| Aluminum toxicity is the most important limiting factor for plant growth and development in acidic soil.It inhibits roots growth and limit its absorption of water and nutrients.In response to aluminum toxicity,plants have evolved both extracellular and intracellular detoxification mechanisms: the former secretes organic acids to chelate extracellular aluminum,and the latter chelates and sequesters the aluminum that enters the cell and regionalizes it into vacuoles.Phosphorus is one of the macronutrient elements necessary for plant growth and various metabolic activities.Studies have shown that aluminum stress can reduce the absorption of phosphorus by plants.So what is the adaptation mechanism of plants to low phosphorus stress caused by aluminum stress? At present,there is no report on the research on the regulation of plant aluminum toxicity response by phosphorus response-related genes.PHR1 is a key transcription factor in low-phosphorus response of the MYB-CC family of plants,and has been reported to regulate the expression of phosphorus starvation genes.In this experiment,it was found that phr1 mutant has a sensitive phenotype under aluminum treatment.Therefore,we use the model plant Arabidopsis as the research material,using plant molecular biology,molecular genetics,plant histochemistry and analytical chemistry to explore what role does PHR1 play in the response of plants to aluminum toxicity.The main contents and results are as follows:1.Loss of PHR1 reduces the aluminum tolerance of plants.Previous studies have shown that Arabidopsis has changes in the expression of low-phosphorus response genes,especially PHR1,which is significantly up-regulated after Al3+ treatment.q-PCR and GUS staining experiments showed that the expression of PHR1 increased significantly after Al3+ stress,which confirmed previous results.In order to focus on whether PHR1 has a certain correlation with the response to aluminum stress,we used T-DNA insertion mutant phr1 and WT to observe the growth phenotype and found that phr1 exhibited a growth-sensitive phenotype with shorter root length under Al3+ stress.Combined with the anion selectivity experiment,it is further confirmed that the phenotype is caused by Al3+ specificity.2.Loss of PHR1 leads to an increase in the accumulation of aluminum in plant cells and a decrease in the phosphorus content in the shoots.In order to analyze the aluminum content of the phr1 and WT,we used hematoxylin staining method to stain the aluminum content of root cell walls,and Morin staining and LMG staining methods were used to stain the aluminum content of root cytoplasm.It was found that hematoxylin staining showed that phr1 cell wall aluminum content was slightly higher than that of WT but not significant,and Morin staining and LMG staining showed that the staining intensity of phr1 root was stronger than WT,and its cytoplasmic aluminum content was higher than WT.Based on this,we speculate that PHR1 can reduce the accumulation of aluminum in the cell after aluminum toxicity.Since PHR1 is a key gene in response to low phosphorus,we analyzed the changes of expression of low phosphorus response genes in phr1 and WT and their phosphorus content determination.The q-PCR results showed that compared with WT,the expression of the transcription factor gene WRKY45,a positive regulation of phosphorus uptake in phr1,and the multi-copper oxidase gene LPR1,which plays an important role in reducing the growth of plant roots in a low-phosphorus environment,increased extremely significantly.Based on this,we speculate that after Al3+ treatment,phr1 is in a phosphorus-deficient state.The above-ground and underground Pi content of WT and phr1 were measured respectively.The results showed that there was no difference in the above-ground and underground phosphorus content of WT before and after Al3+ stress.The above-ground Pi content of phr1 was extremely significantly lower than that of WT.No difference was detected in the underground Pi content of phr1 before and after Al3+ stress.It is speculated that PHR1 may affect the transportation and accumulation of phosphorus in the shoots under aluminum stress.3.No interactions between PHT1;1,ALMT1 and PHR1 in response to plant aluminum toxicity were detected.Since phr1 is in a lower phosphorus state than WT after Al3+ treatment,we used the T-DNA insertion mutant pht1;1 to conduct Al3+ stress growth phenotype experiments to explore PHR1 Is it possible for the downstream PHT1;1 to play a role in the process of Arabidopsis responding to Al3+ poisoning? We found that the growth of pht1;1 and WT,especially the root growth were basically the same,and there was no significant difference in data statistics such as root length and fresh weight.ALMT1 encodes a malate efflux transporter located on the cell plasma membrane,which is critical for plants to respond to aluminum toxicity.At the same time,ALMT1 also played an important role in inhibiting the growth of primary root in response to low phosphorus in plants for root structure remodeling.In the case of aluminum stress and phosphorus deficiency stress,its expression will be induced to up-regulate.In this study,results by q-PCR found that compared with WT,the ALMT1 expression level of phr1 decreased significantly after Al3+ stress,which was nearly 50% lower than that of WT.Therefore,in order to explore whether there may be interactions between ALMT1 and PHR1 in response to aluminum stress,we constructed almt1phr1 double mutant and observed growth phenotype.The results showed that after Al3+ treatment,almt1phr1,almt1,and phr1 all showed growth-sensitive phenotypes,but comparing the sensitivities among the three,no significant differences were found.4.Increased cytoplasmic Pi content helps plants enhance aluminum tolerance;and no interactions between PHR1,ALMT1 and VPT1 in response to plant aluminum toxicity were detected.The members of the vacuolar Pi transporter VPT(also known as PHT5)located on the vacuole membrane perform the function of transporting Pi from the cytoplasm into the vacuole storage.The most critical role for the transport is VPT1,and VPT3 have a "compensation" effect.At the same time,they affect the xylem loading and long-distance transportation of Pi.Therefore,we used mutants to observe the Al3+stress phenotype.It was found that vpt1,vpt2 and vpt3 had no growth phenotype,and vpt1/3 double mutant showed aluminum tolerance phenotype.To explore whether there is a certain interaction between PHR1 and VPT1 in response to Al3+ toxicity,GUS staining was performed on VPT1-GUS/phr1.The results showed that the staining of VPT1-GUS/phr1 with or without Al3+ treatment was consistent with that of VPT1-GUS,and there was no obvious staining change.In addition,in order to explore whether there may be a correlation between the process of Arabidopsis ALMT1 secreting malate in response to Al3+ toxicity and the intracellular Pi content,we constructed almt1vpt1-2 double mutant.It was found that the phenotype of almt1vpt1-2 was basically the same as that of almt1 in the phenotype experiment.In summary,this research found that the PHR1 expression increased significantly in Arabidopsis thaliana after aluminum stress.When the PHR1 is deleted,aluminum tolerance decreases,showing a growth-sensitive phenotype,accompanied by the accumulation of aluminum content in root cells and the decrease of phosphorus content in the shoots.In order to further study the underlying mechanism,we used the transporter materials of plasma membrane location(phosphorus transporter PHT1;1and malate efflux transporter ALMT1)and vacuolar membrane location(vacuolar phosphorus transporter VPTs)to carry out related exploration experiments,and found that the cytoplasm higher phosphorus content is conducive to the enhancement of aluminum tolerance.We explores the physiological mechanism related to the interaction between the aluminum toxicity response and the phosphorus response in plants,in order to provide gene reserves and theoretical guidance for the molecular cultivation of more aluminum-tolerant and phosphorus-efficient crops. |
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