The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.)

The role of arginine metabolic pathway during embryogenesis and germination in maritime pine... Abstract Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis. Introduction Conifers are distributed worldwide and are particularly abundant in the Northern hemisphere, dominating large forest ecosystems and playing essential roles in global carbon fixation as well as the maintenance of biodiversity. Conifers are also of great economic importance since these plants provide a vast range of products of commercial interest, including wood, pulp, biomass and diverse secondary metabolites (Farjon 2010). Maritime pine (Pinus pinaster Ait.) is a broadly planted conifer species in France, Spain and Portugal where it is distributed over ~4 million hectares (Bouffier et al. 2013). Maritime pine is also one of the most advanced model trees for genetic and phenotypic studies (Lamy et al. 2014, Plomion et al. 2016), and a large number of molecular and transcriptomic resources are currently available (Canales et al. 2014, Cañas et al. 2015a, 2015b). In addition, biotechnological tools are in development for the mass propagation of maritime pine via somatic embryogenesis in combination with cryopreservation of embryogenic lines (reviewed in Lelu-Walter et al. 2006, 2016, Trontin et al. 2016a). Efficient vegetative propagation in pine would enable rapid deployment and turnover in multivarietal forestry of selected/tested varieties that are better adapted to a changing climate and also to socio-economic considerations (Lelu-Walter et al. 2016). Efficient protocols are also available in pines to achieve the genetic transformation of somatic embryos (SE) and transgenic plant regeneration for reverse genetics applications (Klimaszewska et al. 2004b, Trontin et al. 2007, 2016b). However, maturation of SE remains a critical step in the production of high-quality SE plants in maritime pine. Furthermore, cotyledonary SE typically have reduced conversion rates to plantlets compared with seeds and a lower performance in the field tests during early growth compared with zygotic seedlings (Trontin et al. 2016a). A better understanding of maturation and germination of SE is therefore of paramount importance to improve embryo quality and generate vigorous SE plants that compete with weeds, particularly during the first season after planting in field. An important process during the maturation phase of embryogenesis is the biosynthesis and deposition of storage proteins. Overall, accumulation of the most abundant storage proteins in maturing and mature SE is much lower than in zygotic embryos (ZE) suggesting an important influence on the quality of SE (Klimaszewska et al. 2004a, 2016, Morel et al. 2014a). In conifer seeds, most of the storage proteins are initially located in the megagametophyte (including in maritime pine, Trontin et al. 2016a) and later, the protein content gradually increases in embryos during maturation. These storage proteins are characterized by nitrogen (N)-rich amino acids (King and Gifford 1997). Arginine has the highest N to carbon ratio and is therefore particularly suitable for N storage and transport in living organisms (Llacer et al. 2008, Winter et al. 2015). Arginine constitutes a large portion of the amino acid pool in storage proteins of conifers and therefore arginine biosynthesis is likely a relevant metabolic pathway during pine embryogenesis (Cantón et al. 2005, Cánovas et al. 2007). Following germination of pine seeds, N reserves are mobilized to support the early stages of plant development until the seedlings initiate autotrophic growth. This mobilization of reserves during germination depends on the activation and synthesis of key enzymes, including those involved in the proteolytic hydrolysation of storage proteins and catabolism of released amino acids, particularly arginine in a process that is closely synchronized with the emergence of the radicle (King and Gifford 1997). The high accumulation of arginine in the embryo is accompanied by increased arginase (ARG) activity to convert arginine to ornithine and urea (Todd et al. 2001, Todd and Gifford 2003). The subsequent hydrolysis of urea by urease is an important source of ammonium for early seedling development, which is reassimilated into glutamine through the catalytic action of cytosolic glutamine synthetases (GS1a and GS1b) (Avila et al. 1998, Cánovas et al. 2007). The metabolism of arginine has been relatively unexplored in plant N metabolism. Most of the genes involved in the arginine pathway have been predicted from bacterial and fungal homologs and subsequently identified in Arabidopsis (Slocum 2005). Metabolic conversion of glutamate to arginine occurs through two well-diferentiated pathways, the ornithine pathway and the arginine pathway (Figure 1). The ornithine pathway begins with the acetylation of glutamate into N-acetylglutamate catalysed by N-acetylglutamate synthase (NAGS). N-acetylglutamate is subsequently phosphorylated, reduced and transaminated to generate N-acetylornithine through the sequential action of the enzymes N-acetylglutamate kinase (NAGK), N-acetylglutamate-5-P-reductase (NAGPR) and N-acetylornithine aminotransferase (NAOAT). Finally, ornithine is produced either by the catalytic activity of N-acetylornithine glutamate acetyltransferase (NAOGACT) or N-acetylornithine deacetylase (NAOD). In the arginine pathway, ornithine is converted to arginine by the sequential action of ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Other key enzymes for arginine metabolism include ARG and δ-ornithine aminotransferase (δ-OAT), which are involved in the metabolic utilization of arginine. Figure 1. View largeDownload slide The arginine metabolic pathway. Schematic representation of the metabolic conversion of glutamate to arginine through two well-differentiated pathways: (i) the ornithine pathway (in blue) and (ii) the arginine pathway (in pink). NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase; CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase. Figure 1. View largeDownload slide The arginine metabolic pathway. Schematic representation of the metabolic conversion of glutamate to arginine through two well-differentiated pathways: (i) the ornithine pathway (in blue) and (ii) the arginine pathway (in pink). NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase; CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase. A limited number of studies have examined the function and molecular regulation of these enzymes. The arginine biosynthetic pathway is primarily located in the plastid; however, many steps still remain poorly characterized, and only a limited number of enzymes have been purified and biochemically characterized (see Winter et al. 2015, for a recent comprehensive review). As arginine is a key amino acid for N storage during embryogenesis and N mobilization during germination, arginine metabolism in conifers deserves special attention. In the present study, the molecular characteristics of enzymes involved in arginine metabolism, as well as their subcellular localization and their transcriptional levels during development and germination of SE and ZE, were investigated in maritime pine. Materials and methods Plant material Somatic embryos The embryogenic cell line PN519 of maritime pine (P. pinaster Ait.) has been used in the present study. This model line initiated at the Institut technologique Forêt, Cellulose, Bois-Construction, Ameublement (FCBA) in 1999 is amenable to both genetic transformation and plant regeneration through somatic embryogenesis and has been extensively characterized during the past 15 years (Trontin et al. 2007, 2016a, 2016b, Lelu-Walter et al. 2016). The cell line was regrown from cryopreserved tissue 2 months prior to the start of these experiments. Proliferation was performed on modified Litvay medium (MLV) with low plant growth regulators (PGRs) as defined by Klimaszewska et al. (2001), 2.2 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 2.2 μM BA (6-benzyladenine), and weekly subcultures onto fresh medium were performed 1 month prior to the maturation experiments. Modified Litvay medium maturation medium is similar to proliferation medium except for the higher the content of sucrose (60 g l−1) and gellan gum (Phytagel, Sigma, Madrid, Spain, 9 g l−1), and PGRs were also replaced with abcisic acid (ABA) at 80 μM. Proliferation and maturation were conducted at 24 °C in darkness inside a culture chamber. Samples were collected at three different stages of maturation: early-stage translucent SE (ES1, after 4–6 weeks), pre-cotyledonary opaque SE (ES2, after 6–10 weeks) and cotyledonary SE (ES3) that were collected after 14 weeks of maturation. ES3 cotyledonary embryos were germinated in darkness at 24 °C. Germination medium was identical to MLV but without PGRs and with 5 g l−1 gellan gum. Samples were collected after 2 days, 4 days and 9 days of germination, frozen in liquid N and stored at −80 °C for RNA and protein extraction. Seeds, zygotic embryos and megagametophytes Zygotic embryos (ZE) were excised from seeds collected from a single maritime pine (P. pinaster Ait.) seed orchard (Picard, Saint-Laurent-Médoc, France) from July to November 2015. Zygotic embryos were sampled at different developmental stages according to de Vega-Bartol et al. (2013): pre-cotyledonary ZE (PC, early to mid-July), early cotyledonary ZE (EC, mid to late-July), cotyledonary immature ZE (C, from early August to early September) and cotyledonary, mature ZE (M, November). The ZE samples were frozen in liquid N and stored at −80 °C until use. Mature seeds of maritime pine (P. pinaster) provided by the Centro de Recursos Genéticos Forestales ‘El Serranillo' (Ministerio de Medio Ambiente y Medio Rural y Marino, Spain) were soaked in destilled water for 2 days with aeration. The megagametophytes were excised and samples of 10 ZE were collected in triplicate (imbibed embryo, EE). Remaining seeds were germinated in vermiculite at 24 °C under a 16 h light/8 h dark photoperiod and samples of 10 embryos were collected in triplicate after 4 (EG4) and 9 (EG9) days of growth. Samples were frozen in liquid N and stored at −80 °C until use. Nicotiana benthamiana L. seeds were sown in pots and cultivated in a controlled growth chamber at 24 °C and 16 h light/8 h dark photoperiod for 5 weeks. This model plant was used for subcellular localization via agroinfiltration. Protein extraction Frozen samples (35–50 mg FW) were homogenized in a lysis buffer with glass beads in a mortar. The buffer contained 50 mM Tris-HCl (pH 8), 2 mM EDTA, 10% (v/v) glycerol, 2% (w/v) SDS, 2% (w/v) PVPP and 5% (v/v) β-mercaptoethanol and it was used in a proportion of 3 μl of buffer per 1 mg of tissue. The homogenates, after incubation 5 min at 95 °C, were centrifuged for 30 min at 18,000g. The supernatants were collected for protein concentration measurements using the Bradford Protein Assay (Bio-Rad, Madrid, Spain). Proteins (20 μg) were analysed by SDS-PAGE in 12.5% (w/v) acrylamide/bisacrylamide gels followed by Coomassie Brilliant Blue staining. Seven protein bands that differentially accumulated were excised using a sterile scalpel and analysed by nano-flow high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry (LC–MS/MS). Protein identification by mass spectrometry The protein bands were washed with acetonitrile in 25 mM ammonium bicarbonate until the bands were completely destained. Afterwards, they were vacuum dried and proteins digested with 12.5 ng μl−1 trypsin in 25 mM ammonium bicarbonate (de la Torre et al. 2007). Then, the samples were treated with 0.5% (v/v) trifluoroacetic acid (TFA), desalted and concentrated by using μC-18 Spin column (Thermo Scientific, Madrid, Spain). The free peptides were vacuum dried and solubilized in 2% (v/v) acetonitrile and 0.05% TFA. The peptides were analysed by LC–MS/MS. The separation was performed using a 300 μm × 5 mm Dionex Acclaim PepMap100 C18 column (Thermo Scientific), followed by ionization with the nanospray ion source, and placed into an Orbitrap Fusion mass spectrometer (Thermo