Fig. 5: An inversion disabled function of tandemly duplicated TPS genes and impacted terpenoid biosynthesis.

a KEGG enrichment of genes within inversions in three Curcuma species and Z. officinale performed with TBtools. Enrichment of KEGG pathways was calculated with the Hypergeometric test. Statistical tests were one-sided and multiple comparisons were adjusted with the Benjamini-Hochberg correction (Q value). b Schematic diagram showing tandemly duplicated TPSs on chromosome 8 (Chr08) of C. alismatifolia. Genes outside of the inversion maintained the intact structure (dark gray rectangle), and genes within the inversion were pseudogenes (hatching chevron). Genes within inversions on different haplotypes lost different domains. ψ: pseudogene (gray font); CaChr8-TPS-TD-INV: Inversion on C. alismatifolia Chr08 with tandem duplicated TPS genes spanning its inversion breakpoint (8-10517073-28214380), shown as the intersecting dashed lines. Light and dark pink boxes indicated inverted regions on two haplotypes. c Compound 1 content detected by GC-MS and floret phenotype at three developmental stages of florets. The bar indicated 1 cm. Error bars represent the standard deviations (SD), and data are presented as mean values ± SD. Three biological replicates were performed. d GC-MS analysis of the main products formed by prokaryotic expression of germacrene synthases, compound 1 (m/z = 93), compound 3 (m/z = 93), and compound 4 (m/z = 93). Enzymes were incubated with compound 5. Reaction products were identified by standard chemicals and comparison of their mass spectra and retention indices with authentic standards and NIST libraries. Empty vector, pCold-TF. p.Gln64Glu, p.His124Leu, p.Ile228Leu, p.Met303Thr, p.Ile374Val, p.Val435Ala, and p.Val513Ile indicated seven different amino acid sites between Chr08HA727 and Chr08HA736 were mutated one by one accordingly. The direction of mutation is from Chr08HA727 to Chr08HA736. DDXXD deletion: DDXXD domain was deleted. Exon deletion (4): The sequence of exon 4 (containing the DDXXD domain) was deleted. All indicated assays were conducted for four or more repetitions. e Relative catalytic activity of germacrene synthase genes in producing compound 1. Box-plot elements are defined as: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, all values. 5–6 biological replicates were performed for each treatment. f GC-MS analysis of the products from prokaryotic expression of Chr08HA727 proteins at different injection temperatures. Reaction products were identified by comparison of their mass spectra and retention indices with authentic standards and NIST libraries. Empty vector, pCold-TF. When the injection temperature was lowered to 150 °C, the content of compound 1 was decreased, and compound 2 was increased significantly (p < 0.01). g Catalytic scheme of germacrene synthase. The triangle represented the cope rearrangement of compound 2 to form compound 1 that easily occurred at high injection temperatures. c, e Statistical tests were two-sided Student’s t-test, and multiple comparisons were adjusted with the Bonferroni correction. Asterisks represented significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, adjusted).