In Angiosperms, the male gametes are delivered to the female gametes through the maternal reproductive tissue by the pollen tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain poorly understood. Here, we describe lorelei, an Arabidopsis thaliana mutant impaired in sperm cell release, reminiscent of the feronia/sirène mutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signaling mechanisms at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of these proteins within a similar biological context.

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.

In plant cells, microtubules (MTs) in the cytokinetic apparatus phragmoplast exhibit an antiparallel array and transport Golgi-derived vesicles toward MT plus ends located at or near the division site. By transmission electron microscopy, we observed that certain antiparallel phragmoplast MTs overlapped and were bridged by electron-dense materials in Arabidopsis thaliana. Robust MT polymerization, reported by fluorescently tagged End Binding1c (EB1c), took place in the phragmoplast midline. The engagement of antiparallel MTs in the central spindle and phragmoplast was largely abolished in mutant cells lacking the MT-associated protein, MAP65-3. We found that endogenous MAP65-3 was selectively detected on the middle segments of the central spindle MTs at late anaphase. When MTs exhibited a bipolar appearance with their plus ends placed in the middle, MAP65-3 exclusively decorated the phragmoplast midline. A bacterially expressed MAP65-3 protein was able to establish the interdigitation of MTs in vitro. MAP65-3 interacted with antiparallel microtubules before motor Kinesin-12 did during the establishment of the phragmoplast MT array. Thus, MAP65-3 selectively crosslinked interdigitating MTs (IMTs) to allow antiparallel MTs to be closely engaged in the phragmoplast. Although the presence of IMTs was not essential for vesicle trafficking, they were required for the phragmoplast-specific motors Kinesin-12 and Phragmoplast-Associated Kinesin-Related Protein2 to interact with MT plus ends. In conclusion, we suggest that the phragmoplast contains IMTs and highly dynamic noninterdigitating MTs, which work in concert to bring about cytokinesis in plant cells.

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A novel kinesin, GhKCH1, has been identified from cotton (Gossypium hirsutum) fibers. GhKCH1 has a centrally located kinesin catalytic core, a signature neck peptide of minus end-directed kinesins, and a unique calponin homology (CH) domain at its N terminus. GhKCH1 and other CH domain-containing kinesins (KCHs) belong to a distinct branch of the minus end-directed kinesin subfamily. To date the KCH kinesins have been found only in higher plants. Because the CH domain is often found in actin-binding proteins, we proposed that GhKCH1 might play a role in mediating dynamic interaction between microtubules and actin microfilaments in cotton fibers. In an in vitro actin-binding assay, GhKCH1's N-terminal region including the CH domain interacted directly with actin microfilaments. In cotton fibers, GhKCH1 decorated cortical microtubules in a punctate manner. Occasionally GhKCH1 was found to be associated with transverse-cortical actin microfilaments, but never with axial actin cables in cotton fibers. Localization of GhKCH1 on cortical microtubules was independent of the integrity of actin microfilaments. Thus, GhKCH1 may play a role in organizing the actin network in coordination with the cortical microtubule array. These data also suggest that flowering plants may employ unique KCHs to coordinate actin microfilaments and microtubules during cell growth.