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Broussonetia papyrifera is widely found in cadmium (Cd) contaminated areas, with an inherent enhanced flavonoids metabolism and inhibited lignin biosynthesis, colonized by lots of symbiotic fungi, such as arbuscular mycorrhizal fungi (AMF). However, the physiological and molecular mechanisms by which Rhizophagus irregularis, an AM fungus, regulates flavonoids and lignin in B. papyrifera under Cd stress remain unclear. Here, a pot experiment of B. papyrifera inoculated and non-inoculated with R. irregularis under Cd stress was carried out. We determined flavonoids and lignin concentrations in B. papyrifera roots by LC-MS and GC-MS, respectively, and measured the transcriptional levels of flavonoids- or lignin-related genes in B. papyrifera roots, aiming to ascertain the key components of flavonoids or lignin, and key genes regulated by R. irregularis in response to Cd stress. Without R. irregularis, the concentrations of eriodictyol, quercetin and myricetin were significantly increased under Cd stress. The concentrations of eriodictyol and genistein were significantly increased by R. irregularis, while the concentration of rutin was significantly decreased. Total lignin and lignin monomer had no alteration under Cd stress or with R. irregularis inoculation. As for flavonoids- or lignin-related genes, 26 genes were co-regulated by Cd stress and R. irregularis. Among these genes, BpC4H2, BpCHS8 and BpCHI5 were strongly positively associated with eriodictyol, indicating that these three genes participate in eriodictyol biosynthesis and were involved in R. irregularis assisting B. papyrifera to cope with Cd stress. This lays a foundation for further research revealing molecular mechanisms by which R. irregularis regulates flavonoids synthesis to enhance tolerance of B. papyrifera to Cd stress., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress., Results: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues., Conclusion: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses., (© 2024. The Author(s).)

Main Conclusion: Genome-wide identification revealed 79 BpNAC genes belonging to 16 subfamilies, and their gene structures and evolutionary relationships were characterized. Expression analysis highlighted their importance in plant selenium stress responses. Paper mulberry (Broussonetia papyrifera), a deciduous arboreal plant of the Moraceae family, is distinguished by its leaves, which are abundant in proteins, polysaccharides, and flavonoids, positioning it as a novel feedstock. NAC transcription factors, exclusive to plant species, are crucial in regulating growth, development, and response to biotic and abiotic stress. However, extensive characterization of the NAC family within paper mulberry is lacking. In this study, 79 BpNAC genes were identified from the paper mulberry genome, with an uneven distribution across 13 chromosomes. A comprehensive, genome-wide analysis of BpNACs was performed, including investigating gene structures, promoter regions, and chromosomal locations. Phylogenetic tree analysis, alongside comparisons with Arabidopsis thaliana NACs, allowed for categorizing these genes into 16 subfamilies in alignment with gene structure and motif conservation. Collinearity analysis suggested a significant homologous relationship between the NAC genes of paper mulberry and those in Morus notabilis, Ficus hispida, Antiaris toxicaria, and Cannabis sativa. Integrating transcriptome data and Se content revealed that 12 BpNAC genes were associated with selenium biosynthesis. Subsequent RT-qPCR analysis corroborated the correlation between BpNAC59, BpNAC62 with sodium selenate, and BpNAC55 with sodium selenite. Subcellular localization experiments revealed the nuclear functions of BpNAC59 and BpNAC62. This study highlights the potential BpNAC transcription factors involved in selenium metabolism, providing a foundation for strategically breeding selenium-fortified paper mulberry., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Broussonetia papyrifera, a valuable feed resource, is known for its fast growth, wide adaptability, high protein content and strong selenium enrichment capacity. Selenomethionine (SeMet), the main selenium form in selenium fortification B. papyrifera, is safe for animals and this enhances its nutritional value as a feed resource. However, the molecular mechanisms underlying SeMet synthesis remain unclear. This study identified three homocysteine S-methyltransferase genes from the B. papyrifera genome. The phylogenetic tree demonstrated that BpHMTs were divided into two classes, and BpHMT2 in the Class 2-D subfamily evolved earlier and possesses more fundamental functions. On the basis of the correlation between gene expression levels and selenium content, BpHMT2 was identified as a key candidate gene associated with selenium tolerance. Subcellular localization experiments confirmed the targeting of BpHMT2 in nucleus, cell membrane and chloroplasts. Moreover, three BpHMT2 overexpression Arabidopsis thaliana lines were confirmed to enhance plant selenium tolerance and SeMet accumulation. Overall, our finding provides insights into the molecular mechanisms of selenium metabolism in B. papyrifera, highlighting the potential role of BpHMT2 in SeMet synthesis. This research contributes to our understanding of selenium-enriched feed resources, with increased SeMet content contributing to the improved nutritional value of B. papyrifera as a feed resource., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Background: Broussonetia papyrifera (L.) L'Hér. ex Vent. has the characteristics of strong stress resistance, high crude protein content, and pruning tolerance. It is an ecological, economic, and medicinal plant. Polyploid plants usually perform better than their corresponding diploid plants in terms of nutrients, active substances, and stress resistance., Results: In this study, the leaves, calli, and seeds of diploid B. papyrifera were used for tetraploid induction by colchicine. The induction effect of colchicine on B. papyrifera was summarized through the early morphology, chromosome count and flow cytometry. It was concluded that the best induction effect (18.6%) was obtained when the leaves of B. papyrifera were treated in liquid MS (Murashige and Skoog) medium containing 450 mg·L -1 colchicine for 3 d. The comparative analysis of the growth characteristics of diploid and tetraploid B. papyrifera showed that tetraploid B. papyrifera has larger ground diameter, larger stomata, thicker palisade tissue and thicker sponge tissue than diploid B. papyrifera. In addition, the measurement of photosynthetic features also showed that tetraploids had higher chlorophyll content and higher photosynthetic rates., Conclusion: This study showed that tetraploid B. papyrifera could be obtained by treating leaves, callus and seeds with liquid and solid colchicine, but the induction efficiency was different. Moreover, there were differences in stomata, leaf cell structure and photosynthetic features between tetraploid B. papyrifera and its corresponding diploid. The induced tetraploid B. papyrifera can provide a technical basis and breeding material for the creation of B. papyrifera germplasm resources in the future., (© 2023. The Author(s).)

