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Paulownias are among the fastest growing trees in the world, but they often suffer tremendous loss of wood production due to infection by Paulownia witches' broom (PaWB) phytoplasmas. In this study, we have sequenced and assembled a high-quality nuclear genome of Paulownia fortunei, a commonly cultivated paulownia species. The assembled genome of P. fortunei is 511.6 Mb in size, with 93.2% of its sequences anchored to 20 pseudo-chromosomes, and it contains 31 985 protein-coding genes. Phylogenomic analyses show that the family Paulowniaceae is sister to a clade composed of Phrymaceae and Orobanchaceae. Higher photosynthetic efficiency is achieved by integrating C3 photosynthesis and the crassulacean acid metabolism pathway, which may contribute to the extremely fast growth habit of paulownia trees. Comparative transcriptome analyses reveal modules related to cambial growth and development, photosynthesis, and defense responses. Additional genome sequencing of PaWB phytoplasma, combined with functional analyses, indicates that the effector PaWB-SAP54 interacts directly with Paulownia PfSPLa, which in turn causes the degradation of PfSPLa by the ubiquitin-mediated pathway and leads to the formation of witches' broom. Taken together, these results provide significant insights into the biology of paulownias and the regulatory mechanism for the formation of PaWB.

Nitazoxanide (NTZ) is a broad‐spectrum antiparasitic and antiviral drug (thiazole). However, although NTZ has been extensively used, there are no reports concerning its toxicology in vertebrates. This study used the zebrafish as a vertebrate model to evaluate the safety of NTZ and to analyse the related molecular mechanisms. The experimental results showed that zebrafish embryos exposed to NTZ had cardiac malformation and dysfunction. NTZ also significantly inhibited proliferation and promoted apoptosis in cardiomyocytes. Transcriptomic analysis used compared gene expression levels between zebrafish embryos in the NTZ treatment and the control groups identified 200 upregulated genes and 232 downregulated genes. Analysis by Kyoto encyclopaedia of genes and genomes (KEGG) and gene ontology (GO) showed that signal pathways on cardiomyocyte development were inhibited while the oxidative stress pathways were activated. Further experiments showed that NTZ increased the content of reactive oxygen species (ROS) in the hearts of zebrafish. Antioxidant gadofullerene nanoparticles (GFNPs) significantly alleviated the developmental toxicity to the heart, indicating that NTZ activated the oxidative stress response to cause embryonic cardiomyocyte injury in zebrafish. This study provides evidence that NTZ causes developmental abnormalities in the cardiovascular system of zebrafish.

To gain insights into the underlying molecular mechanisms of how P. tomentosa (Thunb.) Steud. × P. fortunei (Seem.) Hemsl responds to water-deficit condition at both transcriptional and post-transcriptional levels, we employed mRNA, small RNA and degradome sequencing to identify mRNAs and miRNAs involved in the water-deficit stress response in diploid and autotetraploid P. tomentosa × P. fortunei. Differentially expressed mRNA and miRNA profiles were obtained. The results showed that P. tomentosa × P. fortunei may respond to water-deficit stress through osmotic adjustment, reactive oxygen species (ROS) scavenging and plant hormone signal transduction. Transcription factors (NAC, WRKY, bZIP, ERF, MYB and ZFP) were also involved in the stress response. MiRNAs were found to have a role in P. tomentosa × P. fortunei response to water deficit. This study provides an integral view of the transcriptional and post-transcriptional responses of P. tomentosa × P. fortunei under water-deficit condition. The results will help in better understanding the molecular mechanisms of the water-deficit tolerance in P. tomentosa × P. fortunei, and the candidate water-deficit-responsive genes and miRNAs could be used in genetic engineering for developing Paulownia varieties with enhanced water-deficit tolerance in the future.

Background Paulownia withes’-broom (PaWB) disease caused by phytoplasma is a serious infectious disease for Paulownia. However, the underlying molecular pathogenesis is not fully understood. Recent studies have demonstrated that histone modifications could play a role in plant defense responses to pathogens. But there is still no available genome-wide histone modification data in non-model ligneous species infected with phytoplasma. Results Here, we provided the first genome-wide profiles of three histone marks (H3K4me3, H3K36me3 and H3K9ac) in Paulownia fortunei under phytoplasma stress by using chromatin immunoprecipitation sequencing (ChIP-Seq). We found that H3K4me3, H3K36me3 and H3K9ac were mainly enriched in the genic regions in P. fortunei with (PFI) and without (PF) phytoplasma infection. ChIP-Seq analysis revealed 1738, 986, and 2577 genes were differentially modified by H3K4me3, H3K36me3 and H3K9ac marks in PFI under phytoplasma infection, respectively. The functional analysis of these genes suggested that most of them were mainly involved in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, plant-pathogen interaction and plant hormone signal transduction. In addition, the combinational analysis of ChIP-Seq and RNA-Seq showed that differential histone methylation and acetylation only affected a small subset of phytoplasma-responsive genes. Conclusions Taken together, this is the first report of integrated analysis of histone modifications and gene expression involved in Paulownia-phytoplasma interaction. Our results will provide the valuable resources for the mechanism studies of gene regulation in non-model plants upon pathogens attack. Electronic supplementary material The online version of this article (10.1186/s12864-019-5609-1) contains supplementary material, which is available to authorized users.

Polyploidization is an important feature of speciation that results from genome doubling. Tetraploid plants often have better physical characteristics than their diploid counterparts, and Paulownia is no exception. In our previous studies, several genes related to timber quality and abiotic stress resistance were identified in Paulownia tomentosa and Paulownia fortunei. However, the genes related to disease resistance and high biomass were not confirmed. In this study, small RNA and transcriptome sequencing were performed to analyze changes in microRNA (miRNA) and mRNA expression levels in tetraploid Paulownia tomentosa × Paulownia fortunei and Paulownia australis and their diploid counterparts. A total of 930 common differentially expressed genes and 66 (19 known and 47 novel) common differentially expressed miRNAs were detected in the two tetraploid vs. diploid comparisons. Twenty-two miRNA target genes were predicted, and the regulatory functions of the miRNA–target gene pairs were analyzed, of which the novel miR327- SGT1 HOMOLOG PROTEIN At5g65490 (PAU019930.1) and pau-miR2111a-MYB-RELATED TRANSCRIPTION FACTOR (PAU011118.1) interacting pairs were predicted to co-regulate disease resistance in tetraploid Paulownia. The pau-miR157i-SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 15 (PAU009402.1) and novel miR10-PUTATIVE PENTATRICOPEPTIDE REPEAT-CONTAINING PROTEIN At5g65820 (PAU012281.1) interacting pairs were predicted to co-regulate high biomass in tetraploid Paulownia. The expression trends of the miRNA and candidate target genes were validated by qRT-PCR. The results will help to accelerate genetic gain in Paulownia breeding programs to develop superior varieties.

