CN113884621A - Method for quantitatively absorbing and utilizing bicarbonate by plants - Google Patents
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Abstract
The invention discloses a method for quantitatively absorbing and utilizing bicarbonate by a plant, which comprises the steps of utilizing a bidirectional isotope tracing culture technology, respectively culturing a blank system and a plant to be tested, measuring stable carbon isotope values in the systems at different time, obtaining the share of the bicarbonate added at different time in the total inorganic carbon in a solution and the volume of a culture solution, calculating the consumption of marked bicarbonate in the two systems at different time, and further obtaining the cumulative consumption of the bicarbonate from air dissolution and the total cumulative consumption of the bicarbonate in the systems; and constructing a linear relation model of the total accumulated consumption of the bicarbonate at different time along with time, acquiring the rate of consuming the total bicarbonate in the system, and further acquiring the rate of absorbing and utilizing the bicarbonate by the plant to be detected. The method can rapidly and nondestructively quantify the absorption and utilization capacity of the whole plant to the heavy carbonate, and can lay a foundation for rapidly determining the karst suitable plants.
Description
Technical Field
The invention relates to a method for quantitatively absorbing and utilizing bicarbonate by plants, belonging to the field of plant physiological information detection technology and ecological environment management.
Background
Plants utilize not only carbon dioxide but also bicarbonate ions. However, although the conventional method for determining the bicarbonate ion content of a plant is also a method using bidirectional isotope labeling culture (wu edgefriend, chen germany, li shui, liu ying, a method for obtaining the inorganic carbon source content of a plant using double markers, CN102511362A, 2012), the experimental conditions of this method are relatively harsh, the control difficulty is high, and the determination is made only by assimilating the bicarbonate content in leaves, but not including the bicarbonate absorption and utilization parts of other organs such as roots and stems, so it is difficult to obtain the bicarbonate absorption and utilization capacity of the whole plant. Therefore, a method for rapidly and nondestructively quantifying the capacity of plants to absorb and utilize bicarbonate is urgently needed.
The plant absorbs and utilizes the bicarbonate, on one hand, the carbonate is driven to perform the corrosion action, the formation of corrosion carbon sink is accelerated, and great contribution is made to carbon neutralization, on the other hand, the photosynthesis and the carbon-nitrogen metabolism are promoted, the growth and development of the plant are facilitated, the carbon sink capacity of the plant is increased, and food and energy are provided for an ecosystem. Thus, the ability of plants to take up and utilize bicarbonate can provide new knowledge of the carbon metabolism of plants on the one hand, and basic data for screening karst-suitable plants on the other hand, and finally provide a scheme for 'carbon peak-reaching' and 'carbon neutralization'. The invention is based on an isotope bidirectional labeling culture method, and obtains the capability of plants to absorb and utilize bicarbonate by comparing the consumption rate of labeled bicarbonate in a culture system with plants.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for quantitatively absorbing and utilizing bicarbonate by plants, which overcomes the defects that the prior art is difficult to rapidly and nondestructively determine the capability of absorbing and utilizing the bicarbonate by the plants and is difficult to determine the capability of absorbing and utilizing the bicarbonate by the whole plants.
The invention adopts the following technical scheme: it comprises the following steps:
1. a method for quantitatively absorbing and utilizing bicarbonate by plants is characterized by comprising the following steps:
firstly, the bicarbonate produced by different manufacturers is measured, and two kinds of delta are selected13Bicarbonate with the C value difference larger than 10 per thousand is used as a tracer for isotope labeling 1 and isotope labeling 2;
secondly, the bicarbonate is added into the nutrient solution respectively, the concentration of the bicarbonate in the nutrient solution is set as c, the pH is the pH required by the condition to be measured, and the original volume of the culture solution is v0Isotopically labelled 1 bicarbonate ion delta in solution13C value of deltaC1Isotopically labelled 2 solution of bicarbonate ion delta13C value of deltaC2；
Thirdly, carrying out bidirectional isotope tracing blank culture and bidirectional isotope tracing culture on the prepared solution;
fourthly, measuring the volume of the solution of the bidirectional isotope tracing blank culture system at different time, and obtaining the portion f of the bicarbonate added at different time in the total inorganic carbon in the solution from the bidirectional isotope tracing blank culture0iAnd volume v of culture solution0iWhere i is the number of samples taken at different times; obtaining the portion f of bicarbonate accounting for total inorganic carbon in the solution added at different time from the culture of the plant to be detected by the bidirectional isotope tracingBiAnd volume v of culture broth1iWhere i is the number of samples taken at different times;
fifthly, acquiring the cumulative consumption m of the marked bicarbonate in the bidirectional isotope labeling blank culture system at different time periodsiAnd the consumption p of labelled bicarbonate in a culture system for the two-way isotopic tracing of the plants to be tested at different timesiWhere i is the number of samples taken at different times;
sixthly, according to the portion f of the bicarbonate which is obtained from the bidirectional isotope tracing blank culture system and is added at different time and accounts for the total inorganic carbon in the solution0iAnd cumulative consumption m of labeled bicarbonate in the two-way isotope labeling blank culture system at different time periodsiObtaining the cumulative consumption n of bicarbonate from air dissolution in the bidirectional isotope tracing blank culture system at different time periodsiHere, thei is the sampling times at different times;
seventhly, according to the portion f of the bicarbonate which is obtained from the culture of the plant to be detected by the two-way isotope tracing and is added at different time and accounts for the total inorganic carbon in the solutionBiAnd cumulative consumption p of labeled bicarbonate in the dual isotope labeled blank culture system at different time periodsiObtaining cumulative consumption q of bicarbonate from air dissolution in a two-way isotope-labelled blank culture system at different time periodsiWhere i is the number of samples taken at different times;
eighth, obtaining the cumulative consumption a of total weight carbonate in the bidirectional isotope labeling blank culture system at different time periods0；
Ninthly, obtaining the cumulative consumption b of total weight carbonate in the culture of the bidirectional isotope labeled plant to be detected at different time periods0；
Tenth, construct a at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system0；
Eleventh, construct b at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the plant culture system to be tested by the bidirectional isotope tracing, according to a linear relation model along with time1；
Twelfth, according to the total bicarbonate consumption rate V in the two-way isotope tracing blank culture system0And the rate V of total bicarbonate consumption in the plant culture system to be detected by bidirectional isotope tracing1Obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detectedb；
Thirteenth, determining the plant fresh weight Fw, the root fresh weight Rfw, the overground part fresh weight Sfw, the plant dry weight Dw, the root dry weight Rdw and the overground part dry weight Sdw of the plant to be tested after the culture is finished;
fourteenth, bicarbonate utilization rates V at different unit masses were obtainedjWhere j may denote the weight based on the fresh plant weight Fw, the fresh root weight Rfw, the fresh overground part weight Sfw, the dry plant weight Dw, the dry root weight Rdw and the overground part weight, respectivelyRate of utilization V of bicarbonate per unit mass of dry weight SdwFW、VRFW、VSFW、VDW、VRDWAnd VSDW。
In the third step, adding culture solution prepared by adding the bicarbonate ions marked by the isotope 1 and the isotope 2 respectively into a culture container without plants, and culturing in an environment to be detected; adding a culture solution prepared by adding isotope-labeled 1 bicarbonate into a culture container with a plant to be tested, and culturing in an environment to be tested; then, the plant to be tested is domesticated and cultured for 1 day, and then culture solution prepared by adding the bicarbonate with the isotope labeling 2 is added into a culture container with the plant to be tested and cultured in the environment to be tested;
in the fourth step, in the two-way isotope tracing blank culture system, the stable carbon isotope composition delta in the nutrient solution marked by two isotopes in the blank culture system under the same processing time is respectively measured13C value delta 01iAnd δ 02iValue, test time point more than 5; delta to be measuredC1、δC2And delta 0 at different times1iAnd δ 02iValue substitution equation
Calculating the part f of the remaining added bicarbonate in the blank culture container in the total inorganic carbon in the solution at different times0iWhere i is the number of samples taken at different times; in a culture system of a plant to be detected by bidirectional isotope tracing, stable carbon isotope composition delta in two isotope-labeled nutrient solutions in the culture system of the plant to be detected under the same processing time is respectively measured13C value delta1iAnd delta1iValue, test time point more than 5; delta to be measuredC1、δC2At delta1iAnd delta1iValue substitution equation
Calculating the remaining carbonate occupation in the culture system container of the plant to be detected by the bidirectional isotope tracing at different timeFraction of total inorganic carbon in solution fBiWhere i is the number of samples taken at different times;
in a fifth step, the cumulative consumption m of labeled bicarbonate in the dual isotope labeled blank culture system at different time periodsiIn the method of (a), mi＝(cv0f00-cv0if0i)-d0 iWherein c is the original concentration of the bicarbonate concentration set, v0iFor the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f00The portion of the marked bicarbonate starting from the solution, d0 iAccumulating the consumption for sampling; in a similar way, the consumption p of the labeled bicarbonate in the culture system of the plant to be tested is isotopically traced in two directions at different timesiBy the process of (a) pi＝(cv0fB0-cv1ifBi)-d1 iWherein c is the original concentration of the bicarbonate concentration set, v1iFor the two-way isotopic tracing of the volume of the culture liquid in the culture system of the plant to be tested at different times, fB0The portion of the marked bicarbonate starting from the solution, d1 iAccumulating the consumption for sampling; where i is the number of samples taken at different times; cumulative consumption d of sampling in bidirectional isotope tracing blank culture system at different time periods0 iThe calculation method comprises the following steps: d0 i＝d0 i-1+cvdf0i,vdFor sampling the sample volume for analysis, where i is the number of samples taken at different times and d0 0Is 0, d0 i-1Accumulating consumption for last sampling; similarly, the cumulative consumption d of the two-way isotope tracing to-be-detected plant in the culture system is sampled at different time periods1 iThe calculation method comprises the following steps: d1 i＝d1 i-1+cvdfBi,vdFor sampling the sample volume for analysis, where i is the number of samples taken at different times and d1 0Is 0, d1 i-1Accumulating consumption for last sampling;
in a sixth step, cumulative consumption n of bicarbonate from air dissolution in a bi-directional isotope labeled blank culture system at different time periodsiThe method comprises the following steps:
in a seventh step, cumulative consumption q of bicarbonate from air dissolution in the culture system of the plant to be tested at different time periodsiThe method comprises the following steps:
in the eighth step, the cumulative consumption a of total carbonate in the dual isotope labeled blank culture system at different time periods0Cumulative consumption m of labeled bicarbonateiAnd cumulative bicarbonate consumption n from air dissolutioniSumming;
