characterization of the tomato branched...
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CHARACTERIZATION OF THE TOMATO BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE ENZYME FAMILY MEMBERS
By
GREGORY S. MALONEY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2009
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© 2010 Gregory S. Maloney
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To my parents, James and Maureen Maloney
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ACKNOWLEDGMENTS
I would first like to thank the members of my supervisory committee, Dr. Balasubramani
Rathinasabapathi, Dr. Don McCarty, Dr. Jay Scott for their constructive criticism and helpful
advice. I would next like to thank my advisor, Dr. Harry Klee, for his guidance and for keeping
me focused in my research and for teaching me to write scientifically. I would also like to thank
all of the members of our lab, including Peter Bliss, Mark Taylor, Denise Teiman, and Dawn
Bies, for their assistance with my research. For teaching me fundamental research techniques and
giving great advice I would like to thank Jonathan Vogel, Brian Kevany, Michelle Ziegler,
Sandrine Matheiu, Valeriano Dal Cin, Melissa Hamner, and Charles Goulet from our lab. I am
also obliged to Romain Fouqet for sharing his experience in various techniques, especially
protoplast culture and GFP microscopy. For advice and letting me use his StepOnePlus
instrument I thank Kevin Folta. For helping me with bacterial complementation I would like to
thank Valerie De Crecy-Lagard and Basma El Yacoubi. I also thank fellow members of the Plant
Molecular and Cellular Biology program, in particular Jon Martin, for his help with RT-PCR
work. I also thank the PMCB program, in particular Lindsey Freeman and Eliana Kampf for
keeping my academic life organized. I am also grateful to the University of Florida Alumni
Association for providing me with my Fellowship and the funds to stay afloat through graduate
school.
I give special thanks to all of my family, who have been very supportive throughout my
graduate school process, and to my friends, who have always helped me to relax and keep an
open mind, and to Katherine McGrath for her constant support and for making my time in
Florida so enjoyable.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ...............................................................................................................4
LIST OF TABLES ...........................................................................................................................7
LIST OF FIGURES .........................................................................................................................8
LIST OF ABBREVIATIONS ..........................................................................................................9
ABSTRACT ...................................................................................................................................11
LITERATURE REVIEW ..............................................................................................................13
Tomato as a Research Tool for Flavor ...................................................................................13 Tomato Flavor ........................................................................................................................16 Branched-Chain VOC Metabolism .........................................................................................18 BCAA Metabolism in Plants ..................................................................................................20 Branched-Chain Aminotransferases in Plants ........................................................................23
CHARACTERIZATION OF TOMATO BCATs ..........................................................................31
Cloning of SlBCAT cDNAs ....................................................................................................31 Expression Analysis of SlBCATs ............................................................................................32
Subcellular Localization of SlBCATs ....................................................................................33 Functional Verification of SlBCATs by Bacterial Complementation ....................................34 BCAT Enzyme Assays ...........................................................................................................35 Analysis of SlBCAT1 and SlBCAT3 Transgenic Fruit ............................................................37 Conclusion of Results .............................................................................................................38
BRANCHED-CHAIN VOLATILES IN TOMATO .....................................................................49
Rationale and Background ......................................................................................................49
Results of Substrate Feeding ..................................................................................................50 BCAA and Branched-Chain Volatile Loci .............................................................................56
DISCUSSION OF RESULTS .......................................................................................................63
Diversity of SlBCAT Family ...................................................................................................63 Significance of Substrate Feeding ..........................................................................................66 Concluding Remarks ..............................................................................................................71
MATERIALS AND METHODS ...................................................................................................74
Cloning of SlBCATs...............................................................................................................74
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Constructs ...............................................................................................................................74
Protein Production and Purification........................................................................................75 Enzyme Assays .......................................................................................................................76 Volatile Collection and Analysis ............................................................................................77 Microscopy and Subcellular Localization ..............................................................................78 Metabolite Feeding .................................................................................................................79 GC-MS Analyses of Nonvolatile Plant Metabolites ...............................................................79 Analysis of [U-13C6]Leucine-Labeled Samples .....................................................................79 Expression Analysis ................................................................................................................80 E. coli Complementation ........................................................................................................81 Amino Acid Analysis of Tomato Fruit by GC-MS ................................................................81 Statistical analysis ...................................................................................................................82
LIST OF REFERENCES ...............................................................................................................84
BIOGRAPHICAL SKETCH .........................................................................................................92
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LIST OF TABLES
Table page 1-1 Flavor volatile compounds impacting the perception of ripe tomato fruit flavor ...............30
2-1 Measurement of E. coli cell culture growth rate .................................................................45
2-2 Kinetic parameters of SlBCATs ..........................................................................................46
2-4 Levels of free amino acids in red ripe fruit of M82 and SlBCAT over-expression lines. ...................................................................................................................................48
3-1 Label accumulation in metabolite pools following incubation with [U-13C]leucine ...........61
3-2 Occurrences of loci containing BCAA and branched-chain volatile phenotypes ...............62
4-1 Primer sequences used in this study. ...................................................................................83
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LIST OF FIGURES
Figure page 1-1 BCAA metabolic pathways. ................................................................................................27
1-2 Reported pathway of BCAA catabolism in plants leading to TCA cycle intermediates. ....28
1-3 Schematic of S. lycopersicum and S. pennellii introgression line population and corresponding branched-chain volatile loci. ......................................................................29
2-1 Evolutionary relationships of mature SlBCAT proteins......................................................40
2-2 Evolutionary relationships of mature SlBCAT and AtBCAT proteins ...............................41
2-3 Quantification of SlBCATs RNA in different tissue types. .................................................42
2-5 Growth complementation of E. coli ΔilvE/ΔtyrB mutant cells expressing SlBCAT3 and 4..........................................................................................................................................45
2-6 Analysis of transgenic fruit SlBCAT transcript levels .........................................................47
3-1 Leucine and KIC feeding .....................................................................................................58
3-2 Isoleucine and KMV feeding ...............................................................................................59
3-3 Valine and KIV feeding. ......................................................................................................60
4-1 Hypothesized pathways forming branched-chain volatiles from BCKAs ...........................73
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LIST OF ABBREVIATIONS
AAT Alcohol acetyl transferase
ABA Abscisic acid
ADH Aldehyde dehydrogenase
ALS Acetolactate synthase
At Arabidopsis thaliana
BCAT Branched-chain amino acid aminotransferase
BCAA Branched-chain amino acid
BCKA Branched-chain α-keto acid
BCKADH Branched-chain keto acid dehydrogenase complex
BLAST Basic local alignment search tool
CoA Co-enzyme A
DPA Days post anthesis
EST Expressed sequence tag
GABA Gamma aminobutyric acid
GC-MS Gas chromatography-mass spectrometry
GFP Green fluorescent protein
Hv Hordeum vulgare
IL Intogression line
Kcat Catalyticconstant
KIC α-Ketoisocaproate
KID Keto acid decarboxylase
KIV α-Ketoisovalerate
Km Michealis-Menten constant
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KMV α-Keto-3-methylvalerate
Nb Nicotiana benthamiana
NMR Nuclear magnetic resonance
NADH Nicotinamide adenine dinucleotide (reduced)
PDC Pyruvate decarboxylase
PLP Pyridoxal 5’ phosphate
PMP Pyridoxamine 5’ phosphate
QTL Quantitative trait loci
RFLP Restriction fragment length polymorphism
RT-PCR Reverse transcriptase polymerase chain reaction
Sl Solanum lycopersicum
SNP Single nucleotide polymorphism
TCA Trichloroacetic acid
Vmax Maximum velocity
VOC Volatile organic compound
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF THE TOMATO BRANCHED-CHAIN AMINO ACID
AMINOTRANSFERASE ENZYME FAMILY MEMBERS
By
Gregory S. Maloney
May 2010
Chair: Harry J. Klee Major: Plant Molecular and Cellular Biology
Branched-chain amino acids (BCAAs) are essential to animals and are synthesized in
plants from branched-chain keto acids (BCKAs), but their metabolism in plants is not completely
understood. The interface of BCAA anabolism and catabolism lies with branched-chain
aminotransferases (BCAT). In this study six BCAT genes from the cultivated tomato species
Solanum lycopersicum were identified and characterized. Quantitative RT-PCR showed that
SlBCAT1, 2, 3, and 4 were expressed in multiple plant tissues, while SlBCAT5 and 6 transcripts
were undetectable. SlBCAT2 and 3 were expressed nearly equally in all tissues, while SlBCAT1
was expressed most highly in ripening fruit and SlBCAT4 was expressed primarily in
inflorescences. SlBCAT1 and 2 are located in the mitochondria, SlBCAT3 and 4 in chloroplasts,
while SlBCAT5 and 6 are located in the cytosol and vacuole, respectively. Expression of
SlBCAT3 and 4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, while
expression of SlBCAT1 and 2 were less effective. All SlBCAT enzymes were active in the
forward (BCAA synthesis) and reverse (BCKA synthesis) reactions. SlBCAT3 and SlBCAT4
exhibited a preference for the forward reactions while SlBCAT1 and SlBCAT2 were more active
in the reverse reactions. Over-expression of either SlBCAT1 or SlBCAT3 in tomato fruit did not
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significantly alter amino acid levels or branched-chain volatile emissions. BCAAs and BCKAs
were applied to fruit samples which were then analyzed for branched-chain volatiles. The results
support a model in which these volatile compounds are synthesized primarily from BCKAs and
not BCAAs in tomato fruit, unlike their synthesis from BCAAs in yeast.
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CHAPTER 1 LITERATURE REVIEW
Tomato as a Research Tool for Flavor
The cultivated tomato, Solanum lycopersicum, is the most valuable vegetable crop in
Florida, the leading US producer of fresh market tomatoes (Lucier, 2009). Consumers’ opinions
of the average grocery store tomatoes reflect poor flavor compared to the more flavorful
heirloom tomatoes. This is due in part to the fact that tomato breeding has focused principally on
economically important traits such as yield, appearance, maturity, and disease resistance. It is
difficult and impractical for breeders to select for better flavor in tomatoes because it is a highly
multigenic trait that is greatly influenced by environmental conditions and the composition of a
tasty fruit is not completely understood. Commercial tomatoes are often picked green, stored and
shipped at low temperatures, and bruised during handling, all of which have been shown to
decrease positive flavor compounds (Maul et al., 2000). Tomato varieties targeted towards home
gardeners tend to have superior flavor but are not grown commercially because they yield less
fruit, are softer, ripen too quickly, and do not ship or store well. These issues concerning fresh
tomato flavor quality may have the best chance of being resolved following research that focuses
on the genetics and biochemistry of flavor.
Tomato flavor comes from a blend of three chemical classes: acids, sugars, and volatile
organic compounds (volatiles). Flavor is also determined by the interactions of these chemicals
with alcohols, glycosidic bonds, and other compounds produced during fruit ripening (Tandon et
al., 2000; Ortiz-Serrano and Gil, 2007). Volatiles, which outnumber by far all other classes of
flavor compounds, are sensed by the olfactory system and are important in the diversity and
complexity of fresh uncooked tomato flavor.
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Volatiles are low molecular weight hydrophobic compounds derived from fatty acids,
carotenoids, and amino acids, which diffuse readily through fruit tissue to be released into the
atmosphere. The difficulties in studying ripe tomato flavor include the multitude of metabolic
pathways leading to volatile synthesis, their interconnectivity, and the lack of genes identified in
these pathways. However, some information concerning these pathways is known and serves as a
starting point for research. For instance, the C6-alcohol and aldehyde flavor volatiles are thought
to be derived from fatty acids (Chen et al., 2004), the ketone volatiles from carotenoids (Simkin
et al., 2004), and the amino acid-derived volatiles from alanine, phenylalanine, and branched-
chain amino acids (Tressl and Drawert, 1973; Tieman et al., 2006b).
The study of tomato flavor was previously undertaken (Tieman et al., 2006) by utilizing
the S. pennellii exotic introgression line population created by Dani Zamir in Israel. This
population is a series of 76 near isogenic introgression lines (ILs) that span the whole tomato
genome and overlap to form over 100 bins. It was created from a cross of S. pennellii (accession
code LA716), a wild species, and S. lycopersicum cv. M82, a common processing tomato (Eshed
and Zamir, 1995)
The formation of an introgression population takes about 10 generations of backcrossing.
In brief, the two parent species, in this case S. pennellii and M82, are crossed. The sites of
recombination are determined using existing RFLP markers. Individuals with the best
distribution are backcrossed into the M82 parent, and this is repeated for ten or more generations.
Once lines are found with single small segments of S. pennellii genome in the M82 near-isogenic
background, those plants are self-pollinated until homozygous, creating pure lines. The last step
is to create a map showing the alignment of all of the different introgression lines such that the
whole S. pennellii genome is covered, and finally assignment of bins.
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The importance of these lines is two-fold. First, a large amount of genetic diversity can be
exploited for new traits, given the wide range of evolutionary divergence between the two
species. Second, one can narrow down the genetic location of a phenotype to a particular
chromosomal segment via map-based cloning, eventually leading to gene discovery. This is
possible because S. pennellii has a high enough frequency of single nucleotide polymorphisms
(SNPs) that a dense map of genetic markers has been created and is available for public use
(Mueller et al., 2005).
One argument for the use of introgression populations is that the current cultivated
varieties represent a very narrow genetic base. Many wild species of the Solanaceae family and a
few closely related to tomato exist throughout the world. The genetic backgrounds of these
species are useful for introduction into the tomato varieties that are sold commercially. It is from
these wild species that tomato researchers hope to find the answers to problems with disease and
pest resistance, adaptation tolerance, yield, flavor, postharvest and other tomato qualities.
The method of improving tomato quality by QTL identification has already been used for
traits such as increased sugars and soluble solids (Fridman et al., 2002). By analyzing each
introgression line and comparing its phenotype to that of the M82 parent, the genetic location of
the trait can be immediately localized to a relatively small chromosomal segment. It has already
been found that many of these ILs contain important flavor volatile QTL (Tieman et al., 2006).
Many ripe tomato flavor volatile QTL have also been identified in the IL population of S.
habrochaites, another wild relative of cultivated tomato (Mathieu et al., 2009).
Among the many ILs that show alterations in flavor volatile profiles, twelve of them are
relevant to the research presented here. The volatile profiles of those lines were studied
previously (Tieman et al., 2006). Gas chromatography analysis of fruit from these lines revealed
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elevated levels of the volatiles 2-methylbutanal, 2-methylbutanol, 3-methylbutanal, 3-
methylbutanol, isovaleronitrile, isobutylthiazole, and isobutyl acetate compared to the M82
control. These volatiles are collectively called branched-chain volatiles because of their
structural similarities to BCAAs. The aldehyde and alcohol branched-chain volatiles are known
to attribute unpleasant flavor to tomatoes (Tandon et al., 2000; Baldwin et al., 2008). The
metabolic route leading to these compounds in plants is unknown and presents a challenging
study. Using evidence from microorganisms and predicting putative pathways based on chemical
structures provides a platform to begin studying the synthesis of these compounds and the
enzymatic steps involved.
Tomato Flavor
Much of tomato fruit flavor composition has been worked out qualitatively and
quantitatively (Buttery et al., 1986; Buttery et al., 1987; Buttery et al., 1987; Buttery et al., 1988;
Buttery et al., 1989). The significance of those studies was in the identification of many of the
most important tomato fruit volatiles and determining their odor thresholds, gas chromatography
retention times, and mass spectra. The odor threshold of a volatile compound is the smallest
amount of the compound that can be sensed by human olfaction, and is determined by trained
panelists. If the concentration of a compound exceeds its odor threshold, it is a positive
contributor to flavor. If its concentration is below its odor threshold, it does not contribute to
perceived flavor. Odor units determine the impact of a volatile flavor compound and are
determined by dividing the log of its concentration by its odor threshold. Determination of odor
units is important in choosing compounds to study. For example, in the study by Buttery et. al
(1988), the volatile compound isovaleronitrile was discovered and was present from 10-200 ppb,
depending on the cultivar. They also found that its odor threshold was relatively high, at 150
ppb. Given both figures, this compound probably is not important to tomato aroma. However,
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this compound is structurally related to the volatiles isobutyl cyanide and 2-isobutylthiazole, the
second of which has positive odor units in tomato fruit. On the other hand, the volatile β-
damascenone has an odor threshold of 0.002 ppb and a fresh fruit concentration of 1-3 ppb,
making it a positive contributor to fruit flavor. Table I shows the odor thresholds and
concentrations typically found in ripe fruits for most of the volatiles important in tomato flavor,
adapted from Buttery et al. (1971,1986, 1987,and 1988).
There are many thousands of aroma compounds known today, and over 400 have been
identified in tomatoes. However, only about 16 of these show evidence of being important in
flavor, given their odor units (Buttery and Ling, 1993). Interestingly, some of these compounds
have a pleasant odor in small amounts, but can be perceived as unpleasant when present at higher
concentrations. Examples of these are some of the branched-chain volatiles, which have been
described as pungent and stale, and phenylacetaldehyde, which is responsible for the malodorous
fruit aroma phenotype of IL8-2 (Tadmor et al., 2002). The branched-chain volatiles only seem to
give food a desirable quality when present at low concentration or in certain fermented foods
such as breads, cheeses, beers, and wines, in which they are crucial flavor components. Some
volatiles, however, make a fruit more pleasant when in concentration is increased, such as cis-3-
hexenal, one of the most abundant tomato volatiles, which gives a green aroma (Tandon et al.,
2000).
As mentioned previously, a study was performed in our lab on several of the S. pennellii
introgression lines to find loci important to fruit flavor (Tieman et al., 2006). Twenty-five
different quantitative trait loci (QTL) affecting 23 volatile flavor compounds were identified.
Four QTL that were significantly different in citric acid content were also identified. Other
results in the study are highly relevant to the experiments reported here. First, it was found that
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12 loci had alterations of multiple branched-chain volatile compounds. The fact that this class of
volatiles is altered in so many QTLs suggests that there are many factors or regulatory elements
influencing their metabolism. Second, it was found that isovaleronitrile, whose synthetic
pathway has never been determined, was altered in several QTLs along with other branched-
chain volatiles, suggesting it forms from the same precursors. The authors speculate that the
changes in branched-chain volatile content observed in the IL population could be due to
transcriptional regulators or rate-limiting enzymes affecting the metabolism of any of the
intermediate compounds in their synthesis.
Branched-Chain VOC Metabolism
Largely based on structural considerations and substrate feeding studies, the BCAAs have
been proposed to be precursors for the important flavor volatiles 2-methylbutanol, 3-
methylbutanol, 2-methylbutanal, and 3-methylbutanal in plants (Tressl and Drawert, 1973;
Gonda et al., 2010). These branched-chain volatiles are produced in bacteria, fungi, and plants
(Tressl and Drawert, 1973; Vergnais et al., 1998; Perez et al., 2002; Dickinson et al., 2003;
Nimitkeatkaia et al., 2005) and contribute to flavor in many foods including tomato (Guadagni et
al., 1972; Buttery et al., 1987; Mayer et al., 2003). In microorganisms these volatiles are formed
by the catabolism of BCAAs first by a branched-chain aminotransferase (BCAT, 2.6.1.42) to
form BCKAs. Those acids are subsequently acted upon by pyruvate decarboxylase-like (PDC-
like, 4.1.1.1) enzymes to form branched-chain aldehydes followed by alcohol dehydrogenases
(ADH, 1.1.1.1) to form branched-chain alcohols (Hazelwood et al., 2008). These reactions are
collectively known as the Ehrlich pathway (Ehrlich, 1904) and are illustrated in Figure 1-1A.
BCAAs can also be catabolized by another, more established, primary metabolic pathway
that does not produce volatile compounds. In this pathway, BCKAs are produced from
deamination of BCAAs by BCATs, but are substrates for the branched-chain alpha keto acid
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dehydrogenase (BCKADH) enzyme complex (Fujiki et al., 2000). This step and those that follow
produce acyl-CoA-conjugated compounds, which then yield the intermediate compounds of the
TCA cycle, acetyl-CoA and succinyl-CoA (Taylor et al., 2004). This catabolic pathway is
illustrated in Figure 1-2. It has been shown that high BCAA and BCKA levels induce the
expression of enzymes in this complex as a mechanism for these compounds to regulate their
own concentrations (Fujiki et al., 2001). This pathway has never been shown to produce volatile
compounds. However, when this primary pathway is highly active in yeast, very little branched-
chain alcohols and aldehydes are synthesized, presumably because this pathway uses up
intermediates that would otherwise be used in the volatile-producing pathway.
The volatile synthesis pathways of BCAA catabolism have been best studied in the
budding yeast, Saccharomyces cerevisiae, and the information gained from those studies may be
useful for gene discovery and pathway elucidation in plants. S. cerevisiae makes many long
chain alcohols including 3-methylbutanol from leucine, isobutanol from valine, 2-methylbutanol
from isoleucine, 2-phenylethanol from phenylalanine, and tryptophol from tryptophan.
Isobutanol, 3-methylbutanol, and 2-methylbutanol were shown to be synthesized from valine,
leucine, and isoleucine, respectively, by experiments utilizing 13C-valine and NMR analysis
(Dickinson et al., 1997; Dickinson et al., 1998; Dickinson et al., 2000).
Studies in yeast also demonstrated the enzymatic steps by which the branched-chain
volatiles are synthesized from BCAA catabolism. They also suggest that in some cases the
catabolic pathways of all three BCAAs require different enzymes at each particular step. While
studying leucine catabolism, for example, knocking out one decarboxylase showed that it
contributes to 94% of 3-methylbutanol production, while another decarboxylase contributes 6%.
When both genes are knocked out, no 3-methylbutanol is produced, suggesting the only pathway
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involved in its production in yeast must involve these enzymes. Six of thirteen ADH enzymes in
yeast have been shown through mutant analysis to produce 3-methylbutanol, illustrating the
redundancy of this enzyme group (Dickinson et al., 2003). Yeast mutants lacking BCKADH still
produced wild-type levels of 3-methylbutanol, evidence that the acyl-CoA-mediated pathway of
BCAA catabolism does not contribute to branched-chain volatiles (Dickinson et al., 1997). In
studies of valine catabolism, yeast mutants lacking BCKADH produced wild-type levels of
isobutanol from valine, while PDC triple mutants produced no isobutanol and the PDC-like
mutation had no effect. The evidence together suggests that specificity between valine and
leucine branched-chain alcohol production lies with the carboxylase step. Unlike with leucine
and valine catabolism, in yeast, 2-methylbutanol from isoleucine can be produced from any of
the three PDCs, the PDC-like YDL080, or YDR380. BCKADH does not contribute to 2-
methylbutanol production (Dickinson et al., 2000).
Though it seems the specificity of branched-chain volatile metabolism lies at the
decarboxylase steps, the initiation of these pathways lies with BCATs. Yeast has two
characterized BCAT genes, the products of which are localized in the cytosol or in mitochondria
(Liepman and Olsen, 2004). Mutagenesis or overexpression of these BCATs both resulted in
dramatically decreased and increased values, respectively, of isobutanol emission (Lilly et al.,
2006). A different study found that both yeast BCATs are involved in 3-methylbutanol
production (Schoondermark-Stolk et al., 2006). The function of yeast BCATs in branched-chain
volatile metabolism has encouraged the study of this enzyme family in tomato, the results of
which are described in this work.
BCAA and Volatile Metabolism in Plants
The BCAAs leucine, isoleucine, and valine are primary plant metabolites involved in
many processes. They are synthesized from threonine or pyruvate in plastids of mostly young
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tissues (Schulze-Siebert et al., 1984; Hagelstein et al., 1997). Threonine feeds into the isoleucine
pathway and pyruvate into the valine pathway, and hydroxyethyl-TPP into both, after which the
same four enzymatic steps are shared to form these two amino acids. Leucine is synthesized by a
branch of the valine pathway starting with α-ketoisovalerate in four enzymatic steps (Holmberg
and Petersen, 1988; Kohlhaw, 2003). These steps and their corresponding enzymes are illustrated
in Figure 1-1B.
Although synthesis of BCAAs is well characterized in plants, the catabolic pathways are
not completely understood. Their catabolism is believed to be initiated in mitochondria, where
the BCKDH complex is located (Taylor et al., 2004). The primary fates of BCAAs in plant cells
are peptide elongation, glutamate recycling, glucose and sucrose linked branched-chain ester
synthesis, branched-chain fatty acid synthesis, and respiration through synthesis of TCA cycle
intermediates (Kandra et al., 1990; Walters and Steffens, 1990; Kroumova et al., 1994; Daschner
et al., 1999; Li et al., 2003; Beck et al., 2004; Taylor et al., 2004). BCAA catabolism may be
more central to plant metabolism than previously appreciated. For example, Gu et al. (2010)
showed that a mutation in isovaleryl-CoA dehydrogenase, an enzyme in the BCAA catabolic
pathway, influences the metabolism of many unrelated compounds in Arabidopsis seeds,
including twelve amino acids.
In a banana study, it was shown that leucine and valine levels accumulate during ripening,
while isoleucine levels remain constant (Tressl and Drawert, 1973). Labeling studies in banana
showed that a small but significant amount of [14C]leucine was converted into 3-methylbutanol,
3-methylbutyl esters, 3-methylbutyric acid, and KIC. The largest amount of radioactive label was
found in 3-methylbutanol. Similar results were found for [14C]valine. Banana fruit have a volatile
profile consisting mostly of esters, with small amounts of alcohols, ketones, aldehydes, and
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phenolic ethers (Tressl and Drawert, 1973). Alcohol acetyl transferase (AAT) in banana makes
esters using 2-methylbutanol and 3-methylbutanol as precursors, and the amount of volatile
esters produced is limited by alcohol substrate. Banana also produces 2-methypropyl esters from
valine (Wyllie and Fellman, 2000). In a similar study in strawberry, incubation with isoleucine
resulted in increased 2-methylbutanol and 2-methylbutanal, but levels started to drop off after 48
h, while esters derived from these compounds increased (Perez et al., 2002). A study in
Gypsophila showed that in pre-anthesis flowers, 3-methylbutanol and 3-methylbutanal are
produced from leucine via PDC and ADH enzymes, while leucine is made into 3-methylbutyric
acid via isovaleryl-CoA from the same enzymes in open flowers (Nimitkeatkaia et al., 2005). A
recent study in cucumber fruit provided evidence that some fruit volatiles are derived directly
from amino acids by the action of aminotransferases. Although they showed direct evidence of
labeled phenylalanine incorporation into volatiles, the authors were not able to show this for
volatile formation from BCAAs via BCATs (Gonda et al., 2010), These studies all suggest that
the Ehrlich pathway probably occurs in some plants and tissues, but is not the only route to
branched-chain volatiles. The results in chapter three suggest an alternate route to the synthesis
of these volatiles in tomato which bypasses the BCAT step.
As mentioned previously, there are twelve QTLs in the S. pennellii introgression
population affecting emission of branched-chain volatiles in tomato (Figure 1-3). QTLs from
these lines were generally were altered in multiple branched-chain volatiles, suggesting common
pathways for synthesis. In a study by Schauer et al. (2006), approximately 26 QTLs for BCAA
content of ripe tomato fruits were identified in the S. pennellii introgression population. Of these
QTLs, seven were altered in the levels of all three BCAAs, suggesting that they encode major
BCAA pathway regulatory elements. However, there is no consistent correlation between QTLs
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with increased BCAAs and QTLs with increased branched-chain volatiles. These observations
shed doubt on the likelihood that BCAAs and branched-chain volatiles are metabolically related
in tomato, a thought that is strongly supported by the results in chapter three.
BCAA metabolism is under strict control by multiple regulatory mechanisms, including
gene expression, enzymatic substrate specificity, and feedback inhibition. Most of what is known
about regulation of BCAA metabolism in plants, however, pertains to the BCAA synthesis
enzymes threonine deaminase (TD) and acetolactate synthase (ALS). Product inhibition appears
to be the strongest regulator of plant BCAA metabolism. TD is very specifically feedback-
inhibited by excess isoleucine, but can be reactivated by increased concentrations of valine. ALS
is feedback inhibited by both leucine and valine and to a lesser extent isoleucine. It is the target
of many commercial sulfonylurea-based herbicides. Isopropylmalate synthase (IPMS), the first
enzyme unique to leucine biosynthesis, is feedback inhibited by excess levels of leucine
(Hagelstein and Schultz, 1993; Singh and Shaner, 1995). BCAT enzymes under control of
feedback regulation in plants have not been observed. BCATs may, however, be involved in
regulation of BCAA concentrations by their substrate specificities and expression patterns, as
will be discussed in chapter 2. The feedback mechanisms serve to regulate the levels of BCAAs
in cells, and they may also control the amount of branched-chain volatiles produced in tomato
fruit. This potential regulation will be discussed along with the results in chapter 3.
Branched-Chain Aminotransferases in Plants
BCAT enzymes reversibly catalyze the last reaction in BCAA synthesis and the first
reaction in BCAA catabolism, and therefore are very important targets in elucidating the
pathway to BCAA-derived volatiles. All BCAT enzymes, including those in plants, exhibit ping-
pong kinetics. In the first half reaction, the coenzyme pyridoxal 5’ phosphate (PLP)-bound form
of the enzyme reacts with the amino group of a BCAA. The enzyme then shifts to the
24
pyridoxamine 5’ phosphate (PMP)-bound form, releasing a BCKA. The PMP-bound enzyme
aminates a second α-keto acid, forms the PLP-bound enzyme and releases an amino acid, usually
glutamate (Hutson et al., 2005).
Much of the enzymatic and genetic analyses of the BCAA catabolic pathway in plants
has been done in Arabidopsis, the BCATs being the most characterized to date. In Arabidopsis
there are six BCAT genes that have been studied. Given the localization of AtBCAT1-GFP in
mitochondria, it is thought to be active primarily in BCAA catabolism. The chloroplastic
locations of AtBCATs 2, 3, and 5–GFP suggest they are active primarily in BCAA synthesis.
AtBCATs 4 and 6-GFP locations were not unambiguously determined, but signal peptide
prediction programs suggest cytoplasmic locations for both proteins (Diebold et al., 2002).
Complementation analysis in yeast strains deficient in BCAT activity were also performed
with AtBCATs, confirming the functions of AtBCATs 1, 2, 3, 5, and 6 proteins, but not
AtBCAT4 (Diebold et al., 2002). AtBCAT1 is the primary enzyme candidate for initiating
BCAA catabolism. However, AtBCAT5 has also been isolated from mitochondria fractions in
Arabidopsis cell suspension cultures, suggesting that it may be dually targeted and may have a
catabolic role under certain growth conditions. (Binder et al., 2007). AtBCAT1 catabolizes all
BCAAs to BCKAs in almost all tissues, and its affinity is greatest in the order of isoleucine >
leucine > valine. In the direction of BCAA synthesis, its highest affinity is for KIV (Schuster and
Binder, 2005). AtBCAT2 expression is only observed in flowers, and elevates under stress, while
AtBCAT6 expression is only seen in flowers and siliques. The expression of the other AtBCATs is
not as tissue specific (Liepman and Olsen, 2004). In spinach, only two BCAT genes have been
identified, one with a higher affinity towards KIV and one with a higher affinity towards KIC
and KMV (Binder et al., 2007).
25
Two other studies in Arabidopsis showed that both AtBCAT3 and AtBCAT4 participate in
methionine chain elongation and the production of aliphatic glucosinolates (Schuster et al., 2006;
Knill et al., 2008). In another example of plant BCAT function, a Nicotiana benthamiana
chloroplast-localized BCAT protein was implicated in transcriptional regulation of KNOX genes
that affect levels of gibberellins. The same NbBCAT can restore growth of a BCAT-deficient
yeast strain and is expressed highly in young leaves, suggesting it has a primary role in BCAA
synthesis (Gao et al., 2009). Kochevenko et al. (2010) present evidence that BCATs may also be
partly responsible for increased respiration in ripening tomato fruit. Together, these studies show
that BCATs have functions beyond primary amino acid metabolism, making it important to
understand the characteristics of each isoform in a species.
The BCAAs are considered cytotoxic in mammals when in excess and have been shown
to induce apoptosis in some species (Malatrasi et al., 2006), which suggests BCAT regulation is
very important to cellular health. Leucine toxicity has been reported in the bacteriums
Pseudomonas putida, E. coli, and Hydrogenomonas species. As one might expect, most of the
leucine catabolic pathway genes, including a BCAT, are induced in P. putida grown on leucine
media (Massey et al., 1974). BCAA toxicity has not been studied in detail in plants, but there
have been several studies on their control of BCAT expression. In a BCAA metabolism study in
barley, it was shown that HvBCAT1 expression is induced by drought stress, which the authors
suggest may be part of the cell’s detoxification mechanism to get rid of BCAAs in a stressful
environment (Malatrasi et al., 2006). It is not known for certain what benefits tomato fruit gain
from producing branched-chain volatiles, but it’s possible that they synthesize them as a means
of disposing of excess BCAAs.
26
Not much is known about how BCATs contribute to the regulation of BCAA metabolism
in plants and even less about their contribution to branched-chain volatiles. Nevertheless, they
make interesting candidates for study due to their being the only known BCAA pathway
enzymes with multiple subcellular locations and their position at the interface of BCAA
catabolism and anabolism. Given the importance of these enzymes in primary plant metabolism
and the possibilities of unique secondary metabolic functions, in addition to the immense
commercial value of tomato fruit, there is great justification in studying the BCAT family of
tomato. The experiments and results outlined in Chapter 2 will give insights to the characteristics
of the tomato BCAT family and examine their possible roles in the metabolism of tomato fruit
volatiles. Chapter 3 will describe the experiments and results leading to the elucidation of the
pathways synthesizing some of the branched-chain flavor volatiles. Chapter 4 will discuss the
implications of the experimental results described in this dissertation and how they contribute to
the field of plant metabolism.
