Dopamine alleviates cadmium stress in apple trees by recruiting beneficial microorganisms to enhance the physiological resilience revealed by high-throughput sequencing and soil metabolomics

Abstract Dopamine has demonstrated promise as a stress-relief substance. However, the function of dopamine in Cd tolerance and its mechanism remains largely unknown. The current study was performed to investigate the mechanism of dopamine on alleviating apple Cd stress through regular application of CdCl2 and dopamine solution to potting soil. The results indicated that dopamine significantly reduced reactive oxygen species (ROS) and Cd accumulation and alleviated the inhibitory effect of Cd stress on the growth of apple plants through activation of the antioxidant system, enhancement of photosynthetic capacity, and regulation of gene expression related to Cd absorption and detoxification. The richness of the rhizosphere microbial community increased, and community composition and assembly were affected by dopamine treatment. Network analysis of microbial communities showed that the numbers of nodes and total links increased significantly after dopamine treatment, while the keystone species shifted. Linear discriminant analysis effect size indicated that some biomarkers were significantly enriched after dopamine treatment, suggesting that dopamine induced plants to recruit potentially beneficial microorganisms (Pseudoxanthomonas, Aeromicrobium, Bradyrhizobium, Frankia, Saccharimonadales, Novosphingobium, and Streptomyces) to resist Cd stress. The co-occurrence network showed several metabolites that were positively correlated with relative growth rate and negatively correlated with Cd accumulation, suggesting that potentially beneficial microorganisms may be attracted by several metabolites (L-threonic acid, profenamine, juniperic acid and (3β,5ξ,9ξ)-3,6,19-trihydroxyurs-12-en-28-oic acid). Our results demonstrate that dopamine alleviates Cd stress in apple trees by recruiting beneficial microorganisms to enhance the physiological resilience revealed. This study provides an effective means to reduce the harm to agricultural production caused by heavy metals.


