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Identification of Iridoid Glucoside Transporters in Catharanthus roseus.

ABSTRACT: Monoterpenoid indole alkaloids (MIAs) are plant defense compounds and high-value pharmaceuticals. Biosynthesis of the universal MIA precursor, secologanin, is organized between internal phloem-associated parenchyma (IPAP) and epidermis cells. Transporters for intercellular transport of proposed mobile pathway intermediates have remained elusive. Screening of an Arabidopsis thaliana transporter library expressed in Xenopus oocytes identified AtNPF2.9 as a putative iridoid glucoside importer. Eight orthologs were identified in Catharanthus roseus, of which three, CrNPF2.4, CrNPF2.5 and CrNPF2.6, were capable of transporting the iridoid glucosides 7-deoxyloganic acid, loganic acid, loganin and secologanin into oocytes. Based on enzyme expression data and transporter specificity, we propose that several enzymes of the biosynthetic pathway are present in both IPAP and epidermis cells, and that the three transporters are responsible for transporting not only loganic acid, as previously proposed, but multiple intermediates. Identification of the iridoid glucoside-transporting CrNPFs is an important step toward understanding the complex orchestration of the seco-iridioid pathway.

SUBMITTER: Larsen B

PROVIDER: S-EPMC5921532 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Identification of Iridoid Glucoside Transporters in Catharanthus roseus.

Larsen Bo B Fuller Victoria L VL Pollier Jacob J Van Moerkercke Alex A Schweizer Fabian F Payne Richard R Colinas Maite M O'Connor Sarah E SE Goossens Alain A Halkier Barbara A BA

Plant & cell physiology 20170901 9

Monoterpenoid indole alkaloids (MIAs) are plant defense compounds and high-value pharmaceuticals. Biosynthesis of the universal MIA precursor, secologanin, is organized between internal phloem-associated parenchyma (IPAP) and epidermis cells. Transporters for intercellular transport of proposed mobile pathway intermediates have remained elusive. Screening of an Arabidopsis thaliana transporter library expressed in Xenopus oocytes identified AtNPF2.9 as a putative iridoid glucoside importer. Eigh ...[more]

PMID: 28922750

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Project description:Basic helix-loop-helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup-IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10-hydroxylase (G10H), 8-hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7-deoxyloganetic acid glucosyl transferase (7-DLGT), and 7-deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF-associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G-box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G-box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

Dataset Information

Identification of Iridoid Glucoside Transporters in Catharanthus roseus.

Publications

Identification of Iridoid Glucoside Transporters in Catharanthus roseus.

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