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International audience; The advent of next-generation genome sequencing technologies has allowed approaching the sequencing and analysis of large and complex conifer genomes. Maritime pine (Pinus pinaster Ait.) is an economically and ecologically important conifer species widely distributed in South-West Europe, which shows a significant genetic and adaptive variability. This chapter takes on the task of reviewing the insights into the maritime pine genome sequencing breakthrough and its impact on downstream analysis. Maritime pine genome sequencing and assembly approaches are described along with the impact of related tools. A section of the state-of-the-art research on comparative, functional, structural, and translational genomics aimed at dissecting the genetic basis and the specific regulation of biological processes underlying the expression of traits of interest in maritime pine and other conifers is also described. Perspectives about the impact of these tools as well as additional research approaches are discussed.

Forest trees have access to diverse nitrogenous compounds in the soil such as ammonium, nitrate and amino acids. Recent progress has been made in the identification and characterization of ammonium and nitrate transporters. However, much more limited is our current knowledge of amino acid transport systems despite their relevance to fully understanding nitrogen nutrition in trees. In the present study, we have identified 10 genes encoding putative amino acid permeases of the AAP family in maritime pine (Pinus pinaster Ait.). Four members of this family, PpAAP1, PpAAP2, PpAAP3 and PpAAP4 were phylogenetically related to AtAAP5, involved in arginine transport in Arabidopsis thaliana. One of these genes, PpAAP1, exhibited enhanced expression levels in maritime pine roots when arginine was externally supplied. PpAAP1 was functionally characterized by complementation of a yeast mutant strain defective in the transport of arginine, allowing yeast to take up [14C]-arginine with high affinity. Furthermore, PpAAP1 was able to restore the severely affected root uptake of arginine displayed by AtAAP5 T-DNA mutants. Uptake rates of 15N-labelled arginine were significantly higher in PpAAP1-overexpressing plants when compared to wild-type and AtAAP5 mutant plants. Taken together, our results indicate that PpAAP1 is a high-affinity arginine transporter in maritime pine.

Cytosolic NADP+-isocitrate dehydrogenase (ICDH) is one of the major enzymes involved in the production of 2-oxoglutarate for amino acid biosynthesis in plants. In most plants studied, ICDH is encoded by either one gene or a small gene family, and the protein sequence has been highly conserved during evolution, suggesting it plays different and essential roles in metabolism and differentiation. To elucidate the role of ICDH in hybrid poplar (Populus tremula x P. alba), transgenic plants overexpressing the Pinus pinaster gene were generated. Overexpression of ICDH resulted in hybrid poplar (Populus tremula × P. alba) trees with higher expression levels of the endogenous ICDH gene and higher enzyme content than control untransformed plants. Transgenic poplars also showed an increased expression of glutamine synthetase (GS1.3), glutamate decarboxylase (GAD) and other genes associated with vascular differentiation. Furthermore, these plants exhibited increased growth in height, longer internodes and enhanced vascular development in young leaves and the apical region of stem. Modifications in amino acid and organic acid content were observed in young leaves of the transgenic lines, suggesting an increased biosynthesis of amino acids for building new structures and also for transport to other sink organs, as expanding leaves or young stems. Taken together, these results support an important role of ICDH in plant growth and vascular development.

Summary The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long-lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group, and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the downregulation of several genes associated with cell wall biogenesis, and secondary metabolism. Furthermore, trans-activation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the Arabidopsis SND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and pave the way for applications in conifer biotechnology. This article is protected by copyright. All rights reserved.

Biogenesis of the secondary cell wall in trees involves the massive biosynthesis of the phenylalanine-derived polymer lignin. Arogenate dehydratase (ADT) catalyzes the last, and rate-limiting, step of the main pathway for phenylalanine biosynthesis. In this study, we found that transcript levels for several members of the large ADT gene family, including ADT-A and ADT-D, were enhanced in compression wood of maritime pine, a xylem tissue enriched in lignin. Transcriptomic analysis of maritime pine silenced for PpMYB8 revealed that this gene plays a critical role in coordinating the deposition of lignin with the biosynthesis of phenylalanine. Specifically, it was found that ADT-A and ADT-D were strongly down-regulated in PpMYB8-silenced plants and that they were transcriptionally regulated through direct interaction of this transcription factor with regulatory elements present in their promoters. Another transcription factor, PpHY5, exhibited an expression profile opposite to that of PpMYB8 and also interacted with specific regulatory elements of ADT-A and ADT-D genes, suggesting that it is involved in transcriptional regulation of phenylalanine biosynthesis. Taken together, our results reveal that PpMYB8 and PpHY5 are involved in the control of phenylalanine formation and its metabolic channeling for lignin biosynthesis and deposition during wood formation in maritime pine., Functional genomics unravels the role of two arogenate dehydratases in maritime pine during wood formation, and provides new insights into how lignin biosynthesis and deposition are regulated in conifers.

