Micropylar Pollen Tube Guidance Research Articles

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana.

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase (NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss-of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.

Spatial and temporal localization of homogalacturonans in Hyacinthus orientalis L. ovule cells before and after fertilization.

Key message The composition of homogalacturonans (HGs) in the ovule and the female gametophyte cell walls was shown to be rearranged dynamically during sexual reproduction of H. orientalis.In angiosperms, homogalacturonans (HGs) play an important role in the interaction between the male gametophyte and the pistil transmitting tract, but little is known about the participation of these molecules at the final stage of the progamic phase and fertilization. The aim of our study was to perform immunocytochemical localization of highly (JIM7 MAb) and weakly (JIM5 MAb) methyl esterified and Ca2+-associated HG (2F4 MAb) in the ovule and female gametophyte cells of Hyacinthus orientalis before and after fertilization. It was found that pollination induced the rearrangement of HG in (1) the micropylar canal of the ovule, (2) the filiform apparatus of the synergids, and (3) the region of fusion between sperm cells and their target cells. Fertilization led to further changes in pectin composition of these three regions of the ovule. A new cell wall was synthesized around the zygote with a characteristic pattern of localization of all examined HG fractions, which we called “sporoderm-like”. The developing endosperm prepared for cellularization by synthesizing highly methyl-esterified HG, which was stored in the cytoplasm. Pollination- and fertilization-induced changes in the composition of the HG in the micropyle of the ovule and the apoplast of female gametophyte cells are discussed in the context of: (1) micropylar pollen tube guidance, (2) preparation of the egg cell and the central cells for fusion with sperm cells, and (3) the polyspermy block.

Transcriptome analysis of the Arabidopsis semi-in vivo pollen tube guidance system uncovers a distinct gene expression profile

In flowering plants, pollen tube guidance is an enigmatic process involving multiple cell-to-cell communications. Several important signal molecules associated with this process have been previously identified, such as LUREs, which are pollen tube attractants derived from Torenia fournieri synergid cells. To identify pollen tube receptors of ovule-secreted attractant signals, we constructed an Arabidopsis semi-in vivo pollen tube guidance system and used microarray analysis to characterize the specific transcriptome. A distinct gene expression profile was observed for pollen tubes that had grown through stigma and style tissues of a pistil and communicated with ovules. Genes involved in signal transduction, transcription, and pollen tube growth were overrepresented, with many genes newly detected as expressed in pollen tubes. The roles of some candidate receptor-like kinase genes in micropylar pollen tube guidance were revealed by reverse genetics analysis. Our results provide valuable clues for research on cell-to-cell interactions during fertilization.

Membrane-Bound RLCKs LIP1 and LIP2 Are Essential Male Factors Controlling Male-Female Attraction in Arabidopsis

Successful sexual reproduction in animals and plants requires communication between male and female gametes. In flowering plants, unlike in animals, eggs and sperm cells are enclosed in multicellular embryo sacs and pollen grains, respectively; guided growth of the pollen tube into the ovule is necessary for fertilization. Pollen tube guidance requires accurate perception of ovule-emitted guidance cues by the receptors in pollen tubes. Although several ovule-secreted peptides controlling pollen tube guidance have recently been identified, i.e., maize EGG APPARATUS1 (EA1), Torenia LURE1/LURE2, and Arabidopsis CRP810_1/AtLURE1, little is known about the receptors. Here, we identified two receptor-like kinase (RLK) genes preferentially expressed in Arabidopsis pollen tubes, Lost In Pollen tube guidance 1 (LIP1) and 2 (LIP2), which are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control. Simultaneous inactivation of LIP1 and LIP2 led to impaired pollen tube guidance into micropyle and significantly reduced attraction of pollen tubes toward AtLURE1. Our results suggest that LIP1 and LIP2 represent essential components of the pollen tube receptor complex to perceive the female signal AtLURE1 for micropylar pollen tube guidance.

Overcoming Hybridization Barriers by the Secretion of the Maize Pollen Tube Attractant ZmEA1 from Arabidopsis Ovules

A major goal of plant reproduction research is to understand and overcome hybridization barriers so that the gene pool of crop plants can be increased and improved upon. After successful pollen germination on a receptive stigma, the nonmotile sperm cells of flowering plants are transported via the pollen tube (PT) to the egg apparatus for the achievement of double fertilization. The PT path is controlled by various hybridization mechanisms probably involving a larger number of species-specific molecular interactions. The egg-apparatus-secreted polymorphic peptides ZmEA1 in maize and LURE1 and LURE2 in Torenia fournieri as well as TcCRP1 in T. concolor were shown to be required for micropylar PT guidance, the last step of the PT journey. We report here that ZmEA1 attracts maize PTs in vitro and arrests their growth at higher concentrations. Furthermore, it binds to the subapical region of maize PT tips in a species-preferential manner. To overcome hybridization barriers at the level of gametophytic PT guidance, we expressed ZmEA1 in Arabidopsis synergid cells. Secreted ZmEA1 enabled Arabidopsis ovules to guide maize PT in vitro in a species-preferential manner to the micropylar opening of the ovule. These results demonstrate that the egg-apparatus-controlled reproductive-isolation barrier of PT guidance can be overcome even between unrelated plant families.

