Chapter 1: Introduction

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MIAMI UNIVERSITY The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation

Kerry A. Lucas

Candidate for the Degree:

Doctor of Philosophy

______Director (Dr. John W. Hawes)

______Reader (Dr. Mike W. Crowder)

______Reader (Dr. Gary A. Lorigan)

______Reader (Dr. Chris A. Makaroff)

______Graduate School Representative (Dr. Alfredo J. Huerta)

ABSTRACT

VALINE METABOLISM IN ARABIDOPSIS

By Kerry A. Lucas

Branched-chain amino acids (BCAAs) are essential amino acids, meaning mammals have the inability to synthesize them de novo and must therefore obtain them through dietary intake. They have been well studied in mammalian systems due to their key role in several metabolic and signaling pathways. Plants, on the other hand, have the ability to synthesize and degrade BCAAs, although the metabolism and importance of these amino acids is less well understood. It is known that mutations in the biosynthetic pathway results in herbicide resistant plants, whereas mutations in the degradation pathway result in an array of responses, from resistance to hormones, to embryo lethality. The largest questions concerning BCAA metabolism in plants involve understanding valine and propionyl-CoA metabolic pathways. It is believed they share a common pathway; however, the localization and actual enzymatic reactions remain controversial. In an effort to piece together this pathway, several techniques were employed. NMR spectroscopic results showed the peroxisomal metabolism of exogenous propionate through a pathway similar to valine degradation in the mitochondria. Reverse genetics using several knockout mutants in the valine degradation pathway, as well as measuring mRNA levels under several stress conditions, helped to resolve much of the ambiguity regarding this pathway. Based on the data presented in this dissertation, we are the first to provide evidence for the metabolism of propionyl-CoA by a modified β-oxidation pathway that utilizes both the mitochondria and the peroxisomes to produce acetyl-CoA. In addition, preliminary data are presented regarding the maintenance of BCAA homeostasis and evidence for the stimulation of protein synthesis.

VALINE METABOLISM IN ARABIDOPSIS

A DISSERTATION

Submitted to the Faculty of

Miami University in partial

fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Chemistry and Biochemistry

Kerry A. Lucas

Miami University

Oxford, OH

2008

Dissertation Director: Dr. John W. Hawes

Table of Contents

Chapter 1: Introduction 1 1.1 Branched-chain Amino Acids 1 1.2 BCAA Metabolism in Mammalian Systems 1 1.2.2 BCAAs and Neurotransmitters 3 1.2.3 BCAAs and the Immune System 4 1.2.4 Metabolic Diseases associated with BCAAs 4 1.2.5 Reactive Intermediates 5 1.2.6 Propionyl-CoA Metabolism 5 1.3 BCAA Metabolism in Higher Plants 7 1.3.1 BCAA Synthesis 7 1.3.2 BCAA Degradation 11 1.3.3 Propionyl-CoA Metabolism in Plants 14 1.4 Sections of the Dissertation 16 1.5 References 17 Chapter 2: Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in A. thaliana 27 2.1 Summary 28 2.2 Introduction 29 2.3 Experimental Procedures 31 2.3.1 Materials 31 2.3.2 Cell Culture and Seedling Preparation and NMR Spectroscopy Procedure 31 2.3.3 End Point Assay for Quantitation of β-Hydroxyisobutyrate 32 2.3.4 Valine, Isobutyrate, and Propionate Growth Response 33 2.4 Results 33 2.4.1 Genes for a Modified Propionate β-Oxidation Pathway in Plants 33 2.4.2 Metabolism of 2-13C-Propionate in A. thaliana Seedlings and Suspension Cell Cultures 37 2.4.3 Effect of chy1 Mutation on Metabolism of Propionate and Isobutyrate 40 2.4.4 Metabolism of 2-13C-Propionate in Different Plants 43

2.4.5 Metabolism of U-13C-Valine in A. thaliana Seedlings 43 2.4.6 Effects of Exogenous Isobutyrate, Propionate, and Valine on Seedling Growth of Wild-type and chy1-3 A. thaliana Seedlings 45 2.5 Discussion 49 2.6 Acknowledgements 52 2.7 References 53 Chapter 3: Functional Diversity of Mitochondrial β-Hydroxyacyl-CoA Hydrolases in A. thaliana 59 3.1 Summary 60 3.2 Introduction 61 3.3 Experimental Procedures 63 3.3.1 Materials 63 3.3.2 Plant Materials and Growth Conditions 63 3.3.3 T-DNA Insertion Mutations 64 3.3.4 Bioinformatic Analysis 64 3.3.5 Recombinant Hydrolases 64 3.3.6 Enzyme Activity 65 3.3.7 Meausring mRNA Levels 65 3.3.8 Complementation 66 3.3.9 Microscopy 66 3.4 Results and Discussion 67 3.4.1 Sequence Identity of Putative Mitochondrial Hydrolases 67 3.4.2 Activity of Recombinant Hydrolases 71 3.4.3 Mitochondrial CHY mRNA Levels of Seedling Growth and the Effects of Sucrose 72 3.4.4 Effects of Metabolites and Oxidative Stress on Mitochondrial CHY mRNA Levels 77 3.4.5 Effects of T-DNA Insertion Mutations 79 3.5 Conclusion 85 3.6 Acknowledgements 86 3.7 References 87 Chapter 4: Evidence for Propionate and Isobutyrate Metabolism involving Mitochondrial Methylmalonate Semialdehyde Dehydrogenase in A. thaliana Mitochondria 92

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4.1 Summary 93 4.2 Introduction 94 4.3 Experimental Procedures 97 4.3.1 Plant Materials and Growth Conditions 97 4.3.2 Measuring mRNA Levels 98 4.4 Results 98 4.4.1 AtMMSDH mRNA levels during germination 98 4.4.2 AtMMSDH Knockout Mutant Affects Seed Viability 100 4.4.3 Knockout Mutants Show Sensitivity to Peroxisomal Metabolites 104 4.5 Discussion 107 4.6 Acknowledgements 111 4.7 References 112 Chapter 5: The Biosynthesis of Leucine and Its Effects on Protein Synthesis in A. thaliana 118 5.1 Summary 119 5.2 Introduction 120 5.3.1 Plant Materials and Growth Conditions 124 5.3.2 NMR Sample Preparation and Procedure 124 5.3.3 Measuring mRNA Levels 125 5.4 Results and Discussion 125 5.4.1 The Effects of Sucrose on Leucine Biosynthesis 125 5.4.2 Stimulation of Protein Synthesis by Leucine 126 5.5 Conclusion 129 5.6 Acknowledgements 131 5.7 References 132 Chapter 6: Conclusion 137 6.1 Understanding Branched-Chain Amino Acids 137 6.2 BCAA Metabolism 137 6.3 Valine Degradation 138 6.4 Valine Metabolism in the Chloroplast 140 6.5 Leucine and Protein Synthesis 142 6.6 Future Directions 142

6.6.1 Model for Valine and Propionyl-CoA Metabolism in Plants 142 6.6.2 Stimulation of Protein Synthesis 143 6.7 References 145

List of Tables

Table 1.1 Genes coding for enzymes of valine degradation. 12 Table 2.1 Genes coding for putative enzymes for catabolism of valine and propionyl-CoA in A. thaliana. 35 Table 3.1 Specific Activity of recombinant hydrolases. 73 Table 4.1 Mitochondrial localization prediction scores and potential cleavage sites for AtMMSDH. 109

List of Figures

Figure 1.1 Branched-chain amino acid degradation pathways. 2 Figure 1.2 Structures of reactive intermediates in valine catabolism. 6 Figure 1.3 Propionyl-CoA metabolic pathways for mammals, bacteria, and plants. 8 Figure 1.4 BCAA biosynthetic pathway. 10 Figure 1.5 Possible routes for propionyl-CoA metabolism in plants. 15 Figure 2.1 Proposed pathways for metabolism of valine and propionyl-CoA in mitochondria and peroxisomes in plants. 34 Figure 2.2 13C-NMR analysis of metabolites produced from 2-13C-propionate in A. thaliana. 38 Figure 2.3 Time course of β-hydroxypropionate production. 39 Figure 2.4 HMQC analysis of standard β-hydroxypropionate and A. thaliana seedlings with 2- 13C-propionate. 41 Figure 2.5 β-Hydroxyisobutyrate accumulation in wild-type and chy1-3 A. thaliana seedlings. 42 Figure 2.6 Production of β-hydroxypropionate in various plants. 44 Figure 2.7 Pathway for the metabolism of valine to leucine. 46 Figure 2.8 13C NMR analysis of A. thaliana seedlings with U-13C-valine. 47 Figure 2.9 Effect of exogenous valine, isobutyrate and propionate on seedling growth. 48 Figure 3.1 Valine catabolic pathway. 62 Figure 3.2 Sequence comparison of A. thaliana CHY proteins with human HIBYL-CoA hydrolase. 68 Figure 3.3 Mitochondrial CHY mRNA levels during seedling growth. 74 Figure 3.4 Effects of sucrose on mitochondrial CHY mRNA levels. 76 Figure 3.5 Effect of metabolites and oxidative stress on mitochondrial CHY mRNA levels. 78 Figure 3.6 Location of T-DNA insertion sites for each mitochondrial CHY gene. 80 Figure 3.7 Light microscopy of A. thaliana siliques. 81 Figure 3.8 Confocal microscopy of chy4 seeds from a single heterozygous silique. 82 Figure 3.9 Valine resistance of chy5 mutant seedlings. 83 Figure 4.1 Valine degradation pathway in mitochondria and peroxisomes. 96 Figure 4.2 mRNA levels of MMSDH during germination and seedling establishment. 99 Figure 4.3 MMSDH mRNA levels in the presence and absence of sucrose. 101

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Figure 4.4 MMSDH mRNA levels with GA and 2,4-D. 102 Figure 4.5 Location of T-DNA insertion and confirmation of homozygous knockout. 103 Figure 4.6 Growth development of mmsdh knockout seedlings. 105 Figure 4.7 mmsdh Knockout seedlings grown with valine, isobutyrate, and propionate. 106 Figure 4.8 Sequence alignment of MMSDH from A. thaliana, rice, human, and rat. 108 Figure 5.1 BCAA biosynthesis pathway. 121 Figure 5.2 TOR signaling pathway. 123 Figure 5.3 13C-NMR studies. 127 Figure 5.4 mRNA levels during leucine biosynthesis in response to sucrose. 128 Figure 5.5 GFP response to leucine. 130 Figure 6.1 Final model for valine and propionyl-CoA metabolism in plants. 141

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Acknowledgements

The success of this dissertation would not have been possible without the love and support of several people throughout the last five years. First, I would like to thank my adviser, Dr. John W. Hawes, who taught me a lot about what it means to be a successful educator, advisor, manager, and most of all what it is like to go through the tenure process – best of luck to you John in your future endeavors! Next, I would like to acknowledge my parents, without their encouragement and support throughout my education, none of this would have been possible. To my friends, Andrea, Hyatt, Ashley, Tamara and Casey – thank you for always encouraging me to reach for higher goals and providing me with the means to do so. To Tracy, Jen, Kim, Molly, Betty and most of all, to Meghan – each of you served as my rock at certain times throughout my graudate career, keeping me grounded, sharing in my joys and frustrations, and making sure I kept a smile on my face throughout it all; you ladies have already made a significant impact on my life and those around you – great things await you! To Matt – my life is better because of you and my success is a direct result of your ability to keep me level headed when I was frustrated, lift me up when I was discouraged, make me smile in the midst of tears, and serve as one of my biggest fans over the last couple of years. I could not have finished this without you and I pray that I will be able to do the same for you next year. To Dr. Chris Makaroff, my dissertation committee (Drs. Gary Lorigan, Mike Crowder, and Alfredo Huerta), and Miami University – thank you for providing me an opportunity to learn, grow, and understand what it means to be a scientist in today’s world. Last but not least, to Ellen Wetli – thank you for being excited about chemistry research, it is refreshing and encouraging!

“I can do all things through Him who stregthens me.” Phil. 4:13

Chapter 1: Introduction

1.1 Branched-chain Amino Acids Branched-chain amino acid (BCAA) metabolism is most commonly associated with genetic in-born errors in mammalian systems (see Section 1.2.4). It is well established that the catabolism of these amino acids (valine, leucine, and isoleucine) provides cellular energy and fuels respiration (1-3). BCAAs are broken down in a similar fashion in both plants and mammals as shown in Figure 1.1. In mammalian systems, this pathway occurs strictly in the mitochondria with the first two steps metabolizing the reactions for all three BCAAs. Branched- chain aminotransferase (BCAT, EC 2.6.1.42) converts the three amino acids to their respective α-ketoacids followed by oxidative decarboxylation into CoA esters by the multi-enzyme complex, branched-chain α-ketoacid dehydrogenase (BCKDH, EC 1.2.4.4). BCKDH is believed to be a key regulatory enzyme in BCAA degradation in skeletal muscle (4, 5). Isoleucine and leucine are further metabolized strictly as CoA esters producing propionyl-CoA and acetyl-CoA from isoleucine, and acetyl-CoA only from leucine. The CoA ester formed in valine degradation is first hydrolyzed to a β-hydroxyacid, and then converted back into a CoA ester in the last step of the pathway in the form of propionyl-CoA. The three enzymes responsible for these reactions are β-hydroxyisobutyryl-CoA hydrolase (HIBYL-CoA hydrolase, EC 3.1.2.4), β- hydroxyisobutyrate dehydrogenase (HIBADH, EC 1.1.1.31), and methylmalonate semialdehyde dehydrogenase (MMSDH, EC 1.1.27).

1.2 BCAA Metabolism in Mammalian Systems The inability of mammals to synthesize the BCAAs requires us to obtain them through dietary sources. BCAAs make up as much as 15-20% of our dietary proteins and thus serve as key building blocks for protein synthesis (6). They are also required for branched-chain fatty acid and neurotransmitter synthesis and serve as nitrogen sources for nonessential amino acids (7). BCAA metabolism has long been a topic of interest for researchers, but recent findings related to the promotion of wound healing (8) and protein stimulation in skeletal muscles of the elderly (9) for example, have generated a renewed interest. There is still much to learn about BCAAs, in particular what other metabolic responses they are involved in and how that can be applied to other organisms.

Figure 1.1 Branched-chain amino acid degradation pathways. 1, branched-chain aminotransferase; 2, branched-chain α-ketoacid dehydrogenase; 3,acyl-CoA dehydrogenase/oxidase; 4, methylcrotonyl-CoA carboxylase; 5, methylglutaconyl-CoA hydratase; 6, enoyl-CoA hydratase; 7, 3-hydroxyisobutyryl-CoA hydrolase; 8, 3- hydroxyisobutyryate dehydrogenase; 9, methylmalonate semialdehyde dehydrogenase; 10, 3- hydroxy-2-methylbutyryl-CoA dehydrogenase; 11, 2-methylacetoacetyl-CoA thiolase.

Leucine, Valine, Isoleucine ketoglutarate 1 glutamate α-Ketoisocaproate, α-Ketoisovalerate, α-Ketomethylvalerate CoA, NAD+ 2 CO2, NADH

Isovaleryl-CoA Isobutyryl-CoA 2-Methylbutyryl-CoA FAD+ FAD+ FAD+ 3 3 3 FADH FADH FADH 3-Methylcrotonyl-CoA Methacrylyl-CoA Tiglyl-CoA HCO -, ATP 3 H2O H O 4 6 2 6 4- PO3 , ADP 3-Methylglutaconyl-CoA β-Hydroxyisobutyryl-CoA β-Hydroxy-2-methylbutyryl-CoA

H2O NAD+ H2O 5 7 10 CoA NADH β-Hydroxyisobutyrate 2-Methylacetoacetyl-CoA Acetoacetate NAD+ CoA 11 8 NADH Acetyl-CoA Methylmalonate semialdehyde H O, CoA, NAD+ Acetyl-CoA Propionyl-CoA 2 9 - HCO3 , CO2, NADH Propionyl-CoA

1.2.1. BCAAs and Protein Synthesis Diaphragm muscles of young rats were first discovered to show an increase in protein synthesis and a decrease in protein degradation when incubated with a mixture of BCAAs (10). Subsequent studies showed similar responses in rat skeletal muscles (11). At the time, it was unclear whether the BCAAs themselves or their catabolic metabolites were responsible for the changes in protein levels (10); however, several groups have since determined that at least in part, the response observed is through the signaling of the mammalian target of rapamycin (mTOR) by leucine (as discussed and reviewed by (12-14)). Recently, a review in 2007 highlighted the mTOR pathway and the conclusions reached in leucine stimulation of protein synthesis. The general belief is that leucine stimulation of protein synthesis occurs through an insulin-independent mechanism that may be a result of eIF4G phosphorylation and its association with eIF4E (12). These factors are responsible for initiating mRNA translation by the coordination of ribosomal subunits on the mRNA. Despite great strides made in this field, the exact details regarding leucine stimulation have yet to be determined.

1.2.2 BCAAs and Neurotransmitters Interestingly, a direct correlation between amine neurotransmitter synthesis, such as the synthesis of serotonin, dopamine, and norepinephrine derived from aromatic amino acids, and dietary BCAAs has been documented. Neurotransmitters have a diverse role in the central nervous system, playing a part in cognition, mood, attention, anger, learning, and appetite (15, 16). In relation to BCAAs, following meals a considerable amount of BCAAs are not only metabolized by the liver but also enter the blood stream resulting in a substantial increase in the level of plasma BCAAs (17, 18). These plasma BCAAs compete with aromatic amino acids to cross the blood brain barrier, creating a decrease in aromatic amino acid transport and thus a decrease in the synthesis of these neurotransmitters (19). These theories have been tested with bipolar subjects during periods of mania. It was hypothesized that a treatment of BCAAs would reduce tyrosine uptake, a precursor to catethcholamine, which is known to be elevated during mania (20). Patients received BCAAs daily for seven days and exhibited significant reductions in symptoms common during manic periods (21).

1.2.3 BCAAs and the Immune System Also of particular interest is the relationship of BCAAs and immune response. A study found that feeding mice a diet with limited BCAAs impaired host defense against the bacterium Salmonella typhimurium. The impaired response is partially based on an increased number of viable S. typhimurium in the liver and spleen and an impaired response to vaccination (22, 23). A number of other metabolic responses have been shown to be associated with exogenous and endogenous BCAAs, which were highlighted at the “Symposium on Branched-Chain Amino Acids1” conference in 2005. The current goal of many researchers is to gain a clearer understanding of BCAA metabolism and the implications on cellular metabolism.

1.2.4 Metabolic Diseases associated with BCAAs One common and accepted way to study BCAA metabolism is to investigate metabolic diseases associated with their metabolism. The most commonly known metabolic disease associated with BCAA metabolism is Maple Syrup Urine Disease (1:185,000 births worldwide (24)). It is characterized by severe ketoacidosis, caused by high concentrations of ketone bodies through deamination of the amino acids and a strong maple syrup odor in urine (25, 26). This disease results from a blockage of BCKDH (Fig. 1.1; step 2) and the body’s inability to further metabolize BCAAs. Symptoms are visible within a week after birth and range from anorexia, failure to thrive, hypertonicity, convulsions, and if left untreated, can result in death. The most common therapy used is a synthetic diet containing a reduction in BCAAs and has shown to be remarkably successful (27). Other inborn errors associated with BCAA catabolism are deficiencies in β-ketothiolase (step 11) and 2-methylbutyryl-CoA dehydrogenase (step 3) in isoleucine metabolism, HIBYL -CoA hydrdolase (step 7), HIBADH (step 8), and MMSDH (step 9) in valine metabolism as well as deficiencies in other acyl-CoA dehydrogenases (step 3). Symptoms range from mild defects that can be treated with dietary supplements to severe abnormalities resulting in death shortly after birth (28-32).

1 Published in a supplement to The Journal of Nutrition. Presented at the conference "Symposium on Branched-Chain Amino Acids," held May 23–24, 2005 in Versailles, France. The conference was sponsored by Ajinomoto USA, Inc. The organizing committee for the symposium and guest editors for the supplement were Luc Cynober, Robert A. Harris, Dennis M. Bier, John O. Holloszy, Sidney M. Morris, Jr., and Yoshiharu Shimomura. Guest editor disclosure: L. Cynober, R. A. Harris, D. M. Bier, J. O. Holloszy, S. M. Morris, Y. Shimomura: expenses for travel to BCAA meeting paid by Ajinomoto USA; D. M. Bier: consults for Ajinomoto USA; S. M. Morris: received compensation from Ajinomoto USA for organizing BCAA conference. 4

1.2.5 Reactive Intermediates As mentioned above, valine is degraded unlike leucine and isoleucine. It is believed that valine degradation occurs in this manner to avoid accumulation of the reactive intermediates methacrylyl-CoA and methylmalonate semialdehyde (29). Methyacrylyl-CoA is a substrate in the reversible reaction catalyzed by enoyl-CoA hydratase with β-hydroxyisobutyryl-CoA. The next step is irreversible and catalyzed by HIBYL-CoA hydrolase, most likely used to pull the reaction forward to avoid accumulation of methacrylyl-CoA (Fig. 1.2A). The toxicity of this accumulation is best shown in patients with a deficiency in HIBYL-CoA hydrolase. Patients exhibit severe deformities and retardation and die within the first 3 months of life (29). These symptoms are attributed to the reactive nature of methacrylyl-CoA, as it is known to non- enzymatically react with key cellular thiols in the mitochondria, such as coenzyme A, glutathione, and cysteamine and result in an oxidative environment (29). Methylmalonate semialdehyde (Fig. 1.2B) is also considered to be a highly-reactive intermediate, although genetic defects related to its metabolism are less severe (31-33). Aldehydes are known to be reactive with proteins and nucleic acids, as well as other cellular nucleophiles (34). They are relatively long-lived, allowing them to diffuse or be transported to other organelles, other cells, or even other tissues (35). It is these characteristics, along with several others discussed by Lidahl, that make the proper disposal and use of these aldehydes key to maintaining cellular homeostasis (35).

1.2.6 Propionyl-CoA Metabolism Unlike valine catabolism that is highly-conserved between organisms, the metabolism of the end product of valine degradation, propionyl-CoA, is quite different. In mammals, propionyl-CoA degradation is performed within the peroxisomes following β-oxidation in its conversion to succinyl-CoA, which eventually feeds into the TCA cycle (Fig. 1.3). This biotin- dependent pathway is well characterized, and defects associated with this pathway result in a range of disorders such as ketoacidosis, lethargy, retardation, and in severe cases, death at an early age (36-40).

Figure 1.2 Structures of reactive intermediates in valine catabolism. A, methacrylyl-CoA; B, methylmalonate semialdehyde.

A. B.

CH2 CH3 SCoA O OH H3C O O

Bacterial and plant systems each have their own pathways for metabolizing propionyl- CoA as they do not appear to have the same enzymes available to carry out the reactions as in mammalian systems. Bacteria and yeast utilize a methylcitrate pathway analogous to the TCA and glyoxylate cycles (41). Plants utilize a separate pathway that will be discussed in more detail in section 1.3.3 of this chapter.

1.3 BCAA Metabolism in Higher Plants BCAAs have long been an intriguing area of research for plant scientists, particularly because plants provide a source of essential amino acids required for human and animal diets. Additionally, BCAAs and their subsequent metabolites have also led to significant strides in both agricultural and industrial research. For instance, research conducted on BCAA synthesis has resulted in widely used herbicide resistance crops (42), whereas studies on propionyl-CoA metabolism have resulted in the application of intermediates used in bio-degradable plastics (43, 44).

1.3.1 BCAA Synthesis BCAAs brought about new interest with the discovery of two classes of herbicides that inhibit parts of the BCAA biosynthesis pathway (45, 46). These herbicides changed agricultural practices due to their ability to control weeds found in diverse crops with high efficiency while remaining highly potent, yet exhibiting low toxicity to mammals (42). In an effort to understand this inhibition, numerous reports were published detailing BCAA synthesis in plants (42, 47-49). In plants it is known that synthesis occurs in the chloroplast separate from degradation, which takes place primarily in the mitochondrion (50). Throughout plant growth it was found that the genes required for BCAA synthesis are expressed in almost all tissues; however, the activity and expression is the highest in young tissues associated with rapid growth. (42). The pathway for BCAA synthesis is quite unique in that valine and isoleucine follow four parallel steps, whereas leucine is synthesized from a branch point in the valine pathway (Fig. 1.4).

Figure 1.3 Propionyl-CoA metabolic pathways for mammals, bacteria, and plants.

(S)-Methymalonyl-CoA

(R)-Methylmalonyl-CoA Mammals (Biotin dependent) Succinyl-CoA

TCA Cycle

Acrylyl-CoA Propionyl-CoA

Hydroxypropionyl-CoA

Plants? Hydroxypropionate

Malonate Semialdehyde

Acetyl-CoA 2-methylcitrate

Microbial 2-methyl-cis-aconitate

Methylisocitrate

Pyruvate Succinate

The first step in isoleucine synthesis utilizes threonine dehydratase (TD, EC 4.2.1.16) to convert threonine to α-ketobutyrate with the release of ammonia. This serves as a regulatory point for isoleucine synthesis through feedback inhibition by isoleucine (51). Subsequently, acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyzes the first step of valine synthesis by condensing two pyruvates to form acetolactate as well as condensing α-ketobutyrate with pyruvate in the isoleucine pathway. Like TD, AHAS serves as a regulatory point in valine synthesis through feedback inhibition by valine (52). AHAS has been well studied since the discovery of its inhibition by two classes of herbicides, imidazolinones and sulfonylureas (45, 46). These herbicides function through an uncompetitive binding of the enzyme-pyruvate complex (46, 53). It was then determined that mutations in the gene coding for AHAS in A. thaliana resulted in herbicide-resistant plants. This finding is now applied to canola, corn, soybean, and wheat (reviewed by (42)). Mutations in genes coding for other enzymes in BCAA synthesis have also produced herbicide-resistant plants but not to the same efficacy as those in AHAS mutants (42). The next two steps in valine and isoleucine synthesis include reactions by α-ketoacid reductoisomerase (KARI, EC 1.1.1.86) and dihydroxyacid dehydrogenase (DHAD, 4.2.1.9). Little is known about KARI; however, it is known that DHAD is required for biosynthesis as shown by auxotrophic mutants of N. plumbaginifolia that lack DHAD (54). The last step requires transamination of α-ketoisovalerate and α-ketomethylvalerate to valine and isoleucine, respectively, by branched-chain aminotransferase (BCAT). There are three known BCATs in the chloroplast of A. thaliana, BCAT-2, BCAT-3, and BCAT-5 (50). It is believed one is responsible for valine transamination, while another functions as a leucine/isoleucine transaminase (55); however, it is not known which BCAT enzyme performs each action. Leucine synthesis is the least studied of the three BCAA synthesis pathways. The first step towards leucine synthesis occurs with the condensation of acetyl-CoA and α-ketoisovalerate (found in valine synthesis) by isopropylmalate synthase (IPMS, EC 2.2.3.13). IPMS serves as the regulatory enzyme for leucine synthesis by way of feedback inhibition from leucine (47). There are multiple isoforms of IPMS in A. thaliana, although only two have been characterized to date as true IPMS enzymes, and there seems to be redundancies in their expression and function (56, 57).

Figure 1.4 BCAA biosynthetic pathway. 1, threonine dehydratase; 2, acetolactate synthase; 3, α-ketoacid reductoisomerase; 4, dihydroxyacid dehydrogenase; 5, branched-chain aminotransferase; 6, isopropylmalate synthase; 7, isopropylmalate dehydratase; 8, isopropylmalate dehydrogenase.

Threonine

NH 1 3

2 x Pyruvate Ketobutyrate + Pyruvate HET-PP HET-PP 2 TPP TPP 2-Acetolactate 2-Aceto-2-hydroxybutyrate NADPH, H+ NADPH, H+ 3 NADP+ NADP+ 2,3-dihydroxy-3-isovalerate 2,3-dihydroxy-3-methylvalerate

H O H O 2 4 2 CoA-SH Acetyl-CoA, H2O 2-Isopropylmalate α-Ketoisovalerate α-Ketomethylvalerate 6 7 glutamate 5 glutamate ketoglutarate ketoglutarate 3-Isopropylmalate Valine Isoleucine 8 NAD+ NADH, H+ α-Ketoisocaproate glutamate 5 ketoglutarate Leucine

Very little is known about the steps in leucine synthesis catalyzed by isopropylmalate dehydratase (IPMDHT, EC 4.2.1.33) and then isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85). Ultimately these steps lead to the production of α-ketoisocaproate. As in valine and isoleucine synthesis, the succeeding step is the transamination of α-ketoisocaproate to leucine, by way of BCAT. Leucine is known to play a key signaling role in several metabolic pathways in mammalian systems, however very little is know about its potential role in plants. This potential role is addressed in detail in Chapter 5.

1.3.2 BCAA Degradation The degradation of BCAAs has also been explored in plants due to the role several enzymes also have in fatty acid oxidation. In plants, the valine degradation pathway is similar to that seen in the mammalian systems (Fig. 1.1), however unlike mammals, plants have evolved to code for multiple isoforms for several of the enzymes in the pathway. Table 1.1 summarizes enzymes in the valine degradation pathway, the genes that codes for them, their loci identifier, predicted or known localization, and any functional protein or gene expression data available. It is known that mRNA and protein levels for BCAA degradation are highest throughout germination, in senescing tissues, and during carbon starvation (58-60). High protein turnover during germination is the result of plants switching from consumption of storage proteins to synthesizing new proteins in fast growing tissues (58). Throughout senescence, dying sections of plants will break down proteins for use of amino acids elsewhere, for instance as energy sources to store up lipid reserves during seed development (61, 62). Environmental stresses can limit photosynthetic activity by depleting plants of available carbon sources (60). In order to fuel cellular metabolism, plants utilize the degradation of BCAAs as alternative carbon sources (60, 63-65). In support of BCAA degradation to fuel metabolism, Davies and Humphrey found that the majority of leucine and isoleucine generated from protein turnover is not directly reused in protein synthesis in Lemna minor, but instead further metabolized in the cell (66).

Table 1.1 Genes coding for enzymes of valine degradation. Locus/Gene Enzyme Enzyme Localizationa Gene Expression Data Reference Name Substrate Branched-Chain Aminotransferase BCAT1 At1g10060 M all BCAAs Expressed in all tissues, (50, 67) primarily in seedlings, not sucrose responsive Branched-Chain α-Ketoacid Dehydrogenase BCKDH E1α At1g21400 M Lowest expression in roots, (68) responsive to a number of stress conditions At5g09300 M Highest expression in (68, 69) root tip BCKDH E1β At3g13450 M Highest expression in (68, 69) DIN4 petals, repressed by sucrose BCKDH E2 At3g06850 M Highest expression in (68, 69) DIN3 petals, repressed by sucrose BCKDH E3 At3g17240 M Highest expression in (68, 70) LPD2 root hair At1g48030 M Expressed in most tissues, (70) LPD1 induced by light Acyl-CoA Dehydrogenase/ Oxidase Isovaleryl-CoA At3g45300 M isovaleryl-CoA, Highest expression in (68, 71) Dehydrogenase IVD isobutyryl-CoA petals, repressed by sucrose ATP binding/ At3g06810 P (1) Mutant is IBA responsive (72) acyl-CoA IBR3 Dehydrogenase At4g16760 P (1) medium to long Highest expression in sepal (68, 73-75) ACX1 chain FAs At5g65110 P (2) long chain FAs Highest expression in seed, (68, 73-75) ACX2 pollen and senescent leaves, mutant has delayed germination At1g06290 P (2) medium chain Highest expression in seeds (68, 74, 76, ACX3 FAs and roots, mutant is less 77) sensitive to IBA At3g51840 P (1) short chain FAs Highest expression in petal (68, 74, 78) ACX4 and sepal At2g35690 P (1) Expressed in all tissues and (74) ACX5 throughout development At1g06310 P (2) Highest expression in root (68, 74) ACX6 tip, but low overall Enoyl-CoA Hydratase At3g24360 M Increased expression with (79) Lucas CHY6 sucrose unpublished At4g29010 P (1) short-chain Expressed throughout all (80) AIM1 acyl-CoAs tissues At3g06860 P (1) long chain acyl- Highest expression in (68, 80, 81) MFP2 CoAs seeds, mutants require sucrose for seedling establishment 12

At5g43280 P (1) unsaturated Expressed throughout all (68, 82) ATDCl1 FAs tissues At4g16800 NCS Highest expression in seed (68) Hydroxyacyl-CoA Hydrolase

At4g31810 M β-HIBYL-CoA Highest expression in root (68, 83), CHY4 tip, responds to oxidative Lucas stress, mutant is embryo unpublished lethal At3g60510 M β-HIBYL-CoA Mutant is valine resistant (83), Lucas CHY5 unpublished At5g65940 P (1) β-HIBYL-CoA Expressed in all tissues, (68, 83) CHY1 repressed by sucrose, mutant is IBA resistant At2g30660 P (1) (83) At2g30650 P (1) (83) Hydroxyacid Dehydrogenase At4g20930 M Highest expression in (68) petal/sepal and senescent leaf At4g29120 M MMSDH/MSDH ALDH6B2 At2g14170 M Mutant has multiple (84), Lucas phenotypes and is sensitive unpublished to valine, isobutyrate, propionate aMitochondrial predicted localizations [M] are based on prediction scores from PREDOTAR (85) and TargetP (86) or based on the cited literature. Type 1 peroxisomal predicted localizations [P (1)] are based on the presence of C-terminal SKL or AKL sequences. Type 2 peroxisomal predicted localizations [P (2)] are based on the conserved motif as described in the AraPerox database (87).

