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.
Gehwolf
Renate
R
Institut für Pflanzenphysiologie, University of Salzburg, Hellbrunnerstr. 34, 5020, Salzburg, Austria.
Griessner
Martin
M
Pertl
Heidi
H
Obermeyer
Gerhard
G
D027762:Lilium; D018408:Patch-Clamp Techniques; D011058:Pollen / Q000201:enzymology* / Q000235:genetics; D017494:Proton Pumps / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011523:Protoplasts; D012333:RNA, Messenger / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017494:Proton Pumps; D012333:RNA, Messenger; D006180:Proton-Translocating ATPases