A genome-wide co-expression network analysis revealed ZmNRAMP6-mediated regulatory pathway involved in maize tolerance to lead stress

doi:10.21203/rs.3.rs-2500013/v1

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A genome-wide co-expression network analysis revealed ZmNRAMP6-mediated regulatory pathway involved in maize tolerance to lead stress

https://doi.org/10.21203/rs.3.rs-2500013/v1

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published 04 May, 2023

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Lead (Pb) is one of the most toxic heavy metal pollutants, which can penetrate to plant cells via root absorption and thus cause irreversible damages to the human body through the food chain. To identify the hub gene responsible for Pb tolerance in maize, we performed a trait-associated co-expression network analysis at a genome-wide level, using two maize lines with contrasting Pb tolerances. Finally, ZmNRAMP6 that encodes a metal transporter was identified as the hub gene among the Pb tolerance-associated co-expression module. Heterologous expression of ZmNRAMP6 in yeast verified its role in Pb transport. Combined Arabidopsis overexpression and maize mutant analysis suggested that ZmNRAMP6 conferred plant sensitivity to Pb stress by transporting Pb ion from the roots to shoots. Knockout of ZmNRAMP6 caused Pb retention in the roots and activation of the antioxidant enzyme system, resulting in an increased Pb tolerance in maize. An integration of yeast one-hybrid and dual-luciferase reporter assay uncovered that ZmNRAMP6 was negatively regulated by a known Pb tolerance-related transcript factor ZmbZIP54. Different from the other known NRAMP genes, ZmNRAMP6 acted as a Pb transporter from the roots to shoots and environment. Collectively, knockout of ZmNRAMP6 will aid in the bioremediation of contaminated soil and food safety guarantee of forage and grain corn.

Maize

Co-expression network

ZmNRAMP6

Bioremediation

A metal transporter ZmNRAMP6 was identified by using a trait-associated co-expression network analysis at a genome-wide level. ZmNRAMP6 confers maize sensitivity to Pb by accumulating it to maize shoots. ZmNRAMP6 knockout detains Pb in roots, activates antioxidant enzymes, and improves Pb tolerance.

Lead (Pb) is one of the most toxic heavy metal pollution sources, which has the characteristics of biological accumulation, non-degradability, and persistence (Redwan and Elhaddad 2017). Even a low centration of Pb will poison plants, indirectly causing irreversible damages to the human body through the food chain (Azimi et al. 2017). Pb stress causes the disorder of plant morphological, physiological and biochemical functions, thereby affecting seed germination, seedling growth, root elongation, chlorophyll biosynthesis, biomass accumulation, and yield formation (Jeliazkova and Craker 2003; Fahr et al. 2013; Baruah et al. 2019; Khan et al. 2021). To resist the toxicity of Pb stress, plants have evolved a variety of tolerance and defense mechanisms, involving decrease of uptake, biosorption by cell wall, extracellular precipitation (Shahid et al. 2017), chelation (Pourrut et al. 2011), transport, isolation of vacuoles(Gupta and Lu 2013), and activation of enzyme antioxidant system (Shahid et al. 2014).

Maize is one of the major cereal crops widely grown in the world. Owing to its good adaptability to low-fertility soil, fast growth, and high bioaccumulation, maize has been used as an optimum phytoremediation crop for heavy metal-contaminated soil (Wang et al. 2007). A number of studies have shown that maize accumulates more Pb in its roots and transports only a small amount to its aboveground parts (Dai et al. 2007; Ma et al. 2022; Hou et al. 2022b). This enables maize to aid in soil phytoremediation and energy supply, in consideration of its food security (Li et al. 2023).

In recent years, several gene families have been reported to participate in plant responses to heavy metals. For instance, heavy metal ATP enzymes (HMAs) transport heavy metal ions across the membrane by hydrolyzing ATP in plants (Singh et al. 2016). In Arabidopsis and rice, HMA2, HMA4, and HMA5 are involved in the transport of heavy metal ions from the underground to aboveground parts (Andrés-Colás et al. 2006; Argüello et al. 2007; Takahashi et al. 2012). HMA3 was shown to affect cadmium (Cd) accumulation in maize grains (Tang et al. 2021). ATP-binding cassette (ABC) transporters translocate heavy metals into the central vacuoles to isolate them, thereby alleviating heavy metal toxicity to plant cells (Brunetti et al. 2015). In Arabidopsis, the ABC transporter AtPDR8 serves as an efflux pump of Cd and confers Cd tolerance in Arabidopsis (Kim et al. 2007). The AtPDR8-overexpressed Arabidopsis showed an increased Cd resistance and decreased Cd contents, relative to the wild-type line (Kim et al. 2007). In rice, OsABCG36 that encodes a plasma membrane-targeted ABC transporter protein enables Cd tolerance via exporting Cd from rice root cells (Fu et al. 2019). Knockout of the OsABCG36 gene caused an enhanced Cd accumulation in the cell sap of roots and decreased Cd tolerance. Our previous study revealed a ZmHIPP gene, which encodes a heavy metal-associated isoprenylated plant protein (Ma et al. 2022). ZmHIPP is required for maize seedling tolerance to Pb stress by facilitating Pb deposition in the cell wall and alleviating Pb toxicity (Ma et al. 2022). The natural resistance-associated macrophage protein (NRAMP) family plays crucial roles in heavy metal absorption and transmembrane transport in plants (Bozzi and Gaudet 2021). The previous study reported that the overexpression of pak choi BcNRAMP1 improved the uptake of Cd and manganese (Mn) in yeast and Arabidopsis (Yue et al. 2021). In rice, OsNRAMP5 overexpression significantly reduces Cd concentration in the kernels as a result of decreasing Cd translocation from the roots to abovegrounds (Chang et al. 2020). FeNRAMP5 encodes a heavy metal transporter, which is responsible for Mn and Cd translocation in buckwheat (Yokosho et al. 2021). Expressing FeNRAMP5 in Arabidopsis is able to complement the phenotypes of the mutant atnramp1 under the condition of Mn and Cd accumulation (Yokosho et al. 2021).

