Genetic resources of common ash (Fraxinus excelsior L.) in Poland.

BACKGROUND: Knowledge of genetic structure and the factors that shape it has an impact on forest management practices. European ash (Fraxinus excelsior L.) has declined dramatically throughout its range as a result of a disease caused by the fungus Hymenoscyphus fraxineus. Despite the need for conservation and restoration of the species, genetic data required to guide these efforts at the country level are scarce. Thereofore, we studied the chloroplast and nuclear genetic diversity of 26 natural common ash populations (1269 trees) in Poland. RESULTS: Chloroplast polymorphisms grouped the populations into two geographically structured phylogenetic lineages ascribed to different glacial refugia (the Balkans and the Eastern Alps). However, the populations demonstrated high genetic diversity (mean AR = 12.35; mean Ho = 0.769; mean He = 0.542) but low differentiation based on nuclear microsatellites (FST = 0.045). Significant spatial genetic structure, consistent with models of isolation by distance, was detected in 14 out of 23 populations. Estimated effective population size was moderate-to-high, with a harmonic mean of 57.5 individuals per population. CONCLUSIONS: Genetic diversity was not homogeneously distributed among populations within phylogenetic gene pools, indicating that ash populations are not equal as potential sources of reproductive material. Genetic differences among populations could be related to their histories, including founder effects or gene flow between evolutionary lineages (admixture). Our results suggest that ash stands across Poland could be treated as two main management units (seed zones). Therefore, despite the homogenizing effect of pollen gene flow known for this species, the genetic structure should be taken into account in the management of the genetic resources of the common ash. Although ash dieback poses an additional challenge for the management of genetic resources, efforts should be directed towards protecting populations with high genetic diversity within defined phylogenetic units, as they may be an important source of adaptive variation for future stands.

High quality genomes produced from single MinION flow cells clarify polyploid and demographic histories of critically endangered Fraxinus (ash) species.

With populations of threatened and endangered species declining worldwide, efforts are being made to generate high quality genomic records of these species before they are lost forever. Here, we demonstrate that data from single Oxford Nanopore Technologies (ONT) MinION flow cells can, even in the absence of highly accurate short DNA-read polishing, produce high quality de novo plant genome assemblies adequate for downstream analyses, such as synteny and ploidy evaluations, paleodemographic analyses, and phylogenomics. This study focuses on three North American ash tree species in the genus Fraxinus (Oleaceae) that were recently added to the International Union for Conservation of Nature (IUCN) Red List as critically endangered. Our results support a hexaploidy event at the base of the Oleaceae as well as a subsequent whole genome duplication shared by Syringa, Osmanthus, Olea, and Fraxinus. Finally, we demonstrate the use of ONT long-read sequencing data to reveal patterns in demographic history.

The chromosome-level genome assembly of Fraxinus americana provides insights into the evolution of Oleaceae plants.

White ash (Fraxinus americana linn.) originates from the southeastern United States. It is a tall and fast-growing tree species with strong salt-alkali resistance and cold tolerance, making it an important reforestation species and widely planted worldwide. Here, we completed the chromosome-level reference genome assembly of F. americana based on Illumina, PacBio, and Hi-C reads, with a genome size of 878.98 Mb, an N50 of 3.27 Mb, and a heterozygosity rate of 0.3 %. Based on de novo prediction, transcriptome prediction, and homology-based protein prediction, we obtained 39,538 genes. Approximately 843.21 Mb of the assembly genome was composed of 37,928 annotated protein-coding genes, with a gene function annotation rate of 95.93 %. 99.94 % of the overlap clusters (877.44 Mb) were anchored to 23 chromosomes. Synteny analysis of F. americana and other Oleaceae plants showed that F. americana underwent frequent chromosome rearrangements. The amplification of the Ale transposons effectively promoted the genome size of F. americana. Compared with other Oleaceae plants, the Glutathione S-transferase (GST) gene family in the F. americana genome has undergone significant expansion, which may help F. americana cope with adverse natural environments. Furthermore, we found that key enzyme-coding gene families related to lignin biosynthesis were expanded and highly expressed in F. americana leaves. These key genes drive lignin synthesis and benefit F. americana in fast-growing, as well as resisting biotic and abiotic stress. Overall, the F. americana genome assembly provides insights into the evolution of Oleaceae plants and provides abundant resources for breeding and germplasm conservation of white ash.

Transcriptome profiling of Fraxinus excelsior genotypes infested by emerald ash borer.

European ash, Fraxinus excelsior is facing the double threat of ongoing devastation by the invasive fungal pathogen, Hymenoscyphus fraxineus and the imminent arrival of the non-native emerald ash borer (EAB), Agrilus planipennis. The spread of EAB which is currently moving westwards from European Russia and Ukraine into central Europe, poses an additional substantial threat to European ash, F. excelsior. While the molecular basis for resistance or variation in resistance among European ash genotypes is heavily investigated, comparatively little is known about the molecular ash traits involved in resistance against EAB. In this study we have gathered transcriptomic data from EAB inoculated genotypes of F. excelsior that have previously shown different levels of susceptibility to EAB. Resultant datasets show differential gene expression in susceptible and resistant genotypes in response to EAB infestation. This data will provide important information on the molecular basis of resistance to the EAB and allow the development of management plans to combat a pending threat of a culturally and ecologically important European tree species.

A glimmer of hope - ash genotypes with increased resistance to ash dieback pathogen show cross-resistance to emerald ash borer.

Plants rely on cross-resistance traits to defend against multiple, phylogenetically distinct enemies. These traits are often the result of long co-evolutionary histories. Biological invasions can force naïve plants to cope with novel, coincident pests, and pathogens. For example, European ash (Fraxinus excelsior) is substantially threatened by the emerald ash borer (EAB), Agrilus planipennis, a wood-boring beetle, and the ash dieback (ADB) pathogen, Hymenoscyphus fraxineus. Yet, plant cross-resistance traits against novel enemies are poorly explored and it is unknown whether naïve ash trees can defend against novel enemy complexes via cross-resistance mechanisms. To gain mechanistic insights, we quantified EAB performance on grafted replicates of ash genotypes varying in ADB resistance and characterized ash phloem chemistry with targeted and untargeted metabolomics. Emerald ash borer performed better on ADB-susceptible than on ADB-resistant genotypes. Moreover, changes in EAB performance aligned with differences in phloem chemical profiles between ADB-susceptible and ADB-resistant genotypes. We show that intraspecific variation in phloem chemistry in European ash can confer increased cross-resistance to invasive antagonists from different taxonomic kingdoms. Our study suggests that promotion of ADB-resistant ash genotypes may simultaneously help to control the ADB disease and reduce EAB-caused ash losses, which may be critical for the long-term stability of this keystone tree species.

Transcriptomic and metabolomic analysis of autumn leaf color change in Fraxinus angustifolia.

Fraxinus angustifolia is a type of street tree and shade tree with ornamental value. It has a beautiful shape and yellow or reddish purple autumn leaves, but its leaf color formation mechanism and molecular regulation network need to be studied. In this study, we integrated the metabolomes and transcriptomes of stage 1 (green leaf) and stage 2 (red-purple leaf) leaves at two different developmental stages to screen differential candidate genes and metabolites related to leaf color variation. The results of stage 1 and stage 2 transcriptome analysis showed that a total of 5,827 genes were differentially expressed, including 2,249 upregulated genes and 3,578 downregulated genes. Through functional enrichment analysis of differentially expressed genes, we found that they were involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, pigment metabolism, carotene metabolism, terpenoid biosynthesis, secondary metabolite biosynthesis, pigment accumulation, and other biological processes. By measuring the metabolites of Fraxinus angustifolia leaves, we found the metabolites closely related to the differentially expressed genes in two different periods of Fraxinus angustifolia, among which flavonoid compounds were the main differential metabolites. Through transcriptome and metabolomics data association analysis, we screened nine differentially expressed genes related to anthocyanins. Transcriptome and qRT-PCR results showed that these nine genes showed significant expression differences in different stages of the sample, and we speculate that they are likely to be the main regulatory factors in the molecular mechanism of leaf coloration. This is the first time that we have analyzed the transcriptome combination metabolome in the process of leaf coloration of Fraxinus angustifolia, which has important guiding significance for directional breeding of colored-leaf Fraxinus species and will also give new insights for enriching the landscape.

An updated de novo transcriptome for green ash (Fraxinus pennsylvanica).

De novo transcriptome assembly of next-generation sequencing information has become a powerful tool for the study of non-model species. Transcriptomes generated by this method can have high variability due to endless combinations of user-defined variables and programs available for assembly. Many methods have been developed for evaluating the quality of these assemblies. Here, raw sequencing information for Green ash (Fraxinus pennsylvanica Marshall) that was previously published has been re-evaluated. An updated assembly has been developed by including additional sequencing information not used for the currently accepted transcriptome in combination with more stringent trimming parameters. Input reads were assembled with Trinity and Abyss assembly programs. The resulting Trinity assembly has a 7.3-fold increase in genomic breadth of coverage, a 2.4-fold increase in predicted complete open reading frames, an increased L50 value, and increased Benchmarking Universal Single-Copy Ortholog completeness compared to the earlier published transcriptome. This updated transcriptome can be leveraged to help fight the rapid decline of green ash due to pathogens.

Traces of Hymenoscyphus fraxineus in Northeastern Europe Extend Further Back in History than Expected.

Herbaria are a promising but still poorly applied information source for retrospective microbiological studies. In order to find any evidence of the virulent European origin of ash dieback agent Hymenoscyphus fraxineus and other fungal pathogens, we analyzed 109 leaf samples from three different Estonian botanical herbaria, sampled during 171 years from 20 ash species and cultivars, using a PacBio third-generation sequencing of the fungal internal transcribed spacer ITS1-5.8S-ITS2 ribosomal DNA region. We identified a large amount of saprotrophic fungi naturally colonizing ash leaves. Hymenoscyphus fraxineus colonized a Fraxinus chinensis subsp. rhynchophylla specimen and a F. chinensis specimen collected from Tallinn Botanic Garden in July 1978 and July 1992, respectively. The samples originated from trees grown in this garden from seeds collected from Shamora, Far-East Russia, in 1961 and from a Beijing botanical garden in eastern China in 1985, respectively. Repeated subsequent DNA extraction, real-time quantitative PCR, and Sanger and Illumina sequencing confirmed our findings of these apparently oldest cases of the ash dieback agent in Europe. These results show that H. fraxineus evidently was present in Estonia 19 years earlier than our previous data from fungal herbaria documented and 14 years before the first visible damage of ash trees was registered in Poland. Because we found no evidence of the saprotrophic H. albidus from earlier mycological and botanical herbarium specimens, the presence of H. albidus in Estonia remains questionable.

Genome-wide analysis and molecular dissection of the SPL gene family in Fraxinus mandshurica.

BACKGROUND: SQUAMOSA promoter binding protein-like (SPL) is a unique family of transcription factors in plants, which is engaged in regulating plant growth and development, physiological and biochemical processes. Fraxinus mandshurica is an excellent timber species with a wide range of uses in northeastern China and enjoys a high reputation in the international market. SPL family analysis has been reported in some plants while SPL family analysis of Fraxinus mandshurica has not been reported. RESULTS: We used phylogeny, conserved motifs, gene structure, secondary structure prediction, miR156 binding sites, promoter cis elements and GO annotation to systematically analyze the FmSPLs family. This was followed by expression analysis by subcellular localization, expression patterns at various tissue sites, abiotic stress and hormone induction. Because FmSPL2 is highly expressed in flowers it was selected to describe the SPL gene family of Fraxinus mandshurica by ectopic expression. Among them, 10 FmSPL genes that were highly expressed at different loci were selected for expression analysis under abiotic stress (NaCl and Cold) and hormone induction (IAA and ABA). These 10 FmSPL genes showed corresponding trends in response to both abiotic stress and hormone induction. We showed that overexpression of FmSPL2 in transgenic Nicotiana tabacum L. resulted in taller plants, shorter root length, increased root number, rounded leaves, and earlier flowering time. CONCLUSIONS: We identified 36 SPL genes, which were classified into seven subfamilies based on sequence analysis. FmSPL2 was selected for subsequent heterologous expression by analysis of expression patterns in various tissues and under abiotic stress and hormone induction, and significant phenotypic changes were observed in the transgenic Nicotiana tabacum L. These results provide insight into the evolutionary origin and biological significance of plant SPL. The aim of this study was to lay the foundation for the genetic improvement of Fraxinus mandshurica and the subsequent functional analysis of FmSPL2.

Heterologous expression of a Fraxinus velutina SnRK2 gene in Arabidopsis increases salt tolerance by modifying root development and ion homeostasis.

KEY MESSAGE: FvSnRK2182 is involved in regulating the growth and stress response. SnRK2 family members are positive regulators of downstream signals in the abscisic acid (ABA) signaling pathway, playing key roles in the plant responses to abiotic stresses. Fraxinus velutina Torr. is a candidate phytoremediator of saline-alkali areas, and is a valuable research subject because of its adaptability in saline soil. We identified a SnRK2 gene in F. velutina (named FvSnRK2182), which was significantly upregulated under salt stress. A bioinformatics analysis showed that FvSnRK2182 has a Ser/Thr kinase domain typical of the SnRK2 subfamily. Compared with wild-type (WT) Arabidopsis, its heterologous expression in Arabidopsis resulted in higher auxin content during seed germination and seedling growth, leading to longer primary roots and more lateral roots. The transgenic lines were better able to tolerate treatments with NaCl (100 mM) and/or ABA (0.2 and 0.5 µM), producing a greater biomass than the WT plants. Under NaCl treatment, the shoots of the transgenic lines had lower Na+ contents and higher K+ contents than the WT plants, and the genes encoding the ion transport-related proteins SOS1, HKT1, NHX1, and AKT1 were significantly upregulated. In addition, the expression of the genes functioning downstream of SnRK2 in the ABA signaling pathway (Rboh, AREB4, ABF2, and ABF3) were significantly upregulated in transgenic lines under NaCl stress. These results showed that expressing FvSnRK2182 in Arabidopsis significantly increased their resistance to ABA and salt stress by regulating root development and maintaining ion homeostasis, which suggests that FvSnRK2182 may be involved in regulating the growth and stress response of F. velutina.

Physiological and genomic characterisation of Luteimonas fraxinea sp. nov., a bacterial species associated with trees tolerant to ash dieback.

A group of isolates of the genus Luteimonas was characterised, which represented a specific component of the healthy core microbiome of Fraxinus excelsior in forest districts with a high infection rate of H. fraxineus, the causal agent of ash dieback. Based on phylogenomic and phenotypic analyses, a clear differentiation from related Luteimonas species was shown. Comparisons of the overall genome relatedness indices with the closest phylogenetic neighbours resulted in values below the recommended species cut-off levels. In addition, differences in several physiological and chemotaxonomic traits allowed a clear demarcation from the type strains of closely related species. Conclusively, the strain group was considered to represent a novel species in the genus Luteimonas, for which the name Luteimonas fraxinea sp. nov. is proposed, with strain D4P002T (=DSM 113273T = LMG 32455T) as the type strain. A functional analysis of the genome revealed features particularly associated with attachment, biofilm production and motility, indicating the ability of D4P002T to effectively colonise ash leaves. In nursery trials, ash seedlings inoculated with this strain showed suppression of the pathogen over a period of three years. This effect was accompanied by a significant shift in the bacterial microbiome of the plants. Altogether, the exclusive occurrence in the microbiome of tolerant ash trees, the genetic background and the results of the inoculation experiment suggest that strain D4P002T may suppress the penetration and spreading of H. fraxineus in or on ash leaves via colonisation resistance or trigger a priming effect of plant defences against the pathogen.

First Report of Diplodia fraxini and Diplodia subglobosa Causing Canker and Dieback of Fraxinus excelsior in Slovenia.

In recent decades the vitality and productivity of European ash trees in Slovenia have been reduced by the onset of canker and dieback disease symptoms on young and old trees, identified primarily as ash dieback caused by Hymenoscyphus fraxineus. Given the limited information available about the etiology of this emerging disease, a study was carried out to isolate, identify, and characterize the fungal species involved in the observed ash symptoms. Field surveys were conducted in five forest sites where 50 symptomatic branch samples were collected. All samples were inspected and used for fungal isolation. Based on morphology, colony appearance, and DNA sequence data of the internal transcribed spacer region, 125 fungal colonies belonging to five species were isolated and identified. Only a few symptomatic ash samples yielded colonies of H. fraxineus, whereas Botryosphaeriaceae species were isolated with a high frequency, with Diplodia fraxini as the dominant species. A pathogenicity test proved that all isolated species were pathogenic on European ash, causing bark lesions and wood discoloration. All Botryosphaeriaceae species isolated in this study are reported for the first time on European ash in Slovenia.

A high-quality reference genome for Fraxinus pennsylvanica for ash species restoration and research.

Green ash (Fraxinus pennsylvanica) is the most widely distributed ash tree in North America. Once common, it has experienced high mortality from the non-native invasive emerald ash borer (EAB; Agrilus planipennis). A small percentage of native green ash trees that remain healthy in long-infested areas, termed "lingering ash," display partial resistance to the insect, indicating that breeding and propagating populations with higher resistance to EAB may be possible. To assist in ash breeding, ecology and evolution studies, we report the first chromosome-level assembly from the genus Fraxinus for F. pennsylvanica with over 99% of bases anchored to 23 haploid chromosomes, spanning 757 Mb in total, composed of 49.43% repetitive DNA, and containing 35,470 high-confidence gene models assigned to 22,976 Asterid orthogroups. We also present results of range-wide genetic variation studies, the identification of candidate genes for important traits including potential EAB-resistance genes, and an investigation of comparative genome organization among Asterids based on this reference genome platform. Residual duplicated regions within the genome probably resulting from a recent whole genome duplication event in Oleaceae were visualized in relation to wild olive (Olea europaea var. sylvestris). We used our F. pennsylvanica chromosome assembly to construct reference-guided assemblies of 27 previously sequenced Fraxinus taxa, including F. excelsior. Thus, we present a significant step forward in genomic resources for research and protection of Fraxinus species.

Comparative analyses of the Hymenoscyphus fraxineus and Hymenoscyphus albidus genomes reveals potentially adaptive differences in secondary metabolite and transposable element repertoires.

BACKGROUND: The dieback epidemic decimating common ash (Fraxinus excelsior) in Europe is caused by the invasive fungus Hymenoscyphus fraxineus. In this study we analyzed the genomes of H. fraxineus and H. albidus, its native but, now essentially displaced, non-pathogenic sister species, and compared them with several other members of Helotiales. The focus of the analyses was to identify signals in the genome that may explain the rapid establishment of H. fraxineus and displacement of H. albidus. RESULTS: The genomes of H. fraxineus and H. albidus showed a high level of synteny and identity. The assembly of H. fraxineus is 13 Mb longer than that of H. albidus', most of this difference can be attributed to higher dispersed repeat content (i.e. transposable elements [TEs]) in H. fraxineus. In general, TE families in H. fraxineus showed more signals of repeat-induced point mutations (RIP) than in H. albidus, especially in Long-terminal repeat (LTR)/Copia and LTR/Gypsy elements. Comparing gene family expansions and 1:1 orthologs, relatively few genes show signs of positive selection between species. However, several of those did appeared to be associated with secondary metabolite genes families, including gene families containing two of the genes in the H. fraxineus-specific, hymenosetin biosynthetic gene cluster (BGC). CONCLUSION: The genomes of H. fraxineus and H. albidus show a high degree of synteny, and are rich in both TEs and BGCs, but the genomic signatures also indicated that H. albidus may be less well equipped to adapt and maintain its ecological niche in a rapidly changing environment.

Transcriptional responses in developing lesions of European common ash (Fraxinus excelsior) reveal genes responding to infection by Hymenoscyphus fraxineus.

BACKGROUND: With the expanding ash dieback epidemic that has spread across the European continent, an improved functional understanding of the disease development in afflicted hosts is needed. The study investigated whether differences in necrosis extension between common ash (Fraxinus excelsior) trees with different levels of susceptibility to the fungus Hymenoscyphus fraxineus are associated with, and can be explained by, the differences in gene expression patterns. We inoculated seemingly healthy branches of each of two resistant and susceptible ash genotypes with H. fraxineus grown in a common garden. RESULTS: Ten months after the inoculation, the length of necrosis on the resistant genotypes were shorter than on the susceptible genotypes. RNA sequencing of bark samples collected at the border of necrotic lesions and from healthy tissues distal to the lesion revealed relatively limited differences in gene expression patterns between susceptible and resistant genotypes. At the necrosis front, only 138 transcripts were differentially expressed between the genotype categories while 1082 were differentially expressed in distal, non-symptomatic tissues. Among these differentially expressed genes, several genes in the mevalonate (MVA) and iridoid pathways were found to be co-regulated, possibly indicating increased fluxes through these pathways in response to H. fraxineus. Comparison of transcriptional responses of symptomatic and non-symptomatic ash in a controlled greenhouse experiment revealed a relatively small set of genes that were differentially and concordantly expressed in both studies. This gene-set included the rate-limiting enzyme in the MVA pathway and a number of transcription factors. Furthermore, several of the concordantly expressed candidate genes show significant similarity to genes encoding players in the abscisic acid- or Jasmonate-signalling pathways. CONCLUSIONS: A set of candidate genes, concordantly expressed between field and greenhouse experiments, was identified. The candidates are associated with hormone signalling and specialized metabolite biosynthesis pathways indicating the involvement of these pathways in the response of the host to infection by H. fraxineus.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

