Cloning and bioinformatics analysis of CsMAPEG
CsMAPEG was amplified by PCR using CsMAPEG-F and CsMAPEG-R as the primers and cucumber ‘D0351’ fruit cDNA as the template (Fig. 1a). The CsMAPEG sequence was then confirmed by PCR and repeated sequencing (Fig. 1b). Full-length CsMAPEG is 438 bp in length and encodes 145 amino acids (Fig. 1c). Our sequence was consistent with the coding region of the gene (Csa5G409710.1) obtained from the alignment of sequences in the cucumber genome database (http://www.icugi.org/).
The amino acid sequence alignment of CsMAPEG showed that the protein has one MAPEG domain (Additional file 3: Figure S1) and belongs to the MAPEG superfamily; thus, the protein was named CsMAPEG. The analysis of CsMAPEG revealed a predicted theoretical molecular weight of 16.486 kDa, a theoretical isoelectric point of 9.66, an atomic composition of C775H1193N189O186S11, a total number of atoms of 2354, and a fat coefficient of 104.83. The highest hydrophobicity scores of the protein encoded by this gene (Additional file 4: Figure S2) were found to equal 2.944 at the 16th amino acid position and 2.122 at the 67th amino acid position, and the average hydrophilic coefficient was estimated as − 0.538, which indicated that the protein is hydrophilic. A clear signal peptide cleavage site was not detected for this protein (Additional file 5: Figure S3), which suggested that CsMAPEG might be a cytoplasmic matrix or organelle matrix protein but not a membrane or secretory protein. The prediction of the transmembrane domain of the CsMAPEG protein showed three distinct transmembrane regions (Additional file 6: Figure S4). The secondary structure of the protein (Additional file 7: Figure S5) was analyzed, and the analysis revealed 83 alpha helices, which accounted for 57.24% of the total polypeptide chain, 20 extended main chains, which accounted for 13.79% of the whole polypeptide chain, and 36 random coils, which accounted for 24.83% of the whole polypeptide chain. The protein sequence was submitted to the protein homology modeling program Phyre to predict the three-level structure of the protein (Additional file 8: Figure S6).
The cucumber genome database was searched for Csa5G409710.1 to obtain the complete gene sequence, and a region 2000 bp upstream was selected as the promoter sequence. The analysis results are shown in Additional file 1: Table S1. In addition to the TATA and CAAT elements inherent in the eukaryotic promoter and some common light-responsive elements, such as the 3-AF1-binding site, GT1 motif and SP1-binding site, the promoter also contains an ARE anaerobic induction element, a P-box gibberellin (GA) response element, an ERE ethylene response element, a TC-rich repeat defense and stress response element, three heat shock reaction elements, one TCA-element salicylic acid (SA) response element, one AuxRR-core auxin response element and other cis-elements.
Phylogenetic tree of CsMAPEG
The amino acid sequences of MAPEG family proteins from 10 different specieswere downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/) for homology alignment. A phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA software (version 5.2). The results are shown in Additional file 9: Figure S7, and the scale bar indicates the average number of amino acid substitutions per site. Fifteen family members (Additional file 2: Table S2), including CsMAPEG, were grouped into two groups. The MAPEG protein shows strong conservation among different species, and the proteins from plants in the same family and genus show high similarity. CsMAPEG and the protein from melon Cucumis melo L. (XM008462283.2) form a small evolutionary branch and show the closest relationship; in addition, these proteins exhibit a distance of less than 0.1 from the proteins of balsam pear Momordica charantia L. (XM022285675.1) and pumpkin Cucurbita moschata (XM023137096.1), which belong to Cucurbitaceae. The change in nucleotides among these proteins is small, and their homology is relatively high.
