Response mechanism of Pteris vittata L. under long-term combined heavy metal stress based on transcriptome analysis

ABSTRACT

The response mechanism of plants under long-term heavy metal stress is not fully understood. This study compared and analyzed Pteris vittata L. of the mining areas, which significantly enriched multiple heavy metals (As, Cd, Hg, Pb, Zn, Cu, Sn, Sb, Cr), with those in non mining areas. The transcriptome analysis results showed that compared with non mining areas, there were 1226 genes upregulated and 1801 genes downregulated in the mining area. Functional enrichment analysis of these differentially expressed genes indicates that under long-term heavy metal stress, gene expression in related biological pathways such as Pteris vittata L. phenylpropanoid biosynthesis, starch and sucrose metabolism is inhibited. However, Pteris vittata L. also enhances its resistance to stress by upregulating the expression of genes related to these pathways, such as HCT and POD, enabling it to adapt to heavy metal pollution environments.

KEYWORDS:

• Pteris vittata L
• heavy metals
• transcriptome
• differentially expressed genes
• molecular mechanism

1. Introduction

With the development of modern mining industry, heavy metal pollutants have been discharged into the environment in large quantities and have become a global environmental pollution problem [1–3]. The headwaters of the Diao River basin in Guangxi, China, have been polluted by heavy metals along the coast for nearly a thousand years due to mining development [4], and among the heavy metals that constitute environmental pollution are mainly cadmium (Cd), lead (Pb), arsenic (As), zinc (Zn), and antimony (Sb), which have significant biological toxicity [5,6]. Plants have varying levels of tolerance to different metals. Within a certain concentration range, some metals have a promoting effect on plant growth and development. However, when a certain heavy metal exceeds a certain concentration, it can cause harm to the plant [7]. The toxic effects of heavy metal stress on plants are mainly seen in inhibiting seed germination and seedling growth, affecting the permeability of cell membrane, causing the imbalance of active oxygen metabolism, resulting in oxidative stress, affecting photosynthesis and Cellular respiration [8–11]. This series of injuries will eventually strongly inhibit the growth of plants, and affect the entire ecosystem through the transmission of the biological chain [12].

Pteris vittata L. belongs to the genus Pteris L. of the family Pteridaceae and grows in forest edges, limestone, calcareous soils or roadside rock crevices. Pteris vittata L. grows luxuriantly in the heavy metal pollution area along the Diaojiang River. It is the dominant plant along the Diaojiang River and can be used as an indicator plant for heavy metal pollution in the area [13]. Compared with common plants, Pteris vittata L. is a large biomass and fast-growing super-enriched plant, and is the first known As super-enriched plant that can tolerate the toxicity of high concentrations of As in its body [14–16]. Existing studies have shown that Pteris vittata L. is much more enriched for As and Cd metals than other plants in heavy metal contaminated mines and has the potential to phytoremediate As and Cd metal complex contaminated soils [17,18]. This indicates that the Pteris vittata L. has a certain tolerance to the stress of some heavy metals, but whether its various vital metabolic activities in the body will be impaired as a result, and its response mechanism is not clear. In this study, the transcriptional expression differences of Pteris vittata L. grown under long-term heavy metal complex pollution environment and in non-polluted areas were compared and analyzed by high-throughput sequencing technology to provide a basis for understanding the molecular response mechanism of Pteris vittata L. under long-term heavy metal complex stress.

2. Materials and methods

2.1. Research materials

Pteris vittata L. was studied in two areas, a heavy metal mining area (MA) in the middle reaches of the Diao River basin with hundreds of years of mining history, and a non-metal mining area (NMA) in Rong’an County, Guangxi. Fresh Pteris vittata L. was collected from two sample sites in August 2021. Plant specimens were brought back to the laboratory, and the roots, stems and leaves of Pteris vittata L. were taken as one sample, each sample weighed 4 g. Three samples, i.e. three biological replicates, were prepared from each of the two sample sites for transcriptome sequencing. The transcriptome samples from the mining area sample plots were numbered MA1, MA2 and MA3; the samples from the non-mining area sample plots were numbered NMA1, NMA2 and NMA3. The same fresh tissues from the above two sample plots Pteris vittata L. were also taken over 60 g each for heavy metal content determination. At the same time, soil samples were collected from each of the two sample plots in the mining and non-mining areas for heavy metal content determination in the soil where the Pteris vittata L. was growing.

