An F-Actin Mega-Cable Is Associated With the Migration of the Sperm Nucleus During the Fertilization of the Polarity-Inverted Central Cell of Agave inaequidens.

Asparagaceae's large embryo sacs display a central cell nucleus polarized toward the chalaza, which means the sperm nucleus that fuses with it during double fertilization migrates an atypical long distance before karyogamy. Because of the size and inverted polarity of the central cell in Asparagaceae, we hypothesize that the second fertilization process is supported by an F-actin machinery different from the short-range F-actin structures observed in Arabidopsis and other plant models. Here, we analyzed the F-actin dynamics of Agave inaequidens, a classical Asparagaceae, before, during, and after the central cell fertilization. Several parallel F-actin cables, spanning from the central cell nucleus to the micropylar pole, and enclosing the vacuole, were observed. As fertilization progressed, a thick F-actin mega-cable traversing the vacuole appeared, connecting the central cell nucleus with the micropylar pole near the egg cell. This mega-cable wrapped the sperm nucleus in transit to fuse with the central cell nucleus. Once karyogamy finished, and the endosperm started to develop, the mega-cable disassembled, but new F-actin structures formed. These observations suggest that Asparagaceae, and probably other plant species with similar embryo sacs, evolved an F-actin machinery specifically adapted to support the migration of the fertilizing sperm nucleus within a large-sized and polarity-inverted central cell.

Simple Whole-Mount Staining Protocol of F-Actin for Studies of the Female Gametophyte in Agavoideae and Other Crassinucellate Ovules.

During plant sexual reproduction, F-actin takes part in the elongation of the pollen tube and the movement of sperm cells along with it. Moreover, F-actin is involved in the transport of sperm cells throughout the embryo sac when double fertilization occurs. Different techniques for analysis of F-actin in plant cells have been developed: from classical actin-immunolocalization in fixed tissues to genetically tagged actin with fluorescent proteins for live imaging of cells. Despite the implementation of live cell imaging tools, fixed plant tissue methods for cytoskeletal studies remain an essential tool for genetically intractable systems. Also, most of the work on live imaging of the cytoskeleton has been conducted on cells located on the plant's surface, such as epidermal cells, trichomes, and root hairs. In cells situated in the plant's interior, especially those from plant species with thicker organ systems, it is necessary to utilize conventional sectioning and permeabilization methods to allow the label access to the cytoskeleton. Studies about the role of F-actin cytoskeleton during double fertilization in plants with crassinucellate ovules (e.g., Agave, Yucca, Polianthes, Prochnyantes, and Manfreda) remain scarce due to the difficulties to access the female gametophyte. Here, we have developed a straightforward method for analysis of F-actin in the female gametophyte of different Agavoideae sub-family species. The procedure includes the fixation of whole ovules with formaldehyde, followed by membrane permeabilization with cold acetone, a prolonged staining step with rhodamine-phalloidin, and Hoechst 33342 as a counterstain and two final steps of dehydration of samples in increasing-concentration series of cold isopropanol and clarification of tissues with methyl salicylate. This technique allows the analysis of a large number of samples in a short period, cell positioning relative to neighbor cells is maintained, and, with the help of a confocal microscope, reconstruction of a single 3D image of F-actin structures into the embryo sac can be obtained.

Cytosolic calcium localization and dynamics during early endosperm development in the genus Agave (Asparagales, Asparagaceae).

Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell.

Embryogenesis in Polianthes tuberosa L var. Simple: from megasporogenesis to early embryo development.

The genus Polianthes belongs to the subfamily Agavoideae of the Asparagaceae family formerly known as Agavaceae. The genus is endemic to México and comprises about 15 species, among them is Polianthes tuberosa L. The aim of this work was to study and characterize the embryo sac and early embryo development of this species in order to generate basic knowledge for its use in taxonomy, in vitro fertilization and production of haploid plants and to complement studies already performed in other genera and species belonging to the Agavoideae sub-family. It was found that the normal development of the P. tuberosa var. Simple embryo sac follows a monosporic pattern of the Polygonum type and starts its development from the chalazal megaspore. At maturity, the embryo sac is of a pyriform shape with a chalazal haustorial tube where the antipodals are located, just below the hypostase, which connects the embryo sac with the nucellar tissue of the ovule. The central cell nucleus shows a high polarity, being located at the chalazal extreme of the embryo sac. The position of cells inside the P. tuberosa embryo sac may be useful for in depth studies about the double fertilization. Furthermore, it was possible to make a chronological description of the events that happen from fertilization and early embryo development to the initial development of the endosperm which was classified as of the helobial type.

Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.