STOP1-regulated SMALL AUXIN UP RNA55 (SAUR55) is involved in proton/malate co-secretion for Al tolerance in Arabidopsis.

Proton (H+) release is linked to aluminum (Al)-enhanced organic acids (OAs) excretion from the roots under Al rhizotoxicity in plants. It is well-reported that the Al-enhanced organic acid excretion mechanism is regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1), a zinc-finger TF that regulates major Al tolerance genes. However, the mechanism of H+ release linked to OAs excretion under Al stress has not been fully elucidated. Recent physiological and molecular-genetic studies have implicated the involvement of SMALL AUXIN UP RNAs (SAURs) in the activation of plasma membrane H+-ATPases for stress responses in plants. We hypothesized that STOP1 is involved in the regulation of Al-responsive SAURs, which may contribute to the co-secretion of protons and malate under Al stress conditions. In our transcriptome analysis of the roots of the stop1 (sensitive to proton rhizotoxicity1) mutant, we found that STOP1 regulates the transcription of one of the SAURs, namely SAUR55. Furthermore, we observed that the expression of SAUR55 was induced by Al and repressed in the STOP1 T-DNA insertion knockout (KO) mutant (STOP1-KO). Through in silico analysis, we identified a functional STOP1-binding site in the promoter of SAUR55. Subsequent in vitro and in vivo studies confirmed that STOP1 directly binds to the promoter of SAUR55. This suggests that STOP1 directly regulates the expression of SAUR55 under Al stress. We next examined proton release in the rhizosphere and malate excretion in the T-DNA insertion KO mutant of SAUR55 (saur55), in conjunction with STOP1-KO. Both saur55 and STOP1-KO suppressed rhizosphere acidification and malate release under Al stress. Additionally, the root growth of saur55 was sensitive to Al-containing media. In contrast, the overexpressed line of SAUR55 enhanced rhizosphere acidification and malate release, leading to increased Al tolerance. These associations with Al tolerance were also observed in natural variations of Arabidopsis. These findings demonstrate that transcriptional regulation of SAUR55 by STOP1 positively regulates H+ excretion via PM H+-ATPase 2 which enhances Al tolerance by malate secretion from the roots of Arabidopsis. The activation of PM H+-ATPase 2 by SAUR55 was suggested to be due to PP2C.D2/D5 inhibition by interaction on the plasma membrane with its phosphatase. Furthermore, RNAi-suppression of NtSTOP1 in tobacco shows suppression of rhizosphere acidification under Al stress, which was associated with the suppression of SAUR55 orthologs, which are inducible by Al in tobacco. It suggests that transcriptional regulation of Al-inducible SAURs by STOP1 plays a critical role in OAs excretion in several plant species as an Al tolerance mechanism.

Glucosinolate Catabolism Maintains Glucosinolate Profiles and Transport in Sulfur-Starved Arabidopsis.

Glucosinolates (GSLs) are sulfur (S)-rich specialized metabolites present in Brassicales order plants. Our previous study found that GSL can function as a S source in Arabidopsis seedlings via its catabolism catalyzed by two ß-glucosidases (BGLUs), BGLU28 and BGLU30. However, as GSL profiles in plants vary among growth stages and organs, the potential contribution of BGLU28/30-dependent GSL catabolism at the reproductive growth stage needs verification. Thus, in this study, we assessed growth, metabolic and transcriptional phenotypes of mature bglu28/30 double mutants grown under different S conditions. Our results showed that compared to wild-type plants grown under -S, mature bglu28/30 mutants displayed impaired growth and accumulated increased levels of GSL in their reproductive organs and rosette leaves of before-bolting plants. In contrast, the levels of primary S-containing metabolites, glutathione and cysteine decreased in their mature seeds. Furthermore, the transport of GSL from rosette leaves to the reproductive organs was stimulated in the bglu28/30 mutants under -S. Transcriptome analysis revealed that genes related to other biological processes, such as ethylene response, defense response and plant response to heat, responded differentially to -S in the bglu28/30 mutants. Altogether, these findings broadened our understanding of the roles of BGLU28/30-dependent GSL catabolism in plant adaptation to nutrient stress.

Differences in mineral accumulation and gene expression profiles between two metal hyperaccumulators, Noccaea japonica and Noccaea caerulescens ecotype Ganges, under excess nickel condition.

Here we compare mineral accumulation and global gene expression patterns between two metal hyperaccumulator plants - Noccaea japonica, originating from Ni-rich serpentine soils, and Noccaea caerulescens (ecotype Ganges), originating from Zn/Pb-mine soils - under excess Ni conditions. Significant differences in the accumulation of K, P, Mg, B, and Mo were explained by the expression levels of specific transporters for each mineral. We previously showed that total Ni accumulation in the whole plant is higher in N. caerulescens than in N. japonica. Here we found a similar tendency for Fe under excess Ni; however, the expression of iron-regulated transporter 1 (IRT1), which encodes the primary Fe uptake transporter and causes excess Ni uptake in Arabidopsis thaliana, was higher in N. japonica. NjIRT1 has a point mutation at Asp100, which is essential for Fe transport, and so might lack its Fe and possibly Ni transport function. Noccaea japonica might have lost its IRT1 function, which would prevent excess Ni uptake via IRT1 in Ni-rich soils, and come to rely on other transporters.

High affinity promoter binding of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis.

