Camptothecin (CPT) is a widely known monoterpene indole alkaloid with excellent anticancer activity. CPT has been isolated from different plant species. CPT- producing weedy plant Ophiorrhiza pumila has brought about widespread attention, and the whole genome of O. pumila has been sequenced . Recently, metabolic engineering has been applied in O. pumila to elevate CPT content. For example, individual introduction of G10H or SLS, and co-expression of G10H and SLS significantly enhanced CPT content in transgenic O. pumila hairy roots . Besides, transcription regulation of CPT biosynthesis has been studied. RNA interference of OpERF2 suppressed expression level of genes involved in MEP and secologanin-strictosidine pathways . The transcription repressor OpMYB1 reduced CPT biosynthesis by downregulating expression level of TDC . OpWRKY2 acted as a positive regulator of CPT biosynthesis by directly targeting TDC . OpWRKY1 inhibited CPT biosynthesis by directly downregulating CPR transcription in O. pumila . Nevertheless, transcription regulation of CPT biosynthesis needs further study.
The GATA TF family involved in many aspects of physiology-related processes has been broadly explored in a range of plants including Arabidopsis, rice , grapes , Moso bamboo , and Gossypium sp. . The present study was first reported GATA TFs in O. pumila. Totally, 18 GATA TFs were identified and named OpGATA1–OpGATA18 according to their physical location on the chromosome. The whole OpGATA family in O. pumila could be classified into four groups, similar to those in A. thaliana. In subfamily III, the GATA domain harbored 20 residues in the zinc finger domain, making up a C-X2C-X20-C-X2-C structure, and the other three subfamilies showed that C-X2C-X18-C-X2-C structure, containing 18 residues. The CCT and TIFY domains were specifically identified within subfamily III. The CCT domain was initially found in Arabidopsis Constans protein, which facilitates root and hypocotyl development within A. thaliana and mediates flowering . Previously, the family with a completely conserved TIFY domain was termed TIFY . However, in recent studies, the TIFY domain has been shown to exist extensively in jasmonate ZIM domain protein family and PEAPOD proteins, which are associated with the jasmonic acid pathway .
Motif analysis showed that all OpGATAs contained motif 1 and 5 except OpGATA3, and specific motifs were detected in other groups. For example, motif 4 was only observed in subfamily I, motif 9 was only detected in subfamily II, and motifs 2 and 3 were only detected in subfamily III, suggesting that although some motifs of GATA family genes are highly conserved, new evolutionary motifs may have distinct functions in some plants, and the functions of these new evolutionary motifs need to be further verified. The homology of GATA genes from O. pumila with those from Arabidopsis, rice, soybeans, tomatoes, and grapes was also explored. Notably, the Arabidopsis GATA TFs AtGATA1 (AT3G24050), AtGATA2 (AT3G60530), and AtGATA4 (AT2G45050) have been reported to facilitate light-dependent regulation of gene expression and photomorphogenesis . Accordingly, the homologous genes OpGATA9 (Opuchr03_g0010130-1.1) and OpGATA2 (Opuchr03_g0010130-1.1) may also affect light-dependent regulation of genes. AtGATA22 (AT4G26150), which is homologous with OpGATA12 (Opuchr06_g0009000-1.1), affects the response to cytokinins and hinders root growth in A. thaliana . Additionally, GNC (AT5G56860), which is homologous to OpGATA12, adversely affects seed germination, flowering, and leaf elongation, and overexpression of GNC inhibits the germination, leaf expansion, and flowering of A. thaliana . AtGATA12 (AT5G25830), which is homologous to OpGATA14, is involved in primary dormancy in A. thaliana .
Expression level of most genes encoding the key enzymes in the CPT biosynthesis pathway (i.e., PMK, MDC, SLS, LAMT, TSB, DXR, AACT, 10-HGO, 7-DLGT, G10H, HMGR, STR, TDC, IO, IS, HDS, HDR, IPPI, CMK, HMGS, 8-HGO, and GES) were significantly higher in roots compared with those in other tissues, whereas genes involved in the MVA and MEP pathways (i.e., ASA, MK, DXS, CMS, and MECS) were mostly expressed in leaves. According to the expression patterns of OpGATAs within different tissues, a correlational analysis was conducted. The results showed that OpGATA7, OpGATA14, and OpGATA15 exhibited positive associations with key enzyme genes showing high expression in roots. Moreover, OpGATA15 showed pole-strength correlations with several key genes involved in the biosynthetic pathway (i.e., TDC, HMGS, AACT, PMK, IPPI, DXR, HDS, STR, G10H, LAMT, and SLS). OpGATA14 only had a pole-strength correlation (p < 0.05, r > 0.8) with the HMGR gene and showed strong correlations with G10H, 8-HGO, and LAMT genes. Furthermore, OpGATA7 did not have a pole-strength correlation with any enzyme genes but showed strong correlations (p < 0.05, 0.8 > r > 0.6) with CMK and 8-HGO. OpGATA9, OpGATA12, and OpGATA13, which were highly expressed in leaves, were found to be correlated with ASA, MK, DXS, CMS, and MECS, with Pearson correlation coefficients of greater than 0.6 for OpGATA12 and OpGATA9. Plant terpenoids are synthesized mainly through the MVA and MEP pathways . Genes that are highly expressed in the leaves are typically involved in the MEP and MVA pathways , demonstrating that OpGATA9, OpGATA12, and OpGATA13 may regulate CPT biosynthesis by participating in the upstream pathway. Among the genes mentioned above, GATA motifs were found in the promoters of key enzyme genes (i.e., SLS, MDC, IPPI, HMGR, HDR, GES, G10H, AACT, and 8-HGO) highly expressed in roots and key enzyme genes (i.e., MK, DXS) highly expressed in leaf, demonstrating that these key enzyme genes may be directly regulated by OpGATA genes and then affect the biosynthesis of CPT. Of which, the genes highly expressed in root (i.e., AACT, IPPI, G10H, HMGR) which is pole-strength correlations with OpGATA14 or OpGATA15 is a higher possibility of being directly regulated.
Overall, this comprehensive analysis of GATA family genes from O. pumila provided insights into the characteristics of OpGATA genes and may improve our understanding of the mechanisms regulating CPT biosynthesis in O. pumila.