2.2. Determination of heavy metal content in plants and soil

In this study, the contents of nine heavy metals, As, Cd, Hg, Pb, Zn, Cu, Sn, Sb and Cr, were determined in soil and Pteris vittata L. of two study areas, respectively. Soil samples were air-dried and ground and sieved using a 2 mm stainless steel mesh. Soil heavy metals were determined according to the national standards of soil pH (NY/T 1121.2–2006)，soil Cd, Cu, Cr, Zn, Pb (HJ491–201019), As, Sb, Hg (HJ 680–2013–2013), and Sn (GB/T 37,837–2019). Pteris vittata L. samples were first washed with distilled water to clean the dust attached to the surface of plants, and then the content of heavy metals was determined according to the national standard (GB 5009.268–2016).Microwave digestion and ICP-MS were used to determine the content of nine heavy metals in in Pteris vittata L. The specific steps were as follows: (1) Drying and grinding: The whole plant was completely dried and placed in a mortar to be fully ground into a fine powder, which was stored in dry room temperature. (2) Digestion: Weigh about 0.2 g of the sample for microwave digestion. (3) Acid pick-up: After the completion of the digestion, the acid was picked up in a fume hood for 40 min using an acid pick-up apparatus.(4) Detection: After the temperature of the acid pick-up apparatus was lowered to room temperature, the sample was removed, fixed to 50 mL with ultrapure water, mixed and placed for clarification, and then detected by ICP-MS. At the same time, the national standard was weighed as the analytical blank, with the same standard reference.

2.3. Total RNA extraction

High quality RNA was extracted from each sample of Pteris vittata L. using a plant RNA extraction kit. The size and degradation of RNA were detected by agarose gel electrophoresis, the purity and concentration of RNA were detected by NanoDrop ND-1000 ultra-micro spectrophotometer, and the integrity of RNA was accurately detected by Agilent 2100 Bioanalyzer.

2.4. Transcriptome sequencing, assembly and annotation

In this study, cDNA libraries were constructed using Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, U.S.A.) for a total of six Pteris vittata L. samples from mining and non-mining groups. The libraries were sequenced using Next-Generation Sequencing (NGS) based on the Illumina HiSeq 2500 sequencing platform for Paired-end (PE) sequencing. The sequenced samples will generate Raw Data in FASTQ file format, and then the Raw Data will be filtered to remove Reads containing adapters, N (N means base information cannot be confirmed), fragment length less than 50bp, and average sequence quality score below Q20 to obtain Clean Data. The obtained high-quality sequences were assembled from scratch (De Novo) using Trinity (2.5.1) software based on the DBG (De Bruijn Graph) splicing principle to obtain the transcript sequences, then de-redundant using Tgicl software [19], and finally the data were filtered using CD-HIT software [20] to obtain the final Unigene.

BLAST software was used to link unigene sequences to the NCBI non-redundant protein sequences (Nr), the eukaryotic direct homology protein clustering database (evolutionary genealogy of genes: non-supervised orthologous groups (eggNOG), protein family (Pfam), SwissProt database (a manually annotated and reviewed protein sequence database), Kyoto Encyclopedia of Genes and Genomes (KEGG) (KEGG) and Gene Ontology (GO) to obtain functional annotation information of genes.

2.5. Transcriptome gene expression analysis and differential analysis

Using the transcriptome expression quantification software RSEM, the Clean Reads of each sample were compared to the reference sequence separately, using the transcript sequence as a reference. Then the number of Reads of each sample was counted and the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) value of each gene was calculated, and the valid expressed genes were screened by FPKM ≥ 1. Differentially expressed genes (DEGs) were screened using DEGseq software with qvalue < 0.05 and |log2FoldChange|>1 as criteria.

2.6. GO and KEGG analysis of differential gene expression

GOseq and KOBAS software were used to perform GO functional enrichment analysis of differential gene sets and KEGG pathway enrichment analysis. The enrichment analysis was based on the hypergeometric distribution principle, where the differential gene set was the set of differential genes obtained from the differential significant analysis and annotated to the GO or KEGG database, and the background gene set was the set of all genes for which the differential significant analysis was performed and annotated to the GO or KEGG database. The enrichment analysis results in the enrichment of all differential gene sets, up-regulated differential gene sets, and down-regulated differential gene sets for each differential comparison combination.

2.7. Real-time fluorescence quantitative PCR validation

To verify the authenticity of the sequencing results and data analysis, 11 differentially expressed genes were randomly selected from the SU Pteris vittata L. transcriptome results for qRT-PCR validation, using β-actin as the internal reference gene, and the validated genes and corresponding primer sequences are shown in Table S1. cDNA was synthesized by reverse transcription using a reverse transcription kit, and the reverse transcription system was 40 μL (prepared on ice): 5× PrimeScript^RT Master Mix (Perfect Real-time) 8 μL and RNA template 2000 ng, and finally RNase-free dH₂ O was added to make up to 40 μL. The reaction conditions were: 15 min at 37°C, 5 s at 85°C, and stored in a 4°C refrigerator. ®The PCR reaction system was 20 μL: SYBR Premix Ex Taq (2×) 10 μL, 10 μmol/L upstream and downstream primers 1 μL each, cDNA template 2 μL, and ddH₂ O to 20 μL. The PCR reaction procedure was : 30 s of pre-denaturation at 95°C; 5 s of denaturation at 95°C, 34 s of annealing at 60°C, 40 cycles.