Weight gene co-expression network analysis (WGCNA) that is based on transcriptome data sets has developed to a useful tool for uncovering the gene co-expression module(s) involved in plant responses to environmental stresses (Wang et al. 2018; Ma et al. 2021, 2022; Li et al. 2023). By calculating the correlations between each module and target phenotypes, the traits-associated module(s) was identified (Ma et al. 2022). According to the eigengene-based connectivity (KME) values, the hub gene(s) in the associated module was further selected (Li et al. 2023). Liu et al. used comparative transcriptome-based WGCNA and identified 12 co-expression modules and 277 hub genes for maize response to drought stress(Liu et al. 2021). Using the WGCNA strategy, the regulatory networks for salt response were established in maize, revealing the involvement of ZmIAA1, ZmGRS43, ZmUBE4, and ZmABCB14 in maize tolerance to salt stress (Li et al. 2021; Ma et al. 2021).

In the present study, we performed a comparative transcriptome analysis for a Pb-sensitive line (SCL171) and a Pb-tolerant line (SCL280) during the Pb treatment process. A genome-wide WGCNA was then applied to detect the Pb tolerance-related module(s) by combining the phenotypes of the two lines at different treatment stages. By Gene Ontology (GO) enrichment analysis, two genes in this Pb tolerant-associated module were enriched in the metal ion transport pathway. Finally, the role of ZmNRAMP6 in mediating Pb tolerance was elucidated in yeast, Arabidopsis, and maize by using heterologous expression and mutation strategy. Our study is helpful to understand the metal transporters-mediated regulatory mechanism underlying the Pb tolerance in maize.

Plant materials and phenotypic statistics

A Pb-tolerant line (SCL280) and a sensitive line (SCL177) were subjected to evaluation of Pb-tolerance phenotypes and analysis of comparative transcriptomes. In detail, the seeds of the two maize lines were sterilized with 2% NaClO solutions and washed with distilled water for five times. These seeds were then soaked in saturated calcium sulfate solutions overnight and rinsed with distilled water for five times. These sterilized seeds were shaded and incubated in germination boxes covered with soaked filter paper at 26℃. After germination, these seeds were transplanted to the substrate soils for further culture. At the two-leaf stage, the seedlings with consistent growth were equally divided into two groups. One group was transplanted into a modified Hoagland nutrient solution supplemented with 1.0 mM Pb (NO₃)₂ (Pb treatment) (Ma et al. 2022), and the other was cultured in the Hoagland solution without Pb(NO₃)₂ (control check, CK). The pH of the two solutions were maintained to 4.5 ± 0.1 by titrating with 0.1 M NaOH or HCl solutions if required.

At 0, 24, 48, and 72 h after culture, the plant length (PL) was measured using a ruler. Approximately 0.2 g root and shoot samples were separately collected from each line at each stage of Pb treatment. These samples were completely digested with 10 mL concentrated nitric acid at 120℃. The shoot lead content (SC) and root lead content (RC) were then measured by using an inductively coupled plasma-mass spectrometry (ICP-MS; NexION 2000, PerkinElmer Inc.). The transport coefficient (TC) of Pb was calculated as the ratio of SC and RC. Three independent biological replications were carried out, with each replication containing five seedlings.

Rna Library Construction And Transcriptome Sequencing

The roots were sampled from the lines SCL280 and SCL177 with three biological replicates, respectively, at 0, 24, 48, and 72 h under CK and 1.0 mM Pb treatment. A total of 42 samples were used for transcriptome sequencing, in consideration of the identity of the samples between 0-h CK and 0-h Pb treatment. Using the plant total RNA purification kit (HiPure Plant RNA Mini Kit), total RNA was extracted from the above samples following the manufacturer’s instructions. Transcriptome sequencing was conducted on the HiSeq-PE150 platform (Novogene Co, Ltd.).

Detection And Qrt-pcr Verification Of Differentially Expressed Genes

Clean reads were obtained by removing the low-quality, polyN-containing, and adapter reads from the raw reads with fastp program (Chen et al. 2018). High-quality reads were then aligned to maize B73 genome (RefGen V4) by the HISAT2 software (Kim et al. 2015). The values of FPKM (fragments per kilobase of exon model per million mapped fragments) were used to represent the normalized expression levels for each gene. For per line, the differentially expressed genes (DEGs) were identified by comparing their expression abundances between the three pairs, namely 24 h-CK vs 24 h-Pb, 48 h-CK vs 48 h-Pb, and 72 h-CK vs 72-h Pb. The thresholds of DEG identification were set as the absolute log2fold change (FC) = 2 and false discovery rate (FDR) = 0.05 (Liu et al. 2016). By comparing the expression levels of these DEGs between the two contrasting lines at a given stage (24, 48, or 72 h), we obtained the DEGs differentially responsive to Pb treatment between lines (DEGDs).