Convergent molecular evolution among ash species resistant to the emerald ash borer.

Recent studies show that molecular convergence plays an unexpectedly common role in the evolution of convergent phenotypes. We exploited this phenomenon to find candidate loci underlying resistance to the emerald ash borer (EAB, Agrilus planipennis), the United States' most costly invasive forest insect to date, within the pan-genome of ash trees (the genus Fraxinus). We show that EAB-resistant taxa occur within three independent phylogenetic lineages. In genomes from these resistant lineages, we detect 53 genes with evidence of convergent amino acid evolution. Gene-tree reconstruction indicates that, for 48 of these candidates, the convergent amino acids are more likely to have arisen via independent evolution than by another process such as hybridization or incomplete lineage sorting. Seven of the candidate genes have putative roles connected to the phenylpropanoid biosynthesis pathway and 17 relate to herbivore recognition, defence signalling or programmed cell death. Evidence for loss-of-function mutations among these candidates is more frequent in susceptible species than in resistant ones. Our results on evolutionary relationships, variability in resistance, and candidate genes for defence response within the ash genus could inform breeding for EAB resistance, facilitating ecological restoration in areas invaded by this beetle.

Molecular cloning and functional analysis of 4-Coumarate:CoA ligase 4(4CL-like 1)from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis.

BACKGROUND: Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. RESULTS: In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H2O2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT. CONCLUSIONS: Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.

Comparative transcriptome analysis revealing the potential mechanism of seed germination stimulated by exogenous gibberellin in Fraxinus hupehensis.

BACKGROUND: Fraxinus hupehensis is an endangered tree species that is endemic to in China; the species has very high commercial value because of its intricate shape and potential to improve and protect the environment. Its seeds show very low germination rates in natural conditions. Preliminary experiments indicated that gibberellin (GA3) effectively stimulated the seed germination of F. hupehensis. However, little is known about the physiological and molecular mechanisms underlying the effect of GA3 on F. hupehensis seed germination. RESULTS: We compared dormant seeds (CK group) and germinated seeds after treatment with water (W group) and GA3 (G group) in terms of seed vigor and several other physiological indicators related to germination, hormone content, and transcriptomics. Results showed that GA3 treatment increases seed vigor, energy requirements, and trans-Zetain (ZT) and GA3 contents but decreases sugar and abscisic acid (ABA) contents. A total of 116,932 unigenes were obtained from F. hupehensis transcriptome. RNA-seq analysis identified 31,856, 33,188 and 2056 differentially expressed genes (DEGs) between the W and CK groups, the G and CK groups, and the G and W groups, respectively. Up-regulation of eight selected DEGs of the glycolytic pathway accelerated the oxidative decomposition of sugar to release energy for germination. Up-regulated genes involved in ZT (two genes) and GA3 (one gene) biosynthesis, ABA degradation pathway (one gene), and ABA signal transduction (two genes) may contribute to seed germination. Two down-regulated genes associated with GA3 signal transduction were also observed in the G group. GA3-regulated genes may alter hormone levels to facilitate germination. Candidate transcription factors played important roles in GA3-promoted F. hupehensis seed germination, and Quantitative Real-time PCR (qRT-PCR) analysis verified the expression patterns of these genes. CONCLUSION: Exogenous GA3 increased the germination rate, vigor, and water absorption rate of F. hupehensis seeds. Our results provide novel insights into the transcriptional regulation mechanism of effect of exogenous GA3 on F. hupehensis seed germination. The transcriptome data generated in this study may be used for further molecular research on this unique species.