in a ninth step, the cumulative consumption b of total carbonate in the culture of the bi-isotopically labelled plants to be tested at different time periods0Cumulative consumption p of bicarbonate for labelingiAnd cumulative consumption q of bicarbonate from air dissolutioniSumming;
in the twelfth step, the speed V of the plant to be tested for absorbing and utilizing the bicarbonate is obtainedbThe method of (A) is as followsb＝V1-V0；
In the fourteenth step, the utilization rate V of bicarbonate per unit massFW、VRFW、VSFW、VDW、VRDWAnd VSDWThe calculation method comprises the following steps: vFW＝Vb/Fw，VRFW＝Vb/Rfw，VSFW＝Vb/Sfw，VDW＝Vb/Dw，VRDW＝Vb/Rdw and VSDW＝Vb/Sdw。
The invention has the following advantages:
1) the method can not only determine the capability of the plant to absorb and utilize the added bicarbonate, but also determine the capability of the plant to absorb and utilize the bicarbonateFrom air CO2The converted bicarbonate of (a);
2) the method can rapidly and nondestructively quantify the capability of the plant to absorb and utilize the bicarbonate;
3) the method can rapidly and nondestructively quantify the absorption and utilization capacity of the whole plant to the bicarbonate;
4) the absorption and utilization capacity of the bicarbonate determined by the method not only comprises the inorganic carbon for photosynthetic assimilation but also comprises the inorganic carbon for non-photosynthetic assimilation, thereby providing a technical basis for the research of other physiological actions of the bicarbonate;
5) the method uses a bidirectional isotope tracing culture technology, so that experimental links such as the isotope fractionation value of the bicarbonate absorbed and utilized by the plant are greatly reduced, the characteristic that the bicarbonate ions absorbed and utilized by the plant are rapidly determined under the condition that the complex mechanism of the bicarbonate absorbed and utilized by the plant is not needed to be known, and the basis is laid for rapidly determining the karst suitable plants.
The basic principle of the invention is as follows:
the strongly fractionated character of stable carbon isotopes is the basis for identifying different inorganic carbon sources. Carbon elements in nature have two stable isotopes:12c and13c, their natural average abundances are 98.89% and 1.11%, respectively. The composition of the stable carbon isotope is usually delta13C (‰) represents delta in nature13The change of C is-90 to +20 per mill. The strong fractionation characteristics of stable carbon isotopes facilitate the identification of different inorganic carbon sources. The mass balance principle, isotope mixing model and chemometric method are the basis for quantitatively identifying different inorganic carbon sources.
The isotope mixture model of the two end-members can be expressed as:
δi＝δCa-fBiδa+fBiδci (1)
where delta isiDelta of inorganic carbon in culture solution after a certain period of time for plant cultivation13C value, δaDelta of inorganic carbon dissolved in the culture solution by carbon dioxide in the air13C value, δciIs delta of bicarbonate ion in the initial broth13C value, fBiThe exogenous bicarbonate radical ions in the culture solution account for the total inorganic carbon source in the culture solution after the plants are cultured for a certain time.
For isotopic label 1, equation (1) represents the following formula:
δ1＝δCa-fB1δa+fB1δC1 (2)
where delta is1To add the first known delta13Adding bicarbonate with C value into nutrient solution to culture plants for a certain time, and adding the bicarbonate into the nutrient solution13C value, δaInorganic carbon delta for allowing carbon dioxide in the atmosphere to enter the culture solution during the period13Average value of C, δC1Is delta of the first bicarbonate13C value, fB1The exogenous bicarbonate ion added for the first time in the culture solution for culturing the plant accounts for the share of the total inorganic carbon source in the culture solution.
For isotopic label 2, equation (1) represents the following formula:
δ2＝δCa-fB2δa+fB2δC2 (3)
where delta is2To add a second known delta13Adding bicarbonate with C value into nutrient solution to culture plants for a certain time, and adding the bicarbonate into the nutrient solution13C value, δaInorganic carbon delta for allowing carbon dioxide in the atmosphere to enter the culture solution during the period13Average value of C, δC2Is delta of the second bicarbonate13C value, fB2The exogenous bicarbonate ion added for the second kind in the culture solution after a certain period of time for plant cultivation accounts for the share of the total inorganic carbon source in the culture solution.
(2) And (3) in two equations, fB＝fB1＝fB2(2) and (3) solving simultaneously
F calculated hereBThe value is the share of exogenous bicarbonate ion added in the culture solution in the total inorganic carbon source in the culture solution after the culture solution cultures the plant for a certain time.
In fact, for the same reason, in a plant-free system, the bicarbonate added can also be associated with CO from the atmosphere2The exchange of the converted bicarbonate consumes the added bicarbonate, and after a period of time, the exogenous bicarbonate ions added to the culture solution account for the fraction f of the total inorganic carbon sources in the culture solution0The formula (5) may be used:
here, δ 01And δ 02The values respectively represent the stable carbon isotope composition delta in the nutrient solution marked by two isotopes in the blank culture system without plants under the same treatment time13C value delta 01And δ 02The value is obtained.
Consumption p of labelled bicarbonate in a cultivation system for the bidirectional isotopic tracing of plants to be tested at different timesiBy the process of (a) pi＝(cv0fB0-cv1ifBi)-d1 iWherein c is the original concentration of the bicarbonate concentration set, v1iFor the two-way isotopic tracing of the volume of the culture liquid in the culture system of the plant to be tested at different times, fB0The portion of the marked bicarbonate starting from the solution, d1 iAccumulating the consumption for sampling; where i is the number of samples taken at different times; similarly, the cumulative consumption m of labeled bicarbonate in the two-way isotope labeling blank culture system at different time periodsiIn the method of (a), mi＝(cv0f00-cv0if0i)-d0 iWherein c is the original concentration of the bicarbonate concentration set, v0iFor the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f00For marking by solution initiationProportion of bicarbonate, d0 iThe consumption is accumulated for sampling.
