Predicting the allergenic risk of Phosphite-NAD+-Oxidoreductase and purple acid phosphatase 17 proteins in genetically modified canola using bioinformatic approaches.

Recent advancements in the generation of high-throughput multi-omics data have provided a vast array of candidate genes for the genetic engineering of plants. However, as part of their safety assessment, newly expressed proteins in genetically modified crops must be evaluated for potential cross-reactivity with known allergens. In this study, we developed transgenic canola plants expressing the Arabidopsis thaliana PAP17 gene and a novel selectable marker composed of the ptxD gene from Pseudomonas stutzeri. To evaluate the potential allergenic cross-reactivity of the AtPAP17 and PTXD proteins expressed in transgenic canola, we applied a comprehensive approach utilizing sequence-based, motif-based, and 3D structure-based analyses. Our results demonstrate that the risk of conferring cross-reactivity with known allergens is negligible, indicating that the expression of these proteins in transgenic canola poses a low allergenic risk.

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Functional assessment of AtPAP17; encoding a purple acid phosphatase involved in phosphate metabolism in Arabidopsis thaliana.

PURPOSE: Purple acid phosphatases (PAPs) includ the largest classes of non-specific plant acid phosphatases. Most characterized PAPs were found to play physiological functions in phosphorus metabolism. In this study, we investigated the function of AtPAP17 gene encoding an important purple acid phosphatase in Arabidopsis thaliana. METHODS: The full-length cDNA sequence of AtPAP17 gene under the control of CaMV-35S promoter was transferred to the A. thaliana WT plant. The generated homozygote AtPAP17-overexpressed plants were compared by the types of analyses with corresponding homozygote atpap17-mutant plant and WT in both + P (1.2 mM) and - P (0 mM) conditions. RESULTS: In the + P condition, the highest and the lowest amount of Pi was observed in AtPAP17-overexpressed plants and atpap17-mutant plants by 111% increase and 38% decrease compared with the WT plants, respectively. Furthermore, under the same condition, APase activity of AtPAP17-overexpressed plants increased by 24% compared to the WT. Inversely, atpap17-mutant plant represented a 71% fall compared to WT plants. The comparison of fresh weight and dry weight in the studied plants showed that the highest and the lowest amount of absorbed water belonged to OE plants (with 38 and 12 mg plant-1) and Mu plants (with 22 and 7 mg plant-1) in + P and - P conditions, respectively. CONCLUSION: The lack of AtPAP17 gene in the A. thaliana genome led to a remarkable reduction in the development of root biomass. Thus, AtPAP17 could have an important role in the root but not shoot developmental and structural programming. Consequently, this function enables them to absorb more water and eventually associated with more phosphate absorption.

Critical assessment of genome-scale metabolic models of Arabidopsis thaliana.

Genome-scale metabolic models (GEMs) have enabled researchers to perform systems-level studies of living organisms. Flux balance analysis (FBA), as a constraint-based technique, enables computation of reaction fluxes and prediction of the metabolic phenotypes of a cell under a set of specified conditions. The quality of a GEM is important for obtaining accurate predictions. In this study, we evaluated the quality of five available GEMs for Arabidopsis thaliana from various points of views. To do this, we inspected some of their important features, including the number of reactions with well-defined gene-protein-reaction rules, number of blocked reactions, mass-unbalanced reactions, prediction accuracy in the simulation of key metabolic functions and existence of erroneous energy generating cycles (EGCs). All of the models were found to include some mass-unbalanced reactions. Moreover, four out of five models were found to include EGCs. However, Aracell includes the maximum number of blocked reactions, which suggests the presence of several incomplete pathways. These results clearly show that simulation by using these models may result in erroneous predictions and all of the publicly available GEMs for A. thaliana require extensive curations before being applied in practice.

Comparative proteome analyses of rhizomania resistant transgenic sugar beets based on RNA silencing mechanism.

