Abstract
Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS-PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS-green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI.
Highlights
Pollination in higher plants is a crucial event, required for fertilization and seed set
These in vitro assays confirmed the S-specific activity of PrsS1, as pollen tube growth rates were significantly inhibited when recombinant PrsS1 was added to pollen tubes expressing PrpS1–green fluorescent protein (GFP)
Pollen tubes expressing PrpS3–GFP treated with PrsS1 were not inhibited.These data correspond to the previously reported SI response in PrpS-expressing Arabidopsis pollen, and serve as a baseline to confirm the biological activity of PrpS1 and the S-specific incompatible response triggered
Summary
Pollination in higher plants is a crucial event, required for fertilization and seed set. Unlike most plant cells, which dedifferentiate and lose polarity when put into in vitro culture, pollen maintains its identity and polarity While they do not grow to the lengths observed in vivo, in vitro grown pollen tubes grow relatively uniformly and exhibit highly polarized cytoplasmic organization with several zones, including an apical zone packed with vesicles, a cytoplasmic-rich nuclear zone, and a vacuolar zone further back (Hepler et al, 2001). The accumulated knowledge so far reveals the existence of a complex, self-organizing signalling network, utilizing, amongst others, a tip-localized Rho GTPase, apical cytosolic Ca2+ gradients, reactive oxygen species (ROS), the actin cytoskeleton, and vesicular trafficking to supply cell wall and membrane components for pollen tube growth (see Qin and Yang, 2011 for a review)
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