Plant materials and treatments
Sheepgrass (National certified variety Zhongke 1 from Institute of Botany, the Chinese Academy of Sciences, Beijing, China) was used for this experiment. The sheepgrass seedlings were grown in a plastic pot containing nutrition soil (Pindstrup Bubstrate, Denmark) and vermiculite (2:1, v/v) in the greenhouse at 27/23 ℃, 16 h light/8 h dark for 8 weeks before treatments. Abiotic stresses were performed as follows: the plastic pots containing the seedlings were submerged in 100 µmol/L for ABA treatment, 300 mmol/L mannitol for osmotic stress and 400 mmol/L NaCl for salt stress. Seedlings were transferred to a growth chamber at 4℃ for cold stress. The seedlings were sampled at 0, 1, 3, 8, 12 or 24 h after stress treatments, immediately frozen in liquid nitrogen and stored at -80℃ for RNA isolation. The abiotic stress experiments were excuted at least three times by different people to test the expression patterns of target genes. Stem, leaf, root, bud, panicle and rhizomes were also collected from 2-year-old sheepgrass seedlings for tissue-specific analysis.
Arabidopsis thaliana (A. thaliana; Columbia ecotype) seeds were surface-sterilized with 10% NaClO for 10 min, and then washed 5 times with sterile water. The sterilized seeds were planted on MS solid media (pH 5.8) for germination.
RNA isolation and expression pattern analysis of LcMYB2
Total RNA was extracted using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. qRT-PCR was carried out in triplicate according to the SYBR PremixExTaq™ protocol (TaKaRa, Dalian, China) on a LightCycler480 Real-Time PCR System (Roche, Rotkreuz, Switzerland) with the following program: 95 °C for 5 s and 68 °C for 30 s for 45 cycles. Tissue-specific expression of LcMYB2 was detected using semiquantitative PCR. All primers used in this research are listed in Additional files S4.
Amplification and sequence analysis of LcMYB2
First-strand cDNA for amplification of the 5’ and 3’ ends of LcMYB2 was synthesized with SMARTerTM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The gene-specific primer (GSP-5'RACE), designed according to the 454 high-throughput sequencing results, and universal primers (UPM) were used to amplify the 5’ end of LcMYB2. The putative full-length sequence of LcMYB2 was amplified using gene-specific primers (LcMYB2-F/R) designed according to the sequence assembled from the 5’-RACE and 454 sequencing results. The national center for biotechnology information (NCBI) database was searched for homologs of LcMYB2 using the BLASTX program. Multiple sequence alignment was executed in DNAMAN software (version 7.0) using the selected amino acid sequences. The phylogenetic relationships among the homologs were inferred using the maximum likelihood method based on the JTT matrix-based model in MEGA software version 6.0 [64, 65].
Subcellular localization and transcriptional activity assay of LcMYB2
The ORF of LcMYB2 was ligated into pCAMBIA1302 to form the LcMYB2-GFP fusion protein. The recombinant plasmid was introduced into Agrobacterium tumefaciens EHA105 using a freeze-thaw method and further transformed into A. thaliana using the floral dip method [66]. Positive seedlings were selected on solid MS containing 50 µg/L hygromycin (Roche) and further confirmed by PCR. T3 seeds of the transgenic plants were germinated on MS, and the GFP fluorescence in the roots was observed under a laser confocal scanning microscope (Leica TCS SP5). To assess LcMYB2 transcription activity, the ORF was inserted downstream of GAL-BD in the pBridge vector to obtain pBD-LcMYB2. The recombinant vectors were introduced into the yeast strain AH109, and positive transformants were selected on SD (-Trp) medium and confirmed by PCR. β-galactosidase activity was assayed according to the Yeast Protocols Handbook (Clontech).
Arabidopsis transformation
To reveal the biological function of LcMYB2, the ORF was fused to the p3301-121 vector (modified from vector pCAMBIA3301 and pBI121, donated by the Shen lab) under the control of the CaMV 35S promoter. The recombinant constructs were transformed into A. thaliana by Agrobacterium tumefaciens EHA105 using the floral dip method. Positive transgenic Arabidopsis seeds were screened on MS medium supplemented with 20 µg/L glufosinate ammonium and further confirmed by PCR using DNA extracted from the putative positive seedlings. T3 seeds were used for germination assays under different treatments.
Drought stress tolerance analysis of transgenic seedlings
To reveal the function of LcMYB2 at the germination stage, T3 seeds of transgenic and wild-type A. thaliana were planted on normal solid MS medium and solid MS medium supplemented mannitol (300 m mol/L) or ABA (0.25 µmol/L and 0.5 µmol/L). The Petri dishes were placed in a growth chamber at 22 ℃ with a 16 h / 8 h, light/dark photoperiod. Plants were photographed, and the germination rate, cotyledon greening rate and root length were and measured. Osmotic and ABA stress tolerance experiments were repeated at least three times.
To test the role of LcMYB2 at the seedling stage under drought conditions, 4-day seedlings were transplanted into a pot containing vermiculite and turfy soil (2:1, v/v) and grown in a growth chamber at 22 ℃ with a 16 h / 8 h, light/dark photoperiod for 1 week with sufficient water, then started to natural drought stress without water for the following 42 days. During the drought process, the soil water content was monitored every day in each pot. On the 42nd day, the seedlings were irrigated again, and survival rates were statistically analyzed after three days. There are 60 seedlings for drought stress experiment in each line (WT, L1, L2), respectively.
Measurement of lipid peroxidation and of proline and soluble sugar content
Four-week-old transgenic and WT A. thaliana seedlings were irrigated with 300 mmol/L mannitol. The leaves were sampled at 0 h and 9 h after treatment for gene expression analysis. Two days later, the leaves of transgenic and wild-type lines were harvested for physiological measurements. The level of MDAwas determined by a revised method described by Kramer et al. [67]. SOD content was measured with the nitro-blue tetrazolium (NBT) reduction method as previously described [68]. The contents of proline and soluble sugars were determined according to the protocols previously described by Shan et al [69] and Bailey [70], respectively. Three replicates were carried out for each assay, and the variability was indicated with the standard error (SE).
Chromatin immunoprecipitation assays
Four-week-old seedlings of sheepgrass were treated with 300 mmol/L mannitol for 8 hours and 24 hours to induce the expression of LcMYB2, after which time, the samples were harvested. Six-week-old seedlings of A. thaliana overexpressing LcMYB2 (L1: line 1, L2: line 2) were tested. All samples were fixed with formaldehyde for CHIP analysis. Antibodies against LcMYB2 were prepared by Beijing Protein Innovation Co., Ltd. The EpiQuik Plant ChIP kit (Epigentek, Brooklyn, NY) was used for CHIPanalysis. CHIP DNA was detected with universal PCR (40 cycles of 95℃, 30 S; 68℃, 30 S) and qPCR (45 cycles of 95℃, 5 S; 68℃, 30 S). The method of comparing Ct values was adopted to analyze the qPCR CHIP efficiency.
Statistical analysis
Analysis of variance (ANOVA) and t-test were used to compare the differences between samples. *** indicates p-values < 0.001, ** indicates p-values < 0.01) and * indicates p-values < 0.05.