AtFH5-labeled secretory vesicles-dependent calcium oscillation drives exocytosis and stepwise bulge during pollen germination.

Pollen germination is an essential step for delivering sperm cells to the embryo sac for double fertilization in flowering plants. The cytosolic Ca2+ concentration ([Ca2+]cyt) and vesicle dynamics are critical for pollen germination, but their potential correlation in pollen grains is not fully understood. Here, we report that [Ca2+]cyt oscillates periodically at the prospective germination sites during pollen germination. The [Ca2+]cyt is mainly from extracellular Ca2+ ([Ca2+]ext) influx, which implicates the Ca2+-permeable ion channel cyclic nucleotide-gated channel 18 (CNGC18). The [Ca2+]cyt oscillations spatiotemporally correlate with the accumulation of secretory vesicles labeled by a formin protein AtFH5, and disruption of vesicle accumulation inhibits the [Ca2+]cyt oscillations. In turn, the [Ca2+]cyt oscillations promote exocytosis, which leads to stepwise cell extension during pollen germination. Together, these data provide a timeline of vesicle dynamics, calcium oscillation, and exocytosis during pollen germination and highlight the importance of the correlation of these events for pollen germination.

Profilin promotes formin-mediated actin filament assembly and vesicle transport during polarity formation in pollen.

Pollen germination is critical for the reproduction of flowering plants. Formin-dependent actin polymerization plays vital roles in vesicle trafficking and polarity establishment during this process. However, how formin-mediated actin assembly is regulated in vivo remains poorly understood. Here, we investigated the function of reproductive profilin 4 and 5 (PRF4 and PRF5) in polarity establishment during pollen germination in Arabidopsis thaliana. Our data showed that the actin filament content was reduced in the prf4 prf5 double mutant and substantially increased in both PRF4- and PRF5-overexpressing pollen grains. By contrast, the positive effect of profilin in promoting actin polymerization was abolished in a formin mutant, atfh5. In addition, the interaction between Arabidopsis formin homology 5 (AtFH5) and actin filaments was attenuated and the trafficking of AtFH5-labeled vesicles was slowed in prf4 prf5 pollen grains. Formation of the collar-like structure at the germination pore was also defective in prf4 prf5 pollen grains as the fast assembly of actin filaments was impaired. Together, our results suggest that PRF4 and PRF5 regulate vesicle trafficking and polarity establishment during pollen germination by promoting AtFH5-mediated actin polymerization and enhancing the interaction between AtFH5 and actin filaments.

Cryo-EM Structure of Actin Filaments from Zea mays Pollen.

Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform vital roles in key cellular processes. Although large amounts of data on the biochemical activities, dynamic behaviors, and important cellular functions of plant actin filaments have accumulated, their structural basis remains elusive. Here, we report a 3.9 Å structure of the plant actin filament from Zea mays pollen (ZMPA) using cryo-electron microscopy. The structure shows a right-handed, double-stranded (two parallel strands) and staggered architecture that is stabilized by intra- and interstrand interactions. While the overall structure resembles that of other actin filaments, its DNase I binding loop bends farther outward, adopting an open conformation similar to that of the jasplakinolide- or beryllium fluoride (BeFx)-stabilized rabbit skeletal muscle actin (RSMA) filament. Single-molecule magnetic tweezers analysis revealed that the ZMPA filament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actin filaments have greater stability than animal actin filaments, which might be important to their role as tracks for long-distance vesicle and organelle transportation.plantcell;31/12/2855/FX1F1fx1.

Actin Polymerization Mediated by AtFH5 Directs the Polarity Establishment and Vesicle Trafficking for Pollen Germination in Arabidopsis.

The process of pollen germination is crucial for flowering plant reproduction, but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood. In this study, we report that a formin family protein, AtFH5, is localized to the vesicles and rotates ahead of Lifeact-mEGFP-labeled actin filaments during pollen germination. The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-labeled vesicles and the polymerization of actin filaments: vesicle-localized AtFH5 promotes actin assembly, and the polymerization and elongation of actin filaments, in turn, is essential for the mobility of AtFH5-labeled vesicles in pollen grains. Taken together, our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.

AtFH16, [corrected] an Arabidopsis type II formin, binds and bundles both microfilaments and microtubules, and preferentially binds to microtubules.

Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FH1FH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro. In addition, AtFH16 FH1FH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FH1FH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.

LlSR28 is involved in pollen germination by affecting filamentous actin dynamics.

Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In this study, a serine/argine-rich (SR) protein cDNA with 28 kDa protein (LlSR28) was isolated from a lily (Lilium longiflorum) expression library. Protein domain analysis showed that LlSR28 had similar structures to Arabidopsis SR45 (AtSR45), and LlSR28 could complement the phenotype of loss of AtSR45 function. Therefore, overexpression of LlSR28 and AtSR45 mutant (atsr45-1) were used in the following experiments. Overexpression of LlSR28 in Arabidopsis completely inhibited pollen germination. In contrast, the pollen germination of atsr45-1 was earlier than that of wild-type. In addition, pollen of atsr45-1 contained less F-actin at the corresponding hydration stage during pollen germination compared to that of wild-type. Alternative splicing analysis showed that Arabidopsis villin1 (AtVLN1) transcript encoding the full-length protein was increased, and that encoding the truncated protein was decreased in atst45-1. Moreover, the mRNA expression level of other actin-binding proteins (ABPs) abundant in Arabidopsis pollen was also changed in atsr45-1. In conclusion, we hypothesize that LlSR28 alters F-actin dynamics probably through its alternative splicing activities to affect directly or indirectly the alternative splicing of AtVLN1 and the expression of different ABPs, which then affects the pollen germination.

Cortical microtubule labeling: insight of AFH14 in non-dividing cells.

We recently reported that AFH14 participated in microtubule and actin filament interaction in cell division, and the AFH14 (FH1FH2) was important to the directly binding activity of microtubules and microfilaments. To preliminarily understand the function and localization of AFH14 in non-dividing cells, we overexpressed FH1FH2-RFP in onion epidermal cells, and found a fluorescence labeled filamentous network. The result of double labeling with different cytoskeleton reporter proteins indicated that FH1FH2-RFP co-localized with cortical microtubules. Treatment of cells expressing FH1FH2-RFP with cytoskeleton disrupting drugs confirms that FH1FH2-RFP binds to microtubules. Moreover, the binding of FH1FH2-RFP to microtubules were revealed to be dynamic by fluorescence recovery after photobleaching (FRAP) experiment. Time-lapse confocal microscopy showed that FH1FH2-RFP could display a dynamics similar to the microtubule dynamic instability. These data suggest that FH1FH2 domain may lead AFH14 function on cortical microtubules in non-dividing cells, and FH1FH2-RFP may be utilized as a microtubule reporter protein in living onion epidermal cells.

ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling.

Villin/gelsolin/fragmin superfamily proteins have been shown to function in tip-growing plant cells. However, genes encoding gelsolin/fragmin do not exist in the Arabidopsis thaliana and rice (Oryza sativa) databases, and it is possible that these proteins are encoded by villin mRNA splicing variants. We cloned a 1006-bp full-length cDNA from Lilium longiflorum that encodes a 263-amino acid predicted protein sharing 100% identity with the N terminus of 135-ABP (Lilium villin) except for six C-terminal amino acids. The deduced 29-kD protein, Lilium ACTIN BINDING PROTEIN29 (ABP29), contains only the G1 and G2 domains and is the smallest identified member of the villin/gelsolin/fragmin superfamily. The purified recombinant ABP29 accelerates actin nucleation, blocks barbed ends, and severs actin filaments in a Ca(2+)- and/or phosphatidylinositol 4,5-bisphosphate-regulated manner in vitro. Microinjection of the protein into stamen hair cells disrupted transvacuolar strands whose backbone is mainly actin filament bundles. Transient expression of ABP29 by microprojectile bombardment of lily pollen resulted in actin filament fragmentation and inhibited pollen germination and tube growth. Our results suggest that ABP29 is a splicing variant of Lilium villin and a member of the villin/gelsolin/fragmin superfamily, which plays important roles in rearrangement of the actin cytoskeleton during pollen germination and tube growth.

Reversible protein tyrosine phosphorylation affects pollen germination and pollen tube growth via the actin cytoskeleton.

Phenylarsine oxide (PAO) and genistein are two well-known specific inhibitors of tyrosine phosphatases and kinases, respectively, that have been used in the functional analysis of the status of protein phosphotyrosine in different cell types. Our experiments showed that both PAO and genistein arrested pollen germination and pollen tube growth and led to the malformation of the pollen tubes, although genistein had a lesser effect. The malformations of the pollen tubes caused by PAO and genistein were, however, quite different. In addition, it was found that the rate of pollen germination and tube growth recovered to a certain extent when phalloidin was present during PAO treatment, but not when it was present during genistein treatment. Furthermore, PAO treatment also had a great effect on the dynamic organization of filamentous actin in the pollen grain and pollen tube, while genistein only caused reorganization of actin at the turning point of the pollen tube. Our results suggest that reversible protein tyrosine phosphorylation is a crucial step in pollen germination and pollen tube growth, but that tyrosine kinases and phosphatases may have different effects which may function through the reorganization of the actin cytoskeleton.

Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen.

It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient.