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Characterization of steroid 5?-reductase involved in ?-tomatine biosynthesis in tomatoes.

ABSTRACT: ?-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of ?-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5? reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3?HSD is involved in the three reactions except for C5? reduction, and in the present study, we aimed to elucidate the gene responsible for the C5? reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5?R1 and SlS5?R2, which show high homology with DET2, a brassinosteroid 5? reductase of Arabidopsis thaliana. The expression pattern of SlS5?R2 is similar to those of SGA biosynthetic genes, while SlS5?R1 is ubiquitously expressed, suggesting the involvement of SlS5?R2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5?R1 and SlS5?R2 catalyze the reduction of tomatid-4-en-3-one at C5? to yield tomatid-3-one. Then, SlS5?R1- or SlS5?R2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5?R2-knockout hairy roots, the ?-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of ?-tomatine was observed in the SlS5?R1-knockout hairy root. These results indicate that SlS5?R2 is responsible for the C5? reduction in ?-tomatine biosynthesis and that SlS5?R1 does not significantly contribute to ?-tomatine biosynthesis.

SUBMITTER: Akiyama R

PROVIDER: S-EPMC6978498 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes.

Akiyama Ryota R Lee Hyoung Jae HJ Nakayasu Masaru M Osakabe Keishi K Osakabe Yuriko Y Umemoto Naoyuki N Saito Kazuki K Muranaka Toshiya T Sugimoto Yukihiro Y Mizutani Masaharu M

Plant biotechnology (Tokyo, Japan) 20191201 4

α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (<i>Solanum lycopersicum</i>) and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our ...[more]

PMID: 31983879

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Project description:Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Dataset Information

Characterization of steroid 5?-reductase involved in ?-tomatine biosynthesis in tomatoes.

Publications

Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes.

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