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Genetic and biochemical analysis of serine carboxypeptidase-like proteins in Arabidopsis thaliana
No full textThe Arabidopsis genome encodes a family of 51 proteins that are homologous to known serine carboxypeptidases. Phylogenetic analysis shows that these serine carboxypeptidase-like (SCPL) proteins can be divided into five major clades, with three clades having representatives in plants, animals, and fungi, and two clades having representatives only in plants. RT-PCR analysis demonstrates that the Arabidopsis SCPL genes are diverse in their expression patterns. A subgroup of 21 SCPL proteins includes two that have been shown to function as acyltransferases in plant secondary metabolism: sinapoylglucose: malate sinapoyltransferase (SMT) and sinapoylglucose:choline sinapoyltransferase (SCT). SMT is encoded by one of five SCPL genes arranged in tandem to form a cluster on chromosome 2. Analysis of deletion mutant lines lacking one or more genes in this SCPL gene cluster reveals that three of the genes also encode sinapoylglucose acyltransferases: At2g23000, At2g23010, and At2g22980. At2g23000 encodes sinapoylglucose:anthocyanin sinapoyltransferase (SAT), an enzyme that is required for the synthesis of the sinapoylated anthocyanins in Arabidopsis. At2g23010 encodes an enzyme with sinapoylglucose:sinapoylglucose sinapoyltransferase (SST) activity. SST activity is also exhibited by SMT and the protein encoded by At2g22980. The study of the Arabidopsis SCPL proteins thus provides an opportunity to examine structure-function relationships in enzyme evolution and plant secondary metabolism
Regulation and expression of cinnamate-4 hydroxylase, a cytochrome P450 monooxygenase, in Arabidopsis thaliana
No full textCinnamate-4-hydroxylase is the first cytochrome P450-dependent monooxygenase of the phenylpropanoid pathway. In order to study the expression of this gene in Arabidopsis thaliana, a cinnamate-4-hydroxylase cDNA clone from the Arabidopsis expressed sequence tag database was identified, and was used to isolate its corresponding genomic clone. The entire cinnamate-4-hydroxylase coding sequence plus 2.9 kb of its promoter was isolated on a 5.4 kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of cinnamate-4-hydroxylase was analyzed by RNA blot hybridization analysis, and in transgenic Arabidopsis carrying cinnamate-4-hydroxylase-GUS transcriptional fusions. C4H RNA was present in dark grown seedlings but its level increased after exposure of seedlings to light. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. Although C4H message was found to be light inducible, its expression did not follow a circadian pattern. Cinnamate-4-hydroxylase RNA is widely expressed in various Arabidopsis tissues, particularly in roots and tissues undergoing lignification. The cinnamate-4-hydroxylase promoter-driven GUS expression accurately reflected the tissue specificity and wound-inducibility of the cinnamate-4-hydroxylase promoter indicated by RNA blot hybridization analysis. The analysis of stem cross sections indicates intense staining in lignifying tissues such as the sclerified parenchyma, xylem and hydathodes. In roots, C4H is initially restricted to the vascular tissue and gradually increases in the cortex. Several putative regulatory sequences were found in the C4H promoter that are similar to regulatory sequences found in promoters of other phenylpropanoid genes in Arabidopsis and other species. To analyze the role of putative cis-acting elements in C4H tissue-specificity and environmental-inducibility, eight promoter deletion constructs were generated to drive GUS expression in transgenic Arabidopsis. 5′ deletion of the C4H promoter resulted in a gradual decrease in GUS expression in all organs and resulted in a decrease in the apparent wound inducibility of the promoter in mature leaves
Genetic dissection of phenylpropanoid metabolism in Arabidopsis
No full textPhenylpropanoid biosynthesis has been studied for decades in many different plant species. In the last twenty years, Arabidopsis thaliana has risen to prominence as a useful model plant for studying this secondary metabolic pathway as well as myriad other biochemical and developmental processes. A large number of Arabidopsis mutants have been identified which exhibit altered flavonoid, sinapate ester and lignin phenotypes. The characterization of these plants and the cloning of the genes defective in these mutants has increased our understanding of the enzymes involved in phenylpropanoid metabolism as well as the proteins required for its regulation. A screen for Arabidopsis plants with decreased fluorescence in leaves identified several mutants that are blocked in phenylpropanoid biosynthesis. One of these reduced epidermal fluorescence mutants, ref2, contains reduced levels of a number of phenylpropanoid pathway-derived products, including sinapoylmalate in leaves, sinapoylcholine in seeds, and syringyl lignin in stems. The REF2 gene was cloned and found to encode the