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO 4 treatments), seven different tissues of B. papyrifera , as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB , HSP , NADH , and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera . In addition, HSP , rRNA , NADH , and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera . The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera .

Background: Species of Broussonetia (family Moraceae) are commonly used to make textiles and high-grade paper. The distribution of Broussonetia papyrifera L. is considered to be related to the spread and location of humans. The complete chloroplast (cp) genomes of B. papyrifera , Broussonetia kazinoki Sieb., and Broussonetia kaempferi Sieb. were analyzed to better understand the status and evolutionary biology of the genus Broussonetia ., Methods: The cp genomes were assembled and characterized using SOAPdenovo2 and DOGMA. Phylogenetic and molecular dating analysis were performed using the concatenated nucleotide sequences of 35 species in the Moraceae family and were based on 66 protein-coding genes (PCGs). An analysis of the sequence divergence (pi) of each PCG among the 35 cp genomes was conducted using DnaSP v6. Codon usage indices were calculated using the CodonW program., Results: All three cp genomes had the typical land plant quadripartite structure, ranging in size from 160,239 bp to 160,841 bp. The ribosomal protein L22 gene ( RPL22 ) was either incomplete or missing in all three Broussonetia species. Phylogenetic analysis revealed two clades. Clade 1 included Morus and Artocarpus , whereas clade 2 included the other seven genera. Malaisia scandens Lour. was clustered within the genus Broussonetia . The differentiation of Broussonetia was estimated to have taken place 26 million years ago. The PCGs' pi values ranged from 0.0005 to 0.0419, indicating small differences within the Moraceae family. The distribution of most of the genes in the effective number of codons plot (ENc-plot) fell on or near the trend line; the slopes of the trend line of neutrality plots were within the range of 0.0363-0.171. These results will facilitate the identification, taxonomy, and utilization of the Broussonetia species and further the evolutionary studies of the Moraceae family., Competing Interests: The authors declare there are no competing interests., (©2022 Yang et al.)

