Isolation of ZmNF-YA12
Total RNA was isolated from maize seedlings using TRIzol reagent (CW Biotech, Beijing, China) and reverse-transcribed to complementary DNA (cDNA) using HiScript II Q RT SuperMIX (Vazyme, Nanjing, China). The full ZmNF-YA12cDNA was amplified by PCR using the forward primer (FP) 5′-ATGCTTCTTCCCTCTTCGTCTT-3′ and reserve primer (RP) 5′-TCATCTCATAACTGGAACCCT-3′.
Plant materials, growth conditions, and treatments
For tissue-specific analysis, leaves, stems, and roots were harvested from three-leaf seedlings grown in a greenhouse (28°C, 16/8 h day/night cycle). Mature leaves, roots, silks, tassels, and embryos were harvested at the grain-filling stage from plants grown in the field. All harvested materials were frozen immediately in liquid nitrogen and stored at −80°C.
To determine the ZmNF-YA12 expression patterns under various stress conditions, maize seedlings were grown in 12-cm hydroponic barrels containing nutrient solution. Three-leaf maize seedlings were subjected to dehydration, NaCl, polyethylene glycol (PEG), cold, abscisic acid (ABA), and jasmonic acid (JA) treatments. For dehydration treatment, the whole seedling was removed, washed, and placed on an experimental table for natural dehydration at room temperature (25°C). For salt and PEG treatments, the roots of the seedlings were immersed in solutions containing 200 mM NaCl and 20% PEG, respectively. For cold treatment, seedlings were kept at 4°C. For each of the above four treatments, the shoots and roots were collected at 0, 1, 2, 5, 10, and 24 hours after treatment. For the ABA and JA treatments, the leaves of the seedlings were sprayed with solutions containing 100 μM ABA or 100 μM JA and covered with plastic film. The leaves were then collected at 0, 1, 2, 5, and 10 hours after treatment. The samples were immediately frozen with liquid nitrogen for isolation of RNA.
Arabidopsis thaliana ecotype Columbia (Col-0) was used for transformation in this study. After vernalization treatment, seeds were surface-sterilized in a solution of 0.5% NaClO for 10 minutes, and washed five times with sterile distilled water. Following this treatment, the seeds were germinated and grown on half-strength Murashige and Skoog (1/2 MS) medium (pH 5.8–6.0). The plates were transferred to a growth chamber at 22°C for germination.
Gene expression analysis using qPCR
cDNA samples were obtained as described above. qPCR was performed in a CFX Connect Real-time PCR system (Bio-Rad, Hercules, CA, USA) using a Super Real PreMix (SYBR Green) kit (Tsingke Biotech, Beijing, China) according to the manufacturer’s instructions. The primers used for qPCR were designed according to the ZmNF-YA12 cDNA sequence (FP: 5′-AGCAACCTCCATTTGCGAGTCA-3′ and RP: 5′-GGCTGCCCAAACATCTCCTGAT-3′). Each reaction was performed in triplicate, and the results are expressed relative to the expression levels of Actin (FP: 5′-GGTAACATTGTGCTCAGTGGTGG-3′ and RP: 5′-AACGACCTTAATCTTCATGCTGC-3′) and GAPDH (FP: 5′-CCCTTCATCACCACGGACTAC-3′ and RP: 5′-AACCTTCTTGGCACCACCCT-3′) in each sample using the 2−ΔΔCT method.
Vector construction and transformation of Arabidopsis
The ZmNF-YA12 cDNA was cloned into the pCAMBIA-3301 vector driven by the cauliflower mosaic virus (CaMV) 35S promoter (Supplementary Fig. 1), as confirmed by sequencing. The resulting construct was transformed into Arabidopsis Col-0 by the floral dip method using Agrobacterium tumefaciens GV3101. The first generation (T0) seeds of transgenic Arabidopsis were screened on 1/2 MS medium containing 50 mg/L kanamycin, and transgenic plants were confirmed by PCR and qPCR (FP: 5′-AACTCATCTGCGGCTTGG-3′ and RP: 5′-GTATAATTGCGGGACTCTAATC-3′; and FP: 5′-AGCAACCTCCATTTGCGAGTCA -3′ and RP: 5′-GGCTGCCCAAACATCTCCTGAT -3′, respectively). Homozygous plants of the T3 generation were used for further analysis.
Root growth assay
For root growth assay, transgenic and wild-type (WT) seeds were placed on 1/2 MS agar plates for germination. Seven days later, five germinated seedlings of the same size from each line were carefully transferred to 1/2 MS agar plates supplemented with 150 mM NaCl, 150 mM mannitol, 50 μM JA, or 10 μM ABA. Seedling root lengths were measured using ImageJ software (NIH, Bethesda, MD, USA) after 8 days of upright growth in treatment medium.
Drought and NaCl treatment of transgenic Arabidopsis
Drought and NaCl tolerance assays were performed on seedlings grown in pots in a greenhouse. Transgenic and WT seeds were germinated on 1/2 MS medium. One-week-old seedlings were planted in 7-cm pots containing mixed soil (vermiculite: humus = 1:1) of equal quality and well-watered for 3 weeks. For drought stress treatment, the seedlings were subsequently cultured without watering for 3 weeks and then re-watered for 2 days. For NaCl stress treatment, the plants were irrigated with a solution containing 450 mM NaCl for 1 week. Drought and NaCl tolerance experiments were performed in triplicate.Samples of Arabidopsis leaves were collected after the seedlings exhibited distinct phenotypes under drought and salt treatments. The peroxidase (POD) activity, proline, malondialdehyde (MDA), and chlorophyll contents were measured using a commercial assay kit (Solarbio, Beijing, China) according to the manufacturer’s instructions.
Yeast two-hybrid assays
For yeast two-hybrid analysis, the ZmNF-YA12 cDNA was cloned into the bait plasmid pGBKT7 (pGBKT7-ZmNF-YA12). The full-length cDNAs of ZmNF-YB7 (GRMZM2G169884_T01), ZmNF-YC1 (GRMZM2G089812_T01), ZmNF-YC15 (GRMZM2G124421_T01), ZmNF-YC17 (GRMZM2G311316_T01), ZmCOI1(GRMZM2G151536), and ZmMYC2 (GRMZM2G049229) were separately cloned into the target pGADT7 plasmid. The bait and target plasmids to be tested for interactions were co-transformed into the yeast strain AH109 and plated on synthetic defined (SD) medium lacking leucine and tryptophan (SD/-LT) for screening transformants. The independent transformed colonies were then grown on SD medium lacking leucine, tryptophan, adenine, and histidine (SD/-AHLT). Survival of the yeast colonies on SD/-AHLT medium indicated that the target gene can interact with ZmNF-YA12.
The experiments were repeated three times and the data are presented as the mean ± SEM. The significance of the differences in the data was determined using SPSS statistical software (v. 25.0; SPSS Inc., Chicago, IL, USA). In all analyses, p < 0.05 was taken to indicate statistical significance.