N6-Methyladenosine Modification Changes in the Genome of Paulownia Fortunei Seedlings Infected With Phytoplasma

Background: Phytoplasmas induce diseases in more than 1,000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. N6-methyladenosine sequencing (m6A-seq) has been applied mainly to model plants and not to woody plants. Results: In this study, we applied m6A-seq to study changes in m6A modification in the Paulownia fortunei genome after phytoplasma infection. We found that the m6A modification level in seedlings infected with the phytoplasma that causes Paulownia witches' broom (PaWB) was slightly higher than the m6A modification level in PaWB-infected seedlings treated with 60 mg·L−1 methyl methanesulfonate (MMS). MMS has been shown to restore PaWB-infected seedlings to their normal form and no phytoplasma can be detected in MMS-treated PaWB-infected seedlings. RNA sequencing (RNA-seq) and m6A-seq were used to analyze the expression of genes with m6A peaks and m6A motifs in genes, respectively. 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. The functions of genes related to PaWB 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). The m6A modification levels were higher in PaWB-infected seedlings than they were in MMS-treated seedlings. 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 MMS. RT-PCR verified that the alternative splicing of these two genes was related to m6A modification. Conclusions: In this study, we applied m6A-seq to determine methylation levels in phytoplasma-infected Paulownia, and combined m6A-seq with transcriptome analysis to screen differentially expressed genes associated with PaWB. Also analyzed the effect of m6A methylation on alternative splicing. In future studies, we plan to verify genes directly related to PaWB and methylation-related enzymes in Paulownia to elucidate the pathogenicity mechanism of PaWB caused by phytoplasma invasion.

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, growth and development, 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 healthy and phytoplasma-infected Paulownia fortunei genome. The heatmap results indicated that the strongest interactions between chromosomes were in the telomeres. We confirmed the main structural characteristics, such as A/B compartments, topologically associated domains, and chromatin loops were prominent in Paulownia genome and 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 epigenetic modifications, transcriptional activity might function in combination to shape chromatin packing in healthy and phytoplasm-infected Paulownia. Finally, 11 genes (such as RPN6, Sec61 subunit alpha), commonly located at specific TAD boundaries, A/B compartment switching, specific loops, and 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 non-model plants upon phytopathogen infection and plant disease resistance. Key words: Paulownia witches’ broom; chromatin conformation; histone modification; transcription regulation

Physical Properties of Juvenile Wood of Two Paulownia Hybrids

There is a growing trend in the world of planting fast growing species (rotations 5 to 10 years). Their primary purpose is the production of wood fibers and biomass, but they certainly represent the potential in making solid wood products as well. One of the fast-growing species is Paulownia sp., a species of extremely fast growing wood. Plantation breeding of Paulownia sp. in Croatia is increasing, although there is a little knowledge about the technical properties of Paulownia wood and its end use is questionable. This paper presents preliminary results of some physical properties of juvenile wood of two Paulownia hybrids planted in the area near the town of Glina in the Republic of Croatia. One hybrid is 9501 ((Paulownia fortunei × Paulownia elongata) × (Paulownia fortunei × Paulownia tomentosa)) and the other hybrid is Shan Tong (Paulownia fortunei × Paulownia tomentosa). The aim of this study was to investigate physical properties of juvenile wood of two Paulownia hybrids from one site in Croatia, to determine differences in physical properties of wood between two hybrids and to evaluate the correlation between density and shrinkages of each hybrid. Significant differences in oven dry density, basic density and density at maximum MC, between the two hybrids were determined. There is no statistically significant difference in longitudinal, radial, tangential and volumetric shrinkages between the two hybrids.

Respuesta de material genético de Paulownia spp a tratamiento silvicultural, como estrategia para evaluar su adaptabilidad a condiciones climáticas de estepa espinosa Montano Bajo, Ecuador/Response of Paulownia spp genetic material to silviculture, as a s

El propósito de la investigación consistió en estudiar la respuesta de Paulownia elongata, P. fortunei y un híbrido (Paulownia fortunei x elongata) a un sistema de recepa y evaluar el desempeño de los brotes juveniles, como estrategia para confirmar su adaptabilidad a las condiciones bioclimáticas de estepa espinosa Montano Bajo. El ensayo se instaló en diciembre del 2 015, en una plantación clonal de dos años de edad, plantada por INIAP y ESPOCH en la granja Tunshi, Ecuador. Se establecieron 9 bloques por especie para un total de 27 parcelas experimentales, con 9 observaciones por parcela neta. El tratamiento silvicultural ejecutado fue poda de renovación/recepa, realizando un corte del tallo en bisel a dos centímetros de altura desde el suelo. Las variables evaluadas fueron: Días a la brotación, número de brotes por tocón, diámetro a la base del brote y altura del brote. Los tres materiales estudiados respondieron positivamente a la práctica silvicultural, 90% de tocones brotados a los 61 días de haber realizado el ensayo. A los 29 días de la recepa ya se encontraron brotes epicórmicos en las tres especies de Paulownia, presentando entre 1 y 13 brotes por tocón. En crecimiento dasométrico, P. fortunei experimentó el mayor promedio en altura (81,85 cm), seguida por P. elongata (70,51 cm) y Paulownia hibrido (65,53 cm), siendo 3,5 veces mayor a los promedios de altura y 4 veces mayor en diámetro a la altura de la base del tallo, de los reportados en el primer año de evaluación. Estos resultados avizoran probables comportamientos adecuados y adaptación como una especie forestal-agroforestal de interés para características similares del sitio de estudio. This study is aimed to evaluate the response Paulownia elongata, P. fortunei and hibrid (Paulownia fortunei x elongata) to renewal pruning system and evaluate to develop the juvenile sprout of plants as a strategy to assess its adaptability to the bioclimatic conditions of thorny steppe Montano Bajo, Ecuador. The essay was install in December, 2 015 in one clonal plantation two years old, establish by INIAP and ESPOCH in Tunshi farm, Ecuador. This was established in nine blocks per species for a total of 27 experimental plots with nine observations per net plot. The selviculture treatment executed was renewal pruning, making a bevel cut stem two feet high from the ground. The variables evaluated were: Days sprouting, number of sprouts per stump diameter at the base of the outbreak and height of the outbreak. The three materials studied responded positively to silvicultural practice and 90% of erupted stumps at 61 days responded to development essay. At 29 days from the development the recepa presented epicormics sprout in the three Paulownia species; P. fortunei experienced the greatest average height (81,85 cm), followed by P. elongata (70,51 cm) and hybrid Paulownia (65,53 cm), being 3,5 times greater than the average height and 4 times greater in diameter at the base than those reported in the first year evaluation, these results foresee probable behaviors adapted and adequate as a forestry-agroforestry species of interest from the site of study. Palabras clave: recepa, poda, silvicultura, dendroenergía. Keywords: recepa, pruning, silviculture, wood energy.