Poplar Plants Research Articles

The study of compounds distilled with water vapor of Populus laurifolia Ledeb. leaves

Poplar plants (Populus L.) are one of the promising species for inclusion in the State Pharmacopoeia of Ukraine. To date, extensive experience has been accumulated in the use of extracts from buds, leaves and bark of various types of poplars in folk and official medicine as effective anti-inflammatory, antipyretic, analgetic, antibacterial agents. According to literature data only buds are pharmacopoeial raw materials in some countries of the world.
 Poplar leaves are of interest as a new promising type of medicinal plant raw material, since they have a chemical composition close to the buds, moreover, they are much easier to harvest and the possible volume of raw materials procurement is much larger.
 The laurel-leaf poplar (Populus laurifolia Ledeb.) is assigned to the section of balsamic poplars (Tacamahaca). It is grown in forest culture, used for landscaping and creation of field-protective plantations in Ukraine.
 Continuing a comprehensive pharmacognostic study of raw materials of plants of the genus poplar, purpose of this work was to study composition and content of compounds distilled with water vapor of leaves of laurel-leaf poplar cultivated in Ukraine.
 Leaves for research were harvested in June 2019, from trees growing on the shores of the Murom reservoir in the village of Russkiye Tishki, Kharkiv region (50°09′30″N 36°29′10″E). The composition of compounds distilled in water vapor was investigated on Agilent Technologies 6890N (USA) chromatograph with a 5973N mass spectrometric detector.
 The total content of compounds distilled with water vapor was 2 126.1 mg/kg. 46 compounds were identified. Terpenoids were presented by 20 compounds with a total content 1 444 mg/kg, the remaining 26 compounds were aromatic substances (256.2 mg/kg) and compounds of various groups (216.8 mg/kg). The dominant compounds in content were β-eudesmol (776.4 mg/kg), γ-eudesmol (298 mg/kg), α-bisabolol (125.9 mg/kg), eugenol (70.9 mg/kg).
 The data obtained indicate the rich chemical composition of leaves of laurel-leaf poplar and are the basis for further in-depth study of this raw material for assessment the possibility of using it as a medicine.

Colonization Characteristics of Poplar Fungal Disease Biocontrol Bacteria N6-34 and the Inhibitory Effect on Pathogenic Fungi by Real-Time Fluorescence Quantitative PCR Detection.

Botryosphaeria dothidea is one of the most important diseases which can cause poplar canker. In our previous study, the endophytic Bacillus subtilis N6-34 screened from poplar tissue was found to be an antagonistic strain against B. dothidea. In order to ascertain the colonization rule of B. subtilis N6-34 in poplar plants, colonization of B. subtilis N6-34 labeled with a green fluorescent protein (GFP) was investigated in poplar plants and the rhizosphere soil. To confirm the inhibitory effect of the strain N6-34 on pathogenic fungi, real-time fluorescent quantitative PCR experiment with Fusarium oxysporum as the target strain was carried out. Firstly, a plasmid (pHT01-P43GFPmut3a) containing gfp gene was successfully transformed into wild B. subtilis N6-34, which has the similar characteristics with the strain N6-34 in cell growth and antifungal activity. The poplar pot experiments were carried out to examine the colonization rules and colonization quantity in poplar plants and rhizosphere soil. Observation with a confocal laser scanning microscope showed that GFP-labeled B. subtilis N6-34 (N6-34-GFP) could colonize in primary root, lateral root and adventitious root. With the extension of inoculation time, the colonization quantity of N6-34-GFP in the rhizosphere soil and poplar plants showed a trend of first increasing, then stabilizing for a period of time and then decreasing. The real-time fluorescent quantitative PCR result showed a gradual decrease in the number of F. oxysporum with increasing inoculation time. Therefore, N6-34-GFP exhibited colonization in the rhizosphere soil and different parts of poplar plants. In addition, the strain N6-34 could inhibit the growth of pathogenic fungi. The ability of B. subtilis N6-34 to colonize in the rhizosphere soil and poplar plants and to inhibit fungal growth in vitro suggest a potential application of this strain as a biological control agent.

Enhancement of Zn tolerance and accumulation in plants mediated by the expression of Saccharomyces cerevisiae vacuolar transporter ZRC1

Main conclusionTransgenic Arabidopsis thaliana and Populus alba plants overexpressing the zinc transporter ScZRC1 in shoots exhibit Zn tolerance. Increased Zn concentrations were observed in shoots of P. alba, a species suitable for phytoremediation.Genetic engineering of plants for phytoremediation is worth to consider if genes leading to heavy metal accumulation and tolerance are expressed in high biomass producing plants. The Saccharomyces cerevisiae ZRC1 gene encodes a zinc transporter which is primarily involved in the uptake of Zn into the vacuole. The ZRC1 gene was expressed in the model species A. thaliana and P. alba (cv. Villafranca). Both species were transformed with constructs carrying ScZRC1 under the control of either the CaMV35S promoter for constitutive expression or the active promoter region of the tobacco Rubisco small subunit (pRbcS) to limit the expression to the above-ground tissues. In hydroponic cultures, A. thaliana and poplar ScZRC1-expressing plants accumulated more Zn in vegetative tissues and were more tolerant than untransformed plants. No differences were found between plants carrying the CaMV35::ScZRC1 or pRbcS::ScZRC1 constructs. The higher Zn accumulation in transgenic plants was accompanied by an increased superoxide dismutase (SOD) activity, indicating the activation of defense mechanisms to prevent cellular damage. In the presence of cadmium in addition to Zn, plants did not show symptoms of metal toxicity, neither in hydroponic cultures nor in soil. Zn accumulation increased in shoots, while no differences were observed for Cd accumulation, in comparison to control plants. These data suggest that ectopic expression of ScZRC1 can increase the potential of poplar for the remediation of Zn-polluted soils, although further tests are required to assay its application in remediating multimetal polluted soils.

Interconversion between methoxylated, hydroxylated and sulfated metabolites of PCB 3 in whole poplar plants

Methoxylated polychlorinated biphenyls (MeO-PCBs) are overlooked metabolites of PCBs. In general, they are more toxic to plants than their parent congeners. However, information on the fate of MeO-PCBs and the relationship between methoxylated, hydroxylated and sulfated metabolites of PCBs in plants is scarce. In this work, poplar plants (Populus deltoides × nigra, DN34) were hydroponically and separately exposed to 4′-methoxy-4-monochlorobiphenyl (4′-MeO-PCB 3) and 4′-PCB 3 sulfate for 10 days to investigate the uptake, translocation and metabolism of MeO-PCBs and the relationship between methoxy-PCBs, hydroxyl-PCBs and PCB sulfates within plants. Results showed that 4′-MeO-PCB 3 and 4′-PCB 3 sulfate were taken up by the roots of poplar plants and translocated from roots to shoots and leaves. 4′-OH-PCB 3 and 4′-PCB 3 sulfate were identified as the hydroxylated metabolite and sulfate metabolite of 4′-MeO-PCB 3 in poplar, respectively. In the backward reaction, 4′-OH-PCB 3 and 4′-MeO-PCB 3 were found as metabolites of 4′-PCB 3 sulfate. For exposure groups, the yields of 4′-OH-PCB 3 produced from 4′-MeO-PCB 3 and 4′-PCB 3 sulfate were 1.29% and 0.13% respectively. The yield of 4′-PCB 3 sulfate which originated from 4′-MeO-PCB 3 in wood and root samples of exposure groups was only 0.02%. Only 0.04% of the initial mass of 4′-PCB 3 sulfate was transformed to 4′-MeO-PCB 3 in the exposure groups. The sulfation yield of 4′-OH-PCB 3 was higher than hydrolysis yield of 4′-PCB 3 sulfate, indicating that formation of PCB sulfates was predominant over the reverse reaction, the formation of hydroxy-PCBs. These results provide new perspective on the transport, metabolism, and fate of MeO-PCBs, and also help to better understand sources of OH-PCBs and PCB sulfates in the environment. This study provides the first evidence of interconversion of sulfate metabolites from methoxy-PCBs and methoxy-PCBs from PCB sulfates.

Induction and characterization of tetraploids in poplar

Tetraploid poplar plants were induced by colchicine from tissue-cultured shoots; the induction efficiency varied with colchicine concentration. Morphologically, induced tetraploid plants were significantly distinguishable from diploid plants; their leaves were larger, thicker, and rounder, with larger stomata and more chloroplast numbers but smaller stomata index in the abaxial leaf epidermis. Data from validation with chromosome counting and flow cytometry corresponded well with changes in stomata size and chloroplast number. This ready use protocol would facilitate poplar breeding. Tetraploid induction by colchicine in Populus euroamericana from tissue cultured shoots.

Effect of wastewater sludge treatment on early growth and physiological responses of willow (Salix spp.) and poplar (Populus spp.) pot-grown plants

Combining biomass production from fast-growing woody species with wastewater treatment is an attractive concept from the economic, energy and environmental points of view. A pilot experiment on study-ing the effect of wastewater treatment on the early growth and initial physiological responses of willow and poplar plants was carried out in greenhouse conditions. The survival, height growth, net photosyn-thesis, stomatal conductance and transpiration of two hybrid clones of black poplar (Populus x euroameri-cana (Dode) Guinier) (I-214 and I-45/51) and white willow (S. alba L.) pot-grown plants treated with different doses of agrochemically characterised wastewater sludge were studied during the establishment year. The height growth and number of resprouted willow shoots were also measured during the second year after cutting, at the end of the vegetation season. While the early growth height of poplar clones was markedly affected by the clone origin and wastewater sludge treatment, the latter did not considerably influence the height of the willow plants either during the establishment and the following year, but essentially increased the number of resprouted shoots during the second vegetation season. The rate of CO2 assimilation of willow plants was significantly influenced by the wastewater sludge treatment, with plants grown at the higher doses displaying higher photosynthetic performance. The application of wastewater sludge had no essential impact on the values of the physiological parameters of the poplar plants, but particular responses were found among the clones.

Poplar MYB117 promotes anthocyanin synthesis and enhances flavonoid B-ring hydroxylation by up-regulating the flavonoid 3',5'-hydroxylase gene.

Flavonoids, such as anthocyanins, proanthocyanidins, and flavonols, are widespread plant secondary metabolites and important for plant adaptation to diverse abiotic and biotic stresses. Flavonoids can be variously hydroxylated and decorated; their biological activity is partly dependent on the degree of hydroxylation of the B-ring. Flavonoid biosynthesis is regulated by MYB transcription factors, which have been identified and characterized in a diversity of plants. Here we characterize a new MYB activator, MYB117, in hybrid poplar (Populus tremula×tremuloides). When overexpressed in transgenic poplar plants, MYB117 enhanced anthocyanin accumulation in all tissues. Transcriptome analysis of MYB117-overexpressing poplars confirmed the up-regulation of flavonoid and anthocyanin biosynthesis genes, as well as two flavonoid 3',5'-hydroxylase (F3'5'H) genes. We also identified up-regulated cytochrome b5 genes, required for full activity of F3'5'H . Phytochemical analysis demonstrated a corresponding increase in B-ring hydroxylation of anthocyanins, proanthocyanidins, and flavonols in these transgenics. Similarly, overexpression of F3'5'H1 directly in hybrid poplar also resulted in increased B-ring hydroxylation, but without affecting overall flavonoid content. However, the overexpression of the cytochrome b5 gene in F3'5'H1-overexpressing plants did not further increase B-ring hydroxylation. Our data indicate that MYB117 regulates the biosynthesis of anthocyanins in poplar, but also enhances B-ring hydroxylation by up-regulating F3'5'H1.

Isoprene and β‐caryophyllene confer plant resistance via different plant internal signalling pathways

Isoprene and other terpenoids are important biogenic volatile organic compounds in terms of atmospheric chemistry. Isoprene can aid plant performance under abiotic stresses, but the fundamental biological reasons for the high emissions are not completely understood. Here, we provide evidence of a previously unrecognized ecological function for isoprene and for the sesquiterpene, ß-caryophyllene. We show that isoprene and ß-caryophyllene act as core components of plant signalling networks, inducing resistance against microbial pathogens in neighbouring plants. We challenged Arabidopsis thaliana with Pseudomonas syringae, after exposure to pure volatile terpenoids or to volatile emissions of transformed poplar or Arabidopsis plants. The data suggest that isoprene induces a defence response in receiver plants that is similar to that elicited by monoterpenes and depended on salicylic acid (SA) signalling. In contrast, the sesquiterpene, ß-caryophyllene, induced resistance via jasmonic acid (JA)-signalling. The experiments in an open environment show that natural biological emissions are enough to induce resistance in neighbouring Arabidopsis. Our results show that both isoprene and ß-caryophyllene function as allelochemical components in complex plant signalling networks. Knowledge of this system may be used to boost plant immunity against microbial pathogens in various crop management schemes.

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.

ФЛАВОНОИДЫ РАСТЕНИЙ POPULUS BALSAMIFERA И ИХ БИОЛОГИЧЕСКАЯ АКТИВНОСТЬ

The work is devoted to the chemical studying of the composition of flavonoids of Populus balsamifera, the isolation of individual compounds, and the establishment of their structure. Balsamic poplar plants are a promising source of many biologically active substances, and in particular, flavonoids. From the buds of Populus balsamifera L. identified four flavonoids with antifungal, antimicrobial antioxidant properties and hepatoprotective activity. Data of flavonoids were isolated by extraction on the soxlet apparatus with 95% ethanol by triple thermal extraction at a temperature of 60 °C, followed by thickening on a rotary evaporator. For isolation from the buds of balsamic poplar, the resulting extract was chromatographed on a column with silica gel of the KSK brand 0.31-0.63 microns, using petroleum ether as eluents petroleum ether-ethyl acetate in various ratios and ethyl acetate. The structure of the isolated compounds was determined by IR, UV, NMR 13C, 1H spectroscopy. The biological activity of the isolated flavonoids was also studied.

An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar

BackgroundForest trees have important economic and ecological value. As a model tree, poplar has played a significant role in elucidating the molecular mechanisms underlying tree biology. However, a lack of mutant libraries and time-consuming stable genetic transformation processes severely limit progress into the functional characterization of poplar genes. A convenient and fast transient transformation method is therefore needed to enhance progress on functional genomics in poplar.MethodsA total of 11 poplar clones were screened for amenability to syringe infiltration. Syringe infiltration was performed on the lower side of the leaves of young soil-grown plants. Transient expression was evaluated by visualizing the reporters β-glucuronidase (GUS) and green fluorescent protein (GFP). The experimental parameters of the syringe agroinfiltration were optimized based on the expression levels of the reporter luciferase (LUC). Stably transformed plants were regenerated from transiently transformed leaf explants through callus-induced organogenesis. The functions of Populus genes in secondary cell wall-thickening were characterized by visualizing lignin deposition therein after staining with basic fuchsin.ResultsWe greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar through screening for a suitable poplar clone from a variety of clones and optimizing the syringe infiltration procedure. The selected poplar clone, Populus davidiana × P. bolleana, is amenable to Agrobacterium syringe infiltration, as indicated by the easy diffusion of the bacterial suspension inside the leaf tissues. Using this technique, we localized a variety of poplar proteins in specific intracellular organelles and illustrated the protein–protein and protein–DNA interactions. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes. Furthermore, transdifferentiation of the protoxylem-like vessel element and ectopic secondary wall thickening were induced in the agroinfiltrated leaves via the transient overexpression of genes associated with secondary wall formation.ConclusionsThe application of P. davidiana × P. bolleana in Agrobacterium syringe infiltration provides a foundation for the rapid and high-throughput functional characterization of Populus genes in intact poplar plants, including those involved in wood formation, and provides an effective alternative to Populus stable genetic transformation.

Removal of multi-contaminants from water by association of poplar and Brassica plants in a short-term growth chamber experiment.

The plant association of Populus alba L. 'Villafranca', Brassica oleracea var. acephala sebellica (kale), and B. oleracea var. capitata 'sonsma' (cabbage) was exposed to Zn, Cd, and exogenous caffeine (13CFN)-contaminated water under growth chamber conditions. In the short term of treatment (15days), poplar increased the root dry biomass (+ 25%) and decreased the chlorophyll content in new leaves (- 32%), compared to control. On the contrary, cabbage decreased the root dry biomass, enhancing the shoot dry biomass (+ 50%). Heavy metals were mainly concentrated in plant roots and in poplar reached the highest concentrations of 705 ± 232.6 and 338 ± 85.5μgg-1 DW for Zn and Cd, respectively. The ability of poplar to accumulate more Zn and Cd than kale and cabbage in plant biomass was confirmed by heavy metal contents, following the order: poplar > kale = cabbage. However, poplar and Brassica sp. association was very useful for Zn and Cd decontaminations as reported by the bioconcentration factors (> 1). The concentration of 13CFN was below 2.4ngg-1 FW in poplar and 7.4ngg-1 FW in Brassica species, suggesting the caffeine uptake and degradation by plant association. Under our experimental conditions, the removal efficiency of the system was upper to 79%, indicating the capability of Populus-Brassica association to efficiently remove Zn, Cd, and 13CFN from mixed inorganic-organic-contaminated water in short term.

Plastid Transformation in Poplar: A Model for Perennial Trees.

Poplar (Populus) is an important forest tree and considered model for perennial trees. Here we describe a method for poplar plastid transformation, which involves preparation of explants, vector construction, biolistic bombardment, regeneration and selection of transplastomic poplar plants. The young leaves of 4-week-old poplar plants are used for biolistic bombardment and aadA gene as selectable marker. Homoplasmic transplastomic lines are obtained after regeneration and several rounds of selection with spectinomycin over 10months. Homoplasmy is further confirmed by Southern blot. The establishment of a plastid transformation system for Populus is likely to make a significant contribution to tree genetic improvement.

Ground Beetles (Carabidae) in the Short-Rotation Coppice Willow and Poplar Plants—Synergistic Benefits System

In a short period, we have observed the rapid expansion of bioenergy, resulting in growth in the area of energy crops. In Europe, willow and poplar growing in short-rotation coppices (SRC) are popular bioenergy crops. Their potential impact on biodiversity has not yet been fully investigated. Therefore, there are many uncertainties regarding whether commercial production can cause environmental degradation and biodiversity impoverishment. One of the aspects examined is the impact of these crops on entomofauna and ecosystem services. The best-studied insect group is ground beetles from the Carabidae family. This work gathers data on biodiversity and the functions of carabids in willow and poplar energy plants. The results of these investigations show that energy SRC plants and Carabidae communities can create a synergistic system of mutual benefits. Willow and poplar plants can be a valuable habitat due to the increased biodiversity of entomofauna. Additionally, SRC creates a transitional environment that allows insect migration between isolated populations. On the other hand, ground beetles are suppliers of ecosystem services and make a significant contribution to the building of sustainable agriculture by pest control, thereby ameliorating damage to field crops.

Selective One-Dimensional 13C-13C Spin-Diffusion Solid-State Nuclear Magnetic Resonance Methods to Probe Spatial Arrangements in Biopolymers Including Plant Cell Walls, Peptides, and Spider Silk.

