Masaru Nakayasu

Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway

Silencing two cytochome P450 genes reduces steroidal glycoalkaloid content and stops tuber sprouting. α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.

A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in Steroidal Glycoalkaloid Biosynthesis

The 2-oxoglutarate-dependent dioxygenase 16DOX catalyzes steroid 16α-hydroxylation in the steroidal glycoalkaloid (SGA) pathway and is a suitable target for controlling toxic SGA levels in potato. Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16β-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Jasmonate-induced biosynthesis of steroidal glycoalkaloids depends on COI1 proteins in tomato

In tomato, perception of jasmonates by a receptor complex, which includes the F-box protein CORONATINE INSENSITIVE 1 (COI1), elicits biosynthesis of defensive steroidal glycoalkaloids (SGAs) via a jasmonate-responsive ERF transcription factor, JRE4/GAME9. Although JRE4 is upregulated by jasmonate and induces the expression of many metabolic genes involved in SGA biosynthesis, it is not known whether JRE4 alone is sufficient for increased SGA biosynthesis upon activation of jasmonate signaling. Here, we show that application of methyl jasmonate induces the expression of JRE4 and SGA biosynthesis genes in leaves and hairy roots of wild-type tomato, but not in jasmonic acid insensitive 1 (jai1), a loss-of-function mutant allele of the tomato COI1 gene. Induced overexpression of JRE4 increased the expression of SGA biosynthesis genes in transgenic hairy roots of both wild-type tomato and the jai1 mutant, suggesting that JRE4 is the primary transcription factor that functions downstream of the jasmonate signaling pathway.

JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.

Novel steroidal saponins from Dioscorea esculenta (Togedokoro)

Fifteen steroidal saponins 1–15, which include 4 furostanol glycosides 1–3 and 15, and 11 spirostanol glycosides 4–14, were isolated from the tubers and leaves of lesser yam (Dioscorea esculenta, Togedokoro). Their structures were identified by nuclear magnetic resonance and liquid chromatography mass spectroscopy. Four steroidal saponins 9, 11, 14, and 15 were found to be novel compounds. Steroidal saponins from Dioscorea esculenta.

Formation of a Methylenedioxy Bridge in (+)-Epipinoresinol by CYP81Q3 Corroborates with Diastereomeric Specialization in Sesame Lignans

Plant specialized metabolites are often found as lineage-specific diastereomeric isomers. For example, Sesamum alatum accumulates the specialized metabolite (+)-2-episesalatin, a furofuran-type lignan with a characteristic diastereomeric configuration rarely found in other Sesamum spp. However, little is known regarding how diastereomeric specificity in lignan biosynthesis is implemented in planta. Here, we show that S. alatum CYP81Q3, a P450 orthologous to S. indicum CYP81Q1, specifically catalyzes methylenedioxy bridge (MDB) formation in (+)-epipinoresinol to produce (+)-pluviatilol. Both (+)-epipinoresinol and (+)-pluviatilol are putative intermediates of (+)-2-episesalatin based on their diastereomeric configurations. On the other hand, CYP81Q3 accepts neither (+)- nor (-)-pinoresinol as a substrate. This diastereomeric selectivity of CYP81Q3 is in clear contrast to that of CYP81Q1, which specifically converts (+)-pinoresinol to (+)-sesamin via (+)-piperitol by the sequential formation of two MDBs but does not accept (+)-epipinoresinol as a substrate. Moreover, (+)-pinoresinol does not interfere with the conversion of (+)-epipinoresinol to (+)-pluviatilol by CYP81Q3. Amino acid substitution and CO difference spectral analyses show that polymorphic residues between CYP81Q1 and CYP81Q3 proximal to their putative substrate pockets are crucial for the functional diversity and stability of these two enzymes. Our data provide clues to understanding how the lineage-specific functional differentiation of respective biosynthetic enzymes substantiates the stereoisomeric diversity of lignan structures.

Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene

Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) α-solanine and α-chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16α-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.

Oxidative rearrangement of (+)-sesamin by CYP92B14 co-generates twin dietary lignans in sesame

Abstract(+)-Sesamin, (+)-sesamolin, and (+)-sesaminol glucosides are phenylpropanoid-derived specialized metabolites called lignans, and are rich in sesame (Sesamum indicum) seed. Despite their renowned anti-oxidative and health-promoting properties, the biosynthesis of (+)-sesamolin and (+)-sesaminol remained largely elusive. Here we show that (+)-sesamolin deficiency in sesame is genetically associated with the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme CYP92B14 that constitutes a novel clade separate from sesamin synthase CYP81Q1. Recombinant CYP92B14 converts (+)-sesamin to (+)-sesamolin and, unexpectedly, (+)-sesaminol through an oxygenation scheme designated as oxidative rearrangement of α-oxy-substituted aryl groups (ORA). Intriguingly, CYP92B14 also generates (+)-sesaminol through direct oxygenation of the aromatic ring. The activity of CYP92B14 is enhanced when co-expressed with CYP81Q1, implying functional coordination of CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that contributes to metabolic diversity in nature.Sesame seeds contain phenylpropanoid-derived lignans that are potentially beneficial to human health. Here, the authors clone a cytochrome P450 enzyme that is responsible for the last steps of sesame lignan biosynthesis and show that it acts through a novel oxidative rearrangement mechanism.