All experiments were performed in three biological replications.
Isolation of GmNFYA5
Total RNA was isolated from soybean (Glycine max L. Merr.) cultivar Williams 82 by a previously described method . The full-length cDNA of GmNFYA5 was obtained by PCR with KOD-Plus DNA polymerase (TOYOBO, Japan) and the following primers: F 5’-GTAAGTGCGACTCTAAGCAAGCCT-3’ and R 5’-TAATGTAAATGAGCCAAGGATGACT-3’. The amplified products for sequencing were purified and cloned into the pEASY-Blunt vector (TransGen, China).
Plant growth and treatments
Cultivar Williams 82 was grown in plastic pots (15 cm diameter, 20 cm depth) containing a mixture of peat and vermiculite (1:1, v/v) in a greenhouse at 28/18°C day/night, 70% relative humidity and a 14/10 h light/darkness photoperiod . Twenty-day-old seedlings were used for evaluation of expression patterns. For drought treatment, the whole plant was removed, washed and placed in a laminar flow hood for gradual drought exposure for 12 h . For ABA treatment, the roots were subjected to 100 μM ABA for 12 h. For both treatments, the soybean leaves were collected at 0, 1, 2, 4, 8 and 12 h timepoints and immediately frozen by liquid nitrogen for isolation of RNA. To understand whether ABA was involved in drought-induced transcription of GmNFYA5, detached leaves were placed in H2O for 1 h to eliminate the influence of the wound stress, and then treated with distilled water or 1 mM naproxen (ABA synthesis inhibitor) for 3 h, followed by drought treatment for 2 h . Leaves floated in H2O for the entire period constituted the normal control. The soybean leaves were collected and immediately frozen by liquid nitrogen for isolation of RNA.
To assay transcript levels of GmNFYA5 in various tissues, the roots, stems, cotyledon, leaves and apical buds of 20-day-old soybean seedlings were sampled and immediately frozen by liquid nitrogen for isolation of RNA. Roots, stems, cotyledon, leaves, apical buds, flower buds and flowers of 50-day-old soybean plants were also sampled and immediately frozen for isolation of RNA. All the experiments were carried out in a greenhouse at 28/18°C day/night, 70% relative humidity and a 14/10 h light/darkness photoperiod.
Generation and drought treatment of 35S:GmNFYA5 Arabidopsis lines
The CDS of GmNFYA5 was cloned into the NcoI site of a vector named pCAMBIA1302 and driven by CaMV35S promoter using primer set 5’-GGGACTCTTGACCATGATGAAGAACTTATGTGAG-3’ and 5’-TCAGATCTACCATGGCCATAAGGACTGATAGACG-3’. The pCAMBIA1302:GmNFYA5 construct was introduced into Agrobacterium tumefaciens strain GV3101 which was used to infect Arabidopsis using the floral dip method. Positive transgenic Arabidopsis lines were screened by Hygromycin (Roche, Germany) to select T1 plants and T2 homozygotes.
Transgenic Arabidopsis lines (35S:GmNFYA5-1, 35S:GmNFYA5-2 and 35S:GmNFYA5-5) and ecotype Columbia-0 (WT) seedling were used in this study. Seeds were surface-sterilized with 70% ethanol and thrice washed with sterile water, followed sterilization with 1% sodium hypochlorite for 15 minutes and again washing three times with sterile water. The seeds were sown on half-strength Murashige and Skoog medium (1/2 MS, 2% sucrose, 0.8% agar). After 2 days at 4°C in darkness they were placed in a tissue culture room at 22°C and 70% relative humidity with a 16/8 h light/darkness photoperiod. For drought treatment, 3-week-old seedlings which had been transferred to plastic pots (8 cm in length, width and depth) containing a mixture of peat and vermiculite (1:1, v/v) for 7 days were deprived of water until they became wilted, after which they were irrigated and recovered for 7 days.
To investigate the transcript levels of marker genes under conditions of control, drought and drought + NAP in 35S:GmNFYA5 Arabidopsis plants, leaves of 3-week-old 35S:GmNFYA5 and WT seedlings were placed into H2O for 1 h to eliminate the influence of the wound stress, following by placement in H2O or 1 mM NAP solution for 3 h, and then transferred to a laminar flow hood for 2 h as drought treatment. The leaves of 35S:GmNFYA5 and WT seedlings floated in H2O for the entire period were the normal control. Leaves were sampled and immediately frozen by liquid nitrogen for isolation of RNA. All the treatments were performed in a greenhouse at 22°C and 70% relative humidity with a 16/8 h light/darkness photoperiod.
