Zinc finger protein STOP1 is critical for proton tolerance in Arabidopsis and coregulates a key gene in aluminum tolerance

Abstract

Acid soil syndrome causes severe yield losses in various crop plants because of the rhizotoxicities of ions, such as aluminum (Al(3+)). Although protons (H(+)) could be also major rhizotoxicants in some soil types, molecular mechanisms of their tolerance have not been identified yet. One mutant that was hypersensitive to H(+) rhizotoxicity was isolated from ethyl methanesulfonate mutagenized seeds, and a single recessive mutation was found on chromosome 1. Positional cloning followed by genomic sequence analysis revealed that a missense mutation in the zinc finger domain in a predicted Cys(2)His(2)-type zinc finger protein, namely sensitive to proton rhizotoxicity (STOP)1, is the cause of hypersensitivity to H(+) rhizotoxicity. The STOP1 protein belongs to a functionally unidentified subfamily of zinc finger proteins, which consists of two members in Arabidopsis based on a Blast search. The stop1 mutation resulted in no effects on cadmium, copper, lanthanum, manganese and sodium chloride sensitivitities, whereas it caused hypersensitivity to Al(3+) rhizotoxicity. This stop1 mutant lacked the induction of the AtALMT1 gene encoding a malate transporter, which is concomitant with Al-induced malate exudation. There was no induction of AtALMT1 by Al(3+) treatment in the stop1 mutant. These results indicate that STOP1 plays a critical role in Arabidopsis tolerance to major stress factors in acid soils.

Fig. 1.

Selection of an Arabidopsis thaliana mutant STOP. (A) Growth of Col-0 and stop1 mutant seedlings on various pHs in a root bending assay. Pregrown seedlings were transferred to various pH gelled media and grown upside down. (B) Growth of mutant and WT (Col-0) in hydroponic culture at pH 4.7 and 5.5. Hydroponic culture can enhance rhizotoxicity to a greater degree than a gelled medium. (Scale bar: 1 cm.) (C) Segregation of mutant phenotype among F₂ population derived from a cross between the mutant (Col-0 background) and Ler-0. Arrows indicate the mutant phenotype.

Fig. 2.

Positional cloning of the STOP1 gene and overall domain structure of the STOP1 gene. (A) Schematic representation of the STOP1 region on chromosome I. Numbers on the upper of the chromosome diagrams indicated the distance (in megabases) from the top of the chromosome. The number of recombination events detected in the F₂ progeny crossed with Ler are shown in the lower part of the chromosome diagrams. STOP1 was located on bacterial artificial chromosome clone F7P12. (B) Schematic representation of the overall domain structure and stop1 mutation. The four ZFs (ZF1–ZF4) are indicated. The position of the stop1 mutation and T-DNA insertion of SALK_114108 are also indicated. Asterisks indicate conserved motif of ZF.

Fig. 3.

Complementation test for pH hypersensitivity of the stop1 mutation. WT (Col-0), stop1 (stop1 mutant), STOP1-KO (SALK_114108), and stop1-comp-1 and -2 (transgenic stop1 mutant carrying a CaMV35s-driven WT STOP1 gene) were grown hydroponically at pH 5.5 and 4.7 for 7 days. Mean ± SE of relative root length (%) (pH 4.7/pH 5.5) are shown (n = 5). Asterisks indicate significant difference from stop1 and STOP1-KO (Student's t test, P < 0.05).

Fig. 4.

Multiple alignment of potential ZFs of the STOP1 gene and homologues. Homologues were identified from A. thaliana (At5g22890), Oryza sativa (CT832156) and Z. mays (AY106636) by TBlastN search. Horizontal bars indicate ZFs and asterisks indicate conserved motif of Cys₂His₂ or Cys₂His₂-Cys. The arrow head shows the mutation point of stop1. Conserved amino acids are shaded dark, and the residues that have a positive Blosum62 score (43) are shaded light.

Fig. 5.

Root growth of Col-0 (black bar, WT), stop1 mutant (gray bar), and T-DNA insertion line of the STOP1 gene (white bar; SALK_114108, STOP1-KO) with various rhizotoxic ions in hydroponic culture. Seedlings were grown for 7 days in low-ionic strength nutrient solution that can enhance rhizotoxicity of ions. Seedlings were grown in test solutions containing 3.5 μM CdCl₂, 1.0 μM CuCl₂, 1.0 μM LaCl₃, 8.0 mM NaCl, 100 μM MnSO₄ at pH 5.5, or 4.0 μM AlCl₃ (pH 5.0). Means of relative root length (%) (toxic solution/nontoxic solution) ± SE are shown (n = 5). Asterisks indicate the significant difference from WT (Student's t test, P < 0.05).

Fig. 6.

Response of the stop1 mutation to pH and Al rhizotoxicities. (A) Root growth of the stop1 mutant, homozygous transgenic lines carrying a T-DNA insertion in AtALMT1 (AtALMT1-KO; SALK_009629) and a parental accession Col-0 (WT) with various pH and Al treatments. Seedlings were grown hydroponically for 7 days in a test solution in the presence or absence of 2 μM AlCl₃ at various pH. Mean ± SE values are shown (n = 5). Asterisks indicate the significant difference from root growth at pH 5.5 (Student's t test, P < 0.05). (B) Complementation test for Al hypersensitivity of the stop1 mutation. WT (Col-0), stop1 (stop1 mutant), STOP1-KO (SALK_114108), and stop1-comp-1 and -2 (transgenic stop1 mutant carrying CaMV35s-driven WT STOP1 gene) were grown in hydroponic culture in the presence or absence of 2 μM AlCl₃ at pH 5.5 for 7 days. Mean ± SE values of relative root length (%) (+Al/−Al) are shown (n = 5). Asterisks indicate significant difference between stop1 and STOP1-KO (Student's t test, P < 0.05).

Fig. 7.

Al responsive malate excretion and AtALMT1 expression in the stop1 mutation. (A) The 5-day-old roots of aseptically grown seedlings were incubated in malate collection medium with 10 μM AlCl₃ (solid bar) or without Al (open bar) at pH 5.0 for 24 h. Malate exudation was determined independently from three samples. Mean ± SE values are shown. (B) Analysis of AtALMT1 expression by RT-PCR, using specific primers for AtALMT1. UBQ1 expression is shown as a control.

Fig. 8.

Analysis of STOP1 gene expression by RT-PCR, using specific primers for the STOP1 gene. Col-0 seedlings were grown for 7 days in control medium (pH 5.0) then incubated for 24 h in various conditions (pH 5.0, 4.7, or 4.4 and 4 or 10 μM AlCl₃ at pH 5.0). STOP1 and UBQ1 expression level in the roots was determined by semiquantitative RT-PCR.