Dynamics of Pollen Activation and the Role of H+-ATPase in Pollen Germination in Blue Spruce (Picea pungens)

Abstract

Pollen is a highly specialized structure for sexual plant reproduction. Early stages of pollen germination require the transition from dormant state to active metabolism. In particular, an important role during this early phase of angiosperm pollen germination is played by H+-ATPase. Very little is known about pollen activation in gymnosperm species, and information on the involvement of H+-ATPase is lacking. We tracked four indicators characterizing the physiological state of pollen: membrane potential, intracellular pH, anion efflux and oxygen uptake, in order to monitor the dynamics of activation in Picea pungens. Based on pH dynamics during activation, we assumed the important role of H+-ATPase in spruce pollen germination. Indeed, germination was severely suppressed by P-type ATPase inhibitor orthovanadate. In spruce pollen tubes, a pronounced pH gradient with a maximum in the apical zone was found, which was different from the pollen tubes of flowering plants. Using orthovanadate and fusicoccin, we found that the proton pump is largely responsible for maintaining the gradient. Immunolocalization of the enzyme in pollen tubes showed that the distribution of H+-ATPase generally coincides with the shape of the pH gradient: its maximum accumulation is observed in the apical zone.

Pollen Germination and Pollen Tube Growth in Gymnosperms

“…Later, data on the respiration of pine ( Pinus ponderosa) pollen grain suspension were obtained by infrared spectroscopy: in the first 24 h of incubation, CO 2 release was low, after which it increased rather sharply, the most active increase was observed after 48 h of incubation [ 63 ]. For blue spruce ( Picea pungens ), the pollen of which germinates faster than that of pine, oxygen consumption registered with the Clarke electrode increased sharply from 2 to 9 h of incubation and the maximum coincided with the pollen tube appearance; then, during early stages of pollen tube growth, the level of respiration decreased by about 30% [ 64 ]. In the same species, infrared spectroscopy showed that from 8 h of incubation, the release of carbon dioxide increased sharply and almost linearly [ 63 ].…”

“…These data are difficult to compare, since in the last study, starting at 8 h, respiration was measured once a day, and in the first one, the points of 9 and 14 h were compared. However, 8 and 9 h points, respectively, were marked as tipping points in the studies, which appeared to be associated with the moment of the tube emergence [ 64 ].…”

“…In addition to calcium, protons and membrane potential are important for conifer pollen germination. The study of intracellular pH dynamics during pollen activation in blue spruce showed that both pH and membrane potential change after the first cytological signs of germination (namely, breaks in the exine) [ 64 ]: after 9 h of incubation, there is a significant cytoplasm alkalization and membrane hyperpolarization. Further, when the tube appears, the pH stabilizes, and membrane potential increases slightly (in modulus).…”

“…Further, when the tube appears, the pH stabilizes, and membrane potential increases slightly (in modulus). The first change in ion homeostasis revealed to date is the release of anions from pollen grains; by the time exine ruptures appear, it has already been completed [ 64 ]. It is not yet clear how important these changes are, except for the activity of H + -ATPase: P-type ATPase inhibitor orthovanadate blocked pollen germination, indicating significance of the enzyme for germination.…”

“…A variety of ion transport systems, such as Ca 2+ - and K + -conducting channels, anion channels, and H + -ATPase, are involved in maintaining the gradient, according to inhibitory analysis [ 83 ]. Proton pump is also responsible for pH gradient in pollen tube cytoplasm [ 64 ]. The highest pH values were observed in the tube apex ( Figure 5 ), which corresponds to the proton pump location on the plasmalemma and distinguishes the spruce from angiosperm pollen tubes, in which the alkaline zone was located in the subapex, and H + -ATPase was predominantly localized there [ 86 , 87 , 88 ].…”