A MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis

Our previous studies showed that MAN3-mediated mannose plays an important role in plant responses to cadmium (Cd) stress. However, the underlying mechanisms and signaling pathways involved are poorly understood. In this study, we showed that an Arabidopsis MYB4-MAN3-Mannose-MNB1 signaling cascade is involved in the regulation of plant Cd tolerance. Loss-of-function of MNB1 (mannose-binding-lectin 1) led to decreased Cd accumulation and tolerance, whereas overexpression of MNB1 significantly enhanced Cd accumulation and tolerance. Consistently, expression of the genes involved in the GSH-dependent phytochelatin (PC) synthesis pathway (such as GSH1, GSH2, PCS1, and PCS2) was significantly reduced in the mnb1 mutants but markedly increased in the MNB1-OE lines in the absence or presence of Cd stress, which was positively correlated with Cd-activated PC synthesis. Moreover, we found that mannose is able to bind to the GNA-related domain of MNB1, and that mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance in Arabidopsis. Further analysis showed that MYB4 directly binds to the promoter of MAN3 to positively regulate the transcript of MAN3 and thus Cd tolerance via the GSH-dependent PC synthesis pathway. Consistent with these findings, overexpression of MAN3 rescued the Cd-sensitive phenotype of the myb4 mutant but not the mnb1 mutant, whereas overexpression of MNB1 rescued the Cd-sensitive phenotype of the myb4 mutant. Taken together, our results provide compelling evidence that a MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway.


Introduction
The heavy metal cadmium (Cd) is a toxic trace pollutant for humans, animals and plants, which enters the environment mainly from the burning of fossil fuels and phosphate fertilizers and thus the food chain [1][2][3]. Studies have revealed that Cd can denature proteins by binding to sulphydryl groups and induce oxidative stress, and thereby causes cellular damage by displacing cofactors from a variety of proteins, such as enzymes and transcription factors [4][5][6][7].
It is well known that carbohydrate-binding proteins are commonly referred to as lectins, which are ubiquitous in many plants [31]. Plant mannose-binding lectins are crucial for plant defense signaling during pathogen attack by recognizing specific carbohydrates on pathogen surfaces [32]. All known plant lectins can be divided into 12 lectin families of structurally and evolutionarily related proteins [33]. Among them, monocot mannose-binding and cucurbitaceae phloem lectins were now called the GNA-related and the Nictaba lectins, respectively [34]. It was shown that the pepper mannose-binding lectin CaMBL1 is involved in defense against microbial pathogens [32], and mannose has been observed to bind to plant lectins that possess diverse biological roles [34][35][36]. Interestingly, we found that MAN3-mediated mannose plays a vital role in plant response to Cd stress [37]. Thus, whether the Arabidopsis homolog of CaMBL1 (mannose-binding-lectin, MNB1) is involved in the regulation of MAN3-mediated Cd tolerance remains to be investigated.

