In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.

Lithospermum erythrorhizon(red gromwell; zicao) is a medicinal and economically valuable plant belonging to the Boraginaceae family. Roots fromL. erythrorhizonhave been used for centuries based on the antiviral and wound-healing properties produced from the bioactive compound shikonin and its derivatives. More recently, shikonin, its enantiomer alkannin, and several other shikonin/alkannin derivatives have collectively emerged as valuable natural colorants and as novel drug scaffolds. Despite several transcriptomes and proteomes having been generated fromL. erythrorhizon, a reference genome is still unavailable. This has limited investigations into elucidating the shikonin/alkannin pathway and understanding its evolutionary and ecological significance. In this study, we obtained a de novo genome assembly forL. erythrorhizonusing a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies. The resulting genome is ∼367.41 Mb long, with a contig N50 size of 314.31 kb and 27,720 predicted protein-coding genes. Using theL. erythrorhizongenome, we identified several additionalp-hydroxybenzoate:geranyltransferase (PGT) homologs and provide insight into their evolutionary history. Phylogenetic analysis of prenyltransferases suggests that PGTs originated in a common ancestor of modern shikonin/alkannin-producing Boraginaceous species, likely from a retrotransposition-derived duplication event of an ancestral prenyltransferase gene. Furthermore, knocking down expression ofLePGT1inL. erythrorhizonhairy root lines revealed that LePGT1 is predominantly responsible for shikonin production early in culture establishment. Taken together, the reference genome reported in this study and the provided analysis on the evolutionary origin of shikonin/alkannin biosynthesis will guide elucidation of the remainder of the pathway.

Several members of the Juglandaceae family produce juglone, a specialized 1,4-naphthoquinone (1,4-NQ) natural product that is responsible for the notorious allelopathic effects of black walnut (Juglans nigra). Despite its documented ecological roles and potential for being developed as a novel natural product-based herbicide, none of the genes involved in synthesizing juglone have been identified. Based on classical labeling studies, we hypothesized that biosynthesis of juglone’s naphthalenoid moiety is shared with biochemical steps of the phylloquinone pathway. Here, using comparative transcriptomics in combination with targeted metabolic profiling of 1,4-NQs in various black walnut organs, we provide evidence that phylloquinone pathway genes involved in 1,4-dihydroxynaphthoic acid (DHNA) formation are expressed in roots for synthesis of a compound other than phylloquinone. Feeding experiments using axenic black walnut root cultures revealed that stable isotopically labeled l-glutamate incorporates into juglone resulting in the same mass shift as that expected for labeling of the quinone ring in phylloquinone. Taken together, these results indicate that in planta, an intermediate from the phylloquinone pathway provides the naphthalenoid moiety of juglone. Moreover, this work shows that juglone can be de novo synthesized in roots without the contribution of immediate precursors translocated from aerial tissues. The present study illuminates all genes involved in synthesizing the juglone naphthoquinone ring and provides RNA-sequencing datasets that can be used with functional screening studies to elucidate the remaining juglone pathway genes. Translation of the generated knowledge is expected to inform future metabolic engineering strategies for harnessing juglone as a novel natural product-based herbicide.

Nitrogen nutrient can trigger changes in chromatin modifications, thus affecting RNA processing, metabolism, and growth in plants.

Summary: Peroxisomal β‐oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl‐coenzyme A (BA‐CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating β‐oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl‐CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA‐CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA‐CoA precursor in the cytoplasm, suggesting that acyl‐CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl‐CoA/CoA levels needed to carry out β‐oxidation, (ii) efflux of β‐oxidative products, acyl‐CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal β‐oxidative metabolism.

Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature. We developed a feeding strategy of excised Arabidopsis stems with 13C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, 13C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers. Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates.