Comparative analyses of angiosperm secretomes identify apoplastic pollen tube functions and novel secreted peptides.

KEY MESSAGE: Analyses of secretomes of in vitro grown pollen tubes from Amborella, maize and tobacco identified many components of processes associated with the cell wall, signaling and metabolism as well as novel small secreted peptides. Flowering plants (angiosperms) generate pollen grains that germinate on the stigma and produce tubes to transport their sperm cells cargo deep into the maternal reproductive tissues toward the ovules for a double fertilization process. During their journey, pollen tubes secrete many proteins (secreted proteome or secretome) required, for example, for communication with the maternal reproductive tissues, to build a solid own cell wall that withstands their high turgor pressure while softening simultaneously maternal cell wall tissue. The composition and species specificity or family specificity of the pollen tube secretome is poorly understood. Here, we provide a suitable method to obtain the pollen tube secretome from in vitro grown pollen tubes of the basal angiosperm Amborella trichopoda (Amborella) and the Poaceae model maize. The previously published secretome of tobacco pollen tubes was used as an example of eudicotyledonous plants in this comparative study. The secretome of the three species is each strongly different compared to the respective protein composition of pollen grains and tubes. In Amborella and maize, about 40% proteins are secreted by the conventional "classic" pathway and 30% by unconventional pathways. The latter pathway is expanded in tobacco. Proteins enriched in the secretome are especially involved in functions associated with the cell wall, cell surface, energy and lipid metabolism, proteolysis and redox processes. Expansins, pectin methylesterase inhibitors and RALFs are enriched in maize, while tobacco secretes many proteins involved, for example, in proteolysis and signaling. While the majority of proteins detected in the secretome occur also in pollen grains and pollen tubes, and correlate in the number of mapped peptides with relative gene expression levels, some novel secreted small proteins were identified. Moreover, the identification of secreted proteins containing pro-peptides indicates that these are processed in the apoplast. In conclusion, we provide a proteome resource from three distinct angiosperm clades that can be utilized among others to study the localization, abundance and processing of known secreted proteins and help to identify novel pollen tube secreted proteins for functional studies.

ARMADILLO REPEAT ONLY proteins confine Rho GTPase signalling to polar growth sites.

Polar growth requires the precise tuning of Rho GTPase signalling at distinct plasma membrane domains. The activity of Rho of plant (ROP) GTPases is regulated by the opposing action of guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Whereas plant-specific ROPGEFs have been shown to be embedded in higher-level regulatory mechanisms involving membrane-bound receptor-like kinases, the regulation of GAPs has remained enigmatic. Here, we show that three Arabidopsis ARMADILLO REPEAT ONLY (ARO) proteins are essential for the stabilization of growth sites in root hair cells and trichomes. AROs interact with ROP1 enhancer GAPs (RENGAPs) and bind to the plasma membrane via a conserved polybasic region at the ARO amino terminus. The ectopic spreading of ROP2 in aro2/3/4 mutant root hair cells and the preferential interaction of AROs with active ROPs and anionic phospholipids suggests that AROs recruit RENGAPs into complexes with ROPs to confine ROP signalling to distinct membrane regions.

Transcriptomic and Proteomic Insights into Amborella trichopoda Male Gametophyte Functions.

Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.

Correlative Microscopy and Spectroscopy Workflow for Microplastics.

Microplastics (MPs) have been reported in various environmental compartments and their number is continuously increasing because of degradation into smaller fragments down to nanoplastics. Humans are exposed to these small-sized MPs through food and air with potential health consequences that still need to be determined. This requires, in the first place, efficient and detailed visualization, relocalization, and characterization of the same MPs with complementary analytical methods. Here, we show the first application of a correlative microscopy and spectroscopy workflow to MPs that meets these demands. For this purpose, standard MP particles on aluminum-coated polycarbonate membrane filters were investigated by an optical zoom microscope and a hyphenated scanning electron microscopy (SEM)-Raman system. By merging the obtained data in one software, it is possible to navigate on the entire filters' surface and correlate at identical locations MP morphology at the spatial resolutions of electron (1.6 nm at 1 kV for the used SEM, ∼100 nm minimum MP size in this study) and optical (∼1-10 µm) microscopies with chemical identification by micro-Raman spectroscopy. Moreover, we observed that low-voltage SEM works without a conductive coating of MPs, causes no detectable charging and structural changes, and provides high-resolution surface imaging of single and clustered MP particles, thus enabling subsequent Raman measurements. We believe that further work on the accurate identification and quantification of micro- and nanoplastics in real samples can potentially profit from this workflow.