Genome-wide Co-expression Network Analysis

According to the FPKM values of the above DEGDs at all the stages (0, 24, 48, and 72 h) under CK and Pb treatment conditions, a genome-wide co-expression network analysis was conducted using the WGCNA program in R software to detect the co-expression modules and estimate the gene connections in each module (Langfelder and Horvath 2008). The parameters for co-expression analysis were set as follows: median data of expression > 0; soft threshold = 0.75; max block size = 150; deep split = 2; min module size = 5; and merge cut height = 0.2. The correlations between each characteristic module and the phenotypes of PL, SC, RC, TC, RDW, and SDW were used to evaluate the module-trait association, with the significance threshold set to P = 0.05. The GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis tool (https://www.omicshare.com/) was employed to identify the GO and KEGG terms for all genes in the most significantly associated modules. The Cytoscape software was applied to visualize the co-expression network of the target module (Shannon et al. 2003). Candidate genes for Pb tolerance were identified according to the GO and KEGG terms in the target module.

Subcellular Localization

After removing a termination codon, the full-length coding sequence (CDS) of the candidate gene (ZmNRAMP6) was amplified from the favorable-haplotype line B73 and inserted into the upstream of eGFP under the control of the 35S promoter to generate the recombinant construct p35S:ZmNRAMP6-eGFP. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 and injected into the leaves of Nicotiana benthamiana. The GFP fluorescence signals of the transformed tobacco leaves were detected under a laser confocal microscope 48 hours after Agrobacterium injection.

Expression analysis of ZmNRAMP6 in various maize tissues

A total of twelve tissues were collected from the maize line B73 and subjected to analysis of the relative expressions of ZmNRAMP6, including leaves (six developmental stages), roots (silking stage), stems (silking stage), tassels, female flowers, and kernels (6 days after pollination (DAP) and 12 DAP). Approximately 2 µg of total RNA was used to synthesize the first-strand cDNA with the reverse-transcription kit (Novoprotein, Nanjing, China) based on the manufacturer’s instruction. The expression level of ZmNRAMP6 was quantified by conducting RT-qPCR using SYBR qPCR SuperMix Plus (Novoprotein, Nanjing, China). The ZmActin gene was used as the internal reference to normalize the expression level of ZmNRAMP6 and the 2^-ΔΔCT method was applied to calculate the relative expression levels of ZmNRAMP6 (Hou et al. 2022a). Three biological repetitions were performed for each sample with three technical replicates included in a single biological replication.

Heterologous expression of ZmNRAMP6 in yeast

The full-length CDS of ZmNRAMP6 was inserted into the polyclonal site of the pYES2 vector to construct the recombinant plasmid pYES2-ZmNRAMP6 under the control of the GAL1 promoter. The pYES2-ZmNRAMP6 vector was then introduced into a Pb-sensitive mutant strain (under the background of BY4741) of yeast (Δycf1) to generate the recombinant yeast Δycf1-ZmNRAMP6. The recombinant strain was respectively cultured in YPD (SD-U) mediums containing 0 and 30 µM Pb(NO₃)₂, with different dilution ratios (10^− 1, 10^− 2, 10^− 3, and 10^− 4). The BY4741 strain and Δycf1 mutant were used as the positive and negative controls, respectively. These yeast strains were incubated upside down in an incubator at 30°C for 3–5 days, and the growth conditions of these colonies were observed and photographed (Xia et al. 2010). At 72 h after incubation in 30 µM Pb(NO₃)₂ solutions, the yeast cells were collected for measuring Pb concentration by using a inductively coupled plasma-mass spectrometry (ICP-MS; NexION 2000, PerkinElmer Inc.).

Arabidopsis Transformation And Phenotype Investigation

The full-length CDS of ZmNRAMP6 was amplified from the favorable-haplotype line B73. Using the homologous recombination method, the ZmNRAMP6 CDS was inserted into the pAN101 vector under the control of 35S promoter, generating the ZmNRAMP6-overexpression vector pAN101-ZmNRAMP6. The overexpression plasmid was then transformed into the inflorescences of Arabidopsis thaliana (Col-0) using Agrobacterium injection to produce ZmNRAMP6-overexpressed Arabidopsis lines.

The seeds from the ZmNRAMP6-overexpressed (OE) Arabidopsis and wild-type (WT) Col-0 were individually disinfected with 70% ethanol, washed with distilled water, sterilized in a solution supplemented with 1% sodium hypochlorite, and finally washed with distilled water for five times. The sterilized seeds were then planted on a half-strength MS medium for vernalization at 4°C for three days. After vernalization, these Arabidopsis seeds were vertically cultured at 22°C (16/8 hours, light / dark cycle) under standard conditions. After five days, the seedlings were divided into two groups with one group transferred to a half-strength MS medium without Pb(NO₃)₂ (control check, CK) and the other transferred to that supplemented with 200 mg/L Pb(NO₃)₂ (Pb treatment). After seven days of vertical growth, the seedlings were photographed and measured for root length (RL), rosette diameter (ROD), fresh weight (FW), and rate of wilt leaves (RWL). Furthermore, the sterilized seeds of the OE and WT lines were directly incubated on a half-strength MS medium supplemented with 200 mg/L Pb(NO₃)₂ for three days and subjected to investigation of germination rate.

Evaluation of Pb tolerance in maize mutant zmnramp6

In the present study, the maize mutant zmnramp6 was used to validate the role of ZmNRAMP6 in Pb accumulation and tolerance in maize. zmnramp6 was obtained from the EMS mutant population (http://elabcaas.cn/memd/public/index.html#/). The genetic background of the mutant was B73, and a stop-gain mutation was located in the 13th exon of ZmNRAMP6 and thus led to the ZmNRAMP6-knockout in the mutant. After two generations of self-pollination, the homozygous zmnramp6 was obtained via zmnramp6-specific PCR amplification. At the two-leaf stage, the seedlings of the mutant zmnramp6 and B73 were used to evaluate Pb tolerance and accumulation under 1.0 mM Pb(NO₃)₂ treatment, as described in the “Plant materials and phenotypic statistics” section. At 72 h of Pb treatment, the PL and weight was measured using a ruler and an electronic, respectively. Meanwhile, the total root length (TRL), total root area (TRA), total secondary root length (TSRL), root perimeter (PER), and primary root diameter (DIA) were scanned using the EPSON Expression 10000XL Root Scanner and evaluated by the ARIA software (Pace et al. 2014). A total of three biological replications were carried out.

