Reduced mesophyll conductance under chronic O3 exposure in poplar reflects thicker cell walls and increased subcellular diffusion pathway lengths according to the anatomical model.

Ozone (O3) is one of the most harmful and widespread air pollutants, affecting crop yield and plant health worldwide. There is evidence that O3 reduces the major limiting factor of photosynthesis, namely CO2 mesophyll conductance (gm), but there is little quantitative information of O3-caused changes in key leaf anatomical traits and their impact on gm. We exposed two O3-responsive clones of the economically important tree species Populus × canadensis Moench to 120 ppb O3 for 21 days. An anatomical diffusion model within the leaf was used to analyse the entire CO2 diffusion pathway from substomatal cavities to carboxylation sites and determine the importance of each structural and subcellular component as a limiting factor. gm decreased substantially under O3 and was found to be the most important limitation of photosynthesis. This decrease was mostly driven by an increased cell wall thickness and length of subcellular diffusion pathway caused by altered interchloroplast spacing and chloroplast positioning. By contrast, the prominent leaf integrative trait leaf dry mass per area was neither affected nor related to gm under O3. The observed relationship between gm and anatomy, however, was clone-dependent, suggesting that mechanisms regulating gm may differ considerably between closely related plant lines. Our results confirm the need for further studies on factors constraining gm under stress conditions.

Genome-wide identification and expression analysis of EPF/EPFL gene family in Populus trichocarpa.

The Epidermal Patterning Factor/EPF-like (EPF/EPFL) family encodes a specific type of secreted protein in plants and plays an important role in plant growth and development, especially in the process of morphogenesis. To investigate the characteristics of EPF/EPFL gene family members and their regulatory functions in stomatal development of Populus trichocarpa, a total of 15 EPF/EPFL family genes were identified. Then the gene structure, chromosome location, phylogenetic relationship, protein conserved domain and gene expression profile were analyzed. According to phylogenetic analysis, PtEPF/EPFL can be classified into four groups. The gene structure and protein conservation motifs within the EPF family indicate the high conservation of the PtEPF/EPFL sequence. The promoter region of PtEPF/EPFL was found to contain cis-elements in response to stress and plant hormones. In addition, RT-qPCR results indicated that the PtEPF/EPFL have a differentially expressed in different tissues. Under drought stress treatment, a substantial upregulation was observed in the majority of PtEPF/EPFL members, suggesting their potential involvement in drought response. These results provide a theoretical basis for future exploration of the characteristics and functions of more PtEPF/EPFL genes.

Quantitative and Structural Characterization of Native Lignin in Hardwood and Softwood Bark via Solid-State NMR Spectroscopy.

A major factor limiting bark's industrial use is its greater recalcitrance compared to wood. While lignin is widely recognized as a significant contributor, precise characterization of lignin in bark remains sparse, presenting a crucial gap that impedes understanding of its impact. In this study, we employed advanced solid-state nuclear magnetic resonance (NMR) spectroscopy to analyze bark samples from various species, including willow, poplar, and pine. We established and verified that lignin methoxy peak at 56 ppm serves as a reliable quantitative metric to assess lignin content, with which we calculated the lignin contents in bark are significantly reduced by more than 70% compared to those in wood. Furthermore, in situ characterization revealed significant reduction of ß-ether linkage in bark lignin across species, revealing a more condensed and resistant structural configuration. Our results have substantially advanced our comprehension of the composition and structure of native lignin in tree bark.

Poplar Bud (Populus) Extraction and Chinese Propolis Counteract Oxidative Stress in Caenorhabditis elegans via Insulin/IGF-1 Signaling Pathway.

Poplar buds are characterized by a high content of phenolic compounds, which exhibit a broad spectrum of biological activities. However, the relationship between Chinese propolis and poplar buds based on their antioxidant capacities and underlying mechanisms remains unclear. This study aimed to investigate the antioxidant properties of poplar bud (Populus) extract (PBE) and Chinese propolis (CP) and to elucidate the mechanisms behind their activity. High-performance liquid chromatography (HPLC) analysis revealed that both PBE and CP contain a significant amount of phenolic acids and flavonoids. 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric-reducing antioxidant power (FRAP) assays demonstrated that PBE and CP possess excellent antioxidant activity. Furthermore, administration of PBE and CP improved the survival rate of C. elegans under oxidative stress. They also decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while enhancing the activity of antioxidant enzymes (SOD, CAT). PBE and CP intervention upregulated the expression of key genes daf-16, sod-3, hsp-16.2, and skn-1 in nematodes. This suggests that the antioxidant activity of PBE and CP is dependent on daf-16 and skn-1 signaling pathways. In conclusion, poplar bud extracts ha have the potential to become a substitute for propolis and a potential therapeutic agent for treating diseases associated with oxidative damage.

