International Genetics and Genomics of Diseases: Transgenic Arabidopsis Plants

The Maize Gene ZmGLYI-8 Confers Salt and Drought Tolerance in Transgenic Arabidopsis Plants

Methylglyoxal (MG), a highly reactive and cytotoxic α-oxoaldehyde compound, can over-accumulate under abiotic stress, consequently injuring plants or even causing death. Glyoxalase I (GLYI), the first enzyme of the glyoxalase pathway, plays multiple roles in the detoxification of MG and in abiotic stress responses. However, the GLY1 gene in maize has been little studied in response to abiotic stress. In this study, we screened a glyoxalase I gene (ZmGLYI-8) and overexpressed in Arabidopsis. This gene was localized in the cytoplasm and can be induced in maize seedlings under multiple stress treatments, including salt, drought, MG, ABA, H2O2 and high temperature stress. Phenotypic analysis revealed that after MG, salt and drought stress treatments, overexpression of ZmGLYI-8 increased the tolerance of transgenic Arabidopsis to MG, salt and drought stress. Furthermore, we demonstrated that the overexpression of ZmGLYI-8 scavenges accumulated reactive oxygen species, detoxifies MG and enhances the activity of antioxidant enzymes to improve the resistance of transgenic Arabidopsis plants to salt and drought stress. In summary, this study preliminarily elucidates the molecular mechanism of the maize ZmGLYI-8 gene in transgenic Arabidopsis and provides new insight into the breeding of salt- and drought-tolerant maize varieties.

Materials and Methods

Gene Isolation and Bioinformatics Analysis of ZmGLYI-8
Based on the B73 genome in the MaizeGDB database, Primer 5.0 software was used to design specific primers for ZmGLYI-8. Total RNA was extracted from the leaves of ten-day-old maize seedlings with TRIzol reagent. The concentration and integrity of total RNA were determined by using formaldehyde gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientifi, Shanghai, China). CDNA from B73 leaves was synthesized by using HiScript®II Q Select RT SuperMix (Vazyme, Nanjing, China). The pMD19-T cloning vectors with the PCR products of ZmGLYI-8 were transformed into Escherichia coli DH5α, which was subsequently sequenced by Boshang (Shanghai, China). The molecular weight and isoelectric point of ZmGLYI-8 were evaluated by ProtParam (http://web.expasy.org/protparam/ (accessed on 1 October 2024)), and the conserved functional domains of this gene were predicted by using NCBI CD-Search (https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 1 October 2024)). The similarity of amino acid sequences was determined by using DNAMAN v6 software for multiple sequence alignments. MEGA 4.0 software was used for generating the phylogenetic tree.

Stress Treatment of B73

The maize inbred line B73 was used for investigating the expression pattern of ZmGLYI-8 in different tissues and under various stress treatments. The seeds of inbred line B73 were preserved in our laboratory. Roots, stems and leaves of B73 at the V6 stage were collected for the expression pattern analysis of different tissues. B73 seeds were germinated in quartz sand, and the plants were cultivated at 25 °C under a 14 h light/10 h dark cycle. After ten days, the seedlings were transferred to Hoagland solution supplemented with 150 mM NaCl, 10% (w/v) PEG-6000, 10 mM MG, 0.5 mM H2O2, 50 µM ABA, 50 µM MeJA or 5 mM ET. Other seedlings were subjected to 38 °C for the high-temperature (HT) treatment. Leaf samples were collected 0, 1, 6, 12 and 24 h after the different treatments. Each biological replicate contained three plants, and all ZmGLYI-8 expression pattern experiments were performed by using three biological replicates. All samples were frozen directly in liquid nitrogen and then stored at −80 °C before RNA extraction. The specific gene primers used for expression pattern analysis.

Subcellular Localization

The subcellular localization of ZmGLYI-8 was predicted by the SubLoc program. Moreover, the expression vector PROKII-GFP was used to investigate the subcellular localization of ZmGLYI-8. The coding sequence (CDS) of ZmGLYI-8 without a termination codon was amplified by polymerase chain reaction (PCR) and then fused to the PROKII-GFP vector and driven by the CaMV35S promoter. The ZmGLYI-8 primers used for plasmid construction are listed in Table S1. The Agrobacterium tumefaciens strains GV3101 with the 35S::ZmGLYI-8-GFP plasmid or 35S::GFP plasmid were injected into four-leaf-stage Nicotiana benthamiana plants. Three days after infiltration, GFP fluorescence was observed by using an LSM 880 confocal laser scanning microscope.

Prokaryotic Expression of ZmGLYI-8

The full-length CDS of ZmGLYI-8 was fused to the pET-30a vector and then transformed into Escherichia coli Rosetta. PCR identification and sequencing analysis (Boshang) were used for selecting positive clones. The IPTG-induced bacteria were diluted with LB liquid medium in gradients of 10−2, 10−4 and 10−6. Three microliters of different concentrations of bacteria was added dropwise to LB solid medium supplemented with 0.5 mM MG, 200 mM NaCl or 200 mM mannitol. The growth of the recombinant strain pET-30a-ZmGLYI-8 and the control strain pET-30a was observed after culture at 37 °C for 12 h.

