Mechanisms Of Boron Alleviating Aluminum Toxicity By Promoting Auxin Transport And Cell-Wall Pectin Endocytosis In Root Apices

Plants not only suffer to aluminum（Al）toxicity,but also face to boron（B）deficiency stress on acid soils of South China.Previous study has displayed that B can reduce Al accumulation in root apical transition zone and alleviate Al-induced inhibition of auxin transport in root apices,so as to ameliorate Al-caused inhibition of root growth,but the underlying mechanisms remain elusive.On the basis of previous research work,we have proposed that B mediate the biosynthesis,exocytosis and the degree of pectin methyl-esterase to alleviate Al accumulation in the root apical transition zone,then alleviate Al-induced inhibition of polar auxin transport in root apices and root growth.In this study,model plants pea（Pisum sativum）and Arabidopsis（Arabidopsis thaliana）were used as experimental materials,and histochemical,biochemical,pharmacological,immunolabelling,molecular biology techniques were used to verify the hypothesis.The main results are as below:1.Boron promoted low-esterified pectin to be transported into vacuoles to reduce Al-caused increase of pectin contents in the root apical transition zone,and to alleviate Al stress-induced inhibition via reducing Al accumulation in the root apical transition zoneAl³⁺marked with morin dye and confocal microscopy were employed toanalyze the effects of B on the distribution of Al in the differential root zones of pea lateral root apices.Results revealed that Al mainly located in the transition zone of root apices,especially cell walls of the epidermal and outer cortical cells in the transition zone,and B could reduce Al accumulation in the transition zone of roots.Moreover,Al-morin signals located in the cytosol were detected,and B-supply enhanced Al-morin signals located in the cytosol,demonstrating that B could promote Al translocation from the cell wall into the cytosol for its internalization in the epidermal and outer cortical cells of the root apical transition zone,and finally to sequestrat Al in vacuoles.Immunolabeling techniques were used to analyze the spatial distribution of pectin and the change of pectin esterification degree in differential root zones of pea lateral root apices.Results showed that Al exposure not only elevated pectin content in pea lateral root tips,especially in the transition zone,but also preferentially deployed them to the cell walls of the epidermal and outer cortical cells.In contrast to JIM7-labled high-esterified pectin,the deployment of JIM5-labled low-esterified pectin was more pronounced in this region.B not only alleviated Al-caused increase of pectin contents in root apical transition zones,but also inhibited Al stress-induced increase of low-esterified pectin.Immunolabeling,pharmacology and fluorescence microscopy technologies were used to analyze the effects of B and Al on the endocytosis of pectin and pectin methyl-esterase（PME）in the transition zone lateral root apices of pea.Results displayed that B promoted low-esterified pectin to be transported into vacuoles to alleviate the repression caused by Al stress,and this process was depended on actin filaments but independent on microtubules.In contrast to JIM5-labeled low methyl-esterified pectin,JIM7-labeled high esterified pectin could not endocytic and enter into the cytosol from the cell-wall of root apical transition zone.Moreover,co-localization experiments displayed that PMEs and activity Al co-localized with low esterified pectin in cytosols,implying the endocytosis of low esterified-pectin might couple with Al and PMEs.All these ultimately reduced Al accumulation in the cell walls of the transition zone and Al trafficking for the internal sequestration in vacuoles.2.Boron alleviated Al stress-induced inhibition of the endocytosis and recycling of PIN2 membrane proteins and Al-caused decrease of auxin contentration in the elongation zone of Arabidopsis root apicesTransgenic DⅡ-VENUS and DR5-GFP lines were used to analyze the dynamic changes of auxin distribution patterns in Arabidopsis root apices.Results demonstrated that Al stress resulted in increased auxin accumulation in root apical meristem zones and transition zones while reducing its contents in elongation zone,implying Al stress interfered with polar auxin transport from the meristem zones to elongation zones of root tips.B could alleviate Al-caused auxin decrease in the elongation zones of root apices to alleviate the toxicity of Al.Transgenic PINs-GFP,AUX1-YFP and PIN2-Dendra2 lines were used to visualize the subcellular distribution and expression levels of auxin transport-related proteins.Results displayed that Al induced the increase of PIN1-GFP,PIN2-GFP and AUX1-YFP signals at the plasma membranes,while had no siginificant effect of PIN3-GFP,PIN4-GFP and PIN7-GFP signals.Al exposure influenced on PIN2 posttranscriptional regulation rather than its expression levels.In contrast to another PINs carriers,Al-induced increase of PIN2 membrane signals mainly via repressing PIN2 endocytosis.The endocytosis and recycling experiments demonstrated that B promoted PIN2 proteins endocytosis and recycling.Co-localization experiments of PIN2 proteins and IAA displayed that IAA transport might depend on the endocytosis and recycling of PIN2 endosomes,and B supply could alleviate Al stress-caused repression of the endocytosis of PIN2 endosomes.In summary,B alleviates Al stress-induced increase of pectin contents and the content and activity of pectin methyl-esterase in the root apical transition zone,and promoted the endocytosis of low esterified-pectin,actived Al and PME to enter into vacuoles for sequestration,finally,reduced Al accumulation in root apices.Moreover,B alleviates Al stress-caused repression of the endocytosis and recycling of PIN2 membrane proteins in the root apical transition zone,and finally promoted polar auxin transport in root apices and ameliorated Al-induced inhibition of root growth.