Low nitrogen (N) availability is a major limiting factor for tree growth and development. N uptake, assimilation, storage and remobilization are key processes in the economy of this essential nutrient, and its efficient metabolic use largely determines vascular development, tree productivity and biomass production. Recently, advances have been made that improve our knowledge about the molecular regulation of acquisition, assimilation and internal recycling of N in forest trees. In poplar, a model tree widely used for molecular and functional studies, the biosynthesis of glutamine plays a central role in N metabolism, influencing multiple pathways both in primary and secondary metabolism. Moreover, the molecular regulation of glutamine biosynthesis is particularly relevant for accumulation of N reserves during dormancy and in N remobilization that takes place at the onset of the next growing season. The characterization of transgenic poplars overexpressing structural and regulatory genes involved in glutamine biosynthesis has provided insights into how glutamine metabolism may influence the N economy and biomass production in forest trees. Here, a general overview of this research topic is outlined, recent progress are analyzed and challenges for future research are discussed.

Los programas de educación familiar trabajan con las habilidades parentales, comunicativas, relacionales y organizativas para fomentar el cambio social positivo. Se trata de unos programas donde el rol de los formadores es clave para implicar y motivar a las familias en los procesos de aprendizaje. En el presente artículo se investigan las características de los formadores, para identificar los factores de éxito de su trabajo durante las implementaciones. Se han llevado a cabo entrevistas y grupos de discusión con diferentes formadores del Programa de Competencia Familiar, una experiencia de trabajo socioeducativo con familias. Los resultados apuntan la importancia de las habilidades específicas con padres y con hijos, el rol de la experiencia y el peso de las habilidades comunicativas y de gestión del grupo. Las principales conclusiones señalan que los formadores, a través de la participación en el programa, valoran la necesidad de mejorar su formación y entrenamiento, así como también reconocen el efecto positivo del trabajo en equipo. Family life education programs work with skills in parenting, communication, relationships and organization to promote positive family change. These are programs in which the role of facilitators is key to engage and motivate families in learning processes. In the current article, the characteristics of the trainers are studied, in order to identify the factors of success of their work in the implementations. Interviews and focus groups have been undertaken with several facilitators of the Family Competence Program, an experience of socio-educational work with families. Results underpin the importance of specific skills with parents and with children, the role of experience and the relevance of communication and group management skills. Main conclusions highlight that facilitators, after their participation in the programme, consider improving their training and they underscore the positive effect of team-working.

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.

In this work, the role of the pine transcriptional regulator Dof 5 in carbon and nitrogen metabolism has been examined in poplar trees. The overexpression of the gene and potential effects on growth and biomass production were compared between trees growing in a growth chamber under controlled conditions and trees growing in a field trial during two growth seasons. Ten-week-old transgenic poplars exhibited higher growth than untransformed controls and exhibited enhanced capacity for inorganic nitrogen uptake in the form of nitrate. Furthermore, the transgenic trees accumulated significantly more carbohydrates such as glucose, fructose, sucrose and starch. Lignin content increased in the basal part of the stem likely due to the thicker stem of the transformed plants. The enhanced levels of lignin were correlated with higher expression of the PAL1 and GS1.3 genes, which encode key enzymes involved in the phenylalanine deamination required for lignin biosynthesis. However, the results in the field trial experiment diverged from those observed in the chamber system. The lines overexpressing PpDof5 showed attenuated growth during the two growing seasons and no modification of carbon or nitrogen metabolism. These results were not associated with a decrease in the expression of the transgene, but they can be ascribed to the nitrogen available in the field soil compared to that available for growth under controlled conditions. This work highlights the paramount importance of testing transgenic lines in field trials. This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (BIO2015-69285-R) and Junta de Andalucia (BIO-474).

The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.

