Fusexins, HAP2/GCS1 and Evolution of Gamete Fusion

Gamete fusion is the climax of fertilization in all sexually reproductive organisms, from unicellular fungi to humans. Similarly to other cell-cell fusion events, gamete fusion is mediated by specialized proteins, named fusogens, that overcome the energetic barriers during this process. In recent years, HAPLESS 2/GENERATIVE CELL-SPECIFIC 1 (HAP2/GCS1) was identified as the fusogen mediating sperm-egg fusion in flowering plants and protists, being both essential and sufficient for the membrane merger in some species. The identification of HAP2/GCS1 in invertebrates, opens the possibility that a similar fusogen may be used in vertebrate fertilization. HAP2/GCS1 proteins share a similar structure with two distinct families of exoplasmic fusogens: the somatic Fusion Family (FF) proteins discovered in nematodes, and class II viral glycoproteins (e.g., rubella and dengue viruses). Altogether, these fusogens form the Fusexin superfamily. While some attributes are shared among fusexins, for example the overall structure and the possibility of assembly into trimers, some other characteristics seem to be specific, such as the presence or not of hydrophobic loops or helices at the distal tip of the protein. Intriguingly, HAP2/GCS1 or other fusexins have neither been identified in vertebrates nor in fungi, raising the question of whether these genes were lost during evolution and were replaced by other fusion machinery or a significant divergence makes their identification difficult. Here, we discuss the biology of HAP2/GCS1, its involvement in gamete fusion and the structural, mechanistic and evolutionary relationships with other fusexins.

Studies on the Morphology, Ontogeny and Embryology of the Flowers of Hedycarya Arborea J.R. et G. Forst. (Subfamily Monimioideae) and Laurelia Novae-Zelandiae A. Cunn. (Subfamily Atherospermoideae) of the Family Monimiaceae

<p>The inflorescences, flowers and the vascularization of floral parts of Hedycarya arborea and Laurelia novae-zelandiae were described and comparisons made with other members of the family in an attempt to determine the basic types of inflorescences, flowers and floral vascularization in the family. The vegetative, inflorescence and floral meristems of the two genera were compared. It was concluded that the vegetative apices of both had the tunica-corpus configuration typical of many other woody Ranales and other orders. The inflorescence apices were quite similar to the vegetative ones. The young floral apices are in a state of transition from a tunica-corpus to a mantle-core configuration and older floral apices had the mantle-core configuration, which is typical of the floral apices of many woody Ranales. Unusual features of the floral apices of Hedycarya and Laurelia were the lack of a pronounced rib meristem and the occurrence of relatively frequent divisions within vacuolate cells of the core. The ontogeny of the stamens of Hedycarya and Laurelia was described and comparisons were made. In both genera the micro-sporangium developed in a similar fashions: in Hedycarya 5-6 wall layers are formed inside the epidermis; in Laurelia there are 3-5 layers. Both genera had a typically thickened endothecium and a tapetum of the secretory type in which the tapetal cells become binucleate during the first meiotic division of the pollen mother cells. In Hedycarya the meiotic divisions of the pollen mother cells are of the successive type in which walls form by means of centrifugal cell plates Pollen grains remain in permanent tetrads in this genus. In Laurelia wall formation at the end of meiosis is of a modified simultaneous type, which may not have been hitherto described in the literature. Pollen grains are not in permanent tetrads. When the first division occurs in each microspore in Hedycarya, all four cells of a tetrad are at the same stage of division and the generative cell is cut off towards the distal face of the grain. Each microspore is in the two celled condition when shed. It was deduced that the generative cell is cut off against what represents a radial wall of the grain (with reference to the tetrad stage) in Laurelia. Pollen is shed in either the two or three celled condition. Comparisons were made with the development of microsporangia and male gametophytes in other woody Ranales. A study was made of the ontogeny, structure and function of the staminal appendages of Laurelia. It was found that the appendages function as nectaries, the nectar being predominantly sucrose. After a discussion of the various theories as to the morphological nature of the staminal appendages of the Laurales, it was concluded that they are morphologically staminodes. The carpels of Hedycarya and Laurelia have a basically similar ontogeny in which, as in the Lauraceae, the terminal stigmatic region develops from a solid terminal meristem in contrast to many woody Ranales in which the stigma-consists of crests which surround the external part of the cleft of the carpel. The ovules of Hedycarya and Laurelia resemble those of most other woody Ranales in being bitegmic, crassinucellate and anatropous with a monosporic 8-nucleate embryo sac of the Polygonum type. Both linear and T-shaped megaspore tetrads were found in the two genera. Laurelia has pseudocarps which develop after anthesis and enclose plumose achenes, but in Hedycarya the fruits are drupes. It was concluded that Laurelia and Hedycarya belong to two subfamilies which have been separated from each other for a long time and have undergone considerable evolution in different directions. It was also concluded that the Monimiaceae are closely related to the Lauraceae.</p>

