ABSTRACT
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.
Subject(s)
Aluminum/toxicity , Arabidopsis Proteins , Arabidopsis , Aluminum/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Glutamate Dehydrogenase , Plant Roots/genetics , Plant Roots/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolismABSTRACT
To identify unknown regulatory mechanisms leading to aluminium (Al)-induction of the Al tolerance gene ALS3, we conducted an expression genome-wide association study (eGWAS) for ALS3 in the shoots of 95 Arabidopsis thaliana accessions in the presence of Al. The eGWAS was conducted using a mixed linear model with 145,940 genome-wide single nucleotide polymorphisms (SNPs) and the association results were validated using reverse genetics. We found that many SNPs from the eGWAS were associated with genes related to phosphatidylinositol metabolism as well as stress signal transduction, including Ca2+signals, inter-connected in a co-expression network. Of these, PLC9, CDPK32, ANAC071, DIR1, and a hypothetical protein (AT4G10470) possessed amino acid sequence/ gene expression level polymorphisms that were significantly associated with ALS3 expression level variation. Furthermore, T-DNA insertion mutants of PLC9, CDPK32, and ANAC071 suppressed shoot ALS3 expression in the presence of Al. This study clarified the regulatory mechanisms of ALS3 expression in the shoot and provided genetic evidence of the involvement of phosphatidylinositol-derived signal transduction under Al stress.
Subject(s)
ATP-Binding Cassette Transporters/physiology , Aluminum/toxicity , Arabidopsis Proteins/physiology , Arabidopsis/genetics , Phosphatidylinositols/metabolism , Plant Shoots/metabolism , Signal Transduction , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Arabidopsis/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Genome-Wide Association Study , Malates/metabolism , Plant Shoots/physiology , Polymorphism, Single Nucleotide/genetics , Stress, Physiological , TranscriptomeABSTRACT
To identify the upstream signaling of aluminum-induced malate secretion through aluminum-activated malate transporter 1 (AtALMT1), a pharmacological assay using inhibitors of human signal transduction pathways was performed. Early aluminum-induced transcription of AtALMT1 and other aluminum-responsive genes was significantly suppressed by phosphatidylinositol 4-kinase (PI4K) and phospholipase C (PLC) inhibitors, indicating that the PI4K-PLC metabolic pathway activates early aluminum signaling. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and PI4K reduced aluminum-activated malate transport by AtALMT1, suggesting that both the PI3K and PI4K metabolic pathways regulate this process. These results were validated using T-DNA insertion mutants of PI4K and PI3K-RNAi lines. A human protein kinase inhibitor, putatively inhibiting homologous calcineurin B-like protein-interacting protein kinase and/or Ca-dependent protein kinase in Arabidopsis, suppressed late-phase aluminum-induced expression of AtALMT1, which was concomitant with the induction of an AtALMT1 repressor, WRKY46, and suppression of an AtALMT1 activator, Calmodulin-binding transcription activator 2 (CAMTA2). In addition, a human deubiquitinase inhibitor suppressed aluminum-activated malate transport, suggesting that deubiquitinases can regulate this process. We also found a reduction of aluminum-induced citrate secretion in tobacco by applying inhibitors of PI3K and PI4K. Taken together, our results indicated that phosphatidylinositol metabolism regulates organic acid secretion in plants under aluminum stress.