Efficient acquisition, assimilation and economy of nitrogen are of special importance in trees that must cope with seasonal periods of growth and dormancy over many years. The ability to accumulate nitrogen reserves and to recycle N determine to a great extent the growth and production of forest biomass. The metabolic relevance of two key amino acids, arginine and phenylalanine, as well as other processes potentially involved in the nitrogen economy of conifers are discussed in the current review. During their long life cycles, conifers not only cope with cyclical annual and long-term changes in the environment but also interact with other organisms such as herbivores and symbionts. The interactions of biotic and abiotic factors with conifer nitrogen metabolism will also be outlined in this review.

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs. © 2011 American Society of Plant Biologists., This work was supported by the Ministerio de Ciencia e Innovacio´n, Spain (grant nos. BIO2010–15430 and BFU2007–60300) and the Junta de Andalucı´a, Spain (grant nos. P06–CVI–01578, BIO– 182, and BIO–284).

Summary • In the present work the performance of transgenic poplars expressing a pine glutamine synthetase (GS) transgene was studied in natural conditions. •A field study of eight independent transgenic lines and control plants was carried out for 3 yr in the province of Granada (Spain). •T ransgenic poplars reached average heights that were 21, 36 and 41% greater than control plants after the first, second and third year of growth, respectively. Transgene expression affected plant features with time resulting in increased protein, total GS and ferredoxin-dependent glutamate synthase (Fd-GOGAT) in leaves. However, neither differences in the large subunit of Rubisco (LSU) abundance nor water content were detected between lines. Furthermore, no significant differences were found in total polysaccharide and lignin content in tree trunks. • The analyses of stem diameter, and protein contents in the bark suggest that higher levels of nitrogen reserves accumulated in the stem of transgenics. Our results suggest that modification of GS1 expression may be a useful strategy to complement traditional tree breeding in short rotation plantations.

A model for GABA synthesis in stems of pine seedlings is proposed. The localization of GABA in differentiating tracheids suggests a link between GABA production and vascular development. γ-aminobutyric acid (GABA) is a non-proteinogenic amino acid present in both prokaryotic and eukaryotic organisms. GABA plays a fundamental role as a signal molecule in the central nervous system in animals. In plants, GABA has been correlated with cellular elongation, plant development, gene expression regulation, synthesis of ethylene and other hormones, and signaling. Considering the physiological importance of GABA in plants, the lack of works about GABA localization in this kingdom seems surprising. In this work, the immunolocalization of GABA in root and hypocotyl during seedling development and in bent stem showing compression xylem has been studied. In the seedling root, the GABA signal was very high and restricted to the stele supporting previous evidences indicating a potential role for this amino acid in root growth and nutrient transport. In hypocotyl, GABA was localized in vascular tissues, including differentiating xylem, ray parenchyma and epithelial resin duct cells, drawing also a role for GABA in vascular development, communication and defense. During the production of compression wood, a special lignified wood produced when the stem loss its vertical position, a clear GABA signal was found in the new differentiating xylem cells showing a gradient-like pattern with higher signal in less differentiated elements. The results are in accordance with a previous work indicating that glutamate decarboxylase and GABA production are associated to vascular differentiation in pine Molina-Rueda et al. (Planta 232: 1471–1483, 2010). A model for GABA synthesis in vascular differentiation, communication, and defense is proposed in the stem of pine seedlings.

Plastids are organelles present in photosynthetic and nonphotosynthetic plant tissues. While it is well known that thioredoxin-dependent redox regulation is essential for leaf chloroplast function, little is known of the redox regulation in plastids of nonphotosynthetic tissues, which cannot use light as a direct source of reducing power. Thus, the question remains whether redox regulation operates in nonphotosynthetic plastid function and how it is integrated with chloroplasts for plant growth. Here, we show that NADPH-thioredoxin reductase C (NTRC), previously reported as exclusive to green tissues, is also expressed in nonphotosynthetic tissues of Arabidopsis thaliana, where it is localized to plastids. Moreover, we show that NTRC is involved in maintaining the redox homeostasis of plastids also in nonphotosynthetic organs. To test the relationship between plastids of photosynthetic and nonphotosynthetic tissues, transgenic plants were obtained with redox homeostasis restituted exclusively in leaves or in roots, through the expression of NTRC under the control of organspecific promoters in the ntrc mutant. Our results show that fully functional root amyloplasts are not sufficient for root, or leaf, growth, but fully functional chloroplasts are necessary and sufficient to support wild-type rates of root growth and lateral root formation. © 2012 American Society of Plant Biologists. All rights reserved.

