Method and application of quantitative tracer distribution of trace elements among plant tissues

Abstract

The present invention discloses a method and application for quantifying and tracing the distribution of trace elements between tissues in plants. The method comprises the following steps: S1: culturing the plant to the fruiting stage, inserting one end of a capillary glass tube into the ear of the plant, inserting the other end of the capillary glass tube into a container containing 0.5-1.0 mL of isotope labeled liquid, and delivering the isotope labeled liquid to the ear of the plant; S2: collecting the grains of the ear described in S1, separating the grains, obtaining different tissue samples of the grains, and detecting the content of isotope-labeled trace elements in different tissue samples; wherein the number of isotope labels in the isotope labeled liquid described in S1 is not more than 4. The present invention introduces isotope labels into the grains at the part closest to the grains (spike axis) to trace the distribution of trace elements through a capillary feeding method with less physical damage, which can fully restore the true growth and development state of the plant.

Description

Technical Field

[0001] The invention relates to the technical field of isotope labeling, and in particular to a method and application of quantitatively tracing trace elements distributed among tissues in plants. Background Art

[0002] Trace elements, including essential nutrients (such as zinc and iron) and potentially toxic heavy metal pollutants (such as cadmium), are critical to the growth and development of crops and human health. Wheat is the most widely planted crop in the world and is a representative of cereal crops. As a staple food, wheat grains are the main way for environmental trace elements to enter the human body through diet. Usually, some tissues of wheat grains are removed during processing, and tissues such as the endosperm are retained as the main edible part, that is, the distribution of trace elements among tissues inside the grain is directly related to their levels in food. Therefore, it is crucial to analyze and monitor the trace element content in wheat grains.

[0003] At present, for tracing trace elements inside grains, there are in-situ spatial distribution test methods such as NanoSIMS (nano secondary ion mass spectrometry) or laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to analyze the elemental composition and relative content of each tissue in the cross section of mature wheat or rice grains, and understand the spatial distribution pattern of elements in the grain cross section. However, this method cannot fully reflect or quantify the distribution of elements in the grain cross section. There are also methods that use grinding or grinding to peel off part of the outer tissue of the grain, and then analyze the elemental composition and content of the tissue to understand the distribution pattern of elements between the outer and inner layers of the grain. However, the tissue of the sample obtained by grinding or grinding is not specific enough and is more random.

[0004] Isotope labeling technology is often used to trace the transport and distribution of elements from different sources. For the analysis of immediate distribution patterns, it is necessary to distinguish the elements that have recently entered the grain from the elements that have accumulated in the grain in the previous period, that is, to trace the distribution ratio of the elements that have recently entered the grain among different tissues; for the analysis of dynamic distribution patterns, in contrast to the former, it is necessary to distinguish the elements that have accumulated in the grain at a certain stage from the elements that have subsequently entered the grain, that is, to trace the process of redistribution of the elements after accumulation inside the grain. Traditional labeling technology usually uses radioactive isotopes to label in vitro ears (ears cut from plants are inserted into a solution containing isotopes for culture), combined with tissue dissection, to trace the distribution of trace elements among grain tissues. However, due to the potential hazards of radiation to human health and the environment, related work needs to be carried out in institutions and facilities with relevant qualifications and by personnel with relevant qualifications, which greatly limits the application of isotope labeling technology. In vitro ear labeling destroys the integrity of the original vascular tissue of the plant and cannot reflect the distribution of elements in the grain under natural conditions. In addition, if the research object is an element with large concentration differences among plant species, ion competition will exist during multi-element isotope labeling, and it is impossible to rule out the influence of interactions between elements (such as ion competition) on the observation results.

[0005] Therefore, there is an urgent need for an analytical method that can maintain the natural growth state of plants, has high universality, and can accurately quantify and trace the distribution of trace elements at the internal tissue level of grains. This is very important for ensuring the nutrition and safety of the population's diet. Summary of the invention

[0006] In view of the defects in the prior art, the present invention proposes a method and application for quantifying the distribution of trace elements among tissues in plants.

