Abstract
Plant phenotyping to date typically comprises morphological and physiological profiling in a high-throughput manner. A powerful method that allows for subcellular characterization of organelle stoichiometric/functional characteristics is still missing. Organelle abundance and crosstalk in cell dynamics and signaling plays an important role for understanding crop growth and stress adaptations. However, microscopy cannot be considered a high-throughput technology. The aim of the present study was to develop an approach that enables the estimation of organelle functional stoichiometry and to determine differential subcellular dynamics within and across cultivars in a high-throughput manner. A combination of subcellular non-aqueous fractionation and liquid chromatography mass spectrometry was applied to assign membrane-marker proteins to cell compartmental abundances and functions of Pisum sativum leaves. Based on specific subcellular affiliation, proteotypic marker peptides of the chloroplast, mitochondria and vacuole membranes were selected and synthesized as heavy isotope labeled standards. The rapid and unbiased Mass Western approach for accurate stoichiometry and targeted absolute protein quantification allowed for a proportional organelle abundances measure linked to their functional properties. A 3D Confocal Laser Scanning Microscopy approach was developed to evaluate the Mass Western. Two P. sativum cultivars of varying morphology and physiology were compared. The Mass Western assay enabled a cultivar specific discrimination of the chloroplast to mitochondria to vacuole relations.
Highlights
Modern plant phenotyping is quantitatively linking molecular and biochemical data with the plants morphological, physiological and agronomic parameters
In order to approach the relationship between organelle abundances and functions, we developed a Mass Western kit for organelle stoichiometry investigations of Pisum sativum leaves
non-aqueous fractionation (NAF) gradients were successfully applied to separate proteins belonging to the subcellular compartments chloroplasts, mitochondria and vacuole and turned out to be suitable forannotation of plastidial proteins that have wrongly been annotated to other subcellular localizations before
Summary
Modern plant phenotyping is quantitatively linking molecular and biochemical data with the plants morphological, physiological and agronomic parameters. Cells of living organisms are surrounded by a membrane and contain various sub-structures and organelles. Each of these organelles has a characteristic morphology and processes specific function(s). Upon developmental or environmental changes cells and organelles process adaptation This adaptation includes the re-distribution of metabolites as a crosstalk between the organelles and molecular plasticity of specific organelle functions. One of the best studied examples in plants is the interconnection existing between mitochondria, chloroplasts and peroxisomes (Schnarrenberger and Fock, 1976). They coordinate their activities as a function of photorespiration during drought stress
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