Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus.

Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops.

Broad-spectrum fungal resistance in sorghum is conferred through the complex regulation of an immune receptor gene embedded in a natural antisense transcript.

Sorghum (Sorghum bicolor), the fifth most widely grown cereal crop globally, provides food security for millions of people. Anthracnose caused by the fungus Colletotrichum sublineola is a major disease of sorghum worldwide. We discovered a major fungal resistance locus in sorghum composed of the nucleotide-binding leucine-rich repeat receptor gene ANTHRACNOSE RESISTANCE GENE1 (ARG1) that is completely nested in an intron of a cis-natural antisense transcript (NAT) gene designated CARRIER OF ARG1 (CARG). Susceptible genotypes express CARG and two alternatively spliced ARG1 transcripts encoding truncated proteins lacking the leucine-rich repeat domains. In resistant genotypes, elevated expression of an intact allele of ARG1, attributed to the loss of CARG transcription and the presence of miniature inverted-repeat transposable element sequences, resulted in broad-spectrum resistance to fungal pathogens with distinct virulence strategies. Increased ARG1 expression in resistant genotypes is also associated with higher histone H3K4 and H3K36 methylation. In susceptible genotypes, lower ARG1 expression is associated with reduced H3K4 and H3K36 methylation and increased expression of NATs of CARG. The repressive chromatin state associated with H3K9me2 is low in CARG-expressing genotypes within the CARG exon and higher in genotypes with low CARG expression. Thus, ARG1 is regulated by multiple mechanisms and confers broad-spectrum, strong resistance to fungal pathogens.

MicroRNAs Involved in Regulatory Cytoplasmic Male Sterility by Analysis RNA-seq and Small RNA-seq in Soybean.

Cytoplasmic male sterility (CMS) is an important plant characteristic for exploiting heterosis to enhance crop traits during breeding. However, the CMS regulatory network remains unclear in plants, even though researchers have attempted to isolate genes associated with CMS. In this study, we performed high-throughput sequencing and degradome analyses to identify microRNAs (miRNAs) and their targets in a soybean CMS line (JLCMS9A) and its maintainer line (JLCMS9B). Additionally, the differentially expressed genes during reproductive development were identified using RNA-seq data. A total of 280 miRNAs matched soybean miRNA sequences in miRBase, including mature miRNAs and pre-miRNAs. Of the 280 miRNAs, 30, 23, and 21 belonged to the miR166, miR156, and miR171 families, respectively. Moreover, 410 novel low-abundant miRNAs were identified in the JLCMS9A and JLCMS9B flower buds. Furthermore, 303 and 462 target genes unique to JLCMS9A and JLCMS9B, respectively, as well as 782 common targets were predicted based on the degradome analysis. Target genes differentially expressed between the CMS line and the maintainer line were revealed by an RNA-seq analysis. Moreover, all target genes were annotated with diverse functions related to biological processes, cellular components, and molecular functions, including transcriptional regulation, the nucleus, meristem maintenance, meristem initiation, cell differentiation, auxin-activated signaling, plant ovule development, and anther development. Finally, a network was built based on the interactions. Analyses of the miRNA, degradome, and transcriptome datasets generated in this study provided a comprehensive overview of the reproductive development of a CMS soybean line. The data presented herein represent useful information for soybean hybrid breeding. Furthermore, the study results indicate that miRNAs might contribute to the soybean CMS regulatory network by modulating the expression of CMS-related genes. These findings lay the foundation for future studies on the molecular mechanisms underlying soybean CMS.

Global mRNA and microRNA expression dynamics in response to anthracnose infection in sorghum.