Abbreviations: BCAAs, branched-chain amino acids; FAs, fatty acids; HIBYL-CoA, hydroxyisobutyryl-CoA; IBA, indole-3-butyric acid; MSDH, malonate semialdehyde dehydrogenase; MMSDH, methylmalonate semialdehyde dehydrogenase

It is believed that BCAA metabolism occurs in the mitochondrion of plants; however, with respect to valine catabolism, there is evidence this pathway also exists in peroxisomes. Numerous isoforms for the majority of the enzymes in the valine degradation pathway in A. thaliana are localized to either the mitochondria or the peroxisomes. The only enzymes of this pathway in A. thaliana containing a single isoform are BCAT (67), BCKDH (69), and presumably, methylmalonate semialdehyde dehydrogenase. The presence of multiple isoforms is not limited to A. thaliana alone and has been shown to be the case in many plants including rice and poplar. It is possible that multiple isoforms are needed for different stages of growth and under various stress conditions within the plant.

1.3.3 Propionyl-CoA Metabolism in Plants Of the topics discussed thus far, propionyl-CoA metabolism in plants is the most controversial. Early reports suggested a pathway similar to mammals using propionyl-CoA carboxylase to produce succinyl-CoA (88). Since plants do not appear to have the necessary enzymes to catalyze those reactions, it is believed that plants metabolize propionyl-CoA utilizing a modified β-oxidation pathway (Fig. 1.5). However, localization of this pathway and valine degradation has been controversial. The modified β-oxidation pathway utilizes the enzymes found in valine degradation to convert propionyl-CoA to more metabolically active substrates. Data acquired in support of this pathway used propionate to monitor its progress during degradation as it is well-known that propionate is converted to propionyl-CoA by propionyl-CoA synthase in all organisms (89).

Early reports have confirmed that plants do not require CO2 to degrade propionate, as is the case for mammals suggesting a different pathway (90, 91). Additionally, it was also shown that isolated peroxisomes produced acetyl-CoA from propionyl-CoA using 14C propionate; however, the purity, and in some cases the method for peroxisomal isolation, was not reported (92, 93). The controversy here results from the fact that plants do not appear to have the last two enzymes needed to convert β-hydroxypropionate to acetyl-CoA in the peroxisomes. Based on evidence from the completed A. thaliana genome, the necessary proteins are localized only in mitochondria, suggesting that perhaps the previous reports demonstrating a peroxisomal metabolism of propionyl-CoA to acetyl-CoA may have had samples contaminated with mitochondria.

Figure 1.5 Possible routes for propionyl-CoA metabolism in plants. 1, branched-chain aminotransferase; 2, branched-chain α-ketoacid dehydrogenase; 3, acyl-CoA dehydrogenase/oxidase; 4, enoyl-CoA hydratase; 5, β-hydroxyisobutyryl-CoA hydrolase; 6, β- hydroxyisobutyrate dehydrogenase; 7, methylmalonate semialdehyde dehydrogenase.

Mitochondria Valine

Keto-isovalerate CoASH 2 Peroxizomes Isobutyryl-CoA Propionyl-CoA Propionyl-CoA

3 3 3

Methylacrylyl-CoA Acrylyl-CoA Acrylyl-CoA

4 4 4

Hydroxyisobutyryl-CoA Hydroxypropionyl-CoA Hydroxypropionyl-CoA

5 CoASH 5 CoASH 5 CoASH

Hydroxyisobutyrate Hydroxypropionate Hydroxypropionate

6 6

Methylmalonate Semialdehyde Malonate Semialdehyde

7 7

Propionyl-CoA Acetyl-CoA

Evidence of a transport system between mitochondria and peroxisomes which would support the conversion of propionyl-CoA to acetyl-CoA in plant cells has been shown. Recently, an acyl-CoA transporter was discovered in A. thaliana that lends support for the transport of propionyl-CoA out of mitochondria and into peroxisomes for metabolism (94). Also, evidence for transport of metabolites in and out of the peroxisomes has been shown that would support the transport of β-hydroxypropionate from the peroxisomes back to the mitochondria to be further metabolized (89).

1.4 Sections of the Dissertation BCAAs are a highly studied class of amino acids in mammalian systems due to their association with several metabolic pathways. The same is true for plants; however, much less is known about the localization of the proteins that catalyze the reactions, the process of propionyl- CoA metabolism, and why plants have multiple isoforms in BCAA metabolism. Chapter 2, originally published in the Journal of Biological Chemistry, describes the first evidence for the production of propionyl-CoA metabolic intermediates by 1H and 13C NMR spectroscopy, clearly demonstrating that exogenous propionate and isobutyrate are metabolized in the peroxisomes. Chapter 3, to be submitted to Plant Physiology and Biochemistry, examines three mitochondrial hydroxyacyl-CoA hydrolases (by reverse genetics and mRNA expression studies) which were previously unexplored. Chapter 4, to be submitted to Plant Physiology and Biochemistry, describes phenotypes of a true methylmalonate semialdehyde dehydrogenase knockout in plants and the significance behind this data in regards to how valine is degraded and propionyl-CoA metabolized in plants. Chapter 5, to be submitted as a hypothesis paper to The FASEB Journal, elaborates on data produced in Chapter 2 by investigating the importance of leucine in plant systems. Data from this study resulted in the first findings that support the stimulation of protein synthesis in plants by exogenous leucine. Finally, Chapter 6 summarizes the details presented in this dissertation by illustrating a final model of valine and propionyl-CoA metabolism in plants, as well as future directions with these projects.

1.5 References

1. Robinson, W. G., Nagle, R., Bachhawat, B. K., Kupiecki, F. P., and Coon, M. J. (1957) Coenzyme A Thiol Esters of Isobutyric, Methacrylic, and Beta-Hydroxyisobutyric Acids as Intermediates in the Enzyme Degradation of Valine. J. Biol. Chem. 224, 1-11.

2. Hawes, J. W., Jaskiewicz, J., Shimomura, Y., Huang, B., Bunting, J., Harper, E. T., and Harris, R. A. (1996) Primary Structure and Tissue-Specific Expression of Human Beta- Hydroxyisobutyryl-Coenzyme A Hydrolase. J. Biol. Chem. 271, 26430-26434.

3. Kinnory, D. S., Takeda, Y., and Greenberg, D. M. (1955) Isotope Studies on the Metabolism of Valine. J. Biol. Chem. 212, 385-396.

4. Yeaman, S. J. (1989) The 2-Oxo Acid Dehydrogenase Complexes: Recent Advances. Biochem. J. 257, 625.

5. Shimomura, Y., Fujii, H., Suzuki, M., Murakami, T., Fujitsuka, N., and Nakai, N. (1995) Branched-Chain α-Keto Acid Dehydrogenase Complex in Rat Skeletal Muscle: Regulation of the Activity and Gene Expression by Nutrition and Physical Exercise. J. Nutr. 125, 1762S- 1765.

6. Nutrient Data Laboratory, A.R.S. (2004) USDA Nutrient Database for Standard Reference. Release 17 ed., USDA, Washington, DC.

7. Tom, A., and Nair, K. S. (2006) Assessment of Branched-Chain Amino Acid Status and Potential for Biomarkers. J. Nutr. 136, 324S-330.

8. Zhang, X., Chinkes, D. L., and Wolfe, R. R. (2004) Leucine Supplementation has an Anabolic Effect on Proteins in Rabbit Skin Wound and Muscle. J. Nutr. 134, 3313-3318.

9. Volpi, E., Kobayashi, H., Sheffield-Moore, M., Mittendorfer, B., and Wolfe, R. R. (2003) Essential Amino Acids are Primarily Responsible for the Amino Acid Stimulation of Muscle Protein Anabolism in Healthy Elderly Adults. Am J Clin Nutr. 78, 250-258.

10. Fulks, R. M., Li, J. B., and Goldberg, A. L. (1975) Effects of Insulin, Glucose, and Amino Acids on Protein Turnover in Rat Diaphragm. J. Biol. Chem. 250, 290-298.

11. Garlick, P. J., and Grant, I. (1988) Amino Acid Infusion Increases the Sensitivity of Muscle Protein Synthesis in vivo to Insulin. Effect of Branched-Chain Amino Acids. Biochem. J. 254, 579.

12. Stipanuk, M. H. (2007) Leucine and Protein Synthesis: MTOR and Beyond. Nutr. Rev. 65, 122-129.

13. Kimball, S. R., and Jefferson, L. S. (2006) Signaling Pathways and Molecular Mechanisms through which Branched-Chain Amino Acids Mediate Translational Control of Protein Synthesis. J. Nutr. 136, 227S-231.

14. Norton, L. E., and Layman, D. K. (2006) Leucine Regulates Translation Initiation of Protein Synthesis in Skeletal Muscle After Exercise. J. Nutr. 136, 533S-537.

15. Daikhin, Y., and Yudkoff, M. (2000) Compartmentation of Brain Glutamate Metabolism in Neurons and Glia. J. Nutr. 130, 1026.

16. Fernstrom, J. D. (1990) Aromatic Amino Acids and Monoamine Synthesis in the Central Nervous System: Influence of the Diet. J. Nutr. Biochem. 1, 508.

17. Platell, C., Kong, S. E., McCauley, R., and Hall, J. C. (2000) Branched-Chain Amino Acids. J. Gastroenterol. Hepatol. 15, 706-717.

18. Grimes, M. A., Cameron, J. L., and Fernstrom, J. D. (2000) Cerebrospinal Fluid Concentrations of Tryptophan and 5-Hydroxyindoleacetic Acid in Macaca mulatta: Diurnal Variations and Response to Chronic Changes in Dietary Protein Intake. Neurochem. Res. 25, 413.

19. Fernstrom, M. H., Volk, E. A., and Fernstrom, J. D. (1986) in vivo Inhibition of Tyrosine Uptake into Rat Retina by Large Neutral but Not Acidic Amino Acids. Am. J. Physiol. 251, E393.

20. Scarna, A., Gijsman, H. J., McTavish, S. F. B., Harmer, C. J., Cowen, P. J., and Goodwin, G. M. (2003) Effects of a Branched-Chain Amino Acid Drink in Mania. Br. J. Psychiatry. 182, 210-213.

21. Montgomery, A. J., McTavish, S. F. B., Cowen, P. J., and Grasby, P. M. (2003) Reduction of Brain Dopamine Concentration with Dietary Tyrosine Plus Phenylalanine Depletion: An [11C]Raclopride PET Study. Am. J. Psychiatry. 160, 1887-1889.

22. Petro, T. M., and Bhattacharjee, J. K. (1981) Effect of Dietary Essential Amino Acid Limitations upon the Susceptibility to Salmonella Typhimurium and the Effect upon Humoral and Cellular Immune Responses in Mice. Infect. Immun. 32, 251.

23. Calder, P. C. (2006) Branched-Chain Amino Acids and Immunity. J. Nutr. 136, 288S-293.

24. Nellis, M. M., Kasinski, A., Carlson, M., Allen, R., Schaefer, A. M., Schwartz, E. M., and Danner, D. J. (2003) Relationship of Causative Genetic Mutations in Maple Syrup Urine Disease with their Clinical Expression. Mol. Genet. Metab. 80, 189-195.

25. Dancis, J., Levitz, M., Miller, S., and Westall, R. G. (1959) Maple Syrup Urine Disease. Br. Med. J. 1, 91.

26. Dancis, J., Levitz, M., and Westall, R. G. (1960) Maple Syrup Urine Disease: Branched- Chain Keto-Aciduria. Pediatrics. 25, 72.

27. Snyderman, S. E., Norton, P. M., Roitman, E., and Holt, L. E. Jr. (1964) Maple Syrup Urine Disease, with Particular Reference to Dietotherapy. Pediatrics. 34, 454.

28. Pasquali, M., Monsen, G., Richardson, L., Alston, M., and Longo, N. (2006) Biochemical Findings in Common Inborn Errors of Metabolism. Am. J. Med. Genet. Part C Semin. Med. Genet. 142c, 64.

