Identifying candidate genes involved in Nitric Oxide signaling during cell death

Nitric oxide (NO) is an important signaling molecule that mediates many plant developmental and physiological processes. This small gaseous radical was first found to play a crucial role in mediating hypersensitive response induced cell death together with ROS in plant defense reactions against incompatible bacterial pathogens. Beside this it was also shown to influence numerous physiological processes throughout the entire plant life cycle. To study NO responses in plant we have developed a fumigation chamber which allows to treat plants with a precise amount of NO gas concentration in air. Arabidopsis thaliana plants of different ages were first characterized for their response to different NO treatments. In particular we showed that four weeks old Arabidopsis thaliana plants were insensitive to NO fumigation when treated 8 hours with a gas containing up to100 ppm of NO. Using higher NO gas concentrations appearance of typical cell death symptoms was observed. This NO sensitiveness was shown to be strongly dependent on light conditions. In order to identify candidate genes involved in mediating NO signaling during the cell death process we set up a screening strategy taking advantage of this facility. We fumigated a fast neutron mutant collection of Arabidopsis thaliana Columbia plants and are currently fumigating an EMS mutant collection of Arabidopsis thaliana Landsberg erecta plants. Our first screening showed that this setting was successful for the identification of mutants with an altered response to the NO treatment. We could identify 30 mutant plants (0,35%), among which 15 could be successfully propagated and 10 of them were confirmed as more resistant to NO compared to wild type plants in the following generation. Interestingly among them a candidate showing complete NO resistance as well as an altered growth phenotype was identified and is being further characterized

Separation and identification of bioactive peptides from stem of Tinospora cordifolia (Willd.) Miers

Enzyme hydrolysates (trypsin, papain, pepsin, a-chymotrypsin, and pepsin-pancreatin) of Tinospora cordifolia stem proteins were analyzed for antioxidant efficacy by measuring (1) 1,1-diphenyl-2-picrylhydrazyl (DPPH center dot) radical scavenging activity, (2) 2,20-azinobis(3-ethyl-benzothiazoline- 6-sulfonic acid) (ABTS(+)) radical scavenging capacity, and (3) Fe2+ chelation. Trypsin hydrolysate showed the strongest DPPH center dot scavenging, while alpha-chymotrypsin hydrolysate exhibited the highest ABTS(+) scavenging and Fe2+ chelation. Undigested protein strongly inhibited the gastrointestinal enzymes, trypsin (50% inhibition at enzyme/substrate ratio = 1: 6.9) and a-chymotrypsin (50% inhibition at enzyme/substrate ratio = 1: 1.82), indicating the prolonged antioxidant effect after ingestion. Furthermore, gel filtration purified peptide fractions of papain hydrolysates exhibited a significantly higher ABTS(+) and superoxide radical scavenging as compared to non-purified digests. Active fraction 9 showing the highest radical scavenging ability was further purified and confirmed by MALDI-TOF MS followed by MS/MS with probable dominant peptide sequences identified are VLYSTPVKM-WEPGR, VITVVATAGSETMR, and HIGININSR. The obtained results revealed that free radical scavenging capacity of papain hydrolysates might be related to its consistently low molecular weight hydrophobic peptides

cGMP signaling in plant-pathogen interactions.

Description of the role of cGMP in Arabidopsis thaliana during the hypersensitive respons