Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana.

MAIN CONCLUSION: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

First report of alternate hosts of willow rust disease caused by Melampsora ferrinii in China.

Corydalis acuminata Franch., C. edulis Maxim. and C. racemosa (Thunb.) Pers. of family Papaveraceae are rich in multiple alkaloids and widely used as Chinese medicinal herbs, for treating cough, pruritus, sores tinea and snake venom (Zhang et al. 2008, Iranshahy et al. 2014). In April 2021, orange rust pustules were observed on C. acuminata, C. edulis and C. racemosa in Shaanxi Province (34°4'56'' N, 108°2'9'' E, alt. 770 m), China. Samples were collected and voucher specimens were preserved in the Herbarium Mycologicum Academiae Sinicae (nos. HMAS249947-HMAS249949), China. Consequent geospatial investigations revealed that diseased plants can be observed at an altitude of 400-1000 m, and show an incidence from 40% to 80% varied by altitude. Spermogonia epiphyllous, subcuticular, densely grouped, oval or round, 0.14-0.36 × 0.09-0.30 mm, pale orange-yellow, and type 3 of Cummins and Hiratsuka (1963). Aecia mostly hypophyllous, subepidermal without peridia, Caeoma-type, erumpent, densely grouped, oval or round, 0.27-0.85 × 0.15-0.43 mm, and orange-yellow; hyaline peridial cells produced in a periphery of the sorus under the ruptured epidermis of host plants. Aeciospores globoid or broadly ellipsoid, catenulate with intercalary cells, 15.7-20.1 × 10.8-15.7 µm, yellow to pale orange; walls hyaline, verrucose, 1.7-3.1 µm thick. This fungus was morphologically identified as Melampsora (Melampsoraceae). The rDNA-28S and the internal transcribed spacer (ITS) regions were amplified using primers NL1/NL4 and ITS1/ITS4 (Ji et al. 2020; Wang et al. 2020). Bi-directional sequences were assembled and deposited in GenBank (accession nos. MW990091-MW990093 and MW996576-MW996578). Phylogenetic trees were constructed with the ITS+rDNA-28S dataset based on maximum-likelihood (ML), maximum-parsimony (MP) and Bayesian Inference (BI). ML and MP bootstrap values were calculated by bootstrap analyses of 1,000 replicates using MEGA-X (Kumar et al. 2018), while BI posterior probabilities (Bpps) were calculated using MrBayes ver. 3.1.2 (Ji et al. 2020; Wang et al. 2020). Phylogenetic analyses grouped our specimens and Melampsora ferrinii Toome & Aime into one clade, highly supported by bootstrap values of ML, MP, and Bpps of 100%/100%/1. Inoculations were conducted with 1-year-old plants of original host, Salix babylonica L. (Toome & Aime 2015). Aeciospores suspension with a concentration of 106 spores/ml were sprayed on 20 healthy leaves, with another 20 healthy leaves sprayed with sterile water as the control. The inoculated plants were kept in darkness at 20-25 °C for 2 days and then transferred into greenhouse at 23°C with 16 h light per day. After 8-10 days of inoculation, yellow pustules of uredinia appeared on abaxial surfaces of the inoculated leaves, which were identical to Toome & Aime (2015) reported, while the control leaves remained healthy. Inoculations with the same method were conducted by spraying urediniospores, and the same rust symptoms developed after 8 days. Genus Corydalis was verified as the alternate host of M. chelidonii-pierotii Tak. Matsumoto, M. coleosporioides Dietel, M. idesiae Miyabe and M. yezoensis Miyabe & T. Matsumoto (Shinyama & Yamaoka 2012; Okane et al. 2014; Yamaoka & Okane 2019), and C. incisa (Thunb.) Pers. was speculated as the potential alternate host of M. ferrinii (Toome & Aime 2015). Based on morphology, phylogeny and pathogenicity, we firstly report M. ferrinii in mainland China and verify C. acuminata, C. edulis and C. racemosa instead of C. incisa as its alternate hosts.

Tandem Mass Tag Based Quantitative Proteomics of Developing Sea Buckthorn Berries Reveals Candidate Proteins Related to Lipid Metabolism.

Sea buckthorn ( Hippophae L.) is an economically important shrub or small tree distributed in Eurasia. Most of its well-recognized medicinal and nutraceutical products are derived from its berry oil, which is rich in monounsaturated omega-7 (C16:1) fatty acid and polyunsaturated omega-6 (C18:2) and omega-3 (C18:3) fatty acids. In this study, tandem mass tags (TMT)-based quantitative analysis was used to investigate protein profiles of lipid metabolism in sea buckthorn berries harvested 30, 50, and 70 days after flowering. In total, 8626 proteins were identified, 6170 of which were quantified. Deep analysis results for the proteins identified and related pathways revealed initial fatty acid accumulation during whole-berry development. The abundance of most key enzymes involved in fatty acid and triacylglycerol (TAG) biosynthesis peaked at 50 days after flowering, but TAG synthesis through the PDAT (phospholipid: diacylglycerol acyltransferase) pathway mostly occurred early in berry development. In addition, the patterns of proteins involved in lipid metabolism were confirmed by combined quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and parallel reaction monitoring analyses. Our data on the proteomic spectrum of sea buckthorn berries provide a scientific basic for understanding lipid metabolism and related pathways in the developing berries.

