Myotubularins, PtdIns5P, and ROS in ABA-mediated stomatal movements in dehydrated Arabidopsis seedlings.

Myotubularins (MTMs) are lipid phosphoinositide 3-phosphate phosphatases and the product of their enzyme activity - phosphoinositide 5-phosphate (PtdIns5P) - functions as a signalling molecule in pathways involved in membrane dynamics and cell signalling. Two Arabidopsis genes, AtMTM1 and AtMTM2, encode enzymatically active phosphatases but although AtMTM1 deficiency results in increased tolerance to dehydration stress and a decrease in cellular PtdIns5P, the role of AtMTM2 is less clear, as it does not contribute to the PtdIns5P pool upon dehydration stress. Here we analysed the involvement of AtMTM1, AtMTM2 and PtdIns5P in the response of Arabidopsis seedlings to dehydration stress/ABA, and found that both AtMTM1 and AtMTM2 were involved but affected oppositely stomata movement and the accumulation of reactive oxygen species (ROS, e.g. H2O2). Acting as a secondary messenger in the ABA-induced ROS production in guard cells, PtdIns5P emerges as an evolutionarily conserved signalling molecule that calibrates cellular ROS under stress. We propose the biological relevance of the counteracting AtMTM1 and AtMTM2 activities is to balance the ABA-induced ROS accumulation and cellular homeostasis under dehydration stress.

Pest management through Bacillus thuringiensis (Bt) in a tea-silkworm ecosystem: status and potential prospects.

Bacillus thuringiensis (Bt) is a soil bacterium that forms spores containing crystals comprising one or more Cry or Cyt proteins having potential and specific insecticidal activity. Different strains of Bt produce different types of toxins, affecting a narrow taxonomic group of insects. Therefore, it is used in non-chemical pest management, including inherent pest resistance through GM crops. The specificity of action of Bt toxins reduces the concern of adverse effects on non-target species, a concern which remains with chemical insecticides as well. To make use of Bt more sustainable, new strains expressing novel toxins are actively being sought globally. Since Bt is successfully used against many pests including the lepidopteran pests in different crop groups, the insecticidal activity against Samia cynthia (Drury) (Eri silkworm) and Antheraea assamensis Helfer (Muga silkworm) becomes a concern in the state of Assam in India which is a predominantly tea- and silk-producing zone. Though Bt can be used as an effective non-chemical approach for pest management for tea pests in the same geographical region, yet, it may potentially affect the silk industry which depends on silkworm. There is a need to identify the potentially lethal impact (through evaluating their mortality potential) of local Bt strains on key silkworm species in North Eastern India. This will allow the use of existing Bt for which the silkworms have natural resistance. Through this review, the authors aim to highlight recent progress in the use of Bt and its insecticidal toxins in tea pest control and the potential sensitivity for tea- and silk-producing zone of Assam in India.

Subcellular localizations of Arabidopsis myotubularins MTM1 and MTM2 suggest possible functions in vesicular trafficking between ER and cis-Golgi.

The two Arabidopsis genes AtMTM1 and AtMTM2 encode highly similar phosphoinositide 3-phosphatases from the myotubularin family. Despite the high-level conservation of structure and biochemical activities, their physiological roles have significantly diverged. The nature of a membrane and the concentrations of their membrane-anchored substrates (PtdIns3P or PtdIns3,5P2) and/or products (PtdIns5P and PtdIns) are considered critical for determining the functional specificity of myotubularins. We have performed comprehensive analyses of the subcellular localization of AtMTM1 and AtMTM2 using a variety of specific constructs transiently expressed in Nicotiana benthamiana leaf epidermal cells under the control of 35S promoter. AtMTM1 co-localized preferentially with cis-Golgi membranes, while AtMTM2 associated predominantly with ER membranes. In a stark contrast with animal/human MTMs, neither AtMTM1 nor AtMTM2 co-localizes with early or late endosomes or with TGN/EE compartments, making them unlikely participants in the endosomal trafficking system. Localization of the AtMTM2 is sensitive to cold and osmotic stress challenges. In contrast to animal myotubularins, Arabidopsis myotubularins do not associate with endosomes. Our results suggest that Arabidopsis myotubularins play a role in the vesicular trafficking between ER exit sites and cis-Golgi elements. The significance of these results is discussed also in the context of stress biology and plant autophagy.

Crystal structures of nitroalkane oxidase: insights into the reaction mechanism from a covalent complex of the flavoenzyme trapped during turnover.

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We present the 2.2 A resolution crystal structure of NAO trapped during the turnover of nitroethane as a covalent N5-FAD adduct (ES*). The homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites in an orthorhombic space group. The electron density for the N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The structure of ES was used to solve the crystal structure of oxidized NAO at 2.07 A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A, and there are six subunits (1(1)/(2) holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E(ox)). The active-site structures of E(ox), EI, and ES* reveal a hydrophobic channel that extends from the exterior of the protein and terminates at Asp402 and the N5 position on the re face of the FAD. Thus, Asp402 is in the correct position to serve as the active-site base, where it is proposed to abstract the alpha proton from neutral nitroalkane substrates. The structures for NAO and various members of the ACAD family overlay with root-mean-square deviations between 1.7 and 3.1 A. The homologous region typically spans more than 325 residues and includes Glu376, which is the active-site base in the prototypical member of the ACAD family. However, NAO and the ACADs exhibit differences in hydrogen-bonding patterns between the respective active-site base, substrate molecules, and FAD. These likely differentiate NAO from the homologues and, consequently, are proposed to result in the unique reaction mechanism of NAO.

Nitroalkane oxidase, a carbanion-forming flavoprotein homologous to acyl-CoA dehydrogenase.

While several flavoproteins will oxidize nitroalkanes in addition to their physiological substrates, nitroalkane oxidase (NAO) is the only one which does not require the anionic nitroalkane. This, in addition to the induction of NAO by nitroethane seen in Fusarium oxysporum, suggests that oxidation of a nitroaliphatic species is the physiological role of the enzyme. Mechanistic studies of the reaction with nitroethane as substrate have established many of the details of the enzymatic reaction. The enzyme is unique in being the only flavoprotein to date for which a carbanion is definitively established as an intermediate in catalysis. Recent structural analyses show that NAO is homologous to the acyl-CoA dehydrogenase and acyl-CoA oxidase families of enzymes. In NAO, the glutamate which acts as the active site base in the latter enzymes is replaced by an aspartate.

Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms.

Nitroalkane oxidase (NAO), a flavoprotein cloned and purified from Fusarium oxysporum, catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, with the production of H2O2 and nitrite. In this paper, the crystallization and preliminary X-ray data analysis of three crystal forms of active nitroalkane oxidase are described. The first crystal form belongs to a trigonal space group (either P3(1)21 or P3(2)21, with unit-cell parameters a = b = 103.8, c = 487.0 A) and diffracts to at least 1.6 A resolution. Several data sets were collected using 2theta and kappa geometry in order to obtain a complete data set to 2.07 A resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P2(1)2(1)2(1); a = 147.3, b = 153.5, c = 169.5 A) and crystal form 3 (P3(1) or P3(2); a = b = 108.9, c = 342.5 A) are obtained from slightly different conditions and also contain two homotetramers per asymmetric unit, but have different solvent contents. A three-wavelength MAD data set was collected from selenomethionine-enriched NAO (SeMet-NAO) in crystal form 3 and will be used for phasing.