To further explore the functions of GmNFYA5, 35S:GmNFYA5 Arabidopsis lines were investigated under normal and drought conditions. For the normal control, 3-week-old 35S:GmNFYA5 and WT Arabidopsis plants were planted with well-watered treatment and 0.35 g H2O g-1 dry soil (soil water potential is -70 kPa) was maintained as the soil water content. For drought treatment, Soil water content was deprived of water to 0.20 g H2O g-1 dry soil (soil water potential is -280 kPa). The pots in both treatments were weighed daily and adjusted with water to maintain the target soil water potential until harvest . All the treatments were performed in a greenhouse at 22°C and 70% relative humidity with a 16/8 h light/darkness photoperiod.
The soybean seeds were provided by Dr. Li-Juan Qiu, Institute of Crop Science, Chinese Academy of Agricultural Sciences. The seeds of Arabidopsis were purchased from ABRC (https://abrc.osu.edu/researchers).
The coding sequence (CDS) of GmNFYA5 without the termination codon was fused in frame to the N-terminus of GFP in the vector p16318GFP, and ligated with BamHI site to generate a p16318GFP:GmNFYA5 fusion construct under the control of CaMV35S promoter using primer set 5’-TATCTCTAGAGGATCCATGAAGAACTTATGTGAG-3’ and 5’-TGCTCACCATGGATCCCATAAGGACTGATAGACG-3’. The CDS of GmNFYA3 encoding a nuclear-localized protein  was cloned into the EcoRI site of a vector pLVX-IRES-mCherry using primer set 5’-TCTATTTCCGGTGAATTCATGCAAACTGTTTATCTT-3’ and 5’-ACTAGTCTCGAGGAATTCAACTTTAAGGTTGCAGCA-3’. The GmNFYA3:mCherry fusion protein was used as a nuclear marker. Arabidopsis protoplasts were prepared as described . Transfected protoplasts were incubated in darkness at 22°C for 16-18 h, GFP fluorescence signals were observed with a confocal laser scanning microscope (Zeiss, LSIM 700) .
The promoter of GmNFYA5 was amplified from the DNA of soybean cultivar Williams 82 with primers F 5’-AAGAGGAACACAGAAGTCTATGAGT-3’ and R 5’-GCACATCAGATTCAGAGGAAGTCCC-3’. The products were introduced into a reconstructive pCAMBIA1305 vector (GFP coding region was replaced by GUS coding region) incorporating EcoRI and NcoI sites with the forward primer 5’-CCATGATTACGAATTCAAGAGGAACACAGAAGTC-3’ and reverse primer 5’-CTCAGATCTACCATGGCTCACATAAGTTCTTCAT-3’. The construct was introduced into A. tumefaciens strain GV3101 and transferred into Arabidopsis Col-0 plants by the floral dip method. Positive transgenic Arabidopsis lines were screened by Hygromycin to obtain homozygous lines.
Germination and root growth assays
Sterilized seeds were sown on 1/2 MS medium with 8-10% PEG 6000 (PEG) and 0.5-0.8 μM ABA respectively and placed in a tissue culture room at 22°C and 70% relative humidity with a 16/8 h light/darkness photoperiod after stratification at 4°C for 2 d in darkness. The germination rates were recorded every 12 h until completion. To investigate root growth of the 35S:GmNFYA5 Arabidopsis lines, 3-day-old seedlings were exposed to 1/2 MS medium with 10-12% PEG and 0.5-1 μM ABA. A week later, the root lengths were measured.
