Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.     

Poplar genetic engineering: promoting desirable wood characteristics and pest resistance

Worldwide biomass demand for industrial applications, especially for production of biofuels, is increasing. Extended cultivation of fast growing trees such as poplars may contribute to satisfy the need for renewable resources. However, lignin, which constitutes about 20–30 % of woody biomass, renders poplar wood recalcitrant to saccharification. Genetic engineering of the enzymes of the lignification pathway has resulted in drastic decreases in lignin and greatly improved the carbohydrate yield for ethanol fermentation. While uncovering key enzymes for lignification facilitated rapid biotechnological progress, knowledge on field performance of low-lignin poplars is still lagging behind. The major biotic damage is caused by poplar rust fungi (Melampsora larici-populina), whose defense responses involve lignification and production of phenolic compounds. Therefore, manipulation of the phenylpropanoid pathway may be critical and should be tightly linked with new strategies for improved poplar rust tolerance. Emerging novel concepts for wood improvement are discussed.     

Lignification and structural biomass production in tobacco with suppressed caffeic/5-hydroxy ferulic acid-O-methyl transferase activity under ambient and elevated CO concentrations

To investigate the relationship between growth, biomass partitioning and lignification we used tobacco (Nicotiana tabacum) in which O-methyl transferase (OMT) activity, an enzyme involved in the pathway of sinapyl alcohol formation for lignin synthesis, was suppressed by antisense transformation. To modulate growth, controls and transformed tobacco plants were grown under ambient (approximately 380 p.p.m) or elevated CO(2) (700 p.p.m), respectively. Lignin concentrations and composition were determined with spectrophotometric methods (thioglycolate and acetyl bromide) and Fourier transform infrared (FTIR) spectroscopy, respectively. A comparison of the thioglycolate and acetylbromide method suggested that the thioglycolate method was sensitive to changes in the syringyl/guaiacyl (S/G)-ratio in lignin and therefore not suitable for quantification in tissues with altered lignin composition. Growth under elevated CO(2) increased leaf and stem biomass of both genotypes by 40 and 20%, respectively, compared with ambient CO(2) and had no effect on root biomass. OMT suppression did not affect lignin concentrations in the leaves but caused a shift in biomass partitioning from the structural to the non-structural fraction. Elevated CO(2) caused a shift towards production of structural compounds resulting in decreased foliar lignin concentrations and indicating that the lignin/structural mass ratio is flexible in leaves. By contrast, the lignin concentrations of stems were unaffected by elevated CO(2) or OMT suppression and increased about three-fold from the apex to the base. Antisense-OMT plants produced more stem biomass than controls but showed no changes of the relative partitioning of biomass to the different pools. This indicates that the metabolic control of carbon fluxes to the production of structural versus non-structural fractions is tighter in stems than in leaves. FTIR spectroscopy indicated a relative increase in guaiacyl- as compared with syringyl-units in antisense-OMT tobacco, which was more pronounced under elevated as compared with ambient CO(2). Since there was no evidence for decreased lignin concentrations in the antisense-OMT plants but increased biomass formation we suggest that less methylated lignins are 'cheaper' in biosynthetic carbon and energy demand and, thus, may enable plants to allocate increased resources to growth.     

Condensed lignins are synthesized in poplar leaves exposed to ozone

Poplar (Populus tremula x alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L(-1)). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L(-1)). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.     

Growth parameters and resistance against Drechslera teres of spring barley (Hordeum vulgare L. cv. Scarlett) grown at elevated ozone and carbon dioxide concentrations

Spring barley ( Hordeum vulgare L. cv. Scarlett) was grown at two CO2 levels (400 vs. 700 ppm) combined with two ozone regimes (ambient vs. double ambient) in climate chambers for four weeks, beginning at seedling emergence. Elevated CO2 concentration significantly increased aboveground biomass, root biomass, and tiller number, whereas double ambient ozone significantly decreased these parameters. These ozone-induced reductions in growth parameters were strongly overridden by 700 ppm CO2. The elevated CO2 level increased C : N ratio of the leaf tissue and leaf starch content but decreased leaf protein levels. Exposure to double ambient ozone did not affect protein content and C : N ratio but dramatically increased leaf starch levels at 700 ppm CO2. Resistance against Drechslera teres (Sacc.) Shoemaker was increased in leaves grown at double ambient ozone but was less obvious at 700 ppm than at 400 ppm CO2. Constitutive activities of beta-1,3-glucanase and chitinase were significantly higher in leaves grown at double ambient ozone compared to ambient ozone levels. The sum of methanol-soluble and alkali-released cell wall-bound aromatic metabolites (i.e., C-glycosylflavones and several structurally unidentified metabolites) and lignin contents did not show any treatment-dependent differences.     

