The inhibition of putrescine synthesis affects the in vitro shoot development of Cedrela fissilis Vell. (Meliaceae) by altering endogenous polyamine metabolism and the proteomic profile

Polyamines —(PAs) and proteins have been demonstrated to be fundamental for in vitro shoot development of Cedrela fissilis. We evaluated the influence of 6-benzyladenine (BA) and putrescine (Put) on the growth of shoots, PA metabolism and proteomic profiles of C. fissilis. The longest shoots were obtained under 2.5 µM BA + 2.5 mM Put treatment. The inhibition of Put synthesis by d-arginine (d-arg) reduced the activities of the Put biosynthesis enzymes arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and the endogenous contents of free Put, resulting in reduced shoot growth. The ODC activity was higher than that of ADC, being ODC the main enzyme in the synthesis of Put in C. fissilis. Inhibition of Put synthesis affected the proteomic profile, reducing the accumulation of the ubiquitin receptor RAD23c, peroxidase 15, ADP-ribosylation factor 1, ADP-ribosylation factor-like protein 8a, profilin-4, profilin-2, glucan endo-1,3-beta-glucosidase, and expansin-like B1 and increasing the accumulation of V-type proton ATPase catalytic subunit A and methionine gamma-lyase, highlighting the relevance of these proteins in promoting the length of shoots. Moreover, the transport protein SEC13 homolog B and the basic isoform glucan endo-1,3-beta-glucosidase, unique proteins in shoots treated with BA + Put, were related to the promotion of shoot growth. Our results show that the modulation of endogenous PAs and proteomic profiles is necessary to regulate in vitro morphogenesis in C. fissilis. Moreover, the ODC enzyme is highly involved in the synthesis of Put during in vitro shoot development and is described for the first time in this species. Polyamines metabolism on regulation of in vitro morphogenesis in Cedrela fissilis.


Introduction
Polyamines (PAs) are aliphatic, low-molecular-weight molecules containing positively charged nitrogen atoms that can interact with macromolecules, such as DNA, RNA, phospholipids, cell wall components, and proteins, regulating the growth of plants ( Baron and Stasolla 2008). Studies have shown that PAs are associated with several essential processes in plant growth and development, including cell division and differentiation, growth, regulation of gene expression and cell signaling, stability of nucleic acids, membrane structure and plant survival (Kusano et al. 2008;Chen et al. 2019). In higher plants, the three main PAs are putrescine (Put), spermidine (Spd), and spermine (Spm), which are involved in plant morphogenesis. In PA biosynthesis, Put is synthesized from arginine (Arg) by the action of arginine decarboxylase (ADC), which constitutes the main Put synthesis pathway in plants, and from ornithine (Orn) by the action of ornithine decarboxylase (ODC) (Chen et al. 2019).
The use of PAs as plant growth regulators has been linked to increased rates of in vitro development for some species, such as Bixa orellana and Glycine max (Parimalan et al. 2011;Arun et al. 2014). For Cedrela fissilis Vell. (Meliaceae), an endangered woody species from the Brazilian Atlantic rainforest (IUCN 2020), in vitro propagation studies and ex vitro rooting have been developed (Nunes et al. 2002;Aragão et al. 2016;Aragão et al. 2017;Oliveira et al. 2020;Oliveira et al. 2022;Ribeiro et al. 2022). Studies have shown that the addition of 6-benzyladenine (BA) to culture media increased the endogenous contents of free Put, which is related to shoot growth (Aragão et al. 2016). In addition, the addition of exogenous Put contributes significantly to the elongation of shoots and modulation of both endogenous PAs and proteomic profiles in this species (Aragão et al. 2017). However, the effects of BA and Put, as well as the inhibition of Put synthesis, on shoot development from axillary buds have not yet been studied in C. fissilis but are relevant to understanding the interaction between cytokinins and PAs on in vitro morphogenesis in this species.
The use of inhibitors of PA synthesis can be an important approach to understanding the functions of these substances in developing plants, as PAs are considered biochemical markers of morphogenic competence and plant regeneration ability (Rey et al. 1994;Shoeb et al. 2001). Among the inhibitors of PA synthesis, d-arginine (d-arg) is a competitive inhibitor of ADC enzyme activity, which is responsible for Put synthesis from Arg and has been used in in vitro morphogenesis studies in plants (Königshofer and Lechner 2002;Liu et al. 2009;Cheng et al. 2015). The use of darg inhibited the growth of Malus sylvestris shoots (Hao et al. 2005;Liu et al. 2009). Moreover, the inhibition of Put biosynthesis by d-arg alters protein synthesis during the in vitro germination of pollen (Falasca et al. 2010). Taken together, these studies show that d-arg can be used as a tool for in vitro morphogenesis responses associated with biochemical investigations, such as proteomic profiling.
Proteomics approaches have been applied to understand the qualitative and quantitative changes in proteins during in vitro morphogenesis (Ghosh and Pal 2013;Reis et al. 2016;Aragão et al. 2017), allowing the identification of proteins that are associated with specific in vitro development processes (Heringer et al. 2015;Reis et al. 2016;Passamani et al. 2017Passamani et al. , 2018. The acquisition of morphogenic competence for in vitro shoot development involves specific responsive explants, such as cotyledons obtained from mature seeds or cotyledonary nodal segments obtained from young seedlings (Ghosh and Pal 2013;Aragão et al. 2017). In C. fissilis, the addition of Put affects shoot development from cotyledonary nodal segments, altering the accumulation of proteins, including those related to cell division (Aragão et al. 2017). The identification of possible candidate protein markers associated with morphogenic competence via comparative proteomics is important for understanding in vitro shoot development. In this sense, the inhibition of Put biosynthesis can provide important information about how modulation of PA metabolism and protein accumulation can promote in vitro C. fissilis shoot growth. Thus, the aim of this work was to evaluate the influence of BA and Put on the shoot growth, endogenous PA metabolism and proteomic profiles of C. fissilis.

