Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy.
White spruce is an important commercial species for reforestation. The success in its propagation through somatic embryogenesis is well documented; however the physiological processes involved are poorly understood and remain unoptimized. The variable quality embryos generated in vitro from the same genotype suggest control at the protein and metabolite level. In order to probe metabolic changes, we have conducted a "metabolic footprinting" study, whereby culture media from growing cells was quantitatively analyzed to determine which metabolites were consumed and excreted. Such experiments are advantageous in that there is no need to quench cellular metabolism or extract intracellular metabolites through time-consuming protocols. In this paper we demonstrate the application of the footprinting assay to somatic embryo cells of white spruce (Picea glauca) using 1D (1)H NMR spectroscopy. We have surveyed embryogenesis metabolism in two types of media, maintenance (MN) and maturation (MT). MN medium does not result in shoot apical meristem (SAM) formation, while MT medium induces the necessary changes leading to fully developed somatic embryos. The two types of media were easily distinguished using metabolomics analysis, namely multivariate pattern recognition statistics (orthogonal partial least squares discriminatory analysis). From this analysis, we have identified numerous compounds involved with branched chain amino acid pathways such as valine and isoleucine. These results are explained on the basis of known metabolic pathways implicated in plant and animal developmental processes, and ultimately implicate altered CoA biosynthesis.
Dowlatabadi
Reza
R
Metabolomics Research Centre, University of Calgary, Alberta, Canada.
Weljie
Aalim M
AM
Thorpe
Trevor A
TA
Yeung
Edward C
EC
Vogel
Hans J
HJ
D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D000040:Abscisic Acid / Q000494:pharmacology; D001593:Benzyl Compounds; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000032:analysis / Q000737:chemistry; D007701:Kinetin / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D008954:Models, Biological; D015999:Multivariate Analysis; D028222:Picea / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism*; D011687:Purines
D001593:Benzyl Compounds; D003470:Culture Media; D017077:Culture Media, Conditioned; D011687:Purines; D015084:2,4-Dichlorophenoxyacetic Acid; D000040:Abscisic Acid; C480551:benzylaminopurine; D007701:Kinetin