Ilona Peszlen

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome–based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Hemlock woolly adelgid (Adelges tsugae) infestation affects water and carbon relations of eastern hemlock (Tsuga canadensis) and Carolina hemlock (Tsuga caroliniana)

Hemlock woolly adelgid (HWA) is an exotic insect pest causing severe decimation of native hemlock trees. Extensive research has been conducted on the ecological impacts of HWA, but the exact physiological mechanisms that cause mortality are not known. Water relations, anatomy and gas exchange measurements were assessed on healthy and infested eastern (Tsuga canadensis) and Carolina (Tsuga caroliniana) hemlock trees. These data were then used in a mechanistic model to test whether the physiological responses to HWA infestation were sufficiently significant to induce changes in whole-plant water use and carbon uptake. The results indicated coordinated responses of functional traits governing water relations in infested relative to healthy trees. In response to HWA, leaf water potential, carbon isotope ratios, plant hydraulic properties and stomatal conductance were affected, inducing a reduction in tree water use by > 40% and gross primary productivity by 25%. Anatomical changes also appeared, including the activation of traumatic cells. HWA infestation had a direct effect on plant water relations. Despite some leaf compensatory mechanisms, such as an increase in leaf hydraulic conductance and nitrogen content, tree water use and carbon assimilation were diminished significantly in infested trees, which could contribute to tree mortality.

Down-regulation of glycosyltransferase 8D genes in Populus trichocarpa caused reduced mechanical strength and xylan content in wood

Members of glycosyltransferase protein families GT8, GT43 and GT47 are implicated in the biosynthesis of xylan in the secondary cell walls of Arabidopsis. The Arabidopsis mutant irx8 has a 60% reduction in xylan. However, over-expression of an ortholog of Arabidopsis IRX8, poplar PoGT8D, in Arabidopsis irx8 mutant could not restore xylan synthesis. The functions of tree GT8D genes remain unclear. We identified two GT8 gene homologs, PtrGT8D1 and PtrGT8D2, in Populus trichocarpa. They are the only two GT8D members and are abundantly and specifically expressed in the differentiating xylem of P. trichocarpa. PtrGT8D1 transcript abundance was >7 times that of PtrGT8D2. To elucidate the genetic function of GT8D in P. trichocarpa, the expression of PtrGT8D1 and PtrGT8D2 was simultaneously knocked down through RNAi. Four transgenic lines had 85-94% reduction in transcripts of PtrGT8D1 and PtrGT8D2, resulting in 29-36% reduction in stem wood xylan content. Xylan reduction had essentially no effect on cellulose quantity but caused an 11-25% increase in lignin. These transgenics exhibit a brittle wood phenotype, accompanied by increased vessel diameter and thinner fiber cell walls in stem xylem. Stem modulus of elasticity and modulus of rupture were reduced by 17-29% and 16-23%, respectively, and were positively correlated with xylan content but negatively correlated with lignin quantity. These results suggest that PtrGT8Ds play key roles in xylan biosynthesis in wood. Xylan may be a more important factor than lignin affecting the stiffness and fracture strength of wood.

Dielectrophoresis of cellulose nanocrystals and alignment in ultrathin films by electric field-assisted shear assembly.

Ultrathin films of cellulose nanocrystals (CNCs) are obtained by using a convective assembly setup coupled with a low-strength external AC electric field. The orientation and degree of alignment of the rod-like nanoparticles are controlled by the applied field strength and frequency used during film formation. Calculated dipole moments and Clausius-Mossotti factors allowed the determination of the critical frequencies, the peak dielectrophoresis as well as the principal orientation of the CNCs in the ultrathin films. As a result of the combination of shear forces and low electric field highly ultrathin films with controlled, unprecedented CNC alignment are achieved.