Scientific). Mass spectrometry data (Full Scan) were recorded in the positive ion mode over the 400–1500 m/z range. Data analysis was performed with Proteome Discoverer 2.1 (Thermo Scientific) and combined MASCOT (Matrix Science) and SEQUEST HT searches against the SustainpineDB (Canales et al. 2014). Subcellular localization Full-length cDNAs sequences were obtained in SustainPineDB v.3.0 (http://www.scbi.uma.es/sustainpinedb/sessions/new). Each gene was amplified by PCR using the specific primers listed in Table S1 available as Supplementary Data at Tree Physiology Online. The resulting PCR product was cloned into the pDONR207 (Invitrogen) and transferred for recombination-based cloning to the final gateway vector pGWB5. All constructs were confirmed by sequencing. Empty plasmid pGWB5 was used as a negative control and pGWB6 (p35S-GFP) as positive control. The Agrobacterium tumefaciens strain C58C1 was transformed by electroporation with recombinant plasmids expressing the proteins of interest. Nicotiana benthamiana leaves (5 weeks old) were syringe-infiltrated with cultures containing pGWB5 constructs mixed with cultures containing P19, both with an optical density at 600 nm of 0.5, according to the procedures described previously (Liu et al. 2002). Subcellular localization of proteins was examined by confocal microscopy 36–48 h after agroinfiltration. Mitotracker® Red FM (Thermo Fisher) was used as red-fluorescent dye to confirm the subcellular localization in the mitochondria (see Figure S1 available as Supplementary Data at Tree Physiology Online). A stock solution of the red-fluorescent dye was prepared in DMSO (Sigma). Sections of infiltrated leaves were incubated in 200 nM of the red-fluorescent dye in phosphate buffered saline. Labelling was conducted in the dark at room temperature for 1 h. Confocal microscopy was performed using a Leica SP5 Laser Scanning Confocal Microscope equipped with HyD and PMT detectors, Acousto-Optical beam splitter (AOBS) and a spectral detection system. GFP fluorescence and chloroplast autofluorescence was detected using Argon laser excitation at 488 nm. The mitochondrial labelling was detected at 581 nm. Images were acquired using either Plan APO 40 × 1.30 NA or Plan APO 63 × 1.40 NA oil immersion objectives. Laser intensity and detector settings were optimized according to the imaging conditions and GFP signal intensities. The images were processed using Leica LAS and FIJI ImageJ software (version 4.1.1). RNA extraction, cDNA synthesis and real-time quantitative PCR (qPCR) Extraction of RNA was performed as described by Canales et al. (2012) and quantified using a NanoDrop© ND-1000 spectrophotometer. Synthesis of cDNA was performed with 5X iScriptTM cDNA Synthesis Kit (Bio-Rad). The qPCR analysis was performed in a thermal cycler CFX384 (Bio-Rad). Each reaction proceded in a total volume of 10 μl, 5 μl of SsoFstTM EvaGreen® Supermix (Bio Rad), 2 μl cDNA (5 ng μl−1) and 0.5 μl of 10 mM of a specific primer. Actin-7 (18,113) was used as a reference gene. Sequences of specific primers are listed in Table S2 available as Supplementary Data at Tree Physiology Online. Relative expression profiles for each gene were obtained employing the R package (Ritz and Spiess 2008) and normalized to the reference gene. Results The protein profiles differed in the zygotic and somatic embryos of maritime pine Protein profiles during the embryogenesis and germination of maritime pine SE and ZE were compared (Figure 2). The profiles of soluble polypeptides were resolved using SDS-PAGE during the maturation of SE and ZE (Figure 2a). The most abundant proteins in ZE at the cotyledonary immature (C) and cotyledonary mature (M) stages have apparent molecular sizes of 47, 35, 20 and 15 kDa and were previously identified as vicilin-like and legumin-like storage proteins, globulin and albumin using LC–MS/MS, respectively (Morel et al. 2014a). These major N storage proteins were much less abundant in cotyledonary SE (ES3 collected after 14 weeks maturation) than in cotyledonary immature (C) and mature (M) ZE. The relative abundance of vicilin-like and legumin-like storage proteins, globulin and albumin was considerably lower at the pre-cotyledonary opaque (ES2) stage with barely detectable levels at the early-stage translucent (ES1). Three other minor polypeptides of 29.4, 24.7 and 14.9 kDa were also more abundant in ZE. A polypeptide of 17.5 kDa was primarily present in ZE at the cotyledonary mature (M) stage. By contrast, polypeptides of 16.7, 15.8 and 10.9 kDa were clearly more represented in SE with higher relative abundance before the cotyledonary stages (ES1 and ES2 stages). To further explore the molecular basis of these differences in the protein profiles of SE and ZE, the polypeptides of 29.4, 24.7 16.7, 15.8, 14.9 and 10.9 kDa were excised from polyacrylamide gels and subjected to LC–MS/MS. The most abundant peptides identified in the different analysed gel bands are listed in Table S3 available as Supplementary Data at Tree Physiology Online. Notably, the major protein represented in the ES samples corresponded to late embryogenesis abundant (LEA) proteins, matching polypeptides of 16.7, 15.8 and 10.9 kDa. Moreover, the peptide footprints corresponded to different unigenes encoding LEA proteins in the SustainpineDB (Canales et al. 2014). The profiles of soluble polypeptides during the germination of SE and ZE showed that N storage proteins significantly decreased in ZE after 9 days of germination (Figure 2b). Minor differences in protein profiles were observed between SE and ZE at the last stages of germination examined (4 and 9 days) despite differences in the N storage (Figure 2b). Figure 2. View largeDownload slide Protein profiles in somatic and zygotic embryos during maturation and germination. Total protein (15 μg) from somatic (SE) and zygotic (ZE) embryos during embryogenesis (a) and germination (b) were fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue. Representative gels are depicted. Asterisks and arrowheads indicate the bands excised from ZE and SE, respectively, for mass spectrometry analysis. Molecular masses (kDa) of protein markers are indicated. ES1, early-stage translucent SE; ES2, pre-cotyledonary opaque SE; ES3, cotyledonary SE; C, cotyledonary immature ZE; M, cotyledonary mature ZE; 2D, 4D and 9D, 2, 4 and 9 days in germinating medium, respectively; EE, imbibed zygotic embryos; EG4 and EG9, embryos 4 and 9 days after imbibition. Figure 2. View largeDownload slide Protein profiles in somatic and zygotic embryos during maturation and germination. Total protein (15 μg) from somatic (SE) and zygotic (ZE) embryos during embryogenesis (a) and germination (b) were fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue. Representative gels are depicted. Asterisks and arrowheads indicate the bands excised from ZE and SE, respectively, for mass spectrometry analysis. Molecular masses (kDa) of protein markers are indicated. ES1, early-stage translucent SE; ES2, pre-cotyledonary opaque SE; ES3, cotyledonary SE; C, cotyledonary immature ZE; M, cotyledonary mature ZE; 2D, 4D and 9D, 2, 4 and 9 days in germinating medium, respectively; EE, imbibed zygotic embryos; EG4 and EG9, embryos 4 and 9 days after imbibition. Identification of genes encoding enzymes of the arginine metabolic pathway in maritime pine A first step in the present study was the identification of genes involved in arginine metabolism in the transcriptome of maritime pine through a search in the SustainpineDB (Canales et al. 2014). Seven full-length cDNAs (FLcDNAs) encoding enzymes involved in ornithine biosynthesis were identified, N-acetylglutamate synthase (PpNAGS), N-acetylglutamate kinase (PpNAGK), N-acetylglutamate-5-P reductase (PpNAGPR), N-acetylornithine aminotransferase (PpNAOGACT), N-acetylornithine: glutamate acetyltransferase (PpNAOAT), N-acetylornithine: glutamate acetyltransferase (PpNAOGACT) and N-acetylornithine deacetylase (PpNAOD) (Table 1). An N-terminal sequence for plastid targeting was predicted for PpNAGS, PpNAGK, PpNAGPR and PpNAOAT encoding mature polypeptides with high levels of identity (see Table S4 available as Supplementary Data at Tree Physiology Online) to their Arabidopsis counterparts (Slocum 2005). No pre-sequences for organellar targeting were identified in the open reading frames (ORF) of PpNAOGACT and PpNAOD. Four additional FLcDNAs involved in arginine biosynthesis were identified for enzymes (Table 1), carbamoyl-P synthetase small subunit (PpCPS), ornithine transcarbamoylase (PpOTC), argininosuccinate synthetase (PpASS) and argininosuccinate lyase (PpASL). N-terminal sequences for plastid targeting were predicted in the ORFs for PpASS and PpASL. Two FLcDNAs encoding enzymes involved in arginine catabolism were also identified, arginase (PpARG) and δ-ornithine aminotransferase (Ppδ-OAT). The ORFs from these two sequences contained N-terminal for targeting to mitochondria (Table 1). Table 1. Genes of the arginine metabolic pathway in Pinus pinaster. Name  Gene ID  FLcDNA (bp)  ORF (bp)  Polypeptide (Da)  Subcellular prediction1  Processed protein (Da)  pI  PpNAGS  sp_v3.0_unigene5514  2595  1926  69,406  Plastid (79)  61,065  6.1  PpNAGK  sp_v3.0_unigene15977  1824  1059  36,991  Plastid (51)  31,358  8.3  PpNAGPR  sp_v3.0_unigene5400  1924  1248  45,850  Plastid (49)  40,284  6.7  PpNAOAT  sp_v3.0_unigene5428  2030  1482  53,042  Plastid  –  –  PpNAOGACT  sp_v3.0_unigene7147  2222  1524  52,484  –  –  –  PpNAOD  sp_v3.0_unigene1654  1729  1296  47,427  –  –  –  PpCPS  sp_v3.0_unigene8325  1921  1365  49,210  –  –  –  PpOTC  sp_v3.0_unigene6197  1900  1155  42,011  –  –  –  PpASS  sp_v3.0_unigene6320  2311  1539  56,407  Plastid (37)  52,383  6.0  PpASL  sp_v3.0_unigene5329  2254  1590  58,548  Plastid  –  –  PpARG  sp_v3.0_unigene23824  1554  1026  37,303  Mito  –  –  PpδOAT  sp_v3.0_unigene5775  2230  1407  51,234  Mito  –  –  Name  Gene ID  FLcDNA (bp)  ORF (bp)  Polypeptide (Da)  Subcellular prediction1  Processed protein (Da)  pI  PpNAGS  sp_v3.0_unigene5514  2595  1926  69,406  Plastid (79)  61,065  6.1  PpNAGK  sp_v3.0_unigene15977  1824  1059  36,991  Plastid (51)  31,358  8.3  PpNAGPR  sp_v3.0_unigene5400  1924  1248  45,850  Plastid (49)  40,284  6.7  PpNAOAT  sp_v3.0_unigene5428  2030  1482  53,042  Plastid  –  –  PpNAOGACT  sp_v3.0_unigene7147  2222  1524  52,484  –  –  –  PpNAOD  sp_v3.0_unigene1654  1729  1296  47,427  –  –  –  PpCPS  sp_v3.0_unigene8325  1921  1365  49,210  –  –  –  PpOTC  sp_v3.0_unigene6197  1900  1155  42,011  –  –  –  PpASS  sp_v3.0_unigene6320  2311  1539  56,407  Plastid (37)  52,383  6.0  PpASL  sp_v3.0_unigene5329  2254  1590  58,548  Plastid  –  –  PpARG  sp_v3.0_unigene23824  1554  1026  37,303  Mito  –  –  PpδOAT  sp_v3.0_unigene5775  2230  1407  51,234  Mito  –  –  1Chloroplast transit peptides prediction and amino acid residues (in brackets) using Predotar and ChloroP databases. PpNAGS, N-acetylglutamate synthase; PpNAGK, N-acetylglutamate kinase; PpNAGPR N-acetylglutamate-5-P reductase; PpNAOAT, N2-acetylornithine aminotransferase; PpNAOGACT, N-acetylornithine:glutamate acetyltransferase; PpNAOD, N-acetylornithine deacetylase; PpCPS, carbamoyl-P synthetase small subunit; PpOTC, ornithine transcarbamoylase; PpASS, argininosuccinate synthetase; PpASL, argininosuccinate lyase; PpARG, arginase; PpδOAT, δ-ornithine aminotransferase. Subcellular localization of the arginine metabolic pathway in maritime pine To further characterize the biosynthesis and utilization of arginine, we determined the subcellular localization of the enzymes in the entire pathway. FLcDNAs for all genes identified in the maritime pine transcriptome (Table 1) were PCR-amplified using specific primers (see Table S1 available as Supplementary Data at Tree Physiology Online) and GFP fusions were transiently expressed in N. benthamiana leaves. First, the subcellular location of enzymes involved in ornithine biosynthesis was examined. The transiently expressed constructs for PpNAGS, PpNAGK, PpNAGPR, PpNAOAT and PpNAOGACT displayed GFP flurorescence associated with chloroplasts as revealed in the corresponding images of cholorophyll red