Yasufumi 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.

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.

Molecular and physiological analysis of Al3+ and H+ rhizotoxicities at moderately acidic conditions

Rhizotoxicities of Al3+ and H+ occur at moderately acidic soil conditions (pH [water] = 5–5.5), especially under conditions of low Ca supply. Al3+ and H+ toxicities predicted to occur at moderately acidic conditions (pH [water] = 5–5.5) in low-Ca soils were characterized by the combined approaches of computational modeling of electrostatic interactions of ions at the root plasma membrane (PM) surface and molecular/physiological analyses in Arabidopsis (Arabidopsis thaliana). Root growth inhibition in known hypersensitive mutants was correlated with computed {Al3+} at the PM surface ({Al3+}PM); inhibition was alleviated by increased Ca, which also reduced {Al3+}PM and correlated with cellular Al responses based on expression analysis of genes that are markers for Al stress. The Al-inducible Al tolerance genes ALUMINUM-ACTIVATED MALATE TRANSPORTER1 and ALUMINUM SENSITIVE3 were induced by levels of {Al3+}PM too low to inhibit root growth in tolerant genotypes, indicating that protective responses are triggered when {Al3+}PM was below levels that can initiate injury. Modeling of the H+ sensitivity of the SENSITIVE TO PROTON RHIZOTOXICITY1 knockout mutant identified a Ca alleviation mechanism of H+ rhizotoxicity, possibly involving stabilization of the cell wall. The phosphatidate phosphohydrolase1 (pah1) pah2 double mutant showed enhanced Al susceptibility under low-P conditions, where greater levels of negatively charged phospholipids in the PM occur, which increases {Al3+}PM through increased PM surface negativity compared with wild-type plants. Finally, we found that the nonalkalinizing Ca fertilizer gypsum improved the tolerance of the sensitive genotypes in moderately acidic soils. These findings fit our modeling predictions that root toxicity to Al3+ and H+ in moderately acidic soils involves interactions between both toxic ions in relation to Ca alleviation.

Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes

Tolerance to soil acidity is an important trait for eucalyptus clones that are introduced to commercial forestry plantations in pacific Asian countries, where acidic soil is dominant in many locations. A conserved transcription factor regulating aluminum (Al) and proton (H⁺) tolerance in land-plant species, STOP1 (SENSITIVE TOPROTON RHIZOTOXICITY 1)-like protein, was isolated by polymerase chain reaction-based cloning, and then suppressed by RNA interference in hairy roots produced by Agrobacterium rhizogenes-mediated transformation. Eucalyptus STOP1-like protein complemented proton tolerance in an Arabidopsis thaliana stop1-mutant, and localized to the nucleus in a transient assay of a green fluorescent protein fusion protein expressed in tobacco leaves by Agrobacterium tumefaciens-mediated transformation. Genes encoding a citrate transporting MULTIDRUGS AND TOXIC COMPOUND EXTRUSION protein and an orthologue of ALUMINUM SENSITIVE 3 were suppressed in transgenic hairy roots in which the STOP1 orthologue was knocked down. In summary, we identified a series of genes for Al-tolerance in eucalyptus, including a gene for STOP1-like protein and the Al-tolerance genes it regulates. These genes may be useful for molecular breeding and genomic selection of elite clones to introduce into acid soil regions.

Enhanced salinity tolerance in transgenic mungbean overexpressing Arabidopsis antiporter (NHX1) gene

Efficient compartmentalization of Na+ ions into the vacuole through heterologous overexpression of vacoular antiporter gene NHX1 is a promising approach to develop salt tolerance in plants. Mungbean (Vigna radiata L. Wilczek) is an important grain legume widely cultivated in Southeast Asia for its protein rich grains. Salinity affects growth and productivity of mungbean. In this paper, we report overexpression of an Arabidopsis NHX1 (AtNHX1) in transgenic mungbean plants conferred enhanced salt tolerance. Cotyledonary node explants were transformed via Agrobacterium tumefaciens mediated transformation using pCAMBIA2301 vector that harbours 35S::AtNHX1 in its T-DNA. Putative transformed plants were selected on kanamycin containing medium. Polymerase chain reaction and Southern blot analysis confirmed the presence, integration and copy number of transgenes in T1 transgenic lines. Reverse transcription-PCR analysis showed higher expression of AtNHX1 in transgenic plants as compared to wild type plants (WT). Under salt stress conditions, T2 transgenic lines displayed less damage and stronger growth phenotypes with concurrent physiological changes as compared to WT. In addition, T2 transgenic lines under salt stress accumulated higher K+/Na+ in the aerial parts and higher [Na+] in roots than WT. Moreover, the T2 transgenic lines showed under NaCl treatment reduced membrane lipid peroxidation and H2O2 and O2− accumulation, higher levels of antioxidant enzyme activity and increased accumulation of proline and ascorbate than WT. These results indicated that the activity of heterologous AtNHX1 protein contributing enhanced salt tolerance in transgenic mungbean.

Overexpression of AtALMT1 in the Arabidopsis thaliana ecotype Columbia results in enhanced Al-activated malate excretion and beneficial bacterium recruitment.

