condensation of phosphoenol pyruvate and erythrose-4-phosphate to make. DAHP, which undergoes another six catalytic steps to synthesize chorismate, the.
STRUCTURAL AND FUNCTIONAL INSIGHTS ON REGULATION BY PHENOLIC COMPOUNDS.
By
Dea Shahinas
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Dea Shahinas, 2008
STRUCTURAL AND FUNCTIONAL INSIGHTS ON REGULATION BY PHENOLIC COMPOUNDS. Dea Shahinas 2008 Master of Science Department of Cell and Systems Biology University of Toronto
Abstract The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.
ii
TABLE OF CONTENTS LIST OF FIGURES LIST OF ABBREVIATIONS:
V VI
CHAPTER I: REGULATION OF ARABIDOPSIS DAHP SYNTHASE ISOZYMES IN RESPONSE TO AROMATIC AMINO ACIDS. ........................................................................ 1
SUMMARY INTRODUCTION
1 2
THE SHIKIMATE PATHWAY ............................................................................................................. 3 DAHP SYNTHASE (DAHPS) ........................................................................................................... 8 DAHP SYNTHASES IN ARABIDOPSIS THALIANA ............................................................................ 13 THE PHENYLPOPANOID PATHWAY AND ITS REGULATION. ............................................................ 16 AUXIN BIOSYNTHESIS AND ITS REGULATION. ............................................................................... 19
CHAPTER OBJECTIVES MATERIALS AND METHODS
22 23
PLANT MATERIAL AND GROWTH CONDITIONS............................................................................... 23 GENOMIC DNA EXTRACTION ........................................................................................................ 26 GENERATION OF DAHPS1XDR5::GUS CROSSES ............................................................................ 27 TOTAL RNA EXTRACTION ............................................................................................................ 27 MICROARRAY ANALYSIS ............................................................................................................... 28 VERIFICATION OF DR5::GUS CROSSES WITH PCR ........................................................................ 29 GUS STAINING PROCEDURE........................................................................................................... 29
RESULTS
30
DAHPS2 LACKS A C-TERMINAL ALPHA HELIX WHICH IS PRESENT IN DAHPS1 AND DAHPS3. .. 30 AROMATIC AMINO ACID SUPPLEMENTATION HAS DISTINCTIVE EFFECTS ON THE DHS SEEDLING PHENOTYPE IN ARABIDOPSIS THALIANA. ...................................................................................... 34 TRANSCRIPTIONAL ANALYSIS ....................................................................................................... 43 THE EFFECT OF THE AROMATIC AMINO ACIDS ON DR5::GUS LEVELS. ......................................... 65
DISCUSSION
67
THE ROLE OF THE DAHP SYNTHASES ........................................................................................... 67 THE ROLE OF THE AROMATIC AMINO ACIDS IN REGULATING THE SHIKIMATE PATHWAY. ............ 69 PERSPECTIVES ON THE MODEL AND FUTURE DIRECTIONS. ............................................................ 77 CHAPTER II: THE CRYSTAL STRUCTURE OF A. AEOLICUS PREPHENATE DEHYDROGENASE REVEALS THAT TYROSINE INHIBITION IS MEDIATED BY A SINGLE RESIDUE ..................................................................................................................... 81
ABSTRACT INTRODUCTION MATERIALS AND METHODS
81 82 85
CHEMICALS AND REAGENTS .......................................................................................................... 85 SITE-DIRECTED MUTAGENESIS ...................................................................................................... 86 PROTEIN EXPRESSION AND PURIFICATION ..................................................................................... 86 DETERMINATION OF ENZYME ACTIVITY AND DISSOCIATION CONSTANTS FOR LIGAND BINDING . 86 CRYSTALLIZATION ........................................................................................................................ 87 X-RAY DIFFRACTION AND STRUCTURE DETERMINATION .............................................................. 88
RESULTS AND DISCUSSION
90 iii
CRYSTALLIZATION AND STRUCTURAL SUMMARIES OF Δ19PD-NAD+-HPP, Δ19PD-NAD+HPPROPIONATE AND Δ19PD-NAD+L-TYROSINE ......................................................................... 90 CONFORMATIONAL SHIFTING UPON SUBSTRATE BINDING ............................................................ 96 LOCATION OF A. AEOLICUS PD ACTIVE SITE ................................................................................. 99 ARCHITECTURE OF THE SUBSTRATE BINDING SITE .................................................................... 101 ROLE OF HIS147 IN THE REACTION MECHANISM ........................................................................ 103 ROLE OF SER126 IN THE REACTION MECHANISM ....................................................................... 105 ROLE OF WAT1 IN THE REACTION MECHANISM ........................................................................ 105 ROLE OF ARG250 IN THE REACTION MECHANISM ...................................................................... 106 ROLE OF HIS217 IN THE REACTION MECHANISM ........................................................................ 106 HIS217 AS A DETERMINANT OF LIGAND PREFERENCE ................................................................. 111 STRUCTURAL COMPARISONS OF AD AND PD .............................................................................. 112 BIOLOGICAL AND BIOCHEMICAL RELEVANCE ............................................................................ 113 CHAPTER III: STRUCTURAL INSIGHT ON THE MECHANISM OF REGULATION OF THE MARR FAMILY OF PROTEINS............................................................................ 116
ABSTRACT INTRODUCTION RESULTS AND DISCUSSION
116 117 120
CRYSTALLIZATION AND STRUCTURE DETERMINATION .............................................................. 120 OVERALL STRUCTURE OF MTH313 ............................................................................................ 120 SEQUENCE ANALYSIS................................................................................................................... 124 STRUCTURE ANALYSIS WITH DALI ............................................................................................ 124 BIOPHYSICAL ANALYSIS OF SALICYLATE BINDING TO MTH313 ................................................. 132 SALICYLATE DISRUPTS MTH313 BINDING TO DNA ................................................................... 133 MECHANISM OF INACTIVATION OF MTH313 .............................................................................. 137 BIOLOGICAL RELEVANCE ............................................................................................................ 139
MATERIALS AND METHODS
141