To enlarge the germplasm resource of Paulownia plants, we used colchicine to induce autotetraploid Paulownia tomentosa, as reported previously. Compared with its diploid progenitor, autotetraploid P. tomentosa exhibits better photosynthetic characteristics and higher stress resistance. However, the underlying mechanism for its predominant characteristics has not been determined at the proteome level. In this study, isobaric tag for relative and absolute quantitation coupled with liquid chromatography-tandem mass spectrometry was employed to compare proteomic changes between autotetraploid and diploid P. tomentosa. A total of 1427 proteins were identified in our study, of which 130 proteins were differentially expressed between autotetraploid and diploid P. tomentosa. Functional analysis of differentially expressed proteins revealed that photosynthesis-related proteins and stress-responsive proteins were significantly enriched among the differentially expressed proteins, suggesting they may be responsible for the photosynthetic characteristics and stress adaptability of autotetraploid P. tomentosa. The correlation analysis between transcriptome and proteome data revealed that only 15 (11.5%) of the differentially expressed proteins had corresponding differentially expressed unigenes between diploid and autotetraploid P. tomentosa. These results indicated that there was a limited correlation between the differentially expressed proteins and the previously reported differentially expressed unigenes. This work provides new clues to better understand the superior traits in autotetraploid P. tomentosa and lays a theoretical foundation for developing Paulownia breeding strategies in the future.

MicroRNAs (miRNAs) are small, single-stranded, non-coding endogenous RNAs that play vital roles in the regulation of gene expression at the post-transcriptional level. Several studies of miRNAs in diploid and tetraploid Paulownia plants have been reported; however, miRNAs involved in response to drought stress have not yet been investigated. In this study, six small RNA libraries of Paulownia fortunei, two drought-treated and four controls, were built and then sequenced on an Illumina/Solexa GAIIx platform. A total of 30 conserved miRNAs and 88 novel miRNAs were identified in the sRNA libraries. Among them, 22 miRNAs in common were differentially expressed both in diploid and tetraploid P. fortunei under the drought stress. Degradome sequencing was used to identify the target genes of the miRNAs. Moreover, nine differentially expressed miRNAs and ten target genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This study has provided data for further research into the molecular mechanisms associated with drought stress in diploid and autotetraploid P. fortunei plants.

Paulownia fortunei is a widely cultivated economic forest tree species that is susceptible to infection with phytoplasma, resulting in Paulownia witches’ broom (PaWB) disease. Diseased P. fortunei is characterized by stunted growth, witches’ broom, shortened internodes, and etiolated and smaller leaves. To understand the molecular mechanism of its pathogenesis, we applied isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography coupled with tandem mass spectrometry approaches to study changes in the proteomes of healthy P. fortunei, PaWB-infected P. fortunei, and PaWB-infected P. fortunei treated with 15 mg·L−1 or 75 mg·L−1 dimethyl sulfate. We identified 2969 proteins and 104 and 32 differentially abundant proteins that were phytoplasma infection responsive and dimethyl sulfate responsive, respectively. Based on our analysis of the different proteomes, 27 PaWB-related proteins were identified. The protein-protein interactions of these 27 proteins were analyzed and classified into four groups (photosynthesis-related, energy-related, ribosome-related, and individual proteins). These PaWB-related proteins may help in developing a deeper understanding of how PaWB affects the morphological characteristics of P. fortunei and further establish the mechanisms involved in the response of P. fortunei to phytoplasma.

Background: Paulownia witches' broom (PaWB) is a devastating disease caused by phytoplasma, which can lead to considerable economic losses. Previous studies have shown that 60 mg•L-1 methyl methane sulfonate (MMS) can restore infected Paulownia seedlings to healthy one. To improve the understanding of PaWB, we used single molecule real time (SMRT) sequencing, which can provide long sequences that can be effectively used to identify gene alternative splicing, obtain novel genes, improve gene annotations and comprehensive analysis of the metabolome. Results: Here, the first integrated analysis SMRT sequencing, the Illumina platforms, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) to analyze changes in transcripts and metabolites in Paulownia fortunei. A total of 140,528 non redundant transcripts were identified, which supported 6,209 of novel gene loci that can enrich 70,223 annotation information of Paulownia fortunei （P. fortunei）genome. A total of 5,606 transcripts, 46 lncRNAs, 83 fusion transcripts, 597 transcription factors, and 57 metabolites were related indirectly or directly to PaWB. Subsequent analysis of the transcriptome and metabolome found trans-cinnamate 4-monooxygenase, caffeoyl-CoA-O methyltransferase, ferulic acid and peroxidase were up-regulated in Paulownia fortunei infected with phytoplasma. They were involved in flavonoid, phenylpropane and salicylic acid metabolism, which might be related to Paulownia-phytoplasma interaction. Conclusions: Our results will enrich the understanding of molecular mechanisms of PaWB. This study provides a foundation for better understanding the changes in gene expression, metabolites, and morphology of Paulownia fortunei with PaWB.

MicroRNAs (miRNAs) play major roles in plant responses to various biotic and abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. Paulownia witches' broom (PaWB) disease caused by phytoplasmas reduces Paulownia production worldwide. In this study, we investigated the miRNA-mediated plant response to PaWB phytoplasma by Illumina sequencing and degradome analysis of Paulownia fortunei small RNAs (sRNAs). The sRNA and degradome libraries were constructed from healthy and diseased P. fortunei plants and the plants free from phytoplasma pathogen after 60 mg L(-1) methyl methane sulfonate treatment. A total of 96 P. fortunei-conserved miRNAs and 83 putative novel miRNAs were identified. Among them, 37 miRNAs (17 conserved, 20 novel) were found to be differentially expressed in response to PaWB phytoplasma infection. In addition, 114 target genes for 18 of the conserved miRNA families and 33 target genes for 15 of the novel miRNAs in P. fortunei were detected. The expression patterns of 14 of the PaWB phytoplasma-responsive miRNAs and 12 target genes were determined by quantitative real-time polymerase chain reaction (qPCR) experiments. A functional analysis of the miRNA targets indicated that these targeted genes may regulate transcription, stress response, nitrogen metabolism, and various other activities. Our results will help identify the potential roles of miRNAs involved in protecting P. fortunei from diseases.

Long noncoding RNA (lncRNA), circular RNA (circRNA), and microRNA (miRNA) are important in the regulation of life activities. However, their function is unclear in Paulownia fortunei. To identify lncRNAs, circRNAs, and miRNA, and investigate their roles in the infection progress of Paulownia witches&rsquo, broom (PaWB) disease, we performed RNA sequencing of healthy and infected P. fortunei. A total of 3126 lncRNAs, 1634 circRNAs, and 550 miRNAs were identified. Among them, 229 lncRNAs, 65 circRNAs, and 65 miRNAs were differentially expressed in a significant manner. We constructed a competing endogenous RNA (ceRNA) network, which contains 5 miRNAs, 4 circRNAs, 5 lncRNAs, and 15 mRNAs, all of which were differentially expressed between healthy and infected P. fortunei. This study provides the first catalog of candidate ceRNAs in Paulownia and gives a revealing insight into the molecular mechanism responsible for PaWB.

Paulownia is a tree species grown in many countries. Our previous study reveals that tetraploid Paulownia fortunei is more tolerant to salt stress than its corresponding diploid tree. To investigate the molecular mechanisms of salt stress tolerance in P. fortunei, the transcriptomes of normal and salt-stressed diploid and tetraploid were investigated. After assembling the clean reads, we obtained 130,842 unigenes. The unigenes were aligned against six public databases (Nr, Nt, Swiss-Prot, COG, KEGG, GO) to discover homologs and assign functional annotations. We retrieved 7983 and 15,503 differentially expressed unigenes (DEUs) between the normal and the salt-stressed diploid and tetraploid P. fortunei, respectively. We identified dozens of important DEUs including 3 related to photosynthesis, 10 related to plant growth and development and 11 related to osmolytes. Some of these DEUs were upregulated in tetraploid compared to diploid and others were upregulated under salt stress. Quantitative reverse transcriptase polymerase chain reaction verified the expression patterns of 15 unigenes. Our results provided insights into the molecular aspects why tetraploid is stronger and more energetic than diploid under saline environment. This study provides useful information for further studies on the molecular mechanisms of salt tolerance in other tree plants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-018-0578-4) contains supplementary material, which is available to authorized users.

Paulownia witches&rsquo, broom (PaWB), caused by phytoplasma, is an important disease of Paulownia. To further identify the key miRNAs associated with the formation of PaWB symptoms, miRNA and degradome sequencing were performed to explore important miRNAs&ndash, target regulation in healthy and diseased Paulownia tomentosa, Paulownia fortunei, and P. tomentosa &times, P. fortunei seedlings, and the corresponding diseased seedlings treated with 75 mg L&minus, 1 dimethyl sulfate. A total of 212, 111, and 197 differentially expressed miRNAs (DEMs) were obtained in P. tomentosa, P. fortunei, and P. tomentosa &times, P. fortunei, respectively. Degradome sequencing detected 559, 251, and 568 target genes of the DEMs in P. tomentosa, P. fortunei, and P. tomentosa &times, P. fortunei, respectively. The expression patterns of selected miRNAs and the target genes were verified be qRT-PCR. Through analysis of the expression level of the DEMs in this study, combined with the results in our previous studies, as well as with those reported in other phytoplasma-infected plants, we concluded that miR156 is an important miRNA related to witches&rsquo, broom. According to the functions of the target genes of DEMs, we constructed a co-regulatory network of the DEMs-target genes interaction. These results will help to advance the understanding of the mechanism of PaWB.

Paulownia witches’ broom is arisen from the invasion of an obligatory parasitic bacteria-phytoplasma, which can result in huge economic losses of Paulownia trees. The mechanism of the occurrence of this disease has been studied extensively. However, its pathogenesis is still poorly understood. Here, whole transcriptome strand-specific RNA sequencing was performed to identify long noncoding RNAs (lncRNAs) that referred to phytoplasma infection in Paulownia. In all, 2725 lncRNAs were identified and 748 of them were considered to be differentially expressed. KEGG pathway analysis showed the predicted target genes of these 748 lncRNAs participated mainly in lignin biosynthesis pathway, plant–pathogen interaction pathway and plant hormone signal transduction pathway, which indicated that lncRNAs may be closely related to the occurrence of witches’ broom. The results provide new perspective into the function of lncRNAs in Paulownia and potentially in other trees in answering phytoplasma invasion.

Paulownia is a tree native to China, with important ecological and economic value. Long noncoding RNAs (lncRNAs) are known to play important roles in eukaryotic gene regulation. However, no lncRNAs have been reported in Paulownia so far. We performed RNA sequencing of two Paulownia tomentosa lncRNA libraries constructed from the terminal buds of normal untreated seedlings and 60 mg L(−1) MMS-treated seedlings, and obtained a total of 2531 putative lncRNAs. The average length of the lncRNA transcripts was much less than the average length of the mRNA transcripts in the P. tomentosa libraries. A few of the Paulownia lncRNAs were conserved among ten species tested. We identified seven lncRNAs as precursors of 13 known miRNAs, 15 lncRNAs may act as target mimics of 19 miRNAs, and 351 unique noncoding sequences belonging to 133 conserved lncRNA families. In addition, we identified 220 lncRNAs responsive to methyl methane sulfonate (MMS), including seven phytohormone-related lncRNAs and one lncRNAs involved in base excision repair. This is the first time that lncRNAs have been explored in Paulownia. The lncRNA data may also provide new insights into the MMS-response in P. tomentosa. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-018-0513-8) contains supplementary material, which is available to authorized users.

Paulownia witches’ broom caused by phytoplasma infection affects the production of Paulownia trees worldwide. Emerging evidence showed that long noncoding RNAs (lncRNA) play a protagonist role in regulating the expression of genes in plants. So far, the identification of lncRNAs has been limited to a few model plant species, and their roles in mediating responses to Paulownia tomentosa that free of phytoplasma infection are yet to be characterized. Here, whole-genome identification of lncRNAs, based on strand-specific RNA sequencing, from four Paulownia tomentosa samples, was performed and identified 3689 lncRNAs. These lncRNAs showed low conservation among plant species and some of them were miRNA precursors. Further analysis revealed that the 112 identified lncRNAs were related to phytoplasma infection. We predicted the target genes of these phytoplasma-responsive lncRNAs, and our analysis showed that 51 of the predicted target genes were alternatively spliced. Moreover, we found the expression of the lncRNAs plays vital roles in regulating the genes involved in the reactive oxygen species induced hypersensitive response and effector-triggered immunity in phytoplasma-infected Paulownia. This study indicated that diverse sets of lncRNAs were responsive to Paulownia witches’ broom, and the results will provide a starting point to understand the functions and regulatory mechanisms of Paulownia lncRNAs in the future.

Autopolyploid plants and their organs are larger than their corresponding diploid ancestors, and they attract considerable attention for plant breeding. Paulownia is a fast-growing tree. To identify genes related to the biological characteristics of tetraploid Paulownia, transcriptome and small RNA sequencing were used to identify the key gene expression regulation in tetraploid Paulownia fortunei and tetraploid P. tomentosa and their corresponding diploids. A total of 1977 common differentially expressed genes (DEGs) and 89 differentially expressed miRNAs (DEMs) (38 conserved and 51 novel) were obtained in tetraploid vs. diploid comparisons of the two Paulownia species, and 18 target genes were identified by target prediction. Finally, by analyzing the expression profiles of the DEGs and DEMs and their target genes, we discovered that Pau-miR169, Pau-miR408 and Pau-miR156 interacted with their target gene nuclear transcription factor Y subunit A-9 (NF-YA9), serine/threonine protein phosphatase (PP1) and s-adenosyl-methionine-sterol-c-methyltransfera—se (SAM:SMT) to regulate the abiotic stress tolerance and the timber quality of the tetraploid Paulownia. This study lays a molecular biology foundation for understanding autotetraploid Paulownia and will benefit future breeding work.

Paulownia witches’ broom (PaWB) caused by an obligate biotrophic plant pathogen called phytoplasma, is a devastating disease of paulownia trees over a large part of the world. However, little is known about the molecular mechanisms that underlie phytoplasma pathogenicity in paulownia or about the mode of interactions with host plants. In this study, genome-wide gene expression profiling was used to compare healthy, phytoplasma-infected, and both phytoplasma-infected and 20 mg L−1 methyl methane sulfonate (MMS) treated Paulownia fortunei plants using high-throughput mRNA sequencing analysis. A total of 6571 and 1377 differentially expressed unigenes were identified in the phytoplasma-infected plants versus healthy plants and in 20 mg L−1 MMS-treated plants versus phytoplasma-infected plants, respectively. Expression changes of 16 candidate differentially expressed unigenes were validated by qRT-PCR, indicating significant differences among the three P. fortunei samples. Our analysis showed that dramatic changes occurred in the gene expression profile of P. fortunei after PaWB phytoplasma infection and MMS treatment. The transcription of a large number of genes related to the plant–pathogen interaction, including phenylpropanoid metabolism, hormone biosynthesis and signaling, defense and/or pathogenesis, and signal transduction, were significantly up-regulated in the phytoplasma-infected paulownia and then returned to the levels in the healthy controls after MMS treatment. Our systematic analysis provides comprehensive transcriptomic data about P. fortunei trees infected by PaWB phytoplasma. The findings will help unraveling the molecular mechanisms of plant-phytoplasma interactions and may pave the way for engineering P. fortunei trees with improved properties.

Phytoplasmas are mycoplasma-like pathogens of witches’ broom disease, and are responsible for serious yield losses of Paulownia trees worldwide. The molecular mechanisms of disease development in Paulownia are of considerable interest, but still poorly understood. Here, we have applied transcriptome sequencing technology and a de novo assembly approach to analyze gene expression profiles in Paulownia fortunei infected by phytoplasmas. Our previous researches suggested that methyl methane sulfonated (MMS) could reverse the effects of the infection. In this study, leaf samples from healthy, infected, and both infected and methyl methane sulfonate treated plants were analyzed. The results showed that the gene expression profile of P. fortunei underwent dramatic changes after Paulownia witches’ broom (PaWB) phytoplasma infection. Genes that encoded key enzymes in plant-pathogen interaction processes were significantly up-regulated in the PaWB-infected Paulownia. Genes involved in circadian rhythm and hormone-related genes were also altered in Paulownia after PaWB infection. However, after the PaWB-infected plants were treated with MMS, the expression profiles of these genes returned to the levels in the healthy controls. The data will help identify potential PaWB disease-resistance genes that could be targeted to inhibit the growth and reproduction of the pathogen and to increase plant resistance.

Paulownia is a fast-growing deciduous tree native to China. It has great economic importance for the pulp and paper industries, as well as ecological prominence in forest ecosystems. Paulownia is of much interest to plant breeder keen to explore new plant varieties by selecting on the basis of phenotype. A newly synthesized autotetraploid Paulownia exhibited advanced characteristics, such as greater yield, and higher resistance than the diploid tree. However, tissue-specific transcriptome and genomic data in public databases are not sufficient to understand the molecular mechanisms associated with genome duplication. To evaluate the effects of genome duplication on the phenotypic variations in Paulownia tomentosa×Paulownia fortunei, the transcriptomes of the autotetraploid and diploid Paulownia were compared. Using Illumina sequencing technology, a total of 82,934 All-unigenes with a mean length of 1109 bp were assembled. The data revealed numerous differences in gene expression between the two transcriptomes, including 718 up-regulated and 667 down-regulated differentially expressed genes between the two Paulownia trees. An analysis of the pathway and gene annotations revealed that genes involved in nucleotide sugar metabolism in plant cell walls were down-regulated, and genes involved in the light signal pathway and the biosynthesis of structural polymers were up-regulated in autotetraploid Paulownia. The differentially expressed genes may contribute to the observed phenotypic variations between diploid and autotetraploid Paulownia. These results provide a significant resource for understanding the variations in Paulownia polyploidization and will benefit future breeding work.

Drought is one of the most devastating effects that severely reduce plant growth and development worldwide. In recent years, the availability of a reference Paulownia genome sequence has made it easier to explore gene expression, transcriptional regulation, and posttranscriptional regulation in Paulownia species. Here, we combined the analyses of the transcriptome, small RNAs, and degradome of Paulownia tomentosa seedlings to generate a comprehensive resource to describe the links between key regulatory miRNA-target gene pairs and drought stress. A total of 22,904 differentially expressed genes, 2073 differentially expressed miRNAs, and 198 target genes were identified by deep sequencing. Gene ontology function and KEGG pathway analyses of the differentially expressed genes and the target genes of the differentially expressed miRNAs revealed that momilactone A synthase, 14-3-3 protein, serine/threonine-protein kinase CTR1, and polyphenol oxidase, as well as alternative splicing, were associated directly or indirectly with drought stress in P. tomentosa. Our results will help to pave the way for further genomic studies, not only on P. tomentosa but also on other plants in family Paulowniaceae.

Paulownia witches’ broom (PaWB) disease caused by phytoplasmas is a fatal disease that leads to considerable economic losses. Long non-coding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in posttranscriptional and transcriptional regulation. However, lncRNAs and their functional roles remain poorly characterized in Paulownia. To identify lncRNAs and investigate their roles in the response to PaWB phytoplasmas, RNA sequencing was performed for healthy Paulownia tomentosa, PaWB-infected P. tomentosa, and for healthy and PaWB-infected P. tomentosa treated with 100 mg L−1 rifampicin. A total of 28,614 unique mRNAs and 3693 potential lncRNAs were identified. Comparisons between lncRNAs and coding genes indicated that lncRNAs tended to have shorter transcripts and fewer exon numbers, and displayed significant expression specificity. Based on our comparison scheme, 1063 PaWB-related mRNAs and 110 PaWB-related lncRNAs were identified; among them, 12 PaWB-related candidate target genes that were regulated by nine PaWB-related lncRNAs were characterized. This study provides the first catalog of lncRNAs expressed in Paulownia and gives a revealing insight into the molecular mechanism responsible for PaWB.

Paulownia australis has important economic and ecological values. In this study, the morphological and physiological changes of the leaves in diploid and autotetraploid P. australis under salt stress were analyzed. To detect related genes and gain a comprehensive perspective on the molecular mechanisms underlying salt tolerance in P. australis, transcriptome-wide gene expression profiling was conducted in the leaves of the diploid and autotetraploid P. australis under control and salinity conditions, respectively. Evaluation of the responses against salinity stress revealed the superiority of autotetraploid over diploid in terms of salinity tolerance. Changes in physiological parameters in diploid P. australis (PA2) and tetraploid P. australis (PA4) plants in response to salt stress were measured. Transcriptome data revealed that many of the common unigenes which were involved in accumulation of compatible solutes, oxidative stress detoxification, ion homeostasis, and signal transduction showed significant differences between the two accessions in response to salt stress. A number of salt-responsive unigenes were identified in two accessions of P. australis under salt stress. Furthermore, the differentially expressed unigenes found to be common in both accessions may be useful genetic resources for further genetic improvement of Paulownia using transgenic approaches.

Phytoplasma is an insect-transmitted pathogen that causes witches' broom disease in many plants. Paulownia witches' broom is one of the most destructive diseases threatening Paulownia production. The molecular mechanisms associated with this disease have been investigated by transcriptome sequencing, but changes in protein abundance have not been investigated with isobaric tags for relative and absolute quantitation. Previous results have shown that methyl methane sulfonate (MMS) can help Paulownia seedlings recover from the symptoms of witches' broom and reinstate a healthy morphology. In this study, a transcriptomic-assisted proteomic technique was used to analyze the protein changes in phytoplasma-infected Paulownia tomentosa seedlings, phytoplasma-infected seedlings treated with 20 and 60 mg·L−1 MMS, and healthy seedlings. A total of 2,051 proteins were obtained, 879 of which were found to be differentially abundant in pairwise comparisons between the sample groups. Among the differentially abundant proteins, 43 were related to Paulownia witches' broom disease and many of them were annotated to be involved in photosynthesis, expression of dwarf symptom, energy production, and cell signal pathways.

Understanding the role of miRNAs in regulating the molecular mechanisms responsive to drought stress was studied in Paulownia “yuza 1.” Two small RNA libraries and two degradome libraries were, respectively, constructed and sequenced in order to detect miRNAs and their target genes associated with drought stress. A total of 107 miRNAs and 42 putative target genes were identified in this study. Among them, 77 miRNAs were differentially expressed between drought-treated Paulownia “yuza 1” and the control (60 downregulated and 17 upregulated). The predicted target genes were annotated using the GO, KEGG, and Nr databases. According to the functional classification of the target genes, Paulownia “yuza 1” may respond to drought stress via plant hormone signal transduction, photosynthesis, and osmotic adjustment. Furthermore, the expression levels of seven miRNAs (ptf-miR157b, ptf-miR159b, ptf-miR398a, ptf-miR9726a, ptf-M2153, ptf-M2218, and ptf-M24a) and their corresponding target genes were validated by quantitative real-time PCR. The results provide relevant information for understanding the molecular mechanism of Paulownia resistance to drought and reference data for researching drought resistance of other trees.

MicroRNAs (miRNAs) play profound roles in plant growth and development by regulating gene expression. Tetraploid plants often have better physical characteristics and stress tolerance than their diploid progenitors, but the role of miRNAs in this superiority is unclear. Paulownia tomentosa, (Paulowniaceae) is attracting research attention in China because of its rapid development, wide distribution, and potential economic uses. To identify miRNAs at the transcriptional level in P. tomentosa, Illumina sequencing was used to sequence the libraries of diploid and tetraploid plants. Sequence analysis identified 37 conserved miRNAs belonging to 14 miRNA families and 14 novel miRNAs belonging to seven miRNA families. Among the miRNAs, 16 conserved miRNAs from 11 families and five novel miRNAs were differentially expressed in the tetraploid and diploid; most were more strongly expressed in the former. The miRNA target genes and their functions were identified and discussed. The results showed that several P. tomentosa miRNAs may play important roles in the improved traits seen in tetraploids. This study provides a foundation for understanding the regulatory mechanisms of miRNAs in tetraploid trees.

Paulownia fortunei (family Paulowniaceae) is an economically important tree species indigenous to China. Autotetraploid cultivars of P. fortunei have better growth and wood quality than their diploid counterparts. MicroRNAs (miRNAs) play vital regulatory roles in plant growth, development, and biotic and abiotic stress responses by direct cleavage of transcripts, translational repression, or chromatin modification. Although miRNAs have been identified in various plant species, no reports of miRNAs in P. fortunei have been published so far. To study the functions of miRNAs in the autotetraploid P. fortunei, four sequencing libraries from the autotetraploid and its corresponding diploid plants were constructed. 142 conserved miRNAs grouped into 41 families, and 38 novel miRNAs were obtained. Among these miRNAs, 58 were up-regulated and 30 were down-regulated in the autotetraploid relative to the diploid. MiRNA target genes were identified using a degradome sequencing approach and the differently expressed miRNAs and their target genes were validated by real time PCR analysis. To our knowledge, this is the first study to identify conserved and novel miRNAs and their potential targets from diploid and autotetraploid Paulownia plants using high-throughput sequencing and degradome analysis. Our results provide valuable information on P. fortunei miRNAs and their targets, and will help build a foundation for future studies of the biological functions of miRNA-mediated gene regulation in P. fortunei.

Paulownia witches’ broom (PaWB) is a disastrous epidemic disease in Paulownia tomentosa trees in many parts of the world. Infected Paulownia tomentosa trees are generally characterized by witches’ brooms, proliferating branches, yellowing and withering leaves, and branch dieback. In previous studies, we found that by treating seedlings with a certain concentration of methyl methanesulphonate (MMS), PaWB could be cured. In this study, three small RNA libraries from healthy, PaWB-infected, and PaWB-infected Paulownia tomentosa treated with MMS were constructed and sequenced by Illumina sequencing and identified approximately 50 conserved and novel microRNAs (miRNAs) in each library. We investigated the expression patterns of the miRNAs in three libraries and found that 20 conserved miRNAs were significantly differently expressed in the comparison (healthy vs. PaWB-infected); nine of these conserved miRNAs were also significantly differently expressed in another comparison (PaWB-infected vs. PaWB-infected MMS-treated). The target genes of these identified miRNAs were found through degradome sequencing platform and functionally annotated. Some of the target genes encoded transcription factors, including auxin response factors, which are known regulators of plant defense responses. Further, we found that the expression levels of some of the miRNA sequences changed in response to MMS treatment and were close to their expression levels in the healthy plantlets. Our results indicate possible roles for Paulownia miRNAs associated with plants infected with PaWB and may provide available information for improving the prevention and treatment of PaWB disease in the future.

