Investigating Phragmites Australis Response to Copper Exposure Using Physiologic(cid:0)FTIR and Metabolomic Approaches

Background and aim Phragmites australis is a landscape plant with phytoremediation functions that is widely planted worldwide. However, little is known about the metabolomic background of the resistance mechanisms of Phragmites to heavy metals during its growth and development. Methods Here, we performed copper stress studies on Phragmites and monitored physiological indicators such as malondialdehyde (MDA) and electrolyte leakage (EL). In addition, FTIR was used to study chemical composition changes in the roots, stems and leaves of Phragmites seedlings under excessive copper stress. Furthermore, LC-MS technology combined with metabolomics data processing software was used to analyze the metabolic prole of samples. Result Copper contributed to the accumulation of MDA and EL. And the results of FTIR showed that the antioxidant effects of avonoids and amino acids can be used by Phragmites leaf tissue to improve the tolerance of copper under 5 mg/L concentration. Further, the results of metabolomics reected that Phragmites can improve its resistance to copper by increasing the accumulation of arginine and ayarin in the body. The former is accumulated through two pathways: the citrulline decomposition and conversion pathway and the circular pathway composed of ornithine, citrulline, L-Argininosuccinate and arginine. The latter is synthesized through the quercetin methylation pathway. Conclusion This study provides insights into the resistance mechanism and repair performance of Phragmites and other plant accumulators in response to copper stress. transporters, Galactose metabolism, Tyrosine metabolism, Isoquinoline alkaloid biosynthesis, alpha-Linolenic acid metabolism, Monobactam biosynthesis, Lysine degradation, Aminoacyl-tRNA biosynthesis, and Photosynthesis. Signicantly different metabolic pathways in the negative ion group are: Lysine degradation, Flavone and avonol biosynthesis and Arginine biosynthesis.


Introduction
Although copper is an essential trace element in plants, excessive copper is toxic to plants (Ugulu et al. 2012, Sahin 2016. Recently, copper pollution has attracted increasing attention due copper excessive emissions which are caused by different human activities such as mining, metal processing, fertilizer industry, fungicides, industrial and domestic wastewater and tra c discharges (Dogan,Baslar and Ugulu addition, it is believed that Phragmites has strong heavy metal removal capabilities due to features such as rapid growth to form dense vegetation, higher above-ground biomass, typical tissue systems and defense mechanisms in the body, and higher bioaccumulation and heavy metal removal e ciency than other repair plants (Carricondo et  In this study, we preliminarily assess the degree of plant damage in Phragmites under copper stress during growth and development by measuring the growth index, malondialdehyde (MDA), electrolyte leakage (EL), and copper concentration. Then, we further characterize the functional groups in Phragmites that have changed under copper stress by analyzing infrared data. Finally, we explore the mechanism of the molecular biological changes of internal metabolites of Phragmites leaves under copper stress using the results of LC-MS non-targeted metabolome analysis. The main goal of this study is to analyze the response mechanism of Phragmites to copper stress through metabolomics and further understand the tolerance and resistance mechanism of Phragmites.

Materials And Methods
Experimental Design and Growth Parameters P.australis seedlings used in the experiment were rst hydrocultured for 20 days in pots containing 2 L of Hoagland nutrient solution, before being subjected to copper stress. 0.02 mg·l − 1 nutrient solution acted as the control group and the copper concentration gradient was set to 5,10, and 20 mg·l − 1 . Each treatment was repeated three times, and all solutions replaced every 2 days. The experiment lasted for 21 days. Three seedlings washed with deionized water were randomly selected at each treatment concentration to measure the shoot height, root length, shoot dry weight, and root dry weight.

Determination of Cu Concentrations
The method of analyzing the copper content in Phragmites seedings is shown as follows. Firstly, the seedlings were washed then dried via baking, and then the dried samples were homogenized. Next, they were transferred to a Microwave Digestion System (MARS-5; CEM, Matthews, North Carolina) for mineralization with HNO 3 (67%)-HCl (30%)-HF (49%) acids (5 : 2 : 2, V/V/V). Lastly, analysis of the mineralized samples was conducted using Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) (Perkin Elmer Optima 5300DV, Waltham, Massachusetts).

Physiological Measurements of Phragmites Seedlings under Copper Stress
According to Heath et al. (Heath and Packer 1968), lipid peroxidation was assayed by determining the content of MDA in the functional leaf. A Phragmites leaf sample (0.1g) was homogenized with 5 mL of 10% trichloroacetic acid (TCA) solution and a small amount of quartz sand in the mortar. The homogenate was poured into a glass tube and centrifuged at 4000r/min for 15 min. 2ml of 0.6% Thiobarbituric acid (TBA) replaced 2ml of the supernatant in the centrifuge tube, and the new solution was mixed well and kept in a boiling water bath for 15. The reaction was stopped by cooling and the mixture was centrifuged again. The supernatant was used to measure the absorbance (OD) of the sample at 450nm, 532nm and 600nm wavelengths in a cuvette.
The EL induced by copper was estimated by measuring the electric conductivity (Pang et al. 2003). Plant samples of Phragmites leaves (0.1g) were placed into syringe containing 10 mL of deionized water. Then, the air between the leaves was exhausted by pumping repeatedly for several minutes. Then pour the samples and water into a beaker and wait for about 10 minutes. The electric conductivity was measured at room temperature (EC1). Thereafter, the beakers containing the fronds were placed in a boiling water (100℃) bath for 20 min and then cooled to room temperature, and the electric conductivity was measured again (EC2). The relative electrolyte leakage was calculated using the following formula: EL= (EC1/ EC2) × 100.

FTIR Spectroscopic Analysis
The root, stem, and leaf of Phragmites stressed by copper were separated into different beakers, numbered, and frozen. 4g of frozen plant samples was weighed accurately and grinded into powder. Next, the powdered plant sample was transferred to a sealed centrifuge tube, treated in an ice water bath for 20 minutes, and centrifuged for 10 minutes in a 4000 rpm high-speed centrifuge. The precipitate was washed once each with iced acetone, iced methanol chloroform, and methanol, and centrifuged again at 4000 rpm for 10 min. After the second centrifugation, the precipitate was frozen, vacuum dried, weighed, and numbered. To determine the spectral information, 2 mg of the dried sample prepared above was thoroughly mixed with KBr. The absorbance of each component of Phragmites was then recorded with a FTIR (as per operator procedure).

Metabolomic Analysis
The extraction of metabolites was carried out as follows: Frozen leaf tissues (100 mg) from each sample group were grinded in 1 mL solution of methanol/water (1:1) at a frequency of 60Hz for 2 minutes. The samples were sonicated for 30 min and let stood at -20°C for 20 min. Next, the samples were centrifuged for 10 min (13000 rpm, 4°C) and 200 µL of the supernatant was drawn with a syringe. Finally, the samples were transferred to the LC injection vial and stored at -80°C until LC-MS analysis. Quality control samples (QC) were prepared by mixing extracts of all samples. All extraction reagents in this experiment were pre-cooled at -20°C before use.
The column temperature was kept at 50°C and the injection volume was 10 µL.

Statistical Analysis
All values in this study were the average of three replicate samples. The MDA concentration was calculated according to the following formula: where D532, D600 and D450 represent the optical density values at the wavelengths of 532, 600, and 450 nm, respectively. SPSS (Ver.25.0, US) software was used to perform statistical analyses. The data was analyzed using one-way ANOVAs and least signi cant difference (LSD) test was used to determine the signi cant differences between treatments (P < 0.05).

Distribution of Copper and Effect on P.australis Growth
In this study, the results showed that the copper content in the roots, stems, and leaves of P.australis was constantly increasing as the copper concentration increased (Fig. 1). When the copper concentration changed from 5 mg·l − 1 to 10mg·l − 1 , the copper content in the roots, stems, and leaves of P.australis increased by 44.53%, 113.67%, and 105.32% respectively. Moreover, copper content in roots, stems and leaves increased by 16.57%, 18.7%, and 28.6% respectively when the copper concentration increased from 10 mg·l − 1 to 20 mg·l − 1 . It can be seen that the copper content signi cantly increased in all parts of P.australis as the copper concentration changed from 5 mg·l − 1 to 10 mg·l − 1 , however the accumulation rate slowed down as the copper concentration changed from 10 mg·l − 1 to 20 mg·l − 1 .
As shown in Table 1, in response to copper stress, the shoot height and root length of P.australis had a tendency to decrease gradually from 5mg·l − 1 to 20 mg·l − 1 compared to the control group. Additionally, Table 1 shows that shoot dry weight and root dry weight decreases with increasing copper concentration. Table 1 Effect of copper stress on shoot height, root length, shoot dry weight and root dry weight of P.australis seedlings.

Effect of Copper on EL and MDA in P.australis Leaves
In this study, the MDA content of P.australis leaves increased with an increase in copper concentration (Fig. 2a). As shown in Fig. 2b, a continuous increase in copper concentration led to the increase of EL.
The results above re ect that excess copper can exacerbate the damage of cell tissues. In addition, Fig. 3b shows that the main FTIR changes in P.australis stems were the O-H absorption peak at 3414 cm − 1 -3401 cm − 1 and the N-H amide absorption peak at 1639 cm − 1 -1633 cm − 1 . Figure 3c also shows that the O-H absorption peak of P.australis roots was at 3388 cm − 1 -3403 cm − 1 , and the C-C or C-O group absorption peak was at 1124 cm − 1 -1140 cm − 1 .

Metabolomic Analysis of P.australis under Copper Stress
In the metabolomic results, univariate analysis of fold-change and T statistical were used to test p-value to screen the differentially expressed metabolites. The screening criteria are ratio > = 2 or ratio < = 1/2, p value < 0.05 and the fold change is the ratio of average content of metabolites in the two groups. The results re ected that there were a total of 228 differential metabolites in the positive ion sample group, which can be divided into 64 up-regulated differential metabolites and 164 down-regulated differential metabolites. Meanwhile, a total of 217 differential metabolites were in the negative ion sample group, of which 80 were up-regulated differential metabolites and 137 were down-regulated differential metabolites. In general, pathway analysis is based on the metabolic pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We mapped the differential metabolites to the KEGG database to obtain the enrichment results of their metabolic pathways. By estimating pathway enrichment analysis of differential metabolites, we obtained 73 metabolic pathways and 1609 metabolites in the positive ion group, of which 215 are differential metabolites. At the same time, there were 69 metabolic pathways and 1044 metabolites in the negative ion group, which included 171 differential metabolites. For the screening of signi cant difference metabolic pathways, the signi cant difference metabolic pathway was selected as the pathway with a larger p-value (p < 0.05). Signi cantly different metabolic pathways in the positive ion group are: Starch and sucrose metabolism, Arginine and proline metabolism, Tryptophan metabolism, ABC transporters, Galactose metabolism, Tyrosine metabolism, Isoquinoline alkaloid biosynthesis, alpha-Linolenic acid metabolism, Monobactam biosynthesis, Lysine degradation, Aminoacyl-tRNA biosynthesis, and Photosynthesis. Signi cantly different metabolic pathways in the negative ion group are: Lysine degradation, Flavone and avonol biosynthesis and Arginine biosynthesis.
The Analysis of Arginine Biosynthesis of P.australis under Copper Stress As shown in Fig. 5a, there are three differential metabolites in the arginine biosynthesis pathway. Glutamine and N-Acetyl-L-glutamate 5-semialdehyde are down-regulated metabolites, which are involved in citrulline synthesis and ornithine synthesis respectively. Arginine is an up-regulated metabolite, which is involved in both synthesis processes mentioned. Citrulline and ornithine also participate in the synthesis of arginine. In this study, the synthesis of arginine was divided into two pathways: rstly the direct catabolization of citrulline into arginine, and secondly the circular pathway composed of ornithine, citrulline, L-Argininosuccinate, and arginine (Fig. 5a).

The Analysis of Flavone and Flavonol Biosynthesis of P.australis Copper Stress
In this study, as shown in Fig. 5b, there are 6 differential metabolites in the biosynthesis of avonoids and avonols under copper stress, of which ayarin was the only up-regulated compound. And ayarin was involved in the avonol resistance mechanism of P.australis under copper stress. The result showed that during the biosynthesis of ayarin, quercetin was rstly methylated to form 3-0-methylquercetin, then transformed into 3,7-0-Dimethyquercetagetin and nally obtain ayarin. (Fig. 5b). Moreover,apigenin and kaempferol produced in the process of avonoids biosynthesis. The results also showed that apigenin can be transformed into Cosmosiin, Apin, Isovitexin and Vitexin and they were all down-regulated metabolites. And kaempferol was transformed into quercetin conducted by avonoid 3'-hydroxylase (F3'H) and avonoid 3',5'-hydroxylase (F3'5'H), and quercetin continued to undergo methylation and a series of reactions to nally form ayarin. Meanwhile, kaempferol can also be converted to kaempferin by UDP-glycosyltransferase 78D1 (UGT78D1), kaempferin was down-regulated metabolite.

Discussion
The distribution of copper re ect that P.australis has a great potential to accumulate Cu in their body parts. This is re ective of the addition of copper inhibiting the growth of P.australis due to large amounts of copper accumulation in the cells ( MDA is the oxidation product of lipid peroxidation, indicating the generation of free radicals and re ecting the degree of damaged membranes which are caused by abiotic stress (Riaz et al. 2021, Ohkawa H 1979. Lipid peroxidation is mediated by free radicals (Slater 1984a;b), which is the best measure (Halliwell 1991) to test the damage that is caused by increasing reactive oxygen species (ROS). Judging by the growth trend of MDA, it can be inferred that the increasing copper content will induce a large amount of ROS in P.australis leaves. And ROS can cause lipid peroxidation and cell membrane damage. Cronq seedlings to produce many proteins, amino acids, and other substances, and can also enhance stress resistance, provide nitrogen sources, reduce heavy metal toxicity and stabilize the internal environment by means of osmotic adjustment. Therefore, in this study, the FTIR results demonstrate that the O-H changes in P.australis leaves are related to the production of polyphenols and avonoids, and the content of proteins and amino acids have an in uence on the changes of absorption band near the 1644 cm − 1 under copper stress. According to Yu et al. (Yu et al. 2017), FTIR analysis shows that under Cd stress, the O-H absorption peak of V.zizanioides roots is higher than that of the control group. The O-H of the root cells was complexed with C, which formed stable compounds to improve the plant's resistance to Cd. In this study, it is speculated that the increased O-H in the roots of P.australis are complexed with Cu ions to improve the tolerance of the roots under copper stress.
In this study, infrared spectroscopy detected the changes of the functional groups, some of which were related to amino acids and avonoids in P.australis leaves. By analyzing the metabonomic results, we found that many amino acid-related pathways were signi cantly enriched, one of which being the arginine biosynthesis pathway. The metabolic activity of the compounds in the arginine biosynthesis pathway can not only maintain the balance of citrulline and ornithine in P.australis leaves, but also urge P.australis leaves to accumulate a large amount of arginine. These amino acids can not only chelate heavy metals but can also have antioxidant effects. In addition, the avonoids and avonols biosynthesis pathway in the metabolome revealed the production process of speci c avonoids and avonols and their changes in response to copper stress. Interestingly, these compounds also have high antioxidant activities. Therefore, this project will mainly analyze the arginine biosynthesis pathway and the avonoids and avonols biosynthesis pathway to explain their resistance mechanisms in P.australis leaves under copper stress.
In the arginine biosynthesis pathway, several studies have shown that a large amount of arginine in plants can reduce toxicity by chelating heavy metal ions. At the same time, arginine can also synthesize antioxidant peptides with other substances to inhibit the destruction caused by ROS and the peroxidation of essential fatty acids (Rani, ) have reported that citrulline is a precursor of arginine in the pathway of citrulline catabolizing into arginine. In the second circular pathway, ornithine is rstly converted to citrulline by the catalysis of ornithine transcarbamoylase (OTC). Then, L-Argininosuccinate is formed by the connection of aspartic acid with citrulline conducted via argininosuccinate synthase (ASS1). Finally, the synthesis of arginine is catalyzed by arginosuccinate lyase (ASL), and arginine is sequentially catabolized into ornithine by arginase (Winter et al. 2015, Joshi and Fernie 2017, Monne et al. 2015). According to Fig. 5a, arginine can also be consumed to transformed into citrhlline. Therefore, it is inferred that the second circular pathway dominates the process of arginine accumulation. And the accumulated arginine was used to resist copper stress. glutamine accumulation is a necessary prerequisite for the synthesis of citrulline through carbamoyl phosphate synthetase (CPS). In the second pathway, arginine can be oxidized to citrulline by the catalysis of nitric oxide synthase (NOS) according to Maurya et al. (Maurya and Rani 2017). In this study, glutamine was a down-regulated metabolite, and the biosynthesis of citrulline was reduced in the glutamine synthesis pathway. However, arginine was an up-regulated metabolite, which increased citrulline synthesis. The two pathways jointly maintained the stability of citrulline in P.australis leaves. Citrulline can maintain nitrogen homeostasis by playing a role in plant nitrogen transport under abiotic stress and maintaining cell osmotic pressure, and it is also an effective free radical scavenger (Joshi and Fernie 2017, Breuillard,Cynober and Moinard 2015).
In addition, the synthesis of ornithine is also divided into two pathways: the glutamate synthesis pathway and the arginine synthesis pathway (Monne et al. 2015, Chen et al. 2019). In the rst pathway, Winter et al. (Winter et al. 2015) found that glutamate synthesizes ornithine in a cyclic fashion through several acetylation intermediates. In the second pathway, Singh et al. (Singh et al. 2020) showed that arginine synthesizes ornithine through arginase. In this study, N-Acetyl-L-glutamate 5-semialdehyde, a key acetylation intermediate in the glutamate pathway, was down-regulated, which indicates that the accumulation of ornithine was reduced in this pathway. In the arginine pathway, arginine was an upregulated metabolite, which promoted the accumulation of ornithine. It can be speculated that the two pathways worked together to maintain the balance of ornithine. Studies show that excessive accumulation of ornithine can not only cause the toxicity of plants, but also limit the synthesis of polyamines. Therefore, it is necessary to maintain homeostasis of ornithine in plants. In the avonoids and avonols biosynthesis pathway, avonoids have an important function in many plants, such as pigmentation, preventing dormancy, improving fertility, protecting from ultraviolet rays, defending against plant pathogens, and preventing biological and abiotic stress. Flavone and avonol are avonoids (Iwashina 2003, Jia et al. 2012).
Ayarin is a avonol derived from the gradual methylation of quercetin (Vitalini et al. 2011). Flavonols can act as antioxidants and activate the antioxidant system when plants resist adverse environment and abiotic stresses, and can also eliminate oxidative stress induced by ROS (Zhang et al. 2020, Watkins,Hechler andMuday 2014). Several studies have shown that as a precursor of ayarin, quercetin can also inhibit lipid peroxidation by scavenging ROS and chelating metal ions which can cause the production of ROS (Ishige,Schubert and Sagara 2001, Kato et al. 2016, Je€rey B. Harborne 2000. It is mentioned above that quercetin forms ayarin via the process of 3-Omethylation. Although the oxidation ability of ayarin is weaker than quercetin, the process of 3-Omethylation greatly improves the free radical scavenging ability of ayarin. This is because methylated quercetin is an effective metal chelating agent that will chelate Cu ions to form a complex (Vitalini et al. 2011, Kato et al. 2016, Pekal,Biesaga and Pyrzynska 2011, Bukhari et al. 2009). Therefore, in this study, it is speculated that P.australis resists oxidative stress by exerting a higher antioxidant capacity through the chelation of ayarin.
As a naturally occurring avonoid in plants, apigenin has signi cant antioxidant activity, which can have an effect on scavenging free radicals to inhibit the oxidative stress response of plants (Dou et al. 2020). Studies show that apigenin can also combine with sugar to form Cosmosiin, Apin, Isovitexin and Vitexin and other compounds, and these compounds are glycosides naturally occurring in plants (Meyer et  have reported that F3'H and F3'5'H can promote the accumulation of quercetin, and F3'H plays a leading role in this process. Therefore, it is speculated that kaempferol can be produced in the process of avonoid biosynthesis when kaempferol was consumed by other reactions, which was greatly balance the content of kaempferol in P.australis leaves. By maintaining the content of kaempferol in P.australis leaves and avoiding the decrease of its content, the oxidative stress response caused by copper stress can be resisted.

