Synergetic modulation of plant cadmium tolerance via MYB75-mediated ROS homeostasis and transcriptional regulation

MYB75 enhances plant cadmium tolerance by mediating ROS homeostasis and cadmium tolerance-related genes expression. Cadmium (Cd) is a heavy metal with biological toxicity, which can be detoxified through chelation and compartmentation in plants. Transcriptional regulation mediates plant Cd tolerance by modulating these processes. However, the mechanism remains to be studied. Our results showed a previously unknown function of MYB75 transcription factor in the regulation of Cd tolerance. Cd exposure stimulates anthocyanin accumulation by raising MYB75 expression. Enhanced Cd tolerance was observed in the MYB75-overexpressing plants, whereas increased Cd sensitivity was found in the MYB75 loss-of-function mutants. Under Cd stress conditions, lower reactive oxygen species (ROS) levels were detected in MYB75-overexpressing plants than in wild type plants. In contrast, higher ROS levels were found in MYB75 loss-of-function mutants. Overexpression of MYB75 was associated with increased glutathione (GSH) and phytochelatin (PC) content under Cd exposure. Furthermore, the expression of Cd stress-related gene including ACBP2 and ABCC2 was elevated in MYB75-overexpressing plants, and this upregulation was mediated through the mechanism by which MYB75 directly bind to the promoter of ACBP2 and ABCC2. Our findings reveal an important role for MYB75 in the regulation of plant Cd tolerance via anthocyanin-mediated ROS homeostasis, and through upregulation of Cd stress-related gene at the transcriptional level.


Introduction
Human production processes such as industry and mining can increase concentrations of heavy metals in soil, air, and water. The accumulation of heavy metals in soil can lead to the loss of soil fertility, the reduction of vegetation, and the pollution of water sources (Clemens andMa 2016, Zou et al. 2021). Cadmium (Cd) is widespread heavy metal contamination, which has a serious toxic effect on plants, animals, and humans (Clemens 2019). Cd is a nonessential and toxic element for plant growth and development. It is commonly known that Cd in water can rapidly enter plants via root, resulting in toxicological symptoms (Haider et al. 2021). First, Cd can bind to the sulfhydryl, histidine, and carboxyl of structural protein, thus inhibiting protein function (Huybrechts et al. 2019). Cd also has similar chemical properties to other divalent cations, which hence can replace the metal ion in structural protein and cause impairment of protein function (Cuypers et al. 2010). Further, although Cd has non-redox property, it can indirectly induce the production of reactive oxygen species (ROS) Zhang et al. 2020). ROS-mediated change of redox environment in plant cells can disrupt protein function and destroy cell structure, thus inhibiting physiological metabolic processes such as photosynthesis, respiration, photorespiration, and cell cycle  (Waszczak et al. 2018). Consequently, improvement of Cd tolerance is critical for plant growth and development. Cd in agricultural soil is mainly derived from atmospheric deposition and industrial effluent. Cd accumulation in plant is mostly due to the uptake of soil Cd via root. Previous studies have established that various plant transporters play an important role in uptake of Cd. Cd uptake and transport were implicated in several genes encoding transporters including OsNramp5, HMA2, HMA4, IRT1, and PDR8 (Chang et al. 2020. Cd is transported into and accumulated in plants, which is harmful to plant growth and development. But plants have evolved multiple defense mechanisms to resist and reduce the toxicity of Cd. Initially, Cd 2+ chelated by Glutathione (GSH) and phytochelatin (PC) can sequester into vacuoles (González et al. 2021). Some transcription factors including ZAT6 and WRKY12, regulate Cd tolerance via modulation of GSH and PC biosynthesis at transcriptional level (Chen et al. 2016;Han et al. 2019). Next, plants may mediate the expression of metal uptake transporter genes and efflux transporter genes to compartment Cd distribution . Finally, some membrane protein proteins such as ACBP2 and FP6 can bind Cd 2+ via sulfhydryl groups on cysteine residues to form nontoxic or less toxic compound (Gao et al. 2010(Gao et al. , 2009). PDF2.5 promotes Cd transfer from protoplast to cell wall, thus alleviating Cd toxicity (Luo et al. 2019). In addition, to decrease Cd-induced ROS accumulation, plants enhance ROS scavenging by activating the antioxidant system. Plant Cd tolerance was enhanced by mediating these collective mechanisms at multiple levels, especially at transcriptional level.
MYB family transcription factors play a significant role in plant developmental processes as well as plant tolerance to diverse environmental stresses. Data from several studies suggest that MYB transcription factors participate in regulation of plant Cd tolerance, but details of the regulation mechanism need further research. MYB75, which is also defined as PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1), plays a key role in anthocyanin accumulation (Borevitz et al. 2000;He et al. 2021). However, the possible role of MYB75 in Cd tolerance and the details of the underlying mechanism have not been characterized.
To enrich the regulatory mechanisms under Cd exposure, here we identify MYB75 as a new regulator of plant Cd tolerance. Under Cd stress, a myb75-c mutant shows increased sensitivity to Cd stress and accumulates more ROS, whereas the transgenic lines overexpressing MYB75 decrease ROS levels compared with wild type plants. Furthermore, MYB75 directly binds to the promoter of Cd stress-related genes including ACBP2 and ABCC2, thus raising their transcription. Our study exposes that MYB75 acts as a positive regulator of Cd tolerance by anthocyanin-mediated ROS homeostasis, and through targeting ACBP2 and ABCC2 in Arabidopsis.

Plant materials and growth conditions
The Arabidopsis thaliana pap1-D, 35S: MYB75, and myb75c were described as previously (Zheng et al. 2020a(Zheng et al. , 2019. The 1/2 MS with Arabidopsis seeds were placed at 4 ℃ for 2 days before moving to 22 ℃ under diverse light conditions. For Cd treatment, 100 mM CdCl 2 was added to the 1/2 MS medium (pH 5.8) to a final concentration of 50 μM CdCl 2 , and 75 μM CdCl 2 . Three-day-old wild type and mutant or transgenic seedlings were transferred to 1/2 MS agar plates in the absence or presence of CdCl 2 for the indicated number of days. To reduce variation because of the precipitation of heavy metals, wild type, and mutant or transgenic plants were grown adjacent to each other in the same plate and their growth was compared. After the indicated days of growth, the plants were sampled for root growth and fresh weight assays. Nicotiana benthamiana grown in soil at 22 ℃ under 16-h-light/8-h-dark conditions was used for the transient expression assays.

Chlorophyll measurement
The determination of chlorophyll contents was measured as described . Briefly, a total of 100 mg of plant materials was pulverized with liquid nitrogen, incubated in 1.0 mL of 80% acetone in the dark for 30 min and centrifuged for 5 min at 12,000 rpm. The absorbance of the supernatant was measured at 645 and 663 nm and then total chlorophyll content was calculated.

Analysis of chlorophyll fluorescence
Chlorophyll fluorescence was determined with a pulsemodulated fluorometer (FMS-2, Hansatech, UK). For measurement of F v /F m , plants were dark-adapted for 30 min. Minimal fluorescence (F 0 ) was measured during the weak measuring pulses, and maximal fluorescence (F m ) was measured by 0.8 s pulse of light at about 4000 mmol m −2 s −1 . An actinic light source was then applied to obtain steady-state fluorescence yield (F s ), after which a second saturation pulse was applied for 0.7 s to obtain light-adapted maximum fluo- (Baker 2008).

Ion leakage assay
The determination of ion leakage was measured as described (Ding et al. 2018). Summarily, the injury seedlings were put into 15 ml tubes containing 5 ml deionized water, which were shaken at 22 °C for 15 min, and the conductivity was measured as S1. After detecting S1, the tubes were put into boiled water at 100 ℃ for 15 min and shaken at 22 ℃ for 1 h, and then, S2 was measured. The formula (S1 − S0)/ (S2 − S0) was used to calculate ion leakage (S0: conductivity of deionized water).

Oxidative damage estimation, in situ superoxide, and H 2 O 2 staining
Superoxide and H 2 O 2 levels were visually detected with nitro blue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB), respectively, as described previously (Zou et al. 2016). Seedlings were excised at the base with a razorblade and supplied through the cut ends with NBT (0.5 mg ml −1 ) or DAB (2 mg ml −1 ) solutions for 8 h. Leaves were then decolorized in boiling ethanol (95%) for 20 min. At least three seedlings were used for each treatment. O 2 − and H 2 O 2 content was measured as described . Briefly, the O 2 − reacts with MSDS (hydroxylamine hydrochloride) to generate NO 2 − . Then a red azo compound is generated with the reaction of p-aminobenzenesulfonic acid and α-naphthylamine, which possesses an absorption peak at 530 nm. The yellow titanium peroxide composite generated by the reaction of H 2 O 2 which has an absorption peak at 415 nm is used to calculate the H 2 O 2 content. Each sample has three biological repeats.

Determination of antioxidant enzymes
For the enzyme assays, 0.3 g of leaf were ground with 3 ml ice-cold 25 mM Hepes buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate, and 2% polyvinylpyrrolidone (PVP) (Zou et al. 2016). The homogenates were centrifuged at 4 ℃ for 20 min at 12,000g and the resulting supernatants were used for the determination of enzymatic activity. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD) activities were assayed as described previously.

Real-time PCR analysis
RNA was extracted as described previously, and cDNA synthesis was performed by one microgram (Zhang et al. 2010). qRT-PCR analysis was conducted using SYBR Green PCR Master Mix. Three separate experiments and technical triplicates of each experiment were implemented. Gene expression was standardized to the ACTIN 8 transcript levels.

Cloning and transient expression assay
The 2000 bp promoters of ACBP2 and ABCC2 were PCRamplified from Arabidopsis genomic DNA using specific primers (Supplementary Table S1). These promoters were cloned into the vector YY45 (YFP substitutes for β-glucuronidase [GUS] reporter), named ProACBP2: GUS and ProABCC2: GUS. These two constructs were co-transformed into epidermal cells of Nicotiana benthamiana with the 35S: MYB75-HA construct. Samples were then collected and labeled, and GUS staining was performed as described previously.

LUC reporter transactivation assays
The 2000 bp promoters of ACBP2 and ABCC2 were PCRamplified from Arabidopsis genomic DNA using specific primers (Supplementary Table S1). These promoters were cloned into the vector pGreenII0800-LUC (Li and He 2016), named ProACBP2: LUC and ProABCC2: LUC. These two constructs were co-transformed into wild type protoplasts with the 35S: MYB75-HA construct. For LUC assay, 100 μl of protoplast lysis buffer was added to the frozen protoplasts and mixed with a pipette gun (Yoo et al. 2007). After 5 min incubation on ice, 20 ml of the lysate harvested by centrifugation at 1000g for 2 min and 100 ml LUC mix were used to measure LUC activity.

Electrophoresis mobility shift assay
EMSAs were performed as previously described . Briefly, MBP-MYB75 was expressed in E. coli and purified using MBP Sepharose beads, respectively. The DNA fragment (Supplementary Table S1) was incubated with MBP-MYB75 in EMSA binding buffer [25 mM HEPES-KOH, pH 8.0, 50 mM KCl, 1 mM dithiothreitol (DTT), and 10% glycerol]. An Electrophoretic Mobility Shift Assay (EMSA) Kit with SYBR Green and SYPRO Ruby EMSA stains (Thermo Fisher) were used to detect protein-DNA interactions.

ChIP assays
ChIP assays perform as described (Yu et al. 2008). The 4-week-old plants were collected in 50 mL tubes, and 37 mL 1% formaldehyde solution was used for crosslinked under a vacuum for 20 min. The chromatin was collected and sheared by sonication to reduce the average DNA fragment size to around 500 bps, then the sonicated chromatin complex was immunoprecipitated by specific antibodies anti-HA. After reverse cross-linking, the immunoprecipitated DNA fragment was analysed by qPCR with specific primers shown in Supplementary Table S1.

Statistical analysis
Samples were analyzed in three individual biological replicates, and the data are indicated as the mean ± SD. Two-way ANOVA (LSD's multiple-range test) or Student's t-test were performed at a significance level of p < 0.05.

Accession numbers
The Arabidopsis Genome Initiative identifiers for the genes described in this article are as follows:

MYB75 transcription factor participates in modulation of plant cadmium tolerance
Previous research has proved that plants exposed to Cd stress show transcriptional change of genes involved in phenylpropanoid metabolisms and anthocyanin accumulation (Dai et al. 2012;Herbette et al. 2006), thus we further investigated the mechanism underlying Cd stress-regulated anthocyanin accumulation. To test the impact of Cd stress on anthocyanin accumulation, seeds of the wild type and myb75-c were germinated on one-half-strength Murashige and Skoog (1/2 MS) agar plates containing either 0, 25, 50, 75, 100 μM CdCl 2 for 3 days. As shown in Fig. 1A, anthocyanin accumulation was elevated in the wild type under Cd stress. However, anthocyanin accumulation was minimal in the myb75-c mutant (Fig. 1A). Quantification of anthocyanin also verified that the anthocyanin level increased with the Cd concentration in wild type rather than myb75-c mutant (Fig. 1B). Previous evidence demonstrated that anthocyanin biosynthesis derives from flavonoid biosynthetic pathway and three anthocyanin-specific genes encoding dihydroflavonol 4-reductasae (DFR), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose: flavonoid 3-oglucosyl transferase (UF3GT) have been identified, and expression of these genes is regulated by MYB-bHLH-WD40 (MWB) protein complex (Xu et al. 2015). We hence determined the expression of the regulatory gene in wild type without or with 25, 50, 75, 100 μM CdCl 2 . MYB75 transcription was notably induced in response to Cd stress (Fig. 1B, Fig. S1A). However, Cd treatment would not alter the expression of other regulatory genes (MYB90, TT8, and EGL3) ( Fig. S1B-D). These results suggest that MYB75 participates in Cd stress-induced anthocyanin accumulation.
The above findings led us to conclude that MYB75 transcription factor is involved in the modulation of plant Cd tolerance. Consequently, pap1-D seedlings, the activation tag mutant constitutively overexpresses MYB75/PAP1 and myb75-c that MYB75 knockout mutant using the CRISPR-Cas9 system were used for determining the MYB75 function in plant Cd tolerance. Arabidopsis seedlings were grown vertically in 1/2 MS agar plates without CdCl 2 for 3 days, then were moved to 1/2 MS agar plates without or with 50 or 75 μM CdCl 2 for 7 days. As can be seen from Fig. 1C, when grown on 1/2 MS media without CdCl 2 , pap1-D and myb75-c exhibited no difference compared with wild type. Nevertheless, pap1-D showed higher tolerance in response to Cd stress compared with wild type (Fig. 1C). By comparison, we observed that myb75-c was more sensitive to Cd stress than wild type (Fig. 1C). These results were further verified by quantification of both the root length and fresh weight (Fig. 1D). Overall, these results suggest that MYB75 is involved in the regulation of plant Cd tolerance.
It has previously been observed that Cd exposure weakens the photosystem, we next confirmed whether MYB75 regulated Cd stress-induced photosystem damage. We maintained Arabidopsis seedlings grown in 1/2 MS media without or with 75 μM CdCl 2 for 21 days. From the Fig. 2A, we can see that Cd exposure accelerated chlorophyll degradation. Quantification of chlorophyll verified that pap1-D showed more chlorophyll content compared with wild type (Fig. 2B). Meanwhile, the chlorophyll content of the myb75c mutant was significantly lower than that of the wild type (Fig. 2B). Ion leakage can indicate the degree of damage in plants caused by environmental stress, thus we analyzed the ion leakage under Cd exposure. As shown in Fig. 2C, ion leakage was much higher in the myb75-c mutant than in the wild type. Nonetheless, ion leakage of pap1-D was significantly lower than that of wild type (Fig. 2C).
We further determined the photosystem II (PSII) photochemistry by detecting Chlorophyll fluorescence including F v /F m and ΦPSII. Compared with wild type, F v /F m of pap1-D showed no visible difference without CdCl 2 (Fig. 2D). When exposed to Cd stress, pap1-D exhibited higher levels of F v /F m than that of wild type (Fig. 2D). In contrast, levels of F v /F m were lower in myb75-c than in wild type (Fig. 2D). Similarly, levels of ΦPSII were also higher in pap1-D and lower in myb75-c compared with wild type (Fig. 2E). These results reveal that MYB75 transcription factor alleviates Cd stress-induced photosystem damage. Together with above results, we conclude that MYB75 transcription factor participates in modulation of plant Cd tolerance.

MYB75 positively regulates plant Cd tolerance
To further examine the impact of MYB75 on plant Cd tolerance, we generated transgenic plants expressing MYB75 driven by the constitutive 35S promoter in wild type background. The 35S: MYB75 #7 and 35S: MYB75 #10 transgenic plants exhibited increased tolerance to Cd stress compared with wild type when grown on 1/2 MS media A Three-day-old WT and myb75-c Arabidopsis seedlings grown on plates under different Cd stress. Bars = 0.25 cm. B Anthocyanin levels and MYB75 transcripts in extracts from seedlings in (A). The experiments were performed in biological triplicate (representing anthocyanin content measured from 0.2 g WT and myb75-c plants and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). C Phenotypes of WT, pap1-D and myb75-c mutants under Cd stress. Three-day-old seedlings grown on 1/2 MS medium were transferred to 1/2 MS medium without or with 50 or 75 μM CdCl2. Photographs were taken 7 days after transfer. Bar = 1 cm. D Root length of seedlings described in C. Data are means ± SD; n = 26 biologically independent roots. E Fresh weight of seedlings described in C. Five plants per genotype from one plate were measured for each repeat. Data are means ± SD (n = 3). Different letters represented statistically significant differences (two-way ANOVA, p < 0.05).
with 50 or 75 μM CdCl 2 (Fig. 3A). Quantitative analysis of root length and fresh weight further confirmed these results (Fig. 3B, C), indicating that MYB75 positively regulates plant Cd stress.
Next, the 35S: MYB75 #7 and 35S: MYB75 #10 transgenic plants grown on 1/2 MS media without or with 75 μM CdCl 2 were examined. The chlorophyll content of 35S: MYB75 #7 and 35S: MYB75 #10 was significantly higher than that of wild type (Fig. 4A, B). As can be seen from Fig. 4C, ion leakage of 35S: MYB75 #7 and 35S: MYB75 #10 transgenic plants was significantly lower than that of wild type. When exposed to Cd stress, 35S: MYB75 #7 and 35S: MYB75 #10 transgenic plants exhibited higher levels of F v /F m and ΦPSII than that of wild type (Fig. 4D, E). Taken together, MYB75 transcription factor enhances plant Cd tolerance under Cd exposure.

MYB75 modulates ROS homeostasis via anthocyanin-mediated ROS scavenge in plant tolerance to Cd stress
Much work so far has focused on the key function of MYB75 transcription factor in regulation of anthocyanin accumulation. Given that anthocyanin is critical for scavenging ROS via their antioxidant capability (Li et al. 2017;Nakabayashi et al. 2014), we further examined the ROS levels in MYB75-overexpressing lines and myb75-c mutant under normal or Cd stress conditions. Nitroblue tetrazolium (NBT) staining indicated that the content of superoxide (O 2 − ) was much lower in pap1-D, 35S: MYB75 #7 and 35S: MYB75 #10 plants compared with the wild type under Cd stress (Fig. 5A). On the contrary, the content of O 2 − in myb75-c was much higher than that in the wild type under Cd stress (Fig. 5A). These results were further ascertained by quantification of O 2 − content (Fig. 5B). Moreover, we observed that Data are means ± SD; n = 10 biologically independent seedlings. E ΦPS(II) of seedlings described in A. Data are means ± SD; n = 10 biologically independent seedlings. Different letters represented statistically significant differences (two-way ANOVA, p < 0.05).
pap1-D, 35S: MYB75 #7 and 35S: MYB75 #10 plants accumulated less hydrogen peroxide (H 2 O 2 ) content than wild type under Cd stress (Fig. 5C). In contrast, the content of H 2 O 2 in myb75-c was much higher than that in the wild type under Cd stress (Fig. 5C). Quantification of H 2 O 2 content also confirmed these results (Fig. 5D). Collectively, these results demonstrated that MYB75 transcription factor plays a positive role in the protection of plants from Cd exposure by alleviating oxidative damage. Extensive research has shown that plants have highly effective antioxidant mechanisms involving superoxide dismutase (SOD) and catalase (CAT) to scavenge ROS. Cd exposure led to a decrease in antioxidant enzyme activities (Fig. 5E, F). It is most likely that Cd 2+ inhibits the function of the antioxidant enzyme. Intriguingly, the diminution of SOD and CAT activities in the myb75-c mutant was significantly aggravated under Cd exposure (Fig. 5E, F). Oppositely, SOD and CAT activities of pap1-D, 35S: MYB75 #7 and 35S: MYB75 #10 plants were significantly higher than that of wild type (Fig. 5E, F).
In summary, MYB75 transcription factor reduces ROS accumulation in plant tolerance to Cd stress.

MYB75 transcription factor stimulates GSH-dependent PC synthesis pathway and Cd accumulation
Reduced glutathione (GSH)-oxidized glutathione (GSSG) conversion by ROS homeostasis in plant cells has been intensively investigated (Noctor et al. 2012). To clarify whether ROS homeostasis affects GSH content, we examined catalase-overexpressing plants (35S: CAT2 and 35S: CAT3) under Cd exposure. When compared with wild type, 35S: CAT2 and 35S: CAT3 plants exhibited less H 2 O 2 content, but more GSH and PC content under Cd stress (Fig.  S2). Given that MYB75 transcription factor declined the ROS levels via anthocyanin and antioxidant enzyme, we next determined the GSH levels without or with Cd treatment. As shown in Fig. 6A, no significant difference was detected in Root length of seedlings described in A. Data are means ± SD; n = 26 biologically independent roots. C Fresh weight of seedlings described in A. Five plants per genotype from one plate were measured for each repeat. Data are means ± SD (n = 3). Different letters represented statistically significant differences (two-way ANOVA, p < 0.05) The experiments were performed in biological triplicate (representing chlorophyll content measured from 15 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). C Ion leakage of the plants in A. Error bars denote ± SD (n = 3). D Fv/Fm of seedlings described in A. Data are means ± SD; n = 10 biologically independent seedlings. E ΦPS(II) of seedlings described in A. Data are means ± SD; n = 10 biologically independent seedlings. Different letters represented statistically significant differences (two-way ANOVA, p < 0.05) GSH between the wild type, the pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 plants, and the myb75-c mutants without Cd treatment. Cd exposure significantly depressed GSH concentrations in these plants (Fig. 6A). Nevertheless, compared with wild type, GSH levels were higher in pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 plants, and lower in myb75-c mutants (Fig. 6A) Given the observed change of PC, we further test whether MYB75 affects Cd content through measuring Cd content under Cd stress. As can be seen from Fig. 6C, pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 plants showed higher Cd content in roots and shoots than wild type. On the contrary, Cd content was reduced in myb75-c under Cd stress compared with that in the wild type. These results suggest that The experiments were performed in biological triplicate (representing O 2 − content measured from 10 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). C H 2 O 2 content in extracts from seedlings in A. The experiments were performed in biological triplicate (representing H 2 O 2 content measured from 10 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). D MDA contents in extracts from seedlings in A. The experiments were performed in biological triplicate (representing MDA content measured from 10 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). E SOD activity in extracts from seedlings in A. Error bars denote ± SD (n = 3). F CAT activity in extracts from seedlings in A. Error bars denote ± SD (n = 3). Different letters represented statistically significant differences (two-way ANOVA, p < 0.05).

MYB75 mediates Cd accumulation in roots and shoots under
Cd stress.

