Arabidopsis thaliana ecotype Col-0 and the T-DNA insertion mutants in FKBP15-1 and FKBP15-2 were provided by the European Arabidopsis Stock Centre . Due to the limited number of FKBP15-2 mutant, RNA interference (RNAi) plants of FKBP15-2 were also generated. The FKBP15-2 specific coding region was amplified using primers carrying attB sites (Additional file 6: Table S1) and recombined into the pHellsgate12 vector to generate the RNAi construct . The construct was transformed separately into Col-0 and fkbp15-1 plants by the floral-dip method. Transgenic plants were then self-pollinated to obtain T3-generation homozygotes of the fkbp15-2-silenced plants. The fkbp15-1fkbp15-2 double mutant and fkbp15-1fkbp15-2RNAi plants were then used in the functional analyses.
Two vectors carrying the 35S::FKBP15-1-eYFP::NOS and 35S::FKBP15-2-eYFP::NOS protein fusion cassettes were transformed into Agrobacterium tumefaciens strain GV3101. The resulting strains were then used to transform A. thaliana Col-0 plants to generate the FKBP15-1 and FKBP15-2 overexpression plants, which were self-pollinated to produce the T3-generation homozygotes.
To generate the triple mutant, a plant homozygous for the vin2 gene mutation (SALK_100813) was crossed with the fkbp15-1fkbp15-2 double mutant. After self-pollination of the F1, triple mutant plants (fkbp15-1fkbp15-2vin2) were generated and confirmed by PCR.
RNA extraction and quantitative real-time PCR (qRT-PCR) assays
The tissues (root, stem, leaf, and flower) sampled from 40-day-old seedlings of the Col-0 wild-type and the different mutants and RNAi lines were ground to powder in liquid nitrogen. Total RNA was isolated using the RNA prep-pure Plant Kit as described by the manufacturer (Tiangen, China). RNA samples were quantified by absorbance at 260 nm using the Nanodrop spectrophotometer (Thermo Fisher Scientific, USA), and the relative purity was assessed by the A260/280 ratio. For cDNA synthesis, 1 μg samples of total RNA were treated with DNase I to remove contaminating genomic DNA, and then reverse-transcribed into 1st-strand cDNA using the PrimeScript RT reagent Kit as directed by the manufacturer (Takara, Japan).
The qRT-PCR assays were performed on a LightCycler 96 system (Roche, Switzerland) using the SYBR Premix Ex-Taq kit (Takara, Japan). The amplification conditions were: an initial denaturation at 95°C for 10 min, followed by 15-25 cycles of 95°C for 15 sec, 55°C for 15 sec, and 72°C for 30 sec, after which samples were returned to room temperature. mRNA of the housekeeping gene UBQ10 was used as internal control. Relative transcription levels were calculated using the comparative 2-ΔΔCT method . All experiments were repeated at least three times for reproducibility. The DNA sequences of all primers are given in Additional file 6: Table S1.
Expression profile analysis
To analyse the expression patterns of the FKBP15-1 and FKBP15-2 genes, their promoter regions (1213bp for FKBP15-1 and 1180bp for FKBP15-2) were amplified and inserted into pCAMBIA1305 vector to generate the ProFKBP15-1::GUS::NOS and ProFKBP15-2::GUS::NOS expression constructs. The constructs were transformed into Col-0 plants, and the transgenic plants were verified by PCR.
GUS activity in the transgenic plants was detected histochemically using a previously-described method . Whole 10-day-old seedlings were incubated in GUS staining solution (100 mM phosphate buffer, pH 7.0, 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, 10 mM EDTA, 0.1% Triton X-100, and 2 mM 5-bromo-4-chloro-3-indolyl ρ-o-glucuronic acid) for two hours at 37°C in the dark. The stained samples were washed twice in 70% (v/v) ethanol then examined and photographed using a light microscope (Olympus BX51, Japan).
To determine the expression patterns of FKBP15-1 and FKBP15-2 in roots in detail, GUS-stained roots were fixed overnight in 1% glutaraldehyde and 4% paraformaldehyde (pH 7.4). The samples were dehydrated in a graded ethanol series and embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany). The embedded samples were cut into 10 mm sections with a microtome (Leica EM UC7, Germany), and prepared for observation and imaging with a differential interference contrast (DIC) microscope (NIKON 80i, Japan).
Subcellular localization of FKBP proteins and BIFC analysis
Subcellular localization assays were performed using a previously-described method . Agrobacterium strain GV3101 separately carrying the 35S::FKBP15-1-eYFP::NOS, 35S::FKBP15-2-eYFP::NOS, and ER marker ER-rk expression constructs were grown at 28°C overnight. The ER-rk was created by ﬁrst inserting ER retaining signal at the C-terminus of the mCherry and subsequently adding the signal peptide of AtWAK2 at the N-terminus. The liquid cultures were collected by centrifugation at 2,000 g for 10 min, and the pellets were re-suspended in MS medium and adjusted to OD600=0.5-0.6. A final concentration of 200 μM acetosyringone and 10 mM MES (pH 5.6) were added, and the bacterial suspensions were kept at room temperature for at least 3 hours without shaking prior to infiltration into N. benthamiana leaves. Three days after infiltration, the fluorescence signals in leaf epidermal cells were visualized using a Leica SP5 confocal microscope.
For BiFC assays, full-length FKBP15-1 and FKBP15-2 were fused with C-terminal YFP in the vector pEarleygate202-YC, while full-length VIN2 was cloned into vector pEarleygate201-YN and both were transformed into Agrobacterium strain GV3101. Equal ratio of re-suspended bacterial of FKBP15-1/15-2-cYFP and VIN2-nYFP were co-injected into N. benthamiana leaves as described above. Within 48-72 hours, pieces of the transformed leaves were observed with confocal microscope for fluorescent signal.
