Mechanisms Of Boron-induced Alleviation Of Aluminum Toxicity In Trifoliate Orange

Acid soils comprise up to 40%of the world's potentially arable lands.Aluminum?Al?is the third most abundant element after oxygen and silicon and accounts for 7%of the earth crust.The red soils cover an area accounting for over 20%of the country's total area and mainly distributed in the tropical and subtropical regions of China.In acidic soils,Al toxicity is a major limiting factor for crop development and productivity.Inhibition of root elongation is the major symptom of Al toxicity.Boron?B?is one of the essential micronutrients for the growth and development of higher plants,and its deficiency usually results in the abnormal growth of roots and defects in young and growing parts of the plant.The B defines the cell wall structure and ensures its stability under harsh conditions of the environment.The cell wall of higher plants contains a large number of polysaccharides including rhamnogalacturonan?RG-II?and B aids in the cross-linking of RG-? together through diester bonds.The stable and well-packed network of cells develop stable cell wall with reduced pore sizes and tightens cell wall that restricts entry of large molecules/elements into the cell.The cell wall of plant plays a key role in Al toxicity as it is the primary site of Al accumulation and subsequently toxicity in plants.The primary cell wall with its carboxylate group has been reported the major pool of Al adsorption.There are several agronomic practices to alleviate/lessen the toxic effect of Al including applying lime?liming?.However,liming can induce adverse effects on plant growth by decreasing the availability of essential elements and is not always economically feasible.In some reports,it was suggested that B may alleviate the toxic effects of Al on plant growth and improve the plant performance in acid soils.Till date,none of the studies,however,have been investigated the ameliorative role of B against Al toxicity on trifoliate orange,and the mechanism how B alleviates Al toxicity is not well understood.If this assumption could be verified in citrus,B would be a viable candidate for the amelioration of Al toxicity for citrus in acid soils.Citrus belongs to evergreen subtropical fruit trees cultivated in humid and subhumid tropical,subtropical and temperate regions of the world mainly on acidic soils.In China,citrus is an important fruit crop and usually cultivated in the acidic soils particularly in the areas of Jiangxi,Ganzhou?planting area ranks first in the world?.The high rainfall washes out alkaline and alkaline earth elements from root zones,creating acidic conditions and thus causing severe B deficiency and Al toxicity problems.In this work,we used young seedlings of trifoliate orange as a material to study the role of B in the alleviation of Al toxicity and underlying mechanisms by utilizing high end techniques for assessing biochemical,physiological and structural components.The experiments were carried out in the hydroponic condition at Huazhong Agricultural University,Wuhan.We utilized optimal and extra B beyond the optimal need of plant in a completed randomized design under different concentrations of Al.Transmission electron microscope?TEM?,Fourier-transform infrared spectroscopy?FTIR?,vital staining,assay of antioxidant system,monoclonal antibodies and lumogallion fluorescent molecular probe were extensively used to study the main effects of B on Al toxicity.The main results are summarized below:1-Al toxicity severely hampered the root growth and physiological characteristics of the plant,caused oxidative stress,membrane damage and severe root injury under B deficiency.However,application of B improved root elongation and photosynthic parameters under Al toxicity.Moreover,B supply regulated the activities of antioxidant enzymes?superoxide dismutase,peroxidase,catalase and ascorbate peroxidase?,proline,secondary metabolites?phenylalanine ammonia lyase and polyphenol oxidase?contents,and stabilized integrity of proteins and reduced the root injuries.B concentration was found to be similar either with or without Al indicating a non-significant effect of Al on B adsorption.Results imply that B supply promotes root growth by reducing accumulation of reactive oxygen species?ROS?and Al concentrations in plant parts thus B alleviates Al toxicity and its associated damages in root system;a fact that might be significant for higher productivity of agricultural plants grown in acidic conditions.2-The results showed that Al toxicity considerably accumulated hydrogen peroxide(H₂0₂and superoxide radicals?O2'-?in roots without B.However,treatment of B significantly lowered down the oxidative stress in terms of H₂O₂ and O₂^.-by 48.11%and 44.87%respectively.The supply of B eliminated oxidative stress,membrane peroxidation,membrane leakage,and cell death produced by Al toxicity.In the absence of B,Al toxicity treatment accumulated more Al in both root tips and root cell wall?CW?more than 50%of the total Al in roots.However,B supply resulted in reducing Al concentration in roots and root cell wall by 59%and 58%respectively compared with the Al treated plants without B.3-Accumulation of Al was increased in the cell wall in the absence of B and lumogallion fluorescent molecular probe unequivocally localized mostly Al bound to the periphery of the cell wall?CW?and to the nuclei indicating highly pronounced adsorption of Al to the cell wall as well as cytoplasm.Fluorescence intensities were gradually decreased in the B₁Al⁺,and B₂Al⁺ treatments.The transmission electron microscopy?TEM?micrographs showed that both B₀Al^-and B₀Al⁺resulted in irregular cell characteristics.Remarkable thickened and swollen root cell wall was more intense in B0Al+.The B supply reduced the accumulation of starch grains,as well as a substantial amount of reduced cell wall thickness was observed.Al toxicity showed a disorganized distribution of HG?homogalacturonan?epitopes as revealed with monoclonal antibodies.The spectral results of Fourier-transform infrared spectroscopy?FT-IR?indicated that Al toxicity influenced the intensities of wavelengths especially protein,cellulose,hemicellulose and pectin whereas addition of B at both concentrations showed a significant decrease in peaks values related to pectin and hemicellulose compared to BOAl+.4-Al toxicity induced variations in the CW components through the accumulation of pectin and hemicellulose and increased by 60.04%,and 149.10%respectively without B as compared to control treatment.On the other hand,the optimal and supplementary B treatments reduced the un-methylation of pectin by 37.05%and 46.78%and hemicellulose by 31.34%and 50%in relation to B_OAl⁺ treatment respectively.The treatments of B1A1+and B2AI+increased the degree of methyl esterification of pectin?DM?by 197.18%and 221.61%respectively with respect to the BOAl+treatment.Additionally,B reduced callose deposition and reduced changes in the CW components under A1 toxicity.The results indicate that accumulation of A1 on the cell wall and alteration in the cell wall components might be one of the causes resulting in the quick inhibition of root elongation under B-deficiency by providing susceptible negative charges on pectin matrix for binding of Al.These results imply that B due to its role in the cell wall formation could reduce Al-induced negative effects on plant growth by attenuating apoplastic Al3+ and changes in the CW components which ultimately results in reduced root injury and improved root growth.The results provide a useful understanding of the insight into mechanisms of B-induced mitigation of Al toxicity especially in the trifoliate orange that might be helpful in the production of crops on acidic soils.