REGULATION OF PHENYLPROPANOID METABOLISM IN PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006705
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Biochemistry

Non Technical Summary
The sun is the principle source of energy for our planet, and photosynthesis is the primary mechanism by which that energy is captured and stored in the form of reduced carbon. An outcome of these biochemical events is that plants represent a quantitatively important, sustainable, and carbon-neutral source of energy for humans. In order to maximize the utility of plants for this purpose, it is important that we gain control of the processes associated with energy capture and storage, including the molecular mechanisms that allocate fixed carbon to the myriad biochemical pathways in plants. One of the most significant of these is the phenylpropanoid biosynthetic pathway which leads to the deposition of lignin. Lignin is a cross-linked phenolic polymer that makes the cell walls of specialized plant cells more rigid. Its synthesis represents the single largest metabolic sink for phenylalanine in the biosphere and as such represents a huge metabolic commitment for plant metabolism. Lignin is also a significant barrier to the use of crops for livestock feed, pulp and paper production, and to the generation of cellulosic biofuels. Our objective is to push forward our basic understanding of lignin biosynthesis while simultaneously adding to our ability to engineer plant metabolism so that it can be modified for the improvement of agriculture.Although the enzymes of lignin biosynthesis have now been identified, we know relatively little about how their expression is regulated. Several relevant transcription factors have been isolated, but it is unclear how their expression and activity dictate or contribute to the allocation of photosynthate to lignin as opposed to other plant components such as cellulose, starch, or any other sinks for reduced carbon.We are in a unique position to explore how the amount of lignin in a plant is controlled because we have identified two novel, plant-specific proteins (REF4 and RFR1) that are components of Mediator, a large multi-protein complex that facilitates interactions between DNA-bound transcription factors and RNA polymerase II to activate or repress the expression of downstream genes. Mutants of Arabidopsis that lack REF4 and RFR1 are viable and show little in the way of developmental changes, making them a tractable system in which to examine the function of Mediator. Of particular relevance to this project is that these mutants accumulate more phenylpropanoid end products including lignin. Plants carrying a mutant dominant form of REF4 show the opposite phenotype. Thus, REF4 and RFR1 appear to be components of a system that determines the amount of carbon allocated to the phenylpropanoid biosynthetic pathway. Considering that over 108 gigatons of lignin are synthesized annually in the biosphere, these proteins are important players in the global carbon cycle and represent important new opportunities for the manipulation of lignin synthesis in plants.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2499	1000	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

bioenergy

biofuel

lignin

arabidopsis

cell walls

Goals / Objectives
To elucidate how REF4 and RFR1 function as components of Mediator we will use a robust set of experimental approaches including immunoprecipitation methods to determine the proteins with which REF4 and RFR1 interact in the Mediator complex. These experiments will establish which proteins are relevant to the regulation of phenylpropanoid accumulation in plants and may simultaneously identify additional proteins that are relevant to this process that were not previously known to be part of Mediator. We will then use chromatin immunoprecipitation to identify the targets of the REF4 and RFR1 proteins in the Arabidopsis genome in order to understand which genes must be altered in their expression to divert more or less carbon into the lignin biosynthetic pathway. Finally we will determine whether there are functional differences between REF4 and RFR1 by altering their relative expression levels in different tissues. By completing this entire set of experiments, we will learn how REF4 and RFR1 function to coordinate transcription of genes required for lignin deposition and gain insights into how this pathway can be manipulated for human energy needs.

