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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, p0.05) 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.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.

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the L-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T1 seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H2O2 treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.

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.

SummaryBrassica rapa ssp. chinensis (non-heading Chinese cabbage) is one of the most important vegetables grown in southern China. Autotetraploidy, induced artificially by colchicine treatment, has increased its nutritional value. For the first time, we report the distribution of repeat DNA sequences in autotetraploid B. rapa ssp. chinensis using fluorescence in situ hybridisation (FISH) with ribosomal DNA (rDNA) probes. Comparisons of inbred lines of a diploid (10P15) and its induced autotetraploid (10P11) of B. rapa ssp. chinensis used genomic in situ hybridisation (GISH) by labelling the total genomic DNA as a probe, and using unlabelled genomic DNA of B. oleracea L. var. italica as the blocking reagent. GISH signals appeared at the centromere and/or adjacent regions of each chromosome; therefore, these could be used as alternative centromere markers. In addition, variations in the number and loci of repeat sequences between the diploid and its autotetraploid were observed, suggesting that some deletio...

SummaryNon-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) is one of the most commonly consumed vegetables in China, and has recently been introduced into other countries in Asia, North America, and Europe. In this study, we obtained tetraploid, non-heading Chinese cabbage by treating the apical portion of diploid seedlings with various concentrations of colchicine for different treatment times. Treatments included a total of two, four, or six applications of 1.5 g l–1 or 2.0 g l–1 colchicine, performed twice a day at 09.00 h and 15.00 h. Tetraploid plants were selected and identified based on their morphological, anatomical, agronomic, and cytological characteristics, and on their DNA content, as measured by flow cytometry. The highest rate of tetraploidy (8.6%) occurred in seedlings treated six-times with 1.5 g l–1 colchicine. Compared to diploid plants (untreated controls), the tetraploid plants had wider leaves, larger petals, longer siliques, larger seeds, larger stomata, and fewe...

In Brassicaceae, a self-incompatibility (SI) system mediates pollen-pistil interactions. Self-pollen could be recognized and rejected by incompatible pistils. Several components involved in the SI response have been determined, including S-locus receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11, and arm repeat-containing protein 1 (ARC1). However, the components involved in the SI system of Brassicaceae are not fully understood. Here, we detected expression patterns of 24 SI-related genes in non-heading Chinese cabbage (Brassica campestris ssp chinensis Makino) after compatible and incompatible pollination, and potential interaction relationships of these genes were predicted. SRK and ARC1 transcripts increased initially 0.25 h after incompatible pollination, while kinase-associated protein phosphatase had an expression pattern that was opposite that of SRK transcripts during self-pollination. Plant U-box 8 was not required in the SI response of non-heading Chinese cabbage. Our results showed that the SI signal of non-heading Chinese cabbage could occur within 0.25 h after self-pollination. The hypothetical interaction relationships indicated that plastid-lipid-associated protein and malate dehydrogenase could be negatively regulated by chaperonin 10, glutathione transferase, cytidylate kinase/uridylate kinase, and methionine synthase by indirect interactions. Our findings could be helpful to better understand potential roles of these components in the SI system of non-heading Chinese cabbage.

To improve the efficiency of nitrogen use and to reduce the accumulation of nitrates in vegetables, an improved understanding of the mechanisms that regulate nitrate uptake and signaling is essential. Nitrogen use is regulated largely by the nitrate transporter genes, but few studies have examined the nitrate transporter genes in non-heading Chinese cabbage (Brassica rapa ssp. Chinensis Makino), one of the most important leafy vegetables in East Asia. In this study, the nitrate transporter gene BraNRT2.1 was isolated from non-heading Chinese cabbage. The cDNA for this gene contains an open reading frame of 1593 base pairs and encodes a predicted protein of 530 amino acid residues. Analysis of the BraNRT2.1 showed that BraNRT2.1 was expressed mainly in the roots and that the transcription of the gene was induced following exposure to 250 μM and 25 mM nitrate. In addition, GUS staining revealed that the BraNRT2.1 promoter directed expression to the roots. The BraNRT2.1-YFP fusion protein was observed to be localized to the plasma membrane. Finally, we observed that BraNRT2.1 could restore nitrate uptake in the presence of 200 μM nitrate in Arabidopsis thaliana plants lacking AtNRT2.1 function. Together, these results demonstrate that BraNRT2.1 encodes a high-affinity nitrate transporter that participates in nitrate uptake. These findings provide a foundation for future studies and plant breeding to improve the efficiency of nitrogen use and to reduce the accumulation of nitrates in vegetables.

Evaluation system can encourage and guide entrepreneurs, and impel them to perform well in environment management. An evaluation method based on advantage structure is established. It is used to analyze entrepreneur environment management behavior in China. Entrepreneur environment management behavior evaluation index system is constructed based on empirical research. Evaluation method of entrepreneurs is put forward, from the point of objective programming-theory to alert entrepreneurs concerned to think much of it, which means to take minimized objective function as comprehensive evaluation result and identify disadvantage structure pattern. Application research shows that overall behavior of Chinese entrepreneurs environmental management are good, specially, environment strategic behavior are best, environmental management behavior are second, cultural behavior ranks last. Application results show the efficiency and feasibility of this method.

