Diverse natural products are synthesized in plants by specialized metabolic enzymes, which are often lineage-specific and derived from gene duplication followed by functional divergence. However, ...little is known about the contribution of primary metabolism to the evolution of specialized metabolic pathways.
Betalain pigments, uniquely found in the plant order Caryophyllales, are synthesized from the aromatic amino acid L-tyrosine (Tyr) and replaced the otherwise ubiquitous phenylalanine-derived anthocyanins. This study combined biochemical, molecular and phylogenetic analyses, and uncovered coordinated evolution of Tyr and betalain biosynthetic pathways in Caryophyllales.
We found that Beta vulgaris, which produces high concentrations of betalains, synthesizes Tyr via plastidic arogenate dehydrogenases (TyrAa/ADH) encoded by two ADH genes (BvADHα and BvADHβ). Unlike BvADHβ and other plant ADHs that are strongly inhibited by Tyr, BvADHα exhibited relaxed sensitivity to Tyr. Also, Tyr-insensitive BvADHα orthologs arose during the evolution of betalain pigmentation in the core Caryophyllales and later experienced relaxed selection and gene loss in lineages that reverted from betalain to anthocyanin pigmentation, such as Caryophyllaceae.
These results suggest that relaxation of Tyr pathway regulation increased Tyr production and contributed to the evolution of betalain pigmentation, highlighting the significance of upstream primary metabolic regulation for the diversification of specialized plant metabolism.
Within the angiosperm order Caryophyllales, an unusual class of pigments known as betalains can replace the otherwise ubiquitous anthocyanins. In contrast to the phenylalanine-derived anthocyanins, ...betalains are tyrosine-derived pigments which contain the chromophore betalamic acid. The origin of betalain pigments within Caryophyllales and their mutual exclusion with anthocyanin pigments have been the subject of considerable research. In recent years, numerous discoveries, accelerated by -omic scale data, phylogenetics and synthetic biology, have shed light on the evolution of the betalain biosynthetic pathway in Caryophyllales. These advances include the elucidation of the biosynthetic steps in the betalain pathway, identification of transcriptional regulators of betalain synthesis, resolution of the phylogenetic history of key genes, and insight into a role for modulation of primary metabolism in betalain synthesis. Here we review how molecular genetics have advanced our understanding of the betalain biosynthetic pathway, and discuss the impact of phylogenetics in revealing its evolutionary history. In light of these insights, weexplore our new understanding of the origin of betalains, the mutual exclusion of betalains and anthocyanins, and the homoplastic distribution of betalain pigmentation within Caryophyllales. We conclude with a speculative conceptual model for the stepwise emergence of betalain pigmentation.
• The evolution of L-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that L-DOPA ...4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear.
• To address this, we functionally characterised 23 distinct DODA proteins for L-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of L-DOPA 4,5-dioxygenase activity.
• We find that low L-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated L-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny.
• In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated L-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.
SUMMARY
Cadaverine, a polyamine, has been linked to modification of root growth architecture and response to environmental stresses in plants. However, the molecular mechanisms that govern the ...regulation of root growth by cadaverine are largely unexplored. Here we conducted a forward genetic screen and isolated a mutation, cadaverine hypersensitive 3 (cdh3), which resulted in increased root‐growth sensitivity to cadaverine, but not other polyamines. This mutation affects the BIO3‐BIO1 biotin biosynthesis gene. Exogenous supply of biotin and a pathway intermediate downstream of BIO1, 7,8‐diaminopelargonic acid, suppressed this cadaverine sensitivity phenotype. An in vitro enzyme assay showed cadaverine inhibits the BIO3‐BIO1 activity. Furthermore, cadaverine‐treated seedlings displayed reduced biotinylation of Biotin Carboxyl Carrier Protein 1 of the acetyl‐coenzyme A carboxylase complex involved in de novo fatty acid biosynthesis, resulting in decreased accumulation of triacylglycerides. Taken together, these results revealed an unexpected role of cadaverine in the regulation of biotin biosynthesis, which leads to modulation of primary root growth of plants.
