DTT-Induced Transcriptome Profiling Reveals Divergent ER Proteostasis and Stress-Adaptation Programs in CHO-S/HB8 and CHO-4BGD/DUL Producer Clones

Proteomic Insights into Salt-Stress Tolerance in Zygosaccharomyces rouxii Revealed by Data-Independent Acquisition Mass Spectrometry.

Zygosaccharomyces rouxii is a halotolerant yeast commonly associated with high-salt fermentations, although its proteome-level adaptation mechanisms are little understood. DIA-based quantitative proteomics was used to characterize the salt-stress responses of Z. rouxii CGMCC 3791 grown at 0, 60, and 120 g/L NaCl. Principal component analysis demonstrated high repeatability and a unique proteome state at 120 g/L of NaCl. Differential analysis revealed 251 differentially expressed proteins (DEPs) (148 up, 103 down) at 60 g/L and 798 DEPs (549 up, 249 down) at 120 g/L, demonstrating significant concentration-dependent remodeling. Under severe stress, GO and KEGG enrichment consistently revealed the reinforcement of central carbon and energy metabolism, peroxisome-associated fatty acid turnover, oxidoreductase/redox activities, and translation and nucleotide metabolic pathways. Heatmap clustering and PPI networks revealed additional tightly coordinated modules that connect bioenergetics, redox regulation, and the translational capacity. These findings provide a proteome-scale framework for understanding halotolerance in Z. rouxii and guidance for future high-salt fermentation engineering.

Single-cell transcriptomics and DNA methylomics reveal cell-type specific heat stress response mechanisms in alfalfa.

Heat stress severely limits crop yield and threatens global food security, also significantly influence the growth and development of alfalfa. Although there have been some studies on heat stress at the organ or tissue levels, the heat stress response mechanisms on the cell type level still remains unclear in alfalfa. Here, the single-nucleus RNA sequencing (snRNA-seq) was performed on 61,835 high-quality leaf cells under control and heat-stressed conditions (1 h and 48 h), revealing distinct transcriptional responses across five major cell types identified from 17 cell clusters. A total of 5,098 differentially expressed genes (DEGs) were detected, and cell-type-specific regulators involving lignin biosynthesis (PAL, 4CL, OMT, CCoAOMT), hormone signaling (Aux, IAA, ARP, PIN, ERF, EIN, ASR), and kinase cascades (LRR-RLK, PP2C, WAK, and STPK) in vascular, epidermal, and mesophyll cells were uncovered by pseudo-time trajectory analyses, respectively. The weighted gene co-expression network analysis (WGCNA) identified ten cell-type-specific heat-responsive networks, and some key heat-responsive genes (HSF, HSP, MAPK, CML, CIPK, and WRKY) exhibited opposing expression patterns in different cell types, highlighting the complexity regulatory mechanisms across cell types under heat stress. Furthermore, the whole-genome bisulfite sequencing (WGBS) identified 6,252 differentially methylated regions and revealed a global increase in DNA methylation under heat stress. By integrating snRNA-seq, WGBS, and pseudo-time trajectory analyses, MsHSP90 and MsHSFA5 were core transcription factors appearing repeatedly. Functional studies demonstrated that MsHSP90 and MsHSFA5 significantly influence plant thermotolerance. Overall, our study provided valuable insights into the heat stress response mechanisms of alfalfa at the single-cell level.

Comparative proteomics and metabolomics reveals distinct host protein quality control and metabolic signatures during recombinant IL1-His and IL15-His expression in Nicotiana benthamiana

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.

Selective recruitment of NAT10-catalyzed ac4C-modified mRNAs into stress granules by poly(A)-binding protein promotes mRNA stability and plant heat stress tolerance.

Global warming poses a considerable threat to crop production, making heat stress a pivotal challenge in agriculture. Yet how epitranscriptomic modifications contribute to plant heat stress responses remains to be explored. Here, this study reveals the critical role of phase separation in plant heat stress tolerance and demonstrated that N-acetyltransferase 10 (NAT10), which encodes of the cytosine N4 acetyltransferase protein, contributes to heat resistance. We found that NAT10 interacts with polyadenylate-binding protein (PABP), which contains intrinsically disordered regions (IDRs), thereby facilitating the selective recruitment of ac4C-modified mRNAs into PABP-mediated condensates. Integrative transcriptome-wide analysis, combining ac4C acetylome profiling with SG-enriched transcript sequencing, revealed that detoxification-related mRNAs, including those encoding the cytochrome P450, phenylalanine ammonia-lyase, glutathione S-transferase, and heat shock 70 protein families, preferentially accumulate within these condensates. This accumulation maintains their stability and prevents stress-induced degradation. Conversely, loss of PABP impairs the recruitment of ac4C-modified detoxification-related transcripts into stress granules, thereby promoting their degradation under heat stress. In summary, our findings identify a stress-responsive NAT10-PABP-ac4C axis that promotes phase separation to stabilize ac4C-modified mRNAs under heat stress. By recruiting detoxification-related transcripts into stress granules, this axis ensures mRNA stability and offers insights for enhancing crop resilience under environmental stress.

Transcriptional reprogramming of the stress response and translational-secretory machinery enhances recombinant secreted protein production in Yarrowia lipolytica.

Protein synthesis and secretion processes are non-pathway traits. The genetic complexity makes them very challenging to engineer and genetically optimize. Therefore, we exploit transcriptional reprogramming to create a favorable cellular environment for a trait of interest. In this study, we investigate the effects of transcription factor (TF) engineering on secretory recombinant protein (rProt) production in Y. lipolytica. High-throughput genome-wide pre-screening guided the TFs selection. Here, we run high-throughput functional studies, followed by transcriptome analysis for selected TF-modified strains from semi-steady state. We contrasted the overexpression (OE) vs. deletion (KO) genotypes for their capacity to produce two reporter proteins (fluorescent model and glucoamylase). Despite a wide set of TF-modified strains analyzed here, only several displayed consistent, unbiased phenotypes. Collected data indicated that the known general stress response TFs, Msn4 and Hsf1, elicit their highly beneficial rProt-promoting function specifically towards a "challenging" rProt, while the levels of "simple" rProt remain unaffected. Deletion of the previously identified strong repressor of intracellular rProt synthesis, Azf1, significantly enhanced production of secretory rProt. At the transcriptomic level, deletion of Azf1 was associated with massive upregulation of ribosome biogenesis and cytoplasmic translation, which underwent alternative splicing (global differential exon usage analysis). The results of contrasting the Azf1- and Dep1-driven regulomes (the latter identified as a strong repressor of intracellular rProt synthesis, with no positive effect on the secretory rProt production) suggest that for efficient rProt secretion, upregulation in the vesicle-mediated transportation must co-occur, as in the case of Azf1-KO. In our experimental setting, OE of the general stress response TFs was beneficial for the production of "problematic", UPR-awakening rProt, and deletion of Azf1 for tuning a molecular background beneficial for secretory rProt.

Systems-level discovery of housekeeping and tissue-specific genes reveals core cellular and specialized transcriptional networks in makhana (Euryale ferox Salisb.)