Information regarding the putative MePMEI1 protein of M. esculenta (Manes.06G136000), AtPMEI13 protein of Arabidopsis thaliana (AT5G62360), CbPMEI1 protein of Chorispora bungeana, and OsPMEI36 protein of Oryza sativa (LOC_Os10g36500) was obtained from Phytozome (https://phytozome-next.jgi.doe.gov/). The physicochemical properties of MePMEI1 were predicted using the ProtParam tool in ExPASy (https://web.expasy.org/protparam/). Multiple sequence alignment of the aforementioned proteins was performed using DNAMAN v6 software.

The crantz.cv.M.SC8 variety of M. esculenta was selected from the progeny of the CMR38-120 natural F1 hybrid asexual line introduced from Thailand by the Tropical Crops Genetic Resources Institute of the Chinese Academy of Tropical Agricultural Sciences. The crantz.cv.M.SC8 variety used herein has high yield and extensive adaptability, with strong wind resistance and seedling emergence properties. SC8 cassava seedlings were grown on Murashige Skoog (MS) gar medium from the germplasm preserved in our laboratory, for ~2 months. The tender stems were cut into small segments under sterile conditions, and each segment was ~1 cm long and had two axillary buds. The segments of the stem were then inserted into a fresh MS agar medium for growth, with the axillary buds facing upward. After growing for 45 days at 26°C under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S), the cassava seedlings were subjected to Pb stress. The roots of the cassava seedlings were first rinsed with tap water, without damaging the roots as far as possible. Then, a 1 mM solution of Pb(NO₃)₂ was prepared, and the cassava seedlings were placed in the solution for 0, 2, 6, 12, or 24 h at room temperature. The roots, stems, and leaves of the cassava seedlings were sampled at each of these intervals and immediately frozen in liquid nitrogen and finally stored in an ultra-low temperature refrigerator at −80°C.

The total RNA was extracted from the samples using a Plant Total RNA Isolation Kit (Foregene Biotech, Chengdu, China). The quality of the extracted RNA was evaluated by determining the concentration of the RNA and agarose gel electrophoresis. The residual DNA was removed from the extracted RNA using dsDNase, following which the cDNA was obtained using the MonScript™ RTIII Super Mix with dsDNase (Two-Step) (Monad Biotech, Wuhan, China), according to the manufacturer's instructions.

The transcription level of MePMEI1 in the roots, stems, and leaves of cassava plants under Pb stress was analyzed using a Real-Time PCR EasyTM-SYBR Green I kit (Foregene Biotech, Chengdu, China). Tubulin was considered as the reference gene, and the cDNA was used as the template for RT-qPCR. The specific primers used for RT-qPCR are enlisted in Supplementary Table 1. The RT-qPCR mixture is composed of 10 of 5 μL Real PCR Easy TM Mix-SYBR (2×), 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 4 μL of cDNA template, 0.2 μL of ROX Reference Dye (50×), and 0.4 μL of DNase-free ddH₂O. The conditions of PCR were as follows: initial denaturation at 95°C for 60 s followed by 45 cycles of denaturation at 95°C for 5 s and extension at 60°C for 30 s. The dissolution curve was finally added. Three replicates were performed for each sample. The relative expression of the target gene was calculated using the 2^−ΔΔCt method.

The full-length coding sequence (CDS) of MePMEI1 without the stop codon was obtained by PCR using the Prime STAR HS (Premix), R040 (Takara Biotech, Beijing, China). The amplified products were purified and inserted into the pCAMBIA1300-35s-GFP plant binary expression vector following digestion with the SalI and BamHI restriction enzymes for constructing the pCAMBIA1300-35s-MePMEI1-GFP recombinant vector. The primers used for amplification are enlisted in Supplementary Table 1. The constructed recombinant vector was confirmed by Sanger sequencing.

The full-length CDS of the MePMEI1 gene without a stop codon and signal peptide (1-28 aa) was amplified using the PrimeSTAR enzyme (Takara Biotech, Beijing, China). The amplified products were then purified and inserted into the pET-30a (+) prokaryotic expression vector following digestion with the NdeI and XhoI restriction enzymes, for constructing the PET-30a (+)-MePMEI1 recombinant vector. The primers used for amplification are enlisted in Supplementary Table 1. The constructed recombinant vector was confirmed by Sanger sequencing.

The pCAMBIA1300-35s-MePMEI1-GFP and pCAMBIA1300-35s-GFP recombinant vectors were separately transformed into Nicotiana benthamiana using the leaf disc method, mediated via Agrobacterium tumefaciens. The seeds of wild-type (WT) tobacco were sterilized with 75% ethanol for 20 min and placed on MS agar medium under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) at 22°C for ~1 month for transformation. A. tumefaciens transformed with the pCAMBIA1300-35s-MePMEI1-GFP and pCAMBIA1300-35s-GFP recombinant vectors were cultured in the yeast extract peptone (YEP) liquid medium at 28°C with shaking at 220 rpm to an OD₆₀₀ of 0.8. The cultures were then centrifuged at 4,000 rpm for 10 min, following which the supernatant was discarded and the bacterial cells were resuspended in a 50 mL YEP liquid medium containing 250 μM acetosyringone. The young tobacco leaves were cut into small pieces and soaked in the solution of resuspended bacteria for 10 min. The pieces of leaves were dried on sterile filter paper and cultured in an MS agar medium containing 8 mg/L hygromycin. The MS agar medium was replaced every 2 weeks, and the concentration of hygromycin was gradually increased to 20 mg/L until the positive T0 transgenic plants were obtained. All the experiments were performed under sterile conditions. Transgenic tobacco plants were further identified by leaf PCR using 2 × M5 HiPer Superluminal mix-with blue dye (Mei5bio, Beijing, China), using specific detection primers (Supplementary Table 1). The T0-positive transgenic plants were transferred to vermiculite and cultured under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) at 22°C for obtaining the T0 plants seeds. The seeds of T0-positive transgenic tobacco were continually screened on an MS agar medium containing 20 mg/L hygromycin. The seeds were transferred to vermiculite for obtaining the seeds of T1 plants, and the seeds of transgenic T1 tobacco were germinated to obtain the T2 homozygous lines. The tips of the roots of T2 transgenic tobacco seedlings were used for determining and visualizing the localization of MePMEI1 by laser confocal microscopy.

Expression of recombinant protein: The PET-30a (+)-MePMEI1 recombinant vector was transformed into the BL21 Star (DE3) strain of Escherichia coli. The recombinant bacterial cells were cultured in Luria–Bertani (LB) liquid medium at 37°C with shaking at 220 rpm for 4 h to an OD₆₀₀ of 0.6–0.8. For optimizing the induction conditions, the cultured cells were induced under different conditions, including 0.2 and 1 mM of isopropyl β-D-thiogalactopyranoside (IPTG), temperatures of 16 and 37°C, and shaking for 4 and 16 h. The control setup included bacterial cells that had not been induced. The bacterial cells were centrifuged at 12,000×g for 5 min, and the precipitate was resuspended with a buffer (20 mM Tris and 300 mM NaCl, pH 8.0), following which the recombinant MePMEI1 proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purification of MePMEI1 protein: The expression of the recombinant MePMEI1 protein was induced under optimal induction conditions. The bacterial cells were subsequently collected by centrifugation at 12,000×g for 5 min, following which the precipitate was resuspended with a buffer (20 mM Tris and 300 mM NaCl, pH 8.0). The cells were sonicated on ice by ultrasonic disruption (Φ 3, 15%, 2 s/8 s, 10 min). The lysis solution was centrifuged at 12,000×g for 15 min at 4°C, and the recombinant MePMEI1 proteins in the supernatant and precipitate were detected by SDS-PAGE. The recombinant MePMEI1 proteins obtained from the precipitate solution were purified using a 2 mL Ni-Sepharose column. SDS-PAGE was used for analyzing the purified recombinant MePMEI1 protein. Western blotting: The protein separated by SDS-PAGE was electro-transferred to a polyvinylidene fluoride (PVDF) membrane. After washing three times with PBST (phosphate buffered saline, tween-20), the membrane was blocked with 5% skim milk powder in PBST for 2 h at room temperature. The recombinant MePMEI1 protein was verified using anti-His mouse monoclonal antibodies followed by staining with goat anti-mouse IgG in 5% skim milk powder in PBST for 1 h. The membrane was subsequently visualized with a Western Bright ECL kit, and the signal was detected using a chemiluminescence imaging system. Following renaturation, the purified recombinant MePMEI1 protein was used for analyzing the enzyme activity.

The assay used for determining the activity of PMEI was performed according to the method described by Francis et al. (2006) and Sun (2017), with slight modifications. Briefly, 0.5 μg of MePMEI1 protein was mixed with 0.8 μL of commercial orange peel PME (Sigma p5400) and added to 800 μL of pectin solution [0.1% esterified pectin (Sigma P9561) in 50 mM phosphate buffered saline (PBS), pH 7.0]. The MePMEI1 protein was not added to the control setup. The samples were incubated overnight at 37°C, following which 200 μL of 0.05% ruthenium red solution (Sigma R2751) was added and mixed, and the mixture was incubated for 15 min at room temperature. The samples were added to 200 μL of 0.6 M CaCl₂ for precipitation, and the precipitate was collected by centrifugation at 12,000×g for 10 min. The absorbance of the supernatant was measured at A₅₃₄. The changes in the activity of MePMEI1 were further determined at different pH (4.5, 5.5, 6.5, 7.5, and 8.5) and different temperatures (16, 25, 37, and 95°C). Three replicates were performed for each sample.

The Col-0 ecotype of A. thaliana was used in this study, and the floral dip method was used for transforming the plants. After vernalization at 4°C for 3 days, the seeds of Col-0 Arabidopsis were uniformly sown on vermiculite for germination, and the seedlings were transplanted singly to new vermiculite and cultured at 22°C under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) till the time of flowering. The mature pods and fully opened flowers were cut off, and the Arabidopsis plants were used for transformation. A. tumefaciens containing the pCAMBIA1300-35s-MePMEI1-GFP recombinant vector was activated by streaking the plate, and the obtained single colony was cultured in YEP liquid medium at 28°C with shaking at 220 rpm until the OD₆₀₀ reached 0.8–1.0. The bacterial cells were collected by centrifugation at 4,000 rpm for 10 min and resuspended by adding 1/2 MS liquid medium containing 0.03% L77 to an OD₆₀₀ of 0.8. The flower buds of Arabidopsis were soaked in the resuspended bacterial liquid for 5 min, following which the plants were cultured in the dark for 24 h and subsequently grown at 22°C under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S). The infection was repeated every 2–3 days, for a total of 3–5 times, depending on the number of flowers. The infected Arabidopsis plants were cultured at 22°C under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) to obtain the T0 seeds.

The T0 seeds of Arabidopsis were sterilized three times with 75% ethanol, washed with sterile water, and finally washed with absolute ethanol. Following vernalization at 4°C for 3 days, the seeds were placed on filter papers for drying and were subsequently germinated on 1/2 MS agar medium containing 25 mg/L hygromycin for screening the positive transgenic plants (T1). All the steps were performed under sterile conditions. The transgenic T1 Arabidopsis plants were identified by PCR using the 2 × M5 HiPer Superluminal mix-with blue dye (Mei5bio, Beijing, China). The DNA obtained from the transgenic plants was used as the template, and the specific primers used for PCR detection are enlisted in Supplementary Table 1. Three transgenic Arabidopsis plants with the highest MePMEI1 expression were selected from the transgenic T1 plants by RT-qPCR for subsequent functional verification. The cDNA of the transgenic T1 Arabidopsis plants was used as the template. Actin was considered as the reference gene. The specific primers used for RT-qPCR are enlisted in Supplementary Table 1. The seeds of the three transgenic Arabidopsis plants were further screened on 1/2 MS agar medium containing 25 mg/L hygromycin until the homozygous T2 plants were obtained.

The seeds of the transgenic homozygous T2 plants and WT Arabidopsis were sterilized as previously described and germinated on 1/2 MS agar medium following vernalization at 4°C for 3 days in the dark. The plants were grown in the culture room at 22°C under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) for ~7 days. For each line, seedlings with similar growth status were transferred to 1/2 MS agar medium obtained 750 μM/1 mM Pb(NO₃)₂ or no-Pb(II) (control) and cultured for an additional 14 days. The three Arabidopsis plants from each line were horizontally positioned for observation and comparison.

