Magdy A. Al-Kordy

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.

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.

Suppression of PCD-related genes affects salt tolerance in Arabidopsis.

This work aims at examining a natural exciting phenomenon suggesting that suppression of genes inducing programmed cell death (PCD) might confer tolerance against abiotic stresses in plants. PCD-related genes were induced in tobacco under oxalic acid (OA) treatment (20 mM), and plant cells were characterized to confirm the incidence of PCD. The results indicated that PCD was triggered 24 h after the exposure to OA. Then, RNAs were extracted from tobacco cells 0, 2, 6, 12 and 24 h after treatment for deep sequencing. RNA-Seq analyses were done with a special emphasis to clusters whose PCD-related genes were upregulated after 2 h of OA exposure. Accordingly, 23 tobacco PCD-related genes were knocked down via virus-induced gene silencing (VIGS), whereas our results indicated the influence of five of them on inducing or suppressing PCD. Knockout T-DNA insertion mutants of these five genes in Arabidopsis were tested under salt stress (0, 100, 150, and 200 mM NaCl), and the results indicated that a mutant of an antiapoptotic gene, namely Bax Inhibitor-1 (BI-1), whose VIGS induced PCD in tobacco, was salt sensitive, while a mutant of an apoptotic gene, namely mildew resistance locus O (Mlo), whose VIGS suppressed PCD, was salt tolerant as compared to the WT (Col) control. These data support our hypothesis that retarding PCD-inducing genes can result in higher levels of salt tolerance, while retarding PCD-suppressing genes can result in lower levels of salt tolerance in plants.

Detection of phytochrome-like genes from Rhazya stricta (Apocynaceae) using de novo genome assembly

Phytochrome-like genes in the wild plant species Rhazya stricta Decne were characterized using a de novo genome assembly of next generation sequence data. Rhazya stricta contains more than 100 alkaloids with multiple pharmacological properties, and leaf extracts have been used to cure chronic rheumatism, to treat tumors, and in the treatment of several other diseases. Phytochromes are known to be involved in the light-regulated biosynthesis of some alkaloids. Phytochromes are soluble chromoproteins that function in the absorption of red and far-red light and the transduction of intracellular signals during light-regulated plant development. De novo assembly of the nuclear genome of R. stricta recovered 45,641 contigs greater than 1000bp long, which were used in constructing a local database. Five sequences belonging to Arabidopsis thaliana phytochrome gene family (i.e., AtphyABCDE) were used to identify R. stricta contigs with phytochrome-like sequences using BLAST. This led to the identification of three contigs with phytochrome-like sequences covering AtphyA-, AtphyC- and AtphyE-like full-length genes. Annotation of the three sequences showed that each contig consists of one phytochrome-like gene with three exons and two introns. BLASTn and BLASTp results indicated that RsphyA mRNA and protein sequences had homologues in Wrightia coccinea and and Solanum tuberosum, respectively. RsphyC-like mRNA and protein sequence were homologous to Vitis vinifera and Vitis riparia. RsphyE-like mRNA coding and protein sequences were homologous to Ipomoea nil. Multiple-sequence alignment of phytochrome proteins indicated a homology with 30 sequences from 23 different species of flowering plants. Phylogenetic analysis confirmed that each R. stricta phytochrome gene is related to the same phytochrome gene of other flowering plants. It is proposed that the absence of phyB gene in R. stricta is due to RsphyA gene taking over the role of phyB.

Multifunctional activities of ERF109 as affected by salt stress in Arabidopsis

Transcriptomic analysis was conducted in leaves of Arabidopsis T-DNA insertion ERF109-knocked out (KO) mutant or plants overexpressing (OE) the gene to detect its role in driving expression of programmed cell death- (PCD-) or growth-related genes under high salt (200 mM NaCl) stress. The analysis yielded ~22–24 million reads, of which 90% mapped to the Arabidopsis reference nuclear genome. Hierarchical cluster analysis of gene expression and principal component analysis (PCA) successfully separated transcriptomes of the two stress time points. Analysis indicated the occurrence of 65 clusters of gene expression with transcripts of four clusters differed at the genotype (e.g., WT (wild type), KOERF109 or OEERF109) level. Regulated transcripts involved DIAP1-like gene encoding a death-associated inhibitor of reactive oxygen species (ROS). Other ERF109-regulated transcripts belong to gene families encoding ROS scavenging enzymes and a large number of genes participating in three consecutive pathways, e.g., phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism and plant hormone signal transduction. We investigated the possibility that ERF109 acts as a “master switch” mediator of a cascade of consecutive events across these three pathways initially by driving expression of ASA1 and YUC2 genes and possibly driving GST, IGPS and LAX2 genes. Action of downstream auxin-regulator, auxin-responsive as well as auxin carrier genes promotes plant cell growth under adverse conditions.