cytochrome P450 monooxygenase CYP83A1, a protein with homology to an enzyme involved in glucosinolate biosynthesis. Reinspection of the biochemical phenotypes of ref2 revealed that it accumulates reduced levels of all methionine-derived glucosinolates in seeds and leaves, suggesting that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates. The phenylpropanoid phenotypes of the ref2 mutant, therefore, suggest a novel metabolic link between glucosinolate biosynthesis, a secondary biosynthetic pathway found only in plants in the order Caparalles, and phenylpropanoid metabolism, a pathway found in all plants and considered essential to the survival of terrestrial plant species. During the characterization of the ref2 mutant, it was discovered that Arabidopsis roots exposed to light contain high levels of many soluble phenylpropanoids, including coniferin and syringin (coniferyl and sinapyl-4-O-glycosides) as well as a number of flavonoids. In contrast, roots of etiolated and soil-grown plants contain very low levels of soluble phenylpropanoids. To elucidate the apparent light-dependent regulation of root secondary metabolism, extracts of mutants defective in light perception, including phyA, phyB and hy4, as well as light response, such as hy5, cop9, cop1 and the det mutants, were analysed for phenylpropanoid content. The results of these assays showed that PHYA and PHYB are the primary photoreceptors involved in light-dependent soluble phenylpropanoid accumulation, although CRY1 is also involved in regulating primarily flavonoid biosynthesis. The absence of coniferin, syringin and flavonoids in roots of the hy5 mutant indicates that the HY5 transcription factor is required for phenylpropanoid accumulation. Further, the presence of phenylpropanoids in etiolated roots of det1 and cop9 mutants indicate that these proteins repress root phenylpropanoid biosynthesis in the absence of light. In contrast, the COP1 protein, which is thought to bind HY5 and mediates COPS degradation of HY5 in aerial tissues, does not play a role in regulating phenylpropanoid accumulation in roots. Characterization of this metabolic light response suggests that this is a new high irradiance response in Arabidopsis, and indicates that Arabidopsis roots may offer a novel system for investigating the regulation of phenylpropanoid biosynthesis
SCT: A serine carboxypeptidase-like protein that functions in Arabidopsis secondary metabolism
No full textSerine carboxypeptidase-like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism. The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions. The characterization of the Arabidopsis sng2 (sinapoyl glucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism. The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae. Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, have increased levels of choline, and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT). The cloning of the SNG2 gene by a combination of map-based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism. SCT and the other known SCPL acyltransferases all share the conserved serine, aspartic acid, and histidine residues employed for catalysis by classical serine carboxypeptidases, although the importance of these residues and the mechanism by which this class of SCPL proteins catalyze acyltransferase reactions is unknown. To further characterize SCT and its catalytic mechanism, we have employed the Saccharomyces cerevisiae vacuolar protein localization 1 mutant, which secretes the serine carboxypeptidase, Carboxypeptidase Y, and other proteins normally targeted to the vacuole. When expressed in this strain, SCT is similarly secreted. SCT has been purified from the yeast medium and used for kinetic characterization of the protein. Immunological analysis of SCT has revealed that the expected 50 kDa mature protein is proteolytically processed in yeast and in planta, most likely resulting in the production of a heterodimer derived from a 30 kDa and 17 kDa polypeptide
A functional and comparative study of the evolution of phenylpropanoid metabolism in land plants
No full textPhenylpropanoid metabolism is a hallmark of land plants, and produces a spectrum of secondary metabolites, including lignin, sporopollenin, flavonoids and hydroxycinnamate conjugates. Many of these compounds are essential for plants’ successful survival in a terrestrial environment. Since its origin, phenylpropanoid metabolism has been extensively exploited by plants in adaptation to ecological niches, and this interplay of metabolism, adaptation and ecology has provided a rich system for studying evolutionary biology. The research presented in this dissertation explores phenylpropanoid metabolism in Selaginella moellendorffii, a species that represents a lineage of vascular plants that diverged from the well-studied flowering plants over 400 million years ago. Functional characterization of several key enzymes in the pathway revealed that Selaginella has evolved new metabolic routes within the relatively conserved framework of phenylpropanoid metabolism, leading to the biosynthesis of syringyl lignin, a lignin type traditionally considered to be restricted in flowering plants. By comparing the metabolic pathway of syringyl lignin biosynthesis in Selaginella to the analogous pathway in flowering plants, we have found that the occurrence of syringyl lignin in these two distantly-related lineages is due to convergent evolution, in which distinct biochemical mechanisms were employed. This study not only advances our understanding of the evolution of plant phenylpropanoid metabolism in general, but also provides valuable tools for rerouting lignin biosynthesis and thus modifying lignocellulosic biomass in economically important crops