autofluorescence (Figure 3). Examination of the merged images showed co-localization of chlorophyll with the different protein gene products in the chloroplasts visualized in yellow colour, confirming plastidic localization. While PpNAGS, PpNAGPR, PpNAOAT and PpNAOGACT displayed a diffuse fluorescence pattern throughout the plastids, PpNAGK showed a punctate distribution of the GFP signal (Figure 3, PpNAGK). PpNAOD fluorescence was primarily distributed throughout the cytosol and no GFP signal was detected in the chloroplasts (Figure 3). Figure 3. View largeDownload slide Subcellular localization of enzymes of the ornithine biosynthesis pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localizations were determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows merged images. Scale bar represents 10 μm. Figure 3. View largeDownload slide Subcellular localization of enzymes of the ornithine biosynthesis pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localizations were determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows merged images. Scale bar represents 10 μm. Next, the subcellular localization of enzymes involved in arginine biosynthesis and catabolism was determined (Figure 4). Using a similar approach to that described above, all GFP fusions of PpCPS, PpOTC, PpASS and PpASL were localized to chloroplasts. The co-localization of PpCPS, PpOTC, PpASS and PpASL and chlorophyll in the plastids was indisputably confirmed in the merged images, resulting in chlorophyll and GFP flurorescence. Figure 4 also shows that the GFP-tagged enzymes involved in arginine catabolism, PpARG and PpOAT, were clearly localized outside the chloroplasts but not distributed throughout the cytosol as previously observed for PpNAOD (Figure 3). To identify the precise subcellular localization of PpARG and PpOAT, the mitochondrial prediction derived from the analysis of the ORFs was considered (Table 1). Consequently, the localization of these enzymes was compared with a mitochondrial marker (Mitotracker Red FM), and the results are shown in Figure S1 available as Supplementary Data at Tree Physiology Online. The co-localization of GFP signals and the marker in the merged images confirmed mitochondrial localization. Figure 4. View largeDownload slide Subcellular localization of enzymes of the arginine metabolic pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localization determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows the merged images. Scale bar represent 10 μm. Figure 4. View largeDownload slide Subcellular localization of enzymes of the arginine metabolic pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localization determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows the merged images. Scale bar represent 10 μm. Transcript levels for enzymes of the arginine metabolic pathway in SE and ZE To assess the ability of maritime pine embryos for arginine biosynthesis and metabolic utilization, the expression levels of genes involved in the arginine metabolic pathway were compared during the maturation of SE and ZE. Total RNA was extracted from SE and ZE at several stages of development and the relative transcript abundance for all genes was determined by qPCR analysis using specific primers (see Table S2 available as Supplementary Data at Tree Physiology Online). Overall, similar transcript levels were observed for all genes involved in the ornithine (PpNAGS, PpNAGK, PpNAGPR, PpNAOGACT and PpNAOD) and arginine (PpCPS, PpOTC, PpASS and PpASL) biosynthetic pathways at the three stages (ES1, ES2 and ES3) examined during SE maturation (Figure 5b). By contrast, transcript levels for all genes were generally low at early stages of ZE maturation (PC and EC, which are similar to ES2 and ES3, respectively) but significantly increased at the end of maturation (stages C and M) reaching higher levels than those observed in SE. These expression profiles were conserved for all genes involved in ornithine and arginine biosynthesis. Interestingly, transcript levels of PpARG and PpδOAT, two genes involved in arginine catabolism, were significantly higher during the maturation of SE than during the maturation of ZE (Figure 5b). Figure 5. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during last stages of somatic and zygotic embryogenesis in P. pinaster. (a) Representative images of somatic and zygotic embryos at final stages of embryogenesis (maturation stages). Somatic embryos: ES1, early-stage translucent; ES2, pre-cotyledonary opaque; ES3, cotyledonary. Zygotic embryos: PC, pre-cotyledonary; EC, early cotyledonary; C, cotyledonary immature; M, cotyledonary partially mature. (b) qPCR expression analysis in somatic and zygotic embryos during maturation. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Figure 5. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during last stages of somatic and zygotic embryogenesis in P. pinaster. (a) Representative images of somatic and zygotic embryos at final stages of embryogenesis (maturation stages). Somatic embryos: ES1, early-stage translucent; ES2, pre-cotyledonary opaque; ES3, cotyledonary. Zygotic embryos: PC, pre-cotyledonary; EC, early cotyledonary; C, cotyledonary immature; M, cotyledonary partially mature. (b) qPCR expression analysis in somatic and zygotic embryos during maturation. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Transcript levels for enzymes of the arginine metabolic pathway during the germination of SE and ZE To further explore the arginine metabolic pathway in maritime pine embryos, transcript abundance of genes involved in this pathway was also determined using qPCR analysis during the germination of SE and ZE. Overall, similar transcript levels were observed for all genes involved in ornithine and arginine biosynthesis during SE germination (Figure 6). However, in ZE, the expression levels of most genes were much higher in imbibed embryos and subsequently declined following germination. Exceptions to this general profile were PpNAOD, NAOGACT and PpASL genes for which low transcript levels were observed during germination. With regard to arginine catabolism, significant differences in the transcript levels of PpARG and PpδOAT were observed in SE and ZE at the end of the germination. Figure 6. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during germination of somatic and zygotic embryos of P. pinaster. (a) Representative images of somatic and zygotic embryos at different germination stages. Mature SE in germination media for 2 days (2D), 4 days (4D) and 9 days (9D). Seeds germinated for 2 days (2G), 4 days (4G) and 9 days (9G). (b) qPCR expression analysis in somatic and zygotic embryos during germination. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Figure 6. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during germination of somatic and zygotic embryos of P. pinaster. (a) Representative images of somatic and zygotic embryos at different germination stages. Mature SE in germination media for 2 days (2D), 4 days (4D) and 9 days (9D). Seeds germinated for 2 days (2G), 4 days (4G) and 9 days (9G). (b) qPCR expression analysis in somatic and zygotic embryos during germination. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Discussion Maritime pine (P. pinaster) SE accumulate lower levels of major storage proteins than ZE during the late stages of embryogenesis (Figure 2; Morel et al. 2014a). Moreover, maturation duration (10–14 weeks) is not a critical factor for protein accumulation in cotyledonary embryos (Morel et al. 2014a). Similar results have been described in other pine species such as Pinus strobus (Klimaszewska et al. 2004a), Pinus taeda (Brownfield et al. 2007) and Pinus sylvestris (Lelu-Walter et al. 2008). In fact, storage protein content has been suggested as a suitable molecular marker of SE quality, determining to a greater extent the success of germination and survival of SE plants (Klimaszewska et al. 2004a, Tereso et al. 2007, Miguel et al. 2016). An additional characteristic of maturation process is the acquisition of desiccation tolerance, which in pine SE is externally induced by increasing the osmotic pressure (high sucrose concentration) and lowering the water availability in the culture medium by physical means (high gellan gum concentration). Several extra LEA proteins structurally affiliated with groups 3 and 4 (Amara et al. 2014) were accumulated in SE but were not present in ZE (see Table S3 available as Supplementary Data at Tree Physiology Online). Late embryogenesis abundant proteins are directly involved in the adaptive response of higher plants (including conifers, reviewed in Miguel et al. 2016, Trontin et al. 2016c) to water deficit and may also stabilize a partially unfolded state, preventing protein aggregation (Goyal et al. 2005, Hand et al. 2011, Furuki et al. 2012). The differential accumulation of LEA suggests that SE do not achieve desiccation tolerance to the same degree as ZE during the last stages of maturation. Morel et al. (2014a) reported similar conclusions after a multi-scale analysis (water content, content of various oligosaccharides, total protein content) of cotyledonary SE and maturing ZE. Cotyledonary SE were most similar to cotyledonary ZE at a partially mature, fresh (undessicated) stage. In particular the ratio between stachyose + raffinose (oligosaccharides of the raffinose family, RFOs, involved in desiccation tolerance) and sucrose significantly increased in ZE during maturation but remained low in cotyledonary SE. Interestingly, Morel et al. (2014a) detected various LEAs and other stress-related proteins (heat-shock proteins, HSPs) that were proposed as putative generic markers of the fresh, cotyledonary stage of embryo maturation. In Picea abies, SE developed on the maturation medium supplemented with sucrose as the main carbon source contained high levels of raffinose and LEA and preferentially accumulated starch and lower levels of storage proteins (Businge et al. 2013). It is suggested that acquisition of desiccation tolerance is promoted through a sucrose-based maturation medium and that it could be a concurrent process for the accumulation of storage proteins. Businge et al. (2013) reported that maturation treatment could induce changes in N metabolism in mature embryos of P. abies through differential expression of key enzymes for glutamine, glutamate and arginine synthesis. Consistently, metabolite profiling also revealed significant amino acids associated with N metabolism and polyamine biosynthesis during late embryogeny such as ornithine, arginine and asparagine (Businge et al. 2012). Arginine is very abundant in maritime pine storage proteins (Allona et al. 1992, 1994), and therefore arginine biosynthesis is of paramount importance during embryogenesis. The recent assembly of maritime pine transcriptome (Canales et al. 2014) enabled the identification of genes involved in arginine metabolism. As shown in Table 1, FLcDNA sequences for all enzymes in the ornithine and arginine pathways have been identified. Overall, their molecular characteristics are similar to the corresponding counterparts in Arabidopsis (Slocum 2005, Winter et al. 2015), with minor differences (see Table S4 available as Supplementary Data at Tree Physiology Online). Furthermore, the availability of FLcDNA sequences enabled the prediction of the putative organellar localization of a few but not all enzymes in this pathway. The determination of the precise subcellular compartmentation of the pathway is critical to understand arginine biosynthesis and metabolic utilization in maritime pine. Although the plastidic localization of the ornithine and arginine pathways is assumed through N-terminal prediction of gene sequences, this localization has only been experimentally confirmed for several enzymes (Slocum 2005). Taking advantage of the availability of FLcDNAs for all enzymes of the pathway, their subcellular localization was systematically determined in N. benthamiana. The enzymes of the ornithine and arginine pathways were localized in the plastids, with the exception of PpNAOD, which was localized in the cytosol (Figures 3 and 