Plant material
Cedrela fissilis seeds were obtained in September 2016 from the Caiçara Comércio de Sementes LTDA located in Brejo Alegre, SP, Brazil (21° 10′S and 50° 10′W). The mature seeds were germinated in vitro according to the methods of Aragão et al. (2016) to obtain aseptic seedlings, which were used as explant sources for the experiments.

Effects of exogenous BA and Put on in vitro shoot development
To evaluate the effects of BA and Put on shoot development, cotyledonary and apical nodal segments (explants approximately 2 cm in length) were isolated from 60-dayold seedlings and cultured in Murashige and Skoog (MS) (Murashige and Skoog 1962) media (M519; Phytotechnology Lab, Lenexa, USA) supplemented with 20 g L −1 sucrose (Vetec, Rio de Janeiro, Brazil), 2 g L −1 Phytagel (Sigma-Aldrich, St. Louis, USA), and Put (0 or 2.5 mM) (Sigma-Aldrich) together with different concentrations (0, 0.5, 1.0, 2.5 or 5.0 µM) of BA (Sigma-Aldrich). Together, there were six treatments: a control (absence of BA and Put); 0 µM BA + 2.5 mM Put; 0.5 µM BA + 2.5 mM Put; 1.0 µM BA + 2.5 mM Put; 2.5 µM BA + 2.5 mM Put and 5.0 µM BA + 2.5 mM Put. The pH of the culture media was adjusted to 5.7 before autoclaving at 121 °C and 1.5 atm for 15 min. Put was prepared in water, with the pH adjusted to 5.7, and incorporated into the MS culture media by filter sterilization using a 0.22-µm filter (Biofil, Guangzhou, China). Each culture medium (30 mL) was aliquoted into 150-mL glass culture vessels (8 cm in height × 5 cm in diameter; Aapace, São Paulo, Brazil). The explants were then transplanted into the culture media composing the different treatments and incubated in a growth chamber with a temperature of 25 ± 2 °C and a photoperiod of 16 h of light (55 µmol m −2 s −1 ) for 30 days according to a protocol by Aragão et al. (2017). Each treatment comprised eight replicates, and each replicate consisted of one glass culture vessel with five explants. The length (cm) of the shoots, induction (%) and number of shoots per explant were evaluated after 30 days of incubation.

Effects of the inhibition of put synthesis on in vitro shoot development
To evaluate the effects of the inhibition of Put synthesis on shoot development, the inhibitor d-arg was used according to the methods of Liu et al. (2009), with modifications. Cotyledonary and apical nodal segments were isolated from seedlings and transferred to MS culture media supplemented with 20 g L −1 sucrose, 2 g L −1 Phytagel, BA (0 or 2.5 µM) and Put (0 or 2.5 mM) together with different concentrations (0, 1.0 or 5.0 mM) of d-arg (Sigma-Aldrich). Together, there were six treatments: a control (absence of BA, Put and d-arg); 1.0 mM d-arg, 5.0 mM d-arg; 2.5 µM BA + 2.5 mM Put; 2.5 µM BA + 2.5 mM Put + 1.0 mM d-arg and 2.5 µM BA + 2.5 mM Put + 5.0 mM d-arg. Put and d-arg were prepared in water, with the pH adjusted to 5.7, and were incorporated into the MS culture media after filter sterilization using a 0.22-µm filter. Each treatment was aliquoted into 150-mL culture vessels (30 mL per culture vessel). After they were transplanted into the culture media, the explants were maintained in a growth chamber under the same conditions as those described previously. Each treatment comprised eight replicates, and each replicate consisted of one glass culture vessel with five explants. For all the treatments, the length (cm) of the shoots, induction (%) and number of shoots per explant were evaluated after 30 days of incubation.
Three biological samples of whole shoots (including the stem + leaves and apical meristem) after 30 days of incubation were collected to analyze the endogenous PA content and ADC and ODC enzyme activities. With respect to proteomic analysis, three biological samples of whole shoots from cotyledonary nodal segments were collected from the treatment with the highest shoot length and from the treatment in which d-arg significantly inhibited shoot growth. All biological samples were stored at − 80 °C until analysis.