3. Results

3.1. Heavy metal content in soil and pteris vittata L

As, Sn, Cd, Pb, Zn, Cu, and Cr were about 1569.83, 384.76, 36.71, 27.17, 14.05, 5.32, and 1.43 times higher in the soil of the mining area than in the non-mining area, respectively. Sb was high in the soil of the mining area but not detected in the non-mining area, and Hg was not detected in the soil of the mining area but very low in the non-mining area (Table 1). Similar results were obtained in the assay of Pteris vittata L. samples (Table 2). Compared with non mining areas, the content of Zn, Sn, Hg, Pb, Cu, Cd, and Cr of the Pteris vittata L. in mining areas is 11.22, 6.81, 2.00, 1.60, 1.41, 1.38, and 1.28 times higher, respectively. Correspondingly, Sb has a high content in the Pteris vittata L. in the mining area, but not detected in the non mining area (similar to the content in the soil),The above data indicate that there is a high correlation between the content of heavy metals in the Pteris vittata L. and soil.

Table 1. Chemical properties and heavy metal content (dry weight) of soil samples from mining and non-mining areas.

Collection points	PH	Cd (mg/kg)	Cu (mg/kg)	Cr (mg/kg)	Zn (mg/kg)	Pb (mg/kg)	As (mg/kg)	Sn (mg/kg)	Sb (mg/kg)	Hg (mg/kg)
Non-mining area	8.77 ± 0.13	0.28 ± 0.009	26.34 ±0.268	55.93 ± 0.465	101.62 ± 0.663	33.80 ± 0.224	15.17 ± 0.016	0.06 ± 0.041	0.00 ± 0.000	0.34 ± 0.007
Mining area	7.52 ± 0.01	10.28 ± 0.281	140.15 ± 2.284	79.88 ± 0.148	1428.21 ± 9.282	918.42 ± 15.159	5836.88 ± 18.896	94.19 ± 3.058	668.33 ± 2.282	0.00 ± 0.000

Each value is the mean ± standard deviation (S.D.) of three replicates.

Table 2. Heavy metal content of pteris vittata L. samples from mining and non-mining areas (mg/kg, dry weight).

Collection points	Cd (mg/kg)	Cu (mg/kg)	Cr (mg/kg)	Zn (mg/kg)	Pb (mg/kg)	As (mg/kg)	Sn (mg/kg)	Sb (mg/kg)	Hg (mg/kg)
Non-mining area	0.29 ±0.003	22.60 ±0.203	25.53 ±0.319	13.57 ±0.194	26.65 ±0.100	3.34 ±0.103	0.48 ±0.129	0.00 ±0.000	0.02 ±0.001
Mining area	0.40 ±0.001	31.77 ±0.312	32.67 ±0.333	152.21 ±1.903	42.60 ±0.570	2.11 ±0.107	3.27 ±0.261	16.65 ±0.038	0.04 ±0.000

Each value is the mean ± standard deviation (S.D.) of three replicates.

3.2. Transcriptome sequencing, assembly, and annotation results (summary of sequencing results)

A total of six transcriptomic libraries of mining zone Pteris vittata L. (MA1, MA2, MA3) and non-mining zone Pteris vittata L. (NMA1, NMA2, NMA3) were sequenced by Illumina HiSeq 2500 to obtain an average of 43,976,145 raw sequences (Raw d ata). By removing the Reads contaminated with joints, low quality Reads and Reads containing more than 5% N, the average of 40,911,589 Clean data was obtained (Table S2). The assembly results of the six samples were mixed together and the final 50,632 unigene were obtained after redundancy removal, with a total length of 62,226,503 bp and an average length of 1229 bp, the total number of sequences with lengths greater than N50 was 9,074 and the total number of sequences with lengths greater than N90 was 33,657 (Table S3).

The 50,632 Unigene obtained from the assembly were compared to six databases, and a total of 22,568 genes were annotated in the annotation results, accounting for 44.57% of the total Unigene. The percentage of unsuccessfully annotated Unigene was 55.43%. There were 21,772, 8,765, 9231, 15102, 21112 and 17,955 Unigene annotated to NR, GO, KEGG, Pfam, eggNOG and Swissprot databases, respectively. The NR database contains the gene sequence information of species, and the gene sequences obtained from the annotated Pteris vittata L. in this study were compared with them, and the most matched species were Marchantia polymorpha subsp. polymorpha, small standing bowl moss Physcomitrella patens, southern curly cypress Selaginella moellendorffii, North American spruce Picea sitchensis, oil-free camphor Amborella trichopoda, lotus Nelumbo nucifera and grapevine Vitis vinifera seven plants. In addition, Unigene also matched to some other plants, but the number of matched sequences was low (Figure S1).

A total of 21,112 genes were annotated by eggNOG for eukaryotic direct homologous protein clustering into 26 functional categories, mainly related to general function prediction only, posttranslational modification and translocation, molecular chaperones, translation, ribosomal structure and biosynthesis, signal transduction mechanisms, and ribosomal structure and biosynthesis, modification, protein turnover, chaperones, Signal transduction mechanisms, Translation, ribosomal structure and biogenesis, biogenesis), Replication, recombination and repair (Figure S2). GO annotation classification statistics show that 8765 genes are annotated into 67 functional categories, and the main functions of the annotation classification are cellular process, metabolic process, cell, cell part, catalytic activity and protein binding (Figure S3). KEGG annotation statistics showed that 9231 Unigene were enriched to 35 secondary classification metabolic pathways (Figure S4). Among them, the largest number of genes were enriched to carbohydrate metabolism (748), translation (1110), signal transduction (828), transport and catabolism (544) had the highest number of genes.