Subcellular localization of the CsMAPEG protein
The 35S:CsMAPEG-GFP fusion expression vector and the pGII-EGFP empty vector were introduced into Arabidopsis protoplasts. The subcellular localization of the CsMAPEG fusion protein was observed using a confocal laser microscope, and the results are shown in Fig. 2. Bright green fluorescence was observed in whole cells transfected with the empty vectors, whereas enriched green fluorescence following transfection with the CsMAPEG-pGII-EGFP fusion expression vector was observed only in the cytoplasm. Therefore, the results clearly indicated that CsMAPEG is localized in the cytoplasm.
Expression pattern of CsMAPEG in response to PM treatment
The expression patterns of CsMAPEG in cucumber plants treated with PM showed differences between the low-PM-residue-abundance cultivar ‘D0351’ and the high-PM-residue-abundance cultivar ‘D9320’ (Fig. 3). Exposure to PM increased the expression levels of CsMAPEG in the roots, stems, leaves and fruits of ‘D0351’ over time compared with the expression levels found in the same tissues of ‘D9320’. At all the tested time points, significantly higher CsMAPEG expression was found in ‘D0351’ roots compared with the control group, and specifically, the expression level in the ‘D0351’ roots at 24 h was 2.69-fold higher than that the control level (Fig. 3a). However, CsMAPEG expression in ‘D9320’ was significantly downregulated at 48 h, and the expression levels at the other time points were not significantly different from those of the control group (Fig. 3b). The expression levels of CsMAPEG in the stems of ‘D0351’ were first upregulated and then downregulated. The relative expression of CsMAPEG in the ‘D0351’ stems at each time point was significantly higher than that in the control group and reached its maximal level, which was 3.24-fold higher than that of the control group, at 24 h (Fig. 3c). The levels in ‘D9320’ were decreased significantly compared with those of the control at all the tested time points with the exception of 1 and 6 h, and the overall expression level showed a smooth change and was significantly lower than the expression level in ‘D0351’ (Fig. 3d). The expression of CsMAPEG in the leaves of ‘D0351’ was upregulated at all tested time points compared with that of the control. The relative expression level reached its maximum at 48 h, and this maximal level was 2.61-fold higher than the control level (Fig. 3e). However, CsMAPEG expression in ‘D9320’ leaves was downregulated at all time points; in addition, the change in CsMAPEG expression in ‘D9320’ leaves was stable, and the levels were significantly lower than those in ‘D0351’ (Fig. 3f). The patterns of CsMAPEG expression in ‘D0351’ and ‘D9320’ fruits were basically consistent, characterized by an initial increase and a subsequent decrease, although the gene expression level in ‘D0351’ at all tested time points was significantly higher than that in ‘D9320’. The relative expression of CsMAPEG in ‘D0351’ fruits increased rapidly within 6 h and reached its maximum at 12 h, and this maximal expression level was 5.49-fold higher than that of the control group. After 12 h, the expression level decreased, although the values remained higher than those of the control at all time points (Fig. 3g). The CsMAPEG expression levels in ‘D9320’ fruits were upregulated only at 6 and 24 h, and these values were 1.81- and 1.34-fold higher than those of the control group, respectively. The relative expression of CsMAPEG at the other time points was lower than that of the control group (Fig. 3h). The CsMAPEG expression levels in different organs of the low-pesticide-residue-abundance cultivar ‘D0351’ were ordered as follows: fruit>leaf>stem>root. The expression of CsMAPEG in the stems and leaves of ‘D0351’ was significantly higher than that in the same parts of ‘D9320’. CsMAPEG was specifically expressed in the stems and leaves of ‘D0351’ compared with those of ‘D9320’.
