Altered methionine metabolism impacts phenylpropanoid production and plant development in Arabidopsis thaliana.

Phenylpropanoids are specialized metabolites derived from phenylalanine. Glucosinolates are defense compounds derived mainly from methionine and tryptophan in Arabidopsis. It was previously shown that the phenylpropanoid pathway and glucosinolate production are metabolically linked. The accumulation of indole-3-acetaldoxime (IAOx), the precursor of tryptophan-derived glucosinolates, represses phenylpropanoid biosynthesis through accelerated degradation of phenylalanine-ammonia lyase (PAL). As PAL functions at the entry point of the phenylpropanoid pathway which produces indispensable specialized metabolites such as lignin, aldoxime-mediated phenylpropanoid repression is detrimental to plant survival. Although methionine-derived glucosinolates in Arabidopsis are abundant, any impact of aliphatic aldoximes (AAOx) derived from aliphatic amino acids such as methionine on phenylpropanoid production remains unclear. Here, we investigate the impact of AAOx accumulation on phenylpropanoid production using Arabidopsis aldoxime mutants, ref2 and ref5 . REF2 and REF5 metabolize aldoximes to respective nitrile oxides redundantly, but with different substrate specificities. ref2 and ref5 mutants have decreased phenylpropanoid contents due to the accumulation of aldoximes. As REF2 and REF5 have high substrate specificity toward AAOx and IAOx respectively, it was assumed that ref2 accumulates AAOx, not IAOx. Our study indicates that ref2 accumulates both AAOx and IAOx. Removing IAOx partially restored phenylpropanoid production in ref2 , but not to the wild-type level. However, when AAOx biosynthesis was silenced, phenylpropanoid production and PAL activity in ref2 were completely restored, suggesting an inhibitory effect of AAOx on phenylpropanoid production. Further feeding studies revealed that the abnormal growth phenotype commonly observed in Arabidopsis mutants lacking AAOx production is a consequence of methionine accumulation. Significance Statement: Aliphatic aldoximes are precursors of various specialized metabolites including defense compounds. This study reveals that aliphatic aldoximes repress phenylpropanoid production and that altered methionine metabolism affects plant growth and development. As phenylpropanoids include vital metabolites such as lignin, a major sink of fixed carbon, this metabolic link may contribute to available resource allocation during defense.

Altered methionine metabolism impacts phenylpropanoid production and plant development in Arabidopsis thaliana.

Phenylpropanoids are specialized metabolites derived from phenylalanine. Glucosinolates are defense compounds derived mainly from methionine and tryptophan in Arabidopsis. It was previously shown that the phenylpropanoid pathway and glucosinolate production are metabolically linked. The accumulation of indole-3-acetaldoxime (IAOx), the precursor of tryptophan-derived glucosinolates, represses phenylpropanoid biosynthesis through accelerated degradation of phenylalanine ammonia lyase (PAL). As PAL functions at the entry point of the phenylpropanoid pathway, which produces indispensable specialized metabolites such as lignin, aldoxime-mediated phenylpropanoid repression is detrimental to plant survival. Although methionine-derived glucosinolates in Arabidopsis are abundant, any impact of aliphatic aldoximes (AAOx) derived from aliphatic amino acids such as methionine on phenylpropanoid production remains unclear. Here, we investigate the impact of AAOx accumulation on phenylpropanoid production using Arabidopsis aldoxime mutants, ref2 and ref5. REF2 and REF5 metabolize aldoximes to respective nitrile oxides redundantly, but with different substrate specificities. ref2 and ref5 mutants have decreased phenylpropanoid contents due to the accumulation of aldoximes. As REF2 and REF5 have high substrate specificity toward AAOx and IAOx, respectively, it was assumed that ref2 accumulates AAOx, not IAOx. Our study indicates that ref2 accumulates both AAOx and IAOx. Removing IAOx partially restored phenylpropanoid content in ref2, but not to the wild-type level. However, when AAOx biosynthesis was silenced, phenylpropanoid production and PAL activity in ref2 were completely restored, suggesting an inhibitory effect of AAOx on phenylpropanoid production. Further feeding studies revealed that the abnormal growth phenotype commonly observed in Arabidopsis mutants lacking AAOx production is a consequence of methionine accumulation.

Metabolic link between auxin production and specialized metabolites in Sorghum bicolor.

Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Except for SbCYP79A1, aldoxime biosynthetic enzymes in the cereal sorghum (Sorghum bicolor) have not been characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine but, unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses.

Diastereoselective Indole-Dearomative Cope Rearrangements by Compounding Minor Driving Forces.

Reported herein is the discovery of a diastereoselective indole-dearomative Cope rearrangement. A suite of minor driving forces promote dearomatization: (i) steric congestion in the starting material, (ii) alkylidene malononitrile and stilbene conjugation events in the product, and (iii) an unexpected intramolecular π-π* stack on the product side of the equilibrium. The key substrates are rapidly assembled from simple starting materials, resulting in many successful examples. The products are structurally complex and bear vicinal stereocenters generated by the dearomative Cope rearrangement. They also contain a variety of functional groups for interconversion to complex architectures.

Aldoximes are precursors of auxins in Arabidopsis and maize.

Two natural auxins, phenylacetic acid (PAA) and indole-3-acetic acid (IAA), play crucial roles in plant growth and development. One route of IAA biosynthesis uses the glucosinolate intermediate indole-3-acetaldoxime (IAOx) as a precursor, which is thought to occur only in glucosinolate-producing plants in Brassicales. A recent study showed that overproducing phenylacetaldoxime (PAOx) in Arabidopsis increases PAA production. However, it remains unknown whether this increased PAA resulted from hydrolysis of PAOx-derived benzyl glucosinolate or, like IAOx-derived IAA, is directly converted from PAOx. If glucosinolate hydrolysis is not required, aldoxime-derived auxin biosynthesis may occur beyond Brassicales. To better understand aldoxime-derived auxin biosynthesis, we conducted an isotope-labelled aldoxime feeding assay using an Arabidopsis glucosinolate-deficient mutant sur1 and maize, and transcriptomics analysis. Our study demonstrated that the conversion of PAOx to PAA does not require glucosinolates in Arabidopsis. Furthermore, maize produces PAA and IAA from PAOx and IAOx, respectively, indicating that aldoxime-derived auxin biosynthesis also occurs in maize. Considering that aldoxime production occurs widely in the plant kingdom, aldoxime-derived auxin biosynthesis is likely to be more widespread than originally believed. A genome-wide transcriptomics study using PAOx-overproduction plants identified complex metabolic networks among IAA, PAA, phenylpropanoid and tryptophan metabolism.

Aromatic Cope rearrangements.

Reviewed herein is the aromatic Cope rearrangement, a Cope rearrangement where one (or both) of the alkenes of the 1,5-diene are part of a greater aromatic system. While the Cope rearrangement of 1,5-dienes has seen wide utility, variation, and application in chemical synthesis, the aromatic Cope rearrangement, comparatively, has not. This review summarizes the ∼40 papers dating back to 1956 on this topic and is divided into the following sections: (1) introduction, including kinetic and thermodynamic challenges of the aromatic Cope rearrangement, and (2) key substrate features, of which there are four general types: (i) α-allyl-α-aryl malonates (and related substrates), (ii) 1-aryl-2-vinylcyclopropanes, and (iii) anion-accelerated aromatic oxy-Cope substrates, and (iv) the concept of synchronized aromaticity. Ultimately, we hope this review will draw attention to a potentially valuable transformation for arene functionalization that warrants further studies and development.

Overcoming Kinetic and Thermodynamic Challenges of Classic Cope Rearrangements.

Systematic evaluation of 1,5-dienes bearing 3,3-electron-withdrawing groups and 4-methylation results in the discovery of a Cope rearrangement for Meldrum's acid-containing substrates that have unexpectedly favorable kinetic and thermodynamic profiles. The protocol is quite general due to a concise and convergent synthesis from abundant starting materials. Furthermore, products with an embedded Meldrum's acid moiety are prepared, which, in turn, can yield complex amides under neutral conditions. We have now expanded the scope of the reductive Cope rearrangement, which, via chemoselective reduction, can promote thermodynamically unfavorable [3,3] sigmatropic rearrangements of 3,3-dicyano-1,5-dienes to form reduced Cope rearrangement products. The Cope rearrangement is found to be stereospecific and can yield enantioenriched building blocks when chiral, nonracemic 1,3-disubstituted allylic electrophiles are utilized. We expand further the use of Cope rearrangements for the synthesis of highly valuable building blocks for complex- and drug-like molecular synthesis.