CLONING, PROTEIN EXPRESSION AND PURIFICATION ................................................................. 141 PROTEIN CRYSTALLIZATION ....................................................................................................... 142 X-RAY DIFFRACTION AND STRUCTURE DETERMINATION .......................................................... 143 DNA BINDING STUDY PROBE DESIGN ........................................................................................ 144 GEL SHIFT ASSAY ........................................................................................................................ 144 THERMAL SHIFT SALICYLATE BINDING ASSAY .......................................................................... 145
CONCLUDING DISCUSSION REFERENCES:
147 150
iv
LIST OF FIGURES FIGURE 1- 1: AN OVERVIEW OF THE SHIKIMATE PATHWAY.. ........................................................................... 7 FIGURE 1- 2: THE DAHP FORMATION REACTION MECHANISM.. ......................................................................... 9 FIGURE 1- 3: E.COLI DAHP SYNTHASE CO-CRYSTALLIZED WITH PEP AND PHENYLALANINE.. ............................ 12 FIGURE 1- 4: ORGAN SPECIFIC MAP OF DAHPS ISOZYME TRANSCRIPT EXPRESSION IN A.THALIANA.. ................. 15 FIGURE 1- 5: DIAGRAM OF THE POSITION OF THE T-DNA INSERTION FOR THE DAHP SYNTHASE KNOCKOUT LINES. . ................................................................................................................................................ 25 FIGURE 1- 6: PREDICTED THREE DIMENSIONAL MODELS OF THE ARABIDOPSIS THALIANA DAHP SYNTHASE ISOZYMES.. .......................................................................................................................................... 31 FIGURE 1- 7: MULTIPLE SEQUENCE ALIGNMENT OF DAHP SYNTHASE ISOZYMES FROM SEQUENCED PLANT GENOMES (A-C) AND MICROBIAL SEQUENCES (D).. .......................................................................... 33 FIGURE 1- 8: DIAGRAM OF THE POSITION OF THE PRIMERS USED BY VALERIE CROWLEY FOR GENOTYPING THE DAHP SYNTHASE INSERTION LINES. ........................................................................................... 35 FIGURE 1- 9: RESPONSE OF DAHPS1 TO AROMATIC AMINO ACID SUPPLEMENTATION. ................................ 39 FIGURE 1- 10: RESPONSE OF THE DAHPS1-2 ALLELE TO AROMATIC AMINO ACID SUPPLEMENTATION. ...... 40 FIGURE 1- 11: RESPONSE OF DAHPS2 TO AROMATIC AMINO ACID SUPPLEMENTATION ...................................... 41 FIGURE 1- 12: RESPONSE OF DAHPS3 TO AROMATIC AMINO ACID SUPPLEMENTATION ....................................... 42 FIGURE 1- 13: VENN DIAGRAM REPRESENTATION OF THE PHENYLPROPANOID PATHWAY TRANSCRIPTS SIGNIFICANTLY AFFECTED IN EACH COMPARISON. . ................................................................................ 61 FIGURE 1- 14: VENN DIAGRAM REPRESENTATION OF ALL THE TRANSCRIPTS SIGNIFICANTLY AFFECTED IN EACH COMPARISON.. ...................................................................................................................................... 63 FIGURE 1- 15: EFFECT OF TYROSINE ON THE DR5::GUS MARKER AND DAHPS1XDR5::GUS CROSSES AS OBSERVED AT THE ROOT APEX.. ............................................................................................................. 66 FIGURE 1- 16: ILLUSTRATION OF THE EFFECT OBSERVED ON KEY SHIKIMATE AND PHENYLPROPANOID PATHWAY GENES UPON AROMATIC AMINO ACID SUPPLEMENTATION........................................................................ 75 FIGURE 1- 17: THE MODEL BY WHICH DAHPS ISOZYMES ARE PREDICTED TO INTERACT WITH THE AROMATIC AMINO ACIDS AND AFFECT THE LEVELS OF INTERMEDIATES SHUTTLED TO THE BRANCHING PATHWAYS.. ... 76 FIGURE 2- 1 METABOLIC ROUTES FROM CHORISMATE LEADING TO THE SYNTHESIS OF L-TYROSINE AND LPHENYLALANINE. . ................................................................................................................................ 95 FIGURE 2- 2 CΑ TRACES OF A. AEOLICUS PREPHENATE DEHYDROGENASE SHOWING CONFORMATIONAL CHANGES THAT OCCUR AS A RESULT OF LIGAND BINDING TO THE SUBSTRATE BINDING SITE. .................................... 98 FIGURE 2- 3: A SCHEMATIC OF THE ACTIVE SITE OF A. AEOLICUS PREPHENATE DEHYDROGENASE.. ..................102 FIGURE 2- 4: REPRESENTATIVE ELECTRON DENSITY FOR NAD+, WAT1 AND A BOUND LIGAND IN THE ACTIVE SITE OF A. AEOLICUS PREPHENATE DEHYDROGENASE.. .................................................................................104 FIGURE 2- 5: SUPERIMPOSITION OF LIGANDS IN THE ACTIVE SITE OF PD.........................................................110 FIGURE 3- 1: A RIBBON DIAGRAM OF THE APO PROTEIN MTH313 DIMER.. .....................................................123 FIGURE 3- 2: COMPARISON OF THE TOP DALI STRUCTURAL HOMOLOGUES AT THE HELIX TURN HELIX MOTIF REVEALS CONSERVATION OF THE LIGAND BINDING POCKET IN THREE-DIMENSIONAL SPACE.....................126 FIGURE 3- 3: ELECTROSTATIC SURFACE REPRESENTATION OF MTH313 IN THE APO AND IN COMPLEX WITH SALICYLATE.. ........................................................................................................................................129 FIGURE 3- 4:THE STABILIZING EFFECT OF SALICYLATE ON THE THERMAL DENATURATION AND DNA BINDING OF MTH313. . ....................................................................................................................................135
v
LIST OF ABBREVIATIONS: TyrA Family of enzymes involved in tyrosine biosynthesis MarR Multiple antibiotic resistance repressor DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate PEP Phosphoenol pyruvate E-4-P Erythrose-4-phosphate DAHPS DAHP synthase DHQS Dehydroquinate synthase DHQ Dehydroquinate dehydratase SDH Shikimate dehydrogenase SK Shikimate kinase EPSPS 5-enolpyruvylshikimate-3-phosphate synthase CS Chorismate synthase EPSP 5-enolpyruvylshikimate-3-phosphate 3-DHQ 3-dehydroquinate 3-DHS 3-dehydroshikimate S3P Shikimate-3-phosphate AROM Fungal pentafunctional complex of enzymes 2-6 in the shikimate pathway. UV ultraviolet DAHPS1 DAHP synthase encoded by At4g39980 DAHPS2 DAHP synthase encoded by At4g35510 DAHPS3 DAHP synthase encoded by At1g22410 MS Mushragie and Skoog medium MES morpholino ethane sulfonic acid w/v weight to volume ratio EDTA ethylenediaminetetraacetic acid SDS sodium dodecyl sulfate NaPi Sodium phosphate PHYRE Three-dimensional structure modeling software CLUSTAL W Multiple sequence alignment software F Phenylalanine W Tryptophan Y Tyrosine wt Wild type CM1 Chorismate mutase 1 CM2 Chorismate mutase 2 CM3 Chorismate mutase 3 ICS1 Isochorismate mutase 1 ICS2 Isochorismate mutase 2 ADT2 Arogenate dehydratase 2 ADT3 Arogenate dehydratase 3 ADT5 Arogenate dehydratase 5 4CL 4-Coumarate:CoA ligase PAL Phenylalanine ammonia lyase HPP 4-hydroxyphenylpyruvate PD Prephenate dehydrogenase AD Arogenate dehydrogenase CM-PD Chorismate mutase - prephenate dehydrogenase bifunctional enzyme HPpropionate Hydroxyphenylpropionate Δ19PD Prephenate dehydrogenase construct with 19 amino acid deletion. WAT Water molecule MTH313 Methanobacterium thermoautotrophicus gene 313 MAR Multiple antibiotic resistance MDR Multiple drug resistance SA Salicylic acid
vi
CHAPTER I: REGULATION OF ARABIDOPSIS DAHP SYNTHASE ISOZYMES IN RESPONSE TO AROMATIC AMINO ACIDS.