29. Brown, G. K., Hunt, S. M., Scholem, R., Fowler, K., Grimes, A., Mercer, J. F., Truscott, R. M., Cotton, R. G., Rogers, J. G., and Danks, D. M. (1982) Beta-Hydroxyisobutyryl Coenzyme A Deacylase Deficiency: A Defect in Valine Metabolism Associated with Physical Malformations. Pediatrics. 70, 532-538.

30. Loupatty, F. J., van der Steen, Annemarie, IJlst, L., Ruiter, Jos P. N., Ofman, R., Baumgartner, M. R., Ballhausen, D., Yamaguchi, S., Duran, M., and Wanders, Ronald J. A. (2006) Clinical, Biochemical, and Molecular Findings in Three Patients with 3- Hydroxyisobutyric Aciduria. Mol. Genet. Metab. 87, 243.

31. Gibson, K. M., Lee, C. F., Bennett, M. J., Holmes, B., and Nyhan, W. L. (1993) Combined Malonic, Methylmalonic and Ethylmalonic Acid Semialdehyde Dehydrogenase Deficiencies: An Inborn Error of Beta-Alanine, L-Valine and L-Alloisoleucine Metabolism? J. Inherit. Metab. Dis. 16, 563.

32. Roe, C. R., Struys, E., Kok, R. M., Roe, D. S., Harris, R. A., and Jakobs, C. (1998) Methylmalonic Semialdehyde Dehydrogenase Deficiency: Psychomotor Delay and Methylmalonic Aciduria without Metabolic Decompensation. Mol. Genet. Metab. 65, 35.

33. Gray, R. G. F., Pollitt, R. J., Webley, J. (1987) Methylmalonic Semialdehyde Dehydrogenase Deficiency: Demonstration of Defective Valine and β-Alanine Metabolism and Reduced Malonic Semialdehyde Dehydrogenase Activity in Cultured Fibroblasts. Biochem. Med. Metab. Biol. 38, 121.

34. Schauenstein, E., Esterbauer, H., Zollner, H., and Gore, P. H. (1977) Aldehydes in Biological Systems: Their Natural Occurrence and Biological Activities. Pion, London.

35. Lindahl, R. (1992) Aldehyde Dehydrogenases and their Role in Carcinogenesis. Crit. Rev. Biochem. Mol. Biol. 27, 283.

36. Oberholzer, V. G., Levin, B., Burgess, E. A., and Young, W. F. (1967) Methylmalonic Aciduria. an Inborn Error of Metabolism Leading to Chronic Metabolic Acidosis. Arch. Dis. Child. 42, 492.

37. Stokke, O., Eldjarn, L., Norum, K. R., Steen-Johnsen, J., and Halvorsen, S. (1967) Methylmalonic Acidemia. New Inborn Error of Metabolism which may Cause Fatal Acidosis in the Neonatal Period. Scand. J. Clin. Lab. Invest. 20, 313.

38. Morrow, G., Barness, L. A., Cardinale, G. J., Abeles, R. H., and Flaks, J. G. (1969) Congenital Methylmalonic Acidemia: Enzymatic Evidence for Two Forms of the Disease. Proc. Natl. Acad. Sci. 63, 191-197.

39. Hommes, F. A., Kuipers, J. R., Elema, J. D., Jansen, J. F., and Jonxis, J. H. (1968) Propionicacidemia, a New Inborn Error of Metabolism. Pediatr. Res. 2, 519.

40. Rosenberg, L. E. (1972) Disorders of Propionate, Methylmalonate, and Vitamin B12 Metabolism, in Metab. Basis Inherited Dis.(J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, Eds.) 3rd ed., pp 440-458, McGraw-Hill, New York, N.Y.

41. Horswill, A. R., and Escalante-Semerena, J. C. (2001) In Vitro Conversion of Propionate to Pyruvate by Salmonella enterica Enzymes: 2-Methylcitrate Dehydratase (PrpD) and Aconitase Enzymes Catalyze the Conversion of 2-Methylcitrate to 2-Methylisocitrate. Biochemistry. 40, 4703-4713.

42. Singh, B. K. (1995) Biosynthesis of Branched-Chain Amino Acids: From Test Tube to Field. The Plant Cell Online. 7, 935.

43. Rezzonico, E., Moire, L., and Poirier, Y. (2002) Polymers of 3-Hydroxyacids in Plants. Phytochemistry Rev. 1, 87-92.

44. Snell, K. D., and Peoples, O. P. (2002) Polyhydroxyalkanoate Polymers and their Production in Transgenic Plants. Metab. Eng. 4, 29-40.

45. Ray, T. B. (1984) Site of Action of Chlorsulfuron: Inhibition of Valine and Isoleucine Biosynthesis in Plants. Plant Physiol. 75, 827-831.

46. Shaner, D. L., Anderson, P. C., and Stidham, M. A. (1984) Imidazolinones: Potent Inhibitors of Acetohydroxyacid Synthase. Plant Physiol. 76, 545-546.

47. Singh, B. (1999) Biosynthesis of Valine, Leucine and Isoleucine. in Plant amino acids : Biochemistry and Biotechnology (B. K. Singh, Ed.) 1st ed., pp 227-247, Marcel Dekker, New York.

48. Wittenbach, V. A., and Abell, L. M. (1999) Inhibitors of Valine, Leucine, and Isoleucine Biosynthesis, in Plant Amino Acids (B. K. Singh, Ed.) 1st ed., pp 385-416, Marcel Dekker, Inc., New York.

49. Ellerstrom, M., Josefsson, L. G., Rask, L., and Ronne, H. (1992) Cloning of a cDNA for Rape Chloroplast 3-Isopropylmalate Dehydrogenase by Genetic Complementation in Yeast. Plant Mol. Biol. 18, 557.

50. Diebold, R., Schuster, J., Daschner, K., and Binder, S. (2002) The Branched-Chain Amino Acid Transaminase Gene Family in Arabidopsis Encodes Plastid and Mitochondrial Proteins. Plant Physiol. 129, 540-550.

51. Sharma, R. K., and Mazumder, R. (1970) Purification, Properties, and Feedback Control of l- Threonine Dehydratase from Spinach. J. Biol. Chem. 245, 3008-3014.

52. Borstlap, A. C. (1972) Changes in the Free Amino Acids of Spirodela Polyrhiza during Growth Inhibition by L-Valine, L-Isoleucine, or L-Leucine. Gas Chromatographic Study. Acta Bot. Neerl. 21, 404.

53. Schloss, J. V., and Aulabaugh, A. (1990) Acetolactate Synthase, Mechanism of Action and its Herbicide-Binding Site. Pestic. Sci. 29, 283.

54. Wallsgrove, R. M., Risiott, R., Negrutiu, I., and Bright, Simon W. J. (1986) Biochemical Characterization of Nicotiana plumbaginifolia Auxotrophs that Require Branched-Chain Amino Acids. Plant Cell Rep. 5, 223.

55. Hagelstein, P., Sieve, B., Klein, M., Jans, H., and Schultz, G. (1997) Leucine Synthesis in Chloroplasts. Leucine/isoleucine Aminotransferase and Valine Aminotransferase are Different Enzymes in Spinach Chloroplasts. J. Plant Physiol. 150, 23.

56. de Kraker, J., Luck, K., Textor, S., Tokuhisa, J. G., and Gershenzon, J. (2006) Two Arabidopsis Genes (IPMS1 and IPMS2) Encode Isopropylmalate Synthase, the Branchpoint Step in the Biosynthesis of Leucine. Plant Physiol. 143, 970.

57. Junk, D. J., and Mourad, G. S. (2002) Isolation and Expression Analysis of the Isopropylmalate Synthase Gene Family of Arabidopsis thaliana. J. Exp. Bot. 53, 2453.

58. Anderson, M. D., Che, P., Song, J., Nikolau, B. J., and Wurtele, E. S. (1998) 3- Methylcrotonyl-Coenzyme A Carboxylase is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants. Plant Physiol. 118, 1127-1138.

59. Dunford, R., Kirk, D., and Ap Rees, T. (1990) Respiration of Valine by Higher Plants. Phytochemistry. 29, 41-43.

60. Aubert, S., Alban, C., Bligny, R., and Douce, R. (1996) Induction of β-Methylcrotonyl- Coenzyme A Carboxylase in Higher Plant Cells during Carbohydrate Starvation: Evidence for a Role of MCCase in Leucine Catabolism. FEBS Lett. 383, 175-180.

61. Lohman, K. N., Gan, S., John, M. C., and Amasino, R. M. (1994) Molecular Analysis of Natural Leaf Senescence in Arabidopsis thaliana. Physiol. Plantarum. 92, 322.

62. Simpson, R. J., and Dalling, M. J. (1981) Nitrogen Redistribution during Grain Growth in Wheat (Triticum Aestivum L.). III. Enzymology and Transport of Amino Acids from Senescing Flag Leaves. Planta. 151, 447.

63. Journet, E., Bligny, R., and Douce, R. (1986) Biochemical Changes during Sucrose Deprivation in Higher Plant Cells. J. Biol. Chem. 261, 3193-3199.

64. Dorne, A. J., Bligny, R., Rebeille, F., Roby, C., and Douce, R. (1987) Fatty Acid Disappearance and Phosphorylcholine Accumulation in Higher Plant Cells After a Long Period of Sucrose Deprivation. Plant Physiol. Biochem. 25, 589.

65. Brouquisse, R., James, F., Pradet, A., and Raymond, P. (1992) Asparagine Metabolism and Nitrogen Distribution during Protein Degradation in Sugar-Starved Maize Root Tips. Planta. 188, 384.

66. Davies, D. D., and Humphrey, T. J. (1978) Amino Acid Recycling in Relation to Protein Turnover. Plant Physiol. 61, 54-58.

67. Schuster, J., and Binder, S. (2005) The Mitochondrial Branched-Chain Aminotransferase (AtBCAT-1) is Capable to Initiate Degradation of Leucine, Isoleucine and Valine in almost all Tissues in Arabidopsis thaliana. Plant Mol. Biol. 57, 241-254.

68. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox. Plant Physiol. 136, 2621-2632.

69. Fujiki, Y., Sato, T., Ito, M., and Watanabe, A. (2000) Isolation and Characterization of cDNA Clones for the e1beta and E2 Subunits of the Branched-Chain Alpha-Ketoacid Dehydrogenase Complex in Arabidopsis. J. Biol. Chem. 275, 6007-6013.

70. Lutziger, I., and Oliver, D. J. (2001) Characterization of Two cDNAs Encoding Mitochondrial Lipoamide Dehydrogenase from Arabidopsis. Plant Physiol. 127, 615-623.

71. Daschner, K., Couee, I., and Binder, S. (2001) The Mitochondrial Isovaleryl-Coenzyme a Dehydrogenase of Arabidopsis Oxidizes Intermediates of Leucine and Valine Catabolism. Plant Physiol. 126, 601-612.

72. Zolman, B. K., Nyberg, M., and Bartel, B. (2007) IBR3, a Novel Peroxisomal Acyl-CoA Dehydrogenase-Like Protein Required for Indole-3-Butyric Acid Response. Plant Mol. Biol. 64, 59-72.

73. Hooks, M. A., Kellas, F., and Graham, I. A. (1999) Long-Chain Acyl-CoA Oxidases of Arabidopsis. Plant J. 20, 1-13.

74. Adham, A. R., Zolman, B. K., Millius, A., and Bartel, B. (2005) Mutations in Arabidopsis Acyl-CoA Oxidase Genes Reveal Distinct and Overlapping Roles in Beta-Oxidation. Plant J. 41, 859-874.

