Chemical analysis for metabolites
Sucrose, fructose, glucose, and starch : Sugar contents
were measured by ultra-high performance liquid chromatography system
(UHPLC) as described by Chen et al. (2018a). Sucrose and hexoses were
extracted with 80% (v/v) ethanol for 30 min at 80 °C, followed by
centrifugation at 5000g for 15 min. The supernatant was pooled and
lyophilized using a Maxi-Dry Lyo instrument (Heto-Holten, France), and
resuspended in ultrapure water and filtered through a 0.45 μm pore size
filter. The resulting filtrate was loaded into a Dionex Ultimate 3000
chromatography system. Starch content was measured using anthrone
reagent at wavelength of 620 nm, after extraction by ethanol and
digestion by perchloric acid.
T6P and SnRK1 activity : T6P was extracted and purified
according to the method of Delatte et al. (2009), and quantified by
ultra-performance liquid chromatography (ACQUITY UPLC I-Class PLUS,
Waters, USA) coupled with triple
quadruple mass spectrometry system (Xevo TQ-S micro, Waters, USA),
following the method of Sastre Toraño et al. (2012). SnRK1 was extracted
and its activity was assayed by the method of Zhang et al. (2009), using
AMARA peptide as substrates.
Amino acids and proteins : Samples were boiled with 80%
ethanol until totally bleached. Then, the total volume was kept constant
with distilled water and was centrifuged for 15 min. The supernatant was
pooled and filtered through a 0.45 μm pore size filter, and then used
for amino acid determination by amino acid analyzer (L-8900, Hitachi,
Japan). Protein fractions including albumin, globulin, prolamin and
glutelin were extracted and measured by the methods of Ning et al.
(2010).
Phytohormones and minerals : IAA, ABA, CKs (tZ and tZR)
and GAs (GA1, GA3, GA4,
and GA7) were extracted and purified according to the
method of Wu et al. (2016). IAA, ABA and CKs were quantified by
ultra-performance liquid chromatography (ACQUITY UPLC H-CLASS, Waters,
USA) according to Chou, Chen, Huang, Yu, and Liao (2000). GAs were
quantified by ultra-high performance liquid chromatography (AGLIENT1290,
Agilent Technologies, USA) coupled with mass spectrometry system
(SCIEX-6500Qtrap, AB SCIEX, USA). Minerals, including P, K, Ca, Na, Mg,
Fe, Zn, Mn, and Cu, were quantified by Inductively Coupled Plasma
Optical Emission Spectrometry (ICP-OES 710, Agilent Technologies, USA),
according to the method of Wang et al. (2018).