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Plants produce an array of metabolites (including lignin monomers and soluble UV-protective metabolites) from phenylalanine through the phenylpropanoid biosynthetic pathway. A subset of plants, including many related to Arabidopsis thaliana, synthesizes glucosinolates, nitrogen- and sulfur-containing secondary metabolites that serve as components of a plant defense system that deters herbivores and pathogens. Here, we report that the Arabidopsis thaliana reduced epidermal fluorescence5 (ref5-1) mutant, identified in a screen for plants with defects in soluble phenylpropanoid accumulation, has a missense mutation in CYP83B1 and displays defects in glucosinolate biosynthesis and in phenylpropanoid accumulation. CYP79B2 and CYP79B3 are responsible for the production of the CYP83B1 substrate indole-3-acetaldoxime (IAOx), and we found that the phenylpropanoid content of cyp79b2 cyp79b3 and ref5-1 cyp79b2 cyp79b3 plants is increased compared with the wild type. These data suggest that levels of IAOx or a subsequent metabolite negatively influence phenylpropanoid accumulation in ref5 and more importantly that this crosstalk is relevant in the wild type. Additional biochemical and genetic evidence indicates that this inhibition impacts the early steps of the phenylpropanoid biosynthetic pathway and restoration of phenylpropanoid accumulation in a ref5-1 med5a/b triple mutant suggests that the function of the Mediator complex is required for the crosstalk.

The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana, disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 (ref4-3) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex.

The Mediator complex is a central component of transcriptional regulation in Eukaryotes. The complex is structurally divided into four modules known as the head, middle, tail and kinase modules, and in Arabidopsis thaliana, comprises 28-34 subunits. Here, we explore the functions of four Arabidopsis Mediator tail subunits, MED2, MED5a/b, MED16, and MED23, by comparing the impact of mutations in each on the Arabidopsis transcriptome. We find that these subunits affect both unique and overlapping sets of genes, providing insight into the functional and structural relationships between them. The mutants primarily exhibit changes in the expression of genes related to biotic and abiotic stress. We find evidence for a tissue specific role for MED23, as well as in the production of alternative transcripts. Together, our data help disentangle the individual contributions of these MED subunits to global gene expression and suggest new avenues for future research into their functions.

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Plants produce an array of metabolites (including lignin monomers and soluble UV-protective metabolites) from phenylalanine through the phenylpropanoid biosynthetic pathway. A subset of plants, including many related to Arabidopsis thaliana, synthesizes glucosinolates, nitrogen- and sulfur-containing secondary metabolites that serve as components of a plant defense system that deters herbivores and pathogens. Here, we report that the Arabidopsis thaliana reduced epidermal fluorescence5 (ref5-1) mutant, identified in a screen for plants with defects in soluble phenylpropanoid accumulation, has a missense mutation in CYP83B1 and displays defects in glucosinolate biosynthesis and in phenylpropanoid accumulation. CYP79B2 and CYP79B3 are responsible for the production of the CYP83B1 substrate indole-3-acetaldoxime (IAOx), and we found that the phenylpropanoid content of cyp79b2 cyp79b3 and ref5-1 cyp79b2 cyp79b3 plants is increased compared with the wild type. These data suggest that levels of IAOx or a subsequent metabolite negatively influence phenylpropanoid accumulation in ref5 and more importantly that this crosstalk is relevant in the wild type. Additional biochemical and genetic evidence indicates that this inhibition impacts the early steps of the phenylpropanoid biosynthetic pathway and restoration of phenylpropanoid accumulation in a ref5-1 med5a/b triple mutant suggests that the function of the Mediator complex is required for the crosstalk.

The Mediator complex is a central component of transcriptional regulation in Eukaryotes. The complex is structurally divided into four modules known as the head, middle, tail and kinase modules, and in Arabidopsis thaliana, comprises 28-34 subunits. Here, we explore the functions of four Arabidopsis Mediator tail subunits, MED2, MED5a/b, MED16, and MED23, by comparing the impact of mutations in each on the Arabidopsis transcriptome. We find that these subunits affect both unique and overlapping sets of genes, providing insight into the functional and structural relationships between them. The mutants primarily exhibit changes in the expression of genes related to biotic and abiotic stress. We find evidence for a tissue specific role for MED23, as well as in the production of alternative transcripts. Together, our data help disentangle the individual contributions of these MED subunits to global gene expression and suggest new avenues for future research into their functions.

The Mediator complex is a large, multi-subunit, transcription co-regulator that is conserved across eukaryotes. Studies of the Arabidopsis Mediator complex and its subunits have shown that it functions in nearly every aspect of plant development and fitness. In addition to revealing mechanisms of regulation of plant-specific pathways, studies of plant Mediator complexes have the potential to shed light on the conservation and divergence of Mediator structure and function across Kingdoms and plant lineages. So far, these studies indicate that, despite low sequence similarity between many orthologous subunits, the overall structure and function of Mediator is well conserved between Kingdoms. Several studies have also expanded our knowledge of Mediator to other plant species, opening avenues of investigation into the role of Mediator in plant adaptation and fitness. We summarize these insights to date in Chapter 1. The phenylpropanoid pathway is a major global carbon sink and its regulation is important not only for plant fitness but for the rational engineering of chemical and bioenergy feedstocks. The Arabidopsis Mediator complex subunits MED5a and MED5b are required for phenylpropanoid homeostasis and disruption of both paralogs results in an increase in phenylpropanoid accumulation. In contrast, the semi-dominant MED5b mutant reduced epidermal fluorescence4-3 (ref4-3) is dwarf and has constitutively repressed phenylpropanoid biosynthesis. In Chapter 2, we present the results of a forward genetic screen for suppressors of ref4-3. Whole-genome sequencing of the suppressors revealed that MED2, MED16, MED23, and particular residues in MED5b, are required for the phenotypes associated with ref4-3. Conversely, disruption of MED3 or MED25 has no discernable effect on ref4-3, indicating that the Mediator subunit interactions identified in our screen are specific. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression and that these changes are largely reversed by the suppressors. Our data also show that ref4-3 plants are upregulated in the expression of negative regulators of phenylpropanoid biosynthesis and identifies other pathways that may impinge on plant growth and phenylpropanoid metabolism. Together, our data highlight the functional interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex. In Chapter 3, we explore the functions of MED2, MED5a/b, MED16, and MED23 beyond the phenylpropanoid pathway by comparing the impact of mutations in each on the Arabidopsis transcriptome. We find that these subunits have both overlapping and unique roles in gene expression, leading to the identification of several interesting functional relationships. We also show that, under our growth conditions, the mutants primarily affect the expression of genes in pathways related to biotic and abiotic stress. We also present evidence for a tissue specific role for MED23, as well as evidence for a role for MED23 in the production of alternative transcripts. Together, our data help disentangle the individual contributions of these MED subunits to global gene expression and suggest new avenues for future research into their functions.

In 2000, a large international outbreak of meningococcal disease caused by Neisseria meningitidis serogroup W-135 was identified among pilgrims returning from the Hajj in Saudi Arabia. To assess ongoing risk, we evaluated N. meningitidis carriage among US travelers to the 2001 Hajj. Of 25 N. meningitidis isolates obtained, 15 (60%) were nongroupable and 8 (32%) were serogroup W-135 when tested by standard slide-agglutination techniques. Two additional nongroupable isolates were characterized as serogroup W-135 when tested by polymerase chain reaction. Nine of 10 serogroup W-135 isolates were indistinguishable from the Hajj-2000 clone. None of the departing, but 9 (1.3%) of the returning, pilgrims carried serogroup W-135 (P = .01); all carriers reported previous vaccination. Carriage of N. meningitidis serogroupW-135 increased significantly in pilgrims returning from the Hajj. Although the risk of disease to pilgrims appears to be low, the risk of spread to others of this pathogenic strain remains a concern.

The development and usage of two companion NIH‐funded genetic testing information databases, GeneTests (www.genetests.org) and GeneClinics (www.geneclinics.org), now merged into one web site, reflect the steadily increasing use of genetic testing and the expanding audience for genetic testing information. Established in 1993 as Helix, a genetics laboratory directory of approximately 110 listings, GeneTests has grown into a database of over 900 tests for inherited diseases, a directory of over 500 international laboratories, a directory of over 1,000 U.S. and international genetics clinics, and a resource for educational/teaching materials and reports of summary genetic test data. GeneClinics, founded in 1997 as an expert‐authored, peer‐reviewed, disease‐specific knowledge base relating genetic testing to patient care, has grown steadily, now containing over 130 expert‐authored, peer‐reviewed full‐text entries relating genetic testing information to diagnosis, management, and genetic counseling of specific inherited diseases. In spring 2001 the two databases were merged and in October 2001 the two web sites were merged for the purpose of seamless navigation into the GeneTests‐GeneClinics site (www.genetests.org or www.geneclinics.org); the GeneClinics knowledge base was renamed “GeneReviews” to avoid confusion with the U.S. and international clinic directories. As genetic testing has moved steadily out of research venues and into routine medical practice, the user audience for these databases has become international and expansive and includes healthcare providers, patients, educators, policy makers, and the media. The use of these combined resources has grown to approximately 3,200 visits/day. Hum Mutat 19:501–509, 2002. © 2002 Wiley‐Liss, Inc.