BACKGROUND: Anthracnose is a damaging disease of sorghum caused by the fungal pathogen Colletotrichum sublineolum. Genome-wide mRNA and microRNA (miRNA) profiles of resistant and susceptible sorghum genotypes were studied to understand components of immune responses, and fungal induced miRNA and target gene networks. RESULTS: A total of 18 mRNA and 12 miRNA libraries from resistant and susceptible sorghum lines were sequenced prior to and after inoculation with C. sublineolum. Significant differences in transcriptomes of the susceptible and resistant genotypes were observed with dispersion distance and hierarchical cluster tree analyses. Of the total 33,032 genes predicted in the sorghum genome, 19,593 were induced by C. sublineolum, and 15,512 were differentially expressed (DEGs) between the two genotypes. The resistant line was marked by significant reprogramming of the transcriptome at 24 h post inoculation (hpi), and a decrease at 48 hpi, whereas the susceptible line displayed continued changes in gene expression concordant with elevated fungal growth in the susceptible genotype. DEGs encode proteins implicated in diverse functions including photosynthesis, synthesis of tetrapyrrole, carbohydrate and secondary metabolism, immune signaling, and chitin binding. Genes encoding immune receptors, MAPKs, pentatricopeptide repeat proteins, and WRKY transcription factors were induced in the resistant genotype. In a parallel miRNA profiling, the susceptible line displayed greater number of differentially expressed miRNAs than the resistant line indicative of a widespread suppression of gene expression. Interestingly, we found 75 miRNAs, including 36 novel miRNAs, which were differentially expressed in response to fungal inoculation. The expression of 50 miRNAs was significantly different between resistant and susceptible lines. Subsequently, for 35 differentially expressed miRNAs, the corresponding 149 target genes were identified. Expression of 56 target genes were significantly altered after inoculation, showing inverse expression with the corresponding miRNAs. CONCLUSIONS: We provide insights into genome wide dynamics of mRNA and miRNA profiles, biological and cellular processes underlying host responses to fungal infection in sorghum. Resistance is correlated with early transcriptional reprogramming of genes in various pathways. Fungal induced genes, miRNAs and their targets with a potential function in host responses to anthracnose were identified, opening avenues for genetic dissection of resistance mechanisms.

Identification of resistance loci in Chinese and Canadian canola/rapeseed varieties against Leptosphaeria maculans based on genome-wide association studies.

BACKGROUND: The fungal pathogen Leptosphaeria maculans (Lm). causes blackleg disease on canola/rapeseed in many parts of the world. It is important to use resistant cultivars to manage the disease and minimize yield losses. In this study, twenty-two Lm isolates were used to identify resistance genes in a collection of 243 canola/rapeseed (Brassica napus L.) accessions from Canada and China. These Lm isolates carry different compliments of avirulence genes, and the investigation was based on a genome-wide association study (GWAS) and genotype-by-sequencing (GBS). RESULTS: Using the CROP-SNP pipeline, a total of 81,471 variants, including 78,632 SNPs and 2839 InDels, were identified. The GWAS was performed using TASSEL 5.0 with GLM + Q model. Thirty-two and 13 SNPs were identified from the Canadian and Chinese accessions, respectively, tightly associated with blackleg resistance with P values < 1 × 10- 4. These SNP loci were distributed on chromosomes A03, A05, A08, A09, C01, C04, C05, and C07, with the majority of them on A08 followed by A09 and A03. The significant SNPs identified on A08 were all located in a 2010-kb region and associated with resistance to 12 of the 22 Lm isolates. Furthermore, 25 resistance gene analogues (RGAs) were identified in these regions, including two nucleotide binding site (NBS) domain proteins, fourteen RLKs, three RLPs and six TM-CCs. These RGAs can be the potential candidate genes for blackleg resistance. CONCLUSION: This study provides insights into potentially new genomic regions for discovery of additional blackleg resistance genes. The identified regions associated with blackleg resistance in the germplasm collection may also contribute directly to the development of canola varieties with novel resistance genes against blackleg of canola.

Comparative Analysis of the Metabolic Profiles of Yellow- versus Black-Seeded Rapeseed Using UPLC-HESI-MS/MS and Transcriptome Analysis.

The high levels of secondary metabolites in rapeseed play important roles in determining the oil quality and feeding value. Here, we characterized the metabolic profiles in seeds of various yellow- and black-seeded rapeseed accessions. Two hundred and forty-eight features were characterized, including 31 phenolic acids, 54 flavonoids, 24 glucosinolates, 65 lipid compounds, and 74 other polar compounds. The most abundant phenolic acids and various flavonoids (epicatechin, isorhamnetin, kaempferol, quercetin, and their derivatives) were widely detected and showed significant differences in distribution between the yellow- and black-seeded rapeseed. Furthermore, the related genes (e.g., BnTT3, BnTT18, BnTT10, BnTT12, and BnBAN) involved in the proanthocyanidin pathway had lower expression levels in yellow-seeded rapeseed, strongly suggesting that the seed coat color could be mainly determined by the levels of epicatechin and their derivatives. These results improve our understanding of the primary constituents of rapeseed and lay the foundation for breeding novel varieties with a high nutritional value.