F-actin forms mobile and unwinding ring-shaped structures in germinating Arabidopsis pollen expressing Lifeact.

The flowering plant pollen tube is the fastest elongating plant cell and transports the sperm cells for double fertilization. The highly dynamic formation and reorganization of the actin cytoskeleton is essential for pollen germination and pollen tube growth. To drive pollen-specific expression of fluorescent marker proteins, commonly the strong Lat52 promoter is used. Here we show by quantitative fluorescent analysis that the gametophyte-specific ARO1 promoter from Arabidopsis drives an about 3.5 times weaker transgene expression than the Lat52 promoter. In one third of the pollen of F-actin-labeled ARO1p:tagRFP-T-Lifeact transgenic lines we observed mobile ring-shaped actin structures in pollen grains and pollen tubes. Pollen tube growth, transgene transmission and seed production were not affected by tagRFP-T-Lifeact expression. F-actin rings were able to integrate into emerging actin filaments and they may reflect a particular physiological state of the pollen or a readily available storage form provided for rapid actin network remodeling.

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

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.

Brassinosteroids promote Arabidopsis pollen germination and growth.

Pollen tubes are among the fastest tip-growing plant cells and represent an excellent experimental system for studying the dynamics and spatiotemporal control of polarized cell growth. However, investigating pollen tube tip growth in the model plant Arabidopsis remains difficult because in vitro pollen germination and pollen tube growth rates are highly variable and largely different from those observed in pistils, most likely due to growth-promoting properties of the female reproductive tract. We found that in vitro grown Arabidopsis pollen respond to brassinosteroid (BR) in a dose-dependent manner. Pollen germination and pollen tube growth increased nine- and fivefold, respectively, when media were supplemented with 10 µM epibrassinolide (epiBL), resulting in growth kinetics more similar to growth in vivo. Expression analyses show that the promoter of one of the key enzymes in BR biosynthesis, CYP90A1/CPD, is highly active in the cells of the reproductive tract that form the pathway for pollen tubes from the stigma to the ovules. Pollen tubes grew significantly shorter through the reproductive tract of a cyp90a1 mutant compared to the wild type, or to a BR perception mutant. Our results show that epiBL promotes pollen germination and tube growth in vitro and suggest that the cells of the reproductive tract provide BR compounds to stimulate pollen tube growth.

Male-female communication triggers calcium signatures during fertilization in Arabidopsis.

Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+]cyto) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+]cyto signals during the entire double fertilization process in Arabidopsis. [Ca2+]cyto oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+]cyto transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+]cyto transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+]cyto signature differing by timing and behaviour from [Ca2+]cyto waves reported in mammals.

Same same but different: sperm-activating EC1 and ECA1 gametogenesis-related family proteins.

During double fertilization in Arabidopsis thaliana, the egg cell secretes small cysteine-rich EC1 (egg cell 1) proteins, which enable the arriving sperm pair to rapidly interact with the two female gametes. EC1 proteins are members of the large and unexplored group of ECA1 (early culture abundant 1) gametogenesis-related family proteins, characterized by a prolamin-like domain with six conserved cysteine residues that may form three pairs of disulfide bonds. The distinguishing marks of egg-cell-expressed EC1 proteins are, however, two short amino acid sequence motifs present in all EC1-like proteins. EC1 genes appear to encode the major CRPs (cysteine-rich proteins) expressed by the plant egg cell, and they are restricted to flowering plants, including the most basal extant flowering plant Amborella trichopoda. Many other ECA1 gametogenesis-related family genes are preferentially expressed in the synergid cell. Functional diversification among the ECA1 gametogenesis-related family is suggested by the different patterns of expression in the female gametophyte and the low primary sequence conservation.

Egg cell-secreted EC1 triggers sperm cell activation during double fertilization.

Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple-sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.