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.

乙烯气态激素的信号通路参与植物的繁殖、生长、发育以及应激反应。在繁殖过程中，被子植物雌配子体中的两个助细胞均经历程序性细胞死亡（PCD）/退化，但其方式各异：一个助细胞的PCD/退化促进花粉管破裂，从而释放精子细胞，而另一个助细胞的PCD/退化则阻断额外花粉管的形成。乙烯信号被认为参与某些助细胞功能，例如花粉管的吸引和PCD/退化的诱导。然而，乙烯介导的助细胞PCD/退化诱导以及乙烯本身的作用尚未得到明确证实。本研究中，我们利用CRISPR/Cas9技术敲除五个乙烯生物合成1-氨基环丙烷-1-羧酸氧化酶（ACO）基因，创建了无乙烯生产的拟南芥突变体。无乙烯突变植株在用乙烯而非1-氨基环丙烷-1-羧酸处理时表现出正常的三角反应，但具有增大的侧根密度和花瓣尺寸，这些是乙烯信号通路缺陷突变体的典型表型。利用这些无乙烯植株，我们进一步证明了乙烯的产生并非触发两个助细胞PCD/退化的必要条件，但乙烯信号通路中的某些组分，包括转录因子ETHYLENE-INSENSITIVE 3（EIN3）和EIN3-LIKE 1（EIL1），对于持续助细胞的死亡是必要的。