A cytosolic NAD+-dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance
Plant glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to produce glycerol-3-phosphate (G-3-P), and plays a key role in glycerolipid metabolism as well as stress responses.
In this study, we report the cloning, enzymatic and physiological characterization of a cytosolic NAD+-dependent GPDH from maize. The prokaryotic expression of ZmGPDH1 in E.coli showed that the enzyme encoded by ZmGPDH1 was capable of catalyzing the reduction of DHAP in the presence of NADH. The functional complementation analysis revealed that ZmGPDH1 was able to restore the production of glycerol-3-phosphate and glycerol in AtGPDHc-deficient mutants. Furthermore, overexpression of ZmGPDH1 remarkably enhanced the tolerance of Arabidopsis to salinity/osmotic stress by enhancing the glycerol production, the antioxidant enzymes activities (SOD, CAT, APX) and by maintaining the cellular redox homeostasis (NADH/NAD+, ASA/DHA, GSH/GSSG). ZmGPDH1 OE Arabidopsis plants also exhibited reduced leaf water loss and stomatal aperture under salt and osmotic stresses. Quantitative real-time RT-PCR analyses revealed that overexpression of ZmGPDH1 promoted the transcripts accumulation of genes involved in cellular redox homeostasis and ROS-scavenging system.
Together, these data suggested that ZmGPDH1 is involved in conferring salinity and osmotic tolerance in Arabidopsis through modulation of glycerol synthesis, stomatal closure, cellular redox and ROS homeostasis.
KeywordsGlycerol-3-phosphate dehydrogenase Glycerol Antioxidants Redox homeostasis Salt stress Osmotic stress Maize (Zea mays L.)
Green fluorescent protein
Oxidized pyridine nucleotides
Reduced pyridine nucleotides
quantitative real-time PCR
reverse transcription PCR
Thiobarbituric acid-reactive substances
Water loss rate
It has been shown that glycerol-3-phosphate (G-3-P) serves as a significant intermediary metabolite that connects multiple metabolic pathways, such as gluconeogenes, glycolysis and glycerolipid synthesis [1, 2]. Recent evidences proved that G-3-P also plays a crucial role in adapting to adverse stresses, including salinity, pathogenic microbes, freezing and anaerobic stresses . In higher plants, G-3-P can be biosynthesized through two major pathways. In the first route, G-3-P is generated by NAD+-dependent GPDH (EC 22.214.171.124)-mediated reduction of DHAP; while in the second route, G-3-P is produced from glycerol through phosphorylation catalyzed by glycerol kinase (EC 126.96.36.199) .
Multiple forms of GPDH have been identified from eukaryotes, and most of them are proved to be key regulators in stress responses [5, 6, 7]. There are five GPDH isoforms in Arabidopsis, which are associated with different subcellular organelles: one mitochondrial FAD-dependent GPDH (EC 188.8.131.52), two plastidic NAD+-dependent GPDHs and two cytosolic NAD+-dependent GPDHs [5, 8, 9, 10]. Previous studies demonstrated that the AtGPDHc2 gene encoded a cytosol-targeted GPDH which is involved in pathogen-elicited defense responses in Arabidopsis via its effects on the provision of G-3-P . Plants deficient in plastid-localized GPDH (SFD1/GLY1) exhibited a serious impairment in plastidal glycerolipids pathway of Arabidopsis and overexpression of SFD1/GLY1 could increase the plastidic lipid contents as well as the photosynthetic assimilation rate in transgenic rice plants .
In addition to their pivotal role in lipid metabolism, plants GPDHs also participate in modulating the intracellular redox status through the mitochondrial G-3-P shuttle system [8, 9]. In Arabidopsis thaliana, a mitochondrial FAD-GPDH (EC 184.108.40.206) encoded by the gene AtGPDHm1, along with a cytosolic NAD+-dependent GPDH (EC 220.127.116.11) encoded by the gene AtGPDHc1, was capable of forming the mitochondrial G-3-P shuttle . The operation of G-3-P shuttle is of vital importance to preserve the homeostasis of NADH/NAD+ ratio, which is a prerequisite for cells to keep normal metabolic activities. In previous studies, it has been recognized that the expression of AtGPDHc1 and AtGPDHm1 is dramatically induced under a variety of stress conditions, like oxygen availability, salinity and dehydration [8, 10]. AtGPDHc1 knock-out mutants are more sensitive to abscisic acid (ABA) than wild-type (WT) plants, and have failed to stabilize the balance of NADH/NAD+ . Loss of AtGPDHc1 also affected other metabolic pathway involved in redox shuttling, such as mitochondrial malate/OAA shuttle .
