Aluminum (Al) tolerance in Arabidopsis is a genetically complex trait, yet it is mediated by a single physiological mechanism based on Al-activated root malate efflux. We investigated a possible molecular determinant for Al tolerance involving a homolog of the wheat Al-activated malate transporter, ALMT1 . This gene, named AtALMT1 (At1g08430), was the best candidate from the 14-member AtALMT family to be involved with Al tolerance based on expression patterns and genomic location. Physiological analysis of a transferred DNA knockout mutant for AtALMT1 as well as electrophysiological examination of the protein expressed in Xenopus oocytes showed that AtALMT1 is critical for Arabidopsis Al tolerance and encodes the Al-activated root malate efflux transporter associated with tolerance. However, gene expression and sequence analysis of AtALMT1 alleles from tolerant Columbia (Col), sensitive Landsberg erecta (Ler), and other ecotypes that varied in Al tolerance suggested that variation observed at AtALMT1 is not correlated with the differences observed in Al tolerance among these ecotypes. Genetic complementation experiments indicated that the Ler allele of AtALMT1 is equally effective as the Col allele in conferring Al tolerance and Al-activated malate release. Finally, fine-scale mapping of a quantitative trait locus (QTL) for Al tolerance on chromosome 1 indicated that AtALMT1 is located proximal to this QTL. These results indicate that AtALMT1 is an essential factor for Al tolerance in Arabidopsis but does not represent the major Al tolerance QTL also found on chromosome 1.

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.

The Arabidopsis (Arabidopsis thaliana) mutant stop1 (for sensitive to proton rhizotoxicity1) carries a missense mutation at an essential domain of the histidine-2-cysteine-2 zinc finger protein STOP1. Transcriptome analyses revealed that various genes were down-regulated in the mutant, indicating that STOP1 is involved in signal transduction pathways regulating aluminum (Al)- and H⁺-responsive gene expression. The Al hypersensitivity of the mutant could be caused by down-regulation of AtALMT1 (for Arabidopsis ALUMINUM-ACTIVATED MALATE TRANSPORTER1) and ALS3 (ALUMINUM-SENSITIVE3). This hypothesis was supported by comparison of Al tolerance among T-DNA insertion lines and a transgenic stop mutant carrying cauliflower mosaic virus 35S∷AtALMT1. All T-DNA insertion lines of STOP1, AtALMT1, and ALS3 were sensitive to Al, but introduction of cauliflower mosaic virus 35S∷AtALMT1 did not completely restore the Al tolerance of the stop1 mutant. Down-regulation of various genes involved in ion homeostasis and pH-regulating metabolism in the mutant was also identified by microarray analyses. CBL-INTERACTING PROTEIN KINASE23, regulating a major K⁺ transporter, and a sulfate transporter, SULT3;5, were down-regulated in the mutant. In addition, integral profiling of the metabolites and transcripts revealed that pH-regulating metabolic pathways, such as the γ-aminobutyric acid shunt and biochemical pH stat pathways, are down-regulated in the mutant. These changes could explain the H⁺ hypersensitivity of the mutant and would make the mutant more susceptible in acid soil stress than other Al-hypersensitive T-DNA insertion lines. Finally, we showed that STOP1 is localized to the nucleus, suggesting that the protein regulates the expression of multiple genes that protect Arabidopsis from Al and H⁺ toxicities, possibly as a transcription factor.

The gene for mitochondrial citrate synthase (CS) was isolated from Daucus carota ( DcCS ) and introduced into Arabidopsis thaliana (strain WS) using Agrobacterium tumefaciens -mediated transformation. Characteristics of citrate excretion were compared between T3 transgenic plants, which were derived from the initial transgenic plants by self-fertilization and homozygous for DcCS , and the control plants that had no DcCS . The highest CS activity 0.78 µmol protein min ^–1 exhibited by the transgenic plants was about threefold greater than that found in the control plants (0.23–0.28 µmol protein min ^–1 ). Western analysis of the transgenic plants showed two CS signals corresponding to signals obtained from both D. carota and A. thaliana . Thus, it appears that the CS polypeptides by ectopic expression of DcCS were processed into the mature form and localized in the mitochondria of A. thliana . The signal corresponding to the mature form of DcCS were greater in the transgenic plants having higher levels of CS activity. When the transgenic plants were grown in Al-phosphate media, a correlation between the levels of CS activity and the amounts of citrate excreted into the medium. The highest value (5.1 nmol per plant) was about 2.5-fold greater than that from control plants (1.9 nmol per plant). Both growth and P accumulation were greater in transgenic plants with high CS activity than that in control plants when they were grown on an acid soil where the availability of phosphate was low due to the formation of Al-phosphate. It appears that the overexpression of CS in A. thaliana improves the growth in phosphorous limited soil as a result of enhanced citrate excretion from the roots.

Malate transporters play a critical role in aluminum (Al) tolerance responses for some plant species, such as Arabidopsis (Arabidopsis thaliana). Here, we further characterize AtALMT1, an Arabidopsis aluminum-activated malate transporter, to clarify its specific role in malate release and Al stress responses. Malate excretion from the roots of accession Columbia was sharply induced by Al, which is concomitant with the induction of AtALMT1 gene expression. The malate release was specific for Al among rhizotoxic stressors, namely cadmium, copper, erbium, lanthanum, sodium, and low pH, which accounts for the specific sensitivity of a null mutant to Al stress. Al-specific malate excretion can be explained by a combined regulation of AtALMT1 expression and activation of AtALMT1 protein, which is specific for Al. Although low pH treatment slightly induced gene expression, other treatments did not. In addition, malate excretion in Al-activated seedlings was rapidly stopped by removing Al from the solution. Other rhizotoxic stressors were not effective in maintaining malate release. Protein kinase and phosphatase inhibitor studies indicated that reversible phosphorylation was important for the transcriptional and posttranslational regulation of AtALMT1. AtALMT1 promoter-β-glucuronidase fusion lines revealed that AtALMT1 has restricted expression within the root, such that unnecessary carbon loss is likely minimized. Lastly, a natural nonsense mutation allele of AtALMT1 was identified from the Al-hypersensitive natural accession Warschau-1.

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.