Rhizomania is an economically important disease of sugar beet, which is caused by Beet necrotic yellow vein virus (BNYVV). As previously shown, RNA silencing mechanism effectively inhibit the viral propagation in transgenic sugar beet plants. To investigate possible proteomic changes induced by gene insertion and/or RNA silencing mechanism, the root protein profiles of wild type sugar beet genotype 9597, as a control, and transgenic events named 6018-T3:S6-44 (S6) and 219-T3:S3-13.2 (S3) were compared by two-dimensional gel electrophoresis. The accumulation levels of 25 and 24 proteins were differentially regulated in S3 and S6 plants, respectively. The accumulation of 15 spots were increased or decreased more than 2-fold. Additionally, 10 spots repressed or induced in both, while seven spots showed variable results in two events. All the differentially expressed spots were analyzed by MALDI-TOF-TOF mass spectrometry. The functional analysis of differentially accumulated proteins showed that most of them are related to the metabolism and defense/stress response. None of these recognized proteins were allergens or toxic proteins except for a spot identified as phenylcoumaran benzylic ether reductase, Pyrc5, which was decreased in the genetically modified S6 plant. These data are in favor of substantial equivalence of the transgenic plants in comparison to their related wild type cultivar since the proteomic profile of sugar beet root was not remarkably affected by gene transfer and activation RNA silencing mechanism.

Whole genome sequence of Pantoea agglomerans ANP8, a salinity and drought stress-resistant bacterium isolated from alfalfa (Medicago sativa L.) root nodules.

Environmental abiotic stress conditions, especially drought and salinity, are currently the major factors that reduce crop yields worldwide. It has been reported that plant-associated beneficial bacteria, especially strains resistant to abiotic stresses that could maintain their efficiency under environmental challenging conditions, can contribute to alleviate abiotic stresses of host plants. In this study, we presented the assembly of the whole genome of Pantoea agglomerans ANP8, a plant growth-promoting bacterium resistant to salinity and drought stresses. The draft genome assembly contained 4,713,172 bp with 4586 predicted genes. A primary draft genome with a total of 5,115,548 bp and 1916 contigs was obtained (longest contig length being 485,272 bp and smallest contig being 112 bp). Following assembly upgrades, 68 scaffolds and 70 contigs with lengths ≥ 500 bp and an N50 = 209,657 bp were obtained. Number of 5554 and 5472 open reading frames longer than 50 codons were observed in the direct strand and in the reverse strand, respectively. Due to the multiple plant growth-promoting characteristics of this bacterium, genes involved in various indole-3-acetic acid production pathways, e.g., indole-3-pyruvic acid and indole-3-acetamide pathways, were found in the bacterium's genome. In addition, multiple copies of the gcd gene, most important enzymes involvement in the solubilization of phosphates, glucose dehydrogenase, were also observed in this genome. The study provides new genomic information to help understanding the way of action of a stress-tolerant plant growth-promoting bacterium.

In silico prediction of enzymatic reactions catalyzed by acid phosphatases.

In present work, we describe a methodology for prediction of an enzymatic reaction for which no experimental data are available except for a gene sequence. As a challenging case, we have developed the method for identifying the putative substrates of monoester phosphatases, commonly known as acid phosphatase enzymes, which have no strong substrate specificity. Finding a preferable substrate for each one is an important task to unravel pathways involved in plant phosphate metabolism. Having used an Arabidopsis thaliana haloacid dehalogenase (HAD)-related acid phosphatases, HRP9, with an experimentally known structure and preferred substrate as an instance, we firstly predicted the 3 D-structure of HRP1 for subsequent analysis. Then, molecular docking was used to find the best protein interaction with a ligand existing in a set of possible substrates compiled from genome scale metabolic networks of A. thaliana based on binding energy, binding mode as well as the distance between phosphoric ester and cofactor, Mg2+, localized in the active site of HRP1. Molecular dynamics simulation ratified stable protein-ligand complex model. Our analysis predicted HRP1 preferably bind to pyridoxamine-5'-phosphate (PMP). Thus, it is deduced that the conversion of PMP to pyridoxamine must be catalyzed by HRP1. This procedure is expected to make a reliable pipeline to predict the enzymatic reactions catalyzed by acid phosphatases. Taken as a whole, it could be applicable for discovery of the interacting ligands, inhibitors as well as interacting proteins which limits lab works or used for gap filling in biosystems.Communicated by Ramaswamy H. Sarma.