Studies on the Morphology, Ontogeny and Embryology of the Flowers of Hedycarya Arborea J.R. et G. Forst. (Subfamily Monimioideae) and Laurelia Novae-Zelandiae A. Cunn. (Subfamily Atherospermoideae) of the Family Monimiaceae

<p>The inflorescences, flowers and the vascularization of floral parts of Hedycarya arborea and Laurelia novae-zelandiae were described and comparisons made with other members of the family in an attempt to determine the basic types of inflorescences, flowers and floral vascularization in the family. The vegetative, inflorescence and floral meristems of the two genera were compared. It was concluded that the vegetative apices of both had the tunica-corpus configuration typical of many other woody Ranales and other orders. The inflorescence apices were quite similar to the vegetative ones. The young floral apices are in a state of transition from a tunica-corpus to a mantle-core configuration and older floral apices had the mantle-core configuration, which is typical of the floral apices of many woody Ranales. Unusual features of the floral apices of Hedycarya and Laurelia were the lack of a pronounced rib meristem and the occurrence of relatively frequent divisions within vacuolate cells of the core. The ontogeny of the stamens of Hedycarya and Laurelia was described and comparisons were made. In both genera the micro-sporangium developed in a similar fashions: in Hedycarya 5-6 wall layers are formed inside the epidermis; in Laurelia there are 3-5 layers. Both genera had a typically thickened endothecium and a tapetum of the secretory type in which the tapetal cells become binucleate during the first meiotic division of the pollen mother cells. In Hedycarya the meiotic divisions of the pollen mother cells are of the successive type in which walls form by means of centrifugal cell plates Pollen grains remain in permanent tetrads in this genus. In Laurelia wall formation at the end of meiosis is of a modified simultaneous type, which may not have been hitherto described in the literature. Pollen grains are not in permanent tetrads. When the first division occurs in each microspore in Hedycarya, all four cells of a tetrad are at the same stage of division and the generative cell is cut off towards the distal face of the grain. Each microspore is in the two celled condition when shed. It was deduced that the generative cell is cut off against what represents a radial wall of the grain (with reference to the tetrad stage) in Laurelia. Pollen is shed in either the two or three celled condition. Comparisons were made with the development of microsporangia and male gametophytes in other woody Ranales. A study was made of the ontogeny, structure and function of the staminal appendages of Laurelia. It was found that the appendages function as nectaries, the nectar being predominantly sucrose. After a discussion of the various theories as to the morphological nature of the staminal appendages of the Laurales, it was concluded that they are morphologically staminodes. The carpels of Hedycarya and Laurelia have a basically similar ontogeny in which, as in the Lauraceae, the terminal stigmatic region develops from a solid terminal meristem in contrast to many woody Ranales in which the stigma-consists of crests which surround the external part of the cleft of the carpel. The ovules of Hedycarya and Laurelia resemble those of most other woody Ranales in being bitegmic, crassinucellate and anatropous with a monosporic 8-nucleate embryo sac of the Polygonum type. Both linear and T-shaped megaspore tetrads were found in the two genera. Laurelia has pseudocarps which develop after anthesis and enclose plumose achenes, but in Hedycarya the fruits are drupes. It was concluded that Laurelia and Hedycarya belong to two subfamilies which have been separated from each other for a long time and have undergone considerable evolution in different directions. It was also concluded that the Monimiaceae are closely related to the Lauraceae.</p>

