Effect of aspartic acid on physiological characteristics and gene expression of Tartary buckwheat

doi:10.21203/rs.3.rs-7344052/v1

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Effect of aspartic acid on physiological characteristics and gene expression of Tartary buckwheat

https://doi.org/10.21203/rs.3.rs-7344052/v1

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The salt-tolerant variety Chuanqiao No.1 of Tartary buckwheat was used as the experimental material. 10, 20, 30, 40, 50 and 60 µM aspartic acid treatment was used to determine the germination rate of Tartary buckwheat seeds and growth indexes such as seedling root length, stem diameter, plant height, fresh weight, chlorophyll content and nitrogen content. The expression levels of growth related genes were also measured after treatment with appropriate concentrations of aspartic acid to study the effects of aspartic acid on seed germination and seedling growth of Tartary buckwheat. The results showed that aspartic acid treatment could significantly increase the germination rate of Tartary buckwheat seeds, and 40 µM of aspartic acid was the appropriate concentration to promote seed germination. Spraying appropriate concentration of aspartic acid could increase the root length, stem thickness, plant height, fresh weight, chlorophyll and nitrogen content of Tartary buckwheat seedlings. The appropriate concentration for aspartic acid spraying is 30 µM. After spraying appropriate concentration of aspartic acid, the expression levels of chlorophyll synthesis related genes (FtDCL and FtGLK1) and nitrogen transport related genes (FtAMT1-1 and FtNRT2.3) in Tartary buckwheat significantly increased from 6 h to 24 h, and both reached their maximum expression levels at 6 h of treatment. It indicated that aspartic acid has a promoting effect on seed germination and seedling growth of Tartary buckwheat, which is beneficial for plant growth.

Aspartic acid

Gene expression

Seed germination

Seedling growth

Tartary buckwheat

Buckwheat, belonging to the Polygonaceae family and Fagopyrum, is a dicotyledonous herbaceous plant. Currently, the cultivated species mainly include common buckwheat (Fagopyrum esculentum Moench) and Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) (Yang et al. 2018). Buckwheat is not only rich in eight essential amino acids needed by the human body (Woo et al. 2013), but also contains a large amount of protein, minerals, vitamins, trace elements needed by the human body (Huang et al. 2013; Zhou et al. 2015), and other bioactive flavonoids that are not present in other cereal crops (Kreft et al. 2006), earning it the nickname of the king of grains (Xiang et al. 2013). Buckwheat not only has high nutritional value, but also has high medicinal value. Buckwheat, as a typical representative of medicinal and edible plants, has been recorded as a dietary therapy in ancient books such as "Compendium of Materia Medica" and "Essential Techniques for Qi and Min". It has the functions of "Wide intestine and healthy stomach" (Liu et al. 2021). The nutritional value of Tartary buckwheat is much higher than that of common buckwheat, and its content of eight amino acids and vitamins is higher than that of common buckwheat, especially lysine and rutin (Kreft et al. 2023). Its rutin content is as high as 0.5%~1.2%, which has the effect of maintaining capillary stability, reducing its permeability and fragility, promoting cell proliferation and preventing hemostatic cell aggregation. It also has various pharmacological effects such as anti-inflammatory, anti allergic, diuretic, antispasmodic, cough suppressant, lipid-lowering and cardiotonic (Zhao et al. 2001). Rutin has been proven to have antioxidant, anticancer and anti-inflammatory properties (Gullón et al. 2017; Negahdari et al. 2021). In addition, quercetin also helps to lower blood pressure and blood lipids. Quercetin is a flavonoid derived from quercetin, which is an O-glycoside of quercetin. Quercetin is linked to the alpha-L-sugated portion at position 3(Fabjan et al. 2003; Petrova et al. 2023). Quercetin, especially when combined with other flavonoids in buckwheat, has antioxidant, anti-inflammatory (Chen et al. 2022), and antibacterial properties (Rodríguez-Pérez et al. 2016). Tartary buckwheat is a traditional edible and medicinal plant. Due to its various bioactive compounds, the consumption of Tartary buckwheat is associated with a wide range of health benefits, and its potential as a functional food is receiving increasing attention.

