Faouzi Bekkaoui

Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence

Abstract A synthetic transcribed, untranslated leader sequence from alfalfa mosaic virus RNA4 (AMV leader) has been assessed for its in vivo properties as a cis -active ‘translational activator’ in transient expression assays in protoplasts of Nicotiana tabacum and Picea glauca , as well as in stable expression in transformed Nicotiana tabacum . Levels of GUS enzyme activity produced by chimeric genes with or without the AMV leader sequence, in combination with either a CaMV 35S promoter or a duplicated-enhancer CaMV 35S promoter construct were assessed. In transient assay systems, the presence of a synthetic 40-base leader sequence lead to a 20-fold elevation in GUS activity when the constructs contained a native cauliflower mosaic virus (CaMV) 35S promoter, whereas a 4-fold elevated expression lebel was observed in constructs containing a duplicated-enhancer 35S promoter. Furthermore, elevated expression in chimeric constructs was influenced by the sequence context for translation initiation of the marker gene. In transgenic tobacco plants the mean values for steady-state expression of GUS-containing 35S/AMV constructs were elevated about 8-fold relative to plasmids containing the native 35S promoter alone. A quantitative PCR approach was used to assess relative transcript levels in plants expressing GUS from AMV-containing chimeric constructs. The results showed that elevated expression attributable to the AMV leader sequence was independent of abundance of the corresponding AMV- gus transcript, suggesting a post-transcriptional mechanism of action in vivo. Further, we describe the construction of general-purpose constitutive high expression plant promoter cassettes which incorporate the AMV translational enhancer sequence, as well a duplicated-enhancer 35S promoter in an optimized translational context.

Effects of abscisic acid and analogues on the maturation of white spruce (Picea glauca) somatic embryos

Abstract Somatic embryo-competent cultures of white spruce, Picea glauca (Moench) Voss, were grown on (±)-abscisic acid (ABA) and three analogues known to be biotransformed by intact plants into ABA: (±)-methyl abscisate (MeABA); (±)-ethyl-(2E,4E) and -(2E,4Z)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methylpentadienoate (epoxyester, EE); and (±)-(2E,4E) and -(2E,4Z)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methylpentadienocic acid (epoxyacid, EA). ABA between 8 and 12 μM most efficiently promoted embryo maturation and led to the maximum recovery (4%) of stage 4b embryos when used in the absence of auxin or cytokinin. MeABA in the absence of auxin and cytokinin also promoted embryo maturation, EE promoted only the early stages in maturation, and EA was unable to promote any maturation of somatic embryos. Phytohormone-free medium produced very similar responses to EA, though in rare cases stage 3 embryos were found. Neither ABA nor the analogues promoted embryo maturation when used in combination with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM N6-benzyladenine (BA).

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl−1 2,4-D and 1 mgl−1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss.

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl2.2H2O produced an average of 4.5 × 106 protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 105 protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae.

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.

Isolation, structure and expression of a cDNA for acetolactate synthase from Brassica napus

Acetolactate synthase (EC 4.1.3.18) catalyzes the first enzymatic step in a bifurcated pathway for the synthesis of the branched-chain amino acids. In plants, biochemical and genetic evidence have strongly implicated the involvement of a highly conserved 68 kDa subunit with ALS activity, and several structurally conserved genomic sequences encoding this ALS subunit have been isolated from Saccharomyces cerevisiae [2], Arabidopsis thaliana [ 3 ], Nicotiana tabacum [ 6, 8 ] and Brassica napus [12]. Mutational analyses of these genes have shown they are involved in phenotypic tolerance in vivo and in vitro to three specific classes of herbicides and inhibitors of ALS activity: the sulfonylureas, imidazolinones and triazolopyrimidinesulfonanilides [ 3, 5, 13 ]. On the basis of Southern analysis using an isolated Brassica ALS genomic sequence as probe, we have previously shown that, relative to A. thaliana (one copy) and N. tabacum (two copies), B. napus probably contains a more complex genomic complement of four or more genes [ 12]. We report here the isolation of a second member of the B. napus ALS gene family as a near full-length cDNA, and propose a nomenclature for this gene (als2). A cDNA library was constructed of Brassica napus L. (cv Westar) in 2gt 11 [ 10 ] using poly(A) ÷ m R N A isolated from 5-day seedlings, and was the generous gift of Dr S. Hemmingsen (Plant Biotechnology Institute, Saskatoon). The properties of the library have been previously described [7]. The library was screened for ALShomologous cDNAs using 32p-labelled probes derived from an existing B. napus ALS sequence [12]. Recombinant inserts were recovered from 2gt l l as Eco RI fragments, and subcloned into the phagemid vector pTZ18R (PharmarciaLKB). Approximately 2.0 × 10 5 recombinant plaques from a B. napus 5-day seedling cDNA library were screened through three levels of plaque purification, resulting in the identification fthree hybridization-positive plaques of which one was chosen for further study. The approximate 2.0 kb Eco RI fragment carried by the selected recombinant phage was subcloned into the phagemid pTZ18R in both orientations, yielding pBI-283 and pBI-284. The sequencing strategy and corresponding complete nucleotide sequence for the recombinant pBI-283 insert is shown in Figs 1A and 1B respectively. The recombinant insert

Heat and Drought Stresses in Crops and Approaches for Their Mitigation

Drought and heat are major abiotic stresses that reduce crop productivity and weaken global food security, especially given the current and growing impacts of climate change and increases in the occurrence and severity of both stress factors. Plants have developed dynamic responses at the morphological, physiological and biochemical levels allowing them to escape and/or adapt to unfavorable environmental conditions. Nevertheless, even the mildest heat and drought stress negatively affects crop yield. Further, several independent studies have shown that increased temperature and drought can reduce crop yields by as much as 50%. Response to stress is complex and involves several factors including signaling, transcription factors, hormones, and secondary metabolites. The reproductive phase of development, leading to the grain production is shown to be more sensitive to heat stress in several crops. Advances coming from biotechnology including progress in genomics and information technology may mitigate the detrimental effects of heat and drought through the use of agronomic management practices and the development of crop varieties with increased productivity under stress. This review presents recent progress in key areas relevant to plant drought and heat tolerance. Furthermore, an overview and implications of physiological, biochemical and genetic aspects in the context of heat and drought are presented. Potential strategies to improve crop productivity are discussed.

Differences in developmental morphology of protoplasts from two cell lines of white spruce ( Picea glauca )

Until recently cultured protoplasts of conifers remained recalcitrant (see Dunstan and Thorpe, 1987). The establishment of embryogenic suspension cultures of both white spruce (Picea glauca) (Hakman and Fowke, 1987) and (Pinus taeda) (Gupta and Durzan, 1986) has created a new source of protoplasts capable of somatic embryo regeneration (Attree et al, 1987; Gupta and Durzan; 1987). However, not all embryogenic suspension lines of white spruce have yielded protoplasts capable of regenerating embryos; protoplasts from one line gave rise to green callus but no somatic embryos (Bekkaoui et al, 1987). The ability to regenerate embryos from protoplasts of all cell lines may be crucial to creating improved forest trees using protoplast technology. In this communication a comparison is made between two white spruce embryogenic suspension lines.