First patch, then catch:: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H+ ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch-catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H+ ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments. a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.