Genetic and isotope ratio mass spectrometric evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves

Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous (CO2)-C-13 pulse-chase based studies indicated that starch degradation does not operate in illuminated Arabidopsis leaves. Here we show that leaves of gwd, sex4, bam4, barn1/bam3 and amy3/isa3/Ida starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown dpel plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown mexl/ pglct plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGIcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (delta C-13) in starch of leaves of (CO2)-C-13 pulsed-chased WT and AGP lacking aps1 plants. Notably, the rate of increase of delta C-13 in starch of aps1 leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, delta C-13 decline in starch of aps1 leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.