The Role of 3-Deoxy-D-Arabino-Heptulosonate 7- Phosphate Synthase 1 in Arabidopsis Thaliana Metabolism

THE ROLE OF 3-DEOXY-D-ARABINO-HEPTULOSONATE 7- PHOSPHATE SYNTHASE 1 IN ARABIDOPSIS THALIANA METABOLISM

Jimmy Poulin

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and System Biology University of Toronto

The role of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 1 in Arabidopsis thaliana metabolism

Jimmy Poulin

Master of Science

Cell and System Biology University of Toronto

2011

Abstract

The enzyme 3-deoxy-D-arabino-heptulusonate 7-phosphate synthase (DHS) catalyzes the first step of the shikimate pathway. In bacteria, the regulation of the pathway is mediated by allosteric inhibition of DHS by the aromatic amino acids tyrosine, phenylalanine and tryptophan.

The regulation of the pathway in plants remains elusive but the aromatic amino acids are involved as suggested by the hypersensitivity of dhs1 knockout mutant to tyrosine. In this study the effects of the dhs1 mutation on endogenous levels of aromatic amino acids and of downstream metabolites are explored. HPLC analysis is used to measure levels of tyrosine and phenylalanine and 5-methyltryptophan sensitivity is used to probe levels of tryptophan.

Additionally, the auxin content of whole seedlings was quantified by LC/MS and its local levels at the root apex are visualized with the DR5::GUS reporter system.

Acknowledgements

I could not have completed my master’s degree without the help of many resourceful individuals. First and foremost I would like to thank Dr. Dinesh Christendat for his supervision and guidance. I am also grateful for guidance received from Dr. Darrell Desveaux, Dr. Nicholas

Provart, Dr. Daphne Goring and Dr. Geoff Fucile. I also received valuable advice and support from other members of the Christendat lab including James Peek, Dr. Christel Garcia and Kate

Penney.

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Table of Contents

Abstract...... ii Acknowledgements...... iii Table of Contents ...... iv List of Tables ...... vi List of Figures...... vi List of Abbreviations ...... viii Introduction...... 1 Shikimate Pathway ...... 1 DAHP Synthase ...... 6 Regulation of AroAI DHS ...... 8 Regulation of AroAII DHS...... 9 Regulation of Arabidopsis thaliana DHS ...... 10 Aromatic Amino Acid Biosynthesis...... 13 Auxin Biosynthesis...... 16 Thesis Objective...... 18 Materials and Methods ...... 19 Materials...... 19 Phylogenetic Analysis ...... 19 Plant Growth Conditions and Root Length Assays...... 20 Genomic DNA Extraction...... 20 PCR Genotyping of T-DNA Lines ...... 21 Aromatic Amino Acid Treatment...... 22 Metabolite Extraction and Derivatization ...... 23 HPLC Analysis...... 23 Auxin Quantification by LC-MS...... 24 GUS Staining...... 25 Microarray Data Analysis...... 26 Results...... 28 AroAII-type DHS Gene Duplicate Retention in Higher Plants ...... 28

Conservation of 3D Structure in MtDHS and AtDHS...... 31 Verification of Col-0 and dhs1 Genotypes...... 33 Changes in Intracellular Levels of Aromatic Amino Acids in Atdhs1...... 34 Sensitivity of dhs1 to 5-Methyltryptophan Supplementation...... 39 Auxin Levels in Col-0 and dhs1 Whole Seedlings ...... 45 Auxin Levels in Col-0 and dhs1 Seedling Roots ...... 46 Tyrosine Treatment of dhs1 Elicit Stress Responses...... 49 Discussion...... 54 Diversification in the Regulatory Mechanisms of Bacterial and Plant AroAII Enzymes...... 55 Conservation of Structural Domains between Arabidopsis and M.tuberculosis DHS Enzymes ...... 56 Levels of Aromatic Amino Acids are Disrupted in dhs1 Seedlings...... 57 Changes in Auxin...... 61 Tyrosine treatment of dhs1 knockout turns on Arabidopsis stress response and causes significant transcriptional changes in tryptophan and auxin biosynthetic genes ...... 64 Proposed Model and Future Directions ...... 65 Conclusion ...... 70 References...... 71

List of Tables

Table 1 – Primers used for genotyping ...... 22 Table 2 – PCR program used for genotyping...... 22 Table 3 – Elution profile for HPLC separation of amino acid dansyl derivatives ...... 24 Table 4 – Number of differentially expressed genes in dhs1 following treatment with tyrosine (Y), phenylalanine (F) or tryptophan (W) and between dhs1 and its non-transgenic sibling (NTS) ...... 50

List of Figures

Figure 1 – The 7 enzymatic steps of the shikimate pathway ...... 4 Figure 2 – Shikimate pathway-derived secondary metabolites...... 5 Figure 3 – Condensation reaction catalyzed by DHS ...... 7 Figure 4 – Organ specific map of DHS expression in Arabidopsis thaliana...... 12 Figure 5 – Hypersensitivity of Arabidopsis dhs1 mutants to tyrosine supplementation...... 13 Figure 6 – Aromatic amino acid biosynthesis and regulation...... 16 Figure 7 – DHS1 gene structure and position of T-DNA insertion ...... 21 Figure 8 – Phylogenetic reconstruction of bacterial AroAI DHS ...... 29 Figure 9 – Phylogenetic reconstruction of AroAII DHS ...... 30 Figure 10 – Structural comparison of AroAI and AroAII DHS...... 32 Figure 11 – Multiple sequence alignment of M.tuberculosis DHS and of Arabidopsis DHS1, DHS2 and DHS3 ...... 32 Figure 12 – Verification of homozygosity for Arabidopsis Col-0 and T-DNA mutant dhs1 ...... 33 Figure 13 – Elution profile of standard amino acid dansyl derivatives ...... 35 Figure 14 – Standard curve for the absorbance of dansyl-Tyr derivative as a function of its concentration ...... 36 Figure 15 – Standard curve for the absorbance of dansyl-Phe derivative as a function of its concentration ...... 36 Figure 16 – Intracellular concentration of tyrosine (Tyr) and phenylalanine (Phe) in Arabidopsis seedlings after 8 hr control treatment ...... 38 Figure 17 – Intracellular concentration of tyrosine (Tyr) and phenylalanine (Phe) in Arabidopsis seedlings after 8 hr treatment with 500 µM exogenous tyrosine ...... 38 Figure 18 – Intracellular concentration of tyrosine (Tyr) and phenylalanine (Phe) in Arabidopsis seedlings after 8 hr treatment with 500 µM exogenous tyrosine and phenylalanine ...... 39 Figure 19 – Increased sensitivity of dhs1 seedlings to 5-Methyltryptophan (5-MT)...... 41

Figure 20 – Effect of 5-Methyltryptophan (5-MT) on Arabidopsis dhs1 seedlings...... 42 Figure 21 – Effects of 5-Methyltryptophan and tyrosine on Col-0 root length ...... 43 Figure 22 – Effects of 5-Methyltryptophan and tyrosine on Arabidopsis dhs1 root length...... 44 Figure 23 – Auxin content in whole seedlings...... 46 Figure 24 – Effect of aromatic amino acid supplementation on endogenous auxin levels at the root apex of Arabidopsis seedlings...... 48 Figure 25 – Transcript levels of genes belonging to the shikimate pathway, aromatic amino acid and auxin biosynthesis in dhs1 after treatment with tyrosine (Y), phenylalanine (F) and tryptophan (W) ...... 51 Figure 26 – Functional enrichment analysis of genes with significantly different expression in pairwise comparisons of dhs1 following treatment with tyrosine (Y), phenylalanine (F) or tryptophan (W) and between dhs1 and its non-transgenic sibling (NTS)...... 52 Figure 27 – Transcript levels of stress related genes in dhs1 after treatment with tyrosine (Y), phenylalanine (F) and tryptophan (W) ...... 53 Figure 28 – Proposed model of DHS regulation in Arabidopsis thaliana ...... 68

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List of Abbreviations

5MT 5-methyltryptophan 6MA 6-methylanthranilic acid A.thaliana Arabidopsis thaliana AAAAT aromatic amino acid aminotransferase AAP1 amino acid permease 1 ACS10 ACC synthase 1, At1g62960 ADH arogenate dehydrogenase ADT arogenate dehydratase ADT1 arogenate dehydratase 1, At1g11790 ADT2 arogenate dehydratase 2, At3g07630 ALF1 aberrant lateral root formation 1, At2g20610 AMl1 amidase like protein 1, At1g08980 ATR4 altered tryptophan regulation 4, At4g31500 Bp base pair CaMV cauliflower mosaic virus CM chorismate mutase CM1 Arabidopsis Chorismate mutase 1, At3g29200 CM2 Arabidopsis Chorismate mutase 2, At5g10870 CM3 Arabidopsis Chorismate mutase 3, At1g69370 Co-IP co-immunoprecipitation CS chorismate synthase cTP chloroplast transit peptide Cyp79B3 cytochrome p450, family 79, subfamily b, polypeptide 3, At2g22330 DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate DHQ dehydroquinate dehydratase DHQS dehydroquinate synthase DHS DAHP synthase DHS1 Arabidopsis DAHP synthase 1, At4g39980 DHS2 Arabidopsis DAHP synthase 2, At4g33510 DHS3 Arabidopsis DAHP synthase 3, At4g22410 DNA deoxyribonucleic acid E4P erythrose 4-phosphate EMB1144 embryo defective 1144, At1g48850 EPSPS 5-enolpyruvylshikimate-3-phosphate synthase GPA1 G protein alpha subunit 1, at2g26300 GUS β-glucuronidase IAD indole-3-acetaldehyde IAOX indo-3-acetaldoxime

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IGPS indole-3-glyceraol-phosphate synthase LHT1 lysine histidine transporter 1 MBB18.6 tryptophan synthase beta type 2, At5g38530 MKP11.15 myb-like transcription factor, At5g17300 MS mass spectrometry MYB34 myb domain protein 34, At5g60890 NTS non-transgenic sibling PAI phosphoribosylanthranilate isomerase PAL phenylalanine ammonia lyase PAT prephenate aminotransferase PEP phosphoenolpyruvate Phe phenylalanine PTM post translational modification SDH shikimate dehydrogenase SK shikimate kinase TAA1 tryptophan aminotransferase of Arabidopsis 1, At1g70560 Trp tryptophan TRP3 tryptophan requiring 3, At3g54640 TRP6 transient receptor potential 6, At1g07780 TSB2 tryptophan synthase beta subunit 2, At4g27070 Tyr tyrosine TyrA arogenate dehydrogenase Y2H yeast 2 hybrid αMT α-methyltryptophan

Introduction

Shikimate Pathway

The shikimate pathway is an essential metabolic pathway found in bacteria, fungi, apicomplexan parasites and plants (Herrmann, 1995; Roberts et al., 1998; Herrmann and

Weaver, 1999; Tzin and Galili, 2010). The end product of the pathway chorismate is used as a precursor in the biosynthesis of the aromatic amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) (Figure 1). In bacteria, aromatic amino acids are primarily used for protein biosynthesis while in plants a substantial portion is directed towards secondary or specialized metabolism. In this respect, the shikimate pathway represents the main channel through which carbon flow from primary metabolism is directed towards specialized metabolism. Natural products produced in these specialized metabolic networks include phytoalexins, flavonoids, phenylpropanoids, indole hormones and lignin. These molecules have important biological roles in cellular signaling, pathogen defense, UV protection, and structural support (Herrmann, 1995;

Herrmann and Weaver, 1999).