The transgenic MePMEI1 and WT Arabidopsis seedlings were collected for determining the accumulation of H₂O₂ and O²⁻ by diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining following treatment with 750 μM Pb(NO₃)₂ for 14 days. NBT powder and a DAB Chromogenic Reagent were purchased from Beijing Solarbio Technology Co., Ltd. NBT and DAB were separately prepared to obtain solutions with concentrations of 0.05%. The samples of Arabidopsis seedlings were immersed in the solution for 20–30 min at room temperature under illumination. The staining solution was discarded, and the samples were washed three times with distilled water for terminating the reaction, following which 75% ethanol was added to the centrifuge tube, and the samples were boiled in water for 15 min until the chlorophyll had faded. Following decolorization, the samples were placed under a stereomicroscope for observing the color reaction.

The structures of the cell wall in the roots and leaves of the transgenic and WT Arabidopsis under Pb stress or control conditions were observed by TEM. The plants were developed on 1/2 MS agar medium containing 750 μM Pb(NO₃)₂ or no-Pb(II) for 14 days, following which the roots and leaves of the seedlings were collected and the ultrathin sections were prepared. The sections were prepared as follows: the samples were fixed with 2% glutaraldehyde and 1% osmic acid, dehydrated with 30–50–70–80–90–100% ethanol and acetone, transferred to virgin resin for embedding and sectioning, and the sections were finally stained with uranyl acetate and lead citrate. The ultrathin sections thus prepared were observed under a transmission electron microscope (Hitachi).

The seeds of transgenic MePMEI1 and WT Arabidopsis plants were uniformly sown on vermiculite for germination following vernalization at 4°C. When the plants had grown four true leaves, they were singly transplanted to vermiculite and grown under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) at 22°C for 25 days. All the plants were irrigated with 5 mM Pb(NO₃)₂ solution every 2 days for ~16 days, while the control plants were irrigated with the same volume of tap water at the same frequency. The changes in the phenotype are continuously monitored during treatment. The activities of catalase (CAT) and superoxide dismutase (SOD) and the contents of malondialdehyde (MDA) and H₂O₂ were measured using the appropriate kits from Suzhou Comin Biotechnology Co., Ltd., according to the instructions provided. Three replicates were performed for each sample.

Following vernalization at 4°C for 3 days, the seeds of the transgenic plants with MePMEI1 and the WT Arabidopsis plants were uniformly sown on vermiculite for germination, following which the seedlings were transplanted singly to new vermiculite and cultured under 16 h/8 h light/dark conditions (white LED, 120–150 uM/m²/S) at 22°C for 35 days. The activity of PME was determined using a Pectinesterase (PE) kit from Suzhou Comin Biotechnology Co., Ltd., according to the instructions provided. Three replicates were performed for each sample.

Unless otherwise stated, all the numerical values were presented as the mean ± standard error of mean (SEM) of at least three independent biological replicates. The data were analyzed by a one-way analysis of variance (ANOVA) using SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA). The different letters indicate significantly different means (p < 0.05, Duncan's multiple range tests). Microsoft Excel 2019 was used for data collation. The figures were prepared using GraphPad Prism software (GraphPad Prism 8.0.2, San Diego, CA, USA). The relative expression of the genes was calculated using the 2^−ΔΔCt method.

In our previous study, we identified the MePMEI1 gene with a coding region of 609 bp that encodes a protein of 202 amino acids. The estimated molecular weight and isoelectric point (pI) of the protein were determined to be 21.78 kDa and 5.51, respectively (Zhou et al., 2019).

The AtPMEI13 protein of A. thaliana and the CbPMEI1 protein of C. bungeana are more homologous to the MePMEI1 protein of M. esculenta than with the OsPMEI36 protein of O. sativa, which is possibly attributed to the higher divergence between dicotyledons and monocotyledons. Multiple sequence alignment revealed that the MePMEI1 protein has a typical PMEI domain consisting of four conserved cysteine residues that formed two disulfide bridges (Figure 1).

