The crystal structures of plant class ADH

The crystal structures of plant class ADH3/GSNOR from Arabidopsis thaliana (AtGSNOR) and Solanum lycopersicum cv. Amateur (SlGSNOR) with bound substrates and cofactors revealed important differences between plant and human enzyme, namely within the substrate-binding pocket. GSNOR are unusually homodimeric cysteine-rich proteins, with each subunit consisting of a large catalytic domain with two zinc atoms and coenzyme binding domain [28], [43], [55]. The GSNOR activity has been conclusively demonstrated in many plant species including Arabidopsis, pea, pepper, sunflower, tomato or tobacco and found modulated upon various stress conditions including pathogenesis [1], [2], [4], [8], [19], [21], [28], [29], [31], [54]. A single copy of the GSNOR gene has been identified in most plant genomes, whereas the enzyme has been found in the cytosol, chloroplasts, mitochondria and peroxisomes by immunogold electron microscopy [4], [52], [37], [8], [5]. In Arabidopsis, the GFP-tagged enzyme localized in cytosol and nucleus but not nucleolus. The highest expression of GSNOR was observed in shoot and root apical meristems, in anther filaments, ovary, stigma and petals [55]. GSNOR is involved in NO homeostasis, which is essential for numerous processes including proper auxin and Caspase-4 Colorimetric Assay Kit (ABA) signaling. Mutations in GSNOR gene results in dwarfed plants, stem and trichomes branching defects and increased number of inflorescences [31], [30], impaired auxin signaling and polar auxin transport [47], [53] as well as ABA insensitivity in stomatal regulation. Thus, GSNOR seems necessary for normal plant development [33]. Recent study on six maize inbred lines [56] showed that two lines susceptible to drought display significantly higher GSNOR activities upon drought stress than more resistant lines. GSNOR also contributes to alkaline stress tolerance and its overexpression increases reactive oxygen species (ROS) scavenging efficiency [22]. Lettuce (Lactuca sativa) and cauliflower (Brassica oleracea var. botrytis) represent very important agricultural plants. We used these two model plant systems in previous studies focused on the role of NO and ROS in plant defense mechanisms during pathogenesis and oxidative stress [46], [45], [36]. The main goal of this work was to provide deeper insights into the regulation of NO homeostasis in these plants through the GSNOR activity. We performed a functional characterization of recombinant plant GSNORs in vitro and determined their molecular properties and kinetic parameters. We analyzed GSNOR gene expression and enzyme activity profiles during early developmental stages. Finally, we also performed immunolocalizations of GSNOR and GSNO in leaf sections of both plants.
Materials and methods
Results and discussion
Conclusions Detailed in vitro characterization of purified recombinant GSNOR from lettuce and cauliflower have confirmed their similarity to other plants GSNORs as cysteine-rich proteins showing high catalytic efficiency in GSNOR reduction in presence of NADH. The N6022 compound, as a strong inhibitor of all GSNORs of diverse origins, represents a valuable tool for future studies of GSNOR roles in plants using pharmacological approaches. Thermodynamic data obtained by analysis of purified proteins by isothermal microcalorimetry support the idea that irreversible reduction of GSNO mediated by GSNOR in cytoplasmic compartment with high NADH/NAD+ level ratio is enabled by higher affinity of enzyme to NADH and its tighter binding. Phylogenetic analysis of plant GSNOR sequences uncovered a close relationship of BoGSNOR to AtGSNOR, whereas it indicated that LsGSNOR sequence diverged earlier from other higher plant GSNORs. Striking differences of GSNOR activity values determined in lettuce and cauliflower plants during plant development suggest GSNOR activity and hence S-nitrosothiol levels might differ in several orders among plant species and this should be taken into account when interpreting results obtained in plant model species like Arabidopsis. Similarly, significant differences in GSNOR level among Lactuca genotypes with different degree of resistance to powdery mildew indicate that GSNOR forms a part of plant defense system differentially activated even in non-infected plants, which certainly deserves further detailed studies.