Amir Mousavi

Glycine-rich proteins: a class of novel proteins.

Glycine-rich proteins (GRPs) containing more than 60% glycine have been found in different tissues from many eukaryotic species. Despite the availability of literature on different groups of GRPs, there are few reports in which they are all considered and compared together. Some of these proteins are components of the cell walls of many higher plants. In most cases, it has been shown that they are accumulated in the vascular tissues and that their synthesis is part of the plant’s defense mechanism. Other distinct types of GRPs are characterized by having structures and functions similar to animal cytokeratins or by a domain with typical RNA-binding motifs. The availability of cloned GRP genes facilitates the study of the function of this diverse class of proteins, which is expected to enhance the understanding of cell physiology.

Efficient regeneration of Brassica napus L. hypocotyls and genetic transformation by Agrobacterium tumefaciens

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm−3) and thidiazuron (0.0, 0.15 and 0.30 mg dm−3) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm−3 benzyladenine and 0.3 mg dm−3 thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8 %. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm−3 of indolebutyric acid and 10 mg dm−3 kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.

An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation

The physical barrier imposed by the thick microspore wall constitutes an obstacle for an efficient Agrobacterium-mediated transformation of vacuolate microspores prior to androgenic induction and haploid embryogenic commitment. It is thus necessary to implement additional methods to overcome this drawback. In this study, we focused on the optimization of a protocol to allow for the exogenous DNA to enter the microspore in an efficient manner. We tested different options, based on microprojectile bombardment, to be applied prior to agroinfiltration. From them, the best results were obtained through co-transformation by microspore bombardment with DNA-coated microprojectile particles, followed by Agrobacterium tumefaciens infection. This method provides an efficient means to integrate extraneous DNA into rapeseed microspores prior to androgenesis induction.

Cloning and characterization of the ribosomal protein L3 (RPL3) gene family from Triticum aestivum.

Plant pathogenic fungi of the genus Fusarium can cause severe diseases on small grain cereals and maize. The contamination of harvested grain with Fusarium mycotoxins is a threat to human and animal health. In wheat production of the toxin deoxynivalenol (DON), which inhibits eukaryotic protein biosynthesis, is a virulence factor of Fusarium, and resistance against DON is considered to be part of Fusarium resistance. Previously, single amino acid changes in RPL3 (ribosomal protein L3) conferring DON resistance have been described in yeast. The goal of this work was to characterize the RPL3 gene family from wheat and to investigate the potential role of naturally existing RPL3 alleles in DON resistance by comparing Fusarium-resistant and susceptible cultivars. The gene family consists of three homoeologous alleles of both RPL3A and RPL3B, which are located on chromosomes 4A (RPL3-B2), 4B (RPL3-B1), 4D (RPL3-B3), 5A (RPL3-A3), 5B (RPL3-A2) and 5D (RPL3-A1). Alternative splicing was detected in the TaRPL3-A2 gene. Sequence comparison revealed no amino acid differences between cultivars differing in Fusarium resistance. While using developed SNP markers we nevertheless found that one of the genes, namely, TaRPL3-A3 mapped close to a Fusarium resistance QTL (Qfhs.ifa-5A). The potential role of the RPL3 gene family in DON resistance of wheat is discussed.

New bacterial strain of the genus Ochrobactrum with glyphosate-degrading activity

Thirty bacterial strains with various abilities to utilize glyphosate as the sole phosphorus source were isolated from farm soils using the glyphosate enrichment cultivation technique. Among them, a strain showing a remarkable glyphosate-degrading activity was identified by biochemical features and 16S rRNA sequence analysis as Ochrobactrum sp. (GDOS). Herbicide (3 mM) degradation was induced by phosphate starvation, and was completed within 60 h. Aminomethylphosphonic acid was detected in the exhausted medium, suggesting glyphosate oxidoreductase as the enzyme responsible for herbicide breakdown. As it grew even in the presence of glyphosate concentrations as high as 200 mM, Ochrobactrum sp. could be used for bioremediation purposes and treatment of heavily contaminated soils.

Improved phosphate metabolism and biomass production by overexpression of AtPAP18 in tobacco

Limited availability of phosphate ion (Pi) reduces plant growth in natural ecosystems. Here, we report the functional effects of overexpressing an Arabidopsis thaliana purple acid phosphatase encoding gene, AtPAP18, in Nicotiana tabbacum as a crop model plant. Transgenic tobacco plants exhibited significant increases in acid phosphatase activity, total P and Pi contents leading to improved biomass production in both Pi-deficient and Pi-sufficient conditions. Transient expression of AtPAP18::green fluorescent fusion protein in onion epidermal cells indicated that AtPAP18 is a dual-targeted protein, which is detected mainly in the apoplast of the cells after 24 h and in the vacuole after 72 h. Possibly, AtPAP18 protein confers efficient retrieval of Pi from bonded extracellular compounds as well as expendable intracellular Pi-monoesters and anhydrides. These data clearly indicate that overexpression of AtPAP18 gene offers an effective approach for reducing the consumption of chemical Pi fertilizer through increased acquisition of soil Pi and mobilization of internal resources.

