Light Spectrum and Explant Type Drive Hormonal and Proteomic Reprogramming in Micropropagation of the Endangered Paubrasilia echinata

Light Spectrum and Explant Type Drive Hormonal and Proteomic Reprogramming in Micropropagation of the Endangered Paubrasilia echinata | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Light Spectrum and Explant Type Drive Hormonal and Proteomic Reprogramming in Micropropagation of the Endangered Paubrasilia echinata Júlia Oliveira Schueler, Jociel Nascimento Noronha, Mateus Santana Rodrigues, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8001406/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Paubrasilia echinata , the national tree of Brazil, is critically endangered due to centuries of overexploitation for its high-quality wood and dye-producing sap. Consequently, the development of efficient propagation systems is urgent for its conservation and restoration. In vitro culture represents a viable alternative to conventional propagation, enabling large-scale plantlet production and the preservation of elite genotypes. This study aimed to evaluate the effects of explant type, cytokinin, and light spectrum on in vitro shoot development, alongside changes in polyamine (PA) and plant hormone profiles, and to assess the impact of indole-3-butyric acid (IBA) on ex vitro rooting. This is the first integrated analysis combining explant type, light quality, and physiological–proteomic responses during in vitro development of P. echinata . Cotyledonary nodal segments produced longer shoots than apical nodal explants. The red–blue enriched LED W/mB/dR/fR lamp markedly improved shoot elongation, biomass accumulation, and endogenous levels of BA and putrescine. Proteomic analysis revealed increased accumulation of proteins related to photosynthesis, antioxidant defense (APX, PODs), cytoskeleton organization (tubulin), and stress tolerance (HSPs), indicating enhanced cellular homeostasis and photomorphogenic responses. Ex vitro rooting ranged from 50–65% and was not significantly influenced by explant type or IBA concentration. Overall, these findings establish a physiologically supported micropropagation protocol for large-scale production of P. echinata , providing a strategic tool for the conservation of this culturally and ecologically emblematic Brazilian species. Micropropagation LED lamp Caesalpinia echinata Red light spectrum Polyamines Plant growth hormones Proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Paubrasilia echinata (Lam.) E. Gagnon, H.C. Lima & G.P. Lewis (Fabaceae), commonly known as pau-brasil, is an endemic and emblematic species of the Brazilian Atlantic Forest (SOS Mata Atlântica and INPE 2024; Esser et al. 2019 ). The species holds exceptional historical and cultural significance, as it gave Brazil its name and was officially declared the national tree by Federal Law No. 6,607 on December 7, 1978 (Brasil 1978 ). Originally described by Lamarck in 1785 as Caesalpinia echinata , its taxonomy was later revised based on phylogenetic and morphological evidence. In 2016, this species was reclassified it into a monotypic genus, establishing its current nomenclature as Paubrasilia echinata (Gagnon et al. 2016 ). Historically, this species was subjected to intense exploitation, initially for the extraction of the red dye brazilein, widely used in textile dyeing and ink production. Subsequently, its dense and elastic wood became highly valued for the manufacture of high-quality violin and cello bows (Alves et al. 2008 ). As a result of centuries of overharvesting and habitat loss, P. echinata is currently listed as Endangered on the International Union for Conservation of Nature (IUCN) Red List (IUCN 2025 ). Although the propagation of P. echinata via seeds and cuttings has been reported. Seeds can be stored at low temperatures (-18°C) with water content below 12.7%, remaining viable for five years (Hellmann et al. 2006 ; Mello et al. 2013 ). Vegetative propagation through cuttings using higher concentrations (2,500 mg L⁻¹) of auxins, such as indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) significantly improves rooting, yet overall rooting rates remain low at approximately 16% (Endres et al. 2007 ). Given these constraints, combined with the species’ ecological, economic, and cultural importance and its threatened status, establishing in vitro propagation of shoots from nodal segments emerges as a relevant and efficient biotechnological alternative for propagation and conservation. The morphogenetic response in vitro is influenced by multiple factors, including the species, explant type and age, culture duration, and environmental conditions (Pulianmackal et al. 2014 ). Therefore, it is essential to characterize the specific conditions required for morphogenesis, such as the choice of culture medium, the concentration and type of plant growth regulators (Ivanova et al. 2006 ), and light quality and intensity (Fan et al. 2022 ). Propagation in vitro can be achieved through the development of axillary buds from nodal segments, a strategy widely employed for the micropropagation of various tree species, such as Cedrela fissilis , Cariniana legalis , Dalbergia nigra , Schomburgkia crispa , Pfaffia glomerata , Campomanesia phaea , Actinia deliciosa and Gallesia integrifolia (Aragão et al. 2016 ; Aragão et al. 2017a ; Aragão et al. 2017b ; Arruda et al. 2019 ; Lerin et al. 2019 ; Oliveira et al. 2020 ; Silva et al. 2020 ; Demétrio et al. 2021 ; Oliveira et al. 2022 ; Pessanha et al. 2022 ; Ramos et al. 2023 ; Carrari-Santos et al. 2024 ; Rodrigues et al. 2025 ). The nodal segments derived from the same seedling exhibit different morphogenic potentials for shoot development, with some being more responsive than others along the basal-to-apical axis of the seedling. In some species as D. nigra , C. fissilis and G. integrifolia , cotyledonary nodal segments, from the base of seedling, can be more responsive compared to apical ones when supplied with BA (Aragão et al. 2016 ; Pessanha et al. 2022 ; Rodrigues et al. 2025 ). Besides explant type, the plant growth regulators are often necessary to induce in vitro morphogenesis. Cytokinins, such as N6-benzyladenine (BA), are essential for axillary bud development during shoot multiplication (Aragão et al. 2016 ; Pessanha et al. 2022 ; Rodrigues et al. 2025 ), while auxin could be related to shoot rooting (Lerin et al. 2021 ; Ribeiro et al. 2022 ). In some species, such as C. fissilis , G. integrifolia , and D. nigra (Aragão et al. 2016 ; Pessanha et al. 2022 ; Rodrigues et al. 2025 ), BA enhances shoot elongation. However, in C. legalis , BA alone does not significantly promote shoot growth (Aragão et al. 2017b ), highlighting the need to combine chemical regulation with other strategies to optimize in vitro morphogenesis. One effective approach involves the use of specific light spectra, which serve as physical cues to modulate photomorphogenesis. The light spectrum, particularly combinations of blue, red, and far-red wavelengths, strongly influences in vitro morphogenesis in various wood plant species (Lerin et al. 2019 ; Oliveira et al. 2020 ; Silva et al. 2020 ; Rodrigues 2023 ). In C. fissilis , the use of light-emitting diode (LED) lamp with specific combination of white, medium blue, and red light spectra promoted superior shoot development compared to other LEDs lamps and fluorescent lamp controls (Oliveira et al. 2020 ). Moreover, the light spectrum can modulate the endogenous compounds related to shoot development, such as polyamines (PAs), phytohormones and proteins improving directly the shoot development. Beyond the direct role of PAs in in vitro morphogenesis, studies have shown that light quality can modulate the endogenous PA content, thereby affecting the morphogenetic response in several tree species, as C. fissilis and C. legalis (Lerin et al. 2019 ; Oliveira et al. 2020 ). In shoots of C. legalis , the PAs putrescine (Put) and spermidine (Spd) were more abundant under LED lamp with white spectrum combined with low blue and red spectra than under fluorescent lamp (Lerin et al. 2019 ). Similarly, in C. fissilis , LED containing white light combined with medium blue and red spectra increased the shoot elongation correlated with higher Put levels compared to fluorescent lamp (Oliveira et al. 2020 ). These findings highlight the synergistic effects of chemical signals (cytokinins) and physical cues (light spectrum) in regulating in vitro morphogenesis and optimizing shoot elongation. Moreover, the phytohormones auxins, cytokinins, gibberellins (GA), ethylene, abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) act as essential chemical messengers regulating plant growth and development (Ali et al. 2024 ; Zhang et al. 2024 ). The balance between auxin and cytokinin is particularly important for controlling in vitro morphogenesis and rooting (Skoog and Miller 1957 ; Asghar et al. 2023 ). In C. fissilis shoots derived from cotyledonary nodal segments, a decrease in endogenous levels of 3-indoleacetic acid (IAA), ABA, and 12-oxo-phytodienoic acid (OPDA, an intermediate in JA biosynthesis) in the fourth subculture compared to the first correlated with reduced shoot growth potential and rooting capacity (Oliveira et al. 2022 ). Conversely, the levels of JA, JA-Ile, trans-cinnamic acid (t-CA), and SA increased in the fourth subculture, which was associated with a decline in in vitro development and rooting potential (Oliveira et al. 2022 ). Thus, the spatial distribution and levels of phytohormones are modulated throughout the plant during tissue development and are influenced by external factors such as light, highlighting the relevance of their analysis in elucidating in vitro shoot development under different light spectra. In addition to plant phytohormones, proteomic approaches provide a powerful tool for investigating qualitative and quantitative changes in proteins during in vitro morphogenesis, offering insights into the molecular mechanisms underlying shoot formation (Lerin et al. 2019 ; Oliveira et al. 2020 ; Oliveira et al. 2022 ; Pessanha et al. 2022 ). The type of explant and the light spectrum can positively influence shoot development by modulating protein abundance (Lerin et al. 2019 ; Pessanha et al. 2022 ). In C. legalis , the greatest shoot elongation under LED lamp with white spectra combined with low blue and red (W/lB/dR) spectra was associated with the accumulation of proteins related to cell organization and composition, along with a reduction in proteins linked to stress responses, highlighting the importance of light quality in in vitro morphogenesis abundance (Lerin et al. 2019 ). Moreover, in C. fissilis , the LED lamp with white spectrum combined medium blue and red spectra modulated proteins involved in metabolism, stress, and light response pathways, where the up-accumulation of argininosuccinate synthase, a precursor of Put, and the increased Put content were associated with enhanced shoot elongation compared to fluorescent lamp (Oliveira et al. 2020 ). In addition, shoot rooting is essential for the production of plantlets. Ex vitro rooting offers several advantages over in vitro rooting, including cost reductions of up to 70% (Ranaweera et al. 2013 ), and has been successfully applied to numerous species, including C. fissilis (Ribeiro et al. 2022 )d nigra (Pessanha et al. 2022 ). This method can be highly efficient, often resulting in plantlets with better-developed root systems compared to those rooted in vitro (Yan et al. 2010), which in turn enhances their tolerance to environmental stress (Phulwaria et al. 2013 ). Although numerous studies addressing in vitro morphogenesis and biochemical or molecular mechanisms have been carried out in different tree species, such approaches remain unexplored for P. echinata . Thus, this study aimed to establish the optimal conditions for its in vitro propagation and to characterize the biochemical responses associated with shoot development. Material and Methods Plant material Immature fruits of P. echinata were collected six weeks after flowering from trees located in Campos dos Goytacazes, Rio de Janeiro, Brazil (21°45′43″S, 41°17′28″W), to obtain seeds used for in vitro germination. Specimens of P. echinata were deposited in the Herbarium of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (HUENF 7483, HUENF 7500, HUENF 8126, HUENF 9618; https://jabot.jbrj.gov.br/v3/consulta.php ). Fruit disinfection and seed germination Fruit disinfection and seed germination Fruits were surface disinfected according to Santa-Catarina et al. ( 2001 ). First, fruits were washed in water containing one drop of neutral detergent (Limpol; São Paulo, Brazil). Following, fruits were immersed in 70% ethanol (Tupi; São Paulo, Brazil) for one min, and subsequently treated with 100% commercial bleach (Qboa®; Anhembi, São Paulo - Brazil) containing 1.8–2.5% sodium hypochlorite for 1h. Finally, the fruits were rinsed three times with autoclaved type II deionized water in a flow chamber. Immature seeds were isolated from disinfected fruits and germinated in wood plant medium (WPM; PhytoTech Labs®, Kansas, USA) (Lloyd and McCown 1980 ), both supplied with 20 g L − 1 sucrose (Synth; São Paulo, Brazil), 2 g L − 1 gellan gum (Phytagel®; Sigma‒Aldrich; St Louis, USA). The pH of culture media was adjusted to 5.7 before Phytagel® addition. The culture medium was then distributed into test tubes (10 mL/flask) and autoclaved at 121°C for 20 min. After transferring to the culture medium, seeds were kept in a growth room with a light intensity of 73 µmol.m − 2 .s − 1 , an 16-h light photoperiod, at 25 ± 2°C. Effect of explant type and BA concentration on shoot development Apical and cotyledonary nodal segments (± 1.5 cm) were isolated from 60 day-old-seedlings (Fig. 1 ) and transferred in MS (PhytoTech Labs®) culture medium supplemented with 20 g L − 1 sucrose (Synth), 2 g L − 1 Phytagel® (Sigma‒Aldrich), and different concentrations (0, 0.1, 0.5, and 1.0 µM) of BA (Sigma–Aldrich). The pH of the culture medium was adjusted to 5.7, and then Phytagel® was added. The culture medium was then distributed into culture flasks (30 mL/flask) and autoclaved at 121°C for 20 min. After transferring to the culture medium, the explants were kept in a growth room with a light intensity of 73 µmol.m − 2 .s − 1 , an 8-h photoperiod, at 25 ± 2°C. Each treatment was composed by eight replicates, with five explants per replicate. After 120 days, the induction (%), shoot length (cm), and number of shoots per explant were evaluated. Effects of explant type and light spectra on shoot development Apical and cotyledonary nodal segments (± 1.5 cm) from 60-day-old seedlings in vitro germinated were excised and transferred to WPM (PhytoTech Labs®) culture medium supplemented with 20 g L⁻¹ sucrose (Synth), 0.1 µM BA (Sigma-Aldrich), and 2 g L⁻¹ Phytagel® (Sigma‒Aldrich). The pH of the culture medium was adjusted to 5.7 before adding Phytagel®, dispensed into flasks (30 mL/flask) and autoclaved at 121°C for 20 min. Explant segments were cultured under seven light treatments, being six LED lamps and one fluorescent lamp as a control (Table 1 ). The flasks were covered with four layers of shade cloth, reducing the light intensity from 73 to 16 µmol m⁻²·s⁻¹ at 25°C. Each treatment consisted of eight replicates, with each replicate being one culture flask containing five explants. After 120 days, the induction (%), shoot length (cm), and number of shoots per explant were recorded. Fresh weight (FW), photosynthetic pigments, polyamines (PA), phytohormones, and proteomic analyses were performed on samples from the fluorescent lamp (control) and the LED lamp that promoted the greatest shoot elongation. Samples for PA, phytohormone, and proteomic analyses were collected and stored at − 80°C until further analysis. Table 1 Characteristics of the different LED light wavelengths and tubular fluorescent lamp used in P. echinata shoots development Lamps Lamp wavelenght Luminous intensity at the culture flask (µmol m − 2 s − 1 ) Peak wavelength (nm) Control Tubular fluorescent 16 435/545/580 WmB W/MB 16 450/530 WhB W/HB 16 450/530 WlBdR W/LB/DR 16 450/530/660 WmBdR W/MB/DR 16 450/530/660 WlBdRfR W/LB/DR/FR 16 450/530/660/735 WmBdRfR W/MB/DR/FR 16 450/530/660/735 White (W); low blue (LB) = 8–10% blue; medium blue (MB) = 12–14% blue; high blue (HB) = 16–18% blue; deep red (DR) = 30–50% deep red; far red (FR) = 12% far red Effect of IBA on shoot rooting The rooting of shoots from cotyledonary and apical shoots was carried out ex vitro according to Ribeiro et al. ( 2022 ). Shoot cuttings (1.5 to 2.0 cm) were isolated and the base was immersed in different concentrations (0, 100, 250 and 500 µM) IBA for 3 h. Then, the shoot cuttings were then transferred to plastic cups (50 mL; TotalPlast, Santa Catarina, Brazil) containing the substrate Basaplant® (São Paulo, Brazil) and vermiculite (Basil Minérios; Goiás, Brazil) (1:1;v/v) and maintained in plastic trays (39.4 × 31.9 × 15.4 cm) (Pleion; São Paulo, Brazil) covered with PVC-type plastic film (Lumipam; São Paulo, Brazil) to maintain high relative humidity. The trays were maintained in a growth room at 25 ± 2°C under a photoperiod of 16 h, with a light intensity of 55 µmol m − 2 s − 1 provided by LED lamps (Koninklijke Philips Electronics NV). After 25 days, aiming to reduce the moisture inside trays and stimulate the start of an acclimatization process simultaneously with rooting induction, the PVC parafilm plastic was perforated. This procedure was carried out until the complete removal of the PVC at 100 days after the start of perforation. After 240 days, the rooted plantlets (%) were evaluated. Rooted plantlets were transferred to plastic bags containing soil and maintained in a greenhouse, receiving irrigation three times daily (6:00, 10:00, and 17:00 h) during 210 days. The micropropagated plantlets were transplanted to a field in a biotechnological trial at Instituto Biasse Socioambiental ( https://institutobiasse.org.br/ ) located in Itaocara – RJ – Brazil (21°40′05,18″S, 42°03′32,62″W), for environmental education and extension activities. FW determination The FW of shoots developed under fluorescent and the LED treatment that promoted the higher shoot development from apical and cotyledonary nodal segments was determined using a precision balance. Five repetitions, with a culture flask containing five shoots each repetition, were used for FW analysis. Proteomic analysis For comparative proteomic analysis, samples (300 mg FW each, in triplicate) were collected from apical and cotyledonary nodal segments after 120 days of cultivation in WPM medium supplemented with 0.1 µM BA. The samples were obtained under two lighting conditions: LED, which promoted the greatest shoot elongation, and fluorescent, used as the