Exploring key genes of the alternative respiration pathway, uncoupling, and calcium-binding proteins in methanol-treated rapeseed under drought stress

Key message

The research aimed at studying the impact of drought stress on rapeseed and evaluated the role of methanol foliar spraying in providing a source of carbon dioxide during its deficiency conditions. Further, the focus was on the key genes responsible for the alternative respiration pathway, uncoupling, and calcium-binding proteins.

Abstract

Oilseeds are the largest food reserves in the world after cereal. One of the most significant sources of edible oil is rapeseed. However, environmental stressors, such as drought, play a crucial role in reducing crop yield worldwide. This study was conducted to investigate the impact of drought stress on genes and to analyze the protein-protein interactions among key genes related to alternative respiration, uncoupling, and calcium-binding proteins. Additionally, the research aimed to detect any miRNAs that could potentially modulate target genes in the drought-sensitive (Hyola308) and drought-tolerant (SLM046) rapeseed genotypes. Gene network analysis using STRING v11.5 and cytoHubba (MMC method) identified UCP1, NDB2, NDA2, and AOX1A as major hub genes. To explore their post-transcriptional regulation, we used psRNATarget (expectation threshold ≤ 5) along with Brassicaceae-specific miRNAs from miRBase v22. This analysis predicted 22 microRNAs targeting these four rapeseed genes, primarily from the miR169, miR168, miR319, miR390, miR397, miR393, and miR171 families. These miRNAs are predicted to regulate mitochondrial alternative respiration pathways under drought stress, primarily by modulating key genes involved in ROS scavenging and cellular energy homeostasis. Among the identified miRNAs, bna-miR169a/b/n, bna-miR168a/b, bna-miR403, bna-miR390a/b/c, bna-miR397a/b, bna-miR393, and bna-miR171f/g were found to be rapeseed-specific. For instance, bna-miR169a/b/n, known to regulate NF-YA transcription factors during drought stress, targets NDB2, a gene critical for maintaining NADH oxidation. bna-miR393 is involved in modulating auxin signaling, while bna-miR171f/g targets SCL6 transcripts, influencing genes related to root architecture and contributing to stress adaptation. Furthermore, miR403 is known to modulate responses to both drought and cold stress, and miR390 targets ARF genes, which influence root development and play a crucial role in stress tolerance mechanisms. qRT‐PCR (three biological × three technical replicates) on drought‐stress (DR; 30% field capacity) and methanol‐treated (DM; 20% v/v foliar spray) plants (CL = fully irrigated control) showed that methanol application significantly upregulated UCP1 in tolerant plants at 24 h post‐stress and induced NDA2/AOX1A in sensitive plants at 72 h (DR) and 24 h (DM). Our results demonstrate that foliar methanol enhances drought resilience by boosting alternative respiration and suggest that the identified miRNAs may serve as post‐transcriptional regulators of these hub genes, making them potential targets for genetic or biotechnological improvement of drought tolerance in rapeseed.