Background: Broussonetia × hanjiana has been considered a hybrid owing to its morphology, which is intermediate between that of B. papyrifera (L.) L'Her. ex Vent. and B. kazinoki Siebold. A recent study demonstrated the hybrid origin of B. × hanjiana in Korea using molecular markers. In this study, we developed microsatellite markers for B. × hanjiana using next-generation sequencing and cross-species transferability analysis., Methods and Results: A total of 432 primers were designed from 205,819 contigs. Among them, 24 microsatellite markers showing polymorphisms were used to evaluate the population genetic characteristics. The observed heterozygosity (H O ) and expected heterozygosity (H E ) were 0.835 and 0.628, respectively. The cross-species transferability of these markers was evaluated in two closely related species of Broussonetia; all 24 markers showed cross-species amplification. Using flow cytometry, diploid and triploid individuals were identified in B. × hanjiana. In particular, the BR137 marker showed evidence of two parent species (B. papyripera and B. kazinoki), with a hybrid pattern observed in B. × hanjiana, demonstrating its utility for species identification and ploidy assessment., Conclusions: The new B. × hanjiana microsatellite markers can be useful in genetic studies of closely related B. papyripera, B. kazinoki, and B. × hanjiana., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kōzo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kōzo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kōzo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kōzo appears to have a single origin and the possibility of a Korean origin cannot be ruled out., (© 2022. The Author(s) under exclusive licence to The Botanical Society of Japan.)

YABs play an important role in the leaf development of the paper mulberry ( Broussonetia papyrifera ) and of the heterophylly. Thus, we investigated the function of BpYABs. Gene cloning, phylogenetic analysis, motif identification, subcellular localization, transactivation activity assay, qRT-PCR, in situ hybridization, and ectopic expression were used in our study. Six BpYAB s were isolated, and four of them had transcriptional activity. BpYAB1, BpYAB3, BpYAB4, and BpYAB5 were localized to the nucleus. BpYAB1 was only expressed in the flower, while BpYAB6 was not expressed in any detected tissues; the four remaining BpYAB s were expressed in the bud, leaf and flower, and their expression level decreased with leaf development. Further in situ hybridization showed that BpYAB3 and BpYAB5 were expressed in the vascular tissues and lamina, but neither showed the adaxial-abaxial polarity distribution pattern in the mature leaf lamina. Ectopic expression of BpYAB2 , BpYAB3 , BpYAB4 and BpYAB5 induced increased expression of AtWOX1 and caused the leaf of Arabidopsis to become smaller and curl downwards. Ectopic expression also led to shorter siliques and smaller seeds, but not for BpYAB5 . These results suggest that BpYABs have functional divergency and redundancy in regulating leaf and silique development.

Lysine crotonylation is a newly discovered and reversible posttranslational modification involved in various biological processes, especially metabolism regulation. A total of 5159 lysine crotonylation sites in 2272 protein groups were identified. Twenty-seven motifs were found to be the preferred amino acid sequences for crotonylation sites. Functional annotation analyses revealed that most crotonylated proteins play important roles in metabolic processes and photosynthesis. Bioinformatics analysis suggested that lysine crotonylation preferentially targets a variety of important biological processes, including ribosome, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, proteasome and the TCA cycle, indicating lysine crotonylation is involved in the common mechanism of metabolic regulation. A protein interaction network analysis revealed that diverse interactions are modulated by protein crotonylation. These results suggest that lysine crotonylation is involved in a variety of biological processes. HSP70 is a crucial protein involved in protecting plant cells and tissues from thermal or abiotic stress responses, and HSP70 protein was found to be crotonylated in paper mulberry. This systematic analysis provides the first comprehensive analysis of lysine crotonylation in paper mulberry and provides important resources for further study on the regulatory mechanism and function of the lysine crotonylated proteome.

Broussonetia papyrifera is a multifunctional deciduous tree that is both a food and a source of traditional Chinese medicine for both humans and animals. Further analysis of the UGT gene family is of great significance to the utilization of B. papyrifera . The substrates of plant UGT genes include highly diverse and complex chemicals, such as flavonoids and terpenes. In order to deepen our understanding of this family, a comprehensive analysis was performed. Phylogenetic analysis showed that 155 BpUGTs were divided into 15 subgroups. A conserved motif analysis showed that BpUGT proteins in the same subgroups possessed similar motif structures. Tandem duplication was the primary driving force for the expansion of the BpUGT gene family. The global promoter analysis indicated that they were associated with complex hormone regulatory networks and the stress response, as well as the synthesis of secondary metabolites. The expression pattern analysis showed that the expression level of BpUGTs in leaves and roots was higher than that in fruits and stems. Next, we determined the composition and content of flavonoids, the main products of the BpUGT reaction. A total of 19 compounds were isolated and analyzed by UPLC-ESI-MS/MS in 3 species of Broussonetia including B. kazinoki , B. papyrifera , and B. kazinoki × B. papyrifera , and the number of compounds was different in these 3 species. The total flavonoid content and antioxidant capacities of the three species were analyzed respectively. All assays exhibited the same trend: the hybrid paper mulberry showed a higher total flavonoid content, a higher total phenol content and higher antioxidant activity than the other two species. Overall, our study provides valuable information for understanding the function of BpUGTs in the biosynthesis of flavonoids.

