Application of exogenous glutamine in promotion of enrichment of cadmium in Astera kwangtungensis

Abstract

The invention belongs to the technical field of heavy metal pollution remediation, and particularly relates to application of exogenous glutamine to promotion of enrichment of cadmium in aster brachium. The invention relates to application of exogenous glutamine in promoting enrichment of heavy metals by hyperaccumulators. According to the method, a glutamine aqueous solution is sprayed on leaf surfaces during the growth period of the cadmium hyperaccumulator Aster brachiatum, and the influence of the glutamine aqueous solution on the growth and enrichment of the hyperaccumulator Aster brachiatum is researched. Results show that when the glutamine solution is sprayed to the tatarian aster, the absorption amount of the tatarian aster roots to cadmium is remarkably increased, the biomass of the tatarian aster is improved, the photosynthesis of the tatarian aster is enhanced, meanwhile, the absorption and enrichment amount of the whole tatarian aster to cadmium is increased, and technical support can be provided for strengthening hyperaccumulator restoration.

Description

Technical Field

[0001] The present invention belongs to the technical field of heavy metal pollution remediation, and specifically relates to the application of exogenous glutamine in promoting the cadmium enrichment of Aster subulatus Michx. Background Art

[0002] Heavy metals are divided into natural and anthropogenic sources. Natural sources mainly include bedrock weathering, while human activities such as mining, metal smelting, fertilizer application, and sewage irrigation are the main reasons for the continuous increase of heavy metals in the environment such as water bodies and soil (Xu et al., 2023). Guangxi is a famous "non-ferrous metal" hometown in China, with very rich mineral resources. However, due to the disorderly development of enterprises and the random discharge of waste, serious soil heavy metal pollution has been caused in mining areas, especially the obvious exceeding of elements such as arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg) in the soil (Li Zhaoyi et al., 2024). Since Cd cannot be biodegraded in environmental media, and heavy metal Cd pollution has the characteristics of long-term, cumulative, hidden, and irreversible, it is difficult to treat heavy metal Cd pollution, bringing great potential safety risks to the quality of agricultural products and human health (Diaconu et al., 2020). Therefore, there is an urgent need for effective and economical remediation technologies to treat heavy metal Cd-polluted soil.

[0003] At present, the treatment of heavy metal Cd-polluted soil can mainly be carried out by physical methods, chemical methods, and biological methods (Yang Guodong et al., 2020). Phytoremediation is a kind of bioremediation method, which is a method of directly using green plants to isolate, remove, and degrade organic and inorganic pollutants in soil and water bodies (Pratush et al., 2018; Gomes et al., 2016). Generally speaking, plants can remove heavy metals through methods such as phytoextraction, phytostabilization, rhizoremediation, or phytofiltration (Ashraf et al., 2019). Phytoremediation technology has the characteristics of low remediation cost, good effect, and no secondary pollution, and has high ecological benefits and aesthetic value after reclamation, and can achieve permanent clean remediation of polluted soil, so it is recognized as a green and environmental in-situ remediation technology (Ali et al., 2013; Zhang Jianguang et al., 2023).

[0004] Aster subulatus Michx., also known as Scissor Vegetable, White Chrysanthemum, Wild Bupleurum Root, Nine Dragon Arrow, and Subulate Aster, is a plant of the genus Aster in the Asteraceae family. It grows in moist and saline soil and is distributed in Guangxi, Jiangsu, Zhejiang, Jiangxi, Hunan, Fujian and other places. Aster subulatus Michx. has the advantages of well-developed root systems, large biomass, stable physiological traits, and tolerance to heavy metal pollution stress. Previous studies by the project team found that Aster subulatus Michx. has strong tolerance and accumulation ability to Cd, and confirmed that it is a Cd hyperaccumulator plant, which can be used as an ideal dominant pioneer species for phytoremediation of heavy metal Cd contaminated soil in Guangxi (Chen Wei et al., 2023). Although phytoremediation has shown great advantages in the remediation of heavy metal contaminated environments, there are still some limitations in practical applications, such as long remediation cycles, relatively single enrichment ability, susceptibility to extreme environmental factors, and the treatment of heavy metal-containing biomass remaining after remediation. Therefore, how to strengthen the advantages of phytoremediation and improve the efficiency of phytoremediation has become an important topic of concern for scholars at home and abroad. To improve the remediation efficiency of hyperaccumulator plants, scientists have developed various enhancement technologies such as agronomic measure enhancement, biotechnology enhancement, and chemical-assisted enhancement.