MYB75 transcription factor directly regulates the Cd tolerance-related gene expression
Transcription factors found to be influencing Cd tolerance have been explored in several studies (Agarwal et al. 2020;Zhang et al. 2019). Therefore, we further investigated whether MYB75 regulates Cd tolerance at the transcriptional level. We determined Cd tolerance-related gene expressions such as ACBP2, ABCC2, GSH1, PDR8, ATM3, and PDF2.5. Intriguingly, the transcription levels of GSH1, PDR8, ATM3, and PDF2.5 were induced by Cd stress, but these gene expressions in pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 and myb75-c exhibited no difference compared with wild type (Fig. S3). However, under Cd stress, the transcription levels of ACBP2, an acyl-CoA-binding protein which binds Cd 2+ , were significantly higher in pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 plants than in the wild type, while its expression levels in the myb75-c mutant were significantly lower than that in the wild type (Fig. 7A). We also noticed that expression levels of ABCC2, an ABCC-type phytochelatin transporter, were elevated in pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 plants, but reduced in myb75c mutant without or with Cd stress (Fig. 7B).
Based on the positive impact of MYB75 on ACBP2 and ABCC2 expression, we further surveyed the GUS activity by transient expression analysis in Nicotiana benthamiana. From the Fig. 7C-E and Fig. S4, we can see that MYB75 prompted the expression levels of ProACBP2: GUS and ProABCC2: GUS, indicating that MYB75 has the capacity of prompting reporter activity driven by the promoters of ACBP2 and ABCC2. Moreover, we performed luciferase (LUC) reporter transactivation assays in Arabidopsis protoplasts. Promoters of ACBP2 and ABCC2 were fused with LUC gene to generate promoter-LUC reporter constructs (Fig. 7F). These reporter constructs were co-expressed with empty vector or MYB75-HA in wild type protoplasts treated with MG132, and the reporter gene expression was used to evaluate MYB75 transcriptional activity. We noticed that MYB75 induced ProACBP2-LUC expression compared with empty vector (Fig. 7G). Consistently, MYB75 also induced ProABCC2-LUC expression (Fig. 7H). Based on these findings, we conclude that MYB75 positively regulates ACBP2 and ABCC2 expression.

MYB75 transcription factor directly binds to the promoter of ACBP2 and ABCC2
Taking the above observations into account, we further examined the binding of MYB75 to the promoter regions of ACBP2 and ABCC2 in vitro and in vivo. MYB-recognizing element (MRE) has been identified as the core binding motif of MYB75. We found two MREs within the promoters of ACBP2 and ABCC2 (Fig. 8A, B). To test whether MYB can directly bind to the promoter regions of these target genes, we first performed electrophoretic mobility shift assay (EMSA). The results revealed that MYB75 protein tagged with maltose binding protein (MBP) (MBP-MYB75) bound The experiments were performed in biological triplicate (representing PC content measured from 10 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). C Cd contents in WT, pap1-D, 35S: MYB75 #7, 35S: MYB75 #10 and myb75-c seedlings grown on medium for 2 weeks. The experiments were performed in biological triplicate (representing Cd content measured from 10 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n = 3). Different letters represented statistically significant differences (two-way ANOVA, p < 0.05) Fig. 7 MYB75 regulates ACBP2 and ABCC2 expression. A, B qPCR analysis of ACBP2 and ABCC2 expression levels. Seedlings grown on plates for 2 weeks respectively, then treated with 75 μM CdCl 2 for 4 h, and then their mRNAs were isolated for qPCR analysis. Expression levels were standardized to ACTIN 8, the results of WT under control conditions were set at 1. Error bars denote ± SD (n = 3). Different letters represented statistically significant differences (two-way ANOVA, p < 0.05). C A schematic of the ACBP2promoter and ABCC2-promoter reporter construct, the effector plasmid, and the transfection control plasmid. D, E GUS staining of the ProACBP2: GUS and ProABCC2: GUS reporter after coex-pression of 35S: MYB75-HA in Nicotiana benthamiana. Coexpression of the ProACBP2: GUS and ProABCC2: GUS reporter and 35S empty vector was used as the effector plasmid control. F A schematic of the ACBP2-promoter and ABCC2-promoter reporter construct, the effector plasmid for LUC assays. G, H LUC assays of the ProACBP2: LUC and ProABCC2: LUC reporter after coexpression of 35S: MYB75-HA in Nicotiana benthamiana. Coexpression of the ProACBP2: LUC and ProABCC2: LUC reporter and 35S empty vector was used as the effector plasmid control. Error bars denote ± SD (n = 3). Asterisk represented statistically significant differences (t-test, p < 0.05) to the P1 probe of ACBP2 and C1 probe ABCC2, while the binding was abolished by mutation of MYB75 binding sites in the probes (Fig. 8A, B). Interestingly, MBP-MYB75 weakly bound to the P2 probe in the promoter of ACBP2, but did not bind to the C2 in the promoter of ABCC2 (Fig. 8A,  B). We next employed chromatin immunoprecipitation (ChIP) to further test the affinity of MYB75 for promoters of ACBP2 and ABCC2. We immunoprecipited HA-MYB75 protein from 35S: MYB75 #7 transgenic plants treated with MG132 using anti-GFP antibody. TA3, a retrotransposable element, was used as the internal control. The ChIP-qPCR results revealed that MYB75 significantly enriched the fragments containing P1 and P2 of ProACBP2, C1 of ProABCC2 (Fig. 8C, D). These results indicate that MYB75 directly regulates the transcription of ACBP2 and ABCC2 by binding to their promoters.

