Biosynthesis Of Peroxidase In Peanut Cells In Suspension Culture

Peroxidase (E.C.1.11.1.7) has been widely used as a marker of altered growth and development in plants. However, a definitive role of physiological significance has yet to be ascribed. This may be partially due to studies, that have been performed with a peroxidase molecule neither isolated to purity nor characterized from the system of investigation. A cationic peroxidase molecule (molecular weight 40 kD) had been purified to apparent homogenity and characterized from cultured peanut cells. Antibodies were raised against this peroxidase molecule in rabbits and used for monitoring biosynthetic studies on this peroxidase molecule in cultured peanut cells.;The cationic peroxidase constituted one sixth of the total proteins in the medium of cultured peanut cells. Peanut plant leaves also secreted the same peroxidase in to their intercellular spaces as determined by immunodiffusion assays. Cultured peanut cells synthesized ten fold more peroxidase than the peanut leaves.;By using the technique of differential centrifugation to isolate cell organelles in conjunction with immunoprecipitation it was shown that most of the newly synthesized peroxidase was found to be associated with the microsomal fraction. This peroxidase from the microsomal fraction was only released when microsomes were treated with high salt buffer (phosphate buffer with 0.8 M KCl). This indicated that the intracellular origin of the high ionic extract of peroxidase was from the microsomal pellet. Most of the newly synthesized peroxidase was associated with membrane bound polysomes, as determined by immunoprecipitation of polysomes, by using immunoaffinity purified IgGs against cationic peroxidase.;The heme moiety of peroxidase, was shown to be synthesized from glutamic acid and not from glycine and succinyl CoA, as is the case in animals. The heme moiety was identified as protoheme, based on mass spectrometry, and was synthesized in the mitochondria of cultured peanut cells. The heme was present in equimolar concentration to apoprotein and was found to be essential for peroxidative as well as IAA-oxidase activities of this peroxidase molecule.;No appreciable differences in the enzymatic activities associated with peroxidase could be detected between the purified cationic and anionic peroxidases from cultured peanut cells. Nevertheless, cationic form of peroxidase was the major isozyme that was secreted by cultured peanut cells. The significance of the secretion of the cationic form in relation to its mode of action was discussed

Quantitative expression analysis of selected COR genes reveals their differential expression in leaf and crown tissues of wheat (Triticum aestivum L.) during an extended low temperature acclimation regimen

A number of COR genes (COld-Regulated genes) have been implicated in the acquisition of low temperature (LT) tolerance in wheat (Triticum aestivum L.). This study compared the relative expression patterns of selected COR genes in leaf and crown tissues of wheat near-isogenic lines to increase understanding of the molecular mechanisms underlying LT acclimation. Reciprocal near-isogenic lines were generated such that the dominant Vrn-A1 and recessive vrn-A1 loci were interchanged in a spring cv. Manitou and a winter cv. Norstar. Phenological development, acquisition of LT tolerance, and WCS120 polypeptide accumulation in these genotypes proceeded at rates similar to those previously reported for 6 °C acclimation from 0 to 98 d. However, a differential accumulation of WCS120 polypeptide and expression of the COR genes Wcs120, Wcor410, and Wcor14 was observed in the leaf and crown tissues. COR gene transcript levels peaked at 2 d of the acclimation period in both tissues and differences among genotypes were most evident at this time. COR gene expression was highest for the LT-tolerant and lowest for the tender genotypes. However, expression rates were divergent enough in genotypes with intermediate hardiness that comparisons among tissues and/or times during acclimation often resulted in variable interpretations of the relative expression of the COR genes in the determination of LT tolerance. These observations emphasize the need to pay close attention to experimental conditions, sampling times, and genotype and tissue selection in experiments designed to identify the critical genetic components that interact to determine LT acclimation

Genome-wide gene expression analysis supports a developmental model of low temperature tolerance gene regulation in wheat (Triticum aestivum L.)

<p>Abstract</p> <p>Background</p> <p>To identify the genes involved in the development of low temperature (LT) tolerance in hexaploid wheat, we examined the global changes in expression in response to cold of the 55,052 potentially unique genes represented in the Affymetrix Wheat Genome microarray. We compared the expression of genes in winter-habit (winter Norstar and winter Manitou) and spring-habit (spring Manitou and spring Norstar)) cultivars, wherein the locus for the vernalization gene <it>Vrn-A1 </it>was swapped between the parental winter Norstar and spring Manitou in the derived near-isogenic lines winter Manitou and spring Norstar. Global expression of genes in the crowns of 3-leaf stage plants cold-acclimated at 6°C for 0, 2, 14, 21, 38, 42, 56 and 70 days was examined.</p> <p>Results</p> <p>Analysis of variance of gene expression separated the samples by genetic background and by the developmental stage before or after vernalization saturation was reached. Using gene-specific ANOVA we identified 12,901 genes (at <it>p </it>< 0.001) that change in expression with respect to both genotype and the duration of cold-treatment. We examined in more detail a subset of these genes (2,771) where expression was highly influenced by the interaction between these two main factors. Functional assignments using GO annotations showed that genes involved in transport, oxidation-reduction, and stress response were highly represented. Clustering based on the pattern of transcript accumulation identified genes that were up or down-regulated by cold-treatment. Our data indicate that the cold-sensitive lines can up-regulate known cold-responsive genes comparable to that of cold-hardy lines. The levels of expression of these genes were highly influenced by the initial rate and the duration of the gene's response to cold. We show that the <it>Vrn-A1 </it>locus controls the duration of gene expression but not its initial rate of response to cold treatment. Furthermore, we provide evidence that <it>Ta.Vrn-A1 </it>and <it>Ta.Vrt1 </it>originally hypothesized to encode for the same gene showed different patterns of expression and therefore are distinct.</p> <p>Conclusion</p> <p>This study provides novel insight into the underlying mechanisms that regulate the expression of cold-responsive genes in wheat. The results support the developmental model of LT tolerance gene regulation and demonstrate the complex genotype by environment interactions that determine LT adaptation in winter annual cereals.</p

Genotype and Growing Environment Interaction Shows a Positive Correlation between Substrates of Raffinose Family Oligosaccharides (RFO) Biosynthesis and Their Accumulation in Chickpea (Cicer arietinum L.) Seeds

To develop genetic improvement strategies to modulate raffinose family oligosaccharides (RFO) concentration in chickpea (Cicer arietinum L.) seeds, RFO and their precursor concentrations were analyzed in 171 chickpea genotypes from diverse geographical origins. The genotypes were grown in replicated trials over two years in the field (Patancheru, India) and in the greenhouse (Saskatoon, Canada). Analysis of variance revealed a significant impact of genotype, environment, and their interaction on RFO concentration in chickpea seeds. Total RFO concentration ranged from 1.58 to 5.31 mmol/100 g and from 2.11 to 5.83 mmol/100 g in desi and kabuli genotypes, respectively. Sucrose (0.60−3.59 g/100 g) and stachyose (0.18−2.38 g/ 100 g) were distinguished as the major soluble sugar and RFO, respectively. Correlation analysis revealed a significant positive correlation between substrate and product concentration in RFO biosynthesis. In chickpea seeds, raffinose, stachyose, and verbascose showed a moderate broad sense heritability (0.25−0.56), suggesting the use of a multilocation trials based approach in chickpea seed quality improvement programs

In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions

International audienceExpression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full- length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/ disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues