The immunomodulatory role of the natural biopolymer, chitosan, has already been demonstrated in plants, whilst its nanoparticles have only been examined for biomedical applications. In our present study, we have investigated the possible ability and mechanism of chitosan nanoparticles (CNP) to induce and augment immune responses in plants. CNP-treatment of leaves produced significant improvement in the plant’s innate immune response through induction of defense enzyme activity, upregulation of defense related genes including that of several antioxidant enzymes as well as elevation of the levels of total phenolics. It is also possible that the extracellular localization of CNP may also play a role in the observed upregulation of defense response in plants. Nitric oxide (NO), an important signaling molecule in plant defense, was also observed to increase following CNP treatment. However, such CNP-mediated immuno-stimulation was significantly mitigated when NO production was inhibited, indicating a possible role of NO in such immune induction. Taken together, our results suggest that CNP may be used as a more effective phytosanitary or disease control agent compared to natural chitosan for sustainable organic cultivation.

Neuronal nitric oxide synthase (nNOS) is composed of an oxygenase domain that binds heme, (6R)-tetrahydrobiopterin, and Arg, coupled to a reductase domain that binds FAD, FMN, and NADPH. Activity requires dimeric interaction between two oxygenase domains and calmodulin binding between the reductase and oxygenase domains, which triggers electron transfer between flavin and heme groups. We constructed four different nNOS heterodimers to determine the path of calmodulin-induced electron transfer in a nNOS dimer. A predominantly monomeric mutant of rat nNOS (G671A) and its Arg binding mutant (G671A/E592A) were used as fulllength subunits, along with oxygenase domain partners that either did or did not contain the E592A mutation. The E592A mutation prevented Arg binding to the oxygenase domain in which it was present. It also prevented NO synthesis when it was located in the oxygenase domain adjacent to the full-length subunit. However, it had no effect when present in the full-length subunit (i.e. the subunit containing the reductase domain). The active heterodimer (G671A/E592A full-length subunit plus wild type oxygenase domain subunit) showed remarkable similarity with wild type homodimeric nNOS in its catalytic responses to five different forms and chimeras of calmodulin. This reveals an active involvement of calmodulin in supporting transelectron transfer between flavin and heme groups on adjacent subunits in nNOS. In summary, we propose that calmodulin functions to properly align adjacent reductase and the oxygenase domains in a nNOS dimer for electron transfer between them, leading to NO synthesis by the heme.

We cloned, expressed, and characterized a hemeprotein from Deinococcus radiodurans (D. radiodurans NO synthase, deiNOS) whose sequence is 34% identical to the oxygenase domain of mammalian NO synthases (NOSoxys). deiNOS was dimeric, bound substrate Arg and cofactor tetrahydrobiopterin, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn 2؉ and tetrahydrobiopterin and help form an active dimer. The deiNOS heme accepted electrons from a mammalian NOS reductase and generated NO at rates that met or exceeded NOSoxy. Activity required bound tetrahydrobiopterin or tetrahydrofolate and was linked to formation and disappearance of a typical heme-dioxy catalytic intermediate. Thus, bacterial NOS-like proteins are surprisingly similar to mammalian NOSs and broaden our perspective of NO biochemistry and function.G enes coding for NO synthases (NOSs, EC 4.14.23) are present throughout the plant and animal kingdom. NOS activities are present in plants and lower eukaryotes (1-5). Their primary structures and activities are strikingly similar to the mammalian NOSs, suggesting that NO has been important throughout evolution. All eukaryotic NOSs catalyze the NADPH-and O 2 -dependent oxidation of L-arginine (Arg) to citrulline and NO, with N-hydroxy-L-arginine (NOHA) formed as an enzyme-bound intermediate (6). Structurally, all animal NOSs are bi-domain proteins containing an N-terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R-tetrahydrobiopterin (H 4 B), and Arg and is linked to a C-terminal f lavoprotein domain (NOS reductase domain, NOSred) by a central calmodulin (CaM) binding sequence (7,8). NOSred bears strong sequence and functional similarity to NADPH-cytochrome P450 reductase and related electron transfer flavoproteins (9, 10), and function to transfer NADPHderived electrons to the ferric heme for O 2 activation during NO synthesis. In contrast, NOSoxy and cytochromes P450 have completely different primary, secondary, and tertiary structures, even though both enzyme families use a thiolate-ligated heme for O 2 activation (11, 12). Moreover, unlike P450s, NOSoxy must dimerize to become active (13,14). Dimerization produces functional binding sites for Arg and H 4 B and sequesters the heme catalytic center from solvent. These distinguishing features imply that NOSs evolved separately from other heme-thiolate enzymes and can so provide unique perspectives on their structure-function relationships.Although oxidative (nitrification) or reductive (denitrification) pathways for prokaryote NO biosynthesis are well established (15), there have been few reports on NOS-like proteins in bacteria (16,17). To date no prokaryotic NOS proteins have been completely sequenced, purified, or cloned. However, some have been shown to have nitrite-forming activity that depended on Arg, NADPH, and H 4 B. More recent genome sequencing of Bacillus subtilis, Deinococcus radiodurans, and other bacteria (18)(19)(20) confirm that a subset contain ORFs that code for proteins homo...