The characteristics of GPDH genes in relation to salinity or osmotic tolerance have been described in some halophilic microalga species [6, 12, 13]. A putative phosphoserine phosphatase (PSP) domain has been found in GPDH isoforms from Dunaliella salina (DsGPDH2, G3PDH) and Chlamydomonas reinhardtii (CrGPD2), which can serve as glycerol-3-phosphatase (EC 18.104.22.168.1) enzyme and directly catalyze the conversion of DHAP to glycerol under high osmotic environment [14, 15, 16]. Furthermore, the transcription of mushroom GPDH gene is greatly stimulated by drought and salinity conditions; and overexpression of PsGPD improves the salinity tolerance of transgenic rice by increasing the osmotic potential and stomatal conductance .
Although the importance of GPDH genes in stress responses is well documented in yeast, algae as well as a few plants, there is scarce information about their functions in field crops. Salt and osmotic stresses are the major environmental factors that seriously influence crop growth and productivity . Here, we isolated and characterized a cytosol-localized GPDH (ZmGPDH1) gene from maize, which had functional NAD+-dependent GPDH activity, and apparent transcriptional response to salinity and mannitol treatments. In addition, overexpression of ZmGPDH1 in AtGPDHc-deficient mutant and WT lines enhanced tolerance of transgenic Arabidopsis to salinity and osmotic stresses, with higher glycerol level, lower fluctuation of cellular redox status and stronger ROS antioxidant defense in comparison to both atgpdhc2 mutant and WT plants. The results showed that ZmGPDH1 was pivotal in strengthening salt and osmotic stress tolerance by regulating glycerol production, redox homeostasis and ROS antioxidant defense.
ZmGPDH1 encodes a cytosol-targeted protein with NAD+-dependent GPDH activity
One GPDH gene was originally obtained through BLAST searching against the maize genome utilizing the reported AtGPDHc2 as query , the retrieved gene was designated as ZmGPDH1. The full length CDS of ZmGPDH1 was cloned, which had 458 amino acids and an apparent molecular mass of 51 kDa. The complete CDS sequence of ZmGPDH1 was submitted to GeneBank with the following accession number: MH460963. The sequence alignment revealed that ZmGPDH1 exhibited very high protein sequence identity (77%) to AtGPDHc2, and both proteins consisted of one C-terminal GPD domain (PF07479) that represents DHAP-binding site and one N-terminal NAD-binding domain (PF01210), suggesting that ZmGPDH1 encodes an NAD+-dependent GPDH (Additional file 1: Figure S1).
To further study the catalytic characteristics of ZmGPDH1, the recombinant protein generated by the E. coli Rosetta (DE3) strain expressing plasmid of 6 × His tagged ZmGPDH1 was purified with a Ni-NTA column. A ZmGPDH1-His fusion protein with an expected size of 65 kDa (consisting of target gene and histidine marker) was identified by SDS/PAGE and Western blot (Fig. 1b, c and d). The recombinant ZmGPDH1 protein was assayed for its kinetic properties relative to the substrate DHAP. Using Eadie-Hofstee plot, the Km and Vmax of DHAP were estimated as 2.75 mM and 0.071 umol·min− 1·mg− 1 protein, respectively (Fig. 1e); besides, addition of NADH strongly stimulated the enzyme activity (Fig. 1f). These results indicated that the purified ZmGPDH1 protein was able to catalyze the reduction of DHAP with the assistant of NADH. Furthermore, the optimum pH of the enzyme activity was determined to be pH 7.0 and the optimum temperature was 35 °C, respectively (Additional file 3: Figure S3).
