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).