Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.
- MeSH
- Cell Nucleus MeSH
- Ploidies MeSH
- Flow Cytometry MeSH
- Pollen * MeSH
- Spores * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
SH3P2 (At4g34660), an Arabidopsis thaliana SH3 and Bin/amphiphysin/Rvs (BAR) domain-containing protein, was reported to have a specific role in cell plate assembly, unlike its paralogs SH3P1 (At1g31440) and SH3P3 (At4g18060). SH3P family members were also predicted to interact with formins-evolutionarily conserved actin nucleators that participate in microtubule organization and in membrane-cytoskeleton interactions. To trace the origin of functional specialization of plant SH3Ps, we performed phylogenetic analysis of SH3P sequences from selected plant lineages. SH3Ps are present in charophytes, liverworts, mosses, lycophytes, gymnosperms, and angiosperms, but not in volvocal algae, suggesting association of these proteins with phragmoplast-, but not phycoplast-based cell division. Separation of three SH3P clades, represented by SH3P1, SH3P2, and SH3P3 of A. thaliana, appears to be a seed plant synapomorphy. In the yeast two hybrid system, Arabidopsis SH3P3, but not SH3P2, binds the FH1 and FH2 domains of the formin FH5 (At5g54650), known to participate in cytokinesis, while an opposite binding specificity was found for the dynamin homolog DRP1A (At5g42080), confirming earlier findings. This suggests that the cytokinetic role of SH3P2 is not due to its interaction with FH5. Possible determinants of interaction specificity of SH3P2 and SH3P3 were identified bioinformatically.
Callose is a plant-specific polysaccharide (β-1,3-glucan) playing an important role in angiosperms in many developmental processes and responses to biotic and abiotic stresses. Callose is synthesised at the plasma membrane of plant cells by callose synthase (CalS) and, among others, represents the main polysaccharide in the callose wall surrounding the tetrads of developing microspores and in the growing pollen tube wall. CalS proteins involvement in spore development is a plesiomorphic feature of terrestrial plants, but very little is known about their evolutionary origin and relationships amongst the members of this protein family. We performed thorough comparative analyses of callose synthase family proteins from major plant lineages to determine their evolutionary history across the plant kingdom. A total of 1211 candidate CalS sequences were identified and compared amongst diverse taxonomic groups of plants, from bryophytes to angiosperms. Phylogenetic analyses identified six main clades of CalS proteins and suggested duplications during the evolution of specialised functions. Twelve family members had previously been identified in Arabidopsis thaliana. We focused on five CalS subfamilies directly linked to pollen function and found that proteins expressed in pollen evolved twice. CalS9/10 and CalS11/12 formed well-defined clades, whereas pollen-specific CalS5 was found within subfamilies that mostly did not express in mature pollen vegetative cell, although were found in sperm cells. Expression of five out of seven mature pollen-expressed CalS genes was affected by mutations in bzip transcription factors. Only three subfamilies, CalS5, CalS10, and CalS11, however, formed monophyletic, mostly conserved clades. The pairs CalS9/CalS10, CalS11/CalS12 and CalS3 may have diverged after angiosperms diversified from lycophytes and bryophytes. Our analysis of fully sequenced plant proteins identified new evolutionary lineages of callose synthase subfamilies and has established a basis for understanding their functional evolution in terrestrial plants.
The RNA-directed DNA methylation (RdDM) pathway can be divided into three phases: 1) small interfering RNA biogenesis, 2) de novo methylation, and 3) chromatin modification. To determine the degree of conservation of this pathway we searched for key genes among land plants. We used OrthoMCL and the OrthoMCL Viridiplantae database to analyze proteomes of species in bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms. We also analyzed small RNA size categories and, in two gymnosperms, cytosine methylation in ribosomal DNA. Six proteins were restricted to angiosperms, these being NRPD4/NRPE4, RDM1, DMS3 (defective in meristem silencing 3), SHH1 (SAWADEE homeodomain homolog 1), KTF1, and SUVR2, although we failed to find the latter three proteins in Fritillaria persica, a species with a giant genome. Small RNAs of 24 nt in length were abundant only in angiosperms. Phylogenetic analyses of Dicer-like (DCL) proteins showed that DCL2 was restricted to seed plants, although it was absent in Gnetum gnemon and Welwitschia mirabilis. The data suggest that phases (1) and (2) of the RdDM pathway, described for model angiosperms, evolved with angiosperms. The absence of some features of RdDM in F. persica may be associated with its large genome. Phase (3) is probably the most conserved part of the pathway across land plants. DCL2, involved in virus defense and interaction with the canonical RdDM pathway to facilitate methylation of CHH, is absent outside seed plants. Its absence in G. gnemon, and W. mirabilis coupled with distinctive patterns of CHH methylation, suggest a secondary loss of DCL2 following the divergence of Gnetales.
- MeSH
- Arabidopsis genetics MeSH
- Chromatin metabolism MeSH
- Cycadopsida genetics metabolism MeSH
- Cytosine metabolism MeSH
- DNA-Directed RNA Polymerases metabolism MeSH
- Epigenesis, Genetic MeSH
- Phylogeny MeSH
- Genome, Plant MeSH
- Magnoliopsida enzymology genetics metabolism MeSH
- RNA, Small Interfering metabolism MeSH
- RNA, Small Untranslated chemistry MeSH
- DNA Methylation * MeSH
- Methylation MeSH
- Ribonuclease III classification genetics MeSH
- RNA, Plant chemistry metabolism MeSH
- Genes, Plant * MeSH
- Plant Proteins classification genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