The absence of the shikimate pathway in animals makes it an ideal target for herbicides and antimicrobial agents. The widely used herbicide Round-up® contains the active ingredient glyphosate which acts as a potent inhibitor of the penultimate step of the pathway catalyzed by the enzyme 5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) (Schönbrunn et al., 2001:

Steinrücken and Amrhein, 1980). Glyphosate has been used extensively since its introduction in

1974 as an effective broad spectrum herbicide and no major cases of evolved resistance were reported initially. However, the introduction in the late 1990s of transgenic glyphosate resistant

2 crops allowed the use of the herbicide for selective weed control and was subsequently applied to much wider areas (Powles, 2008). Since then many instances of evolved resistance in weed species have been reported (Powles, 2008; Vila-Aiub et al., 2008) and therefore novel inhibitors of the shikimate pathway will be necessary in the future. Since the pathway is also found in microbes, another potential benefit of shikimate pathway inhibitors could be as antimicrobial agents. These could prove useful in controlling apicomplexan parasites such as malaria causing

Plasmodium (Roberts et al., 2002).

Besides being the origin of many important aromatic molecules, some intermediates in the pathway are important in their own right. For example, shikimate which is produced by the enzyme 3-dehydroquinase/shikimate dehydrogenase in the fourth step of the shikimate pathway, is used as a precursor in the synthesis of the influenza inhibitor oseltamivir phosphate (Tamiflu)

(Karpf and Trussardi, 2009; Rohloff et al., 1998).

In addition to performing essential roles in plant growth and development, the shikimate pathway has important therapeutic and biotechnological applications. Many tyrosine-derived alkaloids such as morphine have medical applications and a variety of shikimate phenylalanine- derived phenylpropanoids have been shown to have wide ranging health benefits (Figure 2). Two prominent examples include the isoflavonoid genistein (Dixon and Ferreira, 2002), which has anti-cancer and cardiovascular disease benefits, and resveratrol a potent stilbene antioxidant

(Crozier et al., 2008; Halls and Yu, 2008). The natural occurrence of these molecules in food crops is often too low to be interesting from a human health point of view and genetic engineering will be necessary to make them viable nutraceuticals. Another example includes chorismate-derived folates (Figure 2). Their biosynthesis has been targeted for genetic engineering with previous attempts at biofortifying food crops with folate mainly focused on

3 increasing the flux of the pathway (Diaz de la Garza et al., 2004; Hossain et al., 2004;

Storozhenko et al., 2007).

Increasing the flux of the shikimate pathway is one promising avenue that can be pursued as being part of a more comprehensive approach to genetic engineering of plant secondary metabolism. Indeed it has been demonstrated that increasing the availability of metabolic precursors upstream of the desired product can be critical for success in plant metabolic engineering. For example, glycerol-3-phosphate synthesis limits the production of triacylglycerol in Brassica napus (Vigeolas and Geigenberger, 2004; Vigeolas et al., 2007). Similarly, the availability of isopentenyl diphosphate and dimethylallyl diphosphate are limiting precursors for the synthesis of plant terpenoids (Aharoni et al., 2005).

The shikimate pathway has already been engineered in several microbes for the production of phenylalanine and tyrosine (Chavez-Bejar et al., 2008; Gosset, 2009). The success of these efforts required an understanding of the regulation of the microbial shikimate pathway at the first enzymatic step, catalyzed by 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase

(DHS; EC 2.5.1.54).

Therefore, a thorough understanding of the shikimate pathway and how it is regulated in plants would seem to be essential for future successes in fields ranging from metabolic engineering to development of new herbicides. Since the enzyme DHS catalyzes the first and committed step of the pathway it is likely to be a key regulatory point. In fact, DHS of many bacteria and fungi have been shown to be subjected to tight regulation. However, the regulation of DHS in plants has remained elusive.

Figure 1 – The 7 enzymatic steps of the shikimate pathway. The plastid-localized pathway starts with the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP). The end product of the pathway is chorismate which is the last common precursor in the biosynthesis of tyrosine, phenylalanine and tryptophan.

Figure 2 – Shikimate pathway-derived secondary metabolites. In plants, chorismate is a precursor in the biosynthesis of folate, tyrosine in the biosynthesis of alkaloids such as morphine, tryptophan in the biosynthesis of indole hormones such as auxin and phenylalanine in the biosynthesis of isoflavanoids such as genistein and stilbenes such as resveratrol.

DAHP Synthase

The shikimate pathway consists of 7 enzymatic steps. The first and committed step of the pathway involves the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate

(PEP) which are predominantly derived from the pentose phosphate pathway and glycolysis respectively (Herrmann and Weaver, 1999) to form 3-Deoxy-D-arabino-heptulosonate-7- phosphate (DAHP) and inorganic phosphate (Figure 3). The reaction is catalyzed by the enzyme

DAHP synthase (DHS) and is a key regulatory point of the pathway in many organisms.

Two homologous families of the enzyme exist, denoted AroAI and AroAII with the

AroAI family divided into subfamilies AroAIα and AroAIβ (Subramaniam et al., 1998; Gosset et al., 2001). While plants species only possess AroAII-type enzymes microorganisms can have

AroAI or AroAII or even both. Additionally although AroAIβ enzymes are functionally DAHP synthases they are evolutionary more closely related to 3-Deoxy-D-manno-octulosonate-8- phosphate (KDOP) synthases (Subramanian et al., 1998).

The low sequence identity between AroAI and AroAII, typically around 10-20%, at first suggested both families may be evolutionary unrelated and the product of convergent evolution.

However, the structure of the first AroAII-type enzyme from Mycobacterium tuberculosis showed a highly similar tertiary structure and active site arrangement of catalytic residues suggesting the two families are the product of divergent evolution and share a common ancestor

(Webby et al., 2005a). The overall structure of DHS enzymes consists of a TIM barrel formed by

8 alternating α-helices and β-strands. Both DHS families are metalloproteins that require divalent metal ions for activity and can be inactivated by chelating agent EDTA (McClandis and

2+ 2+ 2+ 2+ 2+ 2+ 2+ Herrmann, 1978). For example, E.coli DHSs can use Mn , Fe , Cd , Co , Ni , Cu and Zn

7 with Fe2+ and Zn2+ as the preferred metals in vivo (Stephens and Bauerle, 1991) while the metal preference of Arabidopsis DHS seems limited to Mn2+ (Entus et al., 2002).

Figure 3 – Condensation reaction catalyzed by DHS. PEP and E4P cyclization is facilitated by the DHS enzyme. The reaction yields the 6-membered ring DAHP and the release of inorganic phosphate.

Regulation of AroAI DHS

The 3 DHS isozymes from E.coli are the best characterized to date. They belong to the

AroAI class and are allosterically regulated by the aromatic amino acids produced downstream of chorismate. The genes aroF, aroG and aroH code for enzymes that are Tyr-sensitive, Phe- sensitive and Trp-sensitive respectively (Herrmann and Weaver, 1999). In each case the amino acid in question binds the DHS enzyme it regulates at a site distinct from the substrate binding site. This causes a conformational change in the protein leading to rearrangement of active site residues and loss of catalytic activity. More importantly, this non-competitive mode of inhibition is independent from substrate concentration; because they bind at different sites, the substrate cannot out-compete the inhibitor. Additional control of the bacterial shikimate pathway's first step is mediated at the genetic level by Tyr- and Trp-repressors although allosteric regulation seems to be the predominant mode in vivo (Ogino et al., 1982).

Fungal enzymes of the AroAI family follow a similar pattern of regulation.

Saccharomyces cerevisiae, Aspergillus nidulans and Neurospora crassa have two isozymes that are feedback inhibited by tyrosine and phenylalanine, respectively (Schnappauf et al., 1998;

Kunzler et al., 1992). Regulation at the transcriptional level has also been demonstrated in A. nidulans and S. cerevisiae (Hartmann et al., 2001).

In organisms that possess only AroAI enzymes, allosteric feedback inhibition coupled with transcriptional regulation seems sufficient to ensure enough carbon enters the shikimate pathway to meet the organism’s need of aromatic amino acids for protein biosynthesis.

Regulation of AroAII DHS

Our understanding of AroAII-type enzymes is more fragmented. Bacteria possessing

AroAII-type enzymes usually code for a single DHS enzyme. Accordingly, Mycobacterium tuberculosis has only one AroAII-type DHS that is subject to feedback inhibition by the aromatic amino acid tyrosine, phenylalanine and tryptophan. It is moderately inhibited by each aromatic amino acid individually and also synergistically by a combination of these (Webby et al., 2005a;

Webby et al., 2010). The crystal structure of M.tuberculosis DHS shows a homotetramer formed by the association of 2 tightly associated dimers. Co-crystallization also revealed the presence of

4 bound tryptophans, 1 bound to each monomer, as well as 6 phenylalanines, one per monomer as well as another at the interface of each tightly associated dimer (Webby et al., 2010). The phenylalanine binding site at the dimer interface is thought to be the primary binding site and can also accommodate tyrosine (Webby et al., 2010). The combination of tyrosine and tryptophan can strongly inhibit the enzymes although the most significant inhibition is observed with phenylalanine and tryptophan. Conversely, this mode of regulation may not be extendable to all bacterial AroAII-type enzymes as others such as the DHS enzyme from Helicobacter pylori is not feedback inhibited by aromatic amino acids (Webby et al., 2005b).

Although plant DHS enzymes are thought to have evolved from an AroAII-type bacterial ancestor even less is known about their mode of regulation. Allosteric feedback inhibition by the aromatic amino acids has never been demonstrated. Arogenate, an intermediate in Phe and Tyr synthesis, has a capacity to inhibit plant DHS, however, concentration of arogenate in vivo is deemed too low for it to play a physiologically significant role (Rubin and Jensen, 1985).

Notwithstanding this lack of direct effectors of DHS activity, previous studies have showed that plant DHS enzymes do perform an important role in regulating shikimate pathway

10 flux. For example, transcriptional regulation of DHS-encoding genes has been observed in

Arabidopsis thaliana in many instances such as following infiltration with the plant pathogen

Pseudomonas syringae (Keith et al., 1991), after physical wounding and methyl-jasmonate treatment (Devoto et al., 2005; Yan et al., 2007) and UV-B and ABA treatment (Ramani et al.,

2010; Catala et al., 2007; Leonhardt et al., 2004). In addition, DHS isozyme-specific spatio- temporal transcript expression patterns were also reported in tomato (Görlach et al., 1993).

Moreover, contrary to bacteria where the majority of aromatic amino acids are used for protein biosynthesis, plants commit a substantial amount of shikimate pathway flux to the biosynthesis of secondary metabolites many of which have commercial interest. For example, phenylalanine-derived phenylpropanoids alone include more than 8000 different compounds and can make up to 30-45% of plant organic matter (Razal et al., 1996). A good understanding of how plants achieve the partitioning of carbon to meet the demands from these specialized metabolic pathways and how the activity of the shikimate pathway in particular is attuned to those needs will be important in designing new and more comprehensive approach to metabolic engineering.

Regulation of Arabidopsis thaliana DHS

There are 3 loci coding for DHS enzymes in the Arabidopsis thaliana genome annotated

At4g39980, At4g33510 and At1g22410 and referred to as DHS1, DHS2 and DHS3 respectively.

As with the other enzymes of the shikimate pathway, each DHS has an N-terminal chloroplast transit peptide (cTP) which is cleaved upon plastid import (Herrmann and Weaver, 1999). At least one enzyme, DHS1, has been shown to be regulated by photosynthetically-reduced

11 thioredoxin which suggest that light conditions play a prominent role in regulating shikimate pathway flux by redox-regulation of DHS1 (Entus et al., 2002). The 3 DHS isozymes in

Arabidopsis also have different spatio-temporal expression pattern. DHS1 is most highly expressed in rosette leaves and in the quiescent center cells of the root (Figure 4).

Additionally, previous studies in our laboratory have demonstrated the involvement of aromatic amino acids in the regulation of the shikimate pathway at the DHS level. Homozygous

T-DNA insertion lines for the 3 DHS isozymes were used to probe the regulatory mechanism of each. Phenotypically, single DHS knockout lines did not differ from Columbia (Col-0) wild type plants under normal growth conditions but differences appear when plants are grown under stressed conditions (Crowley, 2006).

For example, dhs1 seedlings were shown to be hypersensitive to tyrosine supplementation (Figure 5) (Crowley, 2006). The addition of 150 µM tyrosine did not produce any noticeable phenotypic differences in Col-0, dhs2 or dhs3 but was enough to inhibit growth of dhs1 seedlings (Crowley, 2006). In a similar fashion dhs3 seedlings were shown to be sensitive to tryptophan supplementation (Shahinas, 2008). The phenotypic characteristics of hypersensitivity consisted of stunted growth and reduced root length. Subsequent kinetic experiment showed that none of the 3 Arabidopsis thaliana DHS are directly inhibited by tyrosine in vitro (Crowley, 2006). I therefore suspect a novel mode of regulation for plant DHS enzymes whereby aromatic amino acids regulate the flux of the pathway at its entry point albeit indirectly. Previous experiments have also identified auxin as potentially playing a role in the dhs1 hypersensitivity phenotype (Shahinas, 2008).

Figure 4 – Organ specific map of DHS expression in Arabidopsis thaliana. Expression of DHS1, DHS3 and DHS3 visualized using the Arabidopsis eFP browser at the Bio-Array Resource for Plant Biology (Winter et al., 2007). Yellow represents low and red represents high expression.

Figure 5 – Hypersensitivity of Arabidopsis dhs1 mutants to tyrosine supplementation. 8 days old Arabidopsis thaliana dhs1, dhs2 and dhs3 mutants and wild type Col-0 seedling grown on Murashige-Skoog media supplemented increasing concentration of tyrosine (Crowley, 2006).

Aromatic Amino Acid Biosynthesis

The end product of the shikimate pathway, chorismate, represents the branch point in the biosynthesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan. The first and committing step of tyrosine and phenylalanine synthesis is catalyzed by 3 chorismate mutases

(CM1-3) in Arabidopsis thaliana (Mobley et al., 1999) (Figure 6). The reaction consist of the conversion of chorismate to prephenate and 2 of the 3 isozymes, CM1 and CM3, are feedback inhibited by the end-product of the pathway, tyrosine and phenylalanine (Eberhard et al., 1996;

Mobley et al, 1999). The same 2 isozymes are also activated by tryptophan while CM2 is insensitive to all 3 amino acids (Mobley et al., 1999). From prephenate there are at least 2 possible routes to phenylalanine. One possible route whereby prephenate is converted to phenyl

14 pyruvate by prephenate dehydratase (PDT) could potentially bring a minor contribution to the phenylalanine pool but the conversion of phenyl pyruvate to phenylalanine would require an endogenous aromatic amino acid aminotransferase (AAAAT) and none have so far been identified in plants (Tzin and Galili, 2010). The transamination of prephenate to arogenate by prephenate aminotransferase (PAT) and the subsequent decarboxylation and dehydration of phenylalanine by arogenate dehydratase (ADT) seems to be the predominant phenylalanine biosynthesis pathway in plants (Cho et al., 2007; Maeda et al., 2010)

The main biosynthetic pathway to tyrosine only differs from phenylalanine at the last step where arogenate dehydrogenase (TyrA) catalyzes the decarboxylation of arogenate to tyrosine.

There are 2 isoform of TyrA in Arabidopsis thaliana and both are subject to feedback inhibition by tyrosine (Rippert and Matringe, 2002b). In addition to the feedback inhibition and activation of CM by the end-product of the pathway, CM1 expression is analogous to that of DHS genes and its transcription is elicited in response to wounding or exposure to the plant pathogen

Pseudomonas syringae (Mobley et al., 1999).

Tryptophan biosynthesis is initiated by the conversion of chorismate to anthranilate by the enzyme anthranilate synthase (AS), a heterotetramer formed by 2 α and 2 β subunits. The α subunits are thought to catalyze the aromatization reaction while the β subunits transfer the amino group from glutamine (Li and Last, 1996; Tzin and Galili, 2010). Regulation of tryptophan biosynthesis is achieved through a mix of allosteric feedback inhibition and transcriptional regulation. Tryptophan can bind to the α subunit which allows it to allosterically inhibit the AS complex (Kreps et al., 1996; Spraggon et al., 2001; Kanno et al., 2005). Several plant mutants have been identified with amino acids substitutions at the tryptophan binding site which resulted in insensitivity of AS to feedback inhibition and greater accumulation of

15 tryptophan (Li and Last, 1996; Kanno et al., 2005). These tryptophan insensitive mutants also exhibited increased resistance to tryptophan analogs such as 5MT, 6MA and αMT (Kreps et al.,

1996; Song et al., 1998). Alternatively, many enzymes of the tryptophan biosynthetic pathway are transcriptionally regulated, including AS. For instance, expression of the third enzyme of the pathway, phosphoribosylanthranilate isomerase (PAI) is induced by UV irradiation or the abiotic elicitor silver nitrate (He and Li, 2001) and expression of the fourth enzyme of the pathway, indole-3-glycerol phosphate synthase (IPGS) is controlled by jasmonate (Dombrecht et al.,

2007).

As with the shikimate pathway, aromatic amino acid biosynthesis in plants is thought to occur exclusively in the plastid. In Arabidopsis thaliana the corresponding genes of all enzymes involved in tyrosine, phenylalanine and tryptophan biosynthesis code for a chloroplast transit peptide (cTP) at the N-terminal of the proteins with the exception of ADT1 and CM2. The ADT1 coding gene does contain an extra N-terminal region when compared to bacterial ADTs but it is not recognized as a cTP by bioinformatics analysis. Nevertheless, ADT1 was immunodetected in the chloroplast fraction and ADT1-GFP fusion proteins were localized to the chloroplast (Rippert et al., 1999). On the other hand, it has been demonstrated that CM2 is cytosol-located (d’Amato et al., 1984) but whether or not it contributes to tyrosine and phenylalanine biosynthesis is still debated (Rippert et al., 2009).

Figure 6 – Aromatic amino acid biosynthesis and regulation. The first step of tryptophan biosynthesis is catalyzed by anthranilate synthase (AS) and first step in tyrosine and phenylalanine biosynthesis is catalyzed by chorismate mutase (CM). Arrows represent feedback activation and stopped lines represent feedback inhibition.

Auxin Biosynthesis

Auxin is a phytohormone regulating growth and development and is also involved in responding to environmental signals. The most abundant auxin in higher plants is indole-3-acetic acid (IAA) (Bhalerao and Bennett, 2003). Specifically, auxin plays an important role in directing cellular organization in Arabidopsis thaliana roots and concentration gradients allow it to control cell division, lateral root initiation and elongation, meristem patterning and gravitropism in a dose-dependent manner (Sabatini et al., 1999; Bhalerao and Bennett, 2003; Friml, 2003). The rate of synthesis in source tissue and the rate of transport to the target cells are determining factors in the formation of these gradients. Auxin is synthesized in many parts of the plant with the highest biosynthetic capacity found in young leaves (Ljung et al., 2002). Although the highest capacity for auxin biosynthesis is in the shoot, it is synthesized in many other locations including almost all cell types of the root apex (Petersson et al., 2009). The respective

17 contribution of those 2 sources to the auxin maxima found in the root apical region of

Arabidopsis thaliana seedlings is not well established, but both contribute significantly (Ljung et al., 2005; Ikeda et al., 2010).

The concentration gradients necessary to direct root growth is partially achieved by passive diffusion coupled with influx and efflux carrier dependent active transport between individual cells (Tromas et al., 2010). In Arabidopsis thaliana 8 putative auxin efflux carrier belonging to the PIN family have been identified (Paponov et al., 2005) as well as 4 putative auxin influx carrier, AUX1 and LAX1-3 (Yang et al., 2006; Swarup et al., 2008; Petrášek et al.,

2006).

Local biosynthesis of auxin in the root also contributes significantly to the gradient required for normal root development (Ikeda et al., 2009; Vandenbussche et al., 2010). There are several biosynthetic routes to auxin in plants which can be divided into tryptophan-dependent or tryptophan-independent. It is not clear what are the steps involved in the tryptophan-independent pathway but accumulation of auxin conjugates (Last et al., 1991; Radwansky et al., 1996) in tryptophan synthase mutants trp3-1 and trp2-1 (Normanly et al., 1993; Ouyang et al., 2000) strongly suggests it is present in plants (Tromas and Perrot-Rechenmann, 2010). However, the main contribution to auxin in Arabidopsis thaliana relies on tryptophan-dependent biosynthesis

(Ljung et al., 2005). At least 4 different tryptophan-dependent pathways have been identified as well as many implicated enzymes (Ljung et al., 2002; Woodward and Bartel, 2005). On the other hand, many enzymes still remain unknown and which steps are rate-limiting is also uncertain

(Tromas and Perrot-Rechenmann, 2010). Additionally, our knowledge of how auxin homeostasis is achieved is still imperfect but mechanisms of storage and degradation are involved. Temporary storage of auxin is achieved via reversible conjugation to sugars or amino acids such as alanine

18 or leucine and irreversible conjugation to glutamate and aspartate leads to catabolic degradation

(Bialek and Cohen, 1989; Tam et al., 2000; Kowalczyk et al., 2001). Although auxin homeostasis and the maintenance of an adequate gradient in the root tips of developing plants may be complex, disruption of its biosynthesis, for example by repression or overexpression of auxin biosynthetic genes, does affect it (Cheng et al., 2006, Zhao et al., 2010).

Thesis Objective

The first step of the pathway is a key regulatory point and previous studies in our laboratory have uncovered some of the molecular basis underlying its regulation. For example,

Arabidopsis thaliana dhs1 knockout mutant plants grow normally under control conditions but are hypersensitive to tyrosine supplementation (Crowley, 2006). In addition, preliminary results suggest the hypersensitivity of dhs1 seedlings to tyrosine supplementation may be associated with a decrease in levels of auxin in Arabidopsis root tips (Shahinas, 2008).

The objective of this study is to explore the response of Arabidopsis thaliana associated with the dhs1 mutation at the metabolomics level. More specifically, I investigated the changes in aromatic amino acid in dhs1 mutants and explored the effects of the mutation on downstream metabolites such as the phytohormone auxin. I also looked at the effect of aromatic amino acid treatments on those pathways in the hope that better knowledge of metabolic differences between dhs1 and wild type would help us understand how the shikimate pathway is regulated in plants.

Materials and Methods

Materials

Arabidopsis thaliana plants used in this study were of the Col-0 ecotype and from a T-

DNA insertion line (dhs1-3; salk_055360) from the Salk institute which was purchased from the

Arabidopsis Biological Resources Center. Col-0 plants homozygous for a DR5::GUS reporter cassette were obtained from Dr. Tom Guilfoyle. The Murashige and Skoog (MS media) on which seedlings were grown and the aromatic amino acid tyrosine, phenylalanine and tryptophan that were used in the treatments were from Sigma-Aldrich. PCR primers were purchased from

Integrated DNA Technologies.

Phylogenetic Analysis

DNA sequences coding for plant AroAII DHS were obtained by BLAST (Altschul et al.

1990) analysis using Arabidopsis thaliana DHS1, DHS2 and DHS3 coding sequences as bait.

Sequences from Vitis vinifera, Medicago truncatula, Ricinus communis, Populus trichocarpa,

Zea mays, Sorghum bicolor, Oriza sativa (Japonica) were obtained from phytozome

(www.phytozome.net), Physcomitrella patens from COSMOSS (www.cosmoss.org) and

Mycobacterium tuberculosis from the Broad institute (www.broadinstitute.org). Coding sequences were aligned by codons using ClustalW (Chenna et al., 2003) and manually curated in

MEGA4 (Tamura et al., 2007). The highly divergent N-terminal regions of the sequences coding for chloroplast transit peptides were omitted from the alignment. For bacterial AroAI

20 phylogenetic analysis DHS-coding sequences were retrieved by performing nucleotide BLAST on NCBI website (http://www.ncbi.nlm.nih.gov/) using E.coli DHS sequences as bait. The DHS phylogenies of AroAI and AroAII were reconstructed in MEGA4 (Tamura et al., 2007) using the

Neighbor-Joining method and p-distance model with 1000 bootstrap iterations.

Plant Growth Conditions and Root Length Assays

Seeds were sterilized in 20% commercial bleach with 0.05 % Tween-20 and washed with double distilled water before stratification at 4°C in the dark for 2 days. Seeds were germinated on agar plates containing MS media (Murashige and Skoog, 1962) (Sigma) and 2.5mM morpholino ethane sulfonic acid (MES) (pH 5.7) (Bioshop) under continuous light. For the root length assays tyrosine, phenylalanine, tryptophan or 5-methyltryptophan (5-MT) (Sigma) were autoclaved separately before being added to the agar media. Seedlings were grown vertically under continuous light for 8 days for the aromatic amino acids analysis and 5-MT supplementation assays.

Genomic DNA Extraction

Arabidopsis genomic DNA was extracted from leaves. The leaves were frozen in liquid nitrogen and homogenized to a fine powder using a mortar and pestle. To each sample, 200 µl of extraction buffer (200 mM Tris-HCl pH 8 (BioShop), 25 mM ethylenediaminetetraacetic acid

(EDTA) pH 8 (BioShop) and 0.5 % sodium dodecyl sulfate (SDS, BioShop) was added.

Subsequently, 100 µl phenol (Invitrogen): chloroform (EMD): isoamyl alcohol (BioShop)

(24:24:1) was added. The samples were vortexed and centrifuged at 14000 rpm for 10 minutes.

The aqueous supernatant was transferred to a new tube and DNA was precipitated by addition of an equal volume of isopropanol (EM Science). DNA was pelleted by centrifugation for 10 minutes at 1400 rpm. Supernatant was discarded and DNA pellet was air dried for approximately

30 minutes.

PCR Genotyping of T-DNA Lines

To verify the genotype and homozygosity of T-DNA lines used in subsequent experiments 2 different PCR reactions were used. The first reaction used 2 gene specific primers

(DHS1fwd and DHS1rev) and the other used a left border primer complementary to the T-DNA

(Lba1) and a gene specific primer (DHS1fwd) (Table 1). A pfu polymerase purified in our laboratory was used for the PCR reactions that followed the program outlined in Table 2.

Figure 7 - DHS1 gene structure and position of T-DNA insertion. Exons are shown as black boxes and introns as black lines. The triangle indicates the position of the T-DNA insertion in third exon. Annealing regions for genotyping primers are shown as arrows.

Table 1 – Primers used for genotyping

Primer name Primer sequence Lba1 5'-TGGTTCACGTAGTGGGCCATCG-3' DHS1fwd 5'-GAGCCTTTGCCACTGGAGGTT-3' DHS1rev 5'-TCTCATGTTCTCGGCACCCAT-3'

Table 2 - PCR program used for genotyping

Step temperature (°C) time (min) repeat Denaturation 95 1:00 x1 Denaturation 95 0:30 Annealing 52 0:30 x35 Elongation 72 3:00 . Termination 72 10:00 x1

Aromatic Amino Acid Treatment

Arabidopsis seedlings were grown vertically for 8 days to prevent the root from penetrating the agar and thus become inaccessible to liquid treatment. Plates containing seedlings were put in a horizontal position and treated with aromatic amino acid solutions (6 ml/plate) for

8 hours. The amino acid solution consisted of MS media (Murashige and Skoog, 1962) (Sigma) and 2.5 mM morpholino ethane sulfonic acid (MES) (pH 5.7) (BioShop) and 500 µM of one or a combination of tyrosine, phenylalanine and tryptophan (Sigma). Amino acids were autoclaved

23 separately from MS and MES. After 8 hours, seedlings were thoroughly washed with distilled water and excess water removed with Kimwipe and frozen immediately in liquid nitrogen.

Metabolite Extraction and Derivatization

Frozen seedlings were homogenized to a fine powder in liquid nitrogen using a mortar and pestle. 300 µl Methanol was added and sample was grounded further. 300 µl 50 mM Tris-

HCl pH 7.5 (BioShop) 100 mM NaCl (BioShop) was added. After mixing, sample was put on ice for 10 minutes. 800 µl Chloroform (BioShop) was added and sample was vortexed and incubated on ice for another 10 minutes. After centrifugation at 5000 rpm for 5 minutes, 450 µl aqueous layer was recovered and lyopholyzed until all liquid had evaporated. Dried metabolites were resuspended in 50 µl 40 mM LiCO3 pH 9.5 (BioShop). Resuspended samples were derivatized for 30 minutes by addition of 2 5µl 10 mg/ml Dansyl Chloride (Sigma) in acetonitrile (Caledon

Laboratories).

HPLC Analysis

The separation and quantification of amino acid dansyl derivatives was performed with an Agilent technologies 1200 series HPLC with a 20 µl injection loop and a Zorbax SB-CB 4.6 x

150 mm, 3.5 µm, C-18 column with a guard column of the same material. The mobile phase used was filtered and degassed double distilled water, 0.1% formic acid (A) and HPLC grade acetonitrile (Caledon) (B).

Table 3 – Elution profile for HPLC separation of amino acid dansyl derivatives. Mobile phase consisted of a mixture of double distilled H2O 0.1% formic acid (A) and acetonitrile (B).

Time (min) % A % B 0 95 5 20 0 100 22 0 100 23 95 5 25 95 5

Auxin Quantification by LC-MS

8 days old Arabidopsis seedlings were used for auxin quantification. Typically, 3 seedlings with synchronized growth were selected for each sample to be homogenized. Seedlings were flash frozen in liquid nitrogen and collected in round bottom 2 ml eppendorf tubes to which

750 µl 80 % HPLC-grade methanol (Fisher Scientific), 1 % glacial acetic acid (EMD) was added along with 3 zirconium beads. Samples were homogenized for 5 minutes at 30 cycles/seconds in

TissueLyserII (Qiagen) and stored at 4°C overnight. The following day, 500 µl 80 % methanol, 1

% acetic acid in HPLC-grade water (Caledon laboratories Ltd.) was added to each samples before centrifugation at 12 000 rpm for 5 minutes at 4°C. Supernatant was collected in new tube and pellet was washed twice with 250 µl 80 % methanol, 1 % acetic acid. Supernatant was lyopholyzed and resuspended in 1 ml 1 % acetic acid for Oasis HLB column (Waters) purification. The HLB columns were equilibrated with 1 ml 1 % acetic acid in methanol followed by 1 ml 1 % acetic acid water. Samples were then applied to columns, washed with 1 ml 1 % acetic acid in water twice and eluted with 1 ml 1 % acetic acid in methanol. Elutes were

25 lyopholyzed and resuspended in 500 µl 1 % acetic acid in water before Oasis WAX anion- exchange column (Waters) purification. WAX columns were equilibrated with 1 ml methanol followed by 1 ml 1 % acetic acid in water. Samples were applied to column, washed with 1 % acetic acid in water followed by 1 ml methanol and finally eluted with 1 ml 1 % acetic acid in methanol. Elutes were lyopholyzed and stored in -20°C freezer. Samples were re-suspended in

50 µl methanol and analyzed with LC/MS.

GUS Staining

Seedlings used for GUS staining were grown vertically on standard MS media

(Murashige and Skoog, 1962) for the control. For the different treatments, the MS media was supplemented with the aromatic amino acids. The amino acids were autoclaved separately from

MS and MES and mixed together prior to pouring the media. 8 days old Arabidopsis dhs1xDR5::GUS and DR5::GUS seedlings were incubated with staining solution consisting of

1M Na2HPO4/NaH2PO4 (Bioshop) pH 7.5, 2mM X-Gluc (Bioshop), 5 mM EDTA (Bioshop) pH

8 and 0.01% Triton X-100 (Bioshop). The staining solution also contained potassium ferricyanide and potassium ferrocyanide which facilitate the formation of the final blue-colored product as well and preventing further oxidation which would change its color. Potassium ferri/ferrocyanide also acts as an inhibitor of GUS enzyme so an optimal concentration has to be found experimentally. Usually, a high ferri/ferrocyanide concentration results in a stringent reaction and punctate staining while a lower ferri/ferrocyanide concentration can detect lower auxin concentrations but results in more diffuse staining (Mascarenhas and Hamilton, 1992).

Here, concentrations of 5 and 2.5 mM ferri/ferrocyanide were used for staining. Seedlings were

26 incubated in the staining solution at 37ºC for 16 hours when stained with 5 mM ferri/ferrocyanide or for 8 hours when stained in 2.5 mM ferri/ferrocyanide to prevent excessive diffusion. After staining, seedlings were incubated in 3:1 ethanol: acetic acid for 2 hours at room temperature. Before visualization by differential interference contrast (DIC) microscopy seedlings were washed with 70 % ethanol and transferred into distilled water.

Microarray Data Analysis

Transcriptome analysis data was collected by Dea Shahinas (Shahinas, 2008). Mainly total RNA was extracted from whole-seedlings at 8 days post-germination for transcriptome analysis. Flash frozen plant material was ground to a fine powder in liquid nitrogen and total

RNA was extracted from each sample using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). 10 µg of total RNA was used for whole-genome transcript analysis using the ATH1 Genome Array according to the manufacturer's instructions (Affymetrix) at the

Centre for the Analysis of Genome Evolution & Function at the University of Toronto (CAGEF).

For each treatment, RNA was extracted from three replicate biological samples, and each was hybridized to an ATH1 GeneChip. GeneChip data analysis was performed Geoff Fucile using the

BioConductor suite (Gentleman et al., 2004) in R (R Development Core Team, 2009 - http://www.R-project.org) using the “affy” Bioconductor package (Gautier et al., 2004). Pre- processing of Affymetrix CEL files consisted of background correction using the RMA.2 method

(Wu et al., 2004), normalization by the quantiles method, and summarization using the median polish method (Tukey, 1977). Affymetrix CEL files were pre-processed as two sets of triplicate biological samples for all pairwise tests for significant differential expression. Pre-processed

27 log2 transformed probesets with an interquartile range ≤0.5 were removed using the “genefilter”

Bioconductor package (Gentleman et al., 2004). SAM (Tusher et al., 2001) detection of pairwise significantly differentially expressed genes was conducted in R/Bioconductor as two class unpaired experiments with 500 permutations. SAM delta values were selected based on minimization of the false discovery rate and manual inspection of observed versus expected

SAM scores. The functional annotation and evaluation of significantly differentially expressed genes was conducted using MapMan (Thimm et al., 2004). Functional enrichment analysis of genes with significant differential expression between pairwise comparisons was done using the

Classification Superviewer (Provart and Zhu, 2003).

