The rising world of flow cytometric analysis of pollen grains
2015; Wiley; Volume: 87; Issue: 10 Linguagem: Inglês
10.1002/cyto.a.22700
ISSN1552-4930
Autores Tópico(s)Plant Taxonomy and Phylogenetics
ResumoCytometry Part AVolume 87, Issue 10 p. 885-886 CommentariesFree Access The rising world of flow cytometric analysis of pollen grains João Loureiro, Corresponding Author João Loureiro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, 3000-456 PortugalCorrespondence to: João Loureiro, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456, Portugal. E-mail: [email protected]Search for more papers by this authorSílvia Castro, Sílvia Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, 3000-456 PortugalSearch for more papers by this author João Loureiro, Corresponding Author João Loureiro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, 3000-456 PortugalCorrespondence to: João Loureiro, Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456, Portugal. E-mail: [email protected]Search for more papers by this authorSílvia Castro, Sílvia Castro Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, 3000-456 PortugalSearch for more papers by this author First published: 12 June 2015 https://doi.org/10.1002/cyto.a.22700Citations: 4AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Flow cytometry is nowadays recognized as a vital tool in plant sciences, with more and more applications in both basic and applied research 1. Due to the unique characteristics of plant material, i.e., complex three-dimensional tissues with the presence of rigid cell walls, specific methods had to be developed to produce suspensions of single particles of interest 2. These particles of interest include, nuclei, mitochondria, chloroplasts, and chromosomes, with most of the applications being focused around the estimation of nuclear DNA content (either in absolute or relative amounts), leading to increasing impacts in the fields of plant breeding and population biology. One of the few single particles produced by plants is the pollen grain. Still, most works focused on studying pollen grains have involved the extraction of nuclei or sperm cells, rather than using the intact pollen. This is mostly due to the autofluorescence and/or specific staining of the pollen exine, and to the particle size limitations of some flow cytometers 3. To add to this, isolation of high-quality nuclei from pollen grains is often difficult, at least in comparison with somatic tissues. Together, these difficulties have led to a very minute number of publications involving flow cytometric analysis of pollen grains in the last 25 years. Despite this deficit of studies, four different types of applications using pollen grains have been explored: 1 pollen developmental studies, including evaluation of the nuclear replication stages in mature pollen 4-6 and nuclei development in pollen tubes 7, 8; 2 evaluation of the production of different nuclei types (e.g., male- and female-determining pollen in plants having heteromorphic sex chromosomes) 9, 10; 3 measurements of pollen DNA to detect unreduced (2N) pollen grains, particularly explored in the field of plant breeding 11-13; and, more recently, 4 estimation of the genome size of plants 14, 15. The recent studies of Kron et al. 14, 15 are of utmost relevance to advancements in this area, as they provided improved methodologies and technical approaches toward the use of pollen grains for genome size studies and for the estimation of unreduced gametes. Kron and Husband 14 presented a simple and relatively novel method for extracting pollen nuclei, i.e., the bursting of pollen through a nylon mesh. The authors compared the efficiency of this new approach with some of the methods previously used to isolate nuclei from pollen grains. The filter bursting method was evaluated in a huge dataset comprising 80 species (from 64 genera and 33 families), and yielded better quality histograms, with consistently higher yields of nuclei, than chopping, freeze-chopping, and sonication. This high success rate opens the door for the future use of pollen for estimating genome size in plants. Later, in an ingenious approach, the authors used this protocol to explore the possibility of obtaining information about pollen load composition and foraging behavior based on DNA content, i.e., to test whether pollen loads from single bees could be classified into types based on the genome size of pollen grains, and whether good estimates of proportions of different types could be made 15. Although, the information provided was affected by the complexity of the pollination environments, this approach is promising and constitutes a new tool for examining pollinator behavior and pollen transfer between species or between cytotypes. An application that could greatly benefit from the method of Kron and Husband 14 is the study of unreduced pollen production by flow cytometry. However, these authors indicated that there were still technical challenges related to the estimation of unreduced pollen grains, in particular, the difficulty to distinguish unreduced nuclei (2N nuclei) from other particles with similar fluorescence characteristics, such as 1N doublets. In this issue of Cytometry Part A, Kron and Husband (page 943) explore a very inventive way to address this issue. The authors explored the possibility to apply pulse analysis for doublet correction, as is done already in other flow cytometric applications. As usual, the authors were methodologically very sound and precise and were able to develop a consistent and repeatable pulse analysis protocol, with the establishment of criteria for when the method could be confidently applied for doublet discrimination. The approach involved evaluation of the effect of gating in terms of the relative amount of error after and before gating (I), of the completeness of the gate (pexc), and of the cleanness of the gate (pdbt). With this in mind, the authors defined a set of criteria that needed to be met before considering pulse analysis: accuracy, at a minimum, could not