In 250 and 500 µM treatments, there was no effect on the fresh biomass compared with the control, but the average root length was reduced by nearly 30% and 60%, respectively (Fig. 1). Morphologically, at concentration of 500 µM, Arabidopsis roots were stunted and failed to penetrate the MS medium. In this case, exposure to Ga leads to a common abiotic-stress-induced remodeling of the root system architecture (RSA) characterized by an inhibition of primary root (PR) growth and the simultaneous stimulation of lateral root (LR) formation (Potters et al., 2007; Williams et al., 2011).
This response is also induced following exposure to high concentrations of heavy metals like cadmium, copper, zinc and lead (Pasternak et al., 2005; Potters et al., 2007; Potters et al., 2009) or low concentrations of phosphate (Williamson et al., 2001; López-Bucio et al., 2002; López-Bucio et al., 2003; Desnos, 2008). Several studies have shown the inhibitory effects of other metals on the growth of Arabidopsis. Lequeux et al. (2010) observed a decrease of PR growth from 25 µM Cu2+ with a complete inhibition at 50 µM.
For cadmium, Weber et al. (2006) found that at a Cd2+ concentration of 20 µM caused severe root-growth inhibition and chlorosis of cotyledons. For chromium, Castro et al.
(2007) indicated that treatment with 150 µM of dichromate caused a 50% PR growth inhibition, and primary root growth was totally arrested at 200 µM of dichromate.
Although it seems that the toxicity of Ga is not as strong as other metals, there are the potential risks on ecological environment and human health.
Furthermore, the negative effects of Ga on plant growth may result from bioaccumulation. Most of Ga (60-80%) was sequestered in roots, and the less (20-40%) Ga was transported to shoots. This behavior is similar to other heavy metals that the low accumulation in the aboveground parts of plants could be defined as the principal defense step against heavy metal toxicity (Jun-Yu et al., 2008).
The earliest Ga-toxicity response is the inhibition of root growth, which is associated with gross changes in root morphology and results in inhibited root elongation. Hence, the Ga toxicity study has begun to identify diverse Ga targets in different pathways associated with root growth. Potentially, Ga could exert its harmful effect by interacting with apoplastic (cell wall), plasma membrane, and symplastic (cytosol) targets. A previous study has shown that up to 99% of the total Ga binds to the cell wall in giant algal cells of Chara coralline (Reid et al., 1996), suggesting that the cell wall is an important site for binding of Ga due to its high density of negative charges. The binding of Ga in the cell wall might somehow lead to the inhibition of root elongation in plants. To investigate whether apoplast plays an important role in Ga toxicity, we will further use nano secondary ion mass spectrometry (NanoSIMS) as a subcellular imaging tool to analyze the Ga micro-distibution in Arabidopsis.
Lipid peroxidation is an indicator of membrane damage caused by abiotic stress such as metal toxicity, which can be indirectly measured by MDA formation. Cell membrane damage generally results from reactive oxygen species (ROS) production because ROS is likely to attack on membrane phospholipids, which leads to lipid peroxidation (Ma et al., 2013). The data showed that Ga induced the lipid peroxidation in root, suggesting that Ga might interfere the charges at the surface of plasma membrane and then alter the activities of nearby ions. In addition, this interaction can modify the structure of the plasma membrane as well as the ion channel/pumps in the surface of the cell. These structural modifications can lead to disturbances of ion-transport processes, which subsequently perturb cellular homeostasis.
Our data showed that exposure to Ga can inhibit the accumulation of Fe.
Fe-limited culture media did not significantly alter the toxic response to Ga in plants,
wild-type plants. Then, we monitored the gene expression patterns about iron uptake genes under Ga-treated conditions. The relative expression levels of AtIRT1 and AtFRO2 in 250 and 500 µM Ga treatments were inhibited and significantly lower than
untreated control seedlings. In short, these data reported here demonstrate that in Fe-sufficient condition, Ga inhibits the acquisition of Fe by down-regulating the IRT1 expression, but the IRT1 transporter is not associated with Ga uptake.
It was reported that there is a great similarity in chemical properties between Ga and Fe, which enables Ga to function as an Fe analogue and consequently perturbs cellular Fe homeostasis (Downs, 1993). However, unlike Fe, Ga is unable to undergo redox cycling, so such substitution often leads to the inactivation of the Fe-dependent biomolecules targets (Chitambar et al., 1988). Previous studies have shown that Ga could exert its toxic effect by interfering with the metabolism of Fe in microbes including prokaryotes such as Pseudomonas fluorescens, Escheriehia coli and Mycobacterium tuberculosis (Hubbard et al., 1986; Al-Aoukaty et al., 1992; Olakanmi
et al., 2000), as well as the yeast Saccharomyces cerevisiae (Lesuisse and Labbe, 1989;
Lesuisse et al., 2001). Jakupec and Keppler (2003) reported that in animal studies Ga3+
competed with Fe3+ for binding sites on Fe3+-dependent enzymes such as ribonucleotide reductase thus interfered with cellular DNA replication. In addition, Ga inhibited vacuolar type proton-translocating ATPase as well as Na+-K+-ATPase. However, there was no information available on how Ga perturbs the Fe signaling or the uptake response in Arabidopsis roots. In this study, results show the decreased activity of the two Fe deficiency signaling pathways FIT and PYE. It is implies that Ga perturbs the upstream molecule(s) and interferes the perception of Fe deficiency. .
