Mechanisms Of Cell Cycle Checkpoint Regulator ATR In Aluminium-induced Root Growth Inhibition In Arabidopsis

Acid-soil regions comprise up to 30%of the world's arable land,and aluminum(Al)toxicity is the most severely factor limiting crop growth.Many studies show that aluminum can interact with the cell wall,cell membrane,intracellular components and many other sites,but the role of aluminum and which sites are the most important causes of Al toxicity is still controversial.Previous research has found that the mutation of ATR gene can enhance the resistance to aluminum toxicity,and save the defect of als3 in aluminum toxicity sensitivity.ATR functions as a cell cycle check point is responsible for monitoring single-strand DNA damage and replication fork.Then the cell cycle by activating the switch mechanism to block the cell cycle to the next stage,so that the cells have enough time to repair DNA damage,which ensures the integrity of the genome.Due to the irresponsive to DNA damage stress of atr,cell division and root growth are not inhibited,resulting mutants show more resistant to aluminum toxicity.Meanwhile,the study also indicated that DNA damage induced by aluminum is the main mechanism of aluminum toxicity,but this hypothesis does not adequately explain the phenomena and mechanisms of aluminum toxicity that have been mentioned.To further confirm the mechanism of aluminum resistance of atr,we selected five aluminum sensitive mutants als3,star1,stop1,almtl,alsl lines respectively making a cross with atr line to construct double mutants.Then analyze the phenotypes of each double mutants of aluminum resistance to reveal the upstream and downstream relationships genetically between atr and the sensitive mutants and to prove that atr can save the sensitive mutant of aluminum toxicity in phenotype.The main results are as follows:(1)The experiment based on root growth revealed that atr can not only recover als3 but also star1 resistance to aluminum toxicity.Evans blue staining showed that lowering the degree of cell membrane damage in double mutants reduced which proved that atr can resume als3,starl resistance to aluminum toxicity.However,by measuring the aluminum content in the double mutant star1atr,we found that intracellular accumulation of aluminum has not decreased,and quantitative PCR showed that the expression of anti-aluminum genes between star1atr and WT has no significance.It indicated that atr does not recover als3,star1 resistance to aluminum toxicity through excluding the aluminum actively or up-regulate the anti-aluminum toxicity mechanism.Since atr may have both als3 and star1 repaired,the double mutant starlals3 and the single mutants star1,als3 have the same sensitivity to aluminum toxicity,and did not show an additive effect.atr can restore double mutant star1als3 resistance to aluminum toxicity to the wild-type levels,further indirect verified that ALS3 and STAR1 can interact with each other,and located on the same pathway.(2)By measuring the relative root length we found that with the presence of A1 the root length of almt1atr,stop1 atr and the single mutant almtl,stop1 have no differences,indicating that atr can't restore the aluminum toxicity of almt1 and stopl.Evans blue staining revealed that the double mutant almtlatr,stop1 atr have a greater degree of cell membrane damage.Compared with almtl,stop1,the double mutant have no slightly relief,which also illustrates atr can't save almtl,stop1 sensitivity to aluminum toxicity.Using ICP-MS for aluminum content of each material,found that the double mutant almt1atr,stopl atr have no differences with the corresponding single mutants almtl,stop1.The result has consistent with atr can't restore the aluminum toxicity sensitive of almtl,stop1.ALMT1 mainly through transport organic acid to the extracellular for chelating and relieving aluminum toxicity,and STOP1 mainly by regulating the expression of ALMT1 to mediate resistance to aluminum toxicity.Therefore ALMT1,STOP1 mainly through external detoxicification mechanism to detoxify aluminum.atr can't restore the resistant ability of almt1 and stop1 for aluminum toxicity,indicate that it's not possible for atr to deal with external detoxicification defect.(3)Phenotype analysis and Evans blue staining experiment indicate that atr can recover the aluminum resistance ability of alsl.ALS1 encodes an ABC transporter which localized at tonoplast.Thus,we come to a conclusion that atr have the ability to restore the internal detoxicification defect.Moreover,it remains us that the aluminum sensitive phenotype of alsl may due to DNA damage.(4)To sum up,we can draw a conclusion that atr can alleviate internal aluminum toxicity but give no effect to external aluminum toxicity.In addition,our previous view on how STAR1/ALS3 modify the cell wall to resistance aluminum toxicity is incomplete.In this research,we find atr can restore the aluminum-resistance ability of both star1 and als3,this result shows that internal DNA damage is also an important reason why starl and als3 are sensitive to aluminum.