4). These results are consistent with previous reports describing the plastidic localization of NAGK (Chen et al. 2006) and NAOAT (Fremont et al. 2013) in Arabidopsis and ASL in rice (Xia et al. 2014). Furthermore, these localization studies support the notion that ornithine is synthesized in the chloroplast through the cyclic ornithine pathway and in the cytosol via the enzymatic reaction catalysed by NAOD. These results imply the existence of separate pools of ornithine in two cellular compartments. In the plastids, ornithine can be metabolically used as precursor for arginine biosynthesis, whereas in the cytosol, ornithine would be chanelled for the biosynthesis of polyamines and other nitrogenous compounds, such as alkaloids (Facchini 2001, Majumdar et al. 2013, Tiburcio et al. 2014). Consistently, the downregulation of NAOD in Arabidopsis resulted in decreased levels of ornithine and altered contents of putrescine and spermine (Molesini et al. 2015). Ornithine levels are apparently controlled in plants through the first step in the pathway, a reaction catalysed by NAGS. In fact, the overexpression of NAGS in tomato resulted in the accumulation of high levels of ornithine (Kalamaki et al. 2009). However, arginine biosynthesis is allosterically regulated by feedback inhibition of NAGK (Slocum 2005). From a practical point of view, it is interesting to determine the molecular basis of the lower accumulation and deposition of storage proteins in SE, in spite of the lack of limitation in the availability of N during late embryogenesis (maturation). Is arginine biosynthesis less efficient in SE than in ZE? Are there differences in the expression of genes involved in arginine biosynthesis? To answer these questions, the relative expression levels of all genes involved in the pathway were compared in somatic and zygotic maritime pine embryos. In ZE, gene expression remained low at the initial stages of maturation and increased at the end of this phase, suggesting that arginine biosynthesis is enhanced during the deposition of storage proteins (Figure 5b). By contrast, the observed transcript levels of the ornithine and arginine pathways were high and similar during the three SE maturation stages analysed (Figure 5b). These results suggest that arginine biosynthesis occurs throughout the maturation of SE. One possible explanation for the above results could be that arginine degradation was already and concomitantly activated in SE, consistent with the increased levels observed in SE for PpARG and PpδOAT, two genes involved in arginine catabolism. Moreover, the expression of ARG in pine SE has previously been reported, suggesting an overlap between late embryogenesis and precocious germination (Pérez Rodríguez et al. 2006). Taken together, these results strongly suggest that the pathways for biosynthesis and degradation of arginine simultaneously function in SE and likely reflect the continuous deposition and breakdown of storage proteins. These data support the lower accumulation of storage proteins observed in maritime pine SE compared with ZE (Figure 2a). Consistently, the early mobilization of storage proteins by enhanced protease activity has previously been described in SE of oil palm (Aberlenc-Bertossi et al. 2008). The arginine metabolic pathway is regulated at transcriptional and post-transcriptional levels in yeast (Ljungdahl and Daignan-Fornier 2012). In comparison, little is known about the regulation of the arginine metabolism in plants, particularly at transcriptional level. The results shown in Figure 5 suggest that the expression of genes involved in the ornithine and arginine pathways are coordinately regulated in maritime pine. This assumption is supported by the existence of similar cis-elements in the regulatory region of several genes (see Figure S2 available as Supplementary Data at Tree Physiology Online). However, additional studies are needed to identify specific transcription factors that bind to gene promoters and regulate the transcription of the arginine pathway. Somatic embryos accumulated higher levels of starch and lower levels of storage proteins than ZE (Joy et al. 1991, Tereso et al. 2007). In maritime pine somatic embryogenesis, starch accumulation was promoted by low water availability in the maturation medium (Morel et al. 2014a, 2014b). The reduced water availability promoted the downregulation of genes involved in glucose or pentose metabolisms and was associated with increased embryo dry weight and enhanced starch synthesis (upregulation of glucose-1-phosphate adenylyltransferase). Moreover, it is well documented that sucrose synthase plays an important role in carbohydrate metabolism during maturation (Konrádova et al. 2002) suggesting that SE and ZE exhibit a different carbon/N status, which may explain the differences in the regulation of genes involved in arginine metabolism. The PII protein is a sensor of the carbon/N status in plants mediated by binding to 2-oxoglutarate and modulation of NAGK activity (Chen et al. 2006). Arginine biosynthesis undergoes the feedback regulation of NAGK (Slocum 2005, Llacer et al. 2008). When N is abundant, this inhibition is released through interaction with the N sensor protein PII (Chen et al. 2006, Llacer et al. 2008). In addition, PII controls, in a glutamine-dependent manner, the NAGK enzyme, the key step in arginine biosynthesis (Chellamuthu et al. 2014). It is currently unknown whether PII plays a role in the transcriptional regulation of the arginine biosynthetic pathway in plants, but T-DNA insertional mutants in Arabidopsis exhibited reduced levels of ornithine, citrulline and arginine (Ferrario-Méry et al. 2006). In pine seedlings, the gene encoding PII-like protein is expressed in different organs, and PII-like protein transcripts are particularly abundant in embryos, suggesting a role for PII in the regulation of N metabolism during embryogenesis (F. M. Canovas, C. Avila, unpublished). Therefore, the PII protein in maritime pine deserves special attention, particularly considering that a link between PII and storage protein production has previously been proposed (Uhrig et al. 2009). Nitrogen is a limiting factor for conifer tree growth and development and mobilization of storage proteins during the germination of SE provides substantially lower levels of N-rich amino acids such as arginine, which are essential during early stages of germination (King and Gifford 1997). In fact arginine and proline are predominant amino acids representing more than 60% of the total amino acid content in pine ZE (Cañas et al. 2008). As SE lack the surrounding maternal tissue present around ZE, germination media typically include N supplementation to facilitate germination yield. M.-T. Llebrés, M.-B. Pascual, C. Avila, F. M. Cánovas, K. Klimaszewska (submitted) recently reported that hybrid white pine SE plants germinated on medium without inorganic N developed functional roots and survived at 50% higher rates than those germinated with inorganic and organic N sources or solely inorganic N. These results suggest a critical role for the N source in the germination of pine somatic embryo plants. Figure 7 integrates the compartmentation of the GS-GOGAT cycle in conifer cells (Cánovas et al. 2007) and the results presented here for the subcellular localization of enzymes of the arginine metabolic pathway (Figures 3 and 4). In conifers, glutamine biosynthesis occurs in the cytosol and should be transported into the plastid for glutamate biosynthesis. During the maturation stages, increased levels of GS and GOGAT and enhanced levels of glutamine, glutamate and arginine have been reported in the SE of white spruce (Joy et al. 1997, Stasolla et al. 2003). In addition, the expression of GS1b, a gene encoding cytosolic GS, was induced at the initiation of pine embryo maturation (Pérez-Rodríguez et al. 2006). Glutamine and glutamate are the N donors for the ornithine (Figure 7a) and arginine (Figure 7b) pathways located in the plastid resulting in the biosynthesis of arginine. Arginine is transported outside the plastid for storage protein biosynthesis and deposition into protein vacuoles in the cytosol (Stone and Gifford 1997). During germination, arginine released from storage protein breakdown or directly transferred from the plastid is catabolized in the mitochondria to generate urea and glutamate (Figure 7b). The hydrolysis of urea by urease has been reported in loblolly pine seedlings (Todd and Gifford 2002) and the role of δOAT in the biosynthesis of glutamate from ornithine and 2-oxoglutarate has been described (Cañas et al. 2008). The concerted action of urease and GS in the cytosol channels N from arginine to glutamine and asparagine which are the most abundant amino acids in the seedlings (Cánovas et al. 2007, Cañas et al. 2016). Figure 7. View largeDownload slide Compartmentation of the GS-GOGAT pathway and arginine metabolism in conifer cells. (a) Diagram representing the ornithine biosynthesis pathway in the plastid and the cytosol. GS, glutamine synthetase; GOGAT, glutamate synthase; NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase. (b) Diagram representing the plastidic arginine biosynthetic pathway, deposition of storage proteins in the cytosol and arginine mitochondrial degradation. CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase; ARG, arginase; URE, urease; δOAT, ornithine-δ-aminotransferase; P5CDH, P5C dehydrogenase; GS, glutamine synthetase. Figure 7. View largeDownload slide Compartmentation of the GS-GOGAT pathway and arginine metabolism in conifer cells. (a) Diagram representing the ornithine biosynthesis pathway in the plastid and the cytosol. GS, glutamine synthetase; GOGAT, glutamate synthase; NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase. (b) Diagram representing the plastidic arginine biosynthetic pathway, deposition of storage proteins in the cytosol and arginine mitochondrial degradation. CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase; ARG, arginase; URE, urease; δOAT, ornithine-δ-aminotransferase; P5CDH, P5C dehydrogenase; GS, glutamine synthetase. In summary, the arginine metabolic pathway was characterized in maritime pine. The genes involved in the biosynthesis and metabolic utilization of this amino acid were identified; FLcDNAs were isolated and subsequently used to determine the localization of enzymes in different subcellular compartments. The results derived from gene expression profiling strongly suggest that arginine metabolism is deregulated in SE compared with ZE. These changes in N homeostasis were consistent with the lower accumulation of storage proteins observed during the last steps of somatic embryogenesis. Additional studies are needed to identity the regulatory factors involved in the transcriptional regulation of the pathway and unravel the role of protein sensor PII in arginine metabolism and accumulation of storage proteins. The knowledge acquired from these fundamental studies will help to refine biotechnological procedures for clonal propagation of conifers via somatic embryogenesis. Supplementary Data Supplementary Data for this article are available at Tree Physiology Online. Acknowledgments Authors acknowledge Pierre Alazard for cone sampling in maritime pine seed orchard. Conflict of interest None declared. Funding This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BIO2015-285-R) and Junta de Andalucía (BIO-474). M.-B.P. was supported by a postdoctoral contract from Junta de Andalucía (Proyectos de Excelencia Junta de Andalucía, Spain). ZE sampling at FCBA was partially supported by the French Ministry of Agriculture (DGAL, No. 2014-352, QuaSeGraine project) and benefited from the support of the XYLOBIOTECH technical facility (ANR-10-EQPX-16 XYLOFOREST). References Avila C, García-Gutiérrez A, Crespillo R, Cánovas FM ( 1998) Effects of phosphinothricin treatment on glutamine synthetase isoforms in Scots pine seedlings. Plant Physiol Biochem  36: 857– 863. 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The role of arginine metabolic pathway during embryogenesis and germination in maritime pine (Pinus pinaster Ait.)

