Different heavy metals have various effects on Picea wilsonii pollen germination and tube growth

Abstract

Heavy metal pollution has became one of the realistic matters of globality. Previous reports indicated that heavy metals could significantly inhibit pollen germination and tube growth. In the present study, comparative studies on the effects of different heavy metals (As, Hg, Cd, Cr and Cu) on in-vitro picea wilsonii pollen gernimation and tube growth were carried out. Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As showed the most toxic effects on pollen germination, which was followed by Hg and Cd, while Cr and Cu showed relatively lower toxicity. Besides, pollentubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape were observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes showed numerous vacuole-like acidic organelles, though cytoplasmic vacuolization were also observed in pollen tubes treated with Hg and A. In brief, our data indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in-vitro pollen culture might be used as a competent system for biomonitoring of air pollution.

During the past few decades, heavy metal pollution has became one of the realistic matters of globality. Therefore, the effects of heavy metals on both animal and plant system have received considerable attention. It was reported that pollen grains of cross-pollination species were easy to accumulate high levels of heavy metals during their trips through the polluted air, and thus were proposed as a biomonitor of heavy metal pollution.^1,2 Given that pollen grains of a large number of species can steadily germinate on a simple medium, and that heavy metals, such as Hg (mercury),^3-5 Pb (plumbum),⁶ and Cd (cadmium)^7,8 significantly inhibited pollen germination and tube growth, even at very low concentration, it was reasonable for us to speculate that in-vitro pollen germination and tube growth might be a more competent system for biomonitoring of air pollution. To further validate this speculation, the effects of different heavy metals, including Na₂HAs₄O.12H₂O, HgCl₂, CdCl₂, CrCl₃, and CuCl₂, on in-vitro picea wilsonii pollen gernimation and tube growth were carried out in the present study.^6,9

Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As (arsenic), which is technically not a true metal, but a semi-metal, showed the most toxic effects on P. wilsonii pollen tube development. In fact, the germination rate of pollen grains cultured in standard germination medium for 20 h was nearly 75%. While the data of 2 μM As-treated samples was only 58.6% (p < 0.01). Previous reports indicated that Hg^3-5 and Cd,^8,10-12 strongly inhibited pollen germination and tube growth. In the present study, the germination rates of samples treated with 5 μM Hg and 10 μM Cd were 61.2% and 61.9%, respectively (p < 0.01), confirming the high toxicity of the 2 metals on pollen tube development. Gur and Topdemir¹⁰ reported that, when compared with Cd and Hg, Cu (copper) had the least effects on pollen germination and tube growth of Cydonia oblonga M and Prunus domestica L. Besides, Speranza et al.¹³ reported that effects of Cr (chromium) on pollen tubes of kiwifruit could be observed when 25 or 50 μM Cr were used. Here, our data indicated that, more than 60% pollen grains could still germinate when were treated with as high as 20 μM chromium and copper, respectively (p < 0.01), indicating that Cr and Cu did have relatively lower toxicity.

What more interesting, though pollen grains treated with different concentration of metals (2 μM As, 5 μM Hg, 10 μM Cd, 20 μM Cr, and 20 μM Cu) had similar germination rate, pollen tubes showed varying shapes in response to different heavy metal stress. As shown in Fig. 1, pollen tubes treated with Cd (Fig. 1B), As (Fig. 1D), and Hg (Fig. 1F) were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation, which were seldon observed in untreated tubes (Fig. 1A). These data is similar with previous studies,^8,11 revealing the metal-induced disruption of tip growth in pollen tubes. On the other hand, though diameters of tubes treated with Cu or Cr slightly increased, neither obvious vacuoles, nor swelling tips could be observed in these tubes (Fig. 1C and E), indicating the maintaining of the tip growth in these samples.

Figure 1.

Effects of metals on the tube morphology. Pollen tubes were cultured in standard medium (A), or treated with 10 μM CdCl₂ (B), 20 μM CuCl₂ (C), 2 μM Na₂HAs₄O.12H₂O (D), 20 μM CrCl₃ (E), and 5 μM HgCl₂ (F), respectively. DIC (Differential Interference Contrast) pictures were obtained using a ZEISS Axiovert 200 M microscope equipped with a Q imaging RETIGA-SRV CCD. Bars = 10 μm.

In the previous report, our data indicated that Cd markdly induced the formation of acidic vacuoles in pollen tubes.⁸ To further elucidate the possible relation between metal-induced cytoplasmic vacuolization and acidic organelles, Lyso-Tracker Green was utilized according to previous studies.^8,14 Confocal observation indicated that Lyso-Tracker in untreated tubes was mainly accumulated in some small isolated punctate structures, indicating the existence of some small vacuoles with acid hydrolase (Fig. 2A). Similar results were observed in tubes treated with Cu (Fig. 2C) and Cr (Fig. 2E), indicating that both of the 2 metals did not significantly influence the quantity and distribution of acidic organelles in pollen tubes. On the other hand, numerous vacuole-like organelles with various diameters were labeled with Lyso-Tracker in Cd-treated tubes (Fig. 2B). While hardly any signal were detected in pollen tubes treated with As (Fig. 2D) and Hg (Fig. 2F), though cytoplasmic vacuolization were usually observed in both samples. These data indicated that Cd, but not all kinds of metals, promoted the formation of acidic vacuoles in pollen tubes. We might further speculate that pollen tubes might have multiform mechanisms to survive different metal stress. Given the mutisource of vacuole biogenisis,¹⁵ further researches on the mechanism of metal-induced cytoplasmic vacuolization were required.

Figure 2.

Effects of metals on the formation of acid organelles. Pollen tubes were cultured in standard medium (A), or treated with 10 μM CdCl₂ (B), 20 μM CuCl₂ (C), 2 μM Na₂HAs₄O.12H₂O (D), 20 μM CrCl₃ (E), and 5 μM HgCl₂ (F), respectively, for 20 h. All samples were stained with 10 μM Lyso-Tracker Green (Invitrogen, final concentration) for 30 min, and then were examined using a Zeiss 5 Live laser scanning confocal microscope, with an excitation wavelength of 488 nm and emitted wavelength of BP 494–555 nm. Bars = 10 μm.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by Excellent Talent Training Project of Beijing (Grant No. 2012D005016000006), and the Natural Science Foundation of China (Grant No. 31371387).