Ahmed Bahieldin

Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene.

The ABA-responsive barley gene HVA1, a member of group 3 late embryogenesis abundant (LEA) protein genes, was introduced into spring wheat (Triticum aestivum L.) cv. Hi-Line using the biolistic bombardment method. High levels of expression of the HVA1 gene, regulated by the maize ubi1 promoter, were observed in leaves and roots of independent transgenic wheat plants and were inherited by offspring generations. T(3) progenies of four selected transgenic wheat lines were tested under greenhouse conditions for tolerance of soil water deficit. Potted plants were grown under moderate water deficit and well-watered conditions, respectively. Two homozygous and one heterozygous transgenic lines expressing the HVA1 gene had significantly (P<0.01) higher water use efficiency values, 0.66-0.68 g kg(-1), as compared to 0.57 and 0.53 g kg(-1), respectively, for the non-expressing transgenic and non-transgenic controls under moderate water deficit conditions. The two homozygous transgenic plant lines also had significantly greater total dry mass, root fresh and dry weights, and shoot dry weight compared to the two controls under soil water deficit conditions. Results of this study indicate that growth characteristics were improved in transgenic wheat plants constitutively expressing the barley HVA1 gene in response to soil water deficit.

Transposable elements domesticated and neofunctionalized by eukaryotic genomes.

Whole genome sequencing has provided a massive amount of information about the origin, diversity and genomic impact of repetitive DNA sequences (repDNA). Among the many classes of repDNA, prokaryotic transposable elements (TEs) replicate, move, amplify and accumulate in invaded genomes and thus represent the major force in restructuring host genes and genomes during evolution. Similar to retroviruses, autonomous TEs became part of the host genomes, and after their molecular domestication, they became functional genes (genomic fossils) in eukaryotic genomes. In this review, examples of the domestication events are discussed, some of which are known to be induced by biotic and abiotic stressors.

Whole mitochondrial and plastid genome SNP analysis of nine date palm cultivars reveals plastid heteroplasmy and close phylogenetic relationships among cultivars.

Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.

Expression of Hepatitis B surface Antigen (HBsAg) gene in transgenic banana ( Musa Sp.)

The Hepatitis B virus (HBV) infection is one of the most widespread viral infections of humans and causes acute and chronic hepatitis and hepatocellular carcinoma. The world wide problem of HBV infection has necessitated the development of an effective vaccine. Currently, immunization programs for large segments of the population in many areas of the developing world are very expensive. This limitation led us to attempt the expression of the recombinant Hepatitis B surface antigen (rHBsAg) in plants with the hope of developing a less expensive production system and a way to present the rHBsAg in edible plant tissues in a form that would be useful as an oral vaccine. In this study, we attempted to express the HBsAg in cv. Williams banana plants to be used as an edible vaccine. Using a Biolistic Gene Gun, apical meristem explants of banana were bombarded with plasmid pBHsAg harboring the gene encoding the HBsAg and the bar gene as a selectable marker. Bombarded explants were selected on media containing 3 mg/l Bialaphos. The HBsAg gene was detected using PCR analysis and its expression was tested via western blot analysis using specific polyclonal antibodies directed against human serum derived HBsAg. This study indicates the feasibility of the expression of foreign antigens in plants for possible use as an oral vaccine.

Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter

Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate.

Environmental stress activation of plant long-terminal repeat retrotransposons

Genomic retrotransposons (RTs) are major components of most plant genomes. They spread throughout the genomes by a process termed retrotransposition, which consists of reverse transcription and reinsertion of the copied element into a new genomic location (a copy-and-paste system). Abiotic and biotic stresses activate long-terminal repeat (LTR) RTs in photosynthetic eukaryotes from algae to angiosperms. LTR RTs could represent a threat to the integrity of host genomes because of their activity and mutagenic potential by epigenetic regulation. Host genomes have developed mechanisms to control the activity of the retroelements and their mutagenic potential. Some LTR RTs escape these defense mechanisms, and maintain their ability to be activated and transpose as a result of biotic or abiotic stress stimuli. These stimuli include pathogen infection, mechanical damage, in vitro tissue culturing, heat, drought and salt stress, generation of doubled haploids, X-ray irradiation and many others. Reactivation of LTR RTs differs between different plant genomes. The expression levels of reactivated RTs are influenced by the transcriptional and post-transcriptional gene silencing mechanisms (e.g. DNA methylation, heterochromatin formation and RNA interference). Moreover, the insertion of RTs (e.g. Triticum aestivum L. Wis2-1A) into or next to coding regions of the host genome can generate changes in the expression of adjacent host genes of the host. In this paper, we review the ways that plant genomic LTR RTs are activated by environmental stimuli to affect restructuring and diversification of the host genome.

