Study On Physiological And Molecular Response Of Centipedegrass To High Aluminum Toxicity Stress

Aluminum(Al)is the most abundant metal element and accounts for 7%-8%of the total earth crust.Aluminum toxicity is a major factor limiting crop growth and production in acid soil(pH<5.5).The initial and most easily recognized symptom of Aluminum toxicity is on limiting root growth,and inhibiting root absorption of water and nutrients,thus reducing crop yield.Approximately,40%of the world and 21%of Chinese cultivated land are acid soils,which is severely affected by Aluminum toxicity.Land greening based on ground coverage by planting highly adversity-resistant grass plants is an effective measure for management of acid soils.Therefore,looking for Aluminum tolerant grass species suitable for growth in acidic soil sand revealing the related Aluminum tolerance mechanism are particularly critical.Centipedegrass(Eremochloa ophiuroides(Munro)Hack.)is an important perennial warm-season(C4)grass species,mainly used as a multirole turfgrass and a grazing-purpose grass.It was found that the adaptability of centipedegrass to acid soil was better,but the aluminum resistance of different centipedegrass varied greatly.To explore the morphological,physiological,and molecular differences of different aluminum resistant materials in response to aluminum toxicity,reveal the physiological and molecular mechanism of aluminum resistance of centipedegrass,and to provide theoretical basis and research materials for efficient aluminum resistance breeding of centipedegrass.Accordingly,this study mainly carried out the establishment of an efficient and accurate aluminum resistance evaluation system of centipedegrass and the evaluation and identification of aluminum resistance of representative germplasm materials of centipedegrass.On this basis,the differences of morphology,physiological and biochemical characteristics of different aluminum resistant materials in response to high aluminum toxicity,as well as the role of hormones in regulating the aluminum tolerance of centipedegrass were systematically studied.At the same time,an aluminum ion specific transporter gene EoNRAT1 was cloned in centipedegrass,and its bioinformatics analysis and spatial-temporal expression analysis were carried out.The main research achievements are as follows:1.A set of efficient and accurate evaluation and identification system for aluminum tolerance of centipedegrass was established,that is,the root growth inhibition was observed after three days of treatment with 1.0mM aluminum concentration,and the root tips were dyed with 0.5%concentration Eriochrome Cyanine R for 10min for confirmation.To evaluate and identify the aluminum resistance of the corresponding materials.2.Based on the newly established system method,46 representative germplasm materials from different sources of centipedegrass were systematically evaluated in this study,and two kinds of extremely aluminum resistant materials were successfully selected from them,namely Al-sensitive material(E006)and Al-tolerant material(E105).3.The root micro morphogenesis and leaf physiological characteristics of different genotypes of centipedegrass were studied under continuous high aluminum toxicity stress.The two genotypes showed an obvious difference in root micro morphogenesis after Aluminum treatment,which resulted mainly from the different Aluminum accumulations,with E006 having more Aluminum accumulation.Aluminum toxicity caused abnormalities in root morphogenesis,reductions in photosynthetic performance and relative water content,and increases in electrolyte leakage,malondialdehyde content,and hydrogen peroxide(H2O2)levels in plants.These effects were more pronounced in E006 than in E105.Meanwhile,Aluminum treatment stimulated substantial increases in proline content and enzyme activity as defense responses in E105.Moreover,the significant elevation of endogenous hormone and the differences in the extent of the increase in the hormones abscisic acid(ABA)and salicylic acid(SA)between genotypes implied that the two hormones might mediate the above defense responses under Aluminum toxicity.There are genotypic differences in the changes caused by successive Aluminum treatment.The morphological and physiological damages caused by successive Aluminum exposure in the Al-tolerant genotype were significantly lighter than those in the Al-sensitive genotype,which could be attributed to ABA-and SA-mediated positive defense responses mainly involving the less amount of Aluminum accumulation,and the better-increased osmolytes and elevated antioxidant enzymes activity.4.Exogenous application of 50 μM hormone SA can effectively reduce the uptake and accumulation of aluminum ions in the roots of centipedegrass under high aluminum toxicity stress,which is conducive to the normal or good root growth under aluminum toxicity stress.In addition,it also reduces the production of ROS in vivo under aluminum toxicity stress,which is directly related to the up regulation of the activity level of antioxidant enzymes in vivo.However,inhibition of endogenous SA production by adding 100 μM PAC greatly inhibited the accumulation and elongation of Al3+ in the root system and the outbreak of ROS in the body under high aluminum stress.Therefore,SA plays an important and positive role in alleviating aluminum toxicity and regulating the physiological defense response of centipedegrass to aluminum toxicity.5.The open reading frame(ORF)of the EoNRAT1 gene,a member of the natural resistance-associated macrophage protein(Nramp)family,is 1614 bp and encodes 538 amino acids.The theoretical relative molecular mass of EoNART1 protein is about 58430,the theoretical isoelectric point is pI 7.03,the instability index is 30.98,the hydrophobic amino acids account for 67.2%,and this protein contains 10 transmembrane domains.Moreover,EoNART1 protein is mainly located in plasma membrane,vacuole,and endoplasmic reticulum,its secondary and tertiary structures mainly consist of α-helix and random coil.The multiple alignment result of amino acid sequence homology shows that the sequence identity between EoNRAT1 and NRAT1 from Panicum virgatum Linn.reaches 84.53%.The phylogenetic analysis result shows that EoNRAT1 and NRAT1 from P.virgatum are first clustered together,and clustered into one clade with NRAT1 from 10 species such as,Lolium rigidum Gaud.The qRT-PCR analysis result shows that within 48 h of Al3+stress,EoNRAT1 gene is expressed in root,stem,and leaf of E.ophiuroides;at 12 and 24 h of stress,the relative expression levels of EoNRAT1 gene are significantly higher than that at 0 h of stress;at 48 h of stress,the relative expression level of EoNRAT1 in root still maintains a relatively high level,but that in leaf decreases to the level at 0 h of stress.It is suggested that EoNRAT1 protein is a stable hydrophobic membrane protein,which may be involved in response of E.ophiuroides to aluminum stress.