Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri.

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Surface modification of hollow gold nanoparticles conducted by incorporating cancer cell membrane and AS1411 aptamer, aiming to achieve a dual-targeted therapy for colorectal cancer.

Due to its inherent membrane structure, a nanostructure enveloped by an active cell membrane possesses distinctive characteristics such as prolonged presence in the bloodstream, precise identification capabilities, and evasion of immune responses. This research involved the production of biomimetic nanoparticles, specifically hollow gold nanoparticles (HGNPs) loaded with methotrexate (MTX), which were further coated with cancer cell membrane. These nanoparticles were then adorned with AS1411 aptamer to serve as a targeting agent (Apt-CCM-HG@MTX). The nanoplatform demonstrated precise targeting towards cancer cells due to its dual-targeting characteristic (AS1411 aptamer and C26 cancer cell membrane), exhibiting uniformity in distribution. It also displayed a desirable response to photothermal stimulation, controlled release of drugs, and exceptional properties for fluorescence imaging. The system was composed of spherical HGNPs measuring 51.33 ± 5.70 nm in diameter, which were effectively loaded with MTX using a physical absorption method. The encapsulation efficiency achieved was recorded at 79.54 %, while the loading efficiency reached 38.21 %. The targeted formulation demonstrated a noteworthy mortality of approximately 45 % in the nucleolin positive cell line, C26, as determined by in vitro cytotoxicity assays. As a result of the functionalization process applied to the homologous binding adhesion molecules found in cancer cell membranes and targeting ability of AS1411 aptamer, Apt-CCM-HG@MTX demonstrated a substantial enhancement in targeting tumors and facilitating cellular uptake during in vivo experiments. Furthermore, under NIR radiation the photothermal effect exhibited by Apt-CCM-HG@MTX in the tumor area was notably robust due to the distinctive attributes of HGNPs. The conclusions obtained from this study have the potential to assist in adopting a bioinspired strategy that will significantly improve the effective management of MTX and therapy for individuals with colorectal cancer.

Heterologous Production of Antimicrobial Peptides: Notes to Consider.

Heavy and irresponsible use of antibiotics in the last century has put selection pressure on the microbes to evolve even faster and develop more resilient strains. In the confrontation with such sometimes called "superbugs", the search for new sources of biochemical antibiotics seems to have reached the limit. In the last two decades, bioactive antimicrobial peptides (AMPs), which are polypeptide chains with less than 100 amino acids, have attracted the attention of many in the control of microbial pathogens, more than the other types of antibiotics. AMPs are groups of components involved in the immune response of many living organisms, and have come to light as new frontiers in fighting with microbes. AMPs are generally produced in minute amounts within organisms; therefore, to address the market, they have to be either produced on a large scale through recombinant DNA technology or to be synthesized via chemical methods. Here, heterologous expression of AMPs within bacterial, fungal, yeast, plants, and insect cells, and points that need to be considered towards their industrialization will be reviewed.

Cold-responsive transcription factors in Arabidopsis and rice: A regulatory network analysis using array data and gene co-expression network.

Plant growth and development can be influenced by cold stress. Responses of plants to cold are regulated in part by transcription factors (TFs) and microRNAs, which their determination would be necessary in comprehension of the corresponding molecular cues. Here, transcriptomes of Arabidopsis and rice were analyzed to computationally determine TFs and microRNAs that are differentially responsive to cold treatment, and their co-expression networks were established. Among 181 Arabidopsis and 168 rice differentially expressed TF genes, 37 (26 novel) were up- and 16 (8 novel) were downregulated. Common TF encoding genes were from ERF, MYB, bHLH, NFY, bZIP, GATA, HSF and WRKY families. NFY A4/C2/A10 were the significant hub TFs in both plants. Phytohormone responsive cis-elements such as ABRE, TGA, TCA and LTR were the common cis-elements in TF promoters. Arabidopsis had more responsive TFs compared to rice possibly due to its greater adaptation to ranges geographical latitudes. Rice had more relevant miRNAs probably because of its bigger genome size. The interacting partners and co-expressed genes were different for the common TFs so that of the downstream regulatory networks and the corresponding metabolic pathways. Identified cold-responsive TFs in (A + R) seemed to be more engaged in energy metabolism esp. photosynthesis, and signal transduction, respectively. At post-transcriptional level, miR5075 showed to target many identified TFs in rice. In comparison, the predictions showed that identified TFs are being targeted by diverse groups of miRNAs in Arabidopsis. Novel TFs, miRNAs and co-expressed genes were introduced as cold-responsive markers that can be harnessed in future studies and development of crop tolerant varieties.