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10.1093/treephys/tpx133
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Abstract

Abstract Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis. Introduction Conifers are distributed worldwide and are particularly abundant in the Northern hemisphere, dominating large forest ecosystems and playing essential roles in global carbon fixation as well as the maintenance of biodiversity. Conifers are also of great economic importance since these plants provide a vast range of products of commercial interest, including wood, pulp, biomass and diverse secondary metabolites (Farjon 2010). Maritime pine (Pinus pinaster Ait.) is a broadly planted conifer species in France, Spain and Portugal where it is distributed over ~4 million hectares (Bouffier et al. 2013). Maritime pine is also one of the most advanced model trees for genetic and phenotypic studies (Lamy et al. 2014, Plomion et al. 2016), and a large number of molecular and transcriptomic resources are currently available (Canales et al. 2014, Cañas et al. 2015a, 2015b). In addition, biotechnological tools are in development for the mass propagation of maritime pine via somatic embryogenesis in combination with cryopreservation of embryogenic lines (reviewed in Lelu-Walter et al. 2006, 2016, Trontin et al. 2016a). Efficient vegetative propagation in pine would enable rapid deployment and turnover in multivarietal forestry of selected/tested varieties that are better adapted to a changing climate and also to socio-economic considerations (Lelu-Walter et al. 2016). Efficient protocols are also available in pines to achieve the genetic transformation of somatic embryos (SE) and transgenic plant regeneration for reverse genetics applications (Klimaszewska et al. 2004b, Trontin et al. 2007, 2016b). However, maturation of SE remains a critical step in the production of high-quality SE plants in maritime pine. Furthermore, cotyledonary SE typically have reduced conversion rates to plantlets compared with seeds and a lower performance in the field tests during early growth compared with zygotic seedlings (Trontin et al. 2016a). A better understanding of maturation and germination of SE is therefore of paramount importance to improve embryo quality and generate vigorous SE plants that compete with weeds, particularly during the first season after planting in field. An important process during the maturation phase of embryogenesis is the biosynthesis and deposition of storage proteins. Overall, accumulation of the most abundant storage proteins in maturing and mature SE is much lower than in zygotic embryos (ZE) suggesting an important influence on the quality of SE (Klimaszewska et al. 2004a, 2016, Morel et al. 2014a). In conifer seeds, most of the storage proteins are initially located in the megagametophyte (including in maritime pine, Trontin et al. 2016a) and later, the protein content gradually increases in embryos during maturation. These storage proteins are characterized by nitrogen (N)-rich amino acids (King and Gifford 1997). Arginine has the highest N to carbon ratio and is therefore particularly suitable for N storage and transport in living organisms (Llacer et al. 2008, Winter et al. 2015). Arginine constitutes a large portion of the amino acid pool in storage proteins of conifers and therefore arginine biosynthesis is likely a relevant metabolic pathway during pine embryogenesis (Cantón et al. 2005, Cánovas et al. 2007). Following germination of pine seeds, N reserves are mobilized to support the early stages of plant development until the seedlings initiate autotrophic growth. This mobilization of reserves during germination depends on the activation and synthesis of key enzymes, including those involved in the proteolytic hydrolysation of storage proteins and catabolism of released amino acids, particularly arginine in a process that is closely synchronized with the emergence of the radicle (King and Gifford 1997). The high accumulation of arginine in the embryo is accompanied by increased arginase (ARG) activity to convert arginine to ornithine and urea (Todd et al. 2001, Todd and Gifford 2003). The subsequent hydrolysis of urea by urease is an important source of ammonium for early seedling development, which is reassimilated into glutamine through the catalytic action of cytosolic glutamine synthetases (GS1a and GS1b) (Avila et al. 1998, Cánovas et al. 2007). The metabolism of arginine has been relatively unexplored in plant N metabolism. Most of the genes involved in the arginine pathway have been predicted from bacterial and fungal homologs and subsequently identified in Arabidopsis (Slocum 2005). Metabolic conversion of glutamate to arginine occurs through two well-diferentiated pathways, the ornithine pathway and the arginine pathway (Figure 1). The ornithine pathway begins with the acetylation of glutamate into N-acetylglutamate catalysed by N-acetylglutamate synthase (NAGS). N-acetylglutamate is subsequently phosphorylated, reduced and transaminated to generate N-acetylornithine through the sequential action of the enzymes N-acetylglutamate kinase (NAGK), N-acetylglutamate-5-P-reductase (NAGPR) and N-acetylornithine aminotransferase (NAOAT). Finally, ornithine is produced either by the catalytic activity of N-acetylornithine glutamate acetyltransferase (NAOGACT) or N-acetylornithine deacetylase (NAOD). In the arginine pathway, ornithine is converted to arginine by the sequential action of ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Other key enzymes for arginine metabolism include ARG and δ-ornithine aminotransferase (δ-OAT), which are involved in the metabolic utilization of arginine. Figure 1. View largeDownload slide The arginine metabolic pathway. Schematic representation of the metabolic conversion of glutamate to arginine through two well-differentiated pathways: (i) the ornithine pathway (in blue) and (ii) the arginine pathway (in pink). NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase; CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase. Figure 1. View largeDownload slide The arginine metabolic pathway. Schematic representation of the metabolic conversion of glutamate to arginine through two well-differentiated pathways: (i) the ornithine pathway (in blue) and (ii) the arginine pathway (in pink). NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase; CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase. A limited number of studies have examined the function and molecular regulation of these enzymes. The arginine biosynthetic pathway is primarily located in the plastid; however, many steps still remain poorly characterized, and only a limited number of enzymes have been purified and biochemically characterized (see Winter et al. 2015, for a recent comprehensive review). As arginine is a key amino acid for N storage during embryogenesis and N mobilization during germination, arginine metabolism in conifers deserves special attention. In the present study, the molecular characteristics of enzymes involved in arginine metabolism, as well as their subcellular localization and their transcriptional levels during development and germination of SE and ZE, were investigated in maritime pine. Materials and methods Plant material Somatic embryos The embryogenic cell line PN519 of maritime pine (P. pinaster Ait.) has been used in the present study. This model line initiated at the Institut technologique Forêt, Cellulose, Bois-Construction, Ameublement (FCBA) in 1999 is amenable to both genetic transformation and plant regeneration through somatic embryogenesis and has been extensively characterized during the past 15 years (Trontin et al. 2007, 2016a, 2016b, Lelu-Walter et al. 2016). The cell line was regrown from cryopreserved tissue 2 months prior to the start of these experiments. Proliferation was performed on modified Litvay medium (MLV) with low plant growth regulators (PGRs) as defined by Klimaszewska et al. (2001), 2.2 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 2.2 μM BA (6-benzyladenine), and weekly subcultures onto fresh medium were performed 1 month prior to the maturation experiments. Modified Litvay medium maturation medium is similar to proliferation medium except for the higher the content of sucrose (60 g l−1) and gellan gum (Phytagel, Sigma, Madrid, Spain, 9 g l−1), and PGRs were also replaced with abcisic acid (ABA) at 80 μM. Proliferation and maturation were conducted at 24 °C in darkness inside a culture chamber. Samples were collected at three different stages of maturation: early-stage translucent SE (ES1, after 4–6 weeks), pre-cotyledonary opaque SE (ES2, after 6–10 weeks) and cotyledonary SE (ES3) that were collected after 14 weeks of maturation. ES3 cotyledonary embryos were germinated in darkness at 24 °C. Germination medium was identical to MLV but without PGRs and with 5 g l−1 gellan gum. Samples were collected after 2 days, 4 days and 9 days of germination, frozen in liquid N and stored at −80 °C for RNA and protein extraction. Seeds, zygotic embryos and megagametophytes Zygotic embryos (ZE) were excised from seeds collected from a single maritime pine (P. pinaster Ait.) seed orchard (Picard, Saint-Laurent-Médoc, France) from July to November 2015. Zygotic embryos were sampled at different developmental stages according to de Vega-Bartol et al. (2013): pre-cotyledonary ZE (PC, early to mid-July), early cotyledonary ZE (EC, mid to late-July), cotyledonary immature ZE (C, from early August to early September) and cotyledonary, mature ZE (M, November). The ZE samples were frozen in liquid N and stored at −80 °C until use. Mature seeds of maritime pine (P. pinaster) provided by the Centro de Recursos Genéticos Forestales ‘El Serranillo' (Ministerio de Medio Ambiente y Medio Rural y Marino, Spain) were soaked in destilled water for 2 days with aeration. The megagametophytes were excised and samples of 10 ZE were collected in triplicate (imbibed embryo, EE). Remaining seeds were germinated in vermiculite at 24 °C under a 16 h light/8 h dark photoperiod and samples of 10 embryos were collected in triplicate after 4 (EG4) and 9 (EG9) days of growth. Samples were frozen in liquid N and stored at −80 °C until use. Nicotiana benthamiana L. seeds were sown in pots and cultivated in a controlled growth chamber at 24 °C and 16 h light/8 h dark photoperiod for 5 weeks. This model plant was used for subcellular localization via agroinfiltration. Protein extraction Frozen samples (35–50 mg FW) were homogenized in a lysis buffer with glass beads in a mortar. The buffer contained 50 mM Tris-HCl (pH 8), 2 mM EDTA, 10% (v/v) glycerol, 2% (w/v) SDS, 2% (w/v) PVPP and 5% (v/v) β-mercaptoethanol and it was used in a proportion of 3 μl of buffer per 1 mg of tissue. The homogenates, after incubation 5 min at 95 °C, were centrifuged for 30 min at 18,000g. The supernatants were collected for protein concentration measurements using the Bradford Protein Assay (Bio-Rad, Madrid, Spain). Proteins (20 μg) were analysed by SDS-PAGE in 12.5% (w/v) acrylamide/bisacrylamide gels followed by Coomassie Brilliant Blue staining. Seven protein bands that differentially accumulated were excised using a sterile scalpel and analysed by nano-flow high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry (LC–MS/MS). Protein identification by mass spectrometry The protein bands were washed with acetonitrile in 25 mM ammonium bicarbonate until the bands were completely destained. Afterwards, they were vacuum dried and proteins digested with 12.5 ng μl−1 trypsin in 25 mM ammonium bicarbonate (de la Torre et al. 2007). Then, the samples were treated with 0.5% (v/v) trifluoroacetic acid (TFA), desalted and concentrated by using μC-18 Spin column (Thermo Scientific, Madrid, Spain). The free peptides were vacuum dried and solubilized in 2% (v/v) acetonitrile and 0.05% TFA. The peptides were analysed by LC–MS/MS. The separation was performed using a 300 μm × 5 mm Dionex Acclaim PepMap100 C18 column (Thermo Scientific), followed by ionization with the nanospray ion source, and placed into an Orbitrap Fusion mass spectrometer (Thermo Scientific). Mass spectrometry data (Full Scan) were recorded in the positive ion mode over the 400–1500 m/z range. Data analysis was performed with Proteome Discoverer 2.1 (Thermo Scientific) and combined