The Critical Role of AtPAP17 and AtPAP26 Genes in Arabidopsis Phosphate Compensation Network.

Purple acid phosphatases (PAP)-encoding genes form a complex network that play a critical role in plant phosphate (Pi) homeostasis. Mostly, the functions of PAPs were investigated individually. However, the interactions of most of these genes in response to various concentrations of available Pi remain unknown. In this study, the roles of AtPAP17 and AtPAP26 genes, and their relationship within Pi homeostasis context were investigated. Surprisingly, atpap17 and atpap26 mutants not only showed no obvious developmental defects, but also produced higher biomass in compare to wild type (WT) plants under normal growth conditions. Comparing gene expression patterns of these mutants with WT plant, we identified a set of genes up-regulated in mutant plants but not in WT. Based on these unexpected results and up-regulation of AtPAP17 and AtPAP26 genes by the loss of function of each other, the hypothesis of compensation relationship between these genes in Pi homeostasis was assessed by generating atpap17/atpap26 double mutants. Observation of developmental defects in atpap17/atpap26 mutant but not in single mutants indicated a compensation relationship between AtPAP17 and AtPAP26 genes in Pi homeostasis network. Taken together, these results demonstrate the activation of AtPAP17 and AtPAP26 genes to buffer against the loss of function of each other, and this compensation relationship is vital for Arabidopsis growth and development.

Author Correction: Comprehensive genomic analysis of a plant growth-promoting rhizobacterium Pantoea agglomerans strain P5.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Zinc solubilization characteristics of efficient siderophore-producing soil bacteria.

BACKGROUND AND OBJECTIVES: Iron and zinc are two essential micro-nutrients for plant growth and development. Therefore, isolation of siderophores-producing and zinc-solubilizing rhizobacteria involved in bio-availability of these elements is of great interest. MATERIALS AND METHODS: In this study, soil samples collected from slightly alkaline soil types were screened for high levels of siderophore secretion and zinc solubilization. RESULTS: Among positive colonies, three isolates, named F21A, F37 and F38, were able to secrete siderophore at high levels, ranged between 200 and 300 µM/liter. A close association was observed between siderophore production capability and growth rate as an indicator of active metabolism. Siderophore production was closely correlated with the level of zinc ion released into the medium as well. All three siderophore producing isolates were able to withstand temperature as high as 37°C, high concentration of NaCl (up to 2.5%) and a wide range of initial pH from 6 to 9 while hydrolyzing Zn compounds actively. One of the isolates, F21A, tolerated the presence of 200 mgl-1 of zinc. Biochemical and molecular characteristics are indicative that these isolates are Pseudomonas japonica. As experienced in a greenhouse experiment, inoculation with the F21A and F37 isolates significantly increase the plants height, fresh and dry weight of corn with compared to control. CONCLUSION: These findings demonstrated that the potential of P. japonica strains as plants growth promoting rhizobacteria (PGPR) in iron and zinc deficient soils.

Functional Assessment of an Overexpressed Arabidopsis Purple Acid Phosphatase Gene (AtPAP26) in Tobacco Plants.

