Best practices in plant cytometry

Received: 11 November 2020 Revised: 7 December 2020 Accepted: 14 December 2020 DOI: 10.1002/cyto.a.24295 EDITORIAL Best practices in plant cytometry Flow cytometry (FCM) and flow cytometric sorting (FCS) systems The trigger to develop this virtual issue came from the publica- have developed as experimental tools of remarkable power and are tion, in 2017, of an article entitled “Guidelines for the use of flow enjoying an ever-increasing impact in the general field of biology.1 cytometry and cell sorting in immunological studies” in the European Application of these tools to plant biology has developed more Journal of Immunology.10 As noted in that article, one of the advan- slowly given that the natural form of plants infrequently resembles tages of FCM/FCS systems is that they are relatively simple to imple- that of the single cell suspension, prototypically the hematopoietic ment, with some qualifications, which coupled with the development system that drove the original development of FCM/FCS. Neverthe- of user-friendly devices and software during the last 15 years led to less, these systems have had a profound influence at all levels of increasing applications in other areas, such as plant sciences. How- plant biology, from the study of single cells and subcellular organ- ever, it is also simple to implement and operate the instruments inap- elles, to the behavior of populations of plants, and ultimately to the propriately. This calls for a comprehensive and collective summary of performance of ecosystems. It is safe to say their impact has not the best practices when applying FCM/FCS to plants, as was done for plateaued, as further applications of this unique technology are immunology. increasingly developed by innovative scientists around the world to The first consideration addresses the problem that plants, particu- address questions both in the basic sciences, and to increasingly con- larly the vascular plants, in their commonly recognized and utilized front emerging problems in the applied sector. For example, in forms, exist not as single cell suspensions (typical of immunology) but addressing the challenges of sustainable production of sufficient as complex three-dimensional tissues comprising cells of irregular food resources based on plant breeding involving ploidy-based shapes, different types and functions, that collectively cooperate to approaches (e.g., induction of polyploidy)2 for the needs of our produce the final plant form. Optimal methods for producing suspen- future global citizens, FCM, and FCS systems will play central roles sions of cells, subcellular organelles and other components appropri- in this effort. ate for FCM/FCS from these plant tissues and organs, are therefore The degree to which FCM and FCS systems have impacted plant one of the challenges discussed in this virtual issue. We are fully biology and applied agricultural sciences must not be understated. aware of the mantra that “junk in equals junk out” and having samples The major applications of DNA FCM are ploidy level and genome size of the highest quality prior to FCM/FCS is a critical concern we also estimations, and cell cycle analysis/endoreplication (with the later addressed here. included in a lower percentage of studies). Indeed, FCM is currently/ The second consideration relates to the vast variety of different extensively and almost exclusively employed as the method of choice plant species found globally, and the recognition of the consummate for measurement of plant genome sizes.3,4 Measurements of this type ability of plants to produce secondary metabolites/products, affecting impact agriculture in terms of ploidy estimation, with applications DNA staining and resulting fluorescence. Again, methods for recogniz- ranging from plant biotechnology, breeding and seed quality testing to ing and handling the different challenges provided to FCM/FCS taxonomy and population biology. They also impact the fundamental methods by the biochemistries of the source samples are required. plant sciences in terms of biosystematics, ecology, evolution, geno- The third consideration focuses on the problem of addressing the mics, and conservation, among other applications. One of the most non-critical application of FCM/FCS methods developed for mamma- startling observations of the angiosperms is the bandwidth occupied lian cell systems (typically hematopoietic) to plants without careful by genome size, which spans almost 2400-fold. consideration of their appropriateness. As it will be detailed in this vir- Flow sorting of higher plant chromosomes has provided invalu- tual issue, application of FCM/FCS methods to mammalian cell sys- able information regarding the organization of DNA sequences within tems almost exclusively occurs in the context of analysis of samples plant species. It has also greatly facilitated the process of whole- that comprise a majority, often close to 100%, of single cells in sus- genome sequencing by permitting subdivision of large genomes into pension. For plants, particularly when using these instruments and 5 samples comprising entire chromosomes or chromosome arms. FCS methods for the analysis of organelles in tissue homogenates, the methods applied to wall-less cells (protoplasts) expressing fluorescent objects of interest comprise a very minor subpopulation of the total proteins (FPs) in a cell type-specific manner have allowed elucidation particles passing through the instrument. Concepts such as placing ini- of patterns of co-regulated gene expression and plant hormone gradi- tial gates around populations defined by forward scatter (FS) versus 6,7 ents identification 8,9 within organized tissues, such as roots. Cytometry. 