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a key enzyme in the synthesis of γ-aminobutyric acid (GABA) in higher plants. A complete cDNA encoding glutamate decarboxylase (GAD, EC 4.1.1.15) was characterized from Pinus pinaster Ait, and its expression pattern was studied to gain insight into the role of GAD in the differentiation of the vascular system. Pine GAD contained a C-terminal region with conserved residues and a predicted secondary structure similar to the calmodulin (CaM)-binding domains of angiosperm GADs. The enzyme was able to bind to a bovine CaM-agarose column and GAD activity was higher at acidic pH, suggesting that the pine GAD can be regulated in vivo by Ca(2+)/CaM and pH. A polyclonal antiserum was prepared against the pine protein. GAD expression was studied at activity, protein, and mRNA level and was compared with the expression of other genes during the differentiation of the hypocotyl and induction of reaction wood. In seedling organs, GABA levels closely matched GAD expression, with high levels in the root and during lignification of the hypocotyl. GAD expression was also induced in response to the production of compression wood and its expression matched the pattern of other genes involved in ethylene and 2-oxoglutarate synthesis. The results suggest of a role of GAD in hypocotyl and stem development in pine.

Photosynthesis is a process that inevitably produces reactive oxygen species, such as hydrogen peroxide, which is reduced by chloroplast-localized detoxification mechanisms one of which involves 2-Cys peroxiredoxins (2-Cys Prxs). Arabidopsis chloroplasts contain two very similar 2-Cys Prxs (denoted A and B). These enzymes are reduced by two pathways: NADPH thioredoxin reductase C (NTRC), which uses NADPH as source of reducing power; and plastidial thioredoxins (Trxs) coupled to photosynthetically reduced ferredoxin of which Trx x is the most efficient reductant in vitro. With the aim of establishing the functional relationship between NTRC, Trx x, and 2-Cys Prxs in vivo, an Arabidopsis Trx x knock-out mutant has been identified and a double mutant (denoted Δ2cp) with, This work was supported by grant BIO2007-60644 from Ministerio de Educacion y Ciencia, and grants P06-CVI01578 and BIO-182 from Junta de Andalucia (Spain). K-JD acknowledges support by the DFG (Di346). PP, KK, and MG were supported by pre-doctoral fellowships from Seville University, CSIC, and Junta de Andalucia, respectively.

Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Canton, F.R., Garcia-Gutierrez, A., Canovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Canovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Canovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth of GS transgenic poplar plantlets was 5-fold greater than controls. The response of young leaves to PPT treatment depends on physiological state as indicated by GS and Rubisco (LSU) levels. Young leaves from control plants, typically in a low differentiation state, respond to the herbicide showing up-regulation of GS and LSU. In contrast, young leaves from transgenic lines, with higher initial GS and LSU levels compared to control, display up-regulation of NADP(+)-isocitrate dehydrogenase. Differences between control and GS transgenics in their response to PPT are discussed in relation to their differences in photosynthetic and photorespiratory capacities (El-Khatib et al., 2004).

The biosynthesis of 2-oxoglutarate and glutamate are key steps in the biosynthesis of nitrogen compounds and plant development. The reaction catalyzed by cytosolic isoenzyme of NADP+-linked isocitrate dehydrogenase (IDH) is also considered as the main route in the production of 2-oxoglutarate. According to its expression pattern during development, IDH is also involved in other, yet unknown, processes [1,2]. In addition to the importance of glutamate in the biosynthesis of nitrogen compounds, glutamate also serves as precursor of GABA, a molecule that is currently considered as a signal in higher plants. GABA is produced by the action of glutamate decarboxylase (GAD), a cytosolic enzyme that is regulated by Ca2+/calmodulin and pH. In contrast to IDH, that it is encoded by just one gene in most of plant genomes [2], GAD is encoded by a small family of nuclear genes [3]. The expression of IDH and GAD has been investigated during the differentiation of hypocotyl and stem in tree species. Our results indicate a coordination of the expression of IDH and GAD in developmental processes suggesting a role for 2-oxoglutarate supply and GABA synthesis during early stages of organ differentiation in trees.