Ante- and post-mortem cellular injury dynamics in hybrid poplar foliage as a function of phytotoxic O3 dose.

After reaching phytotoxic levels during the last century, tropospheric ozone (O3) pollution is likely to remain a major concern in the coming decades. Despite similar injury processes, there is astounding interspecific-and sometimes intraspecific-foliar symptom variability, which may be related to spatial and temporal variation in injury dynamics. After characterizing the dynamics of physiological responses and O3 injury in the foliage of hybrid poplar in an earlier study, here we investigated the dynamics of changes in the cell structure occurring in the mesophyll as a function of O3 treatment, time, phytotoxic O3 dose (POD0), leaf developmental stage, and mesophyll layer. While the number of Hypersensitive Response-like (HR-like) lesions increased with higher O3 concentrations and POD0, especially in older leaves, most structural HR-like markers developed after cell death, independent of the experimental factors. The pace of degenerative Accelerated Cell Senescence (ACS) responses depended closely on the O3 concentration and POD0, in interaction with leaf age. Changes in total chlorophyll content, plastoglobuli and chloroplast shape pointed to thylakoid membranes in chloroplasts as being especially sensitive to O3 stress. Hence, our study demonstrates that early HR-like markers can provide reasonably specific, sensitive and reliable quantitative structural estimates of O3 stress for e.g. risk assessment studies, especially if they are associated with degenerative and thylakoid-related injury in chloroplasts from mesophyll.

PtrVCS2 Regulates Drought Resistance by Changing Vessel Morphology and Stomatal Closure in Populus trichocarpa.

Drought has severe effects on plant growth, forest productivity, and survival throughout the world. Understanding the molecular regulation of drought resistance in forest trees can enable effective strategic engineering of novel drought-resistant genotypes of tree species. In this study, we identified a gene, PtrVCS2, encoding a zinc finger (ZF) protein of the ZF-homeodomain transcription factor in Populus trichocarpa (Black Cottonwood) Torr. & A. Gray. ex Hook. Overexpression of PtrVCS2 (OE-PtrVCS2) in P. trichocarpa resulted in reduced growth, a higher proportion of smaller stem vessels, and strong drought-resistance phenotypes. Stomatal movement experiments revealed that the OE-PtrVCS2 transgenics showed lower stomata apertures than wild-type plants under drought conditions. RNA-seq analysis of the OE-PtrVCS2 transgenics showed that PtrVCS2 regulates the expression of multiple genes involved in regulation of stomatal opening and closing, particularly the PtrSULTR3;1-1 gene, and several genes related to cell wall biosynthesis, such as PtrFLA11-12 and PtrPR3-3. Moreover, we found that the water use efficiency of the OE-PtrVCS2 transgenic plants was consistently higher than that of wild type plants when subjected to chronic drought stress. Taken together, our results suggest that PtrVCS2 plays a positive role in improving drought adaptability and resistance in P. trichocarpa.

Contributions of ectomycorrhizal fungi in a reclaimed poplar forest (Populus yunnanensis) in an abandoned metal mine tailings pond, southwest China.

Reclamation using fast-growing trees has great potential for agroforestry development on former non-ferrous metal mining areas. However, the functional traits of ectomycorrhizal fungi (ECMF) and the relationship between ECMF and reclaimed trees remain unknown. Here, the restoration of ECMF and their functions in reclaimed poplar (Populus yunnanensis) growing in a derelict metal mine tailings pond were investigated. We identified ECMF belonging to 15 genera in 8 families, suggesting the occurrence of spontaneous diversification as poplar reclamation progressed. We described a previously unknown ectomycorrhizal relationship between poplar roots and Bovista limosa. Our results showed that B. limosa PY5 alleviated the phytotoxicity of Cd and enhanced poplar heavy metal tolerance, resulting in increased plant growth due to reduced Cd accumulation in host tissues. As part of the improved metal tolerance mechanism, PY5 colonization activated antioxidant systems, enhanced the conversion of Cd into inactive chemical forms, and promoted the compartmentalization of Cd into host cell walls. These results suggest that introducing adaptative ECMF may be an alternative to bioaugmenting reforestation and phytomanagement programs of fast-growing native trees in the barren metal mining and smelting areas.

Genome-Wide Identification of the ERF Transcription Factor Family for Structure Analysis, Expression Pattern, and Response to Drought Stress in Populus alba × Populus glandulosa.

The Ethylene Responsive Factor (ERF) transcription factor family is important for regulating plant growth and stress responses. Although the expression patterns of ERF family members have been reported in many plant species, their role in Populus alba × Populus glandulosa, an important model plant for forest research, remains unclear. In this study, we identified 209 PagERF transcription factors by analyzing the P. alba × P. glandulosa genome. We analyzed their amino acid sequences, molecular weight, theoretical pI (Isoelectric point), instability index, aliphatic index, grand average of hydropathicity, and subcellular localization. Most PagERFs were predicted to localize in the nucleus, with only a few PagERFs localized in the cytoplasm and nucleus. Phylogenetic analysis divided the PagERF proteins into ten groups, Class I to X, with those belonging to the same group containing similar motifs. Cis-acting elements associated with plant hormones, abiotic stress responses, and MYB binding sites were analyzed in the promoters of PagERF genes. We used transcriptome data to analyze the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa, including axillary buds, young leaves, functional leaves, cambium, xylem, and roots, and the results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots. Quantitative verification results were consistent with transcriptome data. When P. alba × P. glandulosa seedlings were treated with 6% polyethylene glycol 6000 (PEG6000), the results of RT-qRCR showed that nine PagERF genes responded to drought stress in various tissues. This study provides a new perspective on the roles of PagERF family members in regulating plant growth and development, and responses to stress in P. alba × P. glandulosa. Our study provides a theoretical basis for ERF family research in the future.

Genome-Wide Analysis of SIMILAR TO RCD ONE (SRO) Family Revealed Their Roles in Abiotic Stress in Poplar.

SIMILAR TO RCD ONE (SRO) gene family is a small plant-specific gene family responsible for growth, development, and stress responses. In particular, it plays a vital role in responding to abiotic stresses such as salt, drought, and heavy metals. Poplar SROs are rarely reported to date. In this study, a total of nine SRO genes were identified from Populus simonii × Populus nigra, which are more similar to dicotyledon SRO members. According to phylogenetic analysis, the nine PtSROs can be divided into two groups, and the members in the same cluster have a similar structure. There were some cis-regulatory elements related to abiotic stress response and hormone-induced factors identified in the promoter regions of PtSROs members. Subcellular localization and transcriptional activation activity of PtSRO members revealed a consistent expression profile of the genes with similar structural profiles. In addition, both RT-qPCR and RNA-Seq results indicated that PtSRO members responded to PEG-6000, NaCl, and ABA stress in the roots and leaves of Populus simonii × Populus nigra. The PtSRO genes displayed different expression patterns and peaked at different time points in the two tissues, which was more significant in the leaves. Among them, PtSRO1c and PtSRO2c were more prominent in response to abiotic stress. Furthermore, protein interaction prediction showed that the nine PtSROs might interact with a broad range of transcription factors (TFs) involved in stress responses. In conclusion, the study provides a solid basis for functional analysis of the SRO gene family in abiotic stress responses in poplar.

PyuARF16/33 Are Involved in the Regulation of Lignin Synthesis and Rapid Growth in Populus yunnanensis.

(1) Background: Lignin is a unique component of the secondary cell wall, which provides structural support for perennial woody plants. ARFs are the core factors of the auxin-signaling pathway, which plays an important role in promoting plant growth, but the specific relationship between auxin response factors (ARFs) and lignin has not been fully elucidated with regard to rapid plant growth in forest trees. (2) Objectives: This study aimed to investigate the relationship between ARFs and lignin with regard to rapid plant growth in forest trees. (3) Methods: We used bioinformatics analysis to investigate the PyuARF family, find genes homologous to ARF6 and ARF8 in Populus yunnanensis, and explore the changes in gene expression and lignin content under light treatment. (4) Results: We identified and characterized 35 PyuARFs based on chromosome-level genome data from P. yunnanensis. In total, we identified 92 ARF genes in P. yunnanensis, Arabidopsis thaliana, and Populus trichocarpa, which were subsequently divided into three subgroups based on phylogenetic analysis and classified the conserved exon-intron structures and motif compositions of the ARFs into the same subgroups. Collinearity analysis suggested that segmental duplication and whole-genome duplication events were majorly responsible for the expansion of the PyuARF family, and the analysis of Ka/Ks indicated that the majority of the duplicated PyuARFs underwent purifying selection. The analysis of cis-acting elements showed that PyuARFs were sensitive to light, plant hormones, and stress. We analyzed the tissue-specific transcription profiles of PyuARFs with transcriptional activation function and the transcription profiles of PyuARFs with high expression under light in the stem. We also measured the lignin content under light treatment. The data showed that the lignin content was lower, and the gene transcription profiles were more limited under red light than under white light on days 1, 7, and 14 of the light treatments. The results suggest that PyuARF16/33 may be involved in the regulation of lignin synthesis, thereby promoting the rapid growth of P. yunnanensis. (5) Conclusions: Collectively, this study firstly reports that PyuARF16/33 may be involved in the regulation of lignin synthesis and in promoting the rapid growth in P. yunnanensis.

