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Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

Despite the ecological and industrial importance of biomass accumulation in wood, the control of carbon (C) allocation to this tissue and to other tree tissues remain poorly understood. We studied sucrose synthase (SUS) to clarify its role in biomass formation and C metabolism at the whole tree level in hybrid aspen (Populus tremula × tremuloides). To this end, we analysed source leaves, phloem, developing wood, and roots of SUSRNAi trees using a combination of metabolite profiling, 13CO2 pulse labelling experiments, and long-term field experiments. The glasshouse grown SUSRNAi trees exhibited a mild stem phenotype together with a reduction in wood total C. The 13CO2 pulse labelling experiments showed an alteration in the C flow in all the analysed tissues, indicating that SUS affects C metabolism at the whole tree level. This was confirmed when the SUSRNAi trees were grown in the field over a 5-yr period; their stem height, diameter and biomass were substantially reduced. These results establish that SUS influences C allocation to developing wood, and that it affects C metabolism at the whole tree level. Fil: Dominguez, Pia Guadalupe. Swedish University of Agricultural Sciences; Suecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina Fil: Donev, Evgeniy. Swedish University of Agricultural Sciences; Suecia Fil: Derba Maceluch, Marta. Swedish University of Agricultural Sciences; Suecia Fil: Bünder, Anne. Swedish University of Agricultural Sciences; Suecia Fil: Hedenström, Mattias. Universidad de Umea; Suecia Fil: Tomásková, Ivana. Czech University Of Life Sciences; República Checa Fil: Mellerowicz, Ewa J.. Swedish University of Agricultural Sciences; Suecia Fil: Niittylä, Totte. Swedish University of Agricultural Sciences; Suecia

Using microbial enzymes in transgenesis is a powerful means to introduce new functionalities in plants. Glucuronoyl esterase (GCE) is a microbial enzyme hydrolyzing the ester bond between lignin and 4-O-methyl-α-D-glucuronic acid present as a side chain of glucuronoxylan. This bond mediates lignin-carbohydrate complex (LCC) formation, considered as crucial factor of lignocellulose recalcitrance to saccharification. Previous studies showed that hybrid aspen (Populus tremula L. x tremuloides Michx.) constitutively expressing Phanerochaete carnosa Burt GCE (PcGCE) had better efficiency of cellulose-to-glucose conversion but were stunned and had lower cellulose content indicating that more studies are needed to design strategy for deployment of this enzyme in planta. Here we report that the transgenic plants exhibit premature leaf senescence, increased accumulation of calcium oxalate crystals, tyloses and necrotic lesions and have strongly activated immune defense reactions as revealed by their altered profiles of transcriptomes, metabolomes and hormones in the leaves. To elucidate if these effects are triggered by damage-associated molecular patterns (DAMPs) or by PcGCE protein perceived as a pathogen-associated molecular pattern (PAMP), we ectopically expressed in aspen an enzymatically inactive PcGCES217A. The mutated PcGCE induced similar growth retardation, leaf necrosis and premature senescence as the active one, providing evidence that PcGCE protein is recognized as PAMP. Transcriptomics analysis of young expanding leaves of 35S:PcGCE plants identified several candidates for receptors of PcGCE, which were not expressed in developing wood tissues. Grafting experiments showed that PcGCE transcripts are not cell-to-cell mobile and that PcGCE expressing leaves augment systemic responses. In agreement, expressing PcGCE in developing wood by using the wood-specific promoter (WP), avoided all off-target effects. Moreover, WP:PcGCE lines grew better than control plants providing evidence that this strategy can be used in transgenic crops dedicated for biorefinery.

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.

