Plant materials and treatments
This study used seeds of salt sensitive and salt tolerant proso millet cultivars (SS 212 and ST 47, respectively). The seeds were surface disinfected (0.1% HgCl2, 5 minutes), rinsed (sterile water, three times), sprouted (3-4 days), and cultured in a hydroponics system until the emergence of three leaves (about 10 days). The culture was done in a seedling pot (12.4 cm × 17.5 cm) under 14h light/ 10h dark cycles, day/night temperatures of 28/18 °C, and relative humidity of 60%. Seedlings were stressed by ½ Hoagland’s nutrient solution containing 0% (control) and 1% NaCl for 7 days and then transplanted into ½ Hoagland’s nutrient solution for another 7 days. Roots were subsequently harvested after 0, ½, 1, 3, and 7 days of salt treatment, and day 7 of subsequent re-watering (RW 7 d). The culture solutions were changed every two days throughout the growth period.
Analysis of root system architecture
Three biological seedling replicates were harvested at 0, ½, 1, 3, and 7 days of watering, and RW 7 d, and then divided into aboveground parts and roots. The root systems of the six sample types were scanned with an Epson Perfection V700 Pro scanner (Seiko Epson Corp, Suwa, Japan). Image data were analyzed by the WinRHIZO software (Reagent Instruments Inc., Quebec, Canada) to quantify root parameters, including the total root length (TRL), root surface area (RSA), and root tip number (RTN).
Measurement of biomass and mortality
To assess effects of salt stress on vegetative growth, the biomass was determined using standard methods recommended by the Organization for Economic Co-operation and Development [20, 21]. The roots and shoots were separated from the seedlings, and dried at 60°C to a constant weight in three biological replicates, with 15 seedlings per replicate to determine biomass. Mortality was determined under 7-day salt stress and subsequent re-watering for 7 days. Seedlings were considered to have dead when they heart leaves lose green.
Determination of growth parameter and root physiological parameters
The levels of root soluble compounds (soluble sugars [SS], soluble proteins [CPr], and proline [PRO]), ascorbate peroxidase (APX), and malondialdehyde (MDA) were determined by the respective Assay Kits (Beijing Solarbio Science & Technology, Beijing, China), according to the manufacturer’s instructions. Root vigor (RV) was determined using a 2,3,5-triphenyl tetrazolium chloride (TTC) assay, as described previously [22].
Determination of Na+, K+, Ca2+, and Mg2+ concentrations
All dry root samples were pulverized using a grinder (A 11 basic Analytical mill, IKA, Staufen, Germany), then 8 ml of nitric acid (HNO3) and 2 ml of 30% (m/m) hydrogen peroxide (H2O2) were added to a 0.20 g sample. Subsequently, the root samples were digested using a microwave digester (Milestone Inc, USA). The contents of Na+, K+, Ca2+, and Mg2+ were detected by an atomic absorption spectrophotometer PinAAcle500 (PerkinElmer, Waltham, USA).
Extraction and determination of cell wall composition
The cell wall was extracted from root, as described previously [23]. The pectin content was determined according to IFJU Method 26. The pectin was coagulated with ethanol in a constant temperature water bath at 85°C for 10 min, centrifuged for 15 min at 4,000 rpm, discarded supernatant, and washed with ethanol repeatedly until there was no sugar left. After coagulation and purification, the pectin mixture was added in 0.5 ml 1M NaOH, and adjusted to 10ml. After the extracting, the pectin mixture was flocculated with carbazole-ethanol, H2SO4, measured at 525 nm using Multiskan GO (THERMO, Boston, USA), and quantified using a D-Galacturonic acid monohydrate (Sigma-Aldrich) standard curve. The hemicellulose and cellulose contents were determined using the anthrone method [24, 25, 26]. The absorbance of the solution is measured with the Multiskan GO (THERMO, Boston, USA) at 620 nm and converted to hemicellulose and cellulose contents using a D-Glucose (Sigma-Aldrich) standard curve. The lignin content was determined using acetyl bromide method [27]. The absorbance of the solution is measured with the 1510-04201 Thermo Fisher Microplate Reader (Waltham, USA) at 280 nm.
