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First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts 1
Author(s) -
Gehwolf Renate,
Griessner Martin,
Pertl Heidi,
Obermeyer Gerhard
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)02246-9
Subject(s) - protoplast , lilium , microbiology and biotechnology , pollen , fusicoccin , biology , pollen tube , atpase , messenger rna , serial dilution , chemistry , botany , biochemistry , enzyme , gene , medicine , alternative medicine , pathology , pollination
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.