[0007] The present invention provides a method for quantifying the distribution of tracer trace elements among tissues in a plant, comprising the following steps:

[0008] S1: Cultivate the plant until the fruiting stage, insert one end of a capillary glass tube into the ear of the plant, insert the other end of the capillary glass tube into a container containing 0.5-1.0 mL of isotope labeling liquid, and transport the isotope labeling liquid to the ear of the plant;

[0009] S2: collecting the grains from the ear described in S1, separating the grains, obtaining different tissue samples of the grains, and detecting the contents of isotope-labeled trace elements in the different tissue samples;

[0010] Among them, the isotope-labeled liquid described in S1 contains stable isotopes, and the number of isotope labels is not more than 4. Since capillary feeding is to directly deliver elements to plants, the toxicity threshold of trace elements is low, and too much isotope labeling will lead to excessive accumulation of element toxicity. In addition, the osmotic potential of the isotope-labeled liquid will be too low, destroying the water balance of plant cells. Moreover, too many isotope labels need to consider factors such as interactions between elements, making it difficult to trace the distribution characteristics of each element.

[0011] In some embodiments, the isotope labeled solution contains a citric acid solution; the concentration of the citric acid is the sum of the molar concentrations of the isotope labeled elements in the isotope labeled solution plus 0.1 to 0.3 mM. In the isotope labeled solution of the present invention, citric acid is added to complex the trace elements, thereby reducing the occurrence ratio of free ion elements, and homogenizing the activities of different ions, thereby avoiding potential toxicity and ion competition. The concentration of citric acid is higher than the concentration of transition metal cations to ensure that the elements can be fully chelated. When the concentration of citric acid is too low, it will lead to insufficient chelation of elements and instability. After labeling, it is easy to interact with substances in the plant body to form precipitation or insoluble complexes, which will be retained at the labeling site and difficult to be absorbed or transported, affecting the test results. When the concentration of citric acid is too high, after high concentrations of citric acid enter the plant body, it will affect the osmotic pressure, pH, and cationic mineral nutrient metabolism of plant tissues, thereby interfering with the normal physiological metabolic activities of the plant.

[0012] In some embodiments, the concentration of the isotope labeled solution is no higher than 1 mM. If the concentration of the isotope labeled solution is too high, there will be a risk of toxicity, and if the concentration is too low, it will be diluted by the elements present in the plant, affecting the accuracy of the detection.

[0013] In some embodiments, the plant is any one of wheat, corn, beans, sorghum, and millet crops.

[0014] In some embodiments, the trace element is a transition metal trace element; preferably any one or more of Cd, Zn, Fe, Ni, and Cu.

[0015] In some embodiments, the detection method of step S2 is one or more of mass spectrometry and isotope dilution method.

[0016] In some embodiments, the mass spectrometry is quadrupole inductively coupled plasma mass spectrometry.

[0017] In some embodiments, the period for collecting the grains in step S2 is 24 hours or more after the ears of the plants in S1 are fed with the isotope labeled liquid.

[0018] In some embodiments, the plant in step S1 is wheat; the isotope labeled in the isotope labeled solution is111 Cd and 67 Zn, 57 Fe, 61 You, 65 Any two, three or four of Cu; preferably 111 Cd and 67 Zn.

[0019] The invention also provides application of the method in analyzing element distribution inside plants.

[0020] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0021] 1. The present invention prepares an isotope-labeled liquid that simulates the chemical environment of plant tissues. Through a capillary feeding method with less physical damage, the isotope label is introduced into the grain at the part closest to the grain (spike axis) to trace the distribution of trace elements, which can fully restore the real growth and development state of the plant, and the influence of human operation is small. The capillary glass tube feeds a fixed volume of labeled liquid, which avoids the randomness of the labeling process and has high repeatability.

[0022] 2. The present invention utilizes citric acid to complex metal trace elements, thereby reducing the occurrence ratio of free ion elements and homogenizing the activities of different ions, thereby avoiding potential toxicity and ion competition and improving the accuracy of observation results.