Summary The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.
1
INTRODUCTION The shikimate pathway is an essential metabolic pathway involved in the synthesis of aromatic compounds in plants, apicomplexan parasites, fungi and microbes (Suzich, Ranjeva et al. 1984; Dyer, Henstrand et al. 1989; Keith, Dong et al. 1991; Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999; Elandalloussi, Rodrigues et al. 2005; Webby, Baker et al. 2005; Webby, Lott et al. 2005). Animals obtain their aromatic compounds from their diet. Starcevic et al. challenge the view that the shikimate pathway is not found in animals by showing its presence in metazoans using phylogenetic analysis (Starcevic, Akthar et al. 2008). However, no functional data has been provided for the identified genes. The interest in the shikimate pathway enzymes as potential targets for non-toxic herbicides and anti-microbial compounds dates back more than 25 years ago. In 1972, the
company
Monsanto
reported
the
discovery
of
glyphosate
(N-
phosphonomethylglycine), which became a billion dollar herbicide, and in the 1980s following Monsanto’s model, more commercially important herbicides that targeted other amino acid biosynthetic pathways became available (Coggins, Abell et al. 2003). Even though glyphosate was discovered as a herbicide, later on it was also tested as an antiparasitic agent. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by glyphosate (Coggins, Abell et al. 2003; Campbell, Richards et al. 2004). This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. However, since its 2
introduction, resistance to glyphosate has been reported, including several species of cyanobacteria that are equipped with a resistant form of 5-enolpyruvylshikimate-3phosphate (EPSP) synthase up to the millimolar range (Forlani, Pavan et al. 2008). Therefore, new efforts have concentrated on more successful drug and herbicide design strategies. Apart from inhibitor discovery, the shikimate pathway is also of high interest for metabolic engineering since the phenolic products of the pathway are extensively used in the industry as antioxidants for processed foods (De Leonardis and Macciola 2004; La Camera, Geoffroy et al. 2005). Shikimic acid, one of the intermediates in the shikimate pathway, is used as a precursor for the synthesis of Tamiflu (Oseltamivir phosphate), the most prescribed flu medicine (Bertelli, Mannari et al. 2008). In plants, the shikimate pathway provides the precursors for the synthesis of the indole hormones and phenylpropanoids, which are currently of high interest due to their potential as nutraceuticals such as ferulic acid and genistein. However, despite its potential the metabolic engineering of the shikimate pathway has encountered a lot of challenges because regulation of the pathway in eukaryotes is much more complex than in microbes due to this secondary metabolism branching (Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999; Delmas, Petit et al. 2003; Singh and Christendat 2006).
The Shikimate Pathway The shikimate pathway consists of seven enzymatic steps that terminate with the production of chorismate (Figure 1-1). The committed step of the shikimate pathway is the aldol condensation of phosphoenol pyruvate (PEP) and erythrose-4-phosphate 3
(E4P) to form 3-deoxy-D-Arabino-Heptulosonate 7-phosphate (DAHP) catalyzed by the enzyme DAHP synthase. The pathway terminates with the synthesis of chorismate, which branches into the production of tyrosine, phenylalanine and tryptophan as well as folates, ubiquinones, salicylic acid, terpenoids, flavonoids, benzylisoquinoline alkaloids (Liscombe and Facchini 2008), indole hormones and lignin in plants (Campbell, Richards et al. 2004). These compounds are involved in signaling, growth, UV protection, pathogen defense and structural support in plants(Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999). Furthermore, benzylisoquinoline alkaloids serve as precursors for semi-synthetic drugs (Liscombe and Facchini 2008). DAHP synthase, the first enzyme of the pathway, interlinks glycolysis and the pentose-phosphate pathway with the shikimate pathway. This enzyme exists in three isoforms in most organisms and as such, it is a key regulatory point for the shikimate pathway. It condenses phosphoenol pyruvate (PEP) from glycolysis, and erythrose 4phosphate (E4P) from the pentose phosphate pathway to form DAHP. 3dehydroquinate (3-DHQ) synthase catalyzes the cyclization of 3-DHQ from DAHP and 3-DHQ dehydratase forms 3-dehydroshikimate (3-DHS), which is converted to shikimate by shikimate dehydrogenase. Shikimate is phosphorylated by shikimate kinase
to
form
shikimate
3-phosphate
(S3P).
S3P
is
converted
to
5-
enolpyruvylshikimate 3-phosphate (EPSP) by EPSP synthase. The seventh and ultimate step of the pathway is catalyzed by chorismate synthase (CS) to form chorismate (Herrmann 1995a). Chorismate is used to synthesize the aromatic amino acids as well as a variety of aromatic compounds such as: folates, ubiquinones, salicylic acid etc. (Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999; Campbell, Richards et al. 2004). 4
In bacteria, a separate enzyme catalyzes each step of the shikimate pathway, but this is not the case in fungi, apicomplexan parasites and plants. Fungi and Taxoplasma gondii have a pentafunctional AROM complex that includes the second to the sixth steps of the pathway (Lumsden and Coggins 1977; Lumsden and Coggins 1978; Campbell, Richards et al. 2004). It has been suggested that arom is the ancient supergene form that was either lost or replaced in several eukaryotic lineages raising interesting questions about the differential regulation of this pathway in the different taxa (Campbell, Richards et al. 2004). This phylogenetic path of the shikimate pathway places plants in an interesting taxonomic context with all the reactions being catalyzed by separate enzymes with the exception of dehydroquinase and shikimate dehydrogenase that form a bifunctional enzyme with two independent active sites (Singh and Christendat 2006). The aromatic amino acids are a direct product of the shikimate pathway, and apart from their role in protein synthesis, they also serve as precursors for a wide range of metabolites such as flavonoids, anthocyanins, lignin and indole hormones, that are involved in signaling, UV protection and structural support in plants (Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999). Shuttling of aromatic amino acids into secondary pathways is generally not observed in microbes, even though there is some evidence for their participation in antibiotic synthesis and other secondary metabolites (Webby, Baker et al. 2005; Webby, Lott et al. 2005). Because the intermediates of the shikimate pathway serve as precursors for many branch pathways, the study of the regulation mechanism of this pathway in plants is both interesting and necessary for the engineering of the pathway as well as for drug, vaccine or herbicide development.
5
All the shikimate pathway enzymes are predicted to be localized in the plant chloroplast using ChloroP (Emanuelsson, Nielsen et al. 1999), which predicts a characteristic N-terminal transit peptide and from data present in the plastid proteome databank which also indicates the localization of the shikimate pathway enzymes in the chloroplast (Friso, Giacomelli et al. 2004). In addition, chorismate synthase has been shown to be inactive in the cytosol in the presence of the cleavable transit peptide and chorismate is not synthesized in the cytosol (Henstrand, Schmid et al. 1995).
6
Figure 1- 1: An overview of the shikimate pathway. This is the pathway by which the aromatic
amino acids are synthesized in plants, fungi, microbes and apicomplexan parasites. The pathway initiates with the condensation of phosphoenol pyruvate (PEP) and erythrose-4phosphate (E-4-P) and through a series of seven enzymatic steps, it terminates with the synthesis of chorismate, which serves as a precursor for aromatic compounds including the aromatic amino acids. In plants, apart from protein synthesis, the aromatic amino acids are also used to synthesize secondary metabolites such as the flavonoids, anthocyanins, lignin and indole hormones. These metabolites are important for signaling, structural support and responding to UV as well as pathogenic stress.
7
DAHP Synthase (DAHPS) DAHP synthase, the first enzyme of the shikimate pathway, catalyzes the condensation of E-4-P and PEP to form DAHP. The substrates for this reaction are derived from primary metabolism. PEP is derived from glycolysis and E-4-P is derived from the pentose phosphate pathway (Figure 1-2). Upon the condensation of PEP and E-4-P into DAHP by DAHP synthase, a phosphate is released (Dyer, Henstrand et al. 1989; Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999; Delmas, Petit et al. 2003; Crowley 2006). From genetic and biochemical studies on the microbial shikimate pathway, it is apparent that DAHPS is the regulatory step of the shikimate pathway. In bacteria, there are three isozymes of DAHPS characterized as aroF, aroG and aroH and they are feedback inhibited respectively by the aromatic amino acids: Tyrosine, Phenylalanine and Tryptophan. Inhibition of the Trp-sensitive isozymes does no exceed 40% to ensure formation of sufficient amount of chorismate to fulfill the need for protein synthesis and for folate biosynthesis (Herrmann 1995; Herrmann 1995; Schmid 1995; Herrmann and Weaver 1999; Gosset, Bonner et al. 2001; Webby, Baker et al. 2005; Webby, Lott et al. 2005).Transcriptional repression by the tyr and trp repressors bound to the aromatic amino acids is also observed, but the main mode of regulation is feedback inhibition at the protein level in vivo (Herrmann 1995; Herrmann 1995; Schmid 1995; Herrmann and Weaver 1999; Gosset, Bonner et al. 2001; Webby, Baker et al. 2005; Webby, Lott et al. 2005).
8
DAHP synthase
-Adapted from (Herrmann and Weaver 1999)Figure 1- 2: The DAHP formation reaction mechanism. DAHP synthase catalyzes the condensation of phosphoenol pyruvate (PEP) and erythrose-4-phosphate (E-4-P) to make DAHP.
9
Structural analyses of bacterial DAHP synthases have revealed the architecture of the active site as well as the allosteric site (Figure 1-3). The PEP and E4P binding site is characterized by a conserved TIM barrell fold consisting of eight alternating alpha helices and beta sheets (Shumilin, Zhao et al. 2002). DAHP synthase enzymes are classified into two homologous clusters: AroAI and AroAII. The AroAI cluster is subdivided into AroAIα and AroAIβ (Gosset, Bonner et al. 2001). Gram negative bacteria contain DAHP synthase isozymes that belong to the AroAIα subcluster. The E.coli DAHP synthase isozymes belonging to this group are feedback regulated by each of the aromatic amino acids. Figure 1-3 is an illustration of the E.coli DAHPS AroG. The allosteric pocket in which a phenylalanine molecule is bound per monomer is located at the proximity of the dimerization domain in the cocrystal structure. This site is distant to the active site, in which a molecule of PEP is bound in each monomer. The active site is found in the center of the TIM barrel (Shumilin, Zhao et al. 2002). The AroAIβ cluster consists of enzymes closely related to KDOP synthases according to the 2001 classification (Gosset, Bonner et al. 2001). These enzymes are involved in the synthesis of lipopolysaccharides in bacteria and phosphorylated KDO in plants. KDO is a rare sugar synthesized by utilizing PEP in plants (Gosset, Bonner et al. 2001; Delmas, Petit et al. 2003). KDOP synthases can function as DAHP synthase with the opposite stereochemistry in vitro but not in vivo (Subramaniam, Xie et al. 1998; Subramanian, Benson et al. 2003). The AroAII family contains enzymes with a high degree of similarity between plants and microbes. A well characterized representative of this family is the Mycobacterium
tuberculosis enzyme which apart from the active site TIM barrel 10
contains two helices that have been inserted between α2 and β3 (Webby, Baker et al. 2005; Webby, Lott et al. 2005). These features distinguish this type II enzyme from the known structures of the Iα or Iβ subtypes. The authors propose that these extra two helices provide two distinct allosteric inhibitor sites that can be regulated by phenylalanine and tyrosine simultaneously, as they observe synergistic inhibition when they assay the enzyme for in vitro activity (Webby, Baker et al. 2005; Webby, Lott et al. 2005).
11
PDB ID: 1KFL Figure 1- 3: E.coli DAHP synthase co-crystallized with PEP and phenylalanine. This microbial enzyme was crystallized as a dimer. The substrate PEP molecule (grey) is located at the centre of the TIM barrel, an α/β barrel.. The allosteric pocket is located in the proximity of the dimerization domain and phenylalanine is bound at this site (cyan).
12
DAHP synthases in Arabidopsis thaliana Arabidopsis thaliana also has three DAHPS: DAHPS1, DAHPS2 and DAHPS3, isozymes encoded by the genes At4g39980, At4g35510 and At1g22410 respectively. Feedback inhibition has not been reported in plants. Since the aromatic amino acids are shunted to different secondary pathways in plants, the regulation mechanism of the shikimate pathway must be more complex than in microbes. It is possible that regulation occurs at several levels including transcriptional and post-translational modifications (Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999; Soll and Schleiff 2004; Winkel 2004). Studies in our lab as well as studies in potato suspension cultures suggest that DAHPS isozymes are regulated in response to wounding, pathogens and exogenous compounds (Dyer, Henstrand et al. 1989; Herrmann 1995; Herrmann 1995; Herrmann and Weaver 1999; Crowley 2006). The EPSP synthase inhibitor, glyphosate, increases DAHP expression in vivo, but not in vitro, in potato suspension cultures (Dyer, Henstrand et al. 1989). Additionally, the DAHPS2 protein levels in plants are redox regulated suggesting that the shikimate pathway is stimulated in the presence of light (Rogers, Dubos et al. 2005). A large number of chloroplast localized proteins are regulated by redox modulation. Post-translational redox regulation does not only apply to light-dark regulation, but it also regulates the plastid response to cytosolic sugar levels (Soll 2002; Geigenberger, Kolbe et al. 2005). Experimental evidence suggests that protein import into the chloroplast can be controlled by redox state and this is a potential mode of regulation for the DAHP synthase isozymes in plants (Soll 2002; Geigenberger, Kolbe et al. 2005; Rogers, Dubos et al. 2005). 13
Studies on the regulation of the shikimate pathway in eukaryotes to determine the mechanism of regulation of this pathway are under way. For example, in yeast, DAHP synthase has been shown to interact with the Smt3p, a SUMO protein, which can affect diverse protein function and has been shown to reduce the activity of several transcription factors (Subramaniam, Xie et al. 1998). However, no validation or functional characterization of such an interaction has been achieved so far. Further evidence that hints towards complex regulation of the pathway in plants shows that DAHPS1 is the only DAHPS affected by wounding and pathogen attack (Keith, Dong et al. 1991) and also, organ specific expression of DAHPS isozymes has been reported in tomato (Gorlach, Beck et al. 1993) and is also observed with DAHPS isozymes in Arabidopsis thaliana (Figure 1- 4). DAHPS1 transcript levels are the highest in the rosette leaves. DAHPS2 is regulated by circadian rhythms and its levels are highest in the dry seed, leaves and floral organs, while DAHPS3 transcript levels are highest in the floral organs and the stem (Schmid, Davison et al. 2005).
