Drought stress occurring during the reproductive growth stage leads to considerable reductions in crop production and has become an important limiting factor for food security globally. In order to explore the possible role of drought priming (pre-exposure of the plants to mild drought stress) on the alleviation of a severe drought stress event later in development, wheat plants were subjected to single or double mild drought episodes (soil relative water content around 35–40%) before anthesis and/or to a severe drought stress event (soil relative water content around 20–25%) 15 d after anthesis. Here, single or double drought priming before anthesis resulted in higher grain yield than in non-primed plants under drought stress during grain filling. The photosynthesis rate and ascorbate peroxidase activity were higher while malondialdehyde content was lower in primed plants than in the non-primed plants under drought stress during grain filling. Proteins in flag leaves differently expressed by the priming and drought stress were mainly related to photosynthesis, stress defence, metabolism, molecular chaperone, and cell structure. Furthermore, the protein abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit, Rubisco activase and ascorbate peroxidase were upregulated in primed plants compared with non-primed plants under drought stress during grain filling. In conclusion, the altered protein expression and upregulated activities of photosynthesis and ascorbate peroxidase in primed plants may indicate their potential roles in alleviating a later-occurring drought stress episode, thereby contributing to higher wheat grain yield under drought stress during grain filling.

Summary: Foods high in amylose content and resistant starch (RS) offer great potential to improve human health and lower the risk of serious noninfectious diseases. Common wheat (Triticum aestivum L.) is a major staple food crop globally. However, the RS contents in the grains of modern wheat varieties are low. Here, we report the generation of high‐amylose wheat through targeted mutagenesis of TaSBEIIa in a modern winter wheat cv Zhengmai 7698 (ZM) and a spring wheat cv Bobwhite by CRISPR/Cas9, respectively. We generated a series of transgene‐free mutant lines either with partial or triple‐null TasbeIIa alleles in ZM and Bobwhite, respectively. Analyses of starch composition, structure and properties revealed that the effects of partial or triple‐null alleles were dosage dependent with triple‐null lines demonstrated more profound impacts on starch composition, fine structures of amylopectin and physiochemical and nutritional properties. The flours of triple‐null lines possessed significantly increased amylose, RS, protein and soluble pentosan contents which benefit human health. Baking quality analyses indicated that the high‐amylose flours may be used as additives or for making cookies. Collectively, we successfully modified the starch composition, structure and properties through targeted mutagenesis of TaSBEIIa by CRISPR/Cas9 in both winter and spring wheat varieties and generated transgene‐free high‐amylose wheat. Our finding provides deep insights on the role of TaSBEIIa in determining starch composition, structure, properties and end‐use quality in different genetic backgrounds and improving RS content with multiple breeding and end‐use applications in cereal crop species through genome editing for health benefits.