Determination of total ribonucleotide pool in plant materials by high-pH anion-exchange high-performance liquid chromatography following extraction with potassium hydroxide.
A new, improved method that only requires a potassium hydroxide extraction procedure is presented for the analysis of a full nucleotide pool in plant materials. Quantification was performed by high-pH anion-exchange chromatography (HPAEC) with UV detection after a potassium hydroxide extraction, and allowed the quantification of 13 linear ribonucleotides in a single run. The method has been validated by comparison of six extraction methods and also by measurement of the intracellular nucleotide levels of three plant species (cell cultures and leaves). The evolution of the nucleotide pool of Nicotiana tabacum cell culture during growth has also been measured, and showed an increase in the pool until the fifth day, where the growth rate reaches a maximum, after which a decrease was observed.
Laboratoire de Physiologie et de Biochimie Végétale, UMR 692 INRA-ENESAD-Université de Bourgogne, ENESAD 26, Boulevard Dr. Petitjean, B.P. 87999, 21079 Dijon, France.
Christophe
C
Riondet
Sylvain
S
Morel
Gérard
G
Alcaraz
D000837:Anion Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000737:chemistry*; D010944:Plants / Q000737:chemistry*; D017680:Potassium Compounds / Q000737:chemistry*; D012015:Reference Standards; D012265:Ribonucleotides / Q000032:analysis*; D013045:Species Specificity
D000837:Anion Exchange Resins; D006878:Hydroxides; D017680:Potassium Compounds; D012265:Ribonucleotides; C029943:potassium hydroxide