Results

AroAII-type DHS Gene Duplicate Retention in Higher Plants

Representative sequences from bacterial species were used to reconstruct the phylogeny of AroAIα-type DHS genes (Figure 8). Every bacterial species used in the analysis contained 3

DHS isogenes that clustered separately in AroF, AroG and AroH orthologous clades which represent sequences coding for tyrosine-sensitive, phenylalanine-sensitive and tryptophan- sensitive enzymes respectively (Figure 8).

Subsequently, the phylogeny of DHS genes belonging to the AroAII family was reconstructed to determine whether a similar conserved topology exists in plants (Figure 9). The coding sequences used in this analysis were obtained from fully sequenced organisms to ensure an accurate representation of the number of DHS-coding genes in each organism. The phylogenetic reconstruction is consistent with previous reports that higher plant DHS genes have a bacterial origin (Gosset et al., 2001) with algal sequences clustering closely to the

M.tuberculosis sequence. The phylogeny produced is also in agreement with the expected topology of speciation of algae, moss, monocot and dicot plant species. Whereas the algal genomes analyzed possess only one DHS locus, the plant genomes possess several. In fact, the number of loci differs substantially between the plant clades. For example, the Physcomitrella patens genome contains seven separate DHS-encoding loci while the dicot Medicago truncatula has two DHS loci and the monocots Oryza sativa and Sorghum bicolor have four. The monocot and dicot DHS sequences clustered separately, suggesting lineage-specific DHS gene duplication and retention. The Arabidopsis thaliana genome contains three DHS loci – At-DHS1, At-DHS2,

29 and At-DHS3. Each of these sequences clusters with other dicot DHS, implying some degree of

DHS gene duplicate conservation in the dicot family.

Figure 8 – Phylogenetic reconstruction of bacterial AroAI DHS. Neighbor-joining phylogenetic tree of DHS nucleotide coding sequences based on amino acid alignment. Consensus scores above 70 from 1000 iteration bootstrap are shown. AroF genes code for Tyr- sensitive, AroG for Phe-sensitive and AroH for Trp-sensitive enzymes.

Figure 9 - Phylogenetic reconstruction of AroAII DHS. Neighbor-joining phylogenetic tree of DHS nucleotide coding sequences based on amino acid alignment. Consensus scores above 70 from 1000 iteration bootstrap are shown.

Conservation of 3D Structure in MtDHS and AtDHS

There is currently no representative AroAII-type DHS protein structures available for the plant kingdom. However, the structure of the M. tuberculosis AroAII-type DHS has been determined (Webby et al., 2005a) and resembles other AroAI-type DHS enzymes (Figure 10). It consists of a TIM barrel core decorated by structural elements that could potentially be involved in allosteric regulation of the enzyme. There are two main structural differences between E.coli and M.tuberculosis DHS. First, the two β-strand β5a and β5b in the E.coli structure are absent in

M.tuberculosis. This structural domain is adjacent to the allosteric binding site of phenylalanine in the E.coli enzyme and residues from the two β-strands take part in the binding interactions

(Shumilin et al., 2002). Phenylalanine can also bind to M.tuberculosis DHS but co-crystallization shows that it binds on the other side of the protein, close to the N-terminal. Secondly, two α- helices, α2a and α2b, from the M.tuberculosis DHS form an additional structure decorating the central TIM barrel that is absent in the E.coli enzyme. This additional structural element has been proposed to have regulatory properties (Webby et al., 2005a). The 2 helices are thought to be part of the tryptophan binding site.

The theoretical structures of the three Arabidopsis isozymes were determined with the

Phyre homology modeling method (Kelley and Sternberg, 2009) using the M.tuberculosis structure as a template [PDD: 2B7O]. The overall folds of the three Arabidopsis DHSs are highly conserved and very similar to the M.tuberculosis structure. Notably, the putative regulatory α- helices, α2a and α2b, flanking the TIM-barrel of the M.tuberculosis structure were resolved in the predicted Arabidopsis enzymes as well (Figure 10). These two helices have been identified as forming part of the putative tryptophan binding site and 3 of the 4 residues interacting with tryptophan in M.tuberculosis DHS are conserved in the Arabidopsis enzymes (Figure 11).

Figure 10 – Structural comparison of AroAI and AroAII DHS. Structural comparison of A) structure of Phe regulated Ec-DHS complexed with Phe B) structure of Mt-DHS complexed with Phe and Trp and C) predicted structure of At-DHS1. Complexed allosteric ligands represented as sphere; Phe is in green and Trp in red.

Figure 11 - Multiple sequence alignment of M.tuberculosis DHS and of Arabidopsis DHS1, DHS2 and DHS3. Only sequences involved in the putative tryptophan binding site of M.tuberculosis and the corresponding Arabidopsis sequences shown. Residues highlighted in grey are conserved among all 4 sequences and residues highlighted in yellow represent the putative tryptophan binding residues in M.tuberculosis DHS.

Verification of Col-0 and dhs1 Genotypes

To investigate the physiological role of DHS1 in Arabidopsis a T-DNA insertion line from the Salk institute was used (dhs1-3; salk_055360). Additionally, to ensure that characteristics such as tyrosine hypersensitivity were specifically caused by mutation in the

DHS1 gene an independent T-DNA insertion line (dhs1-4; salk_117853) has previously been used to confirm it (Crowley, 2006). Two sets of PCR reactions were performed to verify that plants used in subsequent experiments were homozygous wild-type or homozygous dhs1 mutants. DHS1 gene-specific primers were used to verify the presence of the integral DHS1 gene while one DHS1 gene-specific primer and one T-DNA specific primer were used to verify the presence of the TDNA insert in the dhs1 line (Figure 12).

Figure 12 – Verification of homozygosity for Arabidopsis Col-0 and T-DNA mutant dhs1. A) dhs1fwd and dhs1rev are gene specific primers and Lba1 is a T-DNA specific primer. B) DHS1 gene structure showing T-DNA insertion in the third exon.

Changes in Intracellular Levels of Aromatic Amino Acids in Atdhs1

The aromatic amino acids tyrosine, phenylalanine and tryptophan are produced downstream of the shikimate pathway and have as common precursor chorismate. The contents of these 3 aromatic amino acids in Arabidopsis seedlings were quantified in order to understand their role in the physiological differences observed between Col-0 and dhs1 mutants under stressful conditions especially after treatment with exogenous tyrosine. The seedlings were exposed to 3 different treatments; MS solution only (control), MS solution with 500 µM tyrosine and MS solution with 500 µM tyrosine and 500 µM phenylalanine. The metabolite extracts of

Arabidopsis thaliana seedlings that had been subjected to the different treatments were reacted with dansyl-chloride to create primary amine derivatives absorbing strongly at 254 nm. Although aromatic amino acids intrinsically absorb in the UV spectrum, dansyl derivatives were preferred to facilitate their detection. The aqueous phase of the metabolite extract was kept and its molecular components were separated by HPLC using an hydrophobic C18 column and a mobile phase that consisted in a gradient of increasing concentration of acetonitrile (ACN) in water

(Table 3). Standard solutions of tyrosine, phenylalanine and tryptophan dansyl derivatives were used to determine the retention time of each in order to be able to identify the corresponding peaks in metabolite extracts mixtures. The standard tyrosine, phenylalanine and tryptophan dansyl derivatives eluted at 15.62, 16.22 and 19.69 minutes respectively (Figure 13).

Unfortunately, tryptophan levels in Arabidopsis thaliana seedlings metabolite extracts were later found to be too low to be accurately quantified. Therefore, standard samples containing increasing concentrations of tyrosine and phenylalanine dansyl derivatives were used to build standard curves and establish their linear dynamic range for each (Figure 14 and 15).

Figure 13 – Elution profile of standard amino acid dansyl derivatives. HPLC separation achieved with C18 column and mobile phase consisting of ACN and water. Quantification achieved by measuring absorbance at 254 nm.

Figure 14 - Standard curve for the absorbance of dansyl-Tyr derivative as a function of its concentration. Standard solutions of Tyr were reacted with Dansyl chloride were separated and quantified by HPLC.

Figure 15 - Standard curve for the absorbance of dansyl-Phe derivative as a function of its concentration. Standard solutions of Phe were reacted with Dansyl chloride were separated and quantified by HPLC.

The intracellular levels of tyrosine and phenylalanine detected after control treatment were similar in both Col-0 and dhs1 seedlings, at around 15 and 4 nmol/gFW respectively, and no statistically significant differences were observed between the 2 genotypes (Figure 16). As was expected, the levels of tyrosine increased following tyrosine treatment in both Col-0 and dhs1 seedlings, to around 60 nmol/gFW, which represents an approximately 4-fold increase compared to the control. In contrast, levels of phenylalanine following the same treatment remained unchanged in dhs1 seedlings while a 3-fold increase to approximately 15 nmol/gFW was detected in Col-0 (Figure 17). Finally, concentrations of tyrosine and phenylalanine measured in seedlings that had been co-treated with both tyrosine and phenylalanine were higher than those detected in the control seedlings but no significant differences were observed between

Col-0 and dhs1 (Figure 18).

Figure 16 – Concentration of tyrosine (Tyr) and phenylalanine (Phe) in Arabidopsis seedlings after 8 hr control treatment. Amino acid quantified by HPLC analysis of dansyl- derivatives. Error bars represent standard error, n=3.

Figure 17 – Concentration of tyrosine (Tyr) and phenylalanine (Phe) in Arabidopsis seedlings after 8 hr treatment with 500 µM exogenous tyrosine. Amino acid quantified by HPLC analysis of dansyl-derivatives. Error bars represent standard error, n=3, * denotes statistical difference (p=0.01).

Figure 18 – Intracellular concentration of tyrosine (Tyr) and phenylalanine (Phe) in Arabidopsis seedlings after 8 hr treatment with 500 µM exogenous tyrosine and phenylalanine. Amino acid quantified by HPLC analysis of dansyl-derivatives. Error bars represent standard error, n=3.

Sensitivity of dhs1 to 5-Methyltryptophan Supplementation

Direct measurement of tryptophan levels in Arabidopsis seedlings using dansyl chloride derivatization and HPLC analysis was not possible because the endogenous levels of tryptophan are too low. Instead, to investigate how the intracellular pool of tryptophan is affected in

Arabidopsis dhs1 mutants, an indirect assay using sensitivity to 5-methyltryptophan (5-MT) was used (Tzin et al., 2009). 5-MT is a tryptophan analog known to inhibit the enzyme anthranilate synthase which catalyzes the first reaction in tryptophan biosynthesis (Kisaka et al., 1996), and

5-MT resistance has been associated with higher tryptophan levels in plants (Li and Last, 1996).

Seedlings of both Col-0 and dhs1 genotypes were grown on media containing 5-MT varying in concentration from 5 to 20 µM. The dhs1 seedlings were found to be more sensitive to 5-MT and

40 were overall smaller than Col-0 seedlings (Figure 19). Shorter root length was also evident and a statistically significant decrease in root length was observed between Col-0 and dhs1 for all the concentrations of 5-MT tested (Figure 20). The increased 5-MT sensitivity in dhs1 mutants suggests that tryptophan biosynthesis is indeed impaired in those seedlings. To validate the 5-MT sensitivity as a measure of tryptophan availability, seedlings were treated with both 5-MT and tryptophan. The decreased root length observed for both Col-0 and dhs1 seedlings was partially rescued when they were grown on media containing 20 µM 5-MT in combination with 50 µM tryptophan (Figure 20).

To explore the possible link between lower tryptophan levels and tyrosine hypersensitivity in dhs1, seedlings were grown in the presence of both. The decrease in root length observed in Col-0 seedlings grown on 5-MT was completely rescued by addition of 100

µM tyrosine in the media (Figure 21) while no such rescue was observed for dhs1 seedlings

(Figure 22). This suggests that addition of tyrosine in Col-0 seedlings did not affect tryptophan levels which would explain why they are more resistant to 5-MT inhibition. In dhs1 the presence of 100 µM tyrosine was enough to maximal effect on the root length phenotype and 5-MT treatment had no additional effects.

Figure 19 – Increased sensitivity of dhs1 seedlings to 5-Methyltryptophan (5-MT). Phenotypic differences between8 days old Col-0 and dhs1 seedlings grown on MS media supplemented with increasing concentration of 5-MT.

Figure 20 – Effect of exogenous 5-Methyltryptophan (5-MT) on Arabidopsis dhs1 seedlings. Difference in root length between 8 days old Col-0 and dhs1 seedlings when grown on MS media supplemented with 5-MT. Mean values of 9 or more measurements shown with standard error, n≥9. * denotes statistical differences between Col-0 and dhs1 (p=0.001).

Figure 21 - Effects of exogenous 5-Methyltryptophan and tyrosine on Col-0 root length. Root length of 8 days old Col-0 seedlings grown on MS media supplemented with 5-MT (white bars) or 5-MT and 100 µM tyrosine (grey bars). Mean values of 9 or more measurements shown with standard error, n≥9. * denotes statistical difference (p=0.001).

Figure 22 - Effects of exogenous 5-Methyltryptophan and tyrosine on Arabidopsis dhs1 root length. Root length of 8 days old dhs1 seedlings grown on MS media supplemented with 5-MT (white bars) or 5-MT and 100 µM Tyr (grey bars). Mean values of 9 or more measurements shown with standard error, n≥15. * denotes statistical difference (p=0.001).

Auxin Levels in Col-0 and dhs1 Whole Seedlings

Preliminary results (Shahinas, 2008) suggested a correlation between hypersensitivity to tyrosine supplementation and a reduction of auxin levels in the root apical region of Arabidopsis dhs1 seedlings. This could be due to the decrease tryptophan in dhs1 seedlings as deduced from increase 5-MT sensitivity. To further explore the relationship between tyrosine hypersensitivity and the consequent root defects with the intracellular concentration of auxin, direct measurements of the amount of auxin in dhs1 compared to Col-0 seedlings was undertaken. The levels of auxin measured are from metabolite extracts of 8 days old seedlings (Figure 23). Auxin from these extracts was subsequently purified by chromatography and analyzed by LC/MS. A standard of deuterated auxin was added to each sample prior to performing metabolite extractions to control for any loss of metabolites incurred during the extraction process. The concentration of auxin in untreated Col-0 and dhs1 seedlings was around 15 ng/gFW. Upon tyrosine treatment the auxin concentration slightly increased in both genotypes approaching 20 ng/gFW, while treatment with both Tyr and Phe saw a slight decrease and treatment with both

Tyr and Trp saw a small increase in the levels of auxin. However, variability in the data makes it hard to tell if those changes are relevant. Indeed, no statistically significant differences across the treatments or between Col-0 and dhs1 were detected.

Figure 23 – Auxin content in whole seedlings. Concentration of auxin as determined by LC/MS in 8 days old Col-0 and dhs1 seedlings in ng/g fresh weight after amino acid treatment with 50 µM tyrosine (Tyr), 50 µM tyrosine and phenylalanine (Tyr + Phe) and 50 µM tyrosine and tryptophan (Tyr + Trp). Error bars represent standard error, n=3.

Auxin Levels in Col-0 and dhs1 Seedling Roots

The spatial pattern of endogenous auxin distribution at the root apex of Col-0 and dhs1 seedlings was visualized using a DR5::GUS Arabidopsis line. The expression of the reporter gene β-glucuronidase (GUS) is under control of the DR5 synthetic promoter which consists of a

7 tandem repeat of an auxin responsive element (TGTCTC) joined to a minimal 35S CaMV promoter (Ulmasov et al., 1997b; Sabatini et al., 1999). The accumulation of endogenous auxin at the primary root tip of Arabidopsis DR5::GUS line was compared to a dhs1xDR5::GUS cross under different experimental growth conditions. Incubation of DR5::GUS seedlings in a staining

47 solution containing 5 mM ferri/ferrocyanide resulted in localized staining of quiescent center cells and initial columella cells with lesser staining in surrounding areas and especially in the columella root cap (Figure 24A). For the DR5::GUS line, there was a decrease in the intensity of staining in the root tip of seedlings grown in the presence of tyrosine as compared to those grown under control conditions. This decrease could be rescued by supplementing the media with phenylalanine or tryptophan in addition to tyrosine.

On the other hand, the same staining condition only resulted in very faint staining in the dhs1xDR5::GUS lines suggesting that the levels of auxin in this line are much lower than those found in wild-type. Increasing the sensitivity of the staining, by using 2.5 mM instead of 5 mM ferri/ferrocyanide in the staining solution, allowed for a better visualization of GUS activity in the line carrying dhs1 (Figure 24B). In this case the level of GUS activity was the same in all treatments except for the tyrosine and tryptophan treated seedlings where increased staining was observed.

The staining pattern differences observed between the DR5::GUS and dhs1xDR5::GUS lines and those observed in DR5::GUS grown under different conditions were observed in 3 separate experiments. In each experiment, only the primary root tip was considered for comparison and at least 5 primary roots were observed for each genotype and each treatment.

Figure 24 – Effect of aromatic amino acid supplementation on endogenous auxin levels at the root apex of Arabidopsis seedlings. The blue staining represents the activity of the GUS reporter gene in DR5::GUS line and dhs1xDR5::GUS cross line grown on control, 50 µM tyrosine (50 µM Y), 50 µM tyrosine and 50 µM phenylalanine (50 µM YF) or 50 µM tyrosine and 50 µM tryptophan (50 µM YW) media. A) seedling roots stained in solution containing 5 mM ferri/ferrocyanide for 18 hr and B) seedling roots stained in solution containing 2.5 mM ferri/ferrocyanide for 8 hr. n=5. .

Tyrosine Treatment of dhs1 Elicit Stress Responses

Analysis of whole genome transcript expression profiling of 8 days old Arabidopsis thaliana seedlings was used to probe the transcriptional changes associated with the growth defect of tyrosine hypersensitivity in dhs1 and the rescue of this phenotype by the addition of phenylalanine or tryptophan. Transcriptome analysis was conducted on the following combinations of amino acid treatments and genotypes, with abbreviated labels shown in parentheses: untreated dhs1 mutants (dhs1), dhs1 mutants treated with 2 mM tyrosine (dhs1-Y), dhs1 mutants treated with 2 mM tryptophan and 2 mM tyrosine (dhs1-WY), dhs1 mutants treated with 2 mM phenylalanine and 2 mM tyrosine (dhs1-FY) and non-transgenic sibling of dhs1 treated with 2 mM tyrosine (NTS-Y) (Shahinas, 2008).

Comparison of dhs1-Y with NTS-Y reveals that only 60 genes are significantly differentially expressed and suggest that overall gene expression is similar in dhs1 and wild-type seedlings when exposed to Tyr (Table 4). On the other hand, a large number of genes were significantly differentially expressed when dhs1-Y is compared to dhs1 exposing the large transcriptomic changes that are associated with tyrosine treatment of Arabidopsis seedlings.

Notably, several genes involved in aromatic amino acid biosynthesis and auxin biosynthesis and transport are differentially expressed in dhs1 mutants exposed to tyrosine compared to untreated dhs1. Mapping these expression dynamics onto the shikimate pathway and related downstream pathways indicates substantial perturbations of the tryptophan branch leading to auxin biosynthesis (Figure 25) and a large number of stress response annotated genes.

To get a better idea of which plant processes are affected by those transcriptional changes, genes that had significantly different expression between 2 treatments were analyzed with the Classification Superviewer (Provart and Zhu, 2003) for functional enrichment. A

50 positive functional enrichment was uncovered for genes belonging to energy pathways and stress responses when comparing dhs1 with dhs1-Y suggesting that tyrosine treatment of dhs1 seedlings leads to disruption of the plant's metabolic and energy pathways and activates stress responses (Figure 26). There was a similar functional enrichment of stress responses genes in the dhs1-Y vs. dhs1-YF and dhs1-Y vs. dhs1-YW pairwise comparisons implicating stress response in the rescue observed with phenylalanine and tryptophan. In fact, the transcript levels of many of these stress-related genes significantly varied across each of the three pairwise comparisons involving dhs1-Y. Moreover, the increase in transcript levels of many stress-related genes in dhs1 mutants exposed to tyrosine is reversed upon addition of tryptophan or phenylalanine as seen on the heat map representing expression values of stress response related genes across the 4 dhs1 treatments (Figure 27).

Table 4 – Number of differentially expressed genes in dhs1 following treatment with tyrosine (Y), phenylalanine (F) or tryptophan (W) and between dhs1 and its non-transgenic sibling (NTS). Number of significantly differentially expressed genes determined using Significance Analysis of Microarrays (SAM). FDR; mean false discovery rate.

Number of significantly Pairwise comparison SAM FDR differentially expressed genes dhs1-Y vs. NTS-Y 0.05 60 dhs1-Y vs. dhs1 < 0.01 3187 dhs1-FY vs. dhs1-Y < 0.01 1081 dhs1-WY vs. dhs1-Y < 0.01 400

Figure 25 –Transcript levels of genes belonging to the shikimate pathway, aromatic amino acid and auxin biosynthesis in dhs1 after treatment with tyrosine (Y), phenylalanine (F) and tryptophan (W). Heat map representation of differentially expressed genes. Yellow represents above median expression and blue represents below median expression across the 4 datasets.

Figure 26 – Functional enrichment analysis of genes with significantly different expression in pairwise comparisons of dhs1 following treatment with tyrosine (Y), phenylalanine (F) or tryptophan (W) and between dhs1 and its non-transgenic sibling (NTS). Functional annotation based on GO:biological processes classification. P-values ≤ 0.001 of the hypergeometric distributions are shown.

Figure 27 – Transcript levels of stress related genes in dhs1 after treatment with tyrosine (Y), phenylalanine (F) and tryptophan (W). Heat map representation of differentially expressed genes. Yellow represents above median expression and blue represents below median expression across the 4 datasets.

Discussion

In plants the shikimate pathway is the main channel for carbon flow between primary metabolism and secondary metabolic pathways. The regulation of the shikimate pathway therefore needs to be such that the requirements of many downstream pathways are met. This coordination is partly achieved by transcriptional regulation. For instance, the genes coding for

DHS and phenylalanine ammonia lyase (PAL), which catalyze the committed steps of the shikimate pathway and of the phenylpropanoids pathway respectively, are concomitantly induced by wounding or pathogen attack (Dyer et al., 1989; Keith et al., 1991; Gorlach et al.,

1993; Sato et al., 2006). Some level of coordination can thus be achieved at the genetic level but other regulatory mechanisms at the protein level may be involved as suggested by the tyrosine hypersensitivity of dhs1 in Arabidopsis. Beside transcriptional activation of certain genes very little is known about the regulation of the shikimate pathway in plants. The central role played by aromatic amino acids in the regulation of this pathway in bacteria and fungi suggests they may also be involved in regulating the plant pathway. In fact, work in our laboratory provides evidence for aromatic amino acids involvement in the regulation plant DHS. This study further explores the changes associated with the dhs1 mutation in Arabidopsis thaliana by looking at the levels of key downstream metabolites such as aromatic amino acids and auxin.

