Arbuscular mycorrhizal colonization leads to a change of hormone profile in micropropagated plantlet Satureja khuzistanica Jam.

Phytohormones are supposed to contribute to the establishment of mutualistic Arbuscular mycorrhiza (AM) symbioses. However, their role in the acclimation of micropropagated plantlet inoculated with AM is still unknown. To address this question, we performed a hormone profiling during the acclimation of Satureja khuzistanica plantlets inoculated with Rhizoglomus fasciculatum. The levels of indoleacetic acid (IAA), methyl indole acetic acid, cis-zeatin, cis zeatin ribose, jasmonate, jasmonoyl isoleucine, salicylic acid, abscisic acid (ABA) were analyzed. Further, the relative gene expression of AOS (Allene oxide synthase) as a key enzyme of jasmonate biosynthesis, in either inoculated or non-inoculated micropropagated plantlets was evaluated during acclimation period. The concentrations of IAA and cis-zeatin increased in the plantlets inoculated by AM whereas the concentration of ABA decreased upon 60 days acclimation in the whole shoot of plantlets of S. khuzistanica. The relative expression of AOS gene resulted in an increase of isoleucine jasmonate, the bioactive form of jasmonate. Based on our results, IAA and cis-zeatin probably contribute to maintaining growth, and AM reduces transition stress by modifying ABA and jasmonate concentrations.

RAV transcription factor regulatory function in response to salt stress in two Iranian wheat landraces.

Amongst the transcription factor groups, the AP2/ERF (Apetala2/Ethylene Response Factor) superfamily is one of the main groups in plants and plays an essential role in tolerating abiotic and biotic stresses. The AP2/ERF superfamily consists of ERF, AP2, RAV, and Soloist families based on the AP2 domain number. The RAV (Related to ABI3/VP1) family members have been revealed to be stimulate by a number of biotic and abiotic environmental incentives; including pathogen infection, salicylic acid, osmotic stress, cold, high salinity, wounding, and exogenous hormone application. However, limited data are available on the contributions of RAV transcription factors in wheat (Triticum aestivum L.). In the present study, a total of 26 RAV genes were identified in wheat from a genome-wide search against the latest wheat genome data. Phylogenetic and sequence alignment analyses divided the wheat RAV genes into 4 clusters, I, II, III and IV. Chromosomal distribution, gene structure and motif composition were subsequently investigated. The 26 TaRAV genes were unevenly distributed on 21 chromosomes. After cloning and sequencing of 7 TaRAVs candidate genes the expression levels of two TaRAVs, TaRAV4 and TaRAV5, were validated through qPCR analyses in two salt-tolerant Iranian landraces of wheat. Our results showed that the TaRAV4 and TaRAV5 were co-expressed in wheat tissues and were highly correlated to salt tolerance indices such as the K+/Na+ ratio. Protein interaction revealed that the TaRAV4 and TaRAV5 were related to vital proteins such as PK4 and PP2C, and MYB and Zinc finger transcription factors, and Gigantea proteins. This study improved our knowledge of the RAV gene family function in wheat and the probable role of RAVs in salt tolerance mechanisms to improve crop production under changing environments. Also, the two relatively salt-tolerant landraces of wheat that were examined in this study could be suitable candidates for future breeding studies.

The Effect of Endogenous Hormones, Total Antioxidant and Total Phenol Changes on Regeneration of Barley Cultivars.

BACKGROUND: Barley (Hordeum vulgar L.) is a valuable platform for producing recombinant proteins. Before using different barley cultivars as an efficient platform for molecular farming, optimization of cultural conditions and studying the effective factors on the tissue culture are critical. OBJECTIVES: In this study, we evaluated callus induction, plant regeneration and changes in the levels of total antioxidant, total phenol and endogenous hormones of three Iranian barley cultivars (Reyhan, Yousef and Bahman) and Golden Promise cultivar. MATERIALS AND METHODS: We used immature embryos as explants on MS-based medium containing 3 mg.L-1 2,4-D for callus induction. Calluses were transferred to regeneration media with 2 mg.L-1 BAP. The levels of endogenous hormones were measured using High-Performance Liquid Chromatography system and total antioxidant and total phenols were determined using a spectrophotometer. RESULTS: We demonstrated that callus formation was very high in all cultivars (about 91%) and all immature embryo explants had the potential to produce embryogenic calluses. The present study also showed that the regeneration rates among the studied cultivars were very different and the Iranian cultivars showed lower regeneration percentages (about 1.4%) compared to Golden Promise cultivar (about 72.5%). The levels of endogenous hormones in Iranian cultivars and Golden Promise varied distinctly and significant differences in terms of total antioxidants and total phenols were found in the two groups. CONCLUSIONS: Accumulated evidence suggests that for successful regeneration of recalcitrant cultivars, external treatments should be done in a way to reduce the inhibitory effects of internal factors.