2021;99:311–317. wileyonlinelibrary.com/journal/cytoa side scatter (SS), as routinely used to define leukocytes or other © 2021 International Society for Advancement of Cytometry 311 EDITORIAL 312 mammalian cells in culture, are at best meaningless and at worst can mosses, Marchantiophyta—liverworts, and Anthocerotophyta—horn- seriously hamper proper use of the instruments to provide meaningful worts) and three sequentially-splitting lineages of vascular plants: results. Again, plants are sources of many forms of autofluorescence; lycopods (Lycopodiophyta), ferns and horsetails (Monilophyta) and in vascular plants, chloroplasts are intensely fluorescent in the red due seed plants (Spermatophyta). The latter group further splits into gym- to the presence of chlorophyll. Phycoerythrin, a red protein-pigment nosperms (Gymnospermae; i.e., conifers, cycads, Ginkgo, and complex from the light-harvesting phycobiliprotein family found in red gnetophytes) and angiosperms (Angiospermae;).15-17 The current algae and cryptophytes, is commercially employed as a fluorescent review is primarily but not exclusively focused on flow cytometric label for antibodies in cytometry. The presence of autofluorescence applications in flowering plants, as they represent the most diverse can restrict the wavelength bandwidths for fluorescence excitation and economically important, and therefore best studied, group of and emission, and this can affect how best to set up FCM/FCS green plants. However, we mention the other green plant lineages instruments. where necessary and we also include other organisms that are found In order to define and enunciate best practices, we drew together in various parts of the Tree of Life (algae in the traditional sense, a network of volunteer authors, experienced in the application of fungi) and that share certain features of body organization and life FCM/FCS to plants. We have attempted to make this network as style with plants (such as complex tissues or photosynthesis), and comprehensive as possible, to allow recommendations spanning all have for a long time been a subject of Botany in the broadest sense. relevant life-forms, from the simplest photosynthetic microbes, to the The life cycles of algal groups are highly variable and may com- more complex lower and vascular plants, and encompassing also the prise stages only with haplophasic (n) or diplophasic (2n) chromosome fungi. In this endeavor, we gratefully acknowledge the support of numbers, although in other species both stages are present but in sep- Wiley and Attila Tarnok, EIC of Cytometry. arate generations.18 All land plants exhibit a characteristic life cycle 9 As indicated for the Guidelines in Immunology article, we do which alternates between a haplophasic gametophyte and a diplo- wish to keep our recommendations updated. Therefore, please send phasic sporophyte. Still, the relative importance of each stage in the us your critical comments, new ideas, practical suggestions regarding life cycle differs between groups: while the gametophyte stage domi- best practices, and new articles that could be useful for possible nates in bryophytes (and is usually the tissue that is analyzed by future versions of this virtual issue. FCM), the sporophyte stage dominates in the vascular plant groups To end, we would like to remember that this virtual issue reflects and is the main focus of flow cytometric investigations. Despite a sig- the vision and dream of the late Jan Suda. He has been an inspiration nificant reduction in the size of the gametophyte (comprising only up for all of us, and, most certainly, he left us too soon. We are sure that to 3–4 cells/nuclei in flowering plants), there are flow cytometric his legacy will persist, not only in his home country, the Czech Repub- applications focused on either the independent gametophyte or the lic, but also across the world. We sincerely hope this virtual issue of spores of ferns or on pollen grains of seed plants.19 Unlike vascular Cytometry Part A provides an appropriate tribute. plants, fungal life cycles are mostly haplophasic, with a short (often single-celled) diplophasic stage, although most fungi (the Dikarya, i.e., the Ascomycota and the Basidiomycota) are dikaryotic (n + n) in 1 | S E T T I N G T H E S TA G E part of their life cycles. The evolution of plant genomes is dynamic, particularly in angio- Describing how FCCS can be optimally applied to plants requires sperms, encompassing a range of genomic processes including multi- information in two general areas (a) concerning the samples being pre- ple rounds of whole genome duplication (polyploidization,20,21 pared and analyzed, in our case focusing on the relevant physical fea- chromosomal rearrangements22,23 and the turnover and evolution of tures of plants as organisms, and (b) concerning the instrumentation repetitive DNA (including mobile elements and satellite DNA).24,25 being used for this analysis, centering on sample requirements This is mirrored in the tremendous variation in nuclear genome sizes imposed by engineering design and implementation. across green plants in general (c. 11,850-fold; 2) and flowering plants in particular (2,400-fold variation; 3,4). This has crucial implications for flow cytometric applications both with respect to technical issues 2 | VASCULAR AND NONVASCULAR PLANTS (a series of internal standards of different genome size is required) and also as a study topic per se (e.g. what are the mechanisms driving genome size evolution?). Similarly, the relative content of AT versus Green plants (Viridiplantae) constitute a monophyletic clade within 11 the tree of life and comprise oxygenic photosynthetic eukaryotes. GC base pairs is highly variable in green plants, although this variation does not strictly correlate with nuclear DNA-content (e.g., 26). The group encompasses green algae and land plants, and further splits While the algal groups are mostly unicellular, or comprise a rather into major clades: the Chlorophyta,12 comprising only algae, and simple multicellular thallus (e.g., Ulva, Cladophora, or Chara), land Streptophyta as plants form complex tissues and organs. The sporophyte of vascular formed by several algal 13,14 groups (such and the land plants plants typically differentiates into roots, stems and leaves (note that (Embryophyta). Land plants further split into several groups: the possi- the floral parts of flowering plants are derived from the leaves). Similar bly paraphyletic assemblage of three bryophyte lineages (Bryophyta— (yet haplophasic and thus non-homologous) structures are found in Zygnematophyceae and Charophyceae;,) EDITORIAL 313 the gametophytes of bryophytes: rhizoids, cauloids, and phylloids. The distance below the point of laser interception (Figure 1). Based on the specific morphology and anatomy of green plants, as distinct from degree of sample dilution, some of these droplets contain the cells of other eukaryotes, naturally has multiple implications/challenges for interest, and can be selectively displaced into collection vessels by a flow cytometric analysis. Firstly, we encounter cells having thick cell process of charging the droplet at the point of its detachment from walls that render flow cytometric analysis of individual cells impossi- the flow stream followed by passage through a fixed electrostatic ble. Instead, isolated protoplast and, more commonly, nuclear suspen- field. The rates of sorting depend on the size of the cells, which deter- 26–28 sions are used for the analysis of plant tissues. Secondly, two types of endosymbiotic organelles, each with their own genomes, are mines the size of the flow tip, and the rate of flow of the fluid stream.34 present in most plant cells, mitochondria and plastids, and FCM appli- Advances in the area of instrument development have included cations have been designed to analyze those organelles.29–31 Lastly, multiplexed excitation and detection modalities to comprehensively plants present a wide array of chemical compounds, so-called second- cover the excitation and fluorescence emission spectra of the avail- ary metabolites, conferring protection against factors both abiotic able fluorochromes.35 Recently, spectral analysis has been demon- (e.g., UV-light, frost) and biotic (e.g., herbivores, parasites). Some of strated as an alternative to conventional light filters in FCM.36,37 these chemical compounds (for example, tannins) directly co-interact Other advances include the use of flow tips that accommodate cells with the DNA-binding stains used in FCM, and significantly affect the and biological particles that are larger, and sometimes much larger, quality and reliability of such analyses.32 than mammalian blood cells, drastic reductions in overall instrument sizes, footprints, and purchase costs, full replacement of analog by digital signal processing and the use of miniaturized fluidics systems 3 | O V E R V I E W O F I N S T RU M E NT A T I O N AND PRINCIPLES with corresponding improvements in accuracy and reliability, and accompanied by reductions in costs of maintenance. Flow cytometry and cytometric sorting systems are assembled from distinct engineering modules which collectively function to determine 4 | RA T I O N A L E A ND T A RG E T the optical properties of suspensions of biological particles, and selectively isolate these particles, or subsets thereof, for subsequent analy- Flow cytometry and flow cytometric sorting are not new methods. sis constrained However, their use in Plant Biology has grown dramatically in the last hydrodynamically within an aqueous stream to flow singly through decades, and in some cases, such as genome size measurements, regions of intense light, almost exclusively provided by lasers that are these technologies have come to dominate. At the same time, instru- focused on the stream. On illumination, the particles absorb and scat- ments and associated protocols continue to be improved and ter light and, if associated with fluorochromes, subsequently emit expanded (e.g., bead beating, the use of tissues other than leaves, dry fluorescence. The intensities of the scattered and fluorescent light tissue). The literature now includes many resources outlining methods, pulses coming from each particle are then measured. Key