Mutation in SUMO E3 ligase, SIZ1, Disrupts the Mature Female Gametophyte in Arabidopsis

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.

Micropylar pollen tube guidance and burst: adapted from defense mechanisms?

After the first description of fertilization in flowering plants some 125 years ago (Strasburger E: Neue-Untersuchungen über den Befruchtungsvorgang bei den Phanerogamen als Grundlage für eine Theorie der Zeugung. Gustav Fischer; 1884), we are finally beginning to understand the various molecular mechanisms leading to sperm delivery and discharge inside the hidden micropylar region of the female gametophyte (embryo sac). The last phase of pollen tube guidance culminating in tube burst and explosive release of tube contents requires extensive crosstalk between both male and female gametophytes. The first molecules identified that play key roles in these processes represent highly polymorphic proteins, similar to major components of the plant innate immune system. Here we summarize recent advances and briefly discuss the underlying molecular mechanisms also in respect to prezygotic barriers of reproductive isolation.

Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance inArabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.

MAA3 (MAGATAMA3) Helicase Gene is Required for Female Gametophyte Development and Pollen Tube Guidance in Arabidopsis thaliana

The female gametophyte plays a central role in the sexual reproduction of angiosperms. We previously isolated the maa3 (magatama3) mutant of Arabidopsis thaliana, defective in development of the female gametophyte, micropylar pollen tube guidance, and preventing the attraction of multiple pollen tubes. We here observed that the nucleolus of polar nuclei is small, and that the fusion of polar nuclei often did not occur at the time of pollination. The MAA3 gene encodes a homolog of yeast Sen1 helicase, required for RNA metabolism. It is suggested that MAA3 may regulate RNA molecules responsible for nucleolar organization and pollen tube guidance.

GEX3, Expressed in the Male Gametophyte and in the Egg Cell of Arabidopsis thaliana 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 of Arabidopsis 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 Central Cell Plays a Critical Role in Pollen Tube Guidance inArabidopsis

The sperm cell of flowering plants cannot migrate unaided and must be transported by the pollen tube cell of the male gametophyte to achieve successful fertilization. Long-distance pollen tube guidance is controlled by the seven-celled female gametophyte, the embryo sac. Previous reports showed that the synergid cell of the embryo sac is essential for pollen tube guidance. Here, we report the identification of a central cell guidance (ccg) mutant, which is defective in micropylar pollen tube guidance. CCG encodes a nuclear protein with an N-terminal conserved zinc beta-ribbon domain that is functionally interchangeable with that of TFIIB in yeast. This suggests that CCG might act as a transcription regulator for pollen tube guidance. CCG is expressed in the central cell of the female gametophyte. Expression of CCG in the central cell alone is sufficient to restore the normal pollen tube guidance phenotype, demonstrating that the central cell plays a critical role in pollen tube guidance.

Let ZmEA Guide You

In flowering plants, the immotile sperm cells are transported toward the egg through the pollen tube, which navigates its own path from the pollen grain to the female gametophyte. Márton et al. have now identified one of the signals likely to guide the pollen tube. Studying maize, a small egg apparatus-derived protein, ZmEA1, was found to light up the way for the final steps of pollen tube guidance. The ZmEA1 gene was expressed in the egg apparatus, and the protein acted as a short-range signal required for successful fertilization. After fertilization, ZmEA1 expression was rapidly down-regulated. M. L. Márton, S. Cordts, J. Broadhvest, T. Dresselhaus, Micropylar pollen tube guidance by egg apparatus 1 of maize. Science 307 , 573-576 (2005). [Abstract] [Full Text]

Micropylar Pollen Tube Guidance by Egg Apparatus 1 of Maize

Pollen tube guidance precedes the double fertilization of flowering plants. Here, we report the identification of a small maize protein of 94 amino acids involved in short-range signaling required for pollen tube attraction by the female gametophyte. ZmEA1 is exclusively expressed in the egg apparatus, consisting of the egg cell and two synergids. Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. Transgenic down-regulation of the ZmEA1 gene led to ovule sterility caused by loss of close-range pollen tube guidance to the micropyle.