Comprehensive analysis of MAPK gene family in Populus trichocarpa and physiological characterization of PtMAPK3-1 in response to MeJA induction.

Mitogen-activated protein kinases (MAPKs) play important roles in plant growth and development, as well as hormone and stress responses by signaling to eukaryotic cells, through MAPK cascade, the presence of various cues; thereby, regulating various responses. The MAPK cascade consists mainly of three gene families, MAPK, MAPKK, and MAPKKK, which activate downstream signaling pathways through sequential phosphorylation. Although the MAPK cascade gene family has been reported in several species, there is a lack of comprehensive analysis in poplar. We identified 21 MAPK genes, 11 MAPKK genes, and 104 MAPKKK genes in Populus trichocarpa. The phylogenetic classification was supported by conservative motif, gene structure and motif analysis. Whole genome duplication has an important role in the expansion of MAPK cascade genes. Analysis of promoter cis-elements and expression profiles indicates that MAPK cascade genes have important roles in plant growth and development, abiotic and biotic stresses, and phytohormone response. Expression profiling revealed a significant upregulation of PtMAPK3-1 expression in response to drought, salt and disease stresses. Poplar transiently overexpressing PtMAPK3-1 and treated with methyl jasmonic acid (MeJA) had higher catalase and peroxidase levels than non-overexpressing poplar. This work represents the first complete inventory of the MAPK cascade in P. trichocarpa, which reveals that PtMAPK3-1 is induced by the MeJA hormone and participates in the MeJA-induced enhancement of the antioxidant enzyme system.

Transcriptome analysis of Populus × canadensis 'Zhongliao1' in response to low temperature stress.

BACKGROUND: Low temperatures are known to limit the growth and geographical distribution of poplars. Although some transcriptomic studies have been conducted to explore the response of poplar leaves to cold stress, only a few have comprehensively analyzed the effects of low temperature on the transcriptome of poplars and identified genes related to cold stress response and repair of freeze-thaw injury. RESULTS: We exposed the Euramerican poplar Zhongliao1 to low temperatures; after stems were exposed to - 40℃, 4℃, and 20℃, the mixture of phloem and cambium was collected for transcriptome sequencing and bioinformatics analysis. A total of 29,060 genes were detected, including 28,739 known genes and 321 novel genes. Several differentially expressed genes (n = 36) were found to be involved in the Ca2+ signaling pathway, starch-sucrose metabolism pathway, abscisic acid signaling pathway, and DNA repair. They were functionally annotated; glucan endo-1,3-beta-glucosidase and UDP-glucuronosyltransferase genes, for instance, showed a close relationship with cold resistance. The expression of 11 differentially expressed genes was verified by qRT-PCR; RNA-Seq and qRT-PCR data were found to be consistent, which validated the robustness of our RNA-Seq findings. Finally, multiple sequence alignment and evolutionary analysis were performed, the results of which suggested a close association between several novel genes and cold resistance in Zhongliao1. CONCLUSION: We believe that the cold resistance and freeze-thaw injury repair genes identified in this study are of great significance for cold tolerance breeding.

Forest health in the Anthropocene: the emergence of a novel tree disease is associated with poplar cultivation.

Plant domestication and movement are large contributors to the success of new diseases. The introduction of new host species can result in accelerated evolutionary changes in pathogens, affecting long-established coevolutionary dynamics. This has been observed in poplars where severe epidemics of pathogens that were innocuous in their natural pathosystems occurred following host domestication. The North American fungus Sphaerulina musiva is responsible for endemic leaf spots on Populus deltoides. We show that the expansion of poplar cultivation resulted in the emergence of a new lineage of this pathogen that causes stem infections on a new host, P. balsamifera. This suggests a host shift since this is not a known host. Genome analysis of this emerging lineage reveals a mosaic pattern with islands of diversity separated by fixed genome regions, which is consistent with a homoploid hybridization event between two individuals that produced a hybrid swarm. Genome regions of extreme divergence and low diversity are enriched in genes involved in host-pathogen interactions. The specialization of this emerging lineage to a new host and its clonal propagation represents a serious threat to poplars and could affect both natural and planted forests. This work provides a clear example of the changes created by the intensification of tree cultivation that facilitate the emergence of specialized pathogens, jeopardizing the natural equilibrium between hosts and pathogens. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Identification and Functional Prediction of CircRNAs in Leaves of F1 Hybrid Poplars with Different Growth Potential and Their Parents.

