The Characterization of a Novel PrMADS11 Transcription Factor from Pinus radiata Induced Early in Bent Pine Stem.

A novel MADS-box transcription factor from Pinus radiata D. Don was characterized. PrMADS11 encodes a protein of 165 amino acids for a MADS-box transcription factor belonging to group II, related to the MIKC protein structure. PrMADS11 was differentially expressed in the stems of pine trees in response to 45° inclination at early times (1 h). Arabidopsis thaliana was stably transformed with a 35S::PrMADS11 construct in an effort to identify the putative targets of PrMADS11. A massive transcriptome analysis revealed 947 differentially expressed genes: 498 genes were up-regulated, and 449 genes were down-regulated due to the over-expression of PrMADS11. The gene ontology analysis highlighted a cell wall remodeling function among the differentially expressed genes, suggesting the active participation of cell wall modification required during the response to vertical stem loss. In addition, the phenylpropanoid pathway was also indicated as a PrMADS11 target, displaying a marked increment in the expression of the genes driven to the biosynthesis of monolignols. The EMSA assays confirmed that PrMADS11 interacts with CArG-box sequences. This TF modulates the gene expression of several molecular pathways, including other TFs, as well as the genes involved in cell wall remodeling. The increment in the lignin content and the genes involved in cell wall dynamics could be an indication of the key role of PrMADS11 in the response to trunk inclination.

High density linkage maps, genetic architecture, and genomic prediction of growth and wood properties in Pinus radiata.

BACKGROUND: The growing availability of genomic resources in radiata pine paves the way for significant advances in fundamental and applied genomic research. We constructed robust high-density linkage maps based on exome-capture genotyping in two F1 populations, and used these populations to perform quantitative trait locus (QTL) scans, genomic prediction and quantitative analyses of genetic architecture for key traits targeted by tree improvement programmes. RESULTS: Our mapping approach used probabilistic error correction of the marker data, followed by an iterative approach based on stringent parameters. This approach proved highly effective in producing high-density maps with robust marker orders and realistic map lengths (1285-4674 markers per map, with sizes ranging from c. 1643-2292 cM, and mean marker intervals of 0.7-2.1 cM). Colinearity was high between parental linkage maps, although there was evidence for a large chromosomal rearrangement (affecting ~ 90 cM) in one of the parental maps. In total, 28 QTL were detected for growth (stem diameter) and wood properties (wood density and fibre properties measured by Silviscan) in the QTL discovery population, with 1-3 QTL of small to moderate effect size detected per trait in each parental map. Four of these QTL were validated in a second, unrelated F1 population. Results from genomic prediction and analyses of genetic architecture were consistent with those from QTL scans, with wood properties generally having moderate to high genomic heritabilities and predictive abilities, as well as somewhat less complex genetic architectures, compared to growth traits. CONCLUSIONS: Despite the economic importance of radiata pine as a plantation forest tree, robust high-density linkage maps constructed from reproducible, sequence-anchored markers have not been published to date. The maps produced in this study will be a valuable resource for several applications, including the selection of marker panels for genomic prediction and anchoring a recently completed de novo whole genome assembly. We also provide the first map-based evidence for a large genomic rearrangement in radiata pine. Finally, results from our QTL scans, genomic prediction, and genetic architecture analyses are informative about the genomic basis of variation in important phenotypic traits.

Molecular Phylogenomics and Population Structure of Phytophthora pluvialis.

Phytophthora pluvialis is an oomycete that was first isolated from soil, water, and tree foliage in mixed Douglas-fir-tanoak forests of the U.S. Pacific Northwest (PNW). It was then identified as the causal agent of red needle cast of radiata pine (Pinus radiata) in New Zealand (NZ). Genotyping-by-sequencing was used to obtain 1,543 single nucleotide polymorphisms across 145 P. pluvialis isolates to characterize the population structure in the PNW and NZ. We tested the hypothesis that P. pluvialis was introduced to NZ from the PNW using genetic distance measurements and population structure analyses among locations between countries. The low genetic distance, population heterozygosity, and lack of geographic structure in NZ suggest a single colonization event from the United States followed by clonal expansion in NZ. The PNW Coast Range was proposed as a presumptive center of origin of the currently known distribution of P. pluvialis based on its geographic range and position as the central cluster in a minimum spanning network. The Coastal cluster of isolates were located at the root of every U.S. cluster and emerged earlier than all NZ clusters. The Coastal cluster had the highest degree of heterozygosity (Hs = 0.254) and median pairwise genetic distance (0.093) relative to any other cluster. Finally, the rapid host diversification between closely related isolates of P. pluvialis in NZ indicate that this pathogen has the potential to infect a broader range of hosts than is currently recognized.