Project Methods
Objective 1. Determine the components found in REF4- and RFR1-containing Mediator complexes. Hypothesis 1: REF4 and RFR1 are mutually exclusive components of the Mediator complex. Based on the observation that the presence of wild-type RFR1 mitigates the mutant phenotype of the ref4-3 mutant, we will test the hypothesis that REF4 and RFR1 compete with one another for occupancy in Mediator. To do so, we will first generate multiple constructs for both REF4 and RFR1 in which the expressed proteins are fused to the epitope tags HA or FLAG, which have previously been shown to be suitable for use in Arabidopsis (Wood et al., 2006). These expression constructs will then be transformed into ref4 rfr1 null plants, where their functionality can be verified based on their ability to rescue the high phenylpropanoid phenotype of these plants. We will then verify that these tagged proteins are incorporated into Mediator by incubating leaf whole cell extract with the appropriate epitope-directed antibody and protein A sepharose beads, precipitating these beads by centrifugation, and using mass spectrometry to identify co-immunoprecipitating proteins (Axtell and Staskawicz, 2003). We expect to identify known subunits of Mediator and potentially components that have not been previously described. Furthermore, both of these sets may contain proteins that immunoprecipitate with either REF4 or RFR1 but not both. We will next cross plants expressing epitope-tagged REF4 with plants expressing RFR1 that has been tagged with another epitope, and then test whether we can detect tagged RFR1 by protein gel blot analysis when an antibody against tagged REF4 is used for immunoprecipitation, and vice versa. If immunoprecipitation of REF4 can also pull down RFR1, this would show that both REF4 and RFR1 can be present in the same complex, whereas if they are mutually exclusive components of Mediator, immunoprecipitation of either protein will fail to pull down the other.Hypothesis 2: REF4- and RFR1-containing Mediator complexes contain or interact with regulators of phenylpropanoid biosynthesis. Given the well-known role of Mediator in transcriptional co-activation and co-suppression, it seems likely that REF4 and/or RFR1 interact with other proteins that directly or indirectly regulate phenylpropanoid biosynthesis. To identify these putative interacting partners of REF4 and RFR1, we will employ two complementary approaches. First, we will examine the results of the co-immunoprecipitation and mass spectrometry experiments described in Hypothesis 1 above for the presence of any previously uncharacterized components or interacting partners of Mediator. These analyses may be particularly useful for identifying any proteins that are present only in complexes containing REF4 or RFR1, since these would be expected to be enriched in our co-immunoprecipitates. To follow up the analysis of any putative REF4- or RFR1-interacting factors identified by co-immunoprecipitation, we will obtain T-DNA lines of Arabidopsis containing disruptions in the genes encoding interactors, and examine these T-DNA lines for a phenylpropanoid biosynthetic phenotype either alone or in combination with disruptions in REF4 and RFR1.Objective 2. Identify the specific regions in the genome to which REF4- and RFR1-containing Mediator complexes are targeted. Hypothesis 1: REF4 and RFR1 inhibit phenylpropanoid biosynthesis by targeting Mediator to particular sites in the genome and altering the transcription of genes residing at these loci. The experimental observation that REF4 and RFR1 are components of the Mediator complex suggests that their influence on phenylpropanoid biosynthesis may be the result of effects on transcriptional activation or suppression of genes either directly or indirectly involved in the synthesis of phenylpropanoids. To test this hypothesis, we will use the epitope-tagged REF4 and RFR1 expression constructs described in the previous section to carry out chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) to identify the genomic sites where REF4 and RFR1 reside. This approach has been successfully employed previously in Arabidopsis, for example, by Kaufmann et al., to identify the genomic targets of the MADS-domain containing protein SEPALLATA3 (Kaufmann et al., 2009). From this data, a list of candidate loci can be assembled that can be retested via traditional chromatin immunoprecipitation followed by qPCR. If the results of this approach are robust, this technique could also be employed to test whether the ref4-3 protein targets Mediator to different sites in the genome, as well as to test whether deletion of REF4 and RFR1 alters targeting of Mediator, by using another epitope-tagged Mediator subunit expressed in wild-type and ref4 rfr1 null plants. Furthermore, we can compare the gene list obtained from ChIP-seq with the set of genes whose expression is altered in ref4, rfr1, or ref4 rfr1 that we have already obtained from microarray experiments carried out in our laboratory. Specifically, we hypothesize that genes to which REF4, RFR1, or both localize will show altered expression in the ref4, rfr1, and ref4 rfr1 mutants, respectively. Identification of the genomic loci at which REF4 and RFR1 reside, and the directional effect on the transcription of these genes in the absence of REF4 and RFR1 will yield insight into the mechanism of REF4 and RFR1 action, and may provide information on whether they generally are involved in transcriptional co-activation, co-suppression, or either process.Objective 3: Determine whether there are functional differences between REF4 and RFR1. Hypothesis 1: The phenotypic differences seen in ref4 and rfr1 mutants are due only to differences in their expression. We have found that disruption of REF4 alone has little observable effect on phenylpropanoid metabolism. In contrast, rfr1 mutants exhibit a significant increase in lignin deposition and soluble phenylpropanoid accumulation, and this phenotype is enhanced by simultaneous elimination of REF4, indicating that RFR1 likely plays the principle quantitative role in suppressing phenylpropanoid metabolism in wild-type plants. One possible explanation for these observations is that REF4 and RFR1 are functionally interchangeable, but differ in their expression, with RFR1 being expressed either at a higher level than REF4, or in a non-overlapping distribution with REF4. For example, RFR1 may be expressed in phenylpropanoid-producing cells in which REF4 is not expressed (but not vice-versa), such that loss of RFR1 (but not REF4) is sufficient to generate a phenylpropanoid hyper-accumulation phenotype. Alternatively, it is possible that the difference in the phenotype of ref4 and rfr1 mutants arises from intrinsic differences in the two gene products themselves with respect to their binding partners, activity, etc.We will generate constructs in which REF4 expression is driven by the RFR1 promoter and, conversely, in which RFR1 expression is directed by the REF4 promoter. The ability of promoter-swapped constructs to complement the phenotypes of the ref4 rfr1 mutant will be compared to those in which REF4 and RFR1 are driven by their endogenous promoters or by the strong C4H promoter. If phenotypic complementation is more closely associated with the promoter employed, we will conclude that the difference in ref4 and rfr1 mutants is a result of differences in their expression in wild-type plants. On the other hand, if the choice of coding sequence determines to what extent a construct complements the ref4 rfr1 mutant phenotype, we will conclude that some intrinsic difference exists between the REF4 and RFR1 polypeptides