Objectives: To study the possible roles of type-2C protein phosphatases (PP2Cs) which have been confirmed to play roles in the response to diverse abiotic stresses in paper mulberry, we launched a series of genomic and functional studies of BpPP2Cs., Results: Sixty-three PP2C proteins in paper mulberry (Broussonetia papyrifera) were classified into 13 clades. Four BpPP2Cs with kinase domains were verified to be highly conserved in organisms ranging from algae to dicots. Seven pairs of BpPP2C genes were found to be expanding, and 18 BpPP2C genes had orthologues in Arabidopsis. BpPP2Cs showed broad expression in different tissues; the expression levels of 18 BpPP2Cs were changed and the phosphorylation levels of seven BpPP2C proteins increased at low temperature. Cold-response elements were found in the promoter region of 31 BpPP2Cs. Finally, Bp01g0320 was found to act as a hub protein and Bp01g0512 and Bp09g1278 played key roles in the ABA-signaling pathway and MAPK cascades, respectively., Conclusion: These results suggest that the PP2C gene family of paper mulberry is evolutionarily conserved and participates the regulation of the response to cold stress, which will play a vital role in further research on phosphatases in paper mulberry.

The genetic basis of plant local adaptation has been extensively studied, yet the interplay between local adaptation, plant genetic divergence, and the microbial community remains unclear. Our study used the restriction-site associated DNA sequencing (RAD-seq) approach to explore genetic divergence in Broussonetia papyrifera and used internal transcribed spacers (ITS) to characterize fungal community. RAD-seq results show that B. papyrifera individuals could be divided into three genotypes; this genotyping result was consistent with the classification of climate type at the sample site. Most of the 101 highly differentiated genes were related to stress resistance and the microbiome. Moreover, β-diversity results indicated that genetic divergence had a significant effect on fungal community across all compartments ( P <  0.01). At genus and operational taxonomic unit (OTU) level, Mortierella , Hannaella oryzae , OTU81578 ( Mortierella ), and OTU1665209 ( H. oryzae ) were found to be the major OTUs that contribute to differences in fungal community. The properties of cooccurrence networks vary greatly among three genotypes. The results of redundancy analysis (RDA) indicated that B. papyrifera -associated fungal community was significantly related to its local adaptability. Our findings suggest that genetic divergence of B. papyrifera is closely related to local adaptation, with significant effects on the associated fungal community, which in turn would enhance host local adaptability. This improves present understanding about the coevolution of microbial communities and the host plant. IMPORTANCE The coevolution of plants with the associated fungal community and its effect on plant adaptability are not clear, especially for native trees. This study focuses on the genetic basis of local adaptation in plants and the effect of genetic divergence of Broussonetia papyrifera on the associated fungal community. We identified genes related to the microbiome that are important for local adaptation of the host. Our results show that genetic divergence in B. papyrifera significantly affects the fungal community, which has a close connection with local adaptation. This helps us to understand the relationship between local adaptation, genetic divergence, and associated fungal communities. This study highlights the effect of plant genetic divergence on associated fungal community for native trees and establishes a close connection between this effect and local adaptability in the host. In addition, these observations lay a foundation for the research of coevolution of plants and their symbiotic microbiome through genome-wide association study (GWAS)., (Copyright © 2020 American Society for Microbiology.)

Humans introduced paper mulberry (Broussonetia papyrifera) from Taiwan into the Pacific over 5000 years ago as a fiber source to make barkcloth textiles that were, and still are, important cultural artifacts throughout the Pacific. We have used B. papyrifera, a species closely associated to humans, as a proxy to understand the human settlement of the Pacific Islands. We report the first genetic analysis of paper mulberry textiles from historical and archaeological contexts (200 to 50 years before present) and compare our results with genetic data obtained from contemporary and herbarium paper mulberry samples. Following stringent ancient DNA protocols, we extracted DNA from 13 barkcloth textiles. We confirmed that the fiber source is paper mulberry in nine of the 13 textiles studied using the nuclear ITS-1 marker and by statistical estimates. We detected high genetic diversity in historical Pacific paper mulberry barkcloth with a set of ten microsatellites, showing new alleles and specific genetic patterns. These genetic signatures allow tracing connections to plants from the Asian homeland, Near and Remote Oceania, establishing links not observed previously (using the same genetic tools) in extant plants or herbaria samples. These results show that historic barkcloth textiles are cultural materials amenable to genetic analysis to reveal human history and that these artifacts may harbor evidence of greater genetic diversity in Pacific B. papyrifera in the past., Competing Interests: The authors have declared that no competing interests exist.

