Comparative proteomics and metabolomics reveals distinct host protein quality control and metabolic signatures during recombinant IL1-His and IL15-His expression in Nicotiana benthamiana
Findings underscore that the characteristics of the target protein and its interaction with the host's physiology could influence the yield of the recombinant protein production in plants.
Abstract
Agrobacterium-mediated transient expression in Nicotiana benthamiana is widely used for recombinant biopharmaceutical production. To investigate the host plant response upon human cytokine production with contrast accumulation, we expressed codon optimized human IL1 beta, serving as a high expression benchmark with a yield of approximately 80 μg/g leaf fresh weight, and IL15 with undetected signal on western blot, representing protein with low level of accumulation, using the geminiviral vector system. A combined proteomics and metabolomics technique was applied to elucidate underlying cellular mechanisms. Quantitative proteomics revealed that IL1-His was robustly detected (9 unique peptides, 83% coverage), whereas no IL15-His–derived peptides were identified. Based on pathway analysis, the expression of IL15-His induced chaperone expression, with downregulation of photosynthetic and primary metabolism pathways. Additionally, metabolomic pathway analysis revealed that IL1-His preferentially drives the branched chain amino acid biosynthesis, but IL15-His shifts metabolism towards phenylpropanoid biosynthesis routes. These findings underscore that the characteristics of the target protein and its interaction with the host's physiology could influence the yield of the recombinant protein production in plants.
Sorghum seeds accumulate substantial amounts of condensed tannins (CTs), which are also referred to as proanthocyanidins (PAs), contributing to their characteristic astringent taste. Flavan-3-ol polymers, known as PAs, are sequestered within plant vacuoles and become catalytically activated via laccase enzymes. However, the biological roles and regulatory pathways of laccases in sorghum are still largely unclear. Here, integrated transcriptomic and metabolomic profiling of developing sorghum seeds identified 7942 differentially expressed genes between low- and high-CT lines, with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealing flavonoid biosynthesis as a key pathway; weighted gene co-expression network analysis (WGCNA) further pinpointed SbLAC14 as a hub gene within the module most strongly correlated with CT content. We then examined its regulation by microRNA397 (SbmiR397-5p). Dual-luciferase assays confirmed the binding of SbmiR397-5p to SbLAC14 in co-transformed tobacco leaves. Overexpressing SbLAC14 in transgenic Arabidopsis significantly increased CT accumulation while decreasing catechin and epicatechin levels. Furthermore, transgenic plants overexpressing miR397 (OEmiR397-5p) exhibited reduced CT content, accompanied by a lightening of seed color. Conversely, transgenic lines overexpressing a miR397-insensitive laccase transcript exhibited a reversed phenotypic outcome. Our findings indicate that SbmiR397-5p negatively regulates the expression of SbLAC14 in relation to CT biosynthesis, identifying it as a potential target for manipulating CT metabolism in sorghum. Those results provide a genetic entry point for metabolic engineering and breeding efforts aimed at modulating grain phenolic profiles.
Yannan Shi, Yongchao Guo, Jinping Wang et al.· Plants· 0 citations
Blister blight disease, caused by the fungus
Exobasidium vexans
, severely threatens tea production and yield. This study investigates the molecular and metabolic responses of tea to blister blight infection, with a focus on alkaloid metabolism. Here, integrated transcriptomics and metabolomics were employed to investigate the differences between healthy leaves and infected leaves at three disease stages. In total, 79 alkaloid metabolites were identified, with 30 differential metabolites shared across all infection stages. Transcriptome sequencing revealed 846 differentially expressed genes, many of which were enriched in the isoquinoline alkaloid biosynthesis pathway. Key structural genes (e.g.
TAT
,
PPO
) and transcription factors (e.g.,
WRKY
,
GRAS
) were significantly upregulated during infection, correlating with altered alkaloid profiles. Notably, dopamine, a key intermediate, was downregulated, suggesting a potential shift in metabolic flux toward downstream defense-related alkaloid synthesis. qRT-PCR validation confirmed the expression patterns of selected DEGs. Our findings provide the first comprehensive evidence that alkaloid metabolism, particularly the isoquinoline alkaloid pathway, are transcriptionally and metabolically reprogrammed during blister blight infection, suggesting a potential role in tea’s defense against this pathogen. These results offer valuable insights for breeding resistant tea cultivars.
Yanglongyu Chen, Ping Li, Yuqing Ma et al.· Frontiers in Plant Science· 0 citations
Introduction Potato virus S (PVS), is an important member of the genus Carlavirus in the family Betaflexiviridae, is a significant pathogen in potatoes worldwide. Previous studies have found it to be the most frequently detected virus in potatoes in China. However, research data on the response mechanisms of potatoes to PVS remain extremely scarce. Methods Here, comparative transcriptomics and metabolomics were performed on potato (Solanum tuberosum cv. Cooperation 88) leaves to elucidate the response mechanisms underlying the infection of Potato virus S to Solanum tuberosum cv. Cooperation 88 at the molecular level. Results A total of 588 significantly differentially expressed genes (SDEGs) were identified post-infection, including 161 upregulated genes and 427 downregulated genes, primarily enriched in biological processes such as the MAPK signaling pathway, plant hormone signal transduction, phenylpropanoid metabolism, and DNA replication. Metabolomic analysis revealed 1,313 and 880 differential metabolites detected in positive and negative ion modes, respectively, with flavonoids showing a significant accumulation trend. Integrated analysis indicated that the phenylpropanoid metabolic pathway (particularly the flavonoid biosynthesis pathway) plays a central regulatory role in antiviral responses. Real-time quantitative PCR validation further confirmed that the expression patterns of key genes (e.g., PAL, C4H, 4CL) were positively correlated with metabolite accumulation. Conclusion The results suggest that the flavonoid biosynthesis pathway in potatoes plays a key role in responding to PVS infection. This study not only helps to deeply understand the interaction mechanism between PVS and potatoes, but also promotes the selection and utilization of disease-resistant varieties.
