Genome-Wide Characterization of Calmodulin and Calmodulin-like Protein Gene Families in Paulownia fortunei and Identification of Their Potential Involvement in Paulownia Witches' Broom.

As significant Ca2+ sensors, calmodulin (CaM) and calmodulin-like proteins (CML), have been associated with a variety of environmental conditions in plants. However, whether CaMs/CMLs are related to the stress of phytoplasma infection has not been reported in Paulownia fortunei. In the current study, 5 PfCaMs and 58 PfCMLs were detected through a genome-wide investigation. The number of EF-hand motifs in all PfCaMs/CMLs varied. Bioinformatics analyses, including protein characteristics, conserved domain, gene structure, cis-elements, evolutionary relationship, collinearity, chromosomal location, post-translation modification site, subcellular localization and expression pattern analyses, represented the conservation and divergence of PfCaMs/CMLs. Furthermore, some PfCaMs/CMLs might be involved in plants' reaction to phytoplasma infection and exogenous calcium therapy, indicating these genes may play a role in abiotic as well as biotic stress responses. In addition, subcellular localization analysis showed that PfCML10 was located in the cell membrane and nucleus. In summary, these findings establish a stronger platform for their subsequent functional investigation in trees and further characterize their roles in Paulownia witches' broom (PaWB) occurrence.

N6-methyladenosine modification changes during the recovery processes for Paulownia witches' broom disease under the methyl methanesulfonate treatment.

Phytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N 6-methyladenosine (m6A) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the m6A transcriptome map of Paulownia fortunei (P. fortunei) has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on m6A modification of P. fortunei and obtained the whole transcriptome m6A map in P. fortunei by m6A-seq. The m6A-seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of m6A modification of P. fortunei. The correlation analysis between the RNA-seq and m6A-seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. Moreover, the functions of PaWB-related genes were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). In addition, genes F-box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB-infected seedling treated with methyl methanesulfonate, and m6A modification was found in m6A-seq results. Moreover, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) verified that the alternative splicing of these two genes was associated with m6A modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA m6A modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation-related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.

The stability of transcription factor PfSPL1 participates in the response to phytoplasma stress in Paulownia fortunei.

The current understanding of the pathogenesis of phytoplasma is still very limited and challenging. Here, ceRNA regulatory network and degradome sequencing identified a PfmiR156f-PfSPL regulatory module in Paulownia fortunei infected by phytoplasma, and RLM-5'RACE and dual luciferase analyses verified the relationship. The PfmiR156 cleavage site was located at 1104 nt and 1177 nt of PfSPL1 and PfSPL10, respectively. MG132 and epoxomicin, two 26S proteasome inhibitors, significantly increased the accumulation of PfSPL1. PfSPL1 was also the attack target of phytoplasma effectors (Pawb 3/9/16/37/51) after the phytoplasma invaded Paulownia. Moreover, molecular docking implied that the effectors may interact with the conserved SBP domain of the target protein PfSPL1. Basically, these results indicated that the stability of PfSPL1 was regulated by PfmiR156 cleavage activity and/or the 26S proteasome pathway at the post-translation level. The PfSPL1, which is a transcription factor, was also the one of the targets of multiple effectors attacking Paulownia. This study provides a good scope to understand the paulownia phytoplasma infecting mechanism.

Genome-Wide Identification and Expression of the Paulownia fortunei MADS-Box Gene Family in Response to Phytoplasma Infection.

Paulownia witches' broom (PaWB), caused by phytoplasmas, is the most devastating infectious disease of Paulownia. Although a few MADS-box transcription factors have been reported to be involved in the formation of PaWB, there has been little investigation into all of the MADS-box gene family in Paulownia. The objective of this study is to identify the MADS-box gene family in Paulownia fortunei on a genome-wide scale and explore their response to PaWB infection. Bioinformatics software were used for identification, characterization, subcellular localization, phylogenetic analysis, the prediction of conserved motifs, gene structures, cis-elements, and protein-protein interaction network construction. The tissue expression profiling of PfMADS-box genes was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Transcriptome data and the protein interaction network prediction were combined to screen the genes associated with PaWB formation. We identified 89 MADS-box genes in the P. fortunei genome and categorized them into 14 subfamilies. The comprehensive analysis showed that segment duplication events had significant effects on the evolution of the PfMADS-box gene family; the motif distribution of proteins in the same subfamily are similar; development-related, phytohormone-responsive, and stress-related cis-elements were enriched in the promoter regions. The tissue expression pattern of PfMADS-box genes suggested that they underwent subfunctional differentiation. Three genes, PfMADS3, PfMADS57, and PfMADS87, might be related to the occurrence of PaWB. These results will provide a valuable resource to explore the potential functions of PfMADS-box genes and lay a solid foundation for understanding the roles of PfMADS-box genes in paulownia-phytoplasma interactions.

Chemical composition, health benefits and future prospects of Paulownia flowers: A review.

The plants of the genus Paulownia (Scrophulariaceae) have been gaining attention for wood production, and their flowers, which are a seasonal by-product, have been traditionally used in medicinal products. The phytochemistry and pharmacology of Paulownia flowers contribute to their economic uses in medicines, foods, animal feeds, and cosmetics. The chemical composition of Paulownia flowers is mostly flavonoids, phenylpropanoids, terpenoids, volatile components, polysaccharides, lignans, and iridoids, which exhibit various health benefits, such as antioxidant, anti-inflammatory, antibacterial, antiviral, anticancer, hypoglycemic, hypolipidemic, neuroprotective and immunoregulation activities. Moreover, the extracts of the Paulownia flower have been proven safe for animals. These promote the development of new products and technologies using Paulownia flowers, with intellectual property rights. The review presents the current developments on the chemical composition, biological and pharmacological activities, and economic values of Paulownia flowers, and aims to provide a reference for their further utilization.

Comprehensive analyses of the SPL transcription factor family in Paulownia fortunei and their responses to biotic and abiotic stresses.

To study the molecular characteristics, phylogenetic evolution, and gene functions of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes in Paulownia fortunei, a whole genome sequence analysis was carried out, and a total of 23 PfSPL genes were identified. Tandem duplication and fragment replication were the main patterns of gene expansion in the PfSPL family. Phylogenetic analysis showed that the 23 identified PfSPLs formed seven subgroups, and the structures of the proteins in the same subgroup were similar. Functional analysis indicated that PfSPL11 may regulate flowering, PfSPL5 was involved in gibberellin signaling, PfSPL1/4/23 regulated branching, and PfSPL9/16/18 were related to pathogen resistance. Yeast one hybrid technology confirmed that PfSPL4 and PfSP23 can bind to the promoter of PfTCPa. The transcriptome analysis indicated that PfSPL10 was sensitive to both drought and salt stress. Ten PfSPLs that responded to phytoplasma infection were identified. Molecular docking showed that PfSPL10 and PfSPL 4/5/9/10/11/13 formed active pockets in the conserved SBP domain that could bind methyl methane sulfonate (MMS) and rifampicin (Rif) through stable hydrogen bonds, respectively. This study provides a basis for further studies on the functions of the PfSPL transcription factor family, and for genetic improvement and breeding of trees resistant to PaWB disease.

Conformational Changes in Three-Dimensional Chromatin Structure in Paulownia fortunei After Phytoplasma Infection.

Higher-order chromatin structures play important roles in regulating multiple biological processes such as growth and development as well as biotic and abiotic stress response. However, little is known about three-dimensional chromatin structures in Paulownia or about whole-genome chromatin conformational changes that occur in response to Paulownia witches' broom (PaWB) disease. We used high-throughput chromosome conformation capture (Hi-C) to obtain genome-wide profiles of chromatin conformation in both healthy and phytoplasma-infected Paulownia fortunei genome. The heat map results indicated that the strongest interactions between chromosomes were in the telomeres. We confirmed that the main structural characteristics of A/B compartments, topologically associated domains, and chromatin loops were prominent in the Paulownia genome and were clearly altered in phytoplasma-infected plants. By combining chromatin immunoprecipitation sequencing, Hi-C signals, and RNA sequencing data, we inferred that the chromatin structure changed and the modification levels of three histones (H3K4me3/K9ac/K36me3) increased in phytoplasma-infected P. fortunei, which was associated with changes of transcriptional activity. We concluded that for epigenetic modifications, transcriptional activity might function in combination to shape chromatin packing in healthy and phytoplasm-infected Paulownia. Finally, 11 genes (e.g., RPN6, Sec61 subunit-α) that were commonly located at specific topologically associated domain boundaries, A/B compartment switching and specific loops, and had been associated with histone marks were identified and considered as closely related to PaWB stress. Our results provide new insights into the nexus between gene regulation and chromatin conformational alterations in nonmodel plants upon phytopathogen infection and plant disease resistance.

Genome-Wide Analysis of Specific PfR2R3-MYB Genes Related to Paulownia Witches' Broom.

Paulownia witches' broom (PaWB), caused by phytoplasmas, is the most devastating infectious disease of Paulownia. R2R3-MYB transcription factors (TF) have been reported to be involved in the plant's response to infections caused by these pathogens, but a comprehensive study of the R2R3-MYB genes in Paulownia has not been reported. In this study, we identified 138 R2R3-MYB genes distributed on 20 chromosomes of Paulownia fortunei. These genes were classified into 27 subfamilies based on their gene structures and phylogenetic relationships, which indicated that they have various evolutionary relationships and have undergone rich segmental replication events. We determined the expression patterns of the 138 R2R3-MYB genes of P. fortunei by analyzing the RNA sequencing data and found that PfR2R3-MYB15 was significantly up-regulated in P. fortunei in response to phytoplasma infections. PfR2R3-MYB15 was cloned and overexpressed in Populus trichocarpa. The results show that its overexpression induced branching symptoms. Subsequently, the subcellular localization results showed that PfR2R3-MYB15 was located in the nucleus. Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that PfR2R3-MYB15 interacted with PfTAB2. The analysis of the PfR2R3-MYB15 gene showed that it not only played an important role in plant branching, but also might participate in the biosynthesis of photosystem elements. Our results will provide a foundation for future studies of the R2R3-MYB TF family in Paulownia and other plants.

Genomic insights into the fast growth of paulownias and the formation of Paulownia witches' broom.

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.

Whole-genome landscape of H3K4me3, H3K36me3 and H3K9ac and their association with gene expression during Paulownia witches' broom disease infection and recovery processes.

Histone methylation and acetylation participate in the modulation of gene expression. Here, chromatin immunoprecipitation sequencing (ChIP-Seq) was used to determine genome-wide patterns of three histone modifications, H3K4me3, H3K36me3, and H3K9ac (associated with actively expressed genes) and their associations with gene expression in Paulownia fortunei following phytoplasma infection and recovery from Paulownia witches' broom (PaWB) disease after methyl methane sulfonate treatment. The three histone marks were preferentially deposited in genic regions, especially downstream of transcription start sites, and were highly concurrent with gene expression. Genes with all three histone marks exhibited the highest expression levels. Based on the comparison scheme, we detected 365, 2244, and 752 PaWB-associated genes with H3K4me3, H3K36me3, and H3K9ac marks, separately. KEGG pathway analysis showed that these genes were involved in plant-pathogen interaction, plant hormone signal transduction, and starch and sucrose metabolism. A small proportion of differentially modified genes showed changes in expression in response to phytoplasma infection, including genes involved in calcium ion signal transduction, abscisic acid signal transduction, and ethylene biosynthesis. This comprehensive analysis of genome-wide histone modifications and gene expression in Paulownia following phytoplasma infection provides new insights into the epigenetic responses to phytoplasma infection and will be useful for further studies on epigenetic regulation mechanisms in plants under biotic stress.