Drought stress in plants often leads to reduced productivity and limited geographic distribution, which can affect human life and ecosystems. The responses of diploid and tetraploid Paulownia tomentosa × Paulownia fortunei to drought have been reported, but the effects of drought stress on the levels of microRNA (miRNA) expression have not been published so far. Here, we constructed four small RNA (sRNA) libraries and four corresponding degradome libraries of well-watered and severe drought-treated diploid and tetraploid plants to identify the miRNAs and their putative target genes in Paulownia ‘yuza 1’, a P. tomentosa × P. fortunei hybrid clone, by sRNA and degradome sequencing. The putative target genes of miRNAs were annotated with gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. Three conserved and 21 novel miRNAs responsive to drought stress were found, in which 15 were identified as the main drought responsive miRNAs that conferred higher resistance in tetraploid than in diploid of Paulownia ‘yuza 1’. Our results will lay the foundation for investigating the roles of miRNAs in Paulownia and other trees in response to drought.

Polyploidy plays a very important role in speciation and plant evolution by way of genomic merging and doubling. In the process of polyploidy, rapid genomic, and transcriptomic changes have been observed and researched. However, proteomic divergence caused by the effects of polyploidization is still poorly understood. In the present study, we used iTRAQ coupled with mass spectrometry to quantitatively analyze proteomic changes in the leaves of autotetraploid Paulownia and its diploid parent. A total of 2963 proteins were identified and quantified. Among them, 463 differentially abundant proteins were detected between autotetraploid Paulownia and its diploid parent, and 198 proteins were found to be non-additively abundant in autotetraploid Paulownia, suggesting the presence of non-additive protein regulation during genomic merger and doubling. We also detected 1808 protein-encoding genes in previously published RNA sequencing data. We found that 59 of the genes that showed remarkable changes at mRNA level encoded proteins with consistant changes in their abundance levels, while a further 48 genes that showed noteworthy changes in their expression levels encoded proteins with opposite changes in their abundance levels. Proteins involved in posttranslational modification, protein turnover, and response to stimulus, were significantly enriched among the non-additive proteins, which may provide some of the driving power for variation and adaptation in autopolyploids. Quantitative real-time PCR analysis verified the expression patterns of related protein-coding genes. In addition, we found that the percentage of differentially abundant proteins that matched previously reported differentially expressed genes was relatively low.

Paulownia tomentosa is an important economic and greening tree species that is cultivated widely, including salt environment. Our previous studies indicated its autotetraploid induced by colchicine showed better stress tolerance, but the underlying molecular mechanism related to ploidy and salt stress is still unclear. To investigate this issue, physiological measurements and transcriptome profiling of diploid and autotetraploid plants untreated and treated with NaCl were performed. Through the comparisons among four accessions, for one thing, we found different physiological changes between diploid and autotetraploid P. tomentosa; for another, and we detected many differentially expressed unigenes involved in salt stress response. These differentially expressed unigenes were assigned to several metabolic pathways, including “plant hormone signal transduction,” “RNA transporter,” “protein processing in endoplasmic reticulum,” and “plant-pathogen interaction,” which constructed the complex regulatory network to maintain osmotic and intracellular ion homeostasis. Quantitative real-time polymerase chain reaction was used to confirm the expression patterns of 20 unigenes. The results establish the foundation for the genetic basis of salt tolerance in P. tomentosa, which in turn accelerates Paulownia breeding and expands available arable land.

Paulownia is a native fast-growing tree in China that has been introduced into many countries. However, it is often infected by Paulownia witches&rsquo, broom (PaWB) disease, which can lead to large declines in yield. PaWB is caused by a phytoplasma that is an obligate biotrophic plant pathogen. Until now, the molecular mechanisms of interactions between the host plants and the phytoplasma have not been clear. In previous studies, it was reported that PaWB-infected Paulownia exhibited healthy morphology after being treated with methyl methane sulfonate (MMS) at the concentration of 20 mg&middot, L&minus, 1 (for Paulownia tomentosa (PT) and Paulownia fortunei (PF) or 15 mg&middot, 1 (for P. tomentosa &times, P. fortunei) MMS. In this study, the whole transcriptome expression profile of PaWB-infected Paulownia was studied using high-throughput sequencing technology. In total, 74 significantly differentially expressed genes were detected among three species of healthy, PaWB-infected Paulownia, and the Paulownia treated with MMS. We identified and analyzed genes related to the roles of phytohormones and alternative splicing events involved in regulating plant growth. In response to phytoplasma infection, the concentrations of the plants&rsquo, phytohormones were altered, leading to morphology transformation. This research will provide valuable information to detect the molecular mechanisms involved in the Paulownia response to phytoplasma infection.

Paulownia witches’ broom (PaWB) caused by phytoplasma is a destructive disease of Paulownia in China and has been studied over the past 40 years. Recently, our research team found that methyl methanesulfonate (MMS) could help Paulownia plants recover from the symptoms of Paulownia witches’ broom in which the expression levels of genes related to photosynthesis, carbohydrate and nitrogen metabolism, plant-pathogen interaction, circadian rhythm changed. However, the molecular mechanisms underlying the interaction between PaWB and phytoplasma are still not fully understood. Here, high-throughput mRNA sequencing and de novo assembly were conducted in order to investigate gene expression profiles in three Paulownia tomentosa samples (healthy plantlets, diseased plantlets, and diseased plantlets treated with MMS. A total of 85,545 all-unigene were obtained, 2540 of which were significantly differentially expressed among the three samples. Some genes associated with folate and fatty acid synthesis, signal transduction pathways of plant hormones were identified, and their expression patterns were further validated using quantitative real-time PCR. To our knowledge, this is the first transcriptome-based study to demonstrate variations of gene expression related to morphological changes in metabolic pathways of phytoplasma-infected P. tomentosa plants. The information provided in this study will help understand the molecular pathogenesis of the Paulownia plant in response to phytoplasma infection.

Paulownia is a fast-growing tree native to China. It can provide high-quality timber and a green environment for humans and has been planted in many countries. Compared with diploid Paulownia australis, autotetraploid P. australis exhibits predominance in terms of yields, qualities, growth, and resistance to stress. However, the molecular and biologi- cal mechanisms of its predominant characteristics are still unclear. We performed paired-end sequencing of the transcriptomes of diploid and autotetraploidP. australis plants using the Illumina/Solexa Genome Analyzer platform and compared changes in the expression of genes after chromo- somedoubling. We obtainedatotal of48,445,468and46,437, 758 high-quality reads from diploid P. australis (PA2) and autotetraploid P. australis(PA4), respectively. After assembly, 90,881 (PA2) and 88,566 (PA4) unigenes were obtained. The unigenes from both transcriptomes were then assembled into 93,300 all-unigenes, which included 50,892 clusters and 42, 408 singletons. The all-unigene sequences were searched against public protein databases (Nr, Nt, Swiss-Prot, KOG, and KEGG) using BLASTX with a cutoff E-value of 10 −5 . Gene expression levels in the two libraries were compared, and a total of 8851 differentially expressed unigenes were identified. A large number of unigenes were found to be related to carbohydrate and energy metabolism, biosynthesis of cell wall, and stress tolerance. Many of the differentially expressed P. australis genes were attributable to chromosome doubling between the PA2 and PA4 plants. Functional analy- sis revealed variations in the mechanism of growth and stress resistance between PA2 and PA4.

Paulownia tomentosa is a fast-growing tree species with multiple uses. It is grown worldwide, but is native to China, where it is widely cultivated in saline regions. We previously confirmed that autotetraploid P. tomentosa plants are more stress-tolerant than the diploid plants. However, the molecular mechanism underlying P. tomentosa salinity tolerance has not been fully characterized. Using the complete Paulownia fortunei genome as a reference, we applied next-generation RNA-sequencing technology to analyze the effects of salt stress on diploid and autotetraploid P. tomentosa plants. We generated 175 million clean reads and identified 15,873 differentially expressed genes (DEGs) from four P. tomentosa libraries (two diploid and two autotetraploid). Functional annotations of the differentially expressed genes using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed that plant hormone signal transduction and photosynthetic activities are vital for plant responses to high-salt conditions. We also identified several transcription factors, including members of the AP2/EREBP, bHLH, MYB, and NAC families. Quantitative real-time PCR analysis validated the expression patterns of eight differentially expressed genes. Our findings and the generated transcriptome data may help to accelerate the genetic improvement of cultivated P. tomentosa and other plant species for enhanced growth in saline soils.

Paulownia witches’ broom (PaWB) disease caused by phytoplasma is a fatal disease that leads to considerable economic losses. Although there are a few reports describing studies of PaWB pathogenesis, the molecular mechanisms underlying phytoplasma pathogenicity in Paulownia trees remain uncharacterized. In this study, after building a transcriptome database containing 67,177 sequences, we used isobaric tags for relative and absolute quantification (iTRAQ) to quantify and analyze the proteome-level changes among healthyP. fortunei(PF), PaWB-infectedP. fortunei(PFI), and PaWB-infectedP. fortuneitreated with 20 mg L−1or 60 mg L−1methyl methane sulfonate (MMS) (PFI-20 and PFI-60, respectively). A total of 2,358 proteins were identified. We investigated the proteins profiles in PFvs. PFI (infected process) and PFI-20vs. PFI-60 (recovered process), and further found that many of the MMS-response proteins mapped to “photosynthesis” and “ribosome” pathways. Based on our comparison scheme, 36 PaWB-related proteins were revealed. Among them, 32 proteins were classified into three functional groups: (1) carbohydrate and energy metabolism, (2) protein synthesis and degradation, and (3) stress resistance. We then investigated the PaWB-related proteins involved in the infected and recovered processes, and discovered that carbohydrate and energy metabolism was inhibited, and protein synthesis and degradation decreased, as the plant responded to PaWB. Our observations may be useful for characterizing the proteome-level changes that occur at different stages of PaWB disease. The data generated in this study may serve as a valuable resource for elucidating the pathogenesis of PaWB disease during phytoplasma infection and recovery stages.

Timber properties of autotetraploid Paulownia tomentosa are heritable with whole genome duplication, but the molecular mechanisms for the predominant characteristics remain unclear. To illuminate the genetic basis, high-throughput sequencing technology was used to identify the related unigenes. 2677 unigenes were found to be significantly differentially expressed in autotetraploid P. tomentosa. In total, 30 photosynthesis-related, 21 transcription factor-related, and 22 lignin-related differentially expressed unigenes were detected, and the roles of the peroxidase in lignin biosynthesis, MYB DNA-binding proteins, and WRKY proteins associated with the regulation of relevant hormones are extensively discussed. The results provide transcriptome data that may bring a new perspective to explain the polyploidy mechanism in the long growth cycle of plants and offer some help to the future Paulownia breeding.

Paulownia is a fast-growing deciduous hardwood species native to China, which has high ecological and economic value. In an earlier study, we reported ploidy-dependent differences in Paulownia drought tolerance by the microscopic observations of the leaves. Autotetraploid Paulownia has a higher resistance to drought stress than their diploid relatives. In order to obtain genetic information on molecular mechanisms responses of Paulownia plants to drought, Illumina/Solexa Genome sequencing platform was used to de novo assemble the transcriptomes of leaves from diploid and autotetraploid Paulownia tomentosa × Paulownia fortunei seedlings (PTF2 and PTF4 respectively) grown under control conditions and under drought stress and obtained 98,671 nonredundant unigenes. A comparative transcriptome analysis revealed that hundreds of unigenes were predicted to be involved mainly in ROS-scavenging system, amino acid and carbohydrate metabolism, plant hormone biosynthesis and signal transduction, while these unigenes exhibited differential transcript alteration of the two accessions. This study provides a comprehensive map of how P. tomentosa × P. fortunei responds to drought stress at physiological and molecular levels, which may help in understanding the mechanisms involve in water-deficit response and will be useful for further study of drought tolerance in woody plants.

In spite of its economic importance, very little molecular genetics and genomic research has been targeted at the family Paulownia spp. The little genetic information on this plant is a big obstacle to studying the mechanisms of its ability to resist Paulownia Witches’ Broom (PaWB) disease. Analysis of the Paulownia transcriptome and its expression profile data are essential to extending the genetic resources on this species, thus will greatly improves our studies on Paulownia. In the current study, we performed the de novo assembly of a transcriptome on P. tomentosa × P. fortunei using the short-read sequencing technology (Illumina). 203,664 unigenes with a mean length of 1,328 bp was obtained. Of these unigenes, 32,976 (30% of all unigenes) containing complete structures were chosen. Eukaryotic clusters of orthologous groups, gene orthology, and the Kyoto Encyclopedia of Genes and Genomes annotations were performed of these unigenes. Genes related to PaWB disease resistance were analyzed in detail. To our knowledge, this is the first study to elucidate the genetic makeup of Paulownia. This transcriptome provides a quick way to understanding Paulownia, increases the number of gene sequences available for further functional genomics studies and provides clues to the identification of potential PaWB disease resistance genes. This study has provided a comprehensive insight into gene expression profiles at different states, which facilitates the study of each gene’s roles in the developmental process and in PaWB disease resistance.

