Metabolite analysis
Sugar-phosphates were extracted as previously described by Noguchi et
al. (2018). Leaves were sampled under illumination at 1,100 µE
m-2 s-1 in ambient air (40 Pa
CO2, 21kPa O2, 25 oC),
and immediately frozen in liquid N2. The frozen leaves
were ground with a mortar and pestle in liquid N2.
Methanol was added to the homogenized leaves, and same volume of a
solution containing internal standards (100 µM PIPES and 100 µM
methionine sulfone) were mixed after vortex. After centrifugation, the
resulting supernatants were filtered through a 3 kDa cut-off filter
(Millipore) at 16,100×g at 4oC for 30 min. The
sugar-phosphates were separated by capillary electrophoresis-triple
quadrupole-mass spectrometry (CE-QQQ-MS; 7100 Capillary Electrophoresis,
MS; 6420 Triple Quad LC/MS, Agilent Technologies, USA) with multi
reaction monitoring mode as described previously (Miyagi et al. 2010,
2019). All CE-QQQ-MS data were processed using the Agilent MassHunter
software (Agilent Technologies).