CN113884621B - 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 volunteer 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 selected 13 Bicarbonate 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 v 0 Isotopically labelled 1 bicarbonate ion delta in solution 13 C value of delta C1 Isotopically labelled 2 solution of bicarbonate ion delta 13 C value of delta C2 ；
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 culture 0i And volume v of culture solution 0i Where i is the number of samples taken at different times; from two to twoObtaining the portion f of bicarbonate accounting for the total inorganic carbon in the solution added at different time in the culture of the isotope-labeled plant to be detected Bi And volume v of culture broth 1i Where 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 periods i And the consumption p of labelled bicarbonate in a culture system for the two-way isotopic tracing of the plants to be tested at different times i Where 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 solution 0i And cumulative consumption m of labeled bicarbonate in the two-way isotope labeling blank culture system at different time periods i Obtaining the cumulative consumption n of bicarbonate from air dissolution in the bidirectional isotope tracing blank culture system at different time periods i Where 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 solution Bi And cumulative consumption p of labeled bicarbonate in the dual isotope labeled blank culture system at different time periods i Obtaining cumulative consumption q of bicarbonate from air dissolution in a two-way isotope-labelled blank culture system at different time periods i Where 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 periods 0 ；
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 periods 0 ；
Tenth, construct a at different times 0 Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system 0 ；
Eleventh, construct b at different times 0 Obtaining 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 time 1 ；
Twelfth, according to the total bicarbonate consumption rate V in the two-way isotope tracing blank culture system 0 And the rate V of total bicarbonate consumption in the plant culture system to be detected by bidirectional isotope tracing 1 Obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detected b ；
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 obtained j Where 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, respectively FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW 。
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 measured 13 C value delta 0 1i And δ 0 2i Value, test time point more than 5; delta to be measured C1 、δ C2 And delta 0 at different times 1i And δ 0 2i Value substituting 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 times 0i Where 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 measured 13 C value delta 1i And delta 1i Value, test time point more than 5; delta to be measured C1 、δ C2 At delta 1i And delta 1i Value 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 time Bi Where 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 periods i In the method of (a), m i ＝(cv 0 f 00 -cv 0i f 0i )-d 0 i Wherein c is the original concentration of the bicarbonate concentration set, v 0i For the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f 00 The portion of the marked bicarbonate starting from the solution, d 0 i Accumulating 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 times i By the process of (a) p i ＝(cv 0 f B0 -cv 1i f Bi )-d 1 i Wherein c is the original concentration of the bicarbonate concentration set, v 1i For 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, f B0 The portion of the marked bicarbonate starting from the solution, d 1 i Accumulating 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 periods 0 i The calculation method comprises the following steps: d 0 i ＝d 0 i-1 +cv d f 0i ,v d For sampling the sample volume for analysis, where i is the number of samples taken at different times and d 0 0 Is 0, d 0 i-1 Accumulating 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 periods 1 i The calculation method comprises the following steps: d 1 i ＝d 1 i-1 +cv d f Bi ,v d For sampling the sample volume for analysis, where i is the number of samples taken at different times and d 1 0 Is 0, d 1 i-1 Accumulating 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 periods i The 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 periods i The 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 periods 0 Cumulative consumption m of labeled bicarbonate i And cumulative bicarbonate consumption n from air dissolution i Summing;
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 periods 0 Cumulative consumption p of bicarbonate for labeling i And cumulative consumption q of bicarbonate from air dissolution i Summing;
in the twelfth step, the speed V of the plant to be tested for absorbing and utilizing the bicarbonate is obtained b The method of (A) is as follows b ＝V 1 -V 0 ；
In the fourteenth step, the utilization rate V of bicarbonate per unit mass FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW The calculation method comprises the following steps: v FW ＝V b /Fw，V RFW ＝V b /Rfw，V SFW ＝V b /Sfw，V DW ＝V b /Dw，V RDW ＝V b /Rdw and V SDW ＝V b /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 CO from air 2 The 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:
strong fractionation of stable carbon isotopesCharacterization is the basis for identifying different inorganic carbon sources. Carbon elements in nature have two stable isotopes: 12 c and 13 c, their natural average abundances are 98.89% and 1.11%, respectively. The composition of the stable carbon isotope is usually delta 13 C (‰) represents delta in nature 13 The 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:
δ i ＝δ Ca -f Bi δ a +f Bi δ ci (1)
where delta is i Delta of inorganic carbon in culture solution after a certain period of time for plant cultivation 13 C value, δ a Delta of inorganic carbon dissolved in the culture solution by carbon dioxide in the air 13 C value, δ ci Is delta of bicarbonate ion in the initial broth 13 C value, f Bi The 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:
δ 1 ＝δ Ca -f B1 δ a +f B1 δ C1 (2)
where delta is 1 To add the first known delta 13 Adding bicarbonate with C value into nutrient solution to culture plants for a certain time, and adding the bicarbonate into the nutrient solution 13 C value, δ a Inorganic carbon delta for allowing carbon dioxide in the atmosphere to enter the culture solution during the period 13 Average value of C, δ C1 Is delta of the first bicarbonate 13 C value, f B1 The 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:
δ 2 ＝δ Ca -f B2 δ a +f B2 δ C2 (3)
where delta is 2 To add a second known delta 13 Adding bicarbonate with C value into nutrient solution to culture plants for a certain time, and adding the bicarbonate into the nutrient solution 13 C value, δ a Inorganic carbon delta for allowing carbon dioxide in the atmosphere to enter the culture solution during the period 13 Average value of C, δ C2 Is delta of the second bicarbonate 13 C value, f B2 The 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, f B ＝f B1 ＝f B2 (2) and (3) solving simultaneously
F calculated here B The 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 atmosphere 2 The 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 solution 0 The formula (5) may be used:
here, δ 0 1 And δ 0 2 The 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 time 13 C value delta 0 1 And δ 0 2 The value is obtained.
