Figure 1.
Effects of cold acclimation on Camellia sinensis pollen germination and tube growth.
A, Typical serial course of pollen germination at 25°C and 4°C. Pollen germination was inhibited by cold acclimation. B, Typical time courses of mean pollen tube extension growth at 25°C and 4°C. Pollen tube elongation was also inhibited by cold acclimation. All data present means of three replicates ± SD. Means with different letters are significantly different at P<0.05.
Figure 2.
Effects of exogenous DEA NONOate on pollen germination and tube growth.
Pollen germination and tube length were determined after 2 h incubation in 25°C under different concentrations of DEA NONOate, with or without 200 μM cPTIO or 300 μM L-NNA. All data present means of three replicates ± SD. Means with different letters are significantly different at P<0.05.
Figure 3.
Effects of L-NNA, cPTIO, and DEA NONOate on pollen germination and tube growth under cold acclimation.
Pollen germination and tube length were determined after incubating pollen grains from Camellia sinensis for 1–3 h in the control solution alone or containing 300 μM L-NNA, 200 μM cPTIO or 25 μM DEA NONOate, respectively in cold acclimation. All data present means of three replicates ± SD. Means with different letters are significantly different at P<0.05.
Figure 4.
Detection of NO levels in pollen tubes using DAF-FM DA as fluorescent indicator.
Bar = 20 μm. Pollen grains of Camellia sinensis were incubated for 2 h in the control medium alone (A, E) containing 200 μM cPTIO (B, F), 300 μM L-NNA (C, G) or 50 μM DEA NONOate (D, H), under 25°C condition (A–D) and under cold stress (E–H), respectively. Then pollen grains grown on different treatments were loaded with 20 μM DAF-FM DA for 20 min in dark before excess fluorophore was washed out. After loading with indicator, images were acquired by CLSM Leica TCSSL. The corresponding bright field images are shown after the fluorescence images.
Figure 5.
Mean relative intensities of fluorescence from NO specific indicator DAF-FM DA in germinated pollen tubes.
Fluorescence images acquired from CLSM as above described (Figure 4) were analyzed and mean relative intensities of fluorescence were calculated using Image J. A–H shows the corresponding mean relative intensities of fluorescence in pollen tubes treated as above (Figure 4). Data are the means of three replicates ± SD. Means with different small letters are significantly different at P<0.05.
Figure 6.
Cold-induced increase of NO in Camellia sinensis pollen tubes as detected by EPR.
Pollen grains of Camellia sinensis were incubated for 2 h in the control medium alone (A, E) containing 200 μM cPTIO (B, F), 300 μM L-NNA (C, G) or 25 μM DEA NONOate (D, H), under 25°C condition (A–D) and under cold stress (E–H), respectively. Then pollen tubes grown on different treatments were harvested and NO was detected by EPR using the spin trap Fe(II)(DETC)2 as described in ‘Materials and Methods’. I showed typical spectra of Fe(II)(DETC)2 itself. J showed typical spectra of NO-Fe(II)(DETC)2 from 25 μM DEA NONOate. All the representative spectra are from three independent measurements.
Figure 7.
Effect of cold stress and L-NNA on activities of NOS-like enzyme reaction in pollen tubes.
Pollen grains from Camellia sinensis were incubated under control and cold conditions for 2 h with or without 300 μM L-NNA, then the pollen tubes were collected and grounded for crude enzyme preparation according to Zhao et al. [15] with slight modification. For the detection of NOS-like activity, samples were added to a reaction mixture containing the substrate and all cofactors of the NOS reaction, and were incubated for 10 min at 37°C. NO production from NOS-like activity was detected by EPR at 0, 5 and 10 min, respectively. A, NO production from crude extracts of pollen tubes incubated at 25°C. B, NO production from crude extracts of pollen tubes treated by 300 μM L-NNA at 25°C. C, NO production from crude extracts of pollen tubes incubated at 4°C. D, NO production from crude extracts of pollen tubes treated by 300 μM L-NNA at 4°C. E showed the mean relative EPR signal height of NO from NOS-like activity, and data are the means of three replicates ± SD. Means with different letters are significantly different at P<0.05 (E). All the representative spectra are from three independent measurements and were obtained by the accumulation of five recordings.
Figure 8.
Effects of Viagra and ODQ on pollen germination and tube growth in the presence of exogenous NOS inhibitor L-NNA, NO scavenger cPTIO or NO donor DEA NONOate under cold acclimation.
Pollen germination and tube length were determined after 2 h incubation. The concentrations of reagents were as follows, 80 μM Viagra, 200 μM cPTIO, 300 μM L-NNA, 50 μM ODQ and 25 μM DEA NONOate. Data are the means of three replicates ± SD. Means with different letters are significantly different at P<0.05.
Figure 9.
Effects of DEA NONOate and L-NNA on intracellular cGMP level in Camellia sinensis pollen tubes after 2 h cold treatment.
The concentrations of reagents were as follows 25 μM DEA NONOate, 50 μM ODQ and 300 μM L-NNA. Data are the means of three replicates ± SD. Means with different letters are significantly different at P<0.05.
Figure 10.
Effects of cGMP on the regulation of Pro accumulation.
The concentrations of reagents were as follows 80 μM Viagra, 50 μM ODQ, 300 μM L-NNA, 200 μM cPTIO and 25 μM DEA NONOate. All data present means of three replicates ± SD. Means with different letters are significantly different at P<0.05.
Figure 11.
Hypothetical model showing the potential NO signaling events that participate in Camellia sinensis pollen tube tip growth under cold stress.
This simplified model was based on the models proposed by Wang et al. [24]. Cold stress induces an increase in NO through the accumulation of NOS-like activity followed by GC stimulation resulting in cGMP enhancing. As a consequence, the proline (Pro) is stimulated. Together, these pathways lead to tip growth contraction. Black arrows indicate the links established in the induction of pollen tube development; broken arrows represent indirect or still undescribed pathways in pollen tube tip growth. NOS, nitric oxide synthase; GC, guanylyl cyclase; cGMP, cyclic guanosine monophosphate; PDE, phosphodiesterase; P5CS, delta 1-pyrroline-5-carboxylate synthase; P5CR, Pyrroline-5-carboxylate reductase; ProDH, Pro dehydrogenase; ⊥, inhibition.