Introduction
Cadmium (Cd) is a heavy metal pollutant with significantly toxicity [1][2][3]. Moreover, Cd pollution is long-lasting and difficult to remediate [4]. A small amount of Cd can cause abnormalities in plant growth, morphology, and physiology [5]. Cd exposure in plants destroys cell redox homeostasis, which leads to rapid increases in reactive oxygen species (ROS), in turn altering cell membrane function, and allowing the toxicity to spread [6]. Cd stress also causes leaf etiolation, root necrosis, and discoloration, thereby inhibiting key physiological processes such as the absorption and use of water and minerals by plants [5,7]. The process of Cd absorption and detoxification in plants is regulated by several key genes. It is reported that MdNRAMP3, MdHMA4, MdFRO2like, MdHA7 are the key Cd absorption genes, while MdNAS1 and MdCAX2 are the key Cd detoxification genes [8,9]. Cd in the environment is extremely difficult to remove and readily forms organic compounds after absorption by plants [8,9]. In addition, Cd stress can cause imbalance in the functions of the microbial community [10].
The plant rhizosphere is harboring complex microbial communities [11,12]. Microbial diversity is a key factor supporting plant growth and ecological stability [13]. However, plant rhizosphere microbial community could be affected by environmental stimuli [14,15]. Meanwhile, plants seek to cooperate with microbes and recruit beneficial microorganisms from the environment to enhance their capacity to fight adverse conditions [16,17]. Recruitment of microorganisms by plants under stress conditions is reportedly mediated by root exudates, and these changes tend to enhance plant stress resistance [18]. For example, legumes secrete f lavonoids to recruit more nitrogen-fixing bacteria under low-nitrogen conditions [19]; under S-metolachlor stress, wheat attracts potentially beneficial microorganisms with organic acids [20]; and maize enhances its defense responses by releasing benzoxazinoids to attract beneficial microorganisms [21]. Several organic compounds and other molecules such as f lavonoids and coumarins have been identified as plant signals that are involved in shaping the rhizosphere microbiota of plants [22]. In addition, rhizosphere microorganisms can directly use low-molecular-weight compounds secreted by roots as carbon sources [23]. Organic compounds secreted by plant roots further shape soil microbial communities and plant root function [24]. Therefore, plant-metabolite-microbe interactions are essential under abiotic stress conditions [25]. However, studies of the mechanisms through which microorganisms affect plant adaptability to stress remain scarce, and the metabolites that regulate microbial communities are yet to be identified.
In recent years, the risk posed by Cd pollution stress in apple orchards has increased sharply, causing a stress response in apples while also seriously impacting fruit yield and quality [26]. Therefore, a method to alleviate Cd stress production is needed. Dopamine is a highly antioxidant amine and the addition of exogenous dopamine to plants has a wide range of benefits [27,28]. However, elucidating the manner in which dopamine regulates the various processes that confer Cd stress tolerance in plants is also necessary. Some studies have shown potential benefits of dopamine in coordination of beneficial microbes against stress. Dopamine has been widely used in apples to improve resistance to various stresses, including salt [29], drought [30], replant disease [31], waterlogging [28], and nutrient deficiency [32][33][34]. Moreover, studies have shown that dopamine can alleviate Cd stress by improving photosynthesis, enhancing the activity of antioxidant enzymes and regulating Cd absorption and detoxification genes [8]. However, gaps in current research include the effect of exogenous dopamine on rhizosphere microorganisms, and whether plants can regulate rhizosphere microorganisms through control of root exudates to alleviate Cd stress.
In this study, we hypothesize that exogenous dopamine can affect rhizosphere microbial communities and regulate soil metabolites, and relieve Cd stress by recruiting some potentially beneficial microorganisms and metabolites. To test this hypothesis, Malus hupehensis was selected as the model plant. The composition and assembly of microbial communities were analysed and potentially beneficial microorganisms and metabolites were explored by high-throughput sequencing and metabolomics under Cd stress with or without dopamine supplementation. This study provides a new perspective on the stress-relieving properties of dopamine and systematically elucidates the mechanism of dopamine-induced Cd tolerance.
We measured the endogenous levels of dopamine in leaves and roots after 60 days of treatment; compared with CK, the content of dopamine in Cd-stressed roots was significantly elevated. After the application of dopamine, the content of dopamine in root and leaf was significantly increased by 252.6% and 191.7% compared with ST (Fig. 1h), demonstrating that exogenous dopamine appli-cation increased endogenous dopamine levels. In addition, the transpiration rate (Tr), stomatal conductance (Gs) and net photosynthesis rate (Pn) of DST plants were significantly increased by 60.3%, 80.0%, and 64.3% compared to ST, while intercellular CO 2 concentration (Ci) was decreased by 2.3% ( Fig. 1i-l).

Dopamine regulated antioxidant enzymes and ascorbate (AsA)-Glutathione (GSH) cycle-related genes and Cd uptake and detoxification genes
Most antioxidant enzymes and AsA-GSH cycle-related genes showed varying degrees of up-regulation in ST leaves, and no obvious differences were shown in ST roots. However, almost all antioxidant and AsA-GSH-related genes were markedly upregulated after dopamine application. Among them, MdCAT was up-regulated by 1.9 times in leaves, compared to ST, while MdCAT, MdMDHAR, and MdcGR were up-regulated by 3.2, 3.6, and 3.4 times in roots, respectively ( Fig. 4a and

Microbial diversity and community composition
Assessment of bacterial and fungal α-diversity after dopamine treatment indicated that the Simpson index increased markedly compared with ST. Nonmetric multidimensional scaling analysis (NMDS) showed that Cd stress and exogenous dopamine treatment resulted in significant differences among treatments ( Fig. 5a and b). Acidobacteria, Chlorof lexi, Actinobacteria dominated the bacterial communities, while Basidiomycota, Ascomycota dominated the fungal communities in various treatments ( Fig. 5c and d). The dominant bacterial order was Betaproteobacteriales, followed by Rhizobiales and Subgroup_6. Sordariales,

