Wilfred A. Keller

Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses

In addition to an essential role in plant development, brassinosteroids (BRs) appear to have the ability to protect plants against various environmental stresses. However, studies confirming the ability of BRs to modulate plant responses to different environmental stresses are lacking. Earlier we had demonstrated that treatment with 24-epibrassinolide (EBR), a BR, increases the basic thermotolerance of Brassica napus and tomato seedlings [Plant Mol Biol 40:333–342, 1999]. Here we demonstrate that EBR treatment enhances seedling tolerance to drought and cold stresses in both Arabidopsis thaliana and B. napus, and helps to overcome a salt-stress-induced inhibition of seed germination. The ability of EBR to confer tolerance in plants to a variety of stresses was confirmed through analysis of expression of a subset of drought and cold stress marker genes. Transcriptional changes in these genes were more apparent in EBR-treated A. thaliana, in particular during earlier time points of stress. To see if BR is essential for the heat stress (HS) response, we made use of BR-deficient mutants. Both det2-1 and dwf4 mutants still expressed heat shock proteins (hsps) to high levels during HS, indicating that although BR augments thermotolerance in plants, it is not necessary for hsp expression during HS.

Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene.

A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs.

Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

Storage-protein regulation and lipid accumulation in microspore embryos of Brassica napus L.

Embryos derived in vitro from isolated microspores of Brassica napus L. were compared with their zygotic counterparts. Parameters investigated included storage-protein accumulation and gene expression, fattyacid composition, storage-lipid biosynthesis, and the appearance of oil-body proteins. The microspore embryos accumulate storage-protein and show increases in levels of their transcripts during the torpedo stage. These embryos were sensitive to abscisic acid (ABA) with respect to accumulation of storage-protein mRNA and oil-body proteins. Post-transcriptional regulation of cruciferin accumulation is indicated by a disparity between ABA-enhanced transcript accumulation and a less marked effect at the level of protein accumulation. To investigate storage-lipid profiles, two cultivars of Brassica napus, Reston and Topas, were used. The former accumulates major quantities of C20 (11.2%) and C22 (39.9%) fatty acids in its seeds, the latter predominantly C18 fatty acids. The higher-molecular-weight fatty acids (>C18) normally occur only in seeds and were used as biochemical markers for seed-specific metabolism in microspore embryos. Microspore embryos from Reston were found to accumulate C20 (10.6%) and C22 (31.2%) fatty acids after 35 d in culture at levels and proportions comparable to those found in seeds. Similarly, microspore embryos of Topas had a fatty-acid profile similar to that of mature Topas seed. Activities of enzymes involved in the accumulation of storage lipids (erucoyl-CoA synthetase [EC 6.2.1.3], erucoyl-CoA thioesterase [EC 3.1.2.2] and erucoyl-CoA acyltransferase [EC 2.3.1.15 or EC 2.3.1.20]) were detected in torpedostage microspore embryos. Their specific activities were higher than have been reported to date for analogous preparations from zygotic embryos of B. napus. The similarities in storage-lipid and protein composition of these embryos to their zygotic counterparts, along with their sensitivity to ABA, indicate that microspore embryos might be exploited to facilitate studies of biochemistry and gene regulation in oilseeds.

The Arabidopsis BRAHMA chromatin-remodeling ATPase is involved in repression of seed maturation genes in leaves.

Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue.

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata

Abstract Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%).

Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed.

High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts. Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries. Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B. napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%). In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B. napus cultivars, we have focused on two targets using a transgenic approach. We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B. napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B. napus cv. Hero transgenic progeny in the field. Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.

Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line.

Isolated microspore culture techniques are being widely used in Brassica breeding programs to generate haploid and doubled haploid plants. A number of factors influence regeneration response in vitro including genotype. In order to assess the effect of genotype on microspore embryogenesis in B. rapa L. var. oleifera, 17 cultivars and breeding lines were evaluated. Embryos developed from all but one genotype when using NLN medium with 17% sucrose, followed by a reduction in sucrose concentration to 10%, 48 h later. The number of embryos /100 buds differed between genotypes, ranging from 0 to 70. Further studies indicated that sucrose concentration and incubation time influenced embryogenesis. Selection studies carried out with an Agriculture and Agri-Food Canada breeding line have resulted in the identification of a highly embryogenic B. rapa line. This line produced thousands of microspore-derived embryos /100 buds and will be useful in mutant selection and gene transfer as well as biochemical and developmental studies.

Synergistic repression of the embryonic programme by SET DOMAIN GROUP 8 and EMBRYONIC FLOWER 2 in Arabidopsis seedlings

The seed maturation programme occurs only during the late phase of embryo development, and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this programme in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants were made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Analysis of histone methylation status at the chromatin sites of a number of maturation loci revealed a synergistic effect of emf2 and sdg8 on the deposition of the active histone mark which is the trimethylation of Lys4 on histone 3 (H3K4me3). This is consistent with high expression of these genes and formation of somatic embryos in the emf2 sdg8 double mutants. Interestingly, a double mutant of sdg8 and vrn2 (vernalization2), a paralogue of EMF2, grew and developed normally to maturity. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis.

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

SummaryCell 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.