STOP1, a Cys2/His2 type zinc-finger protein, plays critical role in acid soil tolerance in Arabidopsis

Abstract

Under acid soil condition, rhizotoxic ions such as aluminum and protons cause severe yield loss in food and biomass production via inhibition of root growth and/or enhancement of sensitivity to drought stress. Therefore, improvement of crop tolerance to rhizotoxic ions would be an important target for crop breeding, and identification of key genes that regulate tolerance to both aluminum and proton ions should provide a solution to this limitation. We recently isolated a mutant that shows hyper-sensitivity to protons in root growth, namely stop1 (sensitive to proton rhizotoxicity). The stop1 mutant was isolated from an ethyl methanesulfonate mutagenized population by measuring root growth under low pH (pH 4.3). Interestingly, stop1 also shows hypersensitivity to aluminum ion, but not to other rhizotoxic ions. Cloning of the STOP1 gene revealed that it encodes a Cys2/His2 zinc-finger type transcription factor, and a conserved His residue was replaced with Tyr in the predicted amino acid sequence for the mutant gene. This indicates that STOP1 is involved in the signal transduction pathway of proton and aluminum tolerance. Indeed, under acidic condition, stop1 failed to induce the AtALMT1 gene, which encodes one of the major factors for aluminum tolerance. By searching a public database, we have identified genes homologous to STOP1 in rice, maize and some other plants. Thus, the STOP1 family genes may act as a key factor for acid tolerance in a wide variety of plants.

About 40% of world's arable land in developing countries of subtropical and tropical regions of the earth is acidic soil. Under acid soil, the growth of crops is limited because of poor root development by rhizotoxicity caused by ions such as aluminum (Al³⁺) and protons (H⁺).^1–3 Under drought conditions, this would enhance damage of crops because the plant cannot access water in deep soil. Rhizotoxicity can be alleviated by heavy application of lime stone which can neutralize the acidity in soil. However, this is not a practical solution, in particular for local farmers in developing countries because of its high cost. Molecular breeding techniques using DNA markers and/or gene manipulation would be an alternative solution to resolve this limitation.

In this context, several studies have identified key genes that regulate Al³⁺ tolerance.^4–7 For example, a gene encoding Al-activated malate transporter, TaALMT1, was identified using a near isogenic line of wheat, while a gene encoding citrate transporter (Alt_SB) was identified in the major Al-tolerant QTL in sorghum.^6,8 Despite these findings, and even though protons can be considered as one of the major rhizotoxicants in acid soil, the molecular mechanism(s) of plant response against protons had not been identified until we discovered the Arabidopsis stop1 (sensitive to proton rhizotoxicity) mutant.⁹

Using a root-bending assay, we succeeded in isolating the stop1 mutant from an ethyl methanesulfonate-mutagenized population of Arabidopsis as a hypersensitive mutant against proton rhizotoxicity (Fig. 1). Interestingly, the stop1 mutant was also hypersensitive to Al³⁺ ion but not to other rhizotoxic ions, namely cadmium, copper, sodium, manganese and lanthanum ions. Genetic analysis revealed that the stop1 mutant had a single recessive mutation in the gene for the Cys2/His2 type zinc-finger protein that has four zinc-finger domains. A conserved His residue in the first Cys2/His2 zinc-finger domain was replaced by Tyr. This mutation causes dysfunction of STOP1 as the transcriptional factor. As a result, the expression of genes for H⁺ and Al³⁺ tolerance are suppressed in the stop1 mutant even under acid soil conditions. In fact, AtALMT1, which is a key gene for Al tolerance of Arabidopsis, was not induced in the stop1 mutant. This suggests that STOP1 is involved in the signal transduction pathway for Al³⁺ and H⁺ responses as a critical transcriptional regulator (Fig. 2).

Figure 1.

Growth of stop 1 mutant and WT (Col-0) in hydroponic culture at pH 5.5 and 4.7. The growth of stop1 mutant was more sensitive to low pH than that of wild type plants.

Figure 2.

Model for STOP 1 regulation of gene expression in Arabidopsis. STOP1 expression is quite stable under physiological level of H⁺ and Al³⁺ treatments indicating the occurrence of post-translational regulation that activates STOP1, possibly protein phosphorylation. Both Al³⁺ and H+ trigger a typical Al-tolerant gene, AtAlMT1. It was suggested that AtAlMT1 is not involved in H⁺ tolerance by observing the growth of the knock-out mutant. Thus other genes must be induced by STOP1 activation for H⁺ tolerance.

As the expression level of STOP1 is quite stable under physiological Al³⁺ and H⁺ ion concentrations, an activation process such as protein phosphorylation of STOP1 must occur under the acidic condition. This possibility would be supported by a recent study on AtALMT1, in which the involvement of irreversible protein phosphorylation during AtALMT1 induction and expression was revealed.¹⁰ In that study, both H⁺ and Al³⁺ promoted AtALMT1 expression, but this gene is not involved in H⁺ tolerance because its disruption did not affect H⁺ sensitivity. This fact, together with our previous finding of the hyper-sensitivity of the stop1 mutant to H⁺ indicates that other genes may play a critical role in H⁺ tolerance, which is regulated by STOP1. This possibility is supported by a phenotypic clustering analysis of Al³⁺ and H⁺ tolerance among natural accessions, namely JA lines distributed by RIKEN BRC (see. http://www.brc.riken.jp/lab/epd/Eng/species/arabidopsis.shtml), which suggests that the major mechanism in Al³⁺ tolerance and H⁺ tolerance can be distinguished in Arabidopsis.¹¹ It is a promising approach to clarify the mechanism of variation in acid soil tolerance at the molecular level, which may be regulated by the STOP1 system, using these natural accessions.

Our data indicates that STOP1 affects both Al³⁺ and H⁺ tolerance as a key transcriptional factor that regulates acid stress signals. To our knowledge, this is the first report to successfully identify a transcriptional factor that is involved in the signal transduction pathway of Al³⁺ and H⁺ response. A homology search for the highly conserved ZF domain indicates that STOP1 is likely to belong to a small gene family in Arabidopsis. This is often observed for other transcriptional factors including some of the ZF-type transcriptional factors that regulate specific biological systems.^12,13 Interestingly, other plant species such as rice and maize have STOP1 homologues that contain a conserved amino acid sequence in the ZF domain (Fig. 3). A relatively large number of STOP1 family genes in rice may explain its higher acid soil tolerance than Arabidopsis. Further research on this gene family will provide a more holistic picture of plant strategies acquire stress tolerance in acid soil.

Figure 3.

Phylogenic tree of STOP1 homologues in various plant species. Deduced amino acid sequences obtained from mature mRNA or cDNA (accession numbers with AK, NM and NP in NCBI) and those from virtual cDNA (constructed by TIGR cDNA Assemblies project, accession numbers with TA) were used for neighbor-joining phylogenic analysis by CLC Free Workbench ver. 4.0.1. Bootstrap values are indicated each branch. Bar represents 0.5 amino acid substitution per residue.

Addendum to: Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance. Iuchi S, Koyama H, Iuchi A, Kobayashi Y, Kitabayashi S, Kobayashi Y, Ikka T, Hirayama T, Shinozaki K, Kobayashi M. Proc Natl Acad Sci U S A. 2007;104:9900–9905.