control. Initially, samples were ground into a fine powder in liquid nitrogen. Proteins were extracted using a modified trichloroacetic acid (TCA)/acetone method (Damerval et al. 1986 ). Samples were resuspended in 1 mL of protein extraction buffer containing 10% (w/v) TCA (Sigma-Aldrich) in acetone (Sigma-Aldrich) supplemented with 20 mM dithiothreitol (DTT; Bio-Rad, Hercules CA, USA). The resulting pellets were washed three times with cold acetone containing 20 mM DTT. Pellets were air-dried, resuspended in buffer containing 7 M urea (Cytiva; Marlborough, USA), 2 M thiourea (Cytiva), 2% Triton X-100 (Sigma-Aldrich), 1% DTT (Bio-Rad), and 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich). The protein concentration was determined using the 2-D Quant Kit (Cytiva). Before trypsin (V5111; Promega, Madison, USA; final enzyme-to-protein ratio of 1:100) digestion, proteins were precipitated using the methanol/chloroform method (Nanjo et al. 2012 ). Following precipitation, the samples were resuspended in a 7 M urea/2 M thiourea solution. Aliquots containing 100 µg of protein were digested according to the filter-aided sample preparation (FASP) protocol (Wiśniewski et al. 2009 ). Peptides were then resuspended and quantified at 205 nm using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific; Waltham, USA). Samples were transferred to Total Recovery Vials (Waters; Manchester, UK) for mass spectrometry. Mass spectrometry analyses were performed using a nanoACQUITY UPLC system coupled to a Synapt G2-Si HDMS mass spectrometer (Waters) operated in nano-electrospray liquid chromatography tandem mass spectrometry mode (nanoESI-LC–MS/MS). A total of 2 µg of peptides was loaded onto a C18 trap column (180 µm × 20 mm; Waters) at 5 µL min − 1 for 3 min, and subsequently onto a nano-Acquity HSS T3 1.8 µm analytical reverse-phase column (75 µm × 150 mm; Waters) at 400 nL min − 1 , maintained at 45°C. Peptides were eluted using a binary gradient: mobile phase A consisted of water (Tedia; Fairfield, USA) with 0.1% formic acid (Sigma-Aldrich), and mobile phase B consisted of acetonitrile (Sigma-Aldrich) with 0.1% formic acid. The gradient was as follows: 5% B at the start, increased to 40% B by 92 min, ramped to 99.9% B by 96 min, held at 99.9% B until 100 min, decreased to 5% B by 102 min, and maintained at 5% B until 118 min. Mass spectrometry was performed in positive resolution mode (V mode; 35,000 full with at half maximum) with ion mobility separation (IMS) and in data-independent acquisition (DIA) mode (HDMSE). IMS was operated with a wave velocity program from 800 to 500 m s − 1 , helium and IMS gas flows of 180 and 90 mL min − 1 , respectively. Transfer collision energy was ramped from 19 to 55 V in high-energy mode, with cone and capillary voltages of 30 and 2800 V, respectively, and a source temperature of 100°C. Time-of-flight (TOF) parameters included a scan time of 0.5 s in continuum mode, with a mass range of 50–2000 Da. Human [Glu 1 ]-fibrinopeptide B (Sigma-Aldrich) at 100 fmol µL − 1 was used as an external standard, and lock-mass acquisition was performed every 30 s. Data acquisition was carried out for 90 min using MassLynx v.4.1 software. Spectral processing and database searches were performed using ProteinLynx Global Server (PLGS) v.3.0.3 (Waters). Raw data processing settings included a low-energy threshold of 150 counts, a high-energy threshold of 50, and an intensity threshold of 750. Database searches were performed with the following parameters: up to two missed cleavages; minimum of three fragment ions per peptide; minimum of seven fragment ions per protein; minimum of two peptides per protein; carbamidomethylation of cysteine as a fixed modification; oxidation (M) and phosphorylation (STY) as variable modifications; and a maximum false discovery rate (FDR) of 1%. Searches were conducted against the P. echinata (Lam.) Gagnon, H.C.Lima & G.P.Lewis protein database. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2025 ) partner repository with the dataset identifier PXD070163. Label-free quantification analyses were performed using ISOQuant software v.1.7 (Distler et al. 2014 ). Label-free quantification was performed using ISOQuant v.1.8 (Distler et al., 2013). Parameters included an FDR of 1%, a peptide score greater than six, a minimum peptide length of six amino acids, and at least two peptides per protein were considered for label-free quantitation using the TOP3 approach, followed by the multidimensional normalized process within ISOQuant. To ensure robustness, only proteins consistently present or absent across all three biological replicates were considered in differential abundance protein (DAP) analysis. Statistical comparisons were performed using two-tailed Student’s t-tests. Proteins with p-value < 0.05 were classified as up-accumulated if the Log 2 fold-change (FC) exceeded 0.6, and down-accumulated if Log 2 (FC) was below − 0.6. Functional annotations were performed using OmicsBox v.3.0.29 ( https://www.biobam.com ). Protein sequences were queried against the NCBI non-redundant green plant protein database (taxa: 33,090; Viridiplantae) using BLAST. The enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DAPs was performed in STRING version 12.0 ( https://string-db.org ) using Glycine max homologs of P. echinata proteins. To generate a protein‒protein interaction (PPI) network, G. max was considered the reference plant species using a minimum required interaction score of 0.7, and network analysis was performed with Cytoscape ( https://cytoscape.org ) (version 3.10.2) (Shannon et al. 2003 ). Analysis of photosynthetic pigments Photosynthetic pigments chlorophyll (Chl) a, b, and carotenoids were analyzed according to Arnon ( 1949 ). Samples (200 mg FW, in triplicate) of shoots from apical and cotyledonary nodal segments after 120 days of incubation under LED and fluorescent lamps were used. The photosynthetic pigments were extracted with 10 mL of 80% acetone (Sigma-Aldrich). Subsequently, the samples were incubated in the dark at 3°C for 24 h. Absorbance readings were performed at wavelengths of 663 nm for Chl a, 645 nm for Chl b, and 470 nm for carotenoids, using the SoftMax Pro® 6.0 software. Pigment content was calculated using the following equations (Arnon 1949 ): $$\:Chl\:a=\frac{\left(12.7\times\:O{D}_{663}-2.69\times\:O{D}_{645}\right)\times\:V}{1000\times\:W}$$ $$\:Chl\:b=\frac{\left(22.9\times\:O{D}_{645}-4.68\times\:O{D}_{663}\right)\times\:V}{1000\times\:W}$$ $$\:Carotenoids=\frac{\left(1000\times\:O{D}_{470}-3.27\times\:Chl\:a-104\times\:Chl\:b\right)}{229\times\:1000\times\:W}$$ Where: OD represents the optical density, V is the total extract volume (mL), and W is the FW (g) of the shoots used. Determination of free PAs content Shoots originating from apical and cotyledonary nodal segments, obtained under the LED lamp that promoted the highest shoot elongation and under the fluorescent lamp (control) after 120 days, were used. Free PAs were determined following the methodology of Silveira et al. ( 2004 ). Samples (200 mg FW each, in triplicate) were macerated with 1.2 mL of 5% perchloric acid (Merck Millipore; Darmstadt, Germany), incubated for 1h at 4°C, and centrifuged at 20,000 × g for 20 min at 4°C. The supernatant was collected, and free PAs were determined directly from the supernatant by derivatization with dansyl chloride (Merck Millipore). The dansylated PAs were partitioned with toluene (Merck Millipore), which was then evaporated under nitrogen, and the residues were resuspended in absolute acetonitrile (Merck Millipore). Samples were analyzed by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) on a reverse-phase C18 column (Shimadzu Shin-pack CLC ODS). The mobile phase gradient was generated by mixing increasing volumes of absolute acetonitrile with 10% aqueous acetonitrile (pH 5.4, adjusted with HCl). The gradient was programmed from 0 to 65% acetonitrile over the first 10 min, from 65 to 100% between 10 and 13 min, and maintained at 100% from 13 to 21 min, at a flow rate of 1 mL/min and a column temperature of 40°C. PA peaks were detected using a fluorescence detector (excitation 340 nm, emission 510 nm). The peak areas and retention times of the PAs were measured by comparison with those of standard PAs: Put, Spd and Spm (Sigma-Aldrich). Determination of plant hormone The plant hormones IAA, IBA, BA, ABA, GA, SA, JA and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were extracted according to Xavier et al. ( 2025 ),with modifications. Samples (200 mg FW each, in quintuplicate) of 120-day-old shoots from cotyledonary nodal segments were collected, frozen in liquid nitrogen, and grounded into a fine powder. Then, 1.5 mL of extraction buffer composed of 80% ethanol (Sigma-Aldrich) and 1% polyvinylpyrrolidone (PVP-40) (Sigma‒Aldrich) was added. Subsequently, the samples were sonicated for 10 min and centrifuged at 4°C for 10 min. The supernatant was collected and vacuum-dried at 45°C for 140 min until it reached approximately 200 µL. The sample volume was then adjusted with MS water (MS grade; Tedia) to 500 µL/500 mg (using a scale, considering 1:1 mass and volume), and the pH was corrected to 2.5 to 3.2 with 40 µL of acetic acid (Synth, São Paulo, Brazil). To each sample, 1 mL of ethyl ether (Sigma‒Aldrich) was added and incubated on ice for 2 min, collecting the organic phase in a new microtube and placing it in a vacuum concentrator at 45°C until the samples were completely dried. The samples were reconstituted in 500 µL of methanol:MS water (10:90), filtered using a syringe with a PTFE 0.22 µm filter (Merck Millipore; Darmstadt, Germany), and stored in vials (Waters; Manchester, UK). The hormones were separated by liquid chromatography using 10 µL of the solution recovered from the extraction on a heated BEH C18 1.7 µm column (2.1 µm × 50 mm; Waters) at 40°C, with a flow rate of 300 µL min − 1 . The binary gradient consisted of LC-MS grade water with 0.01% formic acid and LC-MS grade methanol with 0.01% formic acid as the eluent. The elution gradient started at 10% methanol and was maintained for two minutes, increasing linearly to 90% over four minutes, then increasing linearly to 100% over one minute, and decreasing to 10% over 50 s, held at 10% for 70 s, totaling a nine-min run. Using a high-performance electrospray ionization (ESI) source with dual orthogonal Z-spray (Waters), the effluents from the Acquity UPLC I-Class FTN (Waters) were introduced into the Xevo TQ-XS triple quadrupole mass spectrometer (Waters). Pressurized nebulization of nitrogen gas at 7 Bar, with a desolvation flow rate of 1000 L h − 1 and a cone gas flow of 150 L h − 1 , was employed. The desolvation gas temperature was set at 650°C, the source temperature at 150°C, and the capillary voltage at 3 kV. For the operation of the tandem dual quadrupole (MS/MS), argon gas was used as the collision gas at a pressure of 0.8 kgf cm² in the collision cell. The spectrometer operated in multiple reaction monitoring (MRM) mode, with cone voltage, collision energy, precursor mass, and fragment mass depending on the hormone under study. MassLynx software v.4.2 (Waters) was used to process the chromatograms. Following acquisition, the spectra were integrated, and hormones were quantified using TargetLynx XS software (Waters). The area under the peak of each hormone was compared with a standard curve (IAA, IBA, BA, ABA, GA, SA, JA, ACC) in pg. Data analysis All data were subjected to analysis of variance (ANOVA). Prior ANOVA, the Shapiro–Wilk test was applied to assess the normality of the data. Mean values for shoot variables, FW, PAs, and photosynthetic pigments were compared using Tukey’s multiple comparison test at a 5% significance level, whereas plant hormone means were compared using a Student’s t -test ( P ≤ 0.05). All statistical analyses were performed in R software version 4.4.2 (R Development Core Team 2024). Results Effects explant type and BA concentrations on shoot development Cotyledonary nodal segments produced shoots with greater elongation, as well as higher shoot induction (%) and number of shoots compared to apical nodal segments (Fig. 2 ). Although 0.1 µM BA promoted greater shoot elongation, no significant difference was observed compared to the control (Fig. 3 a). Higher BA concentrations (0.5 and 1 µM) reduced significantly the shoot induction (Fig. 3 b) and number of shoots (Fig. 3 c). Effects of explant type and light spectra on shoot development LED light treatments had a significant effect on elongation of shoots from both apical and cotyledonary nodal segments compared to shoots grown under a fluorescent lamp (Figs. 4 and 5 ). The greatest elongation of shoots from cotyledonary nodal segments (1.70 cm) was observed under the LED lamp W/mB/dR/fR, which combined White (W), medium blue (mB), deep red (dR), and far red (fR) spectra, compared to the fluorescent lamp (0.85 cm), not differing significantly from shoots maintained under the LED lamp W/lB/dR/fR, with white (W), low blue (lB), deep red (dR), and far red (fR) spectra (Fig. 4 A). Moreover, cotyledonary nodal segments exhibited greater elongation than those from apical segments, except under the LED lamp W/mB/dR with White (W), medium Blue (mB) and deep Red (dR), where no significant difference was observed between explant types. The number (Fig. 4 B) and induction (Fig. 4 C) of shoots were not significantly influenced by either the LED lamp treatment or explant type. Shoots from cotyledonary nodal segments under the LED lamp W/mB/dR/fR exhibited significantly higher FW than shoots from apical segments (Fig. 6 ). However, no significant differences were found between the two explant types under the fluorescent lamp. Additionally, FW did not differ significantly between apical and cotyledonary nodal segments when comparing the two light treatments. Effect of explant type and spectra light on proteomic profile Comparative proteomic analysis was performed using shoots derived from cotyledonary and apical nodal segments grown under LED WmBdRfR and fluorescent lamp (Fig. 7 ). Comparing shoots from cotyledonary nodal segment grown under LED and fluorescent lamp (CLED/CFLU comparison), a total of 31 proteins were identified, in which 25 of which were DAPs, including 1 unique to shoots grown in LED (CLED) and 2 unique proteins in shoots grown under fluorescent lamp (CFLU) (Figs. 7 a and b). Comparing shoots from apical nodal segment grown under LED and fluorescent (FLU) lamp (ALED/AFLU comparison), a total of 39 proteins were identified, 22 of which were DAPs, including 1 unique protein in shoots from Apical segments grown under LED (ALED) and 2 unique proteins in shoots grown under fluorescent lamp (AFLU; Figs. 7 c and d). Comparing shoots from Cotyledonary nodal segments (C) with Apical nodal (A) under LED incubation (comparison CLED/ALED), a total of 20 proteins were identified, 19 of which were DAPs, including 1 unique to shoots from apical segments in LED lamp (ALED; Figs. 7 e and f). In the comparison of shoots from cotyledonary with those from apical nodal segments grown under fluorescent (FLU) lamp (CFLU/AFLU comparison) a total of 20 proteins were identified, 17 of which were DAPs, including 1 unique in shoots from cotyledonary nodal segments and 2 unique to shoots from apical nodal segments (AFLU) (Figs. 7 g and h). In shoots from cotyledonary nodal segments cultured under LED with fluorescent lam (CLED/CFLU comparison) the KEGG pathway enrichment analysis revealed that the DAPs and unique proteins were associated with several biological pathways, including photosynthesis, metabolic pathways, glycolysis/gluconeogenesis, biosynthesis of secondary metabolites, ascorbate and aldarate metabolism, fatty acid degradation, and pentose phosphate pathway (Fig. 8 a). Shoots from apical nodal segments incubated under LED lamp compared to fluorescent lamp (ALED/AFLU comparison), the KEGG pathway enrichment analysis revealed that the DAPs and unique proteins were associated with several biological pathways, including carbon fixation in photosynthetic organisms, carbon metabolism, metabolic pathway, biosynthesis of amino acids, biosynthesis of secondary metabolites, and glycolysis/gluconeogenesis (Fig. 8 b). Comparing the shoots from different explant types grown under the same lamp, only one KEGG pathway was significantly enriched in the CLED/ALED comparison, corresponding to the biosynthesis of amino acids (Fig. 8 c). In contrast, no KEGG pathways were enriched in the CFLU/AFLU comparison. In shoots from cotyledonary nodal segments under LED lamp compared to fluorescent (CLED/CFLU comparison) the DAPs proteins showed a protein-protein interaction network with photosynthesis, biosynthesis of secondary metabolites and ascorbate and aldarate metabolism KEGG pathways enrichment analysis (Fig. S1). The down-accumulated photosystem I reaction center subunit II, chloroplastic-like (paubrasilia_04543) and photosystem I reaction center subunit IV B, chloroplastic-like (paubrasilia_28831) proteins; the chlorophyll a-b binding protein of LHCII type 1-like (paubrasilia_25876) - the unique in shoots from CFLU and the up-accumulated oxygen-evolving enhancer protein 1, chloroplastic (paubrasilia_09643), formed an interaction network related to photosynthesis. Moreover, the proteins down-accumulated glucose-6-phosphate isomerase cytosolic isoform X1 (paubrasilia_34648) and transketolase, chloroplastic (paubrasilia_03703) interact in the biosynthesis of secondary metabolites network. In addition, the down-accumulated aldehyde dehydrogenase family 7 member A1 (paubrasilia_23050) showed an interaction with the up-accumulated protein alcohol dehydrogenase 1 (paubrasilia_51728). Shoots from apical nodal segments under LED compared to fluorescent lamp (ALED/AFLU) shower a protein-protein interaction network of several down-acumulated proteins related with biossynthesis of amino acids and secondary metabolites, carbon metabolism, and glycolysis/gluconeogenesis (Fig. S2). Moreover, a protein-protein interaction network of proteins in shoots from cotyledonar nodal segments, which promoted the best elongation under LED (CLED) compared to apical nodal (ALED) interacted with biosynthesis of amino acids (Fig. S3). Besides no KEGG pathways enriched comparing shoots from different type of explants under fluorescent lamp (CFLU/AFLU comparison), some proteins up-accumulated showed interaction, such as Ei-4; cathepsin B-like protease 2, actin-1-like and elongation factor 2-like isoform X1 (Fig. S4). Some proteins involved in growth and developmental processes specially were highlighted and further discussed. Effects of explant type and spectra on photosynthetic pigments in shoots The analysis of photosynthetic pigments revealed significant effects of explant type and light conditions (Fig. 9 ). Shoots