Transcriptome Analysis Reveals Differentially Expressed Genes Involved in Aluminum, Copper and Cadmium Accumulation in Tea ‘Qianmei 419’ and ‘Qianfu 4’

Tea, as a global nonalcoholic beverage, is widely consumed due to its economic, health and cultural importance. Polyploids have the ability to solve the problems of low yield, cold resistance and insect resistance in tea tree varieties. However, the response mechanism to aluminum and heavy metal remains unclear. In this study, the content of Al, Cu and Cd were measured in the leaves and roots of ‘Qianmei 419’ and ‘Qianfu 4’, respectively. The content of Al, Cd and Cu in the roots of the ‘Qianmei 419’ tea variety were significantly higher than in ‘Qianfu 4’ roots. Only the content of Cu in the leaves of the ‘Qianmei 419’ tea variety was significantly higher than that in the roots of the ‘Qianfu 4’ tea variety. Moreover, we found that the content of Al, Cu and Cd in the soil around the root of ‘Qianfu 4’ were higher than in the soil around the root of ‘Qianmei 419’. RNA-seq was performed to identify the DEGs involved in the accumulation of Al, Cu and Cd between ‘Qianmei 419’ and ‘Qianfu 4’. A total of 23,813 DEGs were identified in the triploid tea variety, including 16,459 upregulated DEGs and 7354 downregulated DEGs. Among them, by analyzing the expression levels of some metal transporter genes, it was found that most of the metal transporter genes were downregulated in the triploid tea plants. In short, through the analysis of transcriptome data and metal content, it was found that changes in metal transporter gene expression affect the accumulation of metals in tea plants. These results provide candidate genes to enhance multi-metal tolerance through genetic engineering technology.


