State Key Laboratory of Tree Genetics and Breeding

MicroRNAs (miRNAs), a type of short (21-23 nucleotides), non-coding RNA molecule, mediate repressive gene regulation through RNA silencing at the post-transcriptional level, and play an important role in defense and response to abiotic and biotic stresses. In the present study, Affymetrix® miRNA Array, real-time quantitative PCR (qPCR) for miRNAs and their targets, and miRNA promoter analysis were used to validate the gene expression patterns of miRNAs in Populus trichocarpa plantlets induced with the poplar stem canker pathogen, Botryosphaeria dothidea. Twelve miRNAs (miR156, miR159, miR160, miR164, miR166, miR168, miR172, miR319, miR398, miR408, miR1448, and miR1450) were upregulated in the stem bark of P. trichocarpa, but no downregulated miRNAs were found. Based on analysis of the miRNAs and their targets, a potential co-regulatory network was developed to describe post-transcriptional regulation in the pathological development of poplar stem canker. There was highly complex cross-talk between diverse miRNA pathway responses to biotic and abiotic stresses. The results suggest that miR156 is probably an integral component of the miRNA response to all environmental stresses in plants. Cis-regulatory elements were binding sites for the transcription factors (TFs) on DNA. Promoter analysis revealed that TC-rich repeats and a W1-box motif were both tightly related disease response motifs in Populus. Promoter analysis and target analysis of miRNAs also revealed that some TFs regulate their activation/repression. Furthermore, a feedback regulatory network in the pathological development of poplar stem canker is provided. The results confirm that miRNA pathways regulate gene expression during the pathological development of plant disease, and provide new insights into understanding the onset and development of poplar stem canker.

Abstract Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

An unbalanced pigment distribution among the sepal and petal segments results in various colour patterns of orchid flowers. Here, we explored this type of mechanism of colour pattern formation in flowers of the Cattleya hybrid 'KOVA'. Our study showed that pigment accumulation displayed obvious spatiotemporal specificity in the flowers and was likely regulated by three R2R3-MYB transcription factors. Before flowering, RcPAP1 was specifically expressed in the epichile to activate the anthocyanin biosynthesis pathway, which caused substantial cyanin accumulation and resulted in a purple-red colour. After flowering, the expression of RcPAP2 resulted in a low level of cyanin accumulation in the perianths and a pale pink colour, whereas RcPCP1 was expressed only in the hypochile, where it promoted α-carotene and lutein accumulation and resulted in a yellow colour. Additionally, we propose that the spatiotemporal expression of different combinations of AP3- and AGL6-like genes might participate in KOVA flower colour pattern formation.

Apigenin-7-O-β-D-glucuronide (AG), an active flavonoid derivative isolated from the agricultural residue of Juglans sigillata fruit husks, possesses multiple pharmacological activities, including anti-oxidant, anti-complement, and aldose reductase inhibitory activities. To date, no report has identified the anti-inflammatory mechanisms of AG. This study was therefore designed to characterize the molecular mechanisms of AG on lipopolysaccharide (LPS)-induced inflammatory cytokines in RAW 264.7 cells and on endotoxin-induced shock in mice. AG suppressed the release of nitric oxide (NO), prostaglandin E2 (PGE2), and tumour necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner without affecting cell viability. Additionally, AG suppressed LPS-induced mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α. AG treatment decreased the translocation of c-Jun into the nucleus, and decreased activator protein-1 (AP-1)-mediated luciferase activity through the inhibition of both p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) phosphorylation. Consistent with the in vitro observations, AG protected mice from LPS-induced endotoxin shock by inhibiting proinflammatory cytokine production. Taken together, these results suggest that AG may be used as a source of anti-inflammatory agents as well as a dietary complement for health promotion.

Lycium barbarum polysaccharide (LBP), is a major active ingredient Lycium barbarum (LB), which exhibits several beneficial effects through NF-κB, PI3K-Akt-mTOR, p38-MAPK, Wnt-β-catenin, PI3K-Akt-GSK-3β, and MyD88 signal pathway, including anti-oxidation, and anti-aging, hypolipidemic and hypoglycemic, radiation, anti-tumor, and neuroprotection. Today, many researching papers are published on the LBP in physiology and pathology; however, the review of the LBP taking part in the signal transduction pathway in physiology and pathology is rear searched. Therefore, this research topic is a collection of reviews and original research articles that focus on the methods of the LBP extraction and its effects on the signal transduction pathway. The aim of this study is to provide theoretical evidence for in-depth analysis of the mechanisms of LBP in clinical clinical research studies.