[0023] 3. The stable isotope labeling and detection work used in the present invention can be carried out in ordinary chemical experiments, and is more universal than traditional radioactive element measurement methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a schematic diagram of feeding with isotope-labeled liquid according to Example 2 of the present invention;

[0026] Figure 2 This is a schematic diagram of the tissue dissection process of wheat grains according to Example 3 of the present invention;

[0027] Figure 3 This is a statistical diagram of the distribution of isotope-labeled cadmium and labeled zinc in various tissues when fed at different stages of grain development (10, 20 and 30 days after anthesis) according to Example 5 of the present invention;

[0028] Figure 4 This is a statistical diagram of the distribution of isotope-labeled cadmium and labeled zinc in various tissues when fed at different stages of grain development (10, 20 and 30 days after anthesis) of Example 5 of the present invention. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0030] The present invention takes wheat ears in the fruiting period as the object, feeds trace elements labeled with stable isotopes through capillaries at the base of the cob, and uses a short-term labeling test (sampling 1 day after isotope feeding) to capture the instant distribution mode of elements in each key period of grain development during the key period of grain development (including cell division period, rapid accumulation period of dry matter and grain maturity period), obtains grain tissue by tissue dissection, determines its isotope ratio and element concentration after digestion, calculates the amount of isotope-labeled elements in each tissue at each stage, and characterizes the preferential distribution direction of labeled elements between different tissues of grains at each stage. A long-term labeling test (sampling at different development stages after isotope feeding once in the early stage of grain development) is used to trace the dynamic distribution process of tracer elements in the fruiting period, obtains grain tissue by tissue dissection, determines its isotope ratio and element concentration after digestion, calculates the change of the amount of isotope-labeled elements in each tissue of grains between each stage, and characterizes the direction and level of redistribution of elements in each stage of grain development. The method of the present invention avoids the damage of the original xylem and phloem system of the plant by in vitro ear culture, does not require the provision of exogenous sucrose in the culture solution to support the dry matter accumulation of the grains, can correctly reflect the growth and development, material loading and trace element distribution of the grains under normal conditions, and is less affected by human manipulation.

[0031] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0032] 111 Cd and 67 Zn isotope: ISOFLEX, USA;

[0033] Sodium citrate: Aladdin (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0034] Example 1 Preparation of isotope labeled solution

[0035] Accurately weigh the high-abundance stable isotope, use 1mL 15.4 M (mol / L) concentrated nitric acid to fully dissolve it, and then dilute it with 50mL pure water to prepare the mother solution of the isotope labeling solution. Use sodium citrate dihydrate (C6H5Na3O7·2H2O) powder to prepare a 0.1M citric acid mother solution. Use 2-morpholineethanesulfonic acid monohydrate (MES; C6H 13 NO4S) powder was used to prepare a 0.01 M MES solution. The pH of the 0.01 M MES solution was adjusted to 6.0 with a 0.1 M KOH solution to prepare a MES-KOH buffer solution.

[0036] The isotope labeling solution is prepared using the mother solution of the isotope labeling element, the mother solution of citric acid and the MES-KOH buffer solution of pH 6.0. The concentration of the stable isotope is determined according to the content of the common trace elements in the wheat cob, and the concentration of citric acid is the sum of the molar concentrations of the isotope labeling elements in the labeling solution plus 0.25mM (millimoles / liter). (Note: the number of isotope labeling elements is generally not more than 4, and the total concentration is generally not more than 1mM).

[0037] In order to study the real-time distribution pattern and dynamics of cadmium and zinc among wheat grain tissues, the prepared isotope labeling solution contained 0.025 mM 111 Cd labeled cadmium ions (Cd 2+ )、0.200mM 67 Zn labeled zinc ion (Zn 2+ ), 0.475mM sodium citrate, 9.217mM MES, 7.834mM potassium ion, 5.652mM nitrate (introduced by isotope labeling mother solution), pH 6.0. Using the Visual MINTEQ model, the occurrence forms of cadmium and zinc in the isotope labeling solution, the proportion of each form, and the negative logarithm of the free ion activity (p(M n+ )Log ionic activity), the larger the value of the logarithm of free ion activity, the smaller the concentration. Under the condition of removing sodium citrate, the model simulation analysis was used again to calculate the above parameters, as shown in Table 1.

