This study provided valuable insights into the heat stress response mechanisms of alfalfa at the single-cell level by integrating snRNA-seq, WGBS, and pseudo-time trajectory analyses, and demonstrated that MsHSP90 and MsHSFA5 were core transcription factors appearing repeatedly.
Abstract
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.
Salt stress is a major abiotic factor limiting plant growth and productivity. Herpetospermum pedunculosum, a medicinal plant adapted to high-altitude environments, offers a unique non-model system for investigating salt stress responses. Here, we combined physiological and biochemical assays with time-course root transcriptome profiling to characterize the responses of H. pedunculosum to 200 mM NaCl treatment. Salt stress induced rapid wilting, oxidative stress-related physiological changes, membrane damage, and significant accumulation of ABA, total lignin, and total lignans. RNA-seq across five time points (1, 3, 24, 48, and 96 h) identified nearly 28,000 differentially expressed genes (DEGs), which formed distinct temporal clusters associated with hormone signaling, transcriptional regulation, stress responses, and phenylpropanoid-related metabolism. Antioxidant enzyme-related genes, ABA biosynthesis/homeostasis genes, ABA signaling components, and lignin/lignan pathway biosynthesis genes (LLPBGs) showed stage-specific expression patterns, with several genes responding rapidly during the early phase of salt stress. WGCNA and co-expression analyses further identified trait-associated modules and candidate links among core TFs, ABA-related genes, and LLPBGs. Subcellular localization, yeast transactivation, Y1H and Dual-LUC assays provided preliminary evidence that the AP2/ERF factor HpERF141 can bind to and activate the promoter of the previously characterized lignan-related gene HpDIR17. Overall, this study provides a root-focused transcriptomic resource and identifies candidate regulatory genes potentially linking ABA-related responses with lignin/lignan-associated metabolism in H. pedunculosum under salt stress.
Yang Tao, Xiao Huang, Enhao Zhang et al.· Plant physiology and biochem...· 1 citation
Salt and heat stresses often occur simultaneously in arid regions, restricting the distribution and productivity of alfalfa (Medicago sativa L.). However, the mechanisms underlying alfalfa responses to combined salt and heat stress remain unclear. Here, we integrated phenotypic, physiological, transcriptomic, and metabolomic analyses to investigate the regulatory mechanisms involved in combined stress responses. Combined stress more strongly inhibited plant height, stem diameter, fresh weight, net photosynthetic rate, and transpiration rate than single stresses. Salt stress had a predominant effect on several agronomic and physiological traits, whereas heat stress mainly affected chloroplast ultrastructure. Multiomics analysis identified flavonoid metabolism, linoleic acid metabolism, and amino acid biosynthesis as key pathways associated with combined stress responses. Moreover, CHS, CHR, P5CS, and LOX genes expression was closely correlated with metabolites such as naringenin, naringenin chalcone, and proline. These findings provide insights into alfalfa adaptation to multiple abiotic stresses.
Lihe Su, Yongcheng Chen, Xudong Zhang et al.· Journal of Agricultural and...· 0 citations
Heat stress (HS) has emerged as a significant environmental factor affecting plant growth and agricultural productivity. Alternative polyadenylation (APA) is a crucial co-transcriptional process that regulates developmental processes and stress responses in plants. However, the distinct roles of its resultant transcripts in plant HS response remain to be investigated. In this study, we employed poly(A) tag sequencing (PAT-seq) to identify over 1500 transcripts whose expression exhibited significant alterations in response to HS, mediated by SIZ1, a SUMO E3 ligase in Arabidopsis. Further analysis revealed that more than 300 switch genes with varying poly(A) site usages were differentially expressed under HS conditions. Compared to non-canonical poly(A) sites, more genes utilize 3’UTR sites to regulate transcript expression by altering the usage of poly(A) signals. Based on the information of APA genes regulated by SIZ1, we selected two genes, Galactinol synthase enzyme (GolS2) and Tetratricopeptide repeat (TPR) like 3 (TTL3), for characterizing their novel functions. Overexpression of the distal transcript of GolS2 or the proximal transcript of TTL3 enhanced heat tolerance in Arabidopsis. Collectively, our current study elucidates the regulation of APA mediated by SIZ1 during HS response and establishes a strategy for identifying specific transcripts arising from APA for plant heat tolerance.