Is not simultaneousConsumption p of labelled bicarbonate in a cultivation system for indirectly bi-directional isotopically tracing plants to be tested i By the process of (a) p i ＝(cv 0 f B0 -cv 1i f Bi )-d 1 i Wherein c is the original concentration of the bicarbonate concentration set, v 1i For 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, f B0 The portion of the marked bicarbonate starting from the solution, d 1 i Accumulating 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 periods i In the method of (a), m i ＝(cv 0 f 00 -cv 0i f 0i )-d 0 i Wherein c is the original concentration of the bicarbonate concentration set, v 0i For the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f 00 The portion of the marked bicarbonate starting from the solution, d 0 i The consumption is accumulated for sampling.
Cumulative consumption d of sampling in bidirectional isotope tracing blank culture system at different time periods 0 i The calculation method comprises the following steps: d 0 i ＝d 0 i-1 +cv d f 0i ,v d For sampling volume in the analysis, where i is the number of samplings at different times and d 0 i Is 0, d 0 i-1 Accumulating 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 periods 1 i The calculation method comprises the following steps: d 1 i ＝d 1 i-1 +cv d f Bi ,v d For sampling the sample volume for analysis, where i is the number of samples taken at different times and d 1 i Is 0, d 1 i-1 Accumulating 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 periods i The 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 periods i The method comprises the following steps:
cumulative consumption b of total carbonate in culture of bi-directional isotopically labeled plant under different time periods 0 Cumulative consumption p of bicarbonate for labeling i And cumulative consumption q of bicarbonate from air dissolution i Summing; similarly, the cumulative consumption a of total carbonate in the dual isotope labeling blank culture system at different time periods 0 Cumulative consumption m of labeled bicarbonate i And cumulative bicarbonate consumption n from air dissolution i And (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 selected 13 Bicarbonate 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 v 0 Isotopically labelled 1 bicarbonate ion delta in solution 13 C value of delta C1 Isotopically labelled 2 solution of bicarbonate ion delta 13 C value of delta C2 ；
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 culture 0i And volume v of culture solution 0i Where 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 tracing Bi And volume v of culture broth 1i Where 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 periods i And the consumption p of labelled bicarbonate in a culture system for the two-way isotopic tracing of the plants to be tested at different times i Where 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 solution 0i And cumulative consumption m of labeled bicarbonate in the two-way isotope labeling blank culture system at different time periods i Obtaining the cumulative consumption n of bicarbonate from air dissolution in the bidirectional isotope tracing blank culture system at different time periods i Where 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 solution Bi And cumulative consumption p of labeled bicarbonate in the dual isotope labeled blank culture system at different time periods i Obtaining cumulative consumption q of bicarbonate from air dissolution in a two-way isotope-labelled blank culture system at different time periods i Where 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 periods 0 ；
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 periods 0 ；
Tenth, construct a at different times 0 Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system 0 ；
Eleventh, construct b at different times 0 Obtaining 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 time 1 ；
Twelfth, according to the total bicarbonate consumption rate V in the two-way isotope tracing blank culture system 0 And the rate V of total bicarbonate consumption in the plant culture system to be detected by bidirectional isotope tracing 1 Obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detected b ；
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 obtained j Where 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, respectively FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW 。
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 measured 13 C value delta 0 1i And δ 0 2i Value, test time point more than 5; delta to be measured C1 、δ C2 And delta 0 at different times 1i And δ 0 2i Value 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 times 0i Where 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 measured 13 C value delta 1i And delta 1i Value, test time point more than 5; delta to be measured C1 、δ C2 At delta 1i And delta 1i Value substituting 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 time Bi Where 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 periods i In the method of (a), m i ＝(cv 0 f 00 -cv 0i f 0i )-d 0 i Wherein c is the original concentration of the bicarbonate concentration set, v 0i For the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f 00 The portion of the marked bicarbonate starting from the solution, d 0 i Accumulating 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 times i By the process of (a) p i ＝(cv 0 f B0 -cv 1i f Bi )-d 1 i Wherein c is the original concentration of the bicarbonate concentration set, v 1i For 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, f B0 The portion of the marked bicarbonate starting from the solution, d 1 i Accumulating 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 periods 0 i The calculation method comprises the following steps: d 0 i ＝d 0 i-1 +cv d f 0i ,v d For sampling the sample volume for analysis, where i is the number of samples taken at different times and d 0 0 Is 0, d 0 i-1 Accumulating 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 periods 1 i The calculation method comprises the following steps: d 1 i ＝d 1 i-1 +cv d f Bi ,v d For sampling the sample volume for analysis, where i is the number of samples taken at different times and d 1 0 Is 0, d 1 i-1 Accumulating 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 periods i The 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 periods i The 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 periods 0 Cumulative consumption m of labeled bicarbonate i And cumulative bicarbonate consumption n from air dissolution i Summing;