Assembly of the microbial community after various treatments
The assembly process (deterministic or stochastic) was evaluated for each treatment. Consistent variable selection was observed in bacterial communities of the CK and DST treatments (|βnearest taxon index (βNTI) | < 2), while various directions were found within ST samples (|βNTI| > 2). Consistent variable selection was also observed within ST and DST samples of rhizosphere fungal communities (|βNTI| > 2), while various directions were found within CK samples (|βNTI| < 2). In addition, the proportions of various processes affecting microbial assembly were assessed. Homogenizing dispersal (RCbray < −0.95) was dominant among stochastic processes in the rhizosphere bacterial communities of CK and ST. Homogeneous selection was of secondary importance in ST samples. In contrast, no dominant process (i.e., ecological drift), homogenizing dispersal, and dispersal limitation accounted for 66.7%, 22.2%, and 11.1%, respectively, of the microbial assembly in bacterial community data from the DST treatment. For rhizosphere fungal communities, no dominant process, dispersal limitation, and homogeneous selection were the most important ecological processes in CK samples. However, homogeneous selection was dominant in ST and DST samples (Fig. 6).

Network analysis of microbial communities
Compared to CK, the quantities of nodes and total links between the bacterial and fungal communities increased significantly.
In the bacterial community of DST sample, quantities of nodes and total links increased significantly compared to ST. For fungal community, quantities of nodes and total links decreased obviously after dopamine treatment compared to ST, and the quantity of total links between the bacterial and fungal communities increased obviously. In addition, network density was higher for bacteria than for fungi. The top five keystone genera in each treatment were identified based on centrality scores illustrating multiple correlations with other microorganisms. The results indicated that Cd stress and dopamine had significant impacts on microbiome structure (Fig. 7).

Exogenous dopamine modulates soil metabolism in Cd-stressed soil
Metabolomics was used to study the soil metabolites in each treatment (VIP > 1, P < 0.05). The partial least squares discrimination analysis (PLS-DA) indicated that the treatments were significantly separated. Fourteen and 18 metabolites markedly changed in abundance in response to Cd stress and the addition of dopamine with Cd treatment, respectively. These results indicate that exogenous dopamine obviously altered the soil metabolite profile under Cd stress. After dopamine application, the relative abundances of 13 soil metabolites were obviously up-regulated and those of five soil metabolites were obviously down-regulated compared to ST (Fig. 11).

