Plant materials and growth conditions
In this study, wild type Arabidopsis was used as a control. The WT and mutant seeds were evenly seeded on 1/2 MS medium with corresponding antibiotics. The culture dish was placed at low temperature for 3 days and cultured in the tissue culture room (22℃-16 h light/18℃-8 h dark), and the screened positive seedlings were transferred to nutrient soil for further cultivation until the seeds mature. The harvested seeds were seeded with the same operation in 1/2 MS medium with salt, and the phenotype of the plants was observed.
Sweet sorghum (Sorghum bicolor (L.) Moench) cultivar M-81E was used in this experiment. The seeds of sweet sorghum were cultured in sand. Tap water was irrigated before emergence and 1/2 Hoagland nutrient solution was poured every day after emergence. When the seedlings grew to three leaves and one heart, Hoagland nutrient solution containing 0, 50, 100, 150 and 200 mM NaCl was applied to sweet sorghum under salt stress.
Cloning, bioinformatics and expression analysis of SbbHLH85
The SbbHLH85 CDS sequence is obtained by comparing the AtRSL2 sequence on the Ensembl website (http://ensembl.gramene.org/). Online website NCBI (https://www.ncbi.nlm.nih.gov/), SMART (http://smart.embl-heidelberg.de/) and software MEGA6 for nucleic acid and protein sequence analysis and the construction of evolutionary trees.
Root tissue of sweet sorghum was used for cloning and expression of SbbHLH85. Samples were immediately frozen in liquid nitrogen and stored -80℃ before analysis. Three biological replicates were performed for RT-qPCR. Sweet sorghum internal reference gene Sbactin-1 as a control, the primers are listed in supplementary table 1.
Subcellular localization of SbbHLH85 protein
Using KpnI and BamHI sites to insert cloned SbbHLH85 CDS into pCAMBIA1300-35S-sGFP vectors to produces 35 S：SbbHLH85-GFP constructs. It was transferred into Agrobacterium tumefaciens GV3101 and used to infect tobacco epidermal cells. GFP signal was observed by two-photon laser scanning confocal microscope (TCS S8MP, leica, germany). 35S: GFP transgenic tobacco was used as a localization control for expression in cytoplasm/nucleus. The primers are listed in supplementary table 1.
Generation of transgenic plants
To produce overexpressed SbbHLH85 of Arabidopsis, SbbHLH85 genes were linked into pROKII vectors with XbaI and KpnI and transferred to Agrobacterium tumefaciens GV3101 to obtain SbbHLH85 overexpressed plants by infecting WT inflorescence. The transgenic Arabidopsis plants were screened with kanamycin (50 g/mL) and verified by RT-PCR. Insert the full-length cDNA of SbbHLH85 into the pMWB110 vector through BamHI and KpnI sites to obtain pMWB110-SbbHLH85 vector. pMWB110-SbbHLH85 vector was introduced into sweet sorghum by Agrobacterium-mediated transformation. PCR, herbicide (glufosinate) spraying and bar rapid detection kit were used to detect transgenic plants. The primers used are listed in supplementary table 1.
Quantification of biomass, MDA content, Na+ and K+ content
For the measurement of biomass, we first take the whole plant out of the pot, wash it, weigh it and record it as fresh weight. Then they were dried in the oven for 7 days and then weighed again as dry weight. Fresh weight and dry weight of each treatment were measured 5 times (Song et al., 2019). The MDA contents were determined as described by Ma(Ma et al., 2013). As follows: the leaves of 0.2 g of each line were weighed, 5 ml 0.1% trichloroacetic acid (TCA) was added for grinding. Mix the homogenate with 5 ml 0.5% thiobarbituric acid (TBA), boil for 10 min and take it out, cool to room temperature, 3000 rpm/min centrifuge 15 min, absorb the supernatant and measure its volume. The absorbance of the solution at wavelength 532 nm and 600 nm was determined by UV spectrophotometer. Blank control was 0.5% TBA solution. For the determination of Na+ and K+, the specific steps are described by Song: each line treated with 0 and 100 mM NaCl. After 10 d, put 0.3 g roots in 5 ml ddH2O, boil 2 h, filter plant residue and volume to 10 ml. The content of Na+ and K+ of each treated lines was determined by flame spectrophotometer (Song et al., 2020).
DAB and NBT staining
When Arabidopsis seedlings growing for about a month, treated with 0 or 100 mM NaCl in 1/2 concentration Hoagland solution for 48 h. Before staining leaves of rosette leaves of WT and overexpressed plants with the same growth, put in DAB or NBT dye solution, immerse the dye in the leaves, placed in the dark for more than 12 hours. Then put in the bleach (3:1:1 ethanol: acetic acid: glycerol), boil in boiling water for 10-15 minutes for decolorization, observe the color change of the blade and take the image.
Root hair experiment
The homologous gene of SbbHLH85 is AtRSL2 and AtRSL4 in Arabidopsis. AtRSL2 is closely related to root hair development and elongation, and it also has a functional redundancy gene AtRSL4. So we selected WT, M-81E, overexpression (At-OX4、At-OX13, Sb-OX1, Sb-OX3, Sb-OX6, Sb-OX7), RSL2 mutant (rsl2-1、rsl2-3), RSL4 mutant (rsl4), and RSL2 and RSL4 double mutant (rsl2rsl4) as experimental subjects, the main root hair development and root hair elongation were tested. Each Arabidopsis seed was on demand in 1/2 MS medium. The sweet sorghum seeds are hydroponic. After 7 d in the lab, development and elongation of root hairs at the root tip 5 mm from the main root of each line were observed under electron microscope. The microscope was magnified by 40 times.
The roots of WT, overexpression, mutant and double mutant were collected and preserved in liquid nitrogen. RNA-seq and differential gene expression analysis were carried out by BMK. Transcriptome analysis of 24 samples was completed, and 156.37 Gb of clean data was obtained. Use the HISAT2 system to sequenced the clean reads of each sample with the designated reference genome, and the reads on was assembled by StringTie comparison.
After the comparison and analysis, the reads on the sample were assembled and quantified by StringTie comparison. Based on the comparison results, the gene expression was analyzed. StringTie uses FPKM (fragments per kilobase of script per million fragments mapped) as an index to measure the expression level of transcripts or genes. The differentially expressed genes were identified according to their expression levels in different samples, and through GO database, KOG database and KEGG database for function annotation and enrichment analysis. KEGG pathway analysis was carried out for the common differential genes in each comparison group, and the heat map clustering analysis was performed for the expression level of selected genes, which were all completed on the platform of BMKCloud.
RT-PCR validation of genes related to plant salt resistance and root hair development
We extracted RNA from root tissues of WT, At-OX13, rsl2-3 and rsl2rsl4 lines which had been growing for about one month, and then reverse them into cDNA and then conduct RT-PCR. The primers used are in supplementary table 2. In sweet sorghum, M-81E and Sb-OX1 were treated with 0 mm and 100 mM NaCl for 48 h. Then RNA was extracted and transformed into cDNA, RT-PCR was performed. The primers used are in supplementary table 3.
Yeast two-hybrid experiment
The CDs of SbbHLH85 were cloned into the bait vector pGBKT7 to obtain SbbHLH85-BD, and then transformed into yeast strain Y2HGold (Clontech). After detecting that the gene has no self-activating activity, we constructed a yeast two-hybrid library of sweet sorghum, and hybridized and screened according to the matching scheme described in Clontech's matchmaker tmgoldyeasttwo hybrid user's manual. After screening, the interaction between SbPHF1 and SbbHLH85 was verified by yeast two-hybrid experiment. The full-length coding sequence (CDS) of SbPHF1 was cloned into pGADT7 to obtain SbPHF1-AD, and SbbHLH85-BD and SbPHF1-AD were co-transformed into Y2HGold. Then the growth of the colony on the corresponding medium was observed. See supplementary table 1 for the primers used in yeast two-hybrid.
The CDs of SbbHLH85 and the N-terminal of pSPYNE-35S were fused to obtain SbbHLH85-N-YFP, and the CDs of SbPHF1 and the C-terminal of pSPYNE-35S were fused to obtain SbPHF1-C-YFP. The obtained plasmid was introduced into Agrobacterium tumefaciens (GV3101), and the method of transient transformation was used to infiltrate the tobacco. After 48 hours of normal culture, the fluorescence was observed under a confocal laser scanning microscope (Olympus). Supplementary table 1 lists the primers of BiFC.
The statistical results are described as mean ± standard deviation. Use the SPSS ver. 17.0 statistical software to analyze the data. One-way ANOVA was used as the designated package. Different letters indicate that there is a significant difference between the average (0.05) of the Duncan test.
The sequence data in this paper can be found in TAIR, NCBI or Ensembl database. The mutant numbers are as follows: rsl2-1 (SALK_048849), rsl2-2 (SALK_ 048857), rsl2-3 (SALK_ 101872), rsl2-4 (SALK_143203). The accession numbers are as follows: SbbHLH85 (SORBI_3008G147800), SbPHF1 (SORBI_3002G060900), SbPYL4 (XM_002465362), SbPIN3 (XM_002436716), SbRSH2 (XM_021465244), SbRSL4 (XM_002464182), SbRHL1 (XM_021450304), SbPER3 (XM_002437414), SbGLO1 (XM_002466296), SbPER4 (XM_002441658), SbPER35 (XM_002466362), SbRLK1 (XM_021446421), SbRLK2 (XM_021466234), SbRLK8 (XM_002455281), AtPIN3 (AT1G70940), AtSAUR50 (AT4G34760), AtPYL6 (AT2G40330), AtPRX33 (PRX33), AtPRX37 (AT4G08770), AtRCI3 (AT1G05260), RSL4 (AT1G27740), RHL1 (AT1G48380), RHS18 (AT5G22410), RSH3 (AT1G54130), RHS15 (AT4G25220), RHS19 (AT5G67400), RHD3 (AT3G13870), RHS17 (AT4G38390), RHD6 (AT1G66470), RHS10 (AT1G70460), RHS12 (AT3G10710), CLH1 (AT1G19670), DGK5 (AT2G20900), CYP97C1 (AT3G53130), OSM34(AT4G11650), PR4(AT3G04720), PDC1(AT4G33070).