Plastic glutamine synthetase(GS2)is a key enzyme responsible for the first step of ammonium assimilation.Although GS2 function has been studied in some diploid species,much less is known about TaGS2 gene function in hexaploid wheat(Triticum aestivum L.)and the contributions of the three TaGS2 homoeologous genes(homoeologs).Through ion beam irradiation mutagenesis of the Kn9204 wheat cultivar,a series of gs2 single mutant was obtained,and double mutants were subsequently generated.TaGS2-A,-B and-D deficiency produced varying degrees of morphological changes,including decreased plant height,spike number and kernel number.Under ambient CO2,the content of chlorophyll,soluble protein and free amino acids decreased in gs2 double mutants.Moreover,the double mutants exhibited a typical photorespiration-deficient phenotype: elevated CO2 rescued the chlorotic phenotype of thegs2double mutants,which developed green leaves under these conditions.Reactive oxygen species(ROS)accumulation and increased antioxidant enzyme activity were also observed in the double mutants.Taken together,the genetic and physiological evidence show that the three TaGS2 homoeologs have varying effects on the photorespiratory nitrogen cycle,withTaGS2-B as the main contributor to yield components rather thanTaGS2-A or-D.ChIP and bisulfite sequencing analysis indicated that the different effects of the three TaGS2 homoeologs on morphological changes reflects differences in their expression levels,which are regulated by histone and DNA methylation.