Wood is an important source of biomass for materials and chemicals, and a target for genetic engineering of its properties for different applications or for research. Wood properties can be altered by using different enzymes acting on cell wall polymers postsynthetically in cell walls. This approach allows for a precise polymer structure modification thanks to the specificity of enzymes used. Such enzymes can originate from all kinds of organisms, or even be modified in a desired way for novel attributes. Here we present a general strategy for expressing a microbial enzyme in aspen and targeting it to cell wall, using an example of fungal glucuronoyl esterase. We describe methods of vector cloning, plant transformation, transgenic line selection and multiplication, testing for the presence of enzymatic activity in different cell compartments, and finally the method of plant transferring from sterile culture to the greenhouse conditions.

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.

Wood is an important source of biomass for materials and chemicals, and a target for genetic engineering of its properties for different applications or for research. Wood properties can be altered by using different enzymes acting on cell wall polymers postsynthetically in cell walls. This approach allows for a precise polymer structure modification thanks to the specificity of enzymes used. Such enzymes can originate from all kinds of organisms, or even be modified in a desired way for novel attributes. Here we present a general strategy for expressing a microbial enzyme in aspen and targeting it to cell wall, using an example of fungal glucuronoyl esterase. We describe methods of vector cloning, plant transformation, transgenic line selection and multiplication, testing for the presence of enzymatic activity in different cell compartments, and finally the method of plant transferring from sterile culture to the greenhouse conditions.

In dicotyledons, xyloglucan is the major hemicellulose of primary walls affecting the load-bearing framework with participation of XTH enzymes. We used loss- and gain-of function approaches to study functions of abundant cambial region expressedXTH4andXTH9in secondary growth. In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. In addition, they stimulated secondary wall thickening, but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition, indicating overall increase in secondary versus primary walls in the mutants, indicative of higher xylem production compared to wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers was increased inxth9and reduced inxth4, whereas the double mutantxth4x9displayed intermediate number of layers. These changes correlated with certain Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers where neitherXTH4norXTH9were expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and in cell wall integrity sensing, includingTHE1andWAK2, were highly induced in the mutants, indicating that deficiency inXTH4andXTH9triggers cell wall integrity signaling, which, we propose, stimulates the xylem cell production and modulates secondary wall thickening. Prominent effects ofXTH4andXTH9on secondary xylem support the hypothesis that altered xyloglucan can affect wood properties both directly andviacell wall integrity sensing.SIGNIFICANCE STATEMENTXyloglucan is a ubiquitous component of primary cell walls in all land plants but has not been so far reported in secondary walls. It is metabolizedin muroby cell wall-residing enzymes - xyloglucan endotransglycosylases/hydrolases (XTHs), which are reportedly abundant in vascular tissues, but their role in these tissues is unclear. Here we report that two vascular expressed enzymes in Arabidopsis, XTH4 and XTH9 contribute to the secondary xylem cell radial expansion and intrusive elongation in secondary vascular tissues.Unexpectedly, deficiency in their activities highly affect chemistry and ultrastructure of secondary cell walls by non-cell autonomous mechanisms, including transcriptional induction of secondary wall-related biosynthetic genes and cell wall integrity sensors. These results link xyloglucan metabolism with cell wall integrity pathways, shedding new light on previous reports about prominent effects of xyloglucan metabolism on secondary walls.One sentence summaryXTH4 and XTH9 positively regulate xylem cell expansion and fiber intrusive tip growth, and their deficiency alters secondary wall formation via cell wall integrity sensing mechanisms.

Glucuronic acids in Arabidopsis thaliana xylans exist in 4-O-methylated (MeGlcA) and non-methylated (GlcA) forms at a ratio of about 3:2. The matrix-assisted laser desorption/ionization mass spectrometry analysis of the endoxylanase liberated acidic oligosaccharides from the Arabidopsis inflorescence stem showed that two peaks with GlcA (GlcA-Xyl4Ac1 and GlcA-Xyl5Ac2) had abnormally high intensities, as well as different tandem mass spectra, than their 4-O-methylated counterparts. These peaks were interestingly enriched in the xylan biosynthesis mutant irx7 and irx9-1. Multi-stages fragmentation analysis using negative ion electrospray-ion trap mass spectrometry indicated that this GlcA was further carrying a pentose residue in the glucuronoxylan-derived oligosaccharide from irx9-1. The structure was also identified in Arabidopsis wild type. The results prove evidence of a new pentose substitution on the GlcA residue of Arabidopsis GX, which is likely present in the primary walls.