Malate efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1) and mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum resistance in Arabidopsis thaliana. Several studies showed that AtALMT1 expression in roots is rapidly observed in response to aluminum; this early induction is an important mechanism to immediately protect roots from aluminum toxicity. Identifying the molecular mechanisms that underlie rapid aluminum resistance responses should lead to a better understanding of plant aluminum sensing and signal transduction mechanisms. In this study, we observed that GFP-tagged STOP1 proteins accumulated in the nucleus soon after aluminum treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1 induction were detected in the presence of a protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2). However STOP1 did not regulate Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for early induction. Finally, we report that GDH1 and 2 which are targets of STOP1, are novel aluminum-resistance genes in Arabidopsis.

Expression GWAS of PGIP1 Identifies STOP1-Dependent and STOP1-Independent Regulation of PGIP1 in Aluminum Stress Signaling in Arabidopsis.

To elucidate the unknown regulatory mechanisms involved in aluminum (Al)-induced expression of POLYGALACTURONASE-INHIBITING PROTEIN 1 (PGIP1), which is one of the downstream genes of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) regulating Al-tolerance genes, we conducted a genome-wide association analysis of gene expression levels (eGWAS) of PGIP1 in the shoots under Al stress using 83 Arabidopsis thaliana accessions. The eGWAS, conducted through a mixed linear model, revealed 17 suggestive SNPs across the genome having the association with the expression level variation in PGIP1. The GWAS-detected SNPs were directly located inside transcription factors and other genes involved in stress signaling, which were expressed in response to Al. These candidate genes carried different expression level and amino acid polymorphisms. Among them, three genes encoding NAC domain-containing protein 27 (NAC027), TRX superfamily protein, and R-R-type MYB protein were associated with the suppression of PGIP1 expression in their mutants, and accordingly, the system affected Al tolerance. We also found the involvement of Al-induced endogenous nitric oxide (NO) signaling, which induces NAC027 and R-R-type MYB genes to regulate PGIP1 expression. In this study, we provide genetic evidence that STOP1-independent NO signaling pathway and STOP1-dependent regulation in phosphoinositide (PI) signaling pathway are involved in the regulation of PGIP1 expression under Al stress.

A single-population GWAS identified AtMATE expression level polymorphism caused by promoter variants is associated with variation in aluminum tolerance in a local Arabidopsis population.

Organic acids (OA) are released from roots in response to aluminum (Al), conferring an Al tolerance to plants that is regulated by OA transporters such as ALMT (Al-activated malate transporter) and multi-drug and toxic compound extrusion (MATE). We have previously reported that the expression level polymorphism (ELP) of AtALMT1 is strongly associated with variation in Al tolerance among natural accessions of Arabidopsis. However, although AtMATE is also expressed following Al exposure and contributes to Al tolerance, whether AtMATE contributes to the variation of Al tolerance and the molecular mechanisms of ELP remains unclear. Here, we dissected the natural variation in AtMATE expression level in response to Al at the root using diverse natural accessions of Arabidopsis. Phylogenetic analysis revealed that more than half of accessions belonging to the Central Asia (CA) population show markedly low AtMATE expression levels, while the majority of European populations show high expression levels. The accessions of the CA population with low AtMATE expression also show significantly weakened Al tolerance. A single-population genome-wide association study (GWAS) of AtMATE expression in the CA population identified a retrotransposon insertion in the AtMATE promoter region associated with low gene expression levels. This may affect the transcriptional regulation of AtMATE by disrupting the effect of a cis-regulatory element located upstream of the insertion site, which includes AtSTOP1 (sensitive to proton rhizotoxicity 1) transcription factor-binding sites revealed by chromatin immunoprecipitation-qPCR analysis. Furthermore, the GWAS performed without the accessions expressing low levels of AtMATE, excluding the effect of AtMATE promoter polymorphism, identified several candidate genes potentially associated with AtMATE expression.

STOP1 regulates the expression of HsfA2 and GDHs that are critical for low-oxygen tolerance in Arabidopsis.

The SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor regulates gene expression associated with multiple stress tolerances in plant roots. In this study, we investigated the mechanism responsible for the sensitivity of the stop1 mutant to low-oxygen stress in Arabidopsis. Transcriptomic analyses revealed that two genes involved in low-oxygen tolerance, namely GLUTAMATE DEHYDROGENASE 1 (GDH1) and GDH2, showed lower expression levels in the stop1 mutant than in the wild-type. Sensitivity of the gdh1gdh2 double-mutant to low-oxygen conditions was partly attributable to the low-oxygen sensitivity of the stop1 mutant. Two transcription factors, STOP2 and HEAT SHOCK FACTOR A2 (HsfA2), were expressed at lower levels in the stop1 mutant. An in planta complementation assay indicated that CaMV35S::STOP2 or CaMV35S::HsfA2 partially rescued the low-oxygen tolerance of the stop1 mutant, which was concomitant with recovered expression of genes regulating low-pH tolerance and genes encoding molecular chaperones. Prediction of cis-elements and in planta promoter assays revealed that STOP1 directly activated the expression of HsfA2. Similar STOP1-dependent low-oxygen sensitivity was detected in tobacco. Suppression of NtSTOP1 induced low-oxygen sensitivity, which was associated with lower expression levels of NtHsfA2 and NtGDHs compared with the wild-type. Our results indicated that STOP1 pleiotropically regulates low-oxygen tolerance by transcriptional regulation.