engleski

Expression of the enzyme myrosinase type 1 in the root, the stem and the leaves of the plant nasturtium (Tropaeolum majus L.)

INTRODUCTION The enzyme myrosinase catalyses the hydrolysis of a group of low molecular weight compounds, the glucosinolates [1, 2]. This two-compound system of secondary products and degradative enzymes is momentous in plant-insect interactions [3]. Tissue disruption brings glucosinolates into contact with myrosinases, resulting in the release of various components with diverse biological activities (anticancer, antbacterial, antifungal…[4, 5]. The myrosinase refers to a certain type of idioblasts called myrosin cells [2]. Myrosin cells are present in several tissues in the plant including the cotyledons and axis of the embryo as well as in the leaves, the stem, the root, the petals, and the siliques of the adult plant [1]. Nasturtium (Tropaeolum majus L.) is a plant that belongs to the Tropaeolaceae family (order Brassicales) and is known for its ornamental and medicinal properties [6].The aim of this study was to investigate the expression of the enzyme myrosinase type 1 in the nasturtium leaf, stem and root regions in the different developmental stages after plant's sprout. MATERIALS AND METHODS Nasturtium tissues were cut into small pieces at different developmental stages after sprouting. The specimens were fixed in 4% paraformaldehyde in phosphate buffer pH 6.8, dehydrated in an ascending series of ethanol, cleared in xylene, and then embedded in paraffin wax. Paraffin sections cut on a rotary microtome were mounted on glass slides [7]. After deparaffinization and rehydration, sections were heated in a citrate buffer (pH 6.0), cooled and a blocking buffer was applied to exclude unspecific staining. Sections were then incubated overnight at RT with TGG1 myrosinase 1 (BGL38) rabbit primary antibody, diluted 1/1000 in PBS. After washing in PBS, Alexa Fluor 488 anti-rabbit IgG secondary antibody, diluted 1/400 in PBS was applied for 1 h and washed in PBS again. Then, nuclei were stained with DAPI. All slides were studied using an Olympus BX51 epifluorescence microscope with digital camera a Nikon DS-Ri1 (Japan). RESULTS The enzyme myrosinase has been found in all investigated organs of the T. majus plant, in idioblasts, also called myrosin cells. The idioblasts are specialized cells that are scattered at low frequency and often as single cells among the other major cells in a tissue. Myrosin cells are anatomically characterized by a high protein content in the vacuole and thus are prone to react cytochemically with certain protein reagents. Immunofluorescence positive myrosinase type 1 enzyme appeared in ground tissue and the vascular tissue in all investigated parts of the plant. In the root, the strongest positive expression of the myrosinase was found in the ground tissue idioblasts in phase 1 and phase 2 of the developmental period. Also, the parenchyma xylem and phloem cells of the root region have shown positive immunoexpression. In the stem, large xylem vessels with empty lumens and tick cell walls in phases 2 and 3 show a distinguished location of the myrosinase, as well as the phloem cells. No significant immunoexpression of the enzyme myrosinase type 1 was observed in all four different developmental phases in the leaf region. Nevertheless the xylem (with pronounced annular thickening) parenchyma cells and through the ground tissue weak signal of enzyme myrosinase type 1 in the leaf region of the nasturtium was observed. CONCLUSION Myrosin cells are specialized cells that are scattered at low frequency associated with the enzyme myrosinase. Myrosinase type 1 enzyme has been found in all investigated organs of T. majus plant. The strongest positive expression of the myrosinase was found in the ground tissue idioblasts of the root. The presence of the glucosinolate-myrosinase system is characteristic of the Brassicales order, and it serves as a defense system against biotic and abiotic stressors. REFERENCES [1] A. S. Höglund, M. Lenman, L. Rask, Plant Sci. 1992, 85, 165. [2] Y. Niu, A. Rogiewicz, C. Wan, M. Guo, F. Huang, A. Bogdan, B. A. Slominski, J. Agric. Food Chem. 2015, 63, 3078. [3] E. Andreasson, L. B. Jørgensen, A.-S. Hoglund, L. Rask, J. Meijer, Plant Phys. 2001, 127, 1750. [4] S. Chen, E. Andreasson, Plant Physiol. Biochem. 2001, 39, 743. [5] I. Vrca, F. Burčul, I. Blažević, T. Bilušić, Croat. J. Food Sci. Tech. 2021, 13, 160. [6] G. A. Garzon, R. E. Wrolstad, Food Chem. 2009, 114, 44. [7] I. Restović, K. Vukojević, A. Paladin, M. Saraga-Babić, I. Bočina, Anat. Rec. 2015, 298, 1700.

Order Brassicales ; Tropaeolum majus L. ; Enzyme myrosinase

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