14
DAHPS1
DAHPS2
DAHPS3
Figure 1- 4: Organ specific map of DAHPS isozyme transcript expression in A.thaliana. The colour intensity bar indicates the relative levels of expression for each isozyme based on the AtGenExpress Consortium Data (Schmid et al, 2005). DAHPS1 is most highly expressed in the rosette leaves. The corresponding organs of highest expression for each of these isozymes are: Rosette leaves for DAHPS1; dry seed, leaves and floral organs for DAHPS2; and, lower stem and floral organs for DAHPS3. The images were generated using the eFP browser at the Bio-Array Resource for Arabidopsis Functional Genomics (Winter, Vinegar et al. 2007).
15
SALK T-DNA insertion lines of each DHS isozyme (Table 1-1) have been used in our lab to elucidate the role of each of these isozymes in the regulation of the shikimate pathway (Crowley 2006). Single homozygous knockout lines have no apparent phenotype when grown under standard growth conditions: 0.5X MS, 2.5mM MES and 1.5% sucrose, standard light and room temperature. However, when these knockout seedlings are stressed, some interesting phenotypes are observed. All these homozygous knockout lines are sensitive to 20 minute UV light exposure compared to wild type plants. They show delayed bolting and dried leaves. In addition, dahps1 plants are sensitive to supplementation with 150 μM tyrosine. These seedlings exhibit arrested growth and leaves do not emerge. This phenotype suggests that tyrosine serves as a modulator of either DAHPS2, DAHPS3 or both. This modulation can be applied directly to these isozymes or it can be mediated. To determine if tyrosine has a direct effect on the function of these proteins, kinetic analysis of each isozyme that measured the release of inorganic phosphate by each of the isozymes in the absence and presence of tyrosine with different substrate concentrations was performed. This analysis showed that there was no difference in the activity of the DAHPS isozymes in the presence of tyrosine (Crowley 2006).
The phenylpopanoid pathway and its regulation. The phenylpropanoid pathway is involved in the synthesis of flavonoids and monolignols (Bate, Orr et al. 1994; Werck-Reichhart 1995; Dixon and Steele 1999; Werck-Reichhart and Feyereisen 2000; Werck-Reichhart, Hehn et al. 2000). Flavonoids are phenolic compounds with anti-oxidant properties and are involved in protecting the plant against ultraviolet and pathogen stress. Monolignols serve as precursors for the 16
synthesis of lignin. The chemical diversity acquired specifically in plants via this pathway is advantageous for large repertoires of pigments, structural and defensive compounds (Bate, Orr et al. 1994; Werck-Reichhart 1995; Dixon and Steele 1999; Werck-Reichhart and Feyereisen 2000; Werck-Reichhart, Hehn et al. 2000). Phenylpropanoids serve as key chemical modulators in defensive phytoalexin responses to infection and herbivory, attraction of insect pollinators via flower color, and induction of root nodulation by symbiotic nitrogen-fixing rhizobial colonies (Ferrer, Austin et al. 2008). Three enzymatic transformations redirect the carbon flow from primary metabolism, transforming phenylalanine into the Coenzyme A (CoA)-activated hydroxycinnamoyl (phenylpropanoid) thioester capable of entering the two major downstream pathways, monolignol and flavonoid biosynthesis. Deamination by phenylalanine ammonia-lyase (PAL) forms the phenylpropanoid skeleton, producing cinnamic acid. Various general phenylpropanoid pathway intermediates are also diverted into biosynthetic pathways for benzoic acid, salicylic acid, and coumarins (Ferrer, Austin et al. 2008). Lignin is an abundant structural polymer formed from monolignol derivatives of the general phenylpropanoid pathway. Together with cellulose, it provides the structural integrity to support plant vertical stature (Ferrer, Austin et al. 2008). It imparts mechanical strength to stems and trunks, and hydrophobicity to water-conducting vascular elements (Dixon, Lamb et al. 1996; Dixon, Howles et al. 1998; Dixon and Steele 1999). Flavonoids provide protection against ultraviolet rays. Chalcone synthase (CHS) is the first enzyme specific for flavonoid biosynthesis. It catalyzes the condensation of three molecules of malonyl CoA with one molecule of 4-coumaroyl CoA (a product of 17
the core phenylpropanoid pathway) to yield a polyketide intermediate which is cyclized on the enzyme to form 2',4,4',6'-tetrahydroxychalcone. Following CHS, several isomerases, reductases, hydroxylases, glycosyltransferases and acyltransferases enrich the basic flavonoid skeleton leading to the variety of flavonoid compound subclasses. The presence of a C2=C3 double bond dinstinguishes flavones and flavonones, making the latter less reactive. Isoflavonoids are characterized by a C2-C3 aryl ring migration and concomitant double bond formation catalyzed by isoflavone synthase. The addition of hydroxyl groups to core flavonoid rings leads to the formation of the flavonols and flavandiols, which serve as precursors of proanthocyanidins and anthocyanins (Dixon, Lamb et al. 1996; Dixon, Howles et al. 1998; Dixon and Steele 1999; Ferrer, Austin et al. 2008). The chemical diversity as well as the multiplicity of roles exhibited by the phenylpropanoid pathway requires tight regulation of these processes. More than 15 P450-dependent reactions have been characterised in this pathway. Several of these reactions constitute important regulatory branching points, which provide clues in the regulation of metabolic pathways in plants. Indirect and direct data indicate that distinct P450s catalyse the different reactions. Cinnamate 4-hydroxylase (C4H), is the most extensively studied plant P450 (Werck-Reichhart 1995; Werck-Reichhart and Feyereisen 2000; Werck-Reichhart, Hehn et al. 2000). The phenylalanine ammonia lyase mediated reaction becomes the dominant rate-determining step in the regulation of lignin deposition at levels 3-4-fold below wild-type (Bate, Orr et al. 1994). There are two phenylalanine ammonial lyases in plants: phenylalanine ammonia lyase 1 (PAL1) and phenylalanine ammonia lyase 2 (PAL2). PAL2 can be induced to localize to the ER by overexpression of cinnamate 4-hydroxylase (C4H), and this localization can be 18
reversed by overexpression of PAL1. This points to protein interactions between the membrane P450 enzyme, C4H, and the two PAL enzymes, and further suggests that the two PAL isoforms play distinct roles in interactions with C4H (Winkel 2004). Experimental evidence exists for the spatial organization of phenylpropanoid enzymes in the cytoplasm of plant cells. Studies in a wide range of species have generated evidence for the co-ordinate expression of genes and enzymes, channeling of labeled intermediates, membrane association of operationally soluble enzymes, and physical interaction of component enzymes making this the pathway with the most diverse regulatory mechanisms at many levels (Winkel 2004). It is possible that these enzymes function in distinct metabolons, each dedicated to producing a particular class of phenylpropanoids.
Auxin biosynthesis and its regulation. Indole-3-acetic acid (IAA) was the first plant growth regulatory substance discovered and ever since, auxins are known to be involved in the regulation of basic growth processes such as cell division and cell elongation. Auxins exhibit pleiotropic physiological effects on tissues, organs and the whole plant in general (Galweiler, Guan et al. 1998; Leyser 1999; Leyser and Berleth 1999; Sabatini, Beis et al. 1999; Geldner, Friml et al. 2001; Leyser 2001; Swarup, Friml et al. 2001; Friml and Palme 2002; Friml 2003; Blakeslee, Peer et al. 2005; Blilou, Xu et al. 2005; Dharmasiri, Dharmasiri et al. 2005; Dhonukshe, Kleine-Vehn et al. 2005; Leyser 2005; Paponov, Teale et al. 2005; Leyser 2006). Auxins are weak acids and at the extracellular pH 5.5, auxin is protonated and can enter by diffusion (Lomax 1997), but this uptake can be enhanced in tissues by 19
auxin influx carriers (Swarup, Friml et al. 2001). However, inside the cell, the higher pH of the cytoplasm results in ionization and auxin is trapped inside the cell. And, therefore, efflux is an active and carrier dependent process mainly carried out by the PIN efflux transporters. PIN proteins cycle between the plasma membrane and the intracellular vesicular compartments (Geldner, Friml et al. 2001) and are frequently localized polarly to specific cell faces (Leyser 2005). This polar localization correlates with the direction of auxin movement. Therefore, PIN transporters are essential for the highly specific patterns of auxin distribution (Paponov, Teale et al. 2005). PIN transcription, accumulation and subcellular localization all seem to be regulated by auxin and in the root tip, there is a strong stabilizing effect in this dynamic (Leyser 2002; Leyser 2005). In a similar fashion, the transcription patterns of the PINs that are required to maintain the auxin distribution pattern are also auxin regulated (Blilou, Xu et al. 2005). Therefore, PIN knockouts exhibit no phenotypic defects because changes in auxin distribution affect the transcription of other PINs to compensate. PIN proteins are members of the major efflux facilitator family of integral membrane proteins, are essential for polarized auxin movement, and align with the vector of auxin transport. In Arabidopsis thaliana, each member of the PIN family displays a unique tissue-specific expression pattern, and pin mutations generally exhibit growth phenotypes that are consistent with the loss of directional auxin transport in the corresponding tissues (Blakeslee, Peer et al. 2005). In the root tip, initially the main source of auxin is from the shoot with auxin transported from the shoot to the root tip through the phloem and the polar transport stream (Swarup, Friml et al. 2001; Bhalerao, Eklof et al. 2002) . The root apical meristem established during embryogenesis occurs in response to local auxin 20
accumulation at the basal end of the embryo (Friml and Palme 2002; Friml 2003). When auxin is low, transcription from auxin response factor (ARF) regulated genes is repressed by dimerization between ARFs and members of the Aux/IAA family of repressors. To activate this transcription, auxin targets Aux/IAAs for degradation by the 26s proteasome (Leyser 2002).
21
Chapter Objectives Previous studies with Arabidopsis. thaliana DAHPS knockout lines in our laboratory have shown that dahps1 seedlings are hypersensitive to tyrosine. This seedlings show arrested growth upon tyrosine concentration at 150μM and this sensitivity is not observed with Columbia up to 750 μM tyrosine (Crowley 2006).
This
has suggested that tyrosine plays an important role in regulating either DAHPS2 or DAHPS3. Furthermore, previous analysis has also shown that dahps1,2,3 knockout lines are hypersensitive to UV stress. In continuation to this investigation, the work presented here has two aims:
1) To establish an understanding of the putative role of the DAHP synthase isozymes in regulating the flux of chorismate into the branching pathways. For this aim, DAHP synthase knockout lines in Arabidopsis thaliana have been used to study the phenotypic and transcriptional changes that occur in these knockout seedlings in the absence of one of the DAHP synthase isozymes. 2) To examine the role of the aromatic amino acids in regulating enzymes of the shikimate pathway and the downstream pathways ie. the phenylpropanoid pathway and the indole hormone biosynthetic pathway. For this aim, the aromatic amino acids: tyrosine, tryptophan and phenylalanine were used to supplement the single dahps knockout lines of Arabidopsis thaliana. Root length was quantified as the most reliable indicator of seedling development. To determine the synergistic role that each aromatic amino acid plays in concert with the DAHP synthases in regulating the partitioning of the chorismate pool, transcriptional analysis of dahps1 seedlings treated with tyrosine and a combination of tyrosine and phenylalanine or
22
tyrosine and tryptophan was done. dahps1 seedlings were chosen because their hypersensitivity to tyrosine, observed by arrest in root growth, was known and transcriptional analysis with one of the other aromatic amino acids revealed which branching pathway was differentially regulated to rescue the tyrosine hypersensitivity which affected transcripts of the phenylpropanoid pathway and expression of auxin response genes. Validation of the rescue of tyrosine hypersensitivity was achieved through crosses with the auxin responsive marker DR5::GUS, which served as an indicator of auxin accumulation at the root tip.
MATERIALS AND METHODS Plant material and growth conditions The transgenic seeds were ordered from the Salk Institute via the Arabidopsis Biological Resource Centre and they were verified as true knockout lines previously in our laboratory (Crowley 2006). These lines are described in table 1-1.
23
Table 1-1: The DAHP synthase insertion lines and the corresponding AGI IDs
Annotation Name Salk Line ID
AGI ID
DAHPS1
dahps1
salk_055360 At4g39980
DAHPS1
dahps1-2 salk_088442 At4g39980
DAHPS2
dahps2
salk_033389 At4g35510
DAHPS3
dahps3
salk_026183 At1g22410
The insertion position of each line is illustrated in figure 1-5.
24
Figure 1- 5: Diagram of the position of the T-DNA insertion for the DAHP synthase knockout lines.
dahps1 contains an insertion in the third exon. dahps2 contains an insertion in the third intron. dahps3 contains an insertion in the second intron.
25
Arabidopsis seeds were sterilized for 10 minutes in 20% (v/v) bleach and 0.05% Tween-20 (BioShop) and were rinsed sequentially with double distilled MilliQ water. Seeds were imbibed for two days on 0.5X Mushragie and Skoog (MS) media (Sigma), 2.5mM morpholino ethane sulfonic acid (pH 7.5) (BioShop) and supplemented with o
1.5% (w/v) sucrose (BioShop) in the dark at 4 C for two days. The same medium was used for their germination and growth in the subsequent 7days under continuous light 2
(45μmol/m /sec) at room temperature. For root quantification and gravitropism responses, the seedlings were grown vertically (ie. perpendicular to the shelf). o
Seedlings that were transferred to soil were grown in a growth chamber at 22 C with 16 hours light and 8 hours dark. Upon seed collection, the seeds were dried for 7 days prior to sterilization. For aromatic amino acid supplementation, all amino acids were obtained from Sigma. Stock solutions of 11mM L-tyrosine, 61mM L-phenylalanine , and 48.9mM L-tryptophan were diluted to the appropriate concentrations in sucrose-supplemented MS-agar medium.
