Comparative transcriptome analysis of alkali stress on rhododendron tolerant and sensitive roots to identify alkali tolerance-related genes

Rhododendrons prefer acidic soil, and soil alkalinity is a critical limiting factor for their growth. However, several varieties of rhododendron demonstrate exceptional adaptabilities to alkaline stress. This study aimed to elucidate the molecular mechanisms underlying alkali tolerance by comparing alkali-tolerant and -sensitive rhododendron varieties at the transcriptomic level, thereby providing insights to facilitate the breeding of improved alkali-tolerant cultivars. Using RNA-Seq, we analyzed differentially expressed genes (DEGs) at 0, 6, and 24 hours following alkaline stress. The results showed that the number of DEGs in the alkali-tolerant variety in response to alkali stress was 1.6 times that of the alkali-sensitive variety. In total, 212 and 781 common DEGs were identified within and between the alkali-tolerant and -sensitive variety, respectively, in response to alkaline stress. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that CYP genes, plant-specific class III peroxidases, and MPK3 play key roles in the alkaline stress response and displayed distinct expression patterns across varieties. Furthermore, numerous transcription factors (TFs) responsive to alkaline stress were identified, with AP2/ERF TFs being the most abundant. Notably, most AP2/ERF TFs were significantly upregulated in the alkali-tolerant variety after 24 hours of alkaline treatment. Functional validation demonstrated that overexpression of RpMPK3 enhanced alkali tolerance in Arabidopsis. In conclusion, this study provided a comprehensive set of candidate genes associated with alkali tolerance in rhododendron and highlighted the positive role of RpMPK3 in conferring this trait. These findings established a theoretical foundation for future molecular breeding of highly alkali-tolerant varieties of rhododendrons.