Cell-to-Eell Communication Involves In Coordinated Development Of Arabidopsis Central Cell And Egg Cell

Arabidopsis female gametophyte development progressed through a serial processes, including cell division, nuclei migration, cellularization, cell fate determination and embryo sac maturation and so on. The female gametes, central cell and egg cell, generated after the female gametophyte cellularization. Then the mature central cell and egg cell fertilized with sperm cells from the pollen tube to trigger embryogenesis and endosperm development. During the femame gamete development and double fertilization, the central cell and egg cell coordinated with each other and become mature female gametes synchronously. However, until now, how to control the female gamete coordinated development is still unknown. Furthermore, after female gamete generation, the mature central and egg cell formed after a maturation process. Until now, the biology impact of this maturation process is still unclear. Whether the maternal mechanism necessary for fertilization and early embryogenesis established in this period should be investigated more carefully. To answer these questions, we took gcdl/GCD1mutant as material. We analyzed the phenotype of gcdl/GCD1during female gamete development and double fertilization, GCD1expression pattern and subcellular location, gcdl female gamete ultrastructure and so on. Also, we use cell-type specific complementary assay and artificial cell-type mutant construction for the purpose. Following is our main research work.1. We have isolated gcdl/GCD1mutant with a single T-DNA insertion. Reciprocal crosses between the mutant heterozygous for the T-DNA insertion and the wild-type showed the mutation dramatically impaired female functioning and completely disrupted male gametophyte functioning. Another T-DNA insertion mutant of the same allele, gcdl-2/GCD1, showed the similar defects in the male and female gametophyte function. A rescue construct introduced into heterozygous gcdl/+mutants was able to revert the mutant phenotypes. These results indicated that gcdl/GCD1defective phenotype was caused by loss function of GCD1. Sequence alignment indicated that GCD1was a conserved novel gene with single copy in Arabidopsis. It’s chemical function is totally unknown. Phylogenetic analysis showed that GCD1is present as a single or duplicated gene in each of the plant species, thus, suggesting a possible conserved function for GCD1throughout the plant lineage.2. pGCD1::GUS signal analysis in transgenic plants suggested universal expression of the GCD1gene with preferential expression in reproductive organs. pGCD1::H2B_GFP was also detected in the female gametophytes at different stages, as well as in the early embryos and endosperms. we next generated a fusion protein construct, GCD1-GFP, comprising the GCD1promoter and the entire GCD1coding region fused with a GFP coding sequence.GCD1-GFP expression was clearly detected in mature female gametophytes, early embryos and endosperms, implying that GCD1may play an important role in these periods.3. gcdl/GCD1mutant analysis indicated that gcdl polar nucleus fusion has been affected. Also, the egg cell size and length was shorted than wild type, indicated that egg cell is immature. Therefore, we can concluded that gdcl mutation affected female gamete maturation. However, the earlier stages of female gametophyte development, including cell division, nuclei migration, embryo sac cellularization and so on, have not been affected. Next we analysis the cell fate determination of gcdl female gametophyte using the central cell specific markers, pDD65::H2B_GFP and AGL80_GFP, egg cell specific marker DD45_GFP, and synergid cell specific marker DD31_GFP. The expressions of all the markers in gcdl/+were comparable to that of the wild-type, suggesting that the cell identity was maintained in the mutants, but that female gamete late maturation was interrupted.4. We pollinated the gcdl/GCD1pistils with wild type pollen and analyzed the ovules after two days. The results showed that all female gametes can be fertilized, indicating that the gcdl immature female gametes can also fuse with sperm cell. However, the embryogenesis initiation and early endosperm development have been affected. To comfirm that the arrested zygote and endosperm were the results of double fertilization, but not autonomous development, we introduced two markers, AGL80_GFP and GRP23_GUS. The paternal expression of these two markers in the gcdl/GCD1is comparable to the wild type, suggested that the aborted zygote and arrested early endosperm is indeed the result of double fertilization. These results also confirmed that gcdl female gametes can fuse with sperm cell, but the embryo initiation and early endosperm development have been affected.5. To understand the behavior of the two unfused polar nuclei during fertilization in the gcdl/+plants, we first measured the size of endosperm nuclei in gcdl/+embryo sacs and found that they were comparable to the wild-type, indicating that the endosperm nuclei in the gcdl/+young seeds were of uniform size and that none of them represented unfused polar nuclei. The aborted ovules containing two endosperm nuclei labeled with paternally derived AGL80-GFP signal were further observed after whole-mount clearing. Only two endosperm nuclei were found, suggesting that both polar nuclei had fused with sperm cell. We also measured the DNA contents of the endosperm nuclei in both gcdl/+and wild-type seeds at2DAP. The DNA contents of all of the endosperm nuclei were between3C-6C, indicating that sperm cell fused with two polar nuclei. To trace the fusion process of male and female gametes during fertilization in gcdl gametophytes, we used our transgenic lines pDD65::H2B_EGFP in the gcdl/+background, in which the central cell was labeled with GFP. The transgenic plants were crossed with wild-type pollen carrying HTR10-monomeric red fluorescent protein1(mRFP1). Unfused polar nuclei were first observed in the gcdl gametophytes, after