Cumulative consumption d of sampling in bidirectional isotope tracing blank culture system at different time periods0 iThe calculation method comprises the following steps: d0 i＝d0 i-1+cvdf0i,vdFor sampling the sample volume for analysis, where i is the number of samples taken at different times and d0 iIs 0, d0 i-1Accumulating consumption for last sampling; similarly, the cumulative consumption d of the two-way isotope tracing to-be-detected plant in the culture system is sampled at different time periods1 iThe calculation method comprises the following steps: d1 i＝d1 i-1+cvdfBi,vdFor sampling the sample volume for analysis, where i is the number of samples taken at different times and d1 iIs 0, d1 i-1Accumulating consumption for last sampling;
cumulative consumption q of bicarbonate from air dissolution in the culture system of the plants to be tested at different time periodsiThe method comprises the following steps:
similarly, cumulative consumption n of bicarbonate from air dissolution in a two-way isotope labeling blank culture system at different time periodsiThe method comprises the following steps:
cumulative consumption b of total carbonate in culture of bi-directional isotopically labeled plant under different time periods0Cumulative consumption p of bicarbonate for labelingiAnd cumulative consumption q of bicarbonate from air dissolutioniSumming; similarly, the cumulative consumption a of total carbonate in the dual isotope labeling blank culture system at different time periods0Cumulative consumption m of labeled bicarbonateiAnd cumulative bicarbonate consumption n from air dissolutioniAnd (4) summing.
From a number of experiments, the results show that the cumulative consumption of total carbonate in the system is directly proportional to time. The difference between the consumption rate of total carbonate in a plant-based system and the consumption rate of total carbonate in a plant-free system is the rate of absorption and utilization of bicarbonate by the plant.
Detailed Description
Examples of the invention: it comprises the following steps:
1. a method for quantitatively absorbing and utilizing bicarbonate by plants is characterized by comprising the following steps:
firstly, the bicarbonate produced by different manufacturers is measured, and two kinds of delta are selected13Bicarbonate with the C value difference larger than 10 per thousand is used as a tracer for isotope labeling 1 and isotope labeling 2;
secondly, the bicarbonate is added into the nutrient solution respectively, the concentration of the bicarbonate in the nutrient solution is set as c, the pH is the pH required by the condition to be measured, and the original volume of the culture solution is v0Isotopically labelled 1 bicarbonate ion delta in solution13C value of deltaC1Isotopically labelled 2 solution of bicarbonate ion delta13C value of deltaC2；
Thirdly, carrying out bidirectional isotope tracing blank culture and bidirectional isotope tracing culture on the prepared solution;
fourthly, measuring the volume of the solution of the bidirectional isotope tracing blank culture system at different time, and obtaining the portion f of the bicarbonate added at different time in the total inorganic carbon in the solution from the bidirectional isotope tracing blank culture0iAnd volume v of culture solution0iWhere i is the number of samples taken at different times; obtaining the portion f of bicarbonate accounting for total inorganic carbon in the solution added at different time from the culture of the plant to be detected by the bidirectional isotope tracingBiAnd volume v of culture broth1iWhere i is the number of samples taken at different times;
fifthly, acquiring the cumulative consumption m of the marked bicarbonate in the bidirectional isotope labeling blank culture system at different time periodsiAnd the weight of the marker in the culture system of the plant to be tested for the two-way isotopic tracing at different timesCarbonate consumption piWhere i is the number of samples taken at different times;
sixthly, according to the portion f of the bicarbonate which is obtained from the bidirectional isotope tracing blank culture system and is added at different time and accounts for the total inorganic carbon in the solution0iAnd cumulative consumption m of labeled bicarbonate in the two-way isotope labeling blank culture system at different time periodsiObtaining the cumulative consumption n of bicarbonate from air dissolution in the bidirectional isotope tracing blank culture system at different time periodsiWhere i is the number of samples taken at different times;
seventhly, according to the portion f of the bicarbonate which is obtained from the culture of the plant to be detected by the two-way isotope tracing and is added at different time and accounts for the total inorganic carbon in the solutionBiAnd cumulative consumption p of labeled bicarbonate in the dual isotope labeled blank culture system at different time periodsiObtaining cumulative consumption q of bicarbonate from air dissolution in a two-way isotope-labelled blank culture system at different time periodsiWhere i is the number of samples taken at different times;
eighth, obtaining the cumulative consumption a of total weight carbonate in the bidirectional isotope labeling blank culture system at different time periods0；
Ninthly, obtaining the cumulative consumption b of total weight carbonate in the culture of the bidirectional isotope labeled plant to be detected at different time periods0；
Tenth, construct a at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system0；
Eleventh, construct b at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the plant culture system to be tested by the bidirectional isotope tracing, according to a linear relation model along with time1；
Twelfth, according to the total bicarbonate consumption rate V in the two-way isotope tracing blank culture system0And the rate V of total bicarbonate consumption in the plant culture system to be detected by bidirectional isotope tracing1Obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detectedb；
Thirteenth, determining the plant fresh weight Fw, the root fresh weight Rfw, the overground part fresh weight Sfw, the plant dry weight Dw, the root dry weight Rdw and the overground part dry weight Sdw of the plant to be tested after the culture is finished;
fourteenth, the utilization rate V of bicarbonate at different unit masses is obtainedjWhere j may denote the utilization rate V of bicarbonate per unit mass based on the plant fresh weight Fw, root fresh weight Rfw, overground part fresh weight Sfw, plant dry weight Dw, root dry weight Rdw and overground part dry weight Sdw, respectivelyFW、VRFW、VSFW、VDW、VRDWAnd VSDW。
