploid Brassica rapa genomes.

BACKGROUND: The Brassicaceae family is an exemplary model for studying plant polyploidy. The Brassicaceae knowledge-base includes the well-annotated Arabidopsis thaliana reference sequence; well-established evidence for three rounds of whole genome duplication (WGD); and the conservation of genomic structure, with 24 conserved genomic blocks (GBs). The recently released Brassica rapa draft genome provides an ideal opportunity to update our knowledge of the conserved genomic structures in Brassica, and to study evolutionary innovations of the mesohexaploid plant, B. rapa. RESULTS: Three chronological B. rapa genomes (recent, young, and old) were reconstructed with sequence divergences, revealing a trace of recursive WGD events. A total of 636 fast evolving genes were unevenly distributed throughout the recent and young genomes. The representative Gene Ontology (GO) terms for these genes were 'stress response' and 'development' both through a change in protein modification or signaling, rather than by enhancing signal recognition. In retention patterns analysis, 98% of B. rapa genes were retained as collinear gene pairs; 77% of those were singly-retained in recent or young genomes resulting from death of the ancestral copies, while others were multi-retained as long retention genes. GO enrichments indicated that single retention genes mainly function in the interpretation of genetic information, whereas, multi-retention genes were biased toward signal response, especially regarding development and defense. In the recent genome, 13,302, 5,790, and 20 gene pairs were multi-retained following Brassica whole genome triplication (WGT) events with 2, 3, and 4 homoeologous copies, respectively. Enriched GO-slim terms from B. rapa homomoelogues imply that a major effect of the B. rapa WGT may have been to acquire environmental adaptability or to change the course of development. These homoeologues seem to more frequently undergo subfunctionalization with spatial expression patterns compared with other possible events including nonfunctionalization and neofunctionalization. CONCLUSION: We refined Brassicaceae GB information using the latest genomic resources, and distinguished three chronologically ordered B. rapa genomes. B. rapa genes were categorized into fast evolving, single- and multi-retention genes, and long retention genes by their substitution rates and retention patterns. Representative functions of the categorized genes were elucidated, providing better understanding of B. rapa evolution and the Brassica genus.

Phenotypic profiling and gene expression analyses for aromatic and volatile compounds in Chamoes (Cucumis melo).

Gotgam chamoe (GgC), a native oriental melon in Korea, is known to possess the aroma of a dried persimmon, an agronomic relevance for melon breeding program. The volatile compounds and the transcript levels of aromatic compound genes in cultivar (Ohbokggul chamoe [OC]) and GgC were profiled. A total of 62 volatile compounds were identified and quantified. Twenty-eight volatile compounds were specific to either the OC or the GgC. The amounts of volatile alcohol, saturated hydrocarbon, and unsaturated hydrocarbon compounds were 2.2, 2.7, and 1.1 times higher in OC, respectively. The amounts of ketone volatiles were 1.2 times higher in GgC, whereas the total amounts of esters were similar. In the shikimate pathway, transcriptional patterns with the fruit parts were different between the two chamoes for CmDAHPS, CmDHD/SDH, and CmEPSPS. The expression levels of all six genes investigated, especially CmCS, were highest in the peel of both chamoes compared to the other parts. The transcript levels of the aromatic amino acid biosynthesis genes demonstrate that phenylalanine and tyrosine are present more in edible parts of the chamoe, while tryptophan may be accumulated low in the chamoe. In addition, phenylalanine and tryptophan are synthesized more in GgC than the OC.

Identification of Yellow Pigmentation Genes in Brassica rapa ssp. pekinensis Using Br300 Microarray.