from cotyledonary nodal segments exhibited higher contents of Chlorophyll a (Fig. 9 a), Chlorophyll b (Fig. 9 b), total chlorophyll (Fig. 9 c) and carotenoids (Fig. 9 d) when cultured under LED W/mB/dR/fR compared to fluorescent lamp, while those from apical nodal segments did not showed significant differences. As cotyledonary nodal segments allowed the development of shoots with greater elongation and higher fresh matter accumulation, as well as exhibited significantly higher levels of chlorophylls and carotenoids, they were selected for the subsequent PAs and hormonal profile analyses. Effect of the light spectrum on the plant hormones contents in shoots from cotyledonary nodal segments The LED lamp with white, medium blue, deep red and far-red (W/mB/dR/fR) light spectra induced a higher content of BA (Fig. 10 a), ACC (Fig. 10 b) and ABA (Fig. 10 c) in shoots from cotyledonary nodal segments incubated compared with those grown under fluorescent lamp. In contrast, the highest SA content was observed in shoots cultivated under fluorescent light (Fig. 10 d). No significant differences were found in IBA (Fig. 10 e), IAA (Fig. 10 f), JA (Fig. 10 g), and GA₃ (Fig. 10 h) contents in shoots comparing the two lamps. Effect of the light spectrum on the content of free PAs in shoots from cotyledonary nodal segments A significantly higher content of total free PAs (Fig. 11 a) and free Put (Fig. 11 b) was observed in shoots from cotyledonary nodal segments grown under under LED W/mB/dR/fR compared with fluorescent lamp. In contrast, no statistically significant differences were detected in Spd (Fig. 11 c) and Spm (Fig. 11 d) contents comparing the lamps. Effect of explant type and IBA on ex vitro rooting The type of explant and IBA concentrations did not affected significantly the rooting (%) of shoots (Fig. 12 a), showing plantlets with similar rooting morphology comparing shoots from apical (Fig. 12 b) and cotyledonary (Fig. 12 c) nodal segments. After 210 days of maintenance on greenhouse, the plantlets showed 100% of survival (data not showed; Fig. 12 d). Rooted plants (Fig. 12 e) were transferred to the field (Fig. 12 f), at the Instituto Biasse Socioambiental in Itaocara – RJ – Brazil. The Fig. 13 summarizes the entire process for plants obtention using in vitro culture and ex vitro rooting of shoots in P. echinata. Discussion In vitro morphogenesis of P. echinata is markedly influenced both the type of explant and the light spectrum, affecting shoot elongation and biomass increase by modulation of differentially accumulated proteins, plant hormones and PAs. Studies carried out with woody species have demonstrated that the position of the explant on the seedlings, such as cotyledonary and apical nodal segments, affects shoot induction and development (Aragão et al. 2017a ; Aragão et al. 2017b ; Lerin et al. 2019 ; Souza et al. 2020a ; Pessanha et al. 2022 ). Among the explant types, cotyledonary nodal segments have consistently promoted shoots with greater elongation, as observed in C. legalis (Aragão et al. 2017b ; Lerin et al. 2019 ), C. fissilis (Aragão et al. 2016 ), D. nigra (Pessanha et al. 2022 ) and also in P. echinata in the present study. The enhanced shoot elongation observed from cotyledonary nodal explants can be associated to a differential accumulation of endogenous levels of growth-promoting hormones such as auxins, cytokinins, gibberellins, and PAs, which are essential for cell division, elongation, and microtubule organization for shoot development. On the other hand, apical nodal segments may be more developmentally mature and subject to apical dominance, with higher levels of auxin transport from the shoot apex that can restrict lateral shoot elongation, as observed in P. echinata. Cytokinin is essential for in vitro shoot development from axillary buds in nodal segments in several species, such as C. fissilis (Nunes et al. 2002 ), D. nigra (Ivanova et al. 2006 ; Aragão et al. 2016 ; Hernández-García et al. 2021 ; Pessanha et al. 2022 ), Actinidia chinensis (Saeiahagh et al. 2019 ), Actinia deliciosa (Arruda et al. 2019 ), Betula oycoviensis (Vítamvas et al. 2020 ), Myrcianthes pungens (Souza et al. 2020b), Campomanesia phaea (Demétrio et al. 2021 ) e Pinus koraiensis (Liang et al. 2022 ). However, in P. echinata , although shoot elongation was observed, the addition of cytokinin BA did not differ significantly from the control treatment. Instead, light quality provided by LED lamp had a predominant effect on shoot elongation. These results are consistent with findings in C. legalis , where BA alone did not improve shoot elongation, indicating that specific light spectra are required to enhance morphogenesis (Aragão et al. 2017b ; Lerin et al. 2019 ). Blue and red wavelengths are crucial in regulating plant development, as their photoreceptors modulate key morphogenetic processes (He et al. 2020 ; Silva et al. 2020 ). LED sources combining red and blue wavelengths have promoted shoot development in various species, such as Camellia oleifera (He et al. 2020 ), hybrids of Corymbia sps (Souza et al. 2020a ), Swertia chirata (Gupta and Karmakar 2017 ), Prunus cerasus and P. canescens (Sarropoulou et al. 2023 ). Blue light is mainly perceived by phototropins and cryptochromes. Phototropins regulate phototropism, chloroplast movement and stomatal opening (Briggs and Christie 2002 ), while cryptochromes participate in flowering, inhibition of etiolation, stomatal regulation, and root elongation (Canamero et al. 2006 ; Bach et al. 2018 ). Red and far-red, in turn, are detected by phytochromes, which control seed germination, stomatal development, flowering transition, leaf movement, senescence, and shade avoidance responses (Franklin and Quail 2009 ). In C. fissilis (Oliveira et al. 2020 ) and Gallesia integrifolia (Rodrigues 2023 ) the W/mB/dR LED lamp enhanced shoot growth, reinforcing the importance of red-enriched spectra for in vitro morphogenesis. In C. legalis , LED lamps emitting white (W), low blue (lB), deep red (dR), and far-red (fR) light (LED W/lB/dR/fR) resulted in greater elongation of shoots from cotyledonary nodal segments compared to fluorescent lamp (Lerin et al. 2019 ). Similarly, in P. echinata , the LED spectrum W/mB/dR/fR—combining white, medium blue, red, and far-red wavelengths—significantly increased shoot length in both cotyledonary and apical nodal explants, suggesting a key role of red and far-red light in shoot elongation. These findings suggest that the combination of low blue and red light induces a shade-avoidance response, enhancing cell elongation and stem extension even in the absence of far-red light. In P. echinata , shoot elongation was more strongly influenced by light quality than by the addition of BA to the culture medium, highlighting the importance of optimizing light spectra to improve in vitro propagation of woody species. In addition, shoots of P. echinata grown under LED W/mB/dR/fR showed endogenous BA, which was not detected in shoots cultivated under fluorescent lamp. In Malus sylvestris , BA is absorbed in the shoot region but remains concentrated in the basal part of the nodal segment, promoting bud development (Nordström and Eliasson 1986 ). In Ulmus campestris , BA is rapidly absorbed (within 30 min), and after 6 h it is degraded into adenine through side-chain cleavage (Biondi et al. 1984 ). In Arabidopsis thaliana , high light intensity reduced cytokinin levels, resulting in increased photooxidative stress and decreased photosynthetic activity (Cortleven et al. 2014 ). Thus, the lower BA contents in P. echinata shoots under fluorescent light may be associated with the higher luminous flux of this lamp compared to LEDs. LED lamps (4.5 W) emit less heat, consume less energy, and have a lower luminous flux (160 lm), while fluorescent lamps (15 W) provide 316 lm (Santos et al. 2015). Similarly, in Triticum aestivum , a low deep red/far-red ratio was shown to reduce cytokinin content (Lei et al. 2022 ). Therefore, the reduced BA concentration in P. echinata shoots under fluorescent light may be due to degradation induced by the higher luminous flux. To further understand how light quality regulates in vitro morphogenesis, proteomic approaches are essential for identifying the proteins and metabolic pathways modulated under these conditions. The light spectrum and explant type also induced differential protein accumulation during shoot development in P. echinata (Supplementary Table S1). Among the differentially accumulated proteins, shoots from cotyledonary nodal segments grown under the WmBdRfR LED spectrum showed exclusive accumulation of 70 kDa heat shock protein (HSP; paubrasilia_14874), which was not detected in shoots grown under fluorescent lamp (CLED/CFLU comparison). A similar response was reported in C. fissilis , where higher accumulation of HSPs were reported in shoots grown under WmBdR LED light compared with those from fluorescent lamp (Oliveira et al. 2020 ). HSP are a conserved family of molecular chaperones that play a central role in plant responses to abiotic and biotic stresses. They prevent protein misfolding, reduce aggregation of denatured proteins, and maintain proteome stability, thereby ensuring cellular homeostasis (Fang et al. 2021 ; Yin et al. 2023 ). Alteration in HSP expression, either suppression or overaccumulation, can impair plant growth and development, as reported in Arabidopsis (Sung and Guy 2003 ; Jungkunz et al. 2011 ). Therefore, the increased abundance of HSPs in shoots grown under the W/mB/dR/fR LED spectrum may be associated with enhanced cellular protection and protein homeostasis, contributing to the greater shoot elongation observed in P. echinata under this light condition. In addition, the proteins ascorbate peroxidase (APX) and peroxidases (PODs) are key enzymes in the plant antioxidant system, acting synergistically to maintain redox homeostasis and protect cells against oxidative damage. APX catalyzes the reduction of hydrogen peroxide (H₂O₂) to water using ascorbate as an electron donor, preventing oxidative damage to proteins, lipids, and cellular membranes (Corpas et al. 2024 ; Yoshimura and Ishikawa 2024 ). In tobacco, overexpression of the StAPX gene significantly reduced H₂O₂ accumulation, strengthening the antioxidant defense system (Sun et al. 2013 ). PODs contribute to H₂O₂ detoxification and participate in lignin biosynthesis and cell wall formation, processes crucial for plant growth and structural integrity (Castillo 2025 ). In Phoenix dactylifera , shoots grown under LED lamp (18 red:2 blue) exhibited enhanced growth, higher multiplication rates, and increased peroxidase activity compared with those grown under fluorescent lamp (Al-Mayahi 2016 ). In the present study, shoots derived from cotyledonary nodal segments of P. echinata grown under the W/mB/dR/fR LED spectrum showed increased accumulation of L-ascorbate peroxidase 2 (paubrasilia_31781) and peroxidase A2-like (paubrasilia_52999) compared to those grown under fluorescent lamp (CLED/CFLU comparison). Similarly, shoots from apical nodal segments exhibited higher levels of peroxidase 73 (paubrasilia_25491) and peroxidase 51-like (paubrasilia_25244) under the same LED spectrum compared to those from fluorescent (ALED/AFLU comparison). Thus, the increased abundance of these antioxidant enzymes under LED conditions suggests a more efficient ROS-scavenging system and improved redox regulation, which may have favored cell expansion and contributed to the increased shoot elongation observed in shoots derived from cotyledonary and apical nodal segments under WmBdRfR LED lamp. Zeaxanthin epoxidase (ZEP) catalyzes the epoxidation of the carotenoid zeaxanthin to violaxanthin in the xanthophyll cycle, providing essential precursors for abscisic acid (ABA) biosynthesis (Wu et al. 2023 ; Yadav et al. 2023 ). Thus, ZEP plays a pivotal role in linking photoprotective mechanisms to hormonal regulation under stress and developmental conditions (Premachandran 2022 ). In C. fissilis , the reduction in shoot length during successive subcultures was associated with a decrease in ABA levels (Oliveira et al. 2021). In our study, the higher accumulation of ZEP (paubrasilia_47510) and carotenoids in shoots from cotyledonary nodal segments grown under LED lamp may induced the increase in ABA levels, which could contribute to the shoot developmental performance in this light condition compared to fluorescent lamp. Moreover, elevated ABA levels are known not only to regulate stress tolerance and developmental processes but also to modulate other hormonal networks. For instance, ABA can induce the expression of ACC synthase and ACC oxidase, enhancing ethylene biosynthesis in several plant species under stress or developmental transitions, as well as ABA-treated tissues show higher ethylene emission (Hansen and Grossmann 2000 ; Sharp 2002 ; Zhang et al. 2009 ). Conversely, ABA often acts antagonistically to SA, suppressing SA biosynthesis genes (ICS1, NPR1), reducing SA accumulation and SA-dependent defense (Yasuda et al. 2008 ; Moeder et al. 2010 ; Alazem et al. 2019 ). Therefore, the higher ZEP–ABA response observed in cotyledonary-derived shoots may contribute indirectly to hormonal reprogramming, favoring ethylene-associated growth responses while down-regulating SA-mediated pathways in P. echinata . This hormonal balance could support enhanced shoot elongation and morphogenetic performance under LED light conditions. In addition, the higher SA content in shoots grown under the fluorescent lamp may have associated with the greater heat emission and higher luminous flux of this lamp compared with LED. These conditions likely induced stress and negatively affected the growth of P. echinata shoots. SA is a key plant hormone involved in responses to stress, acting in plant defense and growth (Seyfferth and Tsuda 2014 ; Ahmad et al. 2019 ; Elsisi et al. 2024 ). Studies emphasize the importance of SA in enhancing plant resistance to both biotic and abiotic stresses (Benjamin et al. 2022 ; Khan et al. 2022 ). In C. fissilis , a significant increase in endogenous SA content in shoots during the fourth subculture, compared with the first, was associated with reduced shoot growth and lower rooting potential (Oliveira et al. 2022 ). This effect may be explained by the antagonistic relationship between SA and auxin, as SA is known to reduce auxin levels, a hormone required for root induction (De Klerk et al. 2011 ). The Oxygen-Evolving Enhancer Protein 1 (OEE1) is an essential extrinsic subunit of photosystem II (PSII), also referred to as the PsbO protein (Carius et al. 2019 ). This protein plays a fundamental role in the light-driven water-splitting reaction, contributing to the release of oxygen and electrons during photosynthesis (Deng et al. 2025 ). In our study, we observed that shoots derived from cotyledonary nodal segments and grown under a white-blue-red-red (WmBdRfR) LED lamp exhibited increased accumulation of OEE1 (paubrasilia_09643), along with higher levels of photosynthetic pigments, including chlorophylls a and b and carotenoids. This suggests that the enhanced presence of OEE1 under LED lighting may contribute to improved photosynthetic efficiency in these shoots. The increase in photosynthetic pigments under LED light can be attributed to the specific light spectrum provided by the LEDs. Red and blue light wavelengths are efficiently absorbed by chlorophylls and carotenoids, leading to enhanced light absorption and energy capture, which are vital for photosynthesis (Liu and Van Iersel 2021 ). Additionally, the presence of OEE1 stabilizes the PSII complex, facilitating efficient electron transport and oxygen evolution, thereby supporting overall photosynthetic activity (Mayfield et al. 1987 ). Thus, the upregulation of OEE1 and the increased accumulation of photosynthetic pigments in shoots grown under LED lamp indicate a synergistic enhancement of photosynthetic efficiency. This combination likely contributes to the observed improved shoot growth under LED conditions. The Tubulin beta chain (paubrasilia_16187) was up-accumulated in shoots derived from cotyledonary segments compared to apical nodal segments grown under the W/mB/dR/fR LED lamp, (CLED/ALED comparison). Tubulin, as the main structural component of microtubules, plays a central role in determining the orientation of cell division and expansion, thereby directly influencing morphogenesis and plant development (Hamada 2014 ; Hsiao and Huang 2023 ). PAs are known to modulate tubulin polymerization and self-association, acting as key regulators of microtubule dynamics (Mechulam et al. 2009 ). In C. legalis , shoots grown under a W/lB/dR LED lamp exhibited higher levels of free Put and increased tubulin accumulation compared with those under fluorescent lighting (Lerin et al. 2019 ). The greater abundance of tubulin in shoots derived from cotyledonary nodal segments, together with their higher endogenous content of total free Put suggests a more active microtubule organization, which may contribute to the enhanced shoot development observed in these shoots. These observations are consistent with additional literature reporting that microtubule stability and orientation are tightly regulated by cytoskeletal-associated proteins and signaling molecules, which influence cell elongation, division plane determination, and organ morphogenesis (Mathur and Hülskamp 2002 ; Nick 2013 ). Together, these findings suggest that the greater abundance of tubulin in cotyledonary nodal segments, in combination with elevated levels of free Put reflects a more active microtubule network. This enhanced cytoskeletal organization likely contributes to the superior shoot elongation and morphogenetic performance observed under LED lighting compared with fluorescent light and apical nodal segments. Moreover, Put is associated with cell cycle progression (G1/S to G2/M) and division activity (Weiger and Hermann 2014 ), and the higher Put levels in P. echinata shoots under LED lighting enriched in red and blue shoots may stimulate cell proliferation and elongation. In C. fissilis , light spectra from LED lamp W/mB/dR increased the endogenous Put content and altered the accumulation profile of proteins related to metabolism, stress responses, biosynthesis, protein modification, and light stimulus, promoting shoot elongation (Oliveira et al. 2020 ). Similarly, in G. integrifolia , where this LED spectrum also increased Put and total free PAs, as well as proteins related with Krebs cycle compared to light from fluorescent lamp (Rodrigues 2023 ). In C. legalis , LED spectra composed of W/lB/dR/fR or W/lB/dR promoted greater shoot elongation from cotyledonary nodal segments compared to fluorescent light, likely due to shade-avoidance responses induced by the combination of low blue and red