In the third step, adding culture solution prepared by adding the bicarbonate ions marked by the isotope 1 and the isotope 2 respectively into a culture container without plants, and culturing in an environment to be detected; adding a culture solution prepared by adding isotope-labeled 1 bicarbonate into a culture container with a plant to be tested, and culturing in an environment to be tested; then, the plant to be tested is domesticated and cultured for 1 day, and then culture solution prepared by adding the bicarbonate with the isotope labeling 2 is added into a culture container with the plant to be tested and cultured in the environment to be tested;
in the fourth step, in the two-way isotope tracing blank culture system, the stable carbon isotope composition delta in the nutrient solution marked by two isotopes in the blank culture system under the same processing time is respectively measured13C value delta 01iAnd δ 02iValue, test time point more than 5; delta to be measuredC1、δC2And delta 0 at different times1iAnd δ 02iValue substitution equation
Calculating the part f of the remaining added bicarbonate in the blank culture container in the total inorganic carbon in the solution at different times0iWhere i is the number of samples taken at different times; respectively measuring the same processing time in a culture system of a plant to be detected by bidirectional isotope tracingThe stable carbon isotope composition delta in the nutrient solution marked by two isotopes in the culture system of the plant to be detected13C value delta1iAnd delta1iValue, test time point more than 5; delta to be measuredC1、δC2At delta1iAnd delta1iValue substitution equation
Calculating the part f of the bicarbonate added into the culture system container of the plant to be detected in the two-way isotope tracing way to account for the total inorganic carbon in the solution at different timeBiWhere i is the number of samples taken at different times;
in a fifth step, the cumulative consumption m of labeled bicarbonate in the dual isotope labeled blank culture system at different time periodsiIn the method of (a), mi＝(cv0f00-cv0if0i)-d0 iWherein c is the original concentration of the bicarbonate concentration set, v0iFor the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f00The portion of the marked bicarbonate starting from the solution, d0 iAccumulating the consumption for sampling; in a similar way, the consumption p of the labeled bicarbonate in the culture system of the plant to be tested is isotopically traced in two directions at different timesiBy the process of (a) pi＝(cv0fB0-cv1ifBi)-d1 iWherein c is the original concentration of the bicarbonate concentration set, v1iFor the two-way isotopic tracing of the volume of the culture liquid in the culture system of the plant to be tested at different times, fB0The portion of the marked bicarbonate starting from the solution, d1 iAccumulating the consumption for sampling; where i is the number of samples taken at different times; cumulative consumption d of sampling in bidirectional isotope tracing blank culture system at different time periods0 iThe calculation method comprises the following steps: d0 i＝d0 i-1+cvdf0i,vdFor sampling the sample volume for analysis, where i is the number of samples taken at different times and d0 0Is 0, d0 i-1Accumulating consumption for last sampling; similarly, the cumulative consumption d of the two-way isotope tracing to-be-detected plant in the culture system is sampled at different time periods1 iThe calculation method comprises the following steps: d1 i＝d1 i-1+cvdfBi,vdFor sampling the sample volume for analysis, where i is the number of samples taken at different times and d1 0Is 0, d1 i-1Accumulating consumption for last sampling;
in a sixth step, cumulative consumption n of bicarbonate from air dissolution in a bi-directional isotope labeled blank culture system at different time periodsiThe method comprises the following steps:
in a seventh step, cumulative consumption q of bicarbonate from air dissolution in the culture system of the plant to be tested at different time periodsiThe method comprises the following steps:
in the eighth step, the cumulative consumption a of total carbonate in the dual isotope labeled blank culture system at different time periods0Cumulative consumption m of labeled bicarbonateiAnd cumulative bicarbonate consumption n from air dissolutioniSumming;
in a ninth step, the cumulative consumption b of total carbonate in the culture of the bi-isotopically labelled plants to be tested at different time periods0Cumulative consumption p of bicarbonate for labelingiAnd cumulative consumption q of bicarbonate from air dissolutioniSumming;
in the twelfth step, the speed V of the plant to be tested for absorbing and utilizing the bicarbonate is obtainedbThe method of (A) is as followsb＝V1-V0；
In the fourteenth step, VFW、VRFW、VSFW、VDW、VRDWAnd VSDWThe calculation method comprises the following steps: vFW＝Vb/Fw，VRFW＝Vb/Rfw，VSFW＝Vb/Sfw，VDW＝Vb/Dw，VRDW＝Vb/Rdw and VSDW＝Vb/Sdw。
Examples
Respectively by delta13Bicarbonate with C of 4.00 per mill and-2707 per mill (PDB) is added into the improved Hoagland nutrient solution to prepare isotope labeled 1 nutrient solution and isotope labeled 2 nutrient solution. Respectively adding the tracer agents of isotope labeling 1 and isotope labeling 2 into the Hoagland nutrient solution, setting the concentration of bicarbonate in the nutrient solution to be 10mM, the pH to be 8.30 and the original volume to be 2000ml, and enabling the hydrogen carbonate ions in the nutrient solution of the isotope labeling 1 to be delta13C value of deltaC1Isotopically labelled 2 nutrient solution13C value of deltaC2(ii) a Adding culture solution prepared by adding isotope labeled 1 bicarbonate ion and isotope labeled 2 bicarbonate ion into plant-free culture container, culturing in environment to be tested, measuring solution volume of blank culture system at the same treatment time, and stable carbon isotope composition delta in two isotope labeled nutrient solutions in the system13C value delta 01iAnd δ 02iThe value is that when the isotope is measured, the sampling volume is 65ml, namely the sampling volume, and the part f of the rest added bicarbonate in the blank culture container, which accounts for the total inorganic carbon in the solution, is calculated0iSubsequently, the cumulative consumption m of labeled bicarbonate in the blank culture system was calculatediAnd cumulative bicarbonate consumption n from air dissolutioniObtaining the cumulative consumption a of carbonate in the blank culture system0Construction of a at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system0。
