Nuclear genomes of many important plant species are tremendously complicated to map and sequence. The ability to isolate single chromosomes, which represent small units of nuclear genome, is priceless in many areas of plant research including cytogenetics, genomics, and proteomics. Flow cytometry is the only technique which can provide large quantities of pure chromosome fractions suitable for downstream applications including physical mapping, preparation of chromosome-specific BAC libraries, sequencing, and optical mapping. Here, we describe step-by-step procedure of preparation of liquid suspensions of intact mitotic metaphase chromosomes and their flow cytometric analysis and sorting.

• Klíčová slova
• Cell cycle synchronization, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Metaphase accumulation, Plants,
• MeSH
• chromozomy rostlin * MeSH
• hybridizace in situ fluorescenční metody   MeSH
• průtoková cytometrie metody   MeSH
• rostliny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.

• Klíčová slova
• DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
• MeSH
• buněčný cyklus MeSH
• chromozomy rostlin * genetika   MeSH
• metafáze MeSH
• průtoková cytometrie MeSH
• rostliny * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Flow cytometry offers a unique way of analyzing and manipulating plant chromosomes. During a rapid movement in a liquid stream, large populations can be classified in a short time according to their fluorescence and light scatter properties. Chromosomes whose optical properties differ from other chromosomes in a karyotype can be purified by flow sorting and used in a range of applications in cytogenetics, molecular biology, genomics, and proteomics. As the samples for flow cytometry must be liquid suspensions of single particles, intact chromosomes must be released from mitotic cells. This protocol describes a procedure for preparation of suspensions of mitotic metaphase chromosomes from meristem root tips and their flow cytometric analysis and sorting for various downstream applications.

• Klíčová slova
• Accumulation of metaphase cells, Chromosome isolation, Cytogenetic stocks, FISH, FISHIS, Flow cytometry and sorting, Hydroponic, Mitotic synchrony, Plants, Seedlings,
• MeSH
• chromozomy rostlin * MeSH
• chromozomy * MeSH
• cytogenetika MeSH
• karyotypizace MeSH
• průtoková cytometrie metody   MeSH
• suspenze MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• suspenze MeSH

Optical mapping-a technique that visualizes short sequence motives along DNA molecules of hundred kilobases to megabase in size-has found an important place in genome research. It is widely used to facilitate genome sequence assemblies and analyses of genome structural variations. Application of the technique is conditional on availability of highly pure ultra-long high-molecular-weight DNA (uHMW DNA), which is challenging to achieve in plants due to the presence of the cell wall, chloroplasts, and secondary metabolites, just as a high content of polysaccharides and DNA nucleases in some species. These obstacles can be overcome by employment of flow cytometry, enabling a fast and highly efficient purification of cell nuclei or metaphase chromosomes, which are afterward embedded in agarose plugs and used to isolate the uHMW DNA in situ. Here, we provide a detailed protocol for the flow sorting-assisted uHMW DNA preparation that has been successfully used to construct whole-genome as well as chromosomal optical maps for 20 plant species from several plant families.

• Klíčová slova
• Bionano genome map, Chromosomes, Flow cytometry and sorting, HMW DNA preparation, Nuclei, Optical mapping, ultralong high-molecular-weight DNA,
• MeSH
• chromozomy rostlin * genetika   MeSH
• genom rostlinný MeSH
• průtoková cytometrie metody   MeSH
• restrikční mapování MeSH
• rostliny * genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.

• Klíčová slova
• cell cycle synchronization, chromosome genomics, chromosome isolation, chromosome sorting, chromosome-specific DNA libraries, flow cytometry, genome sequencing, high-molecular-weight DNA, physical genome mapping, plant chromosomes,
• MeSH
• chromozomy rostlin metabolismus   MeSH
• DNA rostlinná genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• karyotypizace MeSH
• meristém cytologie   účinky léků   MeSH
• metafáze účinky léků   MeSH
• molekulová hmotnost MeSH
• oxid dusný farmakologie   MeSH
• proteomika MeSH
• průtoková cytometrie metody   MeSH
• rostliny genetika   MeSH
• semena rostlinná účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• oxid dusný MeSH

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.

• Klíčová slova
• autofluorescence, best practices, plant flow cytometry and sorting, protoplasts, viability and integrity,
• MeSH
• protoplasty * MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• suspenze MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• suspenze MeSH

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.

• Klíčová slova
• ESCRT machiner, endosomal recycling, multivesicular body/prevacuolar compartment (MVB/PVC), retromer complex, vacuolar trafficking,
• MeSH
• Arabidopsis * metabolismus   MeSH
• endozomální třídící komplexy pro transport metabolismus   MeSH
• endozomy metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• rostliny metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• transportní proteiny metabolismus   MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ALIX protein, Arabidopsis MeSH Prohlížeč
• endozomální třídící komplexy pro transport MeSH
• proteiny huseníčku * MeSH
• transportní proteiny MeSH

Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.

• Klíčová slova
• LAT, PAG, Palmitoylation, Plasma membrane, Protein sorting, Transmembrane domain,
• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• extracelulární prostor chemie   MeSH
• glykosylace MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• Jurkat buňky MeSH
• lidé MeSH
• lipoylace * MeSH
• membránové proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transport proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• membránové proteiny MeSH

The use of flow cytometry for evaluation of plant chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of chromosome suspensions, flow analysis and sorting of chromosomes, and processing of the sorted chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted chromosomes for plant genome mapping.

• MeSH
• buněčný cyklus MeSH
• chromozomy rostlin chemie   genetika   MeSH
• chromozomy ultrastruktura   MeSH
• cytogenetika MeSH
• cytologické techniky metody   MeSH
• DNA rostlinná genetika   MeSH
• fyzikální mapování chromozomů MeSH
• genom MeSH
• genová knihovna MeSH
• průtoková cytometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA rostlinná MeSH

Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.

• MeSH
• buněčný cyklus MeSH
• chromozomy rostlin * MeSH
• cytogenetika MeSH
• DNA rostlinná genetika   MeSH
• hybridizace in situ fluorescenční metody   MeSH
• karyotypizace MeSH
• meristém cytologie   MeSH
• průtoková cytometrie metody   MeSH
• rostlinné buňky MeSH
• rostliny genetika   MeSH
• semena rostlinná růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH