Transgenic Poplars Research Articles

PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathway in a hybrid poplar.

Both anthocyanins and lignins are essential secondary metabolites in plant growth and development. Their biosynthesis is metabolically interconnected and diverges in the central metabolite 4-coumaroyl CoA of the phenylpropanoid pathway. Considerable progress has been made in understanding transcriptional regulation of genes involved in lignin and anthocyanin synthesis pathways, but the concerted regulation of these pathways is not yet fully understood. Here, we functionally characterized PtrMYB120, a R2R3-MYB transcription factor from Populus trichocarpa. Overexpression of PtrMYB120 in a hybrid poplar (i.e., 35S::PtrMYB120) was associated with increased anthocyanin (i.e., cyanidin 3-O-glucoside) accumulation and upregulation of anthocyanin biosynthetic genes. However, transgenic poplars with dominant suppression of PtrMYB120 function achieved by fusing the ERF-associated amphiphilic repression motif to PtrMYB120 (i.e., 35S::PtrMYB120-SRDX) had a dramatic decrease in not only anthocyanin but also Klason lignin content with downregulation of both anthocyanin and lignin biosynthetic genes. Indeed, 35S::PtrMYB120-SRDX poplars had irregularly shaped xylem vessels with reduced S-lignin content in stems, which was proportionally related to the level of the introduced PtrMYB120-SRDX gene. Furthermore, protoplast-based transcriptional activation assay using the PtrMYB120-GR system suggested that PtrMYB120 directly regulates genes involved in both anthocyanin and lignin biosynthesis, including chalcone synthase and ferulate-5 hydroxylase. Interestingly, the saccharification efficiency of line #6 of 35S::PtrMYB120-SRDX poplars, which had slightly reduced lignin content with a normal growth phenotype, was dramatically enhanced (>45%) by NaOH treatment. Taken together, our results suggest that PtrMYB120 functions as a positive regulator of both anthocyanin and lignin biosynthetic pathways and can be targeted to enhance saccharification efficiency in woody perennials.

Rejuvenation remodels transcriptional network to improve rhizogenesis in mature Juglans tree.

Adventitious rooting of walnut species (Juglans L.) is known to be rather difficult, especially for mature trees. The adventitious root formation (ARF) capacities of mature trees can be significantly improved by rejuvenation. However, the underlying gene regulatory networks (GRNs) of rejuvenation remain largely unknown. To characterize such regulatory networks, we carried out the transcriptomic study using RNA samples of the cambia and peripheral tissues on the bottom of rejuvenated and mature walnut (Juglans hindsii × J. regia) cuttings during the ARF. The RNA sequencing data suggested that zeatin biosynthesis, energy metabolism and substance metabolism were activated by rejuvenation, whereas photosynthesis, fatty acid biosynthesis and the synthesis pathways for secondary metabolites were inhibited. The inter- and intra-module GRNs were constructed using differentially expressed genes. We identified 35 hub genes involved in five modules associated with ARF. Among these hub genes, particularly, beta-glucosidase-like (BGLs) family members involved in auxin metabolism were overexpressed at the early stage of the ARF. Furthermore, BGL12 from the cuttings of Juglans was overexpressed in Populus alba × P. glandulosa. Accelerated ARF and increased number of ARs were observed in the transgenic poplars. These results provide a high-resolution atlas of gene activity during ARF and help to uncover the regulatory modules associated with the ARF promoted by rejuvenation.

The transcription factor KNAT2/6b mediates changes in plant architecture in response to drought via down-regulating GA20ox1 in Populus alba × P. glandulosa

Plant architecture is genetically controlled, but is influenced by environmental factors. Plants have evolved adaptive mechanisms that allow changes in their architecture under stress, in which phytohormones play a central role. However, the gene regulators that connect growth and stress signals are rarely reported. Here, we report that a class I KNOX gene, PagKNAT2/6b, can directly inhibit the synthesis of gibberellin (GA), altering plant architecture and improving drought resistance in Populus. Expression of PagKNAT2/6b was significantly induced under drought conditions, and transgenic poplars overexpressing PagKNAT2/6b exhibited shorter internode length and smaller leaf size with short or even absent petioles. Interestingly, these transgenic plants showed improved drought resistance under both short- and long-term drought stress. Histological observations indicated that decreased internode length and leaf size were mainly caused by the inhibition of cell elongation and expansion. GA content was reduced, and the GA20-oxidase gene PagGA20ox1 was down-regulated in overexpressing plants. Expression of PagGA20ox1 was negatively related to that of PagKNAT2/6b under drought stress. ChIP and transient transcription activity assays revealed that PagGA20ox1 was directly targeted by PagKNAT2/6b. Therefore, this study provides evidence that PagKNAT2/6b mediates stress signals and changes in plant architecture via GA signaling by down-regulating PagGA20ox1.

Functional identification and characterization of GST genes in the Asian gypsy moth in response to poplar secondary metabolites

The Asian gypsy moth, Lymantria dispar, as one of the most important forest pests in the world, can feed on more than 500 species of host plants, causing serious damage to the forests. Poplar is one of the favorite host plants of L. dispar. The present study aimed to explore the effects of poplar secondary metabolites on the growth and detoxification function of L. dispar larvae. We also aimed to study the expression of glutathione S-transferase (GST) genes in different developmental stages and in response to treatment with secondary metabolites. Six kinds of main secondary metabolites and three groups of characteristic mixed secondary metabolites were selected as follows: Caffeic acid, salicin, rutin, quercetin, catechol, flavone, mixture 1 (salicin and flavone), mixture 2 (salicin, caffeic acid and catechol), and mixture 3 (flavone, caffeic acid and catechol) according to the content changes of secondary metabolites in poplar. The thirteen GST genes were selected as candidate genes to study the expression of GST genes in different developmental stages and after treatment with secondary metabolites using quantitative real-time reverse transcription PCR. The LdGSTe4 and LdGSTo1 genes could be induced by secondary metabolites and were screened to explore their detoxification function against secondary metabolites using RNA interference technology. The results showed that salicin and rutin significantly induced the expression of LdGSTe4 and LdGSTo1. Under the stress of secondary metabolites, LdGSTe4 silencing affected the adaptability of L. dispar larvae to salicin and rutin. LdGSTe4 silencing resulted in a significant decrease in the body weight of L. dispar, but had little effect on the relative growth rate, relative consumption rate, efficiency of conversion of ingested food, efficiency of conversion of digested food, and approximate digestibility, as well as the survival rate and development time. These results provide a deeper understanding of the adaptive mechanism of L. dispar to host plants, form the foundation for the further research into the host resistance mechanism, and identify target genes for breeding resistant transgenic poplar.

Growth adaptability and foreign gene stability of TaLEA transgenic Populus simonii × nigra

Key message Late-embryogenesis-abundant proteins (LEA) typically accumulate to high levels during seed dehydration and have an important role in the plant response to abiotic stresses. Transgenic hybrid poplars of 9-year-old TaLEA lines showed adaptation to drought and saline–alkali conditions. The exogenous gene was expressed normally, and the T-DNA insertion sites were identified on different chromosomes. ContextImproving the survival rate of afforested species is essential for forest production in saline–alkali areas. Transgenic hybrid poplars could provide potential genetic materials for adaptation to semi-arid areas.AimsWe investigated the growth stability and adaptability of 9-year-old TaLEA transgenic lines of Populus simonii × nigra, as well as the position of T-DNA insertion and the stability of exogenous genes.MethodsTransgenic poplar field trials were carried out using a multi-site joint analysis. Molecular characteristics and the T-DNA insertion sites were identified by genome resequencing.ResultsThe XL-9, XL-7, and XL-13 preferred lines consistently showed the highest and most stable growth across three testing sites. The exogenous gene was integrated into the genome chromosome and was expressed normally in the XL-1, XL-7, and XL-9 lines after 9 years.ConclusionXL-9, XL-7, and XL-13 are the preferred lines for subsequent production tests. Transgenic hybrid poplar lines can be selected for adaptability 2 years after field experiments. The results of this study will provide further guidance for testing of transgenic trees for future forest production.

Galactinol synthase confers salt-stress tolerance by regulating the synthesis of galactinol and raffinose family oligosaccharides in poplar

Plants respond to abiotic stress through a series of transcriptional profile and metabolic process modifications through signaling events. Raffinose family oligosaccharides (RFOs) play a role in the regulatory mechanisms of abiotic stress tolerance and are catalyzed by the key enzyme, galactinol synthase (GolS), in plants. The amino acids sequences of GolS of Populus trichocarpa contained the common features of GolS proteins in other plant species, namely, a hydrophobic pentapeptide (APSAA). The PtrGolS3 promoter region contains several cis-elements for stress responses, including abscisic acid responsive element (ABRE), the dehydration and cold responsive elements (DRE/CRT), the low-temperature responsive element (LTRE), and the transcription factor MYB binding sites. Overexpression of AtGolS2 and PtrGolS3 enhanced abiotic stress tolerance along with the expression of stress-related genes in transgenic poplar. In addition, overexpression of AtGolS2 and PtrGolS3 resulted in higher contents of soluble sugars and other stress-associated metabolites (proline, salicylic acid, phenylalanine, and so forth) in two GolS overexpressing poplar lines compared with wild-type poplar under salt stress. Based on these results, a promising PtrGolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in poplar was identified and it may increase the understanding of RFO metabolism in woody plants under short-term salt treatment.

Microwave-assisted deep eutectic solvents (DES) pretreatment of control and transgenic poplars for boosting the lignin valorization and cellulose bioconversion

Reducing the “biomass recalcitrance” and simultaneously improving the efficiency of deconstruction and conversion is vitally important in a biorefinery process. Herein, a microwave-assisted deep eutectic solvents (DES) pretreatment was developed to deconstruct the control and transgenic (C3H, HCT and C3H + HCT co-inhibition) poplars, and improve the lignin extractability and valorization. NMR and GPC results showed that native lignin (Double Enzymatic Lignin, DEL) in different poplars exhibited slightly different structural characteristics, affecting the subsequent lignin extractability and value-added applications. After two kinds of DES (ethylene glycol-based and glycerol-based DES) pretreatments, the yield of DES lignin was obviously increased to 45.38–53.09 % and 46.38–54.30 % in transgenic poplar woods as compared to control poplar wood (37.25 and 38.86 %). In addition, molecular weight, and abundances of different linkages (β-O-4, β-β, β-5, etc.) in the DES lignin fractions were significantly decreased, suggesting the microwave-assisted DES pretreatment has a strong deconstruction ability towards to poplar wood. Moreover, the rapid and efficient pretreatment facilitated the isolation of the lignin fractions from transgenic poplars, and the regenerated DES lignin fractions exhibited excellent antioxidant properties, which will lay the foundation for the lignin valorization. Furthermore, the enzymatic saccharification ratios of the DES pretreated substrates were significantly increased to 87.36–96.55 % in control and transgenic poplar woods, indicating that microwave-assisted DES pretreatment indeed boosted cellulose bioconversion. In short, the combination of genetic modification and DES pretreatment can significantly reduce the “biomass recalcitrance” and promote the biomass valorization.

Growth and arthropod community characteristics of transgenic poplar 741 in an experimental forest

Use of the Bt insect-resistance gene has provided substantial economic and ecological benefits. Full utilization of transgenic poplars requires a comprehensive understanding of the growth-related phenotypic characteristics and ecological risks associated with of genetically modified trees. In the present study, we assessed the effects of transgenic poplar 741 with different insect-resistant genes on plant growth and arthropod communities in the field. Transgenic lines carrying the Cry1Ac and Cry3A genes were used as model lines, and non-transgenic poplar 741 was used as a control. Over a period of 6 years, we analyzed plant growth and transcriptomic data, and monitored arthropod richness and abundance in an experimental forest comparing transgenic and control lines. Our results indicate that the Pb29 line had a similar plant height to CK, but a significantly lower diameter at breast height (DBH) than CK. The plant height and DBH of the CC84 line were slightly higher than those of CK, but the differences were not significant. Transcriptome data analysis showed downregulated and upregulated expression of growth hormone-related genes and lignin synthesis-related genes, respectively, in the Pb29 line compared with CK and the CC84 line. The survey data obtained in this study showed that annual trends in arthropod numbers in the ecosystem were consistent among transgenic poplar 741 and control lines, gradually decreasing with time since planting. The Cry1Ac line has selective resistance to Lepidoptera and exerted strong toxic effects on Arctiidae, Tortricidae, and Notodontidae, but had no obvious effects on Lyonetiidae or Geometridae. The Cry3A line exhibited significant resistance to coleopterans only in 2016 when the abundance of coleopteran insects was high. Both lines exhibited inhibitory effects on non-target pests. The two transgenic lines were similar to the control with respect to arthropod community structure, and there were no significant differences in arthropod diversity indices between transgenic and control lines, although richness was slightly higher for the Cry3A line than for the control. The results of this study showed that the insertion of Bt genes had no apparent inhibitory effect on the growth of poplar 741. Also, there was no adverse environmental impact of transgenic poplar 741 on ecosystem arthropod communities compared with conventional poplar 741.

Overexpression of DBF-Interactor Protein 6 Containing an R3H Domain Enhances Drought Tolerance in Populus L. (Populus tomentosa).

Drought is the primary disaster that endangers agricultural production, including animal husbandry, and affects the distribution, growth, yield, and quality of crops. Previous study had revealed that DIP, as a potential regulator of DBF activity, played an important role in response to drought stress in maize. In this study, a total of 67 DIPs were identified from seventeen land plants, including six tobacco DIPs (NtDIPs). NtDIP6 gene was further selected as a candidate gene for subsequent experiments based on the phylogenetic analysis and structural analysis. The transgenic tobacco and poplar plants over-expressing NtDIP6 gene were generated using the Agrobacterium- mediated method. Although there was not phenotypic difference between transgenic plants and wild-type plants under normal conditions, overexpression of the NtDIP6 gene in transgenic tobacco and poplar plants enhanced the drought tolerance under drought treatments in comparison with the wild type. The content of antioxidant defense enzymes peroxidase (POD), catalase (CAT), and the photosynthetic rate increased in NtDIP6-Ox transgenic tobacco and poplar plants, while the content of malondialdehyde decreased, suggesting that the overexpression of NtDIP6 enhances the antioxidant capacity of transgenic poplar. Furthermore, the results of qRT-PCR showed that the level of expression of drought-related response genes significantly increased in the NtDIP6-Ox transgenic plants. These results indicated that NtDIP6, as a positive response regulator, improves drought stress tolerance by scavenging superoxide via the accumulation of antioxidant defense enzymes.

Ectopic expression of a Malus hupehensis Rehd. myo- inositol oxygenase gene (MhMIOX2) enhances tolerance to salt stress

Myo-inositol oxygenase (MIOX), an essential monooxygenase enzyme, catalyzes the conversion of myo-inositol (MI) into d-glucuronic acid (D-GlcUA). The MIOX genes have been isolated and studied extensively in model plants and crops, but the characteristics and roles of MIOXs in woody plants remain largely unknown. In this study, two MhMIOXs cloned from Malus hupehensis Rehd. were characterized. Both MhMIOX1 and MhMIOX2 expression were significantly induced throughout the whole ABA and NaCl stress treatment, especially under NaCl treatment. To gain insight into the roles of MhMIOX1/2 in MI metabolism, we heterologously expressed MhMIOX1/2 in poplar and Arabidopsis. The results showed that only MhMIOX2 overexpression caused the decrease of MI content in transgenic plants. Either the transgenic poplar or Arabidopsis lines overexpressing MhMIOX2 showed better growth performance than the wild type (WT) upon salt stress treatment. Furthermore, compared to the WT, the H2O2 and MDA content obviously decreased, and the activity of ROS-scavenging enzymes (POD, APX and CAT) was strongly elevated in transgenic poplar under salt stress conditions, which suggest MhMIOX2 overexpression enhancs the tolerance of the transgenic plants to salt stress. Besides, there was no obvious difference in ascorbate (AsA) content between the WT and transgenic poplar under normal condition, indicating MhMIOX2 is important for MI oxidative metabolism, but not for AsA production in poplar and Arabidopsis.

Poplar Autophagy Receptor NBR1 Enhances Salt Stress Tolerance by Regulating Selective Autophagy and Antioxidant System.

Salt stress is an adverse environmental factor for plant growth and development. Under salt stress, plants can activate the selective autophagy pathway to alleviate stress. However, the regulatory mechanism of selective autophagy in response to salt stress remains largely unclear. Here, we report that the selective autophagy receptor PagNBR1 (neighbor of BRCA1) is induced by salt stress in Populus. Overexpression of PagNBR1 in poplar enhanced salt stress tolerance. Compared with wild type (WT) plants, the transgenic lines exhibited higher antioxidant enzyme activity, less reactive oxygen species (ROS), and higher net photosynthesis rates under salt stress. Furthermore, co-localization and yeast two-hybrid analysis revealed that PagNBR1 was localized in the autophagosome and could interact with ATG8 (autophagy-related gene). PagNBR1 transgenic poplars formed more autophagosomes and exhibited higher expression of ATG8, resulting in less accumulation of insoluble protein and insoluble ubiquitinated protein compared to WT under salt stress. The accumulation of insoluble protein and insoluble ubiquitinated protein was similar under the treatment of ConA in WT and transgenic lines. In summary, our results imply that PagNBR1 is an important selective autophagy receptor in poplar and confers salt tolerance by accelerating antioxidant system activity and autophagy activity. Moreover, the NBR1 gene is an important potential molecular target for improving stress resistance in trees.

Surface characteristics of porous carbon derived from genetically designed transgenic hybrid poplar for electric double-layer capacitors

Wood and wood residues are attractive sources for supercapacitor electrode materials because of their abundance and sustainability. As each lignocellulosic biomass has an intrinsic chemical composition, improvements in the energy storage capability have relied on modified preparation conditions or the introduction of functional groups or foreign materials. However, owing to advances in genetic science, the influence of biomass chemical composition on the surface characteristics and electrochemical behavior of wood-derived porous carbons can be investigated toward the development of high-performance supercapacitor electrodes. Herein, we compare the electrochemical performance of porous carbons derived from genetically designed transgenic and wild poplar following chemical activation with KOH at 800 °C. The transgenic poplar-derived porous carbon shows ~80% higher capacitance than the wild-poplar-derived porous carbon at 0.2 A/g and greater performance at all test rates (0.2–20 A/g). An increased cellulose fraction or decreased lignin fraction in the biomass increases the surface area and the amount of oxygen functional groups on the porous carbon, thus enhancing the electrochemical performance. These findings are expected to advance the use of biomass-derived materials in energy storage applications.

Leaf isoprene emission as a trait that mediates the growth-defense tradeoff in the face of climate stress.

Plant isoprene emissions are known to contribute to abiotic stress tolerance, especially during episodes of high temperature and drought, and during cellular oxidative stress. Recent studies have shown that genetic transformations to add or remove isoprene emissions cause a cascade of cellular modifications that include known signaling pathways, and interact to remodel adaptive growth-defense tradeoffs. The most compelling evidence for isoprene signaling is found in the shikimate and phenylpropanoid pathways, which produce salicylic acid, alkaloids, tannins, anthocyanins, flavonols and other flavonoids; all of which have roles in stress tolerance and plant defense. Isoprene also influences key gene expression patterns in the terpenoid biosynthetic pathways, and the jasmonic acid, gibberellic acid and cytokinin signaling networks that have important roles in controlling inducible defense responses and influencing plant growth and development, particularly following defoliation. In this synthesis paper, using past studies of transgenic poplar, tobacco and Arabidopsis, we present the evidence for isoprene acting as a metabolite that coordinates aspects of cellular signaling, resulting in enhanced chemical defense during periods of climate stress, while minimizing costs to growth. This perspective represents a major shift in our thinking away from direct effects of isoprene, for example, by changing membrane properties or quenching ROS, to indirect effects, through changes in gene expression and protein abundances. Recognition of isoprene's role in the growth-defense tradeoff provides new perspectives on evolution of the trait, its contribution to plant adaptation and resilience, and the ecological niches in which it is most effective.

The PalWRKY77 transcription factor negatively regulates salt tolerance and abscisic acid signaling in Populus.

High salinity, one of the most widespread abiotic stresses, inhibits photosynthesis, reduces vegetation growth, blocks respiration and disrupts metabolism in plants. In order to survive their long-term lifecycle, trees, such as Populus species, recruit the abscisic acid (ABA) signaling pathway to adapt to a saline environment. However, the molecular mechanism behind the ABA-mediated salt stress response in woody plants remains elusive. We have isolated a WRKY transcription factor gene, PalWRKY77, from Populus alba var. pyramidalis (poplar), the expression of which is repressed by salt stress. PalWRKY77 decreases salt tolerance in poplar. Furthermore, PalWRKY77 negatively regulated ABA-responsive genes and relieved ABA-mediated growth inhibition, indicating that PalWRKY77 is a repressor of the ABA response. In vivo and in vitro assays revealed that PalWRKY77 targets the ABA- and salt-induced PalNAC002 and PalRD26 genes by binding to the W-boxes in their promoters. In addition, overexpression of both PalNAC002 and PalRD26 could elevate salt tolerance in transgenic poplars. These findings reveal a novel negative regulation mechanism for the ABA signaling pathway mediated by PalWRKY77 that results in more sensitivity to salt stress in poplar. This deepens our understanding of the complex responses of woody species to salt stress.

Overexpression of a Poplar RING-H2 Zinc Finger, Ptxerico, Confers Enhanced Drought Tolerance via Reduced Water Loss and Ion Leakage in Populus.

Drought stress is one of the major environmental problems in the growth of crops and woody perennials, but it is getting worse due to the global climate crisis. XERICO, a RING (Really Interesting New Gene) zinc-finger E3 ubiquitin ligase, has been shown to be a positive regulator of drought tolerance in plants through the control of abscisic acid (ABA) homeostasis. We characterized a poplar (Populus trichocarpa) RING protein family and identified the closest homolog of XERICO called PtXERICO. Expression of PtXERICO is induced by both salt and drought stress, and by ABA treatment in poplars. Overexpression of PtXERICO in Arabidopsis confers salt and ABA hypersensitivity in young seedlings, and enhances drought tolerance by decreasing transpirational water loss. Consistently, transgenic hybrid poplars overexpressing PtXERICO demonstrate enhanced drought tolerance with reduced transpirational water loss and ion leakage. Subsequent upregulation of genes involved in the ABA homeostasis and drought response was confirmed in both transgenic Arabidopsis and poplars. Taken together, our results suggest that PtXERICO will serve as a focal point to improve drought tolerance of woody perennials.

Effects of Bt-Cry1Ah1 Transgenic Poplar on Target and Non-Target Pests and Their Parasitic Natural Enemy in Field and Laboratory Trials

Increasing areas of artificial afforestation and poplar monoculture in China have led to serious problems with insect pests. The development of genetic engineering technology, such as transgenic modification with Bacillus thuringiensis (Bt) genes, provides novel solutions to the pest problem. We generated a Bt-Cry1Ah1 gene incorporating codon optimization and transferred it into Populus deltoides × P. euramericana cv “Nanlin895” using an Agrobacterium-mediated method. The resulting Bt-Cry1Ah1 transgenic poplars were planted in the field with permission from the State Forestry Administration in 2017. Field and laboratory studies were conducted in Jiangsu, China, to investigate the effects of these transgenic poplars expressing the Cry1Ah1 protein on target and non-target pests and their parasitic natural enemy. Target pests included Hyphantria cunea (Lepidoptera, Arctiidae), Micromelalopha troglodyta (Lepidoptera, Notodontidae), and Clostera anachoreta (Lepidoptera, Notodontidae). Plagiodera versicolora (Coleoptera, Chrysomelidae) served as the non-target pest. Laboratory trials showed that the six transgenic poplar lines exhibited resistance against the target insects. The corrected mortality rates of the target pest larvae fed leaves from the six lines were as high as 87.0%, significantly higher than that of the control. However, the corrected mortality rate of the non-target pest larvae was markedly lower and did not differ significantly from that of the control. Field experiments showed that transgenic poplar exhibited resistance against H. cunea and M. troglodyta. Field mortality rates were slightly higher than laboratory mortality rates. In addition, we investigated Chouioia cunea (Hymenoptera, Eulophidae) as a parasitoid of H. cunea pupae that had been fed transgenic poplar leaves. The emergence time, parasitism rate, and abundance of C. cunea did not differ significantly from those of the control. Therefore, Bt-Cry1Ah1 transgenic poplar can be used to effectively control damage by target insect pests without negatively affecting non-target insects and parasitoids.

Assessment of environmental microbial effects of insect-resistant transgenic Populus × euramericana cv. ‘74/76’ based on high-throughput sequencing

Pests and diseases present major obstacles to poplar growth, which have been overcome via transgenic engineering applications. However, given the long growth cycle of transgenic poplars, long-term environmental safety assessments are needed for transgenic poplars planted in the field. Therefore, we investigated the effects of exogenous genes in transgenic poplar on the diversity of endophytic and rhizosphere soil bacterial communities in poplars. The composition and diversity of bacterial communities in trunk tissue, root tissue, and rhizosphere soil samples of transgenic Populus × euramericana cv. ‘74/76’ containing the high-resistance gene HPb1 (hereafter, HPb1) and non-transgenic Populus × euramericana cv. ‘74/76’ (hereafter, 107) in a 4-year-old experimental forest were analyzed using high-throughput sequencing. In terms of bacterial abundance at the phylum level, in trunk tissue, Bacteroidetes was the dominant phylum in both poplar lines (HPb1: 32.17%; 107: 47.42%). In root tissue, Proteobacteria was the dominant phylum in both lines (HPb1: 67.52%; 107: 65.96%). In rhizosphere soil, Proteobacteria was the dominant phylum in both lines (HPb1: 39.75%; 107: 37.98%). There were no significant differences in the richness and diversity of bacterial communities between HPb1 and 107. Finally, the bacterial community compositions in root tissue and rhizosphere soil were highly similar, both of which differed somewhat from that in trunk samples. This study provides a reference for ecological safety assessments of transgenic poplars and allows for a more systematic understanding of the potential ecosystem risk posed by environmental release of transgenic poplars.

Overexpression of a tonoplast Na+/H+ antiporter from the halophytic shrub Nitraria sibirica improved salt tolerance and root development in transgenic poplar

The sodium/proton exchanger (NHX) mediates Na+ and H+ countertransport in plants and plays an important role in regulating intracellular pH and maintaining ion and osmotic balance. Previously, an NHX1 orthologue was isolated from the halophyte Nitraria sibirica Pall (referred to as NsNHX1), and its role in enhancing salt tolerance of transgenic Arabidopsis was confirmed. To further analyse its features and functions, the 1311-bp sequence of the NsNHX1 promoter was cloned, and histochemical staining showed that β-glucuronidase (GUS) expression driven by the NsNHX1 promoter was strongly induced by abiotic stress and phytohormones, such as salt, drought, gibberellins, and methyl jasmonate, in turn indicating that NsNHX1 might participate in the regulation of various signalling pathways. To determine how NsNHX1 regulates salt tolerance in forestry trees, NsNHX1 was introduced into 84K poplar, and salt tolerance analysis of transgenic poplars showed that overexpression of NsNHX1 increased the overall biomass, survival rate, and plant height, and the contents of chlorophyll, proline and water, all of which are consequences of antioxidant enzyme activity under salt stress conditions. This showed that overexpression of NsNHX1 enhanced the salt tolerance of transgenic poplars as a result of NsNHX1-mediated Na+ compartmentalisation, more efficient photosynthesis, greater activity of antioxidant enzymes, and improved osmotic adjustment. Moreover, overexpression of NsNHX1 enhanced the root development of transgenic poplars; this resulted in increased biomass and height under normal and salt stress conditions, likely due to coupling between NsNHX1 and membrane proton pumps. These results provided a theoretical and experimental basis for further understanding the function and regulatory mechanism of NsNHX1, as well as its application for genetic improvement of forestry trees.