Amino acids are small organic compounds, and they are also essential small multifunctional biomolecules for living organisms (Brosnan 2000). The role of amino acids in plants involves many physiological regulatory processes, which are closely related to plant growth, development and stress resistance (Xing et al. 2018). Research has found that spraying appropriate concentrations of exogenous proline (Pro) can effectively reduce stomatal length, increase stomatal width, density, and area in red sand leaves. At the same time, it has been found that Pro can significantly affect stomatal characteristics and help red sand alleviate the adverse effects of drought stress (Shi et al. 2023). After spraying the appropriate concentration of L-leucine-15N, there was a significant increase in the dry weight and related nitrogen content of rice leaves, stem sheaths and rice grains (Zou et al. 2016). Spraying amino acids in sweet and saline-alkali soil can increase the yield of Tartary buckwheat and facilitate the accumulation of protein, mineral elements and rutin in Tartary buckwheat seeds, while also improving the quality of Tartary buckwheat (Song et al. 2021). Spraying amino acid fertilizer can increase the chlorophyll and carotenoid content of watercress leaves, and promote the accumulation of selenium in watercress (Xiao et al. 2023). The above research indicates that amino acids are excellent cell osmotic regulators that can improve crop yield and quality. This study used the salt-tolerant variety Chuanqiao No.1 of Tartary buckwheat as the experimental material. After treatment with aspartic acid (Asp), the germination rate of Tartary buckwheat seeds and the growth characteristics of seedlings were measured to determine the appropriate concentration of Asp application. The expression levels of chlorophyll synthesis and nitrogen transport related genes in Tartary buckwheat were also measured after applying the appropriate concentration of Asp, so as to providing new evidence for improving the yield and quality of Tartary buckwheat.

Material

Salt-tolerant Tartary buckwheat variety Chuanqiao No.1 was used as the experimental material.

Determination of seed germination rate

Select intact and plump Chuanqiao No.1 seeds, soak and disinfect them in 1 g/L potassium permanganate for 10 minutes, wash the seeds, and place them in a culture dish with two layers of filter paper. For the control group, add water, just enough to cover half of the seeds. For the treatment group, add equal volumes of 10, 20, 30, 40, 50 and 60 µM Asp solution. Cultivate in a constant temperature light incubator, with cultivation conditions set at 26°C, 10 h of light, and 14 h of darkness. After seed germination, count the number of seeds germinated for 5 d. Each treatment has 3 biological replicates. The seed germination rate (%) is equal to the number of germinated seeds divided by the total number of seeds divided by 100%.

Seedling cultivation and treatment

Select intact and plump Chuanqiao No.1 seeds, soak and disinfect them in 1 g/L potassium permanganate for 10 minutes, and evenly sow the seeds in a mixed soil (nutrient soil and vermiculite soil mixed in a ratio of 3 : 1). Under natural light conditions outdoors (15 ~ 28°C), water once a day to keep the soil surface moist, and water once every 2 ~ 3 days after emergence. The seedlings were treated starting at the 3 leaf stage. One group was set as the control (CK), and the Asp treatment group was set with six concentration gradients of 10, 20, 30, 40, 50 and 60 µM, sprayed once a day for 5 consecutive days to measure relevant growth indexes. The chlorophyll content (soil and plant analyzer development value, SPAD value) and N content were measured using the TYS-A chlorophyll content analyzer from Hangzhou Daji Optoelectronic Instrument Co., Ltd. In addition, the expression levels of growth related genes in Tartary buckwheat seedlings were measured after treatment with appropriate concentrations of amino acids. Each treatment has 3 biological replicates.

Methods for measuring gene expression and primer sequences

After spraying a suitable concentration of Asp solution, take Tartary buckwheat leaves at 0 h, 6 h, 12 h, 24 h and 48 h for later use.

