Effect of copper on callus growth and gene expression of in vitro-cultured pith explants of Nicotiana glauca

Abstract

Copper is a vital component of electron transfer reactions mediated by proteins such as superoxide dismutase, cytochrome c oxidase and plastocyanin, but its concentrations in the cells needs to be maintained at low levels. In fact, the same ability of this essential metal ion to transfer electrons can also make it toxic to cells when present in excess. In vitro cultured explants of Nicotiana have been extensively used as a model to analyse metal-DNA interactions. In this report, we examined the effect of copper (1, 10 and 100 μM CuSO₄) on callus growth and protein synthesis of in vitro-cultured pith explants of Nicotiana glauca. In addition, a N. glauca cDNA library from Cu-treated (100 μM CuSO₄) pith explants cultured in vitro for 24 h was analysed by mRNA differential screening. The copper treatments inhibited callus growth of pith explants. The extent of inhibition was directly correlated to metal concentration. One and 10 μM CuSO₄ induced a notable increase of proteins synthesis relative to control explants. By contrast, 100 μM CuSO₄ inhibited protein synthesis relative to control extracts. The SDS-PAGE fluorography of pith proteins revealed, in Cu-treated extracts qualitative and/or quantitative differences in the synthesis of some polypeptides compared with control explants. Copper-modulated patterns of gene expression were also analysed by mRNA differential screening. The N. glauca genes isolated from Cu-treated pith explants shared common identities with other genes known to be elicited by diverse stresses, including pathogenesis and abiotic stress. In particular, the cDNAs were homologues to genes encoding cell wall proteins (i.e., extensin, and arabinogalactan-protein) and pathogenesis-related proteins (i.e., osmotin, endochitinase and a member of the Systemic Acquired Resistance gene family). In addition, an MD-2-related lipid-recognition (ML) domain protein and the enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase appeared involved in the response to copper stress. In animal cells, AdoHcy hydrolase is a copper binding protein in vivo, which suggests that, also in plant tissues, this enzyme may play an important role in regulating the levels and intracellular distribution of copper.