Expression profiles of ZmGPDH1 in response to NaCl or mannitol treatment
It has been proven that GPDH genes are essential for stress adaptations in yeast, marine algae and Arabidopsis [5, 17, 18, 19]. Therefore, to explore its possible involvement in stress responses in maize, we first analyzed the transcripts accumulation of ZmGPDH1 under different stress treatments. The qRT-PCR results showed that ZmGPDH1 was remarkably up-regulated by both salinity and osmotic stresses in maize roots, which reached the highest level at 3 h under both treatments (Fig. 2n and o). Notably, the expression of proZmGPDH1::GUS was also enhanced in the presence of NaCl and mannitol treatments (Fig. 2p), suggested that ZmGPDH1 was involved in the transcriptional response during salt or osmotic adaptions.
Overexpression of ZmGPDH1 enhanced the tolerance of Arabidopsis to salt and osmotic stresses
Additionally, to evaluate the performance of ZmGPDH1 transgenic lines in response to salt or osmotic stresses, the seeds of WT, atgpdhc2, COM and OE lines were germinated on half-strength MS mediums containing 100 mM NaCl or 200 mM mannitol. As shown in Fig. 3d and e, seed germination of atgpdhc2 was severely delayed in comparison to the WT, whereas the germination rate of ZmGPDH1 OE seeds was much higher than that of other lines. The ZmGPDH1 COM seeds displayed stress-sensitive morphologies similarity to the WT, albeit they had a relative higher germination rate (Fig. 3d and e).
Likewise, when the 3-week-old Arabidopsis plants were subjected to 400 mM mannitol or 200 mM NaCl treatment for 9 days, the growth of atgpdhc2 mutants was strongly inhibited compared with the WT (Fig. 4d and e). Conversely, the ZmGPDH1 OE or COM plants showed obviously improved tolerance to salinity or osmotic stress relative to the other lines (Fig. 4d and e). Synchronously, the chlorophyll fluorescence parameter (Fv/Fm) and total chlorophyll content were analyzed in atgpdhc2, COM-1, WT and OE-1 plants. Under standard conditions, chlorophyll content and Fv/Fm had no differences among all four lines (Day 0); however, after exposure to 400 mM mannitol or 200 mM NaCl treatment, atgpdhc2 mutant demonstrated a remarkable reduction in Fv/Fm and chlorophyll content, whereas the Fv/Fm ratio and total chlorophyll content in OE-1 and COM-1 were much higher than that in WT plants (Fig. 4 f and g). These data suggested that overexpression of ZmGPDH1 helped to enhance the photochemical efficiency in transgenic Arabidopsis.
ZmGPDH1 regulates glycerol-3-phosphate and glycerol levels under salt and osmotic stresses
ZmGPDH1 is essential for redox homeostasis under salt and osmotic stresses
To further examine whether overexpression of ZmGPDH1 could influence the other redox couples, the cellular oxidized and reduced pools of ASA and GSH were also determined. Similarly, the contents of ASA, GSH and their oxidized form DHA, GSSG did not change among the six lines under normal growing conditions (Fig. 6b and c). However, when the plants were treated with NaCl or mannitol, the ZmGPDH1 OE lines maintained relative higher ASA and GSH contents compared with the WT, leading to an even higher ASA/DHA or GSH/GSSG ratio. By contrast, there was a noticeable decline in reduced ascorbate or glutathione as well as the redox ratio (ASA/DHA, GSH/GSSG) in atgpdhc2 mutants relative to WT or COM lines (Fig. 6b and c). Collectively, these results implied that the founction of ZmGPDH1 in salinity and osmotic tolerance could partly attribute to sustaining the cellular redox homeostasis.
ZmGPDH1 regulates the ROS level and cell death under salt and osmotic stresses
ZmGPDH1 is involved in stress-induced stomatal closure
The phytohormone ABA has the ability to induce stomatal closure ; however, overexpression of ZmGPDH1 led to ABA insensitivity in stomatal movement (Fig. 9b). In the presence of ABA, the stomatal closure was significantly triggered in atgpdhc2 mutants, moderately triggered in WT or COM and mildly triggered in OE plants, illustrating that ZmGPDH1-mediated stomatal closure was independent of ABA (Fig. 9c). In addition, the H2O2 production in the guard cells was also determined by H2DCF-DA staining. As shown in Fig. 9b, the H2O2 accumulation was less in OE plants and more in atgpdhc2 mutant compared with that in the WT, suggesting that overexpression of ZmGPDH1 contributed to sustain the ROS levels during stomatal movements.
Effects of ZmGPDH1 on expression of genes involved in redox homeostasis and ROS-scavenging system
Maize (Zea mays L.) is an important cereal crops as well as a major source of biofuel, industrial material and animal feed . Although a great deal of research has indicated that glycerol-3-phosphate dehydrogenase (GPDH) plays a pivotal role in plant growth and stress adaptions [3, 17, 21], little is currently known about its functions in field crops including maize. In this study, we isolated a GPDH gene encoding NAD+-dependent GPDH from maize. Similar to other typical NAD+-dependent GPDH [14, 16, 19], ZmGPDH1 protein contained the necessary and specific protein domains (PF07479, PF01210). The conserved GAGAWG motif was found at residues 44–50 of the protein sequence of ZmGPDH1 (Additional file 1: Figure S1), which was similar to the previously reported NAD+-dependent GPDH isoforms with an analogous NAD+-binding fragments corresponding to GXGXXG [8, 10, 32].
Enzymatic assay of recombinant ZmGPDH1 proteins expressed in Escherichia coli Rosetta strain (DE3) (Fig. 1e and f) showed the purified ZmGPDH1 protein had substrate affinity (KmDHAP of 2.75 mM) (Fig. 1e), which compared well with the kinetic parameters previously reported for other GPDH enzymes [10, 33, 34]. The stable or transient expression of a green fluorescent protein (GFP)-tagged ZmGPDH1 in Arabidopsis or wild-type rice were conducted, and both evidenced that ZmGPDH1 proteins were specially targeted to cytosol (Additional file 2: Figure S2 and Fig. 1a). The earlier identified Arabidopsis GPDH proteins (AtGPDHc1 and AtGPDHc2) were predicted to be cytosol-located GPDs owing to the absence of apparent transmembrane regions and subcellular targeting sequences, however, a clear experimental evidence was lacking [5, 8].
In succession, the physiological functions of the cytosolic ZmGPDH1 gene in mediating salinity/osmotic adaption were investigated in this study. The transcript abundance of ZmGPDH1 was markedly increased under NaCl and mannitol treatments (Fig. 2n and o). On the other hand, the transgenic Arabidopsis harboring the ZmGPDH1 promoter fused to a GUS reporter gene also showed relatively higher GUS activity under NaCl or mannitol condition (Fig. 2p), indicating that ZmGPDH1 gene was regulated at the transcription level in response to salt or osmotic stress, which was consistent with the expression patterns of other GPDH genes from A.thaliana (AtGPDHc1, AtGPDHm1), C. reinhardtii (CrGPDH2, CrGPDH3), D. salina (DsGPDH2, G3PDH) and D. viridis (DvGPDH1, DvGPDH2) [6, 8, 9, 13, 19]. Furthermore, overexpression of ZmGPDH1 strongly enhanced the tolerance of Arabidopsis (WT) to salt/osmotic stress and rescued the salt/osmotic sensitivity of atgpdhc2 mutant, as reflected by a pronounced elevation in germination rate, fresh weight, root length, biomass, chlorophyll content and Fv/Fm ratio under salinity or osmotic conditions (Figs. 3 and 4). Similar results have been reported earlier in other species: overexpression of an oyster mushroom GPDH gene (PsGPD) increased the salt tolerance in transgenic potatoes and rice; and overexpression of a black yeast GPDH gene (HwGPD1B) also enhanced NaCl tolerance of Saccharomyces cerevisiae gpd1 mutant [17, 35].
