CN109374586B - Method for identifying cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters - Google Patents
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Abstract
The invention discloses a method for identifying cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters, which comprises the following steps of 1) plant cultivation: treating the plant to be measured with cesium-containing nutrient solution with different concentrations; 2) determination of chlorophyll fluorescence parameters: measuring a rapid chlorophyll fluorescence kinetic curve and fluorescence parameters of the plant after dark adaptation under different cesium treatment concentrations by using a continuous excitation type chlorophyll fluorescence instrument; 3) calculating PI according to the measurement result of the step 2)ABSAnd calculating PI of the cesium treatment groupABSA change value; 4) PI of plant leaves according to step 3)ABSThe variation values identify the cesium enrichment capacity of the plants. The method overcomes the defects of complex treatment, long period, low efficiency and the like of the existing method, has the advantages of nondestructive detection, sensitivity, rapidness and the like, can carry out real-time in-situ measurement on fresh plant samples, and has the characteristics of timeliness, sensitivity, rapidness, high efficiency and the like.
Description
Technical Field
The invention belongs to the field of soil pollution treatment, and particularly relates to a method for identifying cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters.
Background
The development and utilization of nuclear technology bring great economic and social benefits to human beings, and simultaneously generate a large amount of radioactive pollutants. Radioactive contaminants cause radioactive contamination of the ecological environment, particularly soil and water, which is a nuclear pollution. The radionuclide is very difficult to remove after entering the soil and environment. Although the conventional physical and chemical methods can be used for treating the low-level radionuclide in the soil-water system, the cost is high, and secondary pollution is easily caused. The plant restoration technology is an environmental pollution treatment technology for removing pollutants in the environment by using plants and coexisting microbial systems thereof on the basis of the theory that the plants tolerate and excessively accumulate certain or certain chemical elements. The method has the advantages of low investment and maintenance cost, simple and convenient operation, environmental friendliness, cleanness and the like, can obtain certain economic benefit through resource utilization, has wide application prospect, and is considered by scholars at home and abroad to be one of the most effective means for treating the environment polluted by the low-level and extremely low-level nuclides in large-area soil and water. However, the research on the phytoremediation of the soil polluted by the nuclides such as strontium, cesium and uranium at home and abroad is still in the primary stage at present, and is mainly used for screening of hyper-enriched plants. The current research shows that the absorption and accumulation of the nuclides such as strontium, cesium and uranium are different for plants of different families and genera, and the accumulation of strontium, cesium and uranium is different for different organs of the same plant. For the research of phytoremediation technology of nuclide polluted environment, finding and screening more nuclide hyperaccumulator plants is the most important material basis and necessary premise for improving the efficiency of the phytoremediation technology.
At present, the screening and identification of nuclide or heavy metal super-enriched plants at home and abroad are mainly carried out according to the following procedures: collecting a sample, drying the sample, digesting the sample, analyzing the element content of the digested sample liquid by an atomic absorption spectrometer or an inductively coupled plasma emission spectrometer, and finally judging and identifying the enrichment capacity of the plant on nuclides and heavy metals according to the enrichment coefficient and the transport coefficient of the plant on the nuclides or the heavy metals. The whole process almost takes several days, and the sample treatment process is very complicated and the efficiency is very low. Therefore, the method for judging the relative size of the nuclide or heavy metal enrichment capacity of the plant, which is rapid, easy to determine and capable of detecting a large number of samples in a short time, is found to have important application value and prospect for screening nuclide and heavy metal super-enriched plants.
Photosynthesis of plants is a process of absorbing carbon dioxide and water to convert light energy into chemical energy and releasing oxygen. The chloroplast contains two photosynthesis reaction centers of Photosystem I (PS I) and Photosystem II (PS II), and the chlorophyll used for absorbing and transmitting light energy in the chloroplast is a fluorescent substance, and the photosynthesis of green plants is usually accompanied by a fluorescence phenomenon. As shown in fig. 1, the absorbed light energy is mainly used for photochemical reactions in the reaction center by the antenna pigment molecules, the excess excitation energy is dissipated as heat energy, and a small amount of excitation energy is emitted as fluorescence. Since only a small fraction of the light energy absorbed by the plant is dissipated as fluorescence (typically no more than 5% of the incident light energy) and the chlorophyll fluorescence signal is weak, the fluorescence wavelength is longer than the absorption wavelength, Kautsky and Hirsh have recognized the close relationship between the initial photosynthetic response and chlorophyll fluorescence as early as 1931, and they reported that in plants that have been sufficiently dark adapted, their chlorophyll fluorescence rapidly rises to a maximum value under light conditions, then gradually falls, and finally stabilizes at a fixed value.
With the continuous and deep research on chlorophyll fluorescence, the fluorescence induction kinetics curve and the abundant information contained in the rapid induction kinetics are fully known, so that the research on electron transfer on the PS II donor side and the receptor side is more deep. A typical rapid chlorophyll fluorescence induction kinetic curve is shown in FIG. 2 as O, J, I, P etc. Generally, the lowest fluorescence when just exposed to light is defined as the point O, the highest peak value of the fluorescence is defined as the point P, and the fast chlorophyll fluorescence kinetic curve refers to the fluorescence change process from the point O to the point P, and mainly reflects the change conditions of the original photochemical reaction of the psii and the structure and state of the photosynthetic mechanism. The O-J stage is a photochemical reaction stage, which is to transfer electrons to Q through Pheo after the reaction center of PS II is excitedAAnd Q isAReduction to QA -The sensitivity of this stage to light intensity is particularly high; the J-I stage is a PQ library reduction stage, the fluorescence of the J-I stage is caused by the complete reduction of a fast-reduced PQ library in the electron transfer process, and the J-I stage can reflect the heterogeneity of the PQ library; the I-P stage fluorescence is due to the reduction of the slow-reducing PQ pool, which reflects the decrease in redox proteins. When the Oxygen-evolution complex (OEC) of the PS II donor is damaged, the chlorophyll fluorescence intensity is increased, a K phase (about 300 mu s after illumination) appears, and the multiphase fluorescence O-J-I-P is changed into O-K-J-I-P, and even more inflection points appear. From the fast chlorophyll fluorescence kinetic curve, a large amount of raw data can be obtained, and in order to better reflect the relationship between the kinetic curve and the tested sample material, Strasser and Strasser in 1995 are based on the energy flow of biological membrane, and a highly simplified energy flow model diagram (FIG. 3) is established by calculating the energy flow and energy ratio to measure the internal change of the sample material under a given physical state. According to the energy flow model, a part of the absorbed energy (ABS) of the antenna pigment (Chl) is dissipated as heat and fluorescence (F)The other part is captured (TR) by a reaction center (RC, RC being an active reaction center in the JIP-assay), where excitation energy is converted into reduction energy to convert QAReduction of QA -The latter can be re-oxidized to generate Electron Transport (ET), and the transported electrons are used to fix CO2Or other approaches. The data processing developed on this basis (Table 1) is referred to as "JIP-determination". "JIP-assay" can provide a great deal of information about the sample material being tested, especially the structural and functional changes of photosynthetic organs under different environmental conditions.
TABLE 1 terminology and formulas used in Rapid chlorophyll fluorescence-induced kinetic Curve (O-J-I-P) analysis
The photosynthesis of plants is very sensitive to various environmental stresses, and the rapid chlorophyll fluorescence kinetic analysis technology is used as a sensitive probe for evaluating the photosynthesis efficiency of plants and the influence of adverse conditions such as water stress, temperature stress, salt stress and the like on the photosynthesis of plants, and is widely applied to a plurality of fields such as agricultural production, ecological protection, environmental monitoring and the like. The research of the inventor of the invention finds that the nuclides of strontium, cesium and uranium obviously influence the photosynthesis efficiency of plants, the photosynthesis of the plants with different enrichment capacities also shows different response differences to the stress of the nuclides, and the rapid chlorophyll fluorescence kinetic parameters can monitor the change of the response differences very sensitively and rapidly, so the inventor of the application tries to find a method for identifying the difference of the enrichment capacities of the plants on cesium by using the influence of the cesium on the rapid chlorophyll fluorescence kinetic parameters of the leaves of the plants.
Disclosure of Invention
The inventor analyzes the rapid chlorophyll fluorescence dynamics of cesium on different plant leavesThe influence of the number and the establishment of a corresponding database show that the fast chlorophyll fluorescence kinetic parameter PIABSThe change trend of the plant cesium enrichment factor can be in corresponding relation with the plant cesium enrichment capacity, so that the rapid chlorophyll fluorescence kinetic technology can be used for identifying the plant cesium enrichment capacity.
Based on the method, the invention provides a method for identifying the cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters, which comprises the following steps:
1) plant cultivation: treating the plant to be measured with cesium-containing nutrient solution with different concentrations;
2) chlorophyll fluorescence kinetic parameter determination: measuring a rapid chlorophyll fluorescence kinetic curve and fluorescence parameters of the plant after dark adaptation under different cesium treatment concentrations by using a continuous excitation type chlorophyll fluorescence instrument;
3) calculating PI according to the measurement result of the step 2)ABSAnd calculating PI of the cesium treatment groupABSA change value;
4) PI of plant leaves according to step 3)ABSThe variation values identify the cesium enrichment capacity of the plants.
Further, the method for identifying cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters comprises the step 5) of identifying PIABSScreening the plants with the reduction range of more than or equal to 20% into plants with strong cesium enrichment capacity under corresponding concentration; mixing PIABSThe plants with a reduction of less than 20% were selected as plants with a weak cesium enrichment capacity at the corresponding concentration.
Further, in the method for identifying the cesium enrichment capacity of the plant by using the rapid chlorophyll fluorescence kinetic parameters, the step 1) is to treat the plant with cesium-containing nutrient solution with different concentrations when the plant grows to the 6-leaf stage. If the plant is treated by the cesium-containing nutrient solution too early, the plant tolerance is weak, and the plant meeting the requirement is difficult to screen, and if the plant is treated by the cesium-containing nutrient solution too late, the optimal period for the plant to grow and adsorb nuclide is missed, so that the plant in the 6-leaf stage is selected for treatment.
Further, in the method for identifying the cesium enrichment capacity of the plant by using the rapid chlorophyll fluorescence kinetic parameters, step 2) is to perform dark adaptation on the plant for 20-25min before measurement.
According to some embodiments of the present invention, the method for identifying cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters comprises the following steps:
1) plant cultivation: selecting plants to be tested to grow seedlings in a quartz sand and Hoagland nutrient solution cultivation system to 4-leaf stage, selecting healthy plants with consistent growth vigor, transplanting the plants into plastic pots containing 10 kilograms of quartz sand per pot, transplanting 6 plants in each pot, and treating the plants to be tested in the 6-leaf stage by Hoagland nutrient solutions with different cesium concentrations (CsCl) of 0, 0.1, 0.5, 1, 5 mmol.L -15 repeats;
2) fast chlorophyll fluorescence kinetic parameter determination: selecting middle part of just-expanded mature leaf by using continuous excitation type chlorophyll fluorescence instrument (Handy PEA or M-PEA of Hansha corporation in England), measuring dark adaptation for 20-25min, measuring rapid chlorophyll fluorescence kinetic curve of dark adaptation leaf, measuring 6 times for each treatment, and obtaining minimum fluorescence Fo, maximum fluorescence Fm, and K-phase fluorescence (fluorescence at 300 μ s) FKJ-phase fluorescence (fluorescence at 20 ms) FJIso-fluorescence parameters;
3) calculating PI according to the measurement result of the step 2)ABSIs given in Table 1, and the PI of the cesium treatment group is calculatedABSA change value;
4) PI of the plant leaves obtained according to step 3)ABSThe variation value identifies the cesium enrichment capacity, PI, of the plantsABSScreening plants with the reduction range of more than 20% into plants with strong cesium enrichment capacity under corresponding concentration; mixing PIABSThe plants with a reduction of less than 20% were selected as plants with a weak cesium enrichment capacity at the corresponding concentration.
The invention has the beneficial effects that: the method overcomes the defects of complicated sample treatment, long period, low efficiency and the like of the prior method for screening cesium-enriched plants, the rapid chlorophyll fluorescence dynamics analysis technology adopted by the invention has the advantages of nondestructive detection, sensitivity, rapidness and the like, can carry out real-time in-situ measurement on fresh plant samples, does not need sampling, drying and digestion and then carries out instrument analysis and test, and has the characteristics of timeliness, sensitivity, rapidness, high efficiency and the like.
Drawings
FIG. 1 is a schematic of chlorophyll fluorescence generation;
fig. 2 is a typical rapid chlorophyll fluorescence induction kinetic curve, left: linear time axis, right: a logarithmic time axis;
FIG. 3 is a model of energy flow in a photosynthetic organ;
FIG. 4 is a graph comparing the cesium enrichment content of four plants after treatment for 7 d;
FIG. 5 is a graph comparing the enrichment factors for cesium of four plants after treatment for 7 d;
FIG. 6 shows four plant PIs after 7d treatmentABSComparative figures for parameters.
Detailed Description
The present invention is further illustrated by the following specific test examples, but it should not be construed that the scope of the present invention is limited to the following examples, and it is obvious to those skilled in the art that various technical features in the following examples can be appropriately combined, replaced, adjusted, modified, etc. according to the inventive idea and the entire contents of the present invention, and still fall into the scope of the protection of the present invention.