Comparison on Reduction of VOCs Emissions from Radiata Pine (Pinus Radiata D. Don) between Sodium Bicarbonate and Ozone Treatments.

Volatile organic compounds (VOCs) in wood furniture are an important factor that affects indoor air quality. In this study, radiata pine (Pinus radiata D. Don) was treated with sodium bicarbonate and ozone aqueous solution to reduce the VOC contents without sacrificing mechanical properties. The VOCs of radiata pine were identified by gas chromatography-mass spectrometry (GC-MS), and the functional group changes of wood samples were characterized by Fourier-transform infrared spectroscopy (FTIR). The results showed that the main VOCs of radiata pine include alkenes, aldehydes, and esters. The sodium bicarbonate and ozone treatments almost eliminated the VOC contents of radiata pine. The two treatments mentioned above had little effect on compressive strength and surface color of radiata pine.

Genomic selection for non-key traits in radiata pine when the documented pedigree is corrected using DNA marker information.

BACKGROUND: Non-key traits (NKTs) in radiata pine (Pinus radiata D. Don) refer to traits other than growth, wood density and stiffness, but still of interest to breeders. Branch-cluster frequency, stem straightness, external resin bleeding and internal checking are examples of such traits and are targeted for improvement in radiata pine research programmes. Genomic selection can be conducted before the performance of selection candidates is available so that generation intervals can be reduced. Radiata pine is a species with a long generation interval, which if reduced could significantly increase genetic gain per unit of time. The aim of this study was to evaluate the accuracy and predictive ability of genomic selection and its efficiency over traditional forward selection in radiata pine for the following NKTs: branch-cluster frequency, stem straightness, internal checking, and external resin bleeding. RESULTS: Nine hundred and eighty-eight individuals were genotyped using exome capture genotyping by sequencing (GBS) and 67,168 single nucleotide polymorphisms (SNPs) used to develop genomic estimated breeding values (GEBVs) with genomic best linear unbiased prediction (GBLUP). The documented pedigree was corrected using a subset of 704 SNPs. The percentage of trio parentage confirmed was about 49% and about 50% of parents were re-assigned. The accuracy of GEBVs was 0.55-0.75 when using the documented pedigree and 0.61-0.80 when using the SNP-corrected pedigree. A higher percentage of additive genetic variance was explained and a higher predictive ability was observed when using the SNP-corrected pedigree than using the documented pedigree. With the documented pedigree, genomic selection was similar to traditional forward selection when assuming a generation interval of 17 years, but worse than traditional forward selection when assuming a generation interval of 14 years. After the pedigree was corrected, genomic selection led to 37-115% and 13-77% additional genetic gain over traditional forward selection when generation intervals of 17 years and 14 years were assumed, respectively. CONCLUSION: It was concluded that genomic selection with a pedigree corrected by SNP information was an efficient way of improving non-key traits in radiata pine breeding.

Wood Modification by Furfuryl Alcohol Resulted in a Delayed Decomposition Response in Rhodonia (Postia) placenta.

The aim of this study was to investigate differential expression profiles of the brown rot fungus Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point (mass loss, 14%). Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment.IMPORTANCE Fungi are important decomposers of woody biomass in natural habitats. Investigation of the mechanisms employed by decay fungi in their attempt to degrade wood is important for both the basic scientific understanding of ecology and carbon cycling in nature and for applied uses of woody materials. For wooden building materials, long service life and carbon storage are essential, but decay fungi are responsible for massive losses of wood in service. Thus, the optimization of durable wood products for the future is of major importance. In this study, we have investigated the fungal genetic response to furfurylated wood, a commercial environmentally benign wood modification approach that improves the service life of wood in outdoor applications. Our results show that there is a delayed wood decay by the fungus as a response to furfurylated wood, and new knowledge about the mechanisms behind the delay is provided.