Introduction
Tea, as a global nonalcoholic beverage, is widely consumed due to its economic, health and cultural importance. Camellia sinensis' metal pollution of raw tea has always been a food safety concern. Many researchers have performed research on genes related to metal stress [1][2][3]. Metals such as aluminum (Al), copper (Cu), nickel (Ni), cobalt (Co), cadmium (Cd), zinc (Zn), mercury (Hg) and arsenic (As) accumulate in the soil for a long time through human activities such as industrial waste, fertilizer, smelting and sewage treatment [4]. Aluminum and heavy metals are considered important environmental contaminants that induce a wide array of physiological responses in plants [1][2][3]. Cd contamination inhibits the rate of CO 2 fixation, decreases chlorophyll (chl) content and depresses photosynthetic activity [5,6]. Plants grown on Cd-contaminated soil are subjected to osmotic stress by minimizing leaf relative water content and reducing transpiration rate and stomatal conductance [7]. Moreover, Cd can also decrease Fe and Zn uptake, reduce Fe and Zn concentrations and cause more severe leaf chlorosis [8][9][10][11]. Jiang et al. [12] revealed that Cd interacts with mineral nutrients (Ca, Mn, Mg, K and P). Arsenic (As), a toxic metalloid that is ranked 20th in natural abundance, is widely distributed in the environment [13]. Exposure to As (V) can cause numerous stresses in plants, such as respectively; the content of Al, Cd and Cu in the soil samples (5 cm from the main root of the tea tree) of the diploid tea plant were 7341.41, 0.16 and 29.45 mg/kg, respectively ( Figure 1b). There was no significant difference in the content of these three metals in the soil samples (5 cm from the main root of the tea tree) of triploid tea plants and diploid tea plants. However, the Cd, Al, and Cu content in the soil of 'QianFu No.4' (50 cm away from the tea tree) were 5.62, 7943.59, 33.28 mg/kg, and the Cd, Al, and Cu content in the soil of 'Qianmei 419' were 53.4, 7843.27, 32.97 mg/kg, which were higher than the heavy metal content in the soil rhizosphere. It can be inferred that the tea tree roots absorbed some heavy metals. Moreover, we further determined the content of Al and heavy metal in the leaves and roots of the 'Qianmei 419 diploid tea variety and the 'QianFu No.4' triploid tea variety. The content of heavy metal Cd in the root of triploid tea plants (7.29 mg/kg) was 18 times that in the root of diploid tea plants (0.42 mg/kg) but was not detected in the leaves (Figure 1c). The average content of Cu in the leaves and roots of triploid tea plants (13.23 mg/kg in the leaves and 17.93 mg/kg in the roots) was significantly higher than in diploid tea plants (6.93 mg/kg in the leaves and 10.04 mg/kg in roots (Figure 1d)). The content of Al in the roots of triploid tea plants (6760 mg/kg in the roots) was significantly higher than in diploid tea plants (2353.33 mg/kg in the roots) (Figure 1e), and the content of Al in the leaves of triploid tea plants (1640.00 mg/kg in leaves) was not significantly higher than in diploid tea plants (1493.33 mg/kg in leaves) ( Figure 1e). These results indicate that the accumulation of Cd, Al and Cu specifically occurred in the roots of triploid tea plants.
the 'QianFu No.4' triploid tea variety became larger and greener than that of the 'Qianmei 419' diploid tea variety (Figure1a). We measured the contents of Al, Cd and Cu in the soil of 'Qianmei 419' and 'QianFu No.4' tea plants. We found that the content of Al, Cd and Cu in the rhizosphere soil of the triploid tea plant were 7337.12, 0.16 and 29.09 mg/kg, respectively; the content of Al, Cd and Cu in the soil samples (5 cm from the main root of the tea tree) of the diploid tea plant were 7341.41, 0.16 and 29.45 mg/kg, respectively (Fig-ure1b). There was no significant difference in the content of these three metals in the soil samples (5 cm from the main root of the tea tree) of triploid tea plants and diploid tea plants. However, the Cd, Al, and Cu content in the soil of 'QianFu No.4' (50 cm away from the tea tree) were 5.62, 7943.59, 33.28 mg/kg, and the Cd, Al, and Cu content in the soil of 'Qianmei 419′ were 53.4, 7843.27, 32.97 mg/kg, which were higher than the heavy metal content in the soil rhizosphere. It can be inferred that the tea tree roots absorbed some heavy metals. Moreover, we further determined the content of Al and heavy metal in the leaves and roots of the 'Qianmei 419′ diploid tea variety and the 'QianFu No.4' triploid tea variety. The content of heavy metal Cd in the root of triploid tea plants (7.29 mg/kg) was 18 times that in the root of diploid tea plants (0.42 mg/kg) but was not detected in the leaves (Figure1c). The average content of Cu in the leaves and roots of triploid tea plants (13.23 mg/kg in the leaves and 17.93 mg/kg in the roots) was significantly higher than in diploid tea plants (6.93 mg/kg in the leaves and 10.04 mg/kg in roots (Figure 1d)). The content of Al in the roots of triploid tea plants (6760 mg/kg in the roots) was significantly higher than in diploid tea plants (2353.33 mg/kg in the roots) (Figure 1e), and the content of Al in the leaves of triploid tea plants (1640.00 mg/kg in leaves) was not significantly higher than in diploid tea plants (1493.33 mg/kg in leaves) ( Figure 1e). These results indicate that the accumulation of Cd, Al and Cu specifically occurred in the roots of triploid tea plants.    Figure S2). The raw reads were filtered and assembled by the de novo assembly software Trinity. The assembled sequences were redundant, so the software TGICL was used to splice to obtain the longest non-redundant single unigene set and further statistical information about the single unigene set. The data quality is displayed in Table 1.  Figure 2). The E-value threshold was 1 × 10 −5 . Based on the NR annotation results, a total of 45,820 (44.29%) unigenes were annotated in the GO database by Blast2GO based on the NR annotation results ( Figure 2). The cutoff value of the E-value was 1 × 10 −6 . However, due to the lack of complete genomic sequences in tea trees, only 27,030 unigenes were co-annotated into six databases (NR, NT, Swissprot, COG, GO and KEGG), accounting for 26.13% of 103,448 unigenes ( Figure 2).

RNA-Seq Analysis of 'Qianmei 419' and 'QianFu No.4' Tea Varieties
To further investigate the enrichment mechanism of Al, Cu, and Cd in the 'QianFu No.4' triploid tea variety, we performed a comparative analysis of the transcriptome across 'Qianmei 419' and 'QianFu No.4' tea plants using the Illumina HiSeq platform. In total, 58,155,664, 62,062,126, and 48,399,274 raw reads were obtained from triploid tea tree samples (CaS4_1, CaS4_2, CaS4_3), respectively. Diploid tea tree samples (CaS419_1, CaS419_2, CaS419_3) ( Figure S1) obtained 45,512,200,43,924,994, and 60,461,068 raw reads, respectively ( Figure S2). The raw reads were filtered and assembled by the de novo assembly software Trinity. The assembled sequences were redundant, so the software TGICL was used to splice to obtain the longest non-redundant single unigene set and further statistical information about the single unigene set. The data quality is displayed in Table 1.

