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1.  Effects of Zn Fertilization on Hordein Transcripts at Early Developmental Stage of Barley Grain and Correlation with Increased Zn Concentration in the Mature Grain 
PLoS ONE  2014;9(9):e108546.
Zinc deficiency is causing malnutrition for nearly one third of world populations. It is especially relevant in cereal-based diets in which low amounts of mineral and protein are present. In biological systems, Zn is mainly associated with protein. Cereal grains contain the highest Zn concentration during early developmental stage. Although hordeins are the major storage proteins in the mature barley grain and suggested to be involved in Zn binding, very little information is available regarding the Zn fertilization effects of hordein transcripts at early developmental stage and possible incorporation of Zn with hordein protein of matured grain. Zinc fertilization experiments were conducted in a greenhouse with barley cv. Golden Promise. Zn concentration of the matured grain was measured and the results showed that the increasing Zn fertilization increased grain Zn concentration. Quantitative real time PCR showed increased level of total hordein transcripts upon increasing level of Zn fertilization at 10 days after pollination. Among the hordein transcripts the amount of B-hordeins was highly correlated with the Zn concentration of matured grain. In addition, protein content of the matured grain was analysed and a positive linear relationship was found between the percentage of B-hordein and total grain Zn concentration while C-hordein level decreased. Zn sensing dithizone assay was applied to localize Zn in the matured grain. The Zn distribution was not limited to the embryo and aleurone layer but was also present in the outer part of the endosperm (sub-aleurone layers) which known to be rich in proteins including B-hordeins. Increased Zn fertilization enriched Zn even in the endosperm. Therefore, the increased amount of B-hordein and decreased C-hordein content suggested that B-hordein upregulation or difference between B and C hordein could be one of the key factors for Zn biofortification of cereal grains due to the Zn fertilization.
PMCID: PMC4177403  PMID: 25250985
2.  Barley HvHMA1 Is a Heavy Metal Pump Involved in Mobilizing Organellar Zn and Cu and Plays a Role in Metal Loading into Grains 
PLoS ONE  2012;7(11):e49027.
Heavy metal transporters belonging to the P1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.
PMCID: PMC3498361  PMID: 23155447
3.  Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development 
BMC Plant Biology  2012;12:184.
Cereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.
We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and γ-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.
Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences.
PMCID: PMC3492166  PMID: 23043496
SYBR Green; High homology multigene families; Transcript abundance; Hordeins
4.  The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application 
BMC Plant Biology  2010;10:239.
The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell suspension culture from both species.
We established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we chose to study the impact of selected abiotic stress elicitors at the physiological, biochemical and molecular level. We report the results of osmotic stress imposed by NaCl and PEG. As proline is an important osmoprotectant of the cereal cells, colorimetric assay for proline detection was developed for small volumes (200 μl). We performed RT-PCR experiments to study the change in the expression of the genes encoding Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Δ1-pyrroline-5-carboxylate reductase (PC5R) in response to abiotic stress.
We found differences between the wheat and barley suspension cultures, barley being more tolerant to the applied osmotic stresses. We suggested a model to explain the obtained differences in stress tolerance between the two species. The suspension cell cultures have proven useful for determining changes in proline concentration and expression level of genes (P5CS, P5CR) under various treatments and we suggest that the cells can be used as a model host system to study gene expression and regulation in monocots.
PMCID: PMC3017856  PMID: 21054876
5.  A pathway-specific microarray analysis highlights the complex and co-ordinated transcriptional networks of the developing grain of field-grown barley 
Journal of Experimental Botany  2008;60(1):153-167.
The aim of the study was to describe the molecular and biochemical interactions associated with amino acid biosynthesis and storage protein accumulation in the developing grains of field-grown barley. Our strategy was to analyse the transcription of genes associated with the biosynthesis of storage products during the development of field-grown barley grains using a grain-specific microarray assembled in our laboratory. To identify co-regulated genes, a distance matrix was constructed which enabled the identification of three clusters corresponding to early, middle, and late grain development. The gene expression pattern associated with the clusters was investigated using pathway-specific analysis with specific reference to the temporal expression levels of a range of genes involved mainly in the photosynthesis process, amino acid and storage protein metabolism. It is concluded that the grain-specific microarray is a reliable and cost-effective tool for monitoring temporal changes in the transcriptome of the major metabolic pathways in the barley grain. Moreover, it was sensitive enough to monitor differences in the gene expression profiles of different homologues from the storage protein families. The study described here should provide a strong complement to existing knowledge assisting further understanding of grain development and thereby provide a foundation for plant breeding towards storage proteins with improved nutritional quality.
PMCID: PMC3298879  PMID: 19015218
Amino acid metabolism; cDNA microarray; field trial; hordein; Hordeum vulgare; storage proteins
6.  Transformation of Rhizobia with Broad-Host-Range Plasmids by Using a Freeze-Thaw Method 
Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in Mesorhizobium loti without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.
PMCID: PMC1393188  PMID: 16517691
7.  The trans-Acting Protein Interacting with the DNA Motif Proximal to the Transcriptional Start Site of Plant l-Asparaginase Is Bacterial Sarcosine Oxidase 
Journal of Bacteriology  2004;186(3):811-817.
A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the l-asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037). Nodulation tests were performed by using different Fix− Tn5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase α subunit. This was confirmed by expression of the gene encoding the M. loti α subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.
PMCID: PMC321473  PMID: 14729708

Results 1-7 (7)