Untangling metabolic and spatial interactions of stress tolerance in plants. 1. Patterns of carbon metabolism within leaves.

The localization of the key photoreductive and oxidative processes and some stress-protective reactions within leaves of mesophytic C(3) plants were investigated. The role of light in determining the profile of Rubisco, glutamate oxaloacetate transaminase, catalase, fumarase, and cytochrome-c-oxidase across spinach leaves was examined by exposing leaves to illumination on either the adaxial or abaxial leaf surfaces. Oxygen evolution in fresh paradermal leaf sections and CO(2) gas exchange in whole leaves under adaxial or abaxial illumination was also examined.

Untangling metabolic and spatial interactions of stress tolerance in plants. 2. Accelerated method for measuring and predicting stress tolerance. Can we unravel the mysteries of the interactions between photosynthesis and respiration?

A simple method using the O(2) electrode that allows examination of the response of respiration and photosynthesis in leaf slices or algae to anoxia and high light under different temperatures useful for the examination of the interactions among photosynthesis, photorespiration, and respiration is described. The method provides a quantifiable assessment of stress tolerance that also permits us to examine fundamental biochemically and genetically related responses involved in stress tolerance and the cooperation among organelles. Additionally, we demonstrated a role for compounds, such as NO(-)(3) and oxaloacetate, as protective agents against photoinhibition, and we examined the role of dark adaptation in the activation of photosynthesis and NO(-)(3)-dependent O(2) oxygen evolution.

Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry.

Transit peptide cleavage sites of integral thylakoid membrane proteins.

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma.

The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry.

Proteomics seeks to address the entire complement of protein gene products of an organism, but experimental analysis of such complex mixtures is biased against low abundance and membrane proteins. Electrospray-ionization mass spectrometry coupled with reverse-phase chromatography was used to separate and catalogue all detectable proteins in samples of photosystem II-enriched thylakoid membrane subdomains (grana) from pea and spinach. Around 90 intact mass tags were detected corresponding to approximately 40 gene products with variable post-translational covalent modifications.