Graduate Student, University of Wisconsin Madison
I study how tyrosine is produced and how it is regulated for the production of metabolites in plants
Tyrosine (Tyr) is an essential amino acid necessary for protein synthesis in all organisms and also involved in specialized (secondary) metabolites. Tyrosine is known to be produced by two pathways in diverse organisms: by the prephenate dehydrogenase (PDH) enzyme found in most microbes and by the arogenate dehydrogenase (ADH) enzyme found in most plants like Arabidopsis. PDH and ADH enzymes are usually subjected to feedback inhibition by the pathway product, Tyr. However, how the regulation of Tyr biosynhesis contribute to the production of downstream Tyr-derived specialized metabolites is poorly understood. Here we investigated the Tyr biosynthetic pathway and its regulation in the table beet (Beta vulgaris L., order Caryophyllales), which produces high levels of the Tyr-derived pigment, betalain. We found that B. vulgaris have two arogenate dehydrogenases (ADH1 and ADH2) and that ADH2 has relaxed sensitivity to Tyr feedback inhibition. Biochemical and phylogenetic analysis of ADH sequences from various Caryophyllales species further revealed that the ADH duplication and later a neofunctionalization of ADH2 orthologs occurred before the emergence of betalain pigmentation. A sequence comparison of closely-related ADHs but having distinct Tyr sensitivity, together with protein structure model identified several residues potentially involved in Tyr sensitivity. Site-directed mutagenesis further identified three residues that significantly affect ADH Tyr regulation and thus played a key role in the ADH2 neofunctionalization and betalain pigmentation in Caryophyllales. This new finding can be used to modify other ADH enzymes to redirect the primary carbon flux toward Tyr for enhanced production of Tyr-derived natural products in planta.
Abstract: Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.
Pub.: 20 Feb '16, Pinned: 28 Jun '17
Abstract: Flavonoids are secondary metabolites derived from the general phenylpropanoid pathway and are widespread throughout the plant kingdom. The functions of flavonoids are diverse, including defense against phytopathogens, protection against UV light damage and oxidative stress, regulation of auxin transport and allelopathy. One of the most conspicuous functions of flavonoids has long attracted the attention of pollinators and scientist alike: the vivid shades of red, pink, orange, blue and purple on display in the flowers of angiosperms. Thus, flavonoid pigments have perhaps been the most intensely studied phenylpropanoids. From Mendel to McClintock and up to the present, studies centered on flavonoid pigments have resulted in some of the most important scientific discoveries of the last 150 years, including the first examples of transcriptional regulation in plants. Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model for the regulation of the flavonoid pigment pathway. We will survey the history of the MBW model spanning the last three decades, highlighting the major findings that have contributed to our current understanding. In particular, recent discoveries regarding WRKY protein control of the flavonoid pigment pathway and its relationship to the MBW complex will be emphasized. In addition, we will discuss recent findings about the regulation of the beet betalain pigment pathway, and how a MYB member of the MBW complex was co-opted to regulate this chemically unrelated but functionally equivalent pathway.
Pub.: 03 Jun '17, Pinned: 28 Jun '17
Abstract: Caryophyllales are highly diverse in the structure of the perianth and androecium and show a mode of floral development unique in eudicots, reflecting the continuous interplay of gynoecium and perianth and their influence on position, number, and identity of the androecial whorls. The floral development of five species from four genera of a paraphyletic Molluginaceae (Limeum, Hypertelis, Glinus, Corbichonia), representing three distinct evolutionary lineages, was investigated to interpret the evolution of the androecium across Caryophyllales. •Floral buds were dissected, critical-point dried and imaged with SEM. The genera studied are good representatives of the diversity of development of stamens and staminodial petaloids in Caryophyllales. •Sepals show evidence of petaloid differentiation via marginal hyaline expansion. Corbichonia, Glinus, and Limeum also show perianth differentiation via sterilization of outer stamen tiers. In all four genera, stamens initiate with the carpels and develop centrifugally, but subsequently variation is significant. With the exception of Limeum, the upper whorl is complete and alternisepalous, while a second antesepalous whorl arises more or less sequentially, starting opposite the inner sepals. Loss or sterilization of antesepalous stamens occurs in Glinus and Limeum and is caused by altered carpel merism and inhibition by sepal pressures. •Outer stamens of Hypertelis correspond with petaloids of Caryophyllaceae and suggest that staminodial petaloids and outer alternisepalous stamens are interchangeable in the Caryophyllales. We emphasize a switch in the position of first formed stamens from antesepalous to alternisepalous following the divergence of Limeum; thus stamen position is an important synapomorphy for the globular inclusion clade.
Pub.: 07 Sep '13, Pinned: 28 Jun '17