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.

The MATH-BTB Protein TaMAB2 Accumulates in Ubiquitin-Containing Foci and Interacts With the Translation Initiation Machinery in Arabidopsis.

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells.

KEY MESSAGE: A protocol for the isolation of egg apparatus cells from the basal angiosperm Amborella trichopoda to generate RNA-seq data for evolutionary studies of fertilization-associated genes. Sexual reproduction is particularly complex in flowering plants (angiosperms). Studies in eudicot and monocot model species have significantly contributed to our knowledge on cell fate specification of gametophytic cells and on the numerous cellular communication events necessary to deliver the two sperm cells into the embryo sac and to accomplish double fertilization. However, for a deeper understanding of the evolution of these processes, morphological, genomic and gene expression studies in extant basal angiosperms are inevitable. The basal angiosperm Amborella trichopoda is of special importance for evolutionary studies, as it is likely sister to all other living angiosperms. Here, we report about a method to isolate Amborella egg apparatus cells and on genome-wide gene expression profiles in these cells. Our transcriptomics data revealed Amborella-specific genes and genes conserved in eudicots and monocots. Gene products include secreted proteins, such as small cysteine-rich proteins previously reported to act as extracellular signaling molecules with important roles during double fertilization. The detection of transcripts encoding EGG CELL 1 (EC1) and related prolamin-like family proteins in Amborella egg cells demonstrates the potential of the generated data set to study conserved molecular mechanisms and the evolution of fertilization-related genes and their encoded proteins.

Manual Isolation of Living Cells from the Arabidopsis thaliana Female Gametophyte by Micromanipulation.

The few-celled female gametophyte, or embryo sac, of flowering plants is not easily accessible as it is buried within the sporophytic tissues of the ovule. Nevertheless, it has become an attractive model system to study the molecular mechanisms underlying patterning and cell type specification, as well as fertilization of the two female gametes, the egg and the central cell. While female gametes, zygotes, and early embryos can be manually isolated from the embryo sacs in maize, wheat, tobacco, and rice by micromanipulation, this approach had been considered impossible for the much smaller embryo sac of the model plant Arabidopsis thaliana. Here, we describe a method to isolate living cells from the Arabidopsis female gametophyte by micromanipulation. The manual isolation of egg cells, central cells, and synergid cells is a technique that enables a number of important studies such as cell-type-specific transcriptional profiling or the analysis of DNA methylation profiles. It also offers the possibility to use isolated female gametes for in vitro fertilization studies.

Biochemical isolation of Argonaute protein complexes by Ago-APP.

During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.

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.

De novo zygotic transcription in wheat (Triticum aestivum L.) includes genes encoding small putative secreted peptides and a protein involved in proteasomal degradation.

Wheat is one of the world's most important crops, and increasing grain yield is a major challenge for the future. Still, our knowledge about the molecular machineries responsible for early post-fertilization events such as zygotic reprogramming, the initial cell-specification events during embryogenesis, and the intercellular communication between the early embryo and the developing endosperm is very limited. Here, we describe the identification of de novo transcribed genes in the wheat zygote. We used wheat ovaries of defined post-fertilization stages to isolate zygotes and early embryos, and identified genes that are specifically induced in these particular stages. Importantly, we observed that some of the zygotic-induced genes encode proteins with similarity to secreted signaling peptides such as TAPETUM DETERMINANT 1 and EGG APPARATUS 1, and to MATH-BTB proteins which are known substrate-binding adaptors for the Cullin3-based ubiquitin E3 ligase. This suggests that both cell-cell signaling and targeted proteasomal degradation may be important molecular events during zygote formation and the progression of early embryogenesis.

Germline-specific MATH-BTB substrate adaptor MAB1 regulates spindle length and nuclei identity in maize.

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

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.

A general G1/S-phase cell-cycle control module in the flowering plant Arabidopsis thaliana.

The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant Arabidopsis thaliana, which, as a member of the Plantae supergroup, is phylogenetically only distantly related to Opisthokonts such as yeast and animals. This module comprises the Arabidopsis homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2⁺/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network.

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.