Circular RNAs (CircRNAs) regulate plant growth and development; however, their role in poplar heterosis is unclear. We identified 3722 circRNAs in poplar leaves, most of which were intergenic (57.2%) and exonic (40.2%). The expression of circRNAs in F1 hybrids with high growth potential was higher than that in those with low growth potential. Non-additive expression of circRNAs and single-parent expression of circRNAs (SPE-circRNAs) might regulate poplar heterosis through microRNA sponging and protein translation, respectively. DECs among F1 hybrids with different growth potentials might regulate the growth potential of poplar via microRNA sponging. Correlation analysis between circRNA expression and its parent gene expression showed that SPE-M circRNA (circRNAs expressed by male parent only) might regulate poplar heterosis by inhibiting parent gene expression, while other circRNAs might regulate poplar heterosis by enhancing parent gene expression. Weighted correlation network analysis of gene/circRNA expression showed that circRNAs mainly regulate poplar heterosis via carbohydrate metabolism, amino acid metabolism, energy metabolism, and material transport. In addition, we identified seven circRNAs that positively or negatively regulate poplar heterosis. Thus, non-additively expressed circRNAs and SPE circRNAs are involved in regulating poplar heterosis, and DECs among F1 hybrids with different growth potentials were involved in regulating poplar growth potential.

Genome-Wide Comparative Analysis of the Fasciclin-like Arabinogalactan Proteins (FLAs) in Salicacea and Identification of Secondary Tissue Development-Related Genes.

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) containing both AGP-like glycated domains and fasciclin (FAS) domains, which are involved in plant growth and development and synthesis of the cell wall. However, these proteins have not been identified or analyzed in willow, Salix, the sister genus of Populus. In this study, we performed a whole genome study of the FLA gene family of Salix suchowensis and compared it with the FLA gene family of Populus deltoides. The results showed the presence of 40 and 46 FLA genes in P. deltoides and S. suchowensis, distributed on 17 and 16 chromosomes, respectively. Four pairs of tandem repeat genes were found in willow, while poplar had no tandem repeat genes. Twelve and thirteen pairs of duplicated gene fragments were identified in poplar and willow, respectively. The multispecies phylogenetic tree showed that the FLA gene family could be divided into four groups (I-IV), with Group 1 showing significant expansion in woody plants. A gene expression analysis showed that PdeFLA19/27 in Group I of poplar was highly expressed, specifically during the secondary growth period of the stem and the rapid elongation of seed hairs. In the Group I genes of S. suchowensis, SsuFLA25/26/28 was also highly expressed during the secondary growth period, whereas increased expression of SsuFLA35 was associated with seed hair tissue. These results provide important clues about the differences in the FLA gene family during the evolution of herbs and woody plants, and suggest that the FLA gene family may play an essential role in regulating the secondary growth of woody plants. It also provides a reference for further studies on the regulation of secondary growth and seed hair development by FLA genes in poplar and willow.

Relationships between site index and ecological variables of trembling poplar forests (Populus tremula L.) in Türkiye.

This study was carried out to identify relationships between site index (dominant height at a reference age) and ecological variables of trembling poplar forests in Türkiye. Samples were collected from 78 plots differing in elevation, aspect, inclination, slope position, and site class. Physiographic factors of the sample plots were recorded, soil samples were collected from different predefined layers from soil pits, and bedrock samples were collected for identification. From three trees at the stand top height, the tree closest to the arithmetic mean height was felled, and its height and age were determined. Physical and chemical characteristics of the soil samples were analysed. Relationships of the soil properties, physiographic factors, and climate with site index were assessed with correlation, stepwise regression, and regression tree methods. Significant relationships were found between site index at 30 years and elevation from the physiographic factors; the maximum temperature and the number of snowy days of the coldest month from the climate characteristics; fine earth, silt, and pH from the percentage values of soil properties at different depths; and fine earth, silt, and clay from the soil characteristics aggregated on pedon level. The height growth of trembling poplar was 11.8% according to stepwise regression analysis and 18% according to the regression tree method. The models obtained in the current study might help evaluate the potential of sites regarding the growth of trembling poplar.

