Plant Materials and growth conditions
Accessions of CCRI12, CCRI12XW, CCRI12YW, L7, L7XW, TM-1, T582, were provided by Cotton Research Institute, the Chinese Academy of Agricultural Sciences (CAAS) (Anyang, China). CCRI12 (China Cotton Research Institute 12) and L7 (LiaoMian 7) are Upland cotton cultivars with dark-colored pigment glands and high content of gossypol in both plants and seeds. While CCRI12XW, CCRI12YW, and L7XW, which have glandless and low gossypol content in both seeds and plants, are dominant glandless near isogenic lines (NILs) that differ primarily in the gland trait of CCRI12 and L7, respectively[49]. ‘TM-1’, which is widely used as a genetic standard, and it is the glanded accession of the seeds and the whole plant, ‘T582’ is an accession with glandless-stem and glanded-leaf of plant traits. All materials were maintained by self-crossing for several years in our lab.
The seeds were immersed in water and followed by germination in a high humidity environment at 28°C in the dark for 2d. Well-germinated seeds were subsequently planted in 0.3 L pots of 7cm diameter with one seed per pot in a commercially available sand/soil/fertilizer mix and grown for two to three weeks at 28°C (16h light and 8h dark) with LED lamps (Opple lighting Zhongshan China ) in a greenhouse.
Extraction of total RNAs
Samples from different tissues of the cotton plants, including cotyledon, hypocotyl, petiole, true leaf and stem of one or many different gland accessions, served as the source of total RNA were immediately frozen in liquid nitrogen and stored at -80°C. For each sample, total RNAs were isolated from 100 mg of leaf ground with liquid nitrogen using the RNAprep Plant RNA kit (polysaccharides&polyphenolics-rich) (TIANGEN BIOTECH (BEIJING)CO., LTD) according to the manufacturer’s instructions. The quantity and purity of RNAs were assessed according an absorbance ratio of OD 260/280 (1.9-2.1) using a NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi (Thermo Fisher Scientific Inc., Waltham, MA, USA) ultraviolet spectrophotometer, and was confirmed using 1.0% (w/v) denatured formaldehyde agarose gel electrophoresis to investigate its integrality.
Synthesis of the first-strand cDNA
RNA was reversely transcribed into 1st strand cDNA in a 20μL reaction volume using the PrimeSeriptTM1stStrand cDNA Synthesis Kit (TaKaRa Bio, Dalian, China) following the manufacturer’s protocol of Reverse Transcription System. Firstly, two micrograms (2μg) of total RNA was mixed with 1.0μL Oligo dT Primer (50μm), 1.0μL dNTP mixture (10mM each), then RNase free ddH2O was added to make the whole reaction volume up to 10μL, then, A total 10μL reaction volume was incubated at 65℃for 5min and placed on ice for 2 min to denature probable RNA secondary structure. Secondly, the first-strand cDNA synthesis mixture was prepared by adding following components to the above 10μL reaction volume in the indicated order, 4μL 5xPrimeScript II Buffer, 0.5μL RNase Inhibitor (40U/μL), 4.5μL RNase free ddH2O, and1μL Primescript 1I RTase (200U/μL). The first-strand cDNA synthesis mixture was incubated at 30℃for 10 min, 42℃ for 60min and terminated at 95℃for 5 min.
Molecular cloning of GhERF105 gene
The full-length cDNA sequence of GhERF105 was amplified from the leaves was cloned from the leaves of CCRI12. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and GENEWIZ (Suzhou) Co., Ltd. high-fidelity DNA polymerase, dNTPs and other reagents were supplied by TaKaRa Bio, (Dalian) Co., Ltd. PCR amplification of the GhERF105 gene was performed in a reaction volume of 15μL containing 1μL of template cDNA, 1.2μL of dNTP Mixture(2.5mM), 3μL of 5×PrimeSTAR GXL Buffer, 0.3μL of Prime STAR GXL DNA Polymerase, 0.4μL of forward primer and 0.4μL of reverse primer(10μΜ for each), and 8.7μLddH2O. The amplification product was achieved using the following profile: 5 min at 98°C; 35cycles of 10 s at 98 °C, 15s at 55℃,1 min at 68°C and a final cycle of 5 min at 68°C; hold at 10°C. The PCR products were then purified following the instructions in the QIAquick PCR Purification Kit (250) (Qiagen, Düsseldorf, Germany) and eluted in a final sample volume of 35µL of Qiagen EB buffer. three microliters of each PCR product were assessed by size on a 1% agarose gel to select fragments in the range of 700bp ± 50bp. The amplified products were cloned into the pBI121 vector for sequencing (Sangon, Shanghai, China or Genewiz, Suzhou, China). The primers used for GhERF105 cloning were listed in Supplementary Table S1.
Identification of GhERF105 gene and its promoter in Gossypium hirsutum
The amino acid sequences and AP2 domain of GhERF105 were obtained from CottonFGD (https://cottonfgd.org/about/download.html) and Prosite(https://prosite.expasy.org/)respectively, The predicted molecular weight and isoelectric points of GhERF105 protein were calculated using the ExPASy program (http://web.expasy.org/protparam/). The G-box of the promoter of GhERF105 were obtained from PLANTCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Gene Expression Analysis by Quantitative Real-Time PCR
The RNA sequencing samples that were isolated were used to perform real-time quantitative (qRT-PCR) analysis using the ABI Quantstudio 5 Detection System (Applied Biosystems, Carlsbad, CA). Actin (GenBank accession numbers: AY305733) was used as reference gene. The gene-specific primers with about 215bp product size were designed using the Primer 5.0 software or online in NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and listed in Table S1. The specificity of each primer set was validated by melt-curve analysis, and the efficiency was calculated by analyzing the standard curves with a tenfold cDNA dilution series (Bustin et al., 2009). The 20μL qRT-PCR experiment was carried out on the ABI Quantstudio 5 Detection System with TB Green Premix Ex TaqTM (Tli RNaseH Plus) (TaKaRa Bio, Dalian, China). The reaction volume contains 0.5μL of each primer (10μM), 0.4μL ROX Reference DyeII (50x), 1μL above synthesized cDNA template, and 7.6μL of sterilized ddH2O. The qRT-PCR thermal cycling conditions were 95°C for 5 min to pre-denature cDNA template; 40 amplification cycles of 95℃ for 5S, 55℃ for 30S, and 72℃ for 30S; and followed by 15s at 95°C, 1 min at 60°C, and 15 s at 95°C. Each sample was run in triplicate, each biological replicate was assessed three times. The relative expression level of the genes was calculated according to the 2−ΔΔCTmethod [50]. For the reference gene used in this experiment, their geometrical mean was operated at first, and then the relative transcript level of target gene was calculated following the method of one reference gene. Results were generally expressed mean ± standard error (ER) from values three independent tests. The primers used for expression analysis were provided in Supplementary Table S2
VIGS procedure
The virus-induced gene silencing (VIGS) vector tobacco rattle virus (TRV) invades a wide range of hosts and it is able to spread vigorously throughout the entire plant but produces only mild symptoms [51]. Therefore, VIGS system has been proven to be a powerful tool in elucidating gene function and functional genomics in cotton [10, 33, 34-37, 52, 53]. To knockdown the expression of GhERF105, The pTRV-VIGS vectors were constructed using a previously published method [52, 54]. Briefly, cDNA fragments of cotton PDS (GhPDS1, 327bp, GenBank accession numbers: HQ441184) and Pigment gland formation GhERF105 (337bp) were amplified using Prime STAR GXL DNA Polymerase (TaKaRa) from CCRI12 by PCR with gene-specific primers (listed in the table 2). The resulting products were cloned into pTRV2 with BamHI and KpnI to produce recombinant vectors named pTRV2::PDS and pTRV2::GhERF105, respectively. These recombinant vectors and the empty vector (pTRV2::00) were then introduced into the Agrobacterium strain GV3101(Weidi Bio, Shanghai, China) by heat shock method, For the VIGS assay, the transformed Agrobacterium colonies containing pTRV1 , pTRV2-GhPDS, and pTRV2-GhERF105 were grown overnight at 28℃ in an antibiotic selection medium containing rifampicin, gentamicin and kanamycin 50mg/ml. They were subsequently suspended in the solutions (10mM 2-(N-morpholino) ethane sulfonic acid, 10mM MgCl2 and 400µM acetosyringone (AS)) to the final optical densities as OD values of 1.5 at 600nm and then left at 25℃ for 4h without shaking in the dark. Before infiltration, Agrobacterium cultures containing pTRV1 and pTRV2 or its derivatives were mixed in 1:1 ratios. Seedlings with the fully expanded cotyledons but without a visible true leaf of CCRI12 were infiltrated by inserting the Agrobacterium suspension containing pTRV1 and pTRV2, pTRV2-GhPDS, pTRV2-GhERF105 into the cotyledons via a syringe. Plants were grown in the pots at 25℃ in a growth chamber under a 16h light/8h dark photoperiod with 70% humidity. To analyze silencing efficiency, RNA was extracted and qRT-PCR was performed. The Actin (GenBank accession numbers: AY305733) and GhERF105 was amplified as reference gene and target gene, respectively [55]. Leaves were numbered sequentially such that number 1 refers to the first true leaf initiated after the cotyledons. In this study, leaves 2-3 were investigated and collectively referred to as total foliage [56]. All primers used in this experiment were listed in Supplementary Table S3.