which one fused with a sperm nucleus, followed by the other. Thus, in contrast to a secondary nucleus fertilization in GCD1gametophytes, polar nuclei in gcdl successively fused with sperm nucleus during fertilization of central cell.6. we pollinated wild-type pistils with pGCD1::H2B_GFP pollen and found that the paternally derived pGCD1::H2B_GFP was expressed in the egg cell and central cell immediately after fertilization, indicating that there is no epigenetic paternal allele silencing of GCD1. We also pollinated the wild-type pistils with pGCD1::GCD1_GFP pollen and found that paternal GCD1was expressed in zygotes and early endosperm. Furthermore, paternally derived GCD1expression in the arrested zygote and early endosperm was also detected when the gcdl female gametophyte was fertilized with pGCD1::GCD1GFP pollen, but the aborted ovules could not be restored. These data indicate that the paternal cues of GCD1cannot rescue abnormal embryo and endosperm development. However, in the next generation of this crossing line, we were able to detect GCD1-GFP expression in the gcdl/+female gametes and found that there was a significant increase in fertile ovules when pGCD1::GCD1_GFP-gcdl female gametophytes were fertilized with wild-type pollen. These results suggest that GCD1expression in the female gametes before fertilization, as opposed to expression in the zygote and early endosperm immediately after fertilization, rescued the aborted seeds. This indicates that female gamete cells maturity is essential for triggering embryogenesis and early endosperm development.7. We expressed a GCD1protein under the control of a central cell-specific FWA promoter in gcdl/+mutant background to functionally supplement central cell. As expected, the immature central cell has been restored and developed well. To our surprise, the egg cell development has aslo been recovered. This suggests that the normal development of an egg cell can promote fusion of the polar nuclei. In return, expressing pDD45::GCD1in egg cell of the gcdl/+mutant background also recovered the egg cell developmental defect. Similarly, the unfused polar nuclei has also been recovered, indicating that normal egg cells can also promote further maturation of the central cell. These data suggest that central cells and egg cells can send positive signals to one another to promote final maturation of each other. Moreover, we also found that embryo and endosperm developed well after the gcdl female gamete restored, further comfirmed an important role for the female gamete maturation in the embryo initiation. To validate the cell-specific expression driven by the DD45and FWA promoter during female gametophyte development, we generated plasmid constructs in which the expression of the GCD1-GFP fusion protein was driven by the DD45or FWA promoter. Both transgenes caused a significant increase in the seed set of the gcdl/+mutant. Moreover, GCD1-GFP fusion protein expressed specially in central cell or egg cell. These results further confirmed that cell-cell communication between female gametes control their coordinated maturation.8. Subcellular location analysis indicated that GCD1located in mitochodria. Ultrastructural analysis revealed an irregular structure and reduced the number of cristae in the central cell mitochondria of gcdl gametophytes. Therefore, whether the positive signals is related to the mitochodria is still a question. We first introduced pMEA::aac2construct into wild type and specifically destroy central cell mitochodria. As expected, the central cell polar nuclei has been affected. However, the egg cell maturation has also been affected. These data suggest that artificial dysfunction of mitochondria affected the maturation of central cells and blocked the transmission of the positive signal, thereby also influencing the maturation of egg cells. Similarly, the specific expression of aac2A199D in egg cell using the promoter DD45significantly affected egg cell development. Not surprisingly, we observed unfused polar nuclei in the central cell, indicating that immature egg cell can not realse positive signals and thereby central cell development also be affected. These results further confirm the existence of cell-cell communication between the egg cell and central cell, and suggest that the signaling is mitochondria-based.In summary, we isolated gcdl/GCD1mutant. And gcdl mutation dramatically impaired female functioning and completely disrupted male gametophyte functioning. Expression pattern analysis showed that GCD1has a strong expression in the male and female gametophyte, early embryo and endosperm, indicated its important role in these periods. Mutant phenotype analysis showed that gcdl mutation specific affected central cell and egg cell final maturation. The dynamic process of gcdl central cell fertilization showed that the unfused polar nuclei successively fused with sperm cell to form triploid endosperm nuclei, implying fertilization triggered polar fusion. gcdl female gametes are capable of fertilization, but embryogenesis initiation and early endosperm development have been affected. GCD1expression in the female gametes before fertilization instead of expression in the zygote and early endosperm immediately after fertilization, rescued the aborted seeds. This indicates that female gamete cells maturity is essential for triggering embryogenesis and early endosperm development. Central or egg cell-type specific expression of GCD1in the gcdl/GCD1mutant background can restored both female gamete development, indicating cell-to-cell communication between female gametes control their coordinated maturation. We destoryed the central or egg cell mitochodrial and found the abnormal development of the corresponding female gamete. To our surprise, its partner gametic cell development has also been affected. This results further confirmed the signalings between female gametes play an important role in the central cell and egg cell final mutation, and this signaling is mitochodria-dependent.