Characterization of ten date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia using AFLP and ISSR markers.

Date palm is the most economically important plant in the Middle East due to its nutritionally valuable fruit. The development of accurate DNA fingerprints to characterize cultivars and the detection of genetic diversity are of great value for breeding programs. The present study explores the usefulness of ISSR and AFLP molecular markers to detect relationships among 10 date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia. Thirteen ISSR primers and six AFLP primer combinations were examined. The level of polymorphism among cultivars for ISSRs ranged from 20% to 100% with an average of 85%. Polymorphism levels for AFLPs ranged from 63% to 84% with an average of 76%. The total number of cultivar-specific markers was 241, 208 of which were generated from AFLP analysis. AJWA cultivar had the highest number of cultivar-specific ISSR markers, whereas DEK, PER, SUK-Q, SHA and MOS-H cultivars had the lowest. RAB and SHA cultivars had the most and least AFLP cultivar-specific markers, respectively. The highest pairwise similarity indices for ISSRs, AFLPs and combined markers were 84% between DEK (female) and PER (female), 81% between SUK-Q (male) and RAB (male), and 80% between SUK-Q (male) and RAB (male), respectively. The lowest similarity indices were 65% between TAB (female) and SUK-Q (male), 67% between SUK-A (female) and SUK-Q (male), and 67% between SUK-A (female) and SUK-Q (male). Cultivars of the same sex had higher pairwise similarities than those between cultivars of different sex. The Neighbor-Joining (NJ) tree generated from the ISSR dataset was not well resolved and bootstrap support for resolved nodes in the tree was low. AFLP and combined data generated completely resolved trees with high levels of bootstrap support. In conclusion, AFLP and ISSR approaches enabled discrimination among 10 date palm cultivars of from Saudi Arabia, which will provide valuable information for future improvement of this important crop.

Metabolomic response of Calotropis procera growing in the desert to changes in water availability.

Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses.

Control of glycerol biosynthesis under high salt stress in Arabidopsis

Loss-of-function and gain-of-function approaches were utilised to detect the physiological importance of glycerol biosynthesis during salt stress and the role of glycerol in conferring salt tolerance in Arabidopsis. The salt stress experiment involved wild type (WT) and transgenic Arabidopsis overexpressing the yeast GPD1 gene (analogue of Arabidopsis GLY1 gene). The experiment also involved the Arabidopsis T-DNA insertion mutants gly1 (for suppression of glycerol 3-phosphate dehydrogenase or G3PDH), gli1 (for suppression of glycerol kinase or GK), and act1 (for suppression of G3P acyltransferase or GPAT). We evaluated salt tolerance levels, in conjunction with glycerol and glycerol 3-phosphate (G3P) levels and activities of six enzymes (G3PDH, ADH (alcohol dehydrogenase), ALDH (aldehyde dehydrogenase), GK, G3PP (G3P phosphatase) and GLYDH (glycerol dehydrogenase)) involved in the glycerol pathway. The GPD1 gene was used to overexpress G3PDH, a cytosolic NAD+-dependent key enzyme of cellular glycerol biosynthesis essential for growth of cells under abiotic stresses. T2 GPD1-transgenic plants and those of the two mutants gli1 and act1 showed enhanced salt tolerance during different growth stages as compared with the WT and gly1 mutant plants. These results indicate that the participation of glycerol, rather than G3P, in salt tolerance in Arabidopsis. The results also indicate that the gradual increase in glycerol levels in T2 GPD1-transgenic, and gli1 and act1 mutant plants as NaCl level increases whereas they dropped at 200mM NaCl. However, the activities of the G3PDH, GK, G3PP and GLYDH at 150 and 200mM NaCl were not significantly different. We hypothesise that mechanism(s) of glycerol retention/efflux in the cell are affected at 200mM NaCl in Arabidopsis.

Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS -transgenic wheat

The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments.