Proteomic and Metabolic Analysis of Pinus halepensis Mill. Embryonal Masses Induced under Heat Stress.

Understanding the physiological and molecular adjustments occurring during tree stress response is of great importance for forest management and breeding programs. Somatic embryogenesis has been used as a model system to analyze various processes occurring during embryo development, including stress response mechanisms. In addition, "priming" plants with heat stress during somatic embryogenesis seems to favor the acquisition of plant resilience to extreme temperature conditions. In this sense, Pinus halepensis somatic embryogenesis was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) and its effects on the proteome and the relative concentration of soluble sugars, sugar alcohols and amino acids of the embryonal masses obtained were assessed. Heat severely affected the production of proteins, and 27 proteins related to heat stress response were identified; the majority of the proteins with increased amounts in embryonal masses induced at higher temperatures consisted of enzymes involved in the regulation of metabolism (glycolysis, the tricarboxylic acid cycle, amino acid biosynthesis and flavonoids formation), DNA binding, cell division, transcription regulation and the life-cycle of proteins. Finally, significant differences in the concentrations of sucrose and amino acids, such as glutamine, glycine and cysteine, were found.

Testing Explant Sources, Culture Media, and Light Conditions for the Improvement of Organogenesis in Pinus ponderosa (P. Lawson and C. Lawson).

Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15-30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µΜ 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m-2 s-1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m-2 s-1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.

Current status of the cryopreservation of embryogenic material of woody species.

Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen.

Somatic Embryogenesis in Pines.

Among the different in vitro culture techniques, somatic embryogenesis has been one of the most important developments for plant tissue culture; it has enabled mass propagation and the development of biotechnological tools to enhance the productivity and quality of plantation forestry. This propagation technique together with cryopreservation is the base of multivarietal forestry.The development of somatic embryogenesis in forest trees dates from 1985, and in the last years several studies have focused on the development and optimization of the conifer somatic embryogenesis process to make it more efficient in terms of both the quantity and the characteristics of the plants obtained. However, these advances are not sufficiently refined to be implemented commercially for many Pinus spp. due to the high cost of the process derived from hand labor. Nowadays, trying to add value to the plants produced to compensate the high costs of the process, different studies are being developed in order to obtain Pinus somatic plants with better adaptation to environmental stresses prompted by the current situation of climate change.In this chapter, a summary of the recent somatic embryogenesis systems developed to achieve Pinus spp. high quality plants is presented.

Thermopriming-associated proteome and sugar content responses in Pinus radiata embryogenic tissue.

Improving the capacity of plants to face adverse environmental conditions requires a deep understanding of the molecular mechanisms governing stress response and adaptation. Proteomics, combined with metabolic analyses, offers a wide resource of information to be used in plant breeding programs. Previous studies have shown that somatic embryogenesis in Pinus spp. is a suitable tool not only to investigate stress response processes but also to modulate the behaviour of somatic plants. Based on this, the objective of this study was to analyse the protein and soluble sugar profiles of Pinus radiata embryonal masses after the application of high temperatures to unravel the mechanisms involved in thermopriming and memory acquisition at early stages of the somatic embryogenesis process. Results confirmed that heat provokes deep readjustments in the life cycle of proteins, together with a significant reduction in the carbon-flux of central-metabolism pathways. Heat-priming also promotes the accumulation of proteins involved in oxidative stress defence, in the synthesis of specific amino acids such as isoleucine, influences cell division, the organization of the cytoskeleton and cell-walls, and modifies the levels of free soluble sugars like glucose or fructose. All this seems to be regulated by proteins linked with epigenetic, transcriptional and post-transcriptional mechanisms.

Cryopreservation of Holm Oak Embryogenic Cultures for Long-Term Conservation and Assessment of Polyploid Stability.

Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7-63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets.

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL-1 of L-glutamine, 1,050 mgL-1 of L-asparagine, 350 mgL-1 of L-arginine, and 35 mgL-1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.

Heat Stress in Pinus halepensis Somatic Embryogenesis Induction: Effect in DNA Methylation and Differential Expression of Stress-Related Genes.