27
Figure 1-1. BCAA catabolic and anabolic pathways. A. Ehrlich pathway of BCAA catabolism in microbes. 1) branched-chain aminotransferase, 2) α-keto-acid decarboxylase, and 3) aldehyde dehydrogenase. B. Synthetic pathway of BCAAs in plants. 1) threonine deaminase, 2) acetolactate synthase, 3) acetolactate isomeroreductase, 4) dihydroxy-acid dehydratase, 5) branched-chain aminotransferase, 6) 2-isopropylmalate synthase, 7) isopropylmalate isomerase, 8) isopropylmalate dehydrogenase.
28
Figure 1-2. Reported pathway of BCAA catabolism in plants leading to TCA cycle
intermediates. Pathway is adapted from the KEGG database. 1) BCAT, 2) BCKDH complex, 3) Isovaleryl-CoA dehydrogenase, 4) 3-methylcrotonyl-CoA carboxylases, 5) Methylglutaconyl-CoA hydratase, 6) Hydroxymethylglutaryl-CoA lyase, 7) Isobutyryl-CoA dehydrogenase, 8) Enoyl-CoA hydratase, 9) 3-hydroxyisobutyryl-CoA hydrolase, 10) 3-hydroxyisobutyrate dehydrogenase, 11) Malonate-semialdehyde dehydrogenase (acetylating), 12) Methylmalonyl-CoA mutase, 13) Butyryl-CoA dehydrogenase, 14) 3-hydroxyacyl-CoA dehydrogenase, 15) Acetyl-CoA acyltransferase.
29
/
Figure 1-3. Schematic of S. lycopersicum and S. pennellii introgression line population and corresponding branched-chain volatile loci. S. lycopersicum chromosomes are represented by long bars and introgressed genomic fragments of S. pennellii by small bars. Introgression fragments containing loci for branched-chain volatile phenotypes are designated by letters. The letters indicate which volatile phenotypes correspond to a particularintrogression line. Figure was adapted from Tieman et al. (2006).
30
Table 1-1. Flavor volatile compounds impacting the perception of ripe tomato fruit flavor.
Volatile
Conc.
(ppb)
Odor
units* Precursor
Odor
Characteristic
cis-3-Hexenal 12,000 3.7 lipid tomato/green ß-ionone 4 2.8 carotenoid fruity/floral Hexanal 3,100 2.8 lipid green/grassy ß-Damascenone 1 2.7 carotenoid fruity 1-Penten-3-one 520 2.7 lipid fruity floral/green 2-Methylbutanal 27 2.1 BCAAs/BCKAs musty/foot odor 3-Methylbutanal 27 2.1 BCAAs/BCKAs musty/foot odor trans-2-Hexenal 270 1.2 lipid green 2-Isobutylthiazole 36 1 BCAAs/BCKAs tomato vine 1-nitro-2-Phenylethane 17 0.9 phenylalanine musty, earthy trans-2-Heptenal 60 0.7 lipid green Phenylacetaldehyde 15 0.6 phenylalanine floral/alcohol 6-Methyl-5-hepten-2-one 130 0.4 carotenoid fruity, floral cis-3-Hexenol 150 0.3 lipid green 2-Phenylethanol 1,900 0.3 phenylalanine nutty 3-Methylbutanol 380 0.2 BCAAs/BCKAs earthy, musty 2-Methylbutanol 100 0.2 BCAAs/BCKAs earthy, musty Methyl salicylate 48 0.08 phenylalanine wintergreen *Odor unit is defined as the log of the concentration of a volatile divided by its odor threshold Data are from Buttery and Ling (1993).
31
CHAPTER 2 CHARACTERIZATION OF TOMATO BCATS
Cloning of SlBCAT cDNAs
In order to better understand the dynamics of branched-chain amino acid metabolism, six
unique tomato sequences potentially encoding BCAT enzymes were identified by searching the
Sol Genomics Network tomato expressed sequence tag (EST) database
(http://solgenomics.net/index.pl) (Mueller et al., 2005) for unigene sequences similar to those of
BCATs from other species. Unigenes are collections of transcripts that appear to originate from
the same locus. Full-length cDNAs from each unigene were cloned and sequenced, and proteins
were deduced from their open reading frames. The unigene SGN-U569828 (SlBCAT1, 45
members) has the most ESTs of all putative SlBCATs, while the unigene SGN-U569952
(SlBCAT3, 27 members) has the second highest, both far surpassing the numbers of ESTs of the
other putative SlBCATs. Phylogenetic analysis of all putative SlBCATs and comparisons with
Arabidopsis BCATs (Diebold et al., 2002) revealed that SlBCAT1 is most similar to the AtBCAT2
and AtBCAT1 genes from Arabidopsis, respectively. The unigene SGN-U569830 (SlBCAT2,
seven members) is most similar to AtBCAT3. SlBCAT3 and the unigene SGN-U569953
(SlBCAT4, seven members) are highly similar to each other and most similar to AtBCAT5. The
unigenes SGN-U569831 (SlBCAT5, five members) and SGN-U569829 (SlBCAT6, two
members) are most similar to AtBCAT2 and most similar to each other within the putative
SlBCATs (Figures 2-1 and 2-2).
A seventh unigene, SGN-U566152, a putative branched-chain aminotransferase-like
protein, was identified and considered to be a possible member of the tomato BCAT family.
However, after comparing the phylogeny of this gene with the rest of the family, it was decided
that this gene is most likely not a BCAT based on its 29% homology with its closest SlBCAT
32
relative. It is possible that this gene encodes an aminotransferase using amino acids other than
the BCAAs as its substrate.
Another unigene, SGN-U565681, annotated as a branched-chain aminotransferase-like
protein was identified. This gene had only 25% homology with its closest SlBCAT relative, and
was also not considered a BCAT.
The identification of these six BCAT cDNAs gave a preliminary view of the diversity and
depth of this enzyme family in tomatoes. This result parallels the six confirmed BCAT genes in
Arabidopsis, indicating that the gene families in both plants may have similar characteristics and
division of function.
Expression Analysis of SlBCATs
To gain a better understanding of the different roles of each SlBCAT family member,
expression analysis was performed by quantitative RT-PCR on all six SlBCAT cDNAs. As stated
in the introduction, the Arabidopsis BCATs all have unique expression patterns which in some
cases seem to be related to specific functions of those genes. We expected to find similar patterns
with SlBCAT expression in different tissues. The expression patterns observed in most cases
validated the predictions made from the numbers and tissue locations of available ESTs in the
SGN database.
The tissues tested were partially expanded young leaves, inflorescences at one day after
anthesis, and mature green, breaker, turning, and red ripe fruit stages (Figure 2-3). Mature green
is the stage at which the fruit is fully expanded but has not yet started developing color. Breaker
is the stage at which fruits have started to develop external color, but covering no more than 10%
of the fruit. Turning is the stage at which fruits have from 20% to 40% external color coverage.
Red ripe fruit must be at least 95% red in color.
33
Expression of SlBCAT1 is higher in both stages of ripening fruit and in red fruit than all
other SlBCATs, but is very low in leaves, inflorescences, and undetectable in green fruit.
SlBCAT2 is expressed in all tissues at similar levels except much more highly in inflorescences.
SlBCAT3 is expressed nearly equally in all tissues but is most highly expressed in leaves.
Expression of SlBCAT4 is highest in inflorescences, but relatively low in all other tissues
compared to the other SlBCATs. No SlBCAT5 and SlBCAT6 transcripts were detected in this
experiment in the tissues tested. ESTs for these two cDNAs were isolated only from callus
tissue, suggesting they may only be expressed under specific environmental conditions.
The result of these expression analyses helps us define the specificity of the tomato BCAT
family. They show that some isoforms may have functions tailored to particular plant tissues. A
BCAT expressed very highly in ripening fruit, for example, may function in respiration and
catabolism due to the senescent nature of that tissue. A BCAT expressed highly in flowers,
however, may function primarily in BCAA synthesis, given the high demand for primary
metabolites in reproductive tissues.
Subcellular Localization of SlBCATs
Plant organelles have very specific functions that can change during cell and organ
development, as is the case with ripening tomato fruit. Therefore, the subcellular locations of
metabolic enzymes can be important in predicting function. All six SlBCAT cDNAs were cloned
with a C-terminal E-GFP gene fusion, expressed in N. benthamiana leaf protoplasts and
analyzed with confocal microscopy (Figure 2-4).
SlBCAT3 and SlBCAT4 were localized to plastids, consistent with localization algorithm
software and homology with the chloroplast-localized AtBCAT proteins. SlBCAT1 and
SlBCAT2 were localized to mitochondria, consistent with localization prediction software
outputs. Mitochondrial localization was confirmed by overlap of the E-GFP signal with
34
Mitotracker Orange stain. SlBCAT5 appeared to be cytosolic, consistent with the lack of an N-
terminal targeting signal. SlBCAT6 appeared to be localized to the vacuole, based on the E-GFP
signal filling the majority of the space inside the protoplasts, typical of vacuoles in leaf cells. The
subcellular locations of SlBCATs 1 and 4 are consistent with results of Kochevenko et al.
(2010).
These results show that, as in Arabidopsis, SlBCAT enzymes are diverse in their
subcellular locations. The mitochondrial and chloroplast locations of the four expressed
SlBCATs suggest that each may have specific functions in either BCAA synthesis or catabolism,
respectively. Similarly, the cytoplasmic and vacuolar locations of SlBCAT5 and SlBCAT6,
respectively, suggest unique metabolic functions for these two enzymes.
Verification of SlBCAT function by Bacterial Complementation
In order to demonstrate that the isolated SlBCAT cDNA products function as BCATs in
vivo, a complementation assay was performed in E. coli. The E. coli genome contains one BCAT
gene, ilvE. A different gene, tyrB, which encodes an aromatic amino acid aminotransferase
(EC2.6.1.57), can restore BCAT activity in ΔilvE cells (Gelfand and Steinberg, 1977; Powell and
Morrison, 1978; Vartak et al., 1991). The knockout strains for each of these two genes, neither of
which is lethal on minimal medium, were obtained from the Keio collection (Baba et al., 2006).
The double knockout ΔilvE/ΔtyrB was constructed to ensure that cells had no BCAT activity and
were complete branched-chain amino acid auxotrophs. This double knockout did not grow when
streaked on minimal medium.
SlBCAT1, SlBCAT2, SlBCAT3, and SlBCAT4 cDNAs were cloned into the E. coli
expression vector pBAD24 under control of the Pbad promoter (Guzman et al., 1995) and
transformed into the ΔilvE/ΔtyrB double knockout strain. Expression of proteins in the knockout
was confirmed by a protein gel blot of cell extracts, which confirmed that protein concentrations
35
did not vary greatly (data not shown). SlBCAT3 and SlBCAT4 expression restored growth in the
ΔilvE/ΔtyrB knockout in minimal medium lacking branched-chain amino acids, with
approximately half the optical density of wild-type cells when measured after 10 hours of growth
(Table 2-1) (Figure 2-5). SlBCAT1 and SlBCAT2 were able to complement growth in the double
mutant strain, but cultures were much lower in density than those expressing SlBCATs 3 and 4
(Table 2-1). Cells expressing these two cDNAs did not show visible growth on plates after two
days. Restoration of growth by the chloroplastic SlBCAT3 and SlBCAT4 supports the hypothesis
that they are the major branched-chain amino acid synthesizing enzymes in tomato. The less
effective complementation by SlBCAT1 and SlBCAT2 is consistent with mitochondrial
localization, suggesting that these genes are primarily active in BCAA catabolism.
The results of these complementation experiments add further evidence to the potential
activities of these enzymes and support the conclusions formed from the localization
experiments. The mitochondrial locations of SlBCAT1 and 2 suggest they are involved in BCAA
catabolism but not synthesis, which is supported by their poor ability to restore growth in BCAA
auxotrophic cells. The chloroplastic locations of SlBCAT3 and 4 suggest that these enzymes are
primarily involved in BCAA synthesis, which they show a clear ability to do in their restoration
of BCAA auxotrophic cells.
BCAT Enzyme Assays
In order to determine the kinetic characteristics of each SlBCAT, the proteins were
expressed in E. coli cells and purified. Enzyme assays were performed with each recombinant
SlBCAT in both the forward (amino acid forming) and reverse (amino acid degrading)
directions. In the forward direction, product formation was quantified indirectly by measuring
decreasing absorbance upon NADH oxidation in a reaction coupled to glutamate dehydrogenase.
36
In the reverse reaction, product formation was quantified by measuring the absorbance of the
ketone-hydrazone species formed by reaction of the BCKA with 2,4-dinitrophenyl hydrazine.
Table 2-2 shows the Km, Vmax, Kcat and Kcat/Km values determined for each SlBCAT
enzyme with all six branched-chain substrates. All SlBCATs had affinities for all six branched-
chain substrates. SlBCAT3 has a higher affinity for the BCKAs than BCAAs. SlBCAT4 exhibits
slightlyly higher affinities for KMV and isoleucine than other substrates. Like SlBCAT3, its
most closely related isoform, SlBCAT4 has an overall higher affinity for BCKAs than BCAAs,
consistent with the role of chloroplastic BCATs in BCAA synthesis in tomato.
SlBCAT1 has relatively low affinities for BCKAs and much higher affinities for leucine
and isoleucine in the reverse direction. This preference for BCAA substrates, together with its
mitochondrial location, supports a primarily catabolic function for SlBCAT1. SlBCAT2, also
located within mitochondria, has a much higher affinity for the BCAAs than their corresponding
BCKAs, similar to SlBCAT1, suggesting that it also principally functions in BCAA catabolism.
SlBCAT5demonstrates relatively high affinities for BCAAs and BCKAs, with the highest
for KMV and KIC. SlBCAT6 also demonstrates high affinities for BCAAs and BCKAs, but with
a much higher affinity for KIV, which suggests a very specific function for this enzyme in the
valine metabolic pathway.
These kinetic data provide vital information about the functions of particular SlBCATs.
The kinetic evidence suggests that SlBCAT1 and 2 are primarily catabolic in function, given that
they have very low affinities for BCKAs and much higher activity for BCAAs. These data are
consistent with SlBCAT1 expression levels spiking in ripening fruit, a senescent organ which
likely has less demand for protein synthesis than for amino acid catabolism. The kinetic evidence
for SlBCAT3 and 4 is consistent with their role in BCAA synthesis, but also shows they are
37
capable of BCAA catabolism as well. They may have specific enzymatic functions dependent on
environmental conditions, tissue types, or developmental stages. The preference of SlBCAT3 for
BCKAs is consistent with its expression throughout the plant, since BCAAs are needed in
abundance in all tissues for protein synthesis. Since both SlBCATs 2 and 4 are both expressed
highly in flowers, the kinetic data suggests there is a likely a high demand for both BCAA
anabolism and catabolism in reproductive tissues. The specific functions of SlBCAT5 and 6
remain ambiguous even with their kinetic data, although the unusually high specificity of
SlBCAT6 on KIV may indicate that this enzyme has a very specific role in the plant. This is the
only case of a tomato BCAT having a much higher specificity for one substrate over all others.
Analysis of SlBCAT1 and SlBCAT3 Transgenic Fruit
In order to determine if an increase in either a single synthetic or catabolic SlBCAT could
alter fruit metabolism, over-expression constructs of two genes, SlBCAT1 and SlBCAT3, under
control of the FMV constitutive promoter, were created and stably transformed into M82 tomato
plants. These two cDNAs were chosen due to their expression in ripening fruits and because they
represent a primarily catabolic and primarily anabolic enzyme, respectively, given their
localization, kinetic, and growth complementation data.
Plants from the T1 generation were grown in the field and ripe fruits were
analyzed for amino acid content and flavor volatiles in comparison with M82 controls. Three
independent lines from each construct were chosen for this analysis, and RNA from each line
was analyzed by qRT-PCR to show the degree of over-expression. Although there were
significant increases in expression of the transgenes (Figure 2-6), there were no consistent
differences from the control in amino acid content throughout all of the SlBCAT1-OE lines
(Table 2-3). The only significant and consistent change was an increase in isoleucine in all of the
SlBCAT3-OE lines. Some of the lines showed significant increases in emissions of several of the
38
branched-chain volatile (Table 2-4). All lines of SlBCAT1 showed significantly increased
emissions of 3-methylbutanal, while all lines of SlBCAT3 showed significantly increased
emissions of 3-methylbuanal, 2-methylbutanal, and 2-methylbutanol. Though these results were
significant, higher elevation of branched-chain volatiles would have been expected if BCATs are
solely responsible for initiating branched-chain volatile synthesis. In comparison, when the
tomato aromatic amino acid decarboxylase was overexpressed in tomato fruit, 10-fold increases
in 2-phenylethanol and 2-phenylacetaldehyde emissions were observed, demonstrating that
altered expression of a single amino acid metabolic gene can have profound effects on volatile
biosynthesis (Tieman et al., 2006b).
