Transgenic Poplars Research Articles

Characterization, expression, and functional analysis of the pathogenesis-related gene PtDIR11 in transgenic poplar

Lignins and lignans are important for plant resistance to pathogens. Dirigent (DIR) proteins control the regio- and stereo-selectivity of coniferyl alcohol in lignan and lignin biosynthesis. DIR genes have been implicated in defense-related responses in several plant species, but their role in poplar immunity is unclear. We cloned PtDIR11 from Populus trichocarpa; we found that overexpression of PtDIR11 in poplar improved the lignan biosynthesis and enhanced the resistance of poplar to Septotis populiperda. PtDIR11 has a typical DIR domain; it belongs to the DIR-b/d family and is expressed in the cell membrane. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showed that PtDIR11 expression was highest in stems, followed by leaves and roots. Furthermore, PtDIR11 expression was induced by S. populiperda, salicylic acid (SA), jasmonate (JA), and ethylene (ET) stresses. The recombinant PtDIR11 protein inhibited the growth of S. populiperda in vitro. Overexpressing (OE) PtDIR11 in “Nanlin 895” poplar enhanced growth. The OE lines exhibited minimal changes in lignin content, but their total lignan and flavonoid contents were significantly greater than in the wild-type (WT) lines. Overexpression of PtDIR11 affected multiple biological pathways of poplar, such as phenylpropanoid biosynthesis. The methanol extracts of OE-PtDIR11 lines showed greater anti-S. populiperda activity than did lignin extracts from the WT lines. Furthermore, OE-PtDIR11 lines upregulated genes that were related to phenylpropanoid biosynthesis and genes associated with the JA and ET signal transduction pathways; it downregulated genes that were related to SA signal transduction compared with the WT line under S. populiperda stress. Therefore, the OE transgenic plants analysis revealed that PtDIR11 is a good candidate gene for breeding of disease resistant poplar.

Xylem-specific Overexpression of the GIBBERELLIN ACID 20 OXIDASE Gene (GA20-OXIDASE) from Pine in Hybrid Poplar (Populus tremula L. × P. alba L.) Revealed Reliable Increase in Growth and Biomass Production Just in a Single-copy-line

Overexpression of the GA20-OXIDASE gene under control of the constitutive cauliflower mosaic virus 35S promoter in poplar leads to increased shoot growth and biomass production, however, the trees suffer from unstable shoots and poor root growth. Transgenic hybrid poplar (Populus tremula L. × P. alba L.) plants overexpressing the GA20-OXIDASE gene from pine under control of a poplar-xylem-specific DX15-promoter also revealed a superior effect on growth and biomass production but without changing the overall phenotype. We tested seven DX15::GA20-OXIDASE-transgenic lines for growth and biomass production in the glasshouse in 2017, and repeated the experiment in 2018 with the “best-four” lines. Plants from one DX15::GA20-OXIDASE-transgenic line, N457‑4, turned out to be the tallest, with highest biomass, in both years under investigation. In contrast to the other lines tested in 2017 and 2018 carrying two or more copies of the transgene, N457‑4 carries only one copy. We suggest that transcriptional or post-transcriptional transgene silencing in the two- or more copies carrying lines might be responsible for lower GA20-OXIDASE transgene expression and that the single-copy-line N457‑4 has stable GA20-OXIDASE-gene expression.

PeMPK7 is induced in an ROS-dependent manner and confers poplar para-hydroxybenzoic acid stress resistance through the removal of ROS

The continuous cropping of poplar plantations will accumulate allelochemicals such as para-hydroxybenzoic acid (pHBA) in the soil, which restricts the growth and biomass of poplar. Mitogen-activated protein kinase (MAPK) cascades are widely involved in environmental stress responses in plants. However, little is known about the role of MAPK cascade genes in the response to pHBA stress until now. Here, a novel group C MAPK gene, PeMPK7, from Populus × euramericana was characterized. The PeMPK7 transcript was induced by exogenous pHBA and H2O2, and its induced transcription by pHBA was blocked by the reactive oxygen species (ROS) manipulators. Overexpression of PeMPK7 in poplar alleviated the phenotypic injury induced by pHBA stress, reduced H2O2 accumulation and alleviated the oxidative injury in leaf cells. The expression of the encoding genes and enzyme activity of the antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) were upregulated in transgenic poplars under pHBA conditions, which will help to remove excessive ROS. In addition, the enhanced soluble sugar and free proline in transgenic poplar seedlings may contribute to maintaining osmotic homeostasis under pHBA stress. Furthermore, the interaction between PeMPKK3 and PeMPK7 was confirmed. A schematic model was proposed to show the regulatory mechanism of PeMPK7 in response to pHBA stress.

Homologous Drought-Induced 19 Proteins, PtDi19-2 and PtDi19-7, Enhance Drought Tolerance in Transgenic Plants.

Drought-induced 19 (Di19) proteins play important roles in abiotic stress responses. Thus far, there are no reports about Di19 family in woody plants. Here, eight Di19 genes were identified in poplar. We analyzed phylogenetic tree, conserved protein domain, and gene structure of Di19 gene members in seven species. The results showed the Di19 gene family was very conservative in both dicotyledonous and monocotyledonous forms. On the basis of transcriptome data, the expression patterns of Di19s in poplar under abiotic stress and ABA treatment were further studied. Subsequently, homologous genes PtDi19-2 and PtDi19-7 with strong response to drought stress were identified. PtDi19-2 functions as a nuclear transcriptional activator with a transactivation domain at the C-terminus. PtDi19-7 is a nuclear and membrane localization protein. Additionally, PtDi19-2 and PtDi19-7 were able to interact with each other in yeast two-hybrid system. Overexpression of PtDi19-2 and PtDi19-7 in Arabidopsis was found. Phenotype identification and physiological parameter analysis showed that transgenic Arabidopsis increased ABA sensitivity and drought tolerance. PtDi19-7 was overexpressed in hybrid poplar 84K (Populus alba × Populus glandulosa). Under drought treatment, the phenotype and physiological parameters of transgenic poplar were consistent with those of transgenic Arabidopsis. In addition, exogenous ABA treatment induced lateral bud dormancy of transgenic poplar and stomatal closure of transgenic Arabidopsis. The expression of ABA/drought-related marker genes was upregulated under drought treatment. These results indicated that PtDi19-2 and PtDi19-7 might play a similar role in improving the drought tolerance of transgenic plants through ABA-dependent signaling pathways.

Plant biomass fermentation by the extreme thermophile Caldicellulosiruptor bescii for co-production of green hydrogen and acetone: Technoeconomic analysis

A variety of chemical and biological processes have been proposed for conversion of sustainable low-cost feedstocks into industrial products. Here, a biorefinery concept is formulated, modeled, and analyzed in which a naturally (hemi)cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii, is metabolically engineered to convert the carbohydrate content of lignocellulosic biomasses (i.e., soybean hulls, transgenic poplar) into green hydrogen and acetone. Experimental validation of C. bescii fermentative performance demonstrated 82% carbohydrate solubilization of soybean hulls and 55% for transgenic poplar. A detailed technical design, including equipment specifications, provides the basis for an economic analysis that establishes metabolic engineering targets. This robust industrial process leveraging metabolically engineered C. bescii yields 206 kg acetone and 25 kg H2 per metric ton of soybean hull, or 174 kg acetone and 21 kg H2 per metric ton transgenic poplar. Beyond this specific case, the model demonstrates industrial feasibility and economic advantages of thermophilic fermentation.

Chemical and Structural Responses to Downregulated p-Hydroxycinnamoyl-Coenzyme A: Quinate/Shikimate p-Hydroxycinnamoyltransferase in Poplar Cell Walls.

Unraveling the impact of lignin reduction on cell wall construction of poplar stems is important for accurate understanding the regulatory role of biosynthetic genes. However, few cell-level studies have been conducted on the changes in lignin, other important cell wall composition, and the structural properties of transgenic poplar stems at different developmental stages. In this work, the content and microdistributions of cell wall composition as well as the morphological characteristics of cells were studied for p-hydroxycinnamoyl-coenzyme A:quinate/shikimate p-hydroxycinnamoyltransferase (HCT) downregulated transgenic poplar 84K (Populus alba × P. glandulosa cl. ‘84k’) at different developmental stages. Results show that the lignin contents of the upper, middle, and basal parts of HCT transgenic poplar stems were significantly decreased by 10.84, 7.40, and 7.75%, respectively; and the cellulose contents increased by 8.20, 6.45, and 3.31%, respectively, compared with the control group. The cellulose/lignin ratio of HCT transgenic poplars was therefore increased, especially in the upper sections, where it was 23.2% higher. Raman results indicate the appearance of p-hydroxyphenyl units (H) and a decrease in the ratio of syringyl/guaiacyl (S/G) lignin monomers in fiber cell walls of HCT transgenic poplars. In addition, microstructure observations revealed that the fiber and vessel cells of the HCT transgenic poplars exhibited thin cell walls and large lumen diameters. Compared with the control group, the cell wall thickness of fiber and vessel cells decreased by 6.50 and 10.93% on average, respectively. There was a 13.6% decrease in the average ratio of the cell wall thickness to the lumen diameter and an increase in fiber length and width of 5.60 and 6.11%, respectively. In addition, downregulation of HCT did not change the orientation of cellulosic microfibrils, but it led to an 11.1% increase of the cellulose crystallinity in cell walls compared to the control poplars. The information obtained herein could lead to a better understanding of the effects of genetic modifications on wood cell walls.

