Uncoupling Aluminum Toxicity From Aluminum Signals in the STOP1 Pathway.

Aluminum (Al) is a major limiting factor for crop production on acidic soils, inhibiting root growth and plant development. At acidic pH (pH < 5.5), Al3+ ions are the main form of Al present in the media. Al3+ ions have an increased solubility at pH < 5.5 and result in plant toxicity. At higher pH, the free Al3+ fraction decreases in the media, but whether plants can detect Al at these pHs remain unknown. To cope with Al stress, the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) transcription factor induces AL-ACTIVATED MALATE TRANSPORTER1 (ALMT1), a malate-exuding transporter as a strategy to chelate the toxic ions in the rhizosphere. Here, we uncoupled the Al signalling pathway that controls STOP1 from Al toxicity using wild type (WT) and two stop1 mutants carrying the pALMT1:GUS construct with an agar powder naturally containing low amounts of phosphate, iron (Fe), and Al. We combined gene expression [real-time PCR (RT-PCR) and the pALMT1:GUS reporter], confocal microscopy (pSTOP1:GFP-STOP1 reporter), and root growth measurement to assess the effects of Al and Fe on the STOP1-ALMT1 pathway in roots. Our results show that Al triggers STOP1 signaling at a concentration as little as 2 µM and can be detected at a pH above 6.0. We observed that at pH 5.7, 20 µM AlCl3 induces ALMT1 in WT but does not inhibit root growth in stop1 Al-hypersensitive mutants. Increasing AlCl3 concentration (>50 µM) at pH 5.7 results in the inhibition of the stop1 mutants primary root. Using the green fluorescent protein (GFP)-STOP1 and ALMT1 reporters, we show that the Al signal pathway can be uncoupled from the Al toxicity on the root. Furthermore, we observe that Al strengthens the Fe-mediated inhibition of primary root growth in WT, suggesting an interaction between Fe and Al on the STOP1-ALMT1 pathway.

Root responses to aluminium and iron stresses require the SIZ1 SUMO ligase to modulate the STOP1 transcription factor.

STOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (-Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin-like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1-dependent manner. Moreover, loss-of-function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by Western blot analysis in our sumoylation assay in Escherichia coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway.