Combined transcriptomic and metabolomic analyses reveal the phosphorus stresses response mechanisms of Artemisia argyi

doi:10.21203/rs.3.rs-2502781/v1

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Combined transcriptomic and metabolomic analyses reveal the phosphorus stresses response mechanisms of Artemisia argyi

https://doi.org/10.21203/rs.3.rs-2502781/v1

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Purpose

Enzymatic activity in plant signaling, photosynthesis, energy metabolism, and respiratory activities depend on the amount of readily available phosphorus in the soil. Artemisia argyi(A. argyi) is a medicinal plant with antifungal, antiinsect, and anti-inflammatory activities. Our goal was to increase knowledge of the physiological and molecular mechanisms involved in the investigation of the effects of soil phosphorus on important secondary metabolites of A. argyi that regulate growth.

Methods

At various phosphorus levels, A. argyi was examined for its agronomic characteristics, elemental content in the leaves, generation of significant metabolic chemicals, and molecular mechanisms.

Results

A. argyi displayed the highest levels of phenolic acids (chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and neochlorogenic acid) and flavonoids (hispidulin, jaceosidin, eupatilin, and casticin), measured in the leaves, when treated with low levels of phosphorus. P-element levels significantly correlated with the amount of each active ingredient, and transcript Phosphorus levels also affected terpenoid content and the expression of numerous genes involved in the 4-hydroxytoluene pathway (MEP) and the pentadienyl pathway (MVA), which are both necessary for terpenoid production.

Conclusions

Low levels of phosphorus application enhance the accumulation of medicinal chemicals in A. argyi, medium levels of phosphorus application stimulate the growth of A. argyi, and associated genes are often down-regulated with higher levels of phosphorus application.

Artemisia argyi

phosphorus fertilizer

stresses

volatile oil

flavonoids

transcriptome analysis

secondary metabolism

Phosphorus (P) into H₂PO_4, creating an aqueous solution that can be actively absorbed by the root surface through diffusion (Clarkson, 1985). It is thought that cellular phosphorus entry is facilitated by a plasma membrane ATPase-established proton drive with the aid of a protonated phosphate carrier (Lee et al., 1990). Once inside a plant cell, phosphorus can become a component of many essential compounds including nucleic acids, phospholipids, and ATP (Bieleski, 1973). The mineral is involved in various plant processes: it helps regulate the activity of crucial peroxidase and metabolic pathways (Theodorou and Plaxton, 1993); affects mitochondrial electron transport and mechanistic pathways (Van Ray and Van Diest, 1979); and phosphorus is involved in photosynthetic electron transport which can accelerate the photosynthetic rate of leaves. Many growers invest heavily in phosphorus application as it can improve crop water status, increase chlorophyll content (Advances in photosynthesis research. 3, 1984), reduce the photosynthetic CO₂ compensation point (Lauer et al., 1989), and maintain high stomatal conductance and photosynthetic activity. Aside from nitrogen, phosphorus is the most important nutrient for preventing agricultural yield reduction or crop loss due to deficiencies. While phosphorus may be prevalent in soil, the vast majority of its natural forms are insoluble meaning there is very little readily available for plant use (Heckrath et al., 1995). Except for some areas in the Northeast and the Tibetan Plateau, soils with low effective phosphorus are common in China, with many having less than 10 ppm P. While a lack of phosphorus can cause low plant growth, over-fertilization can lead to soil slumping and eutrophication in nearby water bodies. The rational use of phosphorus fertilizer is vital for strong agricultural production and environmental safety (Yan et al., 2013).

Extensive research has been conducted to determine the mechanisms of how P affects the growth and development process in various plant species. Previous studies have shown that a phosphorus deficiency can increase the number of organic acids and phenolics released by the leaves, leading to a delay in plant development (Hermans et al., 2006). In their study of P stress in rice, Cai revealed many different transcriptional responses of roots and shoots to P starvation (Cai et al., 2012); In another study, Stephen L. Byrne derived that perennial ryegrass copes with P deficiency by using glycolytic bypass and replacing phospholipids with sulfolipids (Byrne et al., 2011). Furthermore, Ying Guo's study detailed the mechanisms involved with P regulation of photosynthesis in Ginkgo biloba (Guo et al., 2021). According to research on the growth pattern and changes in secondary metabolites by Na Ding, an increase in switchgrass phosphorus stress led to a content decline in the majority of sugars and sugar alcohols, but under mild and moderate stress, there was an increase in the content of organic and amino acids in stems, leaves, and roots (Ding et al., 2021). The impacts of phosphorus on the metabolic pathways of flavonoids (Winkel, n.d.), phenolic acids, and other chemicals in plants via phenylalanine have been the subject of numerous investigations. A previous study demonstrated that in the hairy roots of Saliva miltiorrhiza, phosphorus deprivation induces the main enzyme genes in a positive feedback system, leading to an increase in the production of phenolic acids (Liu et al., 2016). Pi deficiency has also been found to greatly increase Cyclocarya paliurus's flavonoid accumulation (Zhang et al., 2021). A study on Xiong C illustrated how P shortage affected the tissue-specific distribution of flavonoids in roots, indicating that this accumulation fine-tuned the influence of flavonoids on cluster-root development (Xiong et al., 2022). Finally, the volatile oil content of lavender was also shown to increase with a rising rate in phosphorus application (Peçanha et al., 2021).

Artemisia argyi Level. et Vant (A. argyi)belongs to the Asteraceae family(Bora and Sharma, 2011;Lee et al., 2006).This plant has a long history in China and has been used for many purposes including food and medicine. Its leaves can be boiled in water and added to baths to prevent and control infections in mothers and babies during the puerperium. The whole herb can be used as an insecticide, even having the ability to fumigate whole rooms. The strong effects of this plant have been demonstrated in modern medical experiments and are proven to have anti-inflammatory(Yun et al., 2016; Zimmermann-Klemd et al., 2020), anti-allergic (Lv et al., 2018), anti-microbial (Pertea et al., 2016), and anti-cancerr(Seo et al., 2003) properties. The leaves of A. argyi are rich in volatile oils, flavonoids, terpenoids, and phenolic acids (Guan et al., 2019), which have all been extracted for examination in prior studies(Song et al., 2019)(Bao et al., 2013). The biological activities of several of these extractions, including eupatilin, jaceosidin, apigenin, luteolin, quercetin, and naringin, are known to prevent oxidative damage, inflammation, allergies, and tumors(Pertea et al., 2016; Seo et al., 2003)

Although the effects of phosphorus fertilizer have been well documented in some species, the mechanisms of its impact on the agronomic characteristics and secondary metabolism of A. argyi are not clear. In this study, we measured a variety of growth indicators in A. argyi under various phosphorus fertilization treatments. We also quantified the active ingredients, such as flavonols, phenolic acids, and terpenoids to gain a better understanding of the plants’ biological makeup. Moreover, we conducted transcriptome analyses to identify the intricate molecular processes underlying the production of phenolic acids, flavonoids, and terpenoids in A. argyi leaves under various phosphorus treatments.This study provides a basis for the p induces stress responses as well as the molecular mechanisms involved.

Plant material and growth conditions

This experiment was conducted in the potted plant portion (30 ° 27 ′ N, 114 ° 15 ′ E) of the medicinal botanical garden of Hubei University of Traditional Chinese medicine from January to June in 2022. The tested variety, A. argyi, was collected from Qichun County in Hubei Province. Each pot was filled with 10 kg soil with sand added to improve soil ventilation. There were four duplicates per treatment. Three herbaceous rhizomes were planted in each pot in January 2022, all at a depth of roughly 3 cm. The three phosphorus fertilizer application rates were set at 0 (P1), 1.5 (P2), and 3 (P3) g∙10 kg^− 1. 10 mL of Arnon trace element mixture (Ca(NO₃)₂ 945mg/L,K₂SO₄ 607mg/L,NH₄H₂PO₄ 115mg/L,Mg₂SO₄ 493mg/L) was applied to each basin to supplement the trace phosphorus. The only difference between each pot's agronomic cultivation methods was the quantity of phosphorus used.

Investigation of agronomic characteristics and yield

The selected agronomic traits were measured on June 8, 2022, and included plant height, stem diameter, dead leaf height, leaf number, five-leaf spacing, leaf width, leaf length, and chlorophyll content. The plants were then harvested and dried in the shade. The total dry weight of A. argyi leaves and stems in each pot was measured and then used to calculate the weight per plant.

Determination of volatile components

The content of a plant’s volatile oil was determined according to the method a in general rule 2204 of Chinese Pharmacopoeia (Volume IV), 2020 edition (2020). The steps of identifying volatile components were as follows: weigh and grinding 0.3 g fresh A. argyi leaves, put the grind into a 20 mL headspace bottle with a solid-phase microextraction instrument, insert the head of the manual sampler of pdms-dvb extraction fiber into the bottle, balance at 85 ℃ for 10 min, push out the extraction head, empty the extract for 40 min, remove the grind and immediately insert it into the sample inlet of a chromatograph, desorb for 3 min. The conditions of the gas chromatography-mass spectrometer (GC-MS) were based on a Chen’s study (Chen et al., 2021). The Mainlib library was used to search each chromatographic peak in the total ion flow diagram of volatile oil by GC-MS.Chromatographic peak with a possibility of more than 50% was selected and further analyzed.

Determination of total phenolic acids, total flavonoids, and their components

The contents of total phenolic acids and flavonoids in fresh A. argyi leaves were determined according to the Miao YH (Miao et al., 2022). A 0.3 g sample of fresh A. argyi leaves were collected, crushed, and sieved for the determination of active ingredients. Pure methanol was used to reduce the sieved A. argyi remnants into a powder overnight. The contents of chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cryptochlorogenic acid, and neochlorogenic acid, as well as flavonoids, such as hispidulin, jaceosidin, eupatilin, and casticin were determined by lc-2030c (Shimadzu) conditions as follows: Agilent EClipse XDD-C18 column (4.6mm×250mm, 5µm), a mobile phase of acetonitrile (A)-0.2% aqueous phosphoric acid (B), gradient elution (0-12min, 10%-20%A; 12-25min, 20%-25%A; 25-50min, 25%-65%A; 50 ~ 55min, 65%~10%A) at a flow rate of 0.8mL^{− min−1}; column temperature set on 30°C, detection wavelength 330nm, and detector UV detector.

Total RNA extraction and RNA-sequencing (RNA-seq) analysis

The third to fourth mature leaves of each A. argyi plant were collected for RNA-seq analysis. Three biological replicates were performed for each treatment. The total RNA was extracted by an Invitrogen Trizol kit and the sequencing library was constructed and sequenced on the Illumina novaseq 6000 platform. Clean reads were used for RNA-seq analysis. Differential expression genes (DEGs) were identified using the R language package DESEQ2.0 with a screening threshold of FDR < 0.05 and |log₂FC|>1. Then DEGs were enriched by Kyoto Encyclopedia of Genes (KEGG) biological pathway.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis

Genes involved in phenolic acids, flavonoids, and terpene synthesis in A. argyi leaves were selected for RT-qPCR detection. All primers used are listed in Table S1. Total RNA samples were reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. The ACTIN gene (5'-CGACTATGTTCCCTGGTATTGC-3' and 5'-AGACCCTCCGATCCAGACACT-3') was used as the internal reference. The RT-qPCR reaction procedure was as follows: pre-incubation at 95 ℃ for 15 min; amplification at 95 ℃ for 10 s, 60 ℃ for 30 s (single acquisition mode) for a total for 45 cycles; melting curve generation stage at 95 ℃ for 5 s, 65 ℃ for 1 min, and then keep at 97 ℃. The relative expression levels of the target genes were calculated by the 2^−△△t method.

Statistical analysis

All experiments in this study were performed at least three times and all values are shown as means ± SD. The statistical analysis of the data collected from the control and treatment plant was carried out by a one-way analysis of variance (one-way ANOVA) using the SPSS 20.0 software. A statistically significant difference was determined when the probability value was P > 0.05. The mean and standard deviation (SD) of three replicates are used to represent the data.

Agronomic characteristics and N, P, and K content of A. argyi under the treatment of different phosphorus levels

To explore the effects of phosphate on the growth of A. argyi, we applied different concentrations of phosphate fertilizer and observed changes in agronomic traits. Following harvest, we found that A. argyi plants showed significant variation between the different treatments. As shown in Fig. 1a, the growth of A. argyi was severely inhibited by low phosphorus application levels. The number of seedlings, leaves, stalk thickness, leaf size, plant height, stalk dry weight, and total plant weight under the P1 treatment were significantly lower than treatment P2, as the leaf size, leaf number, stalk dry weight, stalk thickness and emergence, and fresh weight of leaves decreased under P3 treatment compared to that under P2 treatment (Fig. 1b-j). We then assessed the other contents of Mg, Ca, and Zn in A. argyi leaves to further explore how different phosphorus application levels affect other important plant nutrients. The results showed that between the P1 and P3 treatments, the content of N increased from 1.84–2.31%, P increased from 0.16 to 0.2%, and K content declined from 0.73 to 0.47. At moderate (P2) phosphorus application levels, A. argyi leaf Mg content was at its highest, and Ca content was at its lowest. Ca and Zn content were at their highest in P3 conditions (Fig. 1k,l).

Phosphorus affects the flavonoid, phenolic acid, and terpene contents of A. argyi plants

We next tested whether the contents of flavonoids, phenolic acids, and terpene were affected by phosphorus. The results showed that their contents in A. argyi leaves fluctuated significantly under the application of different phosphorus levels (Fig. 2).

With higher treatment levels, the total content of both flavonoids and phenolic acids diminished (Fig. 2c). Moreover, the contents of most flavonoids were negatively correlated with the level of phosphorus: schaftoside content during P2 and P3 treatments were 36.1% and 39.8% of that under P1, respectively (Fig. 2d); hispidulin content during P2 and P3 treatments were 12.5% and 50% of that under P1, respectively(Fig. 2e); and jaceosidin content under P2 and P3 treatments were 20.3% and 68.8% of that under P1, respectively (Fig. 2f). It is worth mentioning that the contents of eupatilin, a specific flavonoids component in A. argyi, broke from this pattern, and declined under P1 and P2 treatment to 18.5% and 13.8% of that under P1 treatment (Fig. 2g). Additionally, when compared with P1, casticin content was reduced by 21.4% under P2 and only 14.29% in P3 (Fig. 2h).

We further detected the contents of several phenolic acids in A. argyi leaves under different conditions. The phenolic acid pattern followed a similar trend as flavonoids: chlorogenic acid content under P2 and P3 treatment were 71.1% and 35.3% of that under P1, respectively (Fig. 2i); isochlorogenic acid A content under P2 and P3 treatment were 28.3% and 54.1% of that under P1, respectively (Fig. 2j); isochlorogenic acid B content under P2 and P3 treatment were 38.2% and 47.3% of that under P1, respectively (Fig. 2k); isochlorogenic acid C content under P2 and P3 treatment were 28.3% and 54.1% of that under P1, respectively (Fig. 2l); and cryptochlorogenic acid contents under P2 and P3 treatment were 44.1% and 67.6% of that under P1, respetivly (Fig. 2m).Conversely, neochlorogenic acid presented a different downward trend, with the contents droping 26.9% and 56.7% (Fig. 2n).

Next, we determined the contents of volatile components in A. argyi leaves under different treatments. The results demonstrated that these contents had greater variation, showing vastly different trends with the increase of phosphate fertilizer application. The contents of endo-borneol, terpinen-4-ol, and camphor increased with the higher levels of phosphate fertilizer (Fig. 2o-q), while the content of caryophyllene decreased sharply (Fig. 2r). Eucalyptol was unique and presented the highest content under P3 treatment and the lowest content under P2 treatment (Fig. 2s). Taken together, these results display that phosphate fertilizer has a range of effects on the accumulation of different volatile components.

Estimation of correlation coefficients for the active ingredient and mineral element content

Following various phosphorus treatments, we analyzed selected ingredient and mineral contents to determine possible changes. We analyzed the content features of the 16 active ingredients and 6 mineral elements selected for study using Pearson correlation. Our results revealed varying degrees of association between each trait (Table 1). Between nitrogen concentration (N) and schaftoside, hispidulin, jaceosidin, casticin, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid B, we found multiple strong and absolute negative connections. Phosphorus elements were found to be related to caryophyllene, isochlorogenic acid, cryptochlorogenic acid, hispidulin, jaceosidin, casticin, isochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C. Chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cryptochlorogenic acid, and caryophyllene were negatively correlated with P as -0.857, -0.975, -0.971, -0.725, -0.971, -0.958, -0.815, -0.749, -0.939, -0.936, and − 0.974, respectively (Table 1). Schaftoside, hispidulin, jaceosidin, eupatilin, casticin, chlorogenic acid, isochlorogenic acid A, B, C, cryptochlorogenic acid, and caryophyllene were all both significantly positively and negatively correlated with endo-borneol, terpinen-4-ol, and camphor (0.860, 0.837, 0.810). We found a strong link between the camphor ratio and Zn. The present study also reflects the strong correlation between the content of N, P, and K and the flavonoids, phenolic acids, terpenoids active ingredients in A. argyi. Research has been conducted in recent years suggesting correlations and heritability in the content of the main mineral elements in various plant species.

Table 1

Estimation of correlation coefficients for active ingredient and mineral element content
Correlation/R	N(%)	P(%)	K(%)	Mg(mg/g)	Ca(mg/g)	Zn(mg/g)
Schaftoside (mg/g)	− .936**	− .857**	.845**	-0.206	0.162	-0.334
Hispidulin (mg/g)	− .680*	− .975**	.676*	0.019	-0.228	-0.581
Jaceosidin (mg/g)	− .749*	− .971**	.710*	0.073	-0.212	-0.575
Eupatilin (mg/g)	-0.627	-0.555	.819**	-0.143	0.074	0.093
Casticin (mg/g)	− .857**	− .725*	.847**	-0.098	0.376	-0.281
Chlorogenic acid (mg/g)	− .847**	− .971**	.837**	0.127	-0.076	-0.517
Isochlorogenic acid A (mg/g)	− .836**	− .958**	.855**	0.161	-0.072	-0.488
Isochlorogenic acid B (mg/g)	− .914**	− .815**	.890**	0.058	0.211	-0.421
Isochlorogenic acid C (mg/g)	− .932**	− .749*	.866**	-0.024	0.273	-0.331
Cryptochlorogenic acid (mg/g)	− .914**	− .939**	.881**	0.033	0.049	-0.478
Neochlorogenic acid (mg/g)	− .870**	− .936**	.759*	0.132	-0.11	-0.529
Endo-borneol (%)	.848**	.971**	− .860**	0.051	0.013	0.503
Terpinen-4-ol (%)	.802**	.931**	− .837**	-0.071	-0.156	0.657
Camphor (%)	.731*	.940**	− .810**	-0.144	-0.094	.701*
Eucalyptol (%)	0.104	0.621	-0.343	-0.399	0.655	0.319
Caryophyllene (%)	− .742*	− .974**	.703*	0.178	-0.298	-0.539

Functional annotation and enrichment analysis of differentially expressed genes (DEGs) in A. argyi leaves under the treatment of different phosphorus levels

To explore the molecular mechanisms of how phosphate fertilizer affects the accumulation of effective components, we performed an RNA-seq analysis of A. argyi leaves during harvest to identify DEGs under different treatments. We performed three biological replicates and found that each had a great correlation coefficient, indicating the high credibility of RNA-seq data. The fragment per kilobase of transcript per million mapped reads value was calculated to quantify the expression abundance and variation using RSEM software (Fig. S1).

We further identified DEGs using a two-by-two comparison (P2-vs-P1, P3-vs-P1, P3-vs-P2) method. The results showed in P2-vs-P1, P3-vs-P1, and P3-vs-P2 treatments of A. argyi leaves, there were a total of 437 DEGs including 252 up-regulated and 185 down-regulated genes, 221 DEGs including 120 up-regulated and 101 down-regulated genes, and 237 DEGs including 101 up-regulated and 136 down-regulated genes, respectively. Our overall distribution of DEGs is presented as a volcano plot (Fig. S2). We subsequently performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification of DEGs in these three groups. the KEGG pathways mainly related to phenylpropanoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and diterpenoid biosynthesis (Fig. 3a-c).

The heatmap of major enzymes in the metabolic pathway of terpenoids, flavonoids, and phenolic acids under different phosphorus treatments