elements in theoretical issues, and limitations of methods (e.g., the “Flow Cyto- these modules are (a) a flow cell, which spatially positions and aligns metry with Plant Cells” book,38 ESACP guidelines http://www. the flow stream containing the particles with the excitation light and classimed.de/esacflow.html). However, despite this progress, it is clear detection axes, (b) light scatter and fluorescence detectors, screened from some recent publications that experimental design and manu- by wavelength-appropriate filters and oriented orthogonally to the script review have not always kept pace with what we know about direction of the flow stream and the excitation light path, the application of FCM and cytometric sorting to plants, and this has (c) electronic circuitry including analog-to-digital converters (ADCs) adversely affected the quality of the results and the conclusions which convert the voltage pulses emerging from the detectors into drawn. and processing. The particles are typically digital values corresponding to the outputs from the individual parti- Contributing factors include: cles, (d) computational architecture to process and store the information from these pulses for further analysis, or to use them 1. The practical need of carrying out experiments at centralized flow immediately for processing sort-related decisions, and (e) mechanisms facilities that are not primarily concerned with, or understand, the to implement individual, high-speed sorting of the particles, based on characteristics of the input plant materials, or the types of ques- preselected combinations of optical characteristics. tions (e.g., the large amounts of samples used in population biol- One of the first implementations of flow sorting, and one of the 33 most influential, was described by Bonner et al. for characterization and isolation of various mammalian cell types including those of the ogy) that are being addressed. 2. Effects of “lab culture,” in which poor practices that have become established in laboratories are taught to uncritical novices. hematopoietic system. To date, immunological applications represent 3. Recommendations for “best practices” being scattered across the the largest fraction of cytometric activities, worldwide. Most flow existing scientific literature: thus, a comprehensive article summa- sorters employ a version of this original implementation, which rizing key rules for the reliable application of FCM and FCS to involves precise conversion of the flow stream into a series of individ- plants is still lacking, even for widely used applications such as ual droplets, electromechanically synchronized to appear at a fixed DNA content measurements (but see 28). EDITORIAL 314 F I G U R E 1 Schematic of the process of droplet formation for a typical droplet-in air flow sorter. Droplet formation is synchronized below the point of interception of the flow stream by the laser illumination. The undulation wavelength (λ) is defined by the velocity of the flow stream, and the drive frequency of the piezo-electric oscillator attached to the flow cell. A constant high DC voltage is maintained across the deflection plates. Precise switching of the charge applied to the flow stream at the time of droplet break-off retains that charge on the droplet, which then can be predictably deflected by the electric field Examples of poor practices and erroneous theories developed as and reviewers, since they should serve to guarantee high-quality a consequence of these practices, identified by Jan Suda in the origi- results through elimination (or, at the very least, minimization) of arti- nal draft, include flax genotrophs and problems with intraspecific vari- factual variation from future research submitted for publication, as ation reports, as reviewed in Greilhuber.35 Recent tendencies in well as providing a means to identify artifacts within the published manuscripts to justify the use of dry tissue based on existing literature literature. frequently lack acknowledgment of necessary precautions from the prior literature. The main objective of this virtual issue is to outline key experi- 5 | T H E SC O P E O F T H I S V I R T U A L I S SU E I S mental issues and associated guidelines (under the heading of “Best Practices”) that researchers are recommended to follow, and to pro- 1. Applications based on the staining of DNA (ploidy, genome size, vide the rationale for these recommendations, such that the guide- AT/GC content, cell cycle, including endoreduplication, nuclei and lines may be modified with confidence as new applications emerge. chromosome sorting) which represent a majority of uses. We also identify those areas where the establishment of clear guide- 2. Applications based on sorting single cells (protoplasts) and organ- lines will require additional empirical data or theoretical work. Such elles (nuclei, mitochondria and plastids), based on use of FPs or guidelines will benefit researchers, facility managers, journal editors, fluorescent dyes in protoplast/organelle sorting for downstream EDITORIAL 315 omics analyses at the cell-type-specific or organelle-specific Petr Bureš6 level. Petr Cápal7 3. A focus predominantly on plants, but with separate sections Mariana Castro3 devoted to algae, and to fungi. However, in many cases, the gen- Sílvia Castro3 eral principles should apply across all organisms; wherever possi- Martin Čertner8,9 ble, we will extrapolate to other organisms. Dora Čertnerová8 Zuzana Chumová8,9 The emphasis will be on providing guidelines for reviewers and Jaroslav Doležel7 for experimental design. This will NOT be a methods virtual issue in Debora Giorgi10 the sense of providing protocols: these are well-covered elsewhere Brian C. Husband11 27 (e.g., 38 “Flow Cytometry with Plant Cells” book, 39 material of Kron et al., Filip Kolář8,9 the supplemental Petr Koutecký12 online resources). Paul Kron11 Ilia J. Leitch13 KE YWORDS best practices, chromosome analysis, cytometry, nuclear suspensions, Karin Ljung4 plant sciences, protoplasts and organelle analysis, single cell Sara Lopes3 Magdalena Lučanová9,12 suspensions Sergio Lucretti10 Wen Ma1,2 CONF LICT OF IN TE RE ST Susanne Melzer14,15 The authors have no conflicts of interest to declare. István Molnár7 AUTHOR CONTRIBUTIONS Ondřej Novák4,16 David Galbraith: Conceptualization; writing-original draft; writing- Nicole Poulton17 review and editing. Jo~ao Loureiro: Conceptualization; writing-original Vladimír Skalický16 draft; writing-review and editing. Ioanna Antoniadi: Writing-review Elwira Sliwinska18 and editing. Jillian Bainard: Writing-review and editing. Petr Bureš: Petr Šmarda6 Writing-review and editing. Petr Cápal: Writing-review and editing. Tyler W. Smith19 Mariana Castro: Writing-review and editing. Sílvia Castro: Writing- Guiling Sun1,2 review and editing. Martin Čertner: Writing-review and editing. Dora Pedro Talhinhas20 Čertnerová: Writing-review and editing. Zuzana Chumová: Writing- Attila Tárnok15,21,22 review and editing. Jaroslav Dolezel: Writing-review and editing. Eva M. Temsch23 Debora Giorgi: Writing-review and editing. Brian Husband: Writing- Pavel Trávníček9 review and editing. Filip Kolar: Writing-review and editing. Petr Tomáš Urfus8 Koutecký: Writing-review and editing. Paul Kron: Writing-review and 1 editing. Ilia Leitch: Writing-review and editing. Karin Ljung: Writing- School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, review and editing. Sara Lopes: Writing-review and editing. Magda- Department of Biomedical Engineering, University of Arizona, Tucson, lena Lučanová: Writing-review and editing. Sergio Lucretti: Writing- Arizona, USA review and editing. Wen Ma: Writing-review and editing. Susanne 2 Melzer: Writing-review and editing. István Molnár: Writing-review Biology, Henan University, School of Life Sciences, State Key Laboratory State Key Laboratory of Cotton Biology, Key Laboratory of Plant Stress and editing. Ondřej Novák: Writing-review and editing. Nicole Poulton: Writing-review and editing. Vladimír Skalický: Writing- of Crop Stress Adaptation and Improvement, Kaifeng, China 3 Centre for Functional Ecology, Department of Life Sciences, University of review and editing. Elwira Sliwinska: Writing-review and editing. Petr Coimbra, Coimbra, Portugal Šmarda: Writing-review and editing. Tyler Smith: Writing-review and 4 editing. Guiling Sun: Writing-review and editing. Pedro Talhinhas: Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden Umeå Plant Science Centre, Department of Forest Genetics and Plant 5 Writing-review and editing. Attila Tárnok: Writing-review and editing. Swift Current Research and Development Centre, Agriculture and Eva Temsch: Writing-review and editing. Pavel Trávnícek: Writing- Agri-Food Canada, Swift Current, Saskatchewan, Canada 6 review and editing. Tomas Urfus: Writing-review and editing. Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, CZ, Czech Republic David Galbraith1,2 7 Jo~ao Loureiro3 Ioanna Antoniadi Institute of Experimental Botany of the Czech Academy of Sciences, Olomouc, Czech Republic 4 Jillian Bainard5 8 Department of Botany, Faculty of Science, Charles University, Prague, Czech Republic EDITORIAL 316 9 Czech Academy of Sciences, Institute of Botany, Průhonice, Czech Republic 10 Green Biotechnology Laboratory, Biotechnology and Agroindustry Division, Casaccia Research Center, ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Rome, Italy 11 Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada 12 Department of Botany, Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 13 Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Richmond, UK 14 Clinical Trial Centre Leipzig, University Leipzig, Leipzig, Germany 15 LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany 16 Laboratory of Growth Regulators, Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Olomouc, Czech Republic 17 Center for Aquatic Cytometry, Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine, USA 18 Laboratory of Molecular Biology and Cytometry, Department of Agricultural Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland 19 Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada 20 LEAF, Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal 21 Department of Precision Instruments, Tsinghua University, Beijing, China 22 Department for Therapy Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany 23 Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria Correspondence David Galbraith, School of Plant Sciences, BIO5 Institute, Arizona Cancer Center, Department of Biomedical Engineering, University of Arizona, Tucson, AZ, 85721. 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