Project description:Flowering plants have immotile sperm that develop within pollen and must be carried to female gametes by a pollen tube. The pollen tube engages in molecular interactions with several cell types within the pistil and these interactions are essential for successful fertilization. We identified a group of three closely related pollen tube-expressed MYB transcription factors (MYB97, MYB101, MYB120), which are required for proper interaction of the pollen tube with the female gametophyte. These transcription factors are transcriptionally induced during growth in the pistil. They regulate a transcriptional network leading to proper differentiation and maturation of the pollen tube, promoting proper pollen tube-ovule interactions resulting in sperm release and double fertilization. We used microarrays to discover genes regulated by the transcription factors MYB97, MYB101 and MYB120 in pollen tubes growing through the pistil at 8 hours after pollination.
Project description:Flowering plants have immotile sperm that develop within pollen and must be carried to female gametes by a pollen tube. The pollen tube engages in molecular interactions with several cell types within the pistil and these interactions are essential for successful fertilization. We identified a group of three closely related pollen tube-expressed MYB transcription factors (MYB97, MYB101, MYB120), which are required for proper interaction of the pollen tube with the female gametophyte. These transcription factors are transcriptionally induced during growth in the pistil. They regulate a transcriptional network leading to proper differentiation and maturation of the pollen tube, promoting proper pollen tube-ovule interactions resulting in sperm release and double fertilization. We used microarrays to discover genes regulated by the transcription factors MYB97, MYB101 and MYB120 in pollen tubes growing through the pistil at 8 hours after pollination. Pistils were collected from ms1 (Male Sterile 1) pistils that were unpollinated, or pollinated with either wild type (Col-0) pollen or myb triple mutant (myb97-1, myb101-4, myb120-3) pollen for 8 hours. We sought to examine transcriptional changes that were taking place in pollen tubes before they reached ovules in wild type pollen tubes, and what portion of this transcriptional regulation was due to MYB97, MYB101 and MYB120. Analysis of growth in the pistil allows discovery of transcriptional changes taking place during pollen tube growth in its native environment, as opposed to mature pollen or in vitro grown pollen, which are essentially naive conditions, as neither have interacted with the pistil environment and any signalling factors found therein.
Project description:In sexual reproduction, a proper communication and cooperation between male and female organs and tissue are essential for male and female gametes to unite. In flowering plants, female sporophytic tissues and gametophyte direct a male pollen tube towards an egg apparatus, which consists of an egg cell and two synergid cells. The cell-cell communication between the pollen tube and the egg apparatus, such as the reception of a signal from the egg apparatus at the pollen tube, makes the tip of pollen tube rapture to release the sperm cell. To isolate male factors involved in the interaction between a pollen tube and an egg apparatus, we focused on receptor-like kinases (RLKs), which are extensively diversified in the flowering plant lineage to comprise a large monophyletic gene family. Approximately 620 members were found in the Arabidopsis thaliana genome. Expression patterns of 558 RLKs were analyzed using an Affymetrix ATH1 microarray of A. thaliana. We focused on two RLKs, ANXUR1 (ANX1) and ANXUR2 (ANX2), and characterized their function. Here we report that pollen tubes of anx1/anx2 ruptured before arriving at the egg apparatus, suggesting that ANX1 and ANX2 are male factors controlling pollen tube behavior with directing rupture at proper timing. Furthermore, ANX1 and ANX2 were the most closely related paralogs to a female factor FERONIA/SIRENE controlling pollen tube behavior expressed in synergid cells. Our finding shows that the coordinated behaviors of female and male reproductive apparatuses are regulated by the sister genes, whose duplication might play a role in the evolution of fertilization system in flowering plants.
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis.
Project description:Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. We also characterized the transcriptome of in vitro-grown pollen tubes (for 0.5hours or 4hours) and dessicated mature pollen in Arabidopsis. Experiment Overall Design: Pollen and pollen tubes were collected as described in the protocols section for RNA extraction and hybridization on Affymetrix ATH1 Genechip microarrays.
Project description:Pollen grains of Arabidopsis thaliana contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein coding genes expressed in pollen, including 289 assayed only by non-specific probe sets. Additional exons and previously unannotated 5’ and 3’ UTRs for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 confirmed by PCR. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of on-going annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.
Project description:In sexual reproduction, a proper communication and cooperation between male and female organs and tissue are essential for male and female gametes to unite. In flowering plants, female sporophytic tissues and gametophyte direct a male pollen tube towards an egg apparatus, which consists of an egg cell and two synergid cells. The cell-cell communication between the pollen tube and the egg apparatus, such as the reception of a signal from the egg apparatus at the pollen tube, makes the tip of pollen tube rapture to release the sperm cell. To isolate male factors involved in the interaction between a pollen tube and an egg apparatus, we focused on receptor-like kinases (RLKs), which are extensively diversified in the flowering plant lineage to comprise a large monophyletic gene family. Approximately 620 members were found in the Arabidopsis thaliana genome. Expression patterns of 558 RLKs were analyzed using an Affymetrix ATH1 microarray of A. thaliana. We focused on two RLKs, ANXUR1 (ANX1) and ANXUR2 (ANX2), and characterized their function. Here we report that pollen tubes of anx1/anx2 ruptured before arriving at the egg apparatus, suggesting that ANX1 and ANX2 are male factors controlling pollen tube behavior with directing rupture at proper timing. Furthermore, ANX1 and ANX2 were the most closely related paralogs to a female factor FERONIA/SIRENE controlling pollen tube behavior expressed in synergid cells. Our finding shows that the coordinated behaviors of female and male reproductive apparatuses are regulated by the sister genes, whose duplication might play a role in the evolution of fertilization system in flowering plants. Experiment Overall Design: Arabidopsis thaliana Col-0 was grown at 18°C for 8 hr in the dark and at 22°C for 16 hr in the light. To collect germinated pollen with pollen tubes, opened flowers were touched to cellophane layered on the pollen germination medium containing 13.5% sucrose, 10 mM CaCl2, 0.4 mM boric acid, 1 mM KCl, 0.001% myo-inositol, and 0.6% agarose. The released pollen was incubated at 25°C for 6 hr in the dark. For collection of seedlings, seeds were sown on Murashige-Skoog medium containing 1x Murashige and Skoog Plant Salt Mixture (Wako), 1x Gamborgâs vitamin solution (Wako), 1% sucrose, and 0.8% agar. Seedlings including roots with one or two rosette leaves were collected for RNA extraction. RNA was extracted with Isogene (Nippon Gene), precipitated with LiCl, and further purified with RNeasy. The ATH1 genechip (Affymetrix) was used for expression analysis using 10 µg each of total RNA following the manufacturer's instructions. Array data were processed and analyzed with the GCOS1.2 program (Affymetrix) and Excel 2004 (Microsoft). Hybridization with germinating pollen RNA was duplicated and the results were compared to that with seedling RNA.
Project description:Sperm cells of seed plants have lost their motility and are transported by the vegetative pollen tube cell for fertilization. The extent to which sperm cells regulate their own transportation is a long-standing debate. By using the novel Arabidopsis double mutant drop1 drop2, we demonstrate here that sperm cells are only passive cargo and that the vegetative tube cell as a vehicle controls the entire journey.