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:The target audience for our work is the general scientific community, with particular emphasis on plant biochemists and plant biologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student Candy Mao explored the relationship between Mediator and phenylpropanoid metabolism. Graduate student Peng Wang led the metabolic flux analysis project. Dr. Jeff Simpson, Fabiola Muro and Courtney Traugh conducted as-yet-unpublished research on the regulation of phenylpropanoid metabolism and its relationship to growth. How have the results been disseminated to communities of interest?Our results have been disseminated through research publications and seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 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. We explored the functions of four Arabidopsis Mediator tail subunits, MED2, MED5a/b, MED16, and MED23, with particular reference to their role in the regulation of phenylpropanoid metabolism, by comparing the impact of mutations in each on the Arabidopsis transcriptome. We found 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 found 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. In other work, we developed a method to conduct metabolic flux analysis in plants. 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. Next, we wanted to measure phenylpropanoid flux in Arabidopsis because lignin is a polymer that significantly inhibitssaccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both beendemonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, itcannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesissuch that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all ligninsubunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yetan in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated andparameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled 13CPhe.Several candidate models were compared by an information theoretic approach to select the one that best matched theexperimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux. Finally, although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5) dependent manner.To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of glucosinolate-deficient mutants using RNAseq and identified mis-regulated genes that are rescued by the disruption of MED5.The expression of a group of Kelch Domain F-Box genes (KFBs) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency in the mutants.Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Kim JI, Zhang X, Pascuzzi PE, Liu CJ, Chapple C (2020) Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL. New Phytol. 225: 154-168
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: University of Nevada  Reno. November 2019. How do plants mediate crosstalk between biochemical pathways?

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:I have communicated my research through seminars at universities and scientific meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student Candy Mao explored the relationship between Mediator and phenylpropanoid metabolism. Post doc Dr. Jeong Im Kim completed the research on the relationship between phenylpropanoid biosynthesis, glucosinolate biosynthesis and Mediator. How have the results been disseminated to communities of interest?Our results have been disseminated through research publications and seminars. What do you plan to do during the next reporting period to accomplish the goals?We are currently working onan inducible gene expression system, which isshedding light on the relationship between phenylpropanoid metabolism and growth. We are also developing a pipeline forthe identification of all phenylpropanoid metabolites in plant extracts by liquid chromatography mass spectrometry.