MASCOT (Matrix Science) and SEQUEST HT searches against the SustainpineDB (Canales et al. 2014). Subcellular localization Full-length cDNAs sequences were obtained in SustainPineDB v.3.0 (http://www.scbi.uma.es/sustainpinedb/sessions/new). Each gene was amplified by PCR using the specific primers listed in Table S1 available as Supplementary Data at Tree Physiology Online. The resulting PCR product was cloned into the pDONR207 (Invitrogen) and transferred for recombination-based cloning to the final gateway vector pGWB5. All constructs were confirmed by sequencing. Empty plasmid pGWB5 was used as a negative control and pGWB6 (p35S-GFP) as positive control. The Agrobacterium tumefaciens strain C58C1 was transformed by electroporation with recombinant plasmids expressing the proteins of interest. Nicotiana benthamiana leaves (5 weeks old) were syringe-infiltrated with cultures containing pGWB5 constructs mixed with cultures containing P19, both with an optical density at 600 nm of 0.5, according to the procedures described previously (Liu et al. 2002). Subcellular localization of proteins was examined by confocal microscopy 36–48 h after agroinfiltration. Mitotracker® Red FM (Thermo Fisher) was used as red-fluorescent dye to confirm the subcellular localization in the mitochondria (see Figure S1 available as Supplementary Data at Tree Physiology Online). A stock solution of the red-fluorescent dye was prepared in DMSO (Sigma). Sections of infiltrated leaves were incubated in 200 nM of the red-fluorescent dye in phosphate buffered saline. Labelling was conducted in the dark at room temperature for 1 h. Confocal microscopy was performed using a Leica SP5 Laser Scanning Confocal Microscope equipped with HyD and PMT detectors, Acousto-Optical beam splitter (AOBS) and a spectral detection system. GFP fluorescence and chloroplast autofluorescence was detected using Argon laser excitation at 488 nm. The mitochondrial labelling was detected at 581 nm. Images were acquired using either Plan APO 40 × 1.30 NA or Plan APO 63 × 1.40 NA oil immersion objectives. Laser intensity and detector settings were optimized according to the imaging conditions and GFP signal intensities. The images were processed using Leica LAS and FIJI ImageJ software (version 4.1.1). RNA extraction, cDNA synthesis and real-time quantitative PCR (qPCR) Extraction of RNA was performed as described by Canales et al. (2012) and quantified using a NanoDrop© ND-1000 spectrophotometer. Synthesis of cDNA was performed with 5X iScriptTM cDNA Synthesis Kit (Bio-Rad). The qPCR analysis was performed in a thermal cycler CFX384 (Bio-Rad). Each reaction proceded in a total volume of 10 μl, 5 μl of SsoFstTM EvaGreen® Supermix (Bio Rad), 2 μl cDNA (5 ng μl−1) and 0.5 μl of 10 mM of a specific primer. Actin-7 (18,113) was used as a reference gene. Sequences of specific primers are listed in Table S2 available as Supplementary Data at Tree Physiology Online. Relative expression profiles for each gene were obtained employing the R package (Ritz and Spiess 2008) and normalized to the reference gene. Results The protein profiles differed in the zygotic and somatic embryos of maritime pine Protein profiles during the embryogenesis and germination of maritime pine SE and ZE were compared (Figure 2). The profiles of soluble polypeptides were resolved using SDS-PAGE during the maturation of SE and ZE (Figure 2a). The most abundant proteins in ZE at the cotyledonary immature (C) and cotyledonary mature (M) stages have apparent molecular sizes of 47, 35, 20 and 15 kDa and were previously identified as vicilin-like and legumin-like storage proteins, globulin and albumin using LC–MS/MS, respectively (Morel et al. 2014a). These major N storage proteins were much less abundant in cotyledonary SE (ES3 collected after 14 weeks maturation) than in cotyledonary immature (C) and mature (M) ZE. The relative abundance of vicilin-like and legumin-like storage proteins, globulin and albumin was considerably lower at the pre-cotyledonary opaque (ES2) stage with barely detectable levels at the early-stage translucent (ES1). Three other minor polypeptides of 29.4, 24.7 and 14.9 kDa were also more abundant in ZE. A polypeptide of 17.5 kDa was primarily present in ZE at the cotyledonary mature (M) stage. By contrast, polypeptides of 16.7, 15.8 and 10.9 kDa were clearly more represented in SE with higher relative abundance before the cotyledonary stages (ES1 and ES2 stages). To further explore the molecular basis of these differences in the protein profiles of SE and ZE, the polypeptides of 29.4, 24.7 16.7, 15.8, 14.9 and 10.9 kDa were excised from polyacrylamide gels and subjected to LC–MS/MS. The most abundant peptides identified in the different analysed gel bands are listed in Table S3 available as Supplementary Data at Tree Physiology Online. Notably, the major protein represented in the ES samples corresponded to late embryogenesis abundant (LEA) proteins, matching polypeptides of 16.7, 15.8 and 10.9 kDa. Moreover, the peptide footprints corresponded to different unigenes encoding LEA proteins in the SustainpineDB (Canales et al. 2014). The profiles of soluble polypeptides during the germination of SE and ZE showed that N storage proteins significantly decreased in ZE after 9 days of germination (Figure 2b). Minor differences in protein profiles were observed between SE and ZE at the last stages of germination examined (4 and 9 days) despite differences in the N storage (Figure 2b). Figure 2. View largeDownload slide Protein profiles in somatic and zygotic embryos during maturation and germination. Total protein (15 μg) from somatic (SE) and zygotic (ZE) embryos during embryogenesis (a) and germination (b) were fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue. Representative gels are depicted. Asterisks and arrowheads indicate the bands excised from ZE and SE, respectively, for mass spectrometry analysis. Molecular masses (kDa) of protein markers are indicated. ES1, early-stage translucent SE; ES2, pre-cotyledonary opaque SE; ES3, cotyledonary SE; C, cotyledonary immature ZE; M, cotyledonary mature ZE; 2D, 4D and 9D, 2, 4 and 9 days in germinating medium, respectively; EE, imbibed zygotic embryos; EG4 and EG9, embryos 4 and 9 days after imbibition. Figure 2. View largeDownload slide Protein profiles in somatic and zygotic embryos during maturation and germination. Total protein (15 μg) from somatic (SE) and zygotic (ZE) embryos during embryogenesis (a) and germination (b) were fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue. Representative gels are depicted. Asterisks and arrowheads indicate the bands excised from ZE and SE, respectively, for mass spectrometry analysis. Molecular masses (kDa) of protein markers are indicated. ES1, early-stage translucent SE; ES2, pre-cotyledonary opaque SE; ES3, cotyledonary SE; C, cotyledonary immature ZE; M, cotyledonary mature ZE; 2D, 4D and 9D, 2, 4 and 9 days in germinating medium, respectively; EE, imbibed zygotic embryos; EG4 and EG9, embryos 4 and 9 days after imbibition. Identification of genes encoding enzymes of the arginine metabolic pathway in maritime pine A first step in the present study was the identification of genes involved in arginine metabolism in the transcriptome of maritime pine through a search in the SustainpineDB (Canales et al. 2014). Seven full-length cDNAs (FLcDNAs) encoding enzymes involved in ornithine biosynthesis were identified, N-acetylglutamate synthase (PpNAGS), N-acetylglutamate kinase (PpNAGK), N-acetylglutamate-5-P reductase (PpNAGPR), N-acetylornithine aminotransferase (PpNAOGACT), N-acetylornithine: glutamate acetyltransferase (PpNAOAT), N-acetylornithine: glutamate acetyltransferase (PpNAOGACT) and N-acetylornithine deacetylase (PpNAOD) (Table 1). An N-terminal sequence for plastid targeting was predicted for PpNAGS, PpNAGK, PpNAGPR and PpNAOAT encoding mature polypeptides with high levels of identity (see Table S4 available as Supplementary Data at Tree Physiology Online) to their Arabidopsis counterparts (Slocum 2005). No pre-sequences for organellar targeting were identified in the open reading frames (ORF) of PpNAOGACT and PpNAOD. Four additional FLcDNAs involved in arginine biosynthesis were identified for enzymes (Table 1), carbamoyl-P synthetase small subunit (PpCPS), ornithine transcarbamoylase (PpOTC), argininosuccinate synthetase (PpASS) and argininosuccinate lyase (PpASL). N-terminal sequences for plastid targeting were predicted in the ORFs for PpASS and PpASL. Two FLcDNAs encoding enzymes involved in arginine catabolism were also identified, arginase (PpARG) and δ-ornithine aminotransferase (Ppδ-OAT). The ORFs from these two sequences contained N-terminal for targeting to mitochondria (Table 1). Table 1. Genes of the arginine metabolic pathway in Pinus pinaster. Name  Gene ID  FLcDNA (bp)  ORF (bp)  Polypeptide (Da)  Subcellular prediction1  Processed protein (Da)  pI  PpNAGS  sp_v3.0_unigene5514  2595  1926  69,406  Plastid (79)  61,065  6.1  PpNAGK  sp_v3.0_unigene15977  1824  1059  36,991  Plastid (51)  31,358  8.3  PpNAGPR  sp_v3.0_unigene5400  1924  1248  45,850  Plastid (49)  40,284  6.7  PpNAOAT  sp_v3.0_unigene5428  2030  1482  53,042  Plastid  –  –  PpNAOGACT  sp_v3.0_unigene7147  2222  1524  52,484  –  –  –  PpNAOD  sp_v3.0_unigene1654  1729  1296  47,427  –  –  –  PpCPS  sp_v3.0_unigene8325  1921  1365  49,210  –  –  –  PpOTC  sp_v3.0_unigene6197  1900  1155  42,011  –  –  –  PpASS  sp_v3.0_unigene6320  2311  1539  56,407  Plastid (37)  52,383  6.0  PpASL  sp_v3.0_unigene5329  2254  1590  58,548  Plastid  –  –  PpARG  sp_v3.0_unigene23824  1554  1026  37,303  Mito  –  –  PpδOAT  sp_v3.0_unigene5775  2230  1407  51,234  Mito  –  –  Name  Gene ID  FLcDNA (bp)  ORF (bp)  Polypeptide (Da)  Subcellular prediction1  Processed protein (Da)  pI  PpNAGS  sp_v3.0_unigene5514  2595  1926  69,406  Plastid (79)  61,065  6.1  PpNAGK  sp_v3.0_unigene15977  1824  1059  36,991  Plastid (51)  31,358  8.3  PpNAGPR  sp_v3.0_unigene5400  1924  1248  45,850  Plastid (49)  40,284  6.7  PpNAOAT  sp_v3.0_unigene5428  2030  1482  53,042  Plastid  –  –  PpNAOGACT  sp_v3.0_unigene7147  2222  1524  52,484  –  –  –  PpNAOD  sp_v3.0_unigene1654  1729  1296  47,427  –  –  –  PpCPS  sp_v3.0_unigene8325  1921  1365  49,210  –  –  –  PpOTC  sp_v3.0_unigene6197  1900  1155  42,011  –  –  –  PpASS  sp_v3.0_unigene6320  2311  1539  56,407  Plastid (37)  52,383  6.0  PpASL  sp_v3.0_unigene5329  2254  1590  58,548  Plastid  –  –  PpARG  sp_v3.0_unigene23824  1554  1026  37,303  Mito  –  –  PpδOAT  sp_v3.0_unigene5775  2230  1407  51,234  Mito  –  –  1Chloroplast transit peptides prediction and amino acid residues (in brackets) using Predotar and ChloroP databases. PpNAGS, N-acetylglutamate synthase; PpNAGK, N-acetylglutamate kinase; PpNAGPR N-acetylglutamate-5-P reductase; PpNAOAT, N2-acetylornithine aminotransferase; PpNAOGACT, N-acetylornithine:glutamate acetyltransferase; PpNAOD, N-acetylornithine deacetylase; PpCPS, carbamoyl-P synthetase small subunit; PpOTC, ornithine transcarbamoylase; PpASS, argininosuccinate synthetase; PpASL, argininosuccinate lyase; PpARG, arginase; PpδOAT, δ-ornithine aminotransferase. Subcellular localization of the arginine metabolic pathway in maritime pine To further characterize the biosynthesis and utilization of arginine, we determined the subcellular localization of the enzymes in the entire pathway. FLcDNAs for all genes identified in the maritime pine transcriptome (Table 1) were PCR-amplified using specific primers (see Table S1 available as Supplementary Data at Tree Physiology Online) and GFP fusions were transiently expressed in N. benthamiana leaves. First, the subcellular location of enzymes involved in ornithine biosynthesis was examined. The transiently expressed constructs for PpNAGS, PpNAGK, PpNAGPR, PpNAOAT and PpNAOGACT displayed GFP flurorescence associated with chloroplasts as revealed in the corresponding images of cholorophyll red autofluorescence (Figure 3). Examination of the merged images showed co-localization of chlorophyll with the different protein gene products in the chloroplasts visualized in yellow colour, confirming plastidic localization. While PpNAGS, PpNAGPR, PpNAOAT and PpNAOGACT displayed a diffuse fluorescence pattern throughout the plastids, PpNAGK showed a punctate distribution of the GFP signal (Figure 