Early Fruit Development Regulation-Related Genes Concordantly Expressed with TCP Transcription Factors in Tomato (Solanum lycopersicum).

The tomato (Solanum lycopersicum L.) is considered one of the most important vegetable crops globally, both agronomically and economically; however, its fruit development regulation network is still unclear. The transcription factors serve as master regulators, activating many genes and/or metabolic pathways throughout the entire plant life cycle. In this study, we identified the transcription factors that are coordinated with TCP gene family regulation in early fruit development by making use of the high-throughput sequencing of RNA (RNAseq) technique. A total of 23 TCP-encoding genes were found to be regulated at various stages during the growth of the fruit. The expression patterns of five TCPs were consistent with those of other transcription factors and genes. There are two unique subgroups of this larger family: class I and class II TCPs. Others were directly associated with the growth and/or ripening of fruit, while others were involved in the production of the hormone auxin. Moreover, it was discovered that TCP18 had an expression pattern that was similar to that of the ethylene-responsive transcription factor 4 (ERF4). Tomato fruit set and overall development are under the direction of a gene called auxin response factor 5 (ARF5). TCP15 revealed an expression that was in sync with this gene. This study provides insight into the potential processes that help in acquiring superior fruit qualities by accelerating fruit growth and ripening.

Stepwise response of MeJA-induced genes and pathways in leaves of C. roseus.

Catharanthus roseus is a perennial herb known for the production of important terpenoid indole alkaloids (TIAs) in addition to a variety of phenolic compounds. The goal of the present work was to detect the prolonged effects of MeJA (6 uM) treatment across time (up to 24 days) in order to detect the stepwise response of MeJA-induced genes and pathways in leaves of C. rouses. Prolonged exposure of plants to MeJA (6 uM) treatment for different time points (6, 12 and 24 days) indicated that genes in the indole alkaloid biosynthesis pathway and upstream pathways were triggered earlier (e.g., 6 days) than those in the anthocyanin biosynthesis pathway and its upstream pathways (e.g., 12 days). Three enzymes, e.g., T16H, OMT, and D4H, in the six-step vindoline biosynthesis and two enzymes, e.g., TDC and STR, acting consecutively in the conversion of tryptophan to strictosidine, were activated after 6 days of MeJA treatment. Two other key enzymes, e.g., TRP and CYP72A1, acting concurrently upstream of the TIA biosynthesis pathway were upregulated after 6 days. The genes encoding TDC and STR might concurrently act as a master switch of the TIA pathway towards the production of the indole alkaloids. On the other hand, we speculate that the gene encoding PAL enzyme also acts as the master switch of phenylpropanoid biosynthesis and the downstream flavonoid biosynthesis and anthocyanin biosynthesis pathways towards the production of several phenolic compounds. PAL and the downstream enzymes were activated 12 days after treatment. Cluster analysis confirmed the concordant activities of the flower- and silique-specific bHLH25 transcription factor and the key enzyme in the TIA biosynthesis pathway, e.g., STR. Due to the stepwise response of the two sets of pathways, we speculate that enzymes activated earlier likely make TIA biosynthesis pathway a more favourable target in C. roseus than anthocyanin biosynthesis pathway.

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), KO ERF109 or OE ERF109 ) 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.

Transcriptomic and metabolic responses of Calotropis procera to salt and drought stress.

BACKGROUND: Calotropis procera is a wild plant species in the family Apocynaceae that is able to grow in harsh, arid and heat stressed conditions. Understanding how this highly adapted plant persists in harsh environments should inform future efforts to improve the hardiness of crop and forage plant species. To study the plant response to droµght and osmotic stress, we treated plants with polyethylene glycol and NaCl and carried out transcriptomic and metabolomics measurements across a time-course of five days. RESULTS: We identified a highly dynamic transcriptional response across the time-course including dramatic changes in inositol signaling, stress response genes and cytokinins. The resulting metabolome changes also involved sharp increases of myo-inositol, a key signaling molecule and elevated amino acid metabolites at later times. CONCLUSIONS: The data generated here provide a first glimpse at the expressed genome of C. procera, a plant that is exceptionally well adapted to arid environments. We demonstrate, through transcriptome and metabolome analysis that myo-inositol signaling is strongly induced in response to drought and salt stress and that there is elevation of amino acid concentrations after prolonged osmotic stress. This work should lay the foundations of future studies in adaptation to arid environments.

Transcriptomic analysis of salt stress responsive genes in Rhazya stricta.