BACKGROUND: Overexpression of known genes encoding key phosphate (Pi)-metabolizing enzymes, such as acid phosphatases (APases), is presumed to help plants with Pi availability and absorption as they are mostly exposed to suboptimal environmental conditions for this vital element. OBJECTIVES: In this study, the overexpression effect of AtPAP26, one of the main contributors in retrieving Pi from intracellular and extracellular compounds, was evaluated from various viewes in tobacco plant. MATERIALS AND METHODS: As a heterologous expression system, the encoding cDNA sequence of AtPAP26 was transferred into tobacco plants. RESULTS: A high growth rate of the transgenic lines was observed which could be due to an increased APase activity, leading to the high total phosphorus as well as the free Pi content of the transgenic plants. Interestingly, a significant increased activity of the other APases was also noticed, indicating a networking among them. These were accompanied by less branched and short primary roots and a decreased lateral root numbers grown in Pi-starvation condition compared to the wild type seedlings. Besides, a delayed germination and dwarf phenotype indicates the possible reduction in gibberellic acid biosynthesis in the transgenic lines. CONCLUSIONS: Such transgenic plants are of interest not only for increased yield but also for the reduced need for chemical fertilizers and removal of excessive Pi accumulation in soils as a consequence of fertilizers' or poultry wastes' over-usage.

Comprehensive genomic analysis of a plant growth-promoting rhizobacterium Pantoea agglomerans strain P5.

In this study, we provide a comparative genomic analysis of Pantoea agglomerans strain P5 and 10 closely related strains based on phylogenetic analyses. A next-generation shotgun strategy was implemented using the Illumina HiSeq 2500 technology followed by core- and pan-genome analysis. The genome of P. agglomerans strain P5 contains an assembly size of 5082485 bp with 55.4% G + C content. P. agglomerans consists of 2981 core and 3159 accessory genes for Coding DNA Sequences (CDSs) based on the pan-genome analysis. Strain P5 can be grouped closely with strains PG734 and 299 R using pan and core genes, respectively. All the predicted and annotated gene sequences were allocated to KEGG pathways. Accordingly,  genes involved in plant growth-promoting (PGP) ability, including phosphate solubilization, IAA and siderophore production, acetoin and 2,3-butanediol synthesis and bacterial secretion, were assigned. This study provides an in-depth view of the PGP characteristics of strain P5, highlighting its potential use in agriculture as a biofertilizer.

Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement.

Hybrid-controlled neurofuzzy networks analysis resulting in genetic regulatory networks reconstruction.

Reverse engineering of gene regulatory networks (GRNs) is the process of estimating genetic interactions of a cellular system from gene expression data. In this paper, we propose a novel hybrid systematic algorithm based on neurofuzzy network for reconstructing GRNs from observational gene expression data when only a medium-small number of measurements are available. The approach uses fuzzy logic to transform gene expression values into qualitative descriptors that can be evaluated by using a set of defined rules. The algorithm uses neurofuzzy network to model genes effects on other genes followed by four stages of decision making to extract gene interactions. One of the main features of the proposed algorithm is that an optimal number of fuzzy rules can be easily and rapidly extracted without overparameterizing. Data analysis and simulation are conducted on microarray expression profiles of S. cerevisiae cell cycle and demonstrate that the proposed algorithm not only selects the patterns of the time series gene expression data accurately, but also provides models with better reconstruction accuracy when compared with four published algorithms: DBNs, VBEM, time delay ARACNE, and PF subjected to LASSO. The accuracy of the proposed approach is evaluated in terms of recall and F-score for the network reconstruction task.

Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.).

Feverfew (Tanacetum parthenium) is a perennial medicinal herb and is a rich source of sesquiterpene lactones. Parthenolide is the main sesquiterpene lactone in feverfew and has attracted attention because of its medicinal potential for treatment of migraine and cancer. In the present work the parthenolide content in different tissues and developmental stages of feverfew was analyzed to study the timing and localization of parthenolide biosynthesis. The strongest accumulating tissue was subsequently used to isolate sesquiterpene synthases with the goal to isolate the gene encoding the first dedicated step in parthenolide biosynthesis. This led to the isolation and charachterization of a germacrene A synthase (TpGAS) and an (E)-ß-caryophyllene synthase (TpCarS). Transcript level patterns of both sesquiterpene synthases were analyzed in different tissues and glandular trichomes. Although TpGAS was expressed in all aerial tissues, the highest expression was observed in tissues that contain high concentrations of parthenolide and in flowers the highest expression was observed in the biosynthetically most active stages of flower development. The high expression of TpGAS in glandular trichomes which also contain the highest concentration of parthenolide, suggests that glandular trichomes are the secretory tissues where parthenolide biosynthesis and accumulation occur.