Genome-wide DNA methylation analysis of paulownia with phytoplasma infection.

DNA methylation, an important epigenetic modification, regulates a wide range of biological processes. Previous MSAP results showed that the occurrence of PaWB related to changes of DNA methylation level; however, the relationship between DNA methylation and gene expression remains obscure in paulownia. Therefore, in the present study, we applied WGBS and RNA-seq techniques to investigate the DNA methylation and gene expression changes between healthy Paulownia fortunei seedlings and the phytoplasma-infected ones. A map of methylated cytosines at the single base pair resolution of paulownia was constructed. Compared to the healthy seedlings, the DNA methylation level increased after phytoplasma infection, and the change of mCHH was the main methylation pattern. DMR analysis showed that 422,662 DMRs in the genome were identified, in which, 27,871 DMR-associated genes were differentially expressed. Finally, 436 genes with significant differences in their methylation levels and mRNA expression profiles were identified through integrated analysis of the DNA methylomic and transcriptomic. KEGG pathway analysis revealed that these genes are mainly involved in plant hormone signal transduction, carbon metabolism, and starch and sucrose metabolism pathways. Two of DMR-associated genes were verified by BS- PCR. Finally, we selected TRP 1 and R2R3-MYB protein were closely related to the occurrence of PaWB. Our findings provide valuable insight into the mechanism of PaWB at the epigenetic level.

A comparison of the transcriptomes between diploid and autotetraploid Paulownia fortunei under salt stress.

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.

ceRNA Cross-Talk in Paulownia Witches' Broom Disease.

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' 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.

Identification and characterization of long noncoding RNA in Paulownia tomentosa treated with methyl methane sulfonate.

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.

Genome-wide expression analysis of salt-stressed diploid and autotetraploid Paulownia tomentosa.

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.

Quantitative proteome-level analysis of paulownia witches' broom disease with methyl methane sulfonate assistance reveals diverse metabolic changes during the infection and recovery processes.

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 healthy P. fortunei (PF), PaWB-infected P. fortunei (PFI), and PaWB-infected P. fortunei treated with 20 mg L-1 or 60 mg L-1 methyl methane sulfonate (MMS) (PFI-20 and PFI-60, respectively). A total of 2,358 proteins were identified. We investigated the proteins profiles in PF vs. PFI (infected process) and PFI-20 vs. 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.

Implications of polyploidy events on the phenotype, microstructure, and proteome of Paulownia australis.

Polyploidy events are believed to be responsible for increasing the size of plant organs and enhancing tolerance to environmental stresses. Autotetraploid Paulownia australis plants exhibit superior traits compared with their diploid progenitors. Although some transcriptomics studies have been performed and some relevant genes have been revealed, the molecular and biological mechanisms regulating the predominant characteristics and the effects of polyploidy events on P. australis remain unknown. In this study, we compared the phenotypes, microstructures, and proteomes of autotetraploid and diploid P. australis plants. Compared with the diploid plant, the leaves of the autotetraploid plant were longer and wider, and the upper epidermis, lower epidermis, and palisade layer of the leaves were thicker, the leaf spongy parenchyma layer was thinner, the leaf cell size was bigger, and cell number was lower. In the proteome analysis, 3,010 proteins were identified and quantified, including 773 differentially abundant proteins. These results may help to characterize the P. australis proteome profile. Differentially abundant proteins related to cell division, glutathione metabolism, and the synthesis of cellulose, chlorophyll, and lignin were more abundant in the autotetraploid plants. These results will help to enhance the understanding of variations caused by polyploidy events in P. australis. The quantitative real-time PCR results provided details regarding the expression patterns of the proteins at mRNA level. We observed a limited correlation between transcript and protein levels. These observations may help to clarify the molecular basis for the predominant autotetraploid characteristics and be useful for plant breeding in the future.

Dynamic expression of novel and conserved microRNAs and their targets in diploid and tetraploid of Paulownia tomentosa.

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.