MNB1 is involved in regulating MAN3-mediated Cd tolerance
It was shown that MAN3-mediated mannose is involved in the regulation of Cd stress response in Arabidopsis [37], and the pepper mannose-binding lectin CaMBL1 has been shown to be involved in defense against microbial pathogens [32]. Therefore, we speculated that the homologous mannose-binding protein may regulate plant responses to Cd stress in Arabidopsis. It has been shown that MNB1 (At1g78830) is a member of mannose-binding lectin family, which is the Arabidopsis homolog of CaMBL1 [32]. The MNB1 gene contains one exon, and encodes a 455-amino acid protein with the GNA-related lectin domain (https:// www.arabidopsis.org/servlets/TairObject?id=31114&type=locus; [32]). BLAST analysis of MNB1 in the Nr (non-redundant) protein database of NCBI revealed that it shares >92% sequence similarity with other proteins such as a predicted epidermis-specific secreted glycoprotein EP1-like (Camelina sativa) protein (accession no.XP_010429723.1), a predicted epidermis-specific secreted glycoprotein EP1-like (Raphanus sativus) protein (accession no. XP_018465133.1), the hypothetical (Arabis alpina) protein (AALP_AA2G232300; accession no.KFK42261.1) and a predicted epidermis-specific secreted glycoprotein EP1-like (Brassica rapa) protein (accession no.XP_009106596.1; S1A and S1B Fig). The transcript of MNB1 was detected in all tissues examined, with higher levels in rosette leaf and root (S1C Fig). In addition, we found that the MNB1 transcript is induced by Cd stress, reaching to the maximum level after 3 hours of Cd treatment and gradually decreasing afterwards (Fig 1A), suggesting that MNB1 may be involved in regulating Cd tolerance in Arabidopsis.
To test this hypothesis, we characterized two transfer (T)-DNA insertion mutants (mnb1-1, SALK_038821C; mnb1-2, SALK_121641) for MNB1 from the SALK T-DNA collection [32,38]; Figs 1B and S1D). These mutants showed similar phenotypes to the growth and development of the Col (S2A Fig). However, under 50 or 75 μM Cd stress, the mnb1 mutants showed decreased Cd tolerance compared with the Col (Fig 1C). Consistent with this result, root length was significantly shorter in the mnb1 mutants than in the Col (Fig 1E). These results suggest that MNB1 is required for Cd tolerance in Arabidopsis. In addition, we also tested the responses of the mnb1 mutants to other stresses, such as Pb(NO 3 ) 2, ZnSO 4 , CuSO 4 , and Na 3 AsO 4 , and found that mnb1 mutants showed enhanced sensitive to Cu and Pb but not to As and Zn (S3 Fig).
To further characterize the role of MNB1 in Cd tolerance, we obtained more than eight transgenic Arabidopsis lines constitutively expressing MNB1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and these lines displayed similar phenotypes to the Col in terms of overall development and flowering time (S4A and S4B Fig). We further chose the OE3 and OE7 lines to analyze their Cd tolerance, and found that the transgenic OE3 and OE7 plants displayed a significantly higher Cd tolerance than wild-type plants (Fig 1D), which is further confirmed by analysis of root length (Fig 1E). Taken together, all these results suggest that MNB1 positively regulates Cd tolerance in Arabidopsis. MNB1 positively regulates Cd tolerance via the GSH-dependent PC synthesis pathway this pathway. First, we analyzed Cd content in the Col, mnb1 mutants, and MNB1-OE plants under Cd stress. Cd contents were lower in roots and shoots of mbn1 mutant plants than in those of the Col; whereas higher Cd contents were detected in the MNB1-OE lines (Fig 2A). These results suggest that MNB1-mediated Cd tolerance may be associated with a sequestration mechanism. Next, we test whether MNB1-mediated Cd tolerance is related to the GSHdependent PC synthesis pathway [17,[39][40][41][42][43][44][45][46][47][48][49]. A GSH synthesis inhibitor buthionine sulfoximine (BSO) was employed to treat the Col and MNB1-overexpressing plants. There were no significant differences between Col and MNB1-overexpressing plants grown on medium containing BSO alone; however, Cd tolerance was enhanced in MNB1-overexpressing plants than in the Col plants in the medium containing CdCl 2 ( Fig 2B). When BSO was added together with CdCl 2 , the Cd-tolerant phenotype in the MNB1-overexpressing plants disappeared ( Fig  2B), which is further confirmed by analysis of root length ( Fig 2C) and fresh weight (Fig 2D), suggesting that the mechanism of MNB1-mediated Cd tolerance is related to the GSH-dependent PC synthesis pathway. We further measured GSH and PCs levels between the Col and the mnb1 mutants or MNB1-OE lines under Cd stress, and found that there were significant differences in GSH ( Fig 2E) and PCs ( Fig 2F) levels between them under Cd stress, suggesting that a GSH-dependent PC synthesis pathway is indeed involved in the mechanism of MNB1-meidated Cd tolerance. Finally, we analyzed the transcript levels of genes involved in the GSHdependent PC synthesis pathway in the Col, mnb1 mutant and MNB1-OE plants in response to Cd stress. Expression of the genes involved in the GSH-dependent PC synthesis pathway (such as GSH1, GSH2, PCS1, and PCS2) was significantly lower in the mnb1 mutants, but was markedly higher in the OE lines than in the Col in the absence or presence of Cd stress ( Fig  2G). In addition, we also analyzed expression of the other Cd stress-related genes in the Col,

Mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance
It was previously shown that CaMBL1 is localized to plasma membrane, and is able to bind to D-Mannose [32]. Consistent with this result, we also found that MNB1 is localized at plasma membrane (S1E Fig).The cell walls have previously been proposed as a major storage location Three independent experiments were done with similar results, each with three biological repeats. Four plants per genotype from one plate were measured for each repeat. Data are presented as means ±SD, n = 3. Bars with different lowercase letters are significantly different at P < 0.05 (Tukey's test). (E, F) Total GSH content and total PC content in Col, mnb1 mutants and MNB1-OE plants in the absence or presence of Cd. Two-week-old plants grown on 1/2 MS medium were treated with or without 50 μM CdCl 2 for 24 h for analysis of glutathione and PC contents. Data are presented as means ± SD, n = 3. Bars with different lowercase letters are significantly different at P < 0.05 (Tukey's test). (G) Transcript levels of genes involved in the PC synthesis pathway in Col, mnb1 mutants and MNB1-OE plants in the absence or presence of Cd. GAPDH was used as an internal control. Two-week-old plants grown on 1/2 MS medium were treated with or without 50 μM CdCl 2 for 6 h. Data are presented as means ± SD, n = 3. Bars with different lowercase letters are significantly different at P < 0.05 (Tukey's test).
https://doi.org/10.1371/journal.pgen.1009636.g002 for metals, and the loss of a cell wall protein might cause pleiotropic effects [50,51]. We performed a cell wall structure observation of Col and mnb1 mutant by scanning electron microscopy (SEM), and found that the cell walls are not seriously affected in the mnb1 mutant (S1F Fig). To test whether MNB1 is also able to bind to D-Mannose, we created a series of MNB1 deletion mutants based on deduced amino acid sequences of MNB1 (Figs 3A and S6). As shown in Fig   . In (C) and (E), three-day-old seedlings grown on 1/2 MS medium were transferred to 1/2 MS medium with or without 50 or 75 μM CdCl 2 for about 2 weeks. Scale bar = 1 cm. In (D) and (F), three independent experiments were done with similar results, each with three biological replicates. Four plants per genotype from one plate were measured for each replicate. Data are presented as means ± SD, n = 3. Bars with different lowercase letters are significantly different at P < 0.05 (Tukey's test).
Based on the fact that MNB1 is able to bind to D-Mannose, we next investigated whether the GNA-related domain of MNB1 is essential for mannose-mediated Cd tolerance. We examined effect of mannose treatment on Cd tolerance of Col, mnb1-1, 35S:MNB1/mnb1-1 and 35S: MNB1 Δ /mnb1-1 seedlings with or without 50 μM CdCl 2 , and found that mannose treatment enhanced Cd tolerance in 35S:MNB1/mnb1-1 seedlings but not 35S:MNB1 Δ /mnb1-1 seedlings

MYB4 regulates expression of MAN3 by directly binding to its promoter
The fact that the MAN3 transcript is induced by Cd stress [37] promoted us to find the transcription factor upstream of MAN3. By searching promoter sequence, we identified four MYB4-binding sites in the promoter region of MAN3 (Fig 4A). It was shown that MYB4 is a member of the MYB transcription factor family and localized in the nucleus [29]. The MYB4 gene contains two exons and one intron, and encodes a 282-amino acid protein located in cell nucleus (http://suba.live/suba-app/factsheet.html?id=AT4G38620). BLAST analysis of MYB4 in the Nr (non-redundant) protein database of NCBI revealed that it shares >91% sequence similarity with other proteins such as a predicted transcription repressor MYB4 (Camelina sativa) protein (accession no.XP_010436960.1), a hypothetical (Arabis alpina) protein (AALP_AA7G264300; accession no.KFK30463.1), a predicted transcription repressor MYB4 (Raphanus sativus) protein (accession no.XP_018480221.1) and a predicted transcription repressor MYB4 (Arabidopsis lyrata subsp. lyrata) protein (accession no.XP_020872408.1; S10A and S10B Fig). The transcript of MYB4 was detected in all tissues examined, with higher levels in silique and root (S10C Fig).
To determine whether MYB4 directly binds to the MAN3 promoter, we performed chromatin immunoprecipitation (ChIP) assay, and found that MYB4 was able to bind to the P1-P4 regions in the promoter of MAN3 (Fig 4B), which is further confirmed by EMSA assay ( Fig  4C). To further determine whether the transcript of MAN3 is regulated by MYB4, we created the MAN3 promoter-driven GUS reporter gene (Fig 4D), and transiently co-expressed with the MYB4 gene in Nicotiana benthamiana leaves and then determined the GUS activity. Coexpression of ProMAN3-GUS with 35S:MYB4 resulted in activation of the GUS reporter ( Fig  4E), suggesting that MYB4 is a transcriptional activator of MAN3.
The data showed that in the absence or presence of Cd stress, the transcript of MAN3 was dramatically downregulated in the myb4-1 mutant (Fig 4F), but was significantly upregulated in MYB4-OE plants ( Fig 4G). Collectively, these data provide evidence that MYB4 regulates the MAN3 transcription by directly binding to its promoter.

MYB4 acts as upstream of MAN3 in the GSH-depenent PC synthesis pathway
Based on the fact that MYB4 directly regulates expression of MAN3, we next determined whether altered expression of MYB4 affects plant Cd tolerance. We identified the myb4-1  Fig 5A), and analyzed its Cd tolerance ( Fig 5B). The myb4-1 mutant showed similar phenotypes to the growth and development of the Ler (S2B Fig). However, Cd tolerance was markedly reduced in myb4-1 mutant plants compared with wild-type plants (Fig 5B), as indicated by root length and fresh weight (Fig 5C  and 5D). We further generated genetically complemented myb4-1 mutant plants, and found that the complemented lines (COM1 and COM2; Fig 5A) reversed the Cd-sensitive phenotype of the myb4-1 mutant to the level of the Ler (Fig 5B-5D), indicating that loss-of-function of MYB4 is responsible for the Cd-sensitive phenotype. We also analyzed the responses of the myb4 mutant to other stresses, including Na 3 AsO 4 , ZnSO 4 , CuSO 4 , and Pb(NO 3 ) 2 , and observed that myb4-1 mutant showed enhanced sensitive to Pb but not to As, Zn, and Cu (S11 Fig). To further determine the role of MYB4 in plant Cd tolerance, we generated more than nine MYB4-overexpression lines, and chose two lines OE1 and OE2 to test their tolerance (S12 Fig). Enhanced Cd tolerance was observed in MYB4-OE plants compared with wild-type plants ( Fig  5B), as demonstrated by root length and fresh weight (Fig 5C and 5D). Taken together, all these results suggest that MYB4 is a positive regulator of plant Cd tolerance. To investigate the genetic epistasis between MYB4 and MAN3, we introduced 35S:MAN3 construct into the wild type Ler and the myb4-1 mutant, and the resulting 35S:MAN3/myb4-1 plants (S5C and S5D Fig) showed similar Cd-tolerant phenotype as 35S:MAN3 plants (Fig 5E), as indicated by root length (Fig 5F). Moreover, we found that Cd-induced transcript of MYB4 is earlier than that of MAN3 (Fig 5G) [37]. These results suggest that MAN3 is epistatic to MYB4.
To further determine whether MYB4 regulates Cd tolerance through MAN3-mediated GSH-dependent pathway, we first measured Cd contents of Ler, myb4-1, Col, MYB4-OE seedlings under Cd tress, and found that Cd contents were significantly lower in root and shoot of myb4-1 plants, but was markedly higher in those of MYB4-OE plants compared with wild-type ( Fig 6A). We then used BSO to treat wild-type and MYB4-overexpressing plants. This result showed that the Cd-tolerant phenotype in MYB4-overexpressing plants disappeared when BSO was added together with CdCl 2 (Fig 6B), as determined by root length (Fig 6C) and fresh weigh (Fig 6D).
Analysis of GSH and PCs levels further showed that significant differences were observed in GSH ( Fig 6E) and PCs (Fig 6F) levels between Ler and myb4-1, Col-0 and MYB4-OE seedlings under Cd tress. Finally, we analyzed the transcript levels of genes GSH1, GSH2, PCS1, and PCS2 in Ler, myb4-1, Col and MYB4-OE seedlings in response to Cd stress. The transcript levels of GSH1, GSH2, PCS1, and PCS2 were significantly lower in the myb4-1 mutants, but were markedly higher in the MYB4-OE lines than in the wild type (Ler and Col, respectively) in the absence or presence of Cd stress (Fig 6G). In addition, we also analyzed the transcript levels of other Cd stress-related genes, including GR1, GR2, ABCC1, ABCC2 and PDR8. The transcript levels of GR1 and PDR8 were consistent with differences of GSH1, GSH2, PCS1, and PCS2 but the transcript levels of GR2, ABCC1 and ABCC2 showed no regular difference in the absence or presence of Cd stress (S13 Fig). Taken together, all these results suggest that MYB4 positively regulates Cd tolerance by the MAN3-mediated GSH-dependent PC synthesis pathway.