Genomic DNA Extraction Leaves or seedlings were frozen in liquid nitrogen and ground to a fine powder. 200μl of fresh extraction buffer consisting of 200mM Tris-HCl (pH 8) (BioShop), 25mM ethylenediaminetetraacetic acid (EDTA) (pH 8) (BioShop) and 0.5% sodium dodecyl sulfate (SDS) (BioShop) was added to the ground tissue. 100μl of phenol (Invitrogen):chloroform (EMD):isoamyl alcohol (BioShop) (24:24:1) was added to the mix. The sample was mixed and centrifuged for 10 minutes at 13000 rpm. The DNA 26
from the supernatant was precipitated with an equal volume of isopropanol (EM Science). The sample was spinned for 10 minutes at 13000 rpm to pellet the DNA. Isopropanol was removed and the excess was evaporated at room temperature for 15 min. DNA samples were resuspended in 50 μL of double distilled water.
Generation of dahps1xDR5::GUS crosses Single mutant flowers were emasculated as soon as the tips of the white petals were visible. The DR5::GUS plant pollen was used to fertilize the dahps1 flowers under the microscope. Hand-pollinated stigmas were wrapped in Saran wrap and left to mature. Seeds were harvested once the siliques turned brown but prior to pod splitting. The F1 plants were allowed to self and the F3 generation was screened via PCR for the maternal dahps1 mutation and through 5mM GUS staining for the paternal DR5::GUS marker. The stained seedlings were visualized under an Olympus bright field microscope at 20x magnification.
Total RNA Extraction For the microarray experiments, the RNA was isolated using the Qiagen Plant RNA preparation kit. Briefly, 100mg of 8-day old seedlings were placed in liquid nitrogen and ground thoroughly. 450 μL of buffer RLT was added to the ground sample and the tube was vortexed for 1 minute. The lysate was transferred to a QIAshredder spin column and spun down for 2 minutes at full speed. The supernatant was transferred to a fresh tube and was precipitated with an equal volume of ethanol and the mixture was transferred to an RNeasy spin column. The sample was spun at 10000 rpm for 15 27
seconds and was washed with 700 μL of buffer RW1. It was spun again at 10000 rpm for 15 seconds and the flow through was discarded. The RNA was washed twice with buffer RPE prior to elution. After spinning for 2 minutes at 10000rpm, the RNA was eluted in 17 μL of RNase-free water.
Microarray Analysis Transgenic dahps1 plants were grown for 8 days under continuous light. For the amino acid supplemented seedlings, 8ml of 2mM tyrosine, tyrosine and phenylalanine or tyrosine and tryptophan was added and the plates were replaced under continous light for the next 8 hours. Seedlings were flash frozen in liquid nitrogen RNA was extracted
as
described
above.
Three
replicates
were
submitted
for
each
treatment.Hybridization to the ATH1 whole genome array (Affymetrix), scanning of the hybridized array and data pre-processing were done at the Department of Cell and Systems Biology Affymetrix Genechip facility headed by Dr. Nicholas Provart. Thanh Nguyen performed the reverse transcription and hybridization with standard oligo dT primers. The signals from the Affymetrix chips were normalized using Microarray Software Suite 2.0 and with an expression average of 500. Statistical analysis was done using the SAM (Significance Analysis of Microarrays) Excel Plug-in (Tusher, Tibshirani et al. 2001) . Significance was determined at 5% false discovery rate based on the number of differentially regulated genes. Overrepresentation of gene groups among the differentially regulated genes was determined using the GOstats tool in Gene Ontology (Beissbarth and Speed 2004). Term enrichment of the GOstats hits was achieved using
28
AmiGO release 2008/08/07. The Venn diagrams were prepared using Venny (Oliveros 2007) and were manipulated using Adobe Photoshop.
Verification of Dr5::GUS crosses with PCR The verification of the parental recessive trait dahps1 in the dahps1xDr5::GUS progeny was achieved through a gene specific and insertion specific reaction. These primers were previously designed in our laboratory (Crowley 2006) and were synthesized
by
Integrated
DNA
Technologies
(IDT):
Forward:
5'-
GAGCCTTTGCCACTGGAGGTT-3' and reverse: 5'-TCTCATGTTCTCGGCACCCAT-3'. To determine if the gene contained T-DNA, the left border primer LBb1 (5'GCGTGGACCGCTTGCTGCAACTC-3') designed by SALK and the gene specific reverse primer were used. All PCR reactions used pfu enzyme purified at home as the polymerizing enzyme. The PCR conditions were an initial 1.5 minute denaturing step at o
o
95 C, followed by 35 cycles of a 1-minute denaturation at 95 C, 1-minute annealing o
o
step at 48-60 C, and a 1.5-3 min extension step at 72 C. This was followed by a final o
extension step for 10 minutes at 72 C.
GUS staining procedure o
Seven day old seedlings were incubated for one hour in -20 C in pre-chilled 90% acetone. After that, the seedlings were washed twice for 5 minutes in 100 mM NaPi o
buffer pH7.7. Then, the seedlings were incubated for 1 hour at 37 C in staining buffer (2mM X-gluc, 5mM ferricyanide/ferrocyanide). The samples were transferred in
29
o
ethanol:acetic acid 3:1 at 4 C for 1 hour. The fixative was replaced with 70% ethanol prior to visualization under the microscope.
RESULTS DAHPS2 lacks a C-terminal alpha helix which is present in DAHPS1 and DAHPS3. To determine the presence of any putative regulatory domains, comparison of Arabidopsis DAHPS three-dimensional models was conducted. Structural modeling using PHYRE (Bennett-Lovsey, Herbert et al. 2008)
selects the Mycobacterium
tuberculosis AroAII structure as the closest relative and as the modeling template for high precision homology modelling. None of the Arabidopsis proteins are predicted to contain the extra α2 and β3 helices that the M.tuberculosis structure contains for allosteric regulation.
Interestingly, the
Arabidopsis DAHPS3 isozyme has a
pronounced hairpin loop that is absent in the microbial protein and the other two Arabidopsis proteins. Additionally, DAHPS2 has a deletion for the last C-terminal helix observed in DAHPS1 and DAHPS3 (Figure 1-6, 1-7). To explore the extent of this deletion in plants and bacteria, multiple sequence alignments were conducted for the best reciprocal hits of each DAHPS isozyme. This Cterminal deletion is also observed in most microbial proteins but in none of the other plant DAHP synthase isozymes (Figures 1-7A,B,C,D). This domain has been identified in the black cottonwood, grape vine, tobacco, European beech, curly leaf parsley, rice, tomato and Indian mulberry
DAHP synthases even though it is partially absent in
potato.