We also found that ZmGPDH1 gene was required for glycerol generation. Glycerol is an important osmo-protectant and its accumulation can compensate for differences between intracellular and extracellular water potentials under hyperosmotic environment [6, 12, 13]. In our study, overexpression of ZmGPDH1 markedly increased the levels of G-3-P and glycerol, demonstrated that ZmGPDH1 played essential roles in plant adaptation to hypersaline or hyperosmotic shock by contributing to the glycerol biosynthesis (Fig. 5). Likewise, of the five GPDH enzymes in Chlamydomonas reinhardtii, CrGPDH2 and CrGPDH3 were shown to be necessary for osmotic-induced glycerol production . Also, loss of AtGPDHc1 gene encoding a cytosol-localized GPDH of Arabidopsis caused the hypersensitivity to salt stress due to the severe impairment in provision of glycerol .
Additionally, our results showed that ZmGPDH1-OE plants exhibited greater stomatal closure compared with other lines, indicating that ZmGPDH1 played a key role in manipulating the stomatal closure under salt or osmotic stress (Fig. 9). However, the ABA-induced stomatal closing was impaired in ZmGPDH1-OE lines but significantly induced in atgpdhc2 mutant, suggesting that the ZmGPDH1-mediated stomatal closure might be independent of ABA. Meanwhile, we found that overexpression of ZmGPDH1 reduced the plant sensitivity to ABA at seed germination and early seedling developmental stages (Additional file 5: Figure S5), which was also observed previously on AtGPDHc1 mutants . Further studies will be needed to interpret the interaction of ZmGPDH1 and ABA signaling pathway.
We reported the characterization of a cytosolic NAD+-dependent GPDH gene from maize, ZmGPDH1, which had profound effects on salt/osmotic tolerance by regulating the glycerol accumulation, cellular redox homeostasis, ROS-scavenging system as well as stomatal movement (Fig. 11).
Plant materials and growth conditions
The maize inbred line accession He-344 (provided by Heilongjiang Academy of Agricultural Sciences, Harbin, China) was used as the plant material in this experiment and normally planted in a growth chamber under controlled photoperiod and temperature (12 h light/12 h dark, 23 ± 2 °C), with a photon flux density of 1000 μmol m− 2 s− 1.
The seeds of T-DNA insertion mutants of AtGPDHc2 (TAIR: At3G07690), namely atgpdhc2 (SALK_033040) were donated by Dr. Pradeep Kachroo (University of Kentucky, USA). The homozygous lines of atgpdhc2 mutant were identified by PCR and reverse transcription PCR (RT-PCR) analysis, and the primers were shown in Additional file 6: Table S1. The plants of Arabidopsis thaliana (ecotype Col) and atgpdhc2 mutant (ecotype Col) were grown in a growth chamber under controlled photoperiod and temperature (16 h light/8 h dark, 21 ± 2 °C), with a photon flux density of 100 μmol m− 2 s− 1.
Plasmid construction and plant transformation
The full length coding region of ZmGPDH1 (1377 bp) was cloned from cultivated maize by the gene specific primers (Additional file 6: Table S1). The PCR product was purified and inserted into the XbaI and SalI sites of the pBI121-GFP vector under control of the CaMV35S promoter. For complementation and over-expression assays, Agrobacterium tumefaciens strain EHA105 carrying the construct pBI121-ZmGPDH1::GFP was used to transform Arabidopsis wild-type or atgpdhc2. T3 homozygous transgenic Arabidopsis were screened by RT-PCR.
Protein subcellular localization and GUS activity assay
To verify the subcellular localization of ZmGPDH1, the mesophyll protoplasts were isolated from T3 homozygous transgenic Arabidopsis harboring pBI121-ZmGPDH1::GFP or pBI121-GFP (control) plasmid, and the subcellular localization of GFP expression was visualized by confocal laser-scanning microscope (Leica, German). The positive control (empty vector) or fusion proteins were also temporarily expressed in rice mesophyll protoplasts according to the methods described previously . For co-localization studies, a far-red fluorescent protein mkate was used as the cytosol marker. To study the promoter activity, a 1642-bp genomic region upstream of the translation initiation codon of ZmGPDH1 gene was cloned into pBI121-GUS at HindIII and XbaI sites (primers see Additional file 1: Table S1). The constitutive proZmGPDH1::GUS transformed WT plants were also generated and T3 homozygous transgenic lines were used for GUS staining according to the reported protocol . The images were visualized by stereo microscope (Olympus, Japan).