Yellowhorn Xso-miR5149-XsGTL1 enhances water-use efficiency and drought tolerance by regulating leaf morphology and stomatal density.

Yellowhorn (Xanthoceras sorbifolium) is a unique edible woody oil tree species in China. Drought stress is the major yield-limiting factor of yellowhorn. MicroRNAs play an important role in regulating the response of woody plants to drought stress. However, the regulatory function of miRNAs in yellowhorn remains unclear. Here, we first constructed coregulatory networks integrated with miRNAs and their target genes. According to GO function and expression pattern analysis, we selected the Xso-miR5149-XsGTL1 module for further study. Xso-miR5149 is a key regulator of leaf morphology and stomatal density by directly mediating the expression of the transcription factor XsGTL1. Downregulation of XsGTL1 in yellowhorn led to increased leaf area and reduced stomatal density. RNA-seq analysis indicated that downregulation of XsGTL1 increased the expression of genes involved in the negative control of stomatal density, leaf morphology, and drought tolerance. After drought stress treatments, the XsGTL1-RNAi yellowhorn plants were less damaged and had higher water-use efficiency than the WT plants, while destruction of Xso-miR5149 or overexpression of XsGTL1 had the opposite effect. Our findings indicated that the Xso-miR5149-XsGTL1 regulatory module plays a critical role in controlling leaf morphology and stomatal density; hence, it's a potential candidate module for engineering enhanced drought tolerance in yellowhorn.

Identification of Camellia oleifera WRKY transcription factor genes and functional characterization of CoWRKY78.

Camellia oleifera Abel is a highly valued woody edible oil tree, which is endemic to China. It has great economic value because C. oleifera seed oil contains a high proportion of polyunsaturated fatty acids. C. oleifera anthracnose caused by Colletotrichum fructicola, poses a serious threat to C. oleifera growth and yield and causes the benefit of the C. oleifera industry to suffer directly. The WRKY transcription factor family members have been widely characterized as vital regulators in plant response to pathogen infection. Until now, the number, type and biological function of C. oleifera WRKY genes are remains unknown. Here, we identified 90 C. oleifera WRKY members, which were distributed across 15 chromosomes. C. oleifera WRKY gene expansion was mainly attributed to segmental duplication. We performed transcriptomic analyses to verify the expression patterns of CoWRKYs between anthracnose-resistant and -susceptible cultivars of C. oleifera. These results demonstrated that multiple candidate CoWRKYs can be induced by anthracnose and provide useful clues for their functional studies. CoWRKY78, an anthracnose-induced WRKY gene, was isolated from C. oleifera. It was significantly down-regulated in anthracnose-resistant cultivars. Overexpression of CoWRKY78 in tobacco markedly reduced resistance to anthracnose than WT plants, as evidenced by more cell death, higher malonaldehyde content and reactive oxygen species (ROS), but lower activities of superoxide dismutase (SOD), peroxidase (POD), as well as phenylalanine ammonia-lyase (PAL). Furthermore, the expression of multiple stress-related genes, which are associated with ROS-homeostasis (NtSOD and NtPOD), pathogen challenge (NtPAL), and pathogen defense (NtPR1, NtNPR1, and NtPDF1.2) were altered in the CoWRKY78-overexpressing plants. These findings increase our understanding of the CoWRKY genes and lay the foundation for the exploration of anthracnose resistance mechanisms and expedite the breeding of anthracnose-resistant C. oleifera cultivars.

Luteodorsum huanglongense (Gomphaceae, Gomphales), a New Genus and Species of Gomphoid Fungus from the Loess Plateau, Northwest China.

During an investigation of the macrofungal flora in the Huanglong Mountains of the Loess Plateau, northwest China, a unique gomphoid fungus was discovered and collected. After morphological identification and molecular phylogenetic analyses, a new genus named Luteodorsum and its type species, L. huanglongense, were proposed. Phylogenetic analyses were conducted using datasets of nuclear ribosomal DNA 28S large subunit (LSU), mitochondrial (mt) adenosine triphosphatase (ATPase) subunit 6 (atp6), and mt small-subunit rDNA (mtSSU). The results confirmed that L. huanglongense forms an independent clade within Gomphales, with full maximum likelihood bootstrap support (MLBS), maximum parsimony bootstrap support (MPBS), and Bayesian posterior probability (BPP). L. huanglongense is characterized by its sandy-brown, orange-brown, or coffee-brown color; clavate to infundibuliform shape; wrinkled and ridged hymenophore; ellipsoid to obovoid warted basidiospores; cylindrical to clavate flexuous pleurocystidia; and crystal basal mycelium. Overall, this study contributes to the growing body of knowledge on the diversity and evolution of Gomphales and provides valuable insights into the unique fungal flora found in the Huanglong Mountains.