Fine mapping of Brassica napus blackleg resistance gene Rlm1 through bulked segregant RNA sequencing.

The fungal pathogen Leptosphaeria maculans causes blackleg disease on canola and rapeseed (Brassica napus) in many parts of the world. A B. napus cultivar, 'Quinta', has been widely used for the classification of L. maculans into pathogenicity groups. In this study, we confirmed the presence of Rlm1 in a DH line (DH24288) derived from B. napus cultivar 'Quinta'. Rlm1 was located on chromosome A07, between 13.07 to 22.11 Mb, using a BC1 population made from crosses of F1 plants of DH16516 (a susceptible line) x DH24288 with bulked segregant RNA Sequencing (BSR-Seq). Rlm1 was further fine mapped in a 100 kb region from 19.92 to 20.03 Mb in the BC1 population consisting of 1247 plants and a F2 population consisting of 3000 plants using SNP markers identified from BSR-Seq through Kompetitive Allele-Specific PCR (KASP). A potential resistance gene, BnA07G27460D, was identified in this Rlm1 region. BnA07G27460D encodes a serine/threonine dual specificity protein kinase, catalytic domain and is homologous to STN7 in predicted genes of B. rapa and B. oleracea, and A. thaliana. Robust SNP markers associated with Rlm1 were developed, which can assist in introgression of Rlm1 and confirm the presence of Rlm1 gene in canola breeding programs.

Comparative transcriptome analysis of flower heterosis in two soybean F1 hybrids by RNA-seq.

Heterosis has been widely exploited as an approach to enhance crop traits during breeding. However, its underlying molecular genetic mechanisms remain unclear. Recent advances in RNA sequencing technology (RNA-seq) have provided an opportunity to conduct transcriptome profiling for heterosis studies. We used RNA-seq to analyze the flower transcriptomes of two F1 hybrid soybeans (HYBSOY-1 and HYBSOY-5) and their parents. More than 385 million high-quality reads were generated and aligned against the soybean reference genome. A total of 681 and 899 genes were identified as being differentially expressed between HYBSOY-1 and HYBSOY-5 and their parents, respectively. These differentially expressed genes (DEGs) were categorized into four major expression categories with 12 expression patterns. Furthermore, gene ontology (GO) term analysis showed that the DEGs were enriched in the categories metabolic process and catalytic activity, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis found that metabolic pathway and biosynthesis of secondary metabolites were enriched in the two F1 hybrids. Comparing the DEGs of the two F1 hybrids by GO term and KEGG pathway analyses identified 26 common DEGs that showed transgressive up-regulation, and which could be considered potential candidate genes for heterosis in soybean F1 hybrids. This identification of an extensive transcriptome dataset gives a comprehensive overview of the flower transcriptomes in two F1 hybrids, and provides useful information for soybean hybrid breeding. These findings lay the foundation for future studies on molecular mechanisms underlying soybean heterosis.

Establishment of the model system between phytochemicals and gene expression profiles in Macrosclereid cells of Medicago truncatula.

Macrosclereid cells, which are a layer in the seed coat of Medicago truncatula, accumulate large amounts of phytochemicals during their development. But little is known about the complex and dynamic changes during macrosclereid cell development. To characterize the phytochemicals and the related gene expression during the development of M. truncatula macrosclereid cells, a high performance liquid chromatography-mass spectrometry (HPLC-MS) assay and microarray study were conducted on transcriptome changes from macrosclereid cell during seed development. A total of 16 flavonoids by HPLC-MS and 4861 genes exhibited significant differences at transcript levels by microarray analysis were identified for macrosclerid cells at six different time points during seed development. 815 abiotic and biotic stress genes, 223 transcriptional factors (TFs), and 155 annotated transporter proteins exhibited differential expression during the development of macrosclereid cells. A total of 102 genes were identified as involved in flavonoid biosynthesis, phenypropanoid biosynthesis, and flavone and flavonol biosynthesis. We performed a weighted gene co-regulatory network (WGCNA) to analyze the gene-flavonoid association and rebuilt the gene regulatory network during macrosclereid cell development. Our studies revealed that macrosclereid cells are, beside as the first barrier of defense against diseases, an excellent model system to investigate the regulatory network that governs flavonoid biosynthesis.