Functional Annotation
BLASTX was used to perform a similarity search to annotate a unigene against various databases. All 103,448 (100%) unigenes were annotated in at least one database. A total of 35,980 (34.78%), 67,980 (65.71%), 90,547 (87.53%), 89,933 (86.94%) and 61,318 (48%) unigenes were similar to the sequences in the COG, KEGG, NR, NT and Swissprot databases, respectively ( Figure 2). The E-value threshold was 1 × 10 −5 . Based on the NR annotation results, a total of 45,820 (44.29%) unigenes were annotated in the GO database by Blast2GO based on the NR annotation results ( Figure 2). The cutoff value of the E-value was 1 × 10 −6 . However, due to the lack of complete genomic sequences in tea trees, only 27,030 unigenes were co-annotated into six databases (NR, NT, Swissprot, COG, GO and KEGG), accounting for 26.13% of 103,448 unigenes ( Figure 2).

Differentially Expressed Genes (DEG) and Enrichment Analysis
Each unigene was assembled by clean reads, and then the number of reads per unigene was calculated by RSEM. The effective length of each unigene and the number of reads on the total alignment were combined, and the expression of each unigene was normalized by the FPKM method. As a result, 16,459 single genes were filtered as upregulated genes in triploid samples, and 7354 genes were calculated as downregulated genes with a cutoff of |log2(foldchange)| > 1 and padj < 0.05 by comparative analysis (Figure 3a). Each unigene was assembled by clean reads, and then the number of reads per unigene was calculated by RSEM. The effective length of each unigene and the number of reads on the total alignment were combined, and the expression of each unigene was normalized by the FPKM method. As a result, 16,459 single genes were filtered as upregulated genes in triploid samples, and 7354 genes were calculated as downregulated genes with a cutoff of |log2(foldchange)| > 1 and padj < 0.05 by comparative analysis ( Figure  3a). To characterize the functions of the DEGs, GO enrichment analysis was performed to categorize potential functions. The DEGs were significantly annotated in the top 30 terms in the GO enrichment. The GO terms included biological process (BP), cellular component (CC) and molecular function (MF). In the BP category, the two main categories of oxidation-reduction process and protein phosphorylation accounted for the highest proportion. Of these, approximately 691 DEG were annotated as related to the oxidationreduction process, and approximately 495 DEG were annotated as related to protein phosphorylation. For the MF category, the main category of ATP binding accounted for the highest proportion of DEGs. In the CC category, the largest category was 'integral component of membrane'. The numbers of DEG enriched in the two categories were 1327 and 2676, respectively, which accounted for 30.61% of the enriched DEG (Figure 3b). To characterize the functions of the DEGs, GO enrichment analysis was performed to categorize potential functions. The DEGs were significantly annotated in the top 30 terms in the GO enrichment. The GO terms included biological process (BP), cellular component (CC) and molecular function (MF). In the BP category, the two main categories of oxidationreduction process and protein phosphorylation accounted for the highest proportion. Of these, approximately 691 DEG were annotated as related to the oxidation-reduction process, and approximately 495 DEG were annotated as related to protein phosphorylation. For the MF category, the main category of ATP binding accounted for the highest proportion of DEGs. In the CC category, the largest category was 'integral component of membrane'. The numbers of DEG enriched in the two categories were 1327 and 2676, respectively, which accounted for 30.61% of the enriched DEG (Figure 3b).
KEGG is a utility database for understanding advanced functions and biological systems (such as cells, organisms and ecosystems), genome sequencing and other highthroughput experimental techniques generated from molecular-level information, especially large molecular data sets. In Figure 3c, there were top 30 pathways that enriched the DEGs; among these pathways, the pathways with the highest richfactor were photosynthesis-antenna proteins and photosynthesis, while the pathways with the largest DEGs enrichment were ribosome and plant-pathogen interaction, with 596 and 533 DEGs enriched, respectively.

