A novel method for determination of the (15) N isotopic composition of Rubisco in wheat plants exposed to elevated atmospheric carbon dioxide.
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Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is mostly known as a key enzyme involved in CO2 assimilation during the Calvin cycle, comparatively little is known about its role as a pool of nitrogen storage in leaves. For this purpose, we developed a protocol to purify Rubisco that enables later analysis of its N-15 isotope composition (N-15) at the natural abundance and N-15-labeled plants. In order to test the utility of this protocol, durum wheat (Triticum durum var. Sula) exposed to an elevated CO2 concentration (700 vs 400 mu mol mol(-1)) was labeled with (KNO3)-N-15 (enriched at 2 atom %) during the ear development period. The developed protocol proves to be selective, simple, cost effective and reproducible. The study reveals that N-15 labeling was different in total organic matter, total soluble protein and the Rubisco fraction. The obtained data suggest that photosynthetic acclimation in wheat is caused by Rubisco depletion. This depletion may be linked to preferential nitrogen remobilization from Rubisco toward grain filling.
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