Abstract
BackgroundThe male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis.ResultsWe developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure.ConclusionsWe provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.
Highlights
The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells
The vegetative cell arrests cell cycle progression upon pollen mitosis I (PM I), while the two sperm cells originated from pollen mitosis II (PM II) are specified into gametes [1]
Studying the expression pattern of both transgenes throughout pollen development revealed that ACT11p::H2B-mRFP is initially expressed in the generative cell (GC) of bicellular pollen, but preferentially expressed in the vegetative cell nucleus (VN) at the mature pollen stage (Figure 1A, B)
Summary
The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. In the male gametophyte (pollen grain), this process requires post-meiotic microspores to undergo two subsequent mitotic divisions, giving rise to the male germ unit (MGU) that is composed of a vegetative cell nucleus (VN) and two sperm cells (SC). Microgametogenesis in Arabidopsis has proven to be an excellent model to identify novel mechanisms controlling cell cycle transitions, cell fate specification and epigenetic reprogramming (reviewed in [12]). Such studies highlighted the importance of analyzing the components of the male germ unit independently, as they activate different transcriptional machineries and establish distinct epigenetic states [2,11,13]. In order to understand epigenetic reprogramming throughout pollen development a simple and powerful method to co-purify the two differentiated types of nuclei in mature pollen, as well as their precursor microspore, was needed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
