Wheat, as a staple crop, its production has to achieve both high yield and good quality due to the demands for food security and life quality. The protein and starch contents vary greatly within different layers of wheat kernels, which provides a potential approach to achieve both high-yield and high-quality of wheat. This study identified how spatiotemporal mechanisms regulate this. With micro Positron Emission Tomography-Computed Tomography (PET-CT), we observed directly and non-destructively that caryopses acquired sugar through the pathway of transfer cells (TC), inner endosperm (IE), outer endosperm (OE), and finally the aleurone (AL). By contrast, glutamine was transported through the pathway of TC, AL, OE and finally IE. The transport pathway of substrate correlated closely to the final spatial distribution of starch and protein. To get an integrated information on spatial distributions of transcripts, developing caryopses were dissected into AL, OE, IE and TC by laser microdissection technology. Genes participated in sucrose metabolism such as sucrose synthase, beta-fructofuranosidase, glucose-1-phosphate adenylyltransferase showed significantly lower expression in AL. Similarly, the expression pattern of genes involved in amino acid metabolism such as argininosuccinate synthase, threonine synthase and homocysteine methyltransferase showed AL>OE>IE. Therefore, substrate supply was crucial for starch and protein gradients formation. Our results imply that transcriptional regulation represents an important means of impacting starch and protein distribution in wheat grains and suggests targets for enhancing specialty pearled wheat with higher quality.