[0038] Table 1 Effect of citric acid addition on the occurrence forms, ratios and logarithms of free ion activities of cadmium and zinc elements in isotope labeling solution

[0039]

[0040]

[0041] As shown in Table 1, under the addition of citric acid, cadmium and zinc free ions (i.e., Cd 2+ and Zn 2+) and ion activity were significantly reduced, indicating that citric acid can effectively complex the cadmium and zinc free ions in the isotope labeled solution and reduce their potential toxicity. In addition, although the concentrations of cadmium and zinc in the isotope labeled solution differ by nearly 10 times, the addition of citric acid adjusts the free ion activity of the two to a similar level, thereby reducing the potential impact of the competition between cadmium and zinc ions on the experimental observation results.

[0042] Example 2 Feeding of isotope labeled liquid

[0043] Wheat was cultured to the fruiting stage, and the date when the spike stalk emerged was recorded as the start of grain development (flowering period). The specific time of each stage of wheat grain development was determined through parallel plants.

[0044] Add 1 mL of isotope labeling solution to a 1.5 mL microcentrifuge tube (EP tube) without a cap. Insert one end of a 10 μL (microliter) capillary glass tube into the base of the spike cob. Figure 1 As shown, the other end is inserted into the bottom of the EP tube, and the EP tube is fixed to the ear handle with paper tape, and the isotope labeled liquid is fed into the ear. The ear is usually the main ear of the plant, that is, the ear that enters the flowering period first. Usually, the isotope labeled liquid can be completely absorbed by the ear within 8 hours.

[0045] Short-term isotope labeling: Feed the grains during the cell division period (about 7 to 10 days after flowering), the rapid dry matter accumulation period (about 20 days after flowering), and the maturity period (about 30 days after flowering). Samples are taken 24 hours after the isotope labeled liquid is completely absorbed by the ear.

[0046] Long-term isotope labeling: Isotope solution is fed only during the cell division period of grain development (approximately 7 to 10 days after anthesis), and samples are taken 24 hours, 10 days, and 20 days after the isotope labeled solution is completely absorbed by the ear.

[0047] Example 3: Dissection of grain tissue after labeling

[0048] Collect the isotope-labeled grains of Example 2, select the grains in the middle of the ear, and select 5 grains of the same size from each ear for tissue dissection. For the grains collected during the cell division period and the rapid accumulation period of dry matter, due to the high water content, they need to be placed in an open plastic culture dish and treated in an oven at 60°C for 30 minutes. For the grains collected during the maturity period, if they have become hard (cannot be cut with fingernails), they need to be soaked in ice-cold pure water for 12 hours.

[0049] Under a stereo microscope, the kernels were dissected using a ceramic scalpel and ceramic or plastic tweezers to separate the kernels into seed coat, ventral groove, embryo, aleurone layer and endosperm. Figure 2As shown. Gently cut the back of the grain and tear off the transparent film-like tissue as the seed coat; then cut the two sides of the ventral groove on the abdomen of the grain and tear off the dark green or dark brown tissue as the ventral groove; cut the two sides of the embryo and peel off the embryo; remove the head and tail of the remaining part and cut it into two sections; cut the central milky white part of each section in a well shape, which is the endosperm; the remaining part and the head and tail cut off before are the aleurone layer. For each tissue, the tissue obtained by mixing 5 grains from the same ear is one sample, which is one biological replicate.