Recombinant ZmGPDH1 protein expression, purification and western blot
The coding region of ZmGPDH1 with the NcoI and XhoI sites was amplified by PCR and then inserted into the pET32a (+) vector containing 6 × His tag. The pET32a-ZmGPDH1 plasmid was transformed into the Escherichia coli (E. coli) Rosetta strain and the expression of ZmGPDH1 was induced with 1 mM IPTG to generate the putative recombinants. Then the His-tagged ZmGPDH1 proteins were extracted and purified under native conditions using Ni-NTA nickel columns (Sigma), and the purified proteins were detected by 12% SDS-PAGE as well as Western blot using 6 × His Tag Antibody as probe.
Phenotypic analyses of the ZmGPDH1 transgenic Arabidopsis under salt or osmotic treatment
For germination analysis, seeds of WT, atgpdhc2, OE and COM lines were plated on half-strength MS plates containing 200 mM mannitol or 100 mM NaCl for 8 days. Germination rates were counted at day 5 after sowing and seed germination was defined as the appearance of visible radicle. To investigate the effects of salinity and osmotic stresses on root length and fresh weight, 7-days-old seedlings of WT, atgpdhc2 mutant, COM and OE lines were transferred into half-strength MS plates supplemented with 150 mM NaCl or 300 mM mannitol. The root length and fresh weights of stress-treated seedlings were determined after 7 day of treatment.
For the stress tolerance test at the adult stage, 3-week-old Arabidopsis plants were irrigated with 200 mM NaCl or 400 mM mannitol solution every 3 days for a total of 9 days. Rosette leaf samples were collected at day 6 of treatments to measure the changes of various physiological and biochemical parameters. All experiments were replicated at least three times with 80–100 plants per treatment. Photographs taken from one representative experiment are shown. Total chlorophyll (chlorophyll a + b) was determined according to the method as previously described  and the fresh young leaf was extracted in 80% (v/v) acetone extract. Photochemical efficiency (Fv/Fm) was examined by using a pulse-modulated fluorometer (FMS2, Hansatech, UK) .
To investigate the water loss rate (WLR), the rosette leaves from 4-week-old Arabidopsis were weighed at specific time points. The decrease in fresh weight was used to calculate WLR. For stomatal closure assays, the strips abaxial epidermis of Arabidopsis leaves were immerged in buffer (10 mM MES/KOH, pH 6.1, 10 mM KCl, 50 μM CaCl2) under light for 1 h to induce the stomatal opening and then treated with 300 mM mannitol, 150 mM NaCl and 20 μM ABA for 3 h. The conformation of stomatal aperture were photographed by confocal microscope and processed with ImageJ software. The experiments were replicated at least three times with 40–50 cells per treatment.
Analysis of GPDH activity, G-3-P and glycerol levels
G-3-P and glycerol contents were measured as previously described with slight modifications . The GPDH activity was examined with regard to the reduction of DHAP by NADH. The total reaction volume of the assay was 1 mL containing 100 mM HEPES buffer, pH 6.9, 4 mM DHAP, 0.2 mM NADH and an appropriate amount of enzyme . The absorbance changes at 340 nm were monitored using an ultraviolet spectrophotometer (U3900, Hitachi High-Technologies, Japan).
Analysis of cellular redox and ROS homeostasis
The reduced pyridine nucleotides (NADH) content, oxidized pyridine nucleotides (NAD+) content and NADH/NAD+ ratio were assayed with an enzymatic cycling procedure . The ascorbate (ASA) content, dehydroascorbate (DHA) content and ASA/DHA ratio were measured following the reported protocols . The glutathione (GSH) content, oxidized glutathione (GSSG) content and GSH/GSSG ratio were assayed as described .