[0005] Glutamine plays an important role in plants, especially in response to environmental stress and adverse conditions. The roles of glutamine in plant stress resistance are as follows: (1) Detoxifying the toxicity of free ammonia When the ammonia content in plants is too high, glutamine, as a storage and transport form of ammonia, can convert the excess ammonia into glutamine or asparagine, thus detoxifying the toxicity of free ammonia; (2) Transport form of nitrogen Glutamine and asparagine are not only the detoxification products of ammonia, but also the transport forms of nitrogen in plants, which is crucial for nitrogen metabolism and distribution in plants under adverse conditions; (3) Antioxidant effect: Glutamine participates in the synthesis of glutathione, which is an important antioxidant that can help plants resist oxidative stress, thereby improving the stress resistance of plants. In summary, the roles of glutamine in plant stress resistance are mainly reflected in detoxifying the toxicity of free ammonia, serving as a transport form of nitrogen, and participating in antioxidant effects. These roles together help plants maintain normal physiological functions in the face of environmental stress and adversity.

[0006] There are no relevant reports on using exogenous glutamine to promote the remediation of heavy metals by hyperaccumulator plants.

[0007] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0008] The purpose of the present invention is to provide the application of exogenous glutamine in promoting the enrichment of cadmium by Aster subulatus Michx.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The first object of the present invention is to provide the application of exogenous glutamine in promoting the enrichment of heavy metals by hyperaccumulator plants.

[0011] The second object of the present invention is to provide a heavy metal repair agent, and the heavy metal repair agent includes the glutamine described above.

[0012] More specifically, the heavy metal repair agent is composed of glutamine and deionized water.

[0013] More specifically, in the heavy metal repair agent, the concentration of glutamine is 0.5 - 20 mg / L.

[0014] The third object of the present invention is to provide the application of the heavy metal repair agent in promoting the enrichment of heavy metals by hyperaccumulator plants.

[0015] More specifically, the hyperaccumulator plant includes *Aster subulatus*; the heavy metal includes cadmium.

[0016] The fourth object of the present invention is to provide the application of the exogenous glutamine or the heavy metal repair agent in improving the stress resistance of *Aster subulatus* in heavy metal polluted areas and promoting the growth of *Aster subulatus*.

[0017] The fifth object of the present invention is to provide the application of the exogenous glutamine or the heavy metal repair agent in increasing the contents of chlorophyll and carotenoid in *Aster subulatus* in heavy metal polluted areas.

[0018] The sixth object of the present invention is to provide a method for promoting the repair of heavy metal pollution by *Aster subulatus* using exogenous glutamine, specifically: during the growth period of *Aster subulatus*, a glutamine solution is sprayed on the leaf surface once every 3 days, for a total of 6 sprays; and *Aster subulatus* is harvested 10 days after the end of the leaf surface spraying.

[0019] More specifically, it is characterized in that the concentration of the glutamine solution is 0.5 - 20 mg / L.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] During the growth period of the cadmium hyperaccumulator *Aster subulatus*, an aqueous glutamine solution is sprayed on the leaf surface to study the effects of the aqueous glutamine solution on the growth and enrichment of the hyperaccumulator plant *Aster subulatus*. The results show that when the glutamine solution is sprayed on *Aster subulatus*, not only the cadmium absorption amount of the roots of *Aster subulatus* is significantly increased, but also the biomass of *Aster subulatus* is increased and its photosynthesis is enhanced. At the same time, the cadmium absorption and enrichment amount of the whole plant of *Aster subulatus* is also increased, which can provide technical support for strengthening the repair of hyperaccumulator plants. Description of the Drawings

[0022] Figure 1 For the biomass (plant height, fresh weight, dry weight) of Aster subulatus under different treatments;

[0023] Figure 2 For the chlorophyll content of Aster subulatus under different treatments;

[0024] Figure 3 For the cadmium content of Aster subulatus under different treatments;

[0025] Figure 4 For the cadmium accumulation amount of Aster subulatus under different treatments. Detailed implementation manners

[0026] The technical solutions of the present invention patent will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those skilled in the art within the scope of the present invention without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0027] 1. Materials and methods

[0028] 1.1 Test materials

[0029] L-glutamine (≥99%, CAS: 56-85-9, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0030] The seeds of Aster subulatus were collected from a lead-zinc mining area about 120 km away from Yulin City, Guangxi.