Discussion
The overuse of metal-based pesticides and fertilizers and the discharge of industrial 'three wastes' lead to widespread Cd pollution, and it is difficult for farmers to completely avoid using Cd-contaminated soil [35,36]. Apple is more susceptible to cadmium stress due to its large planting area, long growth cycle, long time and space span. In addition, cleaning up Cd-contaminated soil is difficult and takes a long time [8,35]. The tolerance to Cd stress can be improved by applying specific exogenous substances [37], but the mechanism through which dopamine induces Cd stress tolerance has not been systematically investigated. In this study, analysis of growth and physiological indicators, high-throughput sequences, and metabolomic data revealed the effect of dopamine on alleviating Cd toxicity.
Dopamine is an ecologically beneficial option for reducing heavy metal stress [37]. In this study, Cd treatment obviously altered the growth of apple plants, with reductions in PL, TD, TDW, and RGR. However, dopamine obviously alleviated the inhibitory effect of Cd stress (Fig. 1). In addition, numerous common physiological indexes, including TCC, Fv/Fm, and NPQ, as well as measures of the antioxidant enzyme and photosynthesis, were severely impacted by Cd stress (Fig. 2). Dopamine obviously decreased the accumulation of ROS and restored some of the physiological damage caused by Cd stress. Dopamine has physiological benefits for plants [34,38]. More importantly, the expression levels of antioxidant enzymes (MdSOD, MdPOD, and MdCAT) and ASA-GSH cycling-related genes (MdcAPX, MdcGR, MdMDHAR, MdDHAR1, and MdDHAR2) were up-regulated (Fig. 4). Studies have shown that dopamine can alleviate plant stress response through modulation of physio-biochemical attributes and antioxidant defense systems, which is consistent with the results of this study [39,40]. Hence, it is assumed that by declining oxidative damage, exogenous dopamine improved the chlorophyll synthesis and photosynthetic activity of plants under Cd stress and restored the growth of plants. In addition, stress conditions can increase the endogenous dopamine content of plants, and the application of exogenous dopamine can further increase endogenous dopamine content, which is the key to dopamine-driven resistance to abiotic stress [27,28,30,40]. This study suggests that dopamine confers tolerance and that plants benefit from dopamine application under Cd stress.
When Cd is present in the cultivated environment, plant roots absorb Cd and transport it upward to aboveground parts, resulting in Cd enrichment of aboveground plant parts [41]. The root system is the essential organ of Cd uptake [8]. Cd accumulation in the aboveground part of the plant is related to Cd accumulation in plant roots [9]. Cd in the roots at much higher levels than in the leaves (Fig. 3). When Cd enters plant cells, it induces the production of ROS, and even cell death [35]. At the same time, plant roots can trap Cd, which weakens their capacity to transport Cd to the aboveground parts [35]. These processes affect Cd transport to the leaves. When dopamine was administered to apples, antioxidant enzymes activity was boosted, resulting in increased ROS scavenging potential and reduced Cd content. MdNRAMP3, MdHMA4, MdFRO2like, and MdHA7 are involved in the absorption and transport of Cd [8]. Higher expression levels of Cd detoxification gene helped to reduce Cd stress [8]. The expression level of Cd absorption genes was obviously decreased in DST plants, while the expression level of Cd detoxification genes was obviously increased (Fig. 4).
Exploration of the rhizosphere microbial community helped clarify the manner in which dopamine relieves Cd stress. The sequencing results showed that exogenous dopamine significantly affected the diversity and composition of microbial communities. We observed that Simpson index increased after exogenous dopamine treatment (Fig. 5). This high α-diversity demonstrates some level of resilience to environmental disturbance [42], allowing microbial community activity to be maintained [43]. Based on analysis of the assembly process of the sampled microbial community, we observed that exogenous dopamine mainly affected the assembly of the bacterial community under Cd stress (|βNTI| < 2). A previous study observed βNTI values between −2 and 2 for healthy rhizosphere soil, and the microbial community of healthy soil samples was assembled through stochastic processes [44]. Four basic processes can be used to show the microbial assembly processes occurring in different environments [45]. In this study, we used this framework to explore the effect of dopamine on the assembly process of the microbial community under Cd stress. Previous worked found that homogeneous selection dominated samples affected by heavy metals [44], while homogenizing dispersal accounted for a larger proportion of healthy soil samples [42]. Network analysis enables better evaluation of the interactions and identifies keystone species that make the largest impacts on microbial communities [46]. Our results showed much greater numbers of nodes and links in the bacterial network than the fungal network. Research has shown that soil bacteria are more active in response to various stress [47]. Microorganisms help their hosts maintain homeostasis and resist abiotic stress through the establishment of dense interaction networks [48]. The more complex the microbial community, the more resistant it is to environmental stress [49,50]. The networks among bacteria and between bacteria and fungi became more complex after exogenous dopamine was applied, as demonstrated by increased numbers of nodes and links.
Exogenous dopamine can improve abiotic stress resistance by altering the rhizosphere microbial community and recruiting beneficial microorganisms [28]. Specific bacterial and fungal groups were obviously enriched to various extents in the treatments of this study. Based on Pearson correlation analysis, potentially beneficial microorganisms that were obviously enriched in treatment DST and were also obviously correlated with RGR and whole-plant Cd accumulation were selected (Fig. 8).
Bacteria have the function of environmental bioremediation [51], and strengthening mutualistic symbioses among bacteria is particularly important to remediating heavy metal pollution [52]. One possible reason that exogenous dopamine alleviates Cd stress is that some taxa have the capacity to adsorb and detoxify Cd pollution, while others have the capacity to promote plant development. Pseudoxanthomonas is a common heterotrophic denitrifying bacterium belonging to the Proteobacteria [53]. Proteobacteria can remove heavy metals, and Pseudoxanthomonas shows effective resistance to chromium (VI) toxicity [54]. In addition, Pseudoxanthomonas has been applied to the removal of the organic pollutants polycyclic aromatic hydrocarbons [55]. Aeromicrobium has been demonstrated to degrade phenanthrene (PHE) in petroleum-contaminated soils, showing potential for use in soil remediation [56]. The increase in the abundance of Bradyrhizobium significantly increased nitrogen accumulation and the yield of maize [57]. Frankia is the main source of nodular symbiotic microorganisms [58]. As an Actinomycete, Frankia not only promotes plant growth but also has functions of heavy metal remediation and environmental protection [59]. LEfSe analysis indicated significant enrichment of Saccharimonadales in Cd-contaminated soils after the application of polymer amendments, which reduced Cd stress to plants [60]. The enrichment of Novosphingobium in clover roots can support complete rhizosphere degradation of PHE [61]. In addition, Novosphingobium has potential functions in disease resistance, soil repair and promotion of plant growth [62,63]. Streptomycetes are widely distributed across diverse habitats and have important ecological functions [64], including persistent growth-promoting, antibacterial, and restorative functions [65].
A variety of beneficial interactions between the root system and the rhizosphere contribute greatly to plant growth [54]. Soil is a storage place for microorganisms as well as a repository of metabolites secreted by plant roots [48,66]. These metabolites act as a bridge and play important roles in the interactions between plants and their environment [48,66]. Different metabolites have different regulatory effects on rhizosphere microbial communities [67]. Soil metabolites provide carbon sources and signaling molecules that can stimulate rhizosphere microorganisms, which in turn help plants resist various stresses and promote plant growth [50]. The present study revealed dopamine-regulated metabolites associated with RGR (positive correlations) and Cd accumulation (negative correlations) (Fig. 12). These metabolites may function as drivers of the rhizosphere bacterial community. The application of exogenous dopamine may alleviate Cd stress by supporting the release of these metabolites, leading to enrichment of potentially beneficial microorganisms. In this study, based on analysis of the co-occurrence network between the relative abundances of differential metabolites and potentially beneficial microorganisms, several metabolites were found to have significant correlations with potentially beneficial microorganisms, suggesting that the recruitment of those microorganisms may be mediated by the differential metabolites. The interactions between bacteria and metabolites are an important factor in alleviating heavy metal toxicity in plants [68].