Adding a culture solution prepared by adding isotope-labeled 1 bicarbonate into a culture container with a plant to be tested, and culturing in an environment to be tested; then, the plant to be tested is acclimatized and cultured for 1 day, and thenAdding culture solution prepared by adding isotope labeled 2 bicarbonate into culture container with the plant to be tested, culturing in environment to be tested, determining solution volume in culture system of plant to be tested at the same treatment time, and stable carbon isotope composition delta in two isotope labeled nutrient solutions in the system13C value delta1iAnd delta1iCalculating the part f of the rest of the added bicarbonate in the culture system of the plant to be detected in the total inorganic carbon in the solutionBiSubsequently, the cumulative consumption p of the marked bicarbonate in the culture system of the plant to be tested is calculatediAnd cumulative consumption q of bicarbonate from air dissolutioniObtaining the cumulative consumption b of total carbonate in the culture system of the plant to be tested0Construction of b at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system1Further obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detectedb(ii) a Determining the plant fresh weight Fw, the root fresh weight Rfw, the overground part fresh weight Sfw, the plant dry weight Dw, the root dry weight Rdw and the overground part dry weight Sdw of the plant to be tested after the culture is finished; calculating the utilization rate V of bicarbonate in unit mass based on the plant fresh weight Fw, the root fresh weight Rfw, the overground part fresh weight Sfw, the plant dry weight Dw, the root dry weight Rdw and the overground part dry weight SdwFW、VRFW、VSFW、VDW、VRDWAnd VSDW。
Example 1 the plant material is broussonetia papyrifera with small root-to-crown ratio, the culture condition is normal illumination, and the culture time is 48 hours;
example 2 the plant material is mulberry, the cultivation condition is normal illumination, the cultivation time is 48 hours;
example 3 the plant material is broussonetia papyrifera with a large root-to-crown ratio, the culture condition is continuous illumination, and the culture time is 24 hours;
example 4 the plant material was broussonetia papyrifera with a large root-to-crown ratio, the cultivation conditions were continuous dark and the cultivation time was 24 hours;
example 5 the plant material was mulberry, the cultivation conditions were continuous light irradiation, cultivation time 24 hours;
example 6 the plant material was mulberry, the cultivation conditions were continuous darkness and the cultivation time was 24 hours.
The process of the present invention is shown below in tabular form.
TABLE 1 indexes of isotope value, cumulative consumption of labeled bicarbonate and cumulative consumption of total carbonate obtained from a two-way isotope labeling blank culture System
Table 2 indexes of isotope value, cumulative consumption of labeled bicarbonate and cumulative consumption of total carbonate obtained from culturing of plant to be tested by two-way isotope labeling in example 1
Table 3 indexes of isotope value, labeled cumulative consumption of labeled bicarbonate and cumulative consumption of total carbonate obtained from culturing of plant to be tested by two-way isotope labeling in example 2
Table 4 indexes of isotope value, labeled cumulative consumption of labeled bicarbonate and cumulative consumption of total carbonate obtained from culturing of plant to be tested by two-way isotope labeling in example 3
Table 5 indexes of isotope value, labeled cumulative consumption of labeled bicarbonate and cumulative consumption of total carbonate obtained from culturing of plant to be tested by two-way isotope labeling in example 4
TABLE 6 indexes of isotope value, cumulative consumption of labeled bicarbonate and cumulative consumption of total bicarbonate obtained from culturing of plants to be tested by bidirectional isotope labeling in example 5
Table 7 indexes of isotope value, cumulative consumption of labeled bicarbonate and cumulative consumption of total carbonate obtained from culturing of plant to be tested by bidirectional isotope labeling in example 6
TABLE 8 cumulative consumption of total carbonate in air and examples (a)0、b0) Model of relationship with time
TABLE 9 Biomass (g) of different samples of different examples
TABLE Ten different examples the utilization rates V of bicarbonate per unit mass for different samplesFW、VRFW、VSFW、VDW、VRDWAnd VSDW(μmol/h.g)
The implementation effect of the invention is as follows:
as can be seen from Table 8, each sample of each example has an excellent linear proportional relationship, and the square values of the correlation coefficients exceed 0.94, indicating that the results obtained by the invention are credible. As can be seen from table 10, the ability of different plant species to assimilate and utilize bicarbonate is different. The ability of broussonetia papyrifera to absorb and utilize bicarbonate is less than that of mulberry, and light can promote the utilization of bicarbonate, which is consistent with the fact that broussonetia papyrifera is a karst plant. The bicarbonate has higher alkalinity, is absorbed too much and is not utilized by plants, and is bound to damage the plants, so that the absorbed bicarbonate of the paper mulberry is smaller than that of the mulberry, and the absorbed bicarbonate can be used as a carbon source for the utilization of photosynthetic organs, thereby further reducing the damage of the bicarbonate to the plants. At the same time, the absorption of the bicarbonate by the plants is promoted under light, which indicates that the absorption of the bicarbonate is transported to the overground part and the leaves along with the transpiration liquid flow through the xylem, and is also consistent with the prior knowledge. The paper mulberry is absorbed in a proper amount and utilized in a large amount, so that the damage is greatly reduced, and meanwhile, the paper mulberry is also used as an inorganic carbon source to be utilized by plants, which is the important embodiment of karst adaptability of the paper mulberry. However, the mulberry tree is difficult to grow in the karst environment, which is related to the characteristic of high absorption and low utilization, and the characteristic is also consistent with the reality. The adaptability of paper mulberry to higher bicarbonate may also be related to its property of proper absorption and large-scale utilization.
