Plant materials
The aseptic seedlings of two diploid landraces CIP-149 (Solanum phureja) and CIP-178 (S. ajanhuiri) from International Potato Center (CIP) were used as experimental materials. The stem segments with one axillary bud were cultured on solid Murashige and Skoog (MS) medium (pH 5.8) with 3% (w/v) sucrose at 25 ± 2 ℃ under a 16 h light photoperiod for 30 d.
Observation of the accumulation of O2·-, H2O2 and total ROS in stolons/tubers during four stages of tuber formation by histochemical staining
Single node seedling stems without leaves were inoculated on solid ½ MS medium containing 5.5% (w/v) sucrose under tuber-inducing conditions [in total darkness (TD) at 18 ± 2°C] for 14 d. The top part (about 1 cm in length) of stolons/tubers including four typical successive morphological stages of tuberization (Aksenova et al. 2012; Lei et al. 2021) was used for histochemical staining.
The amounts of O2·− and H2O2 were assessed using the NBT (nitroblue tetrazolium chloride, BioFroxx) and DAB (3,3ʹ-diaminobenzidine, BioFroxx) staining methods, respectively (Kim et al. 2007; Kumar et al. 2014) with minor modifications. Stolons/tubers were stained by incubating in 50 mM sodium phosphate buffer (pH 7.5) containing 0.2% (w/v) NBT for 0.5 h and DAB staining solution (1 mg/mL DAB solution in 50 mM sodium phosphate buffer, pH 3.8) for 5 h in amber colored bottles at room temperature. The blue precipitate or brownish red precipitate indicate the relative amounts of O2·− and H2O2, respectively. Stained stolon/tuber samples were rinsed by sterile water, and all images were obtained with a dissecting microscope (Olympus, SZX7).
Total ROS accumulation in stolons/tubers was examined using the fluorescent probe H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate, Sigma) staining method (Guan et al. 2019) with minor modifications. Stolons/tubers were incubated in 50 µM H2DCFDA for 3 h. After rinsing with distilled water, all images were obtained with an inverted fluorescence microscope (Olympus, DP73).
Quantitative determination of O2·- production rate and H2O2 levels in stolons/tubers
The O2·− production rate in the tip (about 1 cm in length) of stolons/tubers during the four stages of tuber formation (Lei et al. 2021) were determined using the XTT (Na,3'-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate) method (Li and Gong 2013a) with minor modifications. Briefly, stolons/tubers (1 g) were ground in 3 mL of extracting solution [50 mM Tris-HCl buffer (pH 7.5), containing 1% (w/v) polyvinylpyrrolidone (PVP), 1 mM EDTA, 1.1mM XTT] with a multi-sample tissue grinding machine. The extracts were centrifuged at 10 000 rpm for 20 min; an aliquot of 1.5 mL of supernatant was incubated with 1.5 mL of 50 mM Tris-HCl buffer (pH 7.5) and 300 µL of XTT (final concentration of 100 µM). After reaction at 30 ℃ for 30 min, the absorbance at 470 nm was read with a microplate reader (Naritech Technologies, China) and calculated using the molar extinction coefficient of 2.16 × 104 L mol− 1 cm− 1. The O2·− production rate was expressed as nmol min− 1·g− 1 FW.
The H2O2 content was determined as by the xylenol orange method (Li and Gong 2013b) with some modifications. Stolons/tubers (0.5 g) were homogenized in 2 mL of cold acetone. After centrifugation at 10 000 rpm for 10 min, 1 mL of the supernatant was mixed with 3 mL of extraction agent (CCl4:CHCl3 3:1, v/v). Then, 5 mL of distilled water was added to the mixture and centrifuged at 5000 rpm for 1 min. The absorbance of the upper water phase was measured at 560 nm. The H2O2 content was expressed as µmol·g− 1 FW.
Effects of exogenous H2O2 and the ROS inhibitor DPI or H2O2 scavenger CAT treatments on tuber formation
To investigate the effect of exogenous H2O2 on potato tuberization, the single-node cuttings of CIP-149 were incubated on solid ½ MS medium containing 3% (w/v) sucrose supplemented with 0 (control), 1, 5, 10, 20, 30, 50, and 100 mM H2O2 in TD at 18 ± 2°C for 14 d.
DPI is considered as NADPH oxidase inhibitor and inhibits ROS production, and catalase (CAT) can break H2O2 into H2O and O2 (Hung et al. 2005; Wei et al. 2018). In the present experiments, DPI (Selleckchem) was dissolved in dimethyl sulfoxide (DMSO, BioFroxx), and DPI solution was added into the ½ MS medium with the final concentration of 0 (control), 10 and 20 µM. The control was appropriately treated with an equivalent amount of DMSO. CAT (Sigma- Aldrich) was dissolved in PBS and added into the ½ MS medium with the final concentration of 0 (control), 50, 100, 150, and 200 U mL− 1. Single stem segments of the pre-cultured plantlets of CIP-149 were cut and planted on solid ½ MS medium containing 3% (w/v) sucrose with DPI or CAT in TD at 18 ± 2°C. The tuberization frequency was calculated after 14-day incubation.
Analysis of key tuberization-related genes by RT-qPCR
Single-node explants without leaves from potato CIP-149 were used for tuber induction on solid ½ MS medium containing 3% (w/v) sucrose and supplemented with 0 (control) or 5 mM H2O2 in TD at 18 ± 2°C. At different time points (0, 3, 9, 12, and 16 d), tips (about 1 cm in length) of stolons/tubers were cut off, and RNA was isolated from five typical developmental stages of tuber formation, ranging from axillary buds, slightly larger axillary buds, about 1 cm long stolon tips, swelling stolon tips, and the growing tubers (Fig. 1). The images of different time points (0, 3, 9, 12, and 16 d) were obtained with a dissecting microscope (Olympus, SZX7).
Total RNAs was extracted using a Tiangen RNAprep Pure Plant Plus Kit, and 800 ng total RNAs were reverse-transcribed into first-strand cDNA using a PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, Beijing, China). Quantitative real-time PCR (qPCR) was performed in 10 µL reaction volume (1 µL template cDNA, 0.4 µL each upstream and downstream primer, 5 µL SYBR Green PCR Master Mix and 3.2 µL ddH2O) with TB Green® Premix Ex Taq TM Π (TaKaRa, Beijing, China) on Roche Light Cycler 960 (Roche Diagnostics, Basel, Switzerland). The expression of 9 key tuberization-related genes [StSP6A, Solanum tuberosum sucrose transporter4 (StSUT4), potato homeobox (POTH), Solanum tuberosum calcium-dependent protein kinase1 (StCDPK1), phytochrome B (StPHYB), Solanum tuberosum BEL5 (StBEL5), Solanum tuberosum CONSTANS (StCO), Solanum tuberosum GA 20-oxidase1 (Stga20ox1), and Solanum tuberosum respiratory burst oxidase homolog (Strboh)] selected based on references (Lei et al. 2021; Zierer et al. 2021; Nicolas et al. 2022); Park et al. 2022) was determined. The reference gene L2 (cytoplasmic ribosomal protein) (Nicot et al., 2005) were used to normalize target gene expression. The primers were designed by Primer3 web (https://bioinfo.ut.ee/primer3-0.4.0/). The primer sequences used for RT-qPCR are listed in Supplementary Table S1. The RT-qPCR program was set up for 600 s preincubation at 95 ℃, 2-step amplification of 45 cycles at 95 ℃ for 10 s and 55 ℃ for 15 s, following a 55 to 97 ℃ melting curve analysis at the final step. Three independent biological repetitions and three parallel reactions were conducted in RT-qPCR. And the comparative CT method (2−ΔΔCT method) was used to quantify the RT-qPCR data (Livak and Schmittgen 2001).
Construction of knockout vector for StSP6A null-mutants
Plasmid construct
The binary vector pKESE401 expressing SpCas9 and intermediate vector pCBC-DT1T2 were kindly provided by Qijun Chen (China Agricultural University, Beijing). The gene sequences of StSP6A of potato (gene ID: Soltu.DM.05G026370.1) were downloaded from the Spud DB database (http://solanaceae.plantbiology.msu.edu). Two 20-nt single guide RNA sequence for StSP6A were selected using the CRISPR-P tool 2.0 (http://crispr.hzau.edu.cn/CRISPR2/), and the primers for constructed knockout vector were DT1F, TCGAAGTAGTGATTGGGTTGCATAACAACTTGTGAGTTTTAGAGCTAGAAATAGC and DT2R, TTCTAGCTCTAAAACTTCACTAGGTCTGTTGATCTCAATCTCTTAGTCGACTCTAC. DT1F and DT2R were used to amplify the pCBC-DT1T2. After purification, the PCR product was incorporated in the vector pKESE401 which was digested by BsaI using the Seamless Cloning and Assembly Kit (ClonExpress II One Step Cloning Kit, Vazyme) (Xing et al. 2014). The constructed CRISPR/ Cas9 vector was shown in Fig. 2a.
Agrobacterium tumefaciens-mediated Transformation of potato
CIP-149 was used in this study, and A. tumefaciens transformation followed the protocol reported previously (Ye et al. 2018; Yang et al. 2020) with modifications as follows: after 2 days of pre-culture, the explants were co-cultured with agrobacterium harbouring pKSE401 with the target sequence for 2 days in the presence of 2 mg·L− 1 α-naphthaleneacetic acid and 1 mg·L− 1 zeatin, and followed by callus induction mediated by 0.1 mg·L− 1 α-naphthaleneacetic acid 2 mg·L− 1 zeatin for 2 weeks and regeneration mediated by 0.01 mg·L− 1 α-naphthaleneacetic acid and 1 mg·L− 1 zeatin until shoot proliferation. Transformants were screened based on growth on the medium containing 50 mg·L− 1 kanamycin. The transformants’ DNA were amplified by specific primers spanning the target sites, which were 5’-AGGCGGCATGTCTTCTAGAG-3’ and 5’-TAAACCCCTCTACCCCTCCA-3’. PCR amplicons were cloned into pBM16A Vector (Biomed, Beijing), then transformed E. coli competent. 10 colonies were selected from each transformants’ DNA for sequencing at Sangon Biotech. Geneious 4.8.3 software was used to analysis the sequences.
Homozygous StSP6A null-mutants were obtained
Potato explants were infected by A.tumefaciens containing the constructed CRISPR/ Cas9 vector described above. A total of 125 transgenic plants were obtained. The target sites of each transgenic plant were sequenced with 10 clones per plant, and a total of 3 homozygous StSP6A null-mutants which were display the same mutation type (sp6a85, sp6a107 and sp6a 113) were obtained. The types of mutations were shown in Fig. 2b.
H2O2 treatments of the StSP6A null-mutants sp6a85, sp6a107 and sp6a113
To investigate the effect of exogenous H2O2 on potato tuberization of StSP6A null-mutants, the single-node cuttings of CIP-149 (control) and StSP6A null-mutants (sp6a85, sp6a107 and sp6a113) were incubated on solid ½ MS medium containing 3% (w/v) sucrose supplemented with 0, 5, 20, and 50 mM H2O2 in TD at 18 ± 2°C for 14 d, and tuberization frequency was investigated.