An ABCG-Type Transporter Facilitates ABA Influx and Regulates Camptothecin Biosynthesis in Camptotheca acuminata

Abstract

Camptothecin (CPT) and its derivatives from Camptotheca acuminata have antitumor effects as a DNA topoisomerase I inhibitor. Previous studies have shown that application of exogenous abscisic acid (ABA) significantly promoted the accumulation level of CPT and induced the expression of CPT biosynthetic genes, which revealed that ABA signaling is effectively involved in regulating CPT biosynthesis in C. acuminata. In this study, an ABA transporter, CaABAT, which encodes a plasma membrane protein belonging to the ABCG subfamily, was identified in C. acuminata, and its ABA import activity was confirmed by transport assay in yeast cells. Real-time PCR analysis showed that CaABAT was predominately expressed in C. acuminata leaves and its expression could be significantly upregulated by exogenous ABA treatment. Silencing of CaABAT down-regulated the expression of ABA response genes, which indicated that translocation of ABA by CaABAT should initiate changes in plant physiological status in response to ABA signaling, thus leading to decreased expression of CPT biosynthesis pathway genes and low accumulation levels of CPT in C. acuminata.

Keywords: ABA transporter; Camptotheca acuminata; camptothecin biosynthesis; transport.

Figure 1

CaABAT is a member of the ABC transporter family. (A) Alignment of amino acid sequences of CaABAT with AtABCG40. ABC transporter domain (CaABAT 81–143 aa), ATP binding cassette transporter PDR-like subfamily G domain 1 (CaABAT 152–425 aa) and domain 2 (857–1112 aa), ABC-2 type transporter (504–716 aa, 1185–1399 aa), ABC transporter associated (CaABAT 721–784 aa), ATPase domain (CaABAT 177–409 aa, 897–1089 aa). (B) Phylogenetic relationships of CaABAT with other ABA transporters by MEGA6. AtABCG31: Q7PC88.1; AtABCG30: Q8GZ52.2; GsABCG11: KHN26506; MnABCG11: XP_010094142; AtABCG11: Q8RXN0.1; AtABCG40: Q9M9E1.1; AtABCG25: Q84TH5.1; AtNTL1: Q8H157.1; AtDTX50: Q9FJ87.1; AtABCG22: Q93YS4.1; AhATL11: AQW44869; CaABAT: AUB45106; OsABCG40: Q8GU85. (C) Transmembrane structure of CaABAT was analyzed using TMHMM 2.0 (online at http://www.cbs.dtu.dk/services/, accessed on 2 November 2017). CaABAT protein contains a total of six transmembrane structures. Transmembrane helical structures are represented by amino acid residues 520–542, 555–577, 605–627, 640–659, 664–686, 747–769, 1204–1223, 1236–1253, 1284–1306, 1319–1341, 1351–1370, 1377–1399, and 1428–1450 of the CaABAT protein. Consequently, residues 543–554, 628–639, 687–746, 1224–1235, 1307–1318, 1371–1376, and 1451–1487 are located inside the bilayer (i.e., are intracellular). Amino acid residues 1–519, 578–604, 660–663, 770–1203, 1254–1283, 1342–1350, and 1400–1427 are outside the bilayer.

Figure 2

CaABAT is an ABA influx transporter. (A) The growth of CaABAT yeast transformants in a medium containing ABA. 1 to 4 μL of yeast cultures (OD₆₀₀ = 0.1) were spotted on a 1/2 SG/-URA medium plate for 24 h at 28 °C. (B) ABA import activity of CaABAT. Yeast cells transformed with pDRGAL and pDRGAL-CaABAT were suspended in 1/2 SG/-URA medium supplemented with 100 µM ABA. The error bars represent standard deviations from three biological replicates, and asterisks indicate statistically significant differences compared with pDRGAL. * p < 0.05.

Figure 3

Expression analysis and subcellular localization of CaABAT. (A) Tissue-specific expression analysis of CaABAT in C. acuminata. (B) Tissue-specific expression analysis of CaRD29B in C. acuminata. (C) Subcellular localization of CaABAT in onion epidermal cells. The error bars represent standard deviations from three biological replicates, and asterisks indicate statistically significant differences compared with pDRGAL. ** p < 0.01.

Figure 4

Gene expression and CPT accumulation analysis in response to exogenous ABA treatment in C. acuminata leaves. (A) Relative expression levels of CaABAT and CaRD29B in response to ABA treatments. (B) Accumulation levels of CPT in response to ABA treatments. (C) Relative expression levels of CPT biosynthesis genes in response to ABA treatments. The error bars represent standard deviations from three biological replicates, and asterisks indicate statistically significant differences compared with 0 h. * p < 0.05, ** p < 0.01. (D) Proposed CPT biosynthesis pathway in C. acuminata [22]. G8O, geraniol-8-oxidase; CYC1, Cyclase 1; 7DLS, 7-deoxyloganetic acid synthase; 7-DLGT, 7-deoxyloganetic acid glucosyltransferase; 7-DLH, 7-deoxyloganic acid hydroxylase; SLAS, secologanic acid synthase; AS, anthranilic acid synthetase; TDC, tryptophan decarboxylase; STR, strictosidinic acid synthase; DH, dehydration; RD, reduction.

Figure 5

Virus-induced gene silencing (VIGS) of CaABAT in C. acuminata leaves. (A) Relative expression levels of CaABAT and CaRD29B in pTRV2 control and CaABAT-silenced leaves. (B) CPT accumulation levels in pTRV2 control and CaABAT-silenced leaves. (C) Relative expression levels of CPT biosynthesis genes in pTRV2 control and CaABAT-silenced leaves. The error bars represent standard deviations from three biological replicates, and asterisks indicate statistically significant differences compared with pTRV2 control. * p < 0.05, ** p < 0.01.