Functional analysis of PpAux/IAA2 in Physcomitrella patens (Hedw.) Mitt. during protoplast regeneration

We used wild-type (WT) and auxin-resistant mutant Ppiaa2-87 strains of Physcomitrella patens to explore the regulation mechanism of the early auxin-response gene PpAux/IAA2 in protoplast regeneration. Gene expression, survival rate, cell cycle progression, chromosome remodeling, and DNA methylation during protoplast regeneration were analyzed separately using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), fluorescein diacetate (FDA) staining, flow cytometry of DNA ploidy, 4',6-diamidino-2-phenylindole (DAPI) staining, and methylation-sensitive amplified polymorphism analysis. Results showed that the expression levels of three PpAux/IAA homologous genes in 0-h protoplasts were significantly higher than that in protonemata, 48-h, and 96-h protoplasts; survival rate of Ppiaa2-87 protoplasts decreased significantly with culture time; entry into the S phase of Ppiaa2-87 protoplasts was inhibited; chromosomes of some Ppiaa2-87 0-h protoplasts were not remodeled; expression of four genes in the SWI/SNF protein family in the chromatin remodeling complex decreased in 0-h protoplasts compared with that in protonemata; and methylation of Ppiaa2-87 0-h protoplasts was higher than that of WT protoplasts. This study indicates that the PpAux/IAA2-related auxin signaling pathway plays an important role in protoplast regeneration. Mutation of the PpAux/IAA2 gene affects protoplast DNA methylation and chromatin remodeling, which is involved in cell development reprogramming, leading to protoplast death due to lack of pluripotency.