Two-dimensional (2D) and 3D through-space 13C-13C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13C-enriched biomolecules, but necessarily long acquisition times restrict their applications. In this work, we explore the broad utility and underutilized power of a chemical shift-selective one-dimensional (1D) version of a 2D 13C-13C spin-diffusion solid-state NMR technique. The method, which is called 1D dipolar-assisted rotational resonance (DARR) difference, is applied to a variety of biomaterials including lignocellulosic plant cell walls, microcrystalline peptide fMLF, and black widow dragline spider silk. 1D 13C-13C spin-diffusion methods described here apply in select cases in which the 1D 13C solid-state NMR spectrum displays chemical shift-resolved moieties. This is analogous to the selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment utilized in liquid-state NMR as a faster (1D instead of 2D) and often less ambiguous (direct sampling of the time domain data, coupled with increased signal averaging) alternative to 2D NOESY. Selective 1D 13C-13C spin-diffusion methods are more time-efficient than their 2D counterparts such as proton-driven spin diffusion (PDSD) and dipolar-assisted rotational resonance. The additional time gained enables measurements of 13C-13C spin-diffusion buildup curves and extraction of spin-diffusion time constants TSD, yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13C-enriched hybrid poplar woody stems confirm strong spatial interaction between lignin and acetylated xylan polymers within poplar plant secondary cell walls, and an interpolymer distance of ∼0.45-0.5 nm was estimated. Additionally, Tyr/Gly long-range correlations were observed on isotopically enriched black widow spider dragline silks.

Overexpression of the bamboo sucrose synthase gene (BeSUS5) improves cellulose production, cell wall thickness and fiber quality in transgenic poplar

In plants, sucrose synthase (SUS, EC 2.4.1.13) is widely considered a multifunctional protein involved in modulating sink strength, cellulose biosynthesis, and carbon partitioning. However, supporting genetic evidence regarding the role of SUS from bamboo in fiber development is lacking. Here, we obtained transgenic poplar lines overexpressing the bamboo BeSUS5 gene and conducted functional analysis. We found that overexpression of BeSUS5 enhanced the activity of SUS and significantly promoted the growth of the plants, especially xylem growth. In BeSUS5 overexpressed poplar plants, the total soluble sugar (TSS) and starch contents were decreased in leaves, while the cellulose content was increased in stems, indicating that overexpression of BeSUS5 might enhance the partitioning of carbon to cellulose in poplar. Consistent with these results, the expression of cellulose biosynthesis and phloem loading–related genes, such as cellulose synthase (CesA7), KORRIGAN (KOR), and sucrose transporter (SUT1), was upregulated in transgenic plants. As a result, transgenic poplars displayed not only an increase in cell wall thickness and cell wall crystallinity but also an altered stem fiber phenotype. Taken together, our results imply the vast potential of BeSUS5 for the genetic improvement of wood cellulose production and fiber quality.

Integrated proteomic and metabolomic analyses revealed molecular adjustments in Populus × canescens colonized with the ectomycorrhizal fungus Paxillus involutus, which limited plant host growth.

Ectomycorrhizae (ECMs) are a highly context-dependent interactions that are not always beneficial for the plant host, sometimes leading to a decrease in plant growth. However, the molecular status of these plants remains unknown. We studied Populus × canescens microcuttings characterized by impaired growth in response to colonization by a Paxillus involutus strain via integrative proteomics-metabolomics analyses. The analysed strain was characterized by low compatibility and formed only mantles, not a Hartig net, in the majority of root tips. The increased abundance of photosynthetic proteins and foliar carbohydrates co-occurred with signals of intensified resource exchange via the stems of colonized plants. In the roots, intensified C metabolism resulted in the biosynthesis of secondary C compounds unavailable to the fungal partner but also C skeletons necessary to increase insufficient N uptake from the hyphae. The stress response was also detected in colonized plants but was similar to that reported previously during mutualistic ECM interactions. In colonized poplar plants, mechanisms to prevent imbalanced C/N trade-offs were activated. Root metabolism strongly depended on features of the whole plant, especially the foliar C/N budget. However, despite ECM-triggered growth impairment and the foliar nutrient status, the fungal partner was recognized to be a symbiotic partner.

MYB134-RNAi poplar plants show reduced tannin synthesis in leaves but not roots, and increased susceptibility to oxidative stress.

