PLANT BIOLOGY
ANIMAL BIOLOGY
SUBSCRIPTION
E-SUBSCRIPTION
 
MAP
MAIN PAGE

 

 

 

 

Ilina E.L., Kiryushkin A.S., Demchenko K.N. features of fluorescent protein application to study the root system development of cucurbits (Cucurbitaceae). Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2020, Vol. 55, № 5, p. 1040-1055.
(how to cite this article)

doi: 10.15389/agrobiology.2020.5.1040eng

UDC: 582.98:581.43:57.086:577.21

Acknowledgements:
This study was performed using equipment of the Core Facility of Cell and Molecular Technologies in Plant Science at the Komarov Botanical Institute (St. Petersburg, Russia) and the Core Facility of Genomic Technologies, Proteomics and Cell Biology at the Research Institute for Agricultural Microbiology (St. Petersburg, Pushkin, Russia).
Supported financially by the Russian Science Foundation (grant No. 16-16-00089). We acknowledge the additional support from the Russian Foundation for Basic Research (grant No. 19-04-01079-а) on studies of the LBD expression pattern.

 

FEATURES OF FLUORESCENT PROTEIN APPLICATION TO STUDY THE ROOT SYSTEM DEVELOPMENT OF CUCURBITS (Cucurbitaceae)

E.L. Ilina, A.S. Kiryushkin, K.N. Demchenko ✉

Komarov Botanical Institute, Russian Academy of Sciences, Laboratory of cellular and molecular mechanisms of plant development, 2, ul. Professora Popova, St. Petersburg, 197376 Russia, e-mail eilina@binran.ru, akiryushkin@binran.ru, demchenko@binran.ru (✉ corresponding author)

ORCID:
Ilina E.L. orcid.org/0000-0003-2799-2014
Demchenko K.N. orcid.org/0000-0001-9422-3106
Kiryushkin A.S. orcid.org/0000-0002-9916-4819

Received May 8, 2020

 

Modern studies of detailed processes in plant development would not be possible without using a wide range of fluorescent proteins (R. Day and M. Davidson, 2009; D. Chudakov et al., 2010). However, applications of fluorescent proteins are restricted due to problems with their visualization in plant tissues. Plants are difficult objects for microscopic studies. Indeed, even the most advanced methods have significant limitations regarding the depth of light penetration due to the scattering and absorption of light by cell walls. Therefore, to study the distribution of reporter fluorescent proteins in large organs typical for most plants, it is necessary to fix the plant material and prepare thick histological sections with a vibrating-blade microtome. Chemicals traditionally used for fixing, dehydrating, and embedding of plant tissues samples lead to changes in the structure of fluorescent proteins and, as a result, often to the loss of their fluorescence. Therefore, it is important to optimize the protocols for fixing plant tissues, preparing sections, and studying the distribution of fluorescent proteins by laser scanning confocal microscopy. In this work, we propose a novel, integrated, and potentially universal approach to fixation of tissues of transgenic plants and preparation of sections in the course of studying the patterns of cellular response to auxin and expression of transcription factors using laser scanning confocal microscopy. Our aim was to sum up modern approaches to the application of this technique for visualization of tissue and cellular patterns of fluorescent reporter proteins distribution on sections of large non-model plants. The first step for using fluorescent proteins in plants is the generation of genetic constructs that carry the promoter of the gene of interest fused to a reporter gene encoding a fluorescent protein. For this, a transformation protocol has to be available for the selected plant species. We have described the use of Gateway® cloning technology for the construction of vectors for plant transformation that meet modern experimental requirements. To study auxin localization in vivo we developed a series of vectors with genes encoding various fluorescent proteins (eGFP, tdTomato, mRuby3) under the control of the auxin-sensitive DR5 promoter (E. Ilina et al., 2012). We now demonstrate the advantage of nuclear-targeted fluorescent proteins (mNeonGreen-H2B, tdTomato-H2B, mRuby3-H2B), as well as the possibility of their application for additional visualization of cell nuclei in combination with highly specific cell wall staining using SCRI Renaissance2200. An effective method is presented for constructing vectors to study cell-specific expression patterns of developmental regulators using the transcription factor genes GATA24 (A. Kiryushkin et al., 2019) and LBD16 in some Cucurbitaceae species as examples. We also applied an expression cassette, pAtUBQ10::DsRED1 (E. Limpens et al., 2004), carrying a gene encoding red fluorescent protein under the control of a constitutive promoter, to demonstrate the advantages of the use of fluorescent proteins in screening for transgenic vs. wild type roots. A new method of fixation and clearing of plant tissues containing reporter fluorescent proteins and preparation of sections is presented, using transgenic roots of Cucurbitaceae as an example. The advantage of using melted agarose compared to embedding media for orienting plant parts during the preparation of samples is revealed. The increased photostability of fluorescent proteins in sections due to the use of clearing reagent ClearSee (D. Kurihara et al., 2015) as a mounting medium is demonstrated. In sum, we apply a complex of several modern methodological approaches of sample preparation and laser scanning confocal microscopy that offers significant advantages for studying the developmental biology of large non-model plants.