Diversification in the Regulatory Mechanisms of Bacterial and Plant AroAII Enzymes

The regulation of AroAIα-type bacterial DHS enzymes is mediated primarily by allosteric feedback regulation (Ogino et al., 1982). Bacteria that possess enzymes of this category have 3 DHS isozymes each sensitive to one of the 3 aromatic amino acids produced downstream of the shikimate pathway. Reconstruction of AroAIα DHS ancestry suggests that this mode of regulation is conserved among bacterial species of that group (Figure 8).

On the other hand, phylogenetic reconstruction of AroAII-type DHS shows a more convoluted evolutionary history. Higher plants possess a diverging number of DHS isozymes that arose from moss-, monocot- and dicot-lineage specific gene duplication and retention events

(Figure 9). The topology of the phylogenetic tree of AroAII DHS is consistent with the proposed bacterial origin of plant DHS enzymes (Gosset et al., 2001). The algae C. reinhardtii and of O. lucimarinus possess a single DHS coding gene whose sequence clusters closely to that of M. tuberculosis. Retention of many DHS genes, each presumably allowing the plant a more precise control of its supply of precursor for protein biosynthesis and secondary metabolism reflects plant’s relatively complex lifecycle and suggests that plant DHS enzymes may have evolved different regulatory mechanism to meet those needs. Although monocot and dicot paralogous

DHS isozymes have evolved independently and may therefore have distinct regulatory features, transcriptional induction of at least one DHS gene in response to wounding or fungal elicitors seems to be a recurring regulatory mechanism in the dicots tomato, potato and Arabidopsis (Dyer et al., 1989; Keith et al., 1991; Gorlach et al., 1993) as well as in the monocot O. sativa (Sato et al., 2006). This regulatory feature conserved across both monocot and dicot plant species suggests it was inherited from their last common ancestor. On the other hand, the separate

56 evolutionary origins of monocot and dicot DHS isogenes suggest that findings in the regulation of DHS in one group may not be applicable to the other. For instance, the regulatory of properties of Arabidopsis DHS may not be extendable to enzymes belonging to monocot organisms such as cereals.

Conservation of Structural Domains between Arabidopsis and M.tuberculosis DHS Enzymes

The high level of similarities between the crystal structure of E.coli and M.tuberculosis

DHS enzymes has been used to establish an evolutionary link between AroAI and AroAII enzymes (Webby et al., 2005a). The similarity of the overall fold of the protein does not however extend to the structural motifs decorating the central TIM barrel structure which have been postulated to play regulatory roles (Figure 10). For example, the β-strand β5a and β5b form part of the phenylalanine allosteric binding site in E.coli DHS but are conspicuously absent in the

M.tuberculosis enzyme. In contrast to the E.coli DHS, the co-crystallization of M.tuberculosis

DHS with aromatic amino acids shows phenylalanine to be bound at a different position, next to the amino terminal of the protein thus indicating a different mechanism of allosteric regulation between AroAI and AroAII bacterial DHS. Underlying this different mechanism of allosteric regulation is also the synergistic nature of aromatic amino acid inhibition in M.tuberculosis which is not present in AroAI bacterial enzymes (Webby et al., 2010). Moreover, there are two additional α-helices, α2a and α2b, in the M.tuberculosis enzyme that are adjacent to the putative allosteric binding site of tryptophan. These helices are also present in the modeled structure of

Arabidopsis DHS and 3 of the 4 residues identified as directly interacting with tryptophan in

M.tuberculosis DHS are conserved in the Arabidopsis DHS isozymes (Figure 10). This suggests

57 that although allosteric inhibition by the aromatic amino acids seems unlikely in plants

(Herrmann and Weaver, 1999; Crowley, 2006) this domain may have evolved a different regulatory mechanism.

Levels of Aromatic Amino Acids are Disrupted in dhs1 Seedlings

Before investigating the levels of aromatic amino acids in Col-0 and dhs1 plants, the genotype of each was verified to ensure homozygosity at each genetic locus (Figure 12). HPLC analysis of amino acid dansyl derivatives was important for the quantification of endogenous aromatic amino acid levels in Arabidopsis seedlings. The levels of endogenous tyrosine and phenylalanine found in both Col-0 and dhs1 control seedlings were of approximately 15.5 and 4 nmol/gFW respectively (Figure 16). These levels are comparable to previously reported values for young Arabidopsis plants (Voll et al., 2004). The fact that the endogenous levels of tyrosine and phenylalanine detected were the same in both dhs1 and Col-0 seedlings is consistent with the lack of phenotypic differences between these 2 genotypes when grown under normal laboratory conditions. Indeed, because DHS single knockout plants grow just as well as Col-0 under control conditions it was expected that the loss of one DHS isozymes would not result in major differences in endogenous levels of key metabolic intermediates such as the aromatic amino acids. It other words, under normal condition the loss of one DHS enzyme was expected to be compensated for by the other 2 DHS isozymes.

Similarly, the endogenous levels of tyrosine and phenylalanine were measured in seedlings that were treated with exogenous tyrosine. As anticipated the levels of endogenous tyrosine increased, approximately 4-fold compared to the control (Figure 17). The increase in

58 endogenous tyrosine was observed in both dhs1 and Col-0 and could have resulted from intake of the exogenously supplied tyrosine by amino acid transporters such as amino acid permease 1

(AAP1) or lysine histidine transporter 1 (LHT1) which have both been shown to transport aromatic amino acids into the Arabidopsis root (Hirner et al., 2006; Lee et al., 2007; Sanders et al., 2009).

On the other hand, the levels of endogenous phenylalanine measured in seedlings that were treated with exogenous tyrosine were different between Col-0 and dhs1. The levels found in Col-0 were around 16 nmol/gFW compared to approximately 4 nmol/gFW in dhs1 (Figure

17). In other words, the levels of endogenous phenylalanine in Col-0 seedlings treated with exogenous tyrosine were 4 fold higher than in the control seedlings while the levels in dhs1 after exogenous tyrosine treatment were essentially the same as in the control. This difference between Col-0 and dhs1 seedlings suggests that tyrosine has an inhibitory effect that can be masked by the DHS1 enzyme. In addition, the fact that endogenous levels of phenylalanine are the same in tyrosine-treated as in the control for dhs1 seedlings suggest that it may not play a role in tyrosine hypersensitivity. Whole seedlings were used for these measurements and it is therefore impossible to say what fraction of the increase in endogenous tyrosine resulted from genuine increase in symplastic tyrosine and what fraction resulted from higher tyrosine content in the apoplast. However, the increase in endogenous phenylalanine measured in Col-0 seedlings after exogenous tyrosine treatment is unambiguously the result of increased intracellular phenylalanine since the treatment did not contain any phenylalanine.

The increase in intracellular phenylalanine in Col-0 seedlings after treatment with exogenous tyrosine is probably occasioned by an increase in tyrosine levels. One possibility is that elevated tyrosine feedback inhibits TyrA, the last enzyme in the tyrosine biosynthetic

59 pathway (Figure 6). By blocking its own synthesis, elevated tyrosine levels would decrease the demand on the chorismate pool for tyrosine synthesis, and would allow more of it to be available for phenylalanine or tryptophan biosynthesis. This increased availability of chorismate may explain the 4-fold increase in endogenous phenylalanine measured in Col-0 upon tyrosine treatment. Similarly, the same mechanism would be involved in dhs1 and the tyrosine treatment would increase the availability of chorismate for phenylalanine and tryptophan biosynthesis. But in this instance, I speculated that a decrease in the activity of the shikimate pathway or in the chorismate pool in tyrosine-treated dhs1 seedlings could counter-balance this effect and explain why no increase in endogenous phenylalanine was observed in those seedlings.

Co-treatment of dhs1 seedlings with exogenous tyrosine and phenylalanine partially rescues the tyrosine hypersensitivity of dhs1 seedlings (Crowley, 2006; Shahinas, 2008). The

HPLC analysis revealed that endogenous levels of both amino acids increased upon treatment and that the increase in phenylalanine was even more pronounced than tyrosine, increasing approximately 25-fold compared to the control treatment (Figure 18). This suggests the mechanism underlying the rescue of tyrosine hypersensitivity may involve a large increase the levels of phenylalanine.

The quantification of amino acids by derivatization and HPLC analysis was not sensitive enough to detect endogenous tryptophan levels. Other groups had similar experiences and reflect a limitation of the technique (Voll et al., 2004). Instead, to investigate the effect of the dhs1 mutation on levels of tryptophan, the established 5-methyltryptophan (5-MT) sensitivity of plants was used as an indirect measure of intracellular level of tryptophan (Li and Last, 1996, Celenza et al., 2005). 5-MT is a tryptophan analog that inhibits anthranilate synthase, the first enzyme in tryptophan biosynthesis (Widholm, 1972). Therefore, plants that have high levels of tryptophan

60 are more resistant to 5-MT than plants with low tryptophan levels (Cho et al., 2000; Towaza et al., 2001; Yamada et al., 2008).

The increased sensitivity of dhs1 to 5-MT compared to Col-0 seedlings suggests that tryptophan levels are lower in dhs1 (Figure 19 and 20). This differs from the similar levels of phenylalanine observed in both Col-0 and dhs1. Lower tryptophan levels in dhs1 would result in less of it being available for downstream pathways including auxin biosynthesis.

Surprisingly, co-treatment of Col-0 seedlings with both 5-MT and tyrosine did not result in any growth inhibition (Figure 21). The compensatory effect of tyrosine may be due to elevated levels of tyrosine easing the demand on the chorismate pool and therefore making more of it available for tryptophan biosynthesis.

Alternatively, the increase in endogenous phenylalanine that was measured in Col-0 seedlings upon treatment with exogenous tyrosine may explain why Col-0 seedlings are more resistant to 5-MT when co-treated with tyrosine. Higher phenylalanine content in rice Mtr1 mutant has already been associated with higher tryptophan levels and increase 5-MT resistance, although by what mechanism is still not clear (Yamada et al., 2008). Another explanation, although no concrete evidence exists for it, would be the involvement of DHS1 in tryptophan biosynthesis. Increase 5-MT sensitivity in dhs1 already hint at disruption of tryptophan biosynthesis in those seedlings and formation of a complex between DHS and anthranilate synthase (AS) for example could account for this. Activation of this complex, but not of AS alone, by tyrosine could explain the lack of inhibition in Col-0 seedlings grown on tyrosine and

5-MT. However, this is highly speculative since there is no evidence to suggest that DHS1 and

AS are interacting.

In summary, the previous results suggest that mutation in the dhs1 gene lead to a disruption in tryptophan biosynthesis. This reduction in tryptophan levels could be implicated in the tyrosine hypersensitivity of dhs1 especially considering that it is a precursor for the biosynthesis of indole hormones such as auxin which regulates many developmental processes in the Arabidopsis root (Friml, 2003; Ljung et al., 2005; Mano et al., 2010). Therefore, the next step taken was to investigate if a mutation in dhs1 was associated with a disruption in auxin levels.

Changes in Auxin

Auxin levels were quantified in Col-0 and dhs1 whole seedlings using LC/MS. The concentration of auxin did not differ significantly between both genotypes or between different treatments (Figure 23). This suggests that the putatively lower tryptophan levels in dhs1 do not translate into lower auxin levels and that likewise; treatment with exogenous amino acids does not affect the overall auxin content of Arabidopsis seedlings.

The procedure for the quantification of auxin used whole seedlings that were treated with exogenous amino acid solutions for 8 hr. The rational for this approach was to be consistent with how the microarray data had been collected. However, seedling treatment for a short duration may not be appropriate in this case. Indeed, an 8 hr amino acid treatment may be enough to change gene expression in Arabidopsis but not long enough to significantly alter the endogenous levels of a phytohormone like auxin. In fact, auxin homeostasis is still not fully understood.