MAN3-Mannose-MNB1 is involved in regulation of MYB4-mediated Cd tolerance
Based on the fact that MYB4 acts upstream of MAN3 to positively regulate Cd tolerance through the GSH-dependent PC synthesis pathway, we proposed that MAN3-mediated mannose may function as downstream of MYB4 to modulate Cd tolerance. To test this possibility, we used exogenous mannose to treat the wild-type Ler and the myb4-1 mutant, and found that, when exogenous mannose was added together with CdCl 2 , reduced Cd-tolerance in myb4-1 plants restores to the level of the wild-type Cd-tolerance (Fig 7A), as determined by root length (Fig 7B) and fresh weigh (Fig 7C). To determine whether galactose and glucose have similar protective effects of wild-type and myb4-1 mutant against Cd toxicity, we also tested the effects of galactose and glucose in wild-type and myb4-1 mutant plants to Cd toxicity. We found that these compounds did not confer protective effects of wild-type and myb4-1 mutant plants against Cd toxicity under our experimental conditions (S14A and S14B Fig). These results suggest that mannose is involved in MYB4-mediated Cd tolerance.
To further determine whether MNB1 acts downstream of MYB4 to positively regulate Cd tolerance, we introduced 35S:MNB1 construct into the wild type Ler and the myb4-1 mutant (S5D and S5E Fig), and the resulting 35S:MNB1/myb4-1 plants showed similar Cd-tolerance to 35S:MNB1 plants (Fig 7D), as indicated by root length (Fig 7E) and fresh weight (Fig 7F), suggesting that MNB1 is epistatic to MYB4. In addition, we also constructed the double mutant 35S:MYB4/mnb1-1 and analyzed its cadmium stress phenotype. As shown in S14C and S14D

