Sofía Valenzuela

Validation of reference genes for real-time qRT-PCR normalization during cold acclimation in Eucalyptus globulus

During the last few years, many studies have directed their efforts at elucidating the molecular mechanisms that regulate plant response to cold stress using gene expression analysis. Quantitative real-time qRT-PCR has great advantages compared to traditional transcriptional detection methods due to its high sensibility, reproducibility, and specificity for the detection of low quantities of RNA. However, this technique requires the use of one or several housekeeping genes. In this work, the expression stabilities of six housekeeping genes (EF1α, ACT, α-TUB, PDF, SAND, and UBC) during the cold acclimation of E. globulus plants was analyzed. An ELIP gene that responds to photooxidative stress caused by light and cold stress was used as the target gene to identify the most suitable internal control for normalizing real-time qRT-PCR. Two additional genes involved in the ABA biosynthesis pathway (NCED) and sugar metabolism (GS) were analyzed with the most stable internal control genes in order to check the results found with the ELIP gene. The expressions of UBC, α-TUB and EF1α were the most stable across acclimation and de-acclimation treatments. The expressions of the other housekeeping genes tested varied depending upon the conditions. The relative quantification of ELIP changed according to identities and the number of reference genes used, thus demonstrating the importance of selecting an appropriate number of reference genes in order to achieve an accurate and reliable normalization of gene expression during cold acclimation in E. globulus.

Chemical Composition and Wood Anatomy of Eucalyptus globulus Clones: Variations and Relationships with Pulpability and Handsheet Properties

Abstract Variation and relationships among dasometric parameters, chemical composition, wood anatomy, pulpability (efficacy of kraft delignification), and handsheet properties were studied in fourteen 7-year-old Eucalyptus globulus clones grown in a clonal trial in the Bio-Bio Region, Chile. Three ramets per clone were sampled and all properties were measured for each tree. Results showed variations among clones for parameters associated with wood anatomy, chemical composition, pulpability, and handsheet properties, which are the basis for the improvement of these traits through clonal selection. However, our findings showed that tree size can negatively affect other traits such as basic density and other related parameters as wood consumption, tear index, specific bulk, and Gurley air resistance. Wood density and chemical composition were the most important parameters correlated with pulpability and handsheet properties. Therefore, future efforts in tree improvement should be focused on wood properties such as density, lignin, holocellulose, and α -cellulose contents.

Transgenic trees and forestry biosafety

The benefits from the development of transgenic trees are expected from the improvement of traits as growth and form, wood quality, industrial processes, disease and insect resistance, herbicide tolerance, ecological restoration, rooting ability, etc. One of the first reported field trials with genetically modified forest trees was established in Belgium in 1988 and the characteristic evaluated was herbicide tolerance in poplars. Since then, there have been more than 200 reported trials, involving at least 15 forest species. The majority of the field trials have been carried out in the USA (64%). More than 50% of the field trials are done with Populus species and the main target traits are herbicide tolerance (31%), followed by marker genes (23%) and insect resistance (14%). Until today, there is only one report on commercial-scale production of transgenic forest trees which is Populus nigra with the Bt gene release in China in 2002 and established on commercial plantations in 2003. Operational application of GMOs in forestry depends on technical, economical, political and public aspects, but the development of adequate regulatory frameworks and public acceptance of transgenic trees will define the future of this technology in forestry.

Wood anatomy and biometric parameters variation of Eucalyptus globulus clones

Variation in wood anatomy and biometric variables was studied in 14 7-year-old Eucalyptus globulus clones grown in a clonal trial in Arauco, Bio-Bio Region, Chile. Biometric characteristics were measured for each tree and the anatomical variables were measured at three radial positions by image analysis on transverse microsections cut from radial increment cores sampled at breast height. Results showed, that among clones wide ranges of variation were found for vessel frequency, vessel area and vessel coverage. However, narrow ranges of variation were found for fiber wall thickness, fiber diameter and lumen diameter. From pith to bark, mean vessel area and vessel coverage increased gradually, whereas the vessel frequency decreased. The fiber wall thickness did not differ significantly from pith to bark. Mean fiber and lumen diameters showed the same radial pattern, increasing from pith to the midpoint of the radius to reach a plateau close to the bark. Furthermore, important commercial volume gains that can exceed 100% were found. Considering the commercial importance of tree volume, vessel and fiber anatomy in the pulping and papermaking process, it is suggested that the information given in this study may be used as an additional criteria for the selection of E. globulus.

An efficient and reproducible protocol for production of AFLP markers in tree genomes using fluorescent capillary detection

An optimized protocol for the development and discovery of polymorphic AFLP markers in tree species is described. The protocol was optimized for the production of fluorescently labeled PCR products and analysis using a capillary sequencer. This approach has been demonstrated to be efficient and reproducible for tree species with complex genomes. The most important modification was in the selective amplification step. Instead of using a traditional step down PCR, a fixed and higher annealing temperature was employed, improving the reproducibility and sensitivity of the protocol. The levels of polymorphisms detected with the optimized protocol on three woody species are in agreement with those previously reported in the literature for tree species.

Rescue of syringyl lignin and sinapate ester biosynthesis in Arabidopsis thaliana by a coniferaldehyde 5-hydroxylase from Eucalyptus globulus

Key messageThe gene coding for F5H fromEucalyptus globuluswas cloned and used to transform anf5h-mutant ofArabidopsis thaliana, which was complemented, thus verifying the identity of the cloned gene.AbstractConiferaldehyde 5-hydroxylase (F5H; EC 1.14.13) is a cytochrome P450-dependent monooxygenase that catalyzes the 5-hydroxylation step required for the production of syringyl units in lignin biosynthesis. The Eucalyptusglobulus enzyme was characterized in vitro, and results showed that the preferred substrates were coniferaldehyde and coniferyl alcohol. Complementation experiments demonstrated that both cDNA and genomic constructs derived from F5H from E. globulus under the control of the cinnamate 4-hydroxylase promoter from Arabidopsis thaliana, or a partial F5H promoter from E. globulus, can rescue the inability of the A. thalianafah1-2 mutant to accumulate sinapate esters and syringyl lignin. E. globulus is a species widely used to obtain products that require lignin removal, and the results suggest that EglF5H is a good candidate for engineering efforts aimed at increasing the lignin syringyl unit content, either for kraft pulping or biofuel production.

Transcript abundance of enzymes involved in lignin biosynthesis of Eucalyptus globulus genotypes with contrasting levels of pulp yield and wood density

One hundred genotypes of Eucalyptus globulus were ranked according to specific consumption of wood (cubic meters of wood needed to produce 1 ton of pulp). Ten of the most contrasting genotypes were separated in two groups of five clones each; group 1 (G1) with high wood density, high pulp yield, and low specific consumption, and group 2 (G2) with low density, low pulp yield, and high specific consumption. The contrasting genotypes also had significant differences in lignin content, percent syringyl unit composition, and frequency of β-O-4 linkages. Gene expression for phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate:CoA ligase (4CL) and ferulate 5-hydrolase (F5H) was analyzed in the contrasting genotypes. In both groups, transcript abundance for CAD, PAL, and 4CL were similar and only F5H presented significant differences between groups, with high values in the best ranked genotypes G1 in comparison to G2. Correlations between traits were estimated for lignin content vs. pulp yield (R2 = 0.97), pulp yield vs. syringyl units (R2 = 0.82), β-O-4 linkages vs. pulp yield (R2 = 0.84), and β-O-4 linkages vs. syringyl units (R2 = 0.97). Correlations between chemical composition and transcript abundance for F5H were calculated, finding correlation values with lignin content (R2 = 0.81), syringyl units (R2 = 0.83), and pulp yield (R2 = 0.81). The measurement of transcript abundance of F5H represents a potential genomic tool for tree improvement programs to select trees with high pulp yield.

