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Published on October 13, 2006; 10.1104/pp.106.087882

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Received August 16, 2006
Accepted October 10, 2006

NAD(P)H Oscillates in Pollen Tubes and Is Correlated with Tip-Growth

Luis Cárdenas *, Sylvester T. McKenna , Joseph G. Kunkel , and Peter K. Hepler

Departamento de Biologia Molecular de Plantas Instituto de Biotecnologia, UNAM, Apartado Postal 510-3 Cuernavaca, Morelos 62271, México
Department of Biology Long Island University Brooklyn, NY 11201
Department of Biology and the Plant Biology Graduate Program University of Massachusetts Amherst, MA 01003

* Corresponding author; email: luisc{at}ibt.unam.mx.

The location and changes in NAD(P)H have been monitored during oscillatory growth in pollen tubes of lily using the endogenous fluorescence of the reduced coenzyme (excitation - 360 nm; emission - > 400 nm). The strongest signal resides 20-40 µm 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 7-11 s or 77-1160, whereas the troughs anticipate growth maxima by 5-10 s or 54-1070. 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 µm 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 50-60 µm 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)+) may be coupled to the synthesis of ATP. We also show that diphenyleneiodonium (DPI), 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 (CM-H2DCFDA) indicates that reactive oxygen species (ROS) are most abundant in the region where mitochondria accumulate and where NAD(P)H fluorescence is maximal.




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