The yellow color of inner leaves in Chinese cabbage depends on its lutein and carotene content. To identify responsible genes for yellow pigmentation in leaves, the transcriptome profiles of white (Kenshin) and yellow leaves (Wheessen) were examined using the Br300K oligomeric chip in Chinese cabbage. In yellow leaves, genes involved in carotene synthesis (BrPSY, BrPDS, BrCRTISO, and BrLCYE), lutein, and zeaxanthin synthesis (BrCYP97A3 and BrHYDB) were upregulated, while those associated with carotene degradation (BrNCED3, BrNCED4, and BrNCED6) were downregulated. These expression patterns might support that the content of both lutein and total carotenoid was much higher in the yellow leaves than that in the white leaves. These results indicate that the yellow leaves accumulate high levels of both lutein and ß-carotene due to stimulation of synthesis and that the degradation rate is inhibited. A large number of responsible genes as novel genes were specifically expressed in yellow inner leaves, suggesting the possible involvement in pigment synthesis. Finally, we identified three transcription factors (BrA20/AN1-like, BrBIM1, and BrZFP8) that are specifically expressed and confirmed their relatedness in carotenoid synthesis from Arabidopsis plants.

Development of SSR markers by next-generation sequencing of Korean landraces of chamoe (Cucumis melo var. makuwa).

The oriental melon (Cucumis melo var. makuwa), called 'chamoe' in Korean, is a popular fruit crop cultivated mainly in Asia and a high-market value crop in Korea. To provide molecular breeding resources for chamoe, we developed and characterized genomic SSR markers from the preliminary Illumina read assemblies of Gotgam chamoe (one of the major landraces; KM) and SW3 (the breeding parent). Mononucleotide motifs were the most abundant type of markers, followed by di-, tri-, tetra-, and pentanucleotide motifs. The most abundant dinucleotide was AT, followed by AG and AC, and AAT was the most abundant trinucleotide motif in both assemblies. Following our SSR-marker development strategy, we designed a total of 370 primer sets. Of these, 236 primer sets were tested, exhibiting 93 % polymorphism between KM and SW3. Those polymorphic SSRs were successfully amplified in the netted and Kirkagac melons, which respectively exhibited 81 and 76 % polymorphism relative to KM, and 32 and 38 % polymorphism relative to SW3. Seven selected SSR markers with a total of 17 alleles (2-3 alleles per locus) were used to distinguish between KM, SW3, and four chamoe cultivars. Our results represent the first attempt to provide genomic resources for Korean landraces for the purposes of chamoe breeding, as well as to discover a set of SSR markers capable of discriminating chamoe varieties from Korea and the rest of Asia, which possess little genetic diversity. This study establishes a highly efficient strategy for developing SSR markers from preliminary Illumina assemblies of AT-rich genomes.

Metabolic differentiation of diamondback moth ( Plutella xylostella (L.)) resistance in cabbage ( Brassica oleracea L. ssp. capitata).

The diamondback moth, Plutella xylostella (L.), is a major pest responsible for destroying cabbage and other Brassica vegetable crops. A diamondback moth-resistant cabbage line was studied by comparing its metabolite profiles with those of a susceptible cabbage. Fourier transform infrared spectroscopy analysis revealed that carbohydrates, aromatic compounds, and amides were the major factors that distinguished the resistant and susceptible genotypes. Gas chromatography-time-of-flight mass spectrometry profiled 46 metabolites, including 19 amino acids, 15 organic acids, 8 sugars, 3 sugar alcohols, and 1 amine in two genotypes and F1 hybrid cabbages. The levels of glycolic acid, quinic acid, inositol, fumaric acid, glyceric acid, trehalose, shikimic acid, and aspartic acid were found to be very significantly different between the resistant and susceptible genotypes with a P value of <0.0001. These results will provide a foundation for further studies on diamondback moth resistance in cabbage breeding and for the development of other herbivore-resistant crops.

Comprehensive analysis of genic male sterility-related genes in Brassica rapa using a newly developed Br300K oligomeric chip.

To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage (Brassicarapa ssp. pekinensis), floral bud transcriptome analysis was carried out using a B. rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B. rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility.

MYB transcription factors regulate glucosinolate biosynthesis in different organs of Chinese cabbage (Brassica rapa ssp. pekinensis).

In this study, we investigated the expression of seven MYB transcription factors (a total of 17 genes that included Dof1.1, IQD1-1, MYB28, MYB29, MYB34, MYB51, and MYB122 and their isoforms) involved in aliphatic and indolic glucosinolate (GSL) biosynthesis and analyzed the aliphatic and indolic GSL content in different organs of Chinese cabbage (Brassica rapassp. Pekinensis). MYB28 and MYB29 expression in the stem was dramatically different when compared with the levels in the other organs. MYB34, MYB122, MYB51, Dof1.1, and IQD1-1 showed very low transcript levels among different organs. HPLC analysis showed that the glucosinolates (GSLs) consisted of five aliphatic GSLs (progoitrin, sinigrin, glucoalyssin, gluconapin, and glucobrassicanapin) and four indolic GSLs (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxygluco-brassicin, and neoglucobrassicin). Aliphatic GSLs exhibited 63.3% of the total GSLs content, followed by aromatic GSL (19.0%), indolic GSLs (10%), and unknown GSLs (7.7%) in different organs of Chinese cabbage. The total GSL content of different parts (ranked in descending order) was as follows: seed > flower > young leaves > stem > root > old leaves. The relationship between GSLs accumulation and expression of GSLs biosynthesis MYB TFs genes in different organs may be helpful to understand the mechanism of MYB TFs regulating GSL biosynthesis in Chinese cabbage.

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".

Regulation of the major vacuolar Ca²âº transporter genes, by intercellular Ca²âº concentration and abiotic stresses, in tip-burn resistant Brassica oleracea.

Calcium is an essential plant macronutrient that has unique structural and signaling roles related to tip-burn disorder in Brassica spp. crops. For two types of cabbage inbred lines, tip-burn susceptible and resistant, we measured and compared major macronutrient cations, including Ca(2+), in leaves. In both lines, Ca(2+), Mg(2+), Na(+), and K(+), accumulated more in leaf base than in leaf apex. Ca(2+) and K(+) were >2 times more abundant in the tip-burn resistant line, while Na(+) was higher in the susceptible line. Ca(2+) differences between the two lines resulted from differential accumulation of calcium into cell vacuoles. We profiled major vacuolar Ca(2+) transporters, in both cabbage lines, by growth time and intercellular Ca(2+) concentration. Expression pattern of several Ca(2+) transporter genes differed between tip-burn susceptible and resistant lines by growth time points. We also identified promoter regions of the major Ca(2+) vacuole transporter genes, CAX1, ACA4, and ACA11, which displayed hormonal, light and defense-related cis-acting regulatory elements. Finally, transporter genes in the two cabbage lines responded differently to abiotic stresses, demonstrating diversity in gene regulation among orthologous genes.

Three Brassica rapa metallothionein genes are differentially regulated under various stress conditions.

The expression profiles of three Brassica rapa metallothionein genes (BrMT 1-3) were determined in 7-day-old seedlings exposed to various exogenous factors including plant hormones, heavy metals and abiotic stresses. BrMT1, BrMT2, and BrMT3 were representatives of MT gene type 1, type 2, and type 3, respectively, according to their cysteine alignment. BrMT2 showed a relatively higher basal expression level compared to BrMT1 and BrMT3 under normal conditions. The BrMT1 transcript was markedly increased by various factors including ethephon, polyethylene glycol and hydrogen peroxide, with no down-regulation evident. On the contrary, BrMT2 expression was down-regulated by abscisic acid, salicylic acid, and methyl jasmonate. Heavy metals did not increase BrMT2 expression. BrMT3 expression was only marginally and non-significantly up- and down-regulated by the stress conditions tested. Promoter regions of BrMT1 and BrMT2 display different cis-acting elements supporting the different responses of both genes against various stresses. The results demonstrate the differential regulation of BrMT1-3 by various plant exogenous factors, and indicate the utility of the BrMT1 promoter as a multiple stress inducible promoter.