75. Pinfield-Wells, H., Rylott, E. L., Gilday, A. D., Graham, S., Job, K., Larson, T. R., and Graham, I. A. (2005) Sucrose Rescues Seedling Establishment but not Germination of Arabidopsis Mutants Disrupted in Peroxisomal Fatty Acid Catabolism. Plant J. 43, 861-872.

76. Eastmond, P. J., Hooks, M. A., Williams, D., Lange, P., Bechtold, N., Sarrobert, C., Nussaume, L., and Graham, I. A. (2000) Promoter Trapping of a Novel Medium-Chain Acyl- CoA Oxidase, which is Induced Transcriptionally during Arabidopsis Seed Germination. J. Biol. Chem. 275, 34375-34381.

77. Froman, B. E., Edwards, P. C., Bursch, A. G., and Dehesh, K. (2000) ACX3, a Novel Medium-Chain Acyl-Coenzyme A Oxidase from Arabidopsis. Plant Physiol. 123, 733-742.

78. Hayashi, H., De Bellis, L., Ciurli, A., Kondo, M., Hayashi, M., and Nishimura, M. (1999) A Novel Acyl-CoA Oxidase that can Oxidize Short-Chain Acyl-CoA in Plant Peroxisomes. J. Biol. Chem. 274, 12715-12721.

79. Graham, I. A., and Eastmond, P. J. (2002) Pathways of Straight and Branched Chain Fatty Acid Catabolism in Higher Plants. Prog. Lipid Res. 41, 156-181.

80. Richmond, T. A., and Bleecker, A. B. (1999) A Defect in Beta-Oxidation Causes Abnormal Inflorescence Development in Arabidopsis. Plant Cell. 11, 1911-1924.

81. Rylott, E. L., Eastmond, P. J., Gilday, A. D., Slocombe, S. P., Larson, T. R., Baker, A., and Graham, I. A. (2006) The Arabidopsis thaliana Multifunctional Protein Gene (MFP2) of Peroxisomal Beta-Oxidation is Essential for Seedling Establishment. Plant J. 45, 930-941.

82. Goepfert, S., Vidoudez, C., Rezzonico, E., Hiltunen, J. K., and Poirier, Y. (2005) Molecular Identification and Characterization of the Arabidopsis Delta(3,5),Delta(2,4)-Dienoyl- Coenzyme A Isomerase, a Peroxisomal Enzyme Participating in the Beta-Oxidation Cycle of Unsaturated Fatty Acids. Plant Physiol. 138, 1947-1956.

83. Zolman, B. K., Monroe-Augustus, M., Thompson, B., Hawes, J. W., Krukenberg, K. A., Matsuda, S. P., and Bartel, B. (2001) Chy1, an Arabidopsis Mutant with Impaired Beta- Oxidation, is Defective in a Peroxisomal Beta-Hydroxyisobutyryl-CoA Hydrolase. J. Biol. Chem. 276, 31037-31046.

84. Sweetlove, L. J., Heazlewood, J. L., Herald, V., Holtzapffel, R., Day, D. A., Leaver, C. J., and Millar, A. H. (2002) The Impact of Oxidative Stress on Arabidopsis Mitochondria. Plant J. 32, 891-904. 25

85. Small, I., Peeters, N., Legeai, F., and Lurin, C. (2004) Predotar: A Tool for Rapidly Screening Proteomes for N-Terminal Targeting Sequences. Proteomics. 4, 1581-1590.

86. Emanuelsson, O., Nielsen, H., Brunak, S., and von Heijne, G. (2000) Predicting Subcellular Localization of Proteins Based on their N-Terminal Amino Acid Sequence. J. Mol. Biol. 300, 1005-1016.

87. Reumann, S., Ma, C., Lemke, S., and Babujee, L. (2004) AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes. Plant Physiol. 136, 2587-2608.

88. Wurtele, E. S., and Nikolau, B. J. (1990) Plants Contain Multiple Biotin Enzymes: Discovery of 3-Methylcrotonyl-CoA Carboxylase, Propionyl-CoA Carboxylase and Pyruvate Carboxylase in the Plant Kingdom. Arch. Biochem. Biophys. 278, 179-186.

89. Visser, W. F., Van Roermund, Carlo W. T., Ijlst, L., Waterham, H. R., and Wanders, Ronald J. A. (2007) Metabolite Transport Across the Peroxisomal Membrane. Biochem. J. 401, 365- 375.

90. Giovanelli, J., and Stumpf, P. K. (1958) Fat Metabolism in Higher Plants. X. Modified Beta Oxidation of Propionate by Peanut Mitochondria. J. Biol. Chem. 231, 411-426.

91. Hatch, M. D., and Stumpf, P. K. (1962) Fat Metabolism in Higher Plants. XVIII. Propionate Metabolism by Plant Tissues. Arch. Biochem. Biophys. 96, 193-198.

92. Gerbling, H., and Gerhardt, B. (1989) Peroxisomal Degradation of Branched-Chain 2-Oxo Acids. Plant Physiol. 91, 1387-1392.

93. Gerbling, H., and Gerhardt, B. (1989) Propionyl-CoA Generation and Catabolism in Higher Plant Peroxisomes, in Biological Role of Plant Lipids (P. A. Basics, K. Gruiz, and T. Kremmer, Eds.) pp 21-26, Plenum Publishing, New York.

94. Lawand, S., Dorne, A. J., Long, D., Coupland, G., Mache, R., and Carol, P. (2002) Arabidopsis A BOUT DE SOUFFLE, which is Homologous with Mammalian Carnitine Acyl Carrier, is Required for Postembryonic Growth in the Light. Plant Cell. 14, 2161-2173.

Chapter 2: Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in A. thaliana

Kerry A. Lucas, Jessica R Filley, Jeremy M. Erb, Eric R. Graybill, and John W. Hawes*

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056

*Corresponding author: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, Tel. 513-529-8072; Fax. 513-529-5715, Email: [email protected]

Author contribution: JRF performed initial studies by HMQC, JME collected spectra for wheat by 13C-NMR spectroscopy, ERG was responsible for determining conditions for 13C-NMR spectroscopy, and all other experiments were performed by KAL.

This paper has been published in Journal of Biological Chemistry, 2007, 282, 24980-24989.

2.1 Summary The sub-cellular sites of branched-chain amino acid metabolism in plants have been controversial, particularly with respect to valine catabolism. Potential enzymes for some steps in the valine catabolic pathway are clearly present in both mitochondria and peroxisomes, but the metabolic functions of these isoforms are not clear. The present study examined the possible function of these enzymes in metabolism of isobutyryl-CoA and propionyl-CoA, intermediates in the metabolism of valine and of odd-chain and branched-chain fatty acids. Using 13C-NMR, accumulation of β-hydroxypropionate from 2-13C-propionate was observed in seedlings of Arabidopsis thaliana and a range of other plants including both monocots and dicots. Examination of coding sequences and sub-cellular targeting elements indicated that the completed genome of A. thaliana likely codes for all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, A. thaliana mitochondria may lack some of the key enzymes for metabolism of propionyl-CoA. Known peroxisomal enzymes may convert propionyl-CoA to β-hydroxypropionate by a modified β-oxidation pathway. The chy1-3 mutation, creating a defect in a peroxisomal hydroxyacyl-CoA hydrolase, abolished the accumulation of β-hydroxyisobutyrate from exogenous isobutyrate but not the accumulation of β-hydroxypropionate from exogenous propionate. The chy1-3 mutant also displayed a dramatically increased sensitivity to the toxic effects of excess propionate and isobutyrate but not of valine. 13C-NMR analysis of A. thaliana seedlings exposed to U-13C-valine did not show an accumulation of β-hydroxypropionate. No evidence was observed for modified -oxidation of valine. 13C-NMR analysis showed that valine was converted to leucine through the production of -ketoisovalerate and isopropylmalate. These data suggest that peroxisomal enzymes for a modified β-oxidation of isobutyryl-CoA and propionyl-CoA could function for metabolism of substrates other than valine.

2.2 Introduction Propionate, in the form of propionyl-CoA, is produced from a number of metabolic precursors in higher eukaryotes. It is the final product of odd-chain fatty acid β-oxidation (1). It is also produced during the catabolism of several amino acids including isoleucine, methionine, and valine (1, 2). Propionyl-CoA is also a final product of metabolism of the branched acid, phytanic acid, derived from the degradation of chlorophyll (3). Aside from a basic understanding of metabolic biochemistry, the anabolic and catabolic pathways for propionyl- CoA are also of considerable importance in metabolic engineering of polyhydroxyalkanoates (PHAs) in plants, especially in the production of mixed PHA polymers that have relied on the use of propionyl-CoA as a metabolic intermediate (4, 5). Several pathways have been confirmed for the catabolism of propionyl-CoA (6-8). Bacteria and yeast utilize a 2-methylcitrate pathway with reactions analogous to those of the TCA cycle and glyoxylate cycle (6). Mammals use a well-established biotin and B12-dependent pathway for conversion of propionyl-CoA to succinyl- CoA (7, 8). Although plants have the same capacity described above to produce propionyl-CoA, their catabolic metabolism of propionyl-CoA is not clearly understood, and there are several conflicting reports regarding enzyme activities for different pathways. Examination of plant genomes does not reveal the presence of obvious orthologs corresponding to enzymes from either the bacterial or mammalian pathways. Plant genomes do code for a number of biotin- dependent carboxylases but none with a high level of homology to known propionyl-CoA carboxylases. Nevertheless, propionyl-CoA carboxylase activity has been demonstrated in plant extracts (9), raising some confusion about this enzyme. In the absence of a specific propionyl- CoA carboxylase, this reaction could be catalyzed by either an acetyl-CoA carboxylase or a methylcrotonyl-CoA carboxylase as a side reaction or as a secondary function of such an enzyme. Two separate isoforms of acetyl-CoA carboxylase are present in higher plants (10) as well as methylcrotonyl-CoA carboxylase (9). A problem would then arise as to the subsequent fate of methylmalonyl-CoA derived from carboxylation of propionyl-CoA in the absence of enzymes that could further metabolize methylmalonyl-CoA such as a racemase and a mutase which appear to be absent in plants. Furthermore, whether B12 is involved in plant metabolism is also not clear. Although plants are generally considered to lack B12 (11-13), there are reports in the literature of B12 and other corrin compounds from plant sources (14, 15).