Rhazya stricta is an evergreen shrub that is widely distributed across Western and South Asia, and like many other members of the Apocynaceae produces monoterpene indole alkaloids that have anti-cancer properties. This species is adapted to very harsh desert conditions making it an excellent system for studying tolerance to high temperatures and salinity. RNA-Seq analysis was performed on R. stricta exposed to severe salt stress (500 mM NaCl) across four time intervals (0, 2, 12 and 24 h) to examine mechanisms of salt tolerance. A large number of transcripts including genes encoding tetrapyrroles and pentatricopeptide repeat (PPR) proteins were regulated only after 12 h of stress of seedlings grown in controlled greenhouse conditions. Mechanisms of salt tolerance in R. stricta may involve the upregulation of genes encoding chaperone protein Dnaj6, UDP-glucosyl transferase 85a2, protein transparent testa 12 and respiratory burst oxidase homolog protein b. Many of the highly-expressed genes act on protecting protein folding during salt stress and the production of flavonoids, key secondary metabolites in stress tolerance. Other regulated genes encode enzymes in the porphyrin and chlorophyll metabolic pathway with important roles during plant growth, photosynthesis, hormone signaling and abiotic responses. Heme biosynthesis in R. stricta leaves might add to the level of salt stress tolerance by maintaining appropriate levels of photosynthesis and normal plant growth as well as by the participation in reactive oxygen species (ROS) production under stress. We speculate that the high expression levels of PPR genes may be dependent on expression levels of their targeted editing genes. Although the results of PPR gene family indicated regulation of a large number of transcripts under salt stress, PPR actions were independent of the salt stress because their RNA editing patterns were unchanged.

Transcription factors regulating uspA genes in Catharanthus roseus.

RNA-Seq of the Catharanthus roseus SRA database was done in order to detect putative universal stress proteins (USPs) and their possible controlling factors. Previous analysis indicated the existence and characterization of uspA-like genes. In silico analysis of RNA-Seq database in several plant tissues revealed the possible functions and regulations of some uspA-like transcripts whose transcription factors (TFs) that might drive their expression were detected. BLAST indicated the existence of TF superfamilies erf (ethylene-responsive TF), bHLH (basic helix-loop-helix) and WRKY that might regulate several uspA-like genes. This data was proven via semi-quantitative RT-PCR in four plant tissues. Several of these transcription factor superfamilies are known for their action in the plant defense against biotic and abiotic stresses.

Analysis of transcriptional response to heat stress in Rhazya stricta.

BACKGROUND: Climate change is predicted to be a serious threat to agriculture due to the need for crops to be able to tolerate increased heat stress. Desert plants have already adapted to high levels of heat stress so they make excellent systems for identifying genes involved in thermotolerance. Rhazya stricta is an evergreen shrub that is native to extremely hot regions across Western and South Asia, making it an excellent system for examining plant responses to heat stress. Transcriptomes of apical and mature leaves of R. stricta were analyzed at different temperatures during several time points of the day to detect heat response mechanisms that might confer thermotolerance and protection of the plant photosynthetic apparatus. RESULTS: Biological pathways that were crosstalking during the day involved the biosynthesis of several heat stress-related compounds, including soluble sugars, polyols, secondary metabolites, phenolics and methionine. Highly downregulated leaf transcripts at the hottest time of the day (40-42.4 °C) included genes encoding cyclin, cytochrome p450/secologanin synthase and U-box containing proteins, while upregulated, abundant transcripts included genes encoding heat shock proteins (HSPs), chaperones, UDP-glycosyltransferase, aquaporins and protein transparent testa 12. The upregulation of transcripts encoding HSPs, chaperones and UDP-glucosyltransferase and downregulation of transcripts encoding U-box containing proteins likely contributed to thermotolerance in R. stricta leaf by correcting protein folding and preventing protein degradation. Transcription factors that may regulate expression of genes encoding HSPs and chaperones under heat stress included HSFA2 to 4, AP2-EREBP and WRKY27. CONCLUSION: This study contributed new insights into the regulatory mechanisms of thermotolerance in the wild plant species R. stricta, an arid land, perennial evergreen shrub common in the Arabian Peninsula and Indian subcontinent. Enzymes from several pathways are interacting in the biosynthesis of soluble sugars, polyols, secondary metabolites, phenolics and methionine and are the primary contributors to thermotolerance in this species.

Ethylene responsive transcription factor ERF109 retards PCD and improves salt tolerance in plant.

BACKGROUND: 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). RESULTS: 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). CONCLUSIONS: 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.

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.

Structural identification of putative USPs in Catharanthus roseus.

Nucleotide sequences of the C. roseus SRA database were assembled and translated in order to detect putative universal stress proteins (USPs). Based on the known conserved USPA domain, 24 Pfam putative USPA proteins in C. roseus were detected and arranged in six architectures. The USPA-like domain was detected in all architectures, while the protein kinase-like (or PK-like), (tyr)PK-like and/or U-box domains are shown downstream it. Three other domains were also shown to coexist with the USPA domain in C. roseus putative USPA sequences. These domains are tetratricopeptide repeat (or TPR), apolipophorin III (or apoLp-III) and Hsp90 co-chaperone Cdc37. Subsequent analysis divided USPA-like domains based on the ability to bind ATP. The multiple sequence alignment indicated the occurrence of eight C. roseus residues of known features of the bacterial 1MJH secondary structure. The data of the phylogenetic tree indicated several distinct groups of USPA-like domains confirming the presence of high level of sequence conservation between the plant and bacterial USPA-like sequences.

RNA-Seq analysis of the wild barley (H. spontaneum) leaf transcriptome under salt stress.

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 (500mM 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.

Corrected sequence of the wheat plastid genome.

Wheat is the most important cereal in the world in terms of acreage and productivity. We sequenced and assembled the plastid genome of one Egyptian wheat cultivar using next-generation sequence data. The size of the plastid genome is 133,873 bp, which is 672 bp smaller than the published plastid genome of "Chinese Spring" cultivar, due mainly to the presence of three sequences from the rice plastid genome. The difference in size between the previously published wheat plastid genome and the sequence reported here is due to contamination of the published genome with rice plastid DNA, most of which is present in three sequences of 332, 131 and 131 bp. The corrected plastid genome of wheat has been submitted to GenBank (accession number KJ592713) and can be used in future comparisons.

Construction of naïve camelids VHH repertoire in phage display-based library.

Camelids have unique antibodies, namely HCAbs (VHH) or commercially named Nanobodies(®) (Nb) that are composed only of a heavy-chain homodimer. As libraries based on immunized camelids are time-consuming, costly and likely redundant for certain antigens, we describe the construction of a naïve camelid VHHs library from blood serum of non-immunized camelids with affinity in the subnanomolar range and suitable for standard immune applications. This approach is rapid and recovers VHH repertoire with the advantages of being more diverse, non-specific and devoid of subpopulations of specific antibodies, which allows the identification of binders for any potential antigen (or pathogen). RNAs from a number of camelids from Saudi Arabia were isolated and cDNAs of the diverse vhh gene were amplified; the resulting amplicons were cloned in the phage display pSEX81 vector. The size of the library was found to be within the required range (10(7)) suitable for subsequent applications in disease diagnosis and treatment. Two hundred clones were randomly selected and the inserted gene library was either estimated for redundancy or sequenced and aligned to the reference camelid vhh gene (acc. No. ADE99145). Results indicated complete non-specificity of this small library in which no single event of redundancy was detected. These results indicate the efficacy of following this approach in order to yield a large and diverse enough gene library to secure the presence of the required version encoding the required antibodies for any target antigen. This work is a first step towards the construction of phage display-based biosensors useful in disease (e.g., TB or tuberculosis) diagnosis and treatment.

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.

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.

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.

Thymoquinone causes multiple effects, including cell death, on dividing plant cells.

Thymoquinone (TQ) is a major constituent of Nigella sativa oil with reported anti-oxidative activity and anti-inflammatory activity in animal cells. It also inhibits proliferation and induces programmed cell death (apoptosis) in human skin cancer cells. The present study sought to detect the influence of TQ on dividing cells of three plant systems and on expression of Bcl2-associated athanogene-like (BAG-like) genes that might be involved during the process of cell death. BAG genes are known for the regulation of diverse physiological processes in animals, including apoptosis, tumorigenesis, stress responses, and cell division. Synthetic TQ at 0.1mg/mL greatly reduced wheat seed germination rate, whereas 0.2mg/mL completely inhibited germination. An Evans blue assay revealed moderate cell death in the meristematic zone of Glycine max roots after 1h of TQ treatment (0.2mg/mL), with severe cell death occurring in this zone after 2h of treatment. Light microscopy of TQ-treated (0.2mg/mL) onion hairy root tips for 1h revealed anti-mitotic activity and also cell death-associated changes, including nuclear membrane disruption and nuclear fragmentation. Transmission electron microscopy of TQ-treated cells (0.2mg/mL) for 1h revealed shrinkage of the plasma membrane, leakage of cell lysate, degradation of cell walls, enlargement of vacuoles and condensation of nuclei. Expression of one BAG-like gene, previously associated with cell death, was induced 20 min after TQ treatment in Glycine max root tip cells. Thus, TQ has multiple effects, including cell death, on dividing plant cells and plants may serve as a useful system to further investigate the mechanisms underlying the response of eukaryotic cells to TQ.

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.

Expression, detection of candidate function and homology modeling for Vicia villosa ornithine δ-aminotransferase.

The accumulation of compatible solutes during stress in plant cell is well documented. Proline is one of these solutes which accumulate in the cytosol in response to drought or salinity stress in plants. Proline has several functions during stress just like osmotic adjustment, osmoprotection, free radical scavenger and antioxidant. Ornithine δ-aminotransferase (δ-OAT) is an important enzyme in proline biosynthetic pathway. It catalyzes the transamination of ornithine to pyrroline-5-carboxylate which can be reduced into proline. Expression of ornithine δ-aminotransferase gene isolated from Vicia villosa (VvOAT) showed protein with a molecular mass of 63 KDa which is compatible with the predicted mass and after VvOAT gene delivery into E. coli host HB101, VvOAT gene enhanced its salt tolerance. Homology modeling of VvOAT was performed based on the crystal structure of the ornithine δ-aminotransferase from humans (PDB code 2OATA). With this model, a flexible docking study with the substrate and inhibitors was performed. The results indicated that PHE170 and ASN171 in VvOAT are the important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate and inhibitors. All the obtained results indicated the efficiency of utilizing this gene in conferring salt tolerance.