Significance Statement
Cadaverine is a polyamine produced by plants and microbes, which has been shown to accumulate in plants under stress conditions and to inhibit primary root growth. Here, we show that cadaverine affects Arabidopsis root growth primarily by inhibiting the biotin biosynthesis pathway, thereby affecting primary metabolism and the lipid profile.
SUMMARY
l‐Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate ...dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine‐derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny‐guided TyrA structure–function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non‐active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine‐sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny‐guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.
Significance Statement
Tyrosine is a critical amino acid precursor for biosynthesis of numerous plant natural products, including betalain pigments that are uniquely produced in the plant order Caryophyllales. Here, we identified key residues involved in the evolution of deregulated TyrA enzymes that underlie the overaccumulation of tyrosine and betalains in core Caryophyllales and demonstrated that the combined introduction of the identified residue together with one independently evolved in legume TyrAs converted highly‐regulated Arabidopsis TyrA into a tyrosine‐insensitive enzyme.
L-Tyrosine-derived specialized metabolites perform many important functions in plants, and have valuable applications in human health and nutrition. A necessary step in the overproduction of ...specialised tyrosine-derived metabolites in planta is the manipulation of primary metabolism to enhance the availability of tyrosine. Here, we utilise a naturally occurring de-regulated isoform of the key enzyme, arogenate dehydrogenase, to re-engineer the interface of primary and specialised metabolism, to boost the production of tyrosine-derived pigments in a heterologous plant host. Through manipulation of tyrosine availability, we report a 7-fold increase in the production of tyrosine-derived betalain pigments, with an upper range of 855 mg·kg
·FW, which compare favourably to many in vitro and commercial sources of betalain pigments. Since the most common plant pathway for tyrosine synthesis occurs via arogenate, the de-regulated arogenate dehydrogenase isoform is a promising route for enhanced production of tyrosine-derived pharmaceuticals in diverse plant hosts.
Plant and fungi produce different secondary (or specialized) metabolites. Primary metabolites (i.e. amino acids) support the production of secondary metabolites. However, the mechanism through which ...primary metabolism is regulated for the production of specialized metabolites is poorly understood. To study this process, this work examined the biosynthesis of the primary aromatic amino acid tyrosine (Tyr) in relationship to Tyr-derived metabolites in plants and fungi. Here, we found that primary metabolism is closely related to the regulation and evolution of new metabolites in plants. Tyr is synthesized de novo in plants, microbes, and fungi; but not in animals. Hence, animals need to acquire the amino acid through their diet. In addition to being a building block for protein synthesis, Tyr is also an important precursor of vitamin, lignin, alkaloids, and pigments. Plant pigments like anthocyanins and betalains are good examples of specialized metabolites. Anthocyanins are phenylalanine-derived, vacuole-contained pigments found ubiquitously across the plant kingdom. In contrast, betalains are nitrogen-containing Tyr-derived pigments restricted in the plant order Caryophyllales. Both pigments are believed to have similar, if not the same, function in planta. However, the mystery why betalains are restricted to some families is still unknown but a new hypothesis is introduced in this dissertation. Depending on the species, Tyr can be synthesized by arogenate dehydrogenase (ADH/TyrAa) or prephenate dehydrogenase (PDH/TYR1/TyrAp). Many microbes have exclusively PDH while plants have ADH enzymes. In both cases, ADH and PDH are feedback regulated by Tyr inhibition in a competitive manner. Using extensive biochemical and phylogenetic analysis I found that the Tyr pathway, controlled by the ADH, is unique to the Caryophyllales. ADH duplication occurred early in the ancestor of Caryophyllales producing an enzyme with relaxed sensitivity to Tyr inhibition. Moreover, the ADH enzymes (named ADHα and ADHβ) were duplicated before the emergence of betalain pigments creating a new deregulated enzyme (ADHα). Using phylogenetic, biochemical analaysis, and targeted phylometabolomic, we found that Caryophyllales plants with ADHα have betalains and other Tyr-derived compounds in different plants tissues (i.e. dopamine, tyramine, L-DOPA, norepinephrine) (Chapter 2). The mechanism for the deregulated ADHα enzyme is also resolved by using structure-function analysis, site-directed mutageneisis and large numbers of ADH? and ADHβ sequences (Chapter 3). A single residue is shown to be involved in the relaxed regulation of ADH enzyme in the active site but additional residues are also contributing to the inhibition mechanism. Finally, we elucidated the Tyr biosynthetic pathway in the two main groups of fungi (Ascomycota and Basidiomycota), which were poorly understood (Chapter 4). This dissertation work provides that a certain group of organisms modified their primary metabolism for the evolution and likely efficient production of specialized metabolites. The information presented here will help optimizing the regulation of primary metabolism for the improved production of biological active metabolites for medicine and nutrition.
Betalains are tyrosine-derived pigments that consist of red-violet betacyanins and yellow betaxanthins. These pigments are major sources of natural food dyes in the United States. Decades of table ...beet breeding efforts have increased betalain pigmentation, but yellow betaxanthin accumulation has been lower than that of red betacyanins. To identify possible bottlenecks in betalain production, here we conducted comparative analyses of betalains and their precursor, tyrosine, in various beet genotypes. Consistent with previous studies, red beets had much higher betalain concentrations than yellow beets. Conversely, tyrosine levels were higher in yellow than in red genotypes in both table beet and Swiss chard. Interestingly, increased tyrosine levels were positively correlated with elevated betalain accumulation among red but not yellow genotypes, especially at a later developmental stage, suggesting that yellow beets are not efficiently converting tyrosine into betalain pigments. On the basis of these results, we hypothesize that further increase in tyrosine production will likely enhance betacyanin accumulation in red beets, whereas better utilization of the accumulated tyrosine will be required to further improve betaxanthin production in yellow beets.
L-Tyrosine is an aromatic amino acid necessary for protein synthesis in all living organisms and a precursor of secondary (specialized) metabolites. In fungi, tyrosine-derived compounds are ...associated with virulence and defense (i.e. melanin production). However, how tyrosine is produced in fungi is not fully understood. Generally, tyrosine can be synthesized via two pathways: by prephenate dehydrogenase (TyrAp/PDH), a pathway found in most bacteria, or by arogenate dehydrogenase (TyrAa/ADH), a pathway found mainly in plants. Both enzymes require the cofactor NAD+ or NADP+ and typically are strongly feedback inhibited by tyrosine. Here, we biochemically characterized two TyrA enzymes from two distantly related fungi in the Ascomycota and Basidiomycota, Saccharomyces cerevisiae (ScTyrA/TYR1) and Pleurotus ostreatus (PoTyrA), respectively. We found that both enzymes favor the prephenate substrate and NAD+ cofactor in vitro. Interestingly, while PoTyrA was strongly inhibited by tyrosine, ScTyrA exhibited relaxed sensitivity to tyrosine inhibition. We further mutated ScTyrA at the amino acid residue that was previously shown to be involved in the substrate specificity of plant TyrAs; however, no changes in its substrate specificity were observed, suggesting that a different mechanism is involved in the TyrA substrate specificity of fungal TyrAs. The current findings provide foundational knowledge to further understand and engineer tyrosine-derived specialized pathways in fungi.
•A single copy of TyrA gene is predicted to be present in fungi.•Fungal TryA enzymes prefer the prephenate substrate and NAD+ cofactor.•Only the TyrA enzyme of Saccharomyces cerevisiae exhibits relaxed sensitivity to tyrosine inhibition.•Residue(s) that confers TyrA substrate specificity in fungi is different from that in plants and closely related bacteria.
SUMMARYl‐Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate ...dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine‐derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny‐guided TyrA structure–function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non‐active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine‐sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny‐guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.
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