Root growth measurements
To analyse the root phenotypes in Col-0 and the FKBP mutants, seeds were surface-sterilized for 5 min in 10% (v/v) sodium hypochlorite and washed five times with sterile water, then placed on 0.5× MS agar plates under different treatment conditions. To determine the effects of auxin on root growth, IAA was added to 0.5× MS medium at a concentration 5 nM or 50 nM. The effects of sugars on root growth were determined by adding sucrose, glucose, and fructose separately to 0.5× MS medium at different concentrations (0%, 0.5%, 1%, 2%, 3%, and 5%). Plates were placed vertically in a growth chamber at 22°C at a light/dark cycle of 16/8 h.
After five days of growth, at least 50 plants in each treatment were examined every day. Root lengths were recorded and photographed. All experiments were repeated at least three times. Statistical differences between the different genotypes were calculated using Student’s t-test.
Protein co-immunoprecipitation assays
To determine which proteins interact with FKBP15-1 and FKBP15-2, we performed protein co-immunoprecipitation assays with an anti-GFP antibody . Roots from 12-day-old FKBP15-1-eYFP and FKBP15-2-eYFP transgenic seedlings were collected and ground to powder in liquid nitrogen. Total proteins were extracted in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, pH 7.5, and one complete protease inhibitor cocktail tablet per 25 mL). Following gentle shaking at 4°C for 30 min, the extraction solutions were centrifuged at 15,000 ×g for 10 min. The supernatants were kept on ice before they were used in immunoprecipitation assay. The anti-GFP antibody (Genescript, China) and protein G Sepharose (GE Healthcare, USA) were first mixed and incubated at 4°C for 30 min, and the Sepharose-antibody mixture was then incubated with the supernatant with gentle shaking at 4°C for 1 h. The protein G Sepharose was collected by centrifugation (30 sec, 15,000 ×g) and washed three times before elution with a buffer consisting of 50 mM Tris-HCl, pH 6.8, 50 mM dithiothreitol, 1% SDS,1 mM EDTA, 0.005% bromophenol blue, and 10% glycerol. The eluted proteins were separated by SDS-PAGE for either silver nitrate gel staining, western blotting, or mass spectrometry analysis.
Protein digestion and LC-MS/MS analysis
Protein bands excised from the SDS-PAGE gels were destained by incubation in destaining solution (7.5 mM potassium ferricyanide and 25 mM sodium thiosulfate). The proteins in the gel pieces were reduced by incubation in 10 mM DTT solution at 60°C for 20 min, followed by alkylation in a solution of 25 mM IAM at room temperature for 15 min. The gel pieces were treated with trypsin (Promega, Madison, WI) overnight at 37°C to digest the proteins. The resulting peptides were extracted with 60% acetonitrile containing 5% formic acid, dried in a SpeedVac, and were then re-dissolved in 2% acetonitrile containing 0.1% formic acid for LC-MS/MS analysis.
Peptides were concentrated with a peptide trap column (Thermo Fisher Scientific, USA), and eluted using a solvent system consisting of solvent A (99.9% water, 0.1% formic acid), and solvent B (99.9% acetonitrile, 0.1% formic acid). The peptides were eluted with a gradient of 2-30% solvent B for 80 min, 30-80% solvent B for 10 min, and finally 80% solvent B for 10 min with a constant flow rate of 250 nl/min in a C18 capillary column (Thermo fisher Scientific, USA). The eluted ions were analysed on an ESI-Q-TOF mass spectrometer in data dependent acquisition mode (m/z 350-1500). The Source Capillary was set at 2000-2400 v, the flow rate and temperature of the dry gas were 2.0 L/min and 150°C, respectively. The mass spectrometer was set as one full MS scan followed by ten MS/MS scans on the ten most intense ions from the MS spectrum with the dynamic exclusion duration set at 15 s.
Tandem mass spectra were extracted, and the charge state was de-convoluted and de-isotoped using Compass Data Analysis version 4.1 (Bruker Daltonics). The peak list was directly generated from the raw data using a centroid algorithm with peak width set at 0.1 m/z and intensity above 100. No peak smoothing or filter processing was applied. After the charge states were calculated, the de-isotoped peak lists were exported as mgf files for further Mascot searches. Mascot (version 2.4, Matrix Science) was set up to search the database. The following parameters were considered for the searches: peptide mass tolerance was set to 20 ppm, fragment mass tolerance was set to 0.05 Da, and a maximum of two missed trypsin cleavage sites was chosen. Carbamidomethyl (C) was set as fixed modification, and oxidation (M), was set as variable modifications.
Invertase enzyme activity assays
To assay invertase activity, total proteins were extracted from the roots of 12-day-old Arabidopsis plants in extraction buffer (50 mM Tris-acetate, pH 7.5; 10 mM EDTA; 5 mM DTT). The homogenates were directly used for invertase assays following a previously-described method , and protein concentrations were determined by the Bradford method. For the invertase assays, 100 mg samples of total proteins were incubated for 30 min with 1% sucrose in 50 mM potassium phosphate, pH 7.0, at 37°C, followed by reacting with DNS reagent (100 ºC for 5 min) and reading the absorbance at 540 nm. Tubes without sucrose were used as controls. The initial and final glucose concentrations in each sample were quantified on a calibration curve made with different sucrose concentrations. All experiments were repeated four times.