A pathogenesis related-10 protein CaARP functions as aldo/keto reductase to scavenge cytotoxic aldehydes

Pathogenesis related-10 (PR-10) proteins are present as multigene family in most of the higher plants. The role of PR-10 proteins in plant is poorly understood. A sequence analysis revealed that a large number of PR-10 proteins possess conserved motifs found in aldo/keto reductases (AKRs) of yeast and fungi. We took three PR-10 proteins, CaARP from chickpea, ABR17 from pea and the major pollen allergen Bet v1 from silver birch as examples and showed that these purified recombinant proteins possessed AKR activity using various cytotoxic aldehydes including methylglyoxal and malondialdehyde as substrates and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as co-factor. Essential amino acids for this catalytic activity were identified by substitution with other amino acids. CaARP was able to discriminate between the reduced and oxidized forms of NADP independently of its catalytic activity and underwent structural change upon binding with NADPH. CaARP protein was preferentially localized in cytosol. When expressed in bacteria, yeast or plant, catalytically active variants of CaARP conferred tolerance to salinity, oxidative stress or cytotoxic aldehydes. CaARP-expressing plants showed lower lipid peroxidation product content in presence or absence of stress suggesting that the protein functions as a scavenger of cytotoxic aldehydes produced by metabolism and lipid peroxidation. Our result proposes a new biochemical property of a PR-10 protein

Investigation of Genes Encoding Calcineurin B-Like Protein Family in Legumes and Their Expression Analyses in Chickpea (<i>Cicer arietinum</i> L.)

<div><p>Calcium ion (Ca²⁺) is a ubiquitous second messenger that transmits various internal and external signals including stresses and, therefore, is important for plants’ response process. Calcineurin B-like proteins (CBLs) are one of the plant calcium sensors, which sense and convey the changes in cytosolic Ca²⁺-concentration for response process. A search in four leguminous plant (soybean, <i>Medicago truncatula</i>, common bean and chickpea) genomes identified 9 to 15 genes in each species that encode CBL proteins. Sequence analyses of CBL peptides and coding sequences (CDS) suggested that there are nine original CBL genes in these legumes and some of them were multiplied during whole genome or local gene duplication. Coding sequences of chickpea CBL genes (<i>CaCBL</i>) were cloned from their cDNAs and sequenced, and their annotations in the genome assemblies were corrected accordingly. Analyses of protein sequences and gene structures of CBL family in plant kingdom indicated its diverse origin but showed a remarkable conservation in overall protein structure with appearance of complex gene structure in the course of evolution. Expression of <i>CaCBL</i> genes in different tissues and in response to different stress and hormone treatment were studied. Most of the <i>CaCBL</i> genes exhibited high expression in flowers. Expression profile of <i>CaCBL</i> genes in response to different abiotic stresses and hormones related to development and stresses (ABA, auxin, cytokinin, SA and JA) at different time intervals suggests their diverse roles in development and plant defence in addition to abiotic stress tolerance. These data not only contribute to a better understanding of the complex regulation of chickpea CBL gene family, but also provide valuable information for further research in chickpea functional genomics.</p></div

Comparison of structures of CBL proteins from different species.

<p>Protein structure of calcineurin B of <i>Saccharomyces cerevisiae</i> (A) and CBL1 from, <i>Ostreococcus tauri</i> (B), <i>Chlorella variabilis</i> (C), <i>Physcomitrella patens</i> (D), chickpea (E) and <i>Arabidopsis</i> (F) were generated using Phyre2 remote homology modeling server. All five structures from different species were analysed by pyMOL program. All the structures were superimposed (G) to show presence of extra helix (in boxes) in CBL proteins of <i>Chlorella</i> and chickpea.</p

Relative transcript levels of chickpea <i>CBL</i> genes in response to different abiotic stress treatments.

<p>6 day-old chickpea seedlings were exposed to 250 mM NaCl (A), 20% PEG (B) and Cold at 4°C (C) and samples were harvested at different time interval as mentioned for relative expression studies by qRT-PCR and presented as heatmap. The scale bar represents relative expression values. Hierarchical clustering has been represented at left. Relative fold expression values are presented as bar diagram in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123640#pone.0123640.s004" target="_blank">S4 Fig</a>.</p

Characteristics and chromosomal locations of chickpea <i>CBL</i> genes.

<p>Characteristics and chromosomal locations of chickpea <i>CBL</i> genes.</p

Relative transcript levels of chickpea <i>CBL</i> genes in response to treatment with various phytohormones.

<p>Expression profiles of <i>CBL</i> genes in chickpea seedlings exposed to Abscisic acid (A), BAP (B), IAA (C), Salicylic acid (D) and Methyl Jasmonate (E) for different period by qRT-PCR and presented as heatmap. The scale bar represents relative expression value. Hierarchical clustering was played in data analysis. Relative fold expression values are presented as bar diagram in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123640#pone.0123640.s004" target="_blank">S4 Fig</a>.</p

Tissue specific gene expression analysis of chickpea <i>CBL</i> genes.

<p>For tissue specific expression studies, total RNA was isolated from different tissues of chickpea plants. cDNA were prepared and expression pattern of all chickpea <i>CBL</i> genes were analysed by qRT-PCR and presented as heatmap. Chickpea <i>elongation factor 1-α (EF-1α)</i> mRNA was used as internal control for normalization. The scale bar represents relative expression values. Hierarchical clustering has been shown at left. Relative fold expression values are presented as bar diagram in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123640#pone.0123640.s004" target="_blank">S4 Fig</a>.</p