Genome-wide association mapping and Identification of candidate genes for fatty acid composition in Brassica napus L. using SNP markers.

BACKGROUND: B. napus (oilseed) is an important source of edible vegetable oil, and its nutritional and economic value is determined by its fatty acid composition and content. RESULTS: Using the Brassica 60 K SNP array, we performed a genome-wide association study of fatty acid composition in a population of 520 genetically diverse oilseed accessions. Using the PCA + K model in TASSEL 5.2.1, we identified 62 genomic regions that were significantly associated with the composition of seven fatty acids, and five consensus regions that mapped to the A2, A8, A9, C1, and C3 chromosomes, respectively, of the Brassica napus Darmor-bzh genome. We then identified 24 orthologs of the functional candidate genes involved in fatty acid biosynthesis, excluding BnaA.FAE1 and BnaC.FAE1 on the A8 and C3 homologous genome blocks, which are known to have critical roles in the fatty acid biosynthesis pathway, and potential orthologs of these genes (e.g., LACS9, KCR1, FAB1, LPAT4, KCS17, CER4, TT16, and ACBP5). CONCLUSIONS: Our results demonstrate the power of association mapping in identifying genes of interest in B. napus and provide insight into the genetic basis of fatty acid biosynthesis in B. napus. Furthermore, our findings may facilitate marker-based breeding efforts aimed at improving fatty acid composition and quality in B. napus.

Genome-Wide Survey of Flavonoid Biosynthesis Genes and Gene Expression Analysis between Black- and Yellow-Seeded Brassica napus.

Flavonoids, the compounds that impart color to fruits, flowers, and seeds, are the most widespread secondary metabolites in plants. However, a systematic analysis of these loci has not been performed in Brassicaceae. In this study, we isolated 649 nucleotide sequences related to flavonoid biosynthesis, i.e., the Transparent Testa (TT) genes, and their associated amino acid sequences in 17 Brassicaceae species, grouped into Arabidopsis or Brassicaceae subgroups. Moreover, 36 copies of 21 genes of the flavonoid biosynthesis pathway were identified in Arabidopsis thaliana, 53 were identified in Brassica rapa, 50 in Brassica oleracea, and 95 in B. napus, followed the genomic distribution, collinearity analysis and genes triplication of them among Brassicaceae species. The results showed that the extensive gene loss, whole genome triplication, and diploidization that occurred after divergence from the common ancestor. Using qRT-PCR methods, we analyzed the expression of 18 flavonoid biosynthesis genes in 6 yellow- and black-seeded B. napus inbred lines with different genetic background, found that 12 of which were preferentially expressed during seed development, whereas the remaining genes were expressed in all B. napus tissues examined. Moreover, 14 of these genes showed significant differences in expression level during seed development, and all but four of these (i.e., BnTT5, BnTT7, BnTT10, and BnTTG1) had similar expression patterns among the yellow- and black-seeded B. napus. Results showed that the structural genes (BnTT3, BnTT18, and BnBAN), regulatory genes (BnTTG2 and BnTT16) and three encoding transfer proteins (BnTT12, BnTT19, and BnAHA10) might play an crucial roles in the formation of different seed coat colors in B. napus. These data will be helpful for illustrating the molecular mechanisms of flavonoid biosynthesis in Brassicaceae species.

Molecular Mapping and QTL for Expression Profiles of Flavonoid Genes in Brassica napus.

