Wild salt-tolerant barley ( Hordeum spontaneum ) is the ancestor of cultivated barley ( Hordeum vulgare or H. vulgare ) . Although the cultivated barley genome is well studied, little is known about genome structure and function of its wild ancestor. In the present study, RNA-Seq analysis was performed on young leaves of wild barley treated with salt (500 mM NaCl) at four different time intervals. Transcriptome sequencing yielded 103 to 115 million reads for all replicates of each treatment, corresponding to over 10 billion nucleotides per sample. Of the total reads, between 74.8 and 80.3% could be mapped and 77.4 to 81.7% of the transcripts were found in the H. vulgare unigene database (unigene-mapped). The unmapped wild barley reads for all treatments and replicates were assembled de novo and the resulting contigs were used as a new reference genome. This resulted in 94.3 to 95.3% of the unmapped reads mapping to the new reference. The number of differentially expressed transcripts was 9277, 3861 of which were unigene-mapped. The annotated unigene- and de novo -mapped transcripts (5100) were utilized to generate expression clusters across time of salt stress treatment. Two-dimensional hierarchical clustering classified differential expression profiles into nine expression clusters, four of which were selected for further analysis. Differentially expressed transcripts were assigned to the main functional categories. The most important groups were “response to external stimulus” and “electron-carrier activity”. Highly expressed transcripts are involved in several biological processes, including electron transport and exchanger mechanisms, flavonoid biosynthesis, reactive oxygen species (ROS) scavenging, ethylene production, signaling network and protein refolding. The comparisons demonstrated that mRNA-Seq is an efficient method for the analysis of differentially expressed genes and biological processes under salt stress.

The ultimate goal of this work was to detect the role of transcription factors (TFs) concordantly expressed with genes related to programmed cell death (PCD) during PCD and salt stress. This work was based on the hypothesis that TFs and their driven genes likely co-express under different stimuli. The conserved superfamily ethylene responsive factor (AP2/ERF) draw attention of the present study as it participates in the response to biotic and abiotic stimuli as well as to program cell death (PCD). RNA-Seq analysis was done for tobacco (N. benthamiana) leaves exposed to oxalic acid (OA) at 20 mM for 0, 2, 6, 12 and 24 h to induce PCD. Genes up-regulated after 2 h of OA treatment with known function during PCD were utilized as landmarks to select TFs with concordant expression. Knockdown mutants of these TFs were generated in tobacco via virus induced gene silencing (VIGS) in order to detect their roles during PCD. Based on the results of PCD assay, knockout (KO) T-DNA insertion mutants of Arabidopsis as well as over-expression lines of two selected TFs, namely ERF109 and TFIID5, analogs to those in tobacco, were tested under salt stress (0, 100, 150 and 200 mM NaCl). Results of knockdown mutant tobacco cells confirmed the influence of these two TFs during PCD. Knockout insertion mutants and over-expression lines indicated the role of ERF109 in conferring salt tolerance in Arabidopsis.

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.

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. T2GPD1-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 T2GPD1-transgenic, and gli1 and act1 mutant plants as NaCl level increases whereas they dropped at 200 mM NaCl. However, the activities of the G3PDH, GK, G3PP and GLYDH at 150 and 200 mM NaCl were not significantly different. We hypothesise that mechanism(s) of glycerol retention/efflux in the cell are affected at 200 mM NaCl in Arabidopsis.

The main aim of this study was to improve fungal resistance in bread wheat via transgenesis. Transgenic wheat plants harboring barley chitinase (chi26) gene, driven by maize ubi promoter, were obtained using biolistic bombardment, whereas the herbicide resistance gene, bar, driven by the CaMV 35S promoter was used as a selectable marker. Molecular analysis confirmed the integration, copy number, and the level of expression of the chi26 gene in four independent transgenic events. Chitinase enzyme activity was detected using a standard enzymatic assay. The expression levels of chi26 gene in the different transgenic lines, compared to their respective controls, were determined using qRT-PCR. The transgene was silenced in some transgenic families across generations. Gene silencing in the present study seemed to be random and irreversible. The homozygous transgenic plants of T4, T5, T6, T8, and T9 generations were tested in the field for five growing seasons to evaluate their resistance against rusts and powdery mildew. The results indicated high chitinase activity at T0 and high transgene expression levels in few transgenic families. This resulted in high resistance against wheat rusts and powdery mildew under field conditions. It was indicated by proximate and chemical analyses that one of the transgenic families and the non-transgenic line were substantially equivalent. Transgenic wheat with barley chi26 was found to be resistant even after five generations under artificial fungal infection conditions. One transgenic line was proved to be substantially equivalent as compared to the non-transgenic control.