Fawzy Georges

PROMOTERS FROM KIN1 AND COR6.6, TWO HOMOLOGOUS ARABIDOPSIS THALIANA GENES : TRANSCRIPTIONAL REGULATION AND GENE EXPRESSION INDUCED BY LOW TEMPERATURE, ABA, OSMOTICUM AND DEHYDRATION

The Arabidopsis thaliana genes kin1 and cor6.6 belong to the same family and were expressed at higher levels following low temperature and ABA treatments. In an attempt to elucidate the mechanism of gene regulation by low temperature, the relationship between low-temperature- and abscisic acid (ABA)-induced gene expression and possible differential expression of the two genes, we have cloned a 5.3 kb genomic fragment harboring kin1 and cor6.6 and their respective 5′ sequences. The putative promoters of both genes were fused to the β-glucuronidase (GUS) coding sequence and GUS expression was analysed in transgenic tobacco and Arabidopsis plants. The cor6.6 promoter produced a higher basal level of expression than the kin1 promoter in transgenic tobacco. Enzyme assays of inducible GUS activity in transgenic Arabidopsis and tobacco plants showed that GUS activity directed by both kin1 and cor6.6 promoters was significantly induced by ABA, dehydration and osmoticum, but not by low temperature. Northern analysis revealed, in contrast, that GUS mRNA was significantly induced in these transgenic plants by low temperature. Further analysis showed that, at low temperature, GUS protein synthesis from the induced GUS mRNA was inhibited. Together these results reveal induction of kin1 and cor6.6 transcription by low temperature, exogenous ABA and dehydration. However, low-temperature expression is dramatically reduced at the translational level.

Winter Flounder Antifreeze Protein Improves the Cold Hardiness of Plant Tissues

Summary Exposure of plant tissues to the winter flounder antifreeze protein (AFP) has revealed three novel properties by which plant cold hardiness may be improved. Firstly, vacuum infiltration of the protein into leaves of potato, canola ( Brassica napus ) and Arabidopsis thaliana resulted in a significant depression of the spontaneous freezing temperature relative to water infiltrated controls. In the case of canola, the freezing temperature was decreased by an average of 1.8 °C. These results demonstrated the ability of the AFP to function as an anti-nucleator in plant tissues. Secondly, exposure of suspension cultured cells of bromegrass to the antifreeze protein resulted in a reduction in the amount of freezable water frozen at any given temperature. This showed that the protein could act as a cryoprotectant. Thirdly, the antifreeze protein decreased the rate of ice crystal formation. These results demonstrate the feasibility of improving the cold hardiness of plants by introduction of the antifreeze protein gene.

Over‐expression of Brassica napus phosphatidylinositol‐phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositol-specific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca(2+) modulator which induces distinctive oscillating bursts of cytosolic Ca(2+), resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stress-related genes such as glutathione S-transferase, hormone-regulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)—comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus

The cloning and identification of full-length cDNA fragments coding for the Brassica napus phosphatidylinositol-specific phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase (BnPtdIns S1) is described. In addition, two complementary fragments (120 nucleotides long) corresponding to Arabidopsis PtdIns 4-kinase (PtdIns 4-K) and PtdIns-4-phosphate 5-kinase (PtdIns4P 5-K) sequences were chemically synthesized. These, as well as the cDNA clones, were used as probes to study the corresponding steady state mRNA levels in different tissues and developmental stages of B. napus, as well as in response to different environmental conditions. Transcripts corresponding to BnPLC2, BnPtdIns S1, BnVPS34 and PtdIns 4-K were found constitutively expressed at different levels in most tissues, with young leaves, siliques, and developing seeds showing the lowest levels. No detectable PtdIns4P 5-K transcripts were found in buds or flowers. Up-regulation of BnPLC2 was seen in response to low temperature stress, which was notably accompanied by a parallel down-regulation of BnPtdIns S1, while BnVPS34 and PtdIns 4-K remained at control levels. A moderate increase in PtdIns4P 5-K levels was noted. In high salinity conditions BnPtdIns S1, BnVPS34 and BnPLC2 transcripts had similar responses but at different levels, with no major changes detected for PtdIns 4-K or PtdIns4P 5-K. Significantly, all five transcripts increased under drought stress conditions and all stressed plants clearly showed relatively higher levels of total inositol trisphosphate.

Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells.

A synthetic gene coding for the winter flounder antifreeze protein (AFP) has been constructed. A new strategy for the synthesis has been employed such that one strand of the duplex was chemically synthesized and the other was produced enzymatically by chain extension. The chemically synthesized blocks were constructed so that the second strand was self-priming. The resulting DNA fragment was incorporated into the vector, pGCS1, which contained a translational fusion of the sequence encoding AFP and the N terminus of cat (encoding chloramphenicol acetyltransferase, CAT), under the control of the cauliflower mosaic virus 35S promoter. This plasmid was introduced into protoplasts of corn (var. Black Mexican Sweet) by electroporation. Production of the fusion peptide was monitored by CAT assay and Western blotting with antisera to AFP and CAT.

Development of a Brassica seed cDNA microarray.

Brassica species represent several important crops including canola (Brassica napus). Understanding of genetic elements that contribute to seed-associated functions will impact future improvements in the canola crop. Brassica species share a very close taxonomic and molecular relationship with Arabidopsis thaliana. However, there are several subtle but distinct seed-associated agronomic characteristics that differ among the oil seed crop species. To address these, we have generated 67,535 ESTs predominately from Brassica seeds, analyzed these sequences, and identified 10,642 unigenes for the preparation of a targeted seed cDNA array. A set of 10,642 PCR primer pairs was designed and corresponding amplicons were produced for spotting, along with relevant controls. Critical quality control tests produced satisfactory results for use of this microarray in biological experiments. The microarray was also tested with specific RNA targets from embryos, germinating seeds, and leaf tissues. The hybridizations, signal intensities, and overall quality of these slides were consistent and reproducible. Additionally, there are 429 ESTs represented on the array that show no homology with any A. thaliana annotated gene or any gene in the Brassica genome databases or other plant databases; however, all of these probes hybridized to B. napus transcripts, indicating that the array also will be useful in defining expression patterns for genes so far unique to Brassica species.