Materials and methods
In the embodiment, several plants with high growth speed and large biomass are selected, namely, Shanghai green and Chinese cabbage of Cruciferae and broad bean and pea of Leguminosae respectively. The specific family genus of the plant is shown in Table 1.
TABLE 2 selection of plant names and genera in the experiment
Experimental design and plant cultivation
The marine liriope, Chinese cabbage, broad bean and pea were used as test materials, and the seedlings were grown in soil to 4-leaf stage, and healthy plants with consistent growth were selected and transplanted into plastic pots containing 10 kg/pot of quartz sand, and 6 plants were transplanted per pot.Treating with Hoagland nutrient solution of cesium (CsCl) with different concentrations at 6-leaf stage, wherein the cesium treatment concentrations are 0, 0.1, 0.5, 1, 5 mmol.L-1Each treatment was 5 replicates.
Data acquisition
And (4) carrying out cesium content analysis and rapid chlorophyll fluorescence kinetic analysis on the treated 7 th collected sample.
And (3) measuring the content of cesium: washing the whole plant with tap water, and washing the plant root at 20 mmol.L-1Na of (2)2-soaking in EDTA solution for 30min to chelate cesium adsorbed on the surface of the roots. Then washing with deionized water, draining off water, deactivating enzyme at 105 deg.C for 30min, drying at 75 deg.C to constant weight, grinding, and microwave digesting with 0.1g dry powder by microwave digestion instrument (Mars, CEM corporation). The Cs content of the above-ground and underground parts of the plants was determined by the atomic absorption method (AA700, PE company, USA), and the measurement was repeated 3 times for each sample.
Enrichment factor (BCFs) ═ cesium content in the plants/cesium content in the culture system.
Rapid chlorophyll fluorescence kinetic analysis: the same leaf position of different treated plants was selected, the middle part of the fully developed mature leaf was dark adapted for 20-25min before the measurement, and the rapid fluorescence induction kinetic curve of the leaf was measured using a continuous excitation chlorophyll fluorometer (M-PEA, Hansatech corporation, uk) with 6 replicates per treatment. The JIP-test analysis was performed according to the formula of Table 1, using: fluorescence at 20. mu.s (O phase, F)o) Fluorescence at 300. mu.s (K phase, F)k) Fluorescence at 2ms (J phase, F)J) Fluorescence at 30ms (phase I, FI) and maximum fluorescence (phase P, F)m). Variable fluorescence FkAccount for FJ-FoRatio W of amplitudek(ii) a Variable fluorescence FIAnd FJAccount for Fp-FoRatio V of amplitudeIAnd VJ(ii) a The trapped exciton transfers an electron to Q in the electron transfer chainAProbability Ψ of other electron acceptors downstreamo(ii) a Photochemical performance index PI based on absorbed light energyABSAnd the like.
Data processing and result analysis
Statistical analysis software SPSS PASW Statistics 18.0 was used as an analysis of variance (ANOVA) for the relevant experimental data, comparing the differences between treatments and plotting with Excel 2016.
The cesium content measurement data are shown in table 3, the results of comparing the cesium enrichment contents of the four plants are shown in fig. 4, and the cesium enrichment coefficients of the four plants are shown in fig. 5.
Chlorophyll fluorescence parameter PI of leavesABSAnd the change is shown in Table 4, four kinds of plant chlorophyll fluorescence parameters PIABSThe comparison results are shown in FIG. 6.
In FIGS. 4 to 6, four bar graphs from left to right are a group of the same treatment concentration, which sequentially represent the four plants of Shanghai Qing, cabbage, broad bean and pea, and four repeated bar graphs represent 4 treatment concentrations (0.1, 0.5, 1, 5 mmol. L)-1)。
TABLE 3 enrichment differences for cesium in four plants after 7d treatment
Table 4 fast chlorophyll fluorescence parameter PI of four plant leaves after 7d treatmentABSAnd variations thereof
Traditional method for identifying cesium enrichment capability of four plants
The traditional method generally uses an enrichment factor (the enrichment factor is Cs of overground part or underground part)+Content of (g/kg)/Cs in the culture system+The concentration (g/L)) of the plant is judged to be strong or weak on the enrichment capacity of the plant to cesium, the smaller the enrichment coefficient is, the weaker the adsorption capacity of the plant to nuclides and heavy metals is, the larger the enrichment coefficient is, the absorption and enrichment of the plant to cesium are indicatedThe stronger the collection capacity. According to the data in fig. 4-5 and table 3, it is shown that, in the four plants tested under the same nuclide concentration, the cesium accumulated in the shanghai green body is the most, the enrichment coefficient is the largest, the strongest enrichment capacity is provided, the cesium accumulation amount and the enrichment capacity of the Chinese cabbage are in the middle, while the cesium accumulated in the broad beans and the peas are the least, the enrichment coefficient is also the smallest, and the enrichment capacity is the weakest.