Another fact that the absorption of bicarbonate by broussonetia papyrifera is less is that under the stress of high pH and high bicarbonate, broussonetia papyrifera secretes (produces) more organic acid secretion than mulberry, lowers pH in vascular bundle environment, and causes the absorption of bicarbonate to be reduced.
The fact that the root cap has better karst adaptability than the plant type with the smaller root cap than the big type is also verified by the results measured by the invention, because the root cap absorbs and utilizes less bicarbonate than the plant type with the smaller root cap than the big type, so that the free bicarbonate is less in the body due to the intake of the root cap, and the toxicity of the bicarbonate is reduced to a very small degree. The karst-adaptive property of the plant can be determined by measuring the absorption and utilization of the plant and the assimilation property of inorganic carbon.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. A method for quantitatively absorbing and utilizing bicarbonate by plants is characterized by comprising the following steps:
firstly, the bicarbonate produced by different manufacturers is measured, and two kinds of delta are selected13Bicarbonate with the C value difference larger than 10 per thousand is used as a tracer for isotope labeling 1 and isotope labeling 2;
secondly, the bicarbonate is added into the nutrient solution respectively, the concentration of the bicarbonate in the nutrient solution is set as c, the pH is the pH required by the condition to be measured, and the original volume of the culture solution is v0Isotopically labelled 1 bicarbonate ion delta in solution13C value of deltaC1Isotopically labelled 2 solution of bicarbonate ion delta13C value of deltaC2；
Thirdly, carrying out bidirectional isotope tracing blank culture and bidirectional isotope tracing culture on the prepared solution;
fourthly, measuring the volume of the solution of the bidirectional isotope tracing blank culture system at different time, and obtaining the portion f of the bicarbonate added at different time in the total inorganic carbon in the solution from the bidirectional isotope tracing blank culture0iAnd volume v of culture solution0iI is the sampling times at different times; obtaining the portion f of bicarbonate accounting for total inorganic carbon in the solution added at different time from the culture of the plant to be detected by the bidirectional isotope tracingBiAnd volume v of culture broth1iI is the sampling times at different times;
fifthly, acquiring the cumulative consumption m of the marked bicarbonate in the bidirectional isotope labeling blank culture system at different time periodsiAnd the consumption p of labelled bicarbonate in a culture system for the two-way isotopic tracing of the plants to be tested at different timesiI is the sampling times at different times;
sixthly, according to the portion f of the bicarbonate which is obtained from the bidirectional isotope tracing blank culture system and is added at different time and accounts for the total inorganic carbon in the solution0iAnd bidirectional isotope tracing blank culture system under different time periodsMarked bicarbonate cumulative consumption miObtaining the cumulative consumption n of bicarbonate from air dissolution in the bidirectional isotope tracing blank culture system at different time periodsiI is the sampling times at different times;
seventhly, according to the portion f of the bicarbonate which is obtained from the culture of the plant to be detected by the two-way isotope tracing and is added at different time and accounts for the total inorganic carbon in the solutionBiAnd cumulative consumption p of labeled bicarbonate in the dual isotope labeled blank culture system at different time periodsiObtaining cumulative consumption q of bicarbonate from air dissolution in a two-way isotope-labelled blank culture system at different time periodsiI is the sampling times at different times;
eighth, obtaining the cumulative consumption a of total weight carbonate in the bidirectional isotope labeling blank culture system at different time periods0；
Ninthly, obtaining the cumulative consumption b of total weight carbonate in the culture of the bidirectional isotope labeled plant to be detected at different time periods0；
Tenth, construct a at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system0；
Eleventh, construct b at different times0Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the plant culture system to be tested by the bidirectional isotope tracing, according to a linear relation model along with time1；
Twelfth, according to the total bicarbonate consumption rate V in the two-way isotope tracing blank culture system0And the rate V of total bicarbonate consumption in the plant culture system to be detected by bidirectional isotope tracing1Obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detectedb；
Thirteenth, determining the plant fresh weight Fw, the root fresh weight Rfw, the overground part fresh weight Sfw, the plant dry weight Dw, the root dry weight Rdw and the overground part dry weight Sdw of the plant to be tested after the culture is finished;
fourteenth, obtaining the values at different unit massesBicarbonate utilization rate VjWhere j may denote the utilization rate V of bicarbonate per unit mass based on the plant fresh weight Fw, root fresh weight Rfw, overground part fresh weight Sfw, plant dry weight Dw, root dry weight Rdw and overground part dry weight Sdw, respectivelyFW、VRFW、VSFW、VDW、VRDWAnd VSDW。
2. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: said two-way isotope labeled blank culture in the third step: adding culture solution prepared by adding bicarbonate ions marked by isotope 1 and bicarbonate ions marked by isotope 2 into a plant-free culture container, and culturing in an environment to be detected; the culture method of the bidirectional isotope labeling plant to be tested in the third step is that firstly, a culture solution prepared by adding the bicarbonate with the isotope labeling 1 is added into a culture container with the plant to be tested, and the plant to be tested is cultured in an environment to be tested; then, the plant to be tested is domesticated and cultured for 1 day, and then a culture solution prepared by adding the bicarbonate with the isotope labeling 2 is added into a culture container with the plant to be tested and cultured in the environment to be tested.
3. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: in the fourth step, the part f of the bicarbonate added at different time periods in the total inorganic carbon in the solution is obtained from the bidirectional isotope labeling blank culture0iThe method comprises respectively measuring the stable carbon isotope composition delta in two isotope-labeled nutrient solutions in a blank culture system under the same treatment time in a bidirectional isotope-labeled blank culture system13C value delta 01iAnd δ 02iValue, test time point more than 5; delta to be measuredC1、δC2And delta 0 at different times1iAnd δ 02iValue substitution equation
Calculating the part f of the remaining added bicarbonate in the blank culture container in the total inorganic carbon in the solution at different times0iI is the sampling times at different times; in the fourth step, the part f of the bicarbonate which is added at different time and accounts for the total inorganic carbon in the solution is obtained from the culture of the bidirectional isotope labeled plant to be detectedBiThe method comprises the steps of respectively measuring the stable carbon isotope composition delta in two isotope-labeled nutrient solutions in the culture system of the plant to be detected in the same processing time in the culture system of the plant to be detected by bidirectional isotope tracing13C value delta1iAnd delta1iValue, test time point more than 5; delta to be measuredC1、δC2At delta1iAnd delta1iValue substitution equation
Calculating the part f of the bicarbonate added into the culture system container of the plant to be detected in the two-way isotope tracing way to account for the total inorganic carbon in the solution at different timeBiAnd i is the sampling times at different times.
4. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: obtaining the cumulative consumption m of labeled bicarbonate in the dual isotope labeled blank culture system at different time periods in the fifth stepiIn the method of (a), mi＝(cv0f00-cv0if0i)-d0 iWherein c is the original concentration of the bicarbonate concentration set, v0iFor the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f00The portion of the marked bicarbonate starting from the solution, d0 iAccumulating the consumption for sampling; in a similar way, the consumption p of the labeled bicarbonate in the culture system of the plant to be tested is isotopically traced in two directions at different timesiBy the process of (a) pi＝(cv0fB0-cv1ifBi)-d1 iWherein c is the bicarbonate concentration setOriginal concentration, v1iFor the two-way isotopic tracing of the volume of the culture liquid in the culture system of the plant to be tested at different times, fB0The portion of the marked bicarbonate starting from the solution, d1 iAccumulating the consumption for sampling; i is the number of samples taken at different times.
5. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 4, wherein: cumulative consumption d of sampling in bidirectional isotope tracing blank culture system at different time periods0 iThe calculation method comprises the following steps: d0 i＝d0 i-1+cvdf0i,vdFor sampling volume in sampling analysis, i is the number of sampling times at different times, and d0 0Is 0, d0 i-1Accumulating consumption for last sampling; similarly, the cumulative consumption d of the two-way isotope tracing to-be-detected plant in the culture system is sampled at different time periods1 iThe calculation method comprises the following steps: d1 i＝d1 i-1+cvdfBi,vdFor sampling volume in sampling analysis, i is the number of sampling times at different times, and d1 0Is 0, d1 i-1The consumption is accumulated for the last sampling.
6. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: obtaining cumulative consumption n of bicarbonate from air dissolution in the dual isotope labeled blank culture system at different time periods as described in the sixth stepiThe method comprises the following steps:
cumulative consumption q of bicarbonate from air solubilization in the culture system for obtaining plants to be tested at different time periods as described in the seventh stepiThe method comprises the following steps:
7. the method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: cumulative consumption a of total carbonate in the dual isotope labeled blank culture system at said different time periods in the eighth step0Cumulative consumption m of labeled bicarbonateiAnd cumulative bicarbonate consumption n from air dissolutioniAnd (4) summing.
8. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: cumulative consumption b of total carbonate in the culture of the bi-isotopically labelled test plants at said different time periods of step nine0Cumulative consumption p of bicarbonate for labelingiAnd cumulative consumption q of bicarbonate from air dissolutioniAnd (4) summing.
9. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: in the twelfth step, the speed V of the plant to be tested for absorbing and utilizing the bicarbonate is obtainedbThe method of (A) is as followsb＝V1-V0。
10. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: said obtaining of the rate of utilization of bicarbonate per unit mass V in the fourteenth stepFW、VRFW、VSFW、VDW、VRDWAnd VSDWThe calculation method comprises the following steps: vFW＝Vb/Fw，VRFW＝Vb/Rfw，VSFW＝Vb/Sfw，VDW＝Vb/Dw，VRDW＝Vb/Rdw and VSDW＝Vb/Sdw。
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