Plant materials and growth conditions
B-efficient B. napus ‘QY10’ (winter-type B. napus in China), the RNAi lines of BnaC4.BOR1;1c (Zhang et al., 2017), transgenic Arabidopsis (Col-0 background) were used in this study. For the pot growth of B. napus, seeds were sown in the soil, which includes 0.85 g NH4NO3 kg-1, 383.3 mg KH2PO4·2H2O kg-1, 250 mg MgSO4·7H2O kg-1, 751.1 mg KCl kg-1. Pots were irrigated with 1,500 ml ultrapure water supplemented with micronutrients (3.5 mg 11B, 12.60 mg MnCl2·4H2O, 1.54 mg ZnSO4·7H2O, 0.56 mg CuSO4·5H2O, 0.168 mg Na2MoO4·2H2O, 1.75 mmol Fe-EDTA) to maintain proper humidity. For the inflorescence tracer B experiment, the inflorescences with stem segments about 15 cm in length were cut and cultured two days in a hydroponic box with 5 μM 10B. For the hydroponic growth of B. napus, seeds were surface-sterilized. They germinated one week in ultrapure water and then were transferred into a quarter-strength solution for 5 days growth followed by 5 days half-strength solution treatment and full-strength solution culture. Modified Hoagland’s solution was used in hydroponic growth (Li et al., 2019), in which 100 µM 11B was used. For the tracer B experiment of seedlings, seedlings of B. napus were washed with ultrapure water and then treated in 0.25 µM 11B for 1 day followed by one-day growth in 5 µM 10B. For the Arabidopsis culture, a solid MGRL medium was prepared (Fujiwara et al., 1992) with 1% gellan gum and 1% sucrose. Transgenic Arabidopsis lines were established by the Agrobacterium-mediated in planta method (Clough and Bent, 1998). To generate chimera B. napus plants, a scion cut at 1-2 cm under cotyledon was inserted into a rootstock at the position 1 cm above the cotyledon. A silicon tube was used to support vertical growth. The grafted plants were placed in a transparent box with very high artificial humility. After one-week, grafted plants were successfully established.
To construct ProBOR1;1c (5’-UTR): GUS and ProBOR1;1c (∆5’-UTR): GUS, promoter sequences of BnaC4.BOR1;1c with or without 5’-UTR were amplified from ‘QY10’ DNA by PCR reaction with specific primers (Table S1), then fused with ScaI and SmaI digested PBI121 fragment using In-Fusion Cloning kits (Clontech). To construct Pro35S 5’UTR: GUS, Pro35S 5’UTR∆1-29: GUS, and Pro35S 5’UTR∆1-97: GUS, truncated 5’ UTR sequences were amplified by PCR reaction with specific primers (Table S1) and were fused with XbaI digested PBI121using In-Fusion Cloning kits (Clontech). To generate Pro35S 5’UTR∆1-29: GUS, Pro35S 5’UTR: GUS was used as a template by PCR reaction using specific primers, and the PCR product was self-fused.
Gene expression analysis
To investigate GUS expression, 10-d-old Arabidopsis seedlings grown on 100 μM B were transferred to 0.25 μM B or 250 μM B medium for 2 d growth. Plants were harvested for sampling. RNA was extracted using TRIZOL Reagent (Invitrogen, CA, USA). Reverse transcription was carried out using M-MLVReverse Transcriptase (Promega) according to the manufacturer’s protocol. RT fluorescence quantitative PCR was performed using the SYBR Green Real-Time PCR Master Mix Kit (Toyobo, Japan) and the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Actin and Tubulin were used as internal controls with primers F: 5'-ACAGTGTCTGGATCGGTGGTTC-3', R: 5'-TGCCTCATCATACTCAGCCTTG-3' (ACTIN); F: 5'-CAGCAATACAGTGCCTTGAGTG-3', R: 5'-CCTGTGTACCAATGAAGGAAAGCC-3' (TUBULIN). Each gene’s relative expression level was calculated and normalized based on these two internal controls using the ΔΔCt method (Livak & Schmittgen 2001).
β-Glucuronidase (GUS) histochemical staining and quantification of GUS activity
β-Glucuronidase (GUS) histochemical staining of GUS reporter lines was performed using the GUS Histochemical Kit (bioshap, Cat. BL622A). The sample photos were taken using an Olympus SZX16 stereomicroscope. For the GUS activity assay, GUS line’s total protein was extracted using the GUS extraction buffer (50 mM sodium phosphate buffer (pH 7.0), 10 mM β-mercaptoethanol, 1 mM Na2-EDTA·2H2O, and 0.1% Triton X-100). After centrifugation at 12,000 rpm, the total protein concentration was determined by using the Bradford assay (Bradford, 1976). 50 μg of total protein was mixed with 450 μL MUG buffer incubated at 37°C. 40 μL interaction solution was obtained at 5 min, 15, 25, or 35 min, respectively, and mixed with 160 μL of 0.2 M Na2CO3 to stop the interaction. GUS fluorescence (excitation: 365 nm; emission: 455 nm) was measured using a microplate reader (TECAN Infinite M200). The standard curve was established by mixing 40 μL 4-MU (4-methylumbelliferone) and 160 μL 0.2 M Na2CO3, in which the 4-MU was set as 1/26 mM, 1/27 mM, until to1/212 mM).
