Establishment And Application Of Copper Ion Current Detection System On Plant Root Surface

Extensive and in-depth research work has carried out on the uptake and accumulation of heavy metals in plants.Under physiological conditions,measuring the heavy metal ion fluxes of plant roots is of great significance.Non-incasive Micro-test Technology(NMT)based on ion-selective microelectrode provides a new way to understand the transmembrane transport processes of the heavy metals.Though the technique has been successfully applied to the detection of Cd2+flux in plants,for detection of other heavy metal ions' fluxes still lack research and application.Cu is not only a necessary micronutrient element for plants,but also one of the heavy metals that causes the environment pollution.Therefore,establishing a reliable plant Cu2+flux detection technology has practical significance in studying transport mechanisms of Cu2+.In this thesis,Cu LIX with good performance was selected out.The properties of Cu2+selective microelectrode were investigated through Nernst slope,detection limit and selectivity coefficient.The reliability and stability of Cu2+selective microelectrode were analyzed with artificial ion source test.The effects of commonly used plant nutrition and physiological reagents on the detection of Cu2+flux were also investigated.Thus,a NMT system suitable for plant Cu2+flux detection was established.On this basis,the technique was applied to compare the changes of Cu2+fluxes at different positions of the root tips,and to explored the kinetics of Cu2+fluxes with the different Cu2+supply levels.The effects of K+,Ca2+,P-ATPase,EDTA on Cu2+uptake by white lupin and rice roots and the effects of Si on Cu2+uptake by rice roots were investigated.Furthermore,Cu2+transmembrane transport mechanisms of plants were discussed.The following results were obtained:1.Cu LIX containing 0.36~0.72%N,N,N,N'-tetracyclohexyl-2,2'-thiodiacetamide exhibited a good response to Cu2+,with the Nernst slope of 29.15±0.63~29.22±0.76mV/decade,approaching the the oretical value of 28.5mV/decade;the detection limits of the two kinds of Cu LIX were lower than 1?M Cu2+.The selectivity coefficients for interference ions Ca2+,Mg2+,Zn2+,Cd2+,Pb2+,K+,Na+,NH4+were-2.92~-3.03?-3.23~-3.57?-2.44~-2.58?-1.95~-2.23?-0.56~-,1.38?-4.76~-7.28?-4.95~-6.78?-5.25~-7.18,respectively.In the range of pH 4~7,Cu LIX with 0.36%content had a more stable performance.2.At different distances from the artificial ion source,the measured values of Cu2+flux were close to the predicted values.The measured values maintained stable during 10 minutes of ion flux recording.Therefore.NMT based on Cu2+selective microelectrode provided accurate and stable measurement of Cu2+flux.3.Cu2+flux measurement with NMT system were influenced differently by some chemicals.which were commonly used in plant nutrition and physiological studies.50uM TEA,50?M Na3VO4,200?M LaCl3-200?M GdCl3,200?M EDTA and 200?M NEM had no significant effect on the determination of Cu2+flux,while 200?M citric acid significantly increased the electrical potential.4.Sweep tests of Cu2+fluxes on the root tips of rice,corn and white lupin(within 1000?m from the root apex)showed that Cu2+flux variation with distance from the root apex was different among the plant species.The white lupin obtained the maximum Cu2+flux at 0~50?m from the root apex,while the rice reached the maximum value of Cu2+flux at 100?200?m from the root apex.Corn maintained a high Cu2+influx within 1000 ?m distance from the root apex.5.Cu2+fluxes of root tips increased with the increase of Cu2+concentration from 5?MM to 200 ?M,which was consistent with the characteristics of Michaelis-Menten kinetics.Vmax of white lupin was 199.39±25.43pmol/cm2/s,which was higher than the 155.49± 12.74pmol/cm2/s of Yangdao 6and125.67±7.39pmol/cm2/sof Nipponbare;and Km of Yangdao 6and Nipponbare were 73.49±8.21?M and 79.83±5.17?M,respectively,lower than 84.33± 12.49?M of white lupin.On the whole,the white lupin had the strong Cu2+absorption capacity.6.Under the background of 50 ?M Cu2+.10 mM K+and 5 mM Ca2+significantly inhibited Cu2+uptake by roots of white lupin and rice;200?M EDTA had significant inhibitory effect on Cu2+uptake;50?M Na3VO4 significantly inhibited Cu2+uptake by white lupin roots,but had no significant effect on Cu2+uptake by rice roots.Cu2+flux of rice roots cultured with silicon was lower than that of silicon-free rice.Cu2+uptake of plant was regulated by many factors in the environment and was related to plant species.