Detailed Investigations on the Solid Cell Culture and Antimicrobial Activities of the Iranian Arnebia euchroma

In pursuit of strong shikalkin-producing cell lines, seeds of the Iranian Arnebia euchroma were collected from Dena altitudes in the central Zagross. Chemical analysis showed that the dried root of the plant contained about 8.5% (w/w) shikalkin pigment. The root explants of the young plantlets, obtained from the germinated seeds, were used for establishing callus. Then, parameters effective on proliferation and pigment production of the resulting calli were studied in detail. Accordingly, two modified media called mLS and mM9 were optimized for propagation and pigment production, respectively. Using these media, the biomass of the A.euchroma calli was increased to 600%, and the pigment production reached to a maximum of 16.3 mg per gram of the wet biomass in a period of a subculture (21 days). Parallel to these experiments, the antimicrobial activity of shikalkin pigment was examined on some fungi and Gram-positive and Gram-negative bacteria. Results indicated that the pigment was almost ineffective on fungi and Gram-negative bacteria, but it was meaningfully effective against Micrococcus luteus.

Construction and functional analysis of pathogen-inducible synthetic promoters in Brassica napus

In this study, we selected two known pathogen-inducible cis-acting elements, F and E17, to construct synthetic pathogen-inducible promoters for analysis in transformed canola (Brassica napus L.). The synthetic promoter approach was used, which involved the insertion of dimers and combining two cis-acting elements (E17 and F) upstream of the minimal CaMV 35S promoter. Canola plants were transformed by three constructs, pGEE, pGFF, pGFFEE containing synthetic promoters (SP), SP-EE, SP-FF and SP-FFEE, respectively. Analyses of histochemical and fluorometric GUS expression indicated that synthetic promoters responded to fungal elicitors and phytohormone treatments. The SP-FF promoter showed high responses against methyl jasmonate and Sclerotinia sclerotiorum, while SP-EE demonstrated inducibility only in response to salicylic acid and Rhizoctonia solani. The SP-EE promoter similar to SP-FFEE, did not respond to S. sclerotiorum and methyl jasmonate. However, SP-FFEE was highly induced by R. solani elicitors and showed that the level of GUS expression was greater than that by either of E17 or F elements alone. These three synthetic promoters did not activate the expression of the reporter gene in response to cold, heat, UV and wounding.

Secondary embryogenesis and transient expression of the β-glucuronidase gene in hypocotyls of rapeseed microspore-derived embryos

Secondary embryogenesis from rapeseed microspore-derived embryos (MDEs) was studied in three Brassica napus L. cultivars Global, PF704 and Option. The best results in terms of secondary embryogenesis percentage obtained in cultures of Global and PF704 MDEs (75.88 and 65.97 %, respectively) and PF704 produced the highest number of secondary embryos per each primary embryo (14.91 ± 2.18). After optimization of physical parameters, rapeseed hypocotyls of MDEs were bombarded with microcarriers coated with a plasmid containing GUS reporter gene. The highest levels of transient GUS expression were obtained using bombardment with gold particles of 1.6 µm, at helium pressure of 9.3 MPa, a bombardment distance of 9 cm, chamber vacuum pressure of 7.1 × 10−6 kPa and single bombardment in bombardment medium containing 0.4 M mannitol.

Expression pattern and subcellular localization of Arabidopsis purple acid phosphatase AtPAP9.

Purple acid phosphatase (PAP; EC 3.1.3.2) enzymes are metallophosphoesterases that hydrolysis phosphate ester bonds in a wide range of substrates. Twenty-nine PAP-encoding loci have been identified in the Arabidopsis genome, many of which have multiple transcript variants expressed in response to diverse environmental conditions. Having analyzed T-DNA insertion mutants, we have provided strong pieces of evidence that AtPAP9 locus encodes at least two types of transcripts, designated as AtPAP9-1 and AtPAP9-2. These transcript variants expressed distinctly during the course of growth in medium containing sufficient phosphate or none. Further histochemical analysis by the use of AtPAP9-1 promoter fused to β-glucuronidase reporter gene indicated the expression of this gene is regulated in a tissue-specific manner. AtPAP9-1 was highly expressed in stipule and vascular tissue, particularly in response to fungal infection. Subcellular localization of AtPAP9-1:green fluorescent fusion protein showed that it must be involved in plasma membrane and cell wall adhesion.