Detection of genomic regions associated with tiller number in Iranian bread wheat under different water regimes using genome-wide association study.

Two of the important traits for wheat yield are tiller and fertile tiller number, both of which have been thought to increase cereal yield in favorable and unfavorable environments. A total of 6,349 single nucleotide polymorphism (SNP) markers from the 15 K wheat Infinium array were employed for genome-wide association study (GWAS) of tillering number traits, generating a physical distance of 14,041.6 Mb based on the IWGSC wheat genome sequence. GWAS analysis using Fixed and random model Circulating Probability Unification (FarmCPU) identified a total of 47 significant marker-trait associations (MTAs) for total tiller number (TTN) and fertile tiller number (FTN) in Iranian bread wheat under different water regimes. After applying a 5% false discovery rate (FDR) threshold, a total of 13 and 11 MTAs distributed on 10 chromosomes were found to be significantly associated with TTN and FTN, respectively. Linked single nucleotide polymorphisms for IWB39005 (2A) and IWB44377 (7A) were highly significantly associated (FDR < 0.01) with TTN and FTN traits. Moreover, to validate GWAS results, meta-analysis was performed and 30 meta-QTL regions were identified on 11 chromosomes. The integration of GWAS and meta-QTLs revealed that tillering trait in wheat is a complex trait which is conditioned by the combined effects of minor changes in multiple genes. The information provided by this study can enrich the currently available candidate genes and genetic resources pools, offering evidence for subsequent analysis of genetic adaptation of wheat to different climatic conditions of Iran and other countries.

Different carbon sources and their concentrations change alkaloid production and gene expression in Catharanthus roseus shoots in vitro.

To compare the effects of different carbon sources on physiological aspects, especially medicinal alkaloid biosynthesis and related gene expression in Catharantus roseus (L.) G.Don, we employed sucrose and sorbitol with two concentrations (87.64 mM, the equimolar concentration of sucrose in MS basal medium, and 150 mM) on the plant's shoots in vitro in presence of 100 µM methyl jasmonate. The production of plant alkaloids including vincristine, vinblastine, ajmalicine, vindoline and catharantine and their biosynthetic and regulatory gene expression was measured. Both treatments had incremental effects on alkaloid production, upregulated the mitogen-activated protein kinase3 (MAPK3) and a downstream responsive transcription factor, ORCA3, which resulted in elevated transcript contents of the important genes in terpenoid indol alkaloids biosynthetic pathway including peroxidase1 (PRX1), geissoschizine synthase (GS), strictosidine synthase (STR) and deacetylvindoline acetyltransferase (DAT). Defensive responses such as antioxidant enzymes (catalase, peroxidase and superoxide dismutase) activities and non-enzymatic metabolites (total phenolics, flavonoids and carotenoids) contents increased under both treatments but the effects of sorbitol were stronger. Reduced fresh weight and chlorophylls contents, increased malondialdehyde (MDA) and carotenoid contents were shown after a week under all employed treatments. It seems that replacement of sucrose with sorbitol and also, increased concentrations of both carbon sources via increasing osmotic pressure make stressful conditions for the plant especially in longer times.

Analysis of transcriptional responses in root tissue of bread wheat landrace (Triticum aestivum L.) reveals drought avoidance mechanisms under water scarcity.