The exocyst is a conserved protein complex, and required for vesicles tethering, fusion and polarized exocytosis. Exo70A1, the exocyst subunit, is essential for assembly of the exocyst complex. To better understand potential roles of Exo70A1 in non-heading Chinese cabbage ( Brassica campestris ssp. chinensis ), we obtained the full-length cDNA of Exo70A1 gene, which consisted of 1917 bp and encoded a protein of 638 amino acids. BlastX showed BcExo70A1 shared 94.9% identity with Brassica oleracea var. acephala (AEI26267.1), and clustered into a same group with other homologues in B. oleracea var. acephala and Brassica napus. Subcellular localization analysis showed BcExo70A1 was localized to punctate structures in cytosol of onion epithelial cells. Results showed that BcExo70A1 was widely presented in stamens, young stems, petals, unpollinated pistils, roots and leaves of self compatible and incompatible plants. The transcripts of BcExo70A1 in non-heading Chinese cabbage declined during initial 1.5 h after incompatible pollination, while an opposite trend was presented after compatible pollination. Our study reveals that BcExo70A1 could play essential roles in plant growth and development, and is related to the rejection of self pollen in non-heading Chinese cabbage.

The WRKY transcription factor (TF) plays an important role in plant developmental processes and stress responses. However, little is known about the WRKY TF family in Chinese cabbage (Brassica rapa ssp. pekinensis), although its genome has been completely sequenced. In this study, 145 genes of Chinese cabbage were identified that were anchored onto chromosomes 1–10 and further fractionated into three subgenomes. Organization and syntenic analysis indicated genomic distributions and collinear relationships of the BrWRKYs. Simultaneously, the selection pressures and evolutionary divergence of duplicated gene pairs were analyzed using nonsynonymous substitutions (Ka)/synonymous substitutions (Ks). Phylogenetic analyses showed that 145 BrWRKYs were clustered into three large groups and shared typical characters of WRKY TFs. In addition, Illumina RNA-Seq transcriptome of different tissues (i.e., roots, stems, and leaves) revealed tissue-specific and differential expression profiles of the BrWRKYs, and quantitative real-time polymerase chain reaction analysis showed the distinct and corresponsive expression patterns of the BrWRKYs in response to abiotic and biotic stresses in leaves. This study showed that these gene family members might play several roles in plant development, and abiotic and biotic stress responses might benefit from their functional characterization and utilization in the resistance engineering of Chinese cabbage.

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.

The 70-kDa heat shock proteins (HSP70s) are required for thermotolerance and development in plants and other organisms. We applied the techniques of homology-based cloning and real-time quantitative reverse transcription PCR to identify the functions of BccpHSC70s and BcmtHSC70s—heat shock cognates (HSCs) located in chloroplasts and mitochondria, respectively—in non-heading Chinese cabbage (Brassica campestris ssp. chinensis). cDNA clones of BccpHSC70s and BcmtHSC70s were highly homologous to each other and to those from other individual plants. Unlike other HSP70s, BccpHSC70-1 has only two signature motifs (three classical motifs were found in the predicted amino acid sequence of HSP70s, but not all HSP70s contain all three domains), and was expressed constitutively in three detected cultivars; the expression of BccpHSC70-1 in a thermotolerant cultivar (NHCC001) was greater than in other cultivars (NHCC002 and NHCC004) during development. High expression level of BccpHSC70-2 in leaves might be explained by the fact that chloroplasts are sensitive to temperature stress, and this gene might be a candidate gene for developing thermotolerant cultivars of non-heading Chinese cabbage. Expression of BcmtHSC70-2 was inducible under temperature stress. Expression of BcHSC70s in the sterile line showed it played an important role in the process of apoptosis during development of reproductive organs.

The cDNA clone of ascorbate oxidase gene was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino, cv. Suzhouqing) and characterized. Sequence analysis showed that there was a high similarity between this sequence (named BcAO) and its homologues in other plant species. Southern blotting indicated that more than one nuclear gene encoded this enzyme in non-heading Chinese cabbage. The mRNA level of the BcAO gene in leaves was monitored by real-time PCR at different developmental stages and under different stress conditions. Results showed that the expression of BcAO was upregulated by light, and the BcAO gene responded to copper stress as well. After inoculation with Alternaria brassicae, the expression of BcAO in the leaves was increased in general and peaked at 12 and 72 h post inoculation, with much higher expression at the later date. Cloning the BcAO gene will enable us to further understand its function and would provide useful information for resistance breeding program for non-heading Chinese cabbage.

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.

A gene (named BcFLC1) homologous to the AtFLC gene, which encodes a floral repressor, was isolated from the nonheading Chinese cabbage (Brassica campestris L. ssp. chinensis) cv. NJ074. The gene showed high similarity to AtFLC. For studying the gene function, we designed to introduce the BcFLC1 gene into Arabidopsis thaliana. The results showed that BcFLC1 had effects on flowering time similar to AtFLC. We also found that Arabidopsis cold-tolerance was enhanced by BcFLC1 overexpression. Under low temperature stress, the BcFLC1 transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the BcFLC1 over-expressing plants was also confirmed by the changes of electrolyte leakage and malonyldialdehyde and proline content.

In order to study the defense response to turnip mosaic virus (TuMV) infection in non-heading Chinese cabbage ( Brassica campestris ssp. chinensis Makino), we cloned the LRR II subfamily genes which comprises six members. They were high homologous to the function-known LRR II genes of Arabidopsis . We investigated their expression through quantitative real-time PCR analysis. TuMV infection induced the expression of these genes locally and systematically, and regulated the endogenous accumulation of salicylic acid (SA). Exogenous SA spraying was able to induce resistance to the susceptibility of the TuMV-infected plants, which might function via inhibiting the viral duplication. Though TuMV-induced SA accumulation was not the determinant in regulating gene expression, it mediated the reaction oxygen species (ROS) burst as a channel of defense.

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.

SummarySpecies in the Brassica genus including B. rapa, B. oleracea, and B. napus are cultivated widely around the World as edible vegetables and for their oils. Although progress has been made in identifying molecular markers among Brassica species, expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers have not been applied in the analysis of Brassica species for gene or quantitative trait locus (QTL) mapping. In the present work, 25,631 EST-SSRs were identified among 1,017,392 Brassica ESTs. These SSR-motifs averaged 25 bp in length. Tri-nucleotide SSRs were the most abundant. Among these, AGG/CCT was the dominant motif, accounting for 25% of the total. A total of 20,683 primer pairs were designed successfully. Some of the primer pairs (3,262) from B. napus and B. oleracea were then transferred successfully to B. rapa for comparative sequence analysis and PCR amplification.