Unraveling the lncRNA-miRNA-mRNA Regulatory Network Involved in Poplar Coma Development through High-Throughput Sequencing.

Poplar coma, the fluff-like appendages of seeds originating from the differentiated surface cells of the placenta and funicle, aids in the long-distance dispersal of seeds in the spring. However, it also poses hazards to human safety and causes pollution in the surrounding environment. Unraveling the regulatory mechanisms governing the initiation and development of coma is essential for addressing this issue comprehensively. In this study, strand-specific RNA-seq was conducted at three distinct stages of coma development, revealing 1888 lncRNAs and 52,810 mRNAs. The expression profiles of lncRNAs and mRNAs during coma development were analyzed. Subsequently, potential target genes of lncRNAs were predicted through co-localization and co-expression analyses. Integrating various types of sequencing data, lncRNA-miRNA-TF regulatory networks related to the initiation of coma were constructed. Utilizing identified differentially expressed genes encoding kinesin and actin, lncRNA-miRNA-mRNA regulatory networks associated with the construction and arrangement of the coma cytoskeleton were established. Additionally, relying on differentially expressed genes encoding cellulose synthase, sucrose synthase, and expansin, lncRNA-miRNA-mRNA regulatory networks related to coma cell wall synthesis and remodeling were developed. This study not only enhances the comprehension of lncRNA but also provides novel insights into the molecular mechanisms governing the initiation and development of poplar coma.

Characterisation of New Zealand Propolis from Different Regions Based on Its Volatile Organic Compounds.

Propolis is a bee product mainly consisting of plant resins and is used by bees to maintain the structural integrity of the colony. Propolis is known to contribute to bee health via its antimicrobial activity and is a valued product for human use owing to its nutritional and medicinal properties. Propolis is often characterised into seven categories depending on the resin source. New Zealand propolis is typically assumed as being poplar-type propolis, but few studies have chemically characterised New Zealand propolis to confirm or reject this assumption. Here, for the first time, we characterise propolis originating from different regions in New Zealand based on its volatile organic compounds, using gas chromatography coupled with mass spectrometry (GC-MS). To support this characterisation, we also collected and analysed resin samples from a variety of resin-producing plants (both native to New Zealand and introduced). Our findings suggest that bees mainly use poplar as a resin source, but also utilize native plant species to produce propolis. While regional variation did not allow for clear separation between samples, some patterns emerged, with samples from some regions having more chemical complexity and a higher contribution from native species (as suggested by a higher number of compounds unique to native species resin). Further studies are needed to accurately identify the botanical sources contributing to these samples. It may be also of interest to explore the biological activity of regional propolis samples and their potential nutritional or medicinal benefits.

PagbHLH35 Enhances Salt Tolerance through Improving ROS Scavenging in Transgenic Poplar.

The bHLH transcription factor family plays crucial roles in plant growth and development and their responses to adversity. In this study, a highly salt-induced bHLH gene, PagbHLH35 (Potri.018G141600), was identified from Populus alba × P. glandullosa (84K poplar). PagbHLH35 contains a highly conserved bHLH domain within the region of 52-114 amino acids. A subcellular localization result confirmed its nuclear localization. A yeast two-hybrid assay indicated PagbHLH35 lacks transcriptional activation activity, while a yeast one-hybrid assay indicated it could specifically bind to G-box and E-box elements. The expression of PagbHLH35 reached its peak at 12 h and 36 h time points under salt stress in the leaves and roots, respectively. A total of three positive transgenic poplar lines overexpressing PagbHLH35 were generated via Agrobacterium-mediated leaf disk transformation. Under NaCl stress, the transgenic poplars exhibited significantly enhanced morphological and physiological advantages such as higher POD activity, SOD activity, chlorophyll content, and proline content, and lower dehydration rate, MDA content and hydrogen peroxide (H2O2) content, compared to wild-type (WT) plants. In addition, histological staining showed that there was lower ROS accumulation in the transgenic poplars under salt stress. Moreover, the relative expression levels of several antioxidant genes in the transgenic poplars were significantly higher than those in the WT. All the results indicate that PagbHLH35 can improve salt tolerance by enhancing ROS scavenging in transgenic poplars.