30
DAHPS1
DAHPS2
DAHPS3
Models generated with 100% estimated precision using Phyre: Bennett-Lovsey et al, 2008
Figure 1- 6: Predicted three dimensional models of the Arabidopsis thaliana DAHP synthase isozymes. This prediction shows conservation of the active site TIM-barrel but there is no conservation of the allosteric pocket. Additionally, this analysis identifies the presence of a C-terminal α-helix in DAHP synthase 1 and 3 that is not found in DAHP synthase 2. The C-terminal helix , colored blue, is only present in DAHPS1 and DAHPS3 but not in DAHPS2.
31
A)
CLUSTAL W ALIGNMENT WITH HOMOLOGOUS DAHPS1 SEQUENCES FROM PLANTS SHOWS CONSERVATION OF THE C-TERMINAL REGION.
B)
CLUSTAL W ALIGNMENT WITH HOMOLOGOUS DAHPS2 SEQUENCES FROM PLANTS SHOWS DELETION OF THE C-TERMINAL REGION IN A.THALIANA AND PARTIAL DELETION IN POTATO.
32
C)
CLUSTAL W ALIGNMENT WITH HOMOLOGOUS DAHPS3 SEQUENCES FROM PLANTS SHOWS CONSERVATION OF THE C-TERMINAL REGION. D)
CLUSTAL W ALIGNMENT WITH HOMOLOGOUS DAHPS SEQUENCES FROM BACTERIA SHOWS THE SAME ABSENCE OF THE C-TERMINAL REGION AS A.THALIANA DAHPS2. Figure 1- 7: Multiple sequence alignment of DAHP synthase isozymes from sequenced plant genomes (AC) and microbial sequences (D). This alignment shows the presence of a C-terminal domain in most plant sequences but not in the microbial sequences. A) Multiple sequence alignment of the DAHPS1 closest orthologs based on the best reciprocal match. B) Multiple sequence alignment of the DAHPS2 closest orthologs based on the best reciprocal match. C) Multiple sequence alignment of the DAHPS3 closest orthologs based on the best reciprocal match. D) A) Multiple sequence alignment of the Arabidopsis thaliana DAHPS sequences with the closest microbial orthologs based on the best reciprocal match.
33
Aromatic amino acid supplementation has distinctive effects on the DHS seedling phenotype in Arabidopsis thaliana. To investigate the role of DAHP synthase isozymes in Arabidopsis, T-DNA insertion lines for DAHPS1, DAHPS2, and DAHPS3 were obtained from the Salk Institute. Seeds from the T3 generation were screened via PCR for homozygous mutants. Gene specific and insertion specific PCR were performed to identify the presence of the wild type copy of the gene (Figure 1-8). The extension time was kept 1:30 min and this did not allow polymerization of the T-DNA insertion with gene specific primers. However the insertion was amplified from genomic DNA with a T-DNA specific forward primer designed by the SALK institute and a gene specific reverse primer in order to verify the presence of the T-DNA insertion in the DAHP synthase gene. RTPCR was used to identify true knockout lines for the gene encoding each isozymes based on the lack of transcript levels (Crowley 2006) This analysis does not eliminate the possibility of a T-DNA insertion in another gene of the Arabidopsis thaliana genome.
34
Figure 1- 8: Diagram of the position of the primers used by Valerie Crowley for genotyping the DAHP synthase insertion lines. Polymerization with the gene specific primers in the presence of the insertion would result in a product ~5000 bp. However, due to the limitation of the extension time to 1:30 min, polymerization of this large amplicon was not achieved.
35
The DAHP synthase knockout lines were treated with increasing concentrations of aromatic amino acids in their media ranging from 250 μM to 750 μM tyrosine (Figure 1-9 to 1-12). This experiment was replicated five times with ten seedlings per treatment each time. dahps1 seedlings are hypersensitive to tyrosine. This sensitivity is also observed with the other lines including wild type Columbia but this effect is observed at 750 μM tyrosine as opposed to 250 μM for dahps1 (Figure 1-9). Seedlings grown on tyrosine supplemented media show arrested growth and shorter root length. dahps1 seedlings supplemented with 250-750 μM tyrosine germinate, but do not develop any further. Interestingly, a dose dependent reduction in root growth is observed upon treatment with increasing concentrations of tyrosine both on the dahps1 heterozygous seedlings and on the second dahps1 allele (salk_088442) (Figure 1-10), which is not a true knockout of the gene. However, the arrest in growth in the dahps1 heterozygous seedlings and in the insertion siblings is not as severe as that observed in the true dahps1 knockout line. The tyrosine sensitivity phenotype is alleviated if these lines are grown with a combination of either 500 μM tyrosine and tryptophan or tyrosine and phenylalanine as well as with all three amino acids at the same time. These seedlings show no longer arrested growth and if transferred to soil, they grow to full maturity. Even though there is a significantly different response to aromatic amino acid supplementation for the dahps2 seedlings compared to the no treatment and wild type controls, these seedlings do not exhibit any differential response to amino acid supplementation when compared to the other two knockout lines (Figure 1-10). However, the dahps3 seedlings are hypersensitive to treatment with tryptophan (Figure 1-11). Growth of dahps3 knockout plants in the presence of tryptophan inhibits growth 36
after day 4 and aborts root growth. This is a dose dependent response and 750 μM tryptophan in the media causes the most severe phenotype. These seedlings exhibit arrested growth upon hypocotyl emergence. When the line is treated with 500 μM tryptophan and phenylalanine or tryptophan and tyrosine or with all three amino acids, the sensitivity at embryogenesis is not severe and the seedlings have the potential to grow to maturity. To quantify the dose-dependent effect of the treatment with the aromatic amino acids as well as the degree of recovery by treatment with more than one aromatic amino acid, root length was measured from seedlings grown on aromatic amino acid supplemented media. Germination was synchronized and all the seeds germinated at the same time. The plates were grown vertically and root length was quantified on 5-day old seedlings. In figures 1-9 to 1-12, treatment with phenylalanine also shows a significant effect on root growth on all genotypes and this effect is not as severe as that observed with the tyrosine and tryptophan hypersensitivity. There is no apparent defect on seedling growth and survival upon supplementation with phenylalanine and the seedlings are capable of growing to maturity. Interestingly, treatment with 750μM phenylalanine encourages root growth of wild type seedlings comparable to that observed with the untreated wild type control (Figures 1-9 to 1-12). This response to phenylalanine in the untreated control is not observed with the dahps knockout lines and is also not observed with lower concentrations of phenylalanine. Root length was also quantified from 10-day old seedlings that were grown on plain media for 5 days and then transferred on fresh aromatic amino acid supplemented media. This was done to ensure that the effect of the aromatic amino acids is on growth and not germination. The pattern of sensitivity with the knockout lines and the varied 37
treatments was the same but the highest sensitivity was observed when the seedlings were germinated on amino acid supplemented media. The seedlings were also observed for defects on gravitropism, but no such effect was observed.
38
*: significantly compared to the untreated control, p
