Background

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species.

Results

We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.

Conclusions

This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

As the only source for the low-abundance antitumor agents vinblastine and vincristine, Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement. The biosynthesis of these monoterpenoid indole alkaloids (MIAs) is a complex multistep enzymatic network that is tightly regulated by developmental and environmental factors. Here we review the knowledge achieved in the past 30 years of the MIA pathway in C. roseus, from genetic to metabolic aspects. Two early precursor pathways and a late mono-/bis-indole alkaloid pathway have been largely elucidated and established, as well as their intercellular and subcellular compartmentation. Many genes encoding constitutive structural biosynthetic enzymes, transcription factors, and transporters involved in these pathways have been cloned, characterized and applied in metabolic engineering strategies to improve the MIA production. However, genetic modification in the pathway in C. roseus resulted in complicated changes of both secondary and primary metabolism, affecting not only the MIA pathway but also other pathways. Research at metabolic level is required to increase the knowledge on the genetic regulation of the whole metabolic network connected to the MIA biosynthesis. Nuclear magnetic resonance-based metabolomics (metabolic profiling, fingerprinting and flux analyses) in combination with other “omics” have been implemented in studies of C. roseus for pathway elucidation, including among others, understanding stress response, cross talk between pathways, and diversion of carbon fluxes, with the aim to fully unravel the MIA biosynthesis, its regulation and the function of the alkaloids in the plant from a systems biology point of view.

/pdf/monoterpenoid-indole-alkaloids-biosynthesis-and-its-2yvuebhmqb.pdf

Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites

Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229–403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant.

/pdf/fungal-endophytes-of-catharanthus-roseus-enhance-vindoline-3eg53eizn7.pdf

Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis

The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized. Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors. Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.

/pdf/the-orca2-transcription-factor-plays-a-key-role-in-1u78nu4kbh.pdf

The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway.

Sub-group 4 R2R3-type MYB transcription factors, including MYB3, MYB4, MYB7 and MYB32, act as repressors in phenylpropanoid metabolism. These proteins contain the conserved MYB domain and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) repression domain. Additionally, MYB4, MYB7 and MYB32 possess a putative zinc-finger domain and a conserved GY/FDFLGL motif in their C-termini. The protein 'sensitive to ABA and drought 2' (SAD2) recognizes the nuclear pore complex, which then transports the SAD2-MYB4 complex into the nucleus. Here, we show that the conserved GY/FDFLGL motif contributes to the interaction between MYB factors and SAD2. The Asp → Asn mutation in the GY/FDFLGL motif abolishes the interaction between MYB transcription factors and SAD2, and therefore they cannot be transported into the nucleus and cannot repress their target genes. We found that MYB4(D261N) loses the capacity to repress expression of the cinnamate 4-hydroxylase (C4H) gene and biosynthesis of sinapoyl malate. Our results indicate conservation among MYB transcription factors in terms of their interaction with SAD2. Therefore, the Asp → Asn mutation may be used to engineer transcription factors.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.13008

Changing a conserved amino acid in R2R3‐MYB transcription repressors results in cytoplasmic accumulation and abolishes their repressive activity in Arabidopsis

Catharanthus roseus (L.) is a plant species known for its production of many pharmaceutically terpenoid indole alkaloids (TIAs). The intricate networks of TIAs biosynthesis is regulated by all kinds of molecules such as plant signaling molecules (jasmonate acid, ethylene and nitric oxide), plant growth regulators, prenylated proteins and transcription factors. This fine-tuned regulation is also accompanied with compartmentalization and transportation of various alkaloids in different parts of C. roseus. In this paper, we present an analysis of the state of the art related to these molecules regulation mechanism in the TIAs pathway in C. roseus.

https://www.academicjournals.org/article/article1380619178_Zhou et al.pdf

Molecular regulation of terpenoid indole alkaloids pathway in the medicinal plant, Catharanthus roseus

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Production of catharanthine in cell cultures or in hairy roots established by transformation with Agrobacterium rhizogenes is of interest because catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. Here, we observed a high amount of catharanthine in hairy roots of C. roseus, established by infecting leaf explants with the A. rhizogenes >agropine-type A4 strain carrying plasmid pRi. T-DNA transfer from plasmid pRi into hairy roots was confirmed by PCR for the essential T-DNA genes rolA and rolB and the agropine synthesis gene ags. The results suggest that integration of T-DNA into the plant DNA plays a positive role on the catharanthine pathway in C. roseus hairy roots. Furthermore, co-transformation with the soybean transcription factor GmMYBZ2 indicated that GmMYBZ2 reduces the catharanthine production by alteration of expression of a number of genes linked to the pathway. Transcription levels of the zinc-finger transcription factor 1 gene ZCT1 were high, and the transcription levels of the anthranilate synthase gene ASα, the strictosidine synthase gene STR, and the key transcription factor gene octadecanoid-responsive Catharanthus APETALA2/ethylene response factor were low. In addition, GmMYBZ2 had a negative effect on the gene expression levels of A-type cyclin CYSA and B-type cyclin CYSB, which was correlated with a reduced growth rate of the hairy roots.

Soybean transcription factor GmMYBZ2 represses catharanthine biosynthesis in hairy roots of Catharanthus roseus.

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Molecular regulation of terpenoid indole alkaloids pathway in the medicinal plant, Catharanthus roseus

Soybean transcription factor GmMYBZ2 represses catharanthine biosynthesis in hairy roots of Catharanthus roseus.