Multi-omics analysis of Populus simonii × P. nigra leaves under Hyphantria cunea stress.

Poplar is an important greening and timber tree species in China, which has great economic and ecological values. However, the spread of Hyphantria cunea has become increasingly serious in recent years, resulting in huge economic loss of poplar production. Exploring the molecular mechanism of poplar reponse to H. cunea stress has significant implications for future development of new insect-resistant poplar varieties using genetic engineering technology. In this study, a total of 1039 differentially expressed genes (DEGs), 106 differentially expressed proteins (DEPs) and 212 differentially expressed metabolites (DEMs) were screened from Populus simonii × P. nigra leaves under H. cunea stress by transcriptome, proteomics and metabolomics analysis, respectively. GO and KEGG analysis showed that the DEGs and DEPs are associated with endopeptidase inhibitor activity, stress response, α-linolenic acid metabolism, phenylpropanoid biosynthesis and metabolic pathways, cysteine and methionine metabolism pathways and MAKP signaling pathway. Metabolomics analysis showed the most of DEMs were lipids and lipid molecules, and the pathways associated with transcriptome mainly include plant hormone signal transduction, α-linolenic acid metabolic pathway, amino sugar and nucleotide sugar metabolism, and phenylpropanoid biosynthesis. In particular, multi-omics analysis showed that several pathways such as α-linolenic acid metabolic, phenylpropanoid biosynthesis and metabolic pathway and cysteine and methionine metabolic pathway were significantly enriched in the three omics, which may play an important role in the resistance to pests in poplar.

A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

Effect of Fiber Loading on Mechanical and Flame-Retardant Properties of Poplar-Fiber-Reinforced Gypsum Composites.

Gypsum-based composites were prepared via a slurry casting process using construction gypsum as the binding material and poplar fibers as reinforcing material. The effects of different fiber content and curing time on the mechanical properties, water resistance, and flame retardancy of these composites were investigated, and the influence mechanism was characterized by infrared spectroscopy, scanning electron microscopy, and X-ray diffractometry. The results showed that the best composite mechanical strength was achieved with 10% poplar fiber- content, and the absolute dry flexural and compressive strengths reached 3.59 and 8.06 MPa, respectively. Compared with pure gypsum, the flexural strength and compressive strength increased by 10% and 19%, respectively. The inclusion of fibers somewhat prevented the migration of free water within the composites and enhanced their water resistance. At 10% fiber content, the composite's 24 h water absorption rate was 34.3%, 8% lower than that of pure gypsum, with a softening coefficient of 0.55. However, fiber content increases the porosity of gypsum-based composites. When heated, this increased porosity accelerates' heat conduction within the matrix, raising the peak and total exothermic rates, thereby weakening the composites' inherently flame-retardant properties. Poplar-fiber-reinforced gypsum-based composites offered superior performance in commercial applications, compared to pure gypsum board, providing a sustainable and green alternative for ceilings, partitions, and other applications, while broadening the prospects for gypsum-based composites in the engineering field.

High-resolution methylome analysis uncovers stress-responsive genomic hotspots and drought-sensitive TE superfamilies in the clonal Lombardy poplar.

DNA methylation is environment-sensitive and can mediate stress responses. In long-lived trees, changing environments might cumulatively shape the methylome landscape over their lifetime. However, because high-resolution methylome studies usually focus on single environmental cues, it remains unclear to what extent the methylation responses are generic or stress-specific, and how this relates to their long-term stability. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone that is widespread across Europe. Adult trees from a variety of geographic locations were clonally propagated in a common garden experiment, and the ramets were exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Through comprehensive whole-genome bisulfite sequencing, we analyzed stress-induced and naturally occurring DNA methylation variants. Stress-induced methylation changes predominantly targeted transposable elements. When occurring in CG/CHG contexts, the same regions were often affected by multiple stresses, suggesting a generic response of the methylome. Drought stress caused a distinct CHH hypermethylation response in transposable elements, affecting entire TE superfamilies near drought-responsive genes. Methylation differences in CG/CHG contexts that were induced by stress treatments showed striking overlap with methylation differences observed between trees from distinct geographical locations. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and we identified specific transposable element superfamilies that respond to a specific stress with possible functional consequences. Our results underscore the importance of studying the effects of multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses.

The transcriptional regulation of a putative hemicellulose gene, PtrPARVUS2 in poplar.

The plant cell wall serves as a critical interface between the plant and its environment, offering protection against various stresses and contributing to biomass production. Hemicellulose is one of the major components of the cell wall, and understanding the transcriptional regulation of its production is essential to fully understanding cell wall formation. This study explores the regulatory mechanisms underlying one of the genes involved in hemicellulose biosynthesis, PtrPARVUS2. Six transcription factors (TFs) were identified from a xylem-biased library to negatively regulate PtrPARVUS2 expression. These TFs, belonging to diverse TF families, were confirmed to bind to specific cis-elements in the PtrPARVUS2 promoter region, as validated by Yeast One-Hybrid (Y1H) assays, transient expression analysis, and Chromatin Immunoprecipitation sequencing (ChIP-seq) assays. Furthermore, motif analysis identified putative cis-regulatory elements bound by these TFs, shedding light on the transcriptional regulation of SCW biosynthesis genes. Notably, several TFs targeted genes encoding uridine diphosphate glycosyltransferases (UGTs), crucial enzymes involved in hemicellulose glycosylation. Phylogenetic analysis of UGTs regulated by these TFs highlighted their diverse roles in modulating hemicellulose synthesis. Overall, this study identifies a set of TFs that regulate PARVUS2 in poplar, providing insights into the intricate coordination of TFs and PtrPARVUS2 in SCW formation. Understanding these regulatory mechanisms enhances our ability to engineer plant biomass for tailored applications, including biofuel production and bioproduct development.

The Slow Growth of Adventitious Roots in Tetraploid Hybrid Poplar (Populus simonii × P. nigra var. italica) May Be Caused by Endogenous Hormone-Mediated Meristem Shortening.

Polyploidization produces abundant phenotypic variation. Little is currently known about adventitious root (AR) development variation due to polyploidization. In this study, we analyzed the morphological, cytological, and physiological variations in AR development between tetraploid and diploid Populus plants during in vitro rooting culture. Compared to the diploids, the AR formation times and rooting rates of the tetraploids' stem explants had non-significant changes. However, the tetraploid ARs exhibited significantly slower elongation growth than the diploid ARs. Cytological observation showed that the tetraploid ARs were characterized by shorter root meristems and reduced meristem cell numbers, suggesting the reasons for the slow AR elongation. Analysis of hormones and related metabolites during AR development demonstrated that the total auxin, cytokinin, and jasmonic acid contents were significantly lower in the tetraploid ARs than in those of the diploids, and that the ratio of total auxins to total CKs at 0 h of AR development was also lower in the tetraploids than in the diploids, whereas the total salicylic acid content of the tetraploids was consistently higher than that of the diploids. qPCR analysis showed that the expression levels of several hormone signaling and cell division-related genes in the tetraploid ARs significantly differed from those in the diploids. In conclusion, the slow elongation of the tetraploid ARs may be caused by the endogenous hormone-mediated meristem shortening. Our findings enhance the understanding of polyploidization-induced variation in AR development of forest trees.

Targeted improvement of narrow micropores in porous carbon for enhancing trace benzene vapor removal: Revealing the adsorption mechanism via experimental and molecular simulation.

Porous carbon materials are highly desirable for removing benzene due to their low energy for capture and regeneration. Research has demonstrated that narrow microporous volume is crucial for effective adsorption of benzene at ultra-low concentration. Unfortunately, achieving directional increase in the narrow microporous volume in porous carbon remains a challenge. Here, nitrogen-doped hydrothermal carbon was prepared using urea-assisted hydrothermal method, and then porous carbon (PUC800) was prepared by KOH activation. The resulting material had 180 % higher pore volume and 179 % higher surface area compared to non-nitrogen activation methods. Then, using mechanochemical (mechanical compaction and KOH activation) approach to produce PUC800-3T, which had a 30 % increase in pore volume and a 33 % increase in surface area compared to PUC800. PUC800-3T showed benzene adsorption capacity of 4.2 mmol g-1 at 1 Pa and 5.8 mmol g-1 at 5 Pa. Experimental and molecular simulation indicate that the benzene adsorption at 1 and 5 Pa is determined by pore volume of less than 0.8 and 0.9 nm, respectively. Density functional theory calculations provided insight into the CH⋯X (X = N/O) interactions drive benzene adsorption on the carbon framework. This work provides valuable theoretical and experimental support for designing, preparing, and applying adsorbents for trace removal of benzene vapor.

The adaptability of Ulmus pumila and the sensitivity of Populus sibirica to semi-arid steppe is reflected in the stem and root vascular cambium and anatomical wood traits.