Microstructural analysis of the root
Paraffin sections of the root tips were prepared as described previously [28] to examine the root cell structure. Briefly, 3μm thick root transections were cut and taken through the experimental process, mainly including dehydration, dip waxing, embedding, slicing, and safranin O-fast green staining. Changes in root structure were observed and photographed under epifluorescence microscope (Carl Zeiss, Ober-kochen, Germany). Moreover, root tips were prepared for transmission electron microscope (TEM) using the method described by Meng et al. [29]. Ultrathin sections were stained with uranyl acetate, and lead citrate then examined under a TEM (HT7700, Hitachi, Ltd. Tokyo, Japan).
RNA extraction, cDNA library construction, and digital RNA sequencing
Total RNA was extracted from the root tissues of three replicates for each treatment time points using an RNA Plant Plus Reagent (Tiangen, Beijing, China), according to the manufacturer’s instructions. RNA purity was checked using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, CA, USA), and RNA was resolved on a 1% agarose gel to check for contamination and degradation. The total RNA integrity was assessed on an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and then interrupted into short fragments by a fragmentation buffer. Complementary DNA (cDNA) was synthesized using random hexamer primer, M-MuLV Reverse Transcriptase (RNase H), and DNA Polymerase I. The remaining overhangs were then converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends, NEBNext Adaptors with a hairpin loop structure were ligated to the cDNA to prepare for hybridization and barcoded with a large set of barcode sequences [30]. Then, cDNA fragments (200-250 bp in length) were purified by an AMPure XP system (Beckman Coulter, Beverly, USA), and PCR was performed using a Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer. At last, PCR products were purified by the AMPure XP system, and library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
All the 36 libraries were sequenced on an Illumina HiseqTM 4000 platform (Illumina, CA, USA) using the PE-150 module, and paired-end 150bp reads were generated. The adaptor sequences and low-quality reads were filtered by the Trimmomatic tool (v0.33) to clean raw reads [31]. The clean reads obtained after data processing were mapped to the Panicum miliaceum L. genome sequence using TopHat (v2.1.0), and gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped (FPKM) values.
Analysis of differentially expressed genes (DEGs)
Differentially expressed genes (DEGs) after ½, 1, 3, and 7 days of salt treatment and RW 7 d were identified using the DESeq R package (v1.10.1) by comparing the expression levels at these time points with those at 0 days. In addition, to evaluate for DEGs among the two cultivars, the gene expression levels of SS 212 were compared with those of ST 47. Genes with a minimal 2-fold changes (|log2 (FC) |≥1) and a false discovery rate (FDR) of p ≤0.05 were identified as significantly differentially expressed between the two time points [32].
GO and KEGG pathway enrichment analyses
The GOseq R package (v1.12) and KEGG Orthology Based Annotation System software (KOBAS, v2.0) were used for the analysis of DEGs in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively, and a corrected P-value<0.05 was chosen as the significance cutoff [33, 34].
Validation of RNA-Seq data by RT-qPCR
We conducted RT-qPCR to validate the RNA-Seq results of 10 randomly selected genes from the roots of both proso millet cultivars. The RNA of three independent biological replicates from each treatment time point was isolated using the RNA Plant Plus Reagent (Tiangen, Beijing, China) according to the manufacturer’s protocols. The RNA was reverse transcribed for cDNA synthesis using PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Kusatsu, Japan), following the manufacturers’ protocols. RT-qPCR was conducted using gene-specific primers designed using the Primer Express (v5.0) software and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) according to the manufacturer’s protocols. Relative gene expression was calculated by the comparative CT (2-△△CT) method using the Actin gene as a reference.
Data statistics and analysis
All results were reported as means ± standard error, and experiments were performed in three biological replicates. The data were analyzed by the SPSS software version 23.0 (Inc., Chicago, Ill., USA). Duncan’s multiple comparison test was used at the P < 0.05 level to evaluate significant differences among treatment means.