NAD(P)H oscillates in pollen tubes and is correlated with tip growth

The location and changes in NAD(P) H have been monitored during oscillatory growth in pollen tubes of lily ( Lilium formosanum) using the endogenous fluorescence of the reduced coenzyme ( excitation, 360 nm; emission, > 400 nm). The strongest signal resides 20 to 40 mm behind the apex where mitochondria ( stained with Mitotracker Green) accumulate. Measurements at 3-s intervals reveal that NAD( P) H-dependent fluorescence oscillates during oscillatory growth. Cross-correlation analysis indicates that the peaks follow growth maxima by 7 to 11 s or 77 degrees to 116 degrees, whereas the troughs anticipate growth maxima by 5 to 10 s or 54 degrees to 107 degrees. We have focused on the troughs because they anticipate growth and are as strongly correlated with growth as the peaks. Analysis of the signal in 10-mu m increments along the length of the tube indicates that the troughs are most advanced in the extreme apex. However, this signal moves basipetally as a wave, being in phase with growth rate oscillations at 50 to 60 mm from the apex. We suggest that the changes in fluorescence are due to an oscillation between the reduced ( peaks) and oxidized ( troughs) states of the coenzyme and that an increase in the oxidized state [ NAD( P) 1] may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium, an inhibitor of NAD( P) H dehydrogenases, causes an increase in fluorescence and a decrease in tube growth. Finally, staining with 5-(and-6)-chloromethyl-2',7'-dichlorohydrofluorescein acetate indicates that reactive oxygen species are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.