Development of Transgenic Arabidopsis

The coding sequence of the ZmGLYI-8 gene was cloned and inserted into pBI121 with the CaMV35S promoter. The resulting plasmid was subsequently introduced into Agrobacterium tumefaciens GV3101, which was subsequently used for floral dip transformation of Arabidopsis Columbia-0 (WT) plants. Transgenic Arabidopsis plants were screened on 1/2 MS solid medium supplemented with 50 mg/L kanamycin. The expression level of ZmGLYI-8 in positive transgenic plants was further measured by qRT–PCR. T3 transgenic Arabidopsis plants were used for subsequent experimentation. Fifty seeds of WT and ZmGLYI-8 transgenic Arabidopsis plants were sown on 1/2 MS solid medium supplemented with 5 mM MG. These petri dishes were placed in a growth chamber at 22 °C with a 16-h light/8-h dark cycle. The germination rate of the seeds was calculated every day, and photos were taken on the 7th day after sowing.

Expression Analysis by qRT–PCR

Total RNA from maize and Arabidopsis plants was extracted for qRT–PCR using the TRIzol reagent (TaKaRa, Beijing, China). HiScript®II Q Select RT SuperMix (Vazyme, China) was used for synthesizing cDNA from the total RNA. According to the manufacturers’ protocols, qRT–PCR experiments were performed by using a Bio-Rad CFX96 real-time system with AceQ®qPCR SYBR Green Master Mix (Vazyme, China). The inner reference gene Actin was used for normalizing the expression levels of genes. The gene expression levels were calculated by using the 2−ΔΔCt method. Triplicate independent biological assays of each cDNA sample were performed to ensure accurate statistical analysis.

Assays for Salt and Drought Tolerance

To detect seed germination of ZmGLY1-8 transgenic Arabidopsis lines under MG stress, the sterile seeds of the WT and transgenic Arabidopsis lines (OE3, OE4 and OE5) were sown on 1/2 MS solid medium with MG (0 and 2.5 mM). The number of seed germinations was recorded every day, and the photos were taken on the 7th day after the seeds were sown. For the root length experiment, the sterilized WT and transgenic line seeds were sown on 1/2 MS medium for vernalization at 4 °C for 3 days. Then, these seeds were vertically cultivated at 22 °C under a 16 h light/8 h dark cycle. After five days, the seedlings were transplanted into 1/2 MS solid medium supplemented with 0.5 mM MG, 150 mM NaCl and 200 mM mannitol. The primary root lengths of the transgenic Arabidopsis lines were analyzed by using ImageJ 1.50I software after 7 days of stress treatment. The experiment was repeated with three biological replicates.

Floating Leaf Disc and Chlorophyll Extraction

Leaf discs (1 cm in diameter) were excised from the third and fourth true rosette leaves of the WT and ZmGLY1-8 transgenic plants. The discs were placed in water (simulated control) or under multiple stress conditions (simulated stress treatment) for 48 h, including salt stress in 200 mM and 400 mM NaCl, drought stress in 200 mM and 400 mM mannitol and MG stress in 1 mM and 2.5 mM. The chlorophyll content was determined by an ultraviolet spectrophotometer after extraction of all the leaf discs with 95% ethanol. The experiment was repeated with three biological replicates.

Physiological Analysis and MG Content Determination

Four-week-old Arabidopsis plants were treated with 400 mM NaCl or 200 mM 10% PEG-6000 for 7 days, after which leaves from the same parts were removed for physiological measurements. The contents of O2•− and H2O2 were measured using kits purchased from Suzhou Grace Biotechnology Co., Ltd., Suzhou, China. About 0.1g of the sample was taken, the extract was added, and the supernatant was collected as the solution to be measured after centrifugation. The reaction solution was added according to the kit instructions, and O2•− and H2O2 were measured at 540 nm and 415 nm, respectively. The MDA content was also measured using kits from the same company. MDA was measured according to the kit instructions. First, 0.1 g of sample was taken, 1 mL of extraction solution was added, and the supernatant was collected after centrifuged at 12,000× g for 10 min at 4 °C. Next, 600 mL of working solution was added, and the supernatant was removed from the water bath at 90 °C for 30 min. MDA was measured at 532 nm and 600 nm. The experiment was conducted using three biological replicates. The activities of CAT, POD and SOD were determined by Wu’s method. In order to determine the CAT, POD and SOD, fresh samples were taken, and 5 mL pre-cooled phosphoric acid buffer was added. Then, the mixture was ground mechanically and centrifuged at 10,000× g for 20 min at 4 °C. The collected supernatant was used for subsequent assays. SOD was measured at 560 nm using the nitrogen blue tetrazolium (NBT) method. POD and CAT activities were determined at 470 nm and 240 nm, respectively, with absorbance changes every 10 s. The methylglyoxal content was determined according to previously described methods. MG content was measured from 300 mg of fresh leaf tissue. First, 0.5 M of 3 mL perchloric acid was added, ice-bathed for 15 min and centrifuged at 12,000× g for 10 min at 4 °C. Second, the colored supernatant was transferred to a new centrifuge tube, bleached with charcoal (10 mg/mL) for 15 min at room temperature and centrifuged at 11,000× g for 10 min. Then, the supernatant was transferred to a new centrifuge tube, and the reaction was neutralized by the addition of saturated potassium carbonate at room temperature for 15 min and centrifuged at 11,000× g for 10 min; 650 μL of supernatant was collected. Finally, the reaction mixture (1 mL), containing 250 mL of 7.2 mM 1,2-diaminobenzene, 100 mL of 5 M perchloric acid, 10 mL of 100 mM NaN3 and 650 μL of neutralized supernatant, was incubated for 3 h at room temperature, and the absorbance was measured at 336 nm.