Xylan is one of the main compounds determining wood properties in hardwood species. The xylan backbone is thought to be synthesized by a synthase complex comprising two members of the GT43 family. We downregulated all GT43 genes in hybrid aspen (Populus tremula × tremuloides) to understand their involvement in xylan biosynthesis. All three clades of the GT43 family were targeted for downregulation using RNA interference individually or in different combinations, either constitutively or specifically in developing wood. Simultaneous downregulation in developing wood of the B (IRX9) and C (IRX14) clades resulted in reduced xylan Xyl content relative to reducing end sequence, supporting their role in xylan backbone biosynthesis. This was accompanied by a higher lignocellulose saccharification efficiency. Unexpectedly, GT43 suppression in developing wood led to an overall growth stimulation, xylem cell wall thinning and a shift in cellulose orientation. Transcriptome profiling of these transgenic lines indicated that cell cycling was stimulated and secondary wall biosynthesis was repressed. We suggest that the reduced xylan elongation is sensed by the cell wall integrity surveying mechanism in developing wood. Our results show that wood-specific suppression of xylan-biosynthetic GT43 genes activates signaling responses, leading to increased growth and improved lignocellulose saccharification.

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

Reactive oxygen species (ROS) production and enzymatic antioxidative system [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), alternative oxidase (AOX)] in nonphotosynthesizing pea plant cells were investigated. From the roots of pea plants cultivated hydroponically on a Hoagland medium with the addition of 0.1 and 0.5 mM of Pb(NO3)2, the three following fractions were isolated by means of a Percoll gradient: cytosol, peroxisomal, and mitochondrial. Lead stress caused H2O2 production in these organelles. The mitochondria from pea cell roots were the main site of H2O2 production. Intensive stress caused by 0.5 mM of Pb(NO3)2 brought about a decrease of H2O2 concentration in mitochondria and peroxisomes after 3 days of the exposition, which was due to an increase of CAT activity. The isoenzymatic profile of antioxidative enzymes indicates mitochondrial and peroxisomal localization of MnSOD and cytoplasmic localization of CuSOD. APOX activity was estimated for all three fractions: cytosol, mitochondria, and peroxisomes. Simultaneously, we observed an increased expression of AOX genes on the basis of the amount of mRNA transcript and confirmed it immunologically on the level of synthesized AOX protein (36 kDa). This has been the first evidence of AOX genes expression of which is induced by the treatment of plants with lead ions and it increases along with the concentration of metal.

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9-L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.

Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula × tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.

In order to function in changing environmental conditions, all living organisms need to be equipped with two sets of seemingly contradictory mechanisms; these enable them to (1) integrate themselves in separation from the environment, and (2) sense and communicate with their immediate surrounding. During the course of evolution, several factors—both physical and chemical—have emerged as organismal integrators. Among these, gravity provides a major directional stimulus, while chemical compounds are usually used as internal integratory molecules (Bhalerao and Bennett 2003).

Cell walls are dynamic entities providing the link between cellular and organismal features of the plant. Being external to protoplasts, they provide not only the transportation pathways for signaling molecules, but also constitute a rich source of such molecules. Moreover, their spatial placement also makes them a specific “zone of the first contact” with the surrounding environment. Here, we provide a brief overview of the functions the walls play in plant signaling. We describe the physicochemical properties of the walls and discuss their impact on the type of signaling molecules transported via the apoplast, and the types of extracellular domains of receptor molecules. The role of the walls in the formation and maintenance of the structural and functional continuum between cell walls, the plasma membrane, and the cytoskeleton is then considered, especially with respect to mechanosensing, transduction of mechanical signals, and monitoring of cell wall integrity. Finally, a range of signaling molecules and their activities are reviewed.