Keywords: agrobacterial transformation, fluorescent proteins, confocal microscopy, plant development, transcription factors.

 

REFERENCES

  1. Cesarino I., Ioio R.D., Kirschner G.K., Ogden M.S., Picard K.L., Rast-Somssich M.I., Somssich M. Plant science’s next top models. Annals of Botany, 2020, 126(1): 1-23 CrossRef
  2. Che G., Gu R., Zhao J., Liu X., Song X., Zi H., Cheng Z., Shen J., Wang Z., Liu R., Yan L., Weng Y., Zhang X. Gene regulatory network controlling carpel number variation in cucumber. Development, 2020, 147(7): dev184788 CrossRef
  3. Ilina E.L., Logachov A.A., Laplaze L., Demchenko N.P., Pawlowski K., Demchenko K.N. Composite Cucurbita pepo plants with transgenic roots as a tool to study root development. Annals of Botany, 2012, 110(2): 479-489 CrossRef
  4. Osipowski P., Pawełkowicz M., Wojcieszek M., Skarzyńska A., Przybecki Z., Pląder W. A high-quality cucumber genome assembly enhances computational comparative genomics. Mol. Genet. Genomics, 2020, 295(1): 177-193 CrossRef
  5. Day R.N., Davidson M.W. The fluorescent protein palette: tools for cellular imaging. Chemical Society Reviews, 2009, 38(10): 2887-2921 CrossRef
  6. Chudakov D.M., Matz M.V., Lukyanov S., Lukyanov K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiological Review, 2010, 90(3): 1103-1163 CrossRef
  7. Mathur J. The illuminated plant cell. Trends in Plant Science, 2007, 12(11): 506-513 CrossRef
  8. Berg R.H., Beachy N.R. Fluorescent protein applications in plants. Methods in Cell Biology, 2008, 85: 153-177 CrossRef
  9. Kurihara D., Mizuta Y., Sato Y., Higashiyama T. ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development, 2015, 142(23): 4168-4179 CrossRef
  10. Ursache R., Andersen T.G., Marhavý P., Geldner N. A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J., 2018, 93(2): 399-412 CrossRef
  11. Nagaki K., Yamaji N., Murata M. ePro-ClearSee: a simple immunohistochemical method that does not require sectioning of plant samples. Scientific Reports, 2017, 7: 42203 CrossRef
  12. Truernit E., Bauby H., Dubreucq B., Grandjean O., Runions J., Barthélémy J., Palauqui J.-C. High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell, 2008, 20(6): 1494 CrossRef
  13. Mizuta Y., Kurihara D., Higashiyama T. Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues. Protoplasma, 2015, 252(5): 1231-1240 CrossRef
  14. Feijó J.A., Moreno N. Imaging plant cells by two-photon excitation. Protoplasma, 2004, 223(1): 1-32 CrossRef
  15. Girkin J.M., Carvalho M.T. The light-sheet microscopy revolution. Journal of Optics, 2018, 20(5): 053002 CrossRef
  16. Ovečka M., Vaškebová L., Komis G., Luptovčiak I., Smertenko A., Šamaj J. Preparation of plants for developmental and cellular imaging by light-sheet microscopy. Nature Protocols, 2015, 10: 1234 CrossRef
  17. Valuchova S., Mikulkova P., Pecinkova J., Klimova J., Krumnikl M., Bainar P., Heckmann S., Tomancak P., Riha K. Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy. eLife, 2020, 9: e52546 CrossRef
  18. Ovečka M., von Wangenheim D., Tomančák P., Šamajová O., Komis G., Šamaj J. Multiscale imaging of plant development by light-sheet fluorescence microscopy. Nature Plants, 2018, 4(9): 639-650 CrossRef
  19. Knapp E., Flores R., Scheiblin D., Modla S., Czymmek K., Yusibov V. A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression. BioTechniques, 2012, 52(1): 31-37 CrossRef
  20. Shaner N.C., Lambert G.G., Chammas A., Ni Y., Cranfill P.J., Baird M.A., Sell B.R., Allen J.R., Day R.N., Israelsson M., Davidson M.W., Wang J. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods, 2013, 10(5): 407-409 CrossRef
  21. Bindels D.S., Haarbosch L., van Weeren L., Postma M., Wiese K.E., Mastop M., Aumonier S., Gotthard G., Royant A., Hink M.A., Gadella Jr T.W.J. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods, 2016, 14: 53 CrossRef
  22. Ivanov S., Harrison M.J. A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula. Plant J., 2014, 80(6): 1151-1163 CrossRef
