[Show abstract][Hide abstract] ABSTRACT: Transcriptome profiling was conducted to detect genes whose expression is significantly changed in an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1) during early seedling development when mutant phenotypes could be clearly observed. A total of 2,040 differentially expressed genes were identified, representing approximately 6.7% of the 30,385 DNA oligonucleotide targets on the microarray. Among these differential expressed genes, many were mapped to pathways essential to plant growth and development including those of primary, secondary and hormone metabolisms. A significant proportion of up-regulated genes encoded transposable elements which were mapped to the centromeric and pericentromeric regions of the Arabidopsis chromosomes that were analyzed. A number of down-regulated genes were found to be involved in root hair formation, which might have contributed to the root hair defective phenotype of the mutant. Analysis of genes encoding transposable elements and those associating with root hair development indicated that these genes were highly co-expressed during seedling development. Despite SAHH1 deficiency, the expression of genes encoding methyltransferase remained largely unchanged in the sahh1 mutant. Bisulfite sequencing analysis of the transposable elements and the FWA gene revealed that their sequences in the mutant were deficient of 5-methylcytosines. Analysis of mutant genomic DNA using restriction endonucleases that were unable to cut methylated DNA suggested a genome-wide hypomethylation had occurred in the mutant. These results indicated that SAHH1 plays a critical role in methyl homeostasis, and its deficiency is a major contributing factor to the change of global gene expression, metabolic pathways and activation of transposable elements in the sahh1 mutant.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Experimental Botany 12/2011; 63(3):1391-404. · 5.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative
properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can
be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from
petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various
ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined
after 28days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic
acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced
only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1mg/l kinetin, where the regeneration
frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with
IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25mg/l induce callus and shoot formation
at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived
shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants
were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor
for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.
Hypericum perforatum petals–Plant regeneration
Plant Cell Tissue and Organ Culture 01/2011; 105(1):129-134. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Somatic embryogenesis in Crambe abyssinica, an important industrial oilseed species, was investigated. Cotyledon, hypocotyl and root explants from 8-day-old seedlings
of C. abyssinica cv. Prophet were cultured with levels of 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) ranging
from 2.2 to 39.0μM, combined with 6-benzyladenine (BA) to achieve an auxin:cytokinin ratio of 20:1, and callus formation
assessed. Callus formation frequency for cotyledon and hypocotyl explants was 100% for levels of 2,4-D from 4.5 to 33.9μM.
The response was similar with NAA levels of 13.0 to 39.0μM. Root explants were less responsive. When calluses were transferred
to a medium containing 0.56μM each of thidiazuron and BA with 1.0μM indole-3-butyric acid (IBA), somatic embryos were induced.
Moreover, embryos were induced from calluses grown on media containing either 11.3μM 2,4-D or 13.0μM NAA, or higher. On
a medium without plant growth regulators, embryos were induced but at a much lower frequency. For all three explants, 22.6μM
2,4-D and 26.0μM NAA was optimal for embryogenic callus induction. Hypocotyl-derived calluses were superior to cotyledon-
and root-derived calluses for embryo induction. The best embryo formation response was with medium containing 5.0–6.0% sucrose.
The highest average number of embryos per callus (36) was obtained from hypocotyl calluses from medium with 22.6μM 2,4-D.
Somatic embryos germinated best on half-strength B5 or MS medium with 3% sucrose, and plantlets were successfully established
under greenhouse conditions. The results indicate that high levels of auxins are required for the induction of embryogenic
calluses from explants of C. abyssinicia, while cytokinins are critical for somatic embryo formation.
-Seedling explants-Somatic embryogenesis
Plant Cell Tissue and Organ Culture 01/2011; 104(1):91-100. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Crop development and species diversity are important aspects of the emerging global bioeconomy, as is maximizing crop value through total crop utilization. We advocate development of Brassica carinata as a biorefinery and bioindustrial oils platform using traditional and molecular breeding techniques and tools. We review genetic studies and breeding efforts to develop elite B. carinata germplasm, work involving development of transformation and regeneration protocols, target gene isolation, and transgene expression. Genetic modification strategies using a B. carinata breeding line as a delivery platform for very long-chain fatty acid-enhanced/modified oils are presented as case studies. The target oil products are erucic acid (22:1 Δ13), docosadienoic acid (22:2 Δ5, Δ13) and nervonic acid (24:1 Δ15); in addition transgenic efforts to enhance B. carinata seed oil content are discussed. The overall advantages and current limitations to utilizing this crop are delineated. Other anticipated biobased products from a B. carinata platform may include, but are not limited to, the production of biolubricants, biofuels and biopolymers from the oil, biopesticides, antioxidants, as well as plant gums, and vegetable protein-based bioplastics and novel food and feed products. In summation, this collaborative B. carinata breeding/germplasm development/value-added molecular modification effort will not only contribute to the development of renewable feedstocks for the emerging Canadian bioeconomy (biorefinery/bioproducts), but also promises to generate positive economic and environmental benefits. Published in 2010 by John Wiley & Sons, Ltd.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α-linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The objective of this work was to enhance the quality and quantity of microspore-derived embryos of cruciferous species by
using polyethylene glycol (PEG) to replace sucrose in the culture medium. The main advantage in using PEG is that it produces
embryos that are morphologically more similar to zygotic embryos and have enhanced germination capabilities. When microspores
were cultured in full strength NLN medium supplemented with 25% (w/v) PEG, the addition of 3ml of full strength NLN with 13% (w/v) sucrose at 14d was beneficial for embryo quality and quantity. Experiments showed that this PEG system could be used for
a number of Brassica napus cultivars, as well as a number of other cruciferous species. PEG enhanced microspore embryogenesis in B. nigra, Crambe abyssinica, and Raphanus oleifera. Microspore-derived embryos were obtained from all cruciferous species evaluated (B. alboglabra, B. carinata, B. juncea, B. rapa, B. nigra, R. oleifera, Crambe abyssinica, Sinapis alba) using either sucrose or PEG as the osmoticum. Microspore embryogenesis was induced in B. napus in PEG-based cultures without a 32°C heat shock (i.e., 4, 15, 18, and 24°C). These temperature conditions were non-inductive
when sucrose was used as the osmoticum. Spontaneous chromosome doubling occurred in 64–92% of the regenerated plants when
PEG was used in the NLN culture medium, whereas in culture medium containing sucrose, the spontaneous doubling rate was 2–18%.
[Show abstract][Hide abstract] ABSTRACT: Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the alpha-subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively. Sequence analyses of the genomic DNA flanking the deletion sites have demonstrated that both deletions occurred at the genomic level. These two mutants are referred to as CRBDelta12 and CRCDelta13 with the delta sign indicating a deletion and the number indicating amino acids deleted. Alignment of these two mutant sequences with that of soybean A3B4 subunit, for which the crystal structure was determined recently, have revealed that the CRCDelta13 deletion is located in a hypervariable/disordered region, and will probably not affect the structure of the hexameric globulin. The CRBDelta12 deletion, however, is located in a binding region that is thought to be important for the hexamer formation. However, CRBDelta12 appears to accumulate normally as judged by its band intensity relative to the other SSP subunits on the protein gels. Thus it seems that the seed can, to a certain extent, tolerate some mutations in its storage proteins.
[Show abstract][Hide abstract] ABSTRACT: Experiments were conducted to determine the effects of brassinosteroids on microspore embryogenesis in Brassica species. Two compounds, 24-epibrassinolide (EBR) and brassinolide (BL), were evaluated. An increase in embryogenesis was
observed in all Brassica napus lines evaluated, including Topas 4079 and several recalcitrant cultivars: Garrison, Westar, and Allons. Microspore embryogenesis,
calculated as the number of embryos at 21 d of culture, was increased in the recalcitrant cultivars up to 12 times that of
the control. An increase in microspore embryogenesis was also observed for B. juncea when EBR or BL was added to the culture medium. In constrast, no significant increases in embryogenesis was observed for
several other Brassica species evaluated (i.e. B. carinata, B. nigra, and B. rapa). The addition of brassinosteroids to the induction media did not affect the subsequent conversion of the embryos to plantlets,
but did appear to influence chromosome doubling.
[Show abstract][Hide abstract] ABSTRACT: Erucic acid and its derivatives represent important industrial feedstock compounds, and there is an increasing demand for the production of high erucate oils in this regard. Our goal therefore, is to develop high erucic acid (HEA) Brassicaceae lines with increased proportions of erucic acid and very long-chain fatty acids (VLCFAs). We proposed that oleate availability may be a rate-limiting factor in the biosynthesis of erucic acid. We have tried to address this question by manipulating the expression of the endogenous FAD2 gene in B. carinata using co-supression and antisense approaches. Both methods resulted in transgenic lines exhibiting decreased proportions of polyunsaturated C18 fatty acids (18:2+18:3) and concomitant and significantly increased proportions of 18:1, 22:1 and total VLCFAs. Co-suppressed FAD2 B. carinata lines exhibited 3-18% decreases in 18:2, 22-49% decreases in 18:3 and significantly increased proportions of 18:1 (36-99%), 22:1 (12-27%) and VLCFAs (6-15%). Transgenic B. carinata lines developed using an antisense FAD2 approach exhibited decreased proportions of 18:2 and 18:3 (9-39% and 33-48%, respectively) and significantly increased proportions of 18:1 (54-130%), 22:1 (5-19%) and VLCFAs (6-21%). The possibility of using these approaches to produce prototype transgenic germplasm of the Brassicaceae accumulating seed oils with improved proportions of erucic and other VLCFAs is discussed.
[Show abstract][Hide abstract] ABSTRACT: Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus.
Protein Expression and Purification 07/2003; 29(2):193-201. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T2 transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.
[Show abstract][Hide abstract] ABSTRACT: A major goal of our research is to produce, by genetic manipulation, Brassica napus L. cultivars with higher levels of 22:1 in their seed oil than in present Canadian HEA cultivars developed through traditional breeding. Previously, we reported that transgenic expression of a mutated yeast sn-2 acyltransferase (SLC1-1) in industrial rapeseed cv. Hero resulted in increased seed oil content, increased proportions of erucic acid and increased average seed weight (Zou etal. 1997). Those results were reported only for plants grown in a controlled greenhouse setting. Here we report a summary of the results from two successive years of field trials with T4 and T5 generations of B.napus cv. Hero transformed with the SLC1-1 gene. These trials, conducted at Rosthern, Saskatchewan, in two very different growing seasons, show that the SLC1-1 transgenics clearly and consistently out-performed controls, with much increased oil and 22:1 contents, as well as yield, under varying field conditions.
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochemical Society Transactions 12/2000; 28(6):935-7. · 2.59 Impact Factor