For ROS accumulation analysis, the staining of nitroblue tetrazolium (NBT) and 3,3- diaminobenzidine (DAB) of stress-treated seedlings were performed following the reported protocol . For hydrogen peroxide (H2O2) staining in the guard cells, prepared epidermal peels with NaCl, mannitol or ABA treatment were stained with 2,7-dichlorofluorescin diacetate (H2DCF-DA) for 10 min . Cell death caused by salt or osmotic stress was also estimated by Evan′s blue and PI staining as described . The assays of H2O2 and superoxide (O2.-) were conducted by spectrophotometry as previously described [50, 51]. The lipid peroxidation was measured with reference to the thiobarbituric acid-reactive substances (TBARS) content . Electrolyte leakage (EL) was assessed as described .
To monitor the antioxidant enzyme activities, leaf tissues (0.5 g) were ground in ice bath with 10 mL extraction buffer (K2HPO4-KH2PO4, pH 7.0, 1.5 mM EDTA, 1% PVP, 0.5 mM ASC), and then the homogenate was centrifuged at 12000 rpm for 20 min at 4 °C. The supernatant was used for the determination of enzymes activities. The activities of catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) were determined as described [54, 55], with slight modifications.
Quantitative real-time RT-PCR analysis
To analyze the expression of ZmGPDH1 under osmotic and salt stresses, 3-week-old maize seedlings were treated with 1/2 Hoagland solution containing 400 mM mannitol and 200 mM NaCl solutions for 0, 1, 3, 6, 12 and 24 h, and the roots were sampled to analyze the transcripts of ZmGPDH1. The untreated maize samples from the same time point were used as the controls. ZmGAPDH and ZmACTIN genes served as internal reference in each assay. To examine the tissue-specific expression of ZmGPDH1, total RNA was extracted from rosette leaves (RL), flower buds (FLB), roots (RT), flowers (FL), siliques (SL), stems (ST) and cauline leaves (CL) in proZmGPDH1::GUS transgenic plants. To analyze target genes expression induced by osmotic and salt stresses, 3-week-old WT, atgpdhc2, and OE lines were treated with water (control), 300 mM mannitol or 150 mM NaCl solution. Total RNA was extracted from rosette leaf samples at 24 h after treatments. The expression of target genes in WT plants under control environment was used as a calibrator. ACTIN2 and UBQ7 genes were used as internal reference . The primers used for transcriptional analysis were shown in Additional file 6: Table S1.
Data are presented as Mean ± SD. The Student’s t-test was used to determine the significance levels using SPSS 21.0 software throughout this study. A P-value of < 0.05 was considered statistically significant.
We would like to thank Dr. Pradeep Kachroo (University of Kentucky, USA) for providing us AtGPDHc2-KO mutant seeds.
This work was supported by National Key Research and Development Program of China (2016YFD0101002), Natural Science Foundation of Heilongjiang Province (QC2016036), National Natural Science Foundation of China (31701328), Heilongjiang Bayi Agricultural University Scientific Start-up Found for the Returned Overseas Chinese Scholar (2031011047), Heilongjiang Bayi Agricultural University Key Cultivateing Program (XA2014–01) and Heilongjiang Bayi Agricultural University Graduate Student Innovation Fund Projects (YJSCX2017-Z01). The funding bodies did not play a role in the design of the study and collection, analysis, or interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.
YZ and ML designed and conceived the experiments. YZ, ML, FW, XL, BY and JW performed the experiments, planted materials and collected samples. LH and CZ analyzed the data and interpreted the results. YZ prepared the manuscript. JX and ZL conceived the experiments and revised the manuscript. All authors read and approved the final manuscript.
Ying Zhao, Bowei Yan and Jinpeng Wei, Ph.D. candidates in crop cultivation and geoponics, Heilongjiang Bayi Agricultural University, Daqing City, Heilongjiang Province, China. Meng Liu and Feng Wang, M.S. candidates in crop cultivation and geoponics, Heilongjiang Bayi Agricultural University, Daqing City, Heilongjiang Province, China. Xin Li, research assistant in Heilongjiang Academy of Agricultural Sciences, Harbin City, Heilongjiang Province, China. Changjiang Zhao and Lin He, assistant professors in College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing City, Heilongjiang Province, China. Zuotong Li and Jingyu Xu, professors in College of Agriculture, Heilongjiang Bayi Agricultural University, Daqing City, Heilongjiang Province, China.
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