Flavonoids are secondary metabolites that are extensively distributed in the plant kingdom and contribute to seed coat color formation in rapeseed. To decipher the genetic networks underlying flavonoid biosynthesis in rapeseed, we constructed a high-density genetic linkage map with 1089 polymorphic loci (including 464 SSR loci, 97 RAPD loci, 451 SRAP loci, and 75 IBP loci) using recombinant inbred lines (RILs). The map consists of 19 linkage groups and covers 2775 cM of the B. napus genome with an average distance of 2.54 cM between adjacent markers. We then performed expression quantitative trait locus (eQTL) analysis to detect transcript-level variation of 18 flavonoid biosynthesis pathway genes in the seeds of the 94 RILs. In total, 72 eQTLs were detected and found to be distributed among 15 different linkage groups that account for 4.11% to 52.70% of the phenotypic variance atrributed to each eQTL. Using a genetical genomics approach, four eQTL hotspots together harboring 28 eQTLs associated with 18 genes were found on chromosomes A03, A09, and C08 and had high levels of synteny with genome sequences of A. thaliana and Brassica species. Associated with the trans-eQTL hotspots on chromosomes A03, A09, and C08 were 5, 17, and 1 genes encoding transcription factors, suggesting that these genes have essential roles in the flavonoid biosynthesis pathway. Importantly, bZIP25, which is expressed specifically in seeds, MYC1, which controls flavonoid biosynthesis, and the R2R3-type gene MYB51, which is involved in the synthesis of secondary metabolites, were associated with the eQTL hotspots, and these genes might thus be involved in different flavonoid biosynthesis pathways in rapeseed. Hence, further studies of the functions of these genes will provide insight into the regulatory mechanism underlying flavonoid biosynthesis, and lay the foundation for elaborating the molecular mechanism of seed coat color formation in B. napus.

Global Regulation of Plant Immunity by Histone Lysine Methyl Transferases.

Posttranslational modification of histones modulates gene expression affecting diverse biological functions. We showed that the Arabidopsis thaliana histone methyl transferases SET DOMAIN GROUP8 (SDG8) and SDG25 regulate pep1-, flg22-, and effector-triggered immunity as well as systemic acquired resistance. Genome-wide basal and induced transcriptome changes regulated by SDG8 and/or SDG25 showed that two genes of the SDG-dependent transcriptome, CAROTENOID ISOMERASE2 (CCR2) and ECERIFERUM3 (CER3), were also required for plant immunity, establishing mechanisms in defense functions for SDG8 and SDG25. CCR2 catalyzes the biosynthesis of carotenoids, whereas CER3 is involved in the biosynthesis of cuticular wax. SDG8 and SDG25 affected distinct and overlapping global and locus-specific histone H3 lysine 4 (H3K4) and histone H3 lysine 36 (H3K36) methylations. Loss of immunity in sdg mutants was attributed to altered global and CCR2- and CER3-specific histone lysine methylation (HLM). Loss of immunity in sdg, ccr2, and cer3 mutants was also associated with diminished accumulation of lipids and loss of cuticle integrity. In addition, sdg8 and sdg25 mutants were impaired in H2B ubiquitination (H2Bubn) at CCR2, CER3, and H2Bubn regulated R gene, SNC1, revealing crosstalk between the two types of histone modifications. In summary, SDG8 and SDG25 contribute to plant immunity directly through HLM or indirectly through H2Bubn and by regulating expression of plant immunity genes, accumulation of lipids, biosynthesis of carotenoids, and maintenance of cuticle integrity.

Comparative Transcriptome Analysis of Recessive Male Sterility (RGMS) in Sterile and Fertile Brassica napus Lines.

The recessive genetic male sterility (RGMS) system plays a key role in the production of hybrid varieties in self-pollinating B. napus plants, and prevents negative cytoplasmic effects. However, the complete molecular mechanism of the male sterility during male-gametogenesis in RGMS remains to be determined. To identify transcriptomic changes that occur during the transition to male sterility in RGMS, we examined the male sterile line WSLA and male fertile line WSLB, which are near-isogenic lines (NILs) differing only in the fertility trait. We evaluated the phenotypic features and sterility stage using anatomical analysis. Comparative RNA sequencing analysis revealed that 3,199 genes were differentially expressed between WSLA and WSLB. Many of these genes are mainly involved in biological processes related to flowering, including pollen tube development and growth, pollen wall assembly and modification, and pollen exine formation and pollination. The transcript profiles of 93 genes associated with pollen wall and anther development were determined by quantitative RT-PCR in different flower parts, and classified into the following three major clades: (1) up-regulated in WSLA plants; (2) down-regulated in WSLA plants; and 3) down-regulated in buds, but have a higher expression in stigmas of WSLA than in WSLB. A subset of genes associated with sporopollenin accumulation were all up-regulated in WSLA. An excess of sporopollenin results in defective pollen wall formation, which leads to male sterility in WSLA. Some of the genes identified in this study are candidates for future research, as they could provide important insight into the molecular mechanisms underlying RGMS in WSLA.

