Mature seeds of C. fissilis obtained from Caiçara Comércio de Sementes LTDA located in Brejo Alegre (21°10’S and 50°10’W), São Paulo State, Brazil, were germinated in vitro according to Oliveira et al. (2020). Sixty-day-old seedlings were used as the source of apical and cotyledonary nodal segment explants to obtain the shoots that were used for rooting in each subculture.
In vitro germination
In vitro germination was performed according to Oliveira et al. (2020). First, the seeds were surface disinfected and then transferred to glass test tubes (15 x 2.5 cm; Laborglas; São Paulo, Brazil) containing 10 mL of MS (Murashige and Skoog 1962) culture medium (M519; Phytotechnology Lab; Lenexa, USA) supplemented with 20 g L-1 sucrose (Vetec; Rio de Janeiro, Brazil) and 2 g L-1 Phytagel (Sigma-Aldrich; St. Louis, USA). The pH of the culture medium was adjusted to 5.7 before being autoclaved at 121 °C for 15 min. The seeds were incubated in a growth room at 25 ± 2 °C under a 16-h photoperiod (55 µmol m-2 s-1) for a culture period of 60 days. Sixty-day-old seedlings were used as a source of explants (cotyledonary and apical nodal segments) for the experiment.
In vitro multiplication of shoots during successive subcultures
The explants (apical and cotyledonary nodal segments) obtained from 60-day-old seedlings were inoculated into glass test tubes (15 x 2.5 cm; Laborglas) containing 10 mL of MS culture medium supplemented with 20 g L-1 sucrose, 2 g L-1 Phytagel, and 2.5 μM 6-benzyladenine (BA; Sigma-Aldrich), according to Oliveira et al. (2020). The pH of the culture medium was adjusted to 5.7 before autoclaving at 121 °C for 15 min. The explants were incubated in a growth room at 25 ± 2 °C under a 16-h photoperiod (55 µmol m-2 s-1) for a culture period of 45 days. This step corresponded to the shoots obtained in the first subculture using nodal segments obtained from 60-day-old seedlings as explants. For the second, third, and fourth subcultures, the shoots grown from apical and cotyledonary nodal segments after 45 days of incubation were excised (approximately 2 cm), cutting the leaves and apical meristem to obtain the explants and were then used for the respective subculture. The explants were inoculated in the same culture medium again under the same conditions used for the first subculture. Intervals of 45 days were maintained between each subculture under the same conditions of incubation for the growth of shoots. After 45 days of incubation, at the end of each subculture, shoots were collected and used for ex vitro rooting.
Effect of subculture on the ex vitro adventitious rooting of micropropagated shoots
To study the effect of the number of subcultures on ex vitro rooting, shoots (approximately 2 cm) obtained from apical and cotyledonary nodal segments after 1, 2, 3 and 4 subcultures were used. The shoots were removed from the culture medium and transferred to disposable polypropylene plastic cups (50 mL; Orleans, Santa Catarina, Brazil) containing Basaplant® (Artur Nogueira; São Paulo, Brazil) substrate and vermiculite (Mineração Pedra Lavrada LTDA; Atibaia, Brazil) in a 2:1 (v/v) proportion. The plastic cups were maintained in plastic trays (50 x 60 x 10 cm) covered with plastic film to maintain high humidity for 15 days. Thereafter, the humidity level was gradually reduced through perforation of the plastic film, and after 25 days, the plastic film was removed. The explants were maintained in a growth room at 25 ± 2 °C under a 16 h photoperiod at 55 µmol m-2 s-1 for 45 days.
After rooting, the plantlets were transplanted to disposable polypropylene plastic cups (250 mL) containing Basaplant® substrate and vermiculite in a 2:1 (v/v) proportion and were maintained under a 16 h photoperiod at 55 µmol m-2 s-1 at 25 ± 2 °C for 15 days. Then, the rooted plantlets were transferred to a greenhouse with relative air humidity higher than 85% and temperatures between 20 and 30 °C (measured using an Extech RHT10 USB Datalogger, Extech; Waltham, USA). After 90 days, plantlet survival (%) was analyzed.
From the first to the fourth subcultures, root induction (%), the number of roots per explant, and the length (cm) of roots were evaluated after 45 days of rooting. Each treatment (subculture) was composed of eight replicates, with five shoots per replicate. For PA analyses, shoots from cotyledonary nodal segments with leaves were collected after 45 days of culture across four subcultures. For ADC, ODC, hormones and comparative proteomic analyses, shoots from cotyledonary nodal segments with leaves were collected after 45 days of culture in the first and fourth subcultures. Samples of the shoots used in the proteomic and PA analyses were macerated with liquid nitrogen until a fine powder was obtained, which was then stored at −80 °C until assessment. Samples for hormonal analysis were macerated and lyophilized for storage until assessment.