BcCoi1, a cytoplasmic male sterility related gene, which was isolated from flower buds of Brassica campestris ssp. chinensis Makino using the RACE technology, was characterized and submitted to the NCBI GenBank (accession no. GU263836). The gene encodes a 67.78-kD protein containing 16 leucine-rich repeats and an N-terminal F-box motif and is extremely similar to Arabidopsis thaliana Coi1 gene. The Southern blot showed that BcCoi1 belongs to a multigene family. In A. thaliana, the Coi1 gene is involved in jasmonate signaling, and Coi1 mutant displayed male sterility. In this study, qPCR results demonstrated that BcCoi1 was accumulated in stamens and was significantly higher expressed in flower organs of the maintainer line than in the CMS one. At the microsporocyte development stage, the gene was expressed at a significantly lower extent in the CMS line than in the maintainer line. This expression profile presumes that BcCoi1 plays a role in early microspore development in non-heading Chinese cabbage.

Auxin resistant 1/like aux1 (AUX/LAX), pin-formed (PIN) and ATP binding cassette subfamily B (ABCB/MDR/PGP) are three families of auxin transport genes. The development-related functions of the influx and efflux carriers have been well studied and characterized in model plants. However, there is scant information regarding the functions of auxin genes in Chinese cabbage and the responses of exogenous polar auxin transport inhibitors (PATIs). We conducted a whole-genome annotation and a bioinformatics analysis of BrAUX/LAX, BrPIN, and BrPGP genes in Chinese cabbage. By analyzing the expression patterns at several developmental stages in the formation of heading leaves, we found that most auxin-associate genes were expressed throughout the entire process of leafy head formation, suggesting that these genes played important roles in the development of heads. UPLC was used to detect the distinct and uneven distribution of auxin in various segments of the leafy head and in response to PATI treatment, indicated that the formation of the leafy head depends on polar auxin transport and the uneven distribution of auxin in leaves. This study provides new insight into auxin polar transporters and the possible roles of the BrLAX, BrPIN and BrPGP genes in leafy head formation in Chinese cabbage.

Vernalization-mediated demethylation of BrCKA2 (casein kinase II α-subunit) and BrCKB4 (casein kinase II β-subunit) shorten the period of the clock gene BrCCA1 (circadian clock associated 1) in Brassica rapa. Photoperiod and vernalization are two environmental cues involved in the regulation of floral transition, but the ways in which they interact remain unclear. DNA methylation is one of the main mechanisms involved in controlling the functional state of chromatin and gene expression in response to environmental signals. To study the interaction between photoperiod and vernalization in floral transition, we carried out a comparative genomic analysis of genome-wide DNA methylation profiles in normal (CK) and vernalized (CA) leaves from Brassica rapa using methylated-DNA immunoprecipitation sequencing (MeDIP-seq). Two subunits of casein kinase II (CK2), BrCKA2 (catalytic α-subunit of CK2) and BrCKB4 (regulatory β-subunit of CK2), exhibited gradual DNA demethylation and increased expression in vernalized B. rapa. DNA methylation-defective plants demonstrated the causal link between DNA demethylation changes and changes in gene expression. Virus-induced gene silencing (VIGS) of BrCKA2 and BrCKB4 in B. rapa resulted in no change to the period of BrCCA1 (circadian clock associated 1) and a 1-week late flowering time. Finally, we demonstrated that increased levels of BrCKA2 and BrCKB4 in vernalized B. rapa confer elevated CK2 activity, resulting in a shortened period of the clock gene BrCCA1, which plays an important role in perceiving photoperiod in plants. Thus, our results suggest that there is a direct interaction between photoperiod and vernalization through DNA methylation mechanisms.

Non-heading Chinese cabbage (Brassica rapa ssp. chinensis) is one of the main green leafy vegetables in the world, especially in China, with significant economic value. Hyaloperonospora parasitica is a fungal pathogen responsible for causing downy mildew disease in Chinese cabbage, which greatly affects its production. The objective of this study was to identify transcriptionally regulated genes during incompatible interactions between non-heading Chinese cabbage and H. parasitica using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP). We obtained 129 reliable differential transcript-derived fragments (TDFs) in a resistant line ‘Suzhou Qing’. Among them, 121 upregulated TDFs displayed an expression peak at 24–48 h post inoculation (h.p.i.). Fifteen genes were further selected for validation of cDNA-AFLP expression patterns using quantitative reverse transcription PCR. Results confirmed the altered expression patterns of 13 genes (86.7%) revealed by the cDNA-AFLP. We identified four TDFs related to fungal resistance among the 15 TDFs. Furthermore, comparative analysis of four TDFs between resistant line ‘Suzhou Qing’ and susceptible line ‘Aijiao Huang’ showed that transcript levels of TDF14 (BcLIK1_A01) peaked at 48 h.p.i. and 25.1-fold increased in the resistant line compared with the susceptible line. Similarly, transcript levels of the other three genes, TDF42 (BcCAT3_A07), TDF75 (BcAAE3_A06) and TDF88 (BcAMT2_A05) peaked at 24, 48 and 24 h.p.i. with 25.1-, 100- and 15.8-fold increases, respectively. The results suggested that the resistance genes tended to transcribe at higher levels in the resistance line than in the susceptible line, which may provide resistance against pathogen infections. The present study might facilitate elucidating the molecular basis of the infection process and identifying candidate genes for resistance improvement of susceptible cultivars. The genes activated in a mildew-resistant cabbage suggest possible targets for engineering new, resistant varieties. Non-heading Chinese cabbage (also called bok choy or pak choi) is a widely grown green vegetable, susceptible, like most cabbages, to the fungal disease downy mildew. Chemical control of downy mildew is unreliable, and resistant varieties might be a better means of prevention. Chun-Mei Hu and colleagues of Nanjing Agricultural University, China, used a low-cost genetic method to explore the genes expressed in a resistant Chinese cabbage variety, ‘Suzhou Qing,’ inoculated with downy mildew. Over 120 genes were strongly activated during fungal infection. The authors suggest that the mildew fungus triggers a complex set of metabolic pathways in ‘Suzhou Qing,’; some of the genes involved may prove useful targets for breeding more resistant varieties.