A. rhizogenes-mediated transformation of soybean hairy roots
The CDS of GmNFYA5 was inserted into the pCAMBIA3301 vector incorporating NcoI and BstEII sites with the following primers: F 5’-GGACTCTTGACCATGATGAAGAACTTATGTGAG-3’ and R 5’-ATTCGAGCTGGTCACCCATAAGGACTGATAGACG-3’. A 635 bp synthetic RNAi hairpin fragment (Figure S2) was introduced into the pCAMBIA3301 vector and ligated with the same restriction sites. The pCAMBIA3301:GmNFYA5, pCAMBIA3301 empty vector and pCAMBIA3301:RNAi-GmNFYA5 construct were introduced into A. rhizogenes K599 and used to infect the hypocotyls of 5-day-old soybean seedlings in a tissue culture room at 28/18°C day/night, 70% relative humidity and a 14/10 h light/darkness photoperiod, and then hairy roots were induced for two weeks . The positive hairy roots transformants were screened using a QuickStix kit for PAT/bar (EnviroLogix, America) and qRT-PCR. Transgenic soybean lines having positive hairy roots were named OE-GmNFYA5 (OE), empty vector (EV) and RNAi-GmNFYA5 (RNAi) respectively and transferred to plastic pots (11 cm depth, 13.5 cm diameter) containing a peat and vermiculite mixture (1:1, v/v) to grow for 7 days, followed by deprivation of water until wilting.
To analyze the transcription of drought-related genes under conditions of drought and drought + NAP in transgenic soybean plants, three transgenic hairy roots lines were placed into H2O for 1 h to eliminate the wound stress, following by placement in H2O or 1 mM NAP solution for 3 h, prior to transfer to a laminar flow hood for 2 h as drought treatment. Roots floated in H2O for the entire period were the normal control. All treatments were carried out in a tissue culture room at 28/18°C day/night, 70% relative humidity and a 14/10 h light/darkness photoperiod. Roots were sampled and immediately frozen by liquid nitrogen for isolation of RNA.
Analysis of transcript levels
Transcript levels were measured by qRT-PCR performed with TransStart Top Green qPCR SuperMix (TransGen, China) (20 μl) according to the manufacturer’s instructions on an Applied Biosystems 7500 real-time PCR system. Gene-specific primers designed by https://biodb.swu.edu.cn/qprimerdb/ and used for qRT-PCR assays are listed in Table S1.
Measurement of water loss
Leaves of 3-week-old Arabidopsis lines were detached, and the weight was measured every 30 min in a tissue culture room at 22°C and 70% relative humidity. Percentages of initial fresh weight at 9 timepoints were used to represent water loss in transgenic and WT plants.
ABA concentration and stomatal aperture analysis
Following deprivation of water for 7 day, the ABA concentrations of leaves of soybean and Arabidopsis plants were measured by means of an ABA ELISA assay kit (Jiancheng, China) as described .
Leaves of 3-week-old 35S:GmNFYA5 and WT Arabidopsis seedlings were treated for 3 h in stomatal opening buffer (5 mM MES, 10 mM KCl, 50 mM CaCl2, pH 5.6) as described previously . The leaves were then transferred to H2O or 10 μM ABA solution for 2 h in a greenhouse at 22°C and 70% relative humidity. Stomatal apertures of stomatal complexes with no surrounding mesophyll cells were measured.
Measurement of malondialdehyde (MDA), relative water content (RWC) and ion leakage
RWC and ion leakage were measured in 3-week-old Arabidopsis plants following drought treatment for 14 days. MDA contents, RWC and ion leakage in transgenic soybean plants were measured following drought treatment for 16 days. MDA contents were measured and calculated as described previously [58, 62]. RWC and ion leakage were determined as described previously [63, 64].
Measurement of soil water potential (SWP)
When the transgenic soybean and Arabidopsis plants were shut off water supply, the SWP was measured in drought treatment trials until it reduced to the minimum level using WP4-T dewpoint meter (Decagon Devices, USA) according to the manufacturer’s instructions. WP4-T dewpoint meter had been used in research due to its accuracy in several reports [56, 65].
Transcriptional activation assays
The promoters of GmDREB2 (ABA-related gene) and GmbZIP1 (ABA-unrelated gene) were cloned into the LUC reporter vector pGreen II 0800 containing the Renilla luciferase (REN) gene driven by the CaMV 35S promoter and used as an internal control. The effector and reporter plasmids were extracted and introduced into Arabidopsis protoplasts using PEG4000-mediated transformation. The assays were performed as described .
All the measurements in this study were replicated three times biologically. Variance analyses of all data were preformed using SPSS Statistics 22 (IBM, USA) for a completely randomized design model. Duncan’s tests was used to evaluate differences among plant lines or treatments at P = 0.05.