Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.     

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.     

The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds

Mature seeds of Arabidopsis thaliana and Brassica napus contain a complex mixture of aliphatic monomers derived from the non-extractable lipid polyesters deposited by various seed tissues. Methods of polyester depolymerization of solvent-extracted seeds and analysis of aliphatic monomers were compared. Sodium methoxide-catalyzed depolymerization, followed by GC analysis of the acetylated monomers, was developed for routine quantitative analysis suitable for 0.5g seed samples. In Arabidopsis seeds, the major C16 and C18 monomers identified included omega-hydroxy fatty acids and alpha,omega-dicarboxylic acids derived from palmitate, oleate and linoleate, and 9,10,18-trihydroxyoctadecenoic acid. Among monomers which can collectively be considered likely to be derived from suberin, docosan-1-ol, docosane-1,22-diol, 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid, tetracosane-1,24-dioic acid and ferulic acid were the major species. Compared to Arabidopsis, Brassica seeds showed a roughly similar proportion of monomer classes, with the exception that alkan-1ols were 3-fold higher. Also, there were much less C24 aliphatic species and significant amounts of C14-C16 alkan-1ols, including iso- and anteiso-methyl branched compounds. Dissection and analysis of mature Brassica seeds showed that the trihydroxy C18:1 fatty acid was found mainly in the embryo, while ferulate, fatty alcohols and C22 and C24 species were specific to the seed coat plus endosperm.     

Arabidopsis thaliana chromosome III restores fertility in a cytoplasmic male-sterile Brassica napus line with A. thaliana mitochondrial DNA

Somatic Brassica napus (+) Arabidopsis thaliana hybrids with a cytoplasmic male sterility (CMS)-inducing cytoplasm were screened for fertility-restored plants. One line was selected and recurrently backcrossed with the maintainer line, B. napus, resulting in fertile/sterile segregating populations. Restriction fragment length polymorphism mapping showed the co-segregation of A. thaliana chromosome (chr) III markers with the fertility trait. As it was not possible to stabilise the fertility trait via selfings, a dihaploidisation strategy was assessed. Ninety haploid plants were regenerated and analysed with numerous simple sequence length polymorphism (SSLP) markers. Markers covering both arms of A. thaliana chr III were present in two plants, whereas no A. thaliana DNA could be detected in the other plants. Following colchicine-induced chromosome doubling only these two plants with A. thaliana DNA produced fertile offspring. In one of the two lines, however, the A. thaliana-specific DNA markers and fertility were lost in subsequent generations. The other line remained fertile after repeated selfings. Using genomic in situ hybridisation (GISH) we were able to demonstrate that this latter line possessed a disomic addition of the A. thaliana chromosome. The restored line was comparable to the maintainer line with respect to flower morphology, but the petals and stamens were slightly reduced in size. The homeotic conversion of stamens to pistil-like structures, which is typical for the CMS line, was reversed, and stamens with a normal appearance with viable pollen appeared. Flowering time was as in the CMS line—in both lines it was delayed in comparison to the maintainer line. The introgressed chromosome also contributes to several pleiotropic effects, such as reduced leaf crinkling and shorter stems. The ability to restore fertility through the introgression of nuclear genes from the main cytoplasmic donor species indicates that the CMS trait in this system mainly is due to B. napus/A. thaliana alloplasmic incompatibility and not mitochondrial DNA rearrangements. Further exploitation of the material is discussed.Communicated by C. Möllers     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

Production and characterization of asymmetric somatic hybrids between Arabidopsis thaliana and Brassica napus

Summary Cell suspension-derived protoplasts of a chlorsulfuron-resistant (GH50) strain of Arabidopsis thaliana cv Columbia were X-irradiated at 60 or 90 krad, to facilitate the elimination of GH50 donor chromosomes in fusion products. Irradiated GH50 protoplasts were fused, with polyethylene glycol, to protoplasts derived from stem epidermal strips of Brassica napus cv Westar. Chlorsulfuron-resistant colonies were selected in vitro and then transferred to shoot and root regeneration medium. Seventeen hybrid lines were regenerated in vitro, and eight were successfully established in the greenhouse, where they flowered. These eight asymmetric hybrids were intermediate in vegetative morphology between Arabidopsis and Brassica. The flowers from these hybrids were male-sterile with abnormal petal and pistil structures. Zymograms for phosphoglucomutase, esterase, and peroxidase showed the presence of all parental isozymes in each of the hybrids tested. Nuclear hybridity was also confirmed for the ribosomal RNA genes using a wheat rDNA probe; however, the chloroplast genome in each of the hybrids was derived solely from the Brassica parent. All selected somatic hybrids were capable of rooting at levels of chlorsulfuron which were inhibitory to unfused Brassica plantlets. The degree of herbicide resistance in the hybrid shoots is presently being evaluated.Contribution No. 1428, Plant Research Centre, Agriculture Canada     

Dehiscence-related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus

Pod dehiscence in Arabidopsis thaliana is accompanied by an increase in the expression of a polygalacturonase (PG). The gene encoding this mRNA has been characterized and shown to have extensive homology to a similar PG gene from Brassica napus . The A. thaliana PG promoter was fused to β -glucuronidase (GUS) and the expression of this reporter gene analysed in transgenic B. napus plants. The GUS activity was detected throughout the dehiscence zone of pods from 35 d after anthesis and expression was restricted to those cells that undergo cell separation. Expression was also detectable at the junction between the seed and the funicular tissue and in mature anthers of flowers. Transgenic plants containing the PG promoter fused to barnase were sterile as a consequence of the anthers failing to undergo dehiscence. Fertilization of PG-barnase plants resulted in the development of pods that exhibited a reduced capacity to shatter. The role of PG during cell separation processes in plants is discussed.     

Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana

Over 1000 genetically linked RFLP loci in Brassica napus were mapped to homologous positions in the Arabidopsis genome on the basis of sequence similarity. Blocks of genetically linked loci in B. napus frequently corresponded to physically linked markers in Arabidopsis. This comparative analysis allowed the identification of a minimum of 21 conserved genomic units within the Arabidopsis genome, which can be duplicated and rearranged to generate the present-day B. napus genome. The conserved regions extended over lengths as great as 50 cM in the B. napus genetic map, equivalent to approximately 9 Mb of contiguous sequence in the Arabidopsis genome. There was also evidence for conservation of chromosome landmarks, particularly centromeric regions, between the two species. The observed segmental structure of the Brassica genome strongly suggests that the extant Brassica diploid species evolved from a hexaploid ancestor. The comparative map assists in exploiting the Arabidopsis genomic sequence for marker and candidate gene identification within the larger, intractable genomes of the Brassica polyploids.     

Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana

Gibberellins (GAs) are endogenous hormones that play an important role in regulating plant stature by increasing cell division and elongation in stem internodes. The GA2-oxidase gene from Arabidopsis thaliana (AtGA2ox8) was introduced into Brassica napus L. by Agrobacterium-mediated floral-dip transformation with the aim of decreasing the amount of bioactive GA and hence reducing plant stature. As anticipated, the transgenic plants exhibited dwarf phenotype. Compared with the wild type, the transgenic plants had increased primary branches (by 14.1?C15.3%) and siliques (by 10.8?C15.2%), which resulted in a significant increase in the seed yield (by 9.6?C12.4%). Moreover, the contents of anthocyanin in leaves of 60-day-old transgenic plants was about 9.4-fold higher in winter and about 6.8-fold higher in summer than the wild type. These excellent agronomic traits of the transgenic plants could not only improve the lodging resistance and seed yields, but also protect them against stress. Therefore, the over-expression of AtGA2ox8 might be used to produce dwarf varieties and increase seed yield in Brassica napus L.     

From Arabidopsis thaliana to Brassica napus: development of amplified consensus genetic markers (ACGM) for construction of a gene map

The evolution of genomes can be studied by comparing maps of homologous genes which show changes in nucleic acid sequences and chromosome rearrangements. In this study, we developed a set of 32 amplified consensus gene markers (ACGMs) that amplified gene sequences from Arabidopsis thaliana and Brassica napus. Our methodology, based on PCR, facilitated the rapid sequencing of homologous genes from various species of the same phylogenetic family and the detection of intragenic polymorphism. We found that such polymorphism principally concerned intron sequences and we used it to attribute a Brassica oleracea or Brassica rapa origin to the B. napus sequences and to map 43 rapeseed genes. We confirm that the genetic position of homologous genes varied between B. napus and A. thaliana. ACGMs are a useful tool for genome evolution studies and for the further development of single nucleotide polymorphism suitable for use in genetic mapping and genetic diversity analyses.     

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana

Background

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.

Results

Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.

Conclusion

This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-3) contains supplementary material, which is available to authorized users.     

Isolation of circadian-associated genes in Brassica rapa by comparative genomics with Arabidopsis thaliana

Elucidation of the roles of circadian associated factors requires a better understanding of the molecular mechanisms of circadian rhythms, control of flowering time through photoperiodic pathways, and photosensory signal transduction. In Arabidopsis, the APRR1 quintet, APRRs 1, 3, 5, 7, and 9, are known as central oscillator genes. Other plants may share the molecular mechanism underlying the circadian rhythm. To identify and characterize these circadian response genes in Brassica crops whose genome was triplicated after divergence from Arabidopsis, we identified B. rapa BAC clones containing these genes by BLAST analysis of B. rapa BAC end sequences against the five corresponding Arabidopsis regions. Subsequent fingerprinting, Southern hybridization, and PCR allowed identification of five BAC clones, one for each of the five circadian-related genes. By draft shotgun sequencing of the BAC clones, we identified the complete gene sequences and cloned the five expressed B. rapa circadian-associated gene members, BrPRRs 1, 3, 5, 7, and 9. Phylogenetic analysis revealed that each BrPRR was orthologous to the corresponding APRR at the sequence level. Northern hybridization revealed that the five genes were transcribed at distinct points in the 24 hour period, and Southern hybridization revealed that they are present in 2, 1, 2, 2, and 1 copies, respectively in the B. rapa genome, which was triplicated and then diploidized during the last 15 million years.     