© 2016 American Society of Plant Biologists. All rights reserved. Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize (Zea mays), which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 displays a partially overlapping expression pattern with ZmCCaMK after ABA treatment, and H2O2 is required for ABA-induced ZmNAC84expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates Ser-113 of ZmNAC84 in vitro, and Ser-113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco (Nicotiana tabacum) can improve drought tolerance and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H2O2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK. Subsequently, the activated ZmCCaMK phosphorylates ZmNAC84 at Ser-113, thereby inducing antioxidant defense by activating downstream genes.

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.

Ascorbic acid (AsA) participates in diverse biological processes, is regulated by multiple factors and is a potent antioxidant and cellular reductant. The D-mannose/L-galactose pathway is a major plant AsA biosynthetic pathway that is highly connected within biosynthetic networks, and generally conserved across plants. Previous work has shown that, although most genes of this pathway are expressed under standard growth conditions in Brassica rapa, some paralogs of these genes are not. We hypothesize that regulatory evolution in duplicate AsA pathway genes has occurred as an adaptation to environmental stressors, and that gene retention has been influenced by polyploidation events in Brassicas. To test these hypotheses, we explored the conservation of these genes in Brassicas and their expression patterns divergence in B. rapa. Similar retention and a high degree of gene sequence similarity were identified in B. rapa (A genome), Brassica oleracea (C genome) and Brassica napus (AC genome). However, the number of genes that encode the same type of enzymes varied among the three plant species. With the exception of GMP, which has nine genes, there were one to four genes that encoded the other enzymes. Moreover, we found that expression patterns divergence widely exists among these genes. i) VTC2 and VTC5 are paralogous genes, but only VTC5 is influenced by FLC. ii) Under light treatment, PMI1 co-regulates the AsA pool size with other D-Man/L-Gal pathway genes, whereas PMI2 is regulated only by darkness. iii) Under NaCl, Cu2+, MeJA and wounding stresses, most of the paralogs exhibit different expression patterns. Additionally, GME and GPP are the key regulatory enzymes that limit AsA biosynthesis in response to these treatments. In conclusion, our data support that the conservative and divergent expression patterns of D-Man/L-Gal pathway genes not only avoid AsA biosynthesis network instability but also allow B. rapa to better adapt to complex environments.

Pectins are fundamental polysaccharides in the plant primary cell wall. Polygalacturonases (PGs) and pectin methylesterases (PMEs), major components of the pectin remodeling and disassembly network, are involved in cell separation processes during many stages of plant development. A comprehensive study of these genes in plants could shed light on the evolution patterns of their structural development. In this study, we conducted whole-genome annotation, molecular evolution and gene expression analyses of PGs and PMEs in Brassica rapa and 8 other plant species. A total of 100 PGs and 110 PMEs were identified in B. rapa; they primarily diverged from 12–18 MYA and PMEs were retained more than PGs. Along with another 305 PGs and 348 PMEs in the 8 species, two different expansion or evolution types were discovered: a new branch of class A PGs appeared after the split of gymnosperms and angiosperms, which led to the rapid expansion of PGs; the pro domain was obtained or lost in the proPMEs through comprehensive analyses among PME genes. In addition, the PGs and PMEs exhibit diverged expression patterns. These findings will lead to novel insight regarding functional divergence and conservation in the gene families and provide more support for molecular evolution analyses.

Background Non-heading Chinese cabbage (NHCC, Brassica rapa ssp. chinensis) is an important leaf vegetable grown worldwide. However, little is known about the molecular mechanisms underlying tolerance for extreme temperature in NHCC. The limited availability of NHCC genomic information has greatly hindered functional analysis and molecular breeding. Results Here, we conduct comprehensive analyses of cold and heat treatments in NHCC using RNA-seq. Approximately 790 million paired-end reads representing 136,189 unigenes with N50 length of 1705 bp were obtained. Totally, 14,329 differentially expressed genes (DEGs) were detected. Among which, 10 DEGs were detected in all treatments, including 7 up-regulated and 3 down-regulated. The enrichment analyses showed 25 and 33 genes were enriched under cold and heat treatments, respectively. Additionally, 10,001 LncRNAs were identified, and 9,687 belonged to novel LncRNAs. The expression of miRNAs were more than that of pri-miRNAs and LncRNAs. Furthermore, we constructed a coexpression network for LncRNAs and miRNAs. It showed 67 and 192 genes were regulated by LncRNAs under cold and heat treatments, respectively. We constructed the flowchart for identifying LncRNAs of NHCC using transcriptome. Except conducting the de novo transcriptome analyses, we also compared these unigenes with the Chinese cabbage proteins. We identified several most important genes, and discussed their regulatory networks and crosstalk in cold and heat stresses. Conclusions We presented the first comprehensive characterization for NHCC crops and constructed the flowchart for identifying LncRNAs using transcriptome. Therefore, this study represents a fully characterized NHCC transcriptome, and provides a valuable resource for genetic and genomic studies under abiotic stress. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2625-2) contains supplementary material, which is available to authorized users.