Terpenoids, flavonoids, and phenolic acids are the main active ingredients in A. argyi leaves and their contents showed a significant difference under different phosphorus treatments. We focused on the expression levels of crucial genes participating the metabolic pathways of these ingredients. We combined data obtained in our previous study on the identification of candidate genes with the transcriptome data obtained in this research (Miao et al., 2022), and processed it using ln (fpkm + 1). We then compared the expression of each gene using a heat map (Fig. 4). The results show that between the different treatments of A. argyi leaves, there is a significant difference in the expression levels of genes invololed in the phenylalanine synthesis pathway including AY175483-RA, AY175483-RA, AY239768-RA, and AY239769-RA encoding PAL; AY239164-RA encoding C3H; AY058254-RA and AY067952-RA encoding C4H; AY241117-RA, AY263333-RA, AY273264-RA, AY287354-RA, and AY296163-RA encoding CHI; AY028393-RA and AY165300-RA genes encoding F3H; AY028393-RA and AY165300-RA encoding F3’H; AY030140-RA and AY278182-RA encoding FLS; AY150231-RA encoding FNS, AY067656-RA, and AY296405-RA encoding FNSII. In the terpene synthesis pathway, we also see differential gene expression in the MEP and MVA pathways, including genes encoding 2-C-methyl-D-erythritol-2, 4-cyclodi-phosphate synthase (MDS), 1-deoxy-D-xylulose 5-phosphate synthase(DXS), (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate synthase (HDR), geranyl diphosphate synthase (GPPS), and terpene synthase (TPS).

Validation of DEGs by RT-qPCR

To further investigate the authenticity of our RNA-seq data, we detected the expression levels of several genes related to the metabolic pathways of terpenoids, flavonoids, and phenolic acids. We selected AY175483-RA, AY239769-RA involved in the production of PAL; AY067952-RA and AY058254-RA associated with C4H; AY239164-RA associated with C3H; AY263333-RA and AY241117-RA associated with CHS; AY028393-RA associated with F3'H; AY028393-RA associated with a gene involved in flavone-6-hydroxylase (F6H), AY027448-RA; AY078091-RA involved in the synthesis of DXS in terpene synthesis; AY261936-RA involved in the generation of 2-C-methyl-Derythritol-4-phosphate cytidylyltransferase(MCT); and AY076609-RA involved in MDS generation. The results showed that the relative expression levels based on RT-qPCR were consistent with our RNA-seq analysis (Fig. 5). Taken together, our results indicate that the differential expression of these crucial genes participate in the metabolic pathways of terpenoids, flavonoids, and phenolic acids give rise to the various contents of these gradients in A. argyi leaves under the treatment of different phosphorus concentrations.

In this study, we investigated the effects of different levels of phosphorus on the growth and quality of A. argyi. The results showed great variation between the phosphorus application treatments: low levels inhibited growth but significantly improved the quality of A. argyi leaves; medium levels promoted the growth of A. argyi leaves, producing changes such as taller plants, thicker stalks, and longer leaves; high levels inhibited the normal growth of A. argyi but increased the soil and plant analyzer developrnent (SPAD) value. Related studies conducted on switchgrass (Ding et al., 2021) and alfalfa (Li et al., 2022) showed that low phosphorus treatment produced an impairment of plant physiological functions. Taken together these studies emphasize the importance of selecting the appropriate phosphorus application concentration in accordance with the plant species.

An intricate gene expression network dictates how much phosphorus is allowed in the plant body. Multiple secondary metabolite syntheses in plants may be regulated by their metabolic pathways. In a study analyzing the effects of phosphorus stress on switchgrass, NaDing discovered that levels of the majority of sugars and sugar alcohols decreased as P stress increased. Conversely, it was discovered that the levels of organic and amino acids rose in shoots and roots under mild and moderate P stress, but this trend changed to a decline under severe P stress, particularly in shoots (Ding et al., 2021). According to research by Tingting Sun, the majority of amino acids, their derivatives, organic acids, and flavonoids were present in higher concentrations in the roots of apple seedlings exposed to low phosphorus stress than in the roots of the control (Sun et al., 2021). In this study, we found that low levels of phosphorus encouraged the accumulation of flavonoids and phenolic acids in A. argyi leaves more than in higher levels. Our results demonstrated that low amounts of applied phosphorus caused the formation of flavonoids and phenolic acids which are involved in reducing plant stress. In conclusion, we found that sufficient, but not toxic, levels of phosphorus can increase the accumulation of secondary metabolites leading to an improvement in the yield and quality of A. argyi.

