A-type SGs are formed about 4 days after anthesis (DAA), and ATM inhibitor then continue to enlarge to their maximum at about 19 DAA, with diameters approaching 25–50 μm [7] and [12]. B-type SG formation begins at about 10–19 DAA [13], but these SGs do not enlarge until 21 DAA, with a diameter of only about 9 μm at maturity. The origin of B-type SGs has been debated during the history of starch research in wheat. Badenhuizen [14] demonstrated that B-type SGs are formed in mitochondria; however, many researchers have reported that B-type SGs

form in vesicles budded off from outgrowths of A-type granules [15] or in protrusions emanating from A-type granules containing amyloplasts [9], [13], [16] and [17]. The development and distribution of SGs have been shown to be controlled largely by wheat genotype [18], [19] and [20]. Environmental factors, such as drought or temperature during grain filling, also affect wheat grain development, SG size and SG features [21]. Tester et al. [22] reported that higher temperatures result in smaller SGs, but Hurkman et al. [23] reported

that in conditions with high temperatures the proportion of A granules increases, while that of B granules decreases. Endosperm subjected to drought stress has lower numbers of B-type SGs per cell [24]. After drought and temperature, nitrogen (N) nutrition, an indispensable nutrient for wheat SB431542 production, is considered the third most important environmental factor influencing starch composition and properties [21], [25] and [26]. Blacklow and Incoll [27] showed that a moderate reduction in N leads to small increases in starch content in wheat. Increased N fertilization improves the ratio of A-type SGs while the ratios of B-type SGs in the endosperm of strong-gluten wheat cultivars decreases, but the opposite occurs in the medium-gluten and weak-gluten cultivars [28]. Although N application during endosperm development greatly affects the distribution of SGs and the properties of starch, very little information is available on the microstructure of N-treated wheat relative

to the distribution of SGs in different regions of the endosperm. Visualizing the microstructure of SGs from immature and mature kernels will potentially allow the exploration of the second interior of SGs. In the present study, we used image analysis software to investigate the distribution of both A- and B-type SGs under N treatment. Based on these primary measurements, the reasons for variations in the distribution of SGs in different regions of wheat endosperm are discussed. Wheat (Triticum aestivum L.) cv. Xumai 30, a widely grown hard red winter wheat, was provided by the National Wheat Improvement Center. The experiment was conducted in the research fields of the College of Bioscience and Biotechnology, Yangzhou University, Jiangsu, China from November 1, 2011 to August 10, 2012.