Ahmed Ashoub

Comparative analysis of barley leaf proteome as affected by drought stress

The adaptive response of Egyptian barley land races to drought stress was analyzed using difference gel electrophoresis (DIGE). Physiological measurements and proteome alterations of accession number 15141, drought tolerant, and accession number 15163, drought sensitive, were compared. Differentially expressed proteins were subjected to MALDI-TOF-MS analysis. Alterations in proteins related to the energy balance and chaperons were the most characteristic features to explain the differences between the drought-tolerant and the drought-sensitive accessions. Further alterations in the levels of proteins involved in metabolism, transcription and protein synthesis are also indicated.

Characterization of common and distinctive adjustments of wild barley leaf proteome under drought acclimation, heat stress and their combination

In nature, plants are often exposed to combinations of different stresses at the same time, while in many laboratory studies of molecular stress induction phenomena, single stress responses are analyzed. This study aims to identify the common (i.e. more general stress-responsive) and the stress-specific adjustments of the leaf proteome of wild barley to two often co-occurring stress phenomena, i.e. in response to (long-term) drought acclimation (DA) or to (transient) heat stress (HS). In addition, we analyzed those alterations which are specific for the combination of both stresses. Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry with a 1.5 threshold value of changes in relative protein contents. DA resulted in specific upregulation of proteins with cell detoxification functions, water homeostasis maintenance, amino acids synthesis and lipid metabolism and distinct forms of heat shock proteins (HSPs) and proteins with chaperon functions while proteins related to nitrogen metabolism were downregulated. This response was distinguished from the response to transient HS, which included upregulation of a broad range of HSP products. The common response to both stressors revealed upregulation of additional forms of HSPs and the downregulation of enzymes of the photosynthetic apparatus and chlorophyll binding proteins. The simultaneous exposure to both stress conditions resulted mostly in a combination of both stress responses and to unique abundance changes of proteins with yet unclear functions.

Comparative Analysis of Sorghum bicolor Proteome in Response to Drought Stress and following Recovery

The adaptive response of Sorghum bicolor landraces from Egypt to drought stress and following recovery was analyzed using two-dimensional difference gel electrophoresis, 2D-DIGE. Physiological measurements and proteome alterations of accession number 11434, drought tolerant, and accession number 11431, drought sensitive, were compared to their relative control values after drought stress and following recovery. Differentially expressed proteins were analysed by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry, MALDI-TOF-MS. Alterations in protein contents related to the energy balance, metabolism (sensu Mewes et al. 1997), and chaperons were the most apparent features to elucidate the differences between the drought tolerant and sensitive accessions. Further alterations in the levels of proteins related to transcription and protein synthesis are discussed.

Impact of Drought, Heat, and Their Combination on Chlorophyll Fluorescence and Yield of Wild Barley (Hordeum spontaneum)

The impact of (long-term) drought acclimation and (short-term) heat stress and their combination on fast chlorophyll fluorescence induction curves (OJIP) and grain yield was tested using pot-grown plants of wild barley (Hordeum spontaneum) originating from Northern Egypt. Concerning agronomic traits, the main effect of drought was decreased biomass accumulation and grain yield, while heat specifically affected floral development. The treatments caused specific inhibitions of photosystem II (PSII) functionality. While heat stressed plants showed a reduction of maximum quantum efficiency of PSII (), an indication of effects on oxygen evolving complex (OEC) functionality, and the connectivity of PSII units, these features were entirely missing in drought acclimated plants. Drought caused a reduction of the Performance Index () and of the relative amplitude of the IP-phase of the OJIP induction curve (). Individuals suffering from a combination of drought and heat showed a better ability to recover photosynthetic electron transport after the relief of stress in comparison to heat stressed plants. However, this improved capacity to recover was not accompanied by an increased grain yield. Thus, we conclude that chlorophyll fluorescence measurements provide valuable physiological data; however, their use in agronomic studies for the prediction of agronomic traits should be done with some precaution.

A competent extraction method of plant proteins for 2-D gel electrophoresis

The efficient extraction of high‐quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea‐/thiourea‐ and NP‐40‐containing solution. Protein profiles were examined on pH 3–11 non‐linear IEF strips and SDS‐PAGE and compared with extracts using the established method of acetone‐10% TCA/0.07% 2‐mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1–8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2‐mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250–150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25–15 kDa. The MALDI‐TOF‐MS of differential spots from acetone containing 10% TCA/0.07% 2‐mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.

A Primer-Based Approach to Genome Walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.

Prominent alterations of wild barley leaf transcriptome in response to individual and combined drought acclimation and heat shock conditions

Under field conditions, drought and heat stress typically happen simultaneously and their negative impact on the agricultural production is expected to increase worldwide under the climate change scenario. In this study, we performed RNA-sequencing analysis on leaves of wild barley (Hordeum spontaneum) originated from the northern coastal region of Egypt following individual drought acclimation (DA) and heat shock (HS) treatments and their combination (CS, combined stresses) to distinguish the unique and shared differentially expressed genes (DEG). Results indicated that the number of unique genes that were differentially expressed following HS treatment exceeded the number of those expressed following DA. In addition, the number of genes that were uniquely differentially expressed in response to CS treatment exceeded the number of those of shared responses to individual DA and HS treatments. These results indicate a better adaptation of the Mediterranean wild barley to drought conditions when compared with heat stress. It also manifests that the wild barley response to CS tends to be unique rather than common. Annotation of DEG showed that metabolic processes were the most influenced biological function in response to the applied stresses.

Molecular cloning, expression, sequence analysis and in silico comparative mapping of trehalose 6-phosphate gene from Egyptian durum wheat

Trehalose is a non-reducing disaccharide which consists of two glucose units that functions as a compatible solute to stabilize the membrane structures under heat and desiccation stress. Trehalose-6phosphate synthase (TPS) and trehalose-6phosphate phosphatase (TPP) are the key enzymes for trehalose biosynthesize in the plant kingdom. On the basis of bioinformatics prediction, fragment containing an open reading frame of 945 bp was cloned from durum wheat. Sequence comparison and analysis of conserved domains revealed the presence of a TPP domain. Full length of the gene was isolated using gene race technology. Semi-quantitative RT-PCR and real time quantitative PCR indicated that the expression of this gene is up-regulated in response to drought stress. The biochemical assay of the trehalase activity showed that the enzymes activity decreased under the dehydration stress. The obtained phylogenic tree showed that the isolated TPP protein forms a distinct clad close to the Oryza sativa trehalose-6phosphate phosphatase. In silico and comparative mapping indicated that the isolated TPP gene is localized on rice chromosome 8, durum wheat chromosome 20, bread wheat chromosome 3B, oat linkage group E, sorghum chromosome 4 and barley 5H.