Site-directed mutagenesis of soybean PEAPOD genes using the CRISPR/Cas9 system alters tissue developmental transition

In general, plant organ size is determined using cell number and expansion. In our previous study, we generated soybean (Glycine max) mutants of the PEAPOD (PPD) genes GmPPD1 and GmPPD2 using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 system. Some of these mutants exhibited extremely abnormal phenotypes, such as twisted pods and limited seeds. These phenotypes were attributed to the frameshift mutation in both GmPPD loci. In this study, the physiological and molecular biological properties of mutant plants with two knocked-out GmPPD loci (ppd-KO) were characterized. The ppd-KO mutant exhibited a delayed growth phase from the time of development of the unifoliolate leaves to that of first trifoliolate leaves and a stay-green phenotype, which were not observed in the other mutants of soybean or ppd mutants of other plant species. Gene expression analysis revealed considerably decreased expression of SPIRAL1-LIKE 5 (GmSP1L5), mainly causing the twisted pod phenotype observed in the ppd-KO mutant. The relationship between PPD and SP1L5 has not been previously reported, and in this study, we showed that that loss of PPD functioning affects SP1L5 expression in soybean. In this study, we revealed that the decrease in PPD function contributed to organ enlargement and that complete knockout of PPD has a negative effect on soybean organogenesis.