Oscillatory Growth in Lily Pollen Tubes Does Not Require Aerobic Energy Metabolism

Abstract

Oscillatory tip growth in pollen tubes depends on prodigious amounts of energy. We have tested the hypothesis that oscillations in the electron transport chain lead to growth oscillations in lily (Lilium formosanum). Using three respiratory inhibitors, oligomycin, antimycin A, and cyanide, we find that pollen tube growth is much less sensitive to respiratory inhibition than respiration is. All three block respiration at concentrations severalfold lower than necessary to inhibit growth. Mitochondrial NAD(P)H and potentiometric JC-1 fluorescence, employed as markers for electron transport chain activity, rise rapidly in response to oligomycin, as expected. Pollen tube growth stops for several minutes before resuming. Subsequent growth has a lower mean rate, but continues to oscillate, albeit with a longer period. NAD(P)H fluorescence no longer exhibits coherent oscillations, and mitochondria no longer congregate directly behind the apex: they distribute evenly throughout the cell. Postinhibition growth relies on aerobic fermentation for energy production as revealed by an increase in ethanol in the media. These data suggest that oscillatory growth depends not on a single oscillatory pacemaker but rather is an emergent property arising from a number of stable limit cycles.

Dynamic Adaption of Metabolic Pathways during Germination and Growth of Lily Pollen Tubes after Inhibition of the Electron Transport Chain

“…The time points at which samples were prepared reflected specific physiological states during germination and growth of pollen tubes (Pertl et al, 2009): 0 min corresponds to mature, dehydrated pollen grains, 10 min corresponds to fully rehydrated pollen grains, 30 min corresponds to rehydrated pollen grains just before germination, 60 min corresponds to just-germinated pollen grains, and 75 to 240 min corresponds to growing pollen tubes. A significant decrease in the mean tube length was observed in the presence of 20 mM antimycin A, an inhibitor of complex III (cytochrome b/c 1 ) of the mitochondrial electron transport chain, whereas the germination frequency was not affected, thus confirming previous studies (Rounds et al, 2010). However, the ethanol concentration of the external medium increased significantly after inhibiting the electron transport chain (Fig.…”

“…Previous studies also demonstrated metabolic differences between the germination and the tube growth process. While oligomycin, an inhibitor of the F 0 F 1 -ATP synthase, slowed down tube growth, germination frequencies of tobacco (Nicotiana tabacum) pollen are not affected (Gass et al, 2005), which was also confirmed for lily pollen (Rounds et al, 2010) and, in this study, for antimycin A (Fig. 1).…”

“…However, the ethanol concentration of the external medium increased significantly after inhibiting the electron transport chain (Fig. 1C), indicating a switch to ethanolic fermentation (Tadege et al, 1999;Rounds et al, 2010). To study the dynamics of metabolic pathways in pollen, metabolites were not only determined at specific time points during pollen cultures (0, 10, 30, 60, 75, 90, 120, and 240 min), but the well-documented metabolic switch induced by antimycin A was used as an experimental challenge to test the metabolic flexibility of germinating pollen grains too, as shown in Figure 1D.…”

“…Kuhlemeier and coworkers identified the pyruvate dehydrogenase bypass by which pollen grains produce ethanol to support the tricarbonic acid (TCA) cycle and lipid biosynthesis (Tadege et al, 1999;Mellema et al, 2002;Gass et al, 2005). This aerobic fermentation can be induced by blocking aerobic respiration with inhibitors of the electron transport chain (antimycin A, potassium cyanide) or the mitochondrial F-type ATPase (Rounds et al, 2010). Despite the progress during the last years of pollen research, the present knowledge about the role of metabolic pathways during pollen germination and tube growth is still fragmentary and incomplete, not to mention the interaction between metabolism and molecular as well as cellular processes that determine tube growth.…”

“…With the beginning of germination, the respiration rate decreases but raises again during the phase of steady tube growth (Dickinson, 1965(Dickinson, , 1968, thus, generating all the necessary energy for tube elongation. Furthermore, a direct link between tube growth rates and the activity of oxidative phosphorylation (ATP production) was postulated by monitoring the concentration of cytosolic NAD(P)H + in living pollen tubes (Cárdenas et al, 2006;Rounds et al, 2010).…”