Transcriptome-wide m6A methylation profile reveals tissue specific regulatory networks in switchgrass (Panicum virgatum L.) under cadmium stress.

The heavy metal cadmium (Cd), known for its high toxicity, poses a grave threat to human health through the food chain. N6-methyladenosine (m6A), the most abundant internal modification, regulates plant adaptation to various adversities, yet the panorama of m6A modifications in switchgrass under cadmium stress remains elusive. This study examines the physiological responses of switchgrass roots and shoots exposed to 50 µM CdCl2, alongside an overview of transcriptome-wide m6A methylation patterns. After cadmium treatment, methylation modifications are primarily enriched near stop codons and the 3'UTR region, with a negative correlation between m6A modification and gene expression levels. In shoots, approximately 58 % of DEGs with m6A modifications show upregulation in expression and decrease in m6A peaks, including zinc transporter 4-like (ZIP4). In roots, about 43 % of DEGs with m6A modifications exhibit downregulation in expression and increase in m6A peaks, such as the ABC transporter family member (ABCG25). We further validate the m6A enrichment, gene expression and mRNA stability of ZIP4 in response to Cd treatment. The results suggest that the negative correlation of m6A enrichment and gene expression is due to altered mRNA stability. Our study establishes an m6A regulatory network governing cadmium transport in switchgrass roots and shoots, offering new avenues for candidate gene manipulation in phytoremediation applications of heavy metal pollution.

PvARL1 Increases Biomass Yield and Enhances Alkaline Tolerance in Switchgrass (Panicum virgatum L.).

Switchgrass is an important bioenergy crop valued for its biomass yield and abiotic tolerance. Alkali stress is a major abiotic stress that significantly impedes plant growth and yield due to high salinity and pH; however, the response mechanism of switchgrass to alkali stress remains limited. Here, we characterized PvARL1, an ARF-like gene, which was up-regulated in both the shoot and root tissues under alkali stress conditions. Overexpression of PvARL1 not only improved alkali tolerance but also promoted biomass yield with more tiller and higher plant height in switchgrass. Moreover, PvARL1 overexpression lines displayed higher capacities in the maintenance of water content and photosynthetic stability compared with the controls under alkali treatments. A significant reduction in the ratio of electrolyte leakage, MDA content, and reactive oxygen species (ROS) showed that PvARL1 plays a positive role in protecting cell membrane integrity. In addition, PvARL1 also negatively affected the K+ efflux or uptake in roots to alleviate ion toxicity under alkali treatments. Overall, our results suggest that PvARL1 functions as a positive regulator in plant growth as well as in the plant response to alkali stress, which could be used to improve switchgrass biomass yield and alkali tolerance genetically.

N6-methyladenosine analysis unveils key mechanisms underlying long-term salt stress tolerance in switchgrass (Panicum virgatum).

N6-methyladenosine (m6A) RNA modification is critical for plant growth, development, and environmental stress response. While short-term stress impacts on m6A are well-documented, the consequences of prolonged stress remain underexplored. This study conducts a thorough transcriptome-wide analysis of m6A modifications following 28-day exposure to 200 mM NaCl. We detected 11,149 differentially expressed genes (DEGs) and 12,936 differentially methylated m6A peaks, along with a global decrease in m6A levels. Notably, about 62% of m6A-modified DEGs, including demethylase genes like PvALKBH6_N, PvALKBH9_K, and PvALKBH10_N, showed increased expression and reduced m6A peaks, suggesting that decreased m6A methylation may enhance gene expression under salt stress. Consistent expression and methylation patterns were observed in key genes related to ion homeostasis (e.g., H+-ATPase 1, High-affinity K+transporter 5), antioxidant defense (Catalase 1/2, Copper/zinc superoxide dismutase 2, Glutathione synthetase 1), and osmotic regulation (delta 1-pyrroline-5-carboxylate synthase 2, Pyrroline-5-carboxylate reductase). These findings provide insights into the adaptive mechanisms of switchgrass under long-term salt stress and highlight the potential of regulating m6A modifications as a novel approach for crop breeding strategies focused on stress resistance.

Characterization of the m6A gene family in switchgrass and functional analysis of PvALKBH10 during flowering.

N6-methyladenosine (m6A), a nucleotide modification that is frequently seen in RNA, plays a crucial role in plant growth, development and stress resistance. However, the m6A regulatory machinery in switchgrass (Panicum virgatum L.), a model plant for cellulose-to-ethanol conversion, remains largely unknown. In this study, we identified 57 candidate genes involved in m6A-regulation in the switchgrass genome, and analyzed their chromosomal distribution, evolutionary relationships, and functions. Notably, we observed distinct gene expression patterns under salt and drought stress, with salt stress inducing writer and eraser genes, alongside drought stress predominantly affecting reader genes. Additionally, we knocked out PvALKBH10, an m6A demethylase gene, via CRISPR/Cas9 and found its potential function in controlling flowering time. This study provides insight into the genomic organization and evolutionary features of m6A-associated putative genes in switchgrass, and therefore serves as the basis for further functional studies.