Free PA determination
Free PA contents were determined according to Santa-Catarina et al. (2006) using three biological replicate samples (200 mg fresh matter – FM - each) of shoots in each subculture (1, 2, 3 and 4). The samples were ground in 1.2 mL of 5% perchloric acid (Merck; Darmstadt, Germany), incubated at 4 °C for 1 h, and centrifuged for 20 min at 20,000 × g at 4 °C. The supernatant containing free PAs was obtained, followed by derivatization with dansyl chloride (Merck) and identification by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) using a 5-µm C18 reverse-phase column (Shimadzu Shin-pack CLC ODS). The HPLC column gradient was created by adding increasing volumes of absolute acetonitrile (Merck) to a 10% aqueous acetonitrile solution at pH 3.5, adjusted with hydrochloric acid (Merck). The absolute acetonitrile concentration was maintained at 65% for the first 10 min, increased from 65 to 100% between 10 and 13 min, and maintained at 100% between 13 and 21 min; the mobile phase was added at a flow rate of 1 mL min−1 at 40 °C. The PA concentration was determined using a fluorescence detector at 340 nm (excitation) and 510 nm (emission). The peak areas and retention times of the samples were measured through comparisons with the standard PAs Put, Spd, and Spm (Sigma-Aldrich).
Determination of ADC and ODC activities
ODC and ADC activities were determined according to De Oliveira et al. (2017) with some modifications using three biological replicate samples (500 mg FM per sample) of shoots at the first and fourth subcultures. Each sample was homogenized in an ice-cold mortar with liquid nitrogen and transferred to 500 µL of extraction buffer containing 50 mM Tris–HCl (Invitrogen; Carlsbad, USA) (pH 8.5), 0.5 mM pyridoxal-5-phosphate (Sigma-Aldrich), 0.1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich) and 5 mM dithiothreitol (DTT; Bio-Rad, Hercules, USA). The sample mixtures were vortexed and centrifuged for 20 min at 13,000 × g at 4 °C, and the supernatant was used for the ADC and ODC enzymatic assays. A reaction mixture containing 100 µL of protein extract, 8.3 µL of extraction buffer, 12 mM unlabeled L-Arg or L-Orn, and 25 mCi of either L-[14C(U)]-Arg (specific activity 274.0 mCi mmol-1; PerkinElmer; Waltham, USA) or L-[1-14C]-Orn (specific activity 57.1 mCimmol−1; PerkinElmer) was used. For blank samples, 100 µL of extraction buffer was used. The reaction mixtures were incubated in glass tubes fitted with rubber stoppers and filter paper discs (GE Healthcare; Piscataway, USA) soaked in 2 N KOH (Vetec). The material was incubated at 37 °C at 120 rpm in an orbital shaker for 90 min, and the reaction was stopped by adding 200 µL of 5% (v/v) perchloric acid, followed by further incubation for 15 min under the same conditions. Filter paper containing 14CO2 was immersed in 1 mL of scintillation fluid (PerkinElmer). Radioactivity was then measured using a scintillation counter (Tri-Carb2910TR; PerkinElmer). Protein content was measured using the Bradford method (Bradford 1976) with bovine serum albumin as the standard. The specific enzymatic activities of ADC and ODC were expressed as nmol 14CO2 mg protein-1 h-1.
Plant hormone analysis
Plant hormone analysis was performed according to Durgbanshi et al. (2005) with slight modification using three biological replicate samples (30 mg dry matter (DM) for each sample) of shoots in the first and fourth subcultures. Before extraction, a mixture containing 50 ng of [2H6]-ABA, [C13]-SA, dihydrojasmonic acid and 5 ng of [2H2]-IAA (all from Sigma-Aldrich) was added to each sample as an internal standard. [2H2]-IAA was used to determine IAA contents, [2H6]‐ABA was used to determine ABA contents, [13C]‐SA was used to determine SA and trans-cinnamic acid (t-CA) contents, and dihydrojasmonic acid was used to determine JA, OPDA and jasmonoyl-isoleucine (JA-Ile) contents. The previously macerated and lyophilized samples were immediately homogenized in 2 mL of ultrapure water in a ball mill (MillMix20; Domel; Železniki, Slovenija) at a frequency of 14 Hz for 10 min. After centrifugation at 4,000 × g and 4 °C for 10 min, the supernatants were recovered, and the pH was adjusted to 3.0 with 30% (v/v) acetic acid (Labkem; Barcelona, Spain). The water extract was partitioned twice against 2 mL of diethyl ether (Labkem), and the organic layer was recovered and evaporated under vacuum in a centrifuge concentrator (Speed Vac, Jouan SA; Saint-Herblain, France). Once dried, the samples were resuspended in a 10% (v/v) methanol (Fisher Scientific; Loughborough, UK) solution via gentle sonication. The resulting solution was filtered through 0.22-μm polytetrafluoroethylene membrane syringe filters (PTFE 13-mm diameter; Kinesis Ltda; Cambridgeshire, UK) and directly transferred to vials for mass spectrometry analysis.