It can be concluded that increasing expression of an individual SlBCAT does not greatly
alter amino acid metabolism in tomato fruit, likely due to a tight enzymatic regulation of the
BCAA pathways and/or redundancy of individual SlBCATs in fruit. Interestingly, the study by
Kochevenko et al. (2010) shows that tomato plants constitutively expressing an SlBCAT1
antisense construct have elevated levels of branched-chain amino acids. Their finding adds
support to the hypothesis that SlBCAT1 is primarily catabolic in function.
Conclusion of Results
It is clear from the results presented in this chapter that the BCATs of tomato are diverse
and probably share many different functions among their multiple isoforms. The results show
that there are probably two SlBCATs dedicated primarily to BCAA catabolism and two dedicated
primarily to BCAA anabolism. The subcellular locations of the SlBCATs demonstrate that
anabolic and catabolic BCAA pathways are spatially separated in plant cells. Expression analysis
of all SlBCATs shows that there are isoforms that probably serve as primary metabolic enzymes
throughout the plant, while others have more specific functions. The lack of detectable
expression of the remaining two SlBCATs suggests they are not involved in primary plant
39
metabolism, but likely have more specialized, unidentified secondary functions. The results of
the transgenic experiments indicate that the over-production of either an anabolic or a catabolic
SlBCAT protein is not sufficient to greatly alter BCAA or branched-chain volatile metabolism in
tomato fruit. BCAT protein levels in the overexpression transgenic fruits were not validated.
Therefore, it is possible the proteins were degraded and would not be truly represented by
transcript levels, which could possibly account for the low levels of volatiles.
40
SlBCAT5 1 IDWDNLGFQLMQTDYMYVTK-CSDDGIFRQGQLNRYGNIQLSPSAGVLNYGQGLFEGTKA
SlBCAT6 1 IDWDNLGFQLIQTDYMYMTK-CSDDGIFRKGQLNRYGNINLSPSAGVLNYGQGLFEGTKA
SlBCAT2 1 VDWDKLGFGFTPTDYMYITKSCDVAGNFKQGQLNGYDNIQLSPSAGVLNYGQGLFEGTKA
SlBCAT1 1 FDWDNLGFKLIQTDYMFMTK-SSQNGNFEKGKLNPYGNIELSPSAGVLNYGQGLIEGTKA
SlBCAT4 1 IDWDNIGFAVMPTDYMYSMK-CSQDGNFSKGELQRFGNIELSPASGILNYGQGLFEGLKA
SlBCAT3 1 IDWDNLGFGFMPTDYMYSMK-CSQGENFSKGELQRFGNIELSPSAGILNYGQGLFEGLKA
SlBCAT5 60 YRREDGRIFLFRPDQNAMRMQIGAERMCMPCPSTDQFVEAVKQTAIANKRWIPPFGKGAL
SlBCAT6 60 YRRDDGRVFLFRPEQNAIRMQIGAERMCMPSPTTDQFVDAVKQTALANKRWIPPSGKGSL
SlBCAT2 61 YRQDNGGLSLFRPRENAIRMQIGAERMCMPYPSTDQFVDAVKQTALANKRWIPPPGKGSL
SlBCAT1 60 YRVDDGRIFLFRPQESGIRMQIGAKRMCMPSPSIQQFVDAVKLTTIANKRWIPPAGKGSL
SlBCAT4 60 YKRHDGNILLFRPEENALRMKMGAERICMPSPSVEQFVEAVKATVLANERWIPPPGKGSL
SlBCAT3 60 YRKHDGNILLFRPEENATRLKMGAERMCMPSPSVEQFVEAVKATVLANERWIPPPGKGSL
SlBCAT5 120 YIRPLLIGSGPIFGLAPAPEYTFLVYACPVGYYFKQGTAPLNLYVEEDVHRASRGGAGGV
SlBCAT6 120 YIRPLLIGTGPILGLAPAPEYTFLVYACPVGNYFKQGTAPLNLYVEEDVHRASRGGAGGV
SlBCAT2 121 YIRPLLYGSGSILGLAPAPEYTFLVYACPVGNYFKEGTAPLNLYVDEEFHRASRGGAGGV
SlBCAT1 120 YIRPLLIGNGPILGIAPAPEYTFIVYACPVGNYLRNGTQPLTLYVEEEHHRASQGGAGGV
SlBCAT4 120 YIRPLLMGSGAILGVAPAPEYTFLIYVSPVGNYFKEGMAPINLLIETEMHRATPGGTGGV
SlBCAT3 120 YIRPLLMGSGAVLGLAPAPEYTFLIYVSPVGNYFKEGLAPINLVVETEMHRATPGGTGGV
SlBCAT5 180 KSITNYAPVLKAMKNAKANGYSDVLYLDAVNKKYIEEVSSCNIFLVKGNVLSTPIAKGTI
SlBCAT6 180 KSITNYAPVLKAMKNAKANGYSDVLYLDAVNKKYIEEVSSCNIFLVKGNVLSTPIAKGTI
SlBCAT2 181 KSITNYAPVLRAIRNARERGFSDVLYLDSVNKKYIEEVSSCNIFLVKGKVISTPIACGTI
SlBCAT1 180 KSITNYAPVIKAIQEAKDRGYSDVLYLDSVNKKYIEEVSAANIFLVKGKNISTPIASGTI
SlBCAT4 180 KTIGNYAAVLKAQSAAKAKGYSDVLYLDSVNNRYLEEVSSCNVFIVKGNLIATPAIKGTI
SlBCAT3 180 KTIGNYAAVLKAQSAAKAKGYSDVLYLDCVQKKYLEEVSSCNVFIVKGNLIVTPAIKGTI
SlBCAT5 240 LEGITRKSIMDIAHDLGYTVEERLIEADELFTADEVFCTGTAVGVAPVGSITYKGQRIEY
SlBCAT6 240 LEGITRKSIMDIAHDLGYTVEERLIEADELISADEVFCTGTAVGVAPVGSITYKGQRIDY
SlBCAT2 241 LEGVTRKSIMEIAIDLGYQVEERLIEADELISADEVFCTGTAVGVAPVGSITYKGQRIEY
SlBCAT1 240 LEGVTRKSIIDIAHDLGYKVEERLIEADELFSADEVFCTGTALGVAPVGSITYKNKRINY
SlBCAT4 240 FPGITRKSIIDVALSQGFQVEERQVSVDELLDADEVFCTGTAVVVSPVGSITHLGKRVSY
SlBCAT3 240 LPGITRKSIIDVAISQGFEVEERQVSVDELLDADEVFCTGTAVVVSPVGSITHQGRRVTY
SlBCAT5 300 KIS-SDLSCKQFYSRLVGIQRGVIKDERNWIVEIE
SlBCAT6 300 KIS-SDLSCKQXYSRLVGIQRGVIKDERDWIVEIE
SlBCAT2 301 KIR-SELVCKKLYSTLVGIQKRHIEDKRDWIVDIE
SlBCAT1 300 KVS-SDLISEQLNSRLVAIQKGIIEDKRGWIIEIK
SlBCAT4 300 GSDGVGRVSKQLYSTLTSLQMGLATDNMNWTVELK
SlBCAT3 300 GNDGVGLVSQQLYSALTSLQMGLSEDKMGWIVELK
Figure 2-1. Evolutionary relationships of mature SlBCAT proteins. Mature peptides were predicted by omitting highly divergent N-terminal residues thought to be transit peptides. A, Alignment of amino acid sequences using ClustalW (Larkin et al., 2007). B, The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 1.21366717 is shown. The percentage of replicate trees in which the proteins clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. There were a total of 332 amino acids in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).
SlBCAT5
SlBCAT6
SlBCAT1
SlBCAT2
SlBCAT4
SlBCAT3100
100
83
0.1
A
B
41
SlBCAT5 1 IDWDNLGFQLMQTDYMYVTK-CSDDGIFRQGQLNRYGNIQLSPSAGVLNYGQGLFEGTKA
SlBCAT6 1 IDWDNLGFQLIQTDYMYMTK-CSDDGIFRKGQLNRYGNINLSPSAGVLNYGQGLFEGTKA
SlBCAT2 1 VDWDKLGFGFTPTDYMYITKSCDVAGNFKQGQLNGYDNIQLSPSAGVLNYGQGLFEGTKA
SlBCAT1 1 FDWDNLGFKLIQTDYMFMTK-SSQNGNFEKGKLNPYGNIELSPSAGVLNYGQGLIEGTKA
AtBCAT2 1 LDWDNLGFGLNPADYMYVMK-CSKDGEFTQGELSPYGNIQLSPSAGVLNYGQAIYEGTKA
AtBCAT3 1 IDWDTVGFGLKPADYMYVMK-CNIDGEFSKGELQRFGNIEISPSAGVLNYGQGLFEGLKA
AtBCAT5 1 IDWDKIDFGLKPTDYMYAMK-CSRDGEFSQGQLQPFGNIDINPAAGVLNYGQGLFEGLKA
SlBCAT4 1 IDWDNIGFAVMPTDYMYSMK-CSQDGNFSKGELQRFGNIELSPASGILNYGQGLFEGLKA
SlBCAT3 1 IDWDNLGFGFMPTDYMYSMK-CSQGENFSKGELQRFGNIELSPSAGILNYGQGLFEGLKA
AtBCAT1 1 VDWDNLGFSLVRTDFMFATK-SCRDGNFEQGYLSRYGNIELNPAAGILNYGQGLIEGMKA
AtBCAT6 1 VKWEELGFALTPIDYMYVAK-CRQGESFTQGKIVPYGDISISPCSPILNYGQGLFEGLKA
AtBCAT7 1 VKWDELGFALVPTDYMYVAK-CKQGESFSTGEIVPYGDISISPCAGILNYGQGLFEGLKA
AtBCAT4 1 VKWEELAFKFVRTDYMYVAK-CNHGESFQEGKILPFADLQLNPCAAVLQYGQGLYEGLKA
SlBCAT5 60 YRREDGR-IFLFRPDQNAMRMQIGAERMCMPCPSTDQFVEAVKQTAIANKRWIPPFGKGA
SlBCAT6 60 YRRDDGR-VFLFRPEQNAIRMQIGAERMCMPSPTTDQFVDAVKQTALANKRWIPPSGKGS
SlBCAT2 61 YRQDNGG-LSLFRPRENAIRMQIGAERMCMPYPSTDQFVDAVKQTALANKRWIPPPGKGS
SlBCAT1 60 YRVDDGR-IFLFRPQESGIRMQIGAKRMCMPSPSIQQFVDAVKLTTIANKRWIPPAGKGS
AtBCAT2 60 YRKENGK-LLLFRPDHNAIRMKLGAERMLMPSPSVDQFVNAVKQTALANKRWVPPAGKGT
AtBCAT3 60 YRKKDGNNILLFRPEENAKRMRNGAERMCMPAPTVEQFVEAVTETVLANKRWVPPPGKGS
AtBCAT5 60 YRKQDGN-ILLFRPEENAIRMRNGAERMCMPSPTVEQFVEAVKTTVLANKRWIPPPGKGS
SlBCAT4 60 YKRHDGN-ILLFRPEENALRMKMGAERICMPSPSVEQFVEAVKATVLANERWIPPPGKGS
SlBCAT3 60 YRKHDGN-ILLFRPEENATRLKMGAERMCMPSPSVEQFVEAVKATVLANERWIPPPGKGS
AtBCAT1 60 YRGEDGR-VLLFRPELNAMRMKIGAERMCMHSPSVHQFIEGVKQTVLANRRWVPPPGKGS
AtBCAT6 60 YRTEDDR-IRIFRPDQNALRMQTGAERLCMTPPTLEQFVEAVKQTVLANKKWVPPPGKGT
AtBCAT7 60 YRTEDGR-ITLFRPDQNAIRMQTGADRLCMTPPSPEQFVEAVKQTVLANNKWVPPPGKGA
AtBCAT4 60 YRTEDGR-ILLFRPDQNGLRLQAGADRLYMPYPSVDQFVSAIKQVALANKKWIPPPGKGT
SlBCAT5 119 LYIRPLLIGSGPIFGLAPAPEYTFLVYACPVGYYFKQGTAPLNLYVEEDVHRASRGGAGG
SlBCAT6 119 LYIRPLLIGTGPILGLAPAPEYTFLVYACPVGNYFKQGTAPLNLYVEEDVHRASRGGAGG
SlBCAT2 120 LYIRPLLYGSGSILGLAPAPEYTFLVYACPVGNYFKEGTAPLNLYVDEEFHRASRGGAGG
SlBCAT1 119 LYIRPLLIGNGPILGIAPAPEYTFIVYACPVGNYLRNGTQPLTLYVEEEHHRASQGGAGG
AtBCAT2 119 LYIRPLLMGSGPILGLGPAPEYTFIVYASPVGNYFKEGMAALNLYVEEEYVRAAPGGAGG
AtBCAT3 120 LYVRPLLMGTGAVLGLAPAPEYTFIIYVSPVGNYFKEGVAPINLIVENEFHRATPGGTGG
AtBCAT5 119 LYIRPLLMGTGAVLGLAPAPEYTFLIFVSPVGNYFKEGVAPINLIVETEFHRATPGGTGG
SlBCAT4 119 LYIRPLLMGSGAILGVAPAPEYTFLIYVSPVGNYFKEGMAPINLLIETEMHRATPGGTGG
SlBCAT3 119 LYIRPLLMGSGAVLGLAPAPEYTFLIYVSPVGNYFKEGLAPINLVVETEMHRATPGGTGG
AtBCAT1 119 LYLRPLLFGSGASLGVAAASEYTFLVFGSPVQNYFKEGTAALNLYVEEVIPRAYLGGTGG
AtBCAT6 119 LYIRPLLLGSGATLGVAPAPEYTFLIYASPVGDYHKV-SSGLNLKVDHKYHRAHSGGTGG
AtBCAT7 119 LYIRPLLIGTGAVLGVASAPEYTFLIYTSPVGNYHKA-SSGLNLKVDHNHRRAHFGGTGG
AtBCAT4 119 LYIRPILFGSGPILGSFPIPETTFTAFACPVGRYHKD-NSGLNLKIEDQFRRAFPSGTGG
SlBCAT5 179 VKSITNYAPVLKAMKNAKANGYSDVLYLDAVNKKYIEEVSSCNIFLVKGNVLSTPIAKGT
SlBCAT6 179 VKSITNYAPVLKAMKNAKANGYSDVLYLDAVNKKYIEEVSSCNIFLVKGNVLSTPIAKGT
SlBCAT2 180 VKSITNYAPVLRAIRNARERGFSDVLYLDSVNKKYIEEVSSCNIFLVKGKVISTPIACGT
SlBCAT1 179 VKSITNYAPVIKAIQEAKDRGYSDVLYLDSVNKKYIEEVSAANIFLVKGKNISTPIASGT
AtBCAT2 179 VKSITNYAPVLKALSRAKSRGFSDVLYLDSVKKKYLEEASSCNVFVVKGRTISTPATNGT
AtBCAT3 180 VKTIGNYAAVLKAQSIAKAKGYSDVLYLDCIYKRYLEEVSSCNIFIVKDNVISTPEIKGT
AtBCAT5 179 VKTIGNYAAVLKAQSIAKAKGYSDVLYLDCLHKRYLEEVSSCNIFIVKDNVISTPEIKGT
SlBCAT4 179 VKTIGNYAAVLKAQSAAKAKGYSDVLYLDSVNNRYLEEVSSCNVFIVKGNLIATPAIKGT
SlBCAT3 179 VKTIGNYAAVLKAQSAAKAKGYSDVLYLDCVQKKYLEEVSSCNVFIVKGNLIVTPAIKGT
AtBCAT1 179 VKAISNYGPVLEVMRRAKSRGFSDVLYLDADTGKNIEEVSAANIFLVKGNTIVTPATSGT
AtBCAT6 178 VKSCTNYSPVVKSLLEAKSAGFSDVLFLDAATGRNIEELTACNIFIVKGNIVSTPPTSGT
AtBCAT7 178 VKSCTNYSPVVKSLIEAKSSGFSDVLFLDAATGKNIEEVSTCNIFILKGNIVSTPPTSGT
AtBCAT4 178 VKSITNYCPVWIPLAEAKKQGFSDILFLDAATGKNIEELFAANVFMLKGNVVSTPTIAGT
SlBCAT5 239 ILEGITRKSIMDIAHDLGYTVEERLIEADELFTADEVFCTGTAVGVAPVGSITYKGQRIE
SlBCAT6 239 ILEGITRKSIMDIAHDLGYTVEERLIEADELISADEVFCTGTAVGVAPVGSITYKGQRID
SlBCAT2 240 ILEGVTRKSIMEIAIDLGYQVEERLIEADELISADEVFCTGTAVGVAPVGSITYKGQRIE
SlBCAT1 239 ILEGVTRKSIIDIAHDLGYKVEERLIEADELFSADEVFCTGTALGVAPVGSITYKNKRIN
AtBCAT2 239 ILEGITRKSVMEIASDQGYQVVEKAVHVDEVMDADEVFCTGTAVVVAPVGTITYQEKRVE
AtBCAT3 240 ILPGITRKSMIDVARTQGFQVEERNVTVDELLEADEVFCTGTAVVVSPVGSVTYKGKRVS
AtBCAT5 239 ILPGITRKSIIEVARSQGFKVEERNVTVDELVEADEVFCTGTAVVLSPVGSITYKSQRFS
SlBCAT4 239 IFPGITRKSIIDVALSQGFQVEERQVSVDELLDADEVFCTGTAVVVSPVGSITHLGKRVS
SlBCAT3 239 ILPGITRKSIIDVAISQGFEVEERQVSVDELLDADEVFCTGTAVVVSPVGSITHQGRRVT
AtBCAT1 239 ILGGITRKSIIEIALDLGYKVEERSVPVEELKEAEEVFCTGTAAGVASVGSITFKNTRTE
AtBCAT6 238 ILPGVTRKSISELAHDIGYQVEERDVSVDELLEAEEVFCTGTAVVVKAVETVTFHDKKVK
AtBCAT7 238 ILPGITRKSICELARDIGYEVQERDLSVDELLEAEEVFCTGTAVVIKAVETVTFHDKRVK
AtBCAT4 238 ILPGVTRNCVMELCRDFGYQVEERTIPLVDFLDADEAFCTGTASIVTSIASVTFKDKKTG
SlBCAT5 299 YKISSD-LSCKQFYSRLVGIQRGVIKDERNWIVEIE
SlBCAT6 299 YKISSD-LSCKQXYSRLVGIQRGVIKDERDWIVEIE
SlBCAT2 300 YKIRSE-LVCKKLYSTLVGIQKRHIEDKRDWIVDIE
SlBCAT1 299 YKVSSD-LISEQLNSRLVAIQKGIIEDKRGWIIEIK
AtBCAT2 299 YKTGDE-SVCQKLRSVLVGIQTGLIEDNKGWVTDIN
AtBCAT3 300 YGEGTFGTVSKQLYTVLTSLQMGLIEDNMKWTVNLS
AtBCAT5 299 YGEDGFGTVSKQLYTSLTSLQMGLSEDNMNWTVQLS
SlBCAT4 299 YGSDGVGRVSKQLYSTLTSLQMGLATDNMNWTVELK
SlBCAT3 299 YGNDGVGLVSQQLYSALTSLQMGLSEDKMGWIVELK
AtBCAT1 299 YKVGDG-IVTQQLRSILVGIQTGSIQDTKDWVLQIA
AtBCAT6 298 YRTGEA-ALSTKLHSMLTNIQMGVVEDKKGWMVDID
AtBCAT7 298 YRTGEE-AFSTKLHLILTNIQMGVVEDKKGWMMEID
AtBCAT4 298 FKTGEE-TLAAKLYETLSDIQTGRVEDTKGWTVEID
Figure 2-2. Evolutionary relationships of mature SlBCAT and AtBCAT proteins. Mature peptides were predicted by omitting highly divergent N-terminal residues thought to be transit peptides. A, Alignment of amino acid sequences using ClustalW (Larkin et al., 2007). B, The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 2.55921922 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. There were a total of 331 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).