Enhanced osteogenesis on proantocyanidin-loaded date palm endocarp cellulosic matrices: A novel sustainable approach for guided bone regeneration.

Developing inexpensive, biocompatible natural scaffolds that can support the differentiation and proliferation of stem cells has been recently emphasized by the research community to faster obtain the FDA approvals for regenerative medicine. In this regard, plant-derived cellulose materials are a novel class of sustainable scaffolding materials with high potentials for bone tissue engineering (BTE). However, low bioactivity of the plant-derived cellulose scaffolds restricts cell proliferation and cell differentiation. This limitation can be addressed though surface-functionalization of cellulose scaffolds with natural antioxidant polyphenols, e.g., grape seed proanthocyanidin (PCA)-rich extract (GSPE). Despite the various merits of GSPE as a natural antioxidant, its impact on the proliferation and adhesion of osteoblast precursor cells, and on their osteogenic differentiation is an as-yet unknown issue. Here, we investigated the effects of GSPE surface functionalization on the physicochemical properties of decellularized date (Phoenix dactyliferous) fruit inner layer (endocarp) (DE) scaffold. In this regard, various physiochemical characteristics of the DE-GSPE scaffold such as hydrophilicity, surface roughness, mechanical stiffness, porosity, and swelling, and biodegradation behavior were compared with those of the DE scaffold. Additionally, the impact of the GSPE treatment of the DE scaffold on the osteogenic response of human mesenchymal stem cells (hMSCs) was thoroughly studied. For this purpose, cellular activities including cell adhesion, calcium deposition and mineralization, alkaline phosphatase (ALP) activity, and expression levels of bone-related genes were monitored. Taken together, the GSPE treatment enhanced the physicochemical and biological properties of the DE-GSPE scaffold, thereby raising its potentials as a promising candidate for guided bone regeneration.

In vitro modeling of hepatocellular carcinoma niche on decellularized tomato thorny leaves: a novel natural three-dimensional (3D) scaffold for liver cancer therapeutics.

Liver cancer is now one of the main causes leading to death worldwide. To achieve reliable therapeutic effects, it is crucial to develop efficient approaches to test novel anticancer drugs. Considering the significant contribution of tumor microenvironment to cell's response to medications, in vitro 3D bioinspiration of cancer cell niches can be regarded as an advanced strategy to improve the accuracy and reliability of the drug-based treatment. In this regard, decellularized plant tissues can perform as suitable 3D scaffolds for mammalian cell culture to create a near-to-real condition to test drug efficacy. Here, we developed a novel 3D natural scaffold made from decellularized tomato hairy leaves (hereafter called as DTL) to mimic the microenvironment of human hepatocellular carcinoma (HCC) for pharmaceutical purposes. The surface hydrophilicity, mechanical properties, and topography measurement and molecular analyses revealed that the 3D DTL scaffold is an ideal candidate for liver cancer modeling. The cells exhibited a higher growth and proliferation rate within the DTL scaffold, as verified by quantifying the expression of related genes, DAPI staining, and SEM imaging of the cells. Moreover, prilocaine, an anticancer drug, showed a higher effectiveness against the cancer cells cultured on the 3D DTL scaffold, compared to a 2D platform. Taken together, this new cellulosic 3D scaffold can be confidently proposed for chemotherapeutic testing of drugs on hepatocellular carcinoma.

Genetic dissection of monosaccharides contents in rice whole grain using genome-wide association study.