DNA methylation-mediated phenylpropane and starch metabolism causes male poplars to be more tolerant to nitrogen deficiency than females.

Nitrogen (N) is an essential nutrient for plant growth and development. Dioecious plants, especially perennial plants, are often faced with a shortage of N supply in nature. Poplar is one of the most important dioecious and perennials species. Due to the different ecological functions, female and male poplars adopt different adaptation strategies to N limitation. However, the regulation in epigenetic mechanism is poorly understood on sexes. Here, the integrative analysis of whole-genome bisulfite sequencing (WGBS), RNA sequencing, and plant physiological analysis on female and male Populus cathayana were performed. We found that N deficiency reprograms methylation in both sexes, and the CG and CHH methylation types played critical roles in female and male poplars, respectively. Induced by DNA methylation, N-deficient males had a stronger phenylpropanoid synthesis pathway and less anthocyanin accumulation than females, which not only strengthened the N cycle but also reduced the defense cost of males. In addition, compared with male poplars, females accumulated more starch to expend excess energy under N limited condition. Additionally, DNA methylation also mediated hormone signalling involved in anthocyanin synthesis and starch metabolism. Therefore, our study reveals new molecular evidences that male poplars are more tolerant to N deficiency than females, which provides a reference for ecological adaptability of forest trees.

Over-expression of DREB46 enhances drought tolerance in Populus trichocarpa.

The drought responsive element binding (DREB) gene family has a significant role in plant abiotic stress responses. Here, we cloned a drought-inducible DREB gene, DREB46 (Potri.019G075500), and investigated its function in drought tolerance in Populus trichocarpa. Under treatment with exogenous abscisic acid and 6% PEG6000, DREB46 was rapidly and abundantly expressed. We successfully inserted P. trichocarpa DREB46 constructs into P. trichocarpa. After 11 d of drought stress and 3 d of rehydration treatment, the DREB46 over-expression (OE) lines exhibited significantly increased survival rates relative to the wild type (WT). Histochemical staining showed that the accumulation of reactive oxygen species (ROS) in transgenic plants under drought stress was lower than that in WT plants. Furthermore, OE plants displayed higher superoxide dismutase, peroxidase, and catalase activities and proline content, but lower malondialdehyde content than the WT plants under drought stress. In contrast, DREB46-RNA interference (RNAi) lines exhibited the opposite phenotype. Under PEG-6000 stress, OE plants produced significantly more adventitious roots (ARs) than WT plants. In contrast, RNAi-mediated DREB46-inhibited poplar exhibited fewer ARs. Quantitative real-time PCR indicated that WOX11/12a (Potri.013G066900), a gene related to root growth and development regulation, was significantly increased in OE plants. Additionally, yeast two-hybrid (Y2H) assays showed that DREB46 could interact with protein kinase MPK1 (Potri.002G032100) and protein phosphatase PP2C47 (Potri.007G058700), respectively, and this result was also verified by luciferase complementation assay. Transient co-expression results of leaves showed that PP2C47 and DREB46 Agrobacterium-transformed leaves had strong drought tolerance. These results show that DREB46 plays a key role in drought tolerance by inducing the ROS scavenging system and increasing the number of ARs.

Genome-Wide Identification and Characterization of Auxin Response Factor (ARF) Gene Family Involved in Wood Formation and Response to Exogenous Hormone Treatment in Populus trichocarpa.

Auxin is a key regulator that virtually controls almost every aspect of plant growth and development throughout its life cycle. As the major components of auxin signaling, auxin response factors (ARFs) play crucial roles in various processes of plant growth and development. In this study, a total of 35 PtrARF genes were identified, and their phylogenetic relationships, chromosomal locations, synteny relationships, exon/intron structures, cis-elements, conserved motifs, and protein characteristics were systemically investigated. We also analyzed the expression patterns of these PtrARF genes and revealed that 16 of them, including PtrARF1, 3, 7, 11, 13-17, 21, 23, 26, 27, 29, 31, and 33, were preferentially expressed in primary stems, while 15 of them, including PtrARF2, 4, 6, 9, 10, 12, 18-20, 22, 24, 25, 28, 32, and 35, participated in different phases of wood formation. In addition, some PtrARF genes, with at least one cis-element related to indole-3-acetic acid (IAA) or abscisic acid (ABA) response, responded differently to exogenous IAA and ABA treatment, respectively. Three PtrARF proteins, namely PtrARF18, PtrARF23, and PtrARF29, selected from three classes, were characterized, and only PtrARF18 was a transcriptional self-activator localized in the nucleus. Moreover, Y2H and bimolecular fluorescence complementation (BiFC) assay demonstrated that PtrARF23 interacted with PtrIAA10 and PtrIAA28 in the nucleus, while PtrARF29 interacted with PtrIAA28 in the nucleus. Our results provided comprehensive information regarding the PtrARF gene family, which will lay some foundation for future research about PtrARF genes in tree development and growth, especially the wood formation, in response to cellular signaling and environmental cues.