Discussion
Plants have various strategies in respond to Cd stress for detoxification and tolerance [7,41,42]. The GSH-dependent PCs synthesis pathway is one of the most important mechanisms in Cd detoxification and tolerance in plants [7,41]. In the previous studies, we found that MAN3-mediated mannose triggers GSH-dependent PCs synthesis pathway contributing to the Cd accumulation and detoxification [37]. In this study, we have elucidated the detailed mechanisms of MAN3-mediated Cd tolerance through mannose-binding to the GNA-related domain of MNB1, a homologous mannose-binding lectin in Arabidopsis of CaMBL1 [32], to trigger the GSH-dependent PC synthesis pathway. Moreover, we also demonstrated that, in response to Cd stress, MYB4 acts upstream of MAN3 to positively regulate the Cd accumulation and tolerance. Our results showed that an Arabidopsis MYB4-MAN3-Mannose-MNB1 signaling cascade is involved in the regulation of plant Cd tolerance.
Mannose-binding lectins play an important role in plant defense signaling during pathogen attack [32]. It was found that the pepper mannose-binding lectin CaMBL1 is required to regulate cell death and defense responses to microbial pathogens [32], and mannose has been shown to bind to plant lectins that possess diverse biological roles [34][35][36]. So far, to our knowledge, no plant lectin has been identified to modulate heavy metal detoxification. In this study, we demonstrated that the Arabidopsis mannose-binding lectin MNB1 positively regulates the Cd accumulation and tolerance through the GSH-dependent Cd-activated PC synthesis pathway. First, the expression level of MNB1 was significantly induced by Cd stress, and loss-of-function of MNB1 displayed decreased Cd accumulation and tolerance, whereas overexpression of MNB1 enhanced Cd accumulation and tolerance. Second, we found that mannose is able to bind to the GNA-related domain of MNB1, which is a conserved domain in plant mannose-binding lectins [33,34]. MNB1 deletion mutants that contain full GNA-related lectin domain could bind to D-mannose, whereas MNB1 deletion mutants without GNArelated lectin domain are not able to bind to D-mannose, suggesting that the GNA-related domain of MNB1 is essential for its binding to D-mannose. Because it was shown that MAN3-mediated mannose plays an important role in plant responses to Cd stress [37], we further demonstrated that MNB1 is involved in regulating MAN3-mediated Cd tolerance, and mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance. Finally, we analyzed Cd contents in WT, mnb1 mutants and MNB1-OE lines under Cd stress, and found Cd content was significantly lower in the mnb1 mutants but markedly higher in the MNB1-OE lines than WT under Cd stress, suggesting MNB1-mediated Cd tolerance is involved in a mechanism of Cd accumulation and tolerance. In addition, we also found that MNB1-mediated Cd tolerance phenotype could be eliminated in the presence of BSO, a GSH synthesis inhibitor. The expression of the genes involved in the GSH-dependent PCs synthesis pathway was significantly lower in the mnb1 mutants but markedly higher in the MNB1-OE lines than WT under Cd stress, which was consistent with the differences of GSH content and PC content, suggesting a GSH-dependent PCs synthesis pathway is indeed involved in the mechanism of MNB1-meidated Cd tolerance. Recently, it was found that the potential role of SLIM1 in Cd tolerance of plants by inducing -S responses in the cell caused by depleting the GSH pool [52], and that, in addition to glutathione depletion, an enhanced oxidative state and depletion of upstream thiols, are necessary to induce the transcription of sulfate assimilation genes during early cadmium stress [53]. Therefore, it is of interest to further study to determine whether MNB1 affects sulfate assimilation, thereby altering reactive oxygen species signaling and regulating gene expression of GSH-dependent PCs synthesis pathway. In addition, in this study, we also analyzed whether MNB1 is involved in regulating other stress responses, such as Cu, Pb, As and Zn, and found that mnb1 mutants showed enhanced sensitive to Cu and Pb but not to As and Zn, suggesting that MNB1 may also be involved in the regulation of Cu and Pb stress responses. Therefore, it will be interesting to investigate the mechanisms of MNB1-mediated Cu and Pb tolerance.
Many transcription factors have been showed to be induced by heavy metal stress in plants [23,54]; however, only a few transcriptional factors were identified as the regulators of Cd detoxification and tolerance in plants, including HsfA4a [23], bHLH29, bHLH38, and bHLH39 [24], ZAT6 [25], OXIDATIVE STRESS2 [26], WRKY13 [55], WRKY12 [56], and MYB49 [29]. MYB4 is a member of MYB transcription factor family involved in the regulation of secondary metabolism and cell shape, disease resistance and responds to different stresses [30]. In this study, we demonstrated that MYB4 transcription factor directly binds to the promoter of MAN3 to positively regulate the transcript of MAN3 by several biochemical experiments. Although we failed in yeast one-hybrid, ChIP-qPCR assay showed that MYB4 can specifically bind to the promoter of MAN3 gene, and transient expression experiments and qRT-PCR analysis also showed that MYB4 positively regulates the transcript of MAN3. Moreover, MYB4 positively regulates Cd accumulation and tolerance through the GSH-dependent Cd-activated PC synthesis pathway, and genetics analysis showed that MYB4 act upstream of MAN3 and MNB1 to positively regulates Cd accumulation and tolerance. In addition, we also tested the responses of the myb4-1 mutant to As, Zn, Cu, and Pb stresses. Interestingly, we found that, unlike mnb1 mutants, myb4-1 mutant showed enhanced sensitive to Pb but not to As, Zn, and Cu, suggesting that MYB4 and MNB1 have cross and different functions in regulating heavy metal stress responses.
In summary, we provide compelling evidence supporting the fact that MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway. Cd stress quickly induces the expression of MYB4, which directly activates the expression of MAN3 to increase mannose content, and thus enhances mannose binding to MNB1, which in turn triggers GSH-dependent Cd-activated PC synthesis by coordinated activation of genes expression, and thereby leads to increased Cd accumulation and tolerance (Fig 8).
The Arabidopsis seeds were surface-sterilized and planted on half-strength Murashige-Skoog (1/2 MS) media [57] containing 1% sucrose and 1.2% agar (Sangon Biotech) (pH 5.7). They were vernalized in the dark at 4˚C for 3 d, and then grew in a controlled environment with 16 h light/8 h dark cycles at 22˚C. Three-day-old wild-type and mutant or transgenic seedlings were transferred to 1/2 MS agar plates in the absence or presence of heavy metal or other supplements for the indicated number of days.

Vector construction and plant transformation
The MAN3:GUS construct was generated as described by Chen et al. [25]. To generate MNB1-overexpressing (MNB1-OE), MYB4-overexpressing (MYB4-OE), In response to Cd stress, MYB4 is rapidly induced, and directly activates the expression of MAN3 and then enhances mannose binding to MNB1, which in turn triggers GSH-dependent Cd-activated PC synthesis by coordinated activation of genes expression, and thereby leads to increased Cd accumulation and tolerance.

SEM analysis
Two-week-old plants grown on1/2 MS medium, samples (Col and mnb1 mutant seedlings) were fixed in 2.5% glutaric dialdehyde. The cell wall structure observation of Col and mnb1 mutant by a tungsten filament scanning electron microscope (JSM-6490LV, SEM, Japan), as previously described [60,61]. Micrographs of each sample were observed at× 200 and × 300 magnifications.

Semi-quantitative RT-PCR and quantitative real-time RT-PCR
Plant total RNA was extracted from 2-week-old seedlings using Trizol Reagent (Invitrogen, CA, USA), and the first-stand cDNA was synthesized by total RNA with SuperScript II RNase H2 reverse transcriptase (Invitrogen) using random hexamer primer (Promega). Semi-quantitative RT-PCR was carried out using gene-specific primers. ACTIN8 was used as an internal control. RT-qPCR was performed according to the instructions provided for the Bio-Rad iCycler iQ system (Bio-Rad Laboratories, CA, USA) using platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The fold change of transcripts was calculated based on an efficiency calibrated model [62] and each sample were quantified at least in triplicate and normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal control. Statistical differences between the samples were evaluated by Student's t-test or ANOVA in combination with post-hoc test using delta Ct values [62]. The specific primers used are listed in S1 Table.

Measurement of Cd and GSH/GSSG/PC content
Seeds were grown on 1/2 MS medium with or without 50 μM CdCl 2 for two weeks and their seedlings were then sampled for analysis of Cd content. Cd content was carried out according to the method described by Lee et al. [14] (2003). Digested samples were measured using an atomic absorption spectrometer (Solaar M6;Thermo Fisher). Two-week-old seedlings grown on 1/2 MS medium were treated with or without 50 μM CdCl 2 for 24 h and then sampled for analysis of GSH/GSSG/PC contents. GSH/GSSG/PC contents were quantitated as described by Chen et al. [37].

