Identification of tomato F-box proteins functioning in phenylpropanoid metabolism.

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins.

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.

3-O-glycosylation of kaempferol restricts the supply of the benzenoid precursor of ubiquinone (Coenzyme Q) in Arabidopsis thaliana.

Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and antioxidant in eukaryotes. The recent discovery that kaempferol serves as a precursor for ubiquinone's benzenoid moiety both challenges the conventional view of flavonoids as specialized metabolites, and offers new prospects for engineering ubiquinone in plants. Here, we present evidence that Arabidopsis thaliana mutants lacking kaempferol 3-O-rhamnosyltransferase (ugt78d1) and kaempferol 3-O-glucosyltransferase (ugt78d2) activities display increased de novo biosynthesis of ubiquinone and increased ubiquinone content. These data are congruent with the proposed model that unprotected C-3 hydroxyl of kaempferol triggers the oxidative release of its B-ring as 4-hydroxybenzoate, which in turn is incorporated into ubiquinone. Ubiquinone content in the ugt78d1/ugt78d2 double knockout represented 160% of wild-type level, matching that achieved via exogenous feeding of 4-hydroxybenzoate to wild-type plants. This suggests that 4-hydroxybenzoate is no longer limiting ubiquinone biosynthesis in the ugt78d1/ugt78d2 plants. Evidence is also shown that the glucosylation of 4-hydroxybenzoate as well as the conversion of the immediate precursor of kaempferol, dihydrokaempferol, into dihydroquercetin do not compete with ubiquinone biosynthesis in A. thaliana.

Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear.

Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to 104 cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The Bühlmann-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.