Pontamine fast scarlet 4B bifluorescence and measurements of cellulose microfibril angles.

Pontamine fast scarlet 4B is a red paper and textiles dye that has recently been introduced as a fluorescent probe for plant cell walls. Pontamine exhibits bifluorescence, or fluorescence dependent on the polarization of the excitation light: Because cellulose is aligned within the cell wall, pontamine-labelled cell walls exhibit variable fluorescence as the excitation polarization is modulated. Thus, bifluorescence measurements require polarized excitation that can be directly or indirectly modulated. In our confocal microscopy observations of various cellulose samples labelled with pontamine, we modulated excitation polarization either through sample rotation or by the confocal's scanfield rotation function. This variably rotated laser polarizations on Leica confocal microscopes, but not those from other makers. Beginning with samples with directly observable microfibril orientations, such as purified bacterial cellulose, the velamen of orchid roots and the inner S2 layer of radiata pine compression wood, we demonstrate that modelling the variations in pontamine fluorescence with a sine curve can be used to measure the known microfibril angles. We then measured average local microfibril angles in radiata pine samples, and showed similar microfibril angles in compression and normal (opposite) wood. Significantly, bifluorescence measurements might also be used to understand the degree of local cellulose alignment within the cell wall, as opposed to variations in the overall cellulose angle.

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine).

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.

Modelling the influence of predicted future climate change on the risk of wind damage within New Zealand's planted forests.

Wind is the major abiotic disturbance in New Zealand's planted forests, but little is known about how the risk of wind damage may be affected by future climate change. We linked a mechanistic wind damage model (ForestGALES) to an empirical growth model for radiata pine (Pinus radiata D. Don) and a process-based growth model (cenw) to predict the risk of wind damage under different future emissions scenarios and assumptions about the future wind climate. The cenw model was used to estimate site productivity for constant CO2 concentration at 1990 values and for assumed increases in CO2 concentration from current values to those expected during 2040 and 2090 under the B1 (low), A1B (mid-range) and A2 (high) emission scenarios. Stand development was modelled for different levels of site productivity, contrasting silvicultural regimes and sites across New Zealand. The risk of wind damage was predicted for each regime and emission scenario combination using the ForestGALES model. The sensitivity to changes in the intensity of the future wind climate was also examined. Results showed that increased tree growth rates under the different emissions scenarios had the greatest impact on the risk of wind damage. The increase in risk was greatest for stands growing at high stand density under the A2 emissions scenario with increased CO2 concentration. The increased productivity under this scenario resulted in increased tree height, without a corresponding increase in diameter, leading to more slender trees that were predicted to be at greater risk from wind damage. The risk of wind damage was further increased by the modest increases in the extreme wind climate that are predicted to occur. These results have implications for the development of silvicultural regimes that are resilient to climate change and also indicate that future productivity gains may be offset by greater losses from disturbances.

Nanoscale interactions of polyethylene glycol with thermo-mechanically pre-treated Pinus radiata biofuel substrate.

Non-productive adsorption of cellulose degrading enzymes on lignin is a likely reason for reduced rate and extent of enzymatic conversion of lignocellulosic substrate to sugars. Additives such as polyethyleneglycol (PEG) may act as blocking agents in this non-productive interaction. However, the exact molecular level interactions of PEG with lignin in pre-treated lignocellulosic substrates are not known. We have used confocal fluorescence microscopy combined with Förster resonance energy transfer (FRET) to reveal molecular level interactions between lignin present in thermo-mechanically pre-treated Pinus radiata substrate, and fluorescently labeled PEG. It is demonstrated that PEG interaction with lignin is mainly associated with particles derived from secondary walls, with little or no penetration into fragments derived from the middle lamella. This nanoscale information on the PEG-substrate interaction will assist in rationalizing pre-treatment methods to reduce the recalcitrance of softwood biofuel substrates.

Toxicity evaluation of Pinus radiata D.Don bark wax for potential cosmetic application.