Application of polygene polymerization for insect-resistant poplar breeding

A major goal of poplar breeding is to obtain new insect-resistant poplar varieties through genetic engineering. However, engineering poplars with only a single insect-resistant gene has limitations, such as narrow insecticidal bands and the rapid development of insect tolerance to the gene. To expand insecticidal bands, delay the development of insect tolerance, and improve the insect resistance of transgenic poplars, polygene polymerization is increasingly being applied to cultivate insect-resistant poplars. In the present study, we discuss polygene polymerization, applications of the polygene combination strategy, the current state of research on transgenic poplar with multiple insect-resistant genes, including existing problems, and future research directions. We provide reference data to aid the cultivation and utilization of new polygenic insect-resistant poplar varieties.

PtrHAT22, as a higher hierarchy regulator, coordinately regulates secondary cell wall component biosynthesis in Populus trichocarpa

Homeodomain-leucine zipper (HD-Zip) II transcription factors (TFs) have been reported to play vital roles in diverse biological processes of plants. However, it remains unclear whether HD-Zip II TFs regulate secondary cell wall (SCW) in woody plants. In this study, we performed the functional characterization of a Populus trichocarpa HD-Zip II TF, PtrHAT22, which encodes a nuclear localized transcription repressor predominantly expressing in secondary developing tissues. Overexpression of PtrHAT22 showed arrested growths, including reduced heights and diameters above the ground, small leaves, and decreased biomass. Meanwhile, the contents of lignin, cellulose, and thickness of SCW significantly decreased, whilst the content of hemicellulose obviously increased in PtrHAT22 transgenic poplar. The expressions of some wood-associated TFs and structural genes significantly changed accordingly with the alternations of SCW characteristics in PtrHAT22 transgenic poplar. Furthermore, PtrHAT22 directly repressed the promoter activities of PtrMYB20, PtrMYB28, and PtrCOMT2, and bind two cis-acting elements that were specifically enriched in their promoter regions. Taken together, our results suggested that PtrHAT22, as a higher hierarchy TF like PtrWNDs, exerted coordination regulation of poplar SCW component biosynthesis through directly and indirectly regulating structural genes and different hierarchy TFs of SCW formation network.

Condensed tannins as antioxidants that protect poplar against oxidative stress from drought and UV-B.

Condensed tannins (CTs, proanthocyanidins) are widespread polymeric flavan-3-ols known for their ability to bind proteins. In poplar (Populus spp.), leaf condensed tannins are induced by both biotic and abiotic stresses, suggesting diverse biological functions. Here we demonstrate the ability of CTs to function as physiological antioxidants, preventing oxidative and cellular damage in response to drought and UV-B irradiation. Chlorophyll fluorescence was used to monitor photosystem II performance, and both hydrogen peroxide and malondialdehyde content was assayed as a measure of oxidative damage. Transgenic MYB-overexpressing poplar (Populus tremula × P.tremuloides) with high CT content showed reduced photosystem damage and lower hydrogen peroxide and malondialdehyde content after drought and UV-B stress. This antioxidant effect of CT was observed using two different poplar MYB CT regulators, in multiple independent lines and different genetic backgrounds. Additionally, low-CT MYB134-RNAi transgenic poplars showed enhanced susceptibility to drought-induced oxidative stress. UV-B radiation had different impacts than drought on chlorophyll fluorescence, but all high-CT poplar lines displayed reduced sensitivity to both stresses. Our data indicate that CTs are significant defences against oxidative stress. The broad distribution of CTs in forest systems that are exposed to diverse abiotic stresses suggests that these compounds have wider functional roles than previously realized.