Gossypol detection and analysis
The total gossypol concentration in the leaves from CCRI12, GhERF105-silenced CCRI12 and CCRI12XW plants was determined by high-performance liquid chromatography (HPLC) (Agilent 1100, Agilent, Santa Clara USA). Each 100 mg plant sample, which was freeze-dried and ground into powder with liquid nitrogen. was dissolved with 2ml leaf extraction (acetonitrile/water/phosphoric acid=80:20:0.1). The leaf extraction was centrifuged at 10000rpm for 10 min and then the supernatant was carefully transferred into a new EP tube at room temperature. The eluent was filtered through a 0.45μm nylon filter into a vial for HPLC analysis with Agilent Zorbax Eclipse Plus C18 analytical column (250mm×4.6mm, 5micron). The sample was analyzed at a wavelength of 235nm. The concentration was calculated using Agilent 1100 system by comparing to the gossypol standard curve. A gossypol reference standard was purchased from Sigma Chemical Co. Ltd.
Subcellular Localization of GhERF105 Protein
To study the subcellular localization of GhERF105 protein, the coding regions of GhERF105 was amplified with stop codon removed by Primers listed in Supplementary Table S4, which contained a XbaI and SmaI site (underlined) through polymerase chain reaction (PCR), The resulting fragments were cloned between the XbaI and SmaI site of the transient expression pBI121-GFP vector, which harbors an ORF encoding the green fluorescent protein (GFP) under the control of the CaMV35S promoter, and construct the recombinant plasmid p35S-GhERF105-GFP. p35S-GFP was used as positive control. The plasmids of GFP-GhERF105 and GFP were then introduced into tobacco leaves (Nicotiana benthamiana) respectively via Agrobacterium-mediated transformation and incubated at 25℃ under light for 48-72h. The green fluorescence signals were observed and the localization of the fusion protein was determined using a confocal laser scanning microscope (Leica TCS SP8, Germany).
Transactivation Activity Assay of GhERF105 Protein
To study the transactivation activity of GhERF105 protein, GhERF105 cDNA was amplified with Primers listed in Supplementary Table S5 and cloned into the EcoRI and NotI sites of pGBKT7 vector to generate pGBKT7-GhERF105 construct. This plasmid with empty vector control was then transformed into yeast strain AH109 to analyze the transactivation activity. Yeast transformants with OD600 of 0.1, 0.01and 0.001 were plated on the selective media, SD/-Trp and SD/-Trp/-X-a-gal, and incubated at 30°C for 4 days.
Ethylene treatment
Ethephon (ET), which emits ethylene when dissolved in water, was used as a substitute for ethylene. Leaves from normally grown 3-to 4-week-old plants were used during the trefoil stage. Compared to the leaves sprayed with the water as negative control, Ethylene treatment was performed by spraying the leaves with the mixture of 1mM/L ethephon (Solarbio Bio, Beijing, China). before leaf tissue was sampled, All the control and treated plants were enclosed in plastic bags for different time and place in a sealed chamber at 25℃with a 16-h-light/8-h-dark photoperiod. The whole plants were harvested 0, 2, 4, 6, 8, 10, 12 and 24h after treatments. immediately frozen in liquid nitrogen and stored frozen at -80℃until use.
Statistical analyses
All experiments were repeated at least three times, and the results represent the mean ± standard deviation (SD) of three replicates.Statistical significance of the data was evaluated using one-way ANOVA using GraphPad Prism 8.0 or the SPSS software (version 22.0). A P-value < 0.05 was considered significant. A P-value < 0.01 was considered highly significant.