Cell-type-specific PtrWOX4a and PtrVCS2 form a regulatory nexus with a histone modification system for stem cambium development in Populus trichocarpa.

Stem vascular cambium cells in forest trees produce wood for materials and energy. WOX4 affects the proliferation of such cells in Populus. Here we show that PtrWOX4a is the most highly expressed stem vascular-cambium-specific (VCS) gene in P. trichocarpa, and its expression is controlled by the product of the second most highly expressed VCS gene, PtrVCS2, encoding a zinc finger protein. PtrVCS2 binds to the PtrWOX4a promoter as part of a PtrWOX13a-PtrVCS2-PtrGCN5-1-PtrADA2b-3 protein tetramer. PtrVCS2 prevented the interaction between PtrGCN5-1 and PtrADA2b-3, resulting in H3K9, H3K14 and H3K27 hypoacetylation at the PtrWOX4a promoter, which led to fewer cambium cell layers. These effects on cambium cell proliferation were consistent across more than 20 sets of transgenic lines overexpressing individual genes, gene-edited mutants and RNA interference lines in P. trichocarpa. We propose that the tetramer-PtrWOX4a system may coordinate genetic and epigenetic regulation to maintain normal vascular cambium development for wood formation.

Integration of meta-analysis, machine learning and systems biology approach for investigating the transcriptomic response to drought stress in Populus species.

In Populus, drought is a major problem affecting plant growth and development which can be closely reflected by corresponding transcriptomic changes. Nevertheless, how these changes in Populus are not fully understood. Here, we first used meta-analysis and machine learning methods to identify water stress-responsive genes and then performed a systematic approach to discover important gene networks. Our analysis revealed that large transcriptional variations occur during drought stress. These changes were more associated with the response to stress, cellular catabolic process, metabolic pathways, and hormone-related genes. The differential gene coexpression analysis highlighted two acetyltransferase NATA1-like and putative cytochrome P450 genes that have a special contribution in response to drought stress. In particular, the findings showed that MYBs and MAPKs have a prominent role in the drought stress response that could be considered to improve the drought tolerance of Populus. We also suggest ARF2-like and PYL4-like genes as potential markers for use in breeding programs. This study provides a better understanding of how Populus responses to drought that could be useful for improving tolerance to stress in Populus.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.

Comprehensive estrogenic/anti-estrogenic, anticancer, mutagenic/anti-mutagenic, and genotoxic/anti-genotoxic activity studies on chemically characterized black poplar and Eurasian aspen propolis types.

Propolis is mainly composed of plant resins, and its type is named according to the primary plant origin in its composition. Identification of propolis botanical origin is essential for predicting and repeating its pharmacological activity because of the variations in chemical composition. This study aimed to compare chemical composition of black poplar (Populus nigra L.) type-propolis (PR1 and PR2) and Eurasian aspen (P. tremula L.)-type propolis (PR3) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique and to evaluate their biological activity profiles. According to LC-MS/MS results, in addition to marked caffeic acid phenethyl ester content in PR1 and PR2, flavonoid aglycones such as pinocembrin, chrysin, pinobanksin, and galangin were found to be dominant in these samples. On the other hand, PR3 contained relatively high concentrations of phenolic acids such as ferulic acid, p-coumaric acid, and trans-cinnamic acid. The anti-estrogenic activity test showed that PR2 exerted the highest anti-estrogenic activity by inhibiting cell proliferation by 44.6%. All propolis extracts showed anticancer activity, which was justified by decreasing activity on the 3D spheroid size in a concentration-dependent manner. Besides, all extracts showed moderate or potent antimutagenic activity in Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation, respectively. In addition, the Comet assay results revealed that propolis extracts have a geno-protective effect against H2O2-induced DNA damage in CHO-K1 cells at 0.625 and 1.25 µg/mL concentrations. Overall, the result of this study may help in preparing standardized propolis extracts and developing products with defined pharmacological benefits in the food supplements industry.