SlBCAT5
SlBCAT6
SlBCAT1
AtBCAT2
AtBCAT1
AtBCAT5
SlBCAT4
SlBCAT3
AtBCAT4
AtBCAT6
AtBCAT7
AtBCAT3
SlBCAT2100
100
100
100
100
100
100
100
99
39
0.1
A B
42
Figure 2-3. Quantification of SlBCATs RNA in different tissue types. A, Comparison of
expression of genes relative to each other in each tissue. B, Comparison of expression in tissues within each gene. Analysis was performed on three biological and three technical replicates of tissues for each sample. Values represent percentage of total mRNA per sample ± SD, calculated from a standard curve for each gene. Note differences in y-axes. Expression of SlBCAT5 and SlBCAT6 was below the limit of detection. L = young leaves, F = inflorescences 1dpa, G = mature green fruit, B = breaker fruit, T = turning fruit, R = red ripe fruit.
A
43
Figure 2-3. Continued.
B
44
Figure 2-4. Subcellular localization of SlBCATs. Each gene was fused to E-GFP at the C-
terminal and expressed in N. benthamiana leaf protoplasts. The left column shows GFP fluorescence, the middle column shows marker fluorescence, and the right column shows merging of GFP and marker. Chlorophyll autofluorescence was used to show presence of chloroplasts for SlBCAT-3, 4, 5, and 6. Mitotracker Orange dye was used to show mitochondria for SlBCAT-1 and 2. Scale bars are each 10µm.
45
Figure 2-5. Growth complementation of E. coli ΔilvE/ΔtyrB mutant cells expressing SlBCAT3
and 4. Cells are streaked on media lacking amino acids and SlBCAT cDNAs are expressed under control of the Pbad promoter in mutant cells. The top image shows growth restoration by SlBCAT4, while the bottom image shows restoration by SlBCAT3. Cells expressing the other SlBCATs are not shown, as they did not show visible growth on plates.
Table 2-1. Measurement of E. coli cell culture growth rate Strain BW25113 ΔilvE/ΔtyrB
ΔilvE/ΔtyrB
::SlBCAT1
ΔilvE/ΔtyrB
::SlBCAT2
ΔilvE/ΔtyrB
::SlBCAT3
ΔilvE/ΔtyrB
::SlBCAT4
OD600 1.44 ± 0.021 0.108± 0.004 0.214 ± 0.011 0.197 ± 0.008 0.670 ± 0.010 0.433 ± 0.006
Cultures were grown for 10 h after which OD600 was measured. BW25113 and ΔilvE/ΔtyrB carried an empty pBAD24 vector. SlBCAT cDNAs are under control of the Pbad promoter in the pBAD24 vector (Guzman et al., 1995). Values are averages of two replicates ± SD.
46
Table 2-2. Kinetic parameters of SlBCATs.
Km
(mM) Vmax
(nkatal mg-1) Kcat
(s-1) Kcat/Km
(µM-1 s-1)
KIC SlBCAT1 7.09 ± 0.92 0.5 11.7 0.002
SlBCAT2 7.90 ± 0.80 3.5 84.8 0.011 SlBCAT3 0.35 ± 0.06 1.1 28.1 0.080 SlBCAT4 0.41 ± 0.02 1.4 35.0 0.085 SlBCAT5 0.34 ± 0.06 2.2 54.6 0.160 SlBCAT6 0.22 ± 0.02 1.2 28.7 0.130 KMV SlBCAT1 11.65 ± 1.89 0.7 16.3 0.001 SlBCAT2 12.40 ± 0.90 2.8 67.6 0.006 SlBCAT3 0.19 ± 0.02 1.0 23.6 0.120 SlBCAT4 0.14 ± 0.01 0.8 18.4 0.131 SlBCAT5 0.19 ± 0.02 1.0 23.6 0.120 SlBCAT6 0.16 ± 0.02 1.0 22.7 0.140 KIV SlBCAT1 5.57 ± 0.75 1.0 23.0 0.004 SlBCAT2 5.50 ± 0.60 3.5 84.3 0.015 SlBCAT3 0.65 ± 0.07 1.9 46.5 0.070 SlBCAT4 0.37 ± 0.02 2.5 60.6 0.164 SlBCAT5 1.20 ± 0.10 0.9 22.1 0.020 SlBCAT6 0.15 ± 0.01 4.6 109.8 0.730 Leucine SlBCAT1 0.56 ± 0.04 1.6 39.1 0.070 SlBCAT2 0.20 ± 0.02 0.3 8.1 0.040 SlBCAT3 2.70 ± 0.30 4.8 121.0 0.045 SlBCAT4 0.57 ± 0.03 0.7 17.9 0.031 SlBCAT5 1.80 ± 0.10 4.7 118.0 0.066 SlBCAT6 0.21 ± 0.02 0.6 15.7 0.075 Isoleucine SlBCAT1 0.67 ± 0.09 1.6 40.8 0.061 SlBCAT2 0.31 ± 0.02 0.3 7.6 0.025 SlBCAT3 4.90 ± 0.90 6.9 174.0 0.036 SlBCAT4 0.43 ± 0.03 0.8 20.0 0.047 SlBCAT5 3.20 ± 0.20 6.5 163.0 0.051 SlBCAT6 0.34 ± 0.03 0.8 20.0 0.059 Valine SlBCAT1 1.00 ± 0.10 2.0 50.5 0.050 SlBCAT2 1.40 ± 0.50 0.2 3.8 0.003 SlBCAT3 2.00 ± 0.20 4.4 111.0 0.056 SlBCAT4 1.40 ± 0.10 0.8 20.6 0.015 SlBCAT5 2.60 ± 0.20 4.9 123.0 0.047 SlBCAT6 1.20 ± 0.10 1.0 24.0 0.020
Activities of purified recombinant SlBCAT proteins on all BCAA and BCKA substrates. Km is presented as average ± SE. Km data were obtained using GraphPad Prism5 software. Other parameters were obtained by calculations listed in Materials and Methods.
47
Table 2-3. Volatile emissions of BCAT over-expressing transgenic ripe tomato fruit. Line 3-methyl-
1-butanal 3-methyl-1-butanol
2-methyl-1-butanal
2-methyl-1-butanol
Isovalero nitrile
isobutyl acetate
2-isobutyl thiazole
SlBCAT1-OE-1 190 ± 25 205 ± 33 147 ± 29 123 ± 14 113 ± 22 130 ± 21 115 ± 8 SlBCAT1-OE-2 163 ± 19 126 ± 17 136 ± 8 107 ± 9 199 ± 22 60 ± 7 221 ± 42
SlBCAT1-OE-3 138 ± 16 125 ± 6 129 ± 10 99 ± 10 115 ± 15 102 ± 14 124 ± 16 SlBCAT3-OE-1 148 ± 20 197 ± 34 152 ± 23 227 ± 48 77 ± 7 228 ± 14 109 ± 7 SlBCAT3-OE-2 246 ± 35 275 ± 10 141 ± 22 180 ± 22 184 ± 13 141 ± 10 182 ± 9
SlBCAT3-OE-3 171 ± 32 130 ± 20 141 ± 15 146 ± 7 141 ± 29 205 ± 43 143 ± 22
V Values are percentages compared to 100% of M82 control fruit volatiles, ± SD (n=6). Values in bold are statistically significant as determined by one-way ANOVA followed by Dunnet’s test, (p<0.05).
Figure 2-6. Analysis of transgenic fruit SlBCAT transcript levels. Analysis was performed on three biological and three technical replicates of ripe fruit for each sample. Values represent percentage of total mRNA per sample ± SD, calculated from a standard curve for each gene. Data were analyzed by one-way ANOVA and values of each line are significantly different compared to control as determined by Dunnet’s test (p<0.05).
48
Table 2-4. Levels of free amino acids in red ripe fruit of M82 and SlBCAT over-expression lines.
Line
Amino acid quantity
(ng gFW-1)
Val Leu Ile Ala Pro Met Lys
M82 0.79 ± 0.06 1.12 ± 0.14 2.19 ± 0.26 6.69 ± 0.48 1.91 ± 0.28 0.65 ± 0.10 3.01 ± 0.22 SlBCAT1-OE-1 0.61 ± 0.06 0.95 ± 0.09 2.22 ± 0.18 3.25 ± 0.29 3.73 ± 0.35 0.60 ± 0.05 2.58 ± 0.19 SlBCAT1-OE-2 0.70 ± 0.10 1.06 ± 0.09 2.21 ± 0.18 6.92 ± 1.27 3.45 ± 0.54 0.47 ± 0.05 2.04 ± 0.21
SlBCAT1-OE-3 0.72 ± 0.17 1.13 ± 0.15 2.64 ± 0.40 6.20 ± 1.65 3.32 ± 0.41 0.62 ± 0.04 2.62 ± 0.45 SlBCAT3-OE-1 0.73 ± 0.12 1.19 ± 0.20 3.38 ± 0.60 3.44 ± 0.50 2.88 ± 0.06 0.74 ± 0.16 1.87 ± 0.26
SlBCAT3-OE-2 0.64 ± 0.07 1.08 ± 0.12 3.52 ± 0.42 5.02 ± 0.68 2.61 ± 0.29 0.62 ± 0.06 2.37 ± 0.26 SlBCAT3-OE-3 1.47 ± 0.32 2.61 ± 0.45 7.96 ± 1.81 7.92 ± 1.00 5.57 ± 1.05 1.00 ± 0.03 4.83 ± 0.72
Values are expressed as means ± SD, (n=3).Values in bold are significantly different from control fruit by one-way ANOVA followed by Dunnet’s test (P<0.05).
49
CHAPTER 3 BRANCHED-CHAIN VOLATILES IN TOMATO
Rationale and Background
In order to identify the components involved in the synthesis of branched-chain flavor
compounds in fruit, tomato fruit pericarp discs were supplemented with potential precursors to
those volatiles, including the branched-chain amino acids and their α-keto acid analogs. These
experiments were performed to provide evidence that the pathway leading to branched-chain
volatiles in tomato fruit is either similar or dissimilar to the Ehrlich pathway in yeast. The
experiments may also provide evidence of the rate limiting factors and steps in the putative
pathways, whether they be substrate limited or enzyme limited.
Similar experiments to those described here were performed in yeast with positive results,
showing that such a feeding experiment can yield the downstream branched-chain volatile
compounds as long as the appropriate Ehrlich pathway enzymes are present: branched-chain
amino acid aminotransferase, pyruvate decarboxylase, and aldehydes dehydrogenase (Dickinson
et al., 2003). The yeast studies showed that the flavor volatiles 2-methylbutanal and 2-
methylbutanol are downstream products of isoleucine catabolism and 3-methylbutanal and 3-
methylbutanol are downstream products of leucine catabolism (Dickinson et al., 1997; Dickinson
et al., 2000).
The hypothesis held before performing these experiments was that the branched-chain
volatiles emitted from tomato fruit are synthesized from BCAA substrates. The justification is
that the same volatiles in yeast are products of BCAAs and these substrates share similar
chemical structure with the volatiles. Since BCKAs are synthesized from BCAAs and are
downstream in the Ehrilch pathway, it was predicted that feeding these compounds would also
increase branched-chain volatile emissions. The results from the following experiments,
50
however, were unexpected and required a revision of the current hypothesis that branched-chain
volatiles in tomato are formed via the Ehrlich pathway.
Results of Substrate Feeding
The free amino acids leucine, isoleucine, and valine were applied to M82 red ripe fruit
pericarp discs and incubated for eight hours after which volatiles were quantified by GC-MS.
Emissions of seventeen tomato volatiles having the most impact on flavor were analyzed and
compared to emissions from controls supplemented with only water. Significant changes were
not seen in many of the non-branched-chain volatiles, as was expected due to structural
dissimilarities to the provided substrates.
In tissues supplemented with leucine, small but significant (p<0.05) increases of about 1.5-
fold were observed in 3-methylbutanol and 3-methylbutanal emissions (Figure 3-1). Both of
these volatiles share side-chain structure with leucine. A significant decrease in cis-3-hexenal
and highly variable increase in trans-2-hexenal were also observed in these samples.
The volatile trans-2-hexenal, along with other green leaf volatiles (GLV), is usually
formed from linolenic and linoleic acids via lipoxygenase enzymes (Hatanaka, 1993). GLVs are
thought to exist in low concentrations in healthy plant tissues, but concentrate rapidly in stressed
or wounded tissues, and that tissue laceration provides the lipid substrates and triggers hydrolysis
to form these volatiles (Matsui et al., 2000). It is possible that the tissue injury from creating
pericarp discs and addition of high amounts of branched-chain substrates elicits a stress response
that causes trans-2-hexenal to form.
In tissues supplemented with KIC, significant increases of about nine-fold and ten-fold in
3-methylbutanal and 3-methylbutanol, respectively, were observed (Figure3-1). This result may
be explained by the fact that KIC is one step closer to these volatile compounds than leucine in
the hypothesized Ehrlich pathway. Although 3-methylbutanal is closer in this pathway to KIC
51
than 3-methylbutanol, a greater concentration of 3-methylbutanol was emitted. This could be due
to the fact that 3-methylbutanol is the end product in this pathway causing it to accumulate, and
no reverse reaction exists for conversion back into the aldehyde. Interestingly, a decrease of
almost 50% was seen in 2-methylbutanal in KIC-supplemented tissue. Three possible
explanations include: the steps converting KIC and KMV to 3- and 2-methylbutanal,
respectively, occur via the same enzyme, for which both substrates are in competition, the
unnaturally high levels of substrates applied causes competitive or feedback inhibition of the
isoleucine synthetic pathway, or a several-fold increase in 3-methylbutanal or 3-methylbutanol
signals feedback inhibition of that pathway. In either case, 2-methylbutanal is still catabolized to
cause accumulation of 2-methylbutanol, as shown by the lack of change in 2-methylbutanol
levels from control samples.
When isoleucine was fed to M82 pericarp discs, small but significant increases of about
2.5-fold and 1.5-fold were observed in the levels of 2-methylbutanal and 2-methylbutanol
emissions, respectively (Figure 3-2). This result was expected because these two volatiles share
the same side chain structure as isoleucine and thus predicted to be metabolically related. This
result shows that only a small amount of isoleucine can be converted to branched-chain
volatiles.