Genome-wide systematic characterization of the bZIP transcriptional factor family in tomato (Solanum lycopersicum L.).

BACKGROUND: Transcription factors of the basic leucine zipper (bZIP) family represent exclusively in eukaryotes and have been shown to regulate diverse biological processes in plant growth and development as well as in abiotic and biotic stress responses. However, little is known about the bZIP family in tomato (Solanum lycopersicum L.). METHODS: The SlbZIP genes were identified using local BLAST and hidden Markov model profile searches. The phylogenetic trees, conserved motifs and gene structures were generated by MEGA6.06, MEME tool and gene Structure Display Server, respectively. The syntenic block diagrams were generated by the Circos software. The transcriptional gene expression profiles were obtained using Genevestigator tool and quantitative RT-PCR. RESULTS: In the present study, we carried out a genome-wide identification and systematic analyses of 69 SlbZIP genes that distributes unevenly on the tomato chromosomes. This family can be divided into 9 groups according to the phylogenetic relationship among the SlbZIP proteins. Six kinds of intron patterns (a-f) within the basic and hinge regions are defined. The additional conserved motifs and their presence of the group specificity were also identified. Further, we predicted the DNA-binding patterns and the dimerization property on the basis of the characteristic features in the basic and hinge regions and the leucine zipper, respectively, which supports our classification greatly and helps to classify 24 distinct subfamilies. Within the SlbZIP family, a total of 40 SlbZIP genes are located in the segmental duplicate regions in the tomato genome, suggesting that the segment chromosomal duplications contribute greatly to the expansion of the tomato SlbZIP family. Expression profiling analyses of 59 SlbZIP genes using quantitative RT-PCR and publicly available microarray data indicate that the tomato SlbZIP genes have distinct and diverse expression patterns in different tissues and developmental stages and many of the tomato bZIP genes might be involved in responses to various abiotic and biotic stresses as well as in response to light. CONCLUSIONS: This genome-wide systematic characterization identified a total of 69 members in the SlbZIP family and the analyses of the protein features and gene expression patterns provide useful clues for further functional characterization of the bZIP transcription factors in tomato.

CYCLIN-DEPENDENT KINASE8 differentially regulates plant immunity to fungal pathogens through kinase-dependent and -independent functions in Arabidopsis.

CYCLIN-DEPENDENT KINASE8 (CDK8) is a widely studied component of eukaryotic Mediator complexes. However, the biological and molecular functions of plant CDK8 are not well understood. Here, we provide evidence for regulatory functions of Arabidopsis thaliana CDK8 in defense and demonstrate its functional and molecular interactions with other Mediator and non-Mediator subunits. The cdk8 mutant exhibits enhanced resistance to Botrytis cinerea but susceptibility to Alternaria brassicicola. The contributions of CDK8 to the transcriptional activation of defensin gene PDF1.2 and its interaction with MEDIATOR COMPLEX SUBUNIT25 (MED25) implicate CDK8 in jasmonate-mediated defense. Moreover, CDK8 associates with the promoter of AGMATINE COUMAROYLTRANSFERASE to promote its transcription and regulate the biosynthesis of the defense-active secondary metabolites hydroxycinnamic acid amides. CDK8 also interacts with the transcription factor WAX INDUCER1, implying its additional role in cuticle development. In addition, overlapping functions of CDK8 with MED12 and MED13 and interactions between CDK8 and C-type cyclins suggest the conserved configuration of the plant Mediator kinase module. In summary, while CDK8's positive transcriptional regulation of target genes and its phosphorylation activities underpin its defense functions, the impaired defense responses in the mutant are masked by its altered cuticle, resulting in specific resistance to B. cinerea.

Differential accumulation of phenolic compounds and expression of related genes in black- and yellow-seeded Brassica napus.