Conclusions
The main goal of this report is to evaluate the resistance mechanism of Phragmites to coppr stress during growth and development from the aspect of metabolomics analysis. Here, we studied the variation of the physiological indicators of Phragmites leaves and found that the content of MDA and EL gradually increased with the increase of copper concentration, which re ected that the degree of oxidative damage of Phragmites leaves under copper stress was increasing. Additionally, we used the FTIR to research the change of chemical composition in the roots, stems and leaves of Phragmites seedlings, the results showed that avonoids and amino acids were the main increased substances in Phragmites leaves, which is helpful to explain how plants show tolerance to copper stress. Besides, by analying the metabolomics results to identify signi cant differential metabolic pathways and differential metabolites that were signi cantly up-regulated or down-regulated in the corresponding pathways, including Arginine Biosynthesis,Flavone and Flavonol Biosynthesis arginine and ayarin and so on, which were all related to the resistance of Phragmites leaves. This project is devoted to improve the understanding of the physiological and molecular mechanisms involved the process of growth and development of Phragmites leaves under copper stress, and provided a theoretical basis for improving the resistance mechanism and repair performance of Phragmites to heavy metal.     (a) The volcano graph of differential metabolites. The red origin represents the differential metabolites that are signi cantly up-regulated in the experimental group, and the green origin represents the signi cantly down-regulated differential metabolites. Gray dots represent insigni cantly differential metabolites. (b) The heat map of differential metabolites. Perform Hierarchical Clustering on the expression of signi cantly differential metabolites. (c) General overview of signi cantly differential metabolites in positive ion group and negative ion group. (d) Statistical graph of differential metabolites (Flavone and avonol biosynthesis and Arginine biosynthesis).