Abstract The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors (TFs) are involved in the regulation of specialized terpenoid biosynthesis. However, the AP2/ERF TFs in Litsea cubeba have not been characterized and their role in the biosynthesis of terpenoids is unknown. Here, 174 LcAP2/ERF TFs were identified in L. cubeba and categorized into four subfamilies: 27 AP2, 7 RAV, 1 Soloist, and 139 ERF. Transcriptomic and qRT-PCR assays both showed that the expression levels of LcERF19 were similar to that of terpene synthase LcTPS42 in the pericarp, which is related to the synthesis of geranial and neral in L. cubeba. LcERF19 was further shown to encode a nuclear-localized protein and its expression was strongly induced by jasmonate. Yeast one-hybrid and dual-luciferase assays showed that LcERF19 associated with GCC box elements of the LcTPS42 promoter and promoted its activity. Transient overexpression of LcERF19 in L. cubeba and overexpression of LcERF19 in tomato resulted in a significant increase in geranial and neral. Our findings show that LcERF19 enhances geranial and neral biosynthesis through activation of LcTPS42 expression, which provides a strategy to improve the flavor of tomato and other fruits.

The regulation of crassulacean acid metabolism (CAM) pathway has recently become a topic of intensive research and has been explored in terms of several aspects, including phylogenetics, genomics, and transcriptomics. Orchidaceae, which contains approximately 9,000 CAM species, is one of the largest lineages using this special photosynthetic pathway. However, no comprehensive transcriptomic profiling focused on CAM regulation in orchid species had previously been performed. In this report, we present two Illumina RNA-seq datasets, including a total of 24 mature leaf samples with 844.4 million reads, from Dendrobium catenatum (Orchidaceae), a facultative CAM species. The first dataset was generated from a time-course experiment based on the typical CAM phases in a diel. The second was derived from an experiment on drought stress and stress removal. A series of quality assessments were conducted to verify the reliability of the datasets. These transcriptomic profiling datasets will be useful to explore and understand the essence of CAM regulation.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a fast-growing evergreen conifer with high-quality timber and is an important reforestation and commercial tree species in southern China. Planting density affects the productivity of Chinese fir plantations. To study the effect of five different planting densities and soil depth on soil nutrient contents of a mature C. lanceolata plantation, the soil nutrient contents (soil depths 0–100 cm) of 36-year-old mature Chinese fir plantations under five different planting densities denoted A (1667 trees·ha−1), B (3333 trees·ha−1), C (5000 trees·ha−1), D (6667 trees·ha−1), and E (10,000 trees·ha−1) were measured in Pingxiang county, Guangxi province, China. Samples were collected from the soil surface down to a one meter depth from each of 45 soil profiles, and soil samples were obtained at 10 different soil depths of 0–10, 10–20, 20–30, 30–40, 40–50, 50–60, 60–70, 70–80, 80–90, and 90–100 cm. Twelve soil physical and chemical indicators were analyzed. The results showed that: (1) as planting density increased, the organic matter, organic carbon, total N and P, available N, effective Fe, and bulk density decreased. Soil pH, total K, and effective K increased with increasing planting density. Planting density did not significantly influence the exchangeable Ca and Mg. (2) Soil organic matter; organic carbon; total N and P; effective N, P, and K; exchangeable Ca and Mg; effective Fe content; and bulk density decreased with increasing soil depth. This pattern was particularly evident in the top 30 cm of the soil. (3) Excessively high planting density is not beneficial to the long-term maintenance of soil fertility in Chinese fir plantations, and the planting density of Chinese fir plantations should be maintained below 3333 stems·ha−1 (density A or B) to maintain soil fertility while ensuring high yields.

Real-time Quantitative PCR (RT-qPCR) has become an effective method for accurate analysis of gene expression in several biological systems as well as under different experimental conditions. Although with high sensitivity, specificity and broad dynamic range, this method requires suitable reference genes for transcript normalization in order to guarantee reproducible and meaningful results. In the present study, we evaluated five traditional housekeeping genes and five novel reference genes in Hyperaccumulating ecotype of Sedum alfredii, a well known hyperaccumulator for heavy metals phytoremediation, under Cd, Pb, Zn and Cu stresses of seven different durations. The expression stability of these ten candidates were determined with three programs--geNorm, NormFinder and BestKeeper. The results showed that all the selected reference genes except for SAND could be used for RT-qPCR normalization. Among them UBC9 and TUB were ranked as the most stable candidates across all samples by three programs together. For the least stable reference genes, however, BestKeeper produced different results compared with geNorm and NormFinder. Meanwhile, the expression profiles of PCS under Cd, Pb, Zn and Cu stresses were assessed using UBC9 and TUB respectively, and similar trends were obtained from the results of the two groups. The distinct expression patterns of PCS indicated that various strategies could be taken by plants in adaption to different heavy metals stresses. This study will provide appropriate reference genes for further gene expression quantification using RT-qPCR in Hyperaccumulator S. alfredii.

Heavy metal contamination presents serious threats to living organisms. Functional genes related to cadmium (Cd) hypertolerance or hyperaccumulation must be explored to enhance phytoremediation. Sedum alfredii Hance is a Zn/Cd cohyperaccumulator exhibiting abundant genes associated with Cd hypertolerance. Here, we developed a method for screening genes related to Cd tolerance by expressing a cDNA-library for S. alfredii Hance. Yeast functional complementation validated 42 of 48 full-length genes involved in Cd tolerance, and the majority of them were strongly induced in roots and exhibited diverse expression profiles across tissues. Coexpression network analysis suggested that 15 hub genes were connected with genes involved in metabolic processes, response to stimuli, and metal transporter and antioxidant activity. The functions of a novel SaCTP2 gene were validated by heterologous expression in Arabidopsis, responsible for retarding chlorophyll content decrease, maintaining membrane integrity, promoting reactive oxygen species (ROS) scavenger activities, and reducing ROS levels. Our findings suggest a highly complex network of genes related to Cd hypertolerance in S. alfredii Hance, accomplished via the antioxidant system, defense genes induction, and the calcium signaling pathway. The proposed cDNA-library method is an effective approach for mining candidate genes associated with Cd hypertolerance to develop genetically engineered plants for use in phytoremediation.

Abstract A field survey of native herbaceous plants and associated soil in an antimony mining area of Xikuangshan, Hunan Province, China was conducted to identify species that accumulate heavy metals in their tissues. The results indicate that the soils in the mining area were contaminated mainly by As and Sb, at concentrations of 423.77–526.57 and 228.37–445.20 mg/kg, respectively. Of the herbaceous plants, Miscanthus sinensis and Imperata cylindrica exhibited Hg and Cd phytoextraction, with bioconcentration factors (BCFs) and translocation factors (TFs) greater than 1. Moreover, Phytolacca americana could be used for the phytostabilization of Pb and Cd (BCF = 1.06 and 7.66, respectively), and Cynodon dactylon had considerable potential for As and Sb stabilization (BCF = 2.02 and 6.62, respectively). Boehmeria was capable of accumulating Sb and As in its shoots (TF = 3.12 and 4.86, respectively). Additionally, the concentration of Sb in the roots of C. dactylon reached 2209.3 mg/kg, which is the highest Sb concentration reported in a plant species to date. Our data suggest that native herbaceous plants growing in metal‐contaminated sites have phytoremediation potential.

Resistance to pathogens is essential for survival of wild and cultivated plants. Pathogen susceptibility causes major losses of crop yield and quality. Durable field resistance combined with high yield and other superior agronomic characters are therefore, important objectives in every crop breeding program. Precision and efficacy of resistance breeding can be enhanced by molecular diagnostic tools, which result from knowledge of the molecular basis of resistance and susceptibility. Breeding uses resistance conferred by single R genes and polygenic quantitative resistance. The latter is partial but considered more durable. Molecular mechanisms of plant pathogen interactions are elucidated mainly in experimental systems involving single R genes, whereas most genes important for quantitative resistance in crops like potato are unknown. Quantitative resistance of potato to Phytophthora infestans causing late blight is often compromised by late plant maturity, a negative agronomic character. Our objective was to identify candidate genes for quantitative resistance to late blight not compromised by late plant maturity. We used diagnostic DNA-markers to select plants with different field levels of maturity corrected resistance (MCR) to late blight and compared their leaf transcriptomes before and after infection with P. infestans using SuperSAGE (serial analysis of gene expression) technology and next generation sequencing. We identified 2034 transcripts up or down regulated upon infection, including a homolog of the kiwi fruit allergen kiwellin. 806 transcripts showed differential expression between groups of genotypes with contrasting MCR levels. The observed expression patterns suggest that MCR is in part controlled by differential transcript levels in uninfected plants. Functional annotation suggests that, besides biotic and abiotic stress responses, general cellular processes such as photosynthesis, protein biosynthesis, and degradation play a role in MCR.