The importance of the poplar MYB134 gene in controlling condensed tannin (CT) biosynthesis was tested by suppressing its expression using RNA interference (RNAi). MYB134-RNAi plants grew normally but showed reduced accumulation of stress-induced CTs in leaves. RNA-seq analysis indicated that flavonoid- and CT-related genes, as well as additional CT regulators, were strongly and specifically down-regulated by MYB134 suppression. This confirmed that the primary MYB134 target is the leaf flavonoid and CT pathway. Root CT accumulation was not impacted by MYB suppression, suggesting that additional CT regulators are active in roots and emphasizing the complexity of the regulation of CTs in poplar. To test the effect of CT down-regulation on oxidative stress resistance, leaves of MYB134-RNAi and control plants were exposed to the reactive oxygen species generator methyl viologen. MYB134-RNAi leaves sustained significantly more photosystem II damage, as seen in reduced chlorophyll fluorescence, compared with wild-type leaves. MYB134-RNAi leaves also contained more hydrogen peroxide, a reactive oxygen species, compared with the wild type. Our data thus corroborate the hypothesis that CT can act as an antioxidant in vivo and protect against oxidative stress. Overall, MYB134 was shown to be a central player in the regulation of CT synthesis in leaves.

Simultaneous manipulation of lignin structure and secondary cell wall formation in transgenic poplar

Increasing the wood density to a preferred range contributes to upgrading the value of wood as raw material. Lignin manipulation can also improve wood quality. This study attempted to accelerate secondary cell wall formation in transgenic poplar with an altered lignin structure. To achieve this, OsSWN1, a rice master switch for secondary cell wall formation in fiber cells, was overexpressed in poplar plants in which monolignol biosynthesis was suppressed by the expression of an RNA-interference construct targeted to the gene for cinnamyl alcohol dehydrogenase 1. The generated transgenic poplars successfully overexpressed the chimeric OsSWN1 construct and monolignol biosynthesis remained altered in these plants. Secondary cell wall thickness was increased in the transgenic plants and their wood density was higher compared to the background line. No difference in lignin content was observed, except in one transgenic line. High saccharification characteristics observed in the background line were preserved in the transgenic line with lower OsSWN1 overexpression, but higher OsSWN1 overexpression had a slight negative impact on enzyme saccharification. Our data suggested that fine-tuning of accelerated deposition of the secondary cell wall combined with alteration of monolignol biosynthesis should improve the lignocellulose quality for conventional and future biorefinery uses.

MiR156 regulates anthocyanin biosynthesis through SPL targets and other microRNAs in poplar

Anthocyanins biosynthesized from the flavonoid pathway are types of pigments that are involved in the protection of poplar from biotic and abiotic stresses. Previous researchers studying anthocyanin-related transcription factors and structural genes in poplar have made significant discoveries. However, little is known about the regulatory role of microRNAs in anthocyanin biosynthesis in poplar. Here, we overexpressed miR156 in poplar to study the comprehensive effects of the miR156-SPL module on the biosynthesis of anthocyanins. Small RNA sequencing analysis revealed 228 microRNAs differentially expressed in transgenic poplar plants with dramatically increased miR156 levels. Furthermore, integrated microRNAomic and transcriptomic analysis suggested that two microRNAs, miR160h, and miR858, have the potential to affect anthocyanin accumulation in poplar by regulating auxin response factors and MYB transcription factors, respectively. Additionally, the accumulation of miR160h and miR858 displayed a positive correlation with miR156 levels, suggesting a possible interaction between the miR156-SPL module and these microRNAs in poplar. Last, metabolomics analysis revealed that the levels of anthocyanins, flavones, and flavonols were substantially elevated in transgenic poplar plants overexpressing miR156 compared with the wild type, whereas the total lignin content was reduced in the transgenic plants. Taken together, our results indicate that miR156 can fine tune the anthocyanin biosynthetic pathway via multiple factors, including microRNAs, transcription factors, and the levels of structural genes, in poplar. This provides additional clues for understanding the complex regulatory network of anthocyanin biosynthesis in woody plants.