Ashraf Gholizadeh

Activation of phenylalanine ammonia lyase as a key component of the antioxidative system of salt-challenged maize leaves

Differential antioxidative activities were assessed in the leaves of two maize inbreds (A-180 and A-619) under salt stress and the subsequent recovery period. Total antioxidation test revealed that in both inbreds, this ability was sharply increased during stress period, but was slowly reverted back to the normal level during recovery. The enzymatic antioxidative analysis showed differential patterns in the activities of catalase, peroxidase and polyphenol oxidase in both maize inbreeds. Comparative analysis of the activity of phenylalanine ammonia lyase (PAL), a key enzyme at the gateway of propanoid biosynthetic pathway, suggested that propanoid compounds might be antioxidants of pivotal importance to the salt-challenged maize antioxidation system. As for drought-stressed plants, a PAL-dependent antioxidative strategy is proposed as a promising target for maize salt resistance engineering.

Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.

Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ∼57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome inactivating site. We nominate this short cDNA frag ment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.

Identification of DUF538 cDNA clone from Celosia cristata expressed sequences of nonstressed and stressed leaves.

DUF538 domain-containing protein family consists of several plant proteins of unknown functions. This protein family has already been discovered by genome annotation tools and cloned as an inducible gene product under various environmental stress conditions. For the first time, we presented a full length DUF538 cDNA (encoding 170 amino acid residues) clone, which was randomly isolated from Celosia cristata leaf cDNA library constructed under normal growth conditions and consistently amplified from leaf cDNA populations prepared from nonstressed and drought-stressed leaves. We predicted that a DUF538 gene product can be a putative candidate for common stress-related protein (regulatory factor) in the plant system. The nucleotide and deduced amino acid sequences of the isolated clone have been submitted to EMBL data bases under accession no. AJ535713.

Molecular analysis of maize cystatin expression as fusion product in Escherichia coli

Nowadays, plant cysteine proteinase inhibitors “namely phytocystatins” have attracted researchers towards the identification of their molecular structures and novel physiological functions. Their important roles in plant developmental processes and different stress responses have been well known. In spite of advances in the understanding of phytocystatins, we lack enough data concerning their heterologous expression especially in the forms of fusion products that are most important whether for biochemical, pharmacological or clinical studies. The present work describes an easy method of expression, purification and functional characterization in Escherichia coli of maize cystatin as a part of maltose-binding fusion protein. Assessments revealed that upon expression of fused product the total antioxidation status of the induced recombinant cells is increased. This result leads to question ‘Is there any parallel functional correlation between anti-proteolytic and anti-oxidative systems?’ However, the present research will open a gate for the new studies regarding the putative communicative roles of these systems that may be existing in the biological world.

DUF538 Protein Super Family is Predicted to be the Potential Homologue of Bactericidal/Permeability-Increasing Protein in Plant System

DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.

Induced expression of EcoRI endonuclease as an active maltose-binding fusion protein in Escherichia coli

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.

Effect of RNAi-mediated gene silencing of starch phosphorylase L and starch-associated R1 on cold-induced sweetening in potato

ABSTRACT To decrease the accumulation of reducing and non-reducing sugars in potato tubers stored at low temperature, a multiple gene-silencing vector pBIPhLSAR1-IR possessing a part of starch phosphorylase L and starch-associated R1 genes was constructed and transformed into potato (Solanum tuberosum L.) cultivars Agria and Marfona. Polymerase chain reaction (PCR) and southern blotting indicated that the RNAi construct was transformed successfully into the genome. Real-time reverse transcription PCR (RT-PCR) analysis showed that the expression level of starch phosphorylase L and starch-associated R1 genes was reduced in transgenic microtubers in relation to the non-transgenic plants. The accumulation of total and reducing sugars in transgenic microtubers was significantly decreased (up to 58% in line AM6) compared to the control. Repression of these genes decreased the phosphate content of starch and increased starch content in transgenic microtubers, implying that silencing of starch phosphorylase L and starch-associated R1 genes reduced starch breakdown during cold storage conditions.

Molecular cloning of an actinobacterial-type ClpS gene from Celosia cristata expression library

In proteobacterial cytosol, ClpS protein is known as a molecular adaptor for substrate selectivity and proteolytic activity of the ATP-dependent chaperone-protease complex, ClpAP. ClpA-related ClpS is a small protein usually encoded immediately upstream of ClpA in the genome of proteobacteria. Recent bioinformatics analysis has revealed the presence of cyanobacterial-type ClpS or ClpC-related ClpS in organisms lacking ClpA, including all the plant species sequenced to date. Here we report the identification of an actinobacterial homologue of the ClpS (possibly Clp-related) gene from a plant system. A cDNA, spanning 566 bp with a complete coding region corresponding to 132 amino acids, was isolated from a Celosia cristata expression library constructed on a λ TriplEX2 vector. This cDNA product was considered to be an ATP-dependent Clp protease adaptor and was designated as Celosia actinobacterial-type ClpS, since it contains a highly conserved domain belonging to the ClpS family of proteins from actinobacteria. Celosia ClpS is about 80% identical to actinobacterial ClpS proteins in its overall deduced amino acid sequence. Based on this finding, we may define a novel target of ATP-dependent Clp complex in a plant system or speculate the presence of a second type of molecular chaperone besides ClpC in plants, as predicted for actinobacteria.

Chlorophyll Binding Ability of Non-chloroplastic DUF538 Protein Superfamily in Plants

The type-1 water soluble chlorophyll binding proteins (WSCP1) have been generally known as chlorophyll extractors and transporters from the thylakoid membrane to the chloroplast envelope, where the membrane bound chlorophyllase catabolizes the chlorophyll. Despite the type-2 WSCP, WSCP1 has been known to be located in the chloroplasts of the green plants. In the present study, the non-chloroplastic protein superfamily containing domain of unknown function 538 (DUF538) as functional homologue of type-1 WSCP has been identified in plants. The structural similarities/differences and the cellular locations of Celosia cristata DUF538 and Chenopodium album WSCP1 were predicted by using computational tools and the chlorophyll binding abilities of their purified maltose binding protein-fused forms were estimated by maltose binding affinity method. It was predicted that despite CaWSCP1, CcDUF538 is a non-chloroplastic protein. The chlorophyll binding abilities of the recombinant fusion forms of test WSCP1 and DUF538 were estimated to be about 58 and 56%, respectively. Considering DUF538 as stress-induced protein, it was speculated that they may form complex with chlorophyll molecules or their hydrolyzed products out of chloroplasts to proceed the chlorophyll breakdown and nitrogen/carbon recycling in stress-challenged plants.