|Laclair, Russell -|
|Ohlrogge, John -|
|Shachar-Hill, Yair -|
Submitted to: Plant Cell and Environment
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 22, 2012
Publication Date: June 11, 2012
Repository URL: http://handle.nal.usda.gov/10113/54539
Citation: Allen, D.K., Laclair, R.W., Ohlrogge, J.B., Shachar-Hill, Y. 2012. Isotope labeling of rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments. Plant Cell and Environment. 35(7):1232-1244. Interpretive Summary: This work describes a novel approach to derive information about subcellular metabolism in plant seeds. Plants maintain separate genomes (DNA complement) in the nucleus, mitochondria and chloroplast. The large and small subunits of the enzyme, RuBisCO, are encoded in the chloroplast and nuclear genomes respectively. As a consequence the subunits are synthesized from different amino acid pools specific to either the chloroplast or the cytoplasm (nuclear encoded). We labeled developing green embryos taken from rapeseed with 13C labeled substrates and inspected the amino acids obtained by hydrolyzing purified RuBisCO subunits. Alanine, glycine and serine exhibited different 13C labeling patterns between the two subunits, indicating that their pools are not equally mixed between the chloroplast and cytoplasm. In contrast other amino acids did not show differences across the subcellular locations. The differences in 13C labeling suggest amino acid biosynthesis occurs in multiple locations and involves different pathways. This work is important because there are very few ways to evaluate in vivo metabolism at a subcellular level, yet considerations relating to the compartmentation of enzymes and metabolites are critical to the design of transgenics and successful metabolic engineering. This approach allows evaluation of metabolic differences in at the cellular and subcellular level and should have widespread application to plants as well as other organisms that will enrich our understanding of basic biology and enable metabolic engineering efforts. The information will enable new strategies for seed composition manipulations which will improve nutrative and feed characteristics for our major crops.
Technical Abstract: The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses considerable challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state 13C labeling and analyzed labeling in protein amino acids from plastid and cytosol. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol respectively. Developing Brassica napus embryos were cultured in the presence of [U-13C]-sucrose, [U-13C]-glucose, [U-13C]-glutamine, or [U-13C]-alanine to generate proteins. The large (LSU) and small (SSU) RuBisCO subunits were isolated and the labeling in their constituent amino acids was analyzed by GC mass spectrometry. Amino acids including alanine, glycine and serine exhibited different 13C enrichment in the LSU and SSU, showing that these pools have different metabolic origins and are not equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of the approach to other macromolecules, organelles and cell types of eukaryotes are discussed.