Discussion
As a large number of heavy metal pollutants, Cd pollution has become a global crisis with industrial development, causes serious environmental damage and human disease. High concentration of Cd in the soil also inhibits plant growth and crop yield. In recent years, there has been an increasing interest in studying the mechanism by which plants enrich and detoxify Cd. In this study, we established that MYB75 transcription factor positively regulates plant Cd stress via multiple pathways. Our research revealed that MYB75 alleviates ROS damage via anthocyanin-mediated ROS elimination and activates of Cd tolerance-related genes at the transcriptional level, such as ACBP2 and ABCC2, thus inspiring a new transcription factor function in elevating plant Cd tolerance. A, B EMSA assays of MYB75 binding to ACBP2 and ABCC2 promoter. Wild-type and mutant probes were incubated with MBP-MYB75, and free and bound DNAs were separated on an acrylamide gel. C, D ChIP assays of MYB75 binding to ACBP2 and ABCC2 pro-moter. The 14-day-old seedlings were treated with or without 75 μM CdCl 2 for 4 h respectively, then harvested for ChIP-qPCR assay using anti-HA antibody. TA3 was used as the internal control. Different letters represented statistically significant differences (two-way ANOVA, p < 0.05)

Involvement ROS elimination in MYB75-induced anthocyanin accumulation
It is well established that anthocyanin accumulation is induced by extreme environmental factors, such as high light, drought, cold, thus protecting plants against ROS damage (Allan and Espley 2018). Several studies suggested that Cd stress stimulated anthocyanin accumulation. Anthocyanin biosynthetic genes transcription were induced under Cd exposure (Dobrikova et al. 2021;Mwamba et al. 2020). As we all know, anthocyanin biosynthetic genes were regulated by MBW protein complex. It seems that Cd stress affected anthocyanin regulatory genes expression. Our results illuminated that Cd exposure primarily induces MYB75 expression, thus prompting MBW protein complex and stimulating anthocyanin accumulation (Fig. 1A, B). These findings reveal that Cd exposure aggravated anthocyanin accumulation via transcriptional regulation of MBW protein complex.
Previous studies have shown that ROS plays a significant role in signal recognition and transduction in plant stress response (Mittler 2017). It is well established that ROS may be a secondary messenger in the signal transduction pathway to activate the antioxidant system, but high ROS levels might overwhelm the antioxidant system under stress (Waszczak et al. 2018). In this study, we have provided evidence that O 2 − and H 2 O 2 accumulation is moderate in MYB75 overexpression lines which accumulate high anthocyanin ( Fig. 5A-C). Furthermore, we found that the Cd-inhibited activity of SOD and CAT was aggravated in the myb75-c mutant in which anthocyanin accumulation is extremely inhibited (Fig. 5E, F). These results suggested that MYB75 modulates ROS homeostasis via anthocyaninmediated ROS scavenge in plant tolerance to Cd stress. It is possible, therefore, that MYB75 positively regulates Cd tolerance partly through elevated anthocyanin accumulation, resulting in enhanced protection against ROS.
Several researches demonstrated that there was a negative correlation between the chlorophyll and anthocyanin content under Cd stress (Szopinski et al. 2020;Vazquez et al. 2020). We found that pap1-D, 35S: MYB75 #7 and 35S: MYB75 #10, which accumulated abundant anthocyanin, exhibited higher chlorophyll level and fluorescence than wild type (Figs. 2, 3). These results imply that anthocyanin can protect photosystem from ROS damage under Cd exposure. It has previously been observed that exogenous anthocyanin increased intracellular GSH levels (Norris et al. 2016). We verified that endogenous anthocyanin elevated GSH and PC levels (Fig. 6A, B). Our results are also in accord with previous studies indicating that anthocyanin is beneficial to Cd uptake and hyperaccumulate under Cd exposure (Mwamba et al. 2020;Szopinski et al. 2020) (Fig. 6C). Previous studies showed that anthocyanins and organic acids can facilitate the localization of Cd into the vacuole (Glinska and Gapinska 2013, Sebastian and Prasad 2018, Verbruggen et al. 2009). We speculated that endogenous anthocyanins can benefit Cd 2+ sequestration to reduce Cd toxicity. These observations may support the hypothesis that plants adaptively prompt anthocyanin accumulation during suffering Cd stress thus scavenging ROS and expediting Cd 2+ sequestration to alleviate Cd toxicity.