decrease after the gating was applied; pulse analysis doublet discrimination could only be used if it is possible to, at least qualitatively, estimate I by estimating both pdbt and pexc; and finally, gate placement needs to be repeatable, minimizing or completely removing subjectivity in gate placement. The authors also described the variation in fluorescence properties of pollen nuclei in three of the major plant families (Asteraceae, Brassicaceae and Poaceae), evaluating the applicability of the protocol to different types of pollen grains. All of this was made in comparison with estimates of unreduced gametes obtained visually using microscopy. Despite only in Brassicaceae, nucleus fluorescence height and/or width in the unreduced gametes (2N) region exhibited bimodality, as a reflection of singlets and doublets (when compared with the unimodal distributions of the same parameters in reduced (1N) gametes), this approach constitutes a great improvement on the ability to obtain precise estimations of unreduced male gametes. It was further shown that pulse analysis estimates of doublet proportions were well correlated with estimates obtained with microscopy. In conclusion, this study showed, for the first time, that controlling for pollen grain doublets is very important. The obtained results can also imply that former studies focused in estimating the frequency of unreduced gametes may have overestimated the number of 2N singlets. Although the analysis of pollen grains through flow cytometry is possible with a high precision due to recent advancements (at least in most cases), with potential significant impacts in crop and horticultural science, genome size research, and population and evolutionary biology, there are still some seldom explored applications that could increase the potential of flow cytometry for analyzing pollen grains. In the area of Ecology, in particular in studies of reproductive biology, it is very important to obtain reliable estimates of the number of pollen grains, as a measure of male fitness. Although, flow cytometry can be considered a highly sophisticated particle counter, there are only a few examples in the literature that have used this method to quantify the number of pollen grains produced in an anther, the pollen remaining in the anthers after a pollinator visit or even pollen loads over the stigmas 16, with traditional methods, such as counting pollen grains in a microscopic slide (or in a Neubauer chamber), or in a particle counter, still largely predominating. Although the use of flow cytometry is limited by the size of the pollen grain, in theory it is now possible to employ some reliable methods to make absolute counting of pollen grains. These include the use of reference beads of known concentration which are added to a sample of unknown concentration, or the use of true volumetric absolute counting available in some recent instruments, which more and more reduce the probability of a counting loss for typical event rates to percentages below 2%. Coming back to the beginning, the use of flow cytometry to the analysis of pollen grains (or pollen grain's isolated nuclei) is very promising, leading to the rise of marvelous new applications of flow cytometry and to the proliferation of novel studies. Literature Cited 1 Doležel J, Greilhuber J, Suda J. Flow Cytometry With Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley-Vch Verlag; 2007. 454 p. 2 Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 1983; 220: 1049– 1051. 3 Suda J, Kron P, Husband BC, Trávníček P. Flow cytometry and ploidy: Applications in plant systematics, ecology and evolutionary biology. In: J Doležel, J Greilhuber, J Suda, editors. Flow Cytometry With Plant Cells: Analysis of Genes, Chromosomes and Genomes. Weinheim: Wiley-VCH; 2007. pp 103– 130. 4 Van Tuyl JM, De Vries JN, Bino RJ, Kwakkenbos TAM. Identification of 2n-pollen producing interspecific hybrids of Lilium using flow cytometry. Cytologia 1989; 54: 737– 745. 5 Bino RJ, Van Tuyl JM, De Vries JN. Flow cytometric determination of relative nuclear DNA contents in bicellulate and tricellulate pollen. Ann Bot 1990; 65: 3– 8. 6 Sugiura A, Tao R, Ohkuma T, Tamura M. Pollen nuclear number in four diospyros species. HortScience 1998; 33: 149– 150. 7 Pichot C, El Maâtaoui M. Unreduced diploid nuclei in Cupressus dupreziana A. Camus pollen. Theor Appl Genet 2000; 101: 574– 579. 8 Hirano T. Hoshino Y. Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea. J Plant Res 2009; 122: 225– 234. 9 Błocka-Wandas M, Sliwinska E, Grabowska-Joachimiak A, Musial K, Joachimiak AJ. Male gametophyte development and two different DNA classes of pollen grains in Rumex acetosa L., a plant with an XX/xy1y2 sex chromosome system and a female-biased sex ratio. Sex Plant Rep 2007; 20: 171– 180. 10 Stehlik I, Kron P, Barrett SCH, Husband BC. Sexing pollen reveals female bias in a dioecious plant. New Phytol 2007; 175: 185– 194. 11 Okazaki K, Kurimoto K, Miyajima I, Enami A, Mizuochi H, Matsumoto Y, Ohya H. Induction of 2n pollen in tulips by arresting meiotic process with nitrous oxide gas. Euphytica 2005; 143: 101– 115. 12 Akutsu M, Kitamura S, Toda R, Miyajima I, Okazaki K. Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment. Euphytica 2007; 155: 143– 152. 13 Van Laere K, DeWitte A, Van Huylenbroeck J, Van Bockstaele E. Evidence for the occurrence of unreduced gametes in interspecific hybrids of hibiscus. J Horticultural Sci Biotechnol 2009; 84: 240– 247. 14 Kron P, Husband BC. Using flow cytometry to estimate pollen DNA content: Improved methodology and applications. Ann Bot 2012; 110: 1067– 1078. 15 Kron P, Kwok A, Husband BC. Flow cytometric analysis of pollen grains collected from individual bees provides information about pollen load composition and foraging behaviour. Ann Bot 2014; 113: 191– 197. 16 Rademaker MCJ, De Jong TJ, Klinkhamer PGL. Pollen dynamics of bumble-bee visitation on Echium vulgare. Funct Ecol 1997; 11: 554– 563. Citing Literature Volume87, Issue10October 2015Pages 885-886 ReferencesRelatedInformation
Referência(s)