3.3. Differential expression analysis

A total of 3027 significantly differentially expressed genes were identified in mining area (MA) and non-mining area (NMA) Pteris vittata L.s, including 1226 up-regulated DEGs (qvalue <0.05 and log2FoldChange >1) and 1801 down-regulated DEGs (qvalue <0.05 and log2FoldChange<-1) (Figure 1).

Figure 1. Volcanoes of differentially expressed genes in pteris vittata L.S in mining and non-mining areas.

3.4. Analysis of GO, KEGG enrichment

The results of GO function enrichment analysis of DEGs of Pteris vittata L. under heavy metal stress showed that DEGs were significantly enriched in three major categories: biological process, cellular component and molecular function. MA vs DEGs of NMA annotation The results (Figure 2(a)) showed that among the top 20 most enriched GO Term entries, the DEGs of the mining area (MA) were significantly enriched in the entries of metabolic process, cellular process, biological regulation, and stimulus response in the biological process grouping compared to the non-mining area group (NMA); among the molecular functions, the DEGs were mainly enriched in the entries of catalytic activity, nucleic acid binding transcription factor Among the molecular functions, DEGs were mainly enriched in the entries of catalytic activity and nucleic acid binding transcription factor activity; among the cellular components, DEGs were less enriched.

Figure 2. GO (a) and KEGG (b) differential gene enrichment analysis of pteris vittata L. in mining and non-mining areas. (a) The smallest FDR value, i.e. The top 20 most significantly enriched GO term entries; (b) the smallest FDR value, i.e. The top 20 most significantly enriched KEGG pathways. where term denotes the name of each channel, FDR denotes the enrichment probability, number denotes the number of deg contained in each channel, and rich denotes the degree of enrichment.

KEGG enrichment analysis of the DEGs of MA vs NMA revealed that 864 DEGs were annotated to 107 pathways, and the top 20 most significantly enriched pathways were selected for bubble plots for further analysis (Figure 2(b)). Phenylpropanoid biosynthesis involved the largest number of DEGs with 38, followed by starch and sucrose metabolism with 26, plant hormone signal The highest number of DEGs was 38, followed by 26 for starch and sucrose metabolism, 24 for plant hormone signal transduction, 23 for MAPK signaling pathway – plant, and 22 for plant-pathogen interaction. In addition, further analysis of the pathways significantly enriched in DEGs revealed that thiamine metabolism, sesquiterpenoid and triterpenoid biosynthesis, and photosynthesis – antenna proteins were three significant pathways. The expression of DEGs was down-regulated in all three significantly enriched pathways.

3.5. Real-time fluorescence quantitative PCR validation results

The results of q RT-PCR validation of 11 differentially expressed genes with β-actin as the internal reference gene are shown in Figure 3. The relative expression expression trends of 11 differentially expressed genes showed a very high correlation with the results obtained from RNA-Seq sequencing (R² = 0.8719), which proved that the sequencing results were highly accurate and credible and could be tested in the next step.

Figure 3. RT-qPCR validation of differentially expressed genes.

4. Discussion

Heavy metal pollution in the environment can pose heavy metal stress to plants and affect their growth and development [7]. Heavy metals can accumulate in large quantities in plant roots, stems, leaves and seeds, thus severely affecting plant growth and development [21]. In this study, we found that the soil in heavy metal mining areas was contaminated with heavy metals relative to non-mining areas, while the Pteris vittata L. in mining areas was enriched with relatively high concentrations of heavy metals, suggesting that heavy metals in the environment can accumulate in plants through media such as soil. Subsequently, transcriptome sequencing was performed using transcriptome sequencing technology for the mining Pteris vittata L. and the non-mining Pteris vittata L. Compared with the non-mining Pteris vittata L., there were 1226 up-regulated DEGs and 1801 down-regulated DEGs expressed in the mining Pteris vittata L. The expression of 11 genes related to plant resistance was verified using qRT-PCR. Although there was a slight difference between qRT -PCR results and mRNA sequencing results in terms of ploidy, each gene had the same up- and down-regulation trend, which could indicate the reliability of transcriptome data analysis. GO enrichment of these DEGs revealed that differentially expressed genes were significantly enriched under functional entries of metabolic processes, cellular processes, biological regulation, stimulus response, catalytic activity, and nucleic acid binding transcription factor activity. Also, differentially expressed gene KEGG enrichment analysis revealed a high number of differentially expressed genes enriched in pathways such as phenylpropanoid biosynthesis, starch and sucrose metabolism, phytohormone signaling, MAPK signaling pathway-plant and plant-pathogen interactions (Figure 4). It has been found that gene expression of plant-pathogen interactions, phenylpropanoid biosynthesis, and phytohormone signaling pathways were substantially up-regulated in Broussonetia papyrifera under Cd stress [22]. Tall fescue Tall fescue (Festuca arundinacea Schreb) also exhibited differential expression of genes in the phenylpropanoid biosynthesis pathway under Cd stress [23], suggesting that plants have some similarity in response mechanisms to heavy metal stress and that these pathways may play an important role in Pteris vittata L. in response to heavy metal stress These pathways may play an important regulatory role in Pteris vittata L. in response to heavy metal stress, and the changes in gene expression in these pathways are closely related to this.

Figure 4. The regulatory mechanism of KEGG pathway in the transcriptome of pteris centipede under heavy metal stress. Different colors represent pathways, and the corresponding pathway maps are also represented by their corresponding colors: red circles represent upregulated genes, green circles, and green squares represent downregulated genes; purple triangles represent other substances involved in this process or downstream substance formation; solid arrows represent direct effects, and dashed arrows represent indirect effects.

The benzenepropanoid biosynthetic pathway is one of the three major secondary metabolic pathways in plants [24], and the secondary metabolites produced by this pathway are important for plant growth and development and response to adversity stresses. Secondary metabolites such as flavonoids and proanthocyanidins in this pathway are mainly used to chelate metal ions, which prevent heavy metals from entering the cells and significantly contribute to metal bioavailability and resistance to metal toxicity [25,26]. Phenylalanine ammonia-lyase (PAL), Cinnamate-4-hydroxylase (C4H), also known as trans-cinnamate-4-monooxygenase, is regulated by several genes in the biosynthesis of phenyl propane. 4-monooxygenase) and 4-coumaroyl-CoA ligase (4CL) [27,28]. The phenylalanine-like biosynthetic pathway begins with phenylalanine, and PAL, as the starting and rate-limiting enzyme of this pathway, is able to catalyze the direct deamination of phenylalanine to produce cinnamic acid, which has important roles in plant lignification processes, antibacterial and stress resistance, and is an important defense enzyme in plant tissues [29,30,,31]. C4H is the second key enzyme catalyzing the biosynthesis of metabolites in the phenylpropanoid biosynthetic pathway and is a monooxygenase complex of plant cytochrome P450 [32] that catalyzes the generation of p-hydroxycoumaric acid from cinnamic acid [27], and its gene expression and protein activity affect the in vivo production of plant The gene expression and protein activity affect the biosynthesis of flavonoids and lignans in plants [33,34,35]. The p-hydroxycoumaric acid produced by C4H-catalyzed cinnamic acid is then catalyzed by 4CL to produce 4-coumaric acid coenzyme A, which enters the downstream specific synthesis pathway to produce various phenolpropane metabolites [36]. In the present study, analysis of the pathway map of this pathway revealed that PAL, C4H and 4CL genes were all down-regulated in this reaction system. The results of this study are similar to those of previous studies, such as JańczakPieniążek et al. [37] which showed that heavy metal Cu at 500 ppm and 1000 ppm caused a decrease in the expression of PAL and 4CL genes in wheat ‘Hyvento’ cultivar. The PAL activity of ‘Hyving’ variety at Cu concentrations (200 and 500 ppm) was proportional to the concentration used, but PAL activity decreased when applied at 1000 ppm [37]. In addition, the PAL activity of wheat ‘Hyvento’ variety was significantly reduced under Pb stress [37]. The expression levels of PAL and C4H genes in the biosynthesis process of Althaea officinalis L. were significantly decreased after 96 h of Cu treatment, while 4CL was slowly stimulated [38]. Thus, heavy metal stress inhibits PAL, C4H, and 4CL gene expression in Pteris vittata L., which may directly affect the synthesis of benzenepropanoids in Pteris vittata L. and reduce the content of benzenepropanoids in the plant. However, the shikimate O-hydroxycinnamoyltransferase (HCT), peroxidase (POD), which are involved in the lignin synthesis pathway, a subpathway of the phenylpropanoid biosynthesis pathway [39] gene expression is enhanced so that the content of lignin, which can provide a structural and defensive barrier to the cell wall, is correspondingly increased and is closely linked to plant stress resistance [40]. Plants under biotic and abiotic stresses usually experience accumulation of reactive oxygen species accompanied by lignin accumulation [40], and causing dysregulation of reactive oxygen species metabolism in plants is one of the hazards of heavy metals. Heavy metals can cause damage to plant growing tissues, and the release of this damage needs to be satisfied by the scavenging of reactive oxygen species, which is based on enzymes that can effectively destroy superoxide radicals and hydrogen peroxide, such as peroxidase POD, peroxiredoxin-6, Prx [41], and in the present study expression of these genes were up-regulated in the present study. Thus, it is speculated that the up-regulated expression of these genes may enhance the tolerance of Pteris vittata L. to heavy metals.

Carbohydrates (starch, sucrose, etc.) are important energy providers during plant growth and development, providing the carbon skeleton for the biosynthesis of secondary metabolites, and also play an important regulatory role in the biosynthesis of secondary metabolites [42]. When plants respond to adverse environmental stress, the levels of sugars in the body change rapidly, which in turn affects a variety of physiological and biochemical processes in the plant [43]. In this study, we showed that the starch and sucrose metabolic pathways in Pteris vittata L. under heavy metal stress encode trehalose 6-phosphate synthase/phosphatase (TPS), beta-glucosidase (E3.2.1.21), and beta-amylase (TPS is a key enzyme in the regulation of algal sugar synthesis, which has been found to have an important role in plant development and resistance to various biotic and abiotic stresses [44,45]. The increase in alglucan reserves in plants facilitates their maintenance of an extremely low metabolic state when subjected to adversity stress, acting as a stress protector for plants against adverse factors [46,47]. Plant cell membranes are usually considered as the main site of metal damage, and Fatih et al. [48] showed in their study that 0.5 mM alginate promoted Cd accumulation in aquatic plant phytoplankton, while Cd accumulation decreased significantly at 5 mM, while 2 mM and 5 mM alginate reduced cellular lipid peroxidation. Singer and Lindquist [49], also indicated that alglucan maintained cell membrane and protein stability and reduced aggregation of deformed proteins. Mostofa et al. [50] showed that pretreatment of rice seedlings with alglucan modified their endogenous alglucan content, thereby inhibiting copper uptake and accumulation in rice, contributing to the reduction of Cu toxicity to plants, and by increasing glyoxalase Gly I and Gly II activities, reducing the content of methylglyoxal, a harmful substance induced by heavy metals. These results suggest that alglucan can effectively reduce the oxidative damage induced by heavy metals. Therefore, the upregulation of TPS gene expression in this study should be Pteris vittata L. to enhance the tolerance to heavy metals and make the rapid synthesis of alglucan in the body to protect itself from normal growth. Meanwhile, the gene expression of two hydrolases, β-glucosidase and β-amylase, which are important enzymes of the sugar metabolism pathway and can hydrolyze starch, an important energy substance in plants, was also upregulated [51]. Plants grown in heavy metal environments need to transfer part of their energy to adapt to the adversity environment in addition to using it for normal life activities. Heavy metal stress also inhibits plant respiration [52], which in turn prevents the release of energy from the decomposition of organic matter in plants, resulting in insufficient energy supply and inhibiting plant growth and development. In the present study, the expression of these two enzymes catalyzing starch hydrolysis was up-regulated in Pteris vittata L. plants under heavy metal stress, suggesting that they may release energy by hydrolyzing starch to produce glucose, which is used to resist heavy metal stress.

Similar to stresses by abiotic factors such as heat, salinity, and drought, heavy metal stress can activate signaling pathways in plants. It has been shown that heavy metal stress activates phytohormone signaling pathways such as salicylic acid, ethylene, and jasmonic acid and MAPK signaling pathway [53–56]. Phytohormone signaling is an important pathway that mediates the production of phytohormones, which also play an active role in the heavy metal detoxification signaling cascade response to mitigate the toxicity of heavy metals in plants and support their growth and development [57,58]. The phytohormone signaling pathway upregulated 19 genes in Pteris vittata L. under heavy metal stress, including mitogen-activated protein kinase 6 (MPK6), abscisic acid receptor PYR/PYL (abscisic acid receptor PYR/PYL family. PYR/PYL), brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1), and transcription factor MYC2 (MYC2). The MAPK signaling pathway, which is interconnected with phytohormone biosynthesis and signal transduction, was also significantly enriched, and the genes MPK6, PYR/PYL, BAK1, and MYC2 were also up-regulated in this pathway. In plants such as Arabidopsis, maize, and rice, abiotic factors such as salt stress, low temperature stress, drought stress, trauma, or touch can activate MPK6 [59–61], which can respond to jasmonic acid and ethylene signaling, while the transcription factor MYC2 is a jasmonic acid signaling MYC2 is a positive regulator of the jasmonic acid signaling pathway and can induce a series of jasmonic acid-responsive genes to produce jasmonic acid, a phytohormone that interacts with other hormones in response to biotic stresses. More interestingly, in this study, the expression of abscisic acid (ABA) receptor PYR/PYL gene was up-regulated in the phytohormone signaling and MAPK signaling pathways, while the expression of its downstream 2C-type protein phosphatase (PP2C) and sucrose nonenzymatic kinase 2 (SnRK2) were down-regulated. In contrast, in Arabidopsis, the receptors PYR/PYL, PP2C, and SnRK2 are the main three key proteins for ABA sensing and signaling [62]. Plants have low ABA levels in normal environments, when PP2C interacts with SnRK2s and inhibits their activity as kinases. Unlike the ABA content of plants in normal environments, when external oxidative stress occurs, such as heavy metals entering the plant induces the production of reactive oxygen species, causing a rapid accumulation of ABA, which first binds to the receptor PYR/PYL and then interacts with PP2C to form a ternary complex to release the inhibitory effect of PP2C on SnRK2 so that SnRK2 can phosphorylate the downstream transcription factors, thereby maintaining ion homeostasis, scavenging reactive oxygen species, and promoting plant adaptation to adversity [62,63,64]. Although this is somewhat contradictory to the down-regulation of PP2C and SnRK2 gene expression in Pteris vittata L. in this study, it does not exclude the possibility that it is prolonged heavy metal complex pollution that affects the activity of reactive oxygen scavenging enzymes in the plant body. In addition, oleuropein lactones (Brassinosteroids (BRs)) and abscisic acid can interact, and low concentrations of BRs can promote ABA-regulated stomatal closure via BAK1 phosphorylation of SnRK2 to prevent water evaporation and promote plant resistance to external stresses [65]. Therefore, mine Pteris vittata L. showed upregulated expression of some genes in the phytohormone signaling and MAPK signaling pathways, which also suggests that Pteris vittata L. may respond to heavy metal stress by regulating the expression of genes in the signaling pathway.

Calcium ions play an important role in plant growth and development throughout their life history, serving as important messengers involved in the physiological processes of plants in response to abiotic stresses [66]. Heavy metals and other abiotic stresses can induce significant Ca²⁺ signals in plant cells, which can be recognized and decoded by intracellular Ca²⁺ receptors, which in turn affect the expression of downstream genes or the activity of related channel proteins, ultimately causing different plant-specific responses [67]. In the present study, calmodulin (CALM) and calcium-dependent protein kinase (CPK), two types of sensing proteins that receive calcium signals, were significantly up-regulated genes in the plant-pathogen interaction pathway, suggesting that the entry of heavy metals into Pteris vittata L. cells may have stimulated Ca²⁺ signaling, prompting Pteris vittata L. coordinates resistance to heavy metal stress by regulating the expression of related receptor protein genes. Analysis of plant-pathogen interactions and MAPK signaling pathway maps revealed that up-regulated expression of FLS2 (LRR receptor-like serine-/threonine-protein kinase FLS2) and BAK1 (brassinosteroid insensitive 1-associated receptor kinase 1) genes play important roles in both pathways, with FLS2 and BAK1 interacting in the presence of flg22 stimulation to form the FLS2-flg22-bak1 complex, triggering downstream immune responses such as activation of the MAPK pathway and elevation of reactive oxygen species for plant bacterial defense and immune response [68,69]. Notably, the EF-Tu (elongation factor Tu) gene, which is recognized by the bacterial elongation factor EF-Tu receptor (EFR) in cruciferous plants as a Pathogen – Associated Molecular Pattern (PAMP) to generate a series of immune responses in the plant, was found in the mineral Pteris vittata L.) gene is up-regulated in mineral Pteris vittata L., and elf18, the immunoreactive region in which this gene is located, is able to cause a strong reactive oxygen species burst and ethylene production in Arabidopsis and improve plant disease resistance [70]. Although, this does not directly indicate the role of EF-Tu gene in Pteris vittata L., it can still be speculated that the immune system of Pteris vittata L. is damaged due to the long-term stress of heavy metals, which makes it vulnerable to pathogenic attack in the external environment, and the expression result of this gene can be regarded as the immune regulatory mechanism involved in this gene to initiate ‘self-defense against pathogens’ in Pteris vittata L.”. This shows that plant-pathogen interactions and MAPK signaling pathways may be important defense pathways for Pteris vittata L. in response to heavy metal injury, and also suggests that plants may utilize more diverse and superior pathways to act synergistically in response to adversity stresses.

Although Pteris vittata L. activates a relevant response in vivo to inhibit heavy metal damage to the organism in a chronically compounded heavy metal contaminated environment, making it tolerant to heavy metals, it is inevitable that such stress will diminish certain physiological and biochemical activities of the plant. Rapala-Kozik et al. [71] found that thiamin levels were elevated in Arabidopsis under salt, osmotic stress, and oxidative stress stress, and The expression of THI1, THIC, TH1, and TPK, key enzymes of the thiamin synthesis pathway, was mainly up-regulated at the transcriptional level in the early stages of the stress response. However, the results of the present study showed that the differential genes enriched under the thiamin metabolic pathway were all down-regulated, and two enzyme genes, THI1 and THIC, were significantly down-regulated. Meanwhile, other down-regulated expression genes involved a class of monomeric enzymes, namely adenylate kinase AK, whose role in biology is involved in maintaining the energy homeostasis process of the organism [72], so the down-regulated expression of AK can be said to be consistent with the disturbance of plant energy metabolism caused by heavy metal stress. In addition, the expression of DXS gene, a 1-deoxy-D-xylulose-5-phosphate synthase, was also restricted. Considering that DXS is a key enzyme in the MEP pathway of terpenoid biosynthesis, it can catalyze the first rate-limiting step of the MEP pathway to form two biosynthetic precursors of terpenoid biosynthetic pathway species, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP), which eventually synthesize different terpenoids through the MEP pathway [73]. While overexpression of DXS can increase the content of terpenoids in host plant species [74], if DXS expression is reduced, the content of terpenoids in plants will be affected and some terpenoids involved in essential plant processes such as photosynthesis, respiration, and growth and development will be inhibited [75]. In addition to thiamin metabolic pathway, several genes are significantly down-regulated in other metabolic pathways, such as 22 genes down-regulated in starch and sucrose metabolic pathway, 12 genes down-regulated in cyanogenic amino acid metabolic pathway, and 10 genes down-regulated in terpene skeleton biosynthesis pathway, etc. Seregin and Ivanov [76], showed that lead and cadmium exert non-specific toxic effects on cellular metabolism. This suggests that Pteris vittata L. grown for a long time in soil contaminated with heavy metals will eventually affect the metabolic function of the plant due to the accumulation of heavy metals in its body over time.

Photosynthesis is an important physiological process in which plants use light to produce organic matter and provide material energy for life activities. Light is essential for the photosynthesis process in plants, but photoinhibition occurs when the environment changes, such as water stress, low and high temperature stress, and heavy metal pollution, which can lead to photoinhibition and thus affect the photosynthesis process [77,78]. According to related studies, heavy metal cadmium enters plant cells and firstly acts on mitochondria and chloroplasts, causing disintegration or even disappearance of structures such as mitochondrial periplasm and internal cristae [79], causing damage, crumpling, and disintegration of chloroplast submicroscopic structures such as chloroplast membrane, cystoid, and chloroplast basal granules [80,81], which in turn causes damage to the photosynthetic system and and interferes with electron transfer in the electron transport chain in plants, thus affecting normal photosynthesis in plants [82]. It has been found that Cd stress causes changes in genes regulating photosynthesis in radish shoots [83]. In the present study, the metabolic pathway of photosynthesis-antenna protein was significantly enriched, which is also known as light trapping chlorophyll a/b binding protein (LHC), a photoreceptor that enhances light absorption and excitation energy and transmits it to the photosynthetic system. In the photosynthesis-antennin pathway of the mineral Pteris vittata L., nine genes of the antennin gene family members, light trapping complex II chlorophyll a/b binding protein 1 (LHCB1) and light trapping complex II chlorophyll a/b binding protein 2 (LHCB2), are down-regulated, and as apolipoproteins of the PSII light trapping complex, the down-regulated expression of these genes is detrimental to the normal photosynthesis of the Pteris vittata L. The down-regulated expression of these genes was not conducive to the normal photosynthesis of Pteris vittata L. Meanwhile, this result suggests that the down-regulated expression of photosynthesis-antennal protein pathway genes in Pteris vittata L. is inextricably linked to the stress of heavy metals in its living environment, and the excessive accumulation of heavy metals will damage the plant photosynthetic protein. In addition, we also found in GO enrichment that most of the differentially down-regulated expressed genes were significantly enriched to chloroplasts, chloroplast peroxisomes and chloroplast stroma functional entries, further suggesting that heavy metal complex contamination interferes with the photosynthetic process of Pteris vittata L.

5. Conclusions

The present study showed that the expression of genes in the biological pathways related to phenol propane biosynthesis, starch and sucrose metabolism, thiamine metabolism, signal transduction, plant-pathogen interactions and photosynthesis in Pteris vittata L. was inhibited by long-term heavy metal complex pollution stress. However, the expression of genes related to the above pathways, such as shikimate O-hydroxycinnamoyltransferase (HCT), peroxidase (POD), peroxiredoxin-6 (POD), and peroxiredoxin-6 (POD), was suppressed in Pteris vittata L. by up-regulating the expression of these genes. Peroxiredoxin-6 (Prx), trehalose 6-phosphate synthase/phosphatase (TPS), beta-glucosidase (E3.2.1.21), and beta-amylase (E3.2.1.21), amylase (E3.2.1.2), mitogen-activated protein kinase 6 (MPK6), abscisic acid receptor PYR/PYL (PYR/PYL family), oleuropein lactone insensitive1-related receptor kinase (BAK1), transcription factor MYC2 (MYC2), calmodulin (CALM)) and calcium-dependent protein kinase (CPK), FLS2 (LRR receptor-like serine-/threonine-protein kinase) and elongation factor Tu (EF-Tu). EF-Tu genes to enhance resistance to heavy metal stress and counteract the toxicity of heavy metals to phytoplankton, enabling Pteris vittata L. to adapt to heavy metal complex pollution environment for a long time. These results help to reveal the molecular mechanism of heavy metal stress tolerance in Pteris vittata L. and also provide a theoretical basis for further use of this plant for remediation of soil heavy metal pollution.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The raw RNA-seq sequencing reads used in this study have been uploaded to the Genome Sequence Archive (GSA) database under project ID PRJCA021223.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/26395940.2024.2352412.

Additional information

Funding

This work was supported by the Guangxi Natural Science Foundation Project [2024GXNSFAA010145], the Scientific research project of Guangxi Education Department [Grant No. 2021KY0623], Scientific research project of Hechi University [2021GCC023, 2021GCC017], and Research platform of “Northwest Guangxi characteristic plant resources development and function research center.