Paper mulberry, Broussonetia papyrifera (L.) L'Hér. ex Vent. (Moraceae), a dioecious species, was transported by humans from Taiwan to the islands of Remote Oceania. Its introduction and cultivation in Remote Oceania was intentional due to its cultural importance as a fiber source for barkcloth textiles. The aim of this study was to explore the genetic diversity and structure of paper mulberry populations within Remote Oceania in order to infer dispersal patterns that may reflect past human interaction among island groups. We present the integrated analysis of 380 samples (313 contemporary and 67 herbarium specimens) collected in Near and Remote Oceania. Genetic characterization was based on a set of ten microsatellites developed for B. papyrifera and complemented with the analysis of the ribosomal internal transcribed spacer ITS-1 sequence, a sex marker and the chloroplast ndhF-rpl32 intergenic spacer. Microsatellite data identify a total of 64 genotypes, despite this being a clonally propagated crop, and show three major dispersal hubs within Remote Oceania, centered on the islands of Fiji, Tonga, and Pitcairn. Of 64 genotypes identified, 55 correspond to genotypes associated to female-sexed plants that probably descend from plants introduced by the prehistoric Austronesian-speaking voyagers. The ratio of accessions to genotypes between herbarium and contemporary samples, suggests recent loss of genetic diversity. In addition to the chloroplast haplotypes described previously, we detected two new haplotypes within Remote Oceania both originating in Taiwan. This is the first study of a commensal species to show genetic structuring within Remote Oceania. In spite of the genetic bottleneck, the presence of only one sex, a timespan of less than 5000 years, and asexual propagation of this crop in Remote Oceania, we detect genetic diversity and regional structuring. These observations suggest specific migration routes between island groups within Remote Oceania., Competing Interests: The authors have declared that no competing interests exist.

Paper mulberry (Broussonetia papyrifera) is a well-known woody tree historically used for Cai Lun papermaking, one of the four great inventions of ancient China. More recently, Paper mulberry has also been used as forage to address the shortage of feedstuff because of its digestible crude fiber and high protein contents. In this study, we obtained a chromosome-scale genome assembly for Paper mulberry using integrated approaches, including Illumina and PacBio sequencing platform as well as Hi-C, optical, and genetic maps. The assembled Paper mulberry genome consists of 386.83 Mb, which is close to the estimated size, and 99.25% (383.93 Mb) of the assembly was assigned to 13 pseudochromosomes. Comparative genomic analysis revealed the expansion and contraction in the flavonoid and lignin biosynthetic gene families, respectively, accounting for the enhanced flavonoid and decreased lignin biosynthesis in Paper mulberry. Moreover, the increased ratio of syringyl-lignin to guaiacyl-lignin in Paper mulberry underscores its suitability for use in medicine, forage, papermaking, and barkcloth making. We also identified the root-associated microbiota of Paper mulberry and found that Pseudomonas and Rhizobia were enriched in its roots and may provide the source of nitrogen for its stems and leaves via symbiotic nitrogen fixation. Collectively, these results suggest that Paper mulberry might have undergone adaptive evolution and recruited nitrogen-fixing microbes to promote growth by enhancing flavonoid production and altering lignin monomer composition. Our study provides significant insights into genetic basis of the usefulness of Paper mulberry in papermaking and barkcloth making, and as forage. These insights will facilitate further domestication and selection as well as industrial utilization of Paper mulberry worldwide., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

