Báo cáo khoa học: "A new method for the histochemical localization of laccase in Rhus verniciflua Stokes"

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A new method for the histochemical localization of laccase in Rhus verniciflua Stokes M.R. Li* Department of Forestry and Natural Resources, University of Edinburgh, The King’s Buildings, Ma!eld Road, Edinburgh EH9, 3JU, Scotland, U.K. Intrntimntinn tho. the in!,...tÎB/!+i,"’Bn n.J +4,n enzyme ,during Introduction ..{11..inl"’l inactivation of the Bö ’ o.n"7’/l"’r extraction and purification; its substrate, urushiol, is apparently necessary for main- There has been great interest in laccase taining it in an undenatured state (Guo, for over 100 yr, since Yoshida first de- 1981. ) tected this copper-containing enzyme in Laccase can be demonstrated in vitro 1883. Laccase (EC 1.10.3.2) is found in of biochemical methods, such by means the latex of species of Rhus and is spectrophotometry, oxygen-absorbance as responsible for the oxidation of phenol and polarography from the liquid lacquer urushiol, which is contained in the latex, or from a cell-cultured suspension (Guo, with production of the black resinous lac- 1981; Gan and Gan, 1983; Bligny and quer (Bonner, 1950). Besides Rhus, lacca- Douce, 1983). For the purpose of studying se is also found in numerous other woody its physiological function in the lacquer plants including Aesculus sp., Prunus tree, a simple permanent staining tech- persica, Acer pseudoplatanus and many nique for in situ laccase fixation has been species of the Anacardiaceae family, and developed, which permits its enzymatic in a number of fungi as well as in some activity to be maintained in vivo and the herbaceous plants (Bonner, 1950; Butt, stable product of the catalysis to be distin- 1980; Mayer and Harel, 1979; Bligny and guished under the light microscope. Douce, 1983). Although it is moderately widely distributed in higher plants and there have been many reports concerning its biochemical and biophysical properties, Materials and Methods laccase has benefitted from little or no One year old seedlings of the lacquer tree investigation, and in the literature there (Rhus vernicifltia Stokes cv. Puchengxiaomuy are no speculations as to its physiological were grown from root-cuttings in pots under function in the secretory ducts of Rhus. good growth conditions. Lignified stems were One of the main difficulties encountered in then processed in the experiment as described studying its physiological function may be below. Prør:::&Jnl !rlrlrøcc:. non!rtmont nf P resent address: Ql’Bt!nB1 TrinitBl !I"BIIQ"O tlnivarcitv nf nllhlin Department of Botany, Trinity College, University o Dublin, nithlin 9, IIreland. Dublin 2 ralanri *
substrate solution of 0.05 M phosphate buffer Incubation with 0.01 % (w/v) p (pH 7.4). dihydroxybenzene - Freshly cut blocks (about 1 mm in width) of the In the control treatment, either stem cuttings phloem tissue were incubated in 0.05 M phos- were denatured by leaving them in water at phate buffer (pH 7.4, 5°C) for 1-2 min before 100°C for 5 min before cutting into blocks, or staining for 30 min at 37°C in a newly prepared
freshly prepared blocks were incubated in the Discussion and Conclusion same buffer without the p dihydroxybenzene. - An interesting comparison can be made Prefixation and postfixation between histochemical and biochemical Following incubation, the blocks were rinsed in methods of enzyme demonstration. All lac- 0.05 M phosphate buffer (pH 7.4) and then cases previously described catalyze the transferred into buffered glutaraldehyde (3%) at ’ following reactions: 4°C for 1 h. After fixation, they were rinsed in 3 changes of the 0.05 M phosphate buffer with 1 p-dihydroxybenzene (colorless) laccasep, quin- h for each change. 2% osmic acid in the same buffer was used at 4°C for 2 h. hydrone (dark brown) + H+ (1) urushiol (colorless) laccase quin-urushiol . (light brown) + H+ (2) Embedding and sectioning After fixation, the blocks were rinsed 3 times in Reaction (1 ) is characteristic of laccase distilled water before dehydration in ethanol and and is the main criterion according to embedded in Epon 812. Thin sections (1-2 !m which the enzyme is classified (Mayer and thick) were prepared by using a manually oper- Harel, 1979). Reaction (2) is one of the ated ultramicrotome (LKB Nova V). For com- significant features of laccase in Rhus parison, a parallel study was made using unfixed hand-sections (25-50 pm thick) and fro- species. The brown deposit in the sections zen sections (10-15 5 pm thick). The microtome indicates the localization of active laccase stage and knife were frozen with a semiconduc- in situ. Activity of laccase is also stimu- tor freezer. lated by its natural substrate but the pro- duct does not show enough contrast for observation. Figs. 1-4 show that the distinguishable reaction product of the arti- Results ficial reagent p-dihydroxybenzene does dissolve these during not preparatory A heavy deposit can be seen (Figs. 1-4) steps. in the latex canals and some other cells, Using polyacrylamide gel electrophore- such as sheath cells, epithelial cells and two lacca;se isoenzymes with different sis, some parenchymatous cells. The brown have been isolated and identified by f R deposit in the sections indicates that the several phenols, including p-dihydroxy- reaction product of laccase catalysis is benzene, from the phloem of R. vernici- mainly distributed in the canals and their flua (Li, unpublished). Whether these sheath cells, epithelial cells and the ducts isoenzymes have different physiological with latex droplets. In the control section, functions needs further investigation. The almost no deposit can be seen in the histochemical method described in the canals and surrounding cells (Fig. 5). The present paper may be used to bridge deposit is stable and the embedded mate- these gaps. rial can be stored for at least 3 mo. In In the lacquer tree, laccase may play a comparison with the unfixed section, the role in sealing-off damaged tissue. It could fixed section can be observed more clear- also be involved in a defense mechanism ly under the light microscope because it is thinner than both the hand-section and the against pathogens by oxidizing endo- genous phenols (e.g., urushiol) to the frozen section. The section embedded in resultant toxic quinones (Mayer and Harel, Epon 812 is also suitable for study under 1979; Butt, 1980). Since laccase has been the transmission electron microscope.
shown to be involved in the oxidation of References lignin by fungi and there is extensive R. & Douce R. (1983) Excretion of lac- Bligny excretion of laccase by cultured cells of by sycamore (Acer pseudoplatanus L.) case Acer pseudoplatanus, Bligny and Douce cells. Purification and properties of the enzyme. (1983) suggested that it could play an Biochem. J. 209, 489-496 important role in the synthesis and deposi- Bonner J. (1950) In: Plant Biochemistry. Aca-’ tion of specific wall substances, such as demic Press, New York, p. 182 Butt V.S. (1980) Direct oxidases and related lignin. It should be noted that the allergic enzymes. IV. Laccase. In: The Biochemistry of skin reaction of humans to quin-urushiol is Plants. A Comprehensive Treatise. Vol. 2 still treated as a serious disease in China. (Metabolism and Respiration). (Davies D.D., Evolutionarily, laccase and its substrates ed.), Academic Press, New York, p. 1133 seem to be an adaptation of the lacquer Gan C.J. & Gan J.G. (1983) Laccase. Chinese Lacquer (Xian) 2 (suppl.) 42 . tree for survival in competition with other Guo M.G. (1981) The laccase activity mea- life forms, including insects, fungi, humans sured by oxygen absorbance. Chem. Ind. For. and herbivorous animals. At present, this Prod. (Nanjing) 3, 24 must be regarded as speculation that is Mayer A.M. & Harel E. (1979) Polyphenol worthy of experimental investigation. oxidases in plants. Phytochemistry 18, 198-215 5