In this study, high-throughput sequencing (RNA-Seq) was utilized to evaluate differential expression of transcripts and their related genes involved in response to terminal drought in root tissues of bread wheat landrace (L-82) and drought-sensitive genotype (Marvdasht). Subsets of 460 differentially expressed genes (DEGs) in drought-tolerant genotype and 236 in drought-sensitive genotype were distinguished and functionally annotated with 105 gene ontology (GO) terms and 77 metabolic pathways. Transcriptome profiling of drought-resistant genotype "L-82" showed up-regulation of genes mostly involved in Oxidation-reduction process, secondary metabolite biosynthesis, abiotic stress response, transferase activity and heat shock proteins. On the other hand, down-regulated genes mostly involved in signaling, oxidation-reduction process, secondary metabolite biosynthesis, auxin-responsive protein and lipid metabolism. We hypothesized that the drought tolerance in "L-82" was a result of avoidance strategies. Up-regulation of genes related to the deeper root system and adequate hydraulic characteristics to allow water uptake under water scarcity confirms our hypothesis. The transcriptomic sequences generated in this study provide information about mechanisms of acclimation to drought in the selected bread wheat landrace, "L-82", and will help us to unravel the mechanisms underlying the ability of crops to reproduce and keep its productivity even under drought stress.

Alkaloids production and antioxidant properties in Catharanthus roseus (L.) G. Don. shoots and study of alkaloid biosynthesis-related gene expression levels in response to methyl jasmonate and putrescine treatments as eco-friendly elicitors.

INTRODUCTION: This study aimed to determine the effects of methyl jasmonate (MJ) combined with putrescine as eco-friendly elicitors on secondary metabolism and gene expression of alkaloid biosynthetic pathway in Catharanthus roseus in vifro-propagated shoots. METHODS: The expression of mitogen-activated protein kinase 3 and the transcription factor, octadecanoid-responsive Catharanthus AP2-domain3, upstream of plant alkaloids' biosynthetic pathway, and of key genes in the pathway (CrPRXl, STR, DAT, and GS) are investigated as well using qRt-PCR. Antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities and non-enzymatic antioxidants (phenolics, flavonoids, and carotenoids) contents have studied to determine the stress levels of the plant by spectrophotometer. RESULTS: Results showed increased contents of non-enzymatic antioxidants after 4-8 hr and enzymatic antioxidants activities after 24 hr. Alkaloids contents increased mostly after 1 week. The investigated signaling genes upregulated after 8 hr and biosynthetic genes after 24 hr of treatments. Combined treatments had more positive effects on gene expression levels, antioxidant responses, and secondary metabolite production than MJ individually. DISCUSSION: Increased effects of combined elicitor on genes expression may be due to cross talks between their signaling pathways. Combination of MJ and putrescine can be used as an eco-friendly elicitor for enhancing the production of economically important alkaloids in C. roseus.

A Study to Assess the Role of Gluten Encoded Genes and Their Regulatory Elements in Bread Making Quality of Wheat.

BACKGROUND: Quality of bread baking is affected by gluten genes and balance between their expressions. Hence, it is necessary for a comprehensive research to study and compare all gluten genes and their regulating elements simultaneously. OBJECTIVES: The aim of this study was to evaluate the molecular mechanism of bread quality at the level of coding genes and regulating elements via comparative transcriptome analysis of two extreme wheat cultivars. MATERIALS AND METHODS: RNAs were extracted from the grain of two wheat cultivars with high (Pishtaz) and low (Navid) bread making qualities, collected during endosperm development at five stages. mRNAs were sequenced and gluten transcripts were assessed to find differentially expressed genes. Then, transcription factors interacting with gluten genes were detected and evaluated for expression. RESULTS: Results showed that Ɣ-gliadin and LMW-GS genes had a higher expression in Pishtaz and Navid, respectively. Most identified transcription factors were active at the early stage of growth and it seemed that NAC and ERF transcription factors had significant roles in regulating genes with different expressions. There was no significant difference in the expression level of NACs between two cultivars. It is proposed that the ERF transcription factor which classified as BREB2C transcription factor could control the expression of LMW-GS genes in two cultivars and functionally act as a repressor for their target genes. CONCLUSION: The priority of Pishtaz wheat cultivar in bread quality originated from high expression levels of Ɣ-gliadin gene and ERF transcription factor.

Changes in medicinal alkaloids production and expression of related regulatory and biosynthetic genes in response to silver nitrate combined with methyl jasmonate in Catharanthus roseus in vitro propagated shoots.

