Arabidopsis ACBP1 overexpressors are Pb(II)-tolerant and accumulate Pb(II)

Abstract

In our recent paper in the Plant Journal, we demonstrated that Arabidopsis thaliana acyl-CoA-binding protein ACBP1 binds lead [Pb(II)], its mRNA is induced by Pb(II)-treatment and transgenic Arabidopsis overexpressing ACBP1 are conferred Pb(II) tolerance and accumulate Pb(II). Our results suggest that ACBP1 overexpressors are potentially useful for applications in phytoremediation. Since very few plant proteins that bind and accumulate Pb(II) have been identified, our findings provide a feasible method in phytoremediating Pb(II).

Addendum to: Xiao S, Gao W, Chen QF, Ramalingam S, Chye ML. Overexpression of membrane-associated acyl-CoA-binding protein ACBP1 enhances lead tolerance in Arabidopsis. Plant J 2008; doi:10.1111/j.1365-313X.2008.03402.x

Eukaryotic Acyl-Coenzyme-A-Binding Proteins

In eukaroytes, acyl-CoA-binding proteins (ACBPs) that are conserved at the acyl-CoA-binding domain, facilitate the binding of long-chain acyl-CoA esters. The 10-kDa cytosolic form of ACBP was first identified as a neuropeptide from rat brain and was named diazepam-binding inhibitor.1 This 10-kDa ACBP has been reported to function in acyl-CoA transport, in the maintenance of intracellular acyl-CoA pools, and in protection of cytosolic acyl-CoAs from hydrolysis by cellular acyl-CoA hydrolases.2 Later, larger proteins with acyl-CoA-binding domains were reported in A. thaliana,3–5 Caenorhabditis elegans6 and Cryptosporidium parvum.7

Six genes encode ACBPs in Arabidopsis.5 They are membrane-associated ACBP1 and ACBP2, extracellularly-targeted ACBP3, kelch-motif-containing ACBP4 and ACBP5, and 10-kDa ACBP6.5 The acyl-CoA-binding domain in each of these ACBPs binds long-chain acyl-CoA esters in vitro, suggestive of their roles in plant lipid metabolism.3–5,8,9 Structurally, other than the conserved acyl-CoA-binding domain, ankyrin repeats occur in ACBP1 and ACBP2 and kelch motifs in ACBP4 and ACBP5, indicative that they interact with protein partners.5,10 The N-terminal transmembrane domains in ACBP1 and ACBP2 have been demonstrated to function in targeting to the endoplasmic reticulum and the plasma membrane.11,12

Conservation of the Acyl-Coenzyme-A-Binding Domain in Plant ACBPs Confers Ability to Bind Lead

In humans, only one form of ACBP has been characterized and it is a homologue to Arabidopsis 10-kDa ACBP6. Human ACBP, consisting solely of an acyl-CoA-binding domain, has been identified as a molecular target for Pb(II) in vivo and has been suggested to regulate Pb(II) toxicity in humans.13 We were interested to investigate if Arabidopsis ACBPs bind Pb(II) because this heavy metal pollutant, concentrated along food chains, adversely affects the health and development of humans and animals. Given the paucity of Pb(II)-binding proteins identified so far,14 if Arabidopsis ACBPs can be demonstrated to bind and accumulate Pb(II), they would be useful for Pb(II) phytoremediation.

Interestingly, our results showed that Arabidopsis ACBP6, the homologue to human ACBP, bound Pb(II) less well than ACBP1 and ACBP2. This observation may be explained by the occurrence of a multigene family encoding ACBPs in Arabidopsis, which is not the case in man. It may not be surprising that plasma membrane-associated ACBP1 and ACBP2 bind Pb(II) better because their localization allows them to better interact with environmental Pb(II). They may even have evolved towards this function. Further, Arabidopsis ACBP4 or ACBP5, predicted to be localized in the cytosol like ACBP6,9 could possibly bind Pb(II) in the Arabidopsis cytosol.