Several other possible pathways have been proposed for the metabolism of propionyl- CoA in various organisms. These include conjugation to glyoxylate to produce either hydroxyglutarate (16) or 3-methylmalate (17), and conversion to acrylyl-CoA and β- hydroxypropionyl-CoA by enzymes similar to those used in a modified β-oxidation pathway for valine catabolism (2). Several studies have reported the production of β-hydroxypropionate from either acrylate or propionate by a modified β-oxidation pathway in either whole plants or isolated peroxisomes (2, 12, 13). Although this provides significant evidence for such a pathway, it is still not clear whether this is the primary pathway or the only functional pathway for metabolism of propionyl-CoA in plants. Furthermore it is not clear whether this modified β-oxidation pathway occurs exclusively in peroxisomes or if it is also in mitochondria. This is an important question as propionyl-CoA can be produced by multiple pathways that are expected to occur in both organelles. With the completed genome sequence of A. thaliana and genome databases of numerous other plant species, it is now possible to consider not only the pathways for catabolism but also the genes involved and the sub-cellular localization of the corresponding enzymes. Examination of genes coding for key enzymes of a modified β-oxidation pathway for propionyl-CoA suggests that the catabolic disposal of propionyl-CoA may be exclusively peroxisomal in plants and that β-hydroxypropionate may be the final product of this metabolism. This concept is consistent with previous reports of β-hydroxypropionate production in plants (2, 13). In the present study, we have confirmed the accumulation of β-hydroxypropionate in A. thaliana and other plant species using 2-13C-propionate by 13C NMR spectroscopy. The peroxisomal production of β-hydroxyisobutyrate from exogenous isobutyrate was also examined. Plant genomes code for both mitochondrial and peroxisomal forms of β-hydroxyacyl-CoA hydrolase, a key enzyme in this pathway, which also hydrolyzes branched-chain hydroxyacyl- CoA esters (18-21). It has been suggested that these peroxisomal hydrolases may be functionally important for valine catabolism. The present study shows that β-hydroxypropionate is produced from exogenous propionate but not from propionyl-CoA derived from valine, and that mutation of a peroxisomal hydroxyacyl-CoA hydrolase results in increased sensitivity to propionate but not to valine. This mutation also abolished the accumulation of β-hydroxyisobutyrate from exogenous isobutyrate. 13C NMR studies of A. thaliana seedlings treated with U-13C-valine

showed that the major pathway for valine metabolism was not through a modified β-oxidation pathway, but through the conversion to leucine. These data support a pathway in peroxisomes for the -oxidation of isobutyryl-CoA and propionyl-CoA from metabolic sources other than valine. A better understanding of this pathway may be valuable for the manipulation of this metabolism in the engineered synthesis of polyhydroxyalkanoates containing these acids (4, 5).

2.3 Experimental Procedures

2.3.1 Materials All chemicals were purchased from Sigma-Aldrich unless otherwise noted. 13C-labeled compounds were purchased from Cambridge Isotopes. β-Hydroxypropionate was synthesized according to Herter et al (22). β-Hydroxyisobutyrate was synthesized according to Rougraff et al (23). Malonate semialdehyde was synthesized according to Menon et al (24). chy1-3 Seeds were generously provided by Dr. Bonnie Bartel, Rice University.

2.3.2 Cell Culture and Seedling Preparation and NMR Spectroscopy Procedure Landsberg erecta green cell cultures were grown in 400 mL cultures with Gamborg basal salts ((25) Research Products International Corp.) treated or untreated with 0.1 mM unlabeled propionate. After 6 days growth, cultures were filtered and resuspended in 50 ml of medium with or without 1 mM 2-13C propionate for the specified time. Cells were then centrifuged at 1200 x g for 30 min, treated with perchloric acid (Fisher Scientific, 5% final concentration) to stop metabolism and placed at -80 °C prior to NMR analysis. Wild type A. thaliana ecotype Columbia (Col-0) and chy1-3 mutant seedlings were also used. Approximately 300-400 seedlings were surface sterilized for 30 s with 70% EtOH, then with 10% bleach for 30 min, then rinsed at least 4 times with sterile water. Seeds were dried on filter paper in a sterile hood then sprinkled on plates using ½ -strength Murashige and Skoog medium (MS, (26)) and 1% (w/v) sucrose solidified with 0.8% (w/v) agar (Teknova, Inc). They were placed at 4C for two days to synchronize growth and then transferred to growth chambers. Seedlings were grown under 16-h- light/8-h-dark photoperiods at 21 C-23 C. After 4 days, seedlings were removed from the plates and placed in 50 mL liquid, ½-strength MS media for 10-24 hr with or without 2 mM 2-

13C propionate or U-13C-valine. Seedlings were then rinsed with sterile water, ground to a fine powder in liquid nitrogen, resuspended in 5 mL of 5% perchloric acid and stored at -80°C prior to NMR analysis. Lactuca sativa (lettuce), Pisum sativum (pea), Triticum aestivum (wheat), and Zea mays (corn) seeds were all produced from Burpee®. Seeds were surface sterilized with 0.1% bleach and then rinsed several times with sterile water. Seedlings were dark-grown on paper towels saturated with water for up to 6 days before being placed in MS liquid media for 24 hr with 2-13C-propionate. Seedlings were prepared as above; however, before they were ground in liquid nitrogen, the seed coat was removed. All frozen samples were thawed to room temperature and neutralized with 10 M KOH. Samples were centrifuged at 1200 x g for 15-30 min at 4 °C. The supernatant was lyophilized and resuspended in 1-2 mL of 100% D2O. Samples were syringe-filtered or centrifuged at maximum speed for 10 min before placing in a 200 x 5-mm 535-PP NMR tube. 13C-spectra were acquired at 125.77 MHz with a deuterium lock on a Bruker AVANCE™-500 MHz with a 5 mm TXI probe. Spectra were obtained with a 30° RF pulse, relaxation time of 2 s, 1,024 scans, and the spectra were Fourier transformed using 1-Hz line broadening. Chemical shifts were calibrated to propionate C2 at 30.8 ppm (6) or to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). Heteronuclear multiple quantum correlation (HMQC) spectra were measured in the 1H-detected mode via multiple quantum coherence with proton decoupling in the 13C domain, using a program obtained from Bruker.

2.3.3 End Point Assay for Quantitation of β-Hydroxyisobutyrate Wild-type Col-0 and chy1-3 seedlings were grown as above; however at day 4, seedlings were removed from the agar plates and placed in 10 mL of liquid Gamborg basal salt medium containing neutralized 1 mM isobutyrate. After 10 hr, seedlings were thoroughly rinsed with distilled water and ground to a fine powder in liquid nitrogen; they were weighed and resuspended in 5 mL of 5% perchloric acid, then placed at -80 °C prior to analysis. Frozen samples were thawed to room temperature, neutralized with 10 M KOH, and spun down at 1200 x g for 30 min. The supernatant was then lyophilized before being resuspended in 2 mL of sterile H2O. Any undissolved plant debris was removed by centrifuging the sample for 5 min at maximum speed. The assay conditions were based on Rougraff et al (23) and are as follows:

0.67 M tris (hydroxymethyl) aminomethane, 3.3 mM MgSO4, 1.7 mM EDTA, 0.13 M hydrazine

sulfate, 1 mM NAD+, 50-100 μL extract and 9.6 μg/mL HIB dehydrogenase to a final volume of 1.0 mL. A no-enzyme and no-NAD+ negative control was used for background. Each reaction was initiated with 20 μL of enzyme after baseline was reached (approximately 15 min). The production of NADH was measured at 340 nm on a Varian Cary UV-Vis spectrophotometer with a circulating water jacket at 37 °C.

2.3.4 Valine, Isobutyrate, and Propionate Growth Response Wild-type Col-0 and chy1-3 seedlings were surface sterilized as above and placed on ½- strength MS plates containing 1% sucrose and 0.8% agar, with valine, isobutyrate or propionate as indicated. Seedlings were placed at 4 °C for two days and then moved to growth chambers at 21-23 °C for 7 days. Seedlings were then removed from the agar and the primary roots were measured against untreated seedlings.

2.4 Results

2.4.1 Genes for a Modified Propionate β-Oxidation Pathway in Plants The absence of enzymes for other, more common pathways of propionyl-CoA metabolism has lead to the suggestion that plants metabolize propionyl-CoA by a modified β- oxidation pathway (2, 12, 13). This pathway would be highly analogous to the mitochondrial catabolism of isobutyryl-CoA derived from valine as shown in Figure 2.1. Table 2.1 summarizes A. thaliana genes encoding possible enzymes in this pathway and the putative sub-cellular localizations of the corresponding gene products. These include all the enzymes necessary to convert valine to propionyl-CoA in mitochondria. However, it is not clear whether the same enzymes that convert isobutyryl-CoA to propionyl-CoA can also convert propionyl-CoA to acetyl-CoA in mitochondria as shown in Figure 2.1. Of particular importance here is the apparent lack of a mitochondrial acyl-CoA dehydrogenase (Fig. 2.1; step 3) that may oxidize propionyl-CoA to acrylyl-CoA (27). The only known mitochondrial acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, appears to be highly specific for the branched-chain acyl-group derived from valine (28).

Figure 2.1 Proposed pathways for metabolism of valine and propionyl-CoA in mitochondria and peroxisomes in plants. Enzymes 1-7 can be found in Table 2.1. A, 2-isopropylmalate synthase; B, 3-isopropylmalate dehydratase; C, 3-isopropylmalate dehydrogenase.

Mitochondria Exogenous Exogenous Valine Propionate Isobutyrate

1 A Fatty Acids 2-Isopropylmalate Keto-isovalerate CoASH Propionate Isobutyrate B 2

3-Isopropylmalate Isobutyryl-CoA Propionyl-CoA Propionyl-CoA Isobutyryl-CoA

C 3 3 3 3

2-Isopropyl-3- Methylacrylyl-CoA Acrylyl-CoA Acrylyl-CoA Methylacrylyl-CoA oxosuccinate 4 4 4 Spontaneous ? ? 4 Hydroxyisobutyryl-CoA Hydroxypropionyl-CoA Hydroxy- Hydroxy- 2-Ketoisocaproate propionyl-CoA isobutyryl-CoA 1 5 CoASH 5 CoASH 5 CoASH CHY1 5 CoASH Leucine Hydroxyisobutyrate Hydroxypropionate Hydroxypropionate Hydroxyisobutyrate 6 6 Methylmalonate Semialdehyde Malonate Semialdehyde

Isoleucine 7 7 Methionine Propionyl-CoA Acetyl-CoA Peroxisomes Threonine

Table 2.1 Genes coding for putative enzymes for catabolism of valine and propionyl-CoA in A. thaliana. Enzyme Locus Name Localizationa Reference 1 Branched-chain aminotransferase At1g10060 M (30, 31) Branched-chain α-Ketoacid 2 Dehydrogenase BCKDH E1α At1g21400 M - At5g09300 M (32) BCKDH E1β At3g13450 M (32) BCKDH E2 At3g06850 M (32) BCKDH E3 At3g17240 M (33) Acyl-CoA 3 Dehydrogenase/Oxidase Isovaleryl-CoA Dehydrogenase At3g45300 M (28) ACX1b At4g16760 P (1) (34, 35) ACX2 At5g65110 P (2) (34, 35) ACX3 At1g06290 P (2) (35-37) ACX4 At3g51840 P (1) (35, 38) ACX5 At2g35690 P (1) (35) ACX6 At1g06310 P (2) (35) ATP binding/acyl-CoA At3g06810 P (1) - Dehydrogenase 4 Enoyl-CoA Hydratase At3g24360 M (27) At4g16120 P (1) - At4g29010 P (1) (39) At3g06860 P (1) (39) At5g43280 P (1) (40) At4g16800 NCSc - 5 Hydroxyacyl-CoA Hydrolase At4g31810 M (21) At3g60510 M (21) At2g30660 P (1) (21) At2g30650 P (1) (21) CHY1 At5g65940 P (1) (21) 6 Hydroxyacid Dehydrogenase At4g20930 M - At3g29120 M - At1g17650 M - (Methyl) malonate semialdehyde 7 dehydrogenase At2g14170 M (41)

a Mitochondrial predicted localizations [M] are based on prediction scores from PREDOTAR (42) and TargetP (43) or based on the cited literature. Type 1 peroxisomal predicted localizations (P (1)) are based on the presence of C-terminal SKL or AKL sequences. Type 2

peroxisomal predicted localizations (P (2)) are based on the conserved motif as described in the AraPerox database (29). bACX, acyl-CoA oxidase cNCS, no clear signal was found for the prediction of a mitochondrial or peroxisomal localization

There are also enzymes with putative peroxisomal localization that could catalyze a specific subset of this pathway. These include numerous acyl-CoA oxidases (Fig. 2.1; step 3), enoyl-CoA hydratases (step 4), and hydroxyacyl-CoA hydrolases (Table 2.1; step 5). However, the early enzymes of valine degradation (BCAT, step 1 and BCKDH, step 2) do not appear to have peroxisomal localization signals (Table 2.1). Neither do the enzymes that catalyze the final steps in this pathway, hydroxyisobutyrate dehydrogenase (step 6) and methylmalonate semialdehyde dehydrogenase (step 7). The predicted A. thaliana proteins for these two enzymes each contain sequences that could clearly serve for mitochondrial targeting and lack known peroxisomal targeting sequences (29). Therefore, analysis of the A. thaliana genome raises questions regarding the mitochondrial catabolism of propionyl-CoA and supports a truncated pathway for propionyl-CoA in peroxisomes that could serve primarily in the detoxification of acrylyl-CoA with β-hydroxypropionate as an end product. This would be consistent with the previous reports of β-hydroxypropionate production in isolated peroxisomes (2, 13) and the observed accumulation of 2-13C-hydroxypropionate described in the present study.