Antihypertensive compound ajmalicine and antileukemic vincristine and vinblastine are three important terpenoid indole alkaloids produced by Catharanthus roseus (Apocynaceae). This study has been done to investigate the effects of methyl jasmonate (100 µM) and silver nitrate (50 and 100 µM) individually and simultaneously on the production of mentioned important medicinal alkaloids (vincristine, vinblastine, ajmalicine, vindoline and catharanthine) and the expression profile of related regulatory and biosynthetic genes in micropropagated shoots of C. roseus. The effects of these treatments are also investigated on non-enzymatic defensive metabolites (total phenolics, flavonoids and carotenoids) and antioxidant enzymes activities (peroxidase, EC 1.11.1.7, catalase, EC 1.11.1.6 and superoxide dismutase, EC 1.15.1.1). Changes of dry weight, quantity of lipid peroxidation, and photosynthetic pigments contents have been measured as well. The results showed increased contents of alkaloids and expression levels of investigated regulatory (Mitogen-activated protein kinase3 and Octadecanoid-responsive Catharanthus AP2-domain3) and biosynthetic (strictosidine synthase, geissoschizine synthase, deacetylvindoline acetyltransferase and peroxidase1) genes under the employed treatments. The maximum yields of these alkaloids and the highest levels of the mentioned genes expression were observed under 100 µM methyl jasmonate in combination with 100 µM of AgNO3 after seven days. The employed treatments induced increased lipid peroxidation, higher levels of enzymatic antioxidants activities and more production of non-enzymatic defensive metabolites which shows activity of plant defensive system. The results suggest that silver nitrate and methyl jasmonate signalling pathways may have cross talks and their simultaneous application make an effective combination for elicitation of medicinal alkaloids biosynthesis in C. roseus micropropagated shoots.

Production and Partial Purification of the Grapevine Fanleaf Virus Coat Protein 42 Polyclonal Antibody Against Inclusion Body Expressed in Escherichia coli.

BACKGROUND: Expression of virus coat protein (CP) in Escherichia coli often leads to production of partially folded aggregated proteins which are called inclusion bodies. Grapevine fanleaf virus (GFLV) is one of the most serious and widespread grapevine virus diseases around the world and in Iran. OBJECTIVE: The main objective of this study was to find a simple and brief method for producing polyclonal antibodies (PAbs) to be used for immunodiagnosis of GFLV. MATERIAL AND METHODS: An antigenic determinant in GFLV CP gene was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain BL21 (DE3).The recombinant coat protein of GFLV (CP42) was expressed and characterized by SDS-PAGE and western blot analysis using commercial anti-GFLV antibody. Expression of the CP was detected in the form of inclusion bodies in insoluble cytoplasmic fraction. Then, the inclusion bodies were isolated from the bacterial cells and injected into rabbits for PAbs production. The reaction of the antiserum was checked by ELISA assay. In order to analyze efficiency of the produced PAbs, first the infected and uninfected grapevine samples were confirmed based on morphological symptoms then the indirect plate- trapped antigen Enzyme-linked Immunosorbent Assay (IPTA-ELISA) was applied using the commercial anti GFLV antibody. In the next ELISA assay, efficiency of the raised polyclonal antibody was compared with commercial one. RESULTS: The expression of recombinant CP42 induced by IPTG was confirmed by the band of 42 kDa in SDS-PAGE and western blot. The antiserum of purified inclusion body immunized rabbit was reacted with CP42 and GFLV infected Grapevine samples. The results revealed an acceptable efficacy for prepared antibodies compared to that of commercial antibody. CONCLUSIONS: It was evident that the recombinant coat protein in the form of inclusion bodies can be prepared and used as the antigen for immunizing animals in order to produce PAbs.

Responses of wheat plants to interactions of 24-epibrassinolide and Glomus mosseae in saline condition.