Free PA determination
The free PA contents were determined according to the methods of Aragão et al. (2016). Samples (three biological replicates of 200 mg fresh matter [FM] per sample) from each treatment were ground in 1.3 mL of 5% perchloric acid (Merck, Darmstadt, Germany). After incubation for 1 h at 4 °C, the samples were centrifuged at 20,000×g for 20 min at 4 °C, and free PAs were collected directly from the supernatant. Free PAs were derivatized with dansyl chloride (Merck) and identified by high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) by injection of 20 µL from each sample. The HPLC instrument was equipped with a 5-µm C18 reverse-phase column (Shimadzu Shin-pack CLC ODS). The HPLC column gradient was generated by adding increasing volumes of absolute acetonitrile (Merck) to a 10% acetonitrile solution, and the pH was adjusted to 3.5 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 performed at a flow rate of 1 mL min −1 at 40 °C. A fluorescence detector at 340 nm (excitation) and 510 nm (emission) was used to determine the PA content. The peak areas and retention times of the samples were measured by comparisons with PA Put, Spd, and Spm standards (Sigma-Aldrich) by calibration curves using concentrations from 0 to 25 nmol (data not shown).

ADC and ODC enzyme assays
The activities of the ADC and ODC enzymes were determined according to the methods of de Oliveira et al. (2017), with modifications. Three biological replicates from each treatment were used, consisting of 500 mg FM per sample. Samples were homogenized in liquid nitrogen using an icecold mortar and then transferred to 500 µL of extraction buffer composed of 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 samples were vortexed and centrifuged at 13,000×g for 20 min at 4 °C, and then the supernatant was used for ADC and ODC enzyme assays. For the assays, a reaction mixture containing 100 µL of protein extract, 1 3 8.3 µL of extraction buffer, 12 mM unlabeled l-Arg (Sigma-Aldrich) or l-Orn (Sigma-Aldrich) and 25 nCi of either L-[ l4 C(U)]-Arg (specific activity of 274.0 mCi mmol −1 ) (PerkinElmer, Houten, The Netherlands) or L-[1-14 C]-Orn (specific activity of 57.1 mCi mmol −1 ) (PerkinElmer) was used. Samples containing only 100 µL of extraction buffer were used as blanks. The reaction mixtures were incubated in glass tubes fitted with rubber stoppers and filter paper discs (GE Healthcare, Piscataway, USA). The mixtures were subsequently soaked in 2 N KOH (Vetec). The mixtures were kept at 37 °C with rotation at 120 rpm via an orbital shaker for 90 min. Each reaction was stopped by the addition of 200 µL described above. Filter paper containing 14 CO 2 was immersed in 1 mL of scintillation fluid (PerkinElmer), and the radioactivity was measured using a scintillation counter (Tri-Carb 2910 TR, PerkinElmer). The protein content was measured using the Bradford method (Bradford 1976), with bovine serum albumin (Sigma-Aldrich) as the standard. The activities were expressed as nanomoles of 14 CO 2 per milligram of protein per hour.