Rootstock grafting may improve the resistance of watermelon plants to low temperatures. However, information regarding the molecular responses of rootstock grafted plants to chilling stress is limited. To elucidate the molecular mechanisms of chilling tolerance in grafted plants, the transcriptomic responses of grafted watermelon under chilling stress were analyzed using RNA-seq analysis. Sequencing data were used for digital gene expression (DGE) analysis to characterize the transcriptomic responses in grafted watermelon seedlings. A total of 702 differentially-expressed genes (DEGs) were found in rootstock grafted (RG) watermelon relative to self-grafted (SG) watermelon; among these genes, 522 genes were up-regulated and 180 were down-regulated. Additionally, 164 and 953 genes were found to specifically expressed in RG and SG seedlings under chilling stress, respectively. Functional annotations revealed that up-regulated DEGs are involved in protein processing, plant-pathogen interaction and the spliceosome, whereas down-regulated DEGs are associated with photosynthesis. Moreover, 13 DEGs were randomly selected for quantitative real time PCR (qRT-PCR) analysis. The expression profiles of these 13 DEGs were consistent with those detected by the DGE analysis, supporting the reliability of the DGE data. This work provides additional insight into the molecular basis of grafted watermelon responses to chilling stress.

Cold shock proteins (CSPs) are a kind of evolutionarily conserved nucleic acid-binding protein widely distributed from prokaryotes to eukaryotes. In this study, BcCSP1, a novel CSP, was isolated from a Pak-choi stress-induced cDNA library by the rapid amplification of cDNA end method. This gene had an open-reading frame (ORF) of 822 base pairs encoding 273 amino acids. BcCSP1 contained an N-terminal CSD domain and a glycine-rich region interspersed with seven CCHC-type zinc fingers at its C terminus. Multi-alignment and phylogenetic analyses showed that BcCSP1 shared high similarity to AtCSP1 and AtCSP3. Real-time polymerase chain reaction analysis showed that BcCSP1 was induced and co-expressed under cold stress and abscisic acid treatments. In addition, a BcCSP1-YFP fusion protein was localized to the nucleus and cytoplasm. These results indicated that BcCSP1 plays an important role in responses to cold and ABA treatments in Pak-choi. This work may be useful for future functional analysis of other CSP genes in Pak-choi.

Auxin/indole acetic acids (Aux/IAAs) and auxin response factors (ARFs), major components of the Aux signaling network, are involved in many developmental processes in plants. Investigating their evolution will provide new sight on the relationship between the molecular evolution of these genes and the increasing morphotypes of plants. We constructed comparative analyses of the retention, structure, expansion, and expression patterns of Aux/IAAs and ARFs in Brassica rapa and their evolution in eight other plant species, including algae, bryophytes, lycophytes, and angiosperms. All 33 of the ARFs, including 1 ARF-like (AL) (a type of ARF-like protein) and 53 Aux/IAAs, were identified in the B. rapa genome. The genes mainly diverged approximately 13 Ma. After the split, no Aux/IAA was completely lost, and they were more preferentially retained than ARFs. In land plants, compared with ARFs, which increased in stability, Aux/IAAs expanded more rapidly and were under more relaxed selective pressure. Moreover, BraIAAs were expressed in a more tissue-specific fashion than BraARFs and demonstrated functional diversification during gene duplication under different treatments, which enhanced the cooperative interaction of homologs to help plants adapt to complex environments. In addition, ALs existed widely and had a closer relationship with ARFs, suggesting that ALs might be the initial structure of ARFs. Our results suggest that the rapid expansion and preferential retention of Aux/IAAs are likely paralleled by the increasingly complex morphotypes in Brassicas and even in land plants. Meanwhile, the data support the hypothesis that the PB1 domain plays a key role in the origin of both Aux/IAAs and ARFs.

Figures S1-S21. (PDF 2762 kb)

In Chinese cabbage, leafy head-related traits are directly related to the cabbage yield and marketability, which are often primarily concerned target for breeders. Although intensive studies has been on head formation in Chinese cabbage in the past decade, very scanty information is available on mechanism involved in the head formation under the influence of low temperature at transcriptome and proteome perspective. In this study, quantitative expression profiling based on RNA-Seq transcriptome and iTRAQ proteome were combined to investigate this trait for a global picture of the molecular dynamics. Total of 2931 transcripts and 365 proteins were identified with significantly changed level in abundance from heading and non-heading Chinese cabbage. Related analyses including function annotations, hierarchical categories, as well as the correlation from transcript-to-proteins were performed. The results indicated that the leafy head formation of Chinese cabbage has involved a complex regulatory pattern. The correlated genes that were mapped to the pathway of plant hormone signal transduction suggested that the head formation might be an integrated result of various plant hormones. Our combined analysis will provide a comprehensive approach to understanding the regulation mechanism of leafy head formation in Chinese cabbage. Biological significance This study revealed the direct relation of leafy-heading traits with the yield of the plant. A comprehensive investigation was done by integrating quantitative expression profiling analysis of transcriptome and proteomic to provide crucial information for further research on the molecular mechanism involved in head formation in Chinese cabbage. The correlation of transcript-to-protein in abundance may afford some necessary information of involvement of post-transcriptional factors influencing leafy head formation in Chinese cabbage.

Flowering, transition from vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. FLOWERING LOCUSC (FLC) in Arabidopsis encodes a dosage-dependent repressor of flowering. We have isolated a FLC-related sequence from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and named it as BcFLC1. In this study, we found that fertility was affected by the overexpression of BcFLC1 in Arabidopsis. The floral morphology of shortening anther filaments and the phenotype of reduced or small siliques were observed in BcFLC1 overexpression plants. RT-PCR showed that the reduced fertility in Arabidopsis by the overexpression of FLC was related with the enhanced expression of anther filaments suppressor, such as RGA and RGL genes, and declined expression of SEPALLATA3 (SEP3) gene, encoding a MADS-box transcription factor involved in flower and ovule development.