Genetic and biochemical analyses of glycosyltransferases involved in Arabidopsis sinapate ester metabolism
No full textSinapoylmalate is a major phenylpropanoid accumulated in Arabidopsis thaliana. Its presence causes leaves to fluoresce blue under UV light, and mutations that lead to lower levels of sinapoylmalate decrease UV-induced leaf fluorescence. The Arabidopsis bright trichomes 1 ( brt1) mutant was first identified in a screen for mutants that exhibit a reduced epidermal fluorescence phenotype; however, subsequent examination of the mutant revealed that its trichomes are hyperfluorescent. The results from genetic mapping and complementation analyses showed that BRT1 encodes UGT84A2, a glucosyltransferase previously shown to be capable of using sinapic acid as a substrate. Residual levels of sinapoylmalate and sinapic acid: UDP-glucose glucosyltransferase activity in brt1 leaves suggest that BRT1 is one member of a family of partially redundant glycosyltransferases that function in Arabidopsis sinapate ester biosynthesis. Reverse transcriptase-polymerase chain reaction analysis showed that BRT1 is expressed through all stages of plant life cycle, a result consistent with the impact of the brt1 mutation on both leaf sinapoylmalate levels and seed sinapoylcholine content. Identification of other glycosyltransferases that may be redundant with BRT1 has been initiated by analyses of EMS-mutagenized plants in the brt1-1 background and knockouts of UGT84A3 and UGT88A1. UV screening of EMS-mutagenized M2 plants has identified enhancers and suppressors of the brt1 mutation. A method for isolation of the compound(s) responsible for the hyperfluorescent trichome phenotype is reported
Paralogous genes in Arabidopsis thaliana contribute to diversified phenylpropanoid metabolism
Get PDFSignificant evidence supports the idea that gene duplication drives the evolution of new gene function. Besides being silenced, duplicated genes can either neofunctionalize or subfunctionalize under selective pressure or neutral drift. Understanding the trajectory of how each gene is fixed and presumably provides added fitness remains difficult. Plant specialized metabolism provides an attractive platform to study the fixation of genes post duplication and how this process leads to the chemical diversity seen today. Specialized metabolites by definition are thought to be dispensable under normal growth conditions. Thus deleterious mutations occurring in paralogous genes that otherwise would be selected against in primary metabolism may be more tolerated in specialized metabolism. Using the Arabidopsis phenylpropanoid pathway as a model, in this thesis I describe two cases where neofunctionalization and subfunctionalization of duplicated genes contributed to metabolite diversification. The phenylpropanoid pathway intersects with primary metabolism at phenylalanine. Recently we identified a new set of phenylalanine derived compounds in Arabidopsis which we named arabidopyrones (APs) which include arabidopyl alcohol, iso-arabidopyl alcohol, arabidopic acid and iso-arabidopic acid. CYP84A4 is a paralog of CYP84A1, a well-characterized enzyme in the phenylpropanoid pathway, and CYP84A4 has neofunctionalized relative to its ancestral function. CYP84A4 3-hydroxylates p-coumaraldehyde, a phenylpropanoid intermediate, to generate caffealdehyde. Caffealdehyde can be used by a conserved ring cleavage dioxygenase, AtLigB, in a step required to make the heterocyclic APs. Understanding AP biosynthesis may provide a unique opportunity to learn the broader biological function of LigB homologs. To do so, we tested the hypothesis that enzymes and intermediates in the phenylpropanoid pathway leading to p-coumaraldehyde are involved in AP biosynthesis. The general phenylpropanoid pathway gives rise to p-coumaryl CoA via phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate: CoA ligase (4CL). Cinnamoyl CoA reductase (CCR) then converts p-coumaryl CoA to p-coumaraldehyde, the substrate of CYP84A4. Through the analyses of mutants that are defective in these genes, stable-isotope labeling studies, and chemical complementation experiments, we conclude that the activities of these enzymes are required for AP biosynthesis. In addition, we found that cinnamyl alcohol dehydrogenase C and D (CAD C and D), known enzymes in later steps of the phenylpropanoid pathway, are involved in AP biosynthesis in that they may convert caffealdehyde to caffeoyl alcohol which then can be used by AtLigB to generate arabidopyl alcohol and arabidopic acid. Four isoforms of 4CLs have been identified in Arabidopsis. 4CL generates p-coumaryl CoA and caffeoyl CoA from their respective acids which are required for the major products of this pathway. Phylogenetic analysis reveals that 4CL1, 4CL2 and 4CL4 are more closely related to one another than to 4CL3. Promoter-GUS analysis shows that 4CL1 and 4CL2 are expressed in lignifying cells. In contrast, 4CL3 is expressed in a broad range of cell types, indicating that 4CLs have subfunctionalized with regard to expression patterns. We found that 4cl3 mutants have an over-all reduction in flavonoid biosynthesis, suggesting that 4CL3 has acquired a distinct role in phenylpropanoid metabolism. Sinapoylmalate, the major hydroxycinnamoyl ester found in Arabidopsis is greatly reduced in a 4cl1 4cl3 mutant, showing that 4CL1 and 4CL3 function redundantly in its biosynthesis. The 4cl1 4cl2 double mutant and the 4cl1 4cl2 4cl3 triple mutant are both dwarf and contain less lignin than wild type, indicating that 4CL1 and 4CL2 are important for plant growth and that 4CL3 has a role in lignin biosynthesis in addition to its function in soluble metabolism. We could not find an important role for 4CL4 in any of the organs examined, consistent with its limited expression profile. Together, these data show that the four paralogs of 4CLs in Arabidopsis diverged in their expression patterns, resulting in their overlapping yet distinct roles in phenylpropanoid metabolism
The impact of phenylpropanoid pathway manipulation on lignin deposition and soluble secondary metabolism in Arabidopsis
No full textThe phenylpropanoid pathway, which is conserved throughout land plants, is responsible for the biosynthesis of many compounds that are involved in plants\u27 structural integrity, water transport, UV protection, and defense against herbivores. The major end product of the phenylpropanoid pathway is lignin, a complex polymer of the secondary cell wall. Decreasing or altering lignin structure provides enhanced cell wall digestibility and can greatly increase the utilization of lignin itself or cell wall polysaccharides. Due to the agro-industrial importance of lignin, the genes participating in lignin biosynthesis have been identified and manipulated in many plant species. Here we describe the metabolic changes that result from the stacking of two manipulation strategies to tailor make high aldehyde lignin. We also show that significant metabolic plasticity is observed in both the soluble and cell wall-bound pools in plants with perturbed lignin biosynthesis. Carbon flux which is normally directed toward one or more metabolites in wild-type plants is instead redirected to another biosynthetically related compound or group of compounds. However, this redirection is not always observed. Here we show a specific example of inhibited plasticity that is most likely the result of transcriptional feedback involving the Mediator complex. In addition to metabolic changes, some perturbations in lignin biosynthesis affect development and can result in dwarfism. These observations may indicate a direct impact of altered lignin and cell wall architecture on plant growth, or it may suggest that lignin biosynthesis is tied to other metabolic networks that affect plant growth and development
Genetic Interactions of the Arabidopsis Mediator Complex in the Regulation of Phenylpropanoid Metabolism and Global Gene Expression
No full textThe 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
Elucidating the mechanism of phenylpropanoid regulation by the Arabidopsis mediator complex
Get PDFThe Mediator complex is a multi-protein co-regulator of eukaryotic transcription which plays a role in the expression of many, if not most, genes of the cell. Two Mediator subunits, REF4 and RFR1, were demonstrated to be important for the normal regulation of phenylpropanoid metabolism in Arabidopsis. Phenylpropanoids are a family of specialized plant metabolites derived from the amino acid phenylalanine and are involved in defense against pathogens, UV protection and structural support. In order to understand how Mediator regulates phenylpropanoid metabolism through REF4 and RFR1, a better basic understanding of this protein complex is required. Here I provide data on the initial steps in characterizing the Arabidopsis Mediator complex. First, I performed partial purification of Mediator using ion-exchange chromatography followed by LC-MS analysis of cation-exchange purified fraction. Then we generated and evaluated antibodies against specific Mediator subunits. Lastly, I used yeast two-hybrid to evaluate previously identified putative interactors of REF4, evaluated interaction between Mediator tail subunits with REF4 and RFR1, and identified new interaction candidates for RFR1
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