wavelengths. Under W/lB/dR LEDs, these shoots also showed higher accumulation of Put and Spd, along with proteins involved in metabolic regulation, cellular organization, photosynthesis, and stress responses (Lerin et al. 2019 ). The type of explant and IBA concentrations did not significantly affected ex vitro rooting (%) of shoots, which ranged from 50 to 65% for both explant types. Further studies are need to be performed aiming to improve ex vitro rooting in P. echinata . Although tree species often require an auxin, such as IBA, to promote adventitious rooting, our results indicate that the tested auxins were not effective in enhancing rooting in micropropagated shoots of P. echinata . In C. fissilis , shoots achieved high ex vitro rooting rates (over 90%), without the application of IBA (Ribeiro et al. 2022 ). The authors suggested that this ability is likely due to endogenous IAA metabolism and signaling, which can trigger root induction in these shoots. Ex vitro rooting has been successfully applied in other woody species, but often requires exogenous auxin to promote rooting, as observed for Tecomella undulata (Varshney and Anis 2012 ) and Albizia lebbeck (Perveen et al. 2013 ), both treated with 200 µM IBA for 30 min. Further investigations are necessary to optimize rooting induction in P. echinata . Conclusion The in vitro development of P. echinata is primarily influenced by explant type and light quality. Cotyledonary nodal segments were more efficient in promoting elongated shoots than apical nodal segments. The LED lamp composed of white, medium-blue, red, and far-red (W/mB/dR/fR) light spectra stimulated greater shoot elongation, biomass accumulation, and modulation of key proteins associated with photomorphogenesis, stress responses, hormonal regulation, and cellular organization. LED illumination also promoted higher endogenous accumulation of BA, putrescine (Put), and proteins related to photosynthesis, antioxidant activity (APX and PODs), heat shock proteins (HSPs), and tubulin, suggesting enhanced cellular stability, redox homeostasis, cytoskeletal organization, and photosynthetic efficiency. Ex vitro rooting averaged 50–65%, with no significant effect of IBA or explant type, indicating that further adjustments are needed to optimize the rooting and acclimatization stages of P. echinata . Declarations Conflict of interest The authors of the manuscript have no competing interests to declare that are relevant to the content of this article. Ethics declaration Not applicable Funding This research was supported by the CNPq (309303/2019-2; 312595/2023-9) and the FAPERJ (E26/202.533/2019; E-26/210.088/2022; E-26/200.396/2023). This study was also financed in part by the CAPES—Finance Code 001. Financial interests: The authors declare they have no financial interests. Author contributions CSC conceived the study, designed the experiments, supervised all stages of the work, acquired funding, and contributed to manuscript writing and final revision. JOS, PRC, and LTMM were responsible for in vitro shoot culture, ex vitro rooting, and statistical analyses. RGV and YRSR performed the polyamine analyses. JOS, JNN, and RC-S provided support for the hormone analyses. JNN, MSR, and VS carried out the proteomic analyses. AB, JOS, and CSC were responsible for the field plantation of the first micropropagated plantlet. All authors contributed to manuscript writing and approved the final version. Acknowledgements Funding for this work was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior— Brazil (CAPES) -Finance Code 001. Data Availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD070163. All identified proteins are available in the supplementary material. Code Availability PXD070163. References Ahmad F, Singh A, Kamal A (2019) Chap. 23 - Salicylic Acid–Mediated Defense Mechanisms to Abiotic Stress Tolerance. 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14:12:30","extension":"png","order_by":48,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118716,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/4e23926934621d6f009ee46f.png"},{"id":96918199,"identity":"20e7a841-555e-4ccf-942f-f8014da27eeb","added_by":"auto","created_at":"2025-11-27 14:11:19","extension":"xml","order_by":49,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":264265,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25008160structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/52b6f0093fd074caef3ca937.xml"},{"id":96918393,"identity":"bf756562-4b1e-48c4-9046-61740598feb5","added_by":"auto","created_at":"2025-11-27 14:11:52","extension":"html","order_by":50,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":281227,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/521a7f0aad1ff736d8f3a1be.html"},{"id":96917874,"identity":"3ef6eca5-e21c-405d-adc1-b8a118f73ff1","added_by":"auto","created_at":"2025-11-27 14:10:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6979438,"visible":true,"origin":"","legend":"\u003cp\u003eSixty-day-old seedlings (a) of \u003cem\u003ePaubrasilia echinata\u003c/em\u003ein vitro germinated, showing the apical and cotyledonary nodal segments (b) used as explants for in vitro shoot development. Bars: a = 1 cm; b = 1 cm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/16e0493624aaf4061b541dc3.png"},{"id":96816554,"identity":"de0de902-4fef-45e0-aceb-d43fcfbba0b4","added_by":"auto","created_at":"2025-11-26 11:13:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2181281,"visible":true,"origin":"","legend":"\u003cp\u003eLength (a), induction (b) and number (c) of shoots in \u003cem\u003ePaubrasilia echinata\u003c/em\u003e from apical and cotyledonary nodal segments after 120 days of in vitro incubation. Means followed by different letters show significant differences at the 5% level by Tukey test. CV = Coefficient of variation (n = 8, CV A = 35.98%, CV B = 26.83%, CV C = 32.93%).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/f1c3534d2b25e74de37d3503.png"},{"id":96816556,"identity":"54e3da96-5748-4585-aa77-fd4dd06c084b","added_by":"auto","created_at":"2025-11-26 11:13:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3219857,"visible":true,"origin":"","legend":"\u003cp\u003eLength (a), induction (b) and number (C) of shoots in \u003cem\u003ePaubrasilia echinata\u003c/em\u003e after 120 days of \u003cem\u003ein vitro\u003c/em\u003e incubation in different BA concentrations. Means followed by different letters show significant differences at the 5% level by the Tukey test. CV = coefficient of variation. (n = 8, CV A = 35.98%; CV B = 26.83%; CV C = 32.93%).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/eb8870c5267bedc81495b7d5.png"},{"id":96917902,"identity":"b983e97b-2975-4c73-bf5b-a39c584447f2","added_by":"auto","created_at":"2025-11-27 14:10:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2332344,"visible":true,"origin":"","legend":"\u003cp\u003eLength (A), induction (B), and number (C) of shoots in \u003cem\u003ePaubrasilia echinata\u003c/em\u003e after 120 days of \u003cem\u003ein vitro\u003c/em\u003e incubation under different LED and fluorescent lamps. Means followed by different letters show significant differences at the 5% level by the Tukey test. Capital letters indicate significant differences according to the explant type (apical or cotyledonary nodal segment) in the different lamp treatments. Lowercase letters represent significant differences between the two explant types (apical and cotyledonary nodal segments) in the same lamp treatment. CV = Coefficient of variation (n = 8; CV A = 23.12%, CV B = 8.49%, CV C = 11.33%).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/7399f1d010e53a8ac98090f7.png"},{"id":96816569,"identity":"9db08ed0-f9cf-4c8a-a88b-f0a4b092071c","added_by":"auto","created_at":"2025-11-26 11:13:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9856589,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological aspects of \u003cem\u003ePaubrasilia echinata\u003c/em\u003eshoots from apical and cotyledonary nodal segments after 120 days o incubation under fluorescent and LED - B/Am/V/Vd lamps. Bars = 1 cm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/ebaaa6f11c9411dfc2d74261.png"},{"id":96816560,"identity":"a26d82aa-bbe4-468c-bb22-4eb13a0eedbd","added_by":"auto","created_at":"2025-11-26 11:13:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":938266,"visible":true,"origin":"","legend":"\u003cp\u003eFresh weight (g) of \u003cem\u003ePaubrasilia echinata\u003c/em\u003e shoots from apical and cotyledonary nodal segments after 120 days of incubation under LED B/Am/V/Vd and fluorescent lamps. Means followed by different letters show significant differences at the 5% level using the Tukey test. Capital letters denote differences for each explant type comparing the different lamps. Lowercase letters denote significant differences between the two explant types for the same lamp (n = 8, Coefficient of variation = 28.7%).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/1d9dee60850a75f55560f018.png"},{"id":96816563,"identity":"b8a491f7-8577-4798-bac4-dd7746f77f58","added_by":"auto","created_at":"2025-11-26 11:13:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3344726,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram and volcano plot of differentially accumulated proteins identified in \u003cem\u003ePaubrasilia \u0026nbsp;echinata\u003c/em\u003e shoots from cotyledonary (C) and apical (A) nodal segments at 120 days of grown under WmBdRfR LED and fluorescent (FLU) lamp, in the comparisons of CLED/CFLU (a, b), ALED/AFLU(c, d), CLED/ALED (e, f), and CFLU/AFLU (g, h).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/1d29f8e23b59831fbffd6678.png"},{"id":96816567,"identity":"8213646a-8c5b-45bf-9067-6b256dbc1b8a","added_by":"auto","created_at":"2025-11-26 11:13:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2581199,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway enrichment analysis for \u003cem\u003ePaubrasilia echinata\u003c/em\u003e shoots from cotyledonary (C) and apical (A) nodal segments grown under LED and fluorescent (FLU) lamps comparing CLED/CFLU (a); ALED/AFLU (b) and CLED/ALED (c). The comparison of shoots from cotyledonary under fluorescent lamp compared to apical (CFLU/AFLU) no pathway enrichment was observed.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/b64bed1c9a2577949dfafad4.png"},{"id":96816571,"identity":"17af4aa1-2aab-4ab6-9b94-cdcdee35ae2d","added_by":"auto","created_at":"2025-11-26 11:13:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1682017,"visible":true,"origin":"","legend":"\u003cp\u003eChlorophyll a (A), b (B), total chlorophyll (C) and carotenoid (D) content in \u003cem\u003ePaubrasilia echinata\u003c/em\u003e shoots at 120 days grown under fluorescent and LED lamps. Means followed by the different letter showed significant differences at the 5% level using the Tukey test. Capital letters denote differences for each explant type comparing the different lamps. Lowercase letters denote significant differences between the two explant types for the same lamp. CV = Coefficient of variation (n = 3; CV A = 25.7%, CV B = 20.3%, CV C = 23.8%, CV D = 21.9%).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/6578b2f979da904df8ed0f87.png"},{"id":96918141,"identity":"56156af9-2b78-48c1-803b-aaba091b4b76","added_by":"auto","created_at":"2025-11-27 14:11:12","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":5630588,"visible":true,"origin":"","legend":"\u003cp\u003eEndogenous content (μg g\u003csup\u003e−1\u003c/sup\u003e FW) of BA (A), ACC (B), ABA (C), SA (D), IBA (E), IAA (F), JA (G) and GA\u003csub\u003e3\u003c/sub\u003e (H) in shoots from cotyledonary nodal segments of\u003cem\u003e Paubrasilia echinata\u003c/em\u003e at 120 days of \u003cem\u003ein vitro\u003c/em\u003e growth under LED B/Am/V/Vd and fluorescent lamps. *Indicates significant difference between lamp types at the 5% level using t-test. CV = Coefficient of Variation (n = 5; CV A = 0.10%, CV B = 24.2%, CV C = 5.7%, CV D = 49.1%, CV E = 1.9%, CV F = 1.2%, CV G = 9.5%, CV H = 0.06%).\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/bbcd8701cff4654cc8ddd666.png"},{"id":96917742,"identity":"090c0118-8bc8-439f-91fb-144a4de3ee32","added_by":"auto","created_at":"2025-11-27 14:10:29","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":2075287,"visible":true,"origin":"","legend":"\u003cp\u003eTotal free PAs (A), Put (B), Spd (C) and Spm (D) content (μg g\u003csup\u003e−1\u003c/sup\u003e FW) in \u003cem\u003ePaubrasilia echinata\u003c/em\u003e shoots from cotyledonary nodal segments at 120 days of grown under LED and fluorescent lamp. *Indicates significant differences at 5% of level by t-test. CV = coefficient of variation (n = 3; CV A = 7.7%, CV B = 6.9%, CV C = 25.32%, CV D = 33.6%).\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/ab1f5da77c6d8516cbd02a9a.png"},{"id":96816604,"identity":"2c61ced0-3f80-42bf-ae9c-ab3afb206a32","added_by":"auto","created_at":"2025-11-26 11:13:48","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":28155442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEx vitro\u003c/em\u003e root induction of shoot from apical and cotyledonary nodal segments under different IBA concentrations (a), showing rooted plantlets of shoots from apical (b) and cotyledonary nodal segments, which were transferred to greenhouse (D and E) until to be transferred to the field (f) at Instituto Biasse Socioambiental – Itaocara -RJ – Brazil. Coefficient of variation A = 19.6%. Bars a and b = 1 cm.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/fe1626465900e0ed43969734.png"},{"id":96916189,"identity":"882fb7e6-4a3c-4df9-902e-4b5b009692fb","added_by":"auto","created_at":"2025-11-27 14:08:10","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":14142707,"visible":true,"origin":"","legend":"\u003cp\u003eScheme showing the entire process of \u003cem\u003ein vitro\u003c/em\u003epropagation of \u003cem\u003eP. echinata\u003c/em\u003e, including \u003cem\u003eex vitro\u003c/em\u003e rooting and acclimatization, and transference into the field.\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/5ffdbdacbb664fa22d6924dd.png"},{"id":102234162,"identity":"41c63618-d8a9-4fb4-b285-6bbade7f773b","added_by":"auto","created_at":"2026-02-09 16:06:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":77657873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/16f80e1b-2333-4747-ac75-1f961384b947.pdf"},{"id":96917810,"identity":"18dc5a9b-ea38-48d1-b4c1-a915cc8d694a","added_by":"auto","created_at":"2025-11-27 14:10:35","extension":"tiff","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10136654,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1CLEDCFLU.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/18807ec241f7fed6789b14a7.tiff"},{"id":96916780,"identity":"9028fd81-d71b-4556-97b0-33de3a9bb7a7","added_by":"auto","created_at":"2025-11-27 14:08:52","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9364678,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2ALEDAFLU.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/75abc4df4a49dda9d5e9306b.tiff"},{"id":96917893,"identity":"48d2e726-b78a-458d-9b79-b3e1fefdb551","added_by":"auto","created_at":"2025-11-27 14:10:41","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":282966,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/d4befdc10afc504559a220e2.xlsx"},{"id":96918241,"identity":"418b78db-d66a-4d10-ab91-83118d817490","added_by":"auto","created_at":"2025-11-27 14:11:28","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5717630,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3CLEDALED.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/bb1769581a5c154375182ed2.tiff"},{"id":96816570,"identity":"e4110b87-dfc3-4ed1-a6fe-ec0e4fff1909","added_by":"auto","created_at":"2025-11-26 11:13:47","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4629818,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4CFLUAFLU.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8001406/v1/9192303d35518adb55a04ba2.tiff"}],"financialInterests":"","formattedTitle":"Light Spectrum and Explant Type Drive Hormonal and Proteomic Reprogramming in Micropropagation of the Endangered Paubrasilia echinata","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003ePaubrasilia echinata\u003c/em\u003e (Lam.) E. Gagnon, H.C. Lima \u0026amp; G.P. Lewis (Fabaceae), commonly known as pau-brasil, is an endemic and emblematic species of the Brazilian Atlantic Forest (SOS Mata Atl\u0026acirc;ntica and INPE 2024; Esser et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The species holds exceptional historical and cultural significance, as it gave Brazil its name and was officially declared the national tree by Federal Law No. 6,607 on December 7, 1978 (Brasil \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Originally described by Lamarck in 1785 as \u003cem\u003eCaesalpinia echinata\u003c/em\u003e, its taxonomy was later revised based on phylogenetic and morphological evidence. In 2016, this species was reclassified it into a monotypic genus, establishing its current nomenclature as \u003cem\u003ePaubrasilia echinata\u003c/em\u003e (Gagnon et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistorically, this species was subjected to intense exploitation, initially for the extraction of the red dye brazilein, widely used in textile dyeing and ink production. Subsequently, its dense and elastic wood became highly valued for the manufacture of high-quality violin and cello bows (Alves et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). As a result of centuries of overharvesting and habitat loss, \u003cem\u003eP. echinata\u003c/em\u003e is currently listed as Endangered on the International Union for Conservation of Nature (IUCN) Red List (IUCN \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough the propagation of \u003cem\u003eP. echinata\u003c/em\u003e via seeds and cuttings has been reported. Seeds can be stored at low temperatures (-18\u0026deg;C) with water content below 12.7%, remaining viable for five years (Hellmann et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mello et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Vegetative propagation through cuttings using higher concentrations (2,500 mg L⁻\u0026sup1;) of auxins, such as indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) significantly improves rooting, yet overall rooting rates remain low at approximately 16% (Endres et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Given these constraints, combined with the species\u0026rsquo; ecological, economic, and cultural importance and its threatened status, establishing \u003cem\u003ein vitro\u003c/em\u003e propagation of shoots from nodal segments emerges as a relevant and efficient biotechnological alternative for propagation and conservation.