Previous research on apples found that seedlings control their flavonoid pathways in response to various phosphorus environments (Sun et al., 2021). In studies on Salvia miltiorrhiza, it was found that phosphate starvation response genes (SPX:SYG1, Pho81, and XPR1) can affect the expression of key enzyme genes in response to late (Wu et al., 2003) phenolic acid biosynthesis, leading to the accumulation of phenolic acid. In the present study, we combined the active ingredient content and transcriptome data to elucidate the response mechanisms of A. argyi leaves to different phosphorus levels. GO analysis and KEGG pathway annotation illustrate that different levels of phosphorus application had significant effects on flavonoid and phenolic acid biosynthesis in A. argyi leaves. We found that several genes involved in the phenolic acid and flavonoid biosynthetic pathways were differentially expressed at different phosphorus fertilization levels (Fig. 4), including PAL, 4CL, CHS, and CHI The phenylalanine, tyrosine, and tryptophan biosynthetic pathways regulated by AY259905-RA in P2-VS-P1 were down-regulated by AY055820-RA, AY165300-RA, AY055819-RA, and AY241117-RA in P3-VS-P1. AY241117-RA and AY183661-RA were significantly down-regulated in the biosynthesis of flavonoids. Each gene is responsible for a specific biosynthesis regulation: AY165300-RA regulates the biosynthesis of flavonoids and flavonols; AY240297-RA and AY026693-RA regulate the biosynthetic pathways of phenylalanine, tyrosine, and tryptophan; and AY285346-RA, AY075114-RA, AY174727-RA, and AY149040-RA regulate the biosynthesis of phenylpropanoic acid. Our analyses also revealed that isochlorogenic acid A, neochlorogenic acid, isochlorogenic acid C, cryptochlorogenic acid, isochlorogenic acid B, schaftoside, hispidulin jaceosidin, eupatilin, and casticin all showed a significant decrease In response to high treatment in phenolic acid and flavonoid metabolite content. Plant terpenoids are synthesized independently through two pathways —the MVA located in the cytoplasm and the DXP located in the plastid. In this study, we compared the differential expression of genes encoding related enzymes in the terpene synthesis pathways. While our work provides an excellent foundation, the molecular mechanisms of terpenoid synthesis in A. argyi are complex and still need further investigation.

In this study, we used a combination of HPLC, GC / MS, and RNAs-eq analysis to analyze the growth status, metabolome, and transcriptome of A. argyi leaves treated with different phosphorus levels. Determined by a combination of plant height, leaf size, and stalk thickness, we found that the overall growth state of A. argyi plants was most vigorously affected by treatment with a medium level of phosphorus. The low level of phosphorus treatment presented the worst growth, showing much smaller leaves and more dwarf plants. These results indicate that A. argyi plants exhibited nutrient deficiency in low phosphorus conditions but toxicity in high ones.

When treated with low quantities of phosphorus, A. argyi showed the highest levels of total phenolic acids and flavonoids. This was determined by analysis of six phenolic acid components and five flavonoid components which were measured against standards. The level of P element in measured in the leaves was significantly correlated with the content of each active ingredient. A total of 46,665 differential genes were discovered and annotated by KEGG following a second transcriptome analysis. The mechanisms of phosphorus metabolism in A. argyi was thus determined to be closely related to the flavonoid and phenylalanine metabolic pathways. We discovered that the expression of genes related to the phenylalanine and flavonoid pathways were generally down-regulated with increasing levels of phosphorus application.

Author Contributions: Conceptualization, Liu Dahui; methodology, Wang Zixin and Ma lin; software, Wang zixin and Guo lujuan; validation, Chen changjie; preparation of of plant samples, Wang zixin,Liu Anzheng, and Sun Haodi；collection of data, Wang Zixin, Ma Lin, and Sun Haodi; formal analysis, Wang Zixin and Zhao Tingting; resources, Chen Changjie; writing and original draft preparation, Wang Zixin; writing and editing, Zhao Tingting; supervision, Liu Dahui; funding acquisition, Liu Dahui and Zhao Tingting. All authors have read and agreed to the published version of this manuscript.

Funding: This research was funded by the Young and Middle-aged talents project of the Hubei Provincial Department of Education (Q20212001); the National Natural Science Foundation of China (32270391); the Young Qihuang Scholars of the State Administration of Traditional Chinese Medicine; and the Hubei Provincial Department of Education Excellent Young and Middle-aged Science and Technology Innovation Team Project (T2021008).

Data Availability Statement: The datasets presented in this study can be found in online repositories. The raw data of RNA-seq have been deposited into NCBI under the accession number PRJNA915830.

Ethics approval：Not applicable.

Consent to participate：Not applicable.

Consent for publication: All authors approve the publication of this work.

Conflicts of Interest: The authors declare no conflict of interest.

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Combined transcriptomic and metabolomic analyses reveal the phosphorus stresses response mechanisms of Artemisia argyi

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