Developing yellow-seeded Brassica napus (rapeseed) with improved qualities is a major breeding goal. The intermediate and final metabolites of the phenylpropanoid and flavonoid pathways affect not only oil quality but also seed coat colour of B. napus. Here, the accumulation of phenolic compounds was analysed in the seed coats of black-seeded (ZY821) and yellow-seeded (GH06) B. napus. Using toluidine blue O staining and liquid chromatography-mass spectrometry, histochemical and biochemical differences were identified in the accumulation of phenolic compounds between ZY821 and GH06. Two and 13 unique flavonol derivatives were detected in ZY821 and GH06, respectively. Quantitative real-time PCR analysis revealed significant differences between ZY821 and GH06 in the expression of common phenylpropanoid biosynthetic genes (BnPAL and BnC4H), common flavonoid biosynthetic genes (BnTT4 and BnTT6), anthocyanin- and proanthocyandin-specific genes (BnTT3 and BnTT18), proanthocyandin-specific genes (BnTT12, BnTT10, and BnUGT2) and three transcription factor genes (BnTTG1, BnTTG2, and BnTT8) that function in the flavonoid biosynthetic pathway. These data provide insight into pigment accumulation in B. napus, and serve as a useful resource for researchers analysing the formation of seed coat colour and the underlying regulatory mechanisms in B. napus.

Paraquat Resistant1, a Golgi-localized putative transporter protein, is involved in intracellular transport of paraquat.

Paraquat is one of the most widely used herbicides worldwide. In green plants, paraquat targets the chloroplast by transferring electrons from photosystem I to molecular oxygen to generate toxic reactive oxygen species, which efficiently induce membrane damage and cell death. A number of paraquat-resistant biotypes of weeds and Arabidopsis (Arabidopsis thaliana) mutants have been identified. The herbicide resistance in Arabidopsis is partly attributed to a reduced uptake of paraquat through plasma membrane-localized transporters. However, the biochemical mechanism of paraquat resistance remains poorly understood. Here, we report the identification and characterization of an Arabidopsis paraquat resistant1 (par1) mutant that shows strong resistance to the herbicide without detectable developmental abnormalities. PAR1 encodes a putative l-type amino acid transporter protein localized to the Golgi apparatus. Compared with the wild-type plants, the par1 mutant plants show similar efficiency of paraquat uptake, suggesting that PAR1 is not directly responsible for the intercellular uptake of paraquat. However, the par1 mutation caused a reduction in the accumulation of paraquat in the chloroplast, suggesting that PAR1 is involved in the intracellular transport of paraquat into the chloroplast. We identified a PAR1-like gene, OsPAR1, in rice (Oryza sativa). Whereas the overexpression of OsPAR1 resulted in hypersensitivity to paraquat, the knockdown of its expression using RNA interference conferred paraquat resistance on the transgenic rice plants. These findings reveal a unique mechanism by which paraquat is actively transported into the chloroplast and also provide a practical approach for genetic manipulations of paraquat resistance in crops.

Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds.

The seed oil content in oilseed crops is a major selection trait to breeders. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) are key regulators of fatty acid biosynthesis. Overexpression of AtLEC1 and its orthologs in canola (Brassica napus), BnLEC1 and BnL1L, causes an increased fatty acid level in transgenic Arabidopsis plants, which, however, also show severe developmental abnormalities. Here, we use truncated napin A promoters, which retain the seed-specific expression pattern but with a reduced expression level, to drive the expression of BnLEC1 and BnL1L in transgenic canola. Conditional expression of BnLEC1 and BnL1L increases the seed oil content by 2% to 20% and has no detrimental effects on major agronomic traits. In the transgenic canola, expression of a subset of genes involved in fatty acid biosynthesis and glycolysis is up-regulated in developing seeds. Moreover, the BnLEC1 transgene enhances the expression of several genes involved in Suc synthesis and transport in developing seeds and the silique wall. Consistently, the accumulation of Suc and Fru is increased in developing seeds of the transgenic rapeseed, suggesting the increased carbon flux to fatty acid biosynthesis. These results demonstrate that BnLEC1 and BnL1L are reliable targets for genetic improvement of rapeseed in seed oil production.