[0050] Example 4 Processing, analysis and calculation of element distribution patterns of grain tissue

[0051] After freeze-drying the grain tissue sample of Example 3, weigh it accurately with a one-millionth balance. Pour the sample into a 15mL soluble polytetrafluoroethylene (PFA) sample dissolving tank, add 2.5mL 15.4M nitric acid, shake gently until the sample is fully infiltrated, cover it and pre-digest it on a 40℃ hot plate for more than 12 hours; add 0.5ml 30% hydrogen peroxide, cover it and digest it on a 120℃ hot plate for more than 24 hours; adjust the temperature of the hot plate to 160℃, open the cover and evaporate the liquid in the tank to the size of a bean. After cooling, dilute the remaining liquid in the tank with 0.3M nitric acid and rinse the tank wall to 12mL, and record the volume of the digestion solution by weighing. The element concentration in the digestion solution is determined by graphite furnace atomic absorption spectroscopy (GF-AAS), inductively coupled plasma atomic emission spectroscopy (ICP-OES) and other methods.

[0052] Inductively coupled plasma mass spectrometry (ICP-MS) is used to determine the isotope ratio (molar ratio) in the sample. The numerator in the isotope ratio is the isotope with the highest abundance among the purchased high-abundance stable isotopes, and the denominator should be the isotope with the least interference in the mass spectrometry determination. Taking cadmium as an example, 111 Cd high abundance stable isotope is used as a marker. 110 The mass spectrometric interference of Cd is minimal, so the 111 Cd: 110 Cd ratio. The amount of isotope-labeled elements in the digestion solution is calculated by isotope dilution traceability. Taking cadmium as an example, the amount of labeled cadmium (Cd lab ) lab [%])for:

[0053]

[0054] in 111 AC lab , 110 AC lab (ISOFLEX, USA) is used to mark the abundance of the corresponding isotope in cadmium (provided by the certificate when the isotope is purchased, 111 AC labis 97.21%, 110 AC lab is 1.39%), 111 AC nat , 110 AC nat is the abundance of the corresponding isotope in unlabeled natural cadmium (derived from the IUCN data https: / / www.ciaaw.org / isotopic-abundances.htm, 111 AC nat is 12.80%, 110 AC nat The calculation of zinc is similar, where 67 Zn lab is 89.60%, 66 Zn lab is 3.88%, 67 Zn nat is 4.04%, 66 Zn nat It is 27.74%.

[0055] The cadmium concentration in the tissue sample (CCd [μg g -1 ]) and the dry weight of the tissue (DW[g]), and the Cd lab The amount (QCd lab [μg]):

[0056] QUR lab =Cdf lab ×CCd×DW;

[0057] The dry weight and element content of the kernel tissue sample were divided by 5 to obtain the dry weight and element content of each tissue in a single kernel.

[0058] Calculation of element allocation pattern:

[0059] For short-term labeling experiments, the proportion (%) of the amount of labeled elements in each tissue of the grain at each stage of grain development was calculated, and the preferential distribution destination of the elements newly entering the grain at each stage was analyzed (immediate distribution mode). For long-term labeling experiments, the changes in the amount of labeled elements in each tissue of the grain between different stages of grain development were calculated. The ones with a significant decrease in amount (p<0.05) were the sources of element redistribution, and the degree of decrease (the percentage of the decrease in the amount of labeled elements in the previous stage) was the intensity of redistribution; the ones with a significant increase in amount were sinks that received redistributed elements from other tissues; thus characterizing the direction and level of endogenous element redistribution at each stage of grain development (dynamic distribution mode).

[0060] Example 5 Short-term and long-term labeling experiments of cadmium and zinc in wheat grain tissues

[0061] (1) Short-term labeling test:

[0062] In the instant distribution pattern of cadmium and zinc among wheat grain tissues, capillary feeding with cadmium and zinc isotope labeling was carried out at three key periods of grain development. The cadmium concentration in the isotope labeling solution in the short-term experiment was 0.005mM and the zinc concentration was 0.040mM.