The simplest form of carbohydrates are monosaccharides which are the building blocks for the synthesis of polymers or complex carbohydrates. Monosaccharide contents of 197 rice accessions were quantified by HPAEC-PAD in rice (Oryza sativa L.) whole grain (RWG). A genome-wide association study (GWAS) was carried out using 33,812 single nucleotide polymorphisms (SNPs) to identify corresponding genomic regions influencing neutral monosaccharides contents. In total, 49 GWAS signals contained in 17 genomic regions (quantitative trait loci [QTLs]) on seven chromosomes of rice were determined to be associated with monosaccharides contents of whole grain. The QTLs were found for fucose (1), mannose (1), xylose (2), arabinose (2), galactose (4), and rhamnose (7) contents, all of which are novel. Based on co-location of annotated rice genes in the vicinity of GWAS signals, the constituents of the whole grain were associated with the following candidate genes: arabinose content with α-N-arabinofuranosidase, pectinesterase inhibitor, and glucosamine-fructose-6-phosphate aminotransferase 1; xylose content with ZOS1-10 (a C2H2 zinc finger transcription factor [TF]); mannose content with aldose 1-epimerase-like protein and a MYB family TF; galactose content with a GT8 family member (galacturonosyltransferase-like 3), a GRAS family TF, and a GH16 family member (xyloglucan endotransglucosylase/hydrolase xyloglucan 23); fucose content with gibberellin 20 oxidase and a lysine-rich arabinogalactan protein 19, and finally rhamnose content with myo-inositol-1-phosphate synthase, UDP-arabinopyranose mutase, and COBRA-like protein precursor. The results of this study should improve our understanding of the genetic basis of the factors that might be involved in the biosynthesis, regulation, and turnover of monosaccharides in RWG, aiming to enhance the nutritional value of rice grain and impact the related industries.

Comparative phylogeny and evolutionary analysis of Dicer-like protein family in two plant monophyletic lineages.

BACKGROUND: Small RNAs (sRNAs) that do not get untranslated into proteins exhibit a pivotal role in the expression regulation of their cognate gene(s) in almost all eukaryotic lineages, including plants. Hitherto, numerous protein families such as Dicer, a unique class of Ribonuclease III, have been reported to be involved in sRNAs processing pathways and silencing. In this study, we aimed to investigate the phylogenetic relationship and evolutionary history of the DCL protein family. RESULTS: Our results illustrated the DCL family of proteins grouped into four main subfamilies (DCLs 1-4) presented in either Eudicotyledons or Liliopsids. The accurate observation of the phylogenetic trees supports the independent expansion of DCL proteins among the Eudicotyledons and Liliopsids species. They share the common origin, and the main duplication events for the formation of the DCL subfamilies occurred before the Eudicotyledons/Liliopsids split from their ancestral DCL. In addition, shreds of evidence revealed that the divergence happened when multicellularization started and since the need for complex gene regulation considered being a necessity by organisms. At that time, they have evolved independently among the monophyletic lineages. The other finding was that the combination of DCL protein subfamilies bears several highly conserved functional domains in plant species that originated from their ancestor architecture. The conservation of these domains happens to be both lineage-specific and inter lineage-specific. CONCLUSIONS: DCL subfamilies (i.e., DCL1-DCL4) distribute in their single clades after diverging from their common ancestor and before emerging into higher plants. Therefore, it seems that the main duplication events for the formation of the DCL subfamilies occurred before the Eudicotyledons/Liliopsida split and before the appearance of moss, and after the single-cell green algae. We also observed the same trends among the main DCL subfamilies from functional unit composition and architecture. Despite the long evolutionary course from the divergence of Liliopsida lineage from the Eudicotyledons, a significant diversifying force to domain composition and orientation was absent. The results of this study provide a deeper insight into DCL protein evolutionary history and possible sequence and structural relationships between DCL protein subfamilies in the main higher plant monophyletic lineages; i.e., Eudicotyledons and Liliopsida.

Extracellular vesicles of cannabis with high CBD content induce anticancer signaling in human hepatocellular carcinoma.