Background: Heavy metal contamination is becoming a limitation to the utilization of soil and the distribution of vegetation. In particular, cadmium (Cd) pollution has had a serious impact on the food chain. Broussonetia papyrifera is a widely distributed pioneer tree species of heavy metal contaminated areas with important economic value. However, little is known about the genomic background of the Cd-tolerance mechanism in B. papyrifera., Results: The CdCl 2 responsive physiology was evaluated and proved to be involved in antioxidase activity and active oxygen species (ROS) accumulation. The leaf and root transcriptomes derived from B. papyrifera grown under normal and CdCl 2 stress conditions were systematically investigated using the Illumina HiSeq method. A total of 180,678,660 bp (27.1 GB) clean reads were assembled into 589,487 high-quality unigenes, of which 256,025 (43.43% of the total) and 250,251 (42.45% of the total) were aligned in Gene Ontology (GO) and Protein family (Pfam), respectively. A total of 24,414 differentially expressed genes (DEGs) were GO-annotated into 53, 23, 55, and 60 terms from the transcriptomes of root and leaf tissues under Cd stress and control conditions. A total of 117,547 Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO)-annotated DEGs were enriched in at least 47 KEGG pathway terms among the four comparisons. Many genes encoding important transcription factors (e.g., auxin/indole-3-acetic acid (AUX/IAA), basic helix-loop-helix (bHLH), DNA-binding one zinc finger (Dof), and MYB) and proteins involved in plant-pathogen interactions, phenylpropanoid biosynthesis, plant hormone signal transduction, oxidative phosphorylation, carbon fixation, peroxisomes, flavonoid biosynthesis, and glutathione metabolism, among others, were substantially upregulated under CdCl 2 stress., Conclusions: These genes represent important candidates for studying Cd-response mechanisms and molecular biology of B. papyrifera and related species. Our findings provide a genomic sequence resource for functional genetic assignments in B. papyrifera, which will help elucidate the molecular mechanisms of its Cd-stress responses and facilitate the bioremediation of heavy metal contaminated areas via breeding of new stress-tolerant cultivars., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Written communication plays a crucial role in the history of modern civilizations as manuscripts do not only exist contemporarily, but are passed on to subsequent generations. Besides a document's content, information is stored in the materials used for its production. Analyses of the composition allow, for example, identifying the biological origins of materials, dating, and help to understand degradation patterns. A combination of microscopic and DNA approaches was applied in order to analyze various plant derived writing sheets. Given their diversity and abundance in museum collections, plant based writing supports are yet an underexplored target for DNA studies. DNA retrieval of paper is low compared to raw paper plant material, which is likely due to the loss of organic components during paper production. Optimizing DNA extraction for each respective material drastically increased DNA recovery. Finally, we present a non-invasive DNA sampling method that utilizes nylon membranes, commonly used for bacterial DNA sampling and that is applicable to delicate material. Although bacterial infestation was visible on one sample, as indicated by scanning electron microscopy, endogenous DNA was retrieved. The results presented here are promising as they extend the scope of sources for DNA analyses by demonstrating that DNA molecules can be retrieved from a variety of plant derived writing supports. In future, such analyses can help to explore the biological diversity not only of plants and of additives utilized for producing writing supports, but also of the plenty products made from paper., Competing Interests: The authors have declared that no competing interests exist.

Background and Aims: Paper mulberry or Broussonetia papyrifera (L.) L'Hér. ex Vent. (Moraceae) is a dioecious species native to continental South-east Asia and East Asia, including Taiwan, that was introduced to the Pacific by pre-historic voyagers and transported intentionally and propagated asexually across the full range of Austronesian expansion from Taiwan to East Polynesia. The aim of this study was to gain insight into the dispersal of paper mulberry into Oceania through the genetic analysis of herbaria samples which represent a more complete coverage of the historical geographical range of the species in the Pacific before later introductions and local extinctions occurred., Methods: DNA from 47 herbarium specimens of B. papyrifera collected from 1882 to 2006 from different islands of the Pacific was obtained under ancient DNA protocols. Genetic characterization was based on the ribosomal internal transcribed spacer ITS-1 sequence, a sex marker, the chloroplast ndhF-rpl32 intergenic spacer and a set of ten microsatellites developed for B. papyrifera., Key Results: Microsatellites allowed detection of 15 genotypes in Near and Remote Oceanian samples, in spite of the vegetative propagation of B. papyrifera in the Pacific. These genotypes are structured in two groups separating West and East Polynesia, and place Pitcairn in a pivotal position. We also detected the presence of male plants that carry the Polynesian chloroplast DNA (cpDNA) haplotype, in contrast to findings in contemporary B. papyrifera populations where only female plants bear the Polynesian cpDNA haplotype., Conclusions: For the first time, genetic diversity was detected among paper mulberry accessions from Remote Oceania. A clear separation between West and East Polynesia was found that may be indicative of pulses during its dispersal history. The pattern linking the genotypes within Remote Oceania reflects the importance of central Polynesia as a dispersal hub, in agreement with archaeological evidence., (© The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: Paper mulberry (Broussonetia papyrifera (L.) L'Hér. ex Vent) is a dioecious tree native to East Asia and mainland Southeast-Asia, introduced prehistorically to Polynesia as a source of bark fiber by Austronesian-speaking voyagers. In Oceania, trees are coppiced and harvested for production of bark-cloth, so flowering is generally unknown. A survey of botanical records of paper mulberry revealed a distributional disjunction: the tree is apparently absent in Borneo and the Philippines. A subsequent study of chloroplast haplotypes linked paper mulberry of Remote Oceania directly to a population in southern Taiwan, distinct from known populations in mainland Southeast-Asia., Methodology: We describe the optimization and use of a DNA marker designed to identify sex in paper mulberry. We used this marker to determine the sex distribution in selected localities across Asia, Near and Remote Oceania. We also characterized all samples using the ribosomal internal transcribed spacer sequence (ITS) in order to relate results to a previous survey of ITS diversity., Results: In Near and Remote Oceania, contemporary paper mulberry plants are all female with the exception of Hawaii, where plants of both sexes are found. In its natural range in Asia, male and female plants are found, as expected. Male plants in Hawaii display an East Asian ITS genotype, consistent with modern introduction, while females in Remote Oceania share a distinctive variant., Conclusions: Most paper mulberry plants now present in the Pacific appear to be descended from female clones introduced prehistorically. In Hawaii, the presence of male and female plants is thought to reflect a dual origin, one a prehistoric female introduction and the other a modern male introduction by Japanese/Chinese immigrants. If only female clones were dispersed from a source-region in Taiwan, this may explain the absence of botanical records and breeding populations in the Philippines and Borneo, and Remote Oceania.

