Ameliorative Role And Mechanism Of Boron On Aluminum Toxicity In Trifoliate Orange Roots

Aluminum?Al?is the most abundant element and accounts for about 8%of the total earth crust.Approximately,40%of the world and 21%of China's cultivated land are acid soils,which is severely affected by Al toxicity.Aluminum toxicity is a major factor limiting crop growth and production in acid soils?p H?5?.The initial and most easily recognized symptom of Al toxicity is on limiting root growth,and inhibiting root absorption of water and nutrients,thus reducing crop yield.Boron?B?is an essential microelement for normal growth and development of higher plants.Boron-deficiency and Al toxicity have similar negative effects on root growth,and cell wall is considered to be the main site of B-deficiency and Al toxicity.In recent years,it has been widely reported that B could alleviate Al toxicity in several plants,which is mainly aimed at annual crops,and the research on Al toxicity of perennial plants,especially citrus is relatively less.In China,citrus is an important fruit species and cultivated mainly in acid soils,where Al toxicity accompanied by B-deficiency are common problems in citrus orchards.Trifoliate orange[Poncirus trifoliata?L.?Raf.]is a considered dominant rootstock of citrus in China and is more sensitive to B-deficiency.Keeping this in view,the research was conducted in hydroponics using trifoliate orange rootstock seedlings.By utilizing fluorescence vital staining,fourier transform infrared spectroscopy?FTIR?,¹³C-nuclear magnetic resonance spectroscopy(¹³C-NMR),X-ray diffraction?XRD?,non-target metabolomics?GC-TOF-MS?,X-ray photoelectron spectroscopy?XPS?,scanning/transmission electron microscope-energy dispersive x-ray spectrometer?SEM/TEM-EDS?,and atomic force microscope?AFM?,which to analyze the effects of B on Al-induced root injury,changes in root metabolites and metabolic pathway,enzymatic and non-enzymatic defense system responses,alterations in root cell wall characteristics and structure,distributions of Al in root cell wall components,Al absorption and transportation in root tips of seedlings.The main results are summarized as follows:1 Effects of B on the metabolites and metabolic pathways in roots of trifoliate orange rootstock under Al stressThere were obvious differences in the metabolites content under different B and Al treatments.In this study,60 metabolites with a matching degree of more than 70%were identified and analyzed,including 20 amino acids,17 sugars,12 organic acids,5fatty acids,2 aromatic compounds and 4 other substances.The 17 amino acids and 8sugars were up-regulated in Al-treated roots,while significant down-regulation of 3amino acids?aspartic acid,isoleucine,and glutamic acid?and 6 sugars were indicated under Al stress.Aluminum-induced a decrease of 9 organic acids,especially pyruvic acid,L-malic acid,citric acid,succinic acid,and fumaric acid in the TCA cycle,by98.2%,93.6%,60.4%and 78.6%,respectively.Interestingly,in the presence of Al,B application reduced the 10 amino acids such as asparagine,cycloleucine,citrulline and histidine,as well as 6 sugars such as inositol,raffinose,galactose and 3,6-anhydro-d-galactose.However,there was no obvious difference in the organic acids contents in Al-stressed roots treated with B.Conclusively,our results showed that Al toxicity changed the metabolism of amino acids and carbohydrates and inhibited the TCA cycle.Boron regulated the metabolic patterns of amino acids and carbohydrates and reduced Al toxicity.Nevertheless,B addition did not affect the Al-induced changes in the metabolic pattern of organic acids.2 Effects of B on the enzyme and non-enzyme defense system in roots of trifoliate orange rootstock under Al stressThe results showed that exposure to Al significantly increased the contents of Al and reactive oxygen species?ROS?in roots,and severely impeded plant growth-related parameters.Aluminum stress enhanced the contents of metabolites in the inositol and L-galactose pathway of ascorbic acid?As A?synthesis,and promoted the accumulation of As A.However,B supply decreased the activities of ascorbic acid peroxidase?APX?,dehydroascorbic acid reductase?DHAR?,glutathione reductase?GR?,glutathione peroxidase?GPX?in ascorbic acid glutathione?As A-GSH?cycle and induced the activities of?-glutamylcysteine synthetase??