[0031] 1.2 Pot experiment

[0032] To simulate cadmium-polluted environmental soil, the test was carried out in a sand culture potting method, with 1.5 kg of quartz sand filled in each pot. The Aster subulatus was collected from a lead-zinc mining area about 120 km away from Yulin City, Guangxi. After disinfecting the seeds of Aster subulatus with 95% ethanol, they were placed in a cadmium-free seedling tray for cultivation. After a period of time, Aster subulatus seedlings with similar growth states (similar plant height, number of leaves, and growth vigor) were selected as test materials, and 3 Aster subulatus seedlings were transplanted into each pot, with a total of 15 pots planted.

[0033] The experimental environment was: 12-hour light cycle, 25 °C light cycle / 20 °C dark cycle, relative humidity 70%, and light intensity of 300 μmol / (m 2·s), maintain continuous ventilation for 24 h. Supplement Hoagland nutrient solution every 4 days, and adjust the pH value of the nutrient solution to about 5.6 with 0.1 mmol / L sodium hydroxide or 0.1 mmol / L hydrochloric acid. One week after planting Aster subulatus, add 500 mL of 100 mmol / L Cd solution to 12 of the pots, and set up 1 group (3 pots) of sand culture pots without cadmium solution as the blank control (CK).

[0034] 1.3 Experimental design of foliar spraying

[0035] Based on the above operations, after 3 days, spray aqueous solutions of glutamine at different concentrations. There are a total of 5 treatment groups (Table 1), with the tip of the leaf dripping water as the limit and completely covering the leaf. Spray a total of 6 times, once every 3 days. After all treatments are completed, continue culturing for 10 days and then harvest. Measure the biomass and heavy metal cadmium content of Aster subulatus. Cadmium treatment control group: Spray deionized water instead of the aqueous glutamine solution on the leaves.

[0036] Table 1 Experimental design

[0037]

[0038] 1.4 Sample treatment and analysis

[0039] After the experiment, take out the whole Aster subulatus from the pot, wash it with tap water, and after rinsing it clean and removing the fine sand from the roots, soak the roots in 20 mmol / L Na2-EDTA solution for 20 min to remove the cadmium ions adsorbed on the surface, then rinse it repeatedly with deionized water until clean, dry the water, measure the fresh weight and plant height of the whole plant. Divide Aster subulatus into two parts: aboveground and underground, put it in an oven at 105 °C for 30 min for fixation, then adjust the temperature to 70 °C and dry it to a constant weight to measure the dry matter weight, and grind the sample with stainless steel and pass it through a 40-mesh sieve. Digest the sample with H2O2-HNO3 mixed solvent on an intelligent electric digestion instrument. Use a flame-graphite furnace atomic absorption spectrometer to measure the heavy metal content of the sample digestion solution.

[0040] 1.5 Experimental results

[0041] 1.5.1 Effects on biomass

[0042] The results are shown in Figure 1 .

[0043] As Figure 1 shown in -A, the plant height of Aster subulatus under the treatment showed Cd < 0.5Gln < 5Gln < 20Gln < CK. Among them, Cd decreased by 28.4% compared with CK, while the plant heights of 0.5Gln, 5Gln, and 20Gln increased compared with Cd.

[0044] As Figure 1As shown in Figure - B, the fresh weight of *Aster subulatus* showed 0.5 Gln < Cd < 5 Gln < 20 Gln < CK under the treatments; among them, Cd decreased by 37.3% compared to CK, and compared with Cd, 5 Gln and 20 Gln increased by 15.3% and 63.3% respectively.

[0045] As Figure 1 shown in Figure - C, the dry weight of *Aster subulatus* showed Cd < 0.5 Gln < 5 Gln < CK < 20 Gln under the treatments; among them, Cd decreased by 38.3% compared to CK, and 20 Gln increased by 7.9% compared to CK. Moreover, 0.5 Gln, 5 Gln, and 20 Gln all increased compared to Cd.

[0046] It shows that Cd stress will inhibit the normal growth of *Aster subulatus*, while exogenous addition of glutamine can, to a certain extent, increase the biomass of *Aster subulatus* and alleviate the inhibitory effect of cadmium on the growth of *Aster subulatus*.

[0047] 1.5.2 Effects on chlorophyll content

[0048] The results are shown in Figure 2 .

[0049] As Figure 2 can be seen, the chlorophyll a content of *Aster subulatus* showed Cd < 20 Gln < 5 Gln < 0.5 Gln < CK under the treatments, among which Cd decreased by 51.7% compared to CK, and the chlorophyll a contents of 0.5 Gln, 5 Gln, and 20 Gln increased by 77.7%, 32.6%, and 24.8% respectively compared to Cd.

[0050] The chlorophyll b content of *Aster subulatus* showed Cd < 20 Gln < 5 Gln < 0.5 Gln < CK under the treatments, among which Cd decreased by 19.5% compared to CK, and the chlorophyll b contents of 0.5 Gln, 5 Gln, and 20 Gln increased by 50.8%, 12.6%, and 6.0% respectively compared to Cd.

[0051] The carotenoid content of *Aster subulatus* showed Cd < 20 Gln < 0.5 Gln < 5 Gln < CK under the treatments, among which Cd decreased by 67.3% compared to CK, and the carotenoid contents of 0.5 Gln, 5 Gln, and 20 Gln increased by 118.1%, 124.9%, and 86.1% respectively compared to Cd.

[0052] It shows that exogenous addition of glutamine can alleviate the inhibitory effect of cadmium on the chlorophyll synthesis of *Aster subulatus*, and adding low - concentration glutamine (0.5 or 5 mg / L) has a better promoting effect on the chlorophyll synthesis of *Aster subulatus* under cadmium pollution than high - concentration glutamine (20 mg / L).

[0053] 1.5.3 Effects on the cadmium content of *Aster subulatus*

[0054] The results are shown in Figure 3 .

[0055] It can be seen from Figure 3 that compared with Cd, the cadmium contents in the Ageratina adenophora Sprengel treated with 0.5 Gln, 5 Gln, and 20 Gln increased by 56.4%, 30.9%, and 103.7% respectively, mainly due to the increase in the cadmium content in the underground part (root).

[0056] 1.5.4 Effects on the cadmium accumulation of Ageratina adenophora Sprengel

[0057] The results are shown in Figure 4 .

[0058] It can be seen from Figure 4 that compared with Cd, the cadmium accumulations in the Ageratina adenophora Sprengel treated with 0.5 Gln, 5 Gln, and 20 Gln increased by 21.1%, 56.5%, and 157.4% respectively. This indicates that adding glutamine can enhance the ability of Ageratina adenophora Sprengel to accumulate cadmium, and adding 20 mg / L glutamine has a better effect on enhancing the cadmium accumulation ability of Ageratina adenophora Sprengel.

[0059] In summary, glutamine can improve the stress resistance of Ageratina adenophora Sprengel, enhance its survival ability, promote its growth and development, and strengthen its cadmium accumulation ability in the cadmium stress environment. Compared with single-plant remediation, exogenous addition of glutamine can improve the remediation efficiency of Ageratina adenophora Sprengel for cadmium pollution, and based on this, the cadmium content in the environment can be reduced.

[0060] The foregoing description of the specific exemplary embodiments of the present invention is for the purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is obvious that many changes and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present invention, as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. Application of exogenous glutamine in promoting heavy metal enrichment by hyperaccumulator plants.

2. A heavy metal remediation agent, characterized in that, The heavy metal remediator includes the glutamine described in claim 1.

3. The heavy metal remediator according to claim 2, characterized in that, The heavy metal remediator consists of glutamine and deionized water.

4. The heavy metal remediation agent according to claim 3, wherein In the heavy metal remediator, the concentration of glutamine is 0.5 - 20 mg / L.

5. Application of the heavy metal remediator according to claim 2 in promoting heavy metal enrichment by hyperaccumulator plants.

6. The application according to claim 1 or the application according to claim 5, characterized in that, The hyperaccumulator plants include *Aster subulatus*; the heavy metal includes cadmium.

7. Application of the exogenous glutamine according to claim 1 or the heavy metal remediator according to claim 2 in improving the stress resistance of *Aster subulatus* and promoting the growth of *Aster subulatus* in heavy metal polluted areas.

8. Application of the exogenous glutamine according to claim 1 or the heavy metal remediator according to claim 2 in increasing the contents of chlorophyll and carotenoid in *Aster subulatus* in heavy metal polluted areas.

9. A method for promoting the remediation of heavy metal pollution in Aster subulatus by using exogenous glutamine, characterized in that, Specifically: during the growth period of *Aster subulatus*, spray the glutamine solution on the leaf surface once every 3 days, for a total of 6 sprays; and harvest *Aster subulatus* 10 days after the end of leaf surface spraying.

10. The method for promoting the remediation of heavy metal - polluted Aster subulatus by using exogenous glutamine according to claim 9, wherein, The concentration of the glutamine solution is 0.5 - 20 mg / L.

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