Compartmentation of phenolic compounds and phenylalanine ammonia-lyase in leaves of Phyllanthus tenellus Roxb. and their induction by copper sulphate
2000
Santiago, L.J.M. | Louro, R.P. | Oliveira, D.E. de
The compartmentation of phenolic compounds in mature leaves of Phyllanthus tenellus and their induction by copper sulphate were analysed at histological and subcellular levels. Light and electron microscopy studies demonstrated that the vacuoles of spongy cells were the main sites of phenolic accumulation. Spraying plants with copper sulphate induced punctated lesions formed by groups of necrotic cells which accumulated brownish substances. Histochemical tests and fluorescence microscopy analysis of the sprayed leaves indicated that the phenolic compounds increased in spongy cells within the lesions. Ultrastructural analyses showed that 3 h after elicitation, the organelles of the cells within the lesion started to collapse and the content of phenolic substances increased in the vacuole of spongy cells. Antibody against phenylalanine ammonia-lyase (PAL) from parsley cross-reacted with the crude extract of P. tenellus leaves. Two isoforms, one of 65 kD and the other of 66 kD, were identified. Immunocytochemical studies showed that PAL was synthesized in the palisade and spongy cells, mainly in the cytoplasm and chloroplasts. The phytotoxicity of Cu2+ions induced the accumulation of PAL in sub-cellular compartments of palisade cells. PAL accumulation started to increase 3 h after elicitation and reached a maximum after 6 h, decreasing 12 h post-induction. The increase of PAL was more evident in cells within the necrotic punctated regions than in surrounding cells. Since the vacuole of palisade cells did not accumulate phenolic compounds, the in situ studies suggested that the end products of PAL synthesis play a role in palisade cell wall reinforcement or might accumulate in other tissues. The symptoms induced by copper sulphate suggest that this abiotic elicitor may be a useful tool in the understanding of the regulation of biosynthetic phenolic pathways inP. tenellus.
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