INVESTIGATION OF REGULATORY AND FUNCTIONAL DIVERSIFICATION OF
Arabidopsis thaliana SHIKIMATE KINASE DUPLICATES
Stephen Buranyi
Thesis submitted in conformity with the requirements
For the degree of Master of Science
Department of Cell and Systems Biology
University of Toronto
© Copyright by Stephen Buranyi 2014
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INVESTIGATION OF REGULATORY AND FUNCTIONAL
DIVERSIFICATION OF A rabidopsis thaliana SHIKIMATE KINASE
DUPLICATES
Stephen Buranyi
Master of Science
Department of Cell and Systems Biology
University of Toronto
2014
Abstract
Two Arabidopsis thaliana shikimate kinases, AtSK1 and AtSK2, were previously
identified as having arisen from a duplication event 40-60 million years ago and are both believed
to function in the shikimate pathway. We investigated the homologs have acquired divergent
regulatory or functional roles since duplication. AtSK1 demonstrates different transcript levels
than AtSK2 during heat shock and in floral tissues as determined by RT-PCR. Mining of
publically available microarray datasets identified HSF1 and MYB family transcription factors as
possible regulators of AtSK1 under these conditions. Heat shock response did not appear to be
affected by either sk1 or sk2 knockouts in assays measuring thermotolerance and ROS
production. Floral morphology appears normal during floral stages corresponding with AtSK1
transcript induction however, pollen viability is reduced by 20% in sk1 knockouts as measured
by FDA staining. Thus, this work has identified instances of differential regulation and function
between two recently duplicated shikimate kinases.
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Acknowledgements I would like to thank Dr. Dinesh Christendat for the opportunity to conduct this research
and the members of the Christendat lab over the past two years, James Peek, Stephanie Prezioso,
Daniel Johnson, and Joseph Gemetti, for maintaining such a warm, fun, and helpful research
environment. Thanks to Brandon McCartney for his inspiration and support throughout this work.
I would also like to thank my committee members, Dr. Darrell Desveaux and Dr. Peter McCourt
for their participation and suggestions, and Dr. Wenzislava Ckurshumova and Dr. Tammy Sage
for their advice and help with plant work and microscopy.
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Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ....................................................................................................................... iii List of Tables: ............................................................................................................................... vii List of Figures: ............................................................................................................................ viii List of Abbreviations: .................................................................................................................... ix Chapter 1 -Introduction ................................................................................................................. 1
The Shikimate Pathway ................................................................................................................ 1 Pathway Organization .................................................................................................................. 2 Shikimate Kinase Reaction ........................................................................................................... 4 The Shikimate Pathway in Plants ................................................................................................. 6 Evolutionary Diversification of Shikimate Kinases ..................................................................... 7 Relationship of Arabidopsis Shikimate Kinase Homologs to Bacterial Shikimate Kinases ........ 9
AtSK1 and AtSK2 .................................................................................................................... 9 SKL1 ....................................................................................................................................... 10 SKL2 ....................................................................................................................................... 10
Regulation of the Shikimate Pathway ........................................................................................ 11 Post Translational Regulation ................................................................................................. 11 Transcriptional Regulation ..................................................................................................... 11
Regulation of Shikimate Kinase Gene Duplicates ..................................................................... 12 SKL1 and Plant Chloroplast Biogenesis .................................................................................... 13 Heat Stress Response in Arabidopsis ......................................................................................... 15
Chapter 2 -Thesis Objectives ....................................................................................................... 16 Chapter 3 - Materials and Methods ............................................................................................ 17
Plant Growth Conditions ............................................................................................................ 17 RT-PCR ...................................................................................................................................... 17 Heat Stress Assays ...................................................................................................................... 17
Survival Assay ........................................................................................................................ 17 Hypocotyl Elongation ............................................................................................................. 18 TBARS Assay ........................................................................................................................ 18
Floral Assays .............................................................................................................................. 18 Measurement of Floral Organs ............................................................................................... 18
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Pollen Viability ....................................................................................................................... 19 Seed Count .............................................................................................................................. 19
Cloning of Recombinant Protein Constructs .............................................................................. 19 AtSK1 and AtSK2 .................................................................................................................. 19 CaM Plasmids ......................................................................................................................... 20
Protein Expression: ..................................................................................................................... 21 BL-21 ...................................................................................................................................... 21 Protein Expression in Arctic Express ..................................................................................... 21
Protein Purification ..................................................................................................................... 21 Nickel Column ........................................................................................................................ 21 Phenyl Sepharose Column (Calmodulin Purification) ........................................................... 22 Recovery of Protein From Inclusion Bodies .......................................................................... 22
Far Western (HRP-CaM Overlay) .............................................................................................. 22 Chapter 4 - Results ....................................................................................................................... 25
Expression Analysis of Shikimate Kinase Homologs ................................................................ 25 Arabidopsis thaliana Shikimate Kinase Duplicates ................................................................ 25 Oryza sativa Shikimate Kinase Duplicates ............................................................................. 26 Hordeum vulgare Shikimate Kinase Duplicates ..................................................................... 29 Glycine max Shikimate Kinase Duplicates ............................................................................ 30
Transcriptome Analysis of Heat and Floral Development Transcription Factor Mutants ......... 31 RT-PCR Confirmation of AtSK1 Transcript Induction. ............................................................ 34
Floral Tissue ........................................................................................................................... 34 Heat Stress .............................................................................................................................. 34
sk1 and sk2 During Heat Stress Response ................................................................................. 35 Seedling Survival .................................................................................................................... 35 Reactive Substance Quantification in Heat Stressed sk1 and sk2 Seedlings ......................... 35 Hypocotyl Elongation Following Heat Stress ........................................................................ 36
AtSK Interactions With CaM ..................................................................................................... 39 Cloning and Expression of AtSKs .......................................................................................... 39 Co-Expression and Binding Assays ....................................................................................... 40 Far Western Assay .................................................................................................................. 40
sk1 Stage 13 Flowers .................................................................................................................. 42 Stage 13 Flower Dissection .................................................................................................... 42
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sk1 Pollen Viability ................................................................................................................ 43 Chapter 5 - Discussion .................................................................................................................. 46
Transcript analysis of SK Duplicates Across Species ............................................................... 46 AtSK Duplicates During Heat Stress ......................................................................................... 47
Transcript Analysis ................................................................................................................. 47 sk1 and sk2 Survival and ROS Response During Heat Stress ............................................... 48
AtSK-CaM Interaction Studies ................................................................................................... 49 AtSK Duplicates During Floral Development ........................................................................... 51
Transcript Analysis ................................................................................................................. 51 sk1 Floral Morphology ........................................................................................................... 51 sk1 Pollen Viability and Seed Set .......................................................................................... 52
Pollen Viability During Heat Stress ........................................................................................... 54 Alternative Models ..................................................................................................................... 54
Chapter 6 – Conclusions and Future Directions ....................................................................... 55 References ..................................................................................................................................... 58
List of Tables: Table 1: PCR Conditions 23 Table 2: Primers 23 Table 3: Buffers 24 Table 4: Heat Shock Survival Screen 36
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List of Figures: Figure 1: Enzymes and intermediates of the shikimate pathway. 2 Figure 2: Reaction catalyzed by shikimate kinase. 5 Figure 3: Crystal Structure of AtSK2 6 Figure 4 : pET28-MOD vector map 20 Figure 5 : Transcript profile of Arabidopsis thaliana. 24 Figure 6 : Transcript profile of Oryza sativa. 26 Figure 7 : Transcript profile of Hordeum vulgare. 28 Figure 8 : Transcript profile of Glycine max. 29 Figure 9: Transcript profile of Transcription Factor knockouts. 31 Figure 10: RT-PCR of Col-0 using primers for AtSK1 and AtSK2. 33 Figure 11: sk knockout lines tested for production of ROS at elevated temperatures. 36 Figure 12: Hypocotyl length of seedlings grown at elevated
temperature. 36 Figure 13: Protein purification of AtSK1 and AtSK2. 38 Figure 14: AtSK2 co-expressed in BL-21 DE3 cells with AtCaM8 or
AtCaM9. 39 Figure 15 :Far Western analysis of HRP-CaM and AtSK interaction. 40 Figure 16: sk1 floral morphology. 42 Figure 17: Pollen viability between sk knockouts and Col-0. 43 Figure 18: Seeds counts in sk1 knockouts and Col-0. 44
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List of Abbreviations: ADP Adenosine DiPhosphate ARF Auxin Response Factor At Arabidopsis thaliana ATP Adenosine TriPhosphate BAR BioArray Resource BLAST Basic Local Alignment Search Tool CaM CalModulin CBB Calmodulin Binding Buffer CDS Coding Sequence CEB Calmodulin Elution Buffer CS Chorismate Synthase DAB DiAmino Benzadine DAHP 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate DHQ 3-Dehydroquinate DHS 3-Dehydroshikimate E4P Erythrose-4-Phosphate ECL Enhanced ChemiLuminescence EPSP EnolPyruvylShikimate 3-Phosphate ESB Extended Shikimate Binding FDA Fluorescin DiAcetate GCOS GeneChip Operating Software GEO Gene Expression Omnibus Gm Glycine max HRP HorseRadish Peroxidase HSF Heat Shock Factor HSP Heat Shock Protein HSR Heat Shock Response Hv Hordeum vulgare IPTG Isopropyl β-D-1-thiogalactopyranoside LB Luria-Bertani Brother MS Murashige and Skoog NAD Nicotinamide Adenine Dinucleotide NB Nucleotide Binding NMP Nucleoside MonoPhosphate NMPK Nucleoside Monophosphate Kinase NP-40 Nonyl Phenoxypolyethoxylethanol Os Oryza sativa PAGE PolyAcrylimide Gel Electrophoresis PDB Protein DataBank PEP Phospo-Enol-Pyruvate PGML PhosphoGlycerate Mustase Like PSI/II PhotosystemI/II RC Reduced Core RMSD Root Mean Square Deviation
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ROS Reactive Oxygen Species RPKM Reads Per Kilobase Million SDS Sodium Dodecyl Sulfate SK Shikimate Kinase SKL Shikimate Kinase Like TBARS ThioBarbaturic Reactive Substances TCA TriChloro Acetic acid
1
Chapter 1 -Introduction
The Shikimate Pathway The shikimate pathway consists of seven enzymatic steps (Figure 1), converting
phosphoenol pyruvate from glycolysis and erythrose 4-phosphate from the pentose
phosphate pathway into chorismate, a pre-aromatic compound required for the synthesis
of the aromatic amino acids: tyrosine, tryptophan and phenylalanine. All bacteria, plants,
fungi, and apicomplexia synthesize aromatic amino acids. Chorismate is a primary
metabolite for these organisms. Metazoans obtain the aromatic amino acids from
exogenous sources (Herrmann et al 1999; Maeda et al. 2012). Bacteria and fungi commit
the majority of the shikimate pathway products to protein synthesis, whereas plants
utilize the aromatic amino acids, chorismate, and other intermediates in a variety of
secondary and specialized metabolic pathways contributing to the production of
compounds ranging from phytohoromones such as auxin, to antioxidant phylloquinones,
to lignin (Herrmann et al. 1999; De Luca & Laflamme, 2001; O'Connor & Maresh,
2006). Because the shikimate pathway is a component of central metabolism in non-
metazoans it is an ideal drug target, thus generating much research interest in antibiotic
and herbicide development (McConkey et al. 1990; Pereira et al. 2007; Ducati et al.