Plant-derived extracellular vesicles (EVs) have been the topic of interest in recent years due to their proven therapeutic properties. Intact or manipulated plant EVs have shown antioxidant, anti-inflammatory, and anti-cancerous activities as a result of containing bioactive metabolites and other endogenous molecules. Less is known about the EV efficacy with high levels of bioactive secondary metabolites derived from medicinal or non-edible plants. Numerous data suggest the functionality of Cannabis sativa extract and its phytocannabinoids in cancer treatment. Here, two chemotypes of cannabis with different levels of D-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were selected. EVs were isolated from each chemotype via differential ultracentrifugation. HPLC analysis was illustrative of the absence of THC in EVs derived from both plants. Therefore, two types of EVs were classified according to their CBD content into high- (H.C-EVs) and low-CBD EVs (L.C-EVs). Electron microscopy and DLS showed both cannabis-derived EVs (CDEVs) can be considered as exosome-like nanovesicles. Cytotoxicity assay showed that H.C-EVs strongly decreased the viability of two hepatocellular carcinoma (HCC) cell lines, HepG2 and Huh-7, in a dose and time-dependent manner compared with L.C-EVs. H.C-EVs had no significant effect on HUVECs normal cell growth. The finding showed that the H.C-EVs arrested the G0/G1 phase in the cell cycle and significantly induced cell death by activating mitochondrial-dependent apoptosis signaling pathways in both HCC cell lines. Altogether, the current study highlights that CDEVs can be an ideal natural vehicle for bioactive phytocannabinoids and a promising strategy in cancer management.

Transient expression of an scFvG8 antibody in plants and characterization of its effects on the virulence factor pthA of Xanthomonas citri subsp. citri.

Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major disease of citrus plants, causing a significant loss in the citrus industry. The pthA is a bacterial effector protein mediates protein-protein and protein-DNA interactions and modulates host transcription. Injection of pthA effector protein into the host cell induces the expression of the susceptibility gene CsLOB1 which is required for citrus canker disease development. In this study, we described in planta expression of a specific anti-pthA single-chain variable fragment (scFv) recombinant antibody, scFvG8, and assessed its function using molecular docking, immunoblotting, and indirect enzyme-linked immunosorbent assay (ELISA). Based on the results, homology-based molecular docking suggested that at least eight intermolecular hydrogen bonds are involved in pthA-scFvG8 interactions. Immunoblotting and indirect ELISA results reconfirmed specific binding of scFvG8 to pthA protein. Moreover, gene fragment encoding scFvG8 was cloned into plant expression vector and transiently expressed in leaves of Nicotiana tabacum cv. Samson by agroinfiltration method. Transient expression of scFvG8 (at the expected size of 35 kDa) in N. tabacum leaves was confirmed by western blotting. Also, immunoblotting and indirect ELISA showed that the plant-derived scFvG8 had similar activity to purified scFvG8 antibody in detecting pthA. Additionally, in scFvG8-expressing tobacco leaves challenged with Xcc, a reduction (for up to 70%) of hypersensitive response (HR) possibly via direct interaction with pthA, was observed in the necrotic leaf area compared to control plants infected with empty vector. The results obtained in this study confirm that scFvG8 can suppress the function of pthA effector protein within plant cells, thus the induction of stable expression of scFvG8 in lime trees can be considered as an appropriate approach to confer resistance to Xcc.

Plant RNA-mediated gene regulatory network.

Not all transcribed RNAs are protein-coding. Some non-coding RNAs (ncRNAs) seem to be non-functional and are resulted from spurious transcription. Many others have a significant function in the translation process. Gene expressions depend on complex networks of diverse gene regulatory pathways. Several ncRNAs, as major elements, regulate gene expression in a sequence-specific system either at the transcriptional level or post-transcriptional level. RNA-mediated gene regulation machinery is evolutionarily ancient and pretty complex. In this review, the current knowledge in the field of RNA-mediated gene silencing have been summarized.

Genome-wide association study for lignocellulosic compounds and fermentable sugar in rice straw.

