Observation on Double Fertilization and Early Embryonic Development in Autotetraploid Polyembryonic Rice

The process of double fertilization and the characters of embryo and endosperm development in an autotetraploid polyembryonic mutant rice IR36-Shuang were studied with a laser scanning confocal microscopy. Some abnormalities including degenerated ovary, abortive embryo sac, single fertilization, double-ovule and double-embryo and so on. were found dudng double fertilization and embryo development in IR36-Shuang. The rate of the abnormalities was 46.67% in IR36-Shuang, significantly higher than that in the control, an autotetraploid rice line IR36-4X （33.00%）. Cytological and embryonic evidences were provided for seed setting decline and the initiation of additional embryo in IR36-Shuang.

A one-step rectification of sperm cell targeting ensures the success of double fertilization

Successful fertilization in animals depends on competition among millions of sperm cells, whereas double fertilization in flowering plants usually involves just one pollen tube releasing two immobile sperm cells. It is largely a mystery how the plant sperm cells fuse efficiently with their female targets within an embryo sac. We show that the initial positioning of sperm cells upon discharge from the pollen tube is usually inopportune for gamete fusions and that adjustment of sperm cell targeting occurs through release and re-adhesion of one sperm cell, while the other connected sperm cell remains in stagnation. This enables proper adhesion of each sperm cell to a female gamete and coordinates the gamete fusions. Our 〈br〉 findings reveal inner embryo sac dynamics that ensure the reproductive success of flowering plants and suggest a requirement for sperm cell differentiation as the basis of double fertilization.

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.

Egg cell-secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis

Double fertilization is an innovative phenomenon in angiosperms,in which one sperm cell first fuses with the egg cell to produce the embryo,and then the other sperm fuses with the central cell to produce the endosperm.However,the molecular mechanism of the preferential fertilization of egg cells is poorly understood.In this study,we report that two egg cell-secreted aspartic proteases,ECS1 and ECS2,play an important role in promoting preferential fertilization of egg cells in Arabidopsis.We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20%reduction in fertility,which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2.Detailed phenotypic analysis revealed that the egg cellsperm cell attachment was compromised in ecs1 ecs2 siliques.Limited pollination assays with cyclin-dependent kinase a1(cdka;1)pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant.Taken together,these results demonstrate that egg cells secret two aspartic proteases,ECS1 and ECS2,to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved.This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

Pollen-expressed RLCKs control pollen tube burst

In angiosperms,the pollen tube enters the receptive synergid cell,where it ruptures to release its cytoplasm along with two sperm cells.This interaction is complex,and the exact signal transducers that trigger the bursting of pollen tubes are not well understood.In this study,we identify three homologous receptor-like cytoplasmic kinases(RLCKs)expressed in pollen tubes of Arabidopsis,Delayed Burst 1/2/3(DEB1/2/3),which play a crucial role in this process.These genes produce proteins localized on the plasma membrane,and their knockout causes delayed pollen tube burst and entrance of additional pollen tubes into the embryo sac due to fertilization recovery.We show that DEBs interact with the Ca^(2+)pump ACA9,influencing the dynamics of cytoplasmic Ca^(2+)in pollen tubes through phosphorylation.These results highlight the importance of DEBs as key signal transducers and the critical function of the DEB-ACA9 axis in timely pollen tube burst in synergids.

GEX3, Expressed in the Male Gametophyte and in the Egg Cell of Arabidopsisthaliana, Is Essential for Micropylar Pollen Tube Guidance and Plays a Role during Early Embryogenesis

Double fertilization in flowering plants occurs when the two sperm cells, carried by the pollen tube, are released in a synergid cell of the embryo sac and then fertilize the egg and the central cell. Proteins on the surfaces of the sperm, egg, central, and synergid cells might be important for guidance and recognition/fusion of the gametes. Here, we present functional analyses ofArabidopsis GEX3, which encodes a plasma membrane-localized protein that has homologs in other plants. GEX3 is expressed in both the vegetative and sperm cells of the male gametophyte and in the egg cell of the female gametophyte. Transgenic lines in which GEX3 was down-regulated or overexpressed, using the Arabidopsis GEX2 promoter, had reduced seed set. Reciprocal crosses and imaging after pollination with a reporter line showed that, in both cases, the defect causing reduced seed set occurred in the female. In the antisense lines, micropylar pollen tube guidance failed, In the overexpression lines, fertilization of mutant ovules was mostly blocked because pollen tube guidance failed, although, occasionally, non-viable embryos were formed. We conclude that properly regulated expression of GEX3 in the egg cell of Arabidopsis is essential for pollen tube guidance.

The language of GABA in pollen tube growth and guidance

The achievement of double fertilization in higher plants requires the successful transport of sperm cells to the female gametes,the ovules.Pollen tubes,the tubular structure protruding from pollens,carrying the sperms play an important role in this process.How a pollen tube precisely guides its direction to gain its goal is of mystery.Previous investigation indicated that multiple signal clues from the pistils function as the route signs to regulate the pathway of pollen tube growth.Among the signal clues,c-aminobutyric acid(GABA)seems to be universal.Its gradient concentration has been found both in tobacco and Arabidopsis pollen tube guidance.In the communication of pollens and pistils,what on earth GABA tells pollen tubes is of great interest.The GABA receptors on the pollen membrane are thought to be the hinge in the language conversation.In this review,the mechanism of GABA gradient formation is investigated.The possible GABA receptor on the pollen membrane is examined and its function is discussed.To decipher the possible language of GABA in pollen tube growth and guidance,multiple methods are needed.The combination of transcriptome and proteomics assay is expected to unveil the secret.