Radiata pine bark is a widely available organic waste, requiring alternative uses due to its environmental impact on soil, fauna, and forest fires. Pine bark waxes could be used as cosmetic substitutes, but their toxicity requires evaluation since pine bark may contain toxic substances or xenobiotics, depending on the extraction process. This study evaluates the toxicity of radiata pine bark waxes obtained through various extraction methods on human skin cells grown in vitro. The assessment includes using XTT to evaluate mitochondrial activity, violet crystal dye to assess cell membrane integrity, and ApoTox-Glo triple assay to measure cytotoxicity, viability, and apoptosis signals. Pine bark waxes extracted via T3 (acid hydrolysis and petroleum ether incubation) and T9 (saturated steam cycle, alkaline hydrolysis, and petroleum ether incubation) exhibit non-toxicity up to 2% concentration, making them a potential substitute for petroleum-based cosmetic materials. Integrating the forestry and cosmetic industries through pine bark wax production under circular economy principles could promote development while replacing petroleum-based materials. Extraction methodology affects pine bark wax toxicity in human skin cells due to the retention of xenobiotic compounds including methyl 4-ketohex-5-enoate; 1-naphthalenol; dioctyl adipate; eicosanebioic acid dimethyl ester; among others. Future research will investigate whether the extraction methodology alters the molecular structure of the bark, affecting the release of toxic compounds in the wax mixture.

Pilot-Scale Production of Hemicellulose Ethers from Softwood Hemicelluloses Obtained from Compression Screw Pressate of a Thermo-Mechanical Pulping Plant.

Bio-derived materials are becoming increasingly sought-after as a sustainable alternative to petrochemical-derived polymers. In the present pilot-scale study, a hemicellulose-rich compression screw pressate, obtained from the pre-heating stage of thermo-mechanical pulping (TMP) of radiata pine, was purified by treatment with adsorbent resin (XAD7), then ultrafiltered and diafiltered at 10 kDa to isolate the high-molecular weight (MW) hemicellulose fraction (yield 18.4% on pressate solids), and, finally, reacted with butyl glycidyl ether to plasticise the hemicelluloses. The resulting light brown hemicellulose ethers (yield 102% on the isolated hemicelluloses) contained ca. 0.5 butoxy-hydroxypropyl side chains per pyranose unit and had weight- and number-average MWs of 13,000 Da and 7200 Da, respectively. The hemicellulose ethers may serve as raw material for bio-based products such as barrier films.

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.

Effect of the Longitudinal Tensile Creep on the Stiffness of Radiata Pine (Pinus radiata D. Don).

The influence of load on the cellulose microfibrils of single cells or thin wood foils is known. It can decrease the cellulose microfibril angles and, in turn, increase the stiffness. However, this modification of a piece of wood, which is made up of multiple cells, is unknown. The aim of this research was to study the effect of tensile creep on the longitudinal stiffness of radiata pine wood. The modulus of elasticity of each specimen was determined before and after being subjected to tensile creep. The samples were loaded at 1170 N and 1530 N for 20 min at 70 °C. The load was determined as a function of a percentage of the force at the proportional limit. The moduli of elasticity before and post-tensile creep showed no effect on the stiffness of wood at the macroscopic level, but neither were there damage to the cell structure. It can be assumed that there are changes at the microscopic level, but they are not enough to be reflected at the macro scale. It is also challenging to achieve the modifications that occur at the level of a single cell or in thin wood foils; however, the implications of this would be favorable for the development of stronger wood-based products.

Ultrasonic guided wave measurement in a wooden rod using shear transducer arrays.

Research related to acoustic/ultrasonic guided wave testing in wood is still at an early stage. This paper describes the first study to perform ultrasonic guided wave measurements in a wooden rod using arrays of shear transducers. Enhancement of either longitudinal L(0,1) or torsional T(0,1) wave modes and suppression of other modes was able to be achieved using these arrays. At low frequencies, it was found that the L(0,1) wave mode had a similar speed to that obtained using the traditional resonance and time of flight methods. The torsional T(0,1) wave mode has not been used before for non-destructive testing of wood. Since it is non-dispersive, it would appear to be suitable for wood property estimation and structural health monitoring of wooden structures. These results indicate that ultrasonic guided wave testing techniques have strong potential to be used to provide improved measurement of wood properties and structural health monitoring of wooden structures.

