Principle investigator: Christine Stöhr
Co-supervisor: F. Scholz

Root-specific NO formation at plasma membrane (pm) has been observed in vitro, and in planta for several species [1]. Enzymes similar to bacterial denitrification enzymes have been shown to be exclusively localized at root plasma membranes. Plasma membrane-bound nitrate reductase together with the root specific and plasma membrane-bound Nitrite:NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate or nitrite. The electron donors, menadione, succinate and cytochrome cred. have been proven to be effective. In vivo localization of NO formation with the fluorescent indicator FNOCT [2] clearly demonstrated the specific plasma membrane-localized NO formation under physiological conditions. Although a correlation of NO formation rate with nitrate nutrition of the plant, the oxygen availability of the roots and mycorrhizal inoculation has been demonstrated, the primary targets of the pm localized NO are not clear. In this project, the effect of NO on pm components of root cells will be studied.

1.Possible effects of NO on plasma membrane properties becoming apparent as changes in fluidity will be studied in chronoamperometric measurements of the adhesion-spreading events of liposomes on static Hg electrodes [3]. A potential nitration of unsaturated fatty acids of the plasma membrane lipids by nitric oxide-derived reactive nitrogen species will be investigated by analyzing nitro- and nitrohydroxy adducts by LC-MS/MS analysis in cooperation with B4 (Lalk/Kahlert).
 
2. Proteomic identification of S-nitrosylation of root pm proteins will be analyzed by His-Tag switch and LC–MS/MS (P. Roepstorff and cooperation with C1 (Antelmann)). Pm proteins will be analyzed of plants grown with different nitrogen regimes with and without pretreatment of pm vesicles with NO donor (NO-NOate). The quantification of S-nitrosocysteine (Cys-NO) formation will be determined by triiodide reduction coupled to chemiluminescence detection using a NO analyzer.

3.The effect of NO on enzyme activities will be studied with the main focus on pm proteases. Only plant roots display various pm specific endo- and exoproteases [4]. Some of these enzymes show enhanced activity under physiological conditions as required for high plasma membrane localized NO formation. Preliminary tests have indicated, that NO might be one effector for activity regulation. The effect of NO on enzyme activities and composition of the known set of pm-proteases will be analyzed at root pm vesicles and isolated proteins applying NO donors and scavengers. The studies will be combined with proteomic analysis.

 Literature

1 Eick, M. and Stöhr, C.: Protoplasma (2011) 249, 909.

2 Vandana, S., Sustmann, R., Rauen, U., and Stöhr, C.: Plant Physiol. Biochem. (2012) 59, 80. 

3 Hermes, M., Czesnick, C., Stremlau, S., Stöhr, C., and Scholz, F.: J. Electroanalyt. Chem. (2012) 671, 33.

4 Eick, M. and Stöhr, C.: Plant Physiol. Biochem. (2009) 47, 1003.