\u003c/p\u003e\u003cp\u003eThe morphogenetic response \u003cem\u003ein vitro\u003c/em\u003e is influenced by multiple factors, including the species, explant type and age, culture duration, and environmental conditions (Pulianmackal et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, it is essential to characterize the specific conditions required for morphogenesis, such as the choice of culture medium, the concentration and type of plant growth regulators (Ivanova et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and light quality and intensity (Fan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePropagation \u003cem\u003ein vitro\u003c/em\u003e can be achieved through the development of axillary buds from nodal segments, a strategy widely employed for the micropropagation of various tree species, such as \u003cem\u003eCedrela fissilis\u003c/em\u003e, \u003cem\u003eCariniana legalis\u003c/em\u003e, \u003cem\u003eDalbergia nigra\u003c/em\u003e, \u003cem\u003eSchomburgkia crispa\u003c/em\u003e, \u003cem\u003ePfaffia glomerata\u003c/em\u003e, \u003cem\u003eCampomanesia phaea\u003c/em\u003e, \u003cem\u003eActinia deliciosa\u003c/em\u003e and \u003cem\u003eGallesia integrifolia\u003c/em\u003e (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Arag\u0026atilde;o et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Arag\u0026atilde;o et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Arruda et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dem\u0026eacute;trio et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ramos et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Carrari-Santos et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The nodal segments derived from the same seedling exhibit different morphogenic potentials for shoot development, with some being more responsive than others along the basal-to-apical axis of the seedling. In some species as \u003cem\u003eD. nigra\u003c/em\u003e, \u003cem\u003eC. fissilis\u003c/em\u003e and \u003cem\u003eG. integrifolia\u003c/em\u003e, cotyledonary nodal segments, from the base of seedling, can be more responsive compared to apical ones when supplied with BA (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBesides explant type, the plant growth regulators are often necessary to induce in vitro morphogenesis. Cytokinins, such as N6-benzyladenine (BA), are essential for axillary bud development during shoot multiplication (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), while auxin could be related to shoot rooting (Lerin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ribeiro et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In some species, such as \u003cem\u003eC. fissilis\u003c/em\u003e, \u003cem\u003eG. integrifolia\u003c/em\u003e, and \u003cem\u003eD. nigra\u003c/em\u003e (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rodrigues et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), BA enhances shoot elongation. However, in \u003cem\u003eC. legalis\u003c/em\u003e, BA alone does not significantly promote shoot growth (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e), highlighting the need to combine chemical regulation with other strategies to optimize \u003cem\u003ein vitro\u003c/em\u003e morphogenesis.\u003c/p\u003e\u003cp\u003eOne effective approach involves the use of specific light spectra, which serve as physical cues to modulate photomorphogenesis. The light spectrum, particularly combinations of blue, red, and far-red wavelengths, strongly influences \u003cem\u003ein vitro\u003c/em\u003e morphogenesis in various wood plant species (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rodrigues \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In \u003cem\u003eC. fissilis\u003c/em\u003e, the use of light-emitting diode (LED) lamp with specific combination of white, medium blue, and red light spectra promoted superior shoot development compared to other LEDs lamps and fluorescent lamp controls (Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the light spectrum can modulate the endogenous compounds related to shoot development, such as polyamines (PAs), phytohormones and proteins improving directly the shoot development.\u003c/p\u003e\u003cp\u003eBeyond the direct role of PAs in \u003cem\u003ein vitro\u003c/em\u003e morphogenesis, studies have shown that light quality can modulate the endogenous PA content, thereby affecting the morphogenetic response in several tree species, as \u003cem\u003eC. fissilis\u003c/em\u003e and \u003cem\u003eC. legalis\u003c/em\u003e (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In shoots of \u003cem\u003eC. legalis\u003c/em\u003e, the PAs putrescine (Put) and spermidine (Spd) were more abundant under LED lamp with white spectrum combined with low blue and red spectra than under fluorescent lamp (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, in \u003cem\u003eC. fissilis\u003c/em\u003e, LED containing white light combined with medium blue and red spectra increased the shoot elongation correlated with higher Put levels compared to fluorescent lamp (Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings highlight the synergistic effects of chemical signals (cytokinins) and physical cues (light spectrum) in regulating \u003cem\u003ein vitro\u003c/em\u003e morphogenesis and optimizing shoot elongation.\u003c/p\u003e\u003cp\u003eMoreover, the phytohormones auxins, cytokinins, gibberellins (GA), ethylene, abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) act as essential chemical messengers regulating plant growth and development (Ali et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The balance between auxin and cytokinin is particularly important for controlling \u003cem\u003ein vitro\u003c/em\u003e morphogenesis and rooting (Skoog and Miller \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1957\u003c/span\u003e; Asghar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In \u003cem\u003eC. fissilis\u003c/em\u003e shoots derived from cotyledonary nodal segments, a decrease in endogenous levels of 3-indoleacetic acid (IAA), ABA, and 12-oxo-phytodienoic acid (OPDA, an intermediate in JA biosynthesis) in the fourth subculture compared to the first correlated with reduced shoot growth potential and rooting capacity (Oliveira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Conversely, the levels of JA, JA-Ile, trans-cinnamic acid (t-CA), and SA increased in the fourth subculture, which was associated with a decline in \u003cem\u003ein vitro\u003c/em\u003e development and rooting potential (Oliveira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, the spatial distribution and levels of phytohormones are modulated throughout the plant during tissue development and are influenced by external factors such as light, highlighting the relevance of their analysis in elucidating in vitro shoot development under different light spectra.\u003c/p\u003e\u003cp\u003eIn addition to plant phytohormones, proteomic approaches provide a powerful tool for investigating qualitative and quantitative changes in proteins during in vitro morphogenesis, offering insights into the molecular mechanisms underlying shoot formation (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The type of explant and the light spectrum can positively influence shoot development by modulating protein abundance (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eC. legalis\u003c/em\u003e, the greatest shoot elongation under LED lamp with white spectra combined with low blue and red (W/lB/dR) spectra was associated with the accumulation of proteins related to cell organization and composition, along with a reduction in proteins linked to stress responses, highlighting the importance of light quality in in vitro morphogenesis abundance (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, in \u003cem\u003eC. fissilis\u003c/em\u003e, the LED lamp with white spectrum combined medium blue and red spectra modulated proteins involved in metabolism, stress, and light response pathways, where the up-accumulation of argininosuccinate synthase, a precursor of Put, and the increased Put content were associated with enhanced shoot elongation compared to fluorescent lamp (Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, shoot rooting is essential for the production of plantlets. Ex vitro rooting offers several advantages over in vitro rooting, including cost reductions of up to 70% (Ranaweera et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and has been successfully applied to numerous species, including \u003cem\u003eC. fissilis\u003c/em\u003e (Ribeiro et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)d \u003cem\u003enigra\u003c/em\u003e (Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This method can be highly efficient, often resulting in plantlets with better-developed root systems compared to those rooted in vitro (Yan et al. 2010), which in turn enhances their tolerance to environmental stress (Phulwaria et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough numerous studies addressing in vitro morphogenesis and biochemical or molecular mechanisms have been carried out in different tree species, such approaches remain unexplored for \u003cem\u003eP. echinata\u003c/em\u003e. Thus, this study aimed to establish the optimal conditions for its in vitro propagation and to characterize the biochemical responses associated with shoot development.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant material\u003c/h2\u003e\u003cp\u003eImmature fruits of \u003cem\u003eP. echinata\u003c/em\u003e were collected six weeks after flowering from trees located in Campos dos Goytacazes, Rio de Janeiro, Brazil (21\u0026deg;45\u0026prime;43\u0026Prime;S, 41\u0026deg;17\u0026prime;28\u0026Prime;W), to obtain seeds used for in vitro germination. Specimens of \u003cem\u003eP. echinata\u003c/em\u003e were deposited in the Herbarium of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (HUENF 7483, HUENF 7500, HUENF 8126, HUENF 9618; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jabot.jbrj.gov.br/v3/consulta.php\u003c/span\u003e\u003cspan address=\"https://jabot.jbrj.gov.br/v3/consulta.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFruit disinfection and seed germination\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eFruit disinfection and seed germination\u003c/div\u003e\u003cp\u003eFruits were surface disinfected according to Santa-Catarina et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). First, fruits were washed in water containing one drop of neutral detergent (Limpol; S\u0026atilde;o Paulo, Brazil). Following, fruits were immersed in 70% ethanol (Tupi; S\u0026atilde;o Paulo, Brazil) for one min, and subsequently treated with 100% commercial bleach (Qboa\u0026reg;; Anhembi, S\u0026atilde;o Paulo - Brazil) containing 1.8\u0026ndash;2.5% sodium hypochlorite for 1h. Finally, the fruits were rinsed three times with autoclaved type II deionized water in a flow chamber. Immature seeds were isolated from disinfected fruits and germinated in wood plant medium (WPM; PhytoTech Labs\u0026reg;, Kansas, USA) (Lloyd and McCown \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), both supplied with 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose (Synth; S\u0026atilde;o Paulo, Brazil), 2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e gellan gum (Phytagel\u0026reg;; Sigma‒Aldrich; St Louis, USA). The pH of culture media was adjusted to 5.7 before Phytagel\u0026reg; addition. The culture medium was then distributed into test tubes (10 mL/flask) and autoclaved at 121\u0026deg;C for 20 min. After transferring to the culture medium, seeds were kept in a growth room with a light intensity of 73 \u0026micro;mol.m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an 16-h light photoperiod, at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eEffect of explant type and BA concentration on shoot development\u003c/h3\u003e\n\u003cp\u003eApical and cotyledonary nodal segments (\u0026plusmn;\u0026thinsp;1.5 cm) were isolated from 60 day-old-seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and transferred in MS (PhytoTech Labs\u0026reg;) culture medium supplemented with 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose (Synth), 2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Phytagel\u0026reg; (Sigma‒Aldrich), and different concentrations (0, 0.1, 0.5, and 1.0 \u0026micro;M) of BA (Sigma\u0026ndash;Aldrich). The pH of the culture medium was adjusted to 5.7, and then Phytagel\u0026reg; was added. The culture medium was then distributed into culture flasks (30 mL/flask) and autoclaved at 121\u0026deg;C for 20 min. After transferring to the culture medium, the explants were kept in a growth room with a light intensity of 73 \u0026micro;mol.m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, an 8-h photoperiod, at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Each treatment was composed by eight replicates, with five explants per replicate. After 120 days, the induction (%), shoot length (cm), and number of shoots per explant were evaluated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of explant type and light spectra on shoot development\u003c/h3\u003e\n\u003cp\u003eApical and cotyledonary nodal segments (\u0026plusmn;\u0026thinsp;1.5 cm) from 60-day-old seedlings \u003cem\u003ein vitro\u003c/em\u003e germinated were excised and transferred to WPM (PhytoTech Labs\u0026reg;) culture medium supplemented with 20 g L⁻\u0026sup1; sucrose (Synth), 0.1 \u0026micro;M BA (Sigma-Aldrich), and 2 g L⁻\u0026sup1; Phytagel\u0026reg; (Sigma‒Aldrich). The pH of the culture medium was adjusted to 5.7 before adding Phytagel\u0026reg;, dispensed into flasks (30 mL/flask) and autoclaved at 121\u0026deg;C for 20 min. Explant segments were cultured under seven light treatments, being six LED lamps and one fluorescent lamp as a control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The flasks were covered with four layers of shade cloth, reducing the light intensity from 73 to 16 \u0026micro;mol m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1; at 25\u0026deg;C. Each treatment consisted of eight replicates, with each replicate being one culture flask containing five explants. After 120 days, the induction (%), shoot length (cm), and number of shoots per explant were recorded. Fresh weight (FW), photosynthetic pigments, polyamines (PA), phytohormones, and proteomic analyses were performed on samples from the fluorescent lamp (control) and the LED lamp that promoted the greatest shoot elongation. Samples for PA, phytohormone, and proteomic analyses were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of the different LED light wavelengths and tubular fluorescent lamp used in \u003cem\u003eP. echinata\u003c/em\u003e shoots development\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLamps\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLamp wavelenght\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLuminous intensity at the culture flask\u003c/p\u003e\u003cp\u003e(\u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePeak\u003c/p\u003e\u003cp\u003ewavelength\u003c/p\u003e\u003cp\u003e(nm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTubular fluorescent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e435/545/580\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWmB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW/MB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450/530\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWhB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW/HB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450/530\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWlBdR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW/LB/DR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450/530/660\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWmBdR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW/MB/DR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450/530/660\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWlBdRfR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW/LB/DR/FR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450/530/660/735\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWmBdRfR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eW/MB/DR/FR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e450/530/660/735\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eWhite (W); low blue (LB)\u0026thinsp;=\u0026thinsp;8\u0026ndash;10% blue; medium blue (MB)\u0026thinsp;=\u0026thinsp;12\u0026ndash;14% blue; high blue (HB)\u0026thinsp;=\u0026thinsp;16\u0026ndash;18% blue; deep red (DR)\u0026thinsp;=\u0026thinsp;30\u0026ndash;50% deep red; far red (FR)\u0026thinsp;=\u0026thinsp;12% far red\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eEffect of IBA on shoot rooting\u003c/h3\u003e\n\u003cp\u003eThe rooting of shoots from cotyledonary and apical shoots was carried out \u003cem\u003eex vitro\u003c/em\u003e according to Ribeiro et al. (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Shoot cuttings (1.5 to 2.0 cm) were isolated and the base was immersed in different concentrations (0, 100, 250 and 500 \u0026micro;M) IBA for 3 h. Then, the shoot cuttings were then transferred to plastic cups (50 mL; TotalPlast, Santa Catarina, Brazil) containing the substrate Basaplant\u0026reg; (S\u0026atilde;o Paulo, Brazil) and vermiculite (Basil Min\u0026eacute;rios; Goi\u0026aacute;s, Brazil) (1:1;v/v) and maintained in plastic trays (39.4 \u0026times; 31.9 \u0026times; 15.4 cm) (Pleion; S\u0026atilde;o Paulo, Brazil) covered with PVC-type plastic film (Lumipam; S\u0026atilde;o Paulo, Brazil) to maintain high relative humidity. The trays were maintained in a growth room at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a photoperiod of 16 h, with a light intensity of 55 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e provided by LED lamps (Koninklijke Philips Electronics NV). After 25 days, aiming to reduce the moisture inside trays and stimulate the start of an acclimatization process simultaneously with rooting induction, the PVC parafilm plastic was perforated. This procedure was carried out until the complete removal of the PVC at 100 days after the start of perforation. After 240 days, the rooted plantlets (%) were evaluated.\u003c/p\u003e\u003cp\u003eRooted plantlets were transferred to plastic bags containing soil and maintained in a greenhouse, receiving irrigation three times daily (6:00, 10:00, and 17:00 h) during 210 days. The micropropagated plantlets were transplanted to a field in a biotechnological trial at Instituto Biasse Socioambiental (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://institutobiasse.org.br/\u003c/span\u003e\u003cspan address=\"https://institutobiasse.org.br/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) located in Itaocara \u0026ndash; RJ \u0026ndash; Brazil (21\u0026deg;40\u0026prime;05,18\u0026Prime;S, 42\u0026deg;03\u0026prime;32,62\u0026Prime;W), for environmental education and extension activities.