Ó Springer 2005
Plant Growth Regulation (2005) 46:45–50 DOI 10.1007/s10725-005-6324-2
Eﬀects of heavy metals on seed germination and early seedling growth of Arabidopsis thaliana Weiqiang Li1,2, Mohammad A. Khan3, Shinjiro Yamaguchi1,* and Yuji Kamiya1 1
RIKEN Plant Science Center, Suehiro-cho-1-7-22, Tsurumi-ku Yokohama, Kanagawa 230-0045, Japan; Shijiazhuang Institute of Agricultural Modernization, Chinese Academy of Science, Shijiazhuang 050021, P.R. China; 3Department of Botany, University of Karachi, Karachi 75270, Pakistan; *Author for correspondence (e-mail: [email protected]
; phone: +81-45-503-9663; fax: +81-45-503-9662) 2
Received 17 November 2004; accepted in revised form 25 April 2005
Key words: Arabidopsis thaliana, Heavy metal, Seed germination
Abstract Seed is a developmental stage that is highly protective against external stresses in the plant life cycle. In this study, we analyzed toxicity of essential (Cu2+ and Zn2+) and non-essential heavy metals (Hg2+, Pb2+ and Cd2+) on seed germination and seedling growth in the model species Arabidopsis. Our results show that seedling growth is more sensitive to heavy metals (Hg2+, Pb2+, Cu2+ and Zn2+) in comparison to seed germination, while Cd2+ is the exception that inhibited both of these processes at similar concentrations. To examine if toxicity of heavy metals is altered developmentally during germination, we incubated seeds with Hg2+ or Cd2+ only for a restricted period during germination. Hg2+ displayed relatively strong toxicity at period II (12–24 h after imbibition), while Cd2+ was more eﬀective to inhibit germination at period I (0–12 h after imbibition) rather than at period II. The observed diﬀerences are likely to be due in part to selective uptake of diﬀerent ions by the intact seed, because isolated embryos (without seed coat and endosperm) are more sensitive to both Hg2+ and Cd2+ at period I. We assessed interactive toxicity between heavy metals and non-toxic cations, and found that Ca2+ was able to partially restore the inhibition of seedling growth by Pb2+ and Zn2+.
Introduction Some heavy metals, such as Cu, Zn and Ni, are essential micronutrients for plants, but are toxic to organisms at high concentrations (Munzuroglu and Geckil 2002). Plants are sometimes exposed to non-essential heavy metals, including Hg2+, Cd2+ and Pb2+, that are present in soil and water naturally or as contaminants from human activities. Recent work has started to uncover molecular mechanisms underlying the uptake and transport of some heavy metal species (Howden et al. 1995; Blaudez et al. 2003; Song et al. 2003). However, it remains to be further investigated how essential
and non-essential metal ions aﬀect plant growth at diﬀerent developmental stages under varying environmental conditions. Seed is a stage in the plant life cycle that is well protected against various stresses. However, soon after imbibition and subsequent vegetative developmental processes, they become stress-sensitive in general. Therefore, seeds are thought to carefully monitor such external parameters as light, temperature and nutrient in order to maintain the protective state until external conditions become favorable for following developmental processes (Karssen 1982; Pritchard et al. 1993; Bungard et al. 1997). Although such critical
46 regulatory mechanisms are likely to operate in seeds at the onset of imbibtion, little is known about how stress tolerance is modulated at different phases of germination. Using transcriptomics and proteomics approaches, molecular and biochemical events in imbibed Arabidopsis seeds have recently been analyzed in detail (Gallardo et al. 2001, 2002; Ogawa et al. 2003; Yamauchi et al. 2004). Accumulating knowledge on the regulation of dormancy and germination in this species will allow us to examine how stress tolerance is modiﬁed in imbibed seeds in response to diﬀerent signals. As a ﬁrst step to understand how heavy metals aﬀect the ability of seed germination, we have examined the toxicity of essential (Cu2+ and Zn2+) and non-essential metal ions (Pb2+, Cd2+ and Hg2+) in imbibed Arabidopsis seeds at diﬀerent developmental stages. We have also assessed interactive toxicity between heavy metals and nontoxic cations during germination and seedling growth.
solutions in a 3.5-cm Petri dish. Petri dish were sealed and incubated under continuous white light at 22 °C. When test solutions were changed during incubation, the seeds were collected in a 1.5-ml tube, rinsed with ddH2O, and then washed twice with the new test solution. The seeds were then incubated on ﬁlter papers as described above. Germination tests were carried out using triplicate samples (each containing 50– 60 seeds). Seeds were scored as germinated when the breakage of seed coat was visible. Seedling development was regarded as being inhibited 6 d after imbibition if the seed coat was visibly broken (germination), but the embryo did not grow further. Seed coats were removed carefully using a forceps within 30 min after imbibition on wet ﬁlter papers. The isolated embryos were incubated on ﬁlter papers without or with heavy metals (0–12 h or 12–24 h) under continuous white light. When metal ions were treated for the ﬁrst 12 h, seed coats were removed in the presence of the metal ion.