[0063] The samples were collected 1 day after labeling and the amounts of labeled cadmium and zinc in the grain tissues were measured (Table 2 and Figure 3 ) and relative proportions ( Figure 4 )as follows:

[0064] Table 2 The amount and proportion of isotope-labeled cadmium and zinc in various tissues of grains fed at different stages of grain development

[0065]

[0066] Table 2 reveals the immediate distribution patterns and differences of cadmium and zinc among wheat grain tissues. In the final stage of grain development (30 days after flowering), only trace amounts of cadmium and zinc can enter the grains; cadmium tends to be distributed to the main edible part (endosperm + aleurone layer) on the 10th day after flowering, posing a high risk of affecting food security; on the 20th day after flowering, due to the enhanced barrier effect of the ventral groove, the proportion of cadmium distributed to the endosperm + aleurone layer decreased significantly; at different stages of grain development, the distribution ratio of zinc to different tissues was relatively stable relative to cadmium, and the proportion distributed to the edible part (endosperm + aleurone layer) was always high; although the barrier degree of the ventral groove to zinc was also enhanced on the 20th day after flowering, this barrier effect was weaker than that of cadmium.

[0067] (2) Long-term labeling test

[0068] The isotope labeling solution prepared in Example 1 was used to carry out a long-term labeling experiment.

[0069] Capillary feeding with cadmium and zinc isotope labeling was carried out in the early stage of grain development, and samples were collected on the 1st, 10th and 20th days after labeling, corresponding to different stages of grain development (10th, 20th and 30th days after flowering), and the test results are shown in Table 3. The amount of redistribution was calculated as the decrease in the amount of elements between the two stages; the intensity of redistribution was the percentage of the decrease in the initial amount.

[0070] Table 3 The amount of isotope-labeled cadmium and zinc in each tissue, the tissues where element redistribution occurred between stages, and their amount and intensity

[0071]

[0072]

[0073] Table 3 shows the decrease in the amount of labeled cadmium and zinc in some grain tissues, indicating that these tissues redistributed the accumulated endogenous cadmium and zinc. The redistribution of endogenous cadmium in grains mainly occurred in the testa and endosperm + aleurone layer. The decrease in the amount of labeled cadmium in the two (-0.9 and -1.56) coincided with the increase in the amount of labeled cadmium in the ventral furrow (+1.1) and embryo (+1.52), indicating the source-sink relationship of endogenous cadmium redistribution from the testa to the ventral furrow and from the endosperm + aleurone layer to the embryo; the redistribution of endogenous zinc in grains occurred earlier than that of cadmium and was more intense, and generally showed a redistribution from outer tissues to inner tissues.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for quantifying the distribution of trace elements between tissues in a plant, characterized in that: The steps include: S1: Cultivate the plant until the fruiting stage, insert one end of a capillary glass tube into the ear of the plant, insert the other end of the capillary glass tube into a container containing 0.5-1.0 mL of isotope labeling liquid, and transport the isotope labeling liquid to the ear of the plant; S2: collecting the grains from the ear described in S1, separating the grains, obtaining different tissue samples of the grains, and detecting the contents of isotope-labeled trace elements in the different tissue samples; Wherein, the isotope in the isotope-labeled liquid described in S1 is a stable isotope, and the number of isotope labels is not more than 4; The isotope labeling solution contains a citric acid solution; the concentration of the citric acid is the sum of the molar concentrations of the isotope labeling elements in the isotope labeling solution plus 0.1 to 0.3 mM; The capacity of the capillary glass tube is 10 μL; The plant in step S1 is wheat.

2. The method according to claim 1, characterized in that The concentration of the isotope labeling solution is not higher than 1 mM.

3. The method according to claim 1, characterized in that The trace elements are transition metal trace elements.

4. The method according to claim 1, characterized in that: The detection method of step S2 is one or more of mass spectrometry and isotope dilution method.

5. The method according to claim 4, characterized in that The mass spectrometry is quadrupole inductively coupled plasma mass spectrometry.

6. The method according to claim 1, characterized in that The period for collecting the grains in step S2 is more than 24 hours after the ears of the plants in S1 are fed with the isotope labeled liquid.

7. The method according to claim 1, characterized in that The isotope label in the isotope labeling solution is 111 Cd and 67 Zn, 57 Fe, 61 You, 65 Any two, any three or any four of Cu.

8. Use of the method according to any one of claims 1 to 7 in analyzing the internal element distribution of plants.

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