Broussonetia papyrifera is an important native tree species with high economic value in southwest China. Its resources are drastically reduced because of over-harvesting and habitat fragmentation. In this study, 17 natural populations of B. papyrifera were analyzed using inter-simple sequence repeat (ISSR) markers to assess the genetic diversity and population structure. In total, 100 bands were obtained from 16 ISSR primers. The B. papyrifera populations showed relatively high genetic diversity at the species level [percentage of polymorphic bands (PPB): 96%; Nei's genetic diversity (HE): 0.3074; Shannon's information index (I): 0.4617], while the genetic diversity at the population level was relatively low (PPB: 53.2%; HE: 0.1826; I: 0.2735). Relatively high level of genetic differentiation among populations (41%) was disclosed by analysis of molecular variance, which agrees with the Nei's genetic diversity statistics (40.59%) and Shannon's information measure (40.76%). Gene flow among populations (NM) was only 0.7318. A significant correlation was observed between genetic and geographic distance among the studied populations (r=0.2948). We conjectured that the genetic diversity of B. papyrifera resulted from human disturbance, habitat fragmentation, small effective population size, and geographic barrier. Given the high genetic differentiation among populations, some utilization and conservation strategies were proposed. This study provides a reference for the sustainable use of the species in southwest China.

The paper mulberry is one of the multifunctional tree species in agroforestry systems and is also commonly utilized in traditional medicine in China and other Asian countries. However, little is known about its molecular genetics, which hinders research on and exploitation of this valuable resource. To discern the correlation between gene expression and the essential properties of the paper mulberry, we performed a transcriptomics analysis, assembling a total of 37,725 unigenes from 54,638,676 reads generated by RNA-seq. Among these, 22,692 unigenes showed greater than 60% similarity with genes from other species. The lengths of 13,566 annotated unigenes were longer than 1,000 bp. Functional clustering analysis with COG (Cluster of Orthologous Groups) revealed that 17,184 unigenes are primarily involved in transcription, translation, signal transduction, carbohydrate metabolism, secondary metabolism, and energy metabolism. GO (Gene Ontology) annotation suggests enrichment of genes encoding antioxidant activity, transporter activity, biosynthesis, metabolism and stress response, with a total of 30,659 unigenes falling in these categories. KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway analysis showed that 7,199 unigenes are associated with 119 metabolic pathways. In addition to the basic metabolism, these genes are enriched for plant pathogen interaction, flavonoid metabolism and other secondary metabolic processes. Furthermore, differences in the transcriptomes of leaf, stem and root tissues were analyzed and 7,233 specifically expressed unigenes were identified. This global expression analysis provided novel insights about the molecular mechanisms of the biosynthesis of flavonoid, lignin and cellulose, as well as on the response to biotic and abiotic stresses including the remediation of contaminated soil by the paper mulberry.

