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.

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.

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.

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.

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.

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.

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.

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.

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.

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.