The zinc finger transcription factor MtSTOP1 modulates aluminum resistance and low pH tolerance in Medicago truncatula

Abstract In acidic soils, aluminum (Al) is a primary factor that inhibits plant growth. SENSITIVE-TO-PROTON-RHIZOTOXICITY1 (STOP1), which is accumulated in the nucleus in response to Al or low pH, regulates multiple downstream genes to counteract Al toxicity and acid stress. Here, we discovered four STOP1-like proteins in Medicago truncatula, which are localized in the nucleus and display transcriptional activity. Among them, the expression of MtSTOP1 (the ortholog of AtSTOP1) and MtSTOP2 is slightly induced by Al in the root tips. CRISPR/Cas9-mediated knockout of MtSTOP1 resulted in increased Al and low pH sensitivity. Transcriptomic analysis revealed 110 genes that were differentially downregulated in the Mtstop1 compared to the wild-type under both pH 5.0 and pH 5.0 with Al. qRT-PCR analysis confirmed that MtSTOP1 regulates the expression of several Al-induced genes and MtSTOP2. The mutation of MtSTOP4 significantly decreases the expression of specific Al tolerance genes and compromises Al tolerance, yet less severely than the mutation of MtSTOP1. Furthermore, the double mutant Mtstop1Mtstop4 did not exhibit increased Al sensitivity compared to Mtstop1 alone, suggesting that MtSTOP1 and MtSTOP4 likely function in a cooperative rather than additive manner. Additionally, we found that the C-terminal of F-box protein MtRAE1 interacts with both MtSTOP1 and MtSTOP4. Our study offers significant insights into the transcriptional regulatory mechanisms that respond to Al and acid stress in M. truncatula.