To investigate whether the MePMEI1 gene responds to Pb stress, the samples of the roots, stems, and leaves from the cassava plants treated with Pb(NO₃)₂ were collected for analyses with RT-qPCR. The transcription of MePMEI1 was highest in the roots following treatment with 1 mM Pb(NO₃)₂, was slightly upregulated at first and then downregulated, and was again upregulated and finally reached a peak after 24 h of treatment. The transcription of MePMEI1 was first downregulated in the stems and leaves, then upregulated, and reached a peak after 24 h of treatment (Figure 2). These results indicated that the MePMEI1 gene of cassava responds to Pb stress, especially in the roots.

To assess the subcellular localization of the MePMEI1 protein, the cells in the root tips of tobacco plants transformed with the pCAMBIA1300-35s-MePMEI1-GFP recombinant vector were observed under a laser confocal microscope (Figure 3A). Laser confocal microscopy revealed the absence of fluorescence signals in the WT tobacco plants. In the transgenic tobacco plants containing the pCAMBIA1300-35s-GFP vector, the fluorescence signal was distributed on the cell wall, the cell membrane, the intercellular spaces, and the nuclei. In the transgenic tobacco plants with MePMEI1, the fluorescence signal was observed in the cell wall, the cell membrane, and the intercellular layer. Following plasmolysis with 30% sucrose solution, the fluorescence signal was only observed on the cell wall, but not on the cell membrane, which exhibited a wrinkled morphology. The findings revealed that the MePMEI1 protein was therefore localized in the cell wall (Figure 3B).

The BL21 Star (DE3) E. coli cells containing the PET-30a (+)-MePMEI1 recombinant vector carrying His tags were induced with different concentrations of IPTG (0.2 mM and 1 mM) at different temperatures (15 and 37°C). A specific band for MePMEI1 with a molecular mass of 27 kDa was observed under all induction conditions (Figure 4A, lanes 1–4). It was observed that the expression of the recombinant MePMEI1 protein was higher at 37°C when induced with 1 mM IPTG (Figure 4A, lane 4). Solubility analysis revealed that the induced protein was expressed as an inclusion body (Figure 4A, lane 6). A large proportion of the insoluble protein was purified and examined by SDS-PAGE (Figure 4B). The results of western blotting using an anti-his antibody revealed a specific band at ~27 kDa, which was consistent with the expected results (Figure 4C). The results indicated that the recombinant MePMEI1 protein was successfully expressed in the prokaryotic expression system.

The effect of the recombinant MePMEI1 protein on PME activity was determined by analyzing the enzyme activity. The initial activity value of commercial orange peel PME was 1.47 U/mg at room temperature, which was reduced to 1.2 U/mg following the addition of 0.5 μg of recombinant MePMEI1 protein, indicating that MePMEI1 had an obvious inhibitory effect on commercial orange peel PME (Figure 4D). To explore the effects of pH on the activity of MePMEI1, the recombinant MePMEI1 protein and commercial orange peel PME were incubated with a pectin solution at different pH (4.5, 5.5, 6.5, 7.5, and 8.5). The relative activity of MePMEI1 was represented by subtracting the PME activity value at different pHs when incubated with the recombinant MePMEI1 protein, from the initial activity value of PME at the corresponding pH. We observed that the highest MePMEI1 activity of 31% was observed at pH 5.5 and then decreased with increasing pH. The results demonstrated that the activity of MePMEI1 is higher under acidic conditions, and the optimum pH was determined to be 5.5 (Figure 4E). We also determined the activity of MePMEI1 at different temperatures. The recombinant MePMEI1 protein and commercial orange peel PME were incubated with a pectin solution at 16, 25, 37, and 95°C. The experimental data revealed that the recombinant MePMEI1 protein showed the strongest PME inhibition at 37, 25, and 16°C, and the difference in the activity of recombinant MePMEI1 at 37°C was statistically significant. The activity of MePMEI1 increased with the duration of incubation; however, the protein was inactivated and had no inhibitory effect on PME at 95°C (Figure 4F).

To evaluate the role of MePMEI1 in Arabidopsis, we obtained five Arabidopsis plants with transgenic MePMEI1 by agrobacterium-mediated transformation using the floral dip method. Three transgenic MePMEI1 lines (OE#2, OE#4, and OE#5 from T3) with higher MePMEI1 transcription levels were selected by RT-qPCR for subsequent verification of function (Supplementary Figure 1).