Cellulose and lignin are the two main components of secondary plant cell walls with substantial impact on stalk in the field and on straw during industrial processing. The amount of fermentable sugar that can be accessed is another important parameter affecting various industrial applications. In the present study, genetic variability of rice (Oryza sativa L.) genotypes for cellulose, lignin, and fermentable sugars contents was analyzed in rice straw. A genome-wide association study of 33,484 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) >0.05 was performed. The genome-wide association study identified seven, three, and three genomic regions to be significantly associated with cellulose, lignin, and fermentable sugar contents, respectively. Candidate genes in the associated genomic regions were enzymes mainly involved in cell wall metabolism. Novel SNP markers associated with cellulose were tagged to GH16, peroxidase, GT6, GT8, and CSLD2. For lignin content, Villin protein, OsWAK1/50/52/53, and GH16 were identified. For fermentable sugar content, UTP-glucose-1-phosphate uridylyltransferase, BRASSINOSTEROID INSENSITIVE 1, and receptor-like protein kinase 5 were found. The results of this study should improve our understanding of the genetic basis of the factors that might be involved in biosynthesis, turnover, and modification of major cell wall components and saccharides in rice straw.

Genome-Wide Association Mapping of Mixed Linkage (1,3;1,4)-ß-Glucan and Starch Contents in Rice Whole Grain.

The glucan content of rice is a key factor defining its nutritional and economic value. Starch and its derivatives have many industrial applications such as in fuel and material production. Non-starch glucans such as (1,3;1,4)-ß-D-glucan (mixed-linkage ß-glucan, MLG) have many benefits in human health, including lowering cholesterol, boosting the immune system, and modulating the gut microbiome. In this study, the genetic variability of MLG and starch contents were analyzed in rice (Oryza sativa L.) whole grain, by performing a new quantitative analysis of the polysaccharide content of rice grains. The 197 rice accessions investigated had an average MLG content of 252 µg/mg, which was negatively correlated with the grain starch content. A new genome-wide association study revealed seven significant quantitative trait loci (QTLs) associated with the MLG content and two QTLs associated with the starch content in rice whole grain. Novel genes associated with the MLG content were a hexose transporter and anthocyanidin 5,3-O-glucosyltransferase. Also, the novel gene associated with the starch content was a nodulin-like domain. The data pave the way for a better understanding of the genes involved in determining both MLG and starch contents in rice grains and should facilitate future plant breeding programs.

Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1.

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.

Genotypic diversity of 17 cacti species and application to biosynthesis of gold nanoparticles.

The genotypic diversity of 17 cacti species were examined and grouped in four clusters using seven inter simple sequence repeat (ISSR) markers. Group representatives (five species) were chosen for AuNPs synthesis in the cacti syrups. To synthesize the Gold nanoparticles (AuNPs), reducing and capping potential of five species of cacti rich in the polyphenolics were explored. Based on the synthesized AuNPs traits (concentration, pH, temperature, and synthesis time), Opuntia pycnacantha with the highest absorption peak at 540 nm was chosen for further characterizations. Varieties of diffraction peaks confirmed the successful synthesis of AuNPs. AuNPs functionalization with the phenolic compounds was confirmed by Fourier transform infrared (FTIR) spectroscopy. At the optimum conditions (pH = 5.0 and T = 60 °C), both dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed more than 87% of AuNPs to be 2.5 nm in size with Zeta potential to be equal to -19.9 mV.

Energy Flow from Root to Shoot: A Comprehensive In silico Analysis.

BACKGROUND: Root to shoot connection and transfer of information seems to be taken place mostly via the transmissions of signal molecules, secondary metabolites, amino acids, hormones and proteins, through xylem sap. Examination of earlier reports is indicative of relatively high levels of conservation in xylem sap protein compositions. Apparently these protein molecules are being synthesized in roots in response to environmental changes and get transported to aerial plant parts after secretion into xylem sap. OBJECTIVES: In order to comprehend this so-called passive signaling, some questions need to be answered: 1) Do these proteins have the capability to act as signals? 2) How much energy does root spend for the biosynthesis of the secreted proteins? How similar is the amount of energy that root cells spent for the biosynthesis of intra- and extra-cellular proteins? MATERIALS AND METHODS: Reported xylem sap proteins curated from Arabidopsis, maize and soybean. Their sequences were put under scrutiny in terms of considering their mobility, and physical and chemical properties. Metabolic energy required for their biosynthesis along with the energy hidden in their peptide bonds were calculated and compared with random non-xylem sap proteins as control. RESULTS: Xylem sap proteins were significantly smaller than the root proteins, while they were bigger in size when compared to the leaf group. Xylem protein pIs were significantly higher than the control proteins in different plants. Similarly, the protein stability was higher for xylem sap proteins in comparison with roots and leaves in all analyzed plants, except for soybean that the stability was indifferent between xylem and root. The data were suggestive a significantly lower energy consumption for the synthesis of xylem sap proteins. CONCLUSIONS: Lower energy consumption may suggest an economical route of communication between roots and shoots in plants that mainly rely on symplastic signaling.