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 periods 0 Cumulative consumption p of bicarbonate for labeling i And cumulative consumption q of bicarbonate from air dissolution i Summing;
in the twelfth step, the speed V of the plant to be detected for absorbing and utilizing the bicarbonate is obtained b The method of (A) is as follows b ＝V 1 -V 0 ；
In the fourteenth step, V FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW The calculation method comprises the following steps: v FW ＝V b /Fw，V RFW ＝V b /Rfw，V SFW ＝V b /Sfw，V DW ＝V b /Dw，V RDW ＝V b /Rdw and V SDW ＝V b /Sdw。
Examples
Respectively by delta 13 Bicarbonate 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 delta 13 C value of delta C1 Isotopically labelled 2 nutrient solution 13 C value of delta C2 (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, and testing the sameVolume of solution in blank culture system at treatment time, and composition of stable carbon isotope in two isotope-labeled nutrient solutions in the system delta 13 C value delta 0 1i And δ 0 2i The 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 calculated 0i Subsequently, the cumulative consumption m of labeled bicarbonate in the blank culture system was calculated i And cumulative bicarbonate consumption n from air dissolution i Obtaining the cumulative consumption a of carbonate in the blank culture system 0 Construction of a at different times 0 Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system 0 。
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, 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, the plant to be tested is cultured in the environment to be tested, the volume of the solution in the culture system of the plant to be tested in the same treatment time and the stable carbon isotope composition delta in the nutrient solution marked by two isotopes in the system are measured 13 C value delta 1i And delta 1i Calculating 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 solution Bi Subsequently, the cumulative consumption p of the marked bicarbonate in the culture system of the plant to be tested is calculated i And cumulative consumption q of bicarbonate from air dissolution i Obtaining the cumulative consumption b of total carbonate in the culture system of the plant to be tested 0 Construction of b at different times 0 Obtaining the slope of the model, namely the rate V of consuming total bicarbonate in the bidirectional isotope tracing blank culture system 1 Further obtaining the rate V of the bicarbonate absorption and utilization of the plant to be detected b (ii) a Determining the plant fresh weight Fw, root fresh weight Rfw and overground part fresh weight of the plant to be tested after the culture is finishedWeight Sfw, plant dry weight Dw, root dry weight Rdw and aerial dry weight Sdw; calculating the utilization rate V of the bicarbonate under the 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 Sdw FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW 。
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, labeled carbonate cumulative consumption and total carbonate cumulative consumption 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 、b 0 ) 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 samples FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW (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 (9)
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 selected 13 Bicarbonate 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 set as the pH required by the condition to be measured, and the original volume of the culture solution is v 0 Isotopically labelled 1 bicarbonate ion delta in solution 13 C value of delta C1 Isotopically labelled 2 solution of bicarbonate ion delta 13 C value of delta C2 ；
Thirdly, carrying out bidirectional isotope tracing blank culture and bidirectional isotope tracing culture on the prepared solution;
fourthly, measuring the two-way isotope tracing blank culture under different timeThe volume of the nutrient system solution is obtained from the two-way isotope tracing blank culture, and the portion f of the bicarbonate added at different time to the total inorganic carbon in the solution is obtained 0i And volume v of the culture solution 0i Where 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 tracing Bi And volume v of culture broth 1i Where 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 periods i And the consumption p of labelled bicarbonate in a culture system for the two-way isotopic tracing of the plants to be tested at different times i Where i is the number of samples taken at different times; obtaining the cumulative consumption m of labeled bicarbonate in the dual isotope labeled blank culture system at different time periods in the fifth step i In the method of (a), m i ＝(cv 0 f 00 -cv 0i f 0i )-d 0 i Wherein c is the original concentration of the bicarbonate concentration set, v 0i For the volume of the culture solution in the two-way isotope tracing blank culture system at different times, f 00 The portion of the marked bicarbonate starting from the solution, d 0 i Accumulating 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 times i By the process of (a) p i ＝(cv 0 f B0 -cv 1i f Bi )-d 1 i Wherein c is the original concentration of the bicarbonate concentration set, v 1i For 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, f B0 The portion of the marked bicarbonate starting from the solution, d 1 i Accumulating the consumption for sampling; where i is the number of samples taken at different times;