Root elongation was markedly inhibited in all 7-days-old Arabidopsis seedlings following treatment with 750 μM or 1 mM Pb(NO₃)₂ for 14 days; however, the above-ground parts of Arabidopsis seedlings with transgenic MePMEI1 were larger than those of WT plants, and root growth was also significantly increased in the transgenic plants compared to that of the WT plants. In particular, the WT seedlings failed to grow and tended to die following treatment with 1 mM Pb(NO₃)₂, while the transgenic MePMEI1 plants exhibited increased growth characteristics (Figures 5A,B). One the vermiculite, the 25-days-old transgenic MePMEI1 plants and the WT plants were treated with tap water or 5 mM Pb(NO₃)₂ solution for 16 days. The results demonstrated that the above-ground parts of all the plants remained green and the plants grew well under normal conditions. The edges of the rosette leaves of WT plants turned yellow following treatment with 5 mM Pb(NO₃)₂; however, the rosette leaves of transgenic MePMEI1 plants remained green (Figure 5C). The results indicated that MePMEI1 promotes tolerance to Pb stress. We also evaluated the survival rate of seeds and found that treatment with Pb(NO₃)₂ clearly affected the rooting of Arabidopsis seedlings, but there was no significant difference in rooting between transgenic MePMEI1 plants and WT plants, under normal conditions and Pb stress (Supplementary Figure 2).

The structures of the cell wall in the root tips and leaves of transgenic and WT plants under normal conditions and under 750 μM Pb(NO₃)₂ treatment were observed by TEM. The images revealed that the cell wall in the root tips and the leaves of transgenic MePMEI1 plants were significantly thicker than those of WT plants under normal conditions (red letters indicate the cell wall) in Figure 5DI–IV. The positive effect of MePMEI1 on the thickening of cell walls was more obvious following treatment with 750 μM Pb(NO₃)₂ for 14 days. The cell walls appeared thicker in both transgenic and WT plants following treatment; however, the cell walls in the root tips and the leaves of seedlings with transgenic MePMEI1 were much thicker than those of the WT plants (Figure 5DV–VIII). The results demonstrated that the overexpression of MePMEI1 increased the thickness of the cell wall in Arabidopsis.

Several studies reported that plants usually produce higher quantities of ROS under heavy metal stress. To investigate whether MePMEI1 confers ROS scavenging potential, the accumulation of H₂O₂ and O²⁻ in the root tips and leaves of transgenic MePMEI1 and WT plants following treatment with 750 μM Pb(NO₃)₂ for 14 days was determined by DAB and NBT staining. A higher quantity of brown precipitate was detected in the root tips and leaves of WT plants following DAB staining than in those of the transgenic MePMEI1 lines (Figure 6A), indicating that the accumulation of H₂O₂ was lower in the transgenic plants than in the WT plants. The quantity of O²⁻ was additionally examined by NBT staining. The results demonstrated a higher quantity of blue precipitate in the root tips and leaves of WT Arabidopsis plants than in those of the transgenic MePMEI1 lines (Figure 6B), indicating that the expression of MePMEI1 reduced the accumulation of O²⁻ in Arabidopsis under Pb stress.

The content of MDA and H₂O₂ and the activity of the antioxidant enzymes, CAT and SOD, were further measured in the transgenic and the WT plants under control conditions and following treatment with 5 mM Pb(NO₃)₂ for 16 days. The results demonstrated that the activities of CAT and SOD were higher in all the transgenic MePMEI1 lines than in the WT plants, while the levels of MDA and H₂O₂ were higher in the WT plants than in the transgenic plants under Pb treatment (Figures 6C–F). These findings illustrated that MePMEI1 alleviated oxidative damages under Pb stress by enhancing the activities of CAT and SOD for reducing the excess accumulation of ROS.

To determine the effect of MePMEI1 on the activity of PME in Arabidopsis, we also examined the total PME activity in 35-days-old Arabidopsis plants. The results demonstrated that the PME activity was reduced in transgenic Arabidopsis compared to that of WT plants (Supplementary Figure 3). These findings were consistent with the earlier results and indicated that the MePMEI1 functions as a PMEI and inhibits the activity of plant PMEs.