Proteomic analysis
Protein extraction was performed using the trichloroacetic acid (TCA)/acetone method according to a protocol by Damerval et al. (1986), with modifications. Three biological samples of shoots containing leaves and apical meristems (300 mg FM per sample) were frozen in liquid nitrogen and then ground into a fine powder. Then, 300 milligrams of fine powder per sample was resuspended in 1 mL of chilled solution containing 10% (w/v) TCA (Sigma-Aldrich) in acetone (Sigma-Aldrich) together with 20 mM DTT (GE Healthcare) and vortexed for 5 min at 8 °C. The mixture was maintained at − 20 °C for 1 h before centrifugation at 16,000×g for 30 min at 4 °C. Then, the supernatant was discarded, and the resulting pellets were washed 3 times with cold acetone plus 20 mM DTT and centrifuged for 5 min for each wash. The pellets were air dried and resuspended in buffer consisting of 7 M urea (GE Healthcare), 2 M thiourea (GE Healthcare), 2% Triton X-100 (GE Healthcare), 1% DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich), and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Next, the samples were vortexed for 30 min at 8 °C and then centrifuged at 16,000×g for 20 min. The supernatant from each sample was collected, and the protein concentration was measured by a 2-D Quant Kit (GE Healthcare).
Before the trypsin digestion step, the protein samples were precipitated with methanol/chloroform (Nanjo et al. 2012) and resuspended in a solution of 7 M urea/2 M thiourea. Protein digestion was performed using 100 µg of protein from each sample using the filter-aided sample preparation (FASP) methodology (Reis et al. 2021). Next, the peptides were resuspended in 100 µL solution containing 95% 50 mM ammonium bicarbonate, 5% acetonitrile and 0.1% formic acid. The resulting peptides were quantified according to the A205 nm protein and peptide methodology using a Nan-oDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, USA). The samples were transferred to Total Recovery Vials (Waters) for mass spectrometry analysis.
A nanoACQUITY ultraperformance liquid chromatograph (UPLC) connected to a Q-TOF SYNAPT G2-Si instrument (Waters, Manchester, UK) was used for electrospray-liquid chromatography-tandem mass spectrometry analysis according to (Passamani et al. 2018). For each treatment, three biological replicates of 1 µg of digested peptide per sample were used. For separation, the samples were loaded onto a nanoACQUITY UPLC M-Class Symmetry C18 5 μm trap column (180 μm × 20 mm) at 5 µL min −1 for 3 min and then onto a nanoACQUITY M-Class HSS T3 1.8-µm analytical reversed-phase column (75 μm × 150 mm) at 400 nL min −1 and 45 °C. For peptide elution, a binary gradient was used, with mobile phase A consisting of water (Tedia; Fairfield, Ohio, USA) and 0.1% formic acid and mobile phase B consisting of acetonitrile and 0.1% formic acid. The gradient elution started with 7% B, increased from 7 to 40% B until 91.12 min, increased again from 40 to 99.9% B until 92.72 min, and then remained at 99.9% until 106.00 min, followed by a decrease to 7% B until 106.1 min, and then remained at 7% B until 120 min, the end of the run. Mass spectrometry was performed in positive and resolution mode (V mode), 35,000 full width at half maximum (FWHM), with ion mobility separation (IMS), and in data-independent acquisition mode (HDMS E ). The ion mobility wave was set to 600 m s −1 , with helium and IMS gas flow rates of 180 and 90 mL min −1 , respectively. The external calibrant used was human [Glu1]-fibrinopeptide B (100 fmol µL −1 ). Lock mass acquisition was performed every 30 s, and mass spectra were acquired via MassLynx version 4.1 software.
Spectral processing and comparative analysis were performed according to Passamani et al. (2020). Spectra processing and database searching were performed using ProteinLynx Global Server (PLGS) software version 3.0.2 (Waters), and comparative label-free quantification was performed using ISOQuant software version 1.7 (Distler et al. 2014). For ISOQuant, 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. For protein identification, the obtained data were processed against the content in the nonredundant Cedrela fissilis database (Oliveira et al. 2020). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the dataset identifier PXD036504.
To ensure the quality of the results after data processing, only the proteins present or absent (for unique proteins) in all three runs were considered for differential accumulation analysis via Student's t test (two-tailed; P < 0.05). Proteins with a significant t test result were considered up-accumulated if the log 2 value of the fold change (FC) was greater than 0.60 and down-accumulated if the log 2 value of the FC was less than − 0.60. Finally, the sequences of proteins were used in BLAST searches against the Nonredundant (nr) Green Plants/Viridiplantae Protein Sequences database via OmicsBox software (https:// www. biobam. com/ omics box) for the description of high-throughput functional annotation (Gotz et al. 2008) and UniProtKB (http:// www. unipr ot. org).

Statistical analysis
The data concerning shoot development, free PA content, and the enzymatic activity of ADC and ODC were analyzed by analysis of variance (ANOVA) (P < 0.05) followed by the Tukey test using the R statistical environment (R Core Time 2018).

Effects of exogenous BA and put on in vitro shoot development
The combination of BA and Put significantly affected the development of shoots from both cotyledonary and apical nodal segments (Fig. 1). For cotyledonary nodal segments, a higher shoot length (1.51 cm) was observed under the 2.5 µM BA + 2.5 mM Put treatment, while a lower length (0.47 cm) was observed under the control treatment, without BA and Put supplementation (Figs. 1a and 2). In addition, no significant differences among the treatments were observed in terms of shoot induction, with percentages ranging from 90 to 100% (Fig. 1b), or the number of shoots per explant, with values ranging from 1.62 to 1.85 (Fig. 1c).
With respect to apical nodal segments, the highest shoot lengths were observed for the 1 µM BA + 2.5 mM Put (0.60 cm) and 2.5 µM BA + 2.5 mM Put (0.72 cm) treatments (Fig. 1d). The combination of BA and Put did not significantly affect the induction of shoots from this type of explant, with percentages ranging from 90 to 100% (Fig. 1e). The highest number of shoots was observed for the 2.5 mM Put combined with 1, 2.5 and 5 µM BA treatments (with 1.85, 1.85 and 1.80 shoots per explant, respectively) ( Fig. 1e), while the lowest number of shoots was observed under the 2.5 mM Put treatment (1.09 shoots per explant) (Fig. 1e).

Effects of the inhibition of put synthesis on in vitro shoot development
The effects of the Put synthesis inhibitor d-arg on the growth of shoots from cotyledonary and apical nodal segments of C. fissilis after 30 days of incubation were evaluated (Fig. 3). With respect to the cotyledonary nodal segments, the use of 5 mM d-arg promoted stronger inhibition of shoot length (0.16 cm), while the highest shoot length was obtained under the 2.5 µM BA + 2.5 mM Put treatment (1.51 cm) (Fig. 3a). In addition, the use of 1 or 5 mM d-arg combined with 2.5 µM BA + 2.5 mM Put significantly decreased the shoot length (1.10 and 1 cm, respectively) compared to that under the 2.5 µM BA + 2.5 mM Put treatment (1.51 cm) (Fig. 3a). On the other hand, there were no significant differences in shoot induction, with values ranging from 97 to 100% (Fig. 3b), or the number of shoots per explant, with values ranging from 1.93 to 2.00 (Fig. 3c), among all the treatments involving cotyledonary nodal segments.
With respect to the apical nodal segments, the use of 5 mM d-arg strongly inhibited shoot length (0.15 cm), while the use of 1 or 5 mM d-arg combined with 2.5 µM BA + 2.5 mM Put did not significantly affect the shoot length; the shoot length was greatest under these treatments (Fig. 3d). No significant differences among the treatments tested were observed for shoot induction, with percentages ranging from 95 to 100% (Fig. 3e). However, there was a significant inhibition in the number of shoots per explant under 1 or 5 mM d-arg (1.11 and 1.08 shoots, respectively) ( Fig. 3f), while these d-arg concentrations combined with 2.5 µM BA + 2.5 mM Put resulted in the highest number of shoots per explant (Fig. 3f). Figure 4 shows the morphology of shoots obtained from cotyledonary nodal segments cultured in the presence of 2.5 µM BA + 2.5 mM Put, which yielded the greatest shoot length (Fig. 4a), and in the presence of BA and Put combined with 5 mM d-arg, which significantly inhibited shoot growth (Fig. 4b).