Jun Wang, Xiujuan Wu, Zhou Zhou et al.· Stress Biology· 0 citations
Osmotic stressors impair tissue expansion and slow crop development, which can ultimately reduce yield. The mechanisms through which dividing and differentiating cells perceive, integrate, and respond to stress are not well understood. To explore this knowledge gap, we assessed Asian rice (Oryza sativa) seedling responses to drought and salinity stress by performing single-nucleus transcriptome analyses on developing leaves and by assaying ion distribution using ICP-MS and CoroNa Green staining. With a dataset of > 125 000 high-quality nuclei, we identified transcriptome signatures of developmental trajectories for each of the major tissue systems, from their origin as dividing cells in the shoot apical meristem to mature cells in epidermal, mesophyll, and vascular tissues. We explored the extensive heterogeneity between cell types with respect to their responses to stress by developing an ensemble-based Perturbation Index and a tool for visualizing pseudobulk expression data and characterizing regulatory modules for different stress responses. Transcriptionally, moderate stress perturbs cells early in development and induces regulatory networks that inhibit cell division, whereas mild stressors primarily disrupt gene expression in differentiated tissues. Overall, these results indicate that leaf development occurring in salt and drought stress conditions is the product of a complex interplay of developmental, environmental, and cell-type contexts.
Sean M. Robertson, Obaid Maqsood, C. Dang et al.· New Phytologist· 1 citation
The moss Physcomitrium patens is a well-established model organism for studying plant responses to abiotic stress. As a non-vascular plant, it provides a valuable system for elucidating both conserved and lineage-specific adaptive mechanisms underlying abiotic stress. Previous transcriptomic studies have focused on severe salt stress (350 mM), while the response to moderate salt stress remained largely unexplored. Here, we investigated the transcriptional responses of P. patens to a moderate 150 mM NaCl treatment, using a high-resolution time-series RNA-sequencing approach. Our analysis revealed 2235 previously uncharacterized salt-responsive genes as part of a rapid, dynamic, and multi-layered response. Time-series clustering unveiled salt-specific gene expression patterns overlapping with salt-responsive pathways from angiosperms. We identified novel transcription factors involved in salt-induced transcriptional reprogramming, including PpMYB123, which we experimentally confirmed as a salt-responsive transcription factor. Overexpression of PpMYB123 altered the expression of enzymes involved in salt stress responses, such as those in phenylpropanoid and starch biosynthesis, suggesting a direct or indirect regulatory role. In summary, our findings highlight evolutionarily conserved patterns in stress-specific signaling pathways and in the underlying transcriptional regulation between bryophytes and angiosperms.
Armin Horn, C. Misra, Jose Miguel Sordo et al.· Journal of plant physiology· 0 citations
The powdery mildew pathogen Erysiphe necator poses a major threat to global viticulture, yet the earliest hours of colonization remain transcriptionally uncharacterized at the single-cell resolution. Here, we constructed a single-cell leaf transcriptomic atlas of Vitis vinifera during early E. necator infection by profiling 113,346 cells across five leaf cell types at 0-, 3-, 6-, and 12 h post-inoculation. This revealed cell-type-specific temporal dynamics of defense-related gene expression, with epidermal cells showing delayed transcriptional activation relative to other cell types. Pseudotime trajectory analysis identified four sequential transcriptional states in epidermal cells, and co-expression network analysis uncovered defense-associated gene modules. We identified 230 NLR immune receptor genes exhibiting distinct cell-type-specific expression patterns and 20 small secreted peptide (SSP)-encoding genes differentially expressed in epidermal cells, including four candidates upregulated at 3 or 6 hpi. These findings provide a transcriptomic framework for understanding cell-type-specific defense dynamics and prioritizing candidate genes for functional studies, thereby offering a molecular resource for breeding powdery mildew-resistant grapevine cultivars.
Yasheng Xi, Kai Wu, Bofan Liu et al.· Horticulturae· 0 citations