Afforestation success is measured by the tree establishment and growth capacity which contribute to a range of ecosystem services. In the Mongolian steppe, Populus sibirica and Ulmus pumila have been tested as candidate species for large afforestation programs, by analyzing their response to a combination of irrigation and fertilization treatments. While in temperate and Mediterranean forest ecosystems, xylogenetic studies provide insight into the trees' plasticity and adaptability, this type of knowledge is non-existent in semi-arid regions, whose climatic features are expected to become a global issue. Furthermore, in general, a comparison between the stem and root response is scarce or absent. In the present study, we show that the anatomical traits of the vascular cambium and the xylem, from stem and root microcores, reflect the previously noted dependence of P. sibirica from irrigation - as they proportionally increase and the higher adaptability of U. pumila to drought - due to the reduced impact across all five characteristics. As the first wood anatomy study of these species in semiarid areas, future research is urgently needed, as it could be a tool for quicker understanding of species' suitability under expected to be exacerbated semi-arid conditions.

Highly Efficient Production of Cellulosic Ethanol from Poplar Using an Optimal C6/C5 Co-Fermentation Strain of Saccharomyces cerevisiae.

Cellulosic ethanol is the key technology to alleviate the pressure of energy supply and climate change. However, the ethanol production process, which is close to industrial production and has a high saccharification rate and ethanol yield, still needs to be developed. This study demonstrates the effective conversion of poplar wood waste into fuel-grade ethanol. By employing a two-step pretreatment using sodium chlorite (SC)-dilute sulfuric acid (DSA), the raw material achieved a sugar conversion rate exceeding 85% of the theoretical value. Under optimized conditions, brewing yeast co-utilizing C6/C5 enabled a yield of 35 g/L ethanol from 10% solid loading delignified poplar hydrolysate. We increased the solid loading to enhance the final ethanol concentration and optimized both the hydrolysis and fermentation stages. With 20% solid loading delignified poplar hydrolysate, the final ethanol concentration reached 60 g/L, a 71.4% increase from the 10% solid loading. Our work incorporates the pretreatment, enzymatic hydrolysis, and fermentation stages to establish a simple, crude poplar waste fuel ethanol process, expanding the range of feedstocks for second-generation fuel ethanol production.

Development of binderless fiberboard from poplar wood residue with Trametes hirsuta.

The utilization of agricultural and forestry residues for the development and preparation of green binderless fiberboard (BF) is an effective way to realize high-value utilization of lignocellulose biomass resources. This study focuses on the fabrication of BF with excellent mechanical and waterproof properties, utilizing poplar wood residue (PWR) as raw material and Trametes hirsuta as a pretreatment method. During the fermentation process, lignin-degrading enzymes and biological factors, such as sugars, were produced by T. hirsuta, which activated lignin by depolymerizing lignin bonds and modifying structural functional groups, and forming new covalent bonds between poplar fibers, ultimately enhancing adhesion. Additionally, the activated lignin molecules and sugar molecules coalesce under high temperatures and pressures, forming a dense carbonization layer that bolsters the mechanical properties of the fiberboard and effectively shields it from rapid water infiltration. The bio-pretreated BF for 10 days shows a MOR and MOE of up to 36.1 Mpa and 3704.3 Mpa, respectively, which is 261% and 247.8% higher than that of the bio-untreated fiberboard, and the water swelling ratio (WSR) rate is only 5.6%. Chemical composition analysis revealed that repolymerization occurred among lignin, cellulose, and hemicellulose, especially the molecular weight of lignin changed significantly, with the Mw of lignin increasing from 312066 g/mol to 892362 g/mol, and then decreasing to 825021 g/mol. Mn increased from 277790 g/mol to 316987.5 g/mol and then decreased to 283299.5 g/mol at 21 days. Compared to other artificial fiberboards prepared through microbial pretreatment, the BF prepared by microorganisms in this study exhibited the highest mechanical properties among the poplar wood biobased panels.

Soil methane emissions from plain poplar (Populus spp.) plantations with contrasting soil textures.

