Jiro Hattori

Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth

The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp→Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

SummaryThe Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

SummaryThe acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3′ end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.

A conserved BURP domain defines a novel group of plant proteins with unusual primary structures

Abstract We have identified a new class of plant proteins containing a common C-terminal region, which we have termed the BURP domain. These proteins are defined not only by the BURP domain, but also by the overall similarity in their modular construction. The BURP domain proteins consist of either three or four modules: (i) an N-terminal hydrophobic domain – a presumptive transit peptide, joined to (ii) a short conserved segment or other short segment, (iii) an optional segment consisting of repeated units which is unique to each member, and (iv) the C-terminal BURP domain. These individual modules appear to be combined to form two main classes of BURP domain proteins. The BURP domain proteins, despite the similarities in their primary structural features, show no obvious similarities in the tissues or conditions under which they are expressed. The presence of the conserved BURP domain in diverse plant proteins suggests an important and fundamental functional role for this domain.

In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

SummaryGenes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Assessment of microinjection for introducing DNA into uninuclear microspores of rapeseed

Approximately 2,000 embryogenic uninuclear microspores of rapeseed (Brassica napus) cv. Topas were intranuclearly injected with a chimaeric β-glucuronidase (Escherichia coli Uid A) gene. Stable integration had not occurred among 55 plants that were regenerated. Coinjection of the dye Lucifer Yellow and detection of injected DNA by the polymerase chain reaction revealed high frequencies of transfer. However, the amount of DNA injected was less than 20 copies, which may have been insufficient for stable transformation of microspores.

Bnm1, a Brassica pollen-specific gene

AbstractcDNA and genomic clones of a new pollen-specific gene, Bnm1, have been isolated from Brassica napus cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen. Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv. Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family.

Haplotag: Software for Haplotype-Based Genotyping-by-Sequencing Analysis

Genotyping-by-sequencing (GBS), and related methods, are based on high-throughput short-read sequencing of genomic complexity reductions followed by discovery of single nucleotide polymorphisms (SNPs) within sequence tags. This provides a powerful and economical approach to whole-genome genotyping, facilitating applications in genomics, diversity analysis, and molecular breeding. However, due to the complexity of analyzing large data sets, applications of GBS may require substantial time, expertise, and computational resources. Haplotag, the novel GBS software described here, is freely available, and operates with minimal user-investment on widely available computer platforms. Haplotag is unique in fulfilling the following set of criteria: (1) operates without a reference genome; (2) can be used in a polyploid species; (3) provides a discovery mode, and a production mode; (4) discovers polymorphisms based on a model of tag-level haplotypes within sequenced tags; (5) reports SNPs as well as haplotype-based genotypes; and (6) provides an intuitive visual “passport” for each inferred locus. Haplotag is optimized for use in a self-pollinating plant species.