2007). The basic organization and primary function of the enzymes in the shikimate
pathway are well understood, however, characterizing the diverse branch pathways and
regulation across species and kingdoms is an ongoing process.
2
Figure 1: Enzymes and intermediates of the shikimate pathway. (Adapted from Hermann et al. 1995)
Pathway Organization The initial shikimate pathway step utilizes PEP from glycolysis and E4P from the
pentose phosphate pathway in an aldol condensation catalyzed by 3-Deoxy-d-Arabino-
Heptulosonate 7-Phosphate (DAHP) Synthase (EC 4.1.2.15) to form the cyclic compound
DAHP. The phosphate group attached to the 7 carbon is eliminated from DAHP in the
second step by 3-Dehydroquinate (DHQ) Synthase (EC 4.6.1.3), forming DHQ. In the
third step 3 DHQ-Dehydratase (EC 4.2.1.10) dehydrates 3-dehydroshikimate (3-DHS)
forming the first double bond in the ring structure. In the fourth step shikimate
3
dehydrogenase (EC 1.1.1.25) reduces 3-DHS into shikimate. The fifth and the sixth step
involve transfer of functional groups to the hydroxyl groups at positions 3 and 5. The
fifth step is catalyzed by shikimate kinase (EC 2.7.1.71) which transfers a phosphate
group onto the hydroxyl group at carbon 3 giving shikimate-3-phosphate. In the sixth step
5-Enolpyruvylshikimate 3-Phosphate (EPSP) Synthase (EC 2.5.1.19) transfers the
enolpyruvyl group from PEP, obtained from the same pool as the initial step, the
hydroxyl group at carbon 5 yielding EPSP. In the seventh and final step, Chorismate
Synthase (EC 4.6.1.4) eliminates the phosphate, leaving a double bond and the product
Chorismate. Chorismate is a pre-aromatic compound which is directed to aromatic amino
acid synthesis (Herrmann et al. 1999).
The components of the shikimate pathway exist as separate enzymes in all known
bacterial species although in some cases they are co-transcribed as part of an operon
(Parish et al. 2002). In fungi steps 2-6 of the pathway are catalyzed by a pentafunctional
enzyme, the AroM complex. The complex arose from a fusion of five shikimate pathway
genes that are translated as a single polypeptide (Hawkins 1993) and individually
expressed domains of the AroM can rescue E.Coli strains deficient in the corresponding
enzyme, suggesting that the AroM may be able to act on individual metabolite pools even
when expressed in its full length form (van den Hombergh et al. 1992). Some researchers
however, It has been suggested that the AroM complex facilitates metabolite channeling
to account for the differing rates of each enzymatic step. The AroM complex has also
been identified in the apicomplexian parasite Toxoplasma Gondii (Campbell et al. 2004).
In all plant lineages the third and fourth steps of the shikimate pathway are catalyzed by a
bifunctional DHQ-SDH protein (Bischoff et al. 2001). Structural studies suggest that
4
metabolites may be channeled along the protein for preferential substrate availability
(Singh & Christendat. 2006).
Shikimate Kinase Reaction Shikimate kinase catalyzes the fifth step in the shikimate pathway, transferring a
phosphate group onto the 3-carbon of shikimate (Figure 2). Crystal structures of SKs
from multiple bacterial species have been reported and a consensus on the basic structure
and mechanism of shikimate kinase action has been established (Nasser, 1967; Krell et al
2001; Gu et al. 2002; Hartmann et al. 2006). Shikimate Kinases belong to the nucleoside
monophosphate kinase family (NMP), which includes: adenylate kinase, guanylate
kinase, uridylate kinase, and cytidylate kinase. The family is defined by a tertiary
structural arrangement of central parallel beta sheets with helices arranged on both sides,
and three secondary structural domains; a nucleoside monophosphate (NMP) binding
domain, a CORE domain which contains the nucleotide binding (NB) motifs, and an LID
domain containing the catalytic residues for phosphotransfer (Yan et al. 2006). SKs are
arranged with five central parallel beta sheets flanked by eight α-helices, and contain four
functional domains; an Extended Shikimate Binding (ESB) domain, which corresponds
to the NMP domain in NMPKs, a Nucleotide Binding (NB) domain containing the
nucleotide binding motif and a phosphate binding “P-loop”, an LID catalytic domain, and
a Reduced Core (RC) domain. There is no traditional CORE domain as defined by
NMPK structures, instead, features of the CORE domain are found in the ESB, the NB,
and the RC domains of SK (Gu et al. 2002) (Figure 3). The phosphotransfer reaction
involves conformational changes upon binding of both shikimate and ATP. Kinetic
analysis suggests that either substrate can be bound first with no preference to order,
although binding one appears to enhance affinity for the other (Gu et al. 2002).
5
Phosphotransfer occurs when both Shikimate and ATP are bound and the LID domain is
in the “closed” conformation. SK utilizes Mg2+ as a cofactor to stabilize phosphotransfer
(Hartmann et al. 2006). This mechanism is similar to that used by the NMPK family to
which SK has been assigned. Structural studies on NMPKs have demonstrated the NMP
binding domain and the LID domain rotating independently to form a closed active site
for phosphotransfer (Vonrhein et al. 1995).
Figure 2: Reaction catalyzed by shikimate kinase (EC 2.7.1.71).
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Figure 3: Crystal Structure of AtSK2 (PDB: 3NWJ) A cartoon diagram of AtSK2 with helices and sheets indicated. B cartoon diagram of AtSK2 with functional domains, NB, RC, and ESB, indicated, LID domain is unresolved. C AtSK2 substrate binding pocket (gray) superimposed with MtSK (PDB entry 2IYQ) substrate binding pocket (yellow). Adapted from (Fucile et al. 2011).
The Shikimate Pathway in Plants An estimated 20% of all carbon fixed by plants enters the shikimate pathway and
provides metabolic materials for a wide variety of processes besides protein synthesis
(Hermann et al. 1999; Maeda et al. 2012). With a few exceptions, such as NAD synthesis
(Mattevi. 2006), the majority of aromatic compounds produced by plants contain a
7
chorismate product (Bentley. 1990). For example, phylloquinone and a proposed
salicylate pathway utilize chorismate that is diverted from amino acid synthesis (Garcion
et al. 2004; Gross et al. 2006), Auxin and phenylpropanoids are synthesized from
aromatic amino acids (Bartel et al. 1997; Vogt et al. 2010). The pathway also contains
branch points for the biosynthesis of additional secondary metabolites. DHQ, the second
pathway product, readily converts to quinate which is a precursor for phytoalexins and
UV protectants (Herrmann et al. 1999). In plants, all shikimate pathway enzymes are
encoded by discrete loci with the exception of the third and fourth steps which are
catalyzed by a bifunctional DHQ-SDH. The plant pathway enzymes are believed to
localize to the chloroplast stroma and the majority of identified enzymes contain an N-
terminal chloroplast transit peptide (Richards et al. 2006). Shikimate pathway enzyme
activity has also been detected in the cytosol (Morris et al. 1989; Reinbothe et al. 1994)
and several groups have suggested that a parallel cytosolic pathway may exist; however,
progress toward identifying these cytoplasmic components appears to have stalled.
Evolutionary Diversification of Shikimate Kinases Plant species exhibit a high rate of gene duplication compared to other
eukaryotes. It has been suggested that duplication drives functional diversification of
genes and proteins (Flagelet al. 2007; Blanc et al. 2004). Over evolutionary time
duplicated genes enter one of three categories. Gene duplicates that accrue mutations
deleterious to their original function without providing new functional possibilities are
defined as pseudogenes. Gene duplicates in which the original gene and one or more
duplicates partition functional roles so that each gene now performs a subset of the
original function are subfunctionalized. Gene duplicates where one gene retains the
original function while the other(s) develops an entirely different function are
8
neofunctionalized. The possibility of neofunctionalization is thought to be enhanced by
the events giving rise to gene duplication, which may produce frameshifts, or gene
fusions, allowing rapid addition of new domains or sequence to a gene (Zhang et al.
2003). Phylogenetic analysis revealed four shikimate kinase homologs in Arabidopsis
thaliana; Shikimate Kinase 1 (AtSK1, At2g21940), Shikimate Kinase 2 (AtSK2,
At4g39540), Shikimate Kinase Like 1 (AtSKL1, at2g26900), and Shikimate Kinase Like
2 (AtSKL2, At2g25500). SK1 and SK2 were found to be highly similar to each other and
similar to plant and bacterial SKs. SKL1 and SKL2 genes were found to be more distant
homologs than SK1 and SK2, and are more closely related to each other than to plant or
bacterial SKs based on amino acid substitutions per-site (Fucile et al. 2008). These
relationships are supported by structural and kinetic characterization discussed elsewhere.
The AtSK1 and AtSK2 duplication event occurred 20-60 million years ago and was the
result of a segmental duplication of chromosome 2 to chromosome 4 (Fucile et al. 2008).
The duplication event(s) giving rise to SKL1 and SKL2 are not characterized; the two
homologs have been carried by most plant lineages for at least 400 million years. The
distribution of SK homologs in Arabidopsis is similar to other plant lineages. Many plant
species carry multiple homologs of SK; SKL1 and SKL2 occur in most plant lineages but
no species has been identified as carrying multiple copies of either gene (Fucile et al.
2008).
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Relationship of Arabidopsis Shikimate Kinase Homologs to Bacterial Shikimate Kinases
Kinetic, structural, and sequence analysis suggests that AtSK1 and AtSK2 retain
the domain structure of bacterial SKs and can phosphorylate shikimate while SKL1 and
SKL2 have undergone neofunctionalization, acquiring new domains and cellular roles.
AtSK1 and AtSK2 AtSK1 and AtSK2 retain the domain features of bacterial SKs, the RC, ESB, NB,
and LID domain are present along with identified functionally important residues. A
crystal structure for the apo form of AtSK2 aligns with the apo structure of
Mycobacterium tuberculosis SK with an RMSD of 1.12A. AtSK2 adopts three distinct
regions of secondary structure that does not align with bacterial SK structures, a helical
region in the N-terminus, believed to connect with the chloroplast transit peptide, and two
solvent exposed loop regions; one in the ESB, and the other in the NB domain. Because
the sequence conservation between AtSK1 and AtSK2 is very high (80%) the structure is
assumed to be representative of both Arabidopsis homologs (Figure 3). Kinetic analysis
demonstrates that AtSK1 and AtSK2 can catalyze the phosphorylation of shikimate in
vitro with kinetic parameters similar to bacterial SKs (Fucile et al. 2011). It is believed
that AtSK1 and AtSK2 both function in the shikimate pathway to phosphorylate
shikimate in vivo.
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SKL1 AtSKL1 has substitutions at many functionally important SK residues in the ESB
and LID domains and several arginines in the LID domain required for phosphotransfer
are not present (Gu et al. 200; Leipe et al. 2003; Cheng et al. 2006). Sequence analyses
across plant lineages identified a conserved domain in AtSKL1 not present in SK proteins
(Fucile et al. 2008). The motif, LALLRHG[I/V]S, appears in a predicted helical region
before the LID domain and is similar to phosphoglyerate binding domains identified in
other structures (Jedrzejas et al. 2000). This region has been termed the Phosphoglycerate
Mutase Like (PGML) domain. AtSKL1 is unable to catalyze the phosphorylation of
shikimate in vitro. AtSKL1 is believed to act as an enzyme based on its retention of ATP
binding motifs and the presence of a putative substrate binding site in the PGML domain.
sk1-8 knockout lines demonstrate an albino phenotype and defects in thylakoid
membrane biogenesis (Fucile et al. 2008).
SKL2 AtSKL2 has also undergone substitution at many functionally important SK
residues, in addition to losses in the ESB and LID domains, AtSKL2 retains few of the
residues believed to contribute to ATP binding and stabilization. SKL2 orthologs across
plant lineages contain a CS (CHORD and SGT1) domain in the N-terminal region
downstream of the chloroplast transit peptide. This domain facilitates interaction with
proteins containing HSP90 and CHORD domains in other proteins possessing it. AtSKL2
is unable to catalyze the phosphorylation of shikimate in vitro. SKL2 is believed to be
involved in protein-protein interaction based on the presence of a highly conserved
protein interaction domain and the near-total loss of residues involved in SK enzyme
function (Fucile et al. 2008).
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Regulation of the Shikimate Pathway
Post Translational Regulation Bacterial shikimate pathway enzymes are subject to regulation by downstream
products of aromatic amino acid synthesis. E. coli possess three DHS homologs and each
is negatively regulated by one of the aromatic amino acids (Ogino et al. 1982), similar
feedback systems exist across bacterial species. Several studies have failed to find a
similar regulatory system for the three Arabidopsis DHS homologs however our lab has
determined sensitivity of DHS1 knockout lines to exogenous amino acid supplementation
(Jimmy Poulin, thesis. 2010), suggesting some manner of regulation via the aromatic
amino acids in Arabidopsis. The previously described plant chorismate mutase genes
acting directly downstream of the shikimate pathway are also subject to negative
regulation by the aromatic amino acids (Colquhoun et al. 2010). Shikimate pathway
regulation may also be mediated via protein-protein interaction, a study demonstrated that
Mycobacterium tuberculosis DHS forms a stable complex with CS that changes the
efficiency of the CS enzyme (Sasso et al. 2009). Other such complexes may exist
between shikimate pathway enzymes in other species. The plant DHQ-SDH bifunctional
enzyme has been proposed to limit diffusion of substrates into the quinate pathway via
substrate channeling (Singh & Christendat 2006). The possibility of enzymes forming
complexes to maximize substrate availability and diminish flux into competing pathways
is a fascinating possibility for metabolic regulation.
Transcriptional Regulation Transcriptional control of the entire shikimate pathway has been described in
Arabidopsis in response to wounding (Chen et al. 2006), mediated by the wounding
response factor AtMYB15, and in a variety of biosynthetic processes such as lignin and
12
cell wall synthesis, and phenylpropanoid synthesis (Stracke et al. 2007; Lepiniec et al.
2006). This regulation has been interpreted in the context of provision of materials for
protein synthesis or other downstream products. In addition to instances where the entire
pathway is subject to transcriptional control many enzymes in the shikimate pathway
show differential transcript levels during developmental stages or conditions that do not
affect other enzymes in the pathway.
Regulation of Shikimate Kinase Gene Duplicates
Multiple plant lineages have retained one or more SK homolog after duplication
events and the diversity of conditions giving rise to differential transcript levels of these
homologs when compared to their duplicates or to other shikimate pathway enzymes
provides a strong case for regulatory sub or neo-functionalization. Rice carries three SK
homologs which show markedly different transcript levels during panicle organogenesis,
floral development, and fungal elicitor challenge (Kasai et al. 2005). Studies have
identified a SK homolog that is expressed during cold stress (Sathishkumar et al. 2013).
In Arabidopsis AtSK1 and AtSK2 have markedly different transcript accumulation across
conditions and growth stages. AtSK2 transcript levels are static throughout most
developmental stages but are elevated in mature seeds. They also increase approximately
2-fold during Phytoptera infestans infection. AtSK1 transcript levels remain constant
during development and in vegetative tissues but increase 4 fold during floral maturation
and pollen production. Heat stress at a variety of temperatures and treatment durations
causes AtSK1 transcript level to increase 5-10 fold across all tissues within about six
hours of heat treatment. T-DNA insertion lines have been obtained for AtSK1 and
AtSK2, sk1 and sk2 plants are viable under normal conditions, sk1sk2 double mutant
13
lines could not be generated (Falconer thesis. 2006), suggesting that both enzyme can
function in the shikimate pathway. AtSKL1 transcript reaches its highest levels in leaf
tissues at all developmental stages and is relatively unaffected by abiotic and biotic
perturbations. SKL2 transcripts appear at low levels in all tissues relative to other
shikimate kinase homologs and increase under a variety of pathogen challenges (Fucile et
al. 2008: Fucile et al. 2011).
SKL1 and Plant Chloroplast Biogenesis Plastids are membrane bound compartments derived from an endosymbiotic
engulfment of photosynthetic cyanobacteria. Plastids carry out a variety of metabolic and
storage functions in plant and algal lineages. Plant plastids are bound by two membranes
that are structurally homologous to the inner and outer cyanobacterial membranes (Gould
et al. 2008). Like mitochondrial compartments plastids encode several proteins in their
own compartmentally localized genome that replicates independently of the cellular
genome. Angiosperm plastid genomes encode ~100 proteins, all of which are believed to
localize to the encoding compartment and make up a small portion of the ~4000 proteins
known to be present there. Plastids can follow a variety of developmental routes from an
initial undifferentiated protoplastid, differentiating into specialized compartments
depending on the needs of the plant. Chromoplasts, enriched in floral tissues, are a
differentiated plastid type specialized for pigment synthesis, while amyloplasts are
produced in great numbers by tuber species for starch storage (Neuhaus and Emes. 2000;
Lopez-Juez. 2007). The majority of differentiating protoplasts become chloroplasts, the
site of photosynthetic metabolism (Waters and Langdale. 2009). The protein complexes
that carry out the light harvesting required for photosynthesis, photosystem II (PSII) and
photosystem I (PSI), are anchored in an internal membrane structure known as the
14
thylakoid membrane. Proper function of the photosystem complexes requires the
biogenesis and upkeep of the membrane (Nelson and Ben-Shem. 2004). SKL1 mutants
cannot form thylakoid membranes, instead accumulating vesiculated plastids that are
depleted in photosystem proteins and pigments (Fucile et al. 2008). Other mutants
demonstrating a complete or total loss of thylakoid biogenesis include VIPP1 (Aseeva et
al. 2007), and various members of the FTSH metalloprotease family (Sakamoto et al.
2003). Thylakoid biogenesis is poorly understood and is often presented as separate from
the upkeep of the diverse array of proteins found anchored or associated with the
membrane. Thylakoid associated processes are dynamic, changing with environmental
conditions such as low light or developmental conditions such as transition to flowering.
A variety of transit, insertion, and other protein-protein interaction factors have been
identified which affect these transitions (Kapri-Pardes et al. 2007; Sakamoto et al. 2003).
SKL1’s effect on the thylakoid membrane has been examined in the context of
protoplastids developing into chloroplasts (Fucile et al. 2008), a process that involves
creating a thylakoid membrane de novo. Thylakoid membranes can also be synthesized
or destroyed during plastid interconversion, for example Solanium convert large numbers
of chloroplasts into chromoplasts as they bear fruit, degrading the thylakoid membrane
and removing the associated factors (Simkin et al. 2007). The process can also occur in
reverse, requiring the formation of a new thylakoid membrane (Preberg et al. 2008). It is
not known whether the formation of the membrane during interconversion toward a
chloroplast proceeds in the same manner as it does from a protoplastid (Egea et al. 2010),
however the possibility exists that thylakoid biogenesis factors respond both during the
15
development of new chloroplasts and as part of dynamic maintenance processes over the
life of the plastid.
Heat Stress Response in Arabidopsis As sessile organisms plants must respond to environmental stresses without the
option of changing external conditions through motion. Temperatures above a plant’s
adaptive range can cause damage by denaturing cellular proteins, accumulating Reactive
Oxygen Species (ROS) molecules used for stress signaling to a toxic level, and disrupting
the sensitive photosystem machinery that carries out photosynthesis. The Arabidopsis
Heat Stress Response (HSR) is similar to that of most land plants (Guo et al. 2008). After
temperature elevation is perceived a family of HSF (Heat Shock Factor) transcription
factors co-ordinate the expression of heat stress specific genes, in particular heat shock
protein (HSP) family genes, and shifts in metabolic and housekeeping functions to
maximize Heat Shock Protein (HSP) production and limit non HSR processes (Feder and
Hoffman. 1999; Kotak et al. 2007). There are over 45 identified Arabidopsis HSPs and
characterized members of the group function as molecular chaperones, folding denatured
proteins, preventing toxic protein aggregates, and localizing proteins to aid in the heat
response or to be degraded (Agarwal et al. 2001). ROS species such as hydrogen
peroxide and hydroxyl radicals are involved in stress signaling and regulating the redox
state of the cell or subcellular components, during heat stress ROS can increase to levels
high enough to cause oxidative damage leading to cell death. Proteins that reduce ROS,
or “ROS scavengers”, are produced at a higher rate during heat stress (Dat et al. 1998;
Mittler. 2002). ROS scavengers act at a later stage in heat response than HSPs as their
action in reducing ROS halts the signaling function of ROS that drives the expression of
many HSPs (Panchuk et al. 2002; Volkov et al. 2006). The metabolic response to
16
elevated temperature affects a large proportion of cellular metabolites and includes
increases in shikimate pathway derived compounds such as anthocynanin and
phenylpropanoids, photoprotectants believed to protect PSI and PSII components by
quenching excess light associated with high temperatures (Rivero et al. 2001). Although
studies have found heat stress results in significant changes in the levels of up to 25% of
identified cellular metabolites the overlap between heat and other stress responses is
significant, covering the majority of affected metabolites (Kaplan et al. 2004). Transcript
profiling has also suggested that many HSF and HSP factors act in other stress response
pathways (Larkindale et al. 2005; Swindell et al. 2007) making a heat specific response
difficult to characterize.