LC-electrospray ionization (ESI)-MS/MS analysis was performed using an Acquity Ultra-High Performance Liquid Chromatography (UPLC) system (Waters; Milford, USA) coupled to a tandem Xevo TQ-XS triple quadrupole mass spectrometer (Waters) using an orthogonal Z-Spray ESI interface operated in negative-ion mode. Chromatographic separations were carried out in a reversed-phase C18 column (50 × 2.1 mm, 1.6-μm particle size; Phenomenex Luna Omega; Madrid, Spain) at a flow rate of 300 μL min-1 with a column temperature of 40 °C. A binary gradient was used for elution: mobile phase A consisted of ultrapure water and 0.1% acetic acid, and mobile phase B consisted of 99.9% (v/v) methanol (Fisher Scientific) and 0.1% (v/v) acetic acid. Gradient elution was performed sequentially as follows: maintenance of 10% B for 2 min, followed by ramping from 10 to 90% B at 6 min and a decrease to 10% B at 7 min, after which 10% B was maintained until the end of the run at 8 min. The drying gas and the nebulizing gas were both nitrogen (Praxair; Valencia, Spain). The cone gas flow was set to 250 L h-1, and the desolvation gas flow was set to 1200 L h-1. For operation in tandem MS (MS/MS) mode, the collision gas was 99.995% pure argon (Praxair). The cone voltage and collision energies were adjusted depending on the compound under investigation, as described by Durgbanshi et al. (2005) with few modifications. The desolvation gas temperature was 650 °C, the source temperature was 150 °C, and the capillary voltage was 2 kV. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode. Masslynx v4.1 software was used for mass spectral acquisition, and growth regulators were measured through comparisons with the internal standard for each deuterium-labeled growth regulator.
Protein extraction and digestion
Comparative proteomic analyses were performed with three biological replicate samples (300 mg FM per sample) of shoots from the first and fourth subcultures. Total protein extraction was performed according to Damerval et al. (1986) with some modifications. The samples, previously macerated to a fine powder with liquid nitrogen, were resuspended in 1 mL of chilled solution containing 10% (w/v) trichloroacetic acid (TCA; Sigma-Aldrich) in acetone (Merck) and 20 mM DTT (GE Healthcare, Piscataway, USA), and the mixture was vortexed for 5 min at 4 °C and kept at - 20 °C for 1 h, followed by centrifugation at 16,000 x g for 20 min at 4 °C. The resulting pellets were washed three times with cold acetone containing 20 mM DTT and centrifuged for 5 min in each wash step. The pellets were air dried, resuspended in buffer containing 7 M urea (GE Healthcare), 2 M thiourea (GE Healthcare), 2% Triton X-100 (GE Healthcare), 1% DTT (GE Healthcare), and 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich), vortexed, and incubated on ice for 30 min. Following incubation, the samples were centrifuged at 16,000 × g for 20 min at 4 °C. The supernatants containing total proteins were collected, and the protein concentration was measured using a 2-D Quant Kit (GE Healthcare).
Before the digestion step, the extracted proteins (100 μg from each biological replicate) were first precipitated using methanol/chloroform (Nanjo et al. 2012). Then, the samples were resuspended in a solution of 7 M urea (GE Healthcare) and 2 M thiourea (GE Healthcare), and protein digestion was performed with trypsin (50 ng µL-1; V5111, Promega, Madison, USA) using the filter-aided sample preparation (FASP) method (Reis et al. 2021). The digested samples were transferred to Total Recovery Vials (Waters) for mass spectrometry analysis.
Nano-LC-electrospray ionization (ESI)-MS/MS analysis was performed using a nanoAcquity UPLC system (Waters) coupled to a Synapt G2-Si mass spectrometer (Waters). First, a chromatography step was performed by loading 1 μg of the digested samples according to Oliveira et al. (2020) to normalize the relative protein quantification results. To ensure standardized molar values for all conditions, the normalization among samples was based on stoichiometric measurements of the total ion counts (TICs) from MSE scouting runs prior to the analyses using ProteinLynx Global SERVER v. 3.0 (PLGS; Waters). After sample normalization, the peptide mixtures were separated via liquid chromatography using a nanoAcquity UPLC 5 µm C18 trap column (180 µm × 20 mm; Waters) at 5 µL min-1 for 3 min, followed by a nanoAcquity HSS T3 1.8 µm analytical reverse-phase column (75 µm × 150 mm; Waters) at 400 nL min-1 at 45 °C. A binary gradient of mobile phase A containing water (Tedia; Fairfield, USA) and 0.1% formic acid (Sigma-Aldrich) and mobile phase B containing absolute acetonitrile (Sigma-Aldrich) and 0.1% formic acid was used for peptide elution. Gradient elution started with 7% B, followed by 7 to 40% B until 91.12 min, 40 to 99.9% B until 92.72 min, holding at 99.9% B until 106 min, then a decrease to 7% B until 106.1 min and holding at 7% B until the end of the run at 120 min. Mass spectrometry was performed with the following settings: positive and resolution mode (V mode), 35,000 full width at half maximum ion mobility separation (IMS), and data-independent acquisition (DIA) mode (HDMSE). IMS was performed using a wave velocity of 600 m s-1. The helium and IMS gas flow rates were 180 and 90 mL/min, respectively. The transfer collision energy was ramped from 19 to 55 V in high-energy mode, with cone and capillary voltages of 30 and 2750 V, respectively, and a source temperature of 70 °C. For the time of flight (TOF) parameters, the scan time was set to 0.5 s in continuum mode, with a mass range of 50 to 2000 Da. Human [Glu1]-fibrinopeptide B (Sigma-Aldrich; 100 fmol µL-1) was used as an external standard, and lock mass acquisition was performed every 30 s. Mass spectral acquisition was performed for 90 min using MassLynx v4.0 software.
Proteomic data analysis
Spectral processing and database searching were performed using the ProteinLynx Global Server (PLGS; version 3.0.2) (Waters). The Apex3D parameters were set to a low-energy threshold of 150 counts, an elevated-energy threshold of 50 counts, and an intensity threshold of 750 counts. In addition, the analysis settings included the following: one missed cleavage, minimum fragment ion per peptide equal to 3, minimum fragment ions per protein equal to 7, minimum peptide per protein equal to 2, automatic peptide and fragment tolerance, a fixed modification of carbamidomethyl and variable modifications of oxidation and phosphoryl. The false discovery rate (FDR) was set to a maximum of 1%. Protein identification was performed against a nonredundant protein databank for C. fissilis generated by transcriptome sequencing and de novo assembly (Oliveira et al. 2020). Comparative label-free quantification analysis was performed using ISOQuant software v.1.7 (Distler et al. 2014). The following parameters were used to identify proteins: a 1% FDR, a peptide score greater than six, a minimum peptide length of six amino acids, and at least two peptides per protein were required for label-free quantitation using the TOP3 approach, followed by the multidimensional normalized process within ISOQuant. The mass spectrometry proteomic data have been deposited with ProteomeXchange (Deutsch et al. 2019). Consortium via the PRIDE (Perez-Riverol et al. 2019) partner repository with the dataset identifier PXD023173.
To ensure the quality of the results after data processing, only the proteins that were present or absent (for unique proteins) in all three runs of biological replicates were considered in the differential accumulation analysis using Student’s t-test (two-tailed; P < 0.05). Differentially accumulated proteins were considered to be up-accumulated if the log2 value of their fold change (FC) was greater than 0.6 and down-accumulated if the log2 value of their FC was lower than -0.6 according to Student’s t-test. Finally, the proteins were subjected to BLASTp searches against the Nonredundant (nr) Green Plants/Viridiplantae Protein Sequences database using OmicsBox software (https://www.biobam.com/omicsbox) for high-throughput functional annotation (Götz et al. 2008).
The rooting of shoots was performed using a completely randomized design. The data on root induction, the numbers and lengths of roots, plantlet survival, free PAs, ADC/ODC enzyme activities and plant hormones were analyzed by analysis of variance (ANOVA) (P < 0.05) followed by the Student-Newman-Keuls (SNK) test (Sokal and Rohlf 1995) in the R statistical environment (R Core Team 2017).