Genome-Wide Identification and Characterization of Diterpenoid Pathway CYPs in Andrographis paniculata and Analysis of Their Expression Patterns under Low Temperature Stress

<i>Andrographis paniculata</i> is known for its diterpenoid medicinal compounds with antibacterial and anti-inflammatory properties. However, it faces production and cultivation challenges due to low temperatures (LTs). Cytochrome P450 monooxygenases (CYPs) are key enzymes in diterpenoid accumulation. Nevertheless, the functions and LT-related expression patterns of diterpenoid pathway <i>CYPs</i> in <i>Andrographis paniculata</i> remain poorly understood. In this study, 346 CYPs were discovered in <i>Andrographis paniculata</i>. Among them, 328 CYPs belonged to 42 known subfamilies. The remaining 17 CYPs might have represented novel subfamilies unique to this species. A total of 65 candidate CYPs associated with diterpenoid modification were identified. Of these, 50 were transmembrane proteins, and 57 were localized to chloroplasts. The CYP71 subfamily was the most abundant and had the highest motif diversity. Promoters of all candidate <i>CYPs</i> commonly contained elements responsive to gibberellins (GAs), methyl jasmonate (MeJA), and abiotic stresses. Notably, the XP_051152769 protein, corresponding to a <i>CYP</i> gene over 40,000 bp in length, featured an extraordinarily long intron (40,751 nts). Functional elements within this intron were related to LT, GAs, and dehydration pathways. Based on the promoter element arrangement and subfamily classification, 10 representative candidate CYPs were selected. Under LT stress, significant expression changes were observed in three representative <i>CYPs</i>: <i>CYP71D</i>, <i>ent-kaurenoic acid oxidase</i> (<i>KAO</i>), and <i>ent</i>-<i>kaurene oxidase</i> (<i>KO</i>). <i>KAO</i> and <i>KO</i> were significantly upregulated during early LT stress. KAO and KO interacted with each other and jointly interacted with GA20OX2-like. <i>CYP71D</i> acted as a negative response factor to LT stress. Among the 37 proteins interacting with CYP71D, 95% were CYPs. This study provides a critical preliminary foundation for investigating the functions of diterpenoid pathway CYPs in <i>Andrographis paniculata</i>, thereby facilitating the development of LT-tolerant cultivars.