Conclusion
This study verified that exogenous dopamine could enhance the photosynthesis and activate the ROS scavenging system, thereby alleviating Cd stress in apple trees. Meanwhile, exogenous dopamine application inhibited the expression of Cd absorption genes, promoted the expression of Cd detoxification genes and reduced Cd accumulation in apple plants. Importantly, exogenous dopamine application significantly altered soil metabolites, as well as the diversity and composition of rhizosphere microorganisms associated with apple plants. Some potentially beneficial microorganisms and metabolites were recruited, which showed positive correlations with RGR or significant negative correlations with Cd accumulation. Tighter networks among rhizosphere microorganisms and changes in microbial community assembly processes were also observed when dopamine was applied to rhizosphere soil. Correlation analysis between the relative abundances of potentially beneficial microorganisms and soil differential metabolites indicated that dopamine-induced recruitment of potentially beneficial microorganisms may be driven by soil metabolite changes (Fig. 13). These findings provide the innovative strategies to mitigate the threat of Cd stress to apple production, and the dopamine-related beneficial microorganisms will bring great benefits to the study of plant resistance. However, the actual application effect of dopamine in the field experiments needs to be further studied.

Experimental treatments
One-year-old healthy apple seedlings (M. hupehensis) with heights of approximately 50 cm were used as the experimental material. Healthy seedlings of the same size were selected for 30 days

Measurement of plant growth parameters
The PL and TD in each treatment were measured every 15 days during the experiment. At day 60, 10 seedlings showing consistent growth from each treatment were carefully removed from their   pots and divided into leaves, stems, and roots, and RGR and TDW were calculated [69].