Analysis of Brassica napus dehydrins and their Co-Expression regulatory networks in relation to cold stress.

Dehydrins (DHNs) are plant specific cold and drought stress-responsive proteins that belong to late embryogenesis abundant (LEA) protein families. B. napus DHNs (BnDHNs) were computationally analyzed to establish gene regulatory- and protein-protein interaction networks. Promoter analyses suggested functionality of phytohormones in BnDHNs gene network. The relative expressions of some BnDHNs were analyzed using qRT-PCR in seedling leaves of both cold-tolerant (Zarfam) and -sensitive (Sari Gul) canola treated/untreated by cold. Our expression data were indicative of the importance of BnDHNs in cold tolerance in Zarfam. BnDHNs were classified into three classes according to the expression pattern. Moreover, expression of three BnDHN types, SKn (BnLEA10 and BnLEA18), YnKn (BnLEA90) and YnSKn (BnLEA104) were significantly high in the tolerant cultivar at 12 h of cold treatment. Our findings put forward the possibility of considering these genes as screening biomarker to determine cold-tolerant breeding lines; something that needs to be further corroborated. Furthermore, these genes may have some implications in developing such tolerant lines via transgenesis.

Cold low pressure O2 plasma treatment of Crocus sativus: An efficient way to eliminate toxicogenic fungi with minor effect on molecular and cellular properties of saffron.

In this study cold low pressure radiofrequency oxygen plasma was used for the first time to inactivate toxicogenic fungi proliferation on saffron. Varieties of plasma produced reactive oxygen species which were investigated by optical emission spectroscopy. The data were indicative of the absence of UV radiation. Effects of plasma treatment on antioxidant activity, metabolic content, colour, odour and flavour parameters and physical impact on saffron were investigated. A range of plasma powers and exposure times were assayed in suppression of fungal growth. Amongst which power of 60 W for 15 min was used to eradicate Aspergillus and other microorganisms. The ferric reducing antioxidant power was changed from 1778.21 to 1674.25 mM/g dry weight following plasma treatment. Moreover, crocin ester, picrocrocin and safranal metabolites reduced insignificantly. Additionally, plasma had no significant impact on colour, odour and flavour of saffron.

Computational Identification of MicroRNAs and Their Transcript Target(s) in Field Mustard (Brassica rapa L.).

BACKGROUND: Micro RNAs (miRNAs) are a pivotal part of non-protein-coding endogenous small RNA molecules that regulate the genes involved in plant growth and development, and respond to biotic and abiotic environmental stresses posttranscriptionally. OBJECTIVE: In the present study, we report the results of a systemic search for identification of new miRNAs in B. rapa using homology-based ESTs (Expressed Sequence Tags) analysis and considering a series of fi ltration criteria. MATERIALS AND METHODS: Plant mature miRNA sequences were searched in non-protein coding ESTs registered in NCBI EST database. Zuker RNA folding algorithm was used to generate the secondary structures of the ESTs. Potential sequences were candidate as miRNA genes and characterized evolutionarily only and if only they fi t some described criteria. Also, the web tool psRNATarget was applied to predict candidate B. rapa miRNA targets. RESULTS: In this study, 10 novel miRNAs from B. rapa belonging to 6 miRNA families were identified using EST-based homology analysis by considering a series of fi ltration criteria. All potent miRNAs appropriate fold back structure. Several potential targets with known/unknown functions for these novel miRNAs were identified. The target genes mainly encode transcription factors, enzymes, DNA binding proteins, disease resistance proteins, carrier proteins and other biological processes. CONCLUSIONS: MicroRNA having diverse functions in plant species growth, development and evolution by posttranscriptionally regulating the levels of specific transcriptome so by effecting on their translation products. Research in miRNA led to the identification of many miRNAs and their regulating genes from diverse plant species.

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.

Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.