M82 pericarp discs supplemented with KMV showed significant increases of about 15-fold
and 9-fold in 2-methylbutanal and 2-methylbutanol emissions, respectively, from water fed
controls (Figure 3-2). Like the results from the KIC and leucine feeding experiment, the greater
conversion of KMV than isoleucine to branched-chain volatiles may be explained by a revision
of the putative pathway to branched-chain volatiles. These results suggest BCKAs are more
likely than BCAAs to be the primary precursors to these volatiles. Similar to the KIC feeding
52
results, KMV feeding resulted in decreases of about 60% and 20% in levels of 3-methylbutanal
and 3-methylbutanol emissions. As with KIC, this may be due to high substrate competitive or
feedback inhibition of the pathway leading to these volatiles by the substrate. Similar decreases
of about 20% were seen in the branched-chain volatiles isovaleronitrile and 2-isobutylthiazole,
suggesting high amounts of supplied KMV or the 2-methylbutanal or 2-methylbutanol products
may cause inhibition of the synthetic pathways of those compounds as well. There is also a
significant 1.5-fold increase of isobutyl acetate observed from KMV feeding and significant
increases in trans-2-hexenal in both isoleucine and KMV feeding. Lack of structural similarities
between these volatiles and the substrates suggest they are not related metabolically, but that the
substrates signal a change in these volatiles’ pathways. These changes are not likely due to
metabolic relatedness of volatiles to substrate but rather to regulatory mechanisms.
M82 pericarp discs supplemented with valine did not exhibit significant increases in
branched-chain flavor volatiles (Figure 3-3). In yeast, catabolism of valine results in KIV and the
volatiles isobutanal and isobutanol (Dickinson et al., 1998). Though these volatile compounds
are likely produced in tomato fruit, given that leucine and isoleucine feeding yield their
respective branched-chain volatiles, they are not considered important compounds to flavor
perception and are of less concern in this study. Also, due to their very low molecular weights,
they have a very short retention time on the gas chromatograph and are not detectable with the
methods used in this analysis. The only significant changes observed with valine feeding were a
three-fold increase in trans-2-hexenal and about 0.3-fold decreases in cis-3-hexenal and trans-2-
heptenal.
M82 pericarp discs supplemented with KIV displayed increases of about 4.5-fold and 5-
fold in 3-methylbutanal and 3-methylbutanol emissions, respectively. This result is similar to that
53
observed with leucine feeding and can be explained by the fact that KIV is a precursor to KIC,
therefore it is expected that the same branched-chain volatiles would increase in this experiment.
This explanation is reinforced by results from yeast, in which it was reported that products of
both leucine and valine catabolic pathways are derived from a shared pool of KIV (Dickinson et
al., 1998). It is interesting, however, that although the same amount of leucine and KIV were fed
in each experiment the amount of 3-methylbutanal and 3-methylbutanol increase was greater
with KIV than with leucine. It is possible that a high concentration of leucine, but not KIV, in the
cell exhibits feedback inhibition to steps downstream in the pathway to KIC and volatile
synthesis, such as isopropylmalate synthase. It is also possible that the flux from KIV to KIC is
greater than that of leucine to KIC, since application of KIC yields the greatest increase in 3-
methylbutanal and 3-methylbutanol of all branched-chain substrates. The likely explanation is
that the hypothesis of the Ehrlich pathway occurring in tomatoes is incorrect. These data suggest
that KIC is more likely the primary precursor of 3-methylbutanal and 3-methylbutanol, rather
than leucine. Leucine catabolism may not result in these volatiles at all under biological
conditions, but is observed only when unnaturally high concentrations are fed to fruit. A decrease
of about 40% in 2-methylbutanal emission was observed with KIV feeding, similar to the result
from KIC feeding. KIV feeding also resulted in an increase of about fourteen-fold of the
branched-chain ester isobutyl acetate, making it the only tomato flavor volatile so far known to
be produced predominantly from the valine catabolic pathway. This compound is likely
produced by the reaction of isobutanol with acetyl-CoA and an acetyltransferase enzyme. There
was also an observed 1.5-fold increase in isobutyl acetate in tissues supplemented with KIC,
which may be explained by feeding of KIC into the valine pathway (Figure1-1). As in the other
feeding experiments, feeding of KIV caused significant increases in trans-2-hexenal.
54
[U-13
C]leucine Feeding Reinforces BCAA Catabolic Pathway
The analysis of the feeding experiments above gave evidence for the synthetic pathways of
the volatile end-products of branched-chain catabolism, but did not measure the non-volatile
intermediates upon feeding of BCAAs. Additional evidence is also needed to support the
possibility that the Ehrlich pathway does not occur in tomato fruit as it does in yeast. The above
feeding experiments only provide indirect evidence of a pathway leading to branched-chain
volatiles. To further test and validate the hypothesis of a pathway in tomato fruit, a similar
feeding experiment was performed in which [U-13C]leucine heavy isotope was used as a
substrate. It is likely that the catabolic pathway of leucine is parallel to the pathways of
isoleucine and valine, similar to these pathways in yeast. Leucine was used as a representative of
all three BCAAs and their potential catabolic pathways.
The [U-13C]leucine compound was fed to red ripe fruit pericarp discs and incubated in
Petri dishes. Tissues were immediately frozen after 4 and 8 h time points. Metabolites were
extracted with methanol, then derivitized with methoxyamine hydrochloride in pyrimidines
followed by MSTFA and analyzed by GC-MS. The two time points were used to allow
measurement of 13C label incorporation over time. Analyzing the concentrations of compounds
which have incorporated label should provide evidence of the steps in the leucine catabolic
pathway in tomato fruit, and analyzing them temporally should give an indication of the
sequence of steps in the pathway. 13C-labeled substrate and GC-MS analysis was used in the
experiment to assess this pathway because it has proved useful in previous studies, such as in the
analysis of glycolysis in Arabidopsis (Giege et al., 2003). Molar enrichment of 13C label into
intermediate compounds was determined by comparison to 12C spectral fragments from
55
standards. We specifically analyzed the accumulation of label in putative BCAA catabolic
intermediates and branched-chain volatiles (Table 3-1).
A significant quantity of 13C was observed in KIC, which is the direct catabolic product of
leucine by BCAT (Table 3-1). Significant amounts of label in KIV and KMV were also
observed. The quantities of labeled KIC and KIV were about double that of labeled KMV, which
is likely due to the connection of the leucine metabolic pathway to that of valine but not
isoleucine. It is interesting that the quantity of 13C label in KIV was higher than in KIC after 4 h,
but the opposite is observed after 8 hours. This may be due to a lower rate of carbon transfer into
KIC than KIV, which eventually reaches a steady state while KIC continues to accumulate label.
Of the branched-chain volatiles the incorporation of label was very low, though 3-
methylbutanol and 2-methylbutanol accumulated the most label, while their aldehyde forms had
nearly 10-fold less accumulation. The incorporation of label into these two alcohols about
doubled from 4 h to 8 h, while most other branched-chain intermediates appeared to reach their
isotopic steady-state by 4 h. Both of these observations are evidence that the branched-chain
alcohols are likely the end-products of the pathway in tomato. It also appears from these results
that the conversion of BCKAs from BCAAs occurs rapidly before reaching a steady-state. The
very low concentration of label in the branched-chain volatiles is likely because the pool of
BCKAs with label is used in other more primary pathways such as protein synthesis, leaving
only a small amount to be used in volatile synthesis. There may also be technical difficulties in
detecting such small amounts of volatile compounds.
That there is not a much higher amount of label in the volatiles supports the hypothesis
that BCAAs are not immediate precursors to branched-chain volatiles. It is also supported by the
fact that more label was incorporated into 2-methylbutanol than 3-methylbutanol, while the
56
opposite result would be expected if 3-methylbutanol was produced by a direct route from
leucine. The results of this experiment show that carbon flux from leucine is highly dynamic,
which creates difficulty in interpreting the data. This is evident by the unexpected incorporation
of label into valine and isoleucine, which suggests that much of the carbon was first cycled into
pyruvate and threonine, the precursors to valine and isoleucine.
BCAA and Branched-Chain Volatile Loci
As was mentioned in the introductory chapter, much information can be gained about
tomato traits by identifying loci of interest using the population of S. pennellii introgression
lines. It was previously determined that there are at least 26 genetic loci with significant volatile
emission phenotypes, 12 of which are altered in branched-chain volatile emissions (Tieman et
al., 2006). This high number of branched-chain volatile loci suggests there are many factors
contributing to their synthesis and many points of regulation in their pathways in tomato.
Therefore, it is not surprising that drastic phenotypes were not observed in the transgenic
BCAT over-expression plants, or that the pathway leading to these volatiles may not be as
straightforward as they are in yeast. To further support the theory that branched-chain volatiles
are not derived directly from BCAAs, it is possible to cross-reference volatile loci to loci that are
changed in BCAA quantity in the S. pennellii population. These BCAA loci have already been
established in a previous report in which, like the volatiles, there were a surprisingly high
number of loci with BCAA phenotypes (Schauer et al., 2006). As mentioned in the introduction,
there is no consistent pattern of BCAA loci and branched-chain volatile loci occurring together,
as illustrated in Table 3-2. This observation adds additional evidence to the hypothesized
pathway which suggests branched-chain volatiles are not derived from BCAA catabolism, but
directly from catabolism of BCKAs.
57
The evidence presented in this chapter suggests that branched-chain volatiles are more
likely derived directly from BCKAs, and not from BCAAs as they are in yeast (Dickinson et al.,
2003). Feeding of BCKAs in tomato fruit tissue resulted in much greater production of branched-
chain volatiles than BCAA feeding. The isotopic feeding showed that only a small transfer of
carbon into branched-chain volatiles from leucine occurs. Along with the IL data, these
observations reinforce the possibility of this novel branched-chain catabolic pathway existing in
tomato fruit.
58
Figure 3-1. Leucine and KIC feeding. Levels of relevant flavor volatiles extracted from tomato
pericarp samples supplemented with either leucine or KIC, expressed as a percentage of water-fed control pericarp normalized to 100%. Asterisks mark compounds that were significantly different from control, as determined by one-way ANOVA followed by Dunnet’s test, (p<0.05) (n=3).
59
Figure 3-2. Isoleucine and KMV feeding. Levels of relevant flavor volatiles extracted from
tomato pericarp samples supplemented with either isoleucine or KMV, expressed as a percentage of water-fed control pericarp normalized to 100%. Asterisks mark compounds that were significantly different from control, as determined by one-way ANOVA followed by Dunnet’s test, (p<0.05) (n=3).
60
Figure 3-3. Valine and KIV feeding. Levels of relevant flavor volatiles extracted from tomato
pericarp samples supplemented with either valine or KIV, expressed as a percentage of water-fed control pericarp normalized to 100%. Asterisks mark compounds that were significantly different from control, as determined by one-way ANOVA followed by Dunnet’s test, (p<0.05) (n=3).
61
Table 3-1. Label accumulation in metabolite pools following incubation with [U-13C]leucine.
Metabolite Label accumulation
(nmol gFW-1) 4 h 8 h
BCAAs
Leu 1302 ± 129 1285 ± 106 Iso 412 ± 32.0 461 ± 42.0 Val 256 ± 25.0 267 ± 24.0
BCKAs
KMV 10.9 ± 0.80 10.5 ± 1.00 KIV 21.9 ± 1.05 15.8 ± 1.00 KIC 19.6 ± 1.40 20.0 ± 2.40
Volatiles
3-methylbutanal 0.002 ± 0.000 0.003 ± 0.000 3-methylbutanol 0.025 ± 0.001 0.055 ± 0.003 2-methylbutanal 0.004 ± 0.000 0.004 ± 0.000 2-methylbutanol 0.037 ± 0.001 0.062 ± 0.003 Isovaleronitrile 0.002 ± 0.000 0.003 ± 0.000 Isobutylacetate 0.003 ± 0.001 0.003 ± 0.001
2-isobutylthiazole 0.005 ± 0.000 0.005 ± 0.001 Values are presented as means ± SE from four independent determinants.
62
Table 3-2. Occurrences of loci containing BCAA and branched-chain volatile phenotypes.
BCAA IL
locus
Fold-
change
BCV
locus BCAA
IL
locus
Fold-
change
BCV
locus
L 2-2 0.74 - I 6-2 1.83 - V 0.65 V 1.89 V 2-3 0.61 + L 6-2-2 2.07 - L 2-4 2.15 + I 3.19 I 2.6 V 2.43 V 2-5 0.36 + L 7-1 1.72 + L 2-6-5 1.41 - I 2.16 L 3-1 0.64 - V 1.76 L 3-2 2.49 - L 7-2 2.02 + I 1.96 I 2.37 V 1.69 V 7-3 2.4 - L 3-4 1.94 - I 8-1 2.59 - V 1.87 V 1.99 V 3-5 0.59 + I 8-1-1 2.35 L 4-1 1.76 - V 2.06 I 2.61 L 8-3-1 2.33 - V 1.99 L 9-3 1.78 + L 4-4 1.54 + L 10-1 2.83 + I 2.09 I 2.75 I 5-1 0.55 + V 1.64 V 0.63 L 11-2 2.11 + L 5-2 1.75 - V 1-1-3 0.68 - I 2.6 V 12-1-1 1.51 - V 2.06 L 12-3 2.08 - I 5-3 2.24 - I 2.66 V 1.97
The first column indicates which BCAA is changed. The second column indicates the introgression line containing the phenotype. The third column values indicate fold differences of BCAA concentration in red ripe fruit from each introgression line from M82 control values, as reported by Schauer et al. (2006). The fourth column indicates if an overlapping branched-chain volatile phenotype was observed in the line, according to Tieman et al. (2006). Shaded rows indicate ILs with phenotypes for all three BCAAs. BCAA = branched-chain amino acid, BCV = branched-chain volatile, L = leucine, I = isoleucine, V = valine. Data have been adapted from Schauer et al. (2006) and Tieman et al. (2006).
63
CHAPTER 4 DISCUSSION OF RESULTS
Diversity of SlBCAT Family
In this work we have discovered six BCATs that exist in S. lycopersicum, and expression of
four of these cDNAs was detected in the parts of the plant that were analyzed. The experiments
that followed their discovery revealed important information about this enzyme family and its
individual members in a plant species in which they have not yet been extensively characterized.
It is clear from BCAT studies in other plant species that this is a very important metabolic
enzyme family whose members perform functions in addition to BCAA synthesis and
degradation, making their continued study important to the overall understanding of plant
metabolism. The SlBCATs have very distinct characteristics from each other, suggesting some of
them they may also perform non-primary metabolic processes in the plant.
SlBCAT1 and SlBCAT2 were shown to be located in mitochondria in tobacco leaf cells.
Given that mitochondria are the primary location of BCAA catabolism in plant cells and the
preferences of these two enzymes is primarily catabolic and only minimally restore growth in E.
coli auxotrophs, these are likely to be the primary BCAA-degrading enzymes in tomato. Since
SlBCAT1 is highest in expression in ripening fruit, it may be the primary enzyme for recycling of
BCAAs resulting from protein degradation in senescent fruit tissue. Since SlBCAT2 is expressed
in all green tissues analyzed and very highly in floral tissues, it may be the responsible for
BCAA catabolism throughout. We expect a catabolic mitochondrial gene to exist in all tissues
since BCAAs are known precursors to the TCA cycle intermediates succinyl-CoA and acetyl-
CoA. This gene may also function in the crucial process of maintaining steady-state levels of
BCAAs in cells, which is crucial for compounds of such primary importance in cellular
metabolism.
64
The very low preference of SlBCAT1 for the forward direction was may be explained by
the multiple amino acid substitutions this enzyme has in conserved regions. It has an I101 in its
active site whereas the other SlBCATs all have F101, as do the E. coli, human, and yeast BCAT
proteins at this position. However, AtBCAT1 also contains an isoleucine at this position and is
highly active in the forward direction, making this substitution an unlikely cause (Schuster and
Binder, 2005). More likely are the residues S122, which is an asparagine in all BCATs from
Arabidopsis, human, and yeast, and N175, which is a serine or threonine at the same position in
all Arabidopsis and yeast genes. The human mitochondrial BCAT has this same amino acid
substitution, but humans do not synthesize BCAAs. Future mutagenesis experiments may
determine if forward activity can be enhanced in this tomato enzyme by changing these residues
to their more conserved amino acids. If successful it will provide crucial evidence to the amino
acid configurations of BCAT enzymes that give them reversible catalytic activity.
SlBCAT3 and SlBCAT4 were located in chloroplasts of tobacco leaf cells and were able to
restore growth of E. coli cells lacking the ability to synthesize BCAAs, suggesting that these
enzymes are primarily involved in BCAA synthesis. The substrate affinities of these two
enzymes, especially of SlBCAT3, suggest the same. It is possible, however, that under certain
conditions these two enzymes may have a role in BCAA catabolism as well, which is evident
from these two enzymes’, especially SlBCAT4, ability to use BCAAs as substrates in the kinetic
studies. SlBCAT3 may function as the BCAA synthetic enzyme throughout the plant, since it is
expressed nearly equally in all tissues analyzed, including the four fruit stages. SlBCAT4 appears
to be more specialized in function, given that its expression is by far the highest in flowers and is
relatively low in other tissues compared to the other SlBCATs. An enzyme with such a specific
expression pattern may have a specialized function, possibly in the metabolism of compounds
65
other than BCAAs as is observed with the Arabidopsis AtBCAT3 and AtBCAT4 (Schuster et al.,
2006; Knill et al., 2008). Kochevenko et al. (2010) showed that tomato inflorescences contain
nearly twice the concentration of all three BCAAs as leaves. The same study also revealed that
SlBCAT4 maps to IL3-2 and SlBCAT1 maps to IL12-3, both of which have QTLs for increased
levels of all three BCAAs. A N. benthamiana BCAT, recently found to be involved in hormonal
regulation, is also expressed highly in flowers and located in chloroplasts (Gao F, 2009). The
similarities of SlBCAT4 to this N. benthamiana gene make it an even more interesting subject for
the study of non-primary BCAT functions.
SlBCAT5 and SlBCAT6 transcripts were not detected in any of the tissues analyzed. The
only EST accessions that exist for these two unigenes were isolated from callus tissue cDNA,
therefore they may only be expressed under specific developmental, hormonal, or environmental
conditions. Despite their scarcity of expression, the distinct localization patterns and multi-
substrate kinetics of these two enzymes make them interesting candidates for study. The vacuolar
location of SlBCAT6 suggests that it may function in the recycling of BCAAs from the products
of proteolysis. This function is further supported by its lack of substrate specificity and relatively
high affinity for all branched-chain substrates. Of particular interest is this enzyme’s specificity
on KIV, which is extremely high compared to all other SlBCATs and substrates. It is unclear
why this enzyme has such a strong tendency toward valine synthesis.
The overall results of the SlBCAT kinetic experiments show that these enzymes are
diverse in function and are not entirely specific to individual branched-chain substrates, as might
have been expected from a multi-gene family. SlBCATs tend to have higher overall efficiencies
toward the BCKAs than the BCAAs, although it is assumed that BCATs are generally reversible
enzymes.
66
The lack of striking fruit volatile or BCAA phenotypes in transgenic plants was
unexpected. However, a simple explanation may exist as to why there were no clear phenotypes.
It would be expected that an increase in the expression of SlBCAT1, a catabolic enzyme, would
cause a decrease in BCAAs and thus a decrease in volatiles. As is observed in bacteria (Massey
et al., 1974), the enzymes in the pathway of BCAA synthesis are very tightly regulated by
substrate feedback, therefore if catabolism of BCAAs is increased, one might expect a
simultaneous increase in BCAA synthesis. The opposite effect may occur with overexpression of
SlBCAT3, a primarily anabolic enzyme. Increased BCAA synthesis resulting from an
overproduction of this enzyme might elicit a feedback mechanism to earlier steps in the BCAA
synthesis pathway, such as threonine deaminase or acetolactate synthase, inhibiting excess
BCAAs from being synthesized.
Significance of Substrate Feeding
The substrate feeding experiments were performed to gain insight into the catabolic
pathways of BCAAs and the formation of their respective volatiles in tomato fruit, of which little
is known. It seems the tomato branched-chain volatile synthesis pathways share at least some of
the same steps as in yeast (Dickinson et al., 2003). However, it was particularly interesting that
in the case of isoleucine/KMV and leucine/KIC, feeding of the α-keto-acid resulted in much
greater incorporations into the same branched-chain volatiles than feeding of the amino acid.
These results may be explained by the fact that the α-keto-acids are capable of a greater flux into
these compounds than amino acids. It may also be that the amino acids are quickly metabolized
by other pathways in the cell, such as in protein synthesis, while the α-keto-acids are in less
demand by enzymes other than those forming branched-chain aldehydes. It is likely that BCATs
and BCAAs do not participate in the volatile synthesis pathway, and the BCKAs are converted to
67
volatiles directly following their synthesis via dihydroxy-acid dehydratase and 3-isopropylmalate
dehydrogenase (Figure 4-1).
Tomato fruit cells are capable of disposing a small percentage of excess BCKA substrate
in the form of volatiles. The quantities of 3-methylbutanal and 3-methylbutanol emitted after
KIC feeding were 0.5 and 5.7 nmols/g FW, respectively, corresponding to 0.25 and 2.9 percent,
respectively, of substrate fed. The quantities of 2-methylbutanal and 2-methylbutanol emitted
after KMV feeding were 0.7 and 3.4 nmols/g FW, respectively, which corresponds to 0.35 and
1.7 percent, respectively, of substrate fed. The quantity of isobutyl acetate emitted after KIV
feeding was 0.45 nmols/g FW, corresponding to 0.25 percent of substrate fed. The quantity of
emissions after BCKA feeding compared to control fruit is evidence that these compounds are
limiting factors in the production of branched-chain volatile compounds. The results show that
tomato fruit cells are capable of producing more volatiles than they do under standard conditions
but are limited by the quantity of these intermediate metabolites in the cell. Conversely, it seems
that BCAAs are not limiting to the production of branched-chain volatiles. This is evident from
the lack of correlation between BCAA and volatile phenotypes in the S. pennellii IL population
and from low incorporation of BCAAs into volatiles seen in the feeding experiments.
The slight increase of isobutyl acetate observed with KIC feeding cannot be explained at
present. Since isopropylmalate synthase, the enzyme diverting KIV to the leucine synthetic
pathway, is thought to be irreversible, the conversion of KIC back to KIV and then to isobutyl
acetate is unlikely.
Decreases of 3-methylbutanal and 3-methylbutanol were observed after KMV feeding
and a decrease of 2-methylbutanal after KIC feeding. There are two possible explanations for
these occurrences: the steps in the pathways converting KIC and KMV to 3-methylbutanal and 2-
68
methylbutanal, respectively, may occur via the same enzyme and therefore both substrates would
be in competition for that enzyme, or a great increase in KIC, 3-methylbutanal, or 3-
methylbutanol may cause feedback inhibition of the pathway generating 2-methylbutanal and
vice-versa. In either case the step converting the branched-chain aldehydes to alcohols does not
seem to be affected as much, given the wild-type levels of 2-methylbutanol observed in KIC
feeding and only a 20% decrease of 3-methylbutanol compared with a 60% decrease of 3-
methylbutanal observed in KMV feeding. Also with KMV feeding, decreases of about 20% each
were seen in the branched-chain volatiles isovaleronitrile and 2-isobutylthiazole, suggesting that
KMV, 2-methylbutanal, or 2-methylbutanol may cause feedback inhibition in the pathways
synthesizing those compounds as well.
Feeding of valine and KIV yielded some expected and unexpected results. No significant
changes were seen in branched-chain volatile emissions with valine feeding. This lack of aroma
change was also observed in strawberries supplemented with valine (Perez et al., 2002).We
expected to see an increase in leucine-related volatiles since the valine and leucine pathways are
interrelated. On the other hand, valine is known to be one of the most potent feedback inhibitors
of BCAA synthesis by inhibiting ALS (Borstlap, 1972), so if that enzyme is involved in
branched-chain volatile production it would likely be repressed upon valine feeding (Massey et
al., 1974; Massey et al., 1976). We had not previously seen any evidence of a pathway leading to
isobutyl acetate synthesis, but feeding KIV has provided such evidence. The increase in isobutyl
acetate was greater than any other volatile in these feeding experiments, suggesting that KIV
follows a direct path to the synthesis of this compound. As is the case in microorganisms
(Dickinson et al., 1998), it is likely that this pathway occurs by conversion of KIV to isobutanal,
then to isobutanol, then to isobutyl acetate, this final step being catalyzed by an alcohol acetyl-
69
transferase. Dissection of this putative pathway may provide a new branch of tomato flavor-
related research in our laboratory. Also of interest in KIV feeding was that although the same
concentration of leucine and KIV were supplied in each experiment the amount of 3-
methylbutanal and 3-methylbutanol increases were greater with KIV feeding than with leucine
feeding. These volatiles were expected to increase with KIV feeding, but not to a greater degree
than with leucine, which was predicted to be closer to these volatiles. It could be that a high
concentration of leucine in the cell exhibits feedback inhibition to the downstream steps in the
volatile pathway while KIV does not. It is more likely that the flux from KIV to KIC is greater
than the flux from leucine to KIC, since KIC supplementation yields the greatest increase in
these two volatiles of all substrates supplies. This observation adds strong support to the
hypothesis that branched-chain volatiles are derived from BCKAs and do not involve the activity
of BCATs and BCAA substrates. The facts that only very small amounts of 13C label are
incorporated into branched-chain volatiles and that there is more label in 2-methylbutanol than in
3-methylbutanol also support the hypothesis that leucine is not a direct precursor of 3-
methylbutanal and 3-methylbutanol.
There is no clear indication in current literature that the involvement of BCKAs and
BCAAs in formation of trans-2-hexenal. Those compounds do not share enough structural
similarity to suggest that trans-2-hexenal may be converted from them directly. It is more likely
that the branched-chain compounds induce formation of trans-2-hexenal from long chain fatty
acids. A plausible explanation was given in Chapter 3 for this volatile’s biogenesis. It is known
that trans-2-hexenal and other C6-volatile compounds are synthesized from the degradation of
polyunsaturated fatty acids in plants (Zhuang et al., 1996). It is also known that trans-2-hexenal
and other C6 compounds are induced upon wounding and stress, and that their application to
70
plants induces defense-related genes (Kishimoto et al., 2005). The signaling pathways for these
stress responses are currently unknown. A recent study, however, has implicated γ-aminobutyric
acid (GABA) in this signaling pathway after isolating a GABA-aminotransferase mutant in
Arabidopsis. The authors showed that an increase in GABA elicits a resistance to the effects of
the trans-2-hexenal defense response on the plant (Mirabella et al., 2008). It is possible that
exogenous addition of branched-chain compounds down-regulates GABA formation, which in
turn causes an up-regulation of trans-2-hexenal production. This interaction with the GABA
pathway is feasible because BCAAs and BCKAs share similar structure with GABA, all are
precursors to TCA cycle intermediates, and all are substrates for aminotransferase enzymes and
use glutamate and α-ketoglutarate as donor and acceptor molecules, respectively. An application
of such high amounts of exogenous BCAAs and BCKAs to tomato fruit may have caused
competitive inhibition of GABA-aminotransferase. In addition, high concentrations of branched-
chain compounds may directly trigger a stress response which up-regulates the production of
trans-2-hexenal.
It is important to bear in mind that these feeding experiments are partly artificial in nature,
and the results may not entirely reflect the in vivo characteristics of fruit metabolism, due to the
higher-than-natural concentration of substrate supplementation. In addition, it is not currently
known which subcellular compartments volatile synthesis occurs in, and most of the substrate
presumably pools in the cytosol after application. Nevertheless, the results of these feeding
experiments show convincing evidence that the pathways to branched-chain flavor volatiles in
tomato fruit may share the same steps as in yeast and the Ehrlich pathway (Figure 1-1B),
excluding the first BCAT step. The results also show that the BCKAs are converted into a much
greater amount of their corresponding volatiles than are BCAAs. This may be due to the fact that
71
the BCKAs are only one enzymatic step from their corresponding volatiles while the amino acids
are two steps away, according to the Ehrlich pathway. This could also indicate that the BCAT
step converting BCAAs to BCKAs is much more limiting than the decarboxylase step converting
the BCKAs to aldehydes. Alternatively, and what is now our current hypothesis, BCATs and
BCAAs have only minimal roles in the branched-chain volatile synthetic pathways, while
BCKAs are the primary volatile precursors. BCKAs may be converted to volatiles directly from
their synthesis via dihydroxy-acid dehydratase and 3-isopropylmalate dehydrogenase in plastids
(Figure 4-1).
Concluding Remarks
Taken together, the results of the experiments presented here give much new detail about
BCAA metabolism and to the characteristics of the BCAT family, adding support to the
information that already exists about them in plants. Future work on this family will provide a
full characterization of its members and how they coordinate BCAA anabolism and catabolism
in plant organs. Alternative functions are also expected to be found in more detailed studies of
individual BCATs, particularly in SlBCATs 4, 5, and 6. Transgenic plants over-expressing and
silencing the four SlBCATs not described in this work are in the process of construction and
should soon expand what we know about these enzymes in vivo.
The complexity of tomato flavor, as stated previously, is an interaction of sugars, acids,
and volatile organic compounds. There are only a handful of volatiles that are thought to
contribute to tomato flavor, and of these there are seven believed to be derivatives of the BCAAs
and/or the BCKAs: 3-methylbutanol, 3-methylbutanal, 2-methylbutanol, 2-methylbutanal,
isobutylthiazole, isovaleronitrile, and isobutyl acetate. These compounds were thought to be
directly derived from their most structurally related BCAAs, but the tomato feeding experiments
shown here with valine and KIV suggest that isobutyl acetate derives directly from KIV and not
72
valine, since KIV precedes valine in the pathway of its synthesis. Similar results were found with
the addition of KIC and KMV and labeled metabolite feeding did not indicate a clear pathway
from BCAAs to volatiles, all of which support our new hypothesis that branched-chain volatiles
are the products of BCKAs and not BCAAs, which is a deviation from the Ehrlich pathway.
73
Figure 4-1. Hypothesized pathways forming branched-chain volatiles from BCKAs. 1) threonine
deaminase, 2) acetolactate synthase, 3) acetolactate isomeroreductase, 4) dihydroxy-acid dehydratase, 5) branched-chain aminotransferase, 6) 2-isopropylmalate synthase, 7) isopropylmalate isomerase, 8) isopropylmalate dehydrogenase, 9) α-keto-acid decarboxylase, 10) aldehyde dehydrogenase, 11) alcohol acetyltransferase. Asterisks indicate steps which constitute the Ehrlich pathway of branched-chain volatiles from BCAAs in microbes.
74
CHAPTER 5 MATERIALS AND METHODS
All chemicals and reagents used were purchased from Sigma-Aldrich (St. Louis, MO),
unless otherwise noted. All supplies were purchased from Fisher Scientific (Pittsburgh, PA),
unless otherwise noted. All oligonucleotides were purchased from Integrated DNA Technologies
Inc. (Coralville, IA).
Cloning of SlBCATs
EST sequences of each SlBCAT were found by searching the SGN tomato EST database
(http://solgenomics.net/index.pl) with its BLAST tool for sequences that share homology with
known plant BCATs. The full length 5’ and 3’ ends of each SlBCAT were obtained using RACE
PCR with the SMART RACE cDNA synthesis kit (Clontech Laboratories, Mountain View, CA).
PCR with Advantage HF2 polymerase (Clontech Laboratories, Mountain View, CA) was used to
amplify the full-length open reading frames from cDNA. These were cloned into pGEMT
(Promega, Madison, WI) and sequenced. Alignment of protein sequences were produced using
ClustalW (Larkin et al., 2007) and phylogram trees produced using MEGA4 (Tamura et al.,
2007).
Constructs
Open reading frames for each construct were amplified from cDNA by PCR and cloned
into pGEMT-easy vector (Promega, Madison, WI). Protein expression constructs of each
SlBCAT were made by cloning into the Nhe1 and Sal1 restriction sites of pET-28b, (Invitrogen,
Carlsbad, CA) containing an N-terminal 6xHis tag. Subcellular localization and signal peptides
of each SlBCAT were predicted using outputs from SignalP subcellular localization software
(Emanuelsson et al., 2007). Primers were designed to omit signal peptides, and are listed in
Supplementary Figure S2.
75
Bacterial complementation constructs were made by excising the inserts from pET28b
and inserting them into the pBAD24 (Guzman et al., 1995) using Sal1 and Not1 restriction sites,
resulting in a pBAD24 construct containing a 6xHis tag.
For transgenic plant overexpression constructs, SlBCAT1 and SlBCAT3 cDNAs were
cloned in the sense orientation into pENTR-TOPO and cloned using Gateway LR Recombinase
(Invitrogen, Carlsbad, CA) into a vector containing the figwort mosaic virus promoter (Richins
et al., 1987), a kanamycin resistance gene, and an Agrobacterium tumefaciens nopaline synthase
(nos) 3’terminator. The overexpression constructs were introduced into S. lycopersicum cv. M82
plants by Agrobacterium-mediated transformation using a method previously described
(McCormick et al., 1986). Primary transgenic tomato plants were grown in greenhouses under
standard conditions and supplemented with slow release fertilizer. Subsequent generations of
transgenic and control tomato plants were grown at the North Florida Research and Education
Center (7580 County Road 136, Live Oak, FL 32060).
C-terminal GFP constructs were made by cloning full-length SlBCAT open reading
frames into pDONR221 (Invitrogen, Carlsbad, CA) then cloning into the pK7WGF2 gateway
binary destination vector (Karimi et al., 2002). GFP constructs were transformed into
Agrobacterium strain ABI (Koncz and Schell, 1986).
Protein Production and Purification
Protein expression constructs were transformed into BL21(DE3) competent cells
(Invitrogen, Carlsbad, CA) by heat shock at 42̊ C. Cultures were grown in Luria broth
supplemented with 0.005 mM pyridoxal phosphate at 37̊̊̊ C until reaching an OD600 = 0.5, then
induced with 0.25 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown at 16̊ C for 18
h. All of the following steps were carried out at 4̊ C. Cells were pelleted and lysed by sonication
76
in phosphate buffered saline (PBS), then treated with Histidine-tag Protease Inhibitor Cocktail
(Sigma-Aldrich, St. Louis, MO) according to manufacturer’s directions and 0.1 mM β-
mercaptoethanol. Lysed cells were centrifuged at 10,000 rpm for 10 min and supernatant was
taken off and incubated with 200 µl TALON Affinity Purification Resin (Clontech, Mountain
View, CA) on ice with gentle shaking for 1 h. Resin was pelleted by brief centrifugation on
lowest speed in a 5415C tabletop centrifuge (Eppendorf, Westbury, NY) and put into a 15 ml
gravity flow column (Bio-Rad, Hercules, CA). Resin bed was washed with 50 ml of PBS buffer
containing 10 mM imidizole, then protein was eluted with 3 ml PBS containing 150 mM
imidizole and collected in 0.5 ml fractions. Protein fractions were quantified using the Bradford
method (Bradford, 1976). Protein purity was determined to be at least 95 percent by analysis
with SDS-PAGE and staining with coomassie blue Safestain (Invitrogen, Carlsbad, CA).
Enzyme Assays
The following assays were adapted from known methods (Prohl et al., 2000). The
forward reaction was performed by adding 400 µl of buffer (200 mM Tris-HCl pH 8, 100 mM
NH4Cl), 10 µl 20 mM pyridoxal 5’-phosphate, 10 µl 20 mM NaN3, 40 µl 0.2 M glutamic acid,
10 µl 10 mM NADH, 1U glutamate dehydrogenase (bovine pancreas)(Sigma-Aldrich, St. Louis,
MO), and 1 µg purified SlBCAT protein to a quartz cuvette and left to equilibrate at 25̊ C for 5
min. Sample was read in a SmartSpec (BioRad, Hercules, CA) spectrophotometer at 340 nm
until stable absorbance was reached. Reaction was started by addition of 10 µl branched-chain α-
keto acid, after which a decrease in absorbance of NADH was recorded at 340 nm.
The reverse reaction was performed by adding 400 µl of 75 mM Na4P2O7 pH 8, 10 µl of
BCAA, 5 µl of 20 mM pyridoxal phosphate, and 5 µg of purified protein to a 1.5 ml tube and
incubating at 25̊ C for 5 min. The reaction was started by adding 10 µl 10 mM α-ketoglutarate
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and incubating at 25̊ C for 5 min. To stop the reaction, 100 µl of 60% w/v trichloroacetic acid
was added and mixed. Tubes were centrifuged at 10,000 rpm for 1 min to pellet precipitate.
Reactions were transferred to 15 ml polypropylene tubes and 2 ml 2,4-dinitrophenylhydrazine
(0.5% w/v in 2 N H2SO4) were added to each , mixed thoroughly, and incubated at 25̊ C for 10
min. Five ml of toluene was added to each tube and shaken vigorously for 2 min to separate
branched-chain α-keto acids from α-ketoglutarate. The bottom aqueous layer was taken out and
discarded. Five ml of 0.5 N HCl was added to each tube containing the organic phase, shaken
vigorously for 1 min, and then centrifuged at 4000 rpm for 1 min to pellet precipitate. Two ml of
toluene layer was taken off and put in a clean tube containing 2 ml of 10% w/v Na2CO3 and
shaken vigorously for 1 min. One ml of aqueous layer was taken out and added to a clean tube
containing 1 ml 1.5 N NaOH and mixed by inversion. Samples were transferred to plastic
cuvettes and absorbance at 440 nm was recorded by spectrophotometry. Sample with heat-
denatured enzyme was used to obtain blank reading on the spectrophotometer.
For both assays, reactions lacking substrate, enzyme, or with boiled enzyme were used as
controls. Kinetic data for both forward and reverse reactions were calculated using non-linear
regression on GraphPad Prism 5 software (Graphpad Software, La Jolla, CA).
Volatile Collection and Analysis
The following method has been described previously (Tieman et al., 2006). Fruit were
grown in fields at the North Florida Research and Education Center (Live Oak, FL). Fruit were
picked from several plots of six plants each which were randomly distributed throughout the
growing area. Fruits were picked from all parts of the plant near the time of 1:00 pm once per
week for six weeks. Several ripe tomato fruits from each of six replicates of each construct and
S. lycopersicum cv. M82 control were chopped uniformly and placed in glass tubes. Air was
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filtered through a hydrocarbon trap (Agilent, Palo Alto, CA) before flowing through glass tubes
for 1 h and collected with a Super Q (Alltech, Deerfield, IL) column. After collection, 5 µl nonyl
acetate was added to each column as an internal standard, and volatiles were eluted from
columns by rinsing with 150 µl methylene chloride and forcing into a collection vial with
nitrogen gas. Eluted samples were separated through a DB-5 column (Agilent, Palo Alto, CA)
and analyzed on an Agilent 6890N gas chromatograph. Retention times of volatile compounds
were compared to known standards. An Agilent 5975 MS was used to confirm identity of
volatile peaks. Volatile peaks were analyzed using ChemStation software (Agilent, Palo Alto,
CA). Volatile levels were first calculated in ng g-1 FW h-1, and then reported as a percentage of
M82.
Microscopy and Subcellular Localization
Agrobacterium tumefaciens cultures transformed with SlBCAT GFP constructs were
grown overnight in 10 ml Luria broth, then pelleted by centrifugation at 10,000 rpm for 5 min.
Pellets were resuspended in infiltration solution (10 mM MgCl2, 10 mM MES) to an absorbance
of at OD600=0.4. Agrobacterium solutions were injected into the underside of young fully
expanded N. benthamiana leaves with a 2 ml syringe with needle removed. Plants were grown
for four days after infection. Protoplasts were released from N. benthamiana leaves using the
protocol of Yoo et al. (2007). Protoplasts transformed with SlBCAT1 and SlBCAT2 GFP
constructs were stained with 500 nM MitoTracker Orange, as directed by the manufacturer
(Invitrogen, Carlsbad, CA). Cells were visualized using a Zeiss Pascal LSM5 Confocal Laser
Scanning Microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY) with a 40x objective.
GFP was visualized with an argon laser exciting at 488 nm and detected between 500-530 nm. A
79
HeNe laser, exciting at 543 nm, was used to visualize chlorophyll autofluorescence, detected at
633, and MitoTracker Orange, detected at 576 nm.
Metabolite Feeding
S. lycopersicum cv. M82 plants were grown in fields at the North Florida Research and
Education Center (Live Oak, FL). Fruits were harvested and infiltrated with substrate the same
day. Fruits were cut in half, cored, and pericarp discs were cut out with a 1 cm cork borer. Discs
were trimmed horizontally to a depth of 0.5 cm to insure uniformity. For each sample, forty discs
were placed single-layered in plastic Petri dishes. Thirty µl of 10 mM amino acid, 10 mM α-keto
acid, or deionized water were pipetted onto the surface of each pericarp disc, after which the
plates were sealed and incubated at 25̊ C in darkness for 6 h. Discs from each sample were
weighed and placed in glass tubes and volatiles were collected and analyzed as described above.
GC-MS Analyses of Nonvolatile Plant Metabolites
Metabolite extraction, derivatization, GC-MS analysis and data processing were
performed as described previously (Lisec et al., 2006; Schauer et al., 2006), with the exception
that, for low abundance metabolites, a substantially higher extract concentration was injected
onto the GC-MS. The absolute concentration of metabolites was determined by comparison to
standard concentration curves as defined in Schauer et al. (2005a). Metabolites were identified in
comparison to database entries of authentic standards (Kopka et al., 2005; Schauer et al., 2005b).
In addition, the metabolites KIC, KMV, and KIV for which no MST information was available
were identified by analysis of identically derivatized authentic standards.
Analysis of [U-13
C6]Leucine-Labeled Samples
Isotope was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA).
Tomato pericarp discs were extracted as described above. Uncorrected molar percentage
80
enrichments of metabolites were evaluated as described in Giege et al. (2003) by comparison of
the 12C spectral fragments and the isotopic spectral fractions of non-labeled control incubations
with the fragmentation patterns of the [U-13C]leucine-fed tomato pericarp discs as detailed in
Roessner-Tunali et al. (2004). For the calculation of the total label present in a metabolite pool
the mole fractional enrichment of that metabolite was multiplied by the absolute concentration of
that metabolite.
Expression Analysis
RNA was isolated from tomato fruit tissue using the RNeasy Plant RNA Extraction Kit
(Qiagen, Valencia, CA), followed by DNase treatment to rid samples of contaminating DNA.
RNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham,
MA). Omniscript reverse transcriptase (Qiagen, Valencia, CA) was used with 1 µg of each RNA
sample to synthesize cDNA. SYBR Green Master Mix (Applied Biosystems, Foster City, CA)
was used with 1 µl of each cDNA sample for quantitative RT-PCR on the Applied Biosystems
StepOnePlus real-time PCR machine. Five-point standard curves were made for each SlBCAT to
calculate absolute quantity of transcript. Primer specificity was confirmed with melting curve
analysis on the StepOnePlus real-time PCR machine. Plants for expression analysis were grown
in greenhouses. Leaves were harvested when near completely expanded. Flowers, including
calyx, were harvested when fully opened. Green fruit were harvested when fully expanded, light
green in color, hardened seeds and gelatinous locular tissue. For breaker stage, fruit were picked
with 10% or less color change from green. For turning stage, fruit were picked with 20-30%
color change. For red ripe, fruit were picked with 90% or more color change.
81
E. coli Complementation
E. coli strain BW25113 with knockouts in ilvE (JW5606-1) or tyrB (JW4014-2) were
purchased from the Keio Collection (Baba et al., 2006). Double knockouts were constructed as
previously described (Cherepanov and Wackernagel, 1995; Baba et al., 2006) using the FLP
recombinase plasmid pCP20 to excise the kanamycin resistance gene from Δtyrb and then using
P1vir Le392 phage transduction of the Δilve lesion into the Δtyrb background to create the strain
ΔilvE/ΔtyrB. The knockouts were validated by PCR with primers flanking the sites of the two
genes (Supplementary Table S2). Constructs of SlBCATs in pBAD24 were transformed into
ΔilvE/ΔtyrB cells. Cells were first grown in liquid M9 minimal media supplemented with 0.2%
casamino acids and 1mM thiamine hydrochloride, centrifuged to pellet, and resuspended in
sterile water to OD600=0.6. 50 µl of resuspended cells was transferred to 3 ml liquid M9 minimal
medium lacking amino acids (Sambrook et al., 1989) and supplemented with 0.5% w/v arabinose
for induction, 1.0% w/v glycerol for carbon source, and 50 µg/ml carbenicillin. Cell culture
density was measured by OD600 with a SmartSpec spectrophotometer (Bio-Rad, Hercules, CA)
after 10 hours shaking at 37̊ C. Protein expression levels of SlBCATs were confirmed by protein
gel blotting of cells normalized by OD600, probed with mouse Anti-His Antibody (Invitrogen,
Carlsbad, CA).
Amino Acid Analysis of Tomato Fruit by GC-MS
Amino acid levels in M82 control and SlBCAT1-OE and SlBCAT3-OE transgenic ripe
tomato fruit were determined by derivitization with methyl chloroformate and quantification by
GC-MS according to the method of Chen et al. (2010), using an Agilent 6890N GC and 5975
MS. Three technical replicates of each of three biological replicates were analyzed for each
82
transgenic line. Fruit were grown in fields at the North Florida Research and Education Center
(Live Oak, FL).
Statistical analysis
All statistical analysis of data was performed by algorithms in GraphPad Prism5
software. Data indicated as significant is by Students t-test or one-way ANOVA with p<0.05.
83
Table 4-1. Primer sequences used in this study. All oligonucleotides were ordered from Integrated DNA Technologies, Inc. (Coralville, IA).
Primer name Primer sequence (5'→ 3')
ilvE-FlankF GATGCAACATCAGGTCAATGT ilvE-FlankR CGCAATGGTGTTGAACTCTT tyrB-FlankF CTTATTACGCGCCTGACTTC tyrB-FlankR CACAGGCAATAAGGCAAAGC SlBCAT3-pET28bF GCTAGCGAGAGCGCCGCCGTATTT SlBCAT3-pET28bR GTCGACTTTGAGCTCAACAATCCAACCC SlBCAT1-pET28bF GCTAGCTCTGCACAACCTTCAACTTATAG SlBCAT1-pET28bR GTCGACCTTAATCTCAATAATCCAACCCCT SlBCAT4-pET28bF GCTAGCTTTCAGAAGCAGTCACATTTTGC SlBCAT4-pET28bR GTCGACTCATTTTAGCTCAACAGTCCAATT SlBCAT2-pET28bF GCTAGCTACTACACAGCTCAGGTTG SlBCAT2-pET28bR GTCGACTCATTCAATGTCAACAATCCAATC SlBCAT5-pET28bF GCTAGCGCTTCTTCTCAATCTGTTCTCT SlBCAT5-pET28bR GTCGACTTCGATCTCCACGATCCAATT SlBCAT6-pET28bF GCTAGCTGTTATACAGCTCAGGCGG SlBCAT6-pET28bR GTCGACTCATTCAATCTCCACGATCCAAT SlBCAT3-pDONRF AAAAAGCAGGCTCCATGGAGAGCGCCGCCGTATTT SlBCAT3-pDONRR AGAAAGCTGGGTCTTTGAGCTCAACAATCCAACC SlBCAT1-pDONRF AAAAAGCAGGCTCCATGATCATCCAAAGGGCTTCA SlBCAT1-pDONRR AGAAAGCTGGGTCCTTAATCTCAATAATCCAACCC SlBCAT4-pDONRF AAAAAGCAGGCTCCATGGAGAGCGGCGGCG SlBCAT4-pDONRR AGAAAGCTGGGTCTTTTAGCTCAACAGTCCAATTCA SlBCAT2-pDONRF AAAAAGCAGGCTCCATGATTCAAAGGGCCGCACCT SlBCAT2-pDONRR AGAAAGCTGGGTCTTCAATGTCAACAATCCAATC SlBCAT5-pDONRF AAAAAGCAGGCTCCATGGCTTCTTCTCAATCTGTT SlBCAT5-pDONRR AGAAAGCTGGGTCTTCGATCTCCACGATCC SlBCAT6-pDONRF AAAAAGCAGGCTCCATGATTCGAGGAGCCGCATG SlBCAT6-pDONRR AGAAAGCTGGGTCGTAAAGTGACCCTTTTCCAGAAG Attb1-pDONR GGGGACAAGTTTGTACAAAAAAGCAGGCT Attb2-pDONR GGGGACCACTTTGTACAAGAAAGCTGGGT SlBCAT3-pENTR-TOPOF GAATTCATACTCCCTACAGGAGCAACACCA SlBCAT3-pENTR-TOPOR AGAGCTCATTTGAGCTCAACAATCCAACC SlBCAT1-pENTR-TOPOF CACCATGATCATCCAAAGGGCTTCA SlBCAT1-pENTR-TOPOR TTCCACTAGCAATTGGTGTTGAAATGTTT SlBCAT3-qRT-PCRF GTCACCATAACCACCTTCTGG SlBCAT3-qRT-PCRR GGACTCAACTCAATGTTACCG SlBCAT1-qRT-PCRF AGGGCTCTATTTACTTCTTTTGAG SlBCAT1-qRT-PCRR CATACACATTCTTTTAGCACCAATT SlBCAT4-qRT-PCRF TACTTGCCACCACCTTCCC SlBCAT4-qRT-PCRR TCCATAATTCAATATCCCAGAAGC SlBCAT2-qRT-PCRF AATTGTTTGAATTTTCATCTCTGCG SlBCAT2-qRT-PCRR ATACACATTCTTTCAGCTCCAATC SlBCAT5-qRT-PCRF CTCACCTCTTCTCTACACCAC SlBCAT5-qRT-PCRR AATAGCCGTTTGCTTAACAGCC SlBCAT6-qRT-PCRF GGTGTTATACAGCTCAGGCG SlBCAT6-qRT-PCRR CAGGACGAAATAAAAATACTCTCC
84
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BIOGRAPHICAL SKETCH
Gregory Stephen Maloney was born in Wilmington, Delaware and graduated in 2005from
the University of Delaware with a Bachelors of Science degree in Plant Biology and Landscape
Horticulture. After working at Pioneer Hi-Bred for half a year after college, he started graduate
school at University of Florida, where he earned a Doctor of Philosophy in 2010 in Plant
Molecular and Cellular Biology. From there he went on to a post-doctorate position at Wake
Forest University in Winston-Salem, NC.