SWATH-MS-Based Proteomics Reveals the Regulatory Metabolism of Amaryllidaceae Alkaloids in Three Lycoris Species.

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.

The Effect of Trichoderma harzianum Hypovirus 1 (ThHV1) and Its Defective RNA ThHV1-S on the Antifungal Activity and Metabolome of Trichoderma koningiopsis T-51.

Mycoviruses widely exist in filamentous fungi and sometimes cause phenotypic changes in hosts. Trichoderma harzianum hypovirus 1 (ThHV1) and its defective RNA ThHV1-S were found in T. harzianum and exhibited high transmissibility. In our previous study, ThHV1 and ThHV1-S were transferred to an excellent biological control agent T. koningiopsis T-51 to form a derivative strain 51-13. In this study, we assessed the metabolic changes in strain 51-13 and antifungal activity of its culture filtrate (CF) and volatile organic compounds (VOCs). The antifungal activity of CF and VOCs of T-51 and 51-13 was different. Compared with the CF of T-51, that of 51-13 exhibited high inhibitory activity against B. cinerea, Sclerotinia sclerotiorum, and Stagonosporopsis cucurbitacearum but low inhibitory activity against Leptosphaeria biglobosa and Villosiclava virens. The VOCs of 51-13 exhibited high inhibitory activity against F. oxysporum but low inhibitory activity against B. cinerea. The transcriptomes of T-51 and 51-13 were compared; 5531 differentially expressed genes (DEGs) were identified in 51-13 with 2904 up- and 2627 downregulated genes. In KEGG enrichment analysis, 1127 DEGs related to metabolic pathways (57.53%) and 396 DEGs related to biosynthesis of secondary metabolites (20.21%) were clearly enriched. From the CF of T-51 and 51-13, 134 differential secondary metabolites (DSMs) were detected between T-51 and 51-13 with 39 up- and 95 downregulated metabolites. From these, 13 upregulated metabolites were selected to test their antifungal activity against B. cinerea. Among them, indole-3-lactic acid and p-coumaric acid methyl ester (MeCA) exhibited strong antifungal activity. The IC50 of MeCA was 657.35 µM and four genes possibly related to the synthesis of MeCA exhibited higher expression in 51-13 than in T-51. This study revealed the mechanism underlying the increase in antifungal activity of T-51 because of the mycovirus and provided novel insights in fungal engineering to obtain bioactive metabolites via mycoviruses.

Biological Control and Plant Growth Promotion by Volatile Organic Compounds of Trichoderma koningiopsis T-51.

Trichoderma spp. are widely used in plant disease control and growth promotion due to their high efficacy and multiple biocontrol mechanisms. Trichoderma koningiopsis T-51 is an effective biocontrol agent against gray mold disease by direct contact. However, the indirect physical contact biocontrol potential of Trichoderma spp. is not clear. In this study, the volatile organic compounds (VOCs) produced by T-51 showed high inhibitory activity against plant pathogenic fungi Botrytis cinerea and Fusarium oxysporum. The percentage of B. cinerea and F. oxysporum mycelial growth inhibition by T-51 VOCs was 73.78% and 43.68%, respectively. In both B. cinerea and F. oxysporum, conidial germination was delayed, and germ tube elongation was suppressed when exposed to T-51 VOCs, and the final conidial germination rate of B. cinerea decreased significantly after T-51 treatment. The VOCs from T-51 reduced the Botrytis fruit rot of tomato compared with that noted when using the control. Moreover, the T-51 VOCs significantly increased the size and weight of Arabidopsis thaliana seedlings. Twenty-four possible compounds, which were identified as alkenes, alkanes, and esters, were detected in VOCs of T-51. These results indicate that T. koningiopsis T-51 can exert biological control by integrating actions to suppress plant disease and promote plant growth.

Comprehensive Transcriptome and Metabolic Profiling of Petal Color Development in Lycoris sprengeri.

Lycoris sprengeri (L. sprengeri) is an important ornamental bulbous plant, and its numerous varieties in different color forms are widely planted. Multiple color types of petals in L. sprengeri provide us with possibilities to delineate the complicated metabolic networks underlying the biochemical traits behind color formation in this plant species, especially petal color. In this study, we sequenced and annotated a reference transcriptome of pink and white petals of L. sprengeri and analyzed the metabolic role of anthocyanin biosynthesis in regulating color pigment metabolism. Briefly, white and pink petal samples were sequenced with an Illumina platform, to obtain the reads that could be assembled into 100,778 unique sequences. Sequences expressed differentially between white vs. pink petals were further annotated with the terms of Gene Ontology (GO), Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and eggNOG. Gene expression analyses revealed the repression of anthocyanin and steroid biosynthesis enzymes and R2R3 MYB transcription factor (TF) genes in white petals compared to pink petals. Furthermore, the targeted metabolic profiling of anthocyanins revealed that color-related delphinidin (Del) and cyanidin (Cy) pigments are lower in white petals, which correlate well with the reduced gene expression levels of anthocyanin biosynthesis genes. Taken together, it is hypothesized that anthocyanin biosynthesis, steroid biosynthesis, and R2R3 MYB TFs may play vital regulatory roles in petal color development in L. sprengeri. This work provides a valuable genomic resource for flower breeding and metabolic engineering in horticulture and markers for studying the flower trait evolution of L. sprengeri.

Grafting-responsive miRNAs in cucumber and pumpkin seedlings identified by high-throughput sequencing at whole genome level.

Grafting is an important agricultural technique widely used for improving growth, yields and tolerance of crops to abiotic and biotic stresses. As one type of endogenous, non-coding small RNAs, microRNAs (miRNAs) regulate development and responsiveness to biotic and abiotic stresses by negatively mediating expression of target genes at the post-transcriptional level. However, there have been few detailed studies to evaluate the role of miRNAs in mediation of grafting-induced physiological processes in plants. Cucumis sativus and Cucurbita moschata are important vegetables worldwide. We constructed eight small RNA libraries from leaves and roots of seedlings that were grafted in the following four ways: (1) hetero-grafting, using cucumber as scion and pumpkin as rootstock; (2) hetero-grafting, with pumpkin as scion and cucumber as rootstock; (3) auto-grafting of cucumbers and (4) auto-grafting of pumpkins. High-throughput sequencing was employed, and more than 120 million raw reads were obtained. We annotated 112 known miRNAs belonging to 40 miRNA families and identified 48 new miRNAs in the eight libraries, and the targets of these known and novel miRNAs were predicted by bioinformatics. Grafting led to changes in expression of most miRNAs and their predicted target genes, suggesting that miRNAs may play significant roles in mediating physiological processes of grafted seedlings by regulating the expression of target genes. The potential role of the grafting-responsive miRNAs in seedling growth and long-distance transport of miRNA was discussed. These results are useful for functional characterization of miRNAs in mediation of grafting-dependent physiological processes.