Lack of ethylene does not affect reproductive success and synergid cell death in Arabidopsis

The signaling pathway of the gaseous hormone ethylene is involved in plant reproduction, growth, devel- opment, and stress responses. During reproduction, the two synergid cells of the angiosperm female gametophyte both undergo programmed cell death (PCD)/degeneration but in a different manner: PCD/ degeneration of one synergid facilitates pollen tube rupture and thereby the release of sperm cells, while PCD/degeneration of the other synergid blocks supernumerary pollen tubes. Ethylene signaling was postu- lated to participate in some of the synergid cell functions, such as pollen tube attraction and the induction of PCD/degeneration. However, ethylene-mediated induction of synergid PCD/degeneration and the role of ethylene itself have not been firmly established. Here, we employed the CRISPR/Cas9 technology to knock out the five ethylene-biosynthesis 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes and created Arabidopsis mutants free of ethylene production. The ethylene-free mutant plants showed normal triple responses when treated with ethylene rather than 1-aminocyclopropane-1-carboxylic acid, but had increased lateral root density and enlarged petal sizes, which are typical phenotypes of mutants defective in ethylene signaling. Using these ethylene-free plants, we further demonstrated that production of ethylene is not necessarily required to trigger PCD/degeneration of the two synergid cells, but certain com- ponents of ethylene signaling including transcription factors ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3- LIKE 1 (EIL1) are necessary for the death of the persistent synergid cell.