Starch biosynthesis in plants involves a network of enzymes of which adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 188.8.131.52), and soluble and granule bound starch synthases (SSS and GBSS, E.C. 184.108.40.206) play central roles. Here, we outline the protocol for extraction and assay of these enzymes in developing grains of wheat (Triticum aestivum L.). The principle of the assays outlined is based on a coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be measured easily by spectrophotometer. This protocol does not need expensive labelled chemicals and can be carried out using equipment commonly found in a biochemical laboratory. We applied this protocol to study the dynamics of AGPase, SSS and GBSS activity in developing wheat grains at different time points after anthesis.
[Background] Starch is a carbohydrate polymer made up of amylose, a linear glucan polymer composed of α-1,4-linked glucose molecules, and amylopectin, another glucan polymer composed of α-1,4-linked glucose molecules branched by α-1,6-glycosidic bonds. The enzyme adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 220.127.116.11) catalyzes the first committed step of starch synthesis, converting glucose-1-phosphate and ATP to ADP-glucose and inorganic pyrophosphate (PPi). ADP-glucose is subsequently used by soluble starch synthases (SSS) and granule bound starch synthases (GBSS) (E.C. 18.104.22.168), and starch branching enzymes to elongate and branch the glucan chains of the starch granule.
Initially, AGPase and starch synthase assays were carried out using 14C- and 32P-labelled ADP-glucose (Ghosh and Price, 1966; Vos-Scheperkeuter et al., 1986), which requires the use of expensive chemicals as well as specialized equipment to work with labelled compounds. Here, we outline the method adopted and applied for extracting and assaying AGPase, SSS and GBSS activity in developing wheat (Triticum aestivum L.) grains (Mukherjee et al., 2015). Our protocol is based on the methods reported previously by Nakamura et al. (1989) for measuring AGPase and SSS activities in the endosperm of developing rice grains, and by Schaffer and Petreikov (1997) for measuring the activities of SSS and GBSS in tomato fruits. The method is based on coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be easily measured by spectrophotometers. The protocol can be carried out using the equipment commonly found in biochemical laboratories and does not require the use of labeled compounds.
Because this protocol is an adoption of the methods developed for different tissues of other plant species (endosperm of rice grain and tomato fruit), the reaction mixture composition for all enzymes was optimized for reaction buffer pH and substrate concentration so that enzyme activity was within the linear phase with respect to incubation time and protein concentration. The amounts of enzyme preparation added (PGM, pyruvate kinase, hexokinase, G6PDH) have been adjusted to achieve completion of coupling reaction in expected time frame. This protocol can be used for studying the activities of AGPase, SSS and GBSS in other tissues of wheat as well as in different tissues of other plant species; however, optimization of reaction buffer pH and substrate concentrations is required.
The principle of AGPase assay is presented in Figure 1. AGPase present in plant tissue extract catalyzes the conversion of ADP-glucose and PPi into glucose-1-phosphate and ATP. After the first reaction is stopped by boiling, phosphoglucomutase (PGM) is added to the reaction mixture; PGM converts quantitatively glucose-1-phosphate into glucose-6-phosphate. Subsequently, glucose-6-phosphate dehydrogenase (G6PDH) is added; G6PDH converts glucose-6-phosphate in the presence of NADP into 6-phosphogluconic acid. At the same time, NADP is converted into NADPH and the quantity of NADPH formed is equivalent to the quantity of glucose-6-phosphate oxidized. By measuring NADPH concentration spectrophotometrically at 340 nm, we can quantify the amount of ADP-glucose degraded by AGPase activity.
Figure 1. Principle for ADP-glucose pyrophosphorylase (AGPase) assay
The principle of SSS/GBSS assay is presented in Figure 2. Starch synthases present in plant tissue extract (SSS) or in starch granule suspension (GBSS) catalyze the conversion of ADP-glucose into ADP coupled with the elongation of amylopectin primer for one glucose residue. Pyruvate kinase added to the reaction mixture after the first reaction is stopped by boiling converts quantitatively ADP into ATP in the presence of PEP. Subsequently, addition of glucose together with hexokinase leads to the conversion of ATP into glucose-6-phosphate. In the next step of assay, G6PDH converts glucose-6-phosphate in the presence of NADP into 6-phosphogluconic acid; NADP is converted into NADPH at the same time and the quantity of NADPH formed is equivalent to the quantity of glucose-6-phosphate oxidized. By measuring NADPH concentration spectrophotometrically at 340 nm, we can quantify the amount of ADP-glucose (at the first step of the analysis) degraded by SSS/GBSS activity.
Figure 2. Principle for soluble and granule bound starch synthases (SSS and GBSS) assay
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