Determination Of Antioxidant Enzyme Contents

The roots of the mutant zmnramp6 and wild-type B73 were collected 48 h after 1.0 mM Pb treatment. The activities of SOD and POD were determined using the SOD detection Kit (Solarbio BC0175) and POD detection Kit (Solarbio BC0095), respectively. Three biological repetitions were conducted with a single replication containing three technical replications.

Yeast One-hybrid

The promoter sequence of ZmNRAMP6 was inserted into the polyclonal site of the pBait-AbAi vector. The recombinant construct pAbAi-proZmNRAMP6 was then linearized using the restriction endonuclease BstBI and introduced into the Y1H Gold yeast strain to generate bait clones. The lowest concentration of AbA that inhibited yeast growth was screened on a synthetic dextrose minimal medium without uracil (SD/-Ura medium). Ultimately, the pAbAi-proZmNRAMP6 and pGADT7-ZmbZIP54 vectors were simultaneously transformed into the Y1H Gold strain. The transformed strain was cultured on a SD-Leu/AbA medium for 72 h at 30℃.

Dual-luciferase Reporter Analysis

The ZmbZIP54 CDS was inserted into the pCAMBIA1390 (p1390) vector driven by the 35S promoter. The promoter region (2000 bp upstream) of ZmNRAMP6 was amplified by PCR from the line B73 and cloned into the pGreen II-0800-LUC (p800) plasmid. The two recombinant vectors were co-introduced into tobacco leaves via Agrobacterium-mediated transformation. At 24 h after darkness culture, the transformed tobacco leaves were determined for the REN and LUC activities by using the Dual-Luciferase Assay Reagent (Promega).

Difference in growth phenotypes and Pb contents under Pb treatment between contrasting lines

To evaluate Pb tolerance of two maize lines with contrasting genetic backgrounds, the seedlings of these lines were investigated for the PL, SC, RC, and TC under 1.0 mM Pb(NO₃)₂ treatment and normal conditions, respectively. At all stages of Pb treatment, the line SCL280 indicated a significantly (P < 0.001) larger PL relative to the line SCL177, with increase percentage being 41.38%, 34.26%, 15.58%, and 12.19% at 0, 24, 48, and 72 h of Pb treatment (Fig. 1a). The growth of SCL177 was generally inhibited under Pb treatment, especially at 48 h, compared with CK. However, the growth of SCL280 was promoted at 24 h of Pb treatment and then gradually inhibited with the following stages (Fig. 1a). The promoted growth at the early stage of Pb treatment implicated the rapid adaption of SCL280 to Pb stress.

Moreover, the RC (24 h: 722.5 mg/kg DW; 72 h: 1797.5 mg/kg DW) and SC (24 h: 4.67 mg/kg DW; 72 h: 16.75 mg/kg DW) values were respectively significantly higher and lower in SCL280 than the RC (24 h: 483.50 mg/kg DW; 72 h: 1526.67 mg/kg DW) and SC (24 h: 7.75 mg/kg DW; 72 h: 30.17 mg/kg DW) in SCL177 at both 24 h and 72 h of Pb treatment (Fig. 1b and c). Compared with that in 24-h SCL177, an obviously lower SC in 24-h SCL280 probably explained for the promoted growth of SCL280 at 24 h of Pb treatment (Fig. 1d). Furthermore, the larger RC values in SCL280 indicated that SCL280 had a stronger ability to accumulate Pb in the roots, relative to SCL177 (Fig. 1c). Meanwhile, the TCs (24 h:0.0059; 72 h: 0.0093) were significantly smaller in SCL280 than those (24 h: 0.016; 72 h: 0.020) in SCL177 at the two stages of Pb stress (Fig. 1d), suggesting that SCL177 had a generally stronger capability to transport Pb from the roots to shoots. Nevertheless, no significant differences in the three values were observed between the two maize lines at 48 h (Fig. 1b–d). Collectively, these evidences supported that SCL280 was a Pb-tolerant maize line, which accumulated more Pb in the roots but transported fewer Pb to the shoots.

Comparative transcriptomes revealing differential responses to Pb stress between two lines

To detect the differentially expressed genes (DEGs) in SCL177 and SCL280 under Pb stress, we individually conducted transcriptome sequencing for the root samples collected from the two lines at different stages of Pb treatment. After filtering the raw reads, 6.97 clean reads in average were obtained for each sample, with a range of 6.03 to 8.73 among the 42 samples (Table S1). Based on the FPKM value of each expressed gene, we performed a correlation analysis of all the samples, indicating that the three replicates for a given treatment stage were highly correlated (correlation coefficient > 0.9) and clustered together (Fig. 2a). This supported the high reliability of the RNA-seq data in the present study. According to the threshold values of |log₂(FC)| = 2 and FDR = 0.05, a total of 1021 (752 up- and 269 down-regulated), 1097 (284 up- and 813 down-regulated), and 1130 (153 up- and 977 down-regulated) DEGs were respectively identified in 24 h-, 48 h-, and 72 h- samples of SCL177 under Pb treatment compared with their corresponding CK samples (Fig. 2b and c). In SCL280, 2323 (986 up- and 1337 down-regulated), 622 (276 up- and 346 down-regulated), and 745 (225 up- and 520 down-regulated) DEGs were detected at 24 h, 48 h, and 72 h of Pb treatment, relative to CK conditions (Fig. 2b and c). By comparing the DEGs between the two lines at a given stage of Pb treatment, we respectively obtained 487, 823, and 831 specific DEGs (SDEGs) at 24 h, 48 h, and 72 h in SCL177 and 1789, 348, and 446 DEGs in SCL280 (Fig. 2b). These SDEGs were considered the genes differentially responsive to Pb treatment between the Pb tolerance-contrasting lines, which involved 3643 unique genes (Fig. 2b). Gene ontology enrichment analysis revealed that these SDEGs participated in the biological processes, including cell wall organization or biogenesis, suberin biosynthetic process, water transport, response to endogenous stimulus, response to oxygen-containing compound, and aluminum cation transport (Fig. 2d).

Traits-associated co-expression network construction underscores importance of ZmNRAMP6 in Pb tolerance

To select these SDEGs related to Pb-tolerance phenotypes, we divided the SDEGs into different co-expression modules according to their expression patterns. Subsequently, we calculated the correlation of each module with the four phenotypes (PL, SC, RC, and TC) of the two lines investigated in the present study and the two ones (RDW and SDW) reported in our previous study (Ma et al. 2022), respectively. Finally, these SDEGs were grouped into 67 co-expression modules, and each module included 5–918 genes (Fig. 3a, Figure S1, Table S2). However, a total of 10 genes (grey module) were not classified into any of the above modules (Table S3). A correlation analysis showed that only the royal blue and pink modules were significantly associated with all the six phenotypes above (Fig. 3a). The pink module was significantly (P < 0.05) positively correlated with all the six phenotypes, with the CV ranging from 0.31–0.49 (Fig. 3a). However, the royal blue module was significantly (P < 0.05; CV, -0.53 – -0.34) negatively related to these growth phenotypes (PL, RDW, and SDW), whereas it was significantly (P < 0.05; CV, 0.30–0.62) positively correlated with the Pb-content phenotypes (RC, SC, and TC) (Fig. 3a). The opposite correlations of the royal blue module with the growth and Pb-content traits were consistent with the observation that Pb treatment negatively affected the growth of seedlings in the two maize lines. Collectively, the royal blue module was ultimately designated as the Pb tolerance-associated module in the present study, which contained 42 SDEGs (Fig. 3b) and involved the biological processes of aluminum ion transmembrane transport, response to metal ion, response to cadmium ion, oxidative phosphorylation, response to inorganic substance, and etc (Fig. 3c).

Notably, Zm00001d015133 that encodes Natural Resistance-Associated Macrophage Protein 6 (ZmNRAMP6) was located in the royal blue module (Fig. 3b) and involved in aluminum cation transport according to the GO results. Previous studies indicated that NRAMP6 participated in the translocation of manganese (Mn) and cadmium (Cd) and contributed to heavy metal toxicity in Arabidopsis, rice, and other plant species (Peris-Peris et al. 2017; Meng et al. 2017; Lu et al. 2020; Li et al. 2022). Therefore, ZmNRAMP6 was considered the hub gene among the Pb tolerance-associated module in the present study. Under the threshold of TOM (topological overlap measure) = 0.15, four genes showed significant co-expression relationships with ZmNRAMP6, including Zm00001d048135, Zm00001d047000, Zm00001d034611, and Zm00001d018117 (Fig. 3b, Table S4). Interestingly, ZmNRAMP6 with the four co-expression genes showed higher expression levels under Pb treatment than those under CK conditions (Fig. 3d), illustrating that Pb treatment activated their expressions. Remarkably, under Pb treatment, all the five genes had lower expression abundances in the tolerant line SCL280 than those in the sensitive line SCL177 (Fig. 3d), suggesting that these co-expressed genes negatively mediated Pb tolerance in maize seedlings.

Spatial expression pattern and subcellar localization of ZmNRAMP6 implicating its role in transporting Pb in maize roots

To learn the spatial expression pattern of ZmNRAMP6, we investigated 12 maize tissues for ZmNRAMP6 expression abundances using RT-qPCR. In results, ZmNRAMP6 showed a lower expression level in the leaves (all the stages), stems (silking stage), male flowers, and kernels (6 and 12 days after pollination) (Fig. 4a). In the female flowers, the expression of ZmNRAMP6 was higher, relative to the above tissues (Fig. 4a). Notably, ZmNRAMP6 showed the highest expression in the roots (silking stage), with the expression level being 5.12–318.53 folds higher than those in the other tissues (Fig. 4a). To further detect the subcellar localization of the ZmNRAMP6 protein, we fused the CDS regions of ZmNRAMP6 and eGFP and transformed the recombinant construct into tobacco leaves and maize protoplasts. Under a confocal microscope, the eGFP fluorescence was observed specifically in the plasma membrane of tobacco mesophyll cells and maize protoplasts transformed with the fusion expression construct (Fig. 4b and c). On the contrary, the fluorescence signal was observed in the nucleus, cytoplasm, and plasma membrane in maize and tobacco cells transformed with the negative control vector (p35S:eGFP) (Fig. 4b and c). The plasma membrane localization of ZmNRAMP6 supported its role in metal transport in maize roots.

Heterologous expression in yeast validating role of ZmNRAMP6 in Pb accumulation

To validate the Pb transport activity of ZmNRAMP6, we transformed ZmNRAMP6 to Saccharomyces cerevisiae to investigate the changes in the Pb concentration and growth phenotypes in yeast cells. Relative to the wild-type (WT) strain BY4741, the growth of the Pb-sensitive mutant strain (Δycf1) was obviously inhibited in the medium supplemented with 30 µM Pb (NO₃)₂ (Fig. 5a). However, the heterologously expressed ZmNRAMP6 did not complement the Pb-sensitivity phenotype of Δycf1 (Fig. 5a). Instead, the recombinant Δycf1-ZmNRAMP6 strain showed a more seriously inhibited growth in the Pb-containing medium, compared with Δycf1 (Fig. 5a). Consistently, the yeast growth curve showed that the Δycf1-ZmNRAMP6 strain had a lower growth rate than that of the Δycf1 strain under Pb treatment (Fig. 5c). In contrast, no obvious differences in these growth phenotypes were observed under normal conditions (CK) among the WT, Δycf1, and recombinant Δycf1-ZmNRAMP6 strains (Fig. 5a and b). These suggested that the expression of ZmNRAMP6 decreased Pb tolerance of the mutant. Furthermore, Pb concentration in the Δycf1-ZmNRAMP6 strain was enhanced by 20.44%, compared with that in the Δycf1 mutant under Pb treatment (Fig. 5d). Combined these results demonstrated that ZmNRAMP6 positively contributed to Pb accumulation in yeast cells and thus increased the sensitivity of yeast to Pb stress.

Overexpression of ZmNRAMP6 increasing Arabidopsis sensitivity to Pb

To better understand the role of ZmNRAMP6 in plant tolerance to Pb stress, we constructed two ZmNRAMP6-overexpression (OE) lines in Arabidopsis. qRT-PCR indicated that ZmNRAMP6 was efficiently expressed in the two transgenic lines (Fig. 6a). The seeds of the wild-type (WT) Arabidopsis and ZmNRAMP6-overexpressed lines were separately inoculated on half-strength MS medium containing 200 mg/L Pb(NO₃)₂ (Pb treatment), the medium without Pb(NO₃)₂ acted as the CK conditions. At 72 hours after culture, the seed germination phenotypes were investigated for these Arabidopsis lines. Generally, 200 mg/L Pb(NO₃)₂ seriously inhibited seed germination of the WT and OE lines (Fig. 6b). Under CK, the WT and OE lines germinated normally with the germination rate being approximate 100% (Fig. 6b). Under Pb treatment, the germination rates (44.83–74.30%) of these lines were significantly (P < 0.001) lower than those under CK (Fig. 6b). Remarkably, the germination rates of the OE lines were significantly (P < 0.01) lower than that of the WT under Pb treatment, with the decreasing percentages being 15.52% (OE1) and 39.67% (OE2), respectively (Fig. 6b). On the 7th day after culture, we investigated the root lengths (RLs), indicating that the RLs of all the lines were > 5.0 cm and < 0.75 cm under CK and Pb treatment conditions, respectively (Fig. 6c). Furthermore, the RLs of the OE lines were obviously lower than that of the WT under Pb treatment (Fig. 6c). These evidences supported that the overexpressed ZmNRAMP6 negatively affected the germination-related phenotypes of Arabidopsis seeds.

To investigate the effects of ZmNRAMP6 on the growth of Arabidopsis seedlings, we transferred the seedlings that grew under normal conditions for five days to the Pb-containing medium. On the 7th day after Pb treatment, we determined the RL, fresh weight of seedings (FW), rosette diameter (ROD), and rate of wilted leaves (RWL) for the WT and OE lines. Under CK, the three phenotypes (RL, FW, and ROD) were significantly (P < 0.05) improved in the two OE lines relative to those in the WT, except for the FW of OE1 (Fig. 6d–g). However, under Pb treatment, the RL, FW, and ROD were significantly (P < 0.001) lower in the OE lines than those in the WT (Fig. 6d–g). Moreover, no RWL was found in all the lines under CK, whereas the RWL (OE1, 40.64%; OE2, 31.55%) in the OE lines were significantly (P < 0.001) higher than that (4.83%) in the WT under Pb treatment (Fig. 6d and h). Taken together, overexpressed ZmNRAMP6 increased Arabidopsis sensitivity to Pb stress during the periods of seed germination and seedling growth.

Knockout of ZmNRAMP6 enhancing maize tolerance to Pb stress

To illustrate whether knockout of ZmNRAMP6 contributed to the increased Pb tolerance in maize, we investigated a maize EMS mutant (zmnramp6) that was under the background of B73 and contained a stop-gain variant in the 13th exon of ZmNRAMP6 (Fig. 7a). At the three-leaf stage, the seedlings of zmnramp6 and B73 were treated with 1.0 mM Pb(NO₃)₂ for three days. We investigated the PL, biomass phenotypes, and root system architecture (RSA) traits in B73 and the mutant. Finally, no significant differences in PL, shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW) were observed between zmnramp6 and B73 under CK conditions (Fig. 7b–f). Nevertheless, under the Pb treatment, all the above phenotypes were significantly improved in the mutant relative to B73 (Fig. 7b–f). Furthermore, no significant differences in the RSA traits were found between B73 and zmnramp6 under CK except for TRL (Fig. 7g–l). However, compared with B73, zmnramp6 showed larger TRL, TRA, TSRL, PER, and DIA under the Pb treatment (Fig. 7g–l). These indicated that knockout of ZmNRAMP6 increased maize seedlings to Pb stress and improved the growth of maize shoots and roots under Pb treatment.