Impacts
What was accomplished under these goals? The Mediator complex functions as a hub for transcriptional regulation. MED5, an Arabidopsis Mediator tail subunit, is required for maintaining phenylpropanoid homeostasis. A semi-dominant mutation (ref4-3) that causes a single amino acid substitution in MED5b functions as a strong suppressor of the pathway, leading to decreased soluble phenylpropanoid accumulation, reduced lignin content and dwarfism. In contrast, loss of MED5 results in increased levels of phenylpropanoids. We used a reverse genetic approach to identify suppressors of ref4-3 and found that ref4-3 requires CDK8, a kinase module subunit of Mediator, to repress plant growth. The genetic interaction between MED5 and CDK8 was further characterized using mRNA-sequencing (RNA-seq) and metabolite analysis. Growth inhibition and suppression of phenylpropanoid metabolism can be genetically separated in ref4-3 by elimination of CDK8 kinase activity; however, the stunted growth of ref4-3 is not dependent on the phosphorylation event introduced by the G383S mutation. In addition, rather than perturbation of lignin biosynthesis, mis-regulation of DJC66, a gene encoding a DNAJ protein, is involved in the dwarfism of the med5 mutants. Together, our study reveals genetic interactions between Mediator tail and kinase module subunits and enhances our understanding of dwarfing in phenylpropanoid pathway mutants. Plants produce several hundreds of thousands of secondary metabolites that are important for adaptation to various environmental conditions. Although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5) dependent manner. To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of glucosinolate-deficient mutants using RNAseq and identified mis-regulated genes that are rescued by the disruption of MED5. The expression of a group of Kelch Domain F-Box genes (KFBs) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency in the mutants. Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Mao X, Kim JI, Wheeler MT, Heintzelman AK, Weake VM, Chapple C. (2019) Mutation of Mediator subunit CDK8 counteracts the stunted growth and salicylic acid hyper?accumulation phenotypes of an Arabidopsis MED5 mutant. New Phytologist 223: 233-245.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Yang H, Zhang X, Luo H, Liu B, Shiga TM, Li X, Kim JI, Rubinelli P, Overton JC, Subramanyam V, Cooper BR, Mo H, Abu-Omar MM, Chapple C, Donohoe BS, Makowski L, Mosier NS, McCann MC, Carpita NC, Meilan R. (2019) Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery. Biotechnol Biofuels 12:171.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Kim JI, Zhang X, Pascuzzi PE, Liu CJ, Chapple C (2019) Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL. New Phytologist, in press.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Muro-Villanueva F, Mao X, Chapple C (2019) Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition. Curr Opin Biotech 56: 202-208.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Mao X, Weake VM, Chapple C (2019) Mediator function in plant metabolism revealed by large-scale biology. J. Exp Bot, in press.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:I have communicated my research through seminars at universities and scientific meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student Candy Mao explored the relationship between Mediator and phenylpropanoid metabolism. Graduate student Peng Wang led the metabolic flux analysis project. Dr. Jeff Simpson, Fabiola Muro and Courtney Traugh conducted as-yet-unpublished research on the regulation of phenylpropanoid metabolism and its relationship to growth. How have the results been disseminated to communities of interest?Our results have been disseminated through research publications and seminars. What do you plan to do during the next reporting period to accomplish the goals?We are currently working on new suppressors of the ref4-3 mutant and an inducible gene expression system, both of which are shedding light on the relationship between phenylpropanoid metabolism and growth. We are also developing a pipeline for the identification of all phenylpropanoid metabolites in plant extracts by liquid chromatography mass spectrometry. Finally, we are studying how the phenylpropanoid gene ferulate 5-hydroxylase is regulated at the transcriptional level.

Impacts
What was accomplished under these goals? 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. We explored the functions of four Arabidopsis Mediator tail subunits, MED2, MED5a/b, MED16, and MED23, with particular reference to their role in the regulation of phenylpropanoid metabolism, by comparing the impact of mutations in each on the Arabidopsis transcriptome. We found 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 found 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. In other work, we developed a method to conduct metabolic flux analysis in plants. 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. We wanted to measure phenylpropanoid flux in Arabidopsis because lLignin is a polymer that significantly inhibits saccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both been demonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, it cannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesis such that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all lignin subunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yet an in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated and parameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled 13C-Phe. Several candidate models were compared by an information theoretic approach to select the one that best matched the experimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Guo L, Wang P, Jaini R, Dudareva N, Chapple C, Morgan JA. (2018) Dynamic modeling of subcellular phenylpropanoid metabolism in Arabidopsis lignifying cells. Metab Eng 49: 36-46.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Dolan WL, Chapple C. (2018) Transcriptome Analysis of Four Arabidopsis thaliana Mediator Tail Mutants Reveals Overlapping and Unique Functions in Gene Regulation. G3 8: 3093-3108.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang P, Guo L., Jaini R. Klempien A, McCoy RM, Morgan JA, Dudareva N, Chapple C. (2018) A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems. Plant Methods 14: 51. https://doi.org/10.1186/s13007-018-0318-3
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lignin Gordon Conference. Stonehill College. August 2018. The Mediator Complex is a key player in the modification of lignin content and composition.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:I have communicated my research through seminars at universities and scientific meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Jeong Im Kim worked on multiple projects. She began an independent faculty position at the University of Florida in August of 2017. Whitney Dolan and Candy Mao worked on the Mediator project. Whitney graduated with her Ph.D. in May of 2017. How have the results been disseminated to communities of interest?Our results have been disseminated through research publications and seminars. What do you plan to do during the next reporting period to accomplish the goals?In the future, we hope to purify Mediator from these different Arabidopsis mutant lines so that we can determine how these mutations affect the composition of the complex and its ability to interact with other proteins.

Impacts
What was accomplished under these goals? Plants produce a vast array of compounds from the amino acid phenylalanine, known as phenylpropanoids. Phenylpropanoids are important to plant growth and fitness. Previously, we identified an Arabidopsis mutant named ref4-3 that has reduced levels of phenylpropanoids and stunted growth. The ref4-3 mutation occurs in the MED5 subunit of the Mediator complex. The Mediator complex is made up of approximately 28 different proteins and is an important regulator of gene expression. Mediator impacts gene expression by interacting with other proteins, including transcription factors and RNA Polymerase II. Ultimately, we would like to understand how the ref4-3 mutation alters the function of Mediator and how that leads to a change in plant growth and phenylpropanoid accumulation. To begin, we asked, "What other proteins are required for the function of ref4-3?" To answer this question, we introduced random mutations into ref4-3 seeds and, in following generations, looked for plants that had restored growth and/or phenylpropanoid production. This would indicate that a protein required for the function of ref4-3 had been disrupted. When we sequenced the genomes of these mutants, we found that all of the mutations that suppressed the function of ref4-3 occurred in three other subunits of the Mediator complex. RNAseq analysis of the ref4-3 suppressors, the ref4-3 mutant, and wild-type plants showed that, although in the ref4-3 mutant approximately 30% of the genome was mis-regulated, in the ref4-3 suppressors, normal gene expression was largely restored.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: 1. Dolan WL, Dilkes BP, Stout JM, Bonawitz ND, Chapple C (2017) Mediator Complex Subunits MED2, MED5, MED16, and MED23 genetically interact in the regulation of phenylpropanoid biosynthesis. Plant Cell, in press.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: 2. Jaini R, Wang P, Dudareva N, Chapple C, Morgan HA. (2017) Targeted metabolomics of the phenylpropanoid pathway in Arabidopsis thaliana using reversed phase liquid chromatography coupled with tandem mass spectrometry. Phytochem Anal, 28, 267-276.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: 3. Lee S, Mo H, Kim JI, Chapple C. (2017) Genetic engineering of Arabidopsis to overproduce disinapoyl esters, potential lignin modification molecules. Biotech Biofuels, 10, 40.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:I have communicated my research through seminars at universities and scientific meetings and have communicated science-based knowledge to Purdue University undergraduate students in my class, BCHM 100. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Jeong Im Kim has worked on multiple projects. Whitney Dolan and Candy Mao worked on the Mediator project and Whitney also authored the Mediator review chapter. How have the results been disseminated to communities of interest?Our results have been disseminated through research publications and seminars. What do you plan to do during the next reporting period to accomplish the goals?As described in our proposal, our major focus during the next year will be to gain a greater understanding of the role of the Mediator complex. To elucidate how REF4 and RFR1 function as components of Mediator we will use a robust set of experimental approaches including immunoprecipitation methods to determine the proteins with which REF4 and RFR1 interact in the Mediator complex. These experiments will establish which proteins are relevant to the regulation of phenylpropanoid accumulation in plants and may simultaneously identify additional proteins that are relevant to this process that were not previously known to be part of Mediator. We will then use chromatin immunoprecipitation to identify the targets of the REF4 and RFR1 proteins in the Arabidopsis genome in order to understand which genes must be altered in their expression to divert more or less carbon into the lignin biosynthetic pathway. Finally we will determine whether there are functional differences between REF4 and RFR1 by altering their relative expression levels in different tissues. By completing this entire set of experiments, we will learn how REF4 and RFR1 function to coordinate transcription of genes required for lignin deposition and gain insights into how this pathway can be manipulated for human energy needs.

Impacts
What was accomplished under these goals? While other experiments were in progress, we produced a review article on what is known about the function of the Mediator complex in plants. 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. The majority of insights into plant Mediator function have come from Arabidopsis because it is the only plant from which Mediator has been purified and from which an array of Mediator mutants have been isolated by forward and reverse genetics. 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.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Dolan WL, Chapple C (2016) Conservation and divergence of Mediator structure and function: insights from plants Plant Cell Physiol., in press.