(1) Refer to the instruction manual of SteadyPure Plant RNA Extraction Kit (Aikrui Bioengineering Co., Ltd.) for RNA extraction. (2) Reverse transcription: Reverse transcribe extracted RNA into cDNA, calculate the required RNA volume for 1 µg RNA template in the system based on the measured RNA concentration, and store excess RNA in an 80°C refrigerator. Place the sample on ice and prepare a reverse transcription reaction system according to Table S1. Refer to the instructions of ABScript III RT Master Mix for qPCR with gDNA Remover kit (Wuhan Aibotec Biotechnology Co., Ltd.) for relevant experimental procedures. After preparation, carefully shake and mix it well to prevent the formation of bubbles in the tube. Immediately centrifuge and use it for the next experiment. Perform reverse transcription reaction according to Table S2 using a PCR instrument to obtain cDNA. Dilute 20 µL of cDNA with Nuclease free H₂O to 40 µL, and store the diluted cDNA in a refrigerator at 20°C for the next experiment. (3) RT-qPCR reaction: The real-time fluorescent quantitative PCR primers were FtDCL-F, FtDCL-R, FtGLK1-F, FtGLK1-R, FtAMT1-1-F, FtAMT1-1-R, FtNRT2.3-F and FtNRT2.3-R primer sequences. The real-time fluorescent quantitative PCR primers for the internal reference gene Actin in Tartary buckwheat were designed by Liu et al. (2017) using Actin-F and Actin-R primer sequences (Table S3). The gene expression level was calculated using the 2^−ΔΔT method (Guo et al. 2015; Xu et al. 2016).

Data processing

Microsoft Excel was used to organize the data, and ANOVA and t-test were used to analyze the significance of data differences. Each treatment was replicated three times.

Effect of Asp on seed germination of Tartary buckwheat

Amino acids not only provide nitrogen and energy sources for seed germination, but also participate in regulating the internal metabolic processes of seeds. Studies had shown that certain amino acids such as methionine (Met) can promote seed germination by up-regulating cytosolic Ca²⁺ signaling (Ju et al. 2020). After applying 10, 20, 30, 40, 50 and 60 µM Asp, the germination rate of Tartary buckwheat seeds significantly increased, with increases of 2.01, 4.08, 6.26, 7.78, 5.14 and 2.43 percentage points compared to the control, respectively. Among them, the application of 40 µM Asp had the largest increase, indicating that 40 µM Asp has the best promoting effect on the germination of Tartary buckwheat seeds (Fig. 1).

Effect of Asp spraying on growth of Tartary buckwheat seedlings

Plants absorb nutrients and water from the soil through their roots, and the biomass of aboveground parts is closely related to the number of branches and plant height. Plant height greatly affects the efficiency of photosynthesis in plants, and stem thickness can enhance the plant's ability to resist lodging. Generally, the larger the fresh weight, the higher the accumulation of organic matter in the plant. After spraying 10, 20, 30, 40, 50 and 60 µM Asp, the root length of Tartary buckwheat seedlings significantly increased, which was increased by 9%, 7%, 10%, 14%, 11% and 10% respectively compared to the control. Spraying 40 µM Asp resulted in the largest increase. After spraying 20, 30, 40 and 50 µM Asp, the stem diameter of Tartary buckwheat seedlings significantly increased, which was increased by 16%, 26%, 29% and 21% compared to the control, respectively. Spraying 40 µM Asp resulted in the largest increase. After spraying 20, 30, 40, 50 and 60 µM Asp, the height of Tartary buckwheat seedlings significantly increased, which was increased by 8%, 10%, 9%, 7% and 5% compared to the control, respectively. Spraying 30 µM Asp resulted in the largest increase. After spraying 10, 20, 30, 40, 50 and 60 µM Asp, the fresh weight of Tartary buckwheat seedlings significantly increased, which was increased by 10%, 19%, 28%, 29%, 22% and 23% respectively compared to the control. Spraying 40 µM Asp resulted in the largest increase (Table 1).

