Regulation of Dual Activity of Ascorbate Peroxidase 1 From Arabidopsis thaliana by Conformational Changes and Posttranslational Modifications.

Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS)-scavenging enzyme, which catalyzes the removal of hydrogen peroxide (H2O2) to prevent oxidative damage. The peroxidase activity of APX is regulated by posttranslational modifications (PTMs), such as S-nitrosylation, tyrosine nitration, and S-sulfhydration. In addition, it has been recently reported that APX functions as a molecular chaperone, protecting rice against heat stress. In this study, we attempted to identify the various functions of APX in Arabidopsis and the effects of PTMs on these functions. Cytosol type APX1 from Arabidopsis thaliana (AtAPX1) exists in multimeric forms ranging from dimeric to high-molecular-weight (HMW) complexes. Similar to the rice APX2, AtAPX1 plays a dual role behaving both as a regular peroxidase and a chaperone molecule. The dual activity of AtAPX1 was strongly related to its structural status. The main dimeric form of the AtAPX1 protein showed the highest peroxidase activity, whereas the HMW form exhibited the highest chaperone activity. Moreover, in vivo studies indicated that the structure of AtAPX1 was regulated by heat and salt stresses, with both involved in the association and dissociation of complexes, respectively. Additionally, we investigated the effects of S-nitrosylation, S-sulfhydration, and tyrosine nitration on the protein structure and functions using gel analysis and enzymatic activity assays. S-nitrosylation and S-sulfhydration positively regulated the peroxidase activity, whereas tyrosine nitration had a negative impact. However, no effects were observed on the chaperone function and the oligomeric status of AtAPX1. Our results will facilitate the understanding of the role and regulation of APX under abiotic stress and posttranslational modifications.

Gene Expression and Proteomics Studies Suggest an Involvement of Multiple Pathways Under Day and Day-Night Combined Heat Stresses During Grain Filling in Wheat.

Recent weather fluctuations imposing heat stress at the time of wheat grain filling cause frequent losses in grain yield and quality. Field-based studies for understanding the effect of terminal heat stress on wheat are complicated by the effect of multiple confounding variables. In the present study, the effect of day and day-night combined heat stresses during the grain-filling stage was studied using gene expression and proteomics approaches. The gene expression analysis was performed by using real-time quantitative PCR (RT-qPCR). The expression of genes related to the starch biosynthetic pathway, starch transporters, transcription factors, and stress-responsive and storage proteins, at four different grain developmental stages, indicated the involvement of multiple pathways. Under the controlled conditions, their expression was observed until 28 days after anthesis (DAA). However, under the day stress and day-night stress, the expression of genes was initiated earlier and was observed until 14 DAA and 7 DAA, respectively. The protein profiles generated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS/MS) showed a differential expression of the proteins belonging to multiple pathways that included the upregulation of proteins related to the translation, gliadins, and low-molecular-weight (LMW) glutenins and the downregulation of proteins related to the glycolysis, photosynthesis, defense, and high-molecular-weight (HMW) glutenins. Overall, the defense response to the day heat stress caused early gene expression and day-night heat stress caused suppression of gene expression by activating multiple pathways, which ultimately led to the reduction in grain-filling duration, grain weight, yield, and processing quality.

Functional properties and the oligomeric state of alkyl hydroperoxide reductase subunit F (AhpF) in Pseudomonas aeruginosa.

Alkyl hydroperoxide reductase subunit F (AhpF) is a well-known flavoprotein that transfers electrons from pyridine nucleotides to the peroxidase protein AhpC via redox-active disulfide centers to detoxify hydrogen peroxide. However, study of AhpF has historically been limited to particular eubacteria, and the connection between the functional and structural properties of AhpF remains unknown. The present study demonstrates the dual function of Pseudomonas aeruginosa AhpF (PaAhpF) as a reductase and a molecular chaperone. It was observed that the functions of PaAhpF are closely linked with its structural status. The reductase and foldase chaperone function of PaAhpF predominated for its low-molecular-weight (LMW) form, whereas the holdase chaperone function of PaAhpF was found associated with its high-molecular-weight (HMW) complex. Further, the present study also demonstrates the multiple function of PaAhpF in controlling oxidative and heat stresses in P. aeruginosa resistance to oxidative and heat stress.

Novel functions of peroxiredoxin Q from Deinococcus radiodurans R1 as a peroxidase and a molecular chaperone.

Deinococcus radiodurans R1 is extremely resistant to ionizing radiation and oxidative stress. In this study, we characterized DR0846, a candidate peroxiredoxin in D. radiodurans. DR0846 is a peroxiredoxin Q containing two conserved cysteine residues. DR0846 exists mainly in monomeric form with an intramolecular disulfide bond between the two cysteine residues. We found that DR0846 functions as a molecular chaperone as well as a peroxidase. A mutational analysis indicates that the two cysteine residues are essential for enzymatic activity. A double-deletion mutant lacking DR0846 and catalase DR1998 exhibits decreased oxidative and heat shock stress tolerance with respect to the single mutants or the wild-type cells. These results suggest that DR0846 contributes to resistance against oxidative and heat stresses in D. radiodurans.

Biochemical Adaptations in Zea mays Roots to Short-Term Pb(2+) Exposure: ROS Generation and Metabolism.

The present study investigated the effect of lead (0, 16, 40 and 80 mg L(-1) Pb2+) exposure for 3, 12 and 24 h on root biochemistry in hydroponically grown Zea mays (maize). Pb2+ exposure (80 mg L(-1)) enhanced malondialdehyde content (239%-427%), reactive carbonyl groups (425%-512%) and H2O2 (129%-294%) accumulation during 3-24 h of treatment, thereby indicating cellular peroxidation and oxidative damage. The quantitative estimations were in accordance with in situ detection of ROS generation (using 2',7'-dichlorodihydrofluorescein diacetate dye) and H2O2 accumulation. Pb2+ treatment significantly reduced ascorbate and glutathione content during 3-24 h of exposure. On the contrary, levels of non-protein thiols were enhanced by 3-11.8 time over control in response to 16-80 mg L(-1) Pb2+ treatment, after 24 h. A dose-dependent induction in ascorbate peroxidase and lipoxygenase enzyme activity was observed in Z. mays roots. The activities of ascorbate-recycling enzymes (dehydroascorbate reductase and monodehydroascorbate reductase) were significantly increased in relation to concentration and duration of Pb2+ treatment. The study concludes that Pb2+-exposure induces ROS-mediated oxidative damage during early period of exposure despite the upregulation of enzymes of ascorbate-glutathione cycle.