BACKGROUND: Microtubules are the basic components of the cytoskeleton in eukaryotic cells and are made up of 13 parallel protofilaments, each composed of α- and β-tubulin unit molecules aligned along the longitudinal axis of the microtubule. RESULTS: α-Tubulin gene TUBA2 from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) was expressed at the highest level in stamens and at lower levels in other organs. In addition, it was expressed at a much lower level in the cytoplasmic male sterile (CMS) line than in the maintainer line. Furthermore, at the microsporocyte stage of development in the CMS line the microtubule bundles were knitted together in random organisation, which differed significantly from the radiate microtubule bundles running circumferentially around the nucleus in the maintainer line. Also, large vacuoles appeared within the cytoplasm in the CMS line with no dyed microtubules. CONCLUSION: TUBA2 was very important to pollen development, which might be closely related to male sterility. Large vacuoles might replace the nuclei close to the cell walls and lead to a lack of microtubules when the cells abort. Abnormalities and defects in the organisation and composition of microtubules in the male sterile line highlighted the complex interaction between microtubules and cytoplasmic male sterility. Copyright © 2012 Society of Chemical Industry

BcHSP81-4 gene, a member of heat shock proteins, was identified from a suppression subtractive hybridization cDNA library in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). The deduced amino acid sequence of the BcHSP81-4 cDNA revealed that it has high homology to other plant organelle isoforms and similar homology to both cytoplasmic and prokaryotic HSP90s. To study the regulation of gene expression, BcHSP81-4 genes in maintainer and sterility lines were monitored at different development stages and at different stress treatments. Real-time PCR was used for quantification of BcHSP81-4 mRNA. These results indicate that BcHSP81-4 is not responsive to heat shock at least at 35°C, while it is very responsive to salt and cold stress. And high expression of BcHSP81-4 in the bud of sterile line suggests that it may play prominent roles in sterility of pol CMS in non-heading Chinese cabbage.

Many plants increase in freezing tolerance upon exposure to low non-freezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the inducer of C-repeat/dehydration-responsive element-binding factor (CBF) expression (ICE) transcriptional activators followed by expression of the CBF; subsequently, CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in non-heading Chinese cabbage (Brassica campestris ssp. chinensis L. Makino). We show that non-heading Chinese cabbage encodes ICE1-like gene BrICE1 that bracket an open reading frame of 1,491 bp encoding a protein with a potential bHLH domain, which accumulates rapidly in response to low temperature followed closely by expression of the BrCBF gene, an ortholog of the Arabidopsis CBF3-like gene, and then BrCOR14 gene, an ortholog of the Arabidopsis CBF-targeted COR15b gene. An alignment of the later two genes from Arabidopsis, Brassica napus revealed the presence of conserved CANNTG core element and AP2 domain in BrCBF and a CCG core element in BrCOR14. In addition, BrCBF and BrCOR14 showed increased expression induced by low temperature as well as salt and drought, but not by ABA stress which are similar to those of Arabidopsis. We conclude that components of the CBF cold-response pathway are highly conserved in non-heading Chinese cabbage.

Cytosine DNA methylation is a critical epigenetic mechanism in the silencing of transposable elements, imprinting and regulating gene expression. However, little is known about the potential role of mC in response to heat stress. To determine and explore the functions of the dynamic DNA methylome during heat stress, we characterized single-base resolution methylome maps of Brassica rapa and assessed the dynamic changes of mC under heat stress using whole genome bisulfite sequencing. On average, the DNA methylation levels of CG, CHG and CHH are 39.3%, 15.38% and 5.24% in non-heading Chinese cabbage (NHCC), respectively. We found that the patterns of methylation are similar to other eudicot plants, but with higher CHH methylation levels. Further comparative analysis revealed varying patterns for three sequence contexts (mCG, mCHG and mCHH) under heat stress indicating context- and position-dependent methylation regulation. DNA methylation near the TSS and TES may be closely associated with methylation-dependent transcriptional silencing. Association analysis of differential methylation and differential gene expression revealed a different set of methDEGs involved at early and late stages under heat stress. The systemic characterization of the dynamic DNA methylome during heat stress will improve our understanding of the mechanism of epigenetic regulation under heat stress.

A cDNA clone for the turnip mosaic virus (TuMV)-induced gene in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino cv. Duanbaigeng) was isolated and characterized. The full-length cDNA clone, designated BcTuR3, was isolated during resistance response to TuMV. Sequence analysis of the cDNA clone confirmed that the translation product of the gene is homologous to other plant resistance proteins. Genomic DNA Southern blot analysis indicated that the gene represented a small multi-gene family. Northern hybridizations confirmed its elevated expression in the resistant “Duanbaigeng” and the susceptible “Aijiaohuang.” Upon inoculation with TuMV, the BcTuR3 transcript was rapidly accumulated in the infected leaves and stems. Tissue-specific expression of the gene showed that higher levels of the BcTuR3 transcript were observed in the TuMV-infected leaves, and later in the stems and roots of the resistant and susceptible varieties. These data showed that the BcTuR3 gene may be involved in plant resistance against TuMV pathogen infection.

BACKGROUND: Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen (N) assimilation, catalysing the synthesis of glutamine from ammonium and glutamate. Plants have two types of GS isoenzyme that are localised in different compartments: one in the cytosol (GS1) and the other in the chloroplast (GS2). GS1 is the major form of GS in plant roots and directly converts ammonium taken up by plant roots to glutamine. RESULTS: The GS1 gene cDNA of non-heading Chinese cabbage (Brassica campestrisssp. chinensis Makino) cultivar ‘Suzhouqing’ was isolated by RT-PCR (real-time polymerase chain reaction) and (5′/3′)-RACE (rapid amplification of cDNA ends) techniques. It was classified as GS1 by sequence alignment and motif search and named B. campestris ssp. chinensis Makino GS1 (BcGS1). Subcellular localisation analysis showed that BcGS1 was distributed in the cytoplasm of cells. BcGS1 was expressed in all parts, but mainly in the roots, which was verified by northern blotting analysis. Additionally, its expression was influenced by the N source concentration. CONCLUSION: These results suggest that BcGS1 is a novel member of the GS family in plants. BcGS1 was significantly related to N assimilation in non-heading Chinese cabbage, demonstrating that this gene plays an important role in plant growth and development. Copyright © 2010 Society of Chemical Industry

CMS is induced by the coordinated expression of certain mitochondrial and nuclear genes in flower development. Mitochondrial genes regulate manifestation of CMS, whereas nuclear genes regulate fertility phenotype and thus affect negatively. In this article, the buds of newly bred Ogura CMS of non-heading Chinese cabbage and its maintainer line as plant materials, genes differentially expressed transcripts were analyzed by cDNA-AFLP. Seventeen differently expressed genes were found in new Ogura CMS and nine genes in maintainer line. These genes were involved in energy metabolism, signal transduction, flower development, stress-related metabolism, transcription, etc. Expression patterns of three genes encoding BrCAM6, BrANK, BrTUB3 were verified by qRT-PCR in different organs and various stage flower buds of CMS and its maintainer line. The results revealed that two genes related to signal transduction, BrCAM6 and BrANK, were highly expressed in stamens and microspores of CMS than in maintainer line. As believed, these two genes involved in signal transduction of male sterile in CMS line. In comparison, BrTUB3 gene was accumulated in stamens and was expressed in significantly lower level in CMS line than in maintainer line. It expressed significantly lower in CMS than maintainer line after tetrad stage. This expression profile suggests that BrTUB3 played an important role in the development of the pollen, and may be closely related to male sterility.

Zinc is essential but toxic in excess. A bacterial metallothionein, SmtA from Synechococcus PCC 7942, has high affinity for Zn2+ and the intracellular exclusively handling of Zn2+. In this study, we report a functional analysis of SmtA in Arabidopsis thaliana and its response to zinc stress. After high zinc stress, the transgenic plants over-expressing SmtA showed higher survival rate than the wild type. We also found that over-expression of SmtA in Arabidopsis increased the activities of SOD and POD, and enhanced the tolerance to zinc stress. Together, our results indicate that SmtA may play an important role in the response to zinc stress in Arabidopsis.

The objective of the present work was selection of cultivar and suitable medium for regenerating shoots from leaf segments of non-heading Chinese cabbage. We evaluated six types of supplemented media with 2.0, 5.0 and 10.0 mg l−1 6-BA; 1.0 and 2.0 mg l−1 TDZ; 0.1, 0.3, 0.5, 0.8 and 1.0 mg l−1NAA; 3.0, 5.0 and 7.5 mg l−1AgNO3; 0.01 mg l−1 2–4, D and 4.0 mg l−1 KT for shoot regeneration and six cultivars “Sanchidaye”, “Liuchuandasuomian”, “Qingyou 4”, “Liangbaiye”, “AiKang 5” and “Hanxiao F3”, furthermore for root formation three types of supplemented media with 0.2, 0.3, 0.5 mg l−1 NAA, and for survival rate two types of base media: turf + vermiculite + manure (1:2:0.2) and soil + vermiculite (1:2). Culturing leaf segments on MS medium supplemented with 2 mg l−1 TDZ; 0.5 mg l−1 NAA and 7.5 mg l−1 AgNO3 gave the highest number of shoots per leaf segment (66) while roots were best formed on the medium supplemented with 0.2 mg l−1 NAA. Survival rate was highest (61.6%) in the turf: vermiculite: manure (1:2:0.2) medium. The highest percentage of responding leaf segments, number of shoots per leaf segment, rooting percentage and survival rate were observed in “Liuchuandasuomian”. The plantlets were transferred to the soil and grown into mature plants in pots. These results could be used for preliminary selections of cultivars to transfer disease resistance (Bt) gene through agrobacterium in non-heading Chinese cabbage.

BACKGROUND: Microtubules are prominent components of the cytoskeleton in every eukaryotic cell. Plant microtubules are essential for a wide variety of cellular functions, including generation of cell polarity, intracellular transport, positioning of organelles, cell wall deposition and cell division. The major component of microtubules is tubulin, an α,β heterodimer protein with a molecular mass of each subunit of around 50 kDa. Tubulin exists in cells as a mixture of polypeptides differing in their isoelectric points. Some post-translational modifications of tubulins are thought to modulate the functions and localization of microtubules within the cell. RESULTS: The complete sequence of a single-copy α-tubulin gene Tuba1, belonging to a multiple gene family of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), was obtained. The gene was expressed in high levels in young leaves and stamens, and it was also highly expressed during all stages of microsporogenesis in the maintainer. However, there was a distinct difference in α-tubulin expression between the sterile stage and the normal stages of pollen in a cytoplasmic male sterility line and its maintainer. CONCLUSION:Tuba1 was significantly related to the cell division and elongation of non-heading Chinese cabbage, demonstrating that this gene played an important role in the development of pollen and may be closely related to male sterility. Copyright © 2008 Society of Chemical Industry