Transient expression assays in Nicotiana benthamiana
Transient expression assays were carried out as described in previous studies [25,59,63] ( Sparkes et al., 2006;Chen et al., 2009;Chen et al., 2016). A. tumefaciens cells were obtained by centrifugation and suspended in the solutions containing 50 mM MES, 5 g/L D-Glc, 2 mM Na 3 PO 4 , and 0.1 mM acetosyringone to an optical density (600 nm) value of 0.1. A. tumefaciens cells were incubated at room temperature for 4 h and then co-transformed into epidermal cells of Nicotiana benthamiana using a needle-free syringe. The GUS staining and GUS activity measurements were analyzed at 48 h after injection, according to published protocols [64].

ChIP-qPCR assay
ChIP assay was performed according to the protocol described previously with slight modifications [65]. To determine whether GFP fusion affects the function of MYB4, we analyzed the phenotypes of 35S: MYB4 and 35S:MYB4-GFP plants under Cd stress, and found that both of them were more tolerant to Cd than wild-type plants, and there was no significant difference between them (S15 Fig), indicating that GFP fusion did not affect the function of MYB4. Therefore, we selected 35S:MYB4-GFP plants for CHIP analysis. Briefly, about 3 g of 15-dayold 35S::MYB4:GFP transgenic plants as the experimental group and 35S::GFP plants as the control group were cross-linked by formaldehyde (3%) for 30 min, which was stopped by the addition of glycine to a final concentration of 50mM for 2 min. The purified cross-linked nuclei were sonicated to shear the chromatin into 0.2-1.2 kb fragments, which were divided into three parts, one part was used for input DNA, and the two other parts were incubated with anti-GFP antibody (Abmart, China).The antibody-bound complex was precipitated with Protein A agarose beads. The DNA fragments in the immunoprecipitated complexes were released by reversing the cross-linking at 65˚C for 6 h. Purified immunoprecipitated DNA was analyzed by qPCR using primers listed in Supplementary S1 Table. Electrophoresis mobility shift Assay (EMSA) EMSA was conducted using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, https://www.thermofisher.com/) following the manufacturer's protocol. The recombinant protein MYB4-MBP was expressed and purified from Escherichia coli BL21 (DE3) cell line. Oligonucleotide probes (ProMAN3 P1 and P4) were synthesized and labelled with biotin at the 3 0 hydroxyl end of the sense strand. All oligonucleotide sequences used here are listed in S1 Table. Yeast one-hybrid assay The CDS of MYB4 was ligated into the PJG4-5 vector. The fragment of the MAN3 promoter was cloned into the placZi vector. All primers used are listed in S1 Table. The Y1H assay was conducted as previously described [66].

Expression of the MNB1 Protein, immunoblot analysis and its binding assay with man-agarose
The MNB1 gene was cloned into the vector pET22b to generate pET22b::MNB1, which produced a translational fusion of MNB1 to a His tag at the N terminus. The MNB1 protein construct was expressed in Escherichia coli and purified on a Man-agarose column. The MNB1 deletion series was made by PCR amplification using the primers showed in S1 Table. To express the MNB1 gene, pET22b::MNB1 was transformed into E. coli Rosetta (DE3).Recombinant strains were grown in 200 mL of Luria-Bertani medium at 37˚C to an optical density (600 nm) value of 0.5,induced with 1mM isopropyl-D-thiogalactopyranoside for 12h at 16˚C,and harvested. Cells were collected by centrifugation, resuspended in 20 mL of PBS buffer (137 mM NaCl, pH 7.4, 2.7 mM KCl,10 mM Na 2 HPO 4 ,2mM KH 2 PO 4 ), and disrupted by sonication. Cell debris was removed by centrifugation, and the supernatant was used to a D-Managarose column with purification (Sigma) according to the instructions of the manufacturer. Purified MNB1 proteins (10-50 mg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Sangon Biotech) by electro-blotting. For detection of proteins, an anti-His antibody (Solarbio) was used at 1:3,000 dilution.