In the current context of climate change, plants need to develop different mechanisms of stress tolerance and adaptation to cope with changing environmental conditions. Temperature is one of the most important abiotic stresses that forest trees have to overcome. Recent research developed in our laboratory demonstrated that high temperatures during different stages of conifer somatic embryogenesis (SE) modify subsequent phases of the process and the behavior of the resulting ex vitro somatic plants. For this reason, Aleppo pine SE was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) in order to analyze its effect on the global DNA methylation rates and the differential expression of four stress-related genes at different stages of the SE process. Results showed that a slight decrease of DNA methylation at proliferating embryonal masses (EMs) can correlate with the final efficiency of the process. Additionally, different expression patterns for stress-related genes were found in EMs and needles from the in vitro somatic plants obtained; the DEHYDRATION INDUCED PROTEIN 19 gene was up-regulated in response to heat at proliferating EMs, whereas HSP20 FAMILY PROTEIN and SUPEROXIDE DISMUTASE [Cu-Zn] were down-regulated in needles.

Proteome-Wide Analysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a Combined Response of Sugar Metabolism and Translational Regulation Mechanisms.

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.

Cytokinins are involved in drought tolerance of Pinus radiata plants originating from embryonal masses induced at high temperatures.

Vegetative propagation through somatic embryogenesis is an effective method to produce elite varieties and can be applied as a tool to study the response of plants to different stresses. Several studies show that environmental changes during embryogenesis could determine future plant development. Moreover, we previously reported that physical and chemical conditions during somatic embryogenesis can determine the protein, hormone and metabolite profiles, as well as the micromorphological and ultrastructural organization of embryonal masses and somatic embryos. In this sense, phytohormones are key players throughout the somatic embryogenesis process as well as during numerous stress-adaptation responses. In this work, we first applied different high-temperature regimes (30 °C, 4 weeks; 40 °C, 4 days; 50 °C, 5 min) during induction of Pinus radiata D. Don somatic embryogenesis, together with control temperature (23 °C). Then, the somatic plants regenerated from initiated embryogenic cell lines and cultivated in greenhouse conditions were subjected to drought stress and control treatments to evaluate survival, growth and several physiological traits (relative water content, water potential, photosynthesis, stomatal conductance and transpiration). Based on those preliminary results, even more extreme high-temperature regimes were applied during induction (40 °C, 4 h; 50 °C, 30 min; 60 °C, 5 min) and the corresponding cytokinin profiles of initiated embryonal masses from different lines were analysed. The results showed that the temperature regime during induction had delayed negative effects on drought resilience of somatic plants as indicated by survival, photosynthetic activity and water- use efficiency. However, high temperatures for extended periods of time enhanced subsequent plant growth in well-watered conditions. High-temperature regime treatments induced significant differences in the profile of total cytokinin bases, N6-isopentenyladenine, cis-zeatin riboside and trans-zeatin riboside. We concluded that phytohormones could be potential regulators of stress-response processes during initial steps of somatic embryogenesis and that they may have delayed implications in further developmental processes, determining the performance of the generated plants.

Induction of Radiata Pine Somatic Embryogenesis at High Temperatures Provokes a Long-Term Decrease in DNA Methylation/Hydroxymethylation and Differential Expression of Stress-Related Genes.

Based on the hypothesis that embryo development is a crucial stage for the formation of stable epigenetic marks that could modulate the behaviour of the resulting plants, in this study, radiata pine somatic embryogenesis was induced at high temperatures (23 °C, eight weeks, control; 40 °C, 4 h; 60 °C, 5 min) and the global methylation and hydroxymethylation levels of emerging embryonal masses and somatic plants were analysed using LC-ESI-MS/ MS-MRM. In this context, the expression pattern of six genes previously described as stress-mediators was studied throughout the embryogenic process until plant level to assess whether the observed epigenetic changes could have provoked a sustained alteration of the transcriptome. Results indicated that the highest temperatures led to hypomethylation of both embryonal masses and somatic plants. Moreover, we detected for the first time in a pine species the presence of 5-hydroxymethylcytosine, and revealed its tissue specificity and potential involvement in heat-stress responses. Additionally, a heat shock protein-coding gene showed a down-regulation tendency along the process, with a special emphasis given to embryonal masses at first subculture and ex vitro somatic plants. Likewise, the transcripts of several proteins related with translation, oxidative stress response, and drought resilience were differentially expressed.

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos.

Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate the physical and chemical conditions (water availability) along the process and to study their effect in the final success in terms of quantity of somatic embryos produced. In the last years, our research team has been focused on the study of different aspects of the SE in Pinus spp. One of the main aspects affecting SE is the composition of culture media; in this sense, phytohormones play one of the most crucial roles in this propagation system. Many studies in conifers have shown that different stages of SE and somatic embryo development are correlated with distinct endogenous phytohormone profiles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on this knowledge, the aim of this study was to test the effect of different temperatures (18, 23, and 28°C) and gelling agent concentrations (8, 9, and 10 gL-1) during the maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, phytohormone profile of somatic embryos developed was evaluated. In this sense, the highest gellan gum concentration led to significantly lower water availability. At this gellan gum concentration and 23°C a significantly higher number of somatic embryos was obtained and the overall success of the process increased with respect to other treatments assayed. The somatic embryos produced in these conditions showed the highest concentration of iP-type cytokinins and total ribosides. Although, the different conditions applied during maturation of somatic embryos led to different hormonal profiles, they did not affect the ex vitro survival of the resulting somatic plants, where no significant differences were observed.

Effect of Thermal Stress on Tissue Ultrastructure and Metabolite Profiles During Initiation of Radiata Pine Somatic Embryogenesis.

Climate change will inevitably lead to environmental variations, thus plant drought tolerance will be a determinant factor in the success of plantations and natural forestry recovery. Some metabolites, such as soluble carbohydrates and amino acids, have been described as being the key to both embryogenesis efficiency and abiotic stress response, contributing to phenotypic plasticity and the adaptive capacity of plants. For this reason, our main objectives were to evaluate if the temperature during embryonal mass initiation in radiata pine was critical to the success of somatic embryogenesis, to alter the morphological and ultrastructural organization of embryonal masses at cellular level and to modify the carbohydrate, protein, or amino acid contents. The first SE initiation experiments were carried out at moderate and high temperatures for periods of different durations prior to transfer to the control temperature of 23°C. Cultures initiated at moderate temperatures (30°C, 4 weeks and 40°C, 4 days) showed significantly lower initiation and proliferation rates than those at the control temperature or pulse treatment at high temperatures (50°C, 5 min). No significant differences were observed either for the percentage of embryogenic cell lines that produced somatic embryos, or for the number of somatic embryos per gram of embryonal mass. Based on the results from the first experiments, initiation was carried out at 40°C 4 h; 50°C, 30 min; and a pulse treatment of 60°C, 5 min. No significant differences were found for the initiation or number of established lines or for the maturation of somatic embryos. However, large morphological differences were observed in the mature somatic embryos. At the same time, changes observed at cellular level suggested that strong heat shock treatments may trigger the programmed cell death of embryogenic cells, leading to an early loss of embryogenic potential, and the formation of supernumerary suspensor cells. Finally, among all the differences observed in the metabolic profile, it is worth highlighting the accumulation of tyrosine and isoleucine, both amino acids involved in the synthesis of abiotic stress response-related secondary metabolites.

Somatic Embryogenesis in Pinus spp.

Somatic embryogenesis (SE) has been the most important development for plant tissue culture, not only for mass propagation but also for enabling the implementation of biotechnological tools that can be used to increase the productivity and wood quality of plantation forestry. Development of SE in forest trees started in 1985 and nowadays many studies are focused on the optimization of conifer SE system. However, these advances for many Pinus spp. are not sufficiently refined to be implemented commercially. In this chapter, a summary of the main systems used to achieve SE in Pinus spp. is reported.

Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees.

In Pinus radiata D. Don, the transition from the juvenile to the mature phase is characterized by a reduction in the tree's organogenic potential, which is usually reverted in breeding programs by reinvigoration procedures to enable vegetative propagation. In this work, we have determined the best culture conditions for in vitro reinvigoration of radiata pine buds, tested different cytokinin (CK) types [N6-benzyladenine (BA), meta-topolin (mT) and trans-zeatin] and concentrations (25 and 50 µM), and studied the effect of culture conditions on endogenous CK and indole-3-acetic acid (IAA) levels at different stages of the organogenic process. To this end, the levels of 43 CKs and IAA were determined in P. radiata buds before and during the reinvigoration process. When BA or mT was applied to the induction medium, we did not observe any significant increase or decrease in endogenous isoprenoid CK content. We also report for the first time the presence of O-glucosides in non-treated P. radiata explants from the field and remark the importance of O-glucosides as storage forms.

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels.

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.