Dynamic monitoring of the impact of insect-resistant transgenic poplar field stands on arthropod communities

The safety of genetically modified forest trees is an important ecological issue worldwide, and concerns about genetic modification are one factor hindering in-depth research and large-scale planting of genetically modified trees. This study evaluated the impact of transgenic poplars on the ecosystem by analyzing differences in arthropod communities, target arthropods, and nontarget arthropods in transgenic (BtCryIAc) and non-transgenic experimental forests of Populus × euramericana cv. “74/76” (hereafter, tPE and ntPE, respectively). Five years of dynamic monitoring showed that the tPE had stable and lasting insect resistance to Lepidoptera, especially Arctiidae. However, the insertion of exogenous genes also had a negative impact on certain tPE line. The principle response curves (PRC) indicated that the genetic modification contributed much less to the community difference than the year factor (7% vs. 60%, respectively), and nontarget arthropods may be more sensitive to interannual variations. Nontarget arthropods that respond sharply to the PRC curve include Aphididae and Tenthredinidae (species weight > 0.5), but this difference appears only in certain years or lines. Our research shows that the tPE had no obvious adverse effects on nontarget arthropods. In areas with severe lepidopteran pests, the tPE could help to increase the diversity and stability of arthropod communities, while providing greater ecological and economic benefits than non-transgenic poplars.

Effects of P-Coumarate 3-Hydroxylase Downregulation on the Compositional and Structural Characteristics of Lignin and Hemicelluloses in Poplar Wood (Populus alba × Populus glandulosa).

Elucidating the chemical and structural characteristics of hemicelluloses and lignin in the p-coumarate 3-hydroxylase (C3H) down-regulated poplar wood will be beneficial to the upstream gene validation and downstream biomass conversion of this kind of transgenic poplar. Herein, the representative hemicelluloses and lignin with unaltered structures were prepared from control (CK) and C3H down-regulated 84K poplars. Modern analytical techniques, such as 13C NMR, 2D-HSQC NMR, and gel chromatography (GPC), were performed to better delineate the structural changes of hemicelluloses and lignin caused by transgenesis. Results showed that both the hemicelluloses (H-CK and H-C3H) extracted from control and C3H down-regulated poplar wood have a chain backbone of (1→4)-β-D-Xylan with 4-O-Me-α-D-GlcpA as side chain, and the branch degree of the H-C3H is higher than that of H-CK. With regarding to the lignin macromolecules, NMR results demonstrated that the syringyl/guaiacyl (S/G) ratio and dominant substructure β-O-4 linkages in C3H down-regulated poplar were lower than those of control poplar wood. By contrast, native lignin from C3H down-regulated poplar wood exhibited higher contents of p-hydroxybenzoate (PB) and p-hydroxyphenyl (H) units. In short, C3H down-regulation resulted in the chemical and structural changes of the hemicelluloses and lignin in these poplar wood. The identified structures will facilitate the downstream utilization and applications of lignocellulosic materials in the biorefinery strategy. Furthermore, this study could provide some illuminating results for genetic breeding on the improvement of wood properties and efficient utilization of poplar wood.

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars.

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

OsCYP714D1 improves plant growth and salt tolerance through regulating gibberellin and ion homeostasis in transgenic poplar

Cytochrome P450 monooxygenases (CYP450s) play crucial roles in the regulation of plant growth and response to abiotic stress. However, their functions in woody trees are still largely unknown. Previously, we reported that expression of the rice cytochrome P450 monooxygenase gene OsCYP714D1 increased gibberellic acid (GA) accumulation and shoot growth in transgenic poplar. In this work, we demonstrate that expression of OsCYP714D1 improved the salt tolerance of transgenic poplar plants. Compared to wild type, plant height and K+ content were significantly higher, whereas plant growth inhibition and Na+ content were significantly lower, in transgenic plants grown under high salt stress condition. Transcriptomic analyses revealed that OsCYP714D1 expression up-regulated the expressions of GA biosynthesis, signaling and stress responsive genes in transgenic plants under both normal and high salt stress conditions. Further gene ontology (GO) analyses indicated that genes involved in plant hormone and ion metabolic activities were significantly enriched in transgenic plants. Our findings imply that OsCYP714D1 participated in the regulation of both shoot growth and salt resistance through regulating gibberellin and ion homeostasis in transgenic poplar, and it can be used as a candidate gene for the engineering of new tree varieties with improved biomass production and salt stress resistance.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

Field evaluation of transgenic hybrid poplars with desirable wood properties and enhanced growth for biofuel production by bicistronic expression of PdGA20ox1 and PtrMYB3 in wood-forming tissue

