A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases

Crop breeding schemes can be significantly accelerated by using(doubled)haploid plants.In vivo haploid induction has been applied in plant breeding for decades but is still not available for all crops and genotypes,and haploidization rates are generally very low.Therefore,methodological improvements to and new concepts for haploidization are required.Here,we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases(ECSs).We show that after successful sperm–egg cell fusion,ECSs play a critical role to ensure male and female nucleus fusion after fertilization.The ecs1 ecs2 double mutant can induce haploids by both selfing and hybridization in Arabidopsis and ECS mutation is also capable of producing haploids in rice.In summary,our study develops a novel approach for maternal haploidization and provides new insights into the molecular basis of fertilization.

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.

Do egg cell-secreted aspartic proteases promote gamete attachment?

During the double fertilization process of flowering plants(angiosperms),pollen tubes transport a pair of immobile sperm cells toward and inside the embryo sac for fusion with egg and central cell,respectively.Male–female gamete interactions during fertilization are critical for reproductive success and seed development,and thus have interested biologists for decades.Compared to animals,little is known about gamete interaction mechanisms in angiosperms.

The egg cell is preferentially fertilized in Arabidopsis double fertilization

In flowering plants(angiosperms),fertilization of the egg cell by one sperm cell produces an embryo,whereas fusion of a second sperm cell with the central cell generates the endosperm.In most angiosperms like Arabidopsis,a pollen grain contains two isomorphic sperm cells required for this double fertilization process.A long-standing unsolved question is whether the two fertilization events have any preference.A tool to address this question is the usage of the cyclin-dependent kinase a1(cdka;1)mutant pollen,which produces a single sperm-like cell(SLC).Here,we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC.Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell,precursor of the two sperm cells.Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2%of the ovules,single fertilization of the egg cell occurred.Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery,which fertilized the central cell,resulting in 20.7%double-fertilized ovules.This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell.Taken together,our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.