One unit of SOD activity was defined as the amount of enzyme that inhibits the photochemical reduction of nitroblue tetrazolium to 50%, considering the absorbance of the control mixture as 100%

One unit of SOD activity was defined as the amount of enzyme that inhibits the photochemical reduction of nitroblue tetrazolium to 50%, considering the absorbance of the control mixture as 100%. 2000). When Arabidopsis plants were inoculated with pv (mutant plants showed more developed chlorotic lesions compared with wild-type plants, and LPS-treated plants showed no significant improvement on disease progression both at 3 and 5 dpi. In addition, the numbers of bacteria were significantly reduced by LPS treatment in wild-type plants but not in plants (Fig. 1B). This obtaining correlated with the disease symptoms shown in Physique 1A. For the pathogen-growth assays in Physique 1B, the Arabidopsis plants were more susceptible than wild-type plants and showed obvious lesions at 5 dpi. Such damaged leaves resulted in nutrition loss and limited bacterial growth rate. Therefore, the bacterial numbers in plants were nearly maximal at 3 dpi (Fig. 1B). Open in a separate window Physique 1. Effect of LPS application on disease progression in leaves of wild-type (WT) and plants. A, After spraying with 250 m MgCl2 and 100 m CaCl2 (control answer) or LPS (100 g mL?1 in 250 m MgCl2 and 100 m CaCl2) solution for 24 h, wild-type Arabidopsis and mutant plants were inoculated with pathogen DG3 (OD600 = 0.01 in 10 mm MgCl2). Leaves were infected on their left halves, and samples were collected at 3 and 5 dpi. B, Bacterial growth quantification of BPES1 DG3-inoculated (OD600 = 0.0001) leaves after spraying with control solution or 100 g mL?1 LPS. Samples were collected at 3 and 5 dpi for assay. Each value is the mean se of three replicates. Different letters indicate statistically significant differences between treatments (Duncans multiple range test: 0.05). CFU, Colony-forming models. [See online article for color version of this physique.] Significant Role of NOS-Like Enzyme in Mediating LPS-Induced NO Synthesis in Protoplasts NR and NOS are two key enzymes responsible for herb NO biosynthesis. It seems that herb NOS is not a canonical animal NOS, but it uses the same substrate and cofactors as the animal NOS. Therefore, pharmacological analyses with mammalian NOS inhibitors are often used to study the physiological mechanism of herb NO production. However, the effects of mammalian NOS inhibitors in plants are somewhat difficult to interpret, because the molecular targets and specificity of these compounds are unknown. In this experiment, we combined pharmacological and genetic approaches to investigate the potential source of NO using a mesophyll protoplast system. The freshly isolated Arabidopsis mesophyll protoplasts are similar to those in intact tissues and plants in physiological and cell-autonomous responses (Tena et al., Pentostatin 2001). LPS-induced NO accumulation could be detected after approximately 80 min and leveled off at about 150 min (Supplemental Fig. S1). LPS application resulted in a significant increase of 3-amino,4-aminomethyl-2,7-difluorescein (DAF-FM) fluorescence, which could not be inhibited by the NR inhibitor sodium tungstate. However, the increases were markedly decreased by incubating the protoplasts together with the mammalian NOS inhibitors Arabidopsis that exhibited null NR activity (Zhao et al., 2009), the mutant, the herb (also named mutant (also known as Arabidopsis, in which no significant DAF-FM fluorescence was observed, and mutant protoplasts showed increased endogenous NO levels under normal conditions. Protoplasts of and exhibited significantly high levels of DAF-FM signals under LPS induction (Fig. 2, B and C). Although the basal NO level in and plants was lower than that in wild-type plants, the DAF-FM fluorescence levels of protoplasts from both these mutants were also elevated under LPS treatment. It has been suggested that DAF does not react directly with the NO free radical but does so with NO-derived species (such as N2O3; Mur Pentostatin et al., 2011). To confirm that this changes in fluorescence were caused by NO itself, electron paramagnetic resonance (EPR) analysis was also used. The presented data also exhibited NO production after LPS treatment (Fig. 2D). In addition, an increase in Pentostatin NOS-like enzyme activity was detected during LPS induction, and these increases were dramatically inhibited by l-NNA and l-NAME (Fig. 2E). Moreover, under LPS treatment, we did not observe any increased NR activity in both wild-type and plants. On the contrary, a slight inhibition of NR activity was seen in wild-type plants (Fig. 2F). The results demonstrate that this NOS-like enzyme possibly plays a key role in LPS-elicited NO generation. Open in a separate window Physique 2..