Mechanism such as increase hydrolysis of stored auxin conjugates or decrease in irreversible conjugation and catabolic degradation may be able to compensate short duration disruptions in its biosynthesis and explain why no differences in auxin content were observed following 8 hr

62 amino acid treatment (Bialek and Cohen, 1989; Tam et al., 2000; Kowalczyk et al., 2001). Also, analyzing auxin in whole seedlings may not allow the detection of local variations such as at the root apex. The highest levels of auxin in the root apical region of Arabidopsis seedlings are found in the quiescent center cells which is also where expression levels of DHS1 are highest

(Figure 6) (Brady et al., 2007; Petersson et al., 2009). Therefore, a reduction of auxin at the root apex of dhs1 is still a possibility even if the levels found in whole seedlings seem to be the same in both Col-0 and dhs1.

To investigate the levels of auxin in root tips of dhs1 seedlings and the effect of treatment with aromatic amino acids, a reporter gene experiment with β-glucuronidase (GUS) was used. To examine the level of auxin in planta, dhs1 plants were crossed to the DR5::GUS line. DR5 is a synthetic promoter consisting of a constitutive auxin response element repeated in tandem coupled to a minimal 35S CaMV promoter (Ulmasov et al., 1997; Sabatini et al., 1999).

Activity of the DR5 markers as auxin responsive reporters reflects accumulation of auxin transported from the apical regions of embryos to the root poles (Friml, 2003; Blakeslee et al.,

2005; Dhonukshe et al., 2005; Paponov et al., 2005; Leyser, 2006).

The auxin found at the root tip as visualized by activity of the GUS reporter showed a marked difference between DR5::GUS and dhs1xDR5::GUS. Indeed, after incubation in a staining solution containing 5 mM ferri/ferrocyanide, GUS activity was clearly visible in the

DR5::GUS root apex but barely noticeable in the line carrying dhs1 (Figure 24A). This suggests that the putatively lower tryptophan availability in dhs1 may lead to disruption of auxin in the root tips of Arabidopsis seedlings. Additionally, there was a reduction in GUS activity observed at the root apical region of DR5::GUS seedlings grown on tyrosine which was followed by a recovery in GUS activity for seedlings grown on tyrosine in combination with either

63 phenylalanine or tryptophan. This staining pattern is compatible with the reduction in root length when seedlings are grown on tyrosine and the rescue observed when phenylalanine or tryptophan is also present. However, further experimentation would be required to establish a direct link between the variations in root length following aromatic amino acid treatment and auxin levels at the root tip. For example, auxin supplementation could be used to see if it is sufficient to rescue the root length phenotypes occasioned by tyrosine treatment.

To find out if there was a similar pattern of GUS activity in dhs1 a lower concentration of ferri/ferrocyanide, 2.5 mM instead of 5 mM, was used for the staining (Figure 24B). The big difference in GUS activity between DR5::GUS and dhs1xDR5::GUS was also evident but the staining in the line carrying dhs1 was similar across all conditions except when grown with tyrosine and tryptophan. This increase in GUS activity in the line carrying dhs1 when grown in the presence of tryptophan is consistent with the hypothesis that availability of tryptophan can have an effect on auxin levels.

Furthermore the difference in GUS activity between DR5::GUS and dhs1xDR5::GUS, especially in the control, is in contradiction with the similar auxin levels that were quantified by

LC/MS in Col-0 and dhs1 seedlings (Figure 23). However, the differences that were observed at the root apex of DR5::GUS and dhs1xDR5::GUS seedlings may be localized to that region of the seedlings and may not be detectable when measuring auxin levels in whole seedlings. Therefore, to confirm the results obtain from the GUS reporter experiment, direct quantification of auxin in roots would be required.

Tyrosine treatment of dhs1 knockout turns on Arabidopsis stress response and causes significant transcriptional changes in tryptophan and auxin biosynthetic genes

Tyr treatment of dhs1 seedlings causes transcriptional disruptions in numerous biological processes including photosynthesis, stress, phenylpropanoids metabolism, lignin biosynthesis and sugar metabolism. Of the 3187 genes that are significantly differentially expressed between dhs1 with dhs1-Y (Table 4) functional enrichment analysis reveals genes belonging to energy pathways and stress responses are over represented (Figure 26). Genes belonging to the plant's stress response are also functionally enriched in dhs1-Y vs. dhs1-YW and dhs1-Y vs. dhs1-YF.

Interestingly, the elevated expression of many stress response genes after tyrosine treatment is reversed upon phenylalanine or tryptophan addition (Figure 27). This suggests imbalances in the intracellular levels of the 3 aromatic amino acids activate the plant’s stress response which is alleviated upon addition of either phenylalanine or tryptophan. This is consistent with the central role the shikimate pathway plays in plant growth and development. The addition of tyrosine attenuates the shikimate pathway in the dhs1 mutant line to a level that directly compromised the biosynthesis of growth hormones and structural compounds. This in turn leads to a cascade of stress response gene activation, which can return to more normal expression level with the exogenous application of aromatic amino acids.

An interesting asymmetry of transcriptional changes emerges when mapped onto the shikimate pathway, aromatic amino acid and auxin biosynthesis (Figure 25). There is a greater transcript abundance of tryptophan biosynthesis related genes in dhs1-Y, dhs1-YW and dhs1-YF when compared to untreated dhs1. No such patterns are apparent in phenylalanine and tyrosine biosynthesis. The upregulation of several genes involved in tryptophan biosynthesis is consistent

65 with the increase sensitivity of dhs1 to 5-MT supplementation. Transcriptional changes seen in auxin biosynthesis are more ambiguous. Lower transcript levels of AMI1 in dhs1-Y, dhs1-YW and dhs1-YF suggest that the indole-3-acetamide dependent synthesis of auxin is attenuated when elevated levels of aromatic amino acids are present (Figure 25). On the other hand, two other tryptophan-dependent route to auxin, indo-3-acetaldoxime (IAOX) to indole-3- acetaldehyde (IAD) and indole-3-pyruvic acid to IAD, respond differently. Transcript levels of enzymes belonging to each, CYP79B3 and TAA1 respectively, are similar across dhs1, dhs1-Y and dhs1-YW and slightly higher in dhs1-YF.

Proposed Model and Future Directions

Two speculative models are presented that singly or together could explain the previous findings. The first hypothesis is that tyrosine indirectly inhibits either DHS2 or DHS3 or both at the protein level (Figure 28A). Inhibition of DHS2 or DHS3 would likely have to be mediated by a novel mechanism since allosteric regulation of DHS in plants has never been reported

(Herrmann and Weaver, 1999; Tzin and Galili, 2010) and DHS1, DHS2 and DHS3 from

Arabidopsis are not inhibited by tyrosine in vitro (Crowley, 2006). I speculated that inhibition of

DHS2 and/or DHS3 would not alter the flux of the shikimate pathway in dhs1 under control conditions because the other 2 DHS would compensate for the dhs1 mutation. This would explain why the levels of endogenous tyrosine and phenylalanine measured in dhs1 and Col-0 were comparable (Figure 16).

Additionally, this model could explain the difference in endogenous phenylalanine between Col-0 and dhs1 upon tyrosine treatment. In Col-0, the increase in phenylalanine would be the result of elevated tyrosine which is feedback inhibiting its own synthesis and thus making

66 more chorismate available for phenylalanine biosynthesis. In dhs1, elevated tyrosine would also feedback inhibit its own synthesis but this would be counterbalanced by a reduction in the chorismate pool engendered by tyrosine inhibition of DHS2 and/or DHS3 which in this case could not be compensated for by the activity of DHS1.

Results from 5-MT sensitivity assays are difficult to reconcile with this model alone.

How could it explain the increase sensitivity of dhs1 to 5-MT and the absence of any inhibition in Col-0 when co-treated with 5-MT and tyrosine? This is why a second regulatory mechanism is proposed. Although there is no evidence for it, it seems warranted to fully explain the experimental results presented here and more importantly, to provide testable hypotheses for future studies. This hypothetical regulatory mechanism would be the formation of a non-covalent complex between DHS1 and anthranilate synthase (AS) which would boost the catalytic efficiency of the latter as well as activation of AS when in this complex by tyrosine (Figure

28B).

The increase 5-MT sensitivity of dhs1 suggested a disruption in tryptophan biosynthesis

(Figure 19 and 20). This could be explained by increase catalytic efficiency of AS when in a complex with DSH1. Assuming that decrease in tryptophan would lead to decrease in auxin it would also help explain why there was less GUS activity in dhs1 under normal growth conditions (Figure 24). Moreover, activation of the complex by tyrosine would explain the lack of inhibition from 5-MT in Col-0 co-treated with tyrosine.

An analogous regulatory mechanism is found in M. tuberculosis and lends plausibility to the second model. In M. tuberculosis one chorismate mutase (CM) enzyme is poorly active but is activated by interaction with DHS (Sasso et al., 2009). Furthermore, the CM in question is usually not regulated by aromatic amino acids but the CM activity of the DHS-CM complex is

67 synergistically inhibited by both tyrosine and phenylalanine. Interestingly, the interaction interface between DHS and CM comprises 2 α-helices α2a and α2b that protrude from the central

TIM barrel fold of DHS (Sasso et al., 2009). These 2 extra helices are not present in the E.coli

DHS but are present in the modeled structure of Arabidopsis DHS (Figure 10) suggesting that this mode of regulation may also exist in plant and that in fact it may be a mode of regulation that is specific to the AroAII family of DHS enzymes.

Figure 28 – Proposed model of DHS regulation in Arabidopsis thaliana. Established regulation represented in solid lines. Proposed regulation in dashed lines of A) Indirect inhibition of DHS2 or DHS3 or both by Tyr and B) Complex formation between DHS1 and AS and activation of the complex by Tyr. Arrows represent feedback activation and stopped lines represent feedback inhibition.

The remaining questions that need to be addressed are: 1) does tyrosine inhibit DHS2,

DHS3 or both? If so, what is the molecular mechanism of this inhibition? 2) Does DHS1 interact with AS as proposed? If that were the case, are there other molecular players involved and what is the role of tyrosine? To answer the first question, Arabidopsis thaliana lines overexpressing each DHS2 and DHS3 in a dhs1 background would help to identify which isozyme is inhibited by tyrosine. To elucidate the indirect mechanism of inhibition of Tyr on DHS2 or DHS3, a protein-protein interaction study such as yeast-2-hybrid (Y2H) could help identify which other protein, if any, interact with DHS enzymes. This would also allow testing the proposed interaction of AS with DHS1 and help answer the second question. In addition, antibodies should be raised against each of the 3 DHS isozymes and used for co-immunoprecipitation (Co-IP). This could serve as an additional approach to identify interacting partners of DHS in Arabidopsis and could also be used in tandem with mass spectrometry (MS) to identify if DHS are post- translationally modified in vivo.

Conclusion

The central finding of this study is that the mutation of DHS1 in Arabidopsis thaliana is associated with increased 5-MT sensitivity, which suggests disruption in tryptophan biosynthesis, and decrease GUS activity at the root apex, which suggests lower auxin levels. The disruption of tryptophan and auxin biosynthesis in dhs1 is also supported by microarray data analysis which revealed altered transcript abundance for genes involved in those pathways.

Additionally, analysis of endogenous aromatic amino acids in both Col-0 and dhs1 after exogenous tyrosine treatment has shown in phenylalanine levels. Taken together, these results reveal what effects the mutation of DHS1 has on downstream pathways and will help devise other experiments to uncover the molecular details of the regulation of DHS enzymes in

Arabidopsis.

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