3, PpNAGK). PpNAOD fluorescence was primarily distributed throughout the cytosol and no GFP signal was detected in the chloroplasts (Figure 3). Figure 3. View largeDownload slide Subcellular localization of enzymes of the ornithine biosynthesis pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localizations were determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows merged images. Scale bar represents 10 μm. Figure 3. View largeDownload slide Subcellular localization of enzymes of the ornithine biosynthesis pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localizations were determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows merged images. Scale bar represents 10 μm. Next, the subcellular localization of enzymes involved in arginine biosynthesis and catabolism was determined (Figure 4). Using a similar approach to that described above, all GFP fusions of PpCPS, PpOTC, PpASS and PpASL were localized to chloroplasts. The co-localization of PpCPS, PpOTC, PpASS and PpASL and chlorophyll in the plastids was indisputably confirmed in the merged images, resulting in chlorophyll and GFP flurorescence. Figure 4 also shows that the GFP-tagged enzymes involved in arginine catabolism, PpARG and PpOAT, were clearly localized outside the chloroplasts but not distributed throughout the cytosol as previously observed for PpNAOD (Figure 3). To identify the precise subcellular localization of PpARG and PpOAT, the mitochondrial prediction derived from the analysis of the ORFs was considered (Table 1). Consequently, the localization of these enzymes was compared with a mitochondrial marker (Mitotracker Red FM), and the results are shown in Figure S1 available as Supplementary Data at Tree Physiology Online. The co-localization of GFP signals and the marker in the merged images confirmed mitochondrial localization. Figure 4. View largeDownload slide Subcellular localization of enzymes of the arginine metabolic pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localization determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows the merged images. Scale bar represent 10 μm. Figure 4. View largeDownload slide Subcellular localization of enzymes of the arginine metabolic pathway. Nicotiana benthamiana leaves were infiltrated with A. tumefaciens containing the constructs of interest fused to GFP. Proteins were transiently expressed and their intracellular localization determined by confocal laser scanning microscopy. The GFP signal (green) is shown in the first channel, chlorophyll autofluorescence (red) in the second and the third channel shows the merged images. Scale bar represent 10 μm. Transcript levels for enzymes of the arginine metabolic pathway in SE and ZE To assess the ability of maritime pine embryos for arginine biosynthesis and metabolic utilization, the expression levels of genes involved in the arginine metabolic pathway were compared during the maturation of SE and ZE. Total RNA was extracted from SE and ZE at several stages of development and the relative transcript abundance for all genes was determined by qPCR analysis using specific primers (see Table S2 available as Supplementary Data at Tree Physiology Online). Overall, similar transcript levels were observed for all genes involved in the ornithine (PpNAGS, PpNAGK, PpNAGPR, PpNAOGACT and PpNAOD) and arginine (PpCPS, PpOTC, PpASS and PpASL) biosynthetic pathways at the three stages (ES1, ES2 and ES3) examined during SE maturation (Figure 5b). By contrast, transcript levels for all genes were generally low at early stages of ZE maturation (PC and EC, which are similar to ES2 and ES3, respectively) but significantly increased at the end of maturation (stages C and M) reaching higher levels than those observed in SE. These expression profiles were conserved for all genes involved in ornithine and arginine biosynthesis. Interestingly, transcript levels of PpARG and PpδOAT, two genes involved in arginine catabolism, were significantly higher during the maturation of SE than during the maturation of ZE (Figure 5b). Figure 5. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during last stages of somatic and zygotic embryogenesis in P. pinaster. (a) Representative images of somatic and zygotic embryos at final stages of embryogenesis (maturation stages). Somatic embryos: ES1, early-stage translucent; ES2, pre-cotyledonary opaque; ES3, cotyledonary. Zygotic embryos: PC, pre-cotyledonary; EC, early cotyledonary; C, cotyledonary immature; M, cotyledonary partially mature. (b) qPCR expression analysis in somatic and zygotic embryos during maturation. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Figure 5. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during last stages of somatic and zygotic embryogenesis in P. pinaster. (a) Representative images of somatic and zygotic embryos at final stages of embryogenesis (maturation stages). Somatic embryos: ES1, early-stage translucent; ES2, pre-cotyledonary opaque; ES3, cotyledonary. Zygotic embryos: PC, pre-cotyledonary; EC, early cotyledonary; C, cotyledonary immature; M, cotyledonary partially mature. (b) qPCR expression analysis in somatic and zygotic embryos during maturation. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Transcript levels for enzymes of the arginine metabolic pathway during the germination of SE and ZE To further explore the arginine metabolic pathway in maritime pine embryos, transcript abundance of genes involved in this pathway was also determined using qPCR analysis during the germination of SE and ZE. Overall, similar transcript levels were observed for all genes involved in ornithine and arginine biosynthesis during SE germination (Figure 6). However, in ZE, the expression levels of most genes were much higher in imbibed embryos and subsequently declined following germination. Exceptions to this general profile were PpNAOD, NAOGACT and PpASL genes for which low transcript levels were observed during germination. With regard to arginine catabolism, significant differences in the transcript levels of PpARG and PpδOAT were observed in SE and ZE at the end of the germination. Figure 6. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during germination of somatic and zygotic embryos of P. pinaster. (a) Representative images of somatic and zygotic embryos at different germination stages. Mature SE in germination media for 2 days (2D), 4 days (4D) and 9 days (9D). Seeds germinated for 2 days (2G), 4 days (4G) and 9 days (9G). (b) qPCR expression analysis in somatic and zygotic embryos during germination. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Figure 6. View largeDownload slide Expression patterns of genes involved in arginine biosynthesis and utilization during germination of somatic and zygotic embryos of P. pinaster. (a) Representative images of somatic and zygotic embryos at different germination stages. Mature SE in germination media for 2 days (2D), 4 days (4D) and 9 days (9D). Seeds germinated for 2 days (2G), 4 days (4G) and 9 days (9G). (b) qPCR expression analysis in somatic and zygotic embryos during germination. The expression level for all genes was normalized to that of actin-7 as reference gene. Different letters above bars indicate significant differences between samples at P < 0.05. Bars represent mean values of three assays, with three biological replicates each + standard deviation. Discussion Maritime pine (P. pinaster) SE accumulate lower levels of major storage proteins than ZE during the late stages of embryogenesis (Figure 2; Morel et al. 2014a). Moreover, maturation duration (10–14 weeks) is not a critical factor for protein accumulation in cotyledonary embryos (Morel et al. 2014a). Similar results have been described in other pine species such as Pinus strobus (Klimaszewska et al. 2004a), Pinus taeda (Brownfield et al. 2007) and Pinus sylvestris (Lelu-Walter et al. 2008). In fact, storage protein content has been suggested as a suitable molecular marker of SE quality, determining to a greater extent the success of germination and survival of SE plants (Klimaszewska et al. 2004a, Tereso et al. 2007, Miguel et al. 2016). An additional characteristic of maturation process is the acquisition of desiccation tolerance, which in pine SE is externally induced by increasing the osmotic pressure (high sucrose concentration) and lowering the water availability in the culture medium by physical means (high gellan gum concentration). Several extra LEA proteins structurally affiliated with groups 3 and 4 (Amara et al. 2014) were accumulated in SE but were not present in ZE (see Table S3 available as Supplementary Data at Tree Physiology Online). Late embryogenesis abundant proteins are directly involved in the adaptive response of higher plants (including conifers, reviewed in Miguel et al. 2016, Trontin et al. 2016c) to water deficit and may also stabilize a partially unfolded state, preventing protein aggregation (Goyal et al. 2005, Hand et al. 2011, Furuki et al. 2012). The differential accumulation of LEA suggests that SE do not achieve desiccation tolerance to the same degree as ZE during the last stages of maturation. Morel et al. (2014a) reported similar conclusions after a multi-scale analysis (water content, content of various oligosaccharides, total protein content) of cotyledonary SE and maturing ZE. Cotyledonary SE were most similar to cotyledonary ZE at a partially mature, fresh (undessicated) stage. In particular the ratio between stachyose + raffinose (oligosaccharides of the raffinose family, RFOs, involved in desiccation tolerance) and sucrose significantly increased in ZE during maturation but remained low in cotyledonary SE. Interestingly, Morel et al. (2014a) detected various LEAs and other stress-related proteins (heat-shock proteins, HSPs) that were proposed as putative generic markers of the fresh, cotyledonary stage of embryo maturation. In Picea abies, SE developed on the maturation medium supplemented with sucrose as the main carbon source contained high levels of raffinose and LEA and preferentially accumulated starch and lower levels of storage proteins (Businge et al. 2013). It is suggested that acquisition of desiccation tolerance is promoted through a sucrose-based maturation medium and that it could be a concurrent process for the accumulation of storage proteins. Businge et al. (2013) reported that maturation treatment could induce changes in N metabolism in mature embryos of P. abies through differential expression of key enzymes for glutamine, glutamate and arginine synthesis. Consistently, metabolite profiling also revealed significant amino acids associated with N metabolism and polyamine biosynthesis during late embryogeny such as ornithine, arginine and asparagine (Businge et al. 2012). Arginine is very abundant in maritime pine storage proteins (Allona et al. 1992, 1994), and therefore arginine biosynthesis is of paramount importance during embryogenesis. The recent assembly of maritime pine transcriptome (Canales et al. 2014) enabled the identification of genes involved in arginine metabolism. As shown in Table 1, FLcDNA sequences for all enzymes in the ornithine and arginine pathways have been identified. Overall, their molecular characteristics are similar to the corresponding counterparts in Arabidopsis (Slocum 2005, Winter et al. 2015), with minor differences (see Table S4 available as Supplementary Data at Tree Physiology Online). Furthermore, the availability of FLcDNA sequences enabled the prediction of the putative organellar localization of a few but not all enzymes in this pathway. The determination of the precise subcellular compartmentation of the pathway is critical to understand arginine biosynthesis and metabolic utilization in maritime pine. Although the plastidic localization of the ornithine and arginine pathways is assumed through N-terminal prediction of gene sequences, this localization has only been experimentally confirmed for several enzymes (Slocum 2005). Taking advantage of the availability of FLcDNAs for all enzymes of the pathway, their subcellular localization was systematically determined in N. benthamiana. The enzymes of the ornithine and arginine pathways were localized in the plastids, with the exception of PpNAOD, which was localized in the cytosol (Figures 3 and 4). These results are consistent with previous reports