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFW determination\u003c/h2\u003e\u003cp\u003eThe FW of shoots developed under fluorescent and the LED treatment that promoted the higher shoot development from apical and cotyledonary nodal segments was determined using a precision balance. Five repetitions, with a culture flask containing five shoots each repetition, were used for FW analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProteomic analysis\u003c/h3\u003e\n\u003cp\u003eFor comparative proteomic analysis, samples (300 mg FW each, in triplicate) were collected from apical and cotyledonary nodal segments after 120 days of cultivation in WPM medium supplemented with 0.1 \u0026micro;M BA. The samples were obtained under two lighting conditions: LED, which promoted the greatest shoot elongation, and fluorescent, used as the control. Initially, samples were ground into a fine powder in liquid nitrogen. Proteins were extracted using a modified trichloroacetic acid (TCA)/acetone method (Damerval et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Samples were resuspended in 1 mL of protein extraction buffer containing 10% (w/v) TCA (Sigma-Aldrich) in acetone (Sigma-Aldrich) supplemented with 20 mM dithiothreitol (DTT; Bio-Rad, Hercules CA, USA). The resulting pellets were washed three times with cold acetone containing 20 mM DTT. Pellets were air-dried, resuspended in buffer containing 7 M urea (Cytiva; Marlborough, USA), 2 M thiourea (Cytiva), 2% Triton X-100 (Sigma-Aldrich), 1% DTT (Bio-Rad), and 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich). The protein concentration was determined using the 2-D Quant Kit (Cytiva).\u003c/p\u003e\u003cp\u003eBefore trypsin (V5111; Promega, Madison, USA; final enzyme-to-protein ratio of 1:100) digestion, proteins were precipitated using the methanol/chloroform method (Nanjo et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Following precipitation, the samples were resuspended in a 7 M urea/2 M thiourea solution. Aliquots containing 100 \u0026micro;g of protein were digested according to the filter-aided sample preparation (FASP) protocol (Wiśniewski et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Peptides were then resuspended and quantified at 205 nm using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific; Waltham, USA). Samples were transferred to Total Recovery Vials (Waters; Manchester, UK) for mass spectrometry.\u003c/p\u003e\u003cp\u003eMass spectrometry analyses were performed using a nanoACQUITY UPLC system coupled to a Synapt G2-Si HDMS mass spectrometer (Waters) operated in nano-electrospray liquid chromatography tandem mass spectrometry mode (nanoESI-LC\u0026ndash;MS/MS). A total of 2 \u0026micro;g of peptides was loaded onto a C18 trap column (180 \u0026micro;m \u0026times; 20 mm; Waters) at 5 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 3 min, and subsequently onto a nano-Acquity HSS T3 1.8 \u0026micro;m analytical reverse-phase column (75 \u0026micro;m \u0026times; 150 mm; Waters) at 400 nL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, maintained at 45\u0026deg;C. Peptides were eluted using a binary gradient: mobile phase A consisted of water (Tedia; Fairfield, USA) with 0.1% formic acid (Sigma-Aldrich), and mobile phase B consisted of acetonitrile (Sigma-Aldrich) with 0.1% formic acid. The gradient was as follows: 5% B at the start, increased to 40% B by 92 min, ramped to 99.9% B by 96 min, held at 99.9% B until 100 min, decreased to 5% B by 102 min, and maintained at 5% B until 118 min.\u003c/p\u003e\u003cp\u003eMass spectrometry was performed in positive resolution mode (V mode; 35,000 full with at half maximum) with ion mobility separation (IMS) and in data-independent acquisition (DIA) mode (HDMSE). IMS was operated with a wave velocity program from 800 to 500 m s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, helium and IMS gas flows of 180 and 90 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Transfer collision energy was ramped from 19 to 55 V in high-energy mode, with cone and capillary voltages of 30 and 2800 V, respectively, and a source temperature of 100\u0026deg;C. Time-of-flight (TOF) parameters included a scan time of 0.5 s in continuum mode, with a mass range of 50\u0026ndash;2000 Da. Human [Glu\u003csup\u003e1\u003c/sup\u003e]-fibrinopeptide B (Sigma-Aldrich) at 100 fmol \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used as an external standard, and lock-mass acquisition was performed every 30 s. Data acquisition was carried out for 90 min using MassLynx v.4.1 software.\u003c/p\u003e\u003cp\u003eSpectral processing and database searches were performed using ProteinLynx Global Server (PLGS) v.3.0.3 (Waters). Raw data processing settings included a low-energy threshold of 150 counts, a high-energy threshold of 50, and an intensity threshold of 750. Database searches were performed with the following parameters: up to two missed cleavages; minimum of three fragment ions per peptide; minimum of seven fragment ions per protein; minimum of two peptides per protein; carbamidomethylation of cysteine as a fixed modification; oxidation (M) and phosphorylation (STY) as variable modifications; and a maximum false discovery rate (FDR) of 1%. Searches were conducted against the \u003cem\u003eP. echinata\u003c/em\u003e (Lam.) Gagnon, H.C.Lima \u0026amp; G.P.Lewis protein database. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) partner repository with the dataset identifier PXD070163. Label-free quantification analyses were performed using ISOQuant software v.1.7 (Distler et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLabel-free quantification was performed using ISOQuant v.1.8 (Distler et al., 2013). Parameters included an FDR of 1%, a peptide score greater than six, a minimum peptide length of six amino acids, and at least two peptides per protein were considered for label-free quantitation using the TOP3 approach, followed by the multidimensional normalized process within ISOQuant. To ensure robustness, only proteins consistently present or absent across all three biological replicates were considered in differential abundance protein (DAP) analysis. Statistical comparisons were performed using two-tailed Student\u0026rsquo;s t-tests. Proteins with \u003cem\u003ep-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e were classified as up-accumulated if the Log\u003csub\u003e2\u003c/sub\u003e fold-change (FC) exceeded 0.6, and down-accumulated if Log\u003csub\u003e2\u003c/sub\u003e (FC) was below \u0026minus;\u0026thinsp;0.6. Functional annotations were performed using OmicsBox v.3.0.29 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.biobam.com\u003c/span\u003e\u003cspan address=\"https://www.biobam.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein sequences were queried against the NCBI non-redundant green plant protein database (taxa: 33,090; Viridiplantae) using BLAST. The enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of DAPs was performed in STRING version 12.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003cspan address=\"https://string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using \u003cem\u003eGlycine max\u003c/em\u003e homologs of \u003cem\u003eP. echinata\u003c/em\u003e proteins. To generate a protein‒protein interaction (PPI) network, \u003cem\u003eG. max\u003c/em\u003e was considered the reference plant species using a minimum required interaction score of 0.7, and network analysis was performed with Cytoscape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cytoscape.org\u003c/span\u003e\u003cspan address=\"https://cytoscape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (version 3.10.2) (Shannon et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eAnalysis of photosynthetic pigments\u003c/h3\u003e\n\u003cp\u003ePhotosynthetic pigments chlorophyll (Chl) a, b, and carotenoids were analyzed according to Arnon (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1949\u003c/span\u003e). Samples (200 mg FW, in triplicate) of shoots from apical and cotyledonary nodal segments after 120 days of incubation under LED and fluorescent lamps were used. The photosynthetic pigments were extracted with 10 mL of 80% acetone (Sigma-Aldrich). Subsequently, the samples were incubated in the dark at 3\u0026deg;C for 24 h. Absorbance readings were performed at wavelengths of 663 nm for Chl a, 645 nm for Chl b, and 470 nm for carotenoids, using the SoftMax Pro\u0026reg; 6.0 software. Pigment content was calculated using the following equations (Arnon \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1949\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Chl\\:a=\\frac{\\left(12.7\\times\\:O{D}_{663}-2.69\\times\\:O{D}_{645}\\right)\\times\\:V}{1000\\times\\:W}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Chl\\:b=\\frac{\\left(22.9\\times\\:O{D}_{645}-4.68\\times\\:O{D}_{663}\\right)\\times\\:V}{1000\\times\\:W}$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Carotenoids=\\frac{\\left(1000\\times\\:O{D}_{470}-3.27\\times\\:Chl\\:a-104\\times\\:Chl\\:b\\right)}{229\\times\\:1000\\times\\:W}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere: OD represents the optical density, V is the total extract volume (mL), and W is the FW (g) of the shoots used.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of free PAs content\u003c/h2\u003e\u003cp\u003eShoots originating from apical and cotyledonary nodal segments, obtained under the LED lamp that promoted the highest shoot elongation and under the fluorescent lamp (control) after 120 days, were used. Free PAs were determined following the methodology of Silveira et al. (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Samples (200 mg FW each, in triplicate) were macerated with 1.2 mL of 5% perchloric acid (Merck Millipore; Darmstadt, Germany), incubated for 1h at 4\u0026deg;C, and centrifuged at 20,000 \u0026times; g for 20 min at 4\u0026deg;C. The supernatant was collected, and free PAs were determined directly from the supernatant by derivatization with dansyl chloride (Merck Millipore). The dansylated PAs were partitioned with toluene (Merck Millipore), which was then evaporated under nitrogen, and the residues were resuspended in absolute acetonitrile (Merck Millipore). Samples were analyzed by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) on a reverse-phase C18 column (Shimadzu Shin-pack CLC ODS). The mobile phase gradient was generated by mixing increasing volumes of absolute acetonitrile with 10% aqueous acetonitrile (pH 5.4, adjusted with HCl). The gradient was programmed from 0 to 65% acetonitrile over the first 10 min, from 65 to 100% between 10 and 13 min, and maintained at 100% from 13 to 21 min, at a flow rate of 1 mL/min and a column temperature of 40\u0026deg;C. PA peaks were detected using a fluorescence detector (excitation 340 nm, emission 510 nm). The peak areas and retention times of the PAs were measured by comparison with those of standard PAs: Put, Spd and Spm (Sigma-Aldrich).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of plant hormone\u003c/h2\u003e\u003cp\u003e The plant hormones IAA, IBA, BA, ABA, GA, SA, JA and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were extracted according to Xavier et al. (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2025\u003c/span\u003e),with modifications. Samples (200 mg FW each, in quintuplicate) of 120-day-old shoots from cotyledonary nodal segments were collected, frozen in liquid nitrogen, and grounded into a fine powder. Then, 1.5 mL of extraction buffer composed of 80% ethanol (Sigma-Aldrich) and 1% polyvinylpyrrolidone (PVP-40) (Sigma‒Aldrich) was added. Subsequently, the samples were sonicated for 10 min and centrifuged at 4\u0026deg;C for 10 min. The supernatant was collected and vacuum-dried at 45\u0026deg;C for 140 min until it reached approximately 200 \u0026micro;L. The sample volume was then adjusted with MS water (MS grade; Tedia) to 500 \u0026micro;L/500 mg (using a scale, considering 1:1 mass and volume), and the pH was corrected to 2.5 to 3.2 with 40 \u0026micro;L of acetic acid (Synth, S\u0026atilde;o Paulo, Brazil). To each sample, 1 mL of ethyl ether (Sigma‒Aldrich) was added and incubated on ice for 2 min, collecting the organic phase in a new microtube and placing it in a vacuum concentrator at 45\u0026deg;C until the samples were completely dried. The samples were reconstituted in 500 \u0026micro;L of methanol:MS water (10:90), filtered using a syringe with a PTFE 0.22 \u0026micro;m filter (Merck Millipore; Darmstadt, Germany), and stored in vials (Waters; Manchester, UK).\u003c/p\u003e\u003cp\u003eThe hormones were separated by liquid chromatography using 10 \u0026micro;L of the solution recovered from the extraction on a heated BEH C18 1.7 \u0026micro;m column (2.1 \u0026micro;m \u0026times; 50 mm; Waters) at 40\u0026deg;C, with a flow rate of 300 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The binary gradient consisted of LC-MS grade water with 0.01% formic acid and LC-MS grade methanol with 0.01% formic acid as the eluent. The elution gradient started at 10% methanol and was maintained for two minutes, increasing linearly to 90% over four minutes, then increasing linearly to 100% over one minute, and decreasing to 10% over 50 s, held at 10% for 70 s, totaling a nine-min run.\u003c/p\u003e\u003cp\u003eUsing a high-performance electrospray ionization (ESI) source with dual orthogonal Z-spray (Waters), the effluents from the Acquity UPLC I-Class FTN (Waters) were introduced into the Xevo TQ-XS triple quadrupole mass spectrometer (Waters). Pressurized nebulization of nitrogen gas at 7 Bar, with a desolvation flow rate of 1000 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a cone gas flow of 150 L h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, was employed. The desolvation gas temperature was set at 650\u0026deg;C, the source temperature at 150\u0026deg;C, and the capillary voltage at 3 kV. For the operation of the tandem dual quadrupole (MS/MS), argon gas was used as the collision gas at a pressure of 0.8 kgf cm\u0026sup2; in the collision cell. The spectrometer operated in multiple reaction monitoring (MRM) mode, with cone voltage, collision energy, precursor mass, and fragment mass depending on the hormone under study. MassLynx software v.4.2 (Waters) was used to process the chromatograms. Following acquisition, the spectra were integrated, and hormones were quantified using TargetLynx XS software (Waters). The area under the peak of each hormone was compared with a standard curve (IAA, IBA, BA, ABA, GA, SA, JA, ACC) in pg.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eAll data were subjected to analysis of variance (ANOVA). Prior ANOVA, the Shapiro\u0026ndash;Wilk test was applied to assess the normality of the data. Mean values for shoot variables, FW, PAs, and photosynthetic pigments were compared using Tukey\u0026rsquo;s multiple comparison test at a 5% significance level, whereas plant hormone means were compared using a Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05). All statistical analyses were performed in R software version 4.4.2 (R Development Core Team 2024).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEffects explant type and BA concentrations on shoot development\u003c/h2\u003e\u003cp\u003eCotyledonary nodal segments produced shoots with greater elongation, as well as higher shoot induction (%) and number of shoots compared to apical nodal segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although 0.1 \u0026micro;M BA promoted greater shoot elongation, no significant difference was observed compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Higher BA concentrations (0.5 and 1 \u0026micro;M) reduced significantly the shoot induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and number of shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEffects of explant type and light spectra on shoot development\u003c/h2\u003e\u003cp\u003eLED light treatments had a significant effect on elongation of shoots from both apical and cotyledonary nodal segments compared to shoots grown under a fluorescent lamp (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The greatest elongation of shoots from cotyledonary nodal segments (1.70 cm) was observed under the LED lamp W/mB/dR/fR, which combined White (W), medium blue (mB), deep red (dR), and far red (fR) spectra, compared to the fluorescent lamp (0.85 cm), not differing significantly from shoots maintained under the LED lamp W/lB/dR/fR, with white (W), low blue (lB), deep red (dR), and far red (fR) spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Moreover, cotyledonary nodal segments exhibited greater elongation than those from apical segments, except under the LED lamp W/mB/dR with White (W), medium Blue (mB) and deep Red (dR), where no significant difference was observed between explant types. The number (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) of shoots were not significantly influenced by either the LED lamp treatment or explant type.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eShoots from cotyledonary nodal segments under the LED lamp W/mB/dR/fR exhibited significantly higher FW than shoots from apical segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, no significant differences were found between the two explant types under the fluorescent lamp. Additionally, FW did not differ significantly between apical and cotyledonary nodal segments when comparing the two light treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEffect of explant type and spectra light on proteomic profile\u003c/h2\u003e\u003cp\u003eComparative proteomic analysis was performed using shoots derived from cotyledonary and apical nodal segments grown under LED WmBdRfR and fluorescent lamp (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Comparing shoots from cotyledonary nodal segment grown under LED and fluorescent lamp (CLED/CFLU comparison), a total of 31 proteins were identified, in which 25 of which were DAPs, including 1 unique to shoots grown in LED (CLED) and 2 unique proteins in shoots grown under fluorescent lamp (CFLU) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComparing shoots from apical nodal segment grown under LED and fluorescent (FLU) lamp (ALED/AFLU comparison), a total of 39 proteins were identified, 22 of which were DAPs, including 1 unique protein in shoots from Apical segments grown under LED (ALED) and 2 unique proteins in shoots grown under fluorescent lamp (AFLU; Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and d). Comparing shoots from Cotyledonary nodal segments (C) with Apical nodal (A) under LED incubation (comparison CLED/ALED), a total of 20 proteins were identified, 19 of which were DAPs, including 1 unique to shoots from apical segments in LED lamp (ALED; Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee and f).\u003c/p\u003e\u003cp\u003eIn the comparison of shoots from cotyledonary with those from apical nodal segments grown under fluorescent (FLU) lamp (CFLU/AFLU comparison) a total of 20 proteins were identified, 17 of which were DAPs, including 1 unique in shoots from cotyledonary nodal segments and 2 unique to shoots from apical nodal segments (AFLU) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg and h).\u003c/p\u003e\u003cp\u003eIn shoots from cotyledonary nodal segments cultured under LED with fluorescent lam (CLED/CFLU comparison) the KEGG pathway enrichment analysis revealed that the DAPs and unique proteins were associated with several biological pathways, including photosynthesis, metabolic pathways, glycolysis/gluconeogenesis, biosynthesis of secondary metabolites, ascorbate and aldarate metabolism, fatty acid degradation, and pentose phosphate pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eShoots from apical nodal segments incubated under LED lamp compared to fluorescent lamp (ALED/AFLU comparison), the KEGG pathway enrichment analysis revealed that the DAPs and unique proteins were associated with several biological pathways, including carbon fixation in photosynthetic organisms, carbon metabolism, metabolic pathway, biosynthesis of amino acids, biosynthesis of secondary metabolites, and glycolysis/gluconeogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eComparing the shoots from different explant types grown under the same lamp, only one KEGG pathway was significantly enriched in the CLED/ALED comparison, corresponding to the biosynthesis of amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). In contrast, no KEGG pathways were enriched in the CFLU/AFLU comparison.\u003c/p\u003e\u003cp\u003eIn shoots from cotyledonary nodal segments under LED lamp compared to fluorescent (CLED/CFLU comparison) the DAPs proteins showed a protein-protein interaction network with photosynthesis, biosynthesis of secondary metabolites and ascorbate and aldarate metabolism KEGG pathways enrichment analysis (Fig. S1). The down-accumulated photosystem I reaction center subunit II, chloroplastic-like (paubrasilia_04543) and photosystem I reaction center subunit IV B, chloroplastic-like (paubrasilia_28831) proteins; the chlorophyll a-b binding protein of LHCII type 1-like (paubrasilia_25876) - the unique in shoots from CFLU and the up-accumulated oxygen-evolving enhancer protein 1, chloroplastic (paubrasilia_09643), formed an interaction network related to photosynthesis. Moreover, the proteins down-accumulated glucose-6-phosphate isomerase cytosolic isoform X1 (paubrasilia_34648) and transketolase, chloroplastic (paubrasilia_03703) interact in the biosynthesis of secondary metabolites network. In addition, the down-accumulated aldehyde dehydrogenase family 7 member A1 (paubrasilia_23050) showed an interaction with the up-accumulated protein alcohol dehydrogenase 1 (paubrasilia_51728). Shoots from apical nodal segments under LED compared to fluorescent lamp (ALED/AFLU) shower a protein-protein interaction network of several down-acumulated proteins related with biossynthesis of amino acids and secondary metabolites, carbon metabolism, and glycolysis/gluconeogenesis (Fig. S2). Moreover, a protein-protein interaction network of proteins in shoots from cotyledonar nodal segments, which promoted the best elongation under LED (CLED) compared to apical nodal (ALED) interacted with biosynthesis of amino acids (Fig. S3). Besides no KEGG pathways enriched comparing shoots from different type of explants under fluorescent lamp (CFLU/AFLU comparison), some proteins up-accumulated showed interaction, such as Ei-4; cathepsin B-like protease 2, actin-1-like and elongation factor 2-like isoform X1 (Fig. S4). Some proteins involved in growth and developmental processes specially were highlighted and further discussed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEffects of explant type and spectra on photosynthetic pigments in shoots\u003c/h2\u003e\u003cp\u003eThe analysis of photosynthetic pigments revealed significant effects of explant type and light conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Shoots from cotyledonary nodal segments exhibited higher contents of Chlorophyll a (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), Chlorophyll b (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), total chlorophyll (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003ec) and carotenoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) when cultured under LED W/mB/dR/fR compared to fluorescent lamp, while those from apical nodal segments did not showed significant differences. As cotyledonary nodal segments allowed the development of shoots with greater elongation and higher fresh matter accumulation, as well as exhibited significantly higher levels of chlorophylls and carotenoids, they were selected for the subsequent PAs and hormonal profile analyses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of the light spectrum on the plant hormones contents in shoots from cotyledonary nodal segments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe LED lamp with white, medium blue, deep red and far-red (W/mB/dR/fR) light spectra induced a higher content of BA (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003ea), ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003eb) and ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003ec) in shoots from cotyledonary nodal segments incubated compared with those grown under fluorescent lamp. In contrast, the highest SA content was observed in shoots cultivated under fluorescent light (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003ed). No significant differences were found in IBA (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003ee), IAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003ef), JA (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003eg), and GA₃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e10\u003c/span\u003eh) contents in shoots comparing the two lamps.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of the light spectrum on the content of free PAs in shoots from cotyledonary nodal segments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA significantly higher content of total free PAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e11\u003c/span\u003ea) and free Put (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e11\u003c/span\u003eb) was observed in shoots from cotyledonary nodal segments grown under under LED W/mB/dR/fR compared with fluorescent lamp. In contrast, no statistically significant differences were detected in Spd (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e11\u003c/span\u003ec) and Spm (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e11\u003c/span\u003ed) contents comparing the lamps.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of explant type and IBA on\u003c/b\u003e \u003cb\u003eex vitro\u003c/b\u003e \u003cb\u003erooting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe type of explant and IBA concentrations did not affected significantly the rooting (%) of shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003ea), showing plantlets with similar rooting morphology comparing shoots from apical (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003eb) and cotyledonary (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003ec) nodal segments. After 210 days of maintenance on greenhouse, the plantlets showed 100% of survival (data not showed; Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003ed). Rooted plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003ee) were transferred to the field (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e12\u003c/span\u003ef), at the Instituto Biasse Socioambiental in Itaocara \u0026ndash; RJ \u0026ndash; Brazil. The Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e13\u003c/span\u003e summarizes the entire process for plants obtention using in vitro culture and ex vitro rooting of shoots in \u003cem\u003eP. echinata.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn vitro morphogenesis of \u003cem\u003eP. echinata\u003c/em\u003e is markedly influenced both the type of explant and the light spectrum, affecting shoot elongation and biomass increase by modulation of differentially accumulated proteins, plant hormones and PAs. Studies carried out with woody species have demonstrated that the position of the explant on the seedlings, such as cotyledonary and apical nodal segments, affects shoot induction and development (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Arag\u0026atilde;o et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the explant types, cotyledonary nodal segments have consistently promoted shoots with greater elongation, as observed in \u003cem\u003eC. legalis\u003c/em\u003e (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), C. \u003cem\u003efissilis\u003c/em\u003e (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), D. \u003cem\u003enigra\u003c/em\u003e (Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and also in \u003cem\u003eP. echinata\u003c/em\u003e in the present study. The enhanced shoot elongation observed from cotyledonary nodal explants can be associated to a differential accumulation of endogenous levels of growth-promoting hormones such as auxins, cytokinins, gibberellins, and PAs, which are essential for cell division, elongation, and microtubule organization for shoot development. On the other hand, apical nodal segments may be more developmentally mature and subject to apical dominance, with higher levels of auxin transport from the shoot apex that can restrict lateral shoot elongation, as observed in \u003cem\u003eP. echinata.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCytokinin is essential for in vitro shoot development from axillary buds in nodal segments in several species, such as \u003cem\u003eC. fissilis\u003c/em\u003e (Nunes et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), D. \u003cem\u003enigra\u003c/em\u003e (Ivanova et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hern\u0026aacute;ndez-Garc\u0026iacute;a et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pessanha et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), \u003cem\u003eActinidia chinensis\u003c/em\u003e (Saeiahagh et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eActinia deliciosa\u003c/em\u003e (Arruda et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eBetula oycoviensis\u003c/em\u003e (V\u0026iacute;tamvas et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eMyrcianthes pungens\u003c/em\u003e (Souza et al. 2020b), \u003cem\u003eCampomanesia phaea\u003c/em\u003e (Dem\u0026eacute;trio et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) e \u003cem\u003ePinus koraiensis\u003c/em\u003e (Liang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, in \u003cem\u003eP. echinata\u003c/em\u003e, although shoot elongation was observed, the addition of cytokinin BA did not differ significantly from the control treatment. Instead, light quality provided by LED lamp had a predominant effect on shoot elongation. These results are consistent with findings in \u003cem\u003eC. legalis\u003c/em\u003e, where BA alone did not improve shoot elongation, indicating that specific light spectra are required to enhance morphogenesis (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBlue and red wavelengths are crucial in regulating plant development, as their photoreceptors modulate key morphogenetic processes (He et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Silva et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). LED sources combining red and blue wavelengths have promoted shoot development in various species, such as \u003cem\u003eCamellia oleifera\u003c/em\u003e (He et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), hybrids of \u003cem\u003eCorymbia\u003c/em\u003e sps (Souza et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e), \u003cem\u003eSwertia chirata\u003c/em\u003e (Gupta and Karmakar \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003ePrunus cerasus\u003c/em\u003e and \u003cem\u003eP. canescens\u003c/em\u003e (Sarropoulou et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Blue light is mainly perceived by phototropins and cryptochromes. Phototropins regulate phototropism, chloroplast movement and stomatal opening (Briggs and Christie \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), while cryptochromes participate in flowering, inhibition of etiolation, stomatal regulation, and root elongation (Canamero et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Bach et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Red and far-red, in turn, are detected by phytochromes, which control seed germination, stomatal development, flowering transition, leaf movement, senescence, and shade avoidance responses (Franklin and Quail \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In \u003cem\u003eC. fissilis\u003c/em\u003e (Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and \u003cem\u003eGallesia integrifolia\u003c/em\u003e (Rodrigues \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) the W/mB/dR LED lamp enhanced shoot growth, reinforcing the importance of red-enriched spectra for \u003cem\u003ein vitro\u003c/em\u003e morphogenesis. In \u003cem\u003eC. legalis\u003c/em\u003e, LED lamps emitting white (W), low blue (lB), deep red (dR), and far-red (fR) light (LED W/lB/dR/fR) resulted in greater elongation of shoots from cotyledonary nodal segments compared to fluorescent lamp (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similarly, in \u003cem\u003eP. echinata\u003c/em\u003e, the LED spectrum W/mB/dR/fR\u0026mdash;combining white, medium blue, red, and far-red wavelengths\u0026mdash;significantly increased shoot length in both cotyledonary and apical nodal explants, suggesting a key role of red and far-red light in shoot elongation.\u003c/p\u003e\u003cp\u003eThese findings suggest that the combination of low blue and red light induces a shade-avoidance response, enhancing cell elongation and stem extension even in the absence of far-red light. In \u003cem\u003eP. echinata\u003c/em\u003e, shoot elongation was more strongly influenced by light quality than by the addition of BA to the culture medium, highlighting the importance of optimizing light spectra to improve in vitro propagation of woody species.\u003c/p\u003e\u003cp\u003eIn addition, shoots of \u003cem\u003eP. echinata\u003c/em\u003e grown under LED W/mB/dR/fR showed endogenous BA, which was not detected in shoots cultivated under fluorescent lamp. In \u003cem\u003eMalus sylvestris\u003c/em\u003e, BA is absorbed in the shoot region but remains concentrated in the basal part of the nodal segment, promoting bud development (Nordstr\u0026ouml;m and Eliasson \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). In \u003cem\u003eUlmus campestris\u003c/em\u003e, BA is rapidly absorbed (within 30 min), and after 6 h it is degraded into adenine through side-chain cleavage (Biondi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, high light intensity reduced cytokinin levels, resulting in increased photooxidative stress and decreased photosynthetic activity (Cortleven et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, the lower BA contents in \u003cem\u003eP. echinata\u003c/em\u003e shoots under fluorescent light may be associated with the higher luminous flux of this lamp compared to LEDs. LED lamps (4.5 W) emit less heat, consume less energy, and have a lower luminous flux (160 lm), while fluorescent lamps (15 W) provide 316 lm (Santos et al. 2015). Similarly, in \u003cem\u003eTriticum aestivum\u003c/em\u003e, a low deep red/far-red ratio was shown to reduce cytokinin content (Lei et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the reduced BA concentration in \u003cem\u003eP. echinata shoots\u003c/em\u003e under fluorescent light may be due to degradation induced by the higher luminous flux. To further understand how light quality regulates in vitro morphogenesis, proteomic approaches are essential for identifying the proteins and metabolic pathways modulated under these conditions.\u003c/p\u003e\u003cp\u003eThe light spectrum and explant type also induced differential protein accumulation during shoot development in \u003cem\u003eP. echinata\u003c/em\u003e (Supplementary Table S1). Among the differentially accumulated proteins, shoots from cotyledonary nodal segments grown under the WmBdRfR LED spectrum showed exclusive accumulation of 70 kDa heat shock protein (HSP; paubrasilia_14874), which was not detected in shoots grown under fluorescent lamp (CLED/CFLU comparison). A similar response was reported in \u003cem\u003eC. fissilis\u003c/em\u003e, where higher accumulation of HSPs were reported in shoots grown under WmBdR LED light compared with those from fluorescent lamp (Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). HSP are a conserved family of molecular chaperones that play a central role in plant responses to abiotic and biotic stresses. They prevent protein misfolding, reduce aggregation of denatured proteins, and maintain proteome stability, thereby ensuring cellular homeostasis (Fang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yin et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Alteration in HSP expression, either suppression or overaccumulation, can impair plant growth and development, as reported in Arabidopsis (Sung and Guy \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jungkunz et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Therefore, the increased abundance of HSPs in shoots grown under the W/mB/dR/fR LED spectrum may be associated with enhanced cellular protection and protein homeostasis, contributing to the greater shoot elongation observed in \u003cem\u003eP. echinata\u003c/em\u003e under this light condition.