Analysis of CsMAPEG expression in response to different external factors
The expression patterns of CsMAPEG after ‘D0351’ and ‘D9320’ seedlings at the three-true-leaf stage were treated with the hormones jasmonic acid (JA), SA and GA were analyzed. The results (Fig. 4a and b) showed that SA induction significantly upregulated the expression of CsMAPEG in the low-PM-residue-abundance cultivar ‘D0351’ to a level that was 8.76-fold higher than that of the control. The expression of CsMAPEG in the high-PM residue-abundance cultivar ‘D9320’ was not significantly different from that of the control. The JA-induced expression patterns of CsMAPEG in ‘D0351’ and ‘D9320’ were similar and showed very significant upregulation, although the relative expression of CsMAPEG in ‘D0351’ was higher than that in ‘D9320’. The expression of CsMAPEG in ‘D0351’was 4.47-fold higher than that of the control and 3.03-fold higher than that of the ‘D9320’ cultivar. After GA treatment, CsMAPEG showed different expression patterns in ‘D0351’ and ‘D9320’. Its expression was upregulated significantly in ‘D0351’ to a value that was 6.73-fold higher than that in the control, and this value was only slightly higher than that in ‘D9320’, which presented a relative expression level that was 1.36-fold higher than that in the control. In ‘D0351’, CsMAPEG expression was upregulated after SA, JA and GA induction. In contrast, in ‘D9320’, CsMAPEG expression was upregulated only after JA treatment and did not show significant changes after the SA and GA treatments. However, a significant difference in the CsMAPEG expression pattern was found between ‘D0351’ and ‘D9320’. To study the function of the MAPEG family in stress tolerance, we subjected seedlings of ‘D0351’ and ‘D9320’ at the three-true-leaf stage to drought, Cor and low temperature stress and analyzed the resulting expression patterns of CsMAPEG. As shown in Fig. 4, CsMAPEG expression in ‘D0351’ and ‘D9320’ under PEG stress was upregulated, and these upregulated levels were significantly different from that of the control. The relative expression of CsMAPEG in ‘D0351’ was significantly higher than that in ‘D9320’, and the levels in these strains were 2.63- and 1.83-fold higher than that in the control, respectively. The expression of CsMAPEG was significantly upregulated in ‘D0351’ and ‘D9320’ under Cor stress. The expression of CsMAPEG in these strains was 14.60- and 8.36-fold higher than that in the control, respectively. Under low temperature stress, the expression of CsMAPEG in ‘D0351’ and ‘D9320’ was nearly equivalent, and no significant difference compared with the control was detected.
Construction of the CsMAPEG expression vector and genetic transformation of cucumber
We generated the overexpression vectors CsMAPEG(+)-PCXSN and CsMAPEG(−)-PCXSN under the control of the strong constitutive CaMV35S promoter (Fig. 5a). The overexpression vectors CsMAPEG(+)-PCXSN and CsMAPEG(−)-PCXSN were successfully transferred into ‘D0351’ and ‘D9320’ using cucumber genetic transformation technology (Additional file 10: Figure S8). The total DNA of the resistant plants was used as a template, the pCXSN-CsMAPEG(+) plasmid was used as a positive control, and water was used as a negative control. The primers used for PCR detection were specific for the pCXSN-1250 vector. As shown in Fig. 5b and c, the target fragments were approximately 500 bp in the positive control and some resistant plants, although no specific bands were found in the negative control, which indicated that the pCXSN-CsMAPEG plasmid had been successfully integrated into the cucumber genome. To eliminate false positives in the resistant plants and ensure the integrity and accuracy of the experiment, we extracted RNA using the TRIzol method and identified the sequence by qRT-PCR. The normal expression of CsMAPEG in cucumber was affected by the transfection of exogenous genes, and the expression level was changed. The expression patterns were significantly different. The three CsMAPEG-overexpressing plants with the highest levels of CsMAPEG were selected for the detection of PM residues and the determination of physiological and biochemical indexes. In ‘D0351’, the expression of CsMAPEG was significantly upregulated after the transfer of CsMAPEG(+), and the CsMAPEG expression level in the T0 and T1 plants was 11.12- and 8.72-fold higher than that in the wild-type, respectively. After the transfer of CsMAPEG(−), the expression of CsMAPEG in the T0 and T1 plants was downregulated to levels that were 0.48- and 0.55-fold of the wild-type level (Fig. 5d). In ‘D9320’, the expression of CsMAPEG was upregulated after the transfer of CsMAPEG(+), and the expression levels in the T0 and T1 plants were approximately 6.98- and 5.41-fold higher than that of the wild-type. After the transfer of CsMAPEG(−), the expression levels of CsMAPEG in the T0 and T1 plants were approximately 0.48- and 0.79-fold of the wild-type level (Fig. 5e).
Analysis of PM residues in CsMAPEG-overexpressing plants
‘D9320’ plants with similar expression levels of CsMAPEG were identified by PCR and qRT-PCR, and the PM residues in fruits after treatment with PM were determined (Fig. 6). The levels of residues in CsMAPEG(+)-overexpressing cucumber fruits at six time points from 1 to 72 h were significantly lower in the ‘D9320’ T0-generation fruits compared with the wild-type control fruits, and the effect was extremely significant at 12 and 72 h, with levels that were 0.67- and 0.80-fold of those found in the wild-type fruits, respectively. The average from the six time points obtained from the ‘D9320’ T0-generation fruits was 0.81 mg/kg, which was 0.26 mg/kg lower than the average value found for the wild-type fruits. The results showed that the CsMAPEG(+) transfer could effectively reduce the PM residues in fruits. The residue abundance in the CsMAPEG(−) transgenic plants at four time points from 12 h to 72 h was significantly or extremely significantly higher than that in the wild-type plants. The highest residue level of 1.86 mg/kg was found at 24 h. The residue abundances in the CSMAPEG(−) plants at 24 and 72 h were extremely significantly higher than those of the wild-type, and these values were 1.76- and 1.48-fold higher than those in the wild-type, respectively. The average residue abundance in the CsMAPEG(−) transgenic cucumber fruits at six time points was 1.37 mg/kg, which was 0.29 mg/kg higher than that found for the wild-type plants.
The PM residue abundance in the CsMAPEG(+)-overexpressing, CsMAPEG(−)-overexpressing and wild-type plants at the T1 generation showed the same trend; specifically, the PM residue abundance first increased and then decreased over time. Compared with the T0 plants, these T1 plants showed decreased PM residue abundances at all tested time points. The PM residue abundances in the CsMAPEG(+)-overexpressing plants were lower than those in the wild-type plants at the six tested time points. Specifically, the residue levels in the CsMAPEG(+)-overexpressing plants were 0.69-, 0.79- and 0.52-fold of those of the control plants at 24, 48 and 72 h, respectively, and these differences were significant. The average value from six time points was 0.35 mg/kg, which was 0.12 mg/kg lower than the average value obtained for the wild-type. The PM residue abundance in the CsMAPEG(−)-overexpressing T1 plants first increased significantly, reached a maximum of 0.73 mg/kg at 24 h, and then decreased slowly. The PM residues in these plants were higher than those in the wild-type at all time points with the exception of 1 and 72 h, when slightly lower residue abundances were detected in these plants relative to the wild-type plants. The average residue abundance of the CsMAPEG(−) transgenic cucumber fruit was 0.50 mg/kg, which was 0.03 mg/kg higher than that in the wild-type plants.