Production of asymmetric hybrids between Arabidopsis thaliana and Brassica napus utilizing an efficient protoplast culture system

Application of the protoplast culture method developed for Brassica protoplasts to protoplasts of Arabidopsis thaliana has increased the opportunities for interspecific hybridizations involving Arabidopsis. A more-efficient and much-simpler method was established compared to the earlier-reported protocol developed for A. thaliana protoplasts in which alginate beads were utilized. Mesophyll protoplasts of A. thaliana (ecotypes 'Landsberg erecta' and 'Wassilewskija') were cultured in the modified 8p liquid medium, which had been developed for Brassica protoplasts. For comparison, protoplasts were cultured in sodium alginate beads supplied with B5 medium according to the protocol for A. thaliana. The protoplasts divided with high frequencies in the 8p medium, and calli proliferated more rapidly than in the sodium alginate beads. High frequencies of shoot differentiation and regeneration were observed in calli of both ecotypes, from about 30% in the ecotype 'Wassilewskija' to about 60% for 'Landsberg erecta'. The more-rapidly the calli developed, the higher the regeneration frequencies were. Asymmetric hybrids between A. thaliana and Brassica napus were obtained by treating the protoplasts of A. thaliana with iodoacetamide (IOA) and B. napus protoplasts with UV-irradiation before fusion with polyethylene glycol (PEG). By using the culture procedure developed for Brassica protoplasts, calli developed and plants were regenerated. Although most of the plants regenerated after cell fusion were A. thaliana-like and were judged to be escapes from IOA treatment, more than ten plants showed hybrid features of both morphological and molecular characters. Among the hybrids that have flowered so far, both male-fertile and male-sterile plants have been obtained. Back-crossings to A. thaliana are now in progress as is morphological and molecular characterization of the plants.     

Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis

Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.     

Linkage mapping of genes controlling resistance to white rust (Albugo candida) in Brassica rapa (syn. campestris) and comparative mapping to Brassica napus and Arabidopsis thaliana.

Genes for resistance to white rust (Albugo candida) in oilseed Brassica rapa were mapped using a recombinant inbred (RI) population and a genetic linkage map consisting of 144 restriction fragment length polymorphism (RFLP) markers and 3 phenotypic markers. Young seedlings were evaluated by inoculating cotyledons with A. candida race 2 (AC2) and race 7 (AC7) and scoring the interaction phenotype (IP) on a 0-9 scale. The IP of each line was nearly identical for the two races and the population showed bimodal distributions, suggesting that a single major gene (or tightly linked genes) controlled resistance to the two races. The IP scores were converted to categorical resistant and susceptible scores, and these data were used to map a single Mendelian gene controlling resistance to both races on linkage group 4 where resistance to race 2 had been mapped previously. A quantitative trait loci (QTL) mapping approach using the IP scores detected the same major resistance locus for both races, plus a second minor QTL effect for AC2 on linkage group 2. These results indicate that either a dominant allele at a single locus (Acal) or two tightly linked loci control seedling resistance to both races of white rust in the biennial turnip rape cultivar Per. The map positions of white rust resistance genes in B. rapa and Brassica napus were compared and the results indicate where additional loci that have not been mapped may be located. Alignment of these maps to the physical map of the Arabidopsis genome identified regions to target for comparative fine mapping using this model organism.     

Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

Metallochaperone-like genes in Arabidopsis thaliana

A complete inventory of metallochaperone-like proteins containing a predicted HMA domain in Arabidopsis revealed a large family of 67 proteins. 45 proteins, the HIPPs, have a predicted isoprenylation site while 22 proteins, the HPPs, do not. Sequence comparisons divided the proteins into seven major clusters (I-VII). Cluster IV is notable for the presence of a conserved Asp residue before the CysXXCys, metal binding motif, analogous to the Zn binding motif in E. coli ZntA. HIPP20, HIPP21, HIPP22, HIPP26 and HIPP27 in Cluster IV were studied in more detail. All but HIPP21 could rescue the Cd-sensitive, ycf1 yeast mutant but failed to rescue the growth of zrt1zrt2, zrc1cot1 and atx1 mutants. In Arabidopsis, single and double mutants did not show a phenotype but the hipp20/21/22 triple mutant was more sensitive to Cd and accumulated less Cd than the wild-type suggesting the HIPPs can have a role in Cd-detoxification, possibly by binding Cd. Promoter-GUS reporter expression studies indicated variable expression of these HIPPs. For example, in roots, HIPP22 and HIPP26 are only expressed in lateral root tips while HIPP20 and HIPP25 show strong expression in the root vasculature.     

Overexpression of the nuclear protein gene AtDUF4 increases organ size in Arabidopsis thaliana and Brassica napus

正Organ size is an important trait of many crops that is influenced by internal and environmental signals and controlled by a combination of factors during organogenesis(Krizek,2009).The final size of plant organs is determined by two successive but overlapping processes:cell division,which increases cell number,and cell expansion,which determines final cell size(Anastasiou and Lenhard,