describing the plastidic localization of NAGK (Chen et al. 2006) and NAOAT (Fremont et al. 2013) in Arabidopsis and ASL in rice (Xia et al. 2014). Furthermore, these localization studies support the notion that ornithine is synthesized in the chloroplast through the cyclic ornithine pathway and in the cytosol via the enzymatic reaction catalysed by NAOD. These results imply the existence of separate pools of ornithine in two cellular compartments. In the plastids, ornithine can be metabolically used as precursor for arginine biosynthesis, whereas in the cytosol, ornithine would be chanelled for the biosynthesis of polyamines and other nitrogenous compounds, such as alkaloids (Facchini 2001, Majumdar et al. 2013, Tiburcio et al. 2014). Consistently, the downregulation of NAOD in Arabidopsis resulted in decreased levels of ornithine and altered contents of putrescine and spermine (Molesini et al. 2015). Ornithine levels are apparently controlled in plants through the first step in the pathway, a reaction catalysed by NAGS. In fact, the overexpression of NAGS in tomato resulted in the accumulation of high levels of ornithine (Kalamaki et al. 2009). However, arginine biosynthesis is allosterically regulated by feedback inhibition of NAGK (Slocum 2005). From a practical point of view, it is interesting to determine the molecular basis of the lower accumulation and deposition of storage proteins in SE, in spite of the lack of limitation in the availability of N during late embryogenesis (maturation). Is arginine biosynthesis less efficient in SE than in ZE? Are there differences in the expression of genes involved in arginine biosynthesis? To answer these questions, the relative expression levels of all genes involved in the pathway were compared in somatic and zygotic maritime pine embryos. In ZE, gene expression remained low at the initial stages of maturation and increased at the end of this phase, suggesting that arginine biosynthesis is enhanced during the deposition of storage proteins (Figure 5b). By contrast, the observed transcript levels of the ornithine and arginine pathways were high and similar during the three SE maturation stages analysed (Figure 5b). These results suggest that arginine biosynthesis occurs throughout the maturation of SE. One possible explanation for the above results could be that arginine degradation was already and concomitantly activated in SE, consistent with the increased levels observed in SE for PpARG and PpδOAT, two genes involved in arginine catabolism. Moreover, the expression of ARG in pine SE has previously been reported, suggesting an overlap between late embryogenesis and precocious germination (Pérez Rodríguez et al. 2006). Taken together, these results strongly suggest that the pathways for biosynthesis and degradation of arginine simultaneously function in SE and likely reflect the continuous deposition and breakdown of storage proteins. These data support the lower accumulation of storage proteins observed in maritime pine SE compared with ZE (Figure 2a). Consistently, the early mobilization of storage proteins by enhanced protease activity has previously been described in SE of oil palm (Aberlenc-Bertossi et al. 2008). The arginine metabolic pathway is regulated at transcriptional and post-transcriptional levels in yeast (Ljungdahl and Daignan-Fornier 2012). In comparison, little is known about the regulation of the arginine metabolism in plants, particularly at transcriptional level. The results shown in Figure 5 suggest that the expression of genes involved in the ornithine and arginine pathways are coordinately regulated in maritime pine. This assumption is supported by the existence of similar cis-elements in the regulatory region of several genes (see Figure S2 available as Supplementary Data at Tree Physiology Online). However, additional studies are needed to identify specific transcription factors that bind to gene promoters and regulate the transcription of the arginine pathway. Somatic embryos accumulated higher levels of starch and lower levels of storage proteins than ZE (Joy et al. 1991, Tereso et al. 2007). In maritime pine somatic embryogenesis, starch accumulation was promoted by low water availability in the maturation medium (Morel et al. 2014a, 2014b). The reduced water availability promoted the downregulation of genes involved in glucose or pentose metabolisms and was associated with increased embryo dry weight and enhanced starch synthesis (upregulation of glucose-1-phosphate adenylyltransferase). Moreover, it is well documented that sucrose synthase plays an important role in carbohydrate metabolism during maturation (Konrádova et al. 2002) suggesting that SE and ZE exhibit a different carbon/N status, which may explain the differences in the regulation of genes involved in arginine metabolism. The PII protein is a sensor of the carbon/N status in plants mediated by binding to 2-oxoglutarate and modulation of NAGK activity (Chen et al. 2006). Arginine biosynthesis undergoes the feedback regulation of NAGK (Slocum 2005, Llacer et al. 2008). When N is abundant, this inhibition is released through interaction with the N sensor protein PII (Chen et al. 2006, Llacer et al. 2008). In addition, PII controls, in a glutamine-dependent manner, the NAGK enzyme, the key step in arginine biosynthesis (Chellamuthu et al. 2014). It is currently unknown whether PII plays a role in the transcriptional regulation of the arginine biosynthetic pathway in plants, but T-DNA insertional mutants in Arabidopsis exhibited reduced levels of ornithine, citrulline and arginine (Ferrario-Méry et al. 2006). In pine seedlings, the gene encoding PII-like protein is expressed in different organs, and PII-like protein transcripts are particularly abundant in embryos, suggesting a role for PII in the regulation of N metabolism during embryogenesis (F. M. Canovas, C. Avila, unpublished). Therefore, the PII protein in maritime pine deserves special attention, particularly considering that a link between PII and storage protein production has previously been proposed (Uhrig et al. 2009). Nitrogen is a limiting factor for conifer tree growth and development and mobilization of storage proteins during the germination of SE provides substantially lower levels of N-rich amino acids such as arginine, which are essential during early stages of germination (King and Gifford 1997). In fact arginine and proline are predominant amino acids representing more than 60% of the total amino acid content in pine ZE (Cañas et al. 2008). As SE lack the surrounding maternal tissue present around ZE, germination media typically include N supplementation to facilitate germination yield. M.-T. Llebrés, M.-B. Pascual, C. Avila, F. M. Cánovas, K. Klimaszewska (submitted) recently reported that hybrid white pine SE plants germinated on medium without inorganic N developed functional roots and survived at 50% higher rates than those germinated with inorganic and organic N sources or solely inorganic N. These results suggest a critical role for the N source in the germination of pine somatic embryo plants. Figure 7 integrates the compartmentation of the GS-GOGAT cycle in conifer cells (Cánovas et al. 2007) and the results presented here for the subcellular localization of enzymes of the arginine metabolic pathway (Figures 3 and 4). In conifers, glutamine biosynthesis occurs in the cytosol and should be transported into the plastid for glutamate biosynthesis. During the maturation stages, increased levels of GS and GOGAT and enhanced levels of glutamine, glutamate and arginine have been reported in the SE of white spruce (Joy et al. 1997, Stasolla et al. 2003). In addition, the expression of GS1b, a gene encoding cytosolic GS, was induced at the initiation of pine embryo maturation (Pérez-Rodríguez et al. 2006). Glutamine and glutamate are the N donors for the ornithine (Figure 7a) and arginine (Figure 7b) pathways located in the plastid resulting in the biosynthesis of arginine. Arginine is transported outside the plastid for storage protein biosynthesis and deposition into protein vacuoles in the cytosol (Stone and Gifford 1997). During germination, arginine released from storage protein breakdown or directly transferred from the plastid is catabolized in the mitochondria to generate urea and glutamate (Figure 7b). The hydrolysis of urea by urease has been reported in loblolly pine seedlings (Todd and Gifford 2002) and the role of δOAT in the biosynthesis of glutamate from ornithine and 2-oxoglutarate has been described (Cañas et al. 2008). The concerted action of urease and GS in the cytosol channels N from arginine to glutamine and asparagine which are the most abundant amino acids in the seedlings (Cánovas et al. 2007, Cañas et al. 2016). Figure 7. View largeDownload slide Compartmentation of the GS-GOGAT pathway and arginine metabolism in conifer cells. (a) Diagram representing the ornithine biosynthesis pathway in the plastid and the cytosol. GS, glutamine synthetase; GOGAT, glutamate synthase; NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase. (b) Diagram representing the plastidic arginine biosynthetic pathway, deposition of storage proteins in the cytosol and arginine mitochondrial degradation. CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase; ARG, arginase; URE, urease; δOAT, ornithine-δ-aminotransferase; P5CDH, P5C dehydrogenase; GS, glutamine synthetase. Figure 7. View largeDownload slide Compartmentation of the GS-GOGAT pathway and arginine metabolism in conifer cells. (a) Diagram representing the ornithine biosynthesis pathway in the plastid and the cytosol. GS, glutamine synthetase; GOGAT, glutamate synthase; NAGS, N-acetylglutamate synthase; NAGK, N-acetylglutamate kinase; NAGPR, N-acetylglutamate-5-P reductase; NAOAT, N-acetylornithine aminotransferase; NAOGACT, N-acetylornithine-glutamate acetyltransferase; NAOD, N-acetylornithine deacetylase. (b) Diagram representing the plastidic arginine biosynthetic pathway, deposition of storage proteins in the cytosol and arginine mitochondrial degradation. CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamoylase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase; ARG, arginase; URE, urease; δOAT, ornithine-δ-aminotransferase; P5CDH, P5C dehydrogenase; GS, glutamine synthetase. In summary, the arginine metabolic pathway was characterized in maritime pine. The genes involved in the biosynthesis and metabolic utilization of this amino acid were identified; FLcDNAs were isolated and subsequently used to determine the localization of enzymes in different subcellular compartments. The results derived from gene expression profiling strongly suggest that arginine metabolism is deregulated in SE compared with ZE. These changes in N homeostasis were consistent with the lower accumulation of storage proteins observed during the last steps of somatic embryogenesis. Additional studies are needed to identity the regulatory factors involved in the transcriptional regulation of the pathway and unravel the role of protein sensor PII in arginine metabolism and accumulation of storage proteins. The knowledge acquired from these fundamental studies will help to refine biotechnological procedures for clonal propagation of conifers via somatic embryogenesis. Supplementary Data Supplementary Data for this article are available at Tree Physiology Online. Acknowledgments Authors acknowledge Pierre Alazard for cone sampling in maritime pine seed orchard. Conflict of interest None declared. Funding This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BIO2015-285-R) and Junta de Andalucía (BIO-474). M.-B.P. was supported by a postdoctoral contract from Junta de Andalucía (Proyectos de Excelencia Junta de Andalucía, Spain). ZE sampling at FCBA was partially supported by the French Ministry of Agriculture (DGAL, No. 2014-352, QuaSeGraine project) and benefited from the support of the XYLOBIOTECH technical facility (ANR-10-EQPX-16 XYLOFOREST). References Avila C, García-Gutiérrez A, Crespillo R, Cánovas FM ( 1998) Effects of phosphinothricin treatment on glutamine synthetase isoforms in Scots pine seedlings. Plant Physiol Biochem  36: 857– 863. 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Tree PhysiologyOxford University Press

Published: Mar 1, 2018

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