Knockout of ZmNRAMP6 changing Pb accumulation and activating antioxidant enzyme system in maize roots

To reveal the mechanism underlying increased Pb tolerance in the ZmNRAMP6-knockout line, we measured the Pb concentrations in the shoots and roots of the WT and mutant after 72 h of 1.0 mM Pb(NO₃)₂ treatment. Finally, Pb concentration in the shoots of zmnramp6 was significantly (P < 0.001) lower than that of the WT (B73), with the decrease percentage being 21.59% (Fig. 8a). This explained for the increased growth rate in the mutant compared with that in B73 under Pb stress. Surprisingly, the Pb concentration (4928.18 mg/kg DW) in the roots of the mutant was significantly (P < 0.001) higher than that (2219.97 mg/kg DW) in B73, with the increase percentage being 121.99% (Fig. 8a). Meanwhile, we calculated the TC of the two lines, indicating that the TC was 2.97 folds higher (P < 0.001) in the WT than that in zmnramp6 (Fig. 8b). This suggested that knockout of ZmNRAMP6 reduced Pb transport from the roots to shoots.

To explore the reasons that the increased Pb concentration was associated with improved root development, we compared the activities of the antioxidant enzymes in the roots between the mutant and B73 under Pb stress. In results, no significant difference in the SOD and POD activities was found between the two lines under CK conditions (Fig. 8c and d). However, the two antioxidant enzymes showed significantly (P < 0.05) higher activities in the mutant plants than those in B73 under the Pb treatment (Fig. 8c and d). These observations were consistently with the involvement of Pb tolerance-related DEGs in responses to oxidative stresses (Fig. 2d). These evidences suggested that knockout of ZmNRAMP6 activated antioxidant enzyme system in the roots and thus promoted root growth.

ZmNRAMP6 being regulated by the Pb tolerance-related transcript factor ZmbZIP54

To uncover the upstream factor regulating the ZmNRAMP6 expression, we predicted the transcript factor-binding motifs in the promoter sequence (2000 bp upstream) of ZmNRAMP6 using the on-line tool Plantpan (http://plantpan.itps.ncku.edu.tw). Intriguingly, one ZmbZIP54-binding motif was located in the promoter of ZmNRAMP6 (Fig. 9a). Our previous study demonstrated that ZmbZIP54 was a key transcript factor in responses to Pb stress, which regulated Pb tolerance and accumulation in maize seedlings (Hou et al. 2022b). Subsequently, we used Y1H to test whether ZmbZIP54 bound to the ZmNRAMP6 promoter. The promoter sequence was cloned into the pBait-AbAi vector, generating the recombinant vectors (pAbAi-ProZmNRAMP6). Co-transformation of the pAbAi-proZmNRAMP6 and PGADT7-ZmbZIP54 vectors in the Y1H Gold strain resulted in a normal growth of these transformants on the SD/-Leu medium containing 100 ng/mL AbA (Fig. 9b). However, the negative control led to a growth inhibition of the transformed strain on the medium (Fig. 9b). This verified that the ZmNRAMP6 promoter could be bound by the ZmbZIP54 protein. Dual-luciferase reporter assays were further employed to verify ZmbZIP54 regulating ZmNRAMP6 expression (Fig. 9c). Ultimately, the relative activity of LUC (the ratio of activities between LUC and REN) was significantly (P < 0.05) lower in the tobacco leaves simultaneously transformed with the ZmbZIP54 CDS and ZmNRAMP6 promoter fragment than that in the negative control (Fig. 9c). These suggested that the ZmNRAMP6 expression was negatively regulated by ZmbZIP54 via the ZmbZIP54-binding motif in the ZmNRAMP6 promoter.

Herein, we identified a novel Pb tolerance-related gene ZmNRAMP6 in maize by using a genome-wide co-expression network analysis, which confers the Pb sensitivity on maize by mediating Pb accumulation in maize seedlings. Heterologous expression in yeast revealed the role of ZmNRAMP6 in Pb transport. Overexpression in Arabidopsis and knockout in maize indicated that ZmNRAMP6 positively mediated Pb sensitivity of plant seedlings and Pb accumulation in the shoots. Meanwhile, the decreased ZmNRAMP6 expression contributed to the elevated accumulation in the roots and increased root tolerance to Pb treatment. Knockout of ZmNRAMP6 was associated with the activation of antioxidant enzyme system in maize roots, thus conferring the improved Pb tolerance of the roots. Furthermore, the ZmNRAMP6 expression was negatively regulated the ZmbZIP54 transcription factor, a Pb tolerance-associated gene identified in our previous study.

ZmNRAMP6 belongs to the family of plant NRAMP6 metal transporters that has been previously reported to participate in the transport and toxicity of Cd, iron, and Mn in Arabidopsis, rice, and Sedum alfredii. Presumably, the role of NRAMP6 was relatively conservative across plant species and across various heavy metals. The present study indicated that knockout of ZmNRAMP6 caused significantly decreased and increased Pb contents in the shoots and roots of maize, respectively. Meanwhile, the ZmNRAMP6 transcripts were predominantly expressed in the roots relative to the other tissues, suggesting its crucial role in the roots. Collectively, ZmNRAMP6 probably acted as a transporter that translocated heavy metals from maize roots to shoots and environment. On the other hand, ZmNRAMP6 did not rescue the sensitivity of the yeast mutant Δycf1, instead, the heterologous expression of ZmNRAMP6 caused the decreased growth rate of Δycf1 by increasing Pb content in yeast cells. Therefore, the mechanism underlying Pb tolerance was consistent between yeast and maize. The previous study showed that the overexpression of Arabidopsis NRAMP6 in yeast increased the sensitivity to Cd without affecting Cd content in the cells (Li et al. 2022). Li et al., reported that Arabidopsis NRAMP6 was able to rescue the defective growth of the yeast mutant Δsmf2 that is deficient in Mn endomembrane transport. Combined these findings suggested that the transporting mechanism of NRAMP6 was distinct among various heavy metals.