Improving Air Quality by Nitric Oxide Consumption of Climate-Resilient Trees Suitable for Urban Greening.

Nitrogen oxides (NOx), mainly a mixture of nitric oxide (NO) and nitrogen dioxide (NO2), are formed by the reaction of nitrogen and oxygen compounds in the air as a result of combustion processes and traffic. Both deposit into leaves via stomata, which on the one hand benefits air quality and on the other hand provides an additional source of nitrogen for plants. In this study, we first determined the NO and NO2 specific deposition velocities based on projected leaf area (sV d) using a branch enclosure system. We studied four tree species that are regarded as suitable to be planted under predicted future urban climate conditions: Carpinus betulus, Fraxinus ornus, Fraxinus pennsylvanica and Ostrya carpinifolia. The NO and NO2 sVd were found similar in all tree species. Second, in order to confirm NO metabolization, we fumigated plants with 15NO and quantified the incorporation of 15N in leaf materials of these trees and four additional urban tree species (Celtis australis, Alnus spaethii, Alnus glutinosa, and Tilia henryana) under controlled environmental conditions. Based on these 15N-labeling experiments, A. glutinosa showed the most effective incorporation of 15NO. Third, we tried to elucidate the mechanism of metabolization. Therefore, we generated transgenic poplars overexpressing Arabidopsis thaliana phytoglobin 1 or 2. Phytoglobins are known to metabolize NO to nitrate in the presence of oxygen. The 15N uptake in phytoglobin-overexpressing poplars was significantly increased compared to wild-type trees, demonstrating that the NO uptake is enzymatically controlled besides stomatal dependence. In order to upscale the results and to investigate if a trade-off exists between air pollution removal and survival probability under future climate conditions, we have additionally carried out a modeling exercise of NO and NO2 deposition for the area of central Berlin. If the actually dominant deciduous tree species (Acer platanoides, Tilia cordata, Fagus sylvatica, Quercus robur) would be replaced by the species suggested for future conditions, the total annual NO and NO2 deposition in the modeled urban area would hardly change, indicating that the service of air pollution removal would not be degraded. These results may help selecting urban tree species in future greening programs.

Wood forming tissue-specific expression of PdSuSy and HCHL increases holocellulose content and improves saccharification in Populus

Development of strategies to deconstruct lignocellulosic biomass in tree species is essential for biofuels and biomaterials production. We applied a wood forming tissue-specific system in a hybrid poplar to express both PdSuSy (a sucrose synthase gene from Populus deltoides × P. euramericana that has not been functionally characterized) and HCHL (the hydroxycinnamoyl-CoA hydratase-lyase gene from Pseudomonas fluorescens, which inhibits lignin polymerization in Arabidopsis). The PdSuSy-HCHL overexpression poplars correspondingly driven by the promoters of Arabidopsis AtCesA7 and AtC4H resulted in a significant increase in cellulose (> 8%), xylan (> 12%) and glucose (> 29%) content, accompanying a reduction in galacturonic acid (> 36%) content, compared to control plants. The saccharification efficiency of these overexpression poplars was dramatically increased by up to 27%, but total lignin content was unaffected. These transgenic poplars showed inhibited growth characteristics, including > 16% reduced plant height, > 10% reduced number of internodes, and > 18% reduced fresh weight after growth of 4 months, possibly due to relatively low expression of HCHL in secondary xylem. Our results demonstrate the structural complexity and interaction of the cell wall polymers in wood tissue and outline a potential method to increase biomass saccharification in woody species.