Background

Starch is a carbohydrate widely produced in plants and composed of homopolymers of glucose, consisting mainly of two glucans, amylopectin and amylose [1]. In woody plant species, starch is the main storage carbohydrate [2], which accumulates in dedicated plant organs, covering the long-term glucan needs that fuel different processes such as germination or regrowth after certain periods of dormancy [3]. In addition, starch sustains the development of different plant reproductive tissues [4]. Various waves of amylogenesis and amylolysis have been reported during stamen and anther development [4–7] and during pollen tube growth within the style and the ovary [8,9]. Although the physiological and developmental functions of starch accumulation in reproductive organs are not fully understood, it has been suggested to support the development of reproductive organs and the male and female germline during flower development, the heterotopic growth of pollen tubes during the progamic phase, and the development of the embryo and seed after fertilization [4,8,10] and underpin the likelihood of a flower to become a fruit [11,12].

Different methodologies have been used to quantify starch content, such as high-performance liquid chromatography, gas chromatography, hydrolysis with perchloric or sulfuric acids, and enzymatic methods ([13] and references therein). Although these methods have been applied on leaves, roots, shoots, or bark tissues, they have not been used in developing flower buds harboring one to several tiny flower primordia, probably due to their small size and low starch concentration. Instead, starch quantification in these small reproductive structures has been performed using histochemical microscopy techniques coupled with a specific image analysis system, which can only provide relative values of starch content [14,15]. The enzymatic hydrolysis of starch coupled with the spectrophotometric assay of hexokinase activity, originally used to quantify starch content in fresh leaves [16,17] and later optimized for fresh small flower organs of Arabidopsis [4], has been further optimized in this work to quantify starch content in small developing reproductive organs of cultivated woody plant species like sweet cherry (Prunus avium L.).

Our main objective was to optimize the enzymatic methodology to quantify starch concentration in small developing flower primordia with low starch concentrations. For this purpose, starch was gelatinized and enzymatically digested by amylase and amyloglucosidase into D-glucose. Next, a coupled hexokinase/glucose-6-phosphate dehydrogenase (HK/G6PD) enzyme assay was used to convert D-glucose into a stoichiometric amount of gluconate-6-phosphate and NADH. NADH was finally measured using a microplate reader, as it absorbs UV light at 340 nm. To make it useful for plant research applications where samples are routinely processed for histochemistry, this method is optimized for samples stored in fixative or frozen at -20 °C. This alternative enables developmental time-series sampling and long-term preservation for high-throughput sample processing and measurements. Thus, this protocol has the advantages of being simple, sensitive, and quick, not using harmful reagents, requiring reasonable labor, and not requiring specific laboratory equipment other than a UV spectrophotometer. The protocol has proven to be sensitive to very low sample weights (less than 1 mg) and low starch concentration (less than 0.18 mg/g of frozen or fixed sample), and the same results were obtained with both frozen and fixed samples.

The development of this enzymatic approach, applied for the first time to a fruit species, enables the quantification of starch in very small samples, such as reproductive structures. Previously, starch in these structures could only be assessed through histochemistry combined with image analysis, which allowed for the determination of relative content but not total quantification. Additionally, this method is equally effective with both frozen and fixed material, significantly extending the storage time of the samples.