Chestnut is an important economic crop, which is widely planted in the world including Asia, Europe, North America [1]. Castanea henryi as a native species in China is preferred by consumers due to its high edibility and abundant nutrition [2]. Previous studies reported that chestnuts were rich in various nutrients, such as starch, proteins, vitamins and minerals [3,4] but relatively low in calories and fat, which provided them potential as healthy food [5]. In addition, some functional substances including phenols and carotenoids were also investigated in Castanea henryi, enriching its nutritional and health value [6,7].

Starch, as the major component of chestnuts, accounts for >50 % of dry weight [8]. Compared with other nuts, the unique nutritional value of chestnut mainly depends on its distinctive starch components. Starch biosynthesis is a complex process that mainly performed in chloroplasts and amyloplasts. Starch synthesized in amyloplasts is also called storage starch, which generally accumulates in seeds until seeds mature. The starch in Castanea henryi kernel was mainly synthesized in amyloplasts [9]. The precursor of starch synthesis in amyloplasts is sucrose from photosynthesis. The sucrose is catalyzed in the cytoplasm by sucrose synthetase to convert to UDP-glucose (UDPG) and fructose. UDPG is then converted with pyrophosphate (PPi) to glucose-1-phosphate (G1P), which is further converted to glucose-6-phosphate (G6P) by phosphoglucomutase. After being transported to amyloplast, G6P is converted back to G1P under the action of phosphoglucomutase, and then catalyzed by ADP-glucose pyrophosphorylase (AGPase) to produce ADP-glucose (ADPG), which provides glucose residues for amylose and amylopectin biosynthesis. These glucose residues are catalyzed by starch synthase, starch branching enzyme (SBE) and debranching enzyme (DBE) to synthesize amylose and amylopectin, and eventually form different starch granules. The function of starch synthase is to add glucose residues to the non-reducing ends. Generally speaking, it can be divided into two categories, soluble starch synthase (SSS) and granule bound starch synthase (GBSS). Among them, SSS plays an important role in regulating the synthesis of amylopectin, while GBSS is closely related to the synthesis of amylose [10]. It has been found that SSS contains four isoenzymes (SSS I, SSS II, SSS III, SSS IV), and GBSS contains two isoenzymes (GBSS I and GBSS II) [11]. The function of SBE is to split the α-1, 4-glucoside bond between glucan in amylopectin region and catalyze the formation of α-1, 6-glucoside bond between C1 and C6 of receptor glucan chain, thus forming branching. SBE consists of two isoenzymes (SBE I and SBE II). Two types of DBE exist in plants, isoamylase (ISA) and pullulanase (PUL), and ISA includes three subtypes (ISA I, ISA II, ISA III) [12]. At present, the molecular mechanism of DBE in the synthesis of amylopectin remains unclear. Some studies speculated that DBE might affect the crystal structure of amylopectin by pruning those randomly generated glucan branches with inappropriate distances and thus the overall structure of starch [13].

The functional properties of starch directly affect the quality of starch-based products and their deep processing applications, which are generally determined by starch structure. Chestnut starch had higher swelling power, lower pasting and gelatinization temperature, and better freeze-thaw stability compared with corn starch, which made it a potential alternative in frozen starch-based products [14]. In addition, previous study found that the content of amylose could affect physicochemical properties and gel textural properties of Chinese chestnut starch [15]. Moreover, the digestibility of starch is critical for supporting public health [16]. Starch can be categorized into fast digestible starch (RDS), slow digestible starch (SDS) and resistant starch (RS) according to different digestion rates in human body. RDS can be rapidly absorbed in the small intestine, leading to a rapid rise in blood sugar. SDS and RS have a slow digestion process, which can reduce the rate of blood glucose production. In particular, RS cannot be digested in the small intestine, but it can be fermented in the intestine to produce prebiotics, thus regulating the intestinal microecology.

Although previous studies investigated structural and functional properties of chestnut starches, few studies focused on the variations of starch during chestnut kernel development. The aim of present study was to focus on the changes of structure and digestive properties of starch during the development of Castanea henryi kernel. In addition, transcriptomic analysis and RT-qPCR assays were performed to explore the possible molecular mechanisms affecting starch structure during kernel development. The results provided new insights for starch biosynthetic mechanism of Castanea henryi kernel. Moreover, the study provided valuable information for industrial application by revealing the relationship between the multiscale structure, digestive property and gene expression of starch during the development of Castanea henryi.