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Knockin' on pollen's door: live cell imaging of early polarization events in germinating Arabidopsis pollen.

ABSTRACT: Pollen tubes are an excellent system for studying the cellular dynamics and complex signaling pathways that coordinate polarized tip growth. Although several signaling mechanisms acting in the tip-growing pollen tube have been described, our knowledge on the subcellular and molecular events during pollen germination and growth site selection at the pollen plasma membrane is rather scarce. To simultaneously track germinating pollen from up to 12 genetically different plants we developed an inexpensive and easy mounting technique, suitable for every standard microscope setup. We performed high magnification live-cell imaging during Arabidopsis pollen activation, germination, and the establishment of pollen tube tip growth by using fluorescent marker lines labeling either the pollen cytoplasm, vesicles, the actin cytoskeleton or the sperm cell nuclei and membranes. Our studies revealed distinctive vesicle and F-actin polarization during pollen activation and characteristic growth kinetics during pollen germination and pollen tube formation. Initially, the germinating Arabidopsis pollen tube grows slowly and forms a uniform roundish bulge, followed by a transition phase with vesicles heavily accumulating at the growth site before switching to rapid tip growth. Furthermore, we found the two sperm cells to be transported into the pollen tube after the phase of rapid tip growth has been initiated. The method presented here is suitable to quantitatively study subcellular events during Arabidopsis pollen germination and growth, and for the detailed analysis of pollen mutants with respect to pollen polarization, bulging, or growth site selection at the pollen plasma membrane.

SUBMITTER: Vogler F

PROVIDER: S-EPMC4404733 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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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 18Â°C for 8 hr in the dark and at 22Â°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 25Â°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 Âµg 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.

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Knockin' on pollen's door: live cell imaging of early polarization events in germinating Arabidopsis pollen.

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