BackgroundTo create an ideotype woody bioenergy crop with desirable growth and biomass properties, we utilized the viral 2A-meidated bicistronic expression strategy to express both PtrMYB3 (MYB46 ortholog of Populus trichocarpa, a master regulator of secondary wall biosynthesis) and PdGA20ox1 (a GA20-oxidase from Pinus densiflora that produces gibberellins) in wood-forming tissue (i.e., developing xylem).ResultsTransgenic Arabidopsis plants expressing the gene construct DX15::PdGA20ox1-2A-PtrMYB3 showed a significant increase in both stem fresh weight (threefold) and secondary wall thickening (1.27-fold) relative to wild-type (WT) plants. Transgenic poplars harboring the same gene construct grown in a greenhouse for 60 days had a stem fresh weight up to 2.6-fold greater than that of WT plants. In a living modified organism (LMO) field test conducted for 3 months of active growing season, the stem height and diameter growth of the transgenic poplars were 1.7- and 1.6-fold higher than those of WT plants, respectively, with minimal adverse growth defects. Although no significant changes in secondary wall thickening of the stem tissue of the transgenic poplars were observed, cellulose content was increased up to 14.4 wt% compared to WT, resulting in improved saccharification efficiency of the transgenic poplars. Moreover, enhanced woody biomass production by the transgenic poplars was further validated by re-planting in the same LMO field for additional two growing seasons.ConclusionsTaken together, these results show considerably enhanced wood formation of our transgenic poplars, with improved wood quality for biofuel production.

Kanamycin application features for the selective screening of genetically modified Populus × berolinensis

The study describes the features of the kanamycin application for the effective selection of transgenic poplar expressing nptII gene. It has been shown that kanamycin concentration of 50 mg L−1 in the nutrient medium is the most suitable concentration for the effective selection of transgenic Berlin poplar plants for both regeneration and rooting stages. At the same time, 50 mg L−1 of kanamycin in the nutrient medium may result in the loss of the less resistant transgenic plants, so lower concentrations may be also applied. Kanamycin concentrations of 25 and 35 mg L−1 can give a false positive signal of transgenesis. Transgenic poplars expressing nptII gene can be effectively regenerated on nutrient media with kanamycin at concentrations up to 100 mg L−1.

The U-box E3 ubiquitin ligase PalPUB79 positively regulates ABA-dependent drought tolerance via ubiquitination of PalWRKY77 in Populus.

SummaryThe abscisic acid (ABA) signalling pathway is involved in the plant response to osmotic stress caused by drought and/or salinity. Although the ABA signalling pathway has been elucidated in Arabidopsis, it remains elusive in woody poplars. In this study, genome‐wide analyses of U‐box genes in poplars revealed that a U‐box E3 ubiquitin ligase gene, PalPUB79, is significantly induced following drought, salinity and ABA signalling. PalPUB79 overexpression enhanced drought tolerance in transgenic poplars, while PalPUB79 RNAi lines were more sensitive to drought. PalPUB79 positively regulated ABA signalling pathway. Furthermore, PalPUB79 interacted with PalWRKY77, a negative transcriptional regulator of ABA signalling, and mediated its ubiquitination for degradation, therefore counteracting its inhibitory effect on PalRD26 transcription. However, the finding that PalWRKY77 negatively regulates PalPUB79 expression was indicative of a negative feedback loop between PalWRKY77 and PalPUB79 during ABA signalling in poplar. These findings provide novel insight into the mechanism through which PalPUB79 enhances the ABA‐mediated stress response in woody poplars.

P-Coumaroylation of poplar lignins impacts lignin structure and improves wood saccharification.

The enzymatic hydrolysis of cellulose into glucose, referred to as saccharification, is severely hampered by lignins. Here, we analyzed transgenic poplars (Populus tremula × Populus alba) expressing the Brachypodium (Brachypodium distachyon) p-coumaroyl-Coenzyme A monolignol transferase 1 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (pCA). Several BdPMT1-OE/WT and BdPMT1-OE/AtF5H-OE lines were grown in the greenhouse, and BdPMT1 expression in xylem was confirmed by RT-PCR. Analyses of poplar stem cell walls (CWs) and of the corresponding purified dioxan lignins (DLs) revealed that BdPMT1-OE lignins were as p-coumaroylated as lignins from C3 grass straws. For some transformants, pCA levels reached 11 mg·g−1 CW and 66 mg·g−1 DL, exceeding levels in Brachypodium or wheat (Triticum aestivum) samples. This unprecedentedly high lignin p-coumaroylation affected neither poplar growth nor stem lignin content. Interestingly, p-coumaroylation of poplar lignins was not favored in BdPMT1-OE/AtF5H-OE transgenic lines despite their high frequency of syringyl units. However, lignins of all BdPMT1-OE lines were structurally modified, with an increase of terminal unit with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples improved the saccharification yield of alkali-pretreated CW, demonstrating that the genetically driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to alkaline treatments used during the industrial processing of lignocellulosics.

Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants, particularly for wind-pollinated trees such as poplar. We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE (SVL), a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE (SVP), delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars. Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance, and did not cause morphologically obvious or statistically significant effects on leaf characteristics, tree form, or stem volume. Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific. Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.

RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar.

Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula×Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit. We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.