Plant material and growth conditions
Japonica rice (Oryza sativa L. japonica. cv. Nipponbare) was selected as the WT in this study. Rice seeds were sterilized with 0.1% NaClO for 30 min before being soaked in distilled water for 2 days in the dark and were then transferred to a culture dish containing Hoagland nutrient solution and grown in a climate chamber (Southeast instrument, Ningbo, China) with the temperature of 28°C,the relative humidity of 70%,and the 14-hour light/10-hour dark photoperiod.
To determine the expression pattern of OsWRKY97 under different stress conditions, two-week-old seedlings of Nipponbare rice were subjected to various stress treatments. For drought treatment, seedlings were grown in culture solution containing 20% PEG6000. For salt treatment, NaCl solution was added to achieve a final concentration of 250 mM. ABA treatment was carried out by adding 50 µM ABA to the culture solution. The seedlings were transferred to a 4°C climate chamber for cold treatment. For heat stress, the seedlings were subjected to 42°C heat shock treatment[48, 49]. Samples were collected at 0, 1, 2, 4, 8, 12, 16 and 24 h after treatment. Two-week-old seedings of OsWRKY97 overexpression lines were subjected to drought treatment, and samples were collected at 0h and 10h after treatment respectively.
RNA extraction and real-time PCR
The leaves of 14-day-old plants were sampled, and total RNA was extracted with TRIzol reagent (Invitrogen, Nanjing, China) according to the manufacturer's instructions. One microgram of DNase-treated RNA was reverse-transcribed using a RevertAid RT Reverse Transcription Kit (TaKaRa, Beijing, China) according to the manufacturer’s protocol. Real-time PCR was performed on a Bio-Rad CFX96 real-time PCR system. Each reaction was performed in triplicate, and the reaction procedure was as follows: 95°C for 10 min and, then 39 cycles of 95°C for 10s and, 60°C for 30s. The data of relative expression level were analyzed by the 2 -△△Ct method. The OsActin rice gene was used as an internal control gene, and relevant primer pairs are listed in Additional file 2:Table S1.
Vector construction and gene transformation
To construct the overexpression vector of OsWRKY97, cDNA from rice (Oryza sativa L. japonica. cv. Nipponbare) was used. Total RNA was used to amplify the open reading frame of OsWRKY97, and the relevant primer pairs are listed in Additional file 2:Table S1. The fragments confirmed by sequencing were digested and cloned into the pCAMBIA1300 vector, and the digestion sites were HindΙΙΙ and SalΙ. This gene was driven by the CaMV35S promoter (Additional file 3:Figure S2). All the vectors were transferred into Nipponbare plants via Agrobacterium-mediated transformation.
Determination of stress-associated physiological indicators
Seedlings growing under normal conditions for two weeks were transferred to a solution with or without 20% PEG6000. Samples were collected at 0 h and 10 h, and then, the corresponding physiological indexes were measured. MDA content was qualified by thecreaction method as described by Gao. H2O2 content was determined according to the method of Zhan. For the water loss rate, two-week-old seedlings were selected, exposed to air under the condition of no water supply and weighed and recorded every half hour until the weight was constant. Water loss rate was calculated by comparing the measured weight from each indicated time with the measurement at time zero. It is expressed as percentage of initial fresh weight. Proline was detected by following the reported methods. The activity of active oxygen scavenging enzymes was determined according to previous methods.
Measurement of ABA content
We analyzed the content of ABA in rice by liquid chromatography-mass spectrometry (LC-MS). Briefly, 1 g of frozen leaf tissue was extracted in 10 ml of acetone/water/acetic acid (80:19:1, v/v). The complete homogenate was incubated overnight in darkness at 4°C. After that, they were vortexed and centrifuged at 15,000 rpm, 4°C for 10 minutes, and the crude extract supernatant was collected and dried in in a rotavapor until an aqueous fractions were obtained. Dried samples were resuspended in 1 mL of acetonitrile/water/acetic acid (90:10:0.05, v/v), and ﬁltered through a 0.45 µm PTFE ﬁlter (JET BIOFIL, Guangzhou, China). Quantiﬁcation was done by the standard addition method by spiking control plant samples with ABA solutions.
Subcellular localization of OsWRKY97
The coding region of OsWRKY97 was amplified and cloned into the pAcGFP1-N1 vector to generate the OsWRKY97-GFP (green fluorescent protein) fusion construct, which was inserted into the pCAMBIA1300 vector, and the constructed vector was transferred into Agrobacterium. After sequencing confirmation, the fusion structure and the control (empty body) vector were co-transfected with another NLS-RFP vector respectively into tobacco cells by Agrobacterium mediated transient expression method, and the fluorescence signal was observed by confocal laser scanning microscope.
Analysis of stress tolerance
Two independent T2 over-expression lines and WT seeds were soaked in clear water for two days after being sterilized by 0.1% NaClO, and then, the seeds were transferred to culture solution for growth. When the plant height was approximately 1 cm, seedlings with similar growth potential were selected and transferred to nutrient solution containing 20% PEG, and their growth conditions were observed and recorded. To simulate an arid environment, the two-week-old seedlings were transferred to the sand, and watering was stopped after two days of normal irrigation. The mortality rates were determined. In order to test the sensitivity of ABA sensitivity at germination stage, seeds of OE plants and WT plants were germinated on culture solution containing 5 µM ABA, and the germination rate was calculated on the 6th day after germination.
Results are reported as the mean±standard deviation (SD) values of the three independent biological experiments, all experiments were repeated at least three times, Statistical analysis was performed using the Student’s t-test by SPSS 18.0 (Chicago, IL) sofware package, ∗p < 0.05, ∗∗p < 0.01.