Experiment description
The experiment was carried out in the Key Laboratory of Phytohormones and Growth Development of Hunan Agricultural University, Changsha, Hunan, PR China, in 2014. The tested rice variety was heat tolerant an early indica rice ‘Shenyou 9576’. Rice seeds were washed thoroughly by distilled water and then placed on wetted blotter paper in petridishes for germination. Germinated seeds of rice were pre-grown with complete Kimura B nutrient solution (Yoshida et al., 1976) in a greenhouse for 15 days. Seedlings were then transferred to earthen pots of 30 cm in diameter and 32 cm in depth filled with 7.0 kg of sieved, dry paddy soil (the contents of soil: organic matter, alkaline hydrolytic nitrogen, effective phosphorus, available potassium were 1.8%, 66.2 mg kg-1, 8.5 mg kg-1, and 8.0 mg kg-1, respectively, and soil pH was 5.4) amended with 1.0 g CO(NH2)2, 0.4 g P2O5, and 0.6 g K2O per kg soil to grow. Three earthen pots were placed in different growth chamber as well as in the net house. And two seedlings were transplanted in each earthen pot. Proper management practices were provided as per requirement for proper growth of the seedlings. The treatment consisted of three temperature regimes which are designated as high temperature (35/28oC, 12h light/12h dark, 75-80% relative humidity), low temperature (25/20oC, 12h light/12h dark, 75-80% relative humidity) and natural condition (35/25oC-day/night) as control. The treatments were imposed after anthesis by transferring pots into different growth chambers, but for the control treatment pots were kept in the net house under natural condition. The experiment was laid out in a complete randomized design (CRD) with three replications.
Sampling method
Panicles with rice grains of each treatment were collected at 7-day interval after flowering i.e. 7, 14, 21, 28 and 35 days after flowering (DAF). Samples were collected between 9.00 to 11.00 am and immediately wrapped in aluminum foil and frozen in liquid nitrogen, then placed into a sealed plastic bag and stored at - 60oC until used for different analysis. Rice grains were harvested at 35 DAF and then were sun dried to achieve 14% moisture content. Rough rice (paddy rice) was dehusked by an SBS-80 dehuller, then was polished by a rice polisher for 2 minutes. Milled rice samples were kept in sealed bags under refrigeration (4oC) for later analysis.
Chalkiness and head rice rate measurement
Chalkiness was measured with a system composed of a scanner and a special software Chalkiness 2.0 developed by Hunan Agricultural University (Chen et al., 2011). Head rice refers to the whole grains of milled rice and was computed by using the following equation (Gummert, 2010).
Starch synthesis enzymes assay procedures Preparation of enzymes extraction
The preparation procedure was similar to the method of Nakamura et al., (1989). Three rice grains were de-hulled and then separated from embryo and pericarp. Weight of 3 grains was recorded and then hand-homogenized with a pestle in a pre-cooled mortar (at 0-2o C) with 1 mL of ice-cooled GS buffer (extraction buffer) containing 100 mM Hepes-NaOH, (pH 7.4), 8 mM MgCl2, 2 mMK2HPO4, 2 mM Na2-EDTA, 12.5% (V/V) Glycerol, 5% (W/V) PVP-40, 50 mM 2-mercaptoethanol. After grinding, the liquid was transferred into a 2 mL tube, and then centrifuged at 2oC, 5000 rev/min for 10 minutes. The supernatant portion was transferred into another tube and GS-buffer was added to the sediment, and the operation was repeated again. The supernatant and the sediment were collected separately. The resulting supernatant was used for sucrose synthase (SuSy; EC 1.9.3.1), ADP-glucose pyrophosphorylase (ADPG- Ppase; EC2.7.7.27), soluble starch synthase (SSS, EC 2.4.1.21), starch branching enzyme (SBE; EC2.4.1.18) and starch de-branching enzyme (SDBE; EC3.2.1.70) and the pellet portion was used for granule bound starch synthase (GBSS, EC 2.4.1.21) enzyme assay. Three replications were done for every experiment.
Enzymes assay
The assay of ADP-glucose pyrophosphorylase (ADPG-Ppase; EC2.7.7.27), soluble starch synthase (SSS, EC 2.4.1.21) and starch branching enzyme (SBE; EC2.4.1.18) or Q-enzyme were similar to the procedure of Nakamura et al. (1989). Sediment or pellet was used instead of crude enzyme extraction for the assay of granule bound starch synthase (GBSS, EC 2.4.1.21) and the procedure is same to SSS. The assay of sucrose synthase (SuSy; EC 1.9.3.1), was carried out according to the protocol of Wardlaw and Willenbrink (1994). Starch de- branching enzyme (SDBE; EC3.2.1.70) or R-enzyme activity was assayed according to the protocol of Nelson (1944) and Somogyi (1952). All the assays were carried out at 30°C. Assays were conducted in the range of concentrations of enzyme where the activity increased linearly with increases in the amount of enzyme preparation and the reaction time. The background values were routinely taken as the activities detected with a reaction time of zero (the enzymes were denatured immediately after their addition to the reaction mixtures). One unit of activity of the above enzymes was defined as the amount causing an increase in absorbance of one unit at 340, 480, 520 and 540 nm in one min. All the spectrophotometer readings were taken by using a PGENERAL T6 XinRui spectrophotometer (Persee company, Beijing 101200, China). The activities of ADPG-Ppase, SSS, GBSS were defined as NADPH amount by measuring the increase in absorbance at 340 nm and SBE activity was defined as amount of 1% KI-I2 decreasing at 540 nm. The chemical products were purchased from Sigma- Aldrich, Ruibio and others chemical companies.
Statistical analysis
All experimental data were analyzed following analysis of variance. All statistical analysis was performed by using a statistical software, DPS version 12.01. Mean separation of the treatments was done by using Least Significant Difference (LSD) test at 5% level. Microsoft Excel 2003 (Microsoft, USA) was used to generate graphs. From flowering to harvest, under the natural condition (control), the daily average temperature of the experimental site was within 25oC to 35oC.