Effects of a put synthesis inhibitor on endogenous free PA contents during shoot development
Due to the greater length of shoots from cotyledonary nodal segments than from the other segments, the free PA contents were analyzed in shoots from cotyledonary nodal segments after 30 days of incubation with 2.5 µM BA + 2.5 mM Put and 2.5 µM BA + 2.5 mM Put + 5 mM d-arg (Figs. 5 and 6). The use of d-arg combined with Put and BA significantly reduced the endogenous contents of free Put (Fig. 5a) and free Spd (Fig. 5b) in the shoots. Compared with the other treatments, the treatments with d-arg (0, 1 and 5 mM) but without BA and Put yielded lower contents of free Put and free Spd ( Fig. 5a and b). On the other hand, compared with 1 3 the other treatments, the treatments with 1 or 5 mM d-arg but without BA and Put induced the highest contents of endogenous free Spm (Fig. 5c). Moreover, compared with 2.5 µM BA + 2.5 mM Put, 1 or 5 mM d-arg combined with 2.5 µM BA + 2.5 mM Put significantly decreased the endogenous contents of total free PAs (Fig. 6a) and significantly decreased the PA ratio [Put/(Spd + Spm)] (Fig. 6b).

Effects of the inhibition of put synthesis on ADC and ODC enzyme activities in the shoots
The activities of ADC and ODC were evaluated in the shoots from cotyledonary nodal segments under the 2.5 µM BA + 2.5 mM Put and 2.5 µM BA + 2.5 mM Put + 5 mM d-arg treatments (Fig. 7). Supplementation with a Put synthesis inhibitor (5 mM d-arg) together with BA and Put induced a significant reduction in ADC activity in the shoots from cotyledonary nodal segments compared to shoots under the control and BA + Put treatments (Fig. 7a). On the other hand, the ODC activity increased in shoots incubated under BA + Put treatments compared to the control and BA + Put + d-arg treatments (Fig. 7b). The ODC activity was higher than the ADC activity, indicating that ODC is preferential for Put synthesis during in vitro shoot development in this species (Fig. 7).

Effects of a put synthesis inhibitor on the proteomic profile of the shoots
Proteomic analysis was performed on shoots from cotyledonary nodal segments incubated in the 2.5 µM BA + 2.5 mM Put treatment (BA + Put treatment), which promoted increased shoot length, and in the 5 mM d-arg together with 2.5 µM BA + 2.5 mM Put treatment (BA + Put + d-arg treatment), which reduced the shoot length. The inhibition of Put synthesis by d-arg affects the accumulation of proteins. A total of 926 proteins were identified (Supplementary Table S1). Among them, 701 were unchanged, and 218 differentially accumulated, with 91 proteins down-and 127 up-accumulated in the shoots treated with d-arg (BA + Put + d-arg) compared with the shoots grown in the absence of d-arg (BA + Put). In addition, four proteins were unique in shoots grown under the treatment containing d-arg (BA + Put + d-arg), and three proteins were unique in the treatment without d-arg (BA + Put) (Supplementary Table S1). Among the differentially accumulated proteins (up-or down-accumulated and unique), some are highlighted due to their relationship with plant growth and development processes (Supplementary Table S1).
On the other hand, the use of d-arg (BA + Put + d-arg) induced an up-accumulation of proteins compared with those in shoots grown in the absence of d-arg (BA + Put); these proteins included V-type proton ATPase catalytic subunit A (Ce_fissilis.011699.1) and methionine gammalyase (Ce_fissilis.014973.1) (Supplementary Table S1).

Discussion
The synergistic effect between BA and Put can increase the cellular divisions necessary for efficient shoot growth in C. fissilis, resulting in longer shoots under treatment with 2.5 µM BA + 2.5 mM Put from both types of explants: those from cotyledonary and apical nodal segments. The associations between cytokinins, BA and Put could be relevant to increase the cellular divisions necessary for the growth of shoots, as Put stimulates cell division, promoting the transition through the G1/S and G2/M cell cycle phases (Kuznetsov et al. 2002;Weiger and Hermann 2014). Moreover, BA also promotes cell division, which is necessary for shoot organogenesis and shoot proliferation during in vitro development (Hart et al. 2016). Among the cytokinins, BA has most frequently been used to promote the development of axillary buds, which involves breaking apical dominance and stimulating shoot proliferation, of several tree species (Pijut et al. 2012). Additionally, it has been shown that Put is involved in the growth of M. sylvestris and C. fissilis shoots (Liu et al. 2009;Aragão et al. 2016Aragão et al. , 2017. Moreover, it has been shown that 2.5 µM BA can induce an increase in endogenous free Put contents, which is related to increased shoot development in this species (Aragão et al. 2016). Thus, BA can interact with Put, improving the development of C. fissilis shoots.
The use of the Put inhibitor d-arg reduced both the endogenous free Put contents and the activity of ADC and ODC enzymes, resulting in a reduction in the length of C. fissilis shoots. Thus, the inhibition of Put synthesis could be an important tool for understanding PA metabolism during in vitro morphogenesis in this species. Similarly, the use of d-arg (1-10 mM) has been shown to affect the endogenous contents of PAs, especially Put, in several species, such as Oryza sativa (Sung et al. 1994), M. sylvestris (Hao et al. 2005;Liu et al. 2006), Prunus persica (Liu and Moriguchi 2007) and Pringlea antiscorbutica (Hummel et al. 2002). In Cucumis sativus, the addition of d-arg (1 or 5 mM) significantly decreased adventitious shoot formation from cotyledons of seedlings, reducing endogenous Put contents (Zhu and Chen 2005). In addition, the growth of M. sylvestris shoots was inhibited with the addition of 1 or 5 mM d-arg, which induced a decrease in endogenous Put and Spd contents (Liu et al. 2009). Put is produced by two enzymes in plants: ADC and/or ODC (Chen et al. 2019). Our results show that the addition of Put and BA significantly increased the ODC activity and, consequently, the shoot length, while the use of d-arg resulted in a significant reduction in the activity of ADC and ODC enzymes and consequently reduced the synthesis of free Put, compromising shoot development in this species. Thus, we suggest that both ADC and ODC are required for the development of C. fissilis shoots.
In our work, we observed that the ODC enzyme presented a higher activity than the ADC enzyme in the shoots of C. fissilis, which is the first time Put synthesis-related enzymes have been analyzed in this species. Similarly, in the woody species Araucaria angustifolia, the coexistence of both ADC and ODC has been reported in the Put biosynthesis pathway during zygotic and somatic embryogenesis (de Oliveira et al. 2017;Oliveira et al. 2018), with ODC being the main pathway for Put biosynthesis during somatic embryogenesis in this species (de Oliveira et al. 2018). According to Kevers et al. (2000) and Vuosku et al. (2006), ODCs are particularly active in cell proliferation, whereas ADCs are involved in embryo and organ differentiation. Smith (1985) suggested that ODC is important for Put synthesis in actively dividing cells, while ADC is generally linked to cell elongation. The results described in our work show that the addition of BA + Put significantly increased ODC activity, increasing the endogenous contents of free Put and consequently promoting increased shoot elongation. On the other hand, the use of d-arg reduced the endogenous contents of free Put by reducing ADC and ODC activities, inhibiting the elongation of C. fissilis shoots. Similarly, the use of 5 mM Put increased shoot growth and ODC activity in M. sylvestris, and the addition of 5 mM d-arg resulted in a decrease in shoot length and reduced the activity of both the ADC enzyme and Put synthesis (Liu et al. 2009). Our results demonstrated that Put is an important PA involved in the growth of C. fissilis shoots and that ODC is preferred over ADC for Put synthesis during in vitro shoot development.
In addition, the total free PA content and the PA ratio [Put/(Spd + Spm)] decreased in the presence of the Put synthesis inhibitor. It was previously shown that BA increased the endogenous total free PA content and free Put content in C. fissilis (Aragão et al. 2016), while exogenous Put also increased the total free PA content (Aragão et al. 2017), which was related to the greatest shoot growth in C. fissilis. The PA ratio could be used as a biochemical marker for morphological processes (Shoeb et al. 2001). In our work, we show that a decrease in the PA ratio caused by a decrease in endogenous Put content in the presence of a Put synthesis inhibitor negatively affected the development of C. fissilis shoots. In this sense, an increase in the PA ratio due to the increase in Put content is relevant for proper shoot development in this species. In addition to PAs, the involvement of differentially accumulated proteins in in vitro shoot development was analyzed. Several proteins were down-accumulated when the shoots were incubated with the Put synthesis inhibitor, suggesting that they are important for the growth of C. fissilis shoots. Among the proteins that were down-accumulated are the ubiquitin receptor RAD23c (Ce_fissilis.004398.1 and Ce_ fissilis.004398.2). Rad23 proteins are characterized as DNA repair factors required for nucleotide excision repair and perform a key role in recognizing DNA damage (Shuck et al. 2008) and in transcription and proteolysis (Dantuma et al. 2009). Rad23 proteins were found to play essential roles in the cell cycle and morphology in Arabidopsis thaliana; thus, Rad23 is essential for normal growth (Farmer et al. 2010). Compared with wild-type Pinus sylvestris seedlings, P. sylvestris var. mongolica, a natural dwarf-type mutant, showed decreased accumulation of Rad23 proteins, indicating that these proteins are among the main proteins responsible for the reduction in growth in dwarf-type mutants (Ning et al. 2013). These authors suggested that Rad23 proteins may play an important role in signaling cascades involved in the regulation of plant growth, particularly apical dominance. Indeed, these proteins may be involved in the auxinmediated regulation of apical dominance regulating plant growth (Ning et al. 2013). Thus, this protein may be important for the proper development of C. fissilis shoots, and the decreased accumulation induced by the d-arg treatment (BA + Put + d-arg) could be related to the lower shoot length observed compared to that under the Put + BA treatment.
In addition, peroxidases are important for cell wall extension and cell division necessary for shoot organogenesis (Liszkay et al. 2003;Rajeswari and Paliwal 2008). It has been shown that these enzymes are involved in in vitro growth and differentiation, including direct organogenesis (Viu et al. 2009). In Curcuma longa, the PAs Put, Spd and Spm can induce an increase in peroxidase activity and promote differentiation (Viu et al. 2009). In Centaurium erythraea, peroxidase activity was strongly induced during in vitro morphogenesis, resulting in increased shoot development from the leaves (Filipović et al. 2015). In addition, the inhibition of peroxidase activity can decrease the cell division necessary for differentiation processes in Nicotiana tabacum (Marco and Roubelakis-Angelakis 1996). In this sense, the reduction in the accumulation of peroxidase enzymes (Ce_fissilis.013923.1 and Ce_fissilis.015096.1) in the shoots in the presence of a Put synthesis inhibitor (BA + Put + darg treatment) suggests that these enzymes can be regulated by BA + Put treatment to promote the development of C. fissilis shoots.
ADP-ribosylation factors have a fundamental role in plant growth and development (Naramoto et al. 2010). Studies in plants have revealed that the crucial roles of these factors mainly involve vesicular transport, such as protein trafficking out of the endoplasmic reticulum and other organelles, such as the Golgi apparatus (Pimpl et al. 2003;Stefano et al. 2006;Myung et al. 2013). In addition, these proteins are involved in several plant growth and development processes, including auxin transport, apical dominance, determination of cellular polarity, cell proliferation and elongation, and cell cycle regulation (Gebbie et al. 2005;Xu and Scheres 2005;Naramoto et al. 2010). It is possible that ADP-ribosylation factors are required for normal shoot growth and development in C. fissilis, since the decreased accumulation of these proteins (Ce_fissilis.009793.1 and Ce_fissilis.012754.1) negatively affects the endogenous free Put content and the cell divisions necessary for shoot growth, reducing shoot elongation under d-arg treatment.
The use of a Put synthesis inhibitor decreased the accumulation of two profilin (Ce_fissilis.007056.1 and Ce_fissilis.011671.1) proteins, indicating that profilin proteins are important and required for the in vitro growth of C. fissilis shoots. Profilin is an actin monomer-binding protein that causes either polymerization or depolymerization of actin filaments (Kovar et al. 2000). In higher plants, profilin proteins are involved in cell elongation (Vidali et al. 2007). In shoots of Camellia sinensis multiplied vegetatively by single-node cuttings, a significant increase in the accumulation of profilin occurred to promote growth (Liu et al. 2017).
The most documented function of expansins in plants is their regulation of cell wall extensibility, which promotes the growth and differentiation of plant cells (Cho and Cosgrove 2000). In A. thaliana, expansins play a role in cell enlargement and organ morphogenesis. This fact supports the hypothesis that the cell wall-loosening activity of expansins is an important control point for the regulation of plant cell growth (Cho and Cosgrove 2000). Expansins are also needed for cell wall softening during the growth of the apical meristem, root hairs, shoots, and fruits; for the initiation of leaf primordia; and for the regulation of embryogenesis and germination (Sharova 2007). In our work, the reduction in the accumulation of the expansin protein (Ce_fissilis.010254.1) in shoots treated with d-arg, which ultimately reduced shoot growth, suggests that expansins may play a key role in the control of C. fissilis shoot growth.
Some identified proteins accumulated more in shoots treated with the inhibitor of Put synthesis (BA + Put + d-arg) than in shoots treated with BA + Put, and these proteins, such as the V-type proton ATPase catalytic subunit A (Ce_fissilis.011699.1) protein, could be negatively related to C. fissilis shoot growth. The V-type ATPase plays important roles in the cell growth and development of plants, participating in the uptake and release of solutes across the vacuolar membrane and in the regulation of many cellular processes, including osmoregulation, signal transduction, metabolic regulation and pH (Sze et al. 1992;Kluge et al. 2003). Vacuoles contain V-type ATPases that catalyze the transport of 1 3 H + into the organelle, maintaining cytosolic pH homeostasis, which is important for several enzymes that exhibit their highest activity at a pH of 7 in this compartment (Martinoia et al. 2007). In sugarcane (Saccharum spp.), V-ATPase activity was higher in nonembryogenic callus than in embryogenic callus, suggesting that somatic embryogenesis in this species is negatively affected by greater V-ATPase activity (Passamani et al. 2018). In contrast, in embryogenic suspension cultures of A. angustifolia, exogenous Put treatment stimulated V-ATPase activity and cellular growth, while Spd and Spm addition inhibited the activity of these proton pumps, decreasing cellular growth (Dutra et al. 2013). According to Dutra et al. (2013), the relationship between PAs and proton pump activities in plants and the mechanism of PA action in the regulation of these pumps need to be investigated. Our results suggest that the inhibition of Put synthesis by d-arg modulates endogenous PA, reducing the endogenous content of free Put and consequently increasing the accumulation of V-ATPases in C. fissilis, which may be related to a reduction in shoot length.
In plants, methionine gamma-lyase is a protein that breaks down methionine into methanethiol and 2-ketobutyrate, with 2-ketobutyrate being a precursor for isoleucine biosynthesis (Joshi et al. 2010;Huang et al. 2014). Spd and Spm are produced from Put by the addition of aminopropyl groups, which are provided by methionine (Chen et al. 2019). In our work, the increased accumulation of methionine gammalyase (Ce_fissilis.014973.1) in the shoots grown under the Put synthesis inhibitor treatment (BA + Put + d-arg) suggests a greater degradation of methionine, which may have affected PA metabolism, reflected mainly by the decrease in free Spd content compared to that in the shoots under BA + Put treatment. Our results are in agreement with those reported by Aragão et al. (2015), who found an increase in methionine and Spd contents to promote growth during early seedling growth in C. fissilis.
In addition, some proteins, such as the transport protein SEC13 homolog B (Ce_fissilis.003056.1), were unique in shoots grown under the 2.5 µM BA + 2.5 mM Put treatment, which promoted an increase in the length of C. fissilis shoots. Intracellular protein transport between organelles is mediated by several vesicles (Hino et al. 2011). Among the components of these vesicles is the transport protein SEC13 homolog B, which allows proteins to exit the endoplasmic reticulum toward the Golgi apparatus through a vesicular complex called COPII (Cevher-Keskin 2013). In A. thaliana, this protein contributes to the development of young leaves, the root apical region, and lateral root primordia (Hino et al. 2011). According Brandizzi (2018), the COPII machinery, including the transport protein SEC13 homolog B, may partially account for differences in the metabolic activity of the cells during growth; however, the connection between the endoplasmic reticulum and Golgi apparatus ensures that the protein synthesis necessary for plant growth can occur. Another unique protein in the shoots treated with BA + Put, which increased in length, was the glucan endo-1,3-beta-glucosidase, basic isoform (Ce_fissilis.005316.1). In addition, a glucan endo-1,3-beta-glucosidase 4 (Ce_fissilis.001287.1) protein was down-accumulated in the shoots under d-arg treatment, which ultimately reduced growth, suggesting that these proteins are relevant to the promotion of C. fissilis shoot elongation. The glucan endo-1,3-betaglucosidase enzyme is regulated in part by auxin, cytokinins, and ethylene (Sperisen et al. 1991). In addition, this protein participates in several physiological processes, such as cell division and seed germination (Simmons 1994), and it is involved in cell wall assembly and reorganization (Minic 2008). Thus, our results suggest that these proteins may be important for C. fissilis shoot growth.
In summary, our results indicate that a Put synthesis inhibitor modulates the endogenous metabolism of PAs through the activity of ADC and ODC enzymes and alters the proteomic profile, which affects the development of C. fissilis shoots (Fig. 8).

Conclusion
This manuscript provides new insights into morphogenic competence in C. fissilis regulated by the inhibition of Put synthesis, as well as by BA and Put addition. Our results showed that 2.5 µM BA combined with 2.5 Mm Put increased the shoot length and ODC activity. The use of 1 or 5 mM d-arg combined with BA and Put inhibited shoot elongation by decreasing the endogenous free Put content, the total free PA content, the PA ratio and ADC and ODC activities. The ODC activity was higher than the ADC activity in this species. Moreover, compared with ADC, ODC is preferred for the synthesis of Put during in vitro shoot development and is described for the first time in this species. The inhibition of Put synthesis reduced the abundance of proteins related to plant growth and development, such as the ubiquitin receptor RAD23c, peroxidase 15, ADPribosylation factor 1, ADP-ribosylation factor-like protein 8a, Profilin-4, Profilin-2, expansin-like B1, V-type proton ATPase catalytic subunit A and methionine gamma-lyase, all of which are related to decreased shoot length in this species. In addition, glucan endo-1,3-beta-glucosidase, which was unique to the BA + Put treatment and down-accumulated when BA + Put was combined with d-arg, was associated with an increase in shoot length. These data suggest that Put is essential for morphogenic competence in C. fissilis by modulating the endogenous Put content and proteomic profiles and that effective cell homeostasis at the PA and protein levels is necessary to regulate the growth and development of this species. Proposed scheme of the metabolic modulation of PAs, proteins, ADC and ODC enzymes by the use of a Put synthesis inhibitor, which affected the development of Cedrela fissilis shoots. The blue and red arrows represent proteins whose abundance increased and decreased, respectively. BA benzyladenine, Put putrescine, Spd spermidine, PAs polyamines, ADC arginine decarboxylase, ODC ornithine decarboxylase