Table 1
Effect of Asp spraying on growth main indexes of Tartary buckwheat seedlings
Asp concentration (µM)	Root length (cm)	Stem diameter (cm)	Plant height (cm)	Fresh weight (g)
0	13.53 ± 0.11^d	0.38 ± 0.04^c	26.47 ± 0.29^e	4.76 ± 0.17^d
10	14.75 ± 0.14^c	0.40 ± 0.02^c	27.44 ± 0.23^de	5.23 ± 0.15^c
20	14.53 ± 0.15^c	0.44 ± 0.01^b	28.65 ± 0.15^b	5.66 ± 0.13^b
30	14.95 ± 0.09^b	0.48 ± 0.03^a	29.15 ± 0.14^a	6.09 ± 0.18^a
40	15.45 ± 0.14^a	0.49 ± 0.03^a	28.82 ± 0.16^ab	6.13 ± 0.16^a
50	15.05 ± 0.12^b	0.46 ± 0.03^ab	28.26 ± 0.18^c	5.79 ± 0.16^ab
60	14.88 ± 0.20^bc	0.42 ± 0.02^bc	27.68 ± 0.23^d	5.87 ± 0.14^ab

Effect of Asp spraying on chlorophyll and N content of Tartary buckwheat seedlings

After spraying 10, 20, 30, 40, 50 and 60 µM Asp, the leaf chlorophyll and N content of Tartary buckwheat seedlings significantly increased, with chlorophyll content increasing by 18%, 27%, 36%, 33%, 25% and 21% respectively compared to the control. Spraying 30 µM Asp resulted in the largest increase. The leaf N content increased by 11%, 24%, 39%, 35%, 29% and 27% respectively compared to the control, and spraying 30 µM Asp resulted in the largest increase (Table 2).

Table 2
Effect of Asp spraying on chlorophyll and N content of Tartary buckwheat seedlings
Asp concentration (µM)	Chorophyll content (SPAD value)	N content (mg/g FW)
0	32.66 ± 0.29^f	2.84 ± 0.05^f
10	38.54 ± 0.33^e	3.15 ± 0.02^e
20	41.38 ± 0.32^c	3.52 ± 0.04^d
30	44.26 ± 0.17^a	3.96 ± 0.03^a
40	43.59 ± 0.26^b	3.83 ± 0.04^b
50	40.85 ± 0.28^c	3.65 ± 0.05^c
60	39.65 ± 0.23^d	3.62 ± 0.03^c

Effect of Asp spraying on the expression of chlorophyll synthesis related genes in Tartary buckwheat

After spraying the appropriate concentration of Asp, the expression levels of FtDCL and FtGLK1 gene in Tartary buckwheat were significantly increased at 6 h, 12 h and 24 h. That of FtDCL gene increased by 368%, 1011% and 52% respectively compared to the control, and reached the maximum expression level at 12 h. The expression level of FtGLK1 gene increased by 840%, 178% and 90% respectively compared to the control, and reached the maximum expression level at 6 h (Fig. 2).

Effect of Asp spraying on the expression of N transport related genes in Tartary buckwheat

After spraying the appropriate concentration of Asp, the expression levels of FtAMT1-1 and FtNRT2.3 gene in Tartary buckwheat were significantly increased at 6 h, 12 h and 24 h. The expression level of FtAMT1-1 gene increased by 873%, 430% and 133% respectively compared to the control, and that of FtNRT2.3 gene increased by 1219%, 640% and 532% respectively. Both FtAMT1-1 and FtNRT2.3 genes reached their maximum expression levels at 6 h (Fig. 3).