  23. Curtis M.D., Grossniklaus U. A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology, 2003, 133(2): 462-469 CrossRef
  24. Engler C., Youles M., Gruetzner R., Ehnert T.-M., Werner S., Jones J.D.G., Patron N.J., Marillonnet S. A Golden Gate modular cloning toolbox for plants. ACS Synthetic Biology, 2014, 3(11): 839-843 CrossRef
  25. Karimi M., Inze D., Depicker A. GATEWAY(TM) vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science, 2002, 7(5): 193-195 CrossRef
  26. Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu. Rev. Biochem., 1989, 58(1): 913-941 CrossRef
  27. Hornung E., Krueger C., Pernstich C., Gipmans M., Porzel A., Feussner I. Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds. Biochimica et Biophysica Acta (BBA) — Molecular and Cell Biology of Lipids, 2005, 1738(1-3): 105-114 CrossRef
  28. Op den Camp R.H.M., De Mita S., Lillo A., Cao Q., Limpens E., Bisseling T., Geurts R. A phylogenetic strategy based on a legume-specific whole genome duplication yields symbiotic cytokinin type-A response regulators. Plant Physiology, 2011, 157(4): 2013-2022 CrossRef
  29. Limpens E., Ramos J., Franken C., Raz V., Compaan B., Franssen H., Bisseling T., Geurts R. RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. Journal of Experimental Botany, 2004, 55(399): 983-992 CrossRef
  30. Tsai F.Y., Coruzzi G. Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants. Mol. Cell. Biol., 1991, 11(10): 4966-4972 CrossRef
  31. Ulmasov T., Murfett J., Hagen G., Guilfoyle T.J. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell, 1997, 9(11): 1963-1971 CrossRef
  32. Nam H.-S., Benezra R. High levels of Id1 expression define B1 type adult neural stem cells. Cell Stem Cell, 2009, 5(5): 515-526 CrossRef
  33. Shaner N.C., Steinbach P.A., Tsien R.Y. A guide to choosing fluorescent proteins. Nat. Methods, 2005, 2(12): 905-909 CrossRef
  34. Bajar B.T., Wang E.S., Lam A.J., Kim B.B., Jacobs C.L., Howe E.S., Davidson M.W., Lin M.Z., Chu J. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Scientific Reports, 2016, 6(20889): 1-12 CrossRef
  35. Goh T., Joi S., Mimura T., Fukaki H. The establishment of asymmetry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins. Development, 2012, 139(5): 883-893 CrossRef
  36. De Rybel B., Vassileva V., Parizot B., Demeulenaere M., Grunewald W., Audenaert D., Van Campenhout J., Overvoorde P., Jansen L., Vanneste S., Möller B., Wilson M., Holman T., Van Isterdael G., Brunoud G., Vuylsteke M., Vernoux T., De Veylder L., Inzé D., Weijers D., Bennett M.J., Beeckman T. A novel Aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity. Current Biology, 2010, 20(19): 1697-1706 CrossRef
  37. Kiryushkin A.S., Ilina E.L., Puchkova V.A., Guseva E.D., Pawlowski K., Demchenko K.N. Lateral root initiation in the parental root meristem of cucurbits: Old players in a new position. Front. Plant Sci., 2019, 10: 365 CrossRef
  38. Hostettler L., Grundy L., Käser-Pébernard S., Wicky C., Schafer W.R., Glauser D.A. The bright fluorescent protein mNeonGreen facilitates protein expression analysis in vivo. G3: Genes, Genomes, Genetics, 2017, 7(2): 607-615 CrossRef
  39. Balleza E., Kim J.M., Cluzel P. Systematic characterization of maturation time of fluorescent proteins in living cells. Nat. Methods, 2017, 15: 47 CrossRef
  40. Nagai T., Ibata K., Park E.S., Kubota M., Mikoshiba K., Miyawaki A. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol., 2002, 20(1): 87-90 CrossRef
  41. Yoo S.Y., Bomblies K., Yoo S.K., Yang J.W., Choi M.S., Lee J.S., Weigel D., Ahn J.H. The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta, 2005, 221(4): 523-530 CrossRef
  42. Zheng X.L., Deng W., Luo K.M., Duan H., Chen Y.Q., McAvoy R., Song S.Q., Pei Y., Li Y. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep., 2007, 26(8): 1195-1203 CrossRef
  43. Samac D.A., Tesfaye M., Dornbusch M., Saruul P., Temple S.J. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). Transgenic Res., 2004, 13(4): 349-361 CrossRef
