Yuriko Kobayashi

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

Acid soil syndrome causes severe yield losses in various crop plants because of the rhizotoxicities of ions, such as aluminum (Al3+). 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 Cys2His2-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 Al3+ 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 Al3+ treatment in the stop1 mutant. These results indicate that STOP1 plays a critical role in Arabidopsis tolerance to major stress factors in acid soils.

STOP1 Regulates Multiple Genes That Protect Arabidopsis from Proton and Aluminum Toxicities

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.

Characterization of AtALMT1 Expression in Aluminum-Inducible Malate Release and Its Role for Rhizotoxic Stress Tolerance in Arabidopsis

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.

Comparative transcriptomic characterization of aluminum, sodium chloride, cadmium and copper rhizotoxicities in Arabidopsis thaliana

BackgroundRhizotoxic ions in problem soils inhibit nutrient and water acquisition by roots, which in turn leads to reduced crop yields. Previous studies on the effects of rhizotoxic ions on root growth and physiological functions suggested that some mechanisms were common to all rhizotoxins, while others were more specific. To understand this complex system, we performed comparative transcriptomic analysis with various rhizotoxic ions, followed by bioinformatics analysis, in the model plant Arabidopsis thaliana.ResultsRoots of Arabidopsis were treated with the major rhizotoxic stressors, aluminum (Al) ions, cadmium (Cd) ions, copper (Cu) ions and sodium (NaCl) chloride, and the gene expression responses were analyzed by DNA array technology. The top 2.5% of genes whose expression was most increased by each stressor were compared with identify common and specific gene expression responses induced by these stressors. A number of genes encoding glutathione-S-transferases, peroxidases, Ca-binding proteins and a trehalose-synthesizing enzyme were induced by all stressors. In contrast, gene ontological categorization identified sets of genes uniquely induced by each stressor, with distinct patterns of biological processes and molecular function. These contained known resistance genes for each stressor, such as AtALMT1 (encoding Al-activated malate transporter) in the Al-specific group and DREB (encoding dehydration responsive element binding protein) in the NaCl-specific group. These gene groups are likely to reflect the common and differential cellular responses and the induction of defense systems in response to each ion. We also identified co-expressed gene groups specific to rhizotoxic ions, which might aid further detailed investigation of the response mechanisms.ConclusionIn order to understand the complex responses of roots to rhizotoxic ions, we performed comparative transcriptomic analysis followed by bioinformatics characterization. Our analyses revealed that both general and specific genes were induced in Arabidopsis roots exposed to various rhizotoxic ions. Several defense systems, such as the production of reactive oxygen species and disturbance of Ca homeostasis, were triggered by all stressors, while specific defense genes were also induced by individual stressors. Similar studies in different plant species could help to clarify the resistance mechanisms at the molecular level to provide information that can be utilized for marker-assisted selection.

Amino Acid Polymorphisms in Strictly Conserved Domains of a P-Type ATPase HMA5 Are Involved in the Mechanism of Copper Tolerance Variation in Arabidopsis

Copper (Cu) is an essential element in plant nutrition, but it inhibits the growth of roots at low concentrations. Accessions of Arabidopsis (Arabidopsis thaliana) vary in their tolerance to Cu. To understand the molecular mechanism of Cu tolerance in Arabidopsis, we performed quantitative trait locus (QTL) analysis and accession studies. One major QTL on chromosome 1 (QTL1) explained 52% of the phenotypic variation in Cu tolerance in roots in a Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred population. This QTL regulates Cu translocation capacity and involves a Cu-transporting P1B-1-type ATPase, HMA5. The Cvi allele carries two amino acid substitutions in comparison with the Ler allele and is less functional than the Ler allele in Cu tolerance when judged by complementation assays using a T-DNA insertion mutant. Complementation assays of the ccc2 mutant of yeast using chimeric HMA5 proteins revealed that N923T of the Cvi allele, which was identified in the tightly conserved domain N(x)6YN(x)4P (where the former asparagine was substituted by threonine), is a cause of dysfunction of the Cvi HMA5 allele. Another dysfunctional HMA5 allele was identified in Chisdra-2, which showed Cu sensitivity and low capacity of Cu translocation from roots to shoots. A unique amino acid substitution of Chisdra-2 was identified in another strictly conserved domain, CPC(x)6P, where the latter proline was replaced with leucine. These results indicate that a portion of the variation in Cu tolerance of Arabidopsis is regulated by the functional integrity of the Cu-translocating ATPase, HMA5, and in particular the amino acid sequence in several strictly conserved motifs.

Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis

Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake.

SENSITIVE TO PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2, and Other Transcription Factors Are Involved in ALUMINUM-ACTIVATED MALATE TRANSPORTER1 Expression

A set of unexpected transcription factors affects complex regulatory control of AtALMT1 expression in response to Al stress. In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5′ truncated promoters of different lengths showed that the promoter region between –540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around –297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5′ untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression.

Relative abundance of Δ5-sterols in plasma membrane lipids of root-tip cells correlates with aluminum tolerance of rice

We investigated variations in aluminum (Al) tolerance among rice plants, using ancestor cultivars from the family line of the Al-tolerant and widely cultivated Japonica cultivar, Sasanishiki. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. These two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the plasma membrane (PM) and greater Al uptake within 1 h of Al treatment. This could not be explained by organic acid release. Lipid composition of the PM differed between these cultivars, and may account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 had a lower ratio of phospholipids to Delta(5)-sterols than the sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitors of Delta(5)-sterol synthesis to alter the ratio of phospholipids to Delta(5)-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku. In addition, Rikuu-132 showed a similar level of Al sensitivity when the ratio of phospholipids to Delta(5)-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14alpha-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.

STOP2 Activates Transcription of Several Genes for Al- and Low pH-Tolerance that Are Regulated by STOP1 in Arabidopsis

The zinc-finger protein STOP1 (sensitive to proton rhizotoxicity 1) regulates transcription of multiple genes critical for tolerance to aluminum (Al) and low pH in Arabidopsis. We evaluated the contributions of genes that are suppressed in the stop1 mutant to Al- and low pH-tolerance using T-DNA-inserted disruptants, and transgenic stop1 mutants expressing each of the suppressed genes. STOP2, a STOP1 homolog, partially recovered Al- and low pH-tolerance by recovering the expression of genes regulated by STOP1. Growth and root tip viability under proton stress were partially rescued in the STOP2-complemented line. STOP2 localized in the nucleus and regulated transcription of two genes (PGIP1 and PGIP2) associated with cell wall stabilization at low pH. GUS assays revealed that STOP1 and STOP2 showed similar cellular expression in the root. However, the expression level of STOP2 was much lower than that of STOP1. In a STOP1 promoter::STOP2-complemented line, Al tolerance was slightly recovered, concomitant with the recovery of expression of ALS3 (aluminum sensitive 3) and AtMATE (Arabidopsis thaliana multidrug and toxic compound extrusion), while the expression of AtALMT1 (aluminum-activated malate transporter 1) was not recovered. These analyses indicated that STOP2 is a physiologically minor isoform of STOP1, but it can activate expression of some genes regulated by STOP1.

Characterization of the Complex Regulation of AtALMT1 Expression in Response to Phytohormones and Other Inducers

Complex transcriptional response of AtALMT1 malate transporter could account for its contribution to pleiotropic traits. In Arabidopsis (Arabidopsis thaliana), malate released into the rhizosphere has various roles, such as detoxifying rhizotoxic aluminum (Al) and recruiting beneficial rhizobacteria that induce plant immunity. ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) is a critical gene in these responses, but its regulatory mechanisms remain unclear. To explore the mechanism of the multiple responses of AtALMT1, we profiled its expression patterns in wild-type plants, in transgenic plants harboring various deleted promoter constructs, and in mutant plants with defects in signal transduction in response to various inducers. AtALMT1 transcription was clearly induced by indole-3-acetic acid (IAA), abscisic acid (ABA), low pH, and hydrogen peroxide, indicating that it was able to respond to multiple signals, while it was not induced by methyl jasmonate and salicylic acid. The IAA-signaling double mutant nonphototropic hypocotyls4-1; auxin-responsive factor19-1 and the ABA-signaling mutant aba insensitive1-1 did not respond to auxin and ABA, respectively, but both showed an Al response comparable to that of the wild type. A synthetic microbe-associated molecular pattern peptide, flagellin22 (flg22), induced AtALMT1 transcription but did not induce the transcription of IAA- and ABA-responsive biomarker genes, indicating that both Al and flg22 responses of AtALMT1 were independent of IAA and ABA signaling. An in planta β-glucuronidase reporter assay identified that the ABA response was regulated by a region upstream (−317 bp) from the first ATG codon, but other stress responses may share critical regulatory element(s) located between −292 and −317 bp. These results illustrate the complex regulation of AtALMT1 expression during the adaptation to abiotic and biotic stresses.