DEG of Transporters Related to Cadmium (Cd), Copper (Cu) and Aluminum (Al) in Tea
In order to answer the question of the difference in the content of metal Cd, Cu and Al in the tea plant, we screened out the DEGs associated with their absorption and transport from the differentially expressed genes in the transcriptome. We compared the expression levels of DEGs in the ATM, NRAMP, PCS, PDR, RAN and ZIP related to Cd transport in 'QianFu No.4' tea and 'Qianmei 419' tea. As shown in Figure 4a, most of these genes were downregulated in 'QianFu No.4' tea, and only CL1218.Contig2_All (NRAMP), CL3769.Contig6_All (PCS), and CL6459.Contig2_All (ZIP) were upregulated in 'QianFu No.4' tea (Table S2). transport from the differentially expressed genes in the transcriptome. We compared the expression levels of DEGs in the ATM, NRAMP, PCS, PDR, RAN and ZIP related to Cd transport in 'QianFu No.4' tea and 'Qianmei 419' tea. As shown in Figure 4a, most of these genes were downregulated in 'QianFu No.4' tea, and only CL1218.Contig2_All (NRAMP), CL3769.Contig6_All (PCS), and CL6459.Contig2_All (ZIP) were upregulated in 'QianFu No.4' tea (Table S2).

Real-Time Quantitative PCR Validation
To validate the RNA-seq results in triploid and diploid tea trees, another strategy for single gene regulation was chosen. Four up-and four downregulated unigenes were se-

Real-Time Quantitative PCR Validation
To validate the RNA-seq results in triploid and diploid tea trees, another strategy for single gene regulation was chosen. Four up-and four downregulated unigenes were selected for verification by real-time quantitative PCR (qRT-PCR) with identical RNA samples to those used for RNA-Seq ( Figure 5). Although the expression level of the test gene was not completely consistent, the expression trend of the test gene was similar (R = 0.8569).
Plants 2023, 12, 2580 7 of 12 lected for verification by real-time quantitative PCR (qRT-PCR) with identical RNA samples to those used for RNA-Seq( Figure 5). Although the expression level of the test gene was not completely consistent, the expression trend of the test gene was similar (R = 0.8569).

Discussion
Camellia sinensis is an important global natural resource and a non-alcoholic plant source of healthy drinks [42,43]. The metal content of tea trees has become one of the most concerning food safety issues [44]. Although researchers have also applied transcriptomics to discover genes related to metal stress in tea plants [3,45,46], the enrichment and transport of metals by polyploid tea plants have not been reported on yet.
In this regard, we determined the content of metals such as Cd, Al and Cu in 'Qianfu 4' and 'Qianmei 419'. In order to eliminate the influence of the difference in Cd, Al and Cu content in the soil, this research tested the content of Cd, Al, Cu and other metals in the rhizosphere soil of 'Qianfu 4' and 'Qianmei 419'. There was no significant difference in the content of Cd, Al and Cu in rhizosphere soil, indicating that the two tea plants grew in the same soil conditions. The content of Cd, Cu and Al in the roots of 'Qianfu 4' were significantly higher than in 'Qianmei 419'. This shows that the roots of 'Qianfu 4' had a stronger ability to accumulate enrichment metals. These results are consistent with those of Wang Qiang [47], who discovered that polyploid plants can accumulate cadmium and store it in their roots. However, only the Cu content in the leaves of 'Qianfu 4' was significantly higher than that of 'Qianmei 419'. The concentrations of Cd and Al in the leaves of 'Qianfu 4' were smaller, showing an opposite trend in the roots. The low metal content of tea leaves reduces tea consumption in metal-polluted environments [44].
In this study, RNA-Seq technology was used to construct the transcriptome of 'Qianfu 4' and 'Qianmei 419' leaves. We aimed to obtain complete and accurate comparative transcriptome information of 'Qianfu 4' and 'Qianmei 419' to reveal the regulatory mechanism and related genes that reduce the metal enrichment degree of 'Qianfu 4' tea leaves. RNA-seq is one of the most famous transcriptome research methods. It is sufficient for researchers to understand expression patterns and identify genes [48]. In this study, a total of 23,813 transcripts were identified as differentially expressed genes (DEGs). The qRT-PCR expression level was basically consistent with the change in the transcript abundance identified by RNA-seq, indicating that the RNA-seq data in this study are credible. Through statistical analysis of the terms and pathways of enriched GO related to DEG, we found that the transcripts in triploid tea plants had the most significant and frequently