The forest soil methane (CH4) flux exhibits high spatiotemporal variability. Understanding these variations and their driving factors is crucial for accurately assessing the forest CH4 budget. In this study, we monitored the diurnal and seasonal variations in soil CH4 fluxes in two poplar (Populus spp.) plantations (Sihong and Dongtai) with different soil textures using the static chamber-based method. The results showed that the annual average soil CH4 flux in the Sihong and Dongtai poplar plantations was 4.27 ± 1.37 kg CH4-C ha-1 yr-1 and 1.92 ± 1.07 kg CH4-C ha-1 yr-1, respectively. Both plantations exhibited net CH4 emissions during the growing season, with only weak CH4 absorption (-0.01 to -0.007 mg m-2 h-1) during the non-growing season. Notably, there was a significant difference in soil CH4 flux between the clay loam of the Sihong poplar plantation and the sandy loam of the Dongtai poplar plantation. From August to December 2019 and from July to August and November 2020, the soil CH4 flux in the Sihong poplar plantation was significantly higher than in the Dongtai poplar plantation. Moreover, the soil CH4 flux significantly increased with rising soil temperature and soil water content. Diurnally, the soil CH4 flux followed a unimodal variation pattern at different growing stages of poplars, with peaks occurring at noon and in the afternoon. However, the soil CH4 flux did not exhibit a consistent seasonal pattern across different years, likely due to substantial variations in precipitation and soil water content. Overall, our study emphasizes the need for a comprehensive understanding of the spatiotemporal variations in forest soil CH4 flux with different soil textures. This understanding is vital for developing reasonable forest management strategies and reducing uncertainties in the global CH4 budget.

Evolutionary divergence of CXE gene family in green plants unveils that PtoCXEs overexpression reduces fungal colonization in transgenic Populus.

Plant enzymes significantly contribute to the rapidly diversified metabolic repertoire since the colonization of land by plants. Carboxylesterase is just one of the ubiquitous, multifunctional and ancient enzymes that has particularly diversified during plant evolution. This study provided a status on the carboxylesterase landscape within Viridiplantae. A total of 784 carboxylesterases were identified from the genome of 31 plant species representing nine major lineages of sequenced Viridiplantae and divided into five clades based on phylogenetic analysis. Clade I carboxylesterase genes may be of bacterial origin and then expanded and diversified during plant evolution. Clade II was first gained in the ancestor of bryophytes after colonization of land by plants, Clade III and Clade IV in ferns which were considered the most advanced seedless vascular plants, while Clade V was gained in seed plants. To date, the functions of carboxylesterase genes in woody plants remain unclear. In this study, 51 carboxylesterase genes were identified from the genome of Populus trichocarpa and further divided into eight classes. Tandem and segmental duplication events both contributed to the expansion of carboxylesterase genes in Populus. Although carboxylesterase genes were proven to enhance resistance to pathogens in many herbaceous species, relevant researches on forest trees are still needed. In this study, pathogen incubation assays showed that overexpressing of six Class VI carboxylesterases in Populus tomentosa, to a greater or lesser degree, reduced colonization of detached leaves by fungus Cytospora chrysosperma. A significant difference was also found in functional divergence patterns for genes derived from different gene duplication events. Functional differentiation of duplicated carboxylesterase genes in Populus was proved for the first time by in vivo physiological analysis. The identification of the potentially anti-fungal PtoCXE06 gene also laid a theoretical foundation for promoting the genetic improvement of disease-resistance traits in forest trees.

C2H2 zinc finger protein PagIDD15A regulates secondary wall thickening and lignin biosynthesis in poplar.

Wood production is largely determined by the activity of cambial cell proliferation, and the secondary cell wall (SCW) thickening of xylem cells determines the wood property. In this study, we identified an INDETERMINATE DOMAIN (IDD) type C2H2 zinc finger transcription factor PagIDD15A as a regulator of wood formation in Populus alba × Populus glandulosa. Downregulation of PagIDD15A expression by RNA interference (RNAi) inhibited xylem development and xylem cell secondary wall thickening. RNA-seq analysis showed that PagPAL1, PagCCR2 and PagCCoAOMT1 were downregulated in the differentiating xylem of the PagIDD15A-RNAi transgenic plants, showing that PagIDD15A may regulate SCW biosynthesis through inhibiting lignin biosynthesis. The downregulation of PagVND6-B2, PagMYB10 and PagMYC4 and upregulation of PagWRKY12 in the differentiating xylem of RNAi transgenic plants suggest that PagIDD15A may also regulate these transcription factor (TF) genes to affect SCW thickening. RT-qPCR analysis in the phloem-cambium of RNAi transgenic demonstrates that PagIDD15A may regulate the expression of the genes associated with cell proliferation, including, PagSHR (SHORTROOT), PagSCR (SCARECROW), PagCYCD3;1 (CYCLIN D3;1) and PagSMR4 (SIAMESE-RELATED4), to affect the cambial activity. This study provides the knowledge of the IDD-type C2H2 zinc finger protein in regulating wood formation.