Studies On Physiological Response To Selenite,and Se Absorption,Accumulation And Distribution Characteristics In Alfalfa

Selenium(Se)is an essential element for humans and animals,and plays an important role in physical health.Se supplementation is of paramount importance in Se-deficient countries and regions because it prevents diseases caused by Se-deficiency,strengthens immunity,delays senescence,and promotes the production performance of animals.The main source of human Se consumption is cereals and livestock and poultry meat,and feeding premix with inorganic Se addition is the most common way to improve animal Se deficiency,which is frequently associated with many disadvantages.To overcome the disadvantages,applying Se-enriched forage is safer and more effective than using inorganic Se.Alfalfa(Medicago sativa L.),a kind of high quality pasture,is widely cultivated and the most widely used in livestock production.Hence,this study studied on physiological response to selenite,Se uptake mechanism and transport distribution in alfalfa,and aimed to preliminarily reveal selenite metabolic pathway in alfalfa which provides a theoretical reference for the application of Se in soil.1.Effects of selenite on the growth of alfalfa and related physiological mechanismsPot experiments were conducted in a completely randomized block design in five replications.The levels of selenite application were the conrtol(0 mg kg-1),low level(0.5,1,5 mg kg-1),medium levels(10,20 mg kg-1),and high levels(50 mg kg-1).On the 60th day after sowing,various physiological indexes,biomass and selenium accumulation in the first and second crops were determined.The results are as follows:The Se concentrations of the shoots and roots increased with increasing Se doses.When<20 mg kg-1 Se was applied,shoot biomasses and agronomic characters of the first and second crops were not significantly affected,and the low levels of Se applications(0.5 and 1 mg kg-1)slightly enhanced the DWs of the shoots(P>0.05).At low levels(0.5-5 mg kg-1 selenite),the activities of antioxidants superoxide dismutase,peroxidase,and glutathione peroxidase increased,whereas the MDA content decreased significantly.The carotenoid contents in leaves enhanced by Se applications.Low levels of Se applications(1-5 mg kg-1 selenite)significantly enhanced the net photosynthetic rate and root activities and promoted the absorption of P and K.Meanwhile,1mg kg-1 selenite promoted the absorption of Ca,Fe and Mn.However,high levels of Se applications inhibited the plant growth,antioxidant enzyme activity,photosynthetic parameters,root activity,and P and K absorption.In addition,the proportion of organic Se in the soluble Se of alfalfa was estimated to be about 92.81%.The above results indicated that:Alfalfa growth was not negatively affected by Se application less than 10-20 mg kg-1.Low-dose selenite stimulated plant growth,mainly due to the stimulation of root growth by enhancing root activities and therefore promoting the absorption of P,K,Ca,Fe,and Mn,and improvements of photosynthetic rate and activities of antioxidant enzymes.Considering the safety of animals,5mg kg-1 Se application is suitable for actual production.2.The physiological mechanism of selenite uptake in alfalfaIn this study,hydroponic experiments were conducted to elucidate the physiological mechanisms of selenite absorption in alfalfa using the excised roots and intact seedlings.The results are as follows:The Se concentrations in lateral roots were significant higher than that in taproots(P<0.05).With increasing pH,Se uptake in root dramatically decreased.The relationship between Se uptake rate and selenite concentration in the uptake solution represented a linear correlation(r2=0.9809,P<0.0001)at pH 3,and followed saturation curves at pH 5 and 8.The dose-dependent Se net uptake at pH 5.5 followed the Michaelis-Menten kinetics(r2=0.9940,P<0.0001).Both time-dependent Se net uptake and apoplasmic adsorption in excised roots of alfalfa were not saturated within 210 min.Moreover,inhibitor experiments showed that AgNO3 inhibited selenite uptake at pH 3;DNP,LT,and AgNO3 inhibited the uptake of selenite at pH 5,and DNP and Na2VO4 inhibited selenite uptake at pH 8.Addition of P to the absorption solution significantly decreased Se uptake in the excised roots,and P deficiency significantly increased Se uptake in the roots.The above results indicated that:Lateral roots were the dominant parts of alfalfa root in Se uptake.Selenite uptake in alfalfa root depended on environmental pH due to the different forms of selenite ionization.Alfalfa roots can selectively absorb H2SeO3 and HSeO3-,but the SeO32-is hard to absorb by root.H2SeO3 enters the roots via aquaporins.Partial HSeO3-is absorbed by active transport process and water channels.Lastly,SeO32-is actively absorbed and driven by the change in pH energized by the proton pump(H+-ATPase).P and Se have obvious competitive relationship and may share transporters.3.Accumulation and distribution characteristics of Se in alfalfaPot experiments were conducted in a randomized block design in three replications.The plants were grown in sterilized quartz sand and treated with different selenite levels(0,1,5,10,20 ?M)for 21 days to determine the effect of Se on growth.Plants exposed to 1 and 10 ?M selenite were used to clarify the Se distribution in alfalfa and to conduct the experiment of transpiration inhibition.The results are as follows:Alfalfa growth was significantly stimulated(P<0.05)at 1 ?M Se.Three linear correlations were found between the applied Se doses and Se concentrations in alfalfa roots,stems,and