Discussion
Camellia sinensis is an important global natural resource and a non-alcoholic plant source of healthy drinks [42,43]. The metal content of tea trees has become one of the most concerning food safety issues [44]. Although researchers have also applied transcriptomics to discover genes related to metal stress in tea plants [3,45,46], the enrichment and transport of metals by polyploid tea plants have not been reported on yet.
In this regard, we determined the content of metals such as Cd, Al and Cu in 'Qianfu 4' and 'Qianmei 419'. In order to eliminate the influence of the difference in Cd, Al and Cu content in the soil, this research tested the content of Cd, Al, Cu and other metals in the rhizosphere soil of 'Qianfu 4' and 'Qianmei 419'. There was no significant difference in the content of Cd, Al and Cu in rhizosphere soil, indicating that the two tea plants grew in the same soil conditions. The content of Cd, Cu and Al in the roots of 'Qianfu 4' were significantly higher than in 'Qianmei 419'. This shows that the roots of 'Qianfu 4' had a stronger ability to accumulate enrichment metals. These results are consistent with those of Wang Qiang [47], who discovered that polyploid plants can accumulate cadmium and store it in their roots. However, only the Cu content in the leaves of 'Qianfu 4' was significantly higher than that of 'Qianmei 419'. The concentrations of Cd and Al in the leaves of 'Qianfu 4' were smaller, showing an opposite trend in the roots. The low metal content of tea leaves reduces tea consumption in metal-polluted environments [44].
In this study, RNA-Seq technology was used to construct the transcriptome of 'Qianfu 4' and 'Qianmei 419' leaves. We aimed to obtain complete and accurate comparative transcriptome information of 'Qianfu 4' and 'Qianmei 419' to reveal the regulatory mechanism and related genes that reduce the metal enrichment degree of 'Qianfu 4' tea leaves. RNA-seq is one of the most famous transcriptome research methods. It is sufficient for researchers to understand expression patterns and identify genes [48]. In this study, a total of 23,813 transcripts were identified as differentially expressed genes (DEGs). The qRT-PCR expression level was basically consistent with the change in the transcript abundance identified by RNA-seq, indicating that the RNA-seq data in this study are credible. Through statistical analysis of the terms and pathways of enriched GO related to DEG, we found that the transcripts in triploid tea plants had the most significant and frequently enriched terms and pathways, which are mainly involved in the integral component of membrane of the cellular component. There were also many DEGs involved in the biological oxidationreduction process. Plants develop a variety of metal defense, transport, absorption and other biochemical strategies and mechanisms, all related to cell membranes [49], and the presence of excessive heavy metals and aluminum can increase membrane lipid peroxidation and reduce cell membrane stability [50]. In addition, the KEGG enrichment results of differentially expressed genes (DEGs) showed that the two pathways of photosynthesis-antenna proteins and photosynthesis had the highest degree of enrichment. Plants are highly sensitive to metal ions [51]. Among them, Cd and Cu have multidirectional effects on photosynthesis [52]. Unlike Cd, Cu is an essential plant trace element. Cu participates in photosynthetic electron transport, is a component of plastocyanin and is also important in regulating the function of PSII [53]. This shows that photosynthesis is closely related to the content of metal ions in plants, which is one of the reasons why it is possible to enrich photosynthesis.
The transport of metal ions in plants is critical to the degree of metal enrichment in different plant tissues [41], and metal transporters are responsible for metal transport between plant tissues. For example, the heavy-metal ATPase (HMA) family plays an important role in transition metal transport in plants [54]. Moreover, in 2010, Mills found that Arabidopsis AtHMA4 can transport Cd 2+ [55]. Some transcription factors are also involved in the transcription of metal ions. For example, STOP plays a critical role in tolerance of major stress factors in acid soils, such as proton H+ and aluminum ion Al 3+ , which are required for the expression of genes in response to acidic stress (e.g., ALMT1 and MATE) and Al-activated citrate exudation [56][57][58]. Therefore, we counted the DEGs expressed in some metal transporters and transcription factors ( In summary, 'Qianfu 4' can accumulate more Cd, Cu and Al. However, the downregulation of transport genes related to Cd and Al caused no significant difference in the content of Cd and Al in triploid leaves. This phenomenon may be beneficial to planting polyploid tea trees in areas contaminated with heavy metals and Al and keeping the quality of tea leaves unchanged. Moreover, this molecular mechanism of transport can enrich the theoretical basis of plant metal resistance.

Plant Materials and Growth Conditions
The tea tree 'QianMei 419' is a small-and medium-sized-leaf breed cultivated by the Tea Science Institute of Guizhou Academy of Agricultural Sciences (Guiyang, China). 'Qianfu 4' was obtained using Co60-γ rays to mutate the seeds of 'QianMei 419' and was strictly self-pollinated by this institute. Chen et al. [20]  and fallen leaves were removed and the soil layer was sampled in the 10 cm soil layer using a military shovel. Each mixed-soil sample needs to be collected from 15-20 sample points. After thorough mixing, 500 g of soil samples were retained using the four-part method for detecting heavy metal content.

RNA Extraction and Sequencing
Four tissues (root and leaf from diploid 'Qianmei 419' and triploid 'QianFu No.4') were used to perform RNA-seq analysis. Each sample processed three biological replicates. Total RNA was isolated from samples using an RNA extraction kit (Tiangen, Beijing, China) according to the manufacturer's protocol. Illumina RNA-Seq libraries were prepared and sequenced on an Illumina Novaseq 6000 (san Diego, CA, USA) following the manufacturer's instructions. The sequencing quality of RNA-Seq data was assessed by FastQC software (1.11.4). After removing low-quality reads through SOAPnuke software (2.1), clean reads from each sample were aligned to the Chayote genome by STAR software with default parameters. FPKM (Fragments Per Kilobase per Million) was used to calculate the gene expression values. Htseq software was used to analyze gene expression. The DESeq program was used to perform DEG analysis among the different samples.

Determination of Al, Cu and Cd Content in Root and Leaf
About 0.2~1 g of test sample was placed into the Teflon digestion tank (accurate to 0.1 mg) while adding 5 mL of nitric acid. When the reactions finished, the tanks were sealed with caps and placed into a microwave digestion instrument (WX-8000) (Shanghai Hao Instrument Technology Development Co., Ltd., Shanghai, China). The digestion tanks were taken out and placed in a fume hood with a temperature below 50 • C, which was rinsed with ultrapure water 3~4 times. Then, the tanks were transferred it into a 50 mL volumetric flask, rinsed, diluted to volume with ultrapure water, and the contents of Cu, Al and Cd were measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher Scientific, Waltham, MA, USA).

qRT-PCR Analysis
To verify the accuracy of RNA-Seq results, 10 DEGs were randomly selected for qRT-PCR analysis. Total RNA was extracted from leaf samples of 'QianFu No.4' and 'Qianmei 419' with RNAiso Plus (TaKaRa, Tokyo, Japan). Then, the samples were treated with RNAase-free DNase I (RQ1RNase-Free DNase; Promega, Madison, WI, USA) to remove genomic DNA, after which first-strand cDNA was synthesized (Revert Aid First-strand cDNA Synthesis Kit; Fermentas, Burlington, ON, Canada). The qRT-PCR reactions utilized for gene expression analysis were conducted in a 7500 Fast Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA) using SYBR Premix Ex Taq (TaKaRa Biotech Co., Ltd., Dalian, China) according to the manufacturer's instructions. The amplification cycling conditions were as follows: 5 min at 95 • C, 15 s at 95 • C, and 1 min at 60 • C for 39 cycles. Three biological replicates were performed. EF1 gene was used as an internal control for normalization, and relative gene expression was quantified using the 2 −∆∆Ct method. Gene-specific qRT-PCR primers were designed using primer premier 5.0 software (Table S1).

Conclusions
In this study, a large number of DEGs were obtained from the RNA-seq data of tea plants, which provide more opportunities for studying the molecular regulatory mechanisms of cadmium, aluminum and copper stress responses. In transcriptome data, we identified 25 DEGs related to cadmium transport, 16 DEGs related to Cu transport, and 24 DEGs related to Al transport. This indicates that most of the genes related to Cd and Al transport were downregulated in 'QianFu No.4', resulting in a decrease in the content of Cd and Al transported to the leaves in the triploid root, while the genetic genes related to Cu transport were upregulated in 'QianFu No.4'. In addition, this may be the reason why the copper content in the triploid leaves was higher than that in 'Qianmei 419' leaves. These results will help us understand the mechanisms of cadmium, aluminum and copper stress responses in tea plants and screen key candidate genes for future molecular breeding.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.