AtALMT1 (Arabidopsis thaliana ALuminum activated Malate Transporter 1) encodes an Arabidopsis thaliana malate transporter that has a pleiotropic role in Arabidopsis stress tolerance. Malate released through AtALMT1 protects the root tip from Al rhizotoxicity, and recruits beneficial rhizobacteria that induce plant immunity. To examine whether the overexpression of AtALMT1 can improve these traits, the gene, driven by the cauliflower mosaic virus 35S promoter, was introduced into the Arabidopsis ecotype Columbia. Overexpression of the gene enhanced both Al-activated malate excretion and the recruitment of beneficial bacteria Bacillus subtilis strain FB17. These findings suggest that overexpression of AtALMT1 can be used as an approach to enhance a plants ability to release malate into the rhizosphere, which can enhance plant tolerance to some environmental stress factors.

Characterization of CcSTOP1; a C2H2-type transcription factor regulates Al tolerance gene in pigeonpea

AbstractMain conclusionAl-responsive citrate-transportingCcMATE1function and its regulation byCcSTOP1were analyzed usingNtSTOP1-KD tobacco- and pigeonpea hairy roots, respectively, CcSTOP1 binding sequence ofCcMATE1showed similarity withAtALMT1promoter. The molecular mechanisms of Aluminum (Al) tolerance in pigeonpea (Cajanus cajan) were characterized to provide information for molecular breeding. Al-inducible citrate excretion was associated with the expression of MULTIDRUGS AND TOXIC COMPOUNDS EXCLUSION (CcMATE1), which encodes a citrate transporter. Ectopic expression of CcMATE1-conferred Al tolerance to hairy roots of transgenic tobacco with the STOP1 regulation system knocked down. This gain-of-function approach clearly showed CcMATE1 was involved in Al detoxification. The expression of CcMATE1 and another Al-tolerance gene, ALUMINUM SENSITIVE 3 (CcALS3), was regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (CcSTOP1) according to loss-of-function analysis of pigeonpea hairy roots in which CcSTOP1 was suppressed. An in vitro binding assay showed that the Al-responsive CcMATE1 promoter contained the GGNVS consensus bound by CcSTOP1. Mutation of GGNVS inactivated the Al-inducible expression of CcMATE1 in pigeonpea hairy roots. This indicated that CcSTOP1 binding to the promoter is critical for CcMATE1 expression. The STOP1 binding sites of both the CcMATE1 and AtALMT1 promoters contained GGNVS and a flanking 3′ sequence. The GGNVS region was identical in both CcMATE1 and AtALMT1. By contrast, the 3′ flanking sequence with binding affinity to STOP1 did not show similarity. Putative STOP1 binding sites with similar structures were also found in Al-inducible MATE and ALMT1 promoters in other plant species. The characterized Al-responsive CcSTOP1 and CcMATE1 genes will help in pigeonpea breeding in acid soil tolerance.

Comparative characterization of aluminum responsive transcriptome in Arabidopsis roots: comparison with other rhizotoxic ions at different stress intensities

ABSTRACT Under abiotic stress conditions, plants actively modify the transcriptome either for activation of stress tolerance mechanisms or adjustment of biological processes for adaptation to the stress. Comparative analysis of transcriptome data under different stressors and stress intensities is useful to understand how plants balance growth and survival under stress conditions. In this study, the transcriptome of Arabidopsis roots was investigated in response to four rhizotoxic stressors, namely aluminum (Al3+), cadmium (Cd2+), and copper (Cu2+) ions and sodium chloride (NaCl), under mild stress (50% inhibition of root elongation compared with the control) and severe stress (>90% inhibition of root elongation). Only Al treatment showed similar transcriptome responses between the stress intensities. The shared Al-specific up-regulated genes included known Al-tolerance genes and downstream genes of SENSITIVE TO PROTON1 (STOP1) such as ALUMINUM ACTIVATED MALATE TRANSPORTER1 and malic enzymes, suggesting that Al-defense mechanisms regulated by STOP1 might be activated over a broader range of Al stress intensities. The sulfur metabolism pathway was activated only under severe Al stress, suggesting that rapid activation of sulfur metabolism is crucial for survival of root cells in the early stage of severe Al stress. A large number of genes associated with the response mechanism to reactive oxygen species production were up-regulated by Cd and Cu treatment under both severe and mild stress, but the members of the genes sets differed between the stress severities. The results indicated that Cd and Cu stress trigger common transcriptional responses, but the responses are likely associated with the stress severity. The transcriptional response to mild NaCl stress was relatively smaller compared with those to the other stressors, suggesting that in Arabidopsis different mechanisms other than rapid transcriptomic responses are involved in the response to NaCl stress. Comparison between Al and the other stressors indicated that the STOP1 regulation system and central metabolic pathways contribute to the similarity of the transcriptional response to different intensities of Al stress. The results suggest that robustness of transcriptionally regulated Al tolerance systems to different stress intensities is important for both of growth and survival in plant roots under Al stress.