MYB75 enhances plant Cd tolerance at transcriptional level
Under Cd exposure, plants evolve an adaptive mechanism by which transcription factors regulate the expression of numerous Cd tolerance-related genes at transcriptional level. ZAT6 regulates Cd tolerance by upregulating PC synthesisrelated genes (Chen et al. 2016). MYB49, the R2R3-MYB transcription factor, results in Cd accumulation through inducing HIPP22 and HIPP44 transcription . Cd-induced WRKY13 activates DCD expression to increase the production of H 2 S, bringing about higher Cd tolerance . In our study, LUC and GUS staining assays suggested that MYB75 reinforced the activities of the promoters of ACBP2 and ABCC2 (Fig. 7C-H). EMSA assays demonstrated that MYB75 directly bound to the MYB-recognizing element in the promoter of ACBP2 and ABCC2 (Fig. 8A, B). ChIP-qPCR assays proved that Cd stress strengthened the binding of MYB75 to ACBP2 and ABCC2 (Fig. 8C, D). These results imply that MYB75 directly regulates the ACBP2 and ABCC2 expression.
Previous studies confirmed that ACBP2 alleviates Cd toxicity through binding Cd 2+ (Gao et al. 2010). Additionally, ABA and drought induce ACBP2 transcription thus enhancing drought tolerance (Du et al. 2013). Intriguingly, anthocyanin accumulation is raised by ABA and drought. It seems that MYB75 was activated upon drought stress and then elevated ACBP2 expression to improve drought tolerance. These findings suggested that MYB75 perhaps participated in diversified stress response. Prior reports have shown that ABCC2 exhibited PC transport activity in plant vacuole (Song et al. 2010). Subsequent research observed that anthocyanin is transported to the vacuole via ABCC2 (Behrens et al. 2019). It can thus be suggested that vacuole played an important role in detoxifying Cd via ABCC2-modulated Cd 2+ sequestration. Interestingly, previous studies have revealed that overexpression of ACBP1 (highly homologous with ACBP2) or ABCC1 (highly homologous with ABCC2) resulted in Cd accumulation in the shoot (Park et al. 2012;Xiao et al. 2008). We found that Cd accumulation in shoot and root is elevated in MYB75-overexpressing transgenic plants in this study (Fig. 6C), suggesting that MYB75 perhaps modulates Cd accumulation via transcriptional regulation of ACBP2 and ABCC2. In addition, several reports have shown that Cd hyperaccumulation is accompanied by anthocyanin accumulation (Corso et al. 2018;Szopinski et al. 2019). Together with our results (Fig. 6C), it seems that anthocyanin accumulation occurs to alleviate ROS damage caused by Cd hyperaccumulation in plant response to Cd stress. According to these data, we infer that regulation of MYB75 on Cd 2+ sequestration and compartmentation at transcriptional and metabolic level enhances plant Cd tolerance.

MYB75 balance growth and the stress response
Recent studies suggested that reciprocal regulation between stress response and growth-control pathways occurs at multiple levels. Plants need to strictly limit the allocation of carbon sources to balance development and extreme environmental response. Extensive research has shown that MYB75 functions in the regulation of secondary cell wall formation during plant development (Bhargava et al. 2010). MYB75 transcription facilitates carbon flow into the anthocyanin pathway rather than the lignin pathway under various environmental stresses. Our previous studies demonstrated plant fine-tune anthocyanin accumulation via post-translational regulation of MYB75 through HAT1 transcription factor and SUMO E3 ligase SIZ1 during growth (Zheng et al. 2020b(Zheng et al. , 2019. In general, therefore, it seems that MYB75 primarily regulates secondary cell wall formation under normal conditions. Once plant suffers stress including Cd and drought, the activities of MYB75 is enhanced via multiple levels of regulation, thus promoting anthocyanin accumulation to adapt to the extreme environment. In conclusion, a working model was proposed based on our study (Fig. 9). When the plants are confronted with Cd stress, expression of MYB75 transcription factor could be quickly induced. Cd-induced MYB75 alleviates oxidative damage via anthocyanin-mediated ROS homeostasis. On the other side, MYB75 activates ACBP2 and ABCC2 expression by directly binding to the promoters of ACBP2 and ABCC2, followed by Cd sequestration and compartmentation, thus enhancing plant Cd tolerance.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00299-022-02871-0. When plants were confronted with Cd stress, MYB75 transcription factor could be induced quickly. On the one hand, MYB75 activated anthocyanin accumulation, followed by the ROS scavenge. On the other hand, MYB75 directly bind to the promoter of Cd tolerancerelated gene, such as ACBP2 and ABCC2, thus activating their expression, which brought about Cd sequestration and compartmentation. The synergetic modulation via MYB75 enhanced plant tolerance in response to Cd stress