Nitrate reductase (NR) is a key enzyme that catalyzes the first step in plants nitrogen metabolization. A cDNA of a NR gene from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar ‘Suzhouqing’ was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3049 bp contained an open reading frame of 2733 bp encoding 910 amino acids, a 5′-untranslated region of 113 bp and a 3′-untranslated sequence of 203 bp with a poly (A) tail. This protein shares common structural features with NRs from other higher plants and eukaryotes. It was classified as NR by sequence alignment, motif search and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and consequently nominated Brassica campestris ssp. chinensis Makino NR (BcNR). Southern blot analysis indicated that BcNR gene represented as 3 copies in the non-heading Chinese cabbage genome. Accumulation of BcNR mRNA was transiently induced by nitrate supply and correlated with nitrate concentration, the highest mRNA levels of BcNR was induced by 30 mM NO3− and reached a maximum at 4 h at this optimal concentration treatment, in contrast, NH4+ cannot induce the accumulation of BcNR mRNA. The BcNR was heterologously expressed in Escherichia coli BL21 (DE3) as a fusion protein. There was a specific band at about 110 kDa in size by SDS-PAGE analysis which was according with the expected molecular weight of the recombinant protein. To further explore its biological function, BcNR under regulation of the cauliflower mosaic virus (CaMV) 35S promoter was transferred into Arabidopsis thaliana. The transgenic plants exhibited an enhanced level of NR and nitrate reductase activity (NRA) in leaves under NO3− inducement.

Changes in ascorbic acid content and antioxidant enzyme activities were investigated in non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) leaves of ‘Wutacai’ and ‘Erqing’ exposed to excess copper (Cu). Cu treatment reduced the fresh weight of shoot and root by 57% and 46% in ‘Wutacai’, and 60 and 54% in ‘Erqing’, respectively. The accumulation of copper in leaves was higher in ‘Wutacai’ than that in ‘Erqing’. Compared to the control, ascorbic acid (AsA) contents were significantly decreased after copper treatment in both cultivars, while they were higher in ‘Wutacai’ than in ‘Erqing’, which may explain the higher copper-tolerance of ‘Wutacai’ with higher copper accumulation. The higher AsA contents of ‘Wutacai’ resulted from their lower activities of degrading enzymes, such as ascorbate oxydase (AAO) and ascorbate peroxidase (APX), as well as the increasing activity of dehydroascorbate reductase (DHAR) after copper treatment compared with ‘Erqing’. Copper stimulated superoxide dismutase (SOD) activity in both cultivars, but for catalase (CAT), there was little difference between both cultivars. Peroxidases (POD) activity was decreased after copper treatment in ‘Erqing’, while in ‘Wutacai’, it was significantly increased at 14 days, and POD activity was higher in ‘Wutacai’ than that in ‘Erqing’ at 21 and 28 days. Therefore, the induced increasing activity of POD in ‘Wutacai’ also played an important role in its copper tolerance.

The objective of this article is to study the effect of 5-aminolevulinic acid (ALA) and enhanced chlorophyll content, antioxidative enzymes and photosynthesis rate by foliar application of ALA. We evaluated three concentrations (control-distilled water, T1-50 mg l−1, T2-150 mg l−1, T3-250 mg l−1) of ALA and seven cultivars, “Sanchidaye” (Sa-1), “Lichuandasuomian” (Li-1), “Aijiaohuang” (Ai-1), “Qingyou” No. 4 (Qi-1), “Aikang” No. 5 (Ak-1), “Hanxiao” (Ha-1) and “Shulv” (Sl-1). “Ak-1” showed strongest response of POD (peroxidase) enzyme activity (0.4 U g−1 min−1) in 250 mg l−1 ALA solution. The highest CAT (catalase) activity (0.8 U g−1 min−1) after administration of 250 mg l−1 ALA was observed in “Li-1”. Meanwhile, highest (1.42 mg l−1) total chlorophyll content was also observed in “Ak-1”, when leaves were treated in 50 mg l−1 ALA, “Li-1” and “Ai-1” showed strongest response of specific activity of superoxide dismutase (SOD) in 50 mg l−1 and 50 mg l−1 ALA. Two hundred and fifty milligram per milliliter of ALA-treatment significantly improved the net photosynthetic rate.

Radish (Raphanus sativus L.) is an important vegetable crop worldwide. High nutritional quality was critical in its genetic improvement and production. The nutritional quality of 42 Chinese radish cultivars was analyzed in this study. The contents of six nutritional facts, dry matter (DM), crude fiber (CF), total soluble sugar (TSS), vitamin C (VC), protein, and nitrate, ranged from 29.7 to 88.2, 4.507 to 18.546, 2.233 to 15.457, 0.1416 to 0.3341, 0.34 to 1.15, and 1.81 to 5.89 g•kg^(-1) fresh weight (FW), respectively. Significant differences among the 42 radish cultivars were detected in the contents of nutritional facts. The data were subjected to cross-correlation analysis and principal component analysis (PCA). It was found that DM content was positively correlated with the content of TSS (r=0.7104), V(subscript c) (r=0.4011) and protein (r=0.4120). Vitamin C (V(subscript c)) content of radish showed a positive correlation (r=0.3300) with the protein content. According to the principal component analysis, out of the 42 radish cultivars, Nau-17, Nau-28, Nau-6, Nau-11, Nau-10, Nau-27, and Nau-31 were detected with very high scores in comprehensive evaluation. It could be concluded that abundant diversity of nutritional fact content occurred in different radish genotypes, and PCA analysis was effective for selecting radish germplasm with high quality. The results could contribute useful knowledge of nutritional quality, and provide important germplasms for the elite cultivar development and the inheritance study of nutritional facts in radish.

Simple sequence repeat (SSR) or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an inter-simple sequence repeat (ISSR)-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage ( Brassica rapa ). A total of 190 SSRs were obtained. Among these, AG or CT (54.7%) was the most frequent repeat, followed by AC or GT (31.6%) of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content (PIC) value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.