Immunohistological staining of BnaC4.BOR1;1c
To perform in vivo immunohistological staining of BnaC4.BOR1;1c in B. napus, an antibody against BnaC4.BOR1;1c was obtained by purifying the rabbit antiserum, which was prepared through immunizing rabbits with an artificial peptide (SSTPLNNRSLSSPR). The immunohistological staining method (Ma et al., 2007) was used with modification. Briefly, B. napus seedlings (20-d-old), precultured in 25 μM B, were transferred to 0.25 μM B or 250 μM B for 2 d growth. The roots, basal node, and nodes (petiole junction) from ‘QY10’ and RNAi plants were cut as ~0.5-1 cm thickness and fixed in the solution at 4 ℃ (4% paraformaldehyde, 60 mM sucrose, and 50 mM Cacodylic acid). The samples were centrifuged at 7,000 rpm for 2 min. After exposed to room temperature for 2 h, samples were washed several times using 1 x PBS buffer, then embedded by 5% agar (Nakalai, Tesque, Kyoto, Japan) for solid at 4℃. 100 μm specimens were prepared using a semi-automatic slicer and stored in 1xPBS buffer. Then the specimens were incubated in the 1xPBS buffer supplemented with 0.1%(w/v) pectolyase+0.3%(w/v) Triton X-100 for 2 h. After washed with 1xPBS, the specimens were blocked in 5% (w/v) BSA/PBS for 10 min, followed incubated with primary antibody (anti-BnaC4.BOR1;1c, rabbit, 1:1000 dilution) for overnight. After washed with 1xPBS buffer, the specimens were incubated in secondary antibody (Alexa fluor 488-conjugated Goat anti-rabbit IgG (H+L) AS053, 1:2000 dilution) for 2 h without light. The specimens were then washed 5 times in 1 x PBS and mounted with an antifade polyvinylpyrrolidone mounting medium (Beyotime, Shanghai, China). Fluorescence imaging was examined in the Leica SP8 system (Leica, Mannheim, Germany). GFP was excited by 488 nm, and the signal was collected from 505-545 nm wavelength.
In situ PCR
The in situ PCR method (Athman et al., 2014) was used with modification. Briefly, fresh plant tissues were fixed overnight in FAA solution (63% ethanol, 5% acetic acid, and 2% formalin) without vacuum infiltration step. After three times washes by buffer (63% ethanol and 5% acetic acid) and one-time wash by 1xPBS buffer, the samples were embedded in 5% agar (Nakalai, Tesque, Kyoto, Japan) for solid at 4 ℃. 75 μm specimens were prepared using a semi-automatic slicer and stored in DEPC water containing 100 U RNaseOUT (Takara Bio, Japan), 2.5% DNase I (Promega, China) followed by 45 min incubation at 37ºC. Then 15 mM EDTA was used to stop the reaction at 70ºC for 15 min. After wash by cold DEPC water, the reverse transcription (Promega, China) was performed in the RT mix with BnaC4.BOR1;1c specific primer (4 μl 5 X buffer, 2 μl 10 mM dNTPs, 1 μl 0.1 M DTT, 10 μl H2O and 1 μl reverse primer at 65ºC for 5 min. Then 1 μl M-MLVReverse Transcriptase and 1 μl RNaseOUT were added into the tube on the ice, followed by 1 h incubation at 50ºC and 5 min at 85ºC. The negative control was prepared as above without a specific primer. Specimens were transferred into tube containing PCR mix [5 μl 10 x Taq buffer (Aidlab bio.), 1 μl 10 mM mixed dNTPs , 0.2 μl digoxigenin-11-dUTP (Roche; 4 μM), 2.5 μl 10 mM forward primer, 2.5 μl 10 mM reverse primer, 1 μl Taq polymerase (Aidlab bio), 38.3 μl H2O]. After twice washes by 1 x PBS, specimens were blocked 30 min in 1 x block solution at room temperature (10 mg BSA in 10 ml 1 x PBS). 1.5 U AP-conjugated anti-DIG Fab fragments (Roche) were added for 1 h incubation at room temperature. The specimens were washed twice for 15 min using buffer (0.1 M Tris-Cl; 0.15 M NaCl, pH 9.5) and then incubated in 50 μl BM purple (Roche) for 2 h. Twice washes by washing buffer to remove the blue solution, the specimens were mounted on the microscope slide for imaging.
Measurement of B concentration
All samples were dried at 65°C in an oven for 3-4 d and then ground into fine powders using carnelian mortar. 1 M HCl was used to digest dry powders on the shaker for 2 h at 250 rpm. The solution was filtered, and B concentration was measured by inductively coupled plasma mass spectrometer (ELAN DRC-e; Perkin Elmer, USA).
Statistical Analysis of Data
Data were analyzed using Student’s t-test and Duncan’s test. Significance was defined when P value < 0.05.