Amylopectin

For immunoblotting, total proteins from one mature rice seed and three developing rice seeds (with the embryo removed) were extracted with 10 volumes of denaturing extraction buffer [0.125 M Tris–HCl, pH 6.8, 8 M urea, 4% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol], and then incubated at room temperature for 2 h with rotation according to the method of Crofts et al. (2012) (link). Soluble protein (SP), loosely bound to starch granule protein (LBP), and tightly bound to starch granule protein (TBP) were extracted from eight developing endosperm according to a previously published method (Asai et al., 2014 (link); Crofts et al., 2015 (link)). Immunoblotting was performed as described previously (Crofts et al., 2012 (link)).
Samples used for native-PAGE/activity staining were extracted from three developing endosperm (10–15 DAF) using 1.5 volumes of buffer (for SSIIa zymogram) or 3 volumes of buffer (for the remaining zymograms) relative to seed fresh weight, and equal amounts of protein were loaded from each line (Fujita et al., 1999 (link)). SS activity staining for detecting SSI and SSIIIa was performed using glycogen as primer as described previously (Nishi et al., 2001 (link)), except that 0.5 M citrate was added to the reaction mixture. SSIIa activity staining was performed as described previously (Nishi et al., 2001 (link)) with the following modifications: 9% acrylamide native-PAGE gel containing 0.05% maize amylopectin as primer was electrophoresed at 8 mA for stacking gel and 15 mA for separation gel. The gel was run for an additional 50 min at 15 mA after the dye front reached the bottom, and then incubated with SS reaction buffer containing 0.5 M citrate adjusted to pH 10 with NaOH. BE activity was assessed using gels containing 0.0001% oyster glycogen (Yamanouchi and Nakamura, 1992 (link)). Debranching enzyme (DBE) was assessed as described previously (Fujita et al., 1999 (link)).
A 700-mg aliquot of developing seeds was extracted and fractionated by gel filtration chromatography using Superdex 200 resin according to the method of Crofts et al. (2015) (link). Native-PAGE/SS and native-PAGE/BE activity staining was performed as described above. Native-PAGE/immunoblotting of each fraction was performed as described by Crofts et al. (2015) (link).

Amylose content was measured using a continuous flow analytical system (Skalar San⁺⁺ System, Netherlands). Standards were prepared using potato amylose (Solarbio, China) with a concentration gradient of 2%, 4%, 8%, 12%, 16%, 20%, and 32%. The absorbance of amylose content was determined at 600 nm. Total starch content was measured spectrophotometrically based on the determination of the absorbance of glucose concentration at 540 nm (Li and Peng, 2018 (link)). According to the transformational equation between starch and glucose, total starch content was calculated by the following formula: Total starch content (mg·g^-1) = glucose concentration × 10 × 9.11 ml × 0.9/(50 × 1000). Amylopectin content = total starch content - amylose content (Prathap et al., 2019 (link)).

The total starch content was determined by the optical rotation method of Lu (2011) (link). First, the 2.5-g rice flour sample was added to 40 ml of 85% ethanol, the suspension was centrifuged at 3,000 r/min for 10 min, and then the supernatant was removed. The above operation was repeated four times. Afterward, the residue was transferred into a 100-ml conical flask using 50 ml of 1.128% HCl and heated in a boiling water bath for 15 min. After cooling, the solution was added to 1 ml of 30% ZnSO₄ and 1 ml of 15% potassium ferrocyanide and then was filtrated to obtain a clear liquid. The filtrate was put into a polarimeter (WZZ-2S, China) and the total optical rotation was measured. The starch content was calculated by its total optical rotation according to the method of Lu (2011) (link).
The apparent amylose content (AAC) was determined by the iodine blue colorimetric method (Sandhu and Singh, 2007 (link)). The 100-mg rice flour was transferred to a 100-ml conical flask. Then, 1.0 ml of 95% ethanol and 9.0 ml of 1.0 mol/L NaOH were added. The suspension was mixed and heated in a boiling water bath for 10 min and cooled to room temperature. The solution was transferred to a 100-ml volumetric flask and distilled water was added to a constant volume (100 ml). Then, 0.5 ml of the solution sample was transferred into a covered colorimetric tube with 5 ml water and 0.1 ml of 1 mol/L acetic acid and then 0.2 ml of 0.2% iodine solution was added. Finally, its absorbance at 620 nm was measured with a spectrophotometer (TECAN Infinite M200, Switzerland). The AAC was determined from a standard curve established using a mixed solution of amylose and amylopectin.
The 0.2-g rice flour was transferred into a digestion cube, and 5 ml of concentrated H₂SO₄ was added overnight. The suspension was added with three to four drops of H₂O₂ and then heated at 320°C for 10-15 min by using a digestion oven (Foss Tecator, Hillerod, Denmark). After cooling, two to three drops of H₂O₂ were added into the suspension and heated at 200°C for 5 min again. This step was repeated two to three times until the liquid is transparent. The protein content was determined according to the Kjeldahl method, and a nitrogen conversion factor of 5.95 was used to calculate the crude protein content of the milled rice (Zhang et al., 2016a (link)).