Sirex noctilio infestation led to inevitable pine death despite activating pathways involved in tolerance.

Defense-related metabolome traits in pine species after infestation by Sirex noctilio are largely unknown, despite, in most cases, trees being overwhelmed. Using LC-MS-based untargeted metabolomics, we revealed the systemic metabolic changes induced by this insect in 14-year-old Pinus radiata trees, the most affected species worldwide. An immediate metabolome alteration was expressed in needles after infestation, including the up-regulation of flavonols, flavan-3-ols, oxyneolignans, auxins, proline, and tryptophan, among others. The flavan-3-ols (catechin and procyanidin B1) suggested a rapidly induced photoprotection mechanism aided by diverting proline as an alternative substrate for respiration to compensate for the progressive chlorosis that degrades photosystems. Meanwhile, glutathione, glutamate, and ascorbate levels significantly dropped in needles, which may indicate the critical oxidative stress that trees had to face since the onset of the infestation. They were not fully replenished after long-term infestation, and redox homeostasis was probably not achieved, compromising tree survival. Nevertheless, a huge auxins overexpression detected in needles throughout the infestation may reflect tolerance against the premature senescence caused by the woodwasp venom. In contrast, the metabolome of wood tissues remained initially unchanged, although it seems to collapse after three months. Overall, the metabolomics strategy adopted in this work evidenced its usefulness in uncovering the fundamental roles of plants' chemical defense that govern interactions with specific stressors.

Balancing Breeding for Growth and Fecundity in Radiata Pine (Pinus radiata D. Don) Breeding Programme.

Tree breeding has focused on increasing stem volume growth with a cost to fecundity. However, fecundity is important in maintaining the fitness in natural stands and facilitating cross-pollination to advance breeding populations. Understanding the inheritance of fecundity and the genetic relationship between fecundity and growth is essential to understand the constraints of evolution in natural population and design an optimal selection strategy to balance breeding for growth and fecundity. Inheritance of female fecundity and the genetic relationship between fecundity and growth in radiata pine were investigated using a large Australia-wide progeny test, planted on eight sites involving 279 control-pollinated families. It was found that fecundity of female cones was highly heritable with an estimated heritability of 0.39-0.61, but genetically correlated with growth (-0.30 to -0.39). This indicates that improvement in tree growth alone could reduce the fecundity, thus to break the possible evolutionary constraint in natural population. To maintain fecundity for breeding purposes and minimize the interruption of the evolutionary constraint between fecundity and growth, use of a restraint selection index to impose no change of fecundity is developed in current breeding, while dissecting the genetic basis of adversely correlated traits at loci level is required for optimal long-term strategy.

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.

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.

Pretreatment of pine lignocelluloses by recyclable deep eutectic solvent for elevated enzymatic saccharification and lignin nanoparticles extraction.

This study investigated the process intensification strategies for the pretreatment of Radiata Pine with the green deep eutectic solvent (DES) system composed of benzyltrimethylammonium chloride/formic acid (BTMAC/FA). The results showed that DES pretreatment drastically improved the delignification and hemicelluloses-removal capacity. The conducted process acceptably remained most of the cellulose in pretreated biomass (88.3%-91.8%). Benefiting from the overcoming of recalcitrance, the enzymatic digestibility of pretreated residues reached 92.4%. The efficient conversion was mainly ascribed to the lignin and hemicelluloses co-extraction. Meanwhile, the lignin yield and enzymatic saccharification was still largely maintained after five reuses. Further structural characteristics of lignin nanoparticles revealed that the lignin possessed high purity (95.19-97.51%), medium molecular weight (9600 to 6495 g/mol), and low polydispersity (ca 2.0), which was attributed to cleavage of ether bonds in lignin as well as linkages between lignin and hemicelluloses. Overall, this study illustrated that DES pretreatment was promising to achieve an efficient fractionation of woody biomass into fermentable glucose and high-quality lignin.