The method of the invention identifies the cesium enrichment capacity of four plants
According to FIG. 6 and Table 4, different concentrations of cesium treatment resulted in rapid chlorophyll fluorescence kinetics parameters, i.e., performance parameters PI of the reaction centers based on absorptionABSDecrease in and enrichment of the PI of the Hairhiza virginiana with the strongest capacityABSThe reduction range of the Chinese cabbage is the largest, the reduction range is more than 20 percent, the enrichment capacity of the Chinese cabbage to cesium is smaller than that of the Shanghai green and larger than that of the broad bean and the pea, and the PI of the Chinese cabbage isABSThe reduction range of the cesium-enriched broad beans is between that of the broad beans and the peas, the cesium-enriched broad beans and the peas have the minimum enrichment amount, and the PI of the cesium-enriched broad beans and the peas isABSHas the smallest reduction range, and under the treatment of different concentrations, PIABSThe reduction range of the cesium is less than 20%, the cesium enrichment capacity of the broad beans and the cesium enrichment capacity of the peas are similar, and the PI of the broad beans and the cesium enrichment capacity of the peas are similarABSThe magnitude of the drop is similar. The method of the invention has the same conclusion as the traditional method, and has the advantages of rapidness, high efficiency and no damage.
The method can rapidly and nondestructively screen out the plants with strong cesium enrichment capacity under a certain concentration, and can effectively select the plants according to the cesium concentration of the polluted soil to be treated according to the difference of the enrichment capacities of the plants under different concentrations. For example, within 0.1 to 1.0mmol/L, Shanghai Qing can be selected, and around 5.0mmol/L, Shanghai Qing or Chinese cabbage can be selected.
Claims (2)
1. A method for identifying cesium enrichment capacity of plants by using rapid chlorophyll fluorescence kinetic parameters is characterized by comprising the following steps:
1) plant cultivation: treating plants to be measured, which grow to 6-leaf stage, by using cesium-containing nutrient solutions with different concentrations;
2) measurement of chlorophyll fluorescence parameters: measuring a rapid chlorophyll fluorescence kinetic curve and fluorescence parameters of the plant after dark adaptation under different cesium treatment concentrations by using a continuous excitation type chlorophyll fluorescence instrument; before measurement, dark adaptation is carried out on plants for 20-25 min; selecting the middle part of the mature leaf blade which is just completely unfolded for determination, wherein the fluorescence parameters are the minimum fluorescence Fo, the maximum fluorescence Fm, the K-phase fluorescence FK and the J-phase fluorescence FJ;
3) calculating a PIABS value according to the measurement result of the step 2), and calculating a PIABS change value of the cesium treatment group;
4) identifying the cesium enrichment capacity of the plant according to the PIABS change value of the plant leaf in the step 3); screening plants with fluorescence parameter PIABS decreasing amplitude of more than 20% as plants with strong cesium enrichment capacity at corresponding concentration; and screening the plants with the PIABS reduction amplitude less than 20% into the plants with weak cesium enrichment capacity under the corresponding concentration.
2. The method for identifying cesium enrichment in plants with fast chlorophyll fluorescence kinetic parameters according to claim 1, characterized by comprising the following steps:
1) plant cultivation: selecting plants to be tested, growing seedlings in quartz sand and nutrient solution to 4-leaf stage, selecting healthy plants with consistent growth vigor, transplanting the plants into plastic pots containing 10 kg/pot of quartz sand, transplanting 6 plants in each pot, and treating the plants to be tested in 6-leaf stage by using nutrient solution with different cesium concentrations of 0, 0.1, 0.5, 1.0, 5.0 mmol.L-15 repeats;
2) chlorophyll fluorescence kinetic parameter determination: and (3) selecting the middle part of the just completely unfolded mature leaf for determination by adopting a continuous excitation type chlorophyll fluorescence instrument in the seventh day after the cesium-containing nutrient solution is treated, performing dark adaptation for 20-25min before determination, then determining a rapid chlorophyll fluorescence kinetic curve of the dark adapted leaf, and repeating the determination for 6 times in each treatment and obtaining fluorescence parameters.
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