The dehydration-responsive element binding proteins (DREBs) are important transcription factors in the regulation of plant responses to abiotic stresses. In this study, BpDREB2, an AP2/DREB-type transcription factor gene, was cloned from a woody plant, Broussonetia papyrifera by RACE-PCR. Sequence analyses revealed that BpDREB2 protein has three characteristic domains, including an AP2/EREBP, a nuclear localization signal and an acidic activation domain. Yeast one-hybrid assays showed that BpDREB2 protein specifically binds to the DRE sequence and activates the expression of reporter genes in yeast. These results suggested that BpDREB2 protein could function as a transcription factor of DREB family. The expression of BpDREB2 gene was remarkably induced by dehydration and high-salt treatments, but no significant change was observed under ABA or low-temperature conditions. Importantly, transgenic expression of BpDREB2 gene in Arabidopsis significantly enhanced its tolerance to salt and freezing without causing growth retardation. Taken together, these results suggested that BpDREB2 is a novel member of the AP2/EREBP trans-acting factor family which could enhance salt stress tolerance of plants and has the potential application in the improvement of crops and economical tree species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dehydration-responsive element binding/C-repeat-binding factors (DREB/CBF) control the activity of multiple stress response genes and therefore represent attractive targets for genetic improvement of abiotic stress tolerance. Paper mulberry (Broussonetia papyrifera L. Vent) is well known for its bark fibers and high levels of chalcone and flavonoid derivatives. Transgenic paper mulberry plants expressing a tall fescue (Festuca arundinacea Schreb.) FaDREB1 gene under the control of CaMV 35S were produced to examine the potential utility of FaDREB1 to increase the tolerance of paper mulberry plants to abiotic stress. The overexpressing FaDREB1 plants showed higher salt and drought tolerance than the wild-type plants (WT). After 13 days of withholding water, or 15 days in the presence of 250 mM NaCl, all the WT plants died, while the over-expressing FaDREB1 plants survived. The FaDREB1 plants had higher leaf water and leaf chlorophyll contents, accumulated more proline and soluble sugars, and had less ion leakage (which reflects membrane damage) than the WT plants had under high salt- and water-deficient conditions. The 35S promoter-driven expression of FaDREB1 did not cause growth retardation under normal growth conditions. Therefore, improved tolerance to multiple environmental stresses in paper mulberry might be achieved via genetic engineering through the ectopic expression of an FaDREB1 gene.

Dehydration-responsive element binding (DREB) proteins are a subfamily of AP2/ERF transcription factors that have been shown to improve tolerance to osmotic stresses in plants. To improve the osmotic stress tolerance of paper mulberry (Broussonetia papyrifera L. Vent), an economically important tree, we transformed it with a plasmid carrying tall fescue (Festuca arundinacea Schreb) FaDREB2 under the control of CaMV 35S. The ectopic expression of FaDREB2 did not cause growth retardation, and the paper mulberry seedlings expressing FaDREB2 showed higher salt and drought tolerance than wild-type plants (WT). After 13 d of withholding water, or 15 d in the presence of 250 mM NaCl, all the WT plants died, while the plants expressing FaDREB2 survived. The FaDREB2 transgenic plants had higher leaf water and chlorophyll contents, accumulated more proline and soluble sugars, and had less membrane damage than the WT plants under high salt and water-deficient conditions. Taken together, the results indicate the feasibility of improving tolerance to multiple environmental stresses in paper mulberry seedlings via genetic engineering, by introducing FaDREB2, which promotes the increased accumulation of osmolytes (soluble sugars and proline), to counter osmotic stresses caused by abiotic factors., (© 2011 Institute of Botany, Chinese Academy of Sciences.)

Paper mulberry (Broussonetia papyrifera L. Vent) is well known for its bark fibers, which are used for making paper, cloth, rope, etc. It was found that, in addition to its well-documented role in the enhancement of plant salt tolerance, overexpression of the Na+/H+ antiporter (AtNHX5) gene in paper mulberry plants showed high drought tolerance. After exposure to water deficiency and salt stress, the wild-type (WT) plants all died, while the AtNHX5-overexpressing plants remained alive under high salt stress, and had a higher survival rate (>66%) under drought stress. Measurements of ion levels indicated that Na+ and K+ contents were all higher in AtNHX5-overexpressing leaves than in WT leaves in high saline conditions. The AtNHX5 plants had higher leaf water content and leaf chlorophyll contents, accumulated more proline and soluble sugars, and had less membrane damage than the WT plants under water deficiency and high saline conditions. Taken together, the results indicate that the AtNHX5 gene could enhance the tolerance of paper mulberry plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions, soluble sugars, proline) to counter the osmotic stress caused by abiotic factors.