In Arabidopsis, the physiological mechanisms of Al resistance might provide a
parallelism model system for studies of Ga-toxicity, because of the similar chemical properties between Ga and Al as well as their close toxic effects on the yeast cells.
Currently, Al-activated exudation of organic acid (OA) anions from root apices is the best documented and characterized plant Al-tolerance mechanism. In the rhizosphere, the released OAs from roots such as citrate, malate and oxalate can interact with Al ion to form non-toxic complexes (Delhaize et al., 1993; Delhaize and Ryan, 1995; Kochian et al., 2004; Kinraide et al., 2005; Delhaize et al., 2007). In Arabidopsis, it was reported that AtALMT1-mediated malate exudation and AtMATE-mediated citrate exudation evolved independently to confer Al tolerance (Liu et al., 2009). In the present study, Ga stress rapidly induced the expression of AtALMT1 and AtMATE in the root. It seems that Ga could induce the secretion of organic acids such as citrate and malate, which might be characterized as plant Ga-tolerance mechanism. In general, the chelation of cations such as Al3+, Fe3+, and Ca2+ by tricarboxylates (citrate3−) are stronger than dicarboxylates (malate2−, oxalate2−, malonate2−) and monocarboxylates (acetate−) (Table 7). Therefore, we assumed that the tricarboxylate citrate3- anion might be a better Ga3+ chelator than the dicarboxylate malate2- ion (Ryan et al., 2001). In addition, the toxicity of both Al and Ga ions can be reversed by using citrate in yeast (Ritchie and Raghupathi, 2008).
Here, we demonstrated that supplying exogenous citrate significantly increased Ga tolerance in Arabidopsis and might promote Ga transport from roots to shoots.
In this study, the exogenous application of citrate to the medium containing toxic levels of Ga was shown to protect root growth and reduce Ga toxicity. Ga uptake of Arabidopsis was decreased with the increasing concentration of exogenous citrate.
These results imply that the Ga-citrate complexes may be nonabsorbable or taken up slower than Ga ion in the root. Blocking the entry of excess amounts of metals into
withstand toxic levels of Ga. Additionally, the data also showed that supplying exogenous citrate in some way promoted Ga transport from roots to shoots. The physiological mechanisms underlying this phenomenon are still unclear. Instead of using root sequestration for metal detoxification, the presence of high levels of citrate in plant tissues may increase soluble Ga concentrations within the plant by formation of soluble Ga-citrate, allowing its movement from roots to shoots. This distribution pattern caused by supplying exogenous citrate is thought to be one of the most important characteristics of heavy metal hyperaccumulators. Nowadays, there has been appreciable interest in the use of plants or plant products as a green technology to address recalcitrant environmental contaminants. This technology, termed phytoremediation, uses plants to extract heavy metals from the soil and to concentrate them in the harvestable shoot tissue (Salt et al., 1998; McIntyre, 2003). A potential way for Ga-contaminated soil can be remediated by the application of citrate.
Metal tolerance in plants is generally achieved in two distinct ways, exclusion of excess metals from roots and intracellular tolerance of excess endogenous metals (Clemens, 2001; Lin and Aarts, 2012). From a practical point of view, the secretion of citrate is a mechanism by which Arabidopsis avoids Ga toxicity. To characterize the root exudation of citrate or other organic acids into the rhizosphere in response to excess Ga, the Ga-induced exudation need to be collected and analyzed in the future study. On the other hand, internal detoxification is one of the Ga resistance mechanisms, which involved Ga chelation in the cytosol and subsequent storage of the Ga-ligand complexes in the vacuole. The distribution such as in cytosol or in vacuoles and the form such as free ion and/or complex also need to be addressed in future studies.
Furthermore, the discovery that plant can accumulate Ga to high levels in the shoot by supplying citrate, showing us a way to investigate the physiological aspect of Ga xylem
loading in Arabidopsis. Since metals move acropetally via the xylem fluid (White et al., 1981), it is reasonable to study chemical species and metal complexation in xylem.
Table 7. Formation constants between some commonly exuded organic anions (L) with various cations (M). Except for protons, the values are for a ratios L:M of 1:1 derived in zero ionic strength media at 25oC (Ryan et al., 2001).
Ligands
Metals Citrate2- Oxalat2- Malate2- Malonate2- Fumarate2- Acetate -H+ (M:L)
1:1 6.40 4.27 5.10 5.70 4.49 4.76
2:1 4.76 1.25 3.46 2.85 3.05
3:1 3.13
Al3+ 9.6f 6.1c 5.4g 5.7h - 1.51c
12.3h 6.53h 6.0h
Fe3+ 11.5a,e 7.74d 7.1a,e 7.52b - 3.38a,e
Ca2+ 4.68 3.0e 2.66 2.35 2.0 1.18
Cu+ 5.9a,e 6.23 3.42a,e 5.7 2.51 2.22
Zn2+ 4.98a,e 4.87 2.93a,e 3.84 - 1.57
Mn2+ 4.15a 3.95 2.24a 3.28 0.99a 1.4
Symbols: a0.1 or 0.16M, b0.5M, c1.0M, d3.0M ionic strength; e18oC or 20oC; fFrom (9) ; gFrom (12) ;
hFrom (65) at 31oC. From Martell and Smith (1974) or as shown.