This study was designed to investigate the possible effects of 24-Epibrassinolide (BR), arbuscular mycorrhizal (AM) fungus, Glomus mosseae, singularly and collectively under salt stress in wheat (Triticum aestivum L.) plants. After foliar spraying of mycorrhizal and non-mycorrhizal plants by 5 µM epibrassinolide (24-Epi), they were treated with 0 and 150 mM NaCl for 2 weeks and then harvested. The results showed interactions of G. mosseae and 24-Epi could alleviate the adverse effects of salinity by improving relative water content (RWC) of leaves (62%), relative growth rate (40.74%), shoot fresh weights (39.83%) and shoot phosphorous content (63.93%), stimulating leaf enzymatic antioxidant activities including catalase (2.24 fold) and ascorbate peroxidase (2.18 fold) as well as malondialdehyde (36.17%) and H2O2 concentrations (49.74%) as compared to those of NaCl treatments. Moreover, mycorrhizal dependency of root dry weight (2%) and phosphorus concentration (0.4%) increased with AM infection and 24-Epi application under saline condition. Leaf RWC, also, negatively correlated with membrane electrolyte leakage. Furthermore, the greatest mitigating effects were observed in mycorrhizal plants subjected to NaCl and 24-Epi. This study indicated that 24-Epi application and AM fungi may synergistically mitigate harmful impacts of salinity in wheat plants.

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Polymorphisms in the AOX2 gene are associated with the rooting ability of olive cuttings.

KEY MESSAGE: Different rooting ability candidate genes were tested on an olive cross progeny. Our results demonstrated that only the AOX2 gene was strongly induced. OeAOX2 was fully characterised and correlated to phenotypical traits. The formation of adventitious roots is a key step in the vegetative propagation of trees crop species, and this ability is under strict genetic control. While numerous studies have been carried out to identify genes controlling adventitious root formation, only a few loci have been characterised. In this work, candidate genes that were putatively involved in rooting ability were identified in olive (Olea europaea L.) by similarity with orthologs identified in other plant species. The mRNA levels of these genes were analysed by real-time PCR during root induction in high- (HR) and low-rooting (LR) individuals. Interestingly, alternative oxidase 2 (AOX2), which was previously reported to be a functional marker for rooting in olive cuttings, showed a strong induction in HR individuals. From the OeAOX2 full-length gene, alleles and effective polymorphisms were distinguished and analysed in the cross progeny, which were segregated based on rooting. The results revealed a possible correlation between two single nucleotide polymorphisms of OeAOX2 gene and rooting ability.

Metabolic engineering of morphinan alkaloids by over-expression of codeinone reductase in transgenic hairy roots of Papaver bracteatum, the Iranian poppy.

Papaver bracteatum has a high content of thebaine. It is used as an alternative to P. somniferum for the production of benzylisoquinoline alkaloid. Papaver bracteatum was genetically engineered to over-express codeinone reductase gene in hairy root cultures. Transcript level of the codeinone reductase gene in transgenic hairy root lines increased up to ten- and 24-fold in comparison with hairy roots without CodR over-expression and wild type roots, respectively. Codeine was produced at (0.04 % dry wt) and morphine was at (0.28 % dry wt) in the transgenic hairy root lines. Papaver bracteatum hairy roots expressing CodR gene thus have a high potential to produce morphinan alkaloids.

Penconazole induced changes in photosynthesis, ion acquisition and protein profile of Mentha pulegium L. under drought stress.

Effect of penconazole (PEN) treatment on drought-stressed Mentha pulegium L. plants was investigated. Six weeks after sowing, seedlings were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity (FC) with or without PEN (15 mg l(-1)) for 4 weeks. Results showed that the seedlings at 75 % FC showed maximum growth and water supply lower than 75 % FC was the threshold of drought-initiated negative effects on seedling growth. Drought stress significantly induced proline and carbohydrate contents and the decreased chlorophyll, photosynthesis parameters, soluble proteins and ion accumulations. Exogenous PEN increased the growth parameters, pigments, photosynthesis and ion accumulations in drought stressed and unstressed plants, but the effects of PEN were more significant under water deficit conditions. PEN also reduced the negative effects of drought by osmotic balance and protein accumulations. Electrophoretic patterns indicated that PEN treatment increased the intensity of some protein bands with the molecular weights of 30 kDa in shoot and 31 kDa in roots, and several new protein bands with the molecular masses between 116 and 14 kDa appeared in leaves, shoots and roots. These results suggest that the PEN application can be a useful tool in alleviation of effects of drought stress in M. pulegium plants.