Measurement of leaf physiological indexes and dopamine content
Healthy mature leaves were selected every 15 days during the experiment, and TCC and REL were determined [28]. At day 60, fresh young leaves of the same size were harvested for leaf histochemical staining according to the kit instructions (Servicebio, Wuhan, China). The NPQ, Fv/Fm, H 2 O 2 , O 2 •− , T-AOC, APX, POD, CAT, and SOD were determined according to the methods of Cao et al. [28]. The concentration of dopamine was analysed using highperformance liquid chromatography (HPLC; LC-2010, Shimazu, Japan) [28].

Quantification of gas exchange
The Tr, Pn, Gs, Ci were monitored with a Li-Cor portable photosynthesis system on sunny days. Total 30 leaves were collected for measurement [69].

Determination of cd content
At day 60, samples were exposed to staining solution (acetone, diphenylthiourea, glacial acetic acid) for 2 h. Then, Cd was observed with an optical microscope (BX63, Olympus, Japan) [40]. Cd concentration was determined as described by Huang et al. [70].  [28]. The primer sequences are presented in Table S1 (see online supplementary material).

Microbial community analysis
Loose soil was shaken off. Approximately 30 g of the root system with soil was placed into sterilized NaCl solution. Five plants were mixed into a sample, with five replicates analysed per treatment. Specific primers were used for PCR amplification after total DNA extraction. The 16S rRNA primer sequences were 338F (5 -ACTCCTACGGGAGGCAGCA −3 ) and 806R (5 -GGACTACHVGGGTWTCTAAT −3 ). The internal transcribed spacer (ITS) primer sequences were ITS5 (5 -GGAAGTAAAAGTCG-TAACAAGG −3 ) and ITS2 (5 -GCTGCGTTCTTCATCGATGC -3 ). After amplification, the PCR products were quantified, and then the library construction was constructed. QIIME2 version 2019.4 and the official tutorial were used to modify and improve the process of biological information analysis for microbial groups. The DADA2 plug-in was used for quality filtering, de-noising, concatenation, and chimera removal. The obtained sequences were merged, and characteristic sequence amplicon sequence variants (ASVs) and abundance data tables were generated. The raw sequencing data were submitted to the NCBI Sequence Read Archive (SRA) database under the accession number SRP425616.

Soil metabolome detection
Samples were taken after gently shaking the roots, and all rhizosphere soils within 5 mm of the root surface were collected. The roots of each group of five plants were mixed into a sample with five replicates per treatment. Analyses were performed by Personal Biotechnology Co. Ltd (Shanghai, China). The raw MS data (wiff.scan files) were converted to MzXML files and processed. SIMCA-P 14.1 (Umetrics, Umea, Sweden) was used for PLS-DA [71].

Bioinformatics and statistical analysis
Simpson index values were calculated using QIIME2. The LEfSe was used to determine the types of microorganisms enriched [72]. Then, the βNTI between pairs of samples was calculated, Figure 13. Diagram of the mechanism by which dopamine (DA) enhances cadmium stress tolerance in apple. Cd stress was induced by adding CdCl 2 solution. DA reduced the level of ROS and enhanced photosynthesis under Cd stress. DA could reduce the Cd content by regulating the expression of related genes. In addition, DA could regulate rhizosphere microbial community and soil metabolic profile under Cd stress. Dopamine exerts positive (green arrows) and negative (red arrows) effects. and the phylogenetic pattern of the rhizosphere microbiome was evaluated. Sample pairs with |βNTI| > 2 are expected to result from deterministic processes, while pairs with |βNTI| < 2 are likely governed by stochastic processes. The calculation method was described by Zhang et al. (2022) [72]. A microbial inter-domain network was constructed using filtered ASVs. The correlations between ASVs were determined using R version 3.6.0.
Co-occurrence networks were visualized and the number of nodes and links were calculated using Gephi 9.2 [46]. Differences were determined using SPSS 26.0 (IBM Corp., Armonk, NY, USA) software. The graphs were constructed using GraphPad Prism 9.0 (San Diego, CA, USA). Tukey's multiple range tests were used to check the significant difference between means (P < 0.05).