### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

Lang, Florian, Gillian L. Busch, Markus Ritter, Harald Volkl, Siegfried Waldegger, Erich Gulbins, and Dieter Haussinger. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. ...

Functional Significance of Cell Volume Regulatory Mechanisms

The production of reactive oxygen species (ROS), such as O2- and H2O2, is an unavoidable consequence of aerobic metabolism. In plant cells the mitochondrial electron transport chain (ETC) is a major site of ROS production. In addition to complexes I-IV, the plant mitochondrial ETC contains a non-proton-pumping alternative oxidase as well as two rotenone-insensitive, non-proton-pumping NAD(P)H dehydrogenases on each side of the inner membrane: NDex on the outer surface and NDin on the inner surface. Because of their dependence on Ca2+, the two NDex may be active only when the plant cell is stressed. Complex I is the main enzyme oxidizing NADH under normal conditions and is also a major site of ROS production, together with complex III. The alternative oxidase and possibly NDin(NADH) function to limit mitochondrial ROS production by keeping the ETC relatively oxidized. Several enzymes are found in the matrix that, together with small antioxidants such as glutathione, help remove ROS. The antioxidants are kept in a reduced state by matrix NADPH produced by NADP-isocitrate dehydrogenase and non-proton-pumping transhydrogenase activities. When these defenses are overwhelmed, as occurs during both biotic and abiotic stress, the mitochondria are damaged by oxidative stress.

PLANT MITOCHONDRIA AND OXIDATIVE STRESS: Electron Transport, NADPH Turnover, and Metabolism of Reactive Oxygen Species.

Caffeine and the central nervous system: mechanisms of action, biochemical, metabolic and psychostimulant effects.

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

http://science.sciencemag.org/content/sci/322/5901/590.full.pdf

TMEM16A, A Membrane Protein Associated with Calcium-Dependent Chloride Channel Activity

Exposure to cadmium (Cd2+) can result in cell death, but the molecular mechanisms of Cd2+ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl2 and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl2 treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by NG-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd2+-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd2+ cytotoxicity in plants.

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Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Transgenic tobacco and eggplants expressing the coding region of the iaaM gene from Pseudomonas syringae pv. savastanoi, under the control of the regulatory sequences of the ovule-specific DefH9 gene from Antirrhinum majus, showed parthenocarpic fruit development. Expression of the DefH9-iaaM chimeric transgene occurs during flower development in both tobacco and eggplant. Seedless fruits were produced by emasculated flowers. When pollinated, the parthenocarpic plants produced fruits containing seeds. In eggplant, the genetic manipulation allowed fruit set and growth under environmental conditions prohibitive for fruit setting in the untransformed line, which did not set fruit at all. Under normal environmental conditions, production of marketable fruits took place from pollinated and unpollinated transgenic flowers, while flowers of untransformed control plants did produce fruits of marketable size only from fertilized flowers.

Genetic engineering of parthenocarpic plants

Oxidative stress is a major challenge for all cells living in an oxygen-based world. Among reactive oxygen species, H2O2, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H2O2 in plant cells, it is necessary to have a reliable, specific and non-invasive methodology for its in vivo detection. Hence, the genetically encoded H2O2 sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)-based Cameleon Ca2+ indicator, D3cpv-KVK-SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca2+ increase is followed by Ca2+ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre- and post-bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca2+ rise in vivo stimulates catalase activity, increasing peroxisomal H2O2 scavenging efficiency.

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H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+)-dependent scavenging system.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2802%2902438-9

Nitric oxide affects plant mitochondrial functionality in vivo

The relationship between nitric oxide (NO) and salicylic acid (SA) was investigated in Arabidopsis thaliana. Here it is shown that SA is able to induce NO synthesis in a dose-dependent manner in Arabidopsis. NO production was detected by confocal microscopic analysis and spectrofluorometric assay in plant roots and cultured cells. To identify the metabolic pathways involved in SA-induced NO synthesis, genetic and pharmacological approaches were adopted. The analysis of the nia1,nia2 mutant showed that nitrate reductase activity was not required for SA-induced NO production. Experiments performed in the presence of a nitric oxide synthase (NOS) inhibitor suggested the involvement of NOS-like enzyme activity in this metabolic pathway. Moreover, the production of NO by SA treatment of Atnos1 mutant plants was strongly reduced compared with wild-type plants. Components of the SA signalling pathway giving rise to NO production were identified, and both calcium and casein kinase 2 (CK2) were demonstrated to be involved. Taken together, these results suggest that SA induces NO production at least in part through the activity of a NOS-like enzyme and that calcium and CK2 activity are essential components of the signalling cascade.

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Michela Zottini

Papers

Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Genetic engineering of parthenocarpic plants

H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca(2+)-dependent scavenging system.

Nitric oxide affects plant mitochondrial functionality in vivo

Salicylic acid activates nitric oxide synthesis in Arabidopsis