SUPERVISED PATTERN RECOGNITION TECHNIQUES FOR CLASSIFICATION OF EUCALYPTUS SPECIES FROM LEAVES NIR SPECTRA

Three supervised pattern recognition methods (SPRM) were evaluated to discriminate between Eucalyptus globulus and Eucalyptus nitens species applying near infrared (NIR) spectroscopy on leaves. The methods used were k-nearest neighbor (KNN), soft modeling class analogy (SIMCA) and discriminant partial least squares (PLS-DA). First and second derivatives were used as transform techniques and mean-center (MC) and autoscaling (AS) as preprocessing techniques. The training set was constitued by 288 samples and 20 samples were used as validation set. A significant difference between the assayed methods was not observed, however best results for separation of classes and prediction rate were obtained when first derivative and MC were used for all the recognition pattern methods. Use of leaves and NIR spectroscopy avoids the destructive usual wood analysis in forest industries and facilities the fast classification of these species for forest applications.

Photoperiod, temperature and water deficit differentially regulate the expression of four dehydrin genes from Eucalyptus globulus

During the last few years, considerable effort has been directed toward understanding how Eucalyptus globulus responds and adapts to low temperature due to its low freezing resistance, especially through gene expression analysis. A recent study has confirmed that freezing stress induces the expression of three dehydrin (DHN) genes in E. globulus. This work reports the identification of a new YSK-type DHN gene in E. globulus (EuglDHN3) and examines the responses of this gene and the three previously reported (EuglDHN1, EuglDHN2 and EuglDHN10) under low temperature (LT), short photoperiod and water deficit (WD) in one sensitive and one freezing-resistant genotype of E.globulus, to assign them to a class: low temperature responsive, photoperiod responsive or water deficit responsive. Results indicated that two SKn-type DHNs (EuglDHN1 and EuglDHN2) were strongly induced by LT and WD, the KS-type DHN (EuglDHN10) was strongly induced by LT and the YnSKn-type by WD. The expression patterns under LT and WD were consistent with the regulatory elements identified in the promoter regions of the four DHN genes. The differential accumulation of EuglDHN1, EuglDHN2 and EuglDHN3 transcripts suggests a differential regulation in specific tissues under WD, and EuglDHN1 and EuglDHN10 under LT.

Transcriptome Profile in Response to Frost Tolerance in Eucalyptus globulus

The genetic improvement of trees for freezing tolerance is one of the most important goals to extend the plantations to colder areas. RNA-Seq technology has become a key tool in transcriptome studies. It can quantify overall expression levels for each gene simultaneously with high efficiency and speed through in silico gene expression, where differentially expressed genes can be identified by measuring the reads mapped for each transcript. In this study, the results of ESTs libraries from two Eucalyptus globulus genotypes showing contrasting differences in frost tolerance after cold acclimation using mRNA-Seq and in silico gene expression are discussed. A total of 14,265 non-redundant transcripts were predicted, where 163 corresponded to upregulated and 537 to downregulated genes. Pathway analyses of upregulated transcripts indicated that differences in frost tolerance might be regulated by the tree response to chemical and osmotic stimulus and organic substances, principally by overexpressing proteins that respond to hormone stress. These results suggest that genes coding for dehydrins, outer envelope, and voltage-dependent anion channel proteins are likely to participate in the regulation of the cold acclimation process and may have an important role in frost tolerance. The transcription factor analysis allowed identifying that those most differentially expressed in a resistant genotype were participating in the regulation of transcription, hormone regulation, photosynthesis, and response to stress. Additionally, the screening of polymorphic EST-SSR in silico and the validation of these markers in a reference population lead to identify a polymorphic EST-SSR with potential use for plant breeding and genotype discrimination.