Wood Chemical Composition in Species of Cactaceae: The Relationship between Lignification and Stem Morphology

In Cactaceae, wood anatomy is related to stem morphology in terms of the conferred support. In species of cacti with dimorphic wood, a unique process occurs in which the cambium stops producing wide-band tracheids (WBTs) and produces fibers; this is associated with the aging of individuals and increases in size. Stem support and lignification have only been studied in fibrous tree-like species, and studies in species with WBTs or dimorphic wood are lacking. In this study, we approach this process with a chemical focus, emphasizing the role of wood lignification. We hypothesized that the degree of wood lignification in Cactaceae increases with height of the species and that its chemical composition varies with wood anatomy. To test this, we studied the chemical composition (cellulose, hemicellulose, and lignin content) in 13 species (2 WBTs wood, 3 dimorphic, and 8 fibrous) with contrasting growth forms. We also analyzed lignification in dimorphic and fibrous species to determine the chemical features of WBTs and fibers and their relationship with stem support. The lignin contents were characterized by Fourier transform infrared spectroscopy and high performance liquid chromatography. We found that 11 species have a higher percentage (>35%) of lignin in their wood than other angiosperms or gymnosperms. The lignin chemical composition in fibrous species is similar to that of other dicots, but it is markedly heterogeneous in non-fibrous species where WBTs are abundant. The lignification in WBTs is associated with the resistance to high water pressure within cells rather than the contribution to mechanical support. Dimorphic wood species are usually richer in syringyl lignin, and tree-like species with lignified rays have more guaiacyl lignin. The results suggest that wood anatomy and lignin distribution play an important role in the chemical composition of wood, and further research is needed at the cellular level.     

Ectomycorrhizal Colonization and Diversity in Relation to Tree Biomass and Nutrition in a Plantation of Transgenic Poplars with Modified Lignin Biosynthesis

Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana. The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R² = 97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations.     

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as ‘good neighbors’ in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of ‘good neighbors’ in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.     

Genetic Modification of Short Rotation Popular Wood: Properties for Ethanol Fuel and Fiber Productions

Opportunities for matching wood chemical and physical properties to manufacturing and product requirements via genetic modification have long been recognized. Exploitation is now feasible due to advances in trait measurement, breeding, genetic mapping and marker, and genetic transformation technologies. With respect to classic selection and breeding of short-rotation poplars, genetic parameters are favorable for decreasing lignin content and increasing specific gravity, but less so for increasing cellulose content. Knowledge of functional genomics is expanding, as is that needed for eventual application of marker-aided breeding, trait dissection, candidate gene identification, and gene isolation. Research on gene transfer has yielded transgenic poplars with decreased lignin and increased cellulose contents, but otherwise normal growth and development. Until effective marker-aided breeding technologies become available, the most promising approach for enhancing ethanol fuel and fiber production and processing efficiencies centers on selecting and breeding poplars for high wood substance yields and genetically transforming them for decreased lignin and increased cellulose contents.     

Lignification and lignin topochemistry - an ultrastructural view.

This review discuses the ultrastructural aspects of cell wall lignification and lignin topochemistry. Lignification results from the enzyme mediated polymerization of monolignols initiated by unknown factors (initiation sites) located at the corners of cells and in the middle lamella. Lignification results in the filling of pores within the carbohydrate matrix following a sequence from the outer regions of the wall towards the lumen. The amount and chemical characteristics of lignin vary across the cell wall, with the presence of reaction wood, and among cell types.     

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.     

Growth and Wood/Bark Properties of Abies faxoniana Seedlings as Affected by Elevated CO2

Growth and wood and bark properties of Abies faxoniana seedlings after one year's exposure to elevated CO2 concentration (ambient 350 (=1= 25) μmol/mol) under two planting densities (28 or 84 plants/mz) were investigated in closed-top chambers. Tree height, stem diameter and cross-sectional area, and total biomass were enhanced under elevated CO2 concentration, and reduced under high planting density. Most traits of stem bark were improved under elevated CO2 concentration and reduced under high planting density. Stem wood production was significantly increased in volume under elevated CO2 concentration under both densities, and the stem wood density decreased under elevated CO2 concentration and increased under high planting density. These results suggest that the response of stem wood and bark to elevated CO2 concentration is density dependent. This may be of great importance in a future CO2 enriched world in natural forests where plant density varies considerably. The results also show that the bark/wood ratio in diameter, stem cross-sectional area and dry weight are not proportionally affected by elevated CO2 concentration under the two contrasting planting densities. This indicates that the response magnitude of stem bark and stem wood to elevated CO2 concentration are different but their response directions are the same.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.