  44. An Y.-Q., McDowell J.M., Huang S., McKinney E.C., Chambliss S., Meagher R.B. Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. The Plant Journal, 1996, 10(1): 107-121 CrossRef
  45. Vermeer J.E.M., von Wangenheim D., Barberon M., Lee Y., Stelzer E.H.K., Maizel A., Geldner N. A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis. Science, 2014, 343(6167): 178-183 CrossRef
  46. Chen Z., Wang J., Ye M.-X., Li H., Ji L.-X., Li Y., Cui D.-Q., Liu J.-M., An X.-M. A novel moderate constitutive promoter derived from poplar (Populus tomentosa Carrière). Int. J. Mol. Sci., 2013, 14(3): 6187-6204 CrossRef
  47. Benjamins R., Quint A., Weijers D., Hooykaas P., Offringa R. The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development, 2001, 128(20): 4057-4067.
  48. McLean I.W., Nakane P.K. Periodate-lysine-paraformaldehyde fixative a new fixative for immunoelectron microscopy. Journal of Histochemistry & Cytochemistry, 1974, 22(12): 1077-1083 CrossRef
  49. Kitaeva A.B., Kusakin P.G., Demchenko K.N., Tsyganov V.E. Key methodological features of tubulin cytoskeleton studies in nodules of legume plants. Agricultural Biology [Sel'skokhozyaistvennaya Biologiya], 2018, 53(3): 634-644 CrossRef
  50. Kitaeva A.B., Demchenko K.N., Tikhonovich I.A., Timmers A.C.J., Tsyganov V.E. Comparative analysis of the tubulin cytoskeleton organization in nodules of Medicago truncatula and Pisum sativum: Bacterial release and bacteroid positioning correlate with characteristic microtubule rearrangements. New Phytol., 2016, 210(1): 168-183 CrossRef
  51. Musielak T.J., Schenkel L., Kolb M., Henschen A., Bayer M. A simple and versatile cell wall staining protocol to study plant reproduction. Plant Reprod., 2015, 28(3): 161-169 CrossRef
  52. Zdyb A., Demchenko K., Heumann J., Mrosk C., Grzeganek P., Göbel C., Feussner I., Pawlowski K., Hause B. Jasmonate biosynthesis in legume and actinorhizal nodules. New Phytol., 2011, 189(2): 568-579 CrossRef
  53. Stumpe M., Göbel C., Demchenko K., Hoffmann M., Klösgen R.B., Pawlowski K., Feussner I. Identification of an allene oxide synthase (CYP74C) that leads to formation of α-ketols from 9-hydroperoxides of linoleic and linolenic acid in below-ground organs of potato. The Plant Journal,2006, 47(6): 883-896 CrossRef
  54. Demchenko K., Zdyb A., Feussner I., Pawlowski K. Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules. Plant Biology, 2012, 14(1): 56-63 CrossRef
  55. Ilina E.L., Kiryushkin A.S., Semenova V.A., Demchenko N.P., Pawlowski K., Demchenko K.N. Lateral root initiation and formation within the parental root meristem of Cucurbita pepo: is auxin a key player? Annals of Botany, 2018, 122(5): 873-888 CrossRef
  56. Prunet N., Jack T.P., Meyerowitz E.M. Live confocal imaging of Arabidopsis flower buds. Developmental Biology, 2016, 419(1): 114-120 CrossRef
  57. Dickinson M.E., Bearman G., Tille S., Lansford R., Fraser S.E. Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. BioTechniques, 2001, 31(6): 1272-1278 CrossRef
  58. Kraus B., Ziegler M., Wolff H. Linear fluorescence unmixing in cell biological research. In: Modern research and educational topics in microscopy. A. Méndez-Vilas, J. Díaz (eds.). Formatex, Badajoz, Spain, 2007: 863-872.
  59. Mylle E., Codreanu M.-C., Boruc J., Russinova E. Emission spectra profiling of fluorescent proteins in living plant cells. Plant Meth., 2013, 9(1): 10 CrossRef
  60. Zimmermann T., Rietdorf J., Pepperkok R. Spectral imaging and its applications in live cell microscopy. FEBS Letters, 2003, 546(1): 87-92 CrossRef
  61. Korzhevskii D.E., Kirik O.V., Sukhorukova E.G., Kolos E.A., Karpenko M.N., Sufieva D.A., Nazarenkova A.V. Molekulyarnaya morfologiya. Metody fluorestsentnoi i konfokal'noi lazernoi mikroskopii [Molecular morphology. Methods of fluorescence and confocal laser microscopy]. St. Petersburg, 2014 (in Russ.).

back

 


CONTENTS

 

 

Full article PDF (Rus)

Full article PDF (Eng)