Chapter 2 -Thesis Objectives The aim of this Master’s thesis was to investigate whether AtSK1 and/or AtSK2
had undergone subfunctionalization and acquired new regulatory roles related to heat
stress response or floral development. Three major approaches were utilized:
1) RT-PCR analysis to study AtSK1 transcript induction under heat stress and during
flowering. Querying public microarray databases to establish possible TFs
affecting regulation and to determine whether SKs in other plant lineages were
similarly regulated.
2) In vitro analysis to investigate the interaction between AtSK2 and Calmodulin
proteins, possibly establishing a connection to heat stress response.
3) Phenotypic screening of sk1 and sk2 knockout lines to identify heat stress
response and floral development processes affected by either SK homolog.
17
Chapter 3 - Materials and Methods
Plant Growth Conditions A. thaliana seeds were sterilized by with a mixture of 70% ethanol and 0.05%
triton x-100, and washed 2 times with 100% ethanol. Seeds were dried and suspended in
sterile water in the dark at 4°C for 72 hours. Seeds were plated vertically on MS media
(4.3g/L MS, 0.05g/L MES, 12g/L Agar) (Murashige & Skoog. 1962) and grown in
continuous light conditions (~100 µmol m−2 s−1). For assays requiring mature plants
seedlings were transplanted to soil and placed in a growth chamber set to long day
conditions (8h dark, 16h light ~100 µmol m−2 s−1).
RT-PCR RNA was extracted from either floral tissues or seedlings using NucleoSpin®
RNA Plant Kit (D-Mark) according to the manufacturer’s instructions. cDNA was
prepared using 200 units of SuperScript III (Invitrogen) reverse transcriptase and 1 µg
total RNA. Primers for the full-length AtSK1 and AtSK2 gene products (Table 1) were
used for amplification. PCR was conducted using Pfu polymerase and the cycle
conditions listed in (Table 1). RNA extractions and RT-PCR amplifications were
performed in duplicate yielding similar results.
Heat Stress Assays
Survival Assay This assay was conducted according to (Larkindale et al. 2005). 7-10 day old
seedlings grown using the above conditions were submitted to heat stress for the
durations and temperatures summarized in (Table 4) Heat treatment lasting less than 2
hours was done in a dark incubator. Longer treatments at 37°C were done in a growth
chamber. Seedlings were observed at room temperature for five days following heat
18
treatment and seedlings that remained green and produced new leaves were scored as
surviving.
Hypocotyl Elongation Seedlings were grown using the above conditions for three days before being
moved to a growth chamber at 29°C for four days and then fixed in 500 µL fixing
solution (50% EtOH, 5% Acetic Acid, 4% Formalin). Hypocotyl length measured using a
Leica dissecting scope and ImageJ (Rasband. 1997).
TBARS Assay Seedlings were grown for 7-10 days using the above conditions and submitted to
heat stress at 37°C or 44°C. The TBARS reaction was conducted according to (Hodges et
al. 1999). Green plant tissue was weighed and 0.25 g was homogenized in liquid nitrogen
then suspended in 500 µL 0.1% trichloroacetic acid (TCA) and centrifuged at 16000 x g
for five minutes. 400 µL of supernatant was added to 400 µL of Thiobarbaturic acid
(0.5% W/V in 20% TCA) and boiled in a heat block (85°C) for 30 minutes. The mixture
was cooled, and then centrifuged at 16000 x g for five minutes and transferred into a
cuvette. Absorbance of the supernatant was measured spectrophotometrically at 532nm
and the 600nm background was subtracted.
Floral Assays
Measurement of Floral Organs Stage 13 flowers were harvested in the morning (first 5 hours of 16hr day cycle)
before floral opening and immediately preserved in fixing solution (50% EtOH, 5%
Acetic Acid, 4% Formalin). Flowers were manually dissected and organ size was
measured using a Leica dissecting scope and ImageJ (Rasband. 1997)
19
Pollen Viability Stage 13 flowers were harvested in the morning (first 5 hours of 16hr day cycle)
directly after opening. 6-8 flowers were placed in 300 µL of 7% sucrose solution in a
microcentrifuge tube and vortexed fort thirty seconds to dislodge pollen. Debris was
removed manually using forceps and 3 µL FDA was added (1/100 ratio). After
incubation at room temperature for 10 minutes the FDA solution containing pollen grains
was placed on a slide and viewed using a Zeiss Axioplan microscopy system with an
EXF-O X-Cite 120 fluorescence module.
Seed Count Mature siliques were removed and placed in fixing solution (50% EtOH, 5%
Acetic Acid, 4% Formalin). Siliques were opened manually using forceps and deposited
individually in a 24 well plate. Seeds were counted using a dissecting microscope.
Cloning of Recombinant Protein Constructs
AtSK1 and AtSK2 AtSK1 and AtSK2 were amplified from an Arabidopsis plasmid cDNA library
(Invitrogen) using primers containing restriction sites for molecular cloning (Table 2).
PCR reactions were conducted using Pfu polymerase and cycle conditions listed in (Table
1). Amplified clones were digested with their corresponding Fermentas Fastdigest
Restriction Enzyme (Fermentas) for 1 hour, and ligated into pET28-MOD C-Term His
vector (Figure 4) using 75 ng of insert DNA a molar ratio of 3:1 insert:vector. Ligations
were carried out for 4 hours at room temperature with 1µL T4-Ligase (Fermentas). 80 µL
of chemically competent DH5α E coli cells (Novagen) were transformed by incubation
with 4 µL of ligation mixture on ice for 10 minutes, exposure to a 44°C water bath for 45
20
seconds, and recovered on ice for an additional 5 minutes. Transformed cells were
incubated with shaking at 37°C with an additional 500µL LB for 1 hour before being
plated onto LB Agar plates containing 50 µg/mL Kanamycin and incubated overnight at
37°C. Successful transformants were confirmed via colony PCR using AtSK1 and AtSK2
specific primers. Constructs were sequenced following plasmid extraction by Qiagen
Plasmid Miniprep Kir (Qiagen).
Figure 4: pET28-MOD vector map. pET28-MOD is based on pET28(a) (Novagen) and is modified to exclude the N-terminal 6-HIS tag and include an in-frame C-terminal 6xHIS tag with a TEV cleavage site. pET28MOD was obtained from the Structural Genomics Consortium at the University of Toronto.
CaM Plasmids CaM8 on pET3d and CaM9 on pET5a tagless vectors were a gift from Dr. Park at
Gyuongsang National University. The plasmids were transformed into chemically
competent BL21-CodonPlus (DE3)(Novagen) Codon Plus E coli cells using the same
transformation technique detailed above.
21
Protein Expression:
BL-21 Transformed BL21-CodonPlus (DE3)(Novagen) cells were used to inoculate
50mL LB (Bioshop) OverNight (O/N) at 37°C. The O/N culture was used to inoculate 1L
LB and grown with shaking at 37°C to OD600 0.6 to 0.8. IPTG was added and the culture
was held at 37°C for 4hrs before being lowered to 16°C O/N (Singh & Christendat.
2006).
Protein Expression in Arctic Express Transformed Strategene Arctic Express DE3 Codon Plus E. coli cells (Strategene)
were cultured in LB overnight with appropriate antibiotic at 37°C. The overnight culture
was used to inoculate 1L of LB at a ratio of 1/200. The culture was grown at 30°C for 3
hours before being lowered to 10°C and induced by IPTG. Induced culture was allowed
to express at 10°C for up to 72 hours before being harvested.
Protein Purification Cell cultures were spun at 5500 x g for 10minutes and resuspended in binding
buffer with added protease inhibitor (100 mM benzamidine (BioShop) and 50 mM
phenylmethanesulfonyl fluoride (BioShop) dissolved in 99% ethanol (v/v)). A French
press was used to lyse the cells at ~1000PSI and then the lysate was briefly sonicated.
Protease inhibitor was re-administered and lysate was pelleted at 4°C at 18000 x g for
30minutes and the supernatant was recovered.
Nickel Column 2mL of NiNTA Superflow Resin (Qiagen) added to 10mLcolumn equilibrated
with 20mL binding buffer. Supernatant from previous lysis added to column. Washed
with 200-250mL wash buffer. Eluted with 10 to 20 mL elution buffer until the protein is
22
completely eluted. Protein was collected in one fraction and the elution was monitored
by assaying for protein with a Bradford dye. (Bradford. 1976). (Table 3)
Phenyl Sepharose Column (Calmodulin Purification) 5Mm CaCl was added to cell lysate supernatant and heated to 90°C for 3 minutes.
Heated lysate was pelleted at 4°C at 18000 x g for 30 minutes. 2mL of Phenyl Sepharose
Fastflow Resin (Sigma) was added to 10mL column, washed with 20mL ddH20, 50mL
CBB1, soluble lysate fraction was applied to column, washed with 100mL CBB1, 100mL
CBB2, 50mL CBB1. 10-20mL CEB was used to elute. (Table 3)
Recovery of Protein From Inclusion Bodies Lysed cells in binding buffer (Table 2) containing 0.1%NP40 were centrifuged at
maximum speed for 30 minutes. Supernatant was discarded and visibly viscous bacterial
cell wall components were physically removed from the pellet. The remaining pellet was
suspended in binding buffer with 0.1%NP40 and centrifuged at 18000xG for 30 minutes.
This was repeated four times. Cleaned pellets were suspended in solubilization solution
(6M urea or Guanidinium Chloride, 50Mm NaCl, 100Mm TrisHCl pH 7.5) and incubated
at 4°C with shaking.
Far Western (HRP-CaM Overlay) This assay was conduxted according to (DeFalco et al. 2010). Proteins of interest
and positive control (GmCaMK1 protein provided by Tom DeFalco, Yoshioka Lab,
University of Toronto) were loaded in approximately equal amounts and separated by
SDS-PAGE. Proteins were transferred to nitrocellulose membrane using western transfer
buffer [50 mM tris, 40 mM glycine, 20% methanol, 1.3 mM sds] at 150 mA constant
current and then blocked overnight at 4°C with shaking in 90mL 2% GE Blocking Agent
in CBB1 (Table 2). Membrane was washed once in ~90mL CBB1 and probed with 75nM
23
HRP::CaM/CaM in 1% milk for 1-2 hours at 4°C with shaking. Membrane was washed 3
times in 90mL CBB1 and bound HRP::CaM was detected using a GE life science ECL
detection kit.
Table 1: PCR Conditions for cloning AtSK sequences and RT-PCR. Cloning: Stage Temperature (°C) Time(Minutes) Repetitions Initial Denaturation 92°C 2:00 x1 Denaturation 92°C 1:00 | Annealing 52°C 1:00 | x35 Extension 72°C 1:30 | Final Extension 72°C 5:00 x1 RT-PCR Stage Temperature (°C) Time(Minutes) Repetitions Initial Denaturation 92°C 2:00 x1 Denaturation 92°C 1:00 | Annealing 50°C 1:00 | x22 Extension 72°C 1:15 | Final Extension 72°C 2:00 x1
Table 2 – Primers used to clone AtSK sequences and RT-PCR.
Primer name Primer Sequence SGB1 (SK2 Full length for pET2MOD fwd)
GTG GAC TCA TAT GGA AGC AGC TAC TGT TCA GAG G
SGB2 (SK2 Full length for pET28MOD rev)
CCC TAC TCG AGC TAG AGT CCA TCT GGT CTA GC
SGB7 (SK1 Full length for pET28MODfwd)
GTG GAC TCA TAT GGA AGC AGC TAT TAC TCA GAG GAT T
SGB8 (SK1 Full length for pET28MOD rev)
AGG GAC CTC GAG CTA CTA TTA GAG GTC GCC GTC TGG GAT CTC
24
Table 3 – Buffers used in protein purification.
Binding Buffer Wash Buffer Elution Buffer 500mM Tris pH 7.5 5mM Imidazole 500mM NaCl 5% Glycerol
50mM Tris pH7.5 30mM Imidazole 500mM NaCl 5% Glycerol
50mM Tris pH 7.5 250mM Imidazole 500 mMNaCl 5% Glycerol
CLP Buffer CBB1 CBB2 CEB 50mM Tris-Cl 1.5mM CaCl2
pH 7.5
25mM Tris-Cl 1mM CaCl2
pH 7.5
25mM Tris-Cl 150mM NaCl 1mM CaCl2
pH 7.5
25mM Tris-Cl 2mM EGTA
25
Chapter 4 - Results
Expression Analysis of Shikimate Kinase Homologs We examined transcript levels of SK homologs in various tissues and
developmental states in Arabidopsis thaliana, Oryza sativa, Hordeum vulgare, and
Glycine max. Organisms were selected based on presence of SK duplicates, and
availability of datasets covering multiple tissues and developmental states. SK homologs
in each species were identified by sequence comparison with Arabidopsis SK genes via
BLAST analysis, and verified by manual curation of functional residues conserved within
bacterial SKs.
A rabidopsis thaliana Shikimate Kinase Duplicates Transcript data from Arabidopsis is based off microarray experiments and
represented as normalized mean fluorescent units of samples measured on the Affymetrix
ATH1 Array. There are two Arabidopsis SK duplicates annotated to retain SK activity
AtSK1 (At2g21940) and AtSK2 (At4g39540) (Fucile et al. 2008). AtSK2 appears to
maintain a higher transcript level than AtSK1 in green tissues as seen in cotyledons,
hypocotyls, and cauline leaves (Figure 1). AtSK1 increases approximately ten-fold
following heat stress at 37°C and in stage 15 flower petals and stamens (Figure 1). The
dataset surveyed did not contain measurements covering stages between 12 and 15, AtSK
transcript levels during these stages are discussed elsewhere (Figure 9; Figure 10).
26
Figure 5: Arabidopsis thaliana transcript analysis. Transcript expression profiles are represented as normalized GCOS expression signals after background subtraction. A Transcript levels in root and green tissue. Bars represent standard deviation. B Time course of transcript level in seedlings following heat stress at 37°C. Bars represent standard deviation. C Transcript level in floral organs during flower stages 12 and 15, and mature pollen. Bars represent standard deviation. Gene expression data was retrieved from publically available datasets and accessed via BAR eFP viewer (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi) (Schmid et al. 2005; Killan et al. 2007).
Ory z a sativ a Shikimate Kinase Duplicates Transcript data from rice is based off microarray experiments and represented as
normalized mean fluorescent units of samples measured on the Affymetrix Genechip
Rice Genome Array. There are three rice SK duplicates annotated to retain SK activity
OsSK1 (Os02g51410), OsSK2 (Os06g12150), and OsSK3 (Os04g54800)(Kasai et al.,
2005). These homologs show differential transcript abundance across several tissue types.
OsSK1 is 2 fold higher than OsSK2 in shoots and young leaves and OsSK1 and OsSK2
27
appear at a similar level in mature leaves. OsSK3 transcripts are 15 fold higher than
OsSK1 and OsSK2 in root tissue and appear near background levels in both young and
mature leaves and the shoot apical meristem. In both stigmas and ovaries OsSK3 has a
significantly higher transcript level than the other two homologs. In developing
inflorescences OsSK1 transcripts are consistently low, OsSK2 levels are consistently 3
fold higher than OsSK1. OsSK3 increases across time and by stage 5 is higher than
other homologs. All OsSKs remain relatively constant across time during seed
development with OsSK3 showing the most abundant transcript level. (Figure 6)
28
Figure 6 : Oryza sativa transcript analysis. Transcript expression profiles are represented as normalized GCOS expression signals after background subtraction. A transcript levels in root and shoot tissue. Seedling and Shoot Apical Meristem (SAM) tissue from 7-day old seedlings. Bars represent standard deviation across 3 replicates B Transcript levels in female reproductive tissue, mature stigma and ovaries before pollination. Bars represent standard deviation across 3 replicates. C Developmental course of transcript levels in inflorescence stems binned into P1-P5 based on length. Bars represent standard deviation across 3 replicates. D Time course of transcript levels in seeds binned into S1-S5 based on number of days after pollination. Bars represent standard deviation across 3 replicates. Gene expression data were retrieved from publically available datasets and accessed via BAR eFP viewer (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi) or GEO browser (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) GEO experimental accessions: GSE7951, GSE27988, GSE6893, (Li et al. 2007; Jain et al. 2007; Wei et al. 2010).
29
Hordeum v ulgare Shikimate Kinase Duplicates Transcript data from barley is based off microarray experiments and represented
as normalized mean fluorescent units of samples measured on the Affymetrix Barley1
Genechip Arrary. There are three barley SK duplicates predicted to retain SK activity, of
which the array covered two; Contig9196, and Contig 9177. Some experiments covered
two barley genotypes, Morex, and Golden Promise. In several tissue types differential
transcript level was observed in both genotypes, seedling root tissues had 2 fold higher
levels of Contig9196, while coleoptile and mesocotyl tissues had 2 fold higher levels of
Contig9177. In tissues where only one genotype (Morex) was measured Contig9196
showed higher transcript levels in floral bracts while Contig9177 was approximately 7
fold higher at sites of photosynthesis; awn, palea, and lemmas. Contig9177 had higher
transcript levels across time in caryopsis and embryos. Transcript levels in both
duplicates were unaffected by heat stress at 44°C .(Figure 7)
30
Figure 7: Hordeum vulgare transcript analysis. Transcript expression profiles are represented as normalized GCOS expression signals after background subtraction. A Transcript levels in root and shoot tissue between Morex and Golden Promise genotypes. Bars represent standard deviation across 3 replicates.B Time course of transcript level in caryopsis fruit tissue binned by Days After Pollination (DAP). Bars represent standard deviation across 3 replicates. C Transcript level in developing caryopsis fruit tissue following anthesis, tissues were measured at time points after heat stress at 44°C or control 22°C. Bars represent standard deviation across 2 replicates. Transcript level in floral and photosynthetic tissues. Bars represent standard deviation across 3 replicates. Gene expression data were retrieved from publically available datasets and accessed via BAR eFP viewer (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi) or GEO browser (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) GEO experimental accessions: GSE23896, GSE33394, (Poptkina et al. 2008; Mangelsen et al. 2011).
Gly cine max Shikimate Kinase Duplicates Transcript data from soybean is based of RNA-Seq experiments and is
represented as Reads Per Kilobase Per Million (RPKM). Three soybean SK duplicates
are predicted to retain shikimate kinase activity of which two were identified by the
datasets surveyed, Glyma04g39700, and Glyma05g31370. There are two major RNA-Seq
31
datasets covering soybean tissue and examples of differential transcript abundance occur
in both, the only overlapping instance is in nodule tissue, the site of nitrogen exchange
with symbiotic rhizobia, where Glyma05g31370 was 2 fold higher (figure 8).
Figure 8: Glycine max transcript analysis. Transcript expression profiles are represented as Reads Per Kilobase Per Million (RPKM) A, B Transcript levels in plant tissues across two independent RNA-Seq projects. C Time course of transcript level in seeds binned by Days After Fertilization (DAF) Gene expression data were retrieved from publically available datasets and accessed via BAR eFP viewer (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi) or from the Soybase Sequencing Database (http://www.soybase.org/soyseq/) (Libault et al. 2010; Severin et al. 2010).
Transcriptome Analysis of Heat and Floral Development Transcription Factor Mutants
AtSK1 transcript levels have been reported to reach their highest levels during
floral development and heat stress (Fucile et al. 2008; Fucile et al. 2011). We consulted
32
publicly available microarray data to identify mutant backgrounds affecting AtSK1
transcript levels during these conditions in order to identify regulatory pathways
associated with AtSK1. The transcript microarray data are represented as normalized
mean fluorescent units of samples measured on the Affymetrix ATH1 platform. Two
transcription factor families involved in floral maturation; Auxin Response Factor (ARF),
and MYB (Reeves et al., 2012) knockouts are associated with decreased AtSK1 transcript
levels in floral tissue (Reeves et al., 2012). At floral stage 12 AtSK1 transcript levels in
arf6arf8 (arf6/8) and myb21myb24 (myb21/24) knockout backgrounds are half that of
wild type Col-0. AtSK2 is not affected by the knockouts. At floral stage 13 AtSK1
transcript levels in arf6/8 and myb21/24 knockout backgrounds are ~25% that of Col-0.
Again, AtSK2 does not appear to be affected, The absolute transcript level of AtSK1 also
increases between stage 12 and stage 13 flowers. (Figure 9A).
A Heat Stress Factor (HSF) transcription factor family quadruple knockout in
the Ws background is associated with static AtSK1 transcript level during heat stress.
After exposure to 37°C for 1hr AtSK1 levels in 7-day old Ws seedlings increased 4 fold.
In hsfA1a/hsfA1b/ hsfA1d/ hsfA1e quadruple knockout background exposure to heat
stress was no longer associated with increased AtSK1 transcript, AtSK2 levels were not
significantly affected by the knockout. (Figure 9B).
33
Figure 9: Transcript profile of Transcription Factor knockouts. Transcript expression profiles are represented as GCOS expression signals normalized and background corrected using RMA. A Transcript level of AtSK1 and AtSK2 in Col-0, arf6arf8 knockout background, and myb21myb24 knockout background, during flower stages 12 and 13. Bars indicate standard deviation across 3 replicates. B Transcript level of AtSK1 and AtSK2 in Ws and hsfA1a hsfA1b hsfA1d hsfA1e quadruple knockout background across control (22°C and heat shock (37°C, 1 hour) conditions. Bars indicate standard deviation across 2 replicates. Gene expression data were identified via Genevestigator V3 (Hruz et al., 2008) (http://www.genevestigator.com), and retrieved from publically available datasets using GEO browser (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) GEO experimental accessions: GSE32193, GSE26266. (Reeves et al 2012; Liu, Liao, & Charng, 2010). Raw data was processed and normalized using RMAExpress software.
34
RT-PCR Confirmation of AtSK1 Transcript Induction. Multiple microarray datasets (Figure 5; Figure 9) have reported AtSK1 transcript
induction associated with floral tissue and elevated temperature. We conducted RT-PCR
to ensure that this induction was maintained over our growth and heat stress conditions,
and to determine the flowering stage that induction begins.
Floral Tissue Tissue from Col-0 stage 13 (S13) and stage 15 (S15) flowers was harvested to
determine whether timing of transcript induction was similar to the microarray data for
Arabidopsis (Figure 5). Tissue from stage 9-12 flowers was harvested and pooled (Early)
due to falling outside our stages of interest and difficulty establishing differences between
these stages without dissecting flowers. Leaf tissue was harvested for comparison. AtSK1
transcript was not detected in early stage (9-12) flowers and appeared to increase in stage
13 and stage 15 flowers. AtSK2 doesn’t appear to increase during these stages (Figure
10A).
Heat Stress Col-0 seedlings were exposed to 37°C for 2 hours and then recovered at room
temperature and harvested at 3, 6, or 12 hours after heating. AtSK1 showed elevated
levels of transcript 3 hours, 6 hours, and 12 hours after heating compared to unheated
seedlings. AtSK2 was expected to maintain a steady transcript level following heat stress
and provides a comparison to AtSK1 (Figure 10B).
35
Figure 10: RT-PCR of Col-0 using primers for AtSK1 and AtSK2 A flower tissue and leaf tissue B whole seedlings after various recovery times following 2 hours at 37 °C.
sk1 and sk2 During Heat Stress Response
Seedling Survival sk1 and sk2 seedlings (Falconer thesis. 2006) were submitted to a set of heat
treatments at 37°C and 44°C to test heat stress response. Seedlings were also tested by
heating to 44°C after being acclimated at 37°C for one or more hours to test for defects in
acquired heat tolerance, a response characterized by the buildup of HSPs(J. Larkindale et
al. 2005). Seedlings scored as surviving remained green and continued producing leaves
five days after heat stress. sk knockout lines showed no significant difference from Col-0
in the number of surviving seedlings across the conditions tested. (Table 4)
Reactive Substance Quantification in Heat Stressed sk1 and sk2 Seedlings The buildup of ROS and the resulting oxidative damage in cells during heat stress
can be used to measure the efficacy of the heat stress response and detect more subtle
defects in mutant lines than survival screens. We utilized a Thiobarbaturic Acid Reactive
Substances (TBARS) assay that spectrophotometrically determines the amount of
oxidative membrane lipids via the conversion of thiobarbaturic acid to malondialdehyde.
sk1 and sk2 seedlings were tested for the oxidizing effects of high ROS levels after
36
exposure to 37°C or 44°C. At the two temperatures tested the sk knockout lines showed
similar membrane lipid oxidation levels to the control Col-0 (Figure 11).
Hypocotyl Elongation Following Heat Stress Elevated temperature at a sub-lethal level has been linked to a variety of phenotypic
effects that include faster flowering and senescence in mature plants and rapid hypocotyl
elongation in seedlings (Gray et al. 1998). The hypocotyl elongation phenotype is
sensitive to genetic disturbance and is often used as an indicator for these effects. The
phenotype has been linked to genes involved in auxin synthesis and regulation
(Larkindale et al. 2005). Given the link between the shikimate pathway and the synthesis
of auxin via tryptophan, and the precedent for regulation of pathway by downstream
metabolites we tested the sk knockout lines for defects in hypocotyl elongation at 29°C.
Seedlings were grown for 3 days at 22°C then moved to 29° for 4 days before hypocotyl
length measurement. In both sk knockout lines and Col-0 elevated temperature resulted in
hypocotyls that were ~2mm longer than seedlings grown at 22°C (Figure 12).
37
Table 4: Summary of HS conditions tested with sk1 and sk2 and Col-0.
Table 4: Seedlings were grown on MS media for 7-10 days, exposed to elevated temperature, and observed for a five day period. Seedlings that remained green and continued producing leaves were scored as surviving. 2 hours at 44°C and 24 hours at 37°C represent the upper limit for Col-0 survival under the conditions used. N≥30. Difference in survival rates was tested using t-test for independent samples with dichotomous outcomes.
38
Figure 11: sk1, sk2, and Col-0 tested for production of ROS at elevated temperatures. Charts represent level of TBARS in Col-0, sk1, and sk2 seedlings following heat treatment at 37°C 44°C. TBARS is represented as Absolute absorption at 532nm after background correction. Data reflects two technical replicates each of two biological replicates.
Figure 12: Hypocotyl length of seedlings grown at elevated temperature. Seedling hypocotyls were measured after 7 days at 22°C (Control) or after 3 days at 22°C and 4 days at 29° (29°), measurements were taken using ImageJJ. N≥26 error bars represent standard error.
39
AtSK Interactions With CaM Arabidopsis AtSK2 was previously identified as a possible Calmodulin (CaM)
binding partner by a protein-protein microarray study that demonstrated interactions with
Arabidopsis CaMs 1,2,6,7,8, and 9, but not with the mammalian CaM control (Popescu
SC, 2007). The Calmodulin Target Database Binding Site Search and Analysis software
identified a CaM binding motif for both AtSK1 and AtSK2 in the N-terminal region, with
AtSK2 scoring higher (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.htmL,
accessed 2011). CaMs are well characterized heat response components, acting as
chaperones to localize and alter the function of other cellular proteins(Al-Quraan et al.
2005: McCormack et al 2005). CaM proteins are highly similar to each other and their
target specificity is poorly understood in vivo (Bouche et al. 2005). We attempted to
investigate the reported interaction in vitro using recombinant AtSK and CaM proteins.
Cloning and Expression of AtSKs AtSK1 and AtSK2 full length coding sequences were cloned from an Invitrogen
Arabidopsis CDS library and ligated into pET28-MOD C-Term-HIS vectors for protein
expression. All clones were confirmed by sequencing. Full length forms of AtSK1 and
AtSK2 proteins were insoluble when expressed in BL21-CodonPlus (DE3) cells, failing
to yield soluble product when purified via nickel affinity (Ni-NTA) chomatography. We
recovered small amounts of soluble AtSK2 using Arctic-Express DE3 cells; an E. coli
strain specialized for low temperature protein induction (Figure 13). AtSK2 activity was
confirmed spectrophotometrically using a coupled kinase assay with shikimate. We did
not recover soluble full length AtSK1.
40
Co-Expression and Binding Assays AtSK2 was transformed into BL21-CodonPlus (DE3) cell lines carrying either
AtCAM2 or AtCAM8 for inducible co-expression and purification. Purification via Ni-
NTA failed to yield AtSK2 and purification via phyenyl-sepharose yielded only AtCAM2
or AtCAM8 (Figure 14)
Far Western Assay AtSK1 and AtSK2 proteins were recovered from inclusion bodies and solubilized
in Guanadinium Hydrochloride. GmCaMK1, a Glycine max protein previously
determined to bind CaM in this assay (DeFalco et al. 2010), was included as a control.
The proteins were separated by PAGE and transferred to nitrocellulose membrane. The
immobilized proteins were probed with 75nM solution of Horseradish Peroxidase (HRP)
conjugated CaM81 (HRP::CaM81) and exposed using chemiluminescence reagents.
CaM81 is a petunia calmodulin with 98% sequence identity to AtCaM7 and has been
used to identify CaM binding proteins in multiple plant lineages. AtSK1 and AtSK2 did
not bind the probe (Figure 15A). An additional set of constructs including AtSK2
residues 1-55, containing the putative CaM binding region, and an AtSk2Δ55 construct,
did not bind the probe as well (Figure 15B).
41
Figure 13: Protein purification of AtSK1 and AtSK2. A Soluble AtSK2 from ArcticExpress DE3 cells, arrow indicates location of soluble AtSK2 product (34.6 kD). Nonspecific bands correspond with (top to bottom): Hsp70 (70kD), cpn60 (60kD), and an unknown band (~27kD). B AtSK2 (34.6kD) and AtSK1 (34.0kD) expressed in BL21-CodonPlus (DE3) cells and recovered from inclusion bodies (pellet) after cell lysis.
Figure 14: AtSK2 co-expressed in BL21-CodonPlus (DE3) cells with AtCaM8 or AtCaM9. Arrows denote protein locations in the cell lysate; AtSK2 (34.6kD), AtCaM8 (17.1kD), AtCaM9 (17.0kD). Phenyl sepharose purification for co-expression experiments omitted the 90 °C incubation step and yielded an unknown band at ~19kD, likely representing a small hydrophobic protein interacting with the resin.