Our previous study cloned a Pb accumulation and tolerance-associated gene, ZmHIPP, which confers maize tolerance to Pb by absorbing Pb from the environments and depositing extensive Pb ion to the cell wall. Based on our current knowledge of Pb transport, accumulation, and tolerance in maize, we proposed a model that summarizes the pathway of ZmNRAMP6 affecting Pb sensitivity and accumulation in maize seedlings (Fig. 10). In detail, under Pb treatment, some Pb transporters (including ZmHIPP and other unknown genes) transfer Pb ion from environments to the roots. When recognizing the Pb signal, the expression of ZmNRAMP6 was upregulated to transport Pb ion to the shoots and environments from the roots. Pb accumulation in the shoots caused the toxicity to maize seedlings and thus the growth inhibition (Fig. 10). On the contrary, in the ZmNRAMP6-knockout line, the mutated ZmNRAMP6 was unable to translocate Pb and caused accumulation in the roots when encountering Pb stress (Fig. 10). A number of Pb ion was fixed in the roots, activating the antioxidant enzyme system and leading to root tolerance to Pb stress (Fig. 10). Meanwhile, less Pb content in the shoots alleviated the toxicity to the shoots and caused the enhanced biomass of the mutant (Fig. 10). Therefore, knockout of ZmNRAMP6 in maize was a potential strategy for carrying out phytoremediation of Pb-contaminated soils. Maize has a larger biomass than most of other crops, which has a well-developed root system and potential of absorbing Pb (Li et al. 2023). Compared with the WT, ZmNRAMP6-knockout maize lines accumulate more Pb ion in the roots but translocate less to the shoots. This aids in the bioremediation of contaminated soil and food safety guarantee of forage and grain corn.

In the present study, we creatively used a genome-wide co-expression network analysis to directly identify the key gene involved in maize tolerance to Pb stress based on the comparative transcriptome data involving maize response to Pb treatment. Moreover, we constructed a regulatory network that contained 42 genes and was associated with Pb tolerance in maize. This network pointed out the other valuable genes (Zm00001d048135, Zm00001d047000, Zm00001d034611, and Zm00001d018117) that possibly participate maize response to Pb stress. The regulatory or interaction relationships between these genes in this co-expression network would be revealed to illustrate the molecular mechanisms underlying ZmNRAMP6-mediated Pb tolerance in maize. Map-based cloning is a classic method used for identifying causal genes for complex traits, however this strategy is a time and cost- consuming one. Compared with the map-based approach, transcriptome sequencing is time and cost-saving. However, to clone the key genes, co-expression network analysis based on transcriptome sequencing requires gene expression variation. Thereby, these variations only leading to gene structures could not be effectively excavated using the co-expression network construction.

Supplementary information

Figure S1. The 67 co-expression modules divided using weighted gene co-expression network analysis. The number (outside the bracket) in each colored box shows the correlation coefficient between the module and phenotype; the number inside the bracket represents the P value of correlation. SC, shoot Pb content; RC, root Pb content; TC, transport coefficient of Pb; PL, plant length; SDW, shoot dry weight; RDW, root dry weight.

Table S1. Statistics of the transcriptome sequencing data of the 42 samples collected from SCL177 and SCL280.

Table S2. The 67 co-expression modules constructed by using a weighted gene co-expression network analysis (WGCNA)

Table S3. Unclassified 10 genes in the grey module based on the weighted gene co-expression network analysis (WGCNA).

Table S4. Genes co-expressed with ZmNRAMP6 in the Pb tolerance-associated module.

Author Contribution Statement

Yaou Shen conceived the project. Yaou Shen, Peng Liu, and Langlang Ma supervised the study. Li Jiang, Peng Liu, Ping Long, Qinglin Li, Fengxia Hou, Zhong Chen, Minyan Zhang, and Chaoying Zou conducted the experiments. Peng Liu and Li Jiang analyzed the data. Yaou Shen, Peng Liu, and Li Jiang wrote the manuscript with contributions from Langlang Ma and Guangsheng Yuan. Guangtang Pan provided the maize materials. All authors read and approved the final manuscript.

Acknowledgements

This work is supported by the National Key Research and Development Program of China (2021YFF1000300), the National Natural Science Foundation of China (32072073 and 32101777), and the Sichuan Science and Technology Program (2021JDTD0004 and 2021YJ0476).

Data availability

All raw data generated of 42 samples used in this study were deposited in Genome Sequence Archive (GSA) in National Genomics Data Center (NGDC) database with the accession number CRA004789.

Funding

Competing Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Human and animal rights

This study does not include human or animal subjects.

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A genome-wide co-expression network analysis revealed ZmNRAMP6-mediated regulatory pathway involved in maize tolerance to lead stress

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Journal Publication

Version 1

Abstract

Figures

Key Message

Introduction

Materials And Methods

Plant materials and phenotypic statistics

Rna Library Construction And Transcriptome Sequencing

Detection And Qrt-pcr Verification Of Differentially Expressed Genes

Genome-wide Co-expression Network Analysis

Subcellular Localization

Arabidopsis Transformation And Phenotype Investigation

Determination Of Antioxidant Enzyme Contents

Yeast One-hybrid

Dual-luciferase Reporter Analysis

Results

Difference in growth phenotypes and Pb contents under Pb treatment between contrasting lines

Comparative transcriptomes revealing differential responses to Pb stress between two lines

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1