Transcriptomic Analysis of Differentially Expressed Genes and Alternative Splicing Events Associated with Crassulacean Acid Metabolism in Orchids

Phalaenopsis equestris is an obligate crassulacean acid metabolism (CAM) plant with high ornamental and economic value. CAM photosynthesis is associated with drought tolerance and efficient water utilization, which enhances the survival rate of CAM plants in arid environments. The identification and analysis of CAM-related genes will be helpful to improve our understanding of the regulatory mechanisms of CAM metabolism. In this study, we analyzed RNA-Seq data to identify differentially expressed genes (DEGs) between circadian day and night in P. equestris leaves then performed GO and KEGG functional enrichment analysis. The pathways that were significantly enriched among these DEGs included carbon fixation, circadian clock regulation, glucose metabolism, photosynthesis, and plant hormone signaling. We also used PacBio long-read Iso-Seq technology, which identified many alternative splicing events for key genes in CAM-related pathways, including carbon fixation, circadian clock regulation, and stomatal movement. These findings suggested that alternative splicing events might be involved in CAM metabolism. Many unknown or uncharacterized genes were also found to be potentially involved in CAM metabolism. For example, the Peq000162 gene encodes a protein belonging to the LdpA (light-dependent period) iron-sulfur protein family, and it was found to generate many alternatively spliced products. These findings shed light on CAM metabolic mechanisms in P. equestris along with the molecular functions of key CAM genes. Ultimately, the information may help enhance crop yield and drought tolerance through the introduction of CAM features into C3 crops.