Intersection of two signalling pathways: extracellular nucleotides regulate pollen germination and pollen tube growth via nitric oxide

Abstract

Plant and animal cells release or secrete ATP by various mechanisms, and this activity allows extracellular ATP to serve as a signalling molecule. Recent reports suggest that extracellular ATP induces plant responses ranging from increased cytosolic calcium to changes in auxin transport, xenobiotic resistance, pollen germination, and growth. Although calcium has been identified as a secondary messenger for the extracellular ATP signal, other parts of this signal transduction chain remain unknown. Increasing the extracellular concentration of ATPgammaS, a poorly-hydrolysable ATP analogue, inhibited both pollen germination and pollen tube elongation, while the addition of AMPS had no effect. Because pollen tube elongation is also sensitive to nitric oxide, this raised the possibility that a connection exists between the two pathways. Four approaches were used to test whether the germination and growth effects of extracellular ATPgammaS were transduced via nitric oxide. The results showed that increases in extracellular ATPgammaS induced increases in cellular nitric oxide, chemical agonists of the nitric oxide signalling pathway lowered the threshold of extracellular ATPgammaS that inhibits pollen germination, an antagonist of guanylate cyclase, which can inhibit some nitric oxide signalling pathways, blocked the ATPgammaS-induced inhibition of both pollen germination and pollen tube elongation, and the effects of applied ATPgammaS were blocked in nia1nia2 mutants, which have diminished NO production. The concurrence of these four data sets support the conclusion that the suppression of pollen germination and pollen tube elongation by extracellular nucleotides is mediated in part via the nitric oxide signalling pathway.

Fig. 1.

Pollen germination and pollen tube elongation are inhibited by ATPγS but not AMPS. (A) In vitro germination of Arabidopsis WS pollen treated with various concentrations of ATPγS or AMPS. (B) In vitro germination of Medicago truncatula pollen with various concentrations of ATPγS or AMPS. (C) Arabidopsis WS pollen was germinated in vitro, and, after pollen tubes were visible (1–2 h), various concentrations of ATPγS or AMPS were added and the growth rate of the pollen tubes was measured. Different letters above the bars indicate values that are significantly different from each other (P ≤0.05). Error bars are ±SE.

Fig. 2.

NO signalling agonists promote the effects of ATPγS on pollen germination, and antagonists block the effects of ATPγS on pollen germination and elongation. Different classes of NO signalling agonists and antagonists and various concentrations of ATPγS were added to Arabidopsis WS pollen prior to germination. (A) Water-soluble NO signalling agonists (1 μM Viagra, 20 μM NONOate, 100 μM DibcGMP) shifted the concentration needed for inhibition of germination from 100 μM ATPγS to 50 μM. (B) DMSO-soluble NO signalling agonists (20 μM IBMX, 20 μM SNAP) shifted the concentration needed for inhibition of germination from 100 μM ATPγS to 50 μM. (C) ODQ (100 μM), a DMSO-soluble NO signalling antagonist, reverses pollen germination inhibition by ATPγS. Each agonist or antagonist was used at a concentration that by itself did not affect pollen germination. (D) ODQ also reverses the ATPγS inhibition of WS pollen tube elongation. Treatments were added to growing pollen tubes. Control is PGM+0.5% DMSO, ATPγS concentration is 100 μM+0.5% DMSO and ODQ concentration is 100 μM, which by itself had no effect on pollen tube growth. Different letters above the bars indicate values that are significantly different from each other (P ≤0.05). Error bars are ±SE.

Fig. 3.

Effects of ATPγS on intracellular NO levels, pollen germination, and pollen tube growth in Col-0 wild-type and nia1nia2 pollen, (A) DAF-2D and PGM+0.5% DMSO, 250 μM ATPγS, or 250 μM AMPS were added to elongating pollen tubes, and images were taken using confocal laser microscopy. The emission with added ATPγS or AMPS was compared to the control with DAF-2D only at the 20 min time point. Only in wild-type plants did the addition of ATPγS caused a statistically significant increase in fluorescence (n ≥20). (B) Col-0 wild-type pollen germination is inhibited by ATPγS, while nia1nia2 pollen germination is unchanged by ATPγS. Control is PGM+0.5% DMSO, ATPγS concentration is 500 μM+0.5% DMSO. (C) Col-0 wild-type pollen tube elongation is inhibited by ATPγS, while nia1nia2 pollen tube elongation is promoted by ATPγS. Control is PGM+0.5% DMSO, ATPγS concentration is 250 μM+0.5% DMSO. Different letters above the bars indicate values that are significantly different from each other (P ≤0.05). Error bars are ±SE.

Fig. 4.

Model for eATP signalling in pollen. eATP causes increased cytoplasmic calcium levels which binds to and activates calmodulin (CaM). NO can be produced via nitric oxide synthase and/or nitrate reductase which can be activated by binding the calcium/calmodulin complex. NO binds to and activates guanylate cyclase which converts GTP into cGMP. The cGMP mediates specific cellular responses such as pollen germination and elongation, and is then broken down by phosphodiesterases. NO signalling agonists and antagonists used in this study are indicated in italics.