Abstract
A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with root cells and their visualization under CLSM. DTAF (5-(4,6-dichlorotriazinyl) aminofluorescein) is a fluorescent dye that has been widely used for staining various biological samples for fluorescent microscopy. It reacts with polysaccharides and peptides at ordinary conditions. The possible application and efficiency of DTAF for CLSM studies were examined in various aspects of cyanobacterial-plant interactions. Seedlings of Pisum sativum, Vigna rediata and Triticum aestivum were co-cultivated and stained with DTAF as a fluorochrome. Extracellular and intracellular interactions of cyanobacteria and the plant root surface were observed by CLSM. Results were compared with staining by other commonly used lectins. Advantages of the use of DTAF over other stains are its penetration into root tissues and binding with polysaccharides, mainly the cellulose. The staining was smooth, which clearly showed minute details on the cell of surface and root hairs with higher resolution. The emission wavelength for DTAF is 517 nm, which is highly advantageous as cyanobacteria have auto-fluorescence at 665 nm, and both can be simultaneously used in CLSM by visualizing in different channels. This worked efficiently with all three plants used and with filamentous and unicellular cyanobacterial strains. Cyanobacterial presence was not only clearly observed on the root surface, but also inside the root tissue and epidermal cells. The easy protocol and absence of tissue processing make DTAF a useful probe for studies of cyanobacterial associations with plant roots by CLSM.
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References
Ahmed M, Stal LJ, Hasnain S (2010) Association of non-heterocystous cyanobacteria with crop plants. Plant Soil. doi:10.1007/s11104-010-0488-x
Arregui L, Serrano S, Linares M, Pérez-Uz B, Guinea A (2007) Ciliate contributions to bioaggregation: laboratory assays with axenic cultures of Tetrahymena thermophila. Int Microbiol 10:91–96
Bloem J, Vos A (2004) Fluorescent staining of microbes for total direct counts. In: Kowalchuk GA, De Bruijn FJ, Head IM, Akkermans ADL, Van Elsas JD (eds) Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, pp 861–874
Czymmek KJ, Whallon JH, Klomparens KL (1994) Confocal microscopy in mycological research. Fungal Genet Biol 18:275–293
Grayston SJ, Wang S, Campbell CD, Edwards AC (1998) Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol Biochem 30:369–378
Karthikeyan N, Prasanna R, Sood A, Jaiswal P, Nayak S, Kaushik BD (2009) Physiological characterization and electron microscopic investigation of cyanobacteria associated with wheat rhizosphere. Folia Microbiol 54:43–51
Lerouxel O, Cavalier DM, Liepman AH, Keegstra K (2006) Biosynthesis of plant cell wall polysaccharides—a complex process. Curr Opin Plant Biol 9:621–630
Li Y, Dick WA, Tuovinen OH (2003) Evaluation of fluorochromes for imaging bacteria in soil. Soil Biol Biochem 35:737–744
Mariné MH, Clavero E, Roldán M (2004) Microscopy methods applied to research on cyanobacteria. Limnetica 23:179–186
Neu TR, Lawrence JR (1999) Lectin-binding analysis in biofilm systems. Methods Enzymol 310:145–152
Nilsson M, Bhattacharya J, Rai AN, Bergman B (2002) Colonization of roots of rice (Oryza sativa) by symbiotic Nostoc strains. New Phytol 156:517–525
Pereira S, Zille A, Micheletti E, Moradas-Ferreira P, De Philippis R, Tamagnini P (2009) Complexity of cyanobacterial exopolysaccharides: composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly. FEMS Microbiol Rev 33:917–941
Putt M (1991) Development and evaluation of tracer particles for use in microzooplankton herbivory studies. Mar Ecol Prog Ser 77:27–37
Roldan M, Clavero E, Castel S, Hernandez-Marine M (2004) Biofilms fluorescence and image analysis in hypogean monuments research. Arch Hydrobiol Algol Stud 111:127–143
Schumann R, Rentsch D (1998) Staining particulate organic matter with DTAF-a fluorescence dye for carbohydrates and protein: a new approach and application of a 2D image analysis system. Mar Ecol Prog Ser 163:77–88
Si-Ping Z, Bin C, Xiong G, Wei-Wen Z (2009) Diversity analysis of endophytic bacteria within Azolla microphylla using PCR-DGGE and electron microscopy. Chin J Agri Biotech 5:269–276
Sole A, Diestra E, Esteve I (2009) Confocal laser scanning microscopy image analysis for cyanobacterial biomass determined at microscale level in different microbial mats. Microb Ecol 57:649–656
Stal LJ (2007) Cyanobacteria: diversity and versatility, clues to life in extreme environments. In: Seckbach J (ed) Algae and cyanobacteria in extreme environments. Springer, Dordrecht, pp 659–680
Acknowledgments
The Higher Education Commission of Pakistan is acknowledged for providing funding to Mehboob Ahmed (IRSIP no. 1-8/HEC/HRD/2007/923) to visit The Netherlands Institute of Ecology (NIOO-KNAW) to carry out confocal laser scanning microscopy. We thank Anita Wijnholds for her help with CLSM.
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This article is part of the BioMicroWorld 2009 Special Issue.
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Ahmed, M., Stal, L.J. & Hasnain, S. DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM). J Ind Microbiol Biotechnol 38, 249–255 (2011). https://doi.org/10.1007/s10295-010-0820-8
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DOI: https://doi.org/10.1007/s10295-010-0820-8