Abscisic acid regulates gene expression in cortical fiber cells and silica cells of rice shoots.

Drought-induced growth arrest is a major cause of yield loss in crops and is mediated in part by abscisic acid (ABA). The aim of this study was to identify the cell types targeted by ABA during arrest. As transcription factors ABI3 and ABI5 are essential for ABA-induced growth arrest in Arabidopsis, blast was used to identify OsVP1 and OsABF1 as their structural orthologues in rice (Oryza sativa), and employed RNA in situ hybridization to reveal the cell types accumulating the corresponding transcripts in response to ABA. Exogenous ABA arrested the growth of leaves 1, 2 and 3 in young rice shoots and inhibited secondary cell-wall formation in sclerenchyma, including expression of the cellulose synthase gene OsCesA9. Transcripts for OsVP1, OsABF1 and of the putative target genes OsEm, OsLEA3 and WSI18, increased under ABA, accumulating principally in the cytosol of the major support cells (sclerenchymatous cortical fiber cells and epidermal silica cells) of slowly growing leaf 1. Rapidly growing immature tissues in leaves 2 and 3 accumulated OsABF1, OsEm and WSI18 transcripts in the nuclei of all cells, irrespective of ABA treatment. It is concluded that during arrest of leaf growth, ABA targets support cells in maturing tissues. Target cells in immature tissues remain to be identified.

Differential gene expression between squamous cell carcinoma of esophageus and its normal epithelium; altered pattern of mal, akr1c2, and rab11a expression.

AIM: To identify the altered gene expression patterns in squamous cell carcinoma of esophagus (ESCC) in relation to adjacent normal esophageal epithelium. METHODS: Total RNA was extracted using SV total RNA isolation kit from snap frozen tissues of ESCC samples and normal esophageal epithelium far from the tumor. Radio-labeled cDNA were synthesized from equal quantities of total RNAs of tumor and normal tissues using combinations of 24 arbitrary 13-mer primers and three different anchoring oligo-dT primers and separated on sequencing gels. cDNA with considerable different amounts of signals in tumor and normal tissue were reamplified and cloned. Using southern blot, the clones of each band were controlled for false positive results caused by probable heterogeneity of cDNA population with the same size. Clones that confirmed differential expression by slot blot selected for sequencing and northern analysis. Corresponding full-length gene sequences was predicted using human genome project data, related transcripts were translated and used for various protein/motif searches to speculate their probable functions. RESULTS: The 97 genes showed different levels of cDNA in tumor and normal tissues of esophagus. The expression of mal gene was remarkably down regulated in all 10 surveyed tumor tissues. Akr1c2, a member of the aldo-keto reductase 1C family, which is involved in metabolism of sex hormones and xenobiotics, was up-regulated in 8 out of 10 inspected ESCC samples. Rab11a, RPL7, and RPL28 showed moderate levels of differential expression. Many other cDNAs remained to further studies. CONCLUSION: The mal gene which is switched-off in all ESCC samples can be considered as a tumor suppressor gene that more studies in its regulation may lead to valuable explanations in ESCC development. Akr1c2 which is up-regulated in ESCC probably plays an important role in tumor development of esophagus and may be proposed as a potential molecular target in ESCC treatments. Differential display technique in spite of many disadvantages is still a valuable technique in gene function exploration studies to find new candidates for improved ones like gene chips.