Drought is one of the most devastating effects of global climate change. Leaves contribute significantly to the management of water deficit and plant adaptation to drought stress. In this study, we compared the transcriptomes of leaves of two genotypes of Paulownia fortunei with different drought tolerances. Solexa sequencing and qRT-PCR were used for gene expression analysis and validation. Variations in leaf growth were found between drought-treated and well-watered samples in both genotypes. Drought-treated samples from diploid and autotetraploid P. fortunei cultivars showed inward leaf rolling and smaller blade size compared with the well-watered ones. High throughput transcriptome sequencing generated 266,700,100 high-quality reads representing 110,586 unigenes from the leaves. The drought-treated samples responded to water deficiency by inducing various genes and pathways, such as photosynthesis, carbon fixation in photosynthetic organisms, stress response, plant hormone signal transduction, and flavonoid pathways. Regulatory genes, such as WRKY, and transcription factors, such as NAC, known for leaf development under drought stress, were highly expressed in the drought-treated samples, and so were the genes related to compatible solutes (sugars, sugar alcohols, amino sugars, amino acids, or betaine), hormones, and various transporters. This study illustrates changes in the expression pattern of genes induced in response to drought stress and provides a comprehensive and specific set of drought-responsive genes in P. fortunei. Electronic supplementary material The online version of this article (doi:10.1007/s10142-014-0373-4) contains supplementary material, which is available to authorized users.

An efficient protocol for plant regeneration of Chimonanthus praecox (L.) Link, was developed using leaves from seedlings of seeds. Shoots induction were influenced by cytokinins and nodal positions. The results show that the highest callus induction frequency was obtained on MS medium supplemented with 2.0 mg l −1 6-benzyladenine (6-BA) and 0.9 mg l −1 1-naphthalene-acetic acid (NAA) after 10 days incubation in darkness. The frequency of callus induction of different nodal leaves (from the first to the forth node) varied from 70.0 to 93.5%; the highest frequency of callus induction (93.5%) was observed in the second node group. Through many times subculture of callus, green and compact callus was selected and transferred to regeneration medium (MS, 1/2 MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ) (2.37, 4.74 and 9.48 mg l −1 ) and NAA (0.74, 1.48, and 2.96 mg l −1 ). It was shown that the optimal combination of the three factors was WPM + NAA + TDZ. The highest adventitious shoot regeneration frequency of leaf explants (71.33%) and the highest mean number of shoots per explant (2.23) were obtained on WPM supplemented with 2.37 mg l −1 TDZ and 1.48 mg l −1 NAA. Optimal rooting was seen on 1/2 MS medium with 1.5 mg l −1 IBA for eight days, followed by transfer to hormone-free MS medium. Rooted plantlets were easily acclimated in a glasshouse. Key words: Chimonanthus praecox, in vitro regeneration, adventitious buds.

Paulownia witches' broom (PaWB) caused by a phytoplasma, has caused extensive losses in the yields of paulownia timber and resulted in significant economic losses. However, the molecular mechanisms in Paulownia that underlie the phytoplasma stress are poorly characterized. In this study, we use an Illumina platform to sequence four small RNA libraries and four degradome sequencing libraries derived from healthy, PaWB-infected, and PaWB-infected 15 mg·L-1 and 30 mg·L-1 methyl methane sulfonate (MMS)-treated plants. In total, 125 conserved and 118 novel microRNAs (miRNAs) were identified and 33 miRNAs responsive to PaWB disease were discovered. Furthermore, 166 target genes for 18 PaWB disease-related miRNAs were obtained, and found to be involved in plant-pathogen interaction and plant hormone signal transduction metabolic pathways. Eleven miRNAs and target genes responsive to PaWB disease were examined by a quantitative real-time PCR approach. Our findings will contribute to studies on miRNAs and their targets in Paulownia, and provide new insights to further understand plant-phytoplasma interactions.

Paulownia witches' broom (PaWB) caused by phytoplasma might result in devastating damage to the growth and wood production of Paulownia. To study the effect of phytoplasma on DNA sequence and to discover the genes related to PaWB occurrence, DNA polymorphisms and DNA methylation levels and patterns in PaWB seedlings, the ones treated with various concentration of methyl methane sulfonate (MMS) and healthy seedlings were investigated with amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP). Our results indicated that PaWB seedlings recovered a normal morphology, similar to healthy seedlings, after treatment with more than 20 mg · L-1 MMS; Phytoplasma infection did not change the Paulownia genomic DNA sequence at AFLP level, but changed the global DNA methylation levels and patterns; Genes related to PaWB were discovered through MSAP and validated using quantitative real-time PCR (qRT-PCR). These results implied that changes of DNA methylation levels and patterns were closely related to the morphological changes of seedlings infected with phytoplasmas.

Background Paulownia witches’ broom (PaWB) is a fatal disease of Paulownia caused by a phytoplasma. In previous studies, we found that plants with PaWB symptoms would revert to a healthy morphology after methyl methane sulfonate (MMS) treatment. To completely understand the gene expression profiles of the Paulownia-phytoplasma interaction, three high-throughput sequencing technologies were used to investigate changes of gene expression and microRNAs (miRNAs) in healthy Paulownia tomentosa plantlets, PaWB-infected plantlets, and PaWB-infected plantlets treated with 60 mg · L−1 MMS. Methods Transcriptome, miRNAs and degradome sequencing were performed to explore the global gene expression profiles in the process of Paulownia tomentosa with phytoplasma infection. Results A total of 98,714 all-unigenes, 62 conserved miRNAs, and 35 novel miRNAs were obtained, among which 902 differentially expressed genes (DEGs) and 24 miRNAs were found to be associated with PaWB disease. Subsequently, the target genes of these miRNAs were predicted by degradome sequencing. Interestingly, we found that 19 target genes of these differentially expressed miRNAs were among the 902 DEGs. The targets of pau-miR156g, pau-miR403, and pau-miR166c were significantly up-regulated in the P. tomentosa plantlets infected with phytoplasma. Interaction of miRNA -target genes mediated gene expression related to PaWB were identified. Conclusions The results elucidated the possible roles of the regulation of genes and miRNAs in the Paulownia-phytoplasma interaction, which will enrich our understanding of the mechanisms of PaWB disease in this plant. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2074-3) contains supplementary material, which is available to authorized users.