‘D0351’ plants with similar expression levels of CsMAPEG were identified by PCR and qRT-PCR, and the PM residues in fruits after treatment with PM were determined (Fig. 7). The residue abundances in CsMAPEG(+)-overexpressing cucumber fruits at six time points from 1 to 72 h were lower in the ‘D0351’ T0-generation fruits than in the wild-type fruits. The maximum residue abundance of 0.09 mg/kg was observed at 12 h. The residue abundances at 6, 24 and 72 h were 0.85-, 0.80- and 0.78-fold of those of the wild-type plants, respectively, and these differences were significant. In addition, the residue abundances in the CsMAPEG(+) plants at 1 and 12 h were extremely significantly lower than those in the wild-type plants, and the values obtained for the CsMAPEG(+) plants were 0.71- and 0.67-fold of the wild-type values, respectively. The average value from six time points was 0.07 mg/kg, which was 0.02 mg/kg lower than the average value obtained for the wild-type. The results showed that CsMAPEG(+) transfer could significantly reduce the PM residue abundance in cucumber fruits. The residue abundances in the CsMAPEG(−)-overexpressing cucumber fruits were significantly higher than those in the wild-type plants at six time points from 1 to 72 h, with the exception of 1 and 12 h, when slightly lower values were detected. The average residue abundance in the CsMAPEG(−)-overexpressing plants was 0.09 mg/kg, which was 0.01 mg/kg higher than that obtained for the wild-type. The residue abundances in these plants at 6 and 72 h were 1.11- and 1.28-fold higher compared with the wild-type levels, respectively, and these differences were significant. In addition, the residue abundance in these plants at 48 h was 1.43-fold higher than that in the wild-type plants, and this difference was extremely significant.
The trend found for the variation in the PM residue abundance in CsMAPEG T1-generation transgenic plants was consistent with that found in the T0-generation plants and was characterized by an initial increase followed by a decrease. The residue abundances in the CsMAPEG(+)-overexpressing plants were lower than those in the wild-type plants at the six tested time points. After reaching the maximal value of 0.11 mg/kg at 24 h, the residue abundance showed a significantly decreasing trend. The values found for the CsMAPEG(+)-overexpressing plants at 6 and 48 h were 0.66- and 0.62-fold of the wild-type values, respectively, and these differences were significant; in addition, an extremely significant difference, which corresponded to a 0.52-fold change, was found at 72 h. The average PM residue abundance in CsMAPEG(+)-overexpressing T1-generation plants was 0.07 mg/kg, which was lower than that found for the wild-type plants (0.02 mg/kg). The residue abundance in the CsMAPEG(−)-overexpressing plants was higher than that in the wild-type plants at all tested time points, and significant 1.66- and 1.36-fold changes were detected at 1 and 12 h, respectively. The average residue abundance in the CsMAPEG(−)-overexpressing plants was 0.11 mg/kg, which was 0.02 mg/kg higher than that found for the wild-type plants.
Physiological and biochemical indexes under PM treatment
POD analysis of CsMAPEG-overexpressing plants
As shown in Fig. 8a and b, most treatments of T0 plants resulted in a trend for enzyme activity consisting of an increase followed by a decrease, and the POD enzyme activity reached a maximal level after 6 days. A comparison of the ‘D0351’ and ‘D9320’ wild-type plants treated with distilled water showed that the enzyme activity in ‘D0351’ plants at each time point was higher than that in ‘D9320’ plants, which indicated that POD responds to PM in a low-residue-abundance cucumber cultivar. The enzyme activity of the ‘D0351’ CsMAPEG(−)-overexpressing plants was lower than that of the ‘D0351’ wild-type plants at the various time points after PM treatment. After PM treatment, the ‘D9320’ CsMAPEG(+)-overexpressing plants exhibited higher activity than the ‘D9320’ wild-type plants at all tested time points with the exception of 48 h. These results indicated that the alternation in CsMAPEG expression changed the POD enzyme activity in plants, CsMAPEG overexpression enhanced POD enzyme activity, and antisense CsMAPEG weakened POD enzyme activity.
The change in the POD content in the T1-generation fruits was the same as that found in the T0-generation fruits (Fig. 9a and b). ‘D9320’ CsMAPEG(+)-overexpressing plants showed a maximum POD value of 50.48△OD470•min-1•g-1 FW at 6 days. The maximal difference in activity between the ‘D9320’ CsMAPEG(+)-overexpressing and wild-type plants after PM treatment appeared on day 4, and the POD activity of the CsMAPEG(+)-overexpressing plants was 1.19-fold higher than that of the wild-type plants. The POD activity of the ‘D0351’ CsMAPEG(−)-overexpressing plants was similar to that of the wild-type plants after PM treatment, although the POD content in these fruits was lower than that of the ‘D0351’ CsMAPEG(+)-overexpressing plants, and this difference was highly significant.