leaves.Transpiration inhibition did not significantly affected Se uptake,but significantly inhibited the transport of Se from roots to shoots.The following patterns of Se concentrations were observed:root>Ieaf>stem in the organs;rhizodermis and cortex>stele in the root and stem tissues,epidermis and mesophyll>vascular bundle in the leaf tissues;and younger leaf>mature leaf>older leaf and marginal leaf>midrib>internal leaf in the leaves.In addition,at low-does Se application(1 ?M),the cytoplasm fractions>cell wall fractions>organelle fractions,while at high-does Se application(10 ?M),cell wall fractions>cytoplasm fractions>organelle fractions in root and leaf cells.The above results indicated that:Appropriate selenite addition(1 ?M)benefited alfalfa.Transpiration provides driving force of Se transport in alfalfa,but is not closely related to the Se uptake in roots.Se binding in the rhizodermis and cortex of the root caused the most of Se accumulation in root.Se redistributions may be a possible important factor affecting the distribution of Se in shoot and Se was transported along with the transpiration stream within a single leaf.Vacuolar compartmentalization and cell wall immobilization might be the two major strategies to protect plant organs from potential Se toxicity.4.Cloning and functional verification of Se uptake related genes MsPT2 in alfalfaFull-length cDNAs MsPT2 were cloned from alfalfa in this study.The sequences of gene and amino acid encoded by genes were analysed by bioinformatics method.The green fluorescent protein(GFP)-tagged gene was fused to MsPT2,respectively,to verify the subcellular locations.Meanwhile,the transcript levels of the gene in different tissues were determined.Finally,overexpression vectors were constructed,and the function of MsPT2 was preliminarily verified by Arabidopsis transformation.The results are as follows:The cloned MsPT2 gene had a full length of 1617bp,and encoded 538 amino acid residues.The protein encoded by MsPT2 with the molecular weight of 58.930 kD and isoelectric point of 8.76 had the characteristic sequence of Phtl family protein,and was located on the plasma membrane.Furthermore,the transcript levels of MsPT2 in shoots were higher than that in roots;The overexpression of MsPT2 did not significantly affected P concentration in the roots,but significantly increased the P concentration in shoots of transgenic Arabidopsis thaliana compared with the wild type plants(P<0.05).After 1 week of Se treatment,the Se concentrations in roots,shoots of MsPT2 Arabidopsis transgenic lines(L2 and L9)and the ratio of shoot Se/root Se were significant higher than those of the wild type plants(P<0.05).After 2 weeks of Se treatment,Se concentrations in roots of MsPT2 Arabidopsis transgenic plants were higher than that of the wild type plants,and Se concentrations in shoots and the ratio(shoot Se/root Se)of L2 were 70.42%and 38.89%higher than that of the wild type plants,respectively.The above results indicated that:The MsPT2 is a member of Phtl family,which is associated with the Se transport from root to shoot.The overexpresssion of MsPT2 in Arabidopsis increases the Se uptake in root,and promotes the Se transport from root to shoot.5.Cloning and functional verification of Se uptake related genes MsNIP2:l in alfalfaIn this study,full-length cDNAs were cloned from alfalfa.The bioinformatics,subcellular locations,and the transcript levels of the gene in different tissues were analysed,and the function of MsNIP2:1 was preliminarily verified by Arabidopsis transformation.The results are as follows:The cloned MsNIP2:1 gene had a full length of 825bp,and encoded 274 amino acid residues.The protein encoded by MsNIP2:1 with the molecular weight of 28.825 kD had two characteristic sequences of Asn-Pro-Ala(NPA),and was located on the plasma membrane.Furthermore,the MsNIP2:1 mainly transcripted in roots.After 1 week of Se treatment,Se concentrations in roots of MsNIP2:1 Arabidopsis transgenic lines(L5 and L2)were 32.97%and 27.72%higher than that of the wild type plants,respectively,and the ratio of shoot Se/root Se were higher than that of the wild type plants(P<0.05).After 2 weeks of Se treatment,Se concentrations in roots of lines L5 and L2 were 52.47%and 44.42%higher than those of the wild type plants,respectively,and Se concentrations in shoots and the ratio(shoot Se/root Se)were not significant different from those of the wild type plants.The above results indicated that:MsNIP2:1 is a member of aquaporin family(NOD26-like influx proteins).The overexpresssion of MsNIP2:1 improves the Se uptake and transport,indicating that the aquaporin is one of the ways of selenite entering plant cells.In conclusion,it is feasible to produce Se-enriched alfalfa by applying selenite in soil,and 5mg kg-1 Se application is suitable for actual production considering the forages biomass and animals safety.Lateral roots were the dominant parts of alfalfa root in Se uptake.Selenite uptake mechanisms in alfalfa root depended on the different forms of selenite ionization.The active absorption part may share MsPT2 with phosphate,while the passive part may be related to the aquaporin(MsNIP2:1).The Se in plant roots was mainly transported to the aerial part by transpiration force,which may be mediated by transporters.Selenite immobilization by root epidermis and cortex is a key factor that affect Se transport in plants;Se was transported along with the transpiration stream;The redistribution of Se in old leaves and new leaves is another major factor that affect the distribution of Se in alfalfa;Se prefers to accumulate in cell wall and vacuole.