42
Figure 15 : Far Western analysis of HRP-CaM/AtSK interaction. A Glycine max CaMK1, AtSK2 and AtSK1 full length proteins probed with 75nM HRP::CaM81 and exposed to film after chemilumenescence reaction.B Glycine max CaMK1, AtSK2, AtSK2 1-55, and AtSK2 Δ55, proteins probed with 75nM HRP::CaM81 and exposed to film after chemilumenescence reaction. Loading controls represent Ponceau stain of nitrocellulose membrane after completed exposures.
sk1 Stage 13 Flowers Data from transcriptome analysis (Figure 5; Figure 9) and our own RT-PCR
experiments (Figure 10) suggest that AtSK1 is highly expressed in stage 13 flowers, this
stage is characterized by elongation of the stamen, carpel, and petals prior to flower
opening, and the continued development of pollen grains within the anther (Reeves et al.
2012) . We examined AtSK1 knockout lines for defects in floral organs as well as pollen
viability.
Stage 13 Flower Dissection We dissected stage 13 flowers prior to dehiscence, completely separating the
floral organs, and compared the size of stamens and gynocium in sk1 knockout and Col-0
43
plants (Figure 16A). We determined there is no significant difference in the size of these
structures in sk1 flowers. (Figure 16B).
sk1 Pollen Viability sk1, sk2, and Col-0 flowers were removed at stage 13, just after flowers opened,
and pollen was harvested for FDA staining. FDA identifies viable pollen grains via its
conversion by functional esterases into a fluorescently excitable compound that can be
viewed using fluorescence microscopy (Figure 17A) . Pollen harvested from sk1 flowers
contained 59% viable pollen grains compared to 75% and 77% in Col-0 and AtSK2
flowers respectively (Figure 17B). We examined this decrease under elevated
temperature to determine if the increase in AtSK1 transcript during heat stress is related
to its role in pollen development. sk1, sk2, and Col-0 flowers grown at 32°C after the
first bolt emergence all demonstrated a reduction in viability compared to previous
growth conditions with viable pollen proportions of 23%, 30%, and 31% respectively.
The ratio of nonviable sk1 pollen compared to Col-0 remained similar in both conditions
with a 23% difference during normal conditions and a 25% difference at elevated
temperature. (Figure 17C) We examined mature siliques to determine whether a
reduction in pollen viability produced fewer seeds in sk1 backgrounds however seed
counts didn’t differ significantly between sk1 and Col-0. (Figure 18)
44
Figure 16: Comparison of floral organ size between sk1 and Col-0 flowers. A Representative image of floral dissection. Tissues were cleared using a fixing solution and stamens, gynocium, and petals were separated manually. B Flowers from Col-0 or sk1 plants harvested at stage 13 prior to flower opening and measuremed via ImageJ. Data represents three separate measurements of N>10 stamens and two separate measurements of N=10 gynocium per genotype. Error bars represent standard deviation.
45
Figure 17: Pollen viability in Col-0, sk1, and sk2 stage 13 flowers . A Representative image of FDA staining. Upper panel shows four Col-0 pollen grains stained with FDA and viewed via brightfield microscopy, bottom panel shows same four pollen grains viewed via fluorescence microscopy, viable pollen grains are observed fluorescing. B Pollen from Col-0, sk1, and sk2, backgrounds grown on a 16hr day cycle at 22°C, flowers were harvested at stage 13 before flowers opened. C Pollen from Col-0 sk1, and sk2, backgrounds grown on a 16hr day cycle at 22°C until bolting, then transferred to a 16hr day cycle at 32°. Flowers harvested at stage 13 before flowers opened. For A and B N≥100. Error bars represent standard error. * denotes significant difference between Col-0 and sk1 (p<0.005)
46
Figure 18: Seeds counts in sk1 knockouts and Col-0. Siliques were opened individually and seeds counted. Ten siliques were counted per mature plant and four plants were counted for each genotype. Error bars represent standard error.
Chapter 5 - Discussion
Transcript analysis of SK Duplicates Across Species Tandemly duplicated gene pairs, such as AtSK1 and AtSK2, are more likely to
differ significantly in their expression patterns than those that arose from genome-wide
duplication events (Casneuf et al. 2006). The duplication event that created AtSK2
occurred after Arabidopsis diverged from Oryza sativa (Wolfe et al. 1989) and it is not
known how SK duplicates arose independently in other species. However, each species
represented in our analysis has undergone one or more major genome duplication events
since diverging from the Arabidopsis lineage (Burleigh et al. 2009; Paterson et al. 2004).
Analysis of data from publically available microarray studies demonstrates that
shikimate kinase gene duplicates in multiple species have differential transcript
abundance across tissues, developmental states, and environmental conditions. The
preferential expression of a single homolog in specific tissues is observed in Arabidopsis
thalina green tissues, Hordeum vulgare photosynthetic organs, Glycine max nodules, and
47
Oryza sativa roots and seeds. No single tissue type consistently demonstrated elevated
transcript levels of one homolog across the species examined, suggesting that the
expression patterns observed in these datasets does not represent ancestral regulation of
SK genes. Similarly, the lack of either Hordeum vulgare SK transcript elevation during
heat stress suggests that AtSK1 transcript induction in response to temperature may be a
novel regulatory response acquired since Arabidopsis speciation (Mangelson et al. 2011).
AtSK Duplicates During Heat Stress
Transcript Analysis Induction of AtSK1 transcript following heat stress has been reported in
microarray datasets and suggested by our RT-PCR analysis. The lack of AtSK1 transcript
induction in hsf1a/hsf1b /hsf1d/hsf1e knockouts indicates that AtSK1 is downstream of
these master regulators of Heat Shock Response (HSR) (Fernandes et al. 1994; Liu et al.,
2010). HSFs have been shown to bind Heat Shock Elements (HSE: nGAAnnTTCn)
upstream off the gene promoter in a variety of eukaryotes (Fernandes et al., 1994).
AtSK1 contains one of these elements and AtSK2 contains two in the region -1000bp
upstream of their respective promoters. HSF binding in Arabidopsis is only loosely
correlated with HSE sequences, 33% of total genes contain a HSE sequence and 37-47%
of heat shock regulated genes contain an HSE (Busch et al. 2005). This may be due to the
expansion of HSF genes in land plants compared to other eukaryotes; Arabidopsis carries
21-25 HSFs compared to just 4 in humans (Nover, 2001). The effect of hsf1a/hsf1b
/hsf1d/hsf1e knockout on AtSK1 indicates that it is actively regulated in response to heat
stress however because of the large number of TFs acting downstream of the HSF1
family and the relatively weak specificity of HSF binding elements in Arabidopsis it is
unclear which specific TF is responsible for regulation.
48
sk1 and sk2 Survival and ROS Response During Heat Stress No significant difference in survival after heat shock was found between sk
knockout lines and Col-0. Identification of HSR genes via survival screening is
dependent both on the function of the gene and the network in which it participates.
HSFA1a and HSFA1b are responsible for co-coordinating the regulation of over 2000
genes during heat response however the hsf1a/hsf1b knockout has no discernible
phenotype (Lohmann et al.2004) due to functional redundancy with HSF1d and HSF1e
(Liu et al., 2010). Conversely, 15 of 45 candidate HSR single gene knockouts, identified
by HSF regulation and annotation, had a discernable phenotype when screened for heat
shock survival (Larkindale et al. 2005). AtSK1 may be involved in a HSR pathway with
functional redundancy, or its absence may not perturb HSR to the extent that it increases
cell death under the tested conditions. Measuring ROS oxidation was advanced to
identify subtle defects in HSR that didn’t affect whole organism survival, however
neither tissue staining nor quantification of membrane lipid oxidation via TBARS assay
revealed a difference between sk1, sk2, and Col-0. Similarly, differential hypocotyl
elongation is a widely used HSR screen that failed to identify a phenotype in sk1 and sk2
knockouts. This suggests that AtSK1 may act in a minor role during heat stress that our
assays failed to identify. It is also possible that AtSK1 acts in a functionally redundant
role, either with AtSK2 or additional unknown interactors. In considering additional
interactors expanded protein interaction data would be beneficial; AtSK1 is not included
in the Arabidopsis Y2H interactome and protein interaction data is minimal. Another
possibility is that AtSK1’s role is limited to a specific organ or tissue and that broader
transcript induction is incidental. In this case the screens performed would be largely
ineffective as they examine either the entire organism or leaf tissue. AtSK1 is also
49
expressed during flowering; not only has elevated temperature been linked to more rapid
flowering in Arabidopsis, (Balasubramanian et al. 2006) but HSFs have been implicated
in the regulation of developmental processes in addition to their role in HSR (Kotak et al.
2007; Liu et al., 2010) Our investigation of AtSK1 in HSR and flowering is discussed
elsewhere.
AtSK-CaM Interaction Studies Reports indicating an interaction between AtSKs and CaM complemented our
transcript analysis that placed AtSK1 in a HSR network. Nearly all AtCaM genes are
positively regulated during heat stress (Al-Quraan et al. 2005) and single AtCaMs have
been identified as protein binding partners that influence function and localization during
HSR (Liu et al. 2005; Liu et al., 2010). The protein microarray data that included an
AtSK-CaM interaction implicated AtSK2, however AtSK1 was not included in the array
and bioinformatic analysis of AtSK sequences identified a CaM binding motif on both
proteins (Popescu et al. 2007).
Based on AtSKs’ role in a metabolic pathway we believed CaM might act as a
chaperone to divert it to HSR. We attempted to use this possible chaperone interaction to
increase AtSK2 solubility in BL-21 cells (de Marco, 2007). We also tested protein-
protein interaction via chaperone co-purification (Thain et al. 1996). Although both
proteins expressed well in BL-21 cells neither CaM2 nor CaM8 were able to improve the
solubility of AtSK2. The phenyl sepharose resin used to purify CaM2 and CaM8 captured
a large amount of a single additional unknown protein with a molecular weight of ~19kD
during these tests however this is likely due to the omission of a heat denaturation step in
the protocol designed to precipitate non-thermostable proteins, the step was removed in
order to preserve any AtSK-CaM interaction.
50
The far western approach used to test CaM binding also failed to identify an
interaction between AtSK1, AtSK2, and HRP-CaM81. The overlays were conducted with
large amounts of AtSK protein as bait, and the concentration of HRP-CaM was 75nM,
within the recommended range of 50-100nM. The HRP-CaM overlay method has been
shown to work as reliably as 125I-labeled CaM and biotinylated CaM to identify CaM
binding partners (Lee et al., 1997). It has consistently worked with Arabidopsis proteins
predicted to bind CaM (Reddy et al. 2003; Lee et al. 2008) and as a validation of hits in
high throughput binding studies (Dell'Aglio et al. 2013). The method was especially
attractive for our purpose because the interaction is based entirely on the bait protein’s
primary sequence and utilizes denatured protein, removing the need for soluble AtSK
proteins. The method is more likely to generate a false positive than a false negative due
to exposure of CaM binding domains inaccessible to CaM in a physiological context.
The study that originally identified the interaction tested 20 of their 173 hits via
co-immunoprecipitation and validated 17 (Popescu et al. 2007), suggesting a rough false
positive rate of ~15%. Based on the inability of CaM2 or CaM8 to function as a
chaperone for AtSK2 and the inability of a CaM7 homolog to bind AtSK1 or AtSK2 in a
robust in vitro binding assay we conclude that the AtSK proteins probably do not bind
CaMs in vivo.
51
AtSK Duplicates During Floral Development
Transcript Analysis AtSK1 transcripts are not elevated in floral tissues in arf6/8, and myb21/24
knockout backgrounds. ARF6/8 are auxin responsive TFs that act upstream of
MYB21/24 and regulate their transcription via jasmonic acid signaling (Reeves et al.
2012). AtSK1 is likely downstream of MYB21/24 based on its similar transcript profile
in both arf6/8 and myb21/24 knockout lines. myb21/24 knockouts are male sterile due to
defects in stamen length and anther dehiscence physically hindering pollen deposition,
and pollen development defects resulting in nonviable pollen grains (Yang,et al.2007;
Song et al. 2011). Many genes affected in myb21/24 knockouts are annotated with
metabolic function however AtSK1 is the only shikimate pathway gene with a significant
change in transcript level suggesting that MYB21/24 regulation doesn’t target the entire
shikimate pathway
sk1 Floral Morphology Microarray data and RT-PCR analysis shows that in wild type flowers
AtSK1 transcript increases during stage 12 and 13. During these stages stamens and
gynoecia elongate synchronously in preparation for self-pollination, and anther
dehiscence and floral opening occurs (Goldberg et al. 1993; Reeves et al. 2012). These
developmental events served as a starting point for our analysis of the sk1 knockouts. We
considered that induction of AtSK1 in stage 13 flowers may be related to shikimate
pathway output; rapid tissue development may require increased protein synthesis,
additionally, auxin, a key regulator of floral development (Cheng et al. 2006; Reeves et
al. 2012) is synthesized via a tryptophan dependent pathway. In this case we would
expect disruption of the floral organs undergoing elongation during stage 13. Comparison
52
of sk1 and Col-0 stamens and gynoecium by floral dissection revealed no significant
difference in their development prior to floral opening. By dissecting the flowers and
fully separating the organs we were able to measure from the base, where differentiation
from the floral meristem occurred, more accurately than comparative methods that leave
flowers intact. This led us to conclude that AtSK1 does not affect stamen or gynocieum
elongation.
sk1 Pollen Viability and Seed Set Available expression data doesn’t differentiate between transcripts in the stamen
and transcripts in the anther, the site of pollen development. Anthers in the knockout
contained a lower proportion of viable pollen grains as measured by FDA staining. The
FDA method accounts for cell viability independent of cell wall integrity and is
considered an improvement to other methods such as Alexander’s stain that rely on dye
exclusion for visualization. 59% of pollen grains in the sk1 knockout were viable, which
represents a 21% reduction of viable pollen grains compared to Col-0. Seed counts for
sk1 were similar to wild type however other studies assayed knockout lines with more
severe pollen abortion phenotypes and reported a seed count difference of <4% (Lee et
al. 2008) suggesting that Arabidopsis self pollination in laboratory conditions doesn’t
require a high proportion of viable pollen. Analysis of microarray data indicated that
AtSK1 is not expressed at high levels in pollen (Figure 5), however a number of genes
involved in anther development do not have elevated transcript in pollen but cause pollen
abortion when knocked out, including roxy1roxy2 (Xing et al. 2008) and ems1 (Zhao et
al. 2002). Future investigations should observe pollen development in sk1 plants via
microscopy to determine the specific defect affecting viability. Additionally, manual
53
pollination of emasculated Col-0 flowers with sk1 pollen could confirm that a proportion
of sk1 pollen is sterile and may result in a lower seed yield.
Pollen development proceeds in two phases. The first phase consists of
differentiation of anther tissues and meiotic division of the microspore mother cells to
form tetrads. The second phase consists of pollen grain differentiation and maturation
followed by the degeneration of the anther prior to pollen release (Goldberg, et al.1993).
It is unlikely based on its expression profile that AtSK1 affects pollen development in the
first phase that corresponds to flowering stages 1-7. Events in the second phase
correspond to AtSK1 transcript induction and include degeneration of the tapetum and
cleavage of tissues prior to dehiscence (Sanders et al. 1999). Dehiscence appeared to
proceed normally in sk knockouts, as pollen grains were successfully released. The anther
tapetum is a synthesis and storage site for a wide variety of compounds that are integrated
into the pollen cell as they are released from the tapetum during apoptosis-like cell
destruction (Parish et al. 2010; Phan et al. 2011). These include compounds formed
downstream of the shikimate pathway such as phenylpropanoids and flavonoids, which
make up a significant proportion of the pollen cell wall (Piffanelli et al. 1998; Hsieh et al.
2007). We consider that AtSK1 may play a role in a tapetum related process. Although
no other shikimate pathway components appear to be induced in stamens AtSK1 may
play a sensory or regulatory role monitoring flux through the shikimate pathway during
the transition between metabolite synthesis and release. Determining whether anther or
tapetum specific processes are disrupted in sk1knockouts is a crucial next step in
identifying its role in flowering and quantifying the levels of shikimate and chorismate in
sk1 flowers may help determine whether the observed phenotype is related to shikimate
54
pathway regulation. To confirm that the observed phenotype is associated with knocking
out AtSK1, we proposed to obtain a second TDNA sk1 KO line, and also complement the
sk1 backgrounds with an AtSK1 knock-in to determine whether the native gene restores
pollen viability.
Pollen Viability During Heat Stress sk1 knockouts were assayed for pollen viability after heat stress and while both
sk1 and Col-0 showed a large drop in pollen viability at 32°C the ratio of viable pollen
between the lines remained the same. This experiment was conducted under constant heat
conditions at a sub-lethal level. As discussed earlier it is possible that broader transcript
induction of AtSK1 during heat stress is incidental to its function in the flower, possibly
related to pollen viability during heat shock. Submitting flowers to higher temperatures
than we used, for shorter periods, results in male sterility (Kim et al. 2001) due to pollen
abortion or failure to release pollen. Determining the threshold for heat shock affecting
sterility may identify temperatures at which sk1 knockouts are sterile, but not Col-0.
Alternative Models Inability to generate sk1/sk2 T-DNA insertion lines and the similar in vitro kinetic
parameters of ATK1 and ATSK2 suggests that they are functionally redundant in the
shikimate pathway (Falconer thesis. 2006). Although much of our data is interpreted as
arising from regulatory differences between AtSK1 and AtSK2 it is possible that the
duplicates are fully redundant and the observed transcriptional differences are related to
genomic context and do not impact function. We also consider that the pollen viability
phenotype seen in sk1 flowers could alternatively have arisen from an unrelated genomic
perturbation in the T-DNA line (Tax et al. 2001; Clarke et al. 2010). Several factors lead
us to reject a model in which AtSK1 and AtSK2 are fully redundant, including the length
55
of time each duplicate has been maintained under positive selection since divergence
(Fucile et al. 2008), the lack of a pollen viability defect in sk2, transcriptional evidence
for regulation of AtSK1 by MYB21 and MYB21, and the precedent of Arabidopsis DHS
homologs, in which structurally similar shikimate pathway enzyme homologs
demonstrated differential regulatory responses (Crowley Thesis. 2006; Shahinas Thesis
2008). Several experiments addressing these alternative hypotheses are proposed in our
future directions below.
Chapter 6 – Conclusions and Future Directions The objective of this research was to investigate the regulatory and functional
roles of Arabidopsis thaliana shikimate kinase duplicates. We have established that
AtSK1 and AtSK2 have different transcript levels, with AtSK1 induced under heat stress
and during late stages of flowering. Microarray data detailing the expression of SK
duplicates in other species suggests that the conditions under which AtSK1 is induced are
not related to ancestral regulation of SKs in plants. Further querying of microarray
databases established that AtSK1 is likely regulated downstream of the HSF1 family
under elevated temperature and MYB21/24 in late stage flowers, providing two broad
avenues for investigation: HSR, and floral development. We utilized three major
experimental approaches, assaying sk1 and sk2 knockout lines for HSR phenotypes, in
vitro investigation of CaM binding interaction, and assaying sk knockout lines for floral
development defects. The following conclusions are drawn from our analysis.
(1) Lines lacking AtSK1 had no major defects in HSR, indicating that AtSK1 is
functionally redundant with AtSK2 or another gene, acts in a role outside the responses
we assayed, or has no major effect on HSR. We also consider the possibility that AtSK1
56
induction during HSR is related to its role in floral development and additional tissues are
incidental.
(2) Although AtSK1 and AtSK2 were identified as possible AtCAM interactors in high
throughput studies, we determined they do not bind AtCaM proteins in vitro based on on-
column binding assays and far western analysis.
(3) AtSK1 affects pollen viability through an unknown mechanism. sk1 knockout lines
consistently produced 20% fewer viable pollen grains than Col-0. This reduction doesn’t
affect seed count when Arabidopsis self-pollinates. Moderate temperature increase affects
pollen viability but isn’t exacerbated in lines lacking AtSK1.
The transcriptional differences between AtSK1 and AtSK2 as well as the pollen
viability phenotype associated with sk1 suggests that AtSK1 is both regulated
independently of AtSK2, and affects pollen viability. This leads us to conclude that
AtSK1 has undergone subfunctionalization.
Future research should focus on determining the processes affected by
AtSK1 during pollen development as well as determine the contribution of each duplicate
to the shikimate pathway. To further investigate pollen viability developing pollen grains
should be examined via microscopy to determine whether there are defects in well-
characterized structures such as the pollen cell wall in the sk1 background. Metabolic
analysis via GC/MS may identify perturbations in metabolite levels in sk1 pollen,
possibly establishing a connection to shikimate pathway products. Pollination of
emasculated Col-0 flowers using sk1 pollen should be conducted to confirm defects in
viability and all pollen assays should be repeated with additional T-DNA lines. To
characterize any differences between AtSK1 and AtSK2 in the shikimate pathway
57
generation of sk1sk2 T-DNA KO lines via metabolite supplementation of shikimate-3-
phosphate, or AtSK1 AtSK2 RNAi should be pursued. The effect of each single KO on
the shikimate pathway could be quantified by metabolite profiling to determine
respective contributions. These approaches should determine the degree to which the
duplicates are redundant in the shikimate pathway in vivo.
58
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