GST analysis of CsMAPEG-overexpressing plants
As shown in Fig. 8c and d, the GST content of the ‘D0351’ wild-type plants treated with distilled water first increased, reaching a maximum value of 191.14 U•mg-1 on day 6, and then decreased. However, the GST content of the ‘D9320’ wild-type plants treated with distilled water decreased continuously. The GST contents in the two cucumber genotypes showed different trends; thus, GST might be related to the detoxification of PM in cucumber fruits. After PM treatment, the GST content of ‘D0351’ CsMAPEG(−)-overexpressing plants was lower than that of the wild-type plants at all tested time points with the exception of day 6. In addition, after PM treatment, the GST content of the ‘D9320’ CsMAPEG(+) plants at each time point was significantly higher than that of the ‘D9320’ wild-type plants, which indicated that the GST content was affected by the expression of CsMAPEG. Plants overexpressing CsMAPEG exhibited a high GST content, whereas plants overexpressing antisense CsMAPEG showed a low GST content.
The GST content in the T1 fruits from ‘D0351’ CsMAPEG(−)-overexpressing plants was lower than that in ‘D0351’ wild-type plants at most time points after PM treatment. The GST content of ‘D9320’ CsMAPEG(+)-overexpressing plants was higher than that of the wild-type plants at most tested time points after PM treatment, and the general trend was similar to that found for the T0-generation plants (Fig. 9c and d).
SOD analysis of CsMAPEG-overexpressing plants
As shown in Fig. 8e and f, the SOD content of the ‘D0351’ wild-type plants treated with distilled water increased slightly starting from day 2 and reached a maximum of 264.98 U•g-1 FW on day 4, whereas that of the ‘D9320’ wild-type treated with distilled water showed a continuous decline. These findings indicated that SOD was directly involved in the metabolic response to the pesticide PM, and the difference in its content might be one of the reasons for the difference in PM residue abundance found between fruits of the two varieties. After PM treatment, the SOD contents of the ‘D0351’ CsMAPEG(−)-overexpressing plants were lower than those of the ‘D0351’ wild-type plants at all tested time points. The SOD contents of the ‘D9320’ CsMAPEG(+)-overexpressing plants at all tested time points after PM treatment were higher than those of the ‘D9320’ wild-type plants. These results showed that the expression of CsMAPEG affected the SOD content in cucumbers.
The SOD content in the T1-generation plants was lower compared with that of the T0-generation plants at different time points, although the overall trend remained unchanged. After PM treatment, the SOD content of the ‘D0351’ CsMAPEG(−)-overexpressing plants was lower than that of the ‘D0351’ wild-type plants at each tested time point. In addition, the SOD content of the ‘D9320’ CsMAPEG(+)-overexpressing plants was basically the same as that of the ‘D9320’ wild-type plants after PM treatment (Fig. 9e and f).
MDA analysis of CsMAPEG-overexpressing plants
As shown in Fig. 8g and h, the change in the MDA content after PM exposure showed differences between the ‘D0351’ and ‘D9320’ varieties. The MDA content in ‘D0351’ decreased rapidly, reached the lowest value after 6 days, and then slightly increased. In ‘D9320’ plants, the MDA content decreased slightly during the first 2 days and then increased substantially. ‘D0351’ and ‘D9320’ plants were subjected to four different treatments, namely, the transfection of sense or antisense constructs and the spray administration of distilled water or PM. The results showed that the MDA contents in the same variety were similar and that the differences in the MDA content following the different treatments were very small. The trend obtained for the T1-generation plants was the same as that found for the T0-generation plants, and the MDA content showed slight differences (Fig. 9g and h). These findings indicated that the MDA content in cucumber fruits was only affected by cultivar differences and that CsMAPEG had no effect on the content of MDA. Thus, although MDA is involved in the stress response, its metabolic effect was not significant after PM treatment.