Materials and methods Results Plant materials and chemicals Arabidopsis thaliana ecotype Columbia (Col-0) was used as wild-type in this study. To obtain seeds for germination tests, plants were grown on soil under continuous white light at 22 °C. Harvested seeds were stored at room temperature with 30% relative humidity for 6 months before the start of germination experiments. Heavy metals (provided as chloride salts) were purchased from Nacalai Tesque Inc. (Tokyo, Japan). All solutions were made in doubly distilled water (ddH2O), and ddH2O was used as a control treatment.
Germination tests Dry seeds were washed with 0.02% Triton-X solution, rinsed with water twice (doubly-distilled water was used throughout this study), and then washed with the test solution twice (Yamaguchi et al. 1998). The seeds were placed on doublelayered ﬁlter papers (3 mm, Whatman, Maidstone, UK) wetted with 0.7 ml ddH2O or test
The eﬀect of heavy metals on seed germination and seedling growth Figure 1a shows the eﬀect of heavy metals on germination of after-ripened Arabidopsis seeds under continuous white light at 22 °C. As reported for other plant species, Cu2+, Pb2+ and Zn2+ were not very toxic for seed germination in Arabidopsis (ID50 [50% inhibition] is >50 mM). In comparison, Hg2+ (ID50 = 0.7 mM) and Cd2+ (ID50 = 6 mM) inhibited germination at much lower concentrations. In the presence of heavy metals at certain concentrations, the radicle protruded from testa, but the embryo growth was arrested beyond this point. We examined the arrest of seedling growth by heavy metals 6 d after imbibition, while germination (breakage of seed coat) was scored at 2 d. Figure 1b illustrates that the inhibition of seedling growth occurs at lower concentrations of Cu2+, Pb2+, Zn2+ and Hg2+ that did not inhibit germination. In contrast, Cd2+ exhibited toxicity to seed germination and seedling growth at similar concentrations. Interestingly, two of the essential metals used in this study
47 Heavy metal toxicity on dissected embryos
(a) Seed germination (2 d) (%)
Cu2+ Hg2+ 25
(b) Seedling growth (6 d) (%)
Tissues covering the embryo, such as testa and aleurone (endosperm), may play an important role in protecting the embryo from heavy metal toxicity. To examine this hypothesis, seed coats were mechanically removed shortly (30 min) after imbibition, and the dissected embryos were incubated with heavy metals under continuous white light at period I (0–12 h) or period II (12–24 h) (Figure 3a). In contrast to intact seeds (Figure 2), isolated embryos were more sensitive to Hg2+ at period I (Figure 3b), suggesting a protective role of surrounding tissues against Hg2+ at period I. Other heavy metals, with the exception of Cu2+, were more toxic to the growth of isolated embryos at period I (Figure 3b). Generally, isolated embryos were much more sensitive to heavy metals than intact seeds (Figures 1–3).
0.4 0.6 (mM)
Figure 1. Seed germination (a) and seedling growth (b) of Arabidopsis in the presence of various heavy metals. (a) Seeds were scored as germinated when the breakage of seed coat was visible at 2 d after imbibition. (b) Seedling growth was regarded as being arrested if the seed germinated, but the embryo did not grow further within 6 d after imbibition. For clarity, the results are shown in two separate graphs with diﬀerent concentration ranges in each panel. As a comparison, Cd2+ in (a) and Pb2+ in (b) are shown in both graphs.
(Cu2+ and Zn2+) were not eﬀective to inhibit germination, but did cause strong inhibition of seedling growth at relatively low concentrations (Figure 1b).
Heavy metal toxicity is altered developmentally during germination To examine if sensitivity to heavy metals changes developmentally during seed germination, the seeds were incubated with metal ions at period I (0–12 h) or period II (12–24 h) under continuous white light (Figure 2a). As shown in Figure 2b, seeds were signiﬁcantly more sensitive to Hg2+ at period II than period I, while Cd2+ was evidently more eﬀective in inhibiting germination at period I than at period II.
Interactive eﬀects of non-toxic cations and heavy metals The toxicity of heavy metals to seed germination and seedling growth is known to be aﬀected by other environmental factors, such as pH and availability of other nutrients (Kjar et al. 1998). But, the eﬀect of non-toxic cations, such as Ca2+, Mg2+, K+, Na+, on the toxicity of heavy...