Study of the characteristics of combined insulation in compact pulse transformers.

One of the important research directions in pulse power technology is to increase power density and operational stability of compact high-power pulse transformers. In this paper, the research object is a high-power pulse transformer using combined insulation, and the operating characteristics are obtained using simulation and experiment methods. The experimental results show that the maximum output voltage of the transformer increases by 30% in the combined insulation. At the same time, the combined insulation transformers are safer and have better insulation holding capacity. Moreover, multiple breakdowns have less effect on the subsequent breakdown voltage of the combined insulation transformer. The average subsequent breakdown voltage after 30 breakdowns of combination insulated transformers is still higher than the first breakdown voltage of non-combined insulation transformers. In addition, the energy storage density of combined insulation transformers is ∼40% higher than that of non-combined insulation transformers because of the higher permittivity of Midel 7131 than transformer oil.

Yellowhorn drought-induced transcription factor XsWRKY20 acts as a positive regulator in drought stress through ROS homeostasis and ABA signaling pathway.

Yellowhorn (Xanthoceras sorbifolium) is a peculiar woody edible oil-bearing tree in China. WRKY transcription factors have specific roles in plant multiple abiotic stress responses. However, it is still not clear that the molecular mechanisms of WRKYs involve in drought tolerance in yellowhorn. In this study, we isolated a drought-induced group I WRKY gene from yellowhorn, designated as XsWRKY20. Expression of XsWRKY20 was strongly induced by PEG6000, NaCl, ABA and SA. Virus-induced gene silencing (VIGS) of XsWRKY20 reduced tolerance to drought stress in yellowhorn, as determined through physiological analyses of POD activity, SOD activity and proline content. This susceptibility was coupled with decreased expression of stress-related genes. In contrast, overexpression of XsWRKY20 in tobacco notably improved drought tolerance. Compared with the WT plants, the XsWRKY20-transgenic lines exhibited lower ROS and MDA content and higher antioxidant enzyme activity and proline content after drought treatment. Moreover, overexpression of XsWRKY20 enhanced the expression of several genes associated with encoding these antioxidant enzymes, proline biosynthesis and ABA signaling pathway. Taken together, XsWRKY20 functions as a positive regulator contributing to drought stress tolerance through either ROS homeostasis by antioxidant systems or ABA-dependent/independent gene expression pathway.

Comparative transcriptomic analysis of high- and low-oil Camellia oleifera reveals a coordinated mechanism for the regulation of upstream and downstream multigenes for high oleic acid accumulation.

Tea oil camellia (Camellia oleifera) is an important woody oil tree in southern China. However, little is known regarding the molecular mechanisms that contribute to high oleic acid accumulation in tea oil camellia. Herein, we measured the oil content and fatty acid compositions of high- and low-oil tea oil camellia seeds and investigated the global gene expression profiles by RNA-seq. The results showed that at the early, second and third seed developmental stages, a total of 64, 253, and 124 genes, respectively, were significantly differentially expressed between the high- and low-oil cultivars. Gene ontology (GO) enrichment analysis of the identified differentially expressed transcription factors (TFs; ABI3, FUS3, LEC1, WRI1, TTG2 and DOF4.6) revealed some critical GO terms associated with oil biosynthesis and fatty acid accumulation, including glycolysis, zinc ion binding, positive regulation of fatty acid biosynthetic process, triglyceride biosynthetic process, seed coat development, abscisic acid-mediated signaling pathway and embryo development. Comprehensive comparisons of transcriptomic profiles and expression analysis of multigenes based on qRT-PCR showed that coordinated high expression of the upstream genes HAD, EAR and KASI directly increased the relative levels of C16:0-ACP, which provided enough precursor resources for oleic acid biosynthesis. Continuous high expression of the SAD gene accelerated oleic acid synthesis and accumulation, and coordinated low expression of the downstream genes FAD2, FAD3, FAD7, FAD8 and FAE1 decreased the consumption of oleic acid for conversion. The coordinated regulation of these multigenes ensures the high accumulation of oleic acid in the seeds of tea oil camellia. Our data represent a comprehensive transcriptomic study of high- and low-oil tea oil camellia, not only increasing the number of sequences associated with lipid biosynthesis and fatty acid accumulation in public resource databases but also providing a scientific basis for genetic improvement of the oleic acid content in woody oil trees.