Preliminary Functional Analysis Of An Arabidopsis Aluminum Sensitive Mutant Sta1

Aluminium(Al)toxicity is the most important factor limiting crop production in acidic soils worldwide,and studying how plants respond to Al stress is critical for developing Al-tolerant crops.In recent years,researchers have made a number of important progresses in plant Al-resistant mechanisms,especially in the research of Al signal transduction.The mechanisms of plant Al exclusion and symplastic tolerance have also been well investigated.As DNA is an important target of Al toxicity,studies on how Al stress mediates root growth inhibition by inducing DNA damage are nevertheless rarely reported.In order to identity novel genes involved in plant Al response,we constructed an Arabidopsis EMS mutant library,from which we obtained an Al-sensitive mutant,named sta1(sensitive to aluminum1).Al content determination and hematoxylin staining experiments showed that sta1 accumulated more Al in the root tips than WT.Consistent with this,we found that the root malate secretion was significantly reduced in sta1 mutant versus WT.Surprisingly,the expression levels of Al-responsive genes such as ALMT1,ALS3,GDH1 were all comparable in WT and sta1,indicating that the Al-sensitive phenotype of sta1 is to some extent not related to the absence of known Al-resistant mechanisms.By further comparing the RNA-seq from WT and sta1 roots under Al treatment,we found that there are many metabolism-related genes associated with malate homeostasis(such as NADP-ME1,MDH2,GAPDH)and DNA damage response(DDR)related genes(including NAC103 and GMI1 etc.)differentially expressed in the two genotypes.Moreover,under the treatment of DNA damage inducer zeocin,sta1 had a stronger DDR phenotype;ATM inhibitor KU-55933 repressed the expression of DDR-related genes and largely restored the Al-sensitive phenotype of sta1,but could not restore the Al-induced malate secretion.These results indicate that the Al sensitivity of sta1 is attributed to both the reduced malate secretion and constitutively activation of DDR,and that the increased expression of DDR-related genes in sta1 is partially dependent on ATM-SOG1 module.Further genetic analysis revealed that the Al-response phenotype of sta1 is controlled by a single recessive gene.Although whole genome sequencing and BSA analysis failed to identify the target gene,we found that there is no mutation in the known Al-resistance genes and DDR-related genes,suggesting that the mutation in a potential novel and unknown gene,which may be a new negative DDR regulator,controls sta1 Al-sensitive phenotype.In conclusion,we screened an Arabidopsis aluminum-sensitive mutant sta1,and analyzed the aluminum-sensitive phenotype of the mutant from physiological and molecular levels.This study enriched the DDR network activated by Al stress and established the connection between energy metabolism and DDR under Al stress,which may provide a new strategy for cultivating Al-tolerant crops.