Ri 8020

The method used to assess the composition of carbohydrate content in endosperm was performed as described previously^{34 (link)}. The eluate refractive index was measured with an RI-8020 detector (Tosoh, Japan). Fractions I, II, and III were divided at the minimum values of the refractive index curves. The contents of apparent amylose, true amylose, and extra-long chains in starch were calculated from the ratios of fractions I, II, and III. The contents of each fraction were confirmed by mixing the eluate with iodine and measuring the λ_max value with the spectrophotometer as described previously^{65 (link)}.

Rice flour samples (20 mg) were suspended in 1.6 mL of distilled water in a 15 mL centrifuge tube and gelatinized with 400 µL of 5 M NaOH for 30 min at 37 °C. Then, 2 mL of distilled water and 2 mL of eluent (0.2% NaCl/0.05 M NaOH) were added to each sample, followed by filtration through a 5 µm Durapore polyvinylidene fluoride (PVDF) membrane (Merck, Darmstadt, Germany). A 5 mL aliquot of the filtrate was used in a 4-column system (Toyopearl HW75S × 2-HW65S-HW55S; 2.2 cm diameter, 30 cm × 4; Tosoh Corp., Tokyo, Japan) that was pre-equilibrated with the eluent, and carbohydrates were detected using an RI detector (RI-8020; Tosoh Corp.). The columns were incubated at 40 °C, and the samples were eluted at a flow rate of 1 mL/min using a DP-8020 pump (Tosoh Corp.).

Starch was extracted from mature rice endosperms to assess the amylopectin chain-length distribution as described by Fujita et al.[32 (link)]. The chain-length distributions of endosperm α-glucans were analyzed using capillary electrophoresis methods [32 (link),33 (link)] using a P/ACE MDQ Carbohydrate System (Beckman Coulters, CA, USA).
Gel filtration chromatography of starches and amylopectin was performed as previously described [10 (link),28 (link)] using a Toyopearl HW55S gel filtration column (300 × 20 mm) connected in series to three Toyopearl HW50S columns (300 × 20 mm) equipped with an RI (refractive index) detector (Tosoh RI-8020).

Starch was extracted from mature rice endosperm to assess the amylopectin chain-length distribution according to the method of Fujita et al. (2001) (link). The chain-length distribution of endosperm starch was analysed by capillary electrophoresis (O’Shea and Morell, 1996 (link)) using the P/ACE MDQ Carbohydrate System (Beckman Coulters, CA, USA).
Gel filtration chromatography of starches (from mature and developing endosperm) and amylopectin (from mature endosperm) was performed as described previously (Fujita et al., 2007 (link), 2009 (link)) using a Toyopearl HW55S gel filtration column (300×20mm) connected in series to three Toyopearl HW50S columns (300×20mm) equipped with a refractive index (RI) detector (Tosoh RI-8020).
Estimation of starch content in rice seeds and determination of the amylopectin molecular weight were performed by HPSEC-MALLS-RI according to the method of Fujita et al. (2003) (link). Purified starch granules were coated with gold using a fine coater (JEOL JFC-1200) for 120 s. Starch granule morphology was examined by scanning electron microscopy (SEM; JEOL-500, Tokyo, Japan). SEM was performed in a secondary electron mode at 15kV according to the method of Fujita et al. (2003) (link). Observation of iodine-stained endosperm thin sections was performed according to the method of Matsushima et al. (2010) (link).

The T/H extracts from rubber synthesizing assay were subjected to gel permeation chromatography (GPC) which was carried out with Tosoh GPC-8020 (Tosoh, Tokyo, Japan), equipped in tandem with a series of a TSK gel GPC columns, TSKguardcolumn MP(XL) and two Multipore H_XL-M columns (Tosoh). The chromatography was carried out at 40˚C using THF as eluent, at a flow rate of 1 ml/min. The eluate was monitored by a refractive index detector RI-8020 (Tosoh) and subsequently collected as 1-ml fractions (1-min interval) to measure radioactivity. The molecular mass of the reaction products were estimated by comparison with the retention times of commercially available polyisoprene standards (Mw: 999000, 110000, and 9600, Polymer Standards Service, Mainz, Germany).