sixthly, the bicarbonate added at different times obtained from the two-way isotope tracing blank culture system accounts for the total inorganic carbon in the solutionFraction f 0i And cumulative consumption m of labeled bicarbonate in the two-way isotope labeling blank culture system at different time periods i Obtaining the cumulative consumption n of bicarbonate from air dissolution in the bidirectional isotope tracing blank culture system at different time periods i Where 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 solution Bi And the consumption p of labelled bicarbonate in the culture system of the plant to be tested for bidirectional isotopic tracing at different time periods i Obtaining cumulative consumption q of bicarbonate from air dissolution in a cultivation system of a bi-directional isotopically-labelled plant to be tested at different time periods i Where 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 periods 0 ；
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 periods 0 ；
Tenth, construct a at different times 0 A linear relation model along with time, and the slope of the obtained model is the rate V of total bicarbonate consumption in the bidirectional isotope tracing blank culture system 0 ；
Eleventh, construct b at different times 0 A linear relation model along with time, and the slope of the obtained model is the rate V of total bicarbonate consumption in the bidirectional isotope tracing plant culture system to be detected 1 ；
Twelfth, according to the total bicarbonate consumption rate V in the two-way isotope tracing blank culture system 0 And the rate V of total bicarbonate consumption in the plant culture system to be detected by bidirectional isotope tracing 1 Obtaining the rate V of absorption and utilization of bicarbonate by the plant to be detected b ；
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 obtained j Where j denotes 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, respectively FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW 。
2. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: the bidirectional isotope labeling blank culture in the third step is to add culture solution prepared by adding the bicarbonate ions labeled with the isotope 1 and the bicarbonate ions labeled with the isotope 2 respectively into a plant-free culture container and culture the mixture 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 culture 0i The 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 system 13 C value delta 0 1i And δ 0 2i Value, test time point more than 5; delta to be measured C1 、δ C2 And delta 0 at different times 1i And δ 0 2i Value 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 times 0i Where i is the number of samples taken 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 detected Bi The method comprises respectively measuring stable carbon isotope composition delta in two isotope-labeled nutrient solutions in a culture system of a plant to be detected under the same treatment time in a culture system of the plant to be detected by bidirectional isotope labeling 13 C value delta 1i And delta 1i Value, test time point more than 5; delta to be measured C1 、δ C2 At delta 1i And delta 1i Value substituting 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 time Bi Where i is the number of samples taken at different times.
4. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: cumulative consumption d of sampling in bidirectional isotope tracing blank culture system at different time periods 0 i The calculation method comprises the following steps: d 0 i ＝d 0 i-1 +cv d f 0i ,v d For sampling the sample volume for analysis, where i is the number of samples taken at different times, d 0 0 Is 0, d 0 i-1 Accumulating 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 periods 1 i The calculation method comprises the following steps: d 1 i ＝d 1 i-1 +cv d f Bi ,v d For sampling the sample volume for analysis, where i is the time of the sampleThe number of samplings of d 1 0 Is 0, d 1 i-1 The consumption is accumulated for the last sampling.
5. 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 step i The 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 step i The method comprises the following steps:
6. 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 step 0 Cumulative consumption m of marked bicarbonate i And cumulative bicarbonate consumption n from air dissolution i And (4) summing.
7. 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 nine 0 Cumulative consumption p of bicarbonate for labeling i And cumulative consumption q of bicarbonate from air dissolution i And (4) summing.
8. The method for quantifying the absorption and utilization of bicarbonate by plants according to claim 1, wherein: said obtaining in the twelfth stepThe rate V of absorption and utilization of bicarbonate by the plant to be tested b The method of (A) is b ＝V 1 -V 0 。
9. 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 step FW 、V RFW 、V SFW 、V DW 、V RDW And V SDW The calculation method comprises the following steps: v FW ＝V b /Fw，V RFW ＝V b /Rfw，V SFW ＝V b /Sfw，V DW ＝V b /Dw，V RDW ＝V b /Rdw and V SDW ＝V b /Sdw。
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