Poplar (Populus) is a well-established model system for tree genomics and molecular breeding, and hybrid poplar is widely used in forest plantations. However, distinguishing its diploid homologous chromosomes is difficult, complicating advanced functional studies on specific alleles. In this study, we applied a trio-binning design and PacBio high-fidelity long-read sequencing to obtain haplotype-phased telomere-to-telomere genome assemblies for the 2 parents of the well-studied F1 hybrid "84K" (Populus alba × Populus tremula var. glandulosa). Almost all chromosomes, including the telomeres and centromeres, were completely assembled for each haplotype subgenome apart from 2 small gaps on one chromosome. By incorporating information from these haplotype assemblies and extensive RNA-seq data, we analyzed gene expression patterns between the 2 subgenomes and alleles. Transcription bias at the subgenome level was not uncovered, but extensive-expression differences were detected between alleles. We developed machine-learning (ML) models to predict allele-specific expression (ASE) with high accuracy and identified underlying genome features most highly influencing ASE. One of our models with 15 predictor variables achieved 77% accuracy on the training set and 74% accuracy on the testing set. ML models identified gene body CHG methylation, sequence divergence, and transposon occupancy both upstream and downstream of alleles as important factors for ASE. Our haplotype-phased genome assemblies and ML strategy highlight an avenue for functional studies in Populus and provide additional tools for studying ASE and heterosis in hybrids.

Using the proper means to improve seed germination is of great significance in agriculture and forestry. Here, a meta-analysis was used to examine whether metal nanoparticle treatments have a specific effect on the seed germination and seedling growth of agricultural species. Using the Web of Science (1950–2021), PubMed (1950–2021), and Scopus (1950–2021) databases, a paper search was conducted using the following items (“nanoparticles” and “seed germination”, “nanomaterials” and “seed germination”) to filter the references in the title, abstract, and keywords of the published articles. The results indicated that nanoparticle (NP) treatments had a significantly positive effect on the final germination percentage (FGP), with a mean difference (MD) (that is, the overall effect) of 1.97 (0.96, 2.98) for the silver (Ag)-NP subgroup, 1.21 (0.34, 2.09) for the other-NP subgroup, 1.40 (0.88, 1.92) for the total based on the NP types, 1.47 (0.85, 2.09) for the “Concentrations: <50 mg/L” subgroup, and 1.40 (0.88, 1.92) for the total based on the NP concentrations. Similarly, root length (RL) was positively and significantly affected by NP treatment, with an MD (95% CI) of 0.92 (0.76, 1.09) for the zinc (Zn)-NP subgroup, 0.79 (0.65, 0.92) for the other-NP subgroup, 0.82 (0.72, 0.93) for the total based on the NP types, 0.90 (0.77, 1.04) for the “Concentrations: ≤50 mg/L” subgroup, 0.80 (0.60, 0.99) for the “Concentrations: >50 mg/L” subgroup, and 0.82 (0.72, 0.93) for the total based on the NP concentrations. However, there was no statistical correlation between the nanoparticle concentrations and shoot length (SL), due to the inclusion of zero in the 95% CI of the overall effect. Therefore, Ag-NPs could increase the FGP more than other-NPs, while Zn-NPs enhanced RL more. Moreover, NPs at lower concentrations could improve the FGP and RL of crop species to a larger extent than NPs at higher concentrations. This meta-analysis can provide a reference for the nanoparticle treatment technology utilization in agricultural and forest seeds.

Novel cellulose hydrogels based on the inclusion complex between α-cyclodextrin and binary-drug loaded nanoparticles (carboxymethylcellulose–betulinic acid/hydroxycamptothecine nanoparticles) were prepared in aqueous media for the first time.

Abstract Long noncoding RNAs (lncRNAs) are important in abiotic stress tolerance. Here, we identified salt-responsive genes and lncRNAs in the roots and leaves of Betula platyphylla Suk. (birch), and characterized their lncRNAs functions. In total, 2660 mRNAs and 539 lncRNAs responding to salt treatment were identified using RNA-seq. The salt-responsive genes were substantially enriched in ‘cell wall biogenesis’ and ‘wood development’ in the roots and were enriched in ‘photosynthesis’ and ‘response to stimulus’ in the leaves. Meanwhile, the potential target genes of the salt-responsive lncRNAs in roots and leaves were both enriched in ‘nitrogen compound metabolic process’ and ‘response to stimulus’. We further built a method for quickly identifying abiotic stress tolerance of lncRNAs, which employed transient transformation for overexpression and knock-down of the lncRNA, enabling gain- and loss-of-function analysis. Using this method, 11 randomly selected salt-responsive lncRNAs were characterized. Among them, six lncRNAs confer salt tolerance, two lncRNAs confer salt sensitivity, and the other three lncRNAs are not involved in salt tolerance. In addition, a lncRNA, LncY1, was further characterized, which improves salt tolerance by regulating two transcription factors, BpMYB96 and BpCDF3. Taken together, our results suggested that lncRNAs play important roles in the salt response of birch plants.

• The first reported mitochondrial genome ( Cinnamomum chekiangense ) of the Lauraceae family. • The mitogenome of C. chekiangense retains almost all of the ancestral protein-coding genes and has the highest RNA editing number in angiosperms to date. • Both of the plastid and mitochondrial phylogenetic trees support the magnoliids as a sister group to the clade comprising monocots and eudicots.

Abstract Drought seriously affects the growth and development of plants. MiR159 is a highly conserved and abundant microRNA family that plays a crucial role in plant growth and stress responses. However, studies of its function in woody plants are still lacking. Here, the expression of miR159a was significantly upregulated after drought treatment in poplar, and the overexpression of miR159a (OX159a) significantly reduced the open area of the stomata and improved water-use efficiency in poplar. After drought treatment, OX159a lines had better scavenging ability of reactive oxygen species and damage of the membrane system was less than that in wild-type lines. MYB was the target gene of miR159a, as verified by psRNATarget prediction, RT–qPCR, degradome sequencing, and 5′ rapid amplification of cDNA ends (5′ RACE). Additionally, miR159a–short tandem target mimic suppression (STTM) poplar lines showed increased sensitivity to drought stress. Transcriptomic analysis comparing OX159a lines with wild-type lines revealed upregulation of a series of genes related to response to water deprivation and metabolite synthesis. Moreover, drought-responsive miR172d and miR398 were significantly upregulated and downregulated respectively in OX159a lines. This investigation demonstrated that miR159a played a key role in the tolerance of poplar to drought by reducing stomata open area, increasing the number and total area of xylem vessels, and enhancing water-use efficiency, and provided new insights into the role of plant miR159a and crucial candidate genes for the molecular breeding of trees with tolerance to drought stress.

In order to study the characteristics of soil nutrient elements and the changes in biomass under different densities and soil layers of forest stand, this paper considers Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) density test forests with five densities (A: 1667 trees·ha−1; B: 3333 trees·ha−1; C: 5000 trees·ha−1; D: 6667 trees·ha−1; E: 10,000 trees·ha−1) as the research objects, located in Naxi District, Sichuan Province, China. Eleven soil physical and chemical property indicators, understory vegetation, and litter biomass were measured. The results were as follows: (1) The stand density had a significant effect on the soil nutrient content, understory vegetation, and litter biomass. A low density is conducive to the accumulation of soil organic matter, hydrolytic N, available P, available K, and total Ca. (2) With the increase in soil depth, the contents of soil organic matter, total N, hydrolytic N, and total P decreased gradually; pH and total Ca decreased gradually; and available P showed a trend of decrease-up-decrease. The soil layers had no significant effect on the total K, total Fe, and total Mg concentrations. (3) Low density (density A or B) was found to be beneficial to the growth of undergrowth vegetation and forest trees, the return of nutrients, long-term soil maintenance, and the stable high yield of Chinese fir plantations.

BACKGROUND: Betulaceae is a relatively small but morphologically diverse family, with many species having important economic and ecological values. Although plastome structure of Betulaceae has been reported sporadically, a comprehensive exploration for plastome evolution is still lacking. Besides, previous phylogenies had been constructed based on limited gene fragments, generating unrobust phylogenetic framework and hindering further studies on divergence ages, biogeography and character evolution. Here, 109 plastomes (sixteen newly assembled and 93 previously published) were subject to comparative genomic and phylogenomic analyses to reconstruct a robust phylogeny and trace the diversification history of Betulaceae. RESULTS: > 0.035), were identified as candidate DNA barcodes for phylogenetic analysis and species delimitation. Phylogenomic analyses yielded high-resolution topology that supported reciprocal monophyly between Betula and Alnus within Betuloideae, and successive divergence of Corylus, Ostryopsis, and Carpinus-Ostrya within Coryloideae. Incomplete lineage sorting and hybridization may be responsible for the mutual paraphyly between Ostrya and Carpinus. Betulaceae ancestors originated from East Asia during the upper Cretaceous; dispersals and subsequent vicariance accompanied by historical environment changes contributed to its diversification and intercontinental disjunction. Ancestral state reconstruction indicated the acquisition of many taxonomic characters was actually the results of parallel or reversal evolution. CONCLUSIONS: Our research represents the most comprehensive taxon-sampled and plastome-level phylogenetic inference for Betulaceae to date. The results clearly document global patterns of plastome structural evolution, and established a well-supported phylogeny of Betulaceae. The robust phylogenetic framework not only provides new insights into the intergeneric relationships, but also contributes to a perspective on the diversification history and evolution of the family.