Kuo Wu, Xia Liu, Yu Li et al.· Frontiers in Microbiology· 0 citations
Rhododendron yedoense var. poukhanense is an important medicinal plant, but still little is known about how its bioactive flavonoids are made in different organs. Comprehensive metabolomic profiling was performed using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS), coupled with reference-based RNA sequencing (RNA-seq), to analyze root, stem, and leaf tissues from three independent biological replicates. Subsequently, we performed two-way orthogonal partial least squares (O2PLS) regression, canonical correlation analysis (CCA), and Pearson correlation analyses. A total of 2182 metabolites were detected. Among these, 1116, 896, and 1264 metabolites exhibited differential accumulation across the three pairwise comparisons. Concurrently, 9836, 5457, and 8007 genes were differentially expressed across the three pairwise comparisons, yielding a total of 13,019 unique differentially expressed genes (DEGs). The bifunctional flavanone 3-hydroxylase/flavonol synthase (F3H/FLS) enzyme exhibited the highest expression level in roots, consistent with root-preferential biosynthesis of flavonoid skeletons and the subsequent accumulation of flavonol glycosides in this tissue. In contrast, dihydroflavonol 4-reductase (DFR) exhibited predominant activity in roots, consistent with its role in farrerol biosynthesis. The multi-omics integration model demonstrated excellent goodness-of-fit to the experimental data. Canonical correlation analysis (CCA) further revealed a robust positive association between dihydrokaempferol accumulation and kaempferol biosynthesis. These findings collectively support flavanone 3-hydroxylase (F3H) as a candidate regulatory node governing organ-specific flavonoid partitioning. However, functional validation is required to substantiate this inference.
R. Fei, Si-Yu Duan, Xiuting Zhao et al.· Plants· 0 citations
Rice false smut caused by Ustilaginoidea virens is a major fungal disease of rice in rice-growing regions throughout the world. However, the key genes and key metabolites related to U. virens resistance in rice remain unclear. Here, we used transcriptomics and metabolomics to determine the gene expression and metabolite accumulation changes in rice at 5, 7, and 9 d after inoculation with U. virens. By comparing the transcriptomes of IR27 (resistant cultivar) and 9311 (susceptible cultivar) spikelets, variable transcriptional responses under control and infection conditions were revealed. In total, 11,235 and 13,453 differentially expressed genes (DEGs) were identified in IR27 and 9311, respectively. The results of Kyoto Encyclopedia of Genes and Genomes and co‑expression analyses showed that the DEGs involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and plant hormone signal transduction responded to disease resistance. Several WRKY transcription factors were also differentially regulated in the resistant and susceptible cultivars. The metabolome analysis identified 343 and 303 differentially accumulated metabolites in IR27 and 9311, respectively, including salicylic acid, jasmonic acid, gibberellin, d-pantothenic acid, and p-coumaric acid. Many of these are primarily involved in plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis pathways. Furthermore, the combined transcriptome and metabolome analysis revealed that plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis were significantly enriched in resistant rice varieties. Therefore, these results provide valuable information on the molecular mechanisms by which rice defends against U. virens infection, and they will facilitate the development of disease‑resistant rice cultivars.
Rongtao Fu, Huan Li, Xi Luo et al.· BMC Plant Biology· 0 citations
Deciphering the metabolic basis of high-yield antibiotic production in Streptomyces is crucial for strain optimization. Atmospheric and room-temperature plasma (ARTP) mutagenesis of Streptomyces xinghaiensis sf106 generated a mutant with a 30% increase in tylosin-equivalent concentration (μg/mL). 4D-FastDIA quantitative proteomics identified 279 differentially abundant proteins enriched in the Type I polyketide synthase (PKS) pathway, with increased abundance of key macrolide-biosynthesis-related proteins. Lysine-acetylome profiling identified 1152 differentially abundant acetylation sites and revealed altered acetylation of enzymes involved in fatty acid metabolism and the tricarboxylic acid (TCA) cycle, suggesting adjustments in central metabolism associated with acyl-CoA precursor availability and energy generation. Integration of proteomic and acetylomic data suggests coordinated changes in protein abundance and lysine acetylation associated with the increased tylosin-equivalent concentration. These results highlight candidate nodes for rational metabolic engineering of S. xinghaiensis.
C. Jia, Cilang Ma, Yuting Jiang et al.· Microbiology Research· 0 citations