\u003c/p\u003e\u003cp\u003eIn addition, the proteins ascorbate peroxidase (APX) and peroxidases (PODs) are key enzymes in the plant antioxidant system, acting synergistically to maintain redox homeostasis and protect cells against oxidative damage. APX catalyzes the reduction of hydrogen peroxide (H₂O₂) to water using ascorbate as an electron donor, preventing oxidative damage to proteins, lipids, and cellular membranes (Corpas et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yoshimura and Ishikawa \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In tobacco, overexpression of the \u003cem\u003eStAPX\u003c/em\u003e gene significantly reduced H₂O₂ accumulation, strengthening the antioxidant defense system (Sun et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). PODs contribute to H₂O₂ detoxification and participate in lignin biosynthesis and cell wall formation, processes crucial for plant growth and structural integrity (Castillo \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In \u003cem\u003ePhoenix dactylifera\u003c/em\u003e, shoots grown under LED lamp (18 red:2 blue) exhibited enhanced growth, higher multiplication rates, and increased peroxidase activity compared with those grown under fluorescent lamp (Al-Mayahi \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the present study, shoots derived from cotyledonary nodal segments of \u003cem\u003eP. echinata\u003c/em\u003e grown under the W/mB/dR/fR LED spectrum showed increased accumulation of L-ascorbate peroxidase 2 (paubrasilia_31781) and peroxidase A2-like (paubrasilia_52999) compared to those grown under fluorescent lamp (CLED/CFLU comparison). Similarly, shoots from apical nodal segments exhibited higher levels of peroxidase 73 (paubrasilia_25491) and peroxidase 51-like (paubrasilia_25244) under the same LED spectrum compared to those from fluorescent (ALED/AFLU comparison). Thus, the increased abundance of these antioxidant enzymes under LED conditions suggests a more efficient ROS-scavenging system and improved redox regulation, which may have favored cell expansion and contributed to the increased shoot elongation observed in shoots derived from cotyledonary and apical nodal segments under WmBdRfR LED lamp.\u003c/p\u003e\u003cp\u003eZeaxanthin epoxidase (ZEP) catalyzes the epoxidation of the carotenoid zeaxanthin to violaxanthin in the xanthophyll cycle, providing essential precursors for abscisic acid (ABA) biosynthesis (Wu et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yadav et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, ZEP plays a pivotal role in linking photoprotective mechanisms to hormonal regulation under stress and developmental conditions (Premachandran \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eC. fissilis\u003c/em\u003e, the reduction in shoot length during successive subcultures was associated with a decrease in ABA levels (Oliveira et al. 2021). In our study, the higher accumulation of ZEP (paubrasilia_47510) and carotenoids in shoots from cotyledonary nodal segments grown under LED lamp may induced the increase in ABA levels, which could contribute to the shoot developmental performance in this light condition compared to fluorescent lamp. Moreover, elevated ABA levels are known not only to regulate stress tolerance and developmental processes but also to modulate other hormonal networks. For instance, ABA can induce the expression of ACC synthase and ACC oxidase, enhancing ethylene biosynthesis in several plant species under stress or developmental transitions, as well as ABA-treated tissues show higher ethylene emission (Hansen and Grossmann \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sharp \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Conversely, ABA often acts antagonistically to SA, suppressing SA biosynthesis genes (ICS1, NPR1), reducing SA accumulation and SA-dependent defense (Yasuda et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Moeder et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Alazem et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, the higher ZEP\u0026ndash;ABA response observed in cotyledonary-derived shoots may contribute indirectly to hormonal reprogramming, favoring ethylene-associated growth responses while down-regulating SA-mediated pathways in \u003cem\u003eP. echinata\u003c/em\u003e. This hormonal balance could support enhanced shoot elongation and morphogenetic performance under LED light conditions. In addition, the higher SA content in shoots grown under the fluorescent lamp may have associated with the greater heat emission and higher luminous flux of this lamp compared with LED. These conditions likely induced stress and negatively affected the growth of \u003cem\u003eP. echinata\u003c/em\u003e shoots. SA is a key plant hormone involved in responses to stress, acting in plant defense and growth (Seyfferth and Tsuda \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Elsisi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies emphasize the importance of SA in enhancing plant resistance to both biotic and abiotic stresses (Benjamin et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eC. fissilis\u003c/em\u003e, a significant increase in endogenous SA content in shoots during the fourth subculture, compared with the first, was associated with reduced shoot growth and lower rooting potential (Oliveira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This effect may be explained by the antagonistic relationship between SA and auxin, as SA is known to reduce auxin levels, a hormone required for root induction (De Klerk et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Oxygen-Evolving Enhancer Protein 1 (OEE1) is an essential extrinsic subunit of photosystem II (PSII), also referred to as the PsbO protein (Carius et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This protein plays a fundamental role in the light-driven water-splitting reaction, contributing to the release of oxygen and electrons during photosynthesis (Deng et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In our study, we observed that shoots derived from cotyledonary nodal segments and grown under a white-blue-red-red (WmBdRfR) LED lamp exhibited increased accumulation of OEE1 (paubrasilia_09643), along with higher levels of photosynthetic pigments, including chlorophylls a and b and carotenoids. This suggests that the enhanced presence of OEE1 under LED lighting may contribute to improved photosynthetic efficiency in these shoots. The increase in photosynthetic pigments under LED light can be attributed to the specific light spectrum provided by the LEDs. Red and blue light wavelengths are efficiently absorbed by chlorophylls and carotenoids, leading to enhanced light absorption and energy capture, which are vital for photosynthesis (Liu and Van Iersel \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, the presence of OEE1 stabilizes the PSII complex, facilitating efficient electron transport and oxygen evolution, thereby supporting overall photosynthetic activity (Mayfield et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Thus, the upregulation of OEE1 and the increased accumulation of photosynthetic pigments in shoots grown under LED lamp indicate a synergistic enhancement of photosynthetic efficiency. This combination likely contributes to the observed improved shoot growth under LED conditions.\u003c/p\u003e\u003cp\u003eThe Tubulin beta chain (paubrasilia_16187) was up-accumulated in shoots derived from cotyledonary segments compared to apical nodal segments grown under the W/mB/dR/fR LED lamp, (CLED/ALED comparison). Tubulin, as the main structural component of microtubules, plays a central role in determining the orientation of cell division and expansion, thereby directly influencing morphogenesis and plant development (Hamada \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hsiao and Huang \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). PAs are known to modulate tubulin polymerization and self-association, acting as key regulators of microtubule dynamics (Mechulam et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In \u003cem\u003eC. legalis\u003c/em\u003e, shoots grown under a W/lB/dR LED lamp exhibited higher levels of free Put and increased tubulin accumulation compared with those under fluorescent lighting (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The greater abundance of tubulin in shoots derived from cotyledonary nodal segments, together with their higher endogenous content of total free Put suggests a more active microtubule organization, which may contribute to the enhanced shoot development observed in these shoots. These observations are consistent with additional literature reporting that microtubule stability and orientation are tightly regulated by cytoskeletal-associated proteins and signaling molecules, which influence cell elongation, division plane determination, and organ morphogenesis (Mathur and H\u0026uuml;lskamp \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Nick \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Together, these findings suggest that the greater abundance of tubulin in cotyledonary nodal segments, in combination with elevated levels of free Put reflects a more active microtubule network. This enhanced cytoskeletal organization likely contributes to the superior shoot elongation and morphogenetic performance observed under LED lighting compared with fluorescent light and apical nodal segments.\u003c/p\u003e\u003cp\u003eMoreover, Put is associated with cell cycle progression (G1/S to G2/M) and division activity (Weiger and Hermann \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and the higher Put levels in \u003cem\u003eP. echinata\u003c/em\u003e shoots under LED lighting enriched in red and blue shoots may stimulate cell proliferation and elongation. In \u003cem\u003eC. fissilis\u003c/em\u003e, light spectra from LED lamp W/mB/dR increased the endogenous Put content and altered the accumulation profile of proteins related to metabolism, stress responses, biosynthesis, protein modification, and light stimulus, promoting shoot elongation (Oliveira et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, in \u003cem\u003eG. integrifolia\u003c/em\u003e, where this LED spectrum also increased Put and total free PAs, as well as proteins related with Krebs cycle compared to light from fluorescent lamp (Rodrigues \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In \u003cem\u003eC. legalis\u003c/em\u003e, LED spectra composed of W/lB/dR/fR or W/lB/dR promoted greater shoot elongation from cotyledonary nodal segments compared to fluorescent light, likely due to shade-avoidance responses induced by the combination of low blue and red wavelengths. Under W/lB/dR LEDs, these shoots also showed higher accumulation of Put and Spd, along with proteins involved in metabolic regulation, cellular organization, photosynthesis, and stress responses (Lerin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe type of explant and IBA concentrations did not significantly affected ex vitro rooting (%) of shoots, which ranged from 50 to 65% for both explant types. Further studies are need to be performed aiming to improve ex vitro rooting in \u003cem\u003eP. echinata\u003c/em\u003e. Although tree species often require an auxin, such as IBA, to promote adventitious rooting, our results indicate that the tested auxins were not effective in enhancing rooting in micropropagated shoots of \u003cem\u003eP. echinata\u003c/em\u003e. In \u003cem\u003eC. fissilis\u003c/em\u003e, shoots achieved high ex vitro rooting rates (over 90%), without the application of IBA (Ribeiro et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The authors suggested that this ability is likely due to endogenous IAA metabolism and signaling, which can trigger root induction in these shoots. Ex vitro rooting has been successfully applied in other woody species, but often requires exogenous auxin to promote rooting, as observed for \u003cem\u003eTecomella undulata\u003c/em\u003e (Varshney and Anis \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and \u003cem\u003eAlbizia lebbeck\u003c/em\u003e (Perveen et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), both treated with 200 \u0026micro;M IBA for 30 min. Further investigations are necessary to optimize rooting induction in \u003cem\u003eP. echinata\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e development of \u003cem\u003eP. echinata\u003c/em\u003e is primarily influenced by explant type and light quality. Cotyledonary nodal segments were more efficient in promoting elongated shoots than apical nodal segments. The LED lamp composed of white, medium-blue, red, and far-red (W/mB/dR/fR) light spectra stimulated greater shoot elongation, biomass accumulation, and modulation of key proteins associated with photomorphogenesis, stress responses, hormonal regulation, and cellular organization. LED illumination also promoted higher endogenous accumulation of BA, putrescine (Put), and proteins related to photosynthesis, antioxidant activity (APX and PODs), heat shock proteins (HSPs), and tubulin, suggesting enhanced cellular stability, redox homeostasis, cytoskeletal organization, and photosynthetic efficiency. \u003cem\u003eEx vitro\u003c/em\u003e rooting averaged 50\u0026ndash;65%, with no significant effect of IBA or explant type, indicating that further adjustments are needed to optimize the rooting and acclimatization stages of \u003cem\u003eP. echinata\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cp\u003eThe authors of the manuscript have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics declaration\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the CNPq (309303/2019-2; 312595/2023-9) and the FAPERJ (E26/202.533/2019; E-26/210.088/2022; E-26/200.396/2023). This study was also financed in part by the CAPES\u0026mdash;Finance Code 001. Financial interests: The authors declare they have no financial interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eCSC conceived the study, designed the experiments, supervised all stages of the work, acquired funding, and contributed to manuscript writing and final revision. JOS, PRC, and LTMM were responsible for in vitro shoot culture, ex vitro rooting, and statistical analyses. RGV and YRSR performed the polyamine analyses. JOS, JNN, and RC-S provided support for the hormone analyses. JNN, MSR, and VS carried out the proteomic analyses. AB, JOS, and CSC were responsible for the field plantation of the first micropropagated plantlet. All authors contributed to manuscript writing and approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eFunding for this work was provided by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and the Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Amparo \u0026agrave; Pesquisa do Estado do Rio de Janeiro (FAPERJ). This study was also financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior\u0026mdash; Brazil (CAPES) -Finance Code 001.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD070163. All identified proteins are available in the supplementary material.\u003c/p\u003e\u003ch2\u003eCode Availability\u003c/h2\u003e\u003cp\u003ePXD070163.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmad F, Singh A, Kamal A (2019) Chap. 23 - Salicylic Acid\u0026ndash;Mediated Defense Mechanisms to Abiotic Stress Tolerance. In: Khan MIR, Reddy PS, Ferrante A \u0026amp; Khan NA (eds) Plant Signaling Molecules. Woodhead Publishing, p 355\u0026ndash;369\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Mayahi AMWJ (2016) Effect of red and blue light emitting diodes CRB-LED on in vitro organogenesis of date palm (\u003cem\u003ePhoenix dactylifera\u003c/em\u003e L.) cv. Alshakr. 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Plant Cell Rep 43(4):99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00299-024-03189-9\u003c/span\u003e\u003cspan address=\"10.1007/s00299-024-03189-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Micropropagation, LED lamp, Caesalpinia echinata, Red light spectrum, Polyamines, Plant growth hormones, Proteomics","lastPublishedDoi":"10.21203/rs.3.rs-8001406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8001406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003ePaubrasilia echinata\u003c/em\u003e, the national tree of Brazil, is critically endangered due to centuries of overexploitation for its high-quality wood and dye-producing sap. Consequently, the development of efficient propagation systems is urgent for its conservation and restoration. In vitro culture represents a viable alternative to conventional propagation, enabling large-scale plantlet production and the preservation of elite genotypes. This study aimed to evaluate the effects of explant type, cytokinin, and light spectrum on in vitro shoot development, alongside changes in polyamine (PA) and plant hormone profiles, and to assess the impact of indole-3-butyric acid (IBA) on ex vitro rooting. This is the first integrated analysis combining explant type, light quality, and physiological\u0026ndash;proteomic responses during in vitro development of \u003cem\u003eP. echinata\u003c/em\u003e. Cotyledonary nodal segments produced longer shoots than apical nodal explants. The red\u0026ndash;blue enriched LED W/mB/dR/fR lamp markedly improved shoot elongation, biomass accumulation, and endogenous levels of BA and putrescine. Proteomic analysis revealed increased accumulation of proteins related to photosynthesis, antioxidant defense (APX, PODs), cytoskeleton organization (tubulin), and stress tolerance (HSPs), indicating enhanced cellular homeostasis and photomorphogenic responses. Ex vitro rooting ranged from 50\u0026ndash;65% and was not significantly influenced by explant type or IBA concentration. Overall, these findings establish a physiologically supported micropropagation protocol for large-scale production of \u003cem\u003eP. echinata\u003c/em\u003e, providing a strategic tool for the conservation of this culturally and ecologically emblematic Brazilian species.\u003c/p\u003e","manuscriptTitle":"Light Spectrum and Explant Type Drive Hormonal and Proteomic Reprogramming in Micropropagation of the Endangered Paubrasilia echinata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 11:13:42","doi":"10.21203/rs.3.rs-8001406/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-14T14:22:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-14T14:19:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-12T04:40:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-11-10T12:54:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"18f10473-ad87-4fda-9ede-64f29fac2c11","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:03:01+00:00","versionOfRecord":{"articleIdentity":"rs-8001406","link":"https://doi.org/10.1007/s11240-026-03347-9","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2026-02-07 15:59:22","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-11-26 11:13:42","video":"","vorDoi":"10.1007/s11240-026-03347-9","vorDoiUrl":"https://doi.org/10.1007/s11240-026-03347-9","workflowStages":[]},"version":"v1","identity":"rs-8001406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8001406","identity":"rs-8001406","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}