-GCS?,and reduced the metabolism in L-galactose pathway of ASA synthesis such as D-mannose-1-P,L-galactose-1-P,L-galactose,and L-galactono-1,4-lactose.Boron application decreased the contents of As A and oxidized glutathione?GSSG?while increased the content of GSH.Meanwhile,B increased superoxide dismutase?SOD?activity and decreased the activities of peroxidase?POD?,catalase?CAT?and polyphenol oxidase?PPO?during Al exposure.B reduced the Al-induced H₂O₂accumulation in roots as evidenced by lower fluorescence intensity of H₂O₂staining,improved plant biomass,root activity and relative root elongation.Overall,our results suggested that B could protect roots against Al-induced oxidative stress possibly by reducing metabolites accumulation in the L-galactose pathway of As A synthesis and regulating the As A-GSH cycle and antioxidant enzyme defense system.3 Effects of B on the pectin and cellulose contents and characteristics in roots of trifoliate orange rootstock under Al stressThe results showed that Al toxicity adversely inhibited root elongation and exhibited higher oxidative stress in terms of H₂O₂and O₂.^-under B-deficiency.Al-induced the cell wall thicking and changed the contents and characteristics of pectin and cellulose.Interestingly,B supply reduced the content of alkali-soluble pectin and increased the degree methyl-esterification of alkali-soluble pectin under Al stress,reducing the demethylation and hydrolysis of alkali soluble pectin to free carboxyl group.Boron application increased the content of 2-keto-3-deoxyoctonic acid?KDO?in two pectin forms under Al toxicity.The results of FTIR?Fourier transform infrared spectroscopy?and ¹³C-NMR(¹³C-nuclear magnetic resonance)spectra revealed the decrease of carboxyl groups and cellulose contents by B application during Al exposure,additionally,the X-ray diffraction?XRD?analysis confirmed the increase of cellulose crystallinity in the cell wall.Boron significantly reduced the accumulation of callose,ROS and Al in root,and reduced the thickness of cell wall.The study concluded that B could mitigate Al phytotoxicity by reducing the carboxyl group in pectin and shielding potential Al binding sites.Moreover,B can reduce cellulose content and increase its crystallinity,and increase cell wall rigidity,facilitate cell wall extension,and then promote root elongation.4 Effects of B on the distribution of Al in cell wall and the adsorption and transportation of Al in root tips of trifoliate orange rootstock under Al stressAl treatment significantly accumulated high Al content in the root and root cell wall,and the Al content in the cell wall accounted for a large proportion of the total Al in root,79.60-87.33%.Most of the root cell wall Al was bound to hemicellulose 1,followed by hemicellulose 2 and pectin,respectively,and the Al content in cellulose was the lowest.Boron supply attenuated Al-induced accumulation of Al in the cell wall.X-ray photoelectron spectroscopy?XPS?in conjunction with scanning electron microscope-energy dispersive x-ray spectrometer?SEM-EDS?revealed that B reduced the atomic ratio of Al in root cell wall.Boron supply mainly reduced the Al content of pectin,especially alkali-soluble pectin under Al stress.Correspondingly,with the increase of B level,the alkali-soluble pectin content and the activity of alkali-soluble methylesterase?PME?decreasd gradiently,while the degree methyl-esterification of alkali-soluble pectin increases gradually,thus reducing the carboxyl group in pectin.Atomic force microscope?AFM?images depicted that alkali-soluble pectin molecules were orderly and closely distributed,indicating that alkali-soluble pectin had better cross-linking combination,formed a stable network structure of cell wall,and further reduced the fixation of Al in cell wall.Furthermore,B application inhibited NRAT1 gene expression while increased ALS1 gene expression during Al exposure.The proportion of Al contents in intercellular space and vacuole were significantly increased,while in cytoplasm decreased by B application under Al stress as observed by transmission electron microscope-energy dispersive x-ray spectrometer?TEM-EDS?analysis.Taken together,our results indicated that B mainly reduced the enrichment of Al in the cell wall by regulating the content and characteristics of alkali-soluble pectin.More importantly,B alleviated some of the toxic effects of Al by decreasing the deposition of Al in the cytoplasm and compartmentalizes Al into vacuoles.