Comparison of morphological and chemical properties between juvenile wood and compression wood of loblolly pine

Abstract In conifers, juvenile wood (JW) is always associated with compression wood (CW). Due to their similar properties, there is a common belief that JW is the same as CW. To resolve whether JW is identical to CW, 24 rooted cuttings of one loblolly pine clone were planted in growth chambers under normal, artificial bending, and windy environments. The results show that the morphology of JW is significantly different from CW. Furthermore, chemical analyses revealed that JW and CW are significantly different in chemical composition. Our results indicate that JW is different from CW, and the wood formed under a controlled windy environment is a mild type of compression wood.

Preliminary tests to evaluate the mechanical properties of young trees with small diameter

Abstract This paper describes methods developed to test the mechanical properties of small-diameter (3–10 mm) 1-year-old trees. Special test fixtures and procedures were designed to accommodate the small diameter and uneven shape of these young trees. The modulus of elasticity and strength in tension parallel to the grain, compression parallel to the grain, and bending were measured.

Effect of Lignin Genetic Modification on Wood Anatomy of Aspen Trees

The directed modification of specific traits of trees through genetic engineering provides opportunities for making significant genetic improvements to wood properties in matter of years instead of extended time frames required for traditional natural selection. An attractive target of forest- tree engineering is the modification of lignin content and lignin structure. While lower lignin content improves pulping efficiency, a decrease in lignin content could affect wood characteristics that are critical for solid wood use.After one year of growth in a greenhouse, a total of forty transgenic aspen (Populus tremuloides Michx.) with reduced lignin content and increased syringyl to guaiacyl ratio were harvested and diameter growth and cell morphology were investigated using quantitative wood anatomy and fiber quality analysis techniques. Comparing genetic groups to the wild-type as the control, similar radial growth and quantitative anatomical properties were observed for the genetic group with reduced lignin content. The genetic group with increased S/G ratio had lower diameter growth, lower vessel lumen diameter, but more numerous vessels. The combined effect of changes in lignin content and structure on radial growth and cell morphology seems to be more complex and gave inconsistent results.

Distribution of wood polymers within the cell wall of transgenic aspen imaged by Raman microscopy

Abstract Little is known about the effect of genetic modification on the chemical composition and structure of wood, which could have a significant effect on reactivity during chemical and enzymatic processing. In this study, information was collected by confocal Raman microscopy (CRM) on the spatial distribution of lignin and polysaccharides in the cell wall of young transgenic aspen with reduced lignin content, increased syringyl/guaiacyl (S/G) ratio, and simultaneously reduced lignin content and increased S/G ratio. CRM revealed that the lignin content of the cell wall and compound middle lamella was reduced by the genetic modification. A higher volume of water was also found in the cell wall of transgenic aspen compared with wild-type aspen, indicating an increase in the hydrophilicity of the cell wall.

Fungal biodegradation of genetically modified and lignin-altered quaking aspen (Populus tremuloides Michx.)

Abstract Better access to wood carbohydrates as a result of reduced, or altered, lignin is a goal of biopulping, as well as biofuel research. In the present article, woods from three transgenic trees and one wild-type quaking aspen (Populus tremuloides Michx.) were analyzed in terms of mass loss of cellulose and lignin after incubation with lignocellulolytic fungi. The transgenic trees had reduced lignin content through transfer of an antisense -4CL gene, elevated syringyl/guaiacyl (S/G) ratio through insertion of a sense CAld5H gene and low lignin content and elevated S/G ratio through simultaneous insertion of -4CL and CAld5H genes, respectively. The lignocellulolytic fungi employed were a lignin-selective white rot fungus Ceriporiopsis subvermispora, a simultaneous white rot fungus Trametes versicolor and a brown rot fungus Postia placenta. Reduced lignin degradation was observed in woods with increased S/G ratios indicating that this analytical feature influences decay resistance, regardless of the fungal decay mechanism.

Elastic Modulus Determination of Transgenic Aspen Using a Dynamic Mechanical Analyzer in Static Bending Mode

The applicability of a dynamic mechanical analyzer (DMA) in determining the modulus of elasticity (MOE) of 2.5-year-old transgenic aspen (Populus tremuloides Michx.) was investigated. Fifty sample ...