With the rapid development of the social economy, people's living standards have been greatly improved, but the resulting health problems have become increasingly apparent, and in recent years there has even been a trend towards younger age groups. At present, people are paying more and more attention to health issues, and their requirements for diet are also increasing. They are no longer just satisfied with food and clothing, but pay more attention to the nutritional value and health functions of food. Therefore, nutritious and natural buckwheat has gradually become the mainstream in the market, attracting more and more attention and favor from people. Buckwheat is a characteristic crop that is both medicinal and edible. Research has found that it has extremely high nutritional value and good health benefits (Zhu et al. 2003; Shi et al. 2019). Buckwheat is a medicinal and edible plant with rich nutritional components, functional substances, and various pharmacological effects. It has high development and utilization value in the fields of food processing and medicine. Its nutritional components include protein, starch, fat, dietary fiber, vitamins and minerals. Buckwheat not only contains high levels of protein, but also various trace elements such as iron, zinc, copper, manganese, etc., which have a good effect on promoting human health (Yu et al. 2023). Research has shown that buckwheat is rich in nutrients such as vitamins, fats, proteins, and mineral elements, and its content is significantly higher than other crops (Sytar et al. 2016). The nutritional value of Tartary buckwheat is much higher than that of common buckwheat. The flavonoid content of Tartary buckwheat is much higher than that of common buckwheat. The main components of its flavonoids are rutin (accounting for 70%~90% of the total flavonoids) and quercetin (Fabjan et al. 2003). Rutin has multiple physiological activities and can be used to prevent cerebral hemorrhage caused by capillary fragility, pulmonary hemorrhage, hemorrhagic nephritis, gastritis, gastric ulcer and gingival bleeding (Ladan et al. 2017; Zhang et al. 2017). The study also showed that rutin can inhibit the carcinogenic effect of benzopyrene on mouse skin (Woch et al. 2016). Corticosteroids protect cell membranes by inhibiting lipid peroxidation. Quercetin also significantly inhibits platelet aggregation and selectively binds to blood clots on the vascular wall, thereby preventing thrombus formation (Sadauskiene et al. 2018). At very low concentrations, it can directly block the proliferation of cancer cells (Ren et al. 2001). Buckwheat is the only crop that combines seven nutrients, including carbohydrates (sugar), protein, fat, minerals, fiber, vitamins, and water. Therefore, it has excellent nutritional and health value and good therapeutic effects (Ren et al. 2016). Buckwheat can also be fried and made into tea. Drinking this tea every day has therapeutic effects on patients with hypertension, blood sugar and blood lipids (Zhu 2016).

In recent years, buckwheat has been recognized by people all over the world because of its unique health care value, and its sales continue to rise in the international grain market (Zhou 2022). Therefore, exploring how to promote the growth of Tartary buckwheat is quite important, and amino acids, as exogenous substances, are also a very good cell osmotic regulator that can improve the physiological characteristics of crops, increase crop yield and quality.

Chlorophyll is a photosynthetic pigment located on the thylakoid membrane of chloroplasts. Chlorophyll binds to proteins in a non covalent form, forming pigment protein complexes. DCL protein is a nuclear protein, and the DCL gene is related to the formation and development of chloroplasts. The DCL gene is expressed in plant tissues such as roots, stems and leaves (Guo 2017). GLK (golden like), also known as Golden2 Like transcription factor, is a transcription factor widely present in plants and capable of affecting chloroplast development. Research has found that GLK transcription factors can affect the formation and development of chloroplasts in plant cells by influencing chlorophyll synthesis, ultimately affecting plant photosynthesis. GLK can also enhance plant disease resistance (Li et al. 2020). Related studies have shown that the expression level of AbGLK1 gene in green tissues is significantly higher than that in leaf edge albino tissues (Mao et al. 2022). Nitrogen (N) is an essential element for plant growth and is the main component of living substances such as nucleic acids and proteins in living organisms. Plants mainly absorb and transport NH4 through ammonium transporters (AMT), which are encoded by the AMT gene family. There are two main ammonium nitrogen transporters in plants: AMT1 and AMT2 family. AMT1 belongs to the high affinity transport system (Liu et al. 2021). AMT is responsible for the absorption and transport of ammonium nitrogen, and the AMT1 gene plays a role in regulating plant growth and development (Zhao et al. 2022). The JrAMT gene in walnuts can promote chlorophyll synthesis in plants, which helps walnuts absorb ammonium ammonia (Liu 2019). The BvAMT1-3 gene of beet ammonium transporter may play a role in plant response to nitrogen stress (Wei et al. 2021). Under nitrogen deficiency conditions, over-expression of OsAMT1;1 gene can significantly improve the nitrogen nutrition status of rice plants, which is beneficial for biomass accumulation. Under nitrogen reduction conditions, OsAMT1;1 gene can increase the growth rate of rice and facilitate its biomass accumulation (Chen et al. 2023). The expression level of nitrate transporter gene NRT1.3 in alfalfa roots is significantly positively correlated with nitrogen application and soil nitrogen concentration. The expression of NRT1.3 gene in alfalfa is beneficial for increasing fresh weight, chlorophyll and nitrate content, and can promote plant growth (Jiang et al. 2018). FtNRT2.3 gene is a member of the NRT2 gene family, and the expression of NRT2 family genes is beneficial for the transport of ammonia in plants (Chen et al. 2021). The study on the expression level of TaNRT2 and its absorption efficiency of NO in wheat under NO treatment conditions confirms the important role of TaNRT2 in wheat's absorption of NO (Zhao et al. 2004). Under low nitrogen treatment conditions, the significant increase in the expression levels of PbNRT2.4 and PbNRT2.5 in Phoebe bournei is likely related to nitrogen absorption and transport (Li et al. 2023). The expression of NRT2 gene in peanuts can respond to low ammonia stress, and the expression of AhNRT2.7a can improve nitrogen utilization efficiency and enhance nitrogen and carbon metabolism processes (Wang et al. 2022).