ACBP1 Overexpressors Accumulate Lead in Shoots

We observed that the expression in root of ACBP1 and ACBP2, but not ACBP6, is induced by Pb(II) nitrate treatment. ACBP1 was subsequently demonstrated to bind Pb(II) comparatively better in in vitro Pb(II)-binding assays. Later, we found that transgenic Arabidopsis lines overexpressing ACBP1 were more tolerant to Pb(II)-induced stress, with significantly higher Pb(II) accumulation in shoots, when compared to wild type. Furthermore, we observed that the acbp1 mutant showed enhanced sensitivity to Pb(II) when germinated and grown in the presence of Pb(II) nitrate, with restoration of tolerance upon complementation using an ACBP1 cDNA. Our findings support a role for ACBP1 in mediating Pb(II) tolerance in Arabidopsis. Our observations of Pb(II) accumulation, rather than Pb(II) extrusion, in the ACBP1-overexpressors suggest that ACBP1 is suitable for Pb(II) phytoremediation.

Lead Phytoremediation

Phytoremediation is an environmentally-friendly, low-cost (solar-driven) and efficient process whereby plants are used in situ to decontaminate the environment.15,16 Transgenic plants that can better tolerate and accumulate heavy metals, would effectively eliminate toxic pollutants from the soil.17 However, many genes currently used for phytoremediation are derived from bacteria or yeast and few originate from plants.18–20

Song et al. (2003) has shown that transgenic Arabidopsis overexpressing the Saccharomyces vacuolar transporter YCF1 are Pb(II)- and Cd(II)-tolerant and accumulate these heavy metals in shoots.14 However, transgenic tobacco overexpressing the calmodulin-binding transporter NtCBP4 were hypersensitive to Pb(II) although they accumulated Pb(II).21 The Arabidopsis ATPase binding cassette (ABC) transporters, AtATM3, AtPDR12 and AtPDR8, confer Pb(II) tolerance in transgenic plants but are deemed unsuitable for applications in phytoremediation as Pb(II) accumulation, a pre-requisite for their eventual harvest and disposal, was lacking in these transgenic lines.22–24

In general, the mechanism of Pb(II)-binding and uptake is much less understood when compared to those of other heavy metals. Many more putative proteins that are functionally associated with other heavy metals have been identified from the Arabidopsis Information Resources database, e.g., 1272 putative proteins for Zn(II), 108 for Cu(II) and 4 for Ni(II).25 Our search of the same database yielded only 1 putative protein, STA1/ATM3 (GenBank Accession: AF286795) that may be associated with Pb(II). Efforts have been made to identify Pb(II)-responsive genes from Sesbania drummondii and 63 such genes showed ≥2.5-fold differential expression in response to Pb(II) treatment.26 Further work on some of these Pb(II)-associated proteins is expected to yield potential candidates that can be harnessed for future applications in phytoremediation.

Conclusions and Perspectives

We suggest that ACBP1 is involved in plasma membrane-associated trafficking of Pb(II) ions into Arabidopsis tissues. Our findings here are consistent with our previous observations that ACBP1 is highly-expressed in Arabidopsis roots on Western blot analysis.3 ACBP1 has been previously proposed to be involved in acyl-CoA transfer originating from the endoplasmic reticulum to the plasma membrane and in the formation of a membrane-associated acyl-CoA pool.3,11,12 ACBP1 may play a role in lipid bilayer membrane repair at the plasma membrane as a consequence of Pb(II)-induced stress, other than being directly associated with Pb(II) trafficking due to its ability to bind Pb(II). The presence of ankyrin repeats in ACBP1 permits it to interact with protein partners in Pb(II) transport and/or lipid bilayer membrane repair. Future experiments need to be carried out to address these issues and to further understand the role of plant ACBPs in relation to Pb(II)-mediated stress.

Addendum to: