Enzymatic Starch Quantification in Developing Flower Primordia of Sweet Cherry

Starch is a carbohydrate widely used in the plant kingdom as a fuel for different physiological processes. While different techniques are available for the quantification of starch stored in seeds and bark tissues, they have hardly been used to quantify starch content in developing flower buds, where starch has been reported to accumulate in different reproductive organs. Here, we detail a quantitative enzymatic method to measure starch concentration in developing flower primordia in sweet cherry (Prunus avium L.). First, starch is enzymatically hydrolyzed to D-glucose, which was then quantified by an enzyme-coupled assay involving hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) and spectrophotometric quantification of NADH absorbance at 340 nm. This method is a sensitive, rapid, and affordable protocol specifically optimized for tiny flower buds with low starch content. The technique is revealed to successfully determine starch content in non-freshly harvested samples-frozen and stored at -20 °C or stored in fixatives-allowing a temporal separation of sampling and quantification and making the protocol suitable for high-throughput experimental designs in different fields of plant research. Key features • Sensitive protocol to quantify starch content in developing flower primordia of sweet cherry. • Starch quantification based on enzymatic hydrolysis of starch and spectrophotometric quantification of NADH absorbance at 340 nm using the hexokinase and glucose-6-phosphate dehydrogenase coupled assay. • Accurately detects starch in very small samples with low starch concentrations in both fixed and frozen plant material.