Aspartic acid treatment can significantly improve the germination rate of Tartary buckwheat seeds, and 40 µM Asp is the appropriate concentration to promote seed germination. Spraying aspartic acid can increase the root length, stem diameter, plant height, fresh weight, chlorophyll and nitrogen content of Tartary buckwheat seedlings, and the appropriate concentration for aspartic acid spraying is 30 µM Asp.

After spraying appropriate concentrations of aspartic acid, the expression levels of chlorophyll synthesis related genes (FtDCL and FtGLK1) and nitrogen transport related genes (FtAMT1-1 and FtNRT2.3) in Tartary buckwheat significantly increased, reaching their maximum expression levels at 6 h. Asp has a promoting effect on seed germination and seedling growth of Tartary buckwheat, which is beneficial for plant growth.

AMT Ammonium transporters

Asp Aspartic acid

GLK Golden like

Met Methionine

Pro Proline

SD Standard deviation

SPAD Soil and plant analyzer development value

Ethical Approval

This study does not involve ethical issues.

Informed Consent

All the authors agreed on the contents of the paper and post no conflicting interest.

Conflict of Interest

There is no conflict exists among all the authors, and the contribution of the authors is clear and unquestionable. All of them declare that they have no conflict of interest. Therefore, all authors are allowed to publish the article.

Author Contributions

Xiangqin Wu analyzed the data and drafted the manuscript; Meng Zhang determined the physiological indexes and analyzed the gene expression of Tartary buckwheat; Hong-Bing Yang designed the study and helped draft the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (31371552).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Summary statement

Aspartic acid on physiological characteristics and gene expression

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TableS1.docx
Table S1 Reverse transcription reaction system
TableS2.docx
Table S2 Reverse transcription reaction program
TableS3.docx
Table S3 Fluorescence quantitative PCR primer sequence

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First submitted to journal
11 Aug, 2025

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Effect of aspartic acid on physiological characteristics and gene expression of Tartary buckwheat

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Version 1

Abstract

Figures

Introduction

Material and methods

Material

Determination of seed germination rate

Seedling cultivation and treatment

Methods for measuring gene expression and primer sequences

Data processing

Results

Effect of Asp on seed germination of Tartary buckwheat

Effect of Asp spraying on growth of Tartary buckwheat seedlings

Effect of Asp spraying on chlorophyll and N content of Tartary buckwheat seedlings

Effect of Asp spraying on the expression of chlorophyll synthesis related genes in Tartary buckwheat

Effect of Asp spraying on the expression of N transport related genes in Tartary buckwheat

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1