The RST and PARP-like domain containing SRO protein family : Analysis of protein structure, function and conservation in land plants

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Transcriptomics and Functional Genomics of ROS-Induced Cell Death Regulation by RADICAL-INDUCED CELL DEATH1

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Not Available

Not AvailableSingle Nucleotide Polymorphisms in Alpha-Lactalbumin gene in Murrah and South Kanara Buffaloes (Bubalus bubalis).Not Availabl

Biochemical and molecular characterization of stress-induced β-carbonic anhydrase from a C<SUB>4</SUB> plant, Pennisetum glaucum

Genes encoding for many &#946;-carbonic anhydrases and their functions in various developmental processes are well established in lower plants, however, similar studies are limited in higher plants. We report the cloning and characterization of cDNA encoding for a &#946;-carbonic anhydrase (PgCA) from Pennisetum glaucum, a C4 crop plant. cDNA encoding 249 amino acids and its deduced amino acid sequence analysis revealed that is related to other plant &#946;-CA family members with an over all conserved architecture of a typical &#946;-CA protein. Phylogenetic analysis revealed that PgCA is evolutionarily very close to chloroplast &#946;-CA isoform. Signal sequence predicting programs identify a N-terminus putative chloroplast targeting sequence. Heterologous Escherichia coli expression system was utilized to overexpress recombinant PgCA, which showed high thermostability, an alkaline pH optima and dual activity with both reversible CO2 hydration and esterase activities. The &#946;-CAs studied so far possessed only CO2 hydration activity with no detectable esterase activity. Recombinant PgCA esterase activity is inhibited by standard CA inhibitors acetazolamide, methazolamide and azide. Subcellular immunostaining studies revealed a chloroplastic localization of PgCA protein. Expression of PgCA transcript is differentially up regulated in response to various abiotic stresses wherein its accumulation in Pennisetum leaves positively correlated with the intensity and duration of stress. Biochemical and transcript analyses suggest that PgCA may play a significant role in plant's adaptation to different abiotic stresses in addition to the previously recognized role of replenishing the CO2 supply within plant cells

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation

Cluster analysis of gene expression in clean air and O₃-treated plants.

<p>Three-week-old plants were treated with 6 h of O₃ (350 nL L⁻¹) and samples harvested at 2 and 8 h after the start of the O₃ exposure. Expression of selected marker genes in each genotype was studied with qPCR and bootstrapped Bayesian hierarchical clustering was applied to log₂-transformed expression values in arbitrary units (A). Gene expression in mutant genotypes was compared to the respective Col-0 sample at each time point (2 h, 8 h) and log₂-transformed fold changes were calculated for O₃ treatment (B) and control plants (C). Asterisks mark statistical significance according to the linear model (P<0.10:“.”; P<0.05:“*”; P<0.01:“**”; P<0.001:“***”.).</p

Expression of selected marker genes in lesion mimic mutants.

<p>Samples from three lesion mimic genotypes (<i>acd2</i>, <i>acd5</i> and <i>lsd1</i>) 3 d after the transfer to LD were classified as samples from plants with no lesions (labelled 0); leaves with no lesions from plants with lesions (labelled −); leaves with lesions (labelled +). Averages of qPCR results (arbitrary units) from three biological replicates are shown; error bars depict standard deviation. Asterisks depict statistical significance (P<0.05) between to Col-0 (within 0 samples) or to 0 samples within the respective genotype (− and + samples).</p

Cluster analysis of genes differentially regulated in <i>rcd1</i> compared to Col-0.

<p>Bootstrapped Bayesian hierarchical clustering of 423 genes with at least two-fold changed expression (log₂ ratio ±1, q<0.05) in clean air or O₃-treated <i>rcd1</i> is shown. Data sets used were <i>rcd1</i> mutant grown in control conditions, O₃-treated Col-0, O₃-treated <i>rcd1</i> and several other available experiments related to stress signaling and cell death (see “<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004112#s3" target="_blank">Materials and Methods</a>” for the complete set of experiments). Six main clusters (I to VI) with subclusters (marked with a or b) were identified. GO and promoter element enrichment results are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004112#pgen.1004112.s004" target="_blank">Table S1</a>. Magenta and green indicate increased and decreased expression as log₂ ratio compared with untreated or wild type plants, respectively.</p