Proteomic Analysis of Generative and Vegetative Nuclei Reveals Molecular Characteristics of Pollen Cell Differentiation in Lily

In plants, the cell fates of a vegetative cell (VC) and generative cell (GC) are determined after the asymmetric division of the haploid microspore. The VC exits the cell cycle and grows a pollen tube, while the GC undergoes further mitosis to produce two sperm cells for double fertilization. However, our understanding of the mechanisms underlying their fate differentiation remains limited. One major advantage of the nuclear proteome analysis is that it is the only method currently able to uncover the systemic differences between VC and GC due to GC being engulfed within the cytoplasm of VC, limiting the use of transcriptome. Here, we obtained pure preparations of the vegetative cell nuclei (VNs) and generative cell nuclei (GNs) from germinating lily pollens. Utilizing these high-purity VNs and GNs, we compared the differential nucleoproteins between them using state-of-the-art quantitative proteomic techniques. We identified 720 different amount proteins (DAPs) and grouped the results in 11 fate differentiation categories. Among them, we identified 29 transcription factors (TFs) and 10 cell fate determinants. Significant differences were found in the molecular activities of vegetative and reproductive nuclei. The TFs in VN mainly participate in pollen tube development. In comparison, the TFs in GN are mainly involved in cell differentiation and male gametogenesis. The identified novel TFs may play an important role in cell fate differentiation. Our data also indicate differences in nuclear pore complexes and epigenetic modifications: more nucleoporins synthesized in VN; more histone variants and chaperones; and structural maintenance of chromosome (SMC) proteins, chromatin remodelers, and DNA methylation-related proteins expressed in GN. The VC has active macromolecular metabolism and mRNA processing, while GC has active nucleic acid metabolism and translation. Moreover, the members of unfolded protein response (UPR) and programmed cell death accumulate in VN, and DNA damage repair is active in GN. Differences in the stress response of DAPs in VN vs. GN were also found. This study provides a further understanding of pollen cell differentiation mechanisms and also a sound basis for future studies of the molecular mechanisms behind cell fate differentiation.

Unequal contribution of two paralogous CENH3 variants in cowpea centromere function

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

Unequal contribution of two paralogous centromeric histones to function the cowpea centromere

The legume cowpea (Vigna unguiculata, 2n=2x=22) has significant tolerance to drought and heat stress. Here we analysed and manipulated cowpea centromere-specific histone H3 (CENH3) genes, aiming to establish a centromere-based doubled-haploid method for use in genetic improvement of this dryland crop in future. Cowpea encodes two functional CENH3 variants (CENH3.1 and CENH3.2) and two CENH3 pseudogenes. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of V. unguiculata and the related V. mungo without a genome duplication event. Both functional cowpea CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the outer kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. Differences between both CENH3 paralogs are restricted to the N-terminus. The complete CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development, suggesting that the gene is an early stage of subfunctionalization or pseudogenization.One-sentence summaryThe two paralogous centromeric histones (CENH3) of cowpea contribute unequal to the function of the centromere.

Isolation of Sperm and Egg Cells from Pepper

Pepper (Capsicum annuum) pollen is bicellular and contains a vegetative cell and a generative cell, which divides in pollen tubes to form two sperm cells. Sperm cells of pepper were isolated using an in vivo–in vitro method. Hand-pollinated styles were first grown in vivo for several hours, then cut from their base and cultured in vitro until pollen tubes grew from the cut end. When the pollen tubes were transferred to a breaking solution, sperm cells were released from broken tubes. Viable embryo sac cells of pepper were isolated using enzymatic digestion and mechanical dissection. Isolated ovules were digested using cellulase and pectinase for 40 minutes and then transferred to an enzyme-free solution for mechanical dissection. Three cells of the egg apparatus and a central cell were released from a cut at the chalazal end of each ovule by pressing on the micropylar area of the ovule with a microneedle. Optimal isolation conditions included 11% mannitol, 0.04% CaCl2, 1% bovine serum albumin (BSA), 1% cellulase, 1% pectinase, and 0.3% pectolyase. Using this protocol, populations of pepper egg cells, synergids, and central cells were isolated.