Esterase Isozymes as Markers of Somatic Embryogenesis in Cultured Carrot Cells

Summary Two major groups of esterase isozymes were identified in extracts of carrot ( Daucus carota L. var. Royal Chantaney) cells cultured in vitro using polyacrylamide gel electrophoresis. In the slow moving group, an isozyme marked «y» was absent and another marked «x» was present in the embryogenic cell line (Ca-7) as compared to the non-embryogenic cell line (Ca-4). The pattern of a slow moving esterase was also maintained in different stages of embryo development. The substrate specificity for esterase isozymes suggested that the slow moving group to which isozyme «x» and «y» belongs is the aryl esterase (3.1.1.2). The potential use of these isozymes as markers of competence of cells to undergo somatic embryogenesis is discussed.

A 5.3-kilobase genomic fragment from Arabidopsis thaliana containing kin1 and cor6.6.

Cloning: acclimation have been identified in recent years (Lee and Chen, 1993). The gene k in l from Arabidopsis thaliana is particularly interesting because it codes for a 6.5-kD polypeptide that bears some compositional similanty to the fish Alarich antifreeze proteins (Kurkela and Franck, 1990). This similarity as well as its increased expression during cold acclimation has led to the speculation that the product of kinl might be involved in enhancing plant cold tolerance. A homologous gene was more recently reported by Gilmour et al. (1992) as cor6.6 and by Kurkela and Borg-Franck (1992) as kin2. We are interested in how the expression of stress-related genes is regulated. Therefore, we set out to clone sequences containing the promoters of k in l and cor6.6/kin2. Using nonradioactive approaches, we identified genomic clones, and a 5.3-kb region was sequenced that contained both proteincoding sequences and extensive sequences 5’ and 3’ to the transcribed regions (Table I). In the 5311-bp genomic fragment, almost 4 kb consists of two homologous sequences (direct repeats) (nucleotides 6722628 and 2665-4597) with a short spacer (nucleotides 26292664). From previous reports, k in l (Kurkela and Franck, 1990) can be located in the region 1946 to 2758, and cor6.6 (Gilmour et al., 1992) lies between 4006 and 4739. There are three mismatches between the sequence presented here and that reported previously (Kurkela and Franck, 1990), which has an A (instead of T) at 2065 and an A (instead of C) at 2562 and misses 2 As at 2175 and 2176. The same ecotype (Columbia) was used for both studies. The sequence coinciding with cor6.6 is identical. A sequence comparison of the transcribed regions was made previously by Gilmour et al. (1992) for cor6 6. From the present sequence it is interesting to note that the longest transcribed k in l (Kurkela and Franck, 1990) ends at 2743, which is 79 nucleotides into the 5’ of the second homologous sequence. This region thus has homology to the upper 5’ region of k in l (nucleotides 672-751). The 1.4-kb upstream sequences of both genes are mostly homologous. The regions proximal to the transcriptional start

Perturbing the metabolic dynamics of myo-inositol in developing Brassica napus seeds through in vivo methylation impacts its utilization as phytate precursor and affects downstream metabolic pathways

Backgroundmyo-Inositol (Ins) metabolism during early stages of seed development plays an important role in determining the distributional relationships of some seed storage components such as the antinutritional factors, sucrose galactosides (also known as raffinose oligosaccharides) and phytic acid (PhA) (myo-inositol 1,2,3,4,5,6-hexakisphosphate). The former is a group of oligosaccharides, which plays a role in desiccation at seed maturation. They are not easily digested by monogastric animals, hence their flatulence-causing properties. Phytic acid is highly negatively charged, which chelates positive ions of essential minerals and decreases their bioavailability. It is also a major cause of phosphate-related water pollution. Our aim was to investigate the influence of competitive diversion of Ins as common substrate on the biosynthesis of phytate and sucrose galactosides.ResultsWe have studied the initial metabolic patterns of Ins in developing seeds of Brassica napus and determined that early stages of seed development are marked by rapid deployment of Ins into a variety of pathways, dominated by interconversion of polar (Ins phosphates) and non-polar (phospholipids) species. In a time course experiment at early stages of seed development, we show Ins to be a highly significant constituent of the endosperm and seed coat, but with no phytate biosynthesis occurring in either tissue. Phytate accumulation appears to be confined mainly within the embryo throughout seed development and maturation. In our approach, the gene for myo-inositol methyltransferase (IMT), isolated from Mesembryanthemum crystallinum (ice plant), was transferred to B. napus under the control of the seed-specific promoters, napin and phaseolin. Introduction of this new metabolic step during seed development prompted Ins conversion to the corresponding monomethyl ether, ononitol, and affected phytate accumulation. We were able to produce homozygous transgenic lines with 19% - 35% average phytate reduction. Additionally, changes in the raffinose content and related sugars occurred along with enhanced sucrose levels. Germination rates, viability and other seed parameters were unaffected by the IMT transgene over-expression.ConclusionsCompetitive methylation of Ins during seed development reduces seed antinutritional components and enhances its nutritional characteristics while maintaining adequate phosphate reserves. Such approach should potentially raise the canola market value and likely, that of other crops.

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.