2.4.2 Metabolism of 2-13C-Propionate in A. thaliana Seedlings and Suspension Cell Cultures Previous studies of propionate metabolism in higher plants utilized a number of different plant species but not the model plant A. thaliana. In order to determine if propionate is metabolized via a β-oxidation pathway in A. thaliana, we performed 13C NMR analyses of A. thaliana seedlings and suspension cell cultures exposed to 2-13C-propionate. When A. thaliana seedlings (4 days after imbibition) were incubated with 2-13C-propionate in liquid MS medium with the addition of 2-13C-propionate and analyzed by 13C-NMR, a unique spectral peak at 40.1 ppm accumulated, corresponding to β-hydroxypropionate C2 (Fig. 2.2B). This peak was not observed in natural abundance spectra of control samples (Fig. 2.2A) and was persistent in samples incubated with the labeled propionate for various time periods ranging from several hours to over 24 hr. A dramatic and persistent accumulation of β-hydroxypropionate, as a spectral peak at 40.1 ppm, was also observed in A. thaliana suspension cell cultures. As with seedlings, this was the only major peak observed to accumulate. This peak arose after several hours of incubation with 2-13C-propionate and persisted at relatively high signal strengths for time periods greater than 24 hr (Fig. 2.3).

Figure 2.2 13C-NMR analysis of metabolites produced from 2-13C-propionate in A. thaliana. Four-day-old seedlings were incubated with 2 mM 2-13C-propionate in MS medium for 24 hr and analyzed by 13C NMR as described in Experimental Procedures. A, Control spectrum from wild- type seedlings with no labeled propionate. B, Spectrum from wild-type seedlings incubated with 2-13C-propionate. C, Spectrum from chy1-3 seedlings incubated with 2-13C-propionate. Each spectrum was adjusted to the same scale and expanded to show the region of β- hydroxypropionate. Spectrum was calibrated to propionate C2 (6).

Figure 2.3 Time course of β-hydroxypropionate production. Lansberg erecta cell cultures were incubated with 1 mM 2-13C-propionate for 2, 5, 10, 15, and 30 hr and then analyzed by 13C NMR as described in Experimental Procedures. Each spectrum was adjusted to the same scale and expanded to show region of β-hydroxypropionate.

40 ppm 40 ppm 40 ppm 40 ppm 40 ppm

2 h 5 h 10 h 15 h 30 h

Several unique peaks with weaker signal strengths appeared at specific time points but with much less abundance compared to β-hydroxypropionate. Comparison of these peaks to those produced by relevant standard compounds indicated that none of these peaks correspond to those of other metabolites in this pathway such as acrylate or malonate semialdehyde, nor do they correspond to peaks of acetate, acetyl-CoA, or succinate (11, 13). These minor peaks currently remain unassigned. Assignment of the peak at 40.1 ppm as that of β- hydroxypropionate C2 was based on analysis of standard β-hydroxypropionate by carbon NMR analysis and by 1H(13C) Hetero-nuclear Multiple Quantum Correlation (HMQC) analysis. 13C- NMR spectra of standard β-hydroxypropionate showed peaks at 181.1 ppm, 40.1 ppm and 59.3 ppm, corresponding to carbons 1, 2, and 3, respectively. HMQC analysis of standard β- hydroxypropionate showed a peak at 40.1 ppm with correlation to 1H resonance at 2.38 ppm (Fig. 2.4B). HMQC analysis of A. thaliana samples showed that the unique peak at 40.1 ppm correlated to 1H peaks with shifts identical to that of standard β-hydroxypropionate (Fig. 2.4A).

2.4.3 Effect of chy1 Mutation on Metabolism of Propionate and Isobutyrate Although NMR analysis revealed a dramatic accumulation of β-hydroxypropionate from exogenous propionate in wild-type seedlings, it is not clear whether this was the result of mitochondrial or peroxisomal metabolism, or both. To address this question, identical NMR analyses were performed using 2-13C-propionate with the well-characterized chy1-3 mutant A. thaliana line. The CHY1 gene codes for a peroxisomal form of β-hydroxyacyl-CoA hydrolase, which could potentially catalyze the hydrolysis of β-hydroxypropionyl-CoA in peroxisomes (21, 44). The dramatic accumulation of 2-13C-hydroxypropionate that was observed in wild-type plants was also observed in the chy1 mutant (Fig. 2.2C). In contrast, the metabolism of exogenous isobutyrate was dramatically altered in chy1-3 seedlings. When wild-type A. thaliana seedlings were incubated with exogenous isobutyrate, an accumulation of β-hydroxyisobutyrate could be observed using an enzyme end-point assay known to be specific for β- hydroxyisobutyrate (23) (Fig. 2.5). Only very low concentrations of β-hydroxyisobutyrate were present in seedlings without the addition of exogenous isobutyrate. The production of β- hydroxyisobutyrate from isobutyrate presumably occurs through the conversion of isobutyryl- CoA to methylacrylyl-CoA, hydration to hydroxyisobutyryl-CoA, and the subsequent hydrolysis of the hydroxyisobutyryl-CoA ester.

Figure 2.4 HMQC analysis of standard β-hydroxypropionate and A. thaliana seedlings with 2-13C-propionate. A, Four-day-old wild-type seedlings were incubated with 2-13C-propionate for 24 hr in liquid MS medium before performing HMQC analysis. B, Standard β-hydroxypropionate was analyzed by HMQC using the same protocol as for seedlings.

Figure 2.5 β-Hydroxyisobutyrate accumulation in wild-type and chy1-3 A. thaliana seedlings. Four-day-old wild-type and chy1-3 seedlings were incubated with 2 mM isobutyrate for 24 hr. Accumulation of β-HIBA was measured as described in Experimental Procedures and expressed as μmol/mg fresh weight. Error bars indicate the standard error of the mean.

2.5

2.0

1.5

1.0

0.5 Hydroxyisobutyrateumol/mg weight fresh

0.0 WT WT chy1-3 chy1-3 +Iso -Iso +Iso -Iso

These reactions are identical to those that occur during mitochondrial valine metabolism (Fig. 2.1). However, the accumulation of β-hydroxyisobutyrate from exogenous isobutyrate was abolished in the chy1-3 mutant, suggesting that these reactions occurred in the peroxisomes (Fig. 2.5). That the accumulation of β-hydroxypropionate was not so dramatically affected suggests that different enzymes may be involved in the hydrolysis of β-hydroxypropionyl-CoA vs. β- hydroxyisobutyryl-CoA. Furthermore, assuming that exogenous isobutyrate is activated to its CoA thioester, these data indicate that metabolism of isobutyryl-CoA in plants can occur in peroxisomes, producing -hydroxyisobutyrate. This peroxisomal metabolism may utilize similar enzymes as that for isobutyryl-CoA derived from valine in mitochondria, but may end with the production of β-hydroxyisobutyrate, resulting in its accumulation.

2.4.4 Metabolism of 2-13C-Propionate in Different Plants To determine if metabolism of 2-13C-propionate in A. thaliana seedlings and cell cultures is consistent with that in other plants, we performed similar 13C NMR analyses using seedlings from a variety of species including both monocots and dicots. Figure 2.6 shows carbon NMR spectra of metabolites produced from 2-13C-propionate in a range of plants including Lactuca sativa (lettuce), Pisum sativum (pea), Triticum aestivum (wheat), and Zea mays (corn). Each showed a dramatic accumulation of β-hydroxypropionate (unique peak at 40.1 ppm), which was very similar to that observed in A. thaliana, and a notable absence of any other accumulating species as compared to natural abundance spectra. Thus, there appeared to be no major differences in the metabolism of exogenous propionate in these varied plant species as observed by carbon NMR analysis.

2.4.5 Metabolism of U-13C-Valine in A. thaliana Seedlings The major known pathway for valine catabolism is a modified -oxidation pathway producing propionyl-CoA (45). We considered whether propionyl-CoA produced from mitochondrial metabolism of valine would also lead to an accumulation of β-hydroxypropionate. Incubation of A. thaliana seedlings with uniformly labeled 13C-valine failed to reveal the production of 13C-hydroxypropionate as measured by both 1D-carbon NMR and HMQC analysis.

Figure 2.6 Production of β-hydroxypropionate in various plants. Seedlings were dark grown on paper towels saturated with sterile water for approximately 6 days. They were then incubated with 1 mM 2-13C-propionate in MS medium for 24 hr and then analyzed by 13C NMR as described in Experimental Procedures. A, Pea; B, Lettuce; C, Wheat; D, Corn.

A B Hydroxypropionate C2 Propionate C2

Hydroxypropionate C2

? ? Propionate C2 ?

48 46 44 42 40 38 36 34 32 30 28 26 24 ppm 48 46 44 42 40 38 36 34 32 30 28 26 24 ppm

C Hydroxypropionate C2 D Propionate C2 Propionate C2

Hydroxypropionate C2

48 46 44 42 40 38 36 34 32 30 28 26 24 ppm 48 46 44 42 40 38 36 34 32 30 28 26 24 ppm

13C-hydroxypropionate was not produced from valine under a variety of conditions including various concentrations of valine and various times of incubation with the 13C-label. Instead, the major product observed from the metabolism of U-13C-valine in growing A. thaliana seedlings was leucine. The label from U-13C-valine was incorporated into carbons 3, 4, 5, and 6 of leucine, observed as unique spectral peaks with shifts at 42.6, 26.7, 24.7, and 23.6 ppm, respectively (Fig. 2.7). These data are consistent with the conversion of valine to - ketoisovalerate by the branched-chain transaminase and then entry into the known pathway for leucine biosynthesis (Fig. 2.1). This pathway would result in exactly the labeling pattern observed as shown in Figure 2.8.

2.4.6 Effects of Exogenous Isobutyrate, Propionate, and Valine on Seedling Growth of Wild- type and chy1-3 A. thaliana Seedlings Wild-type A. thaliana seedlings have been shown to display a characteristic sensitivity to valine during germination and seedling growth (46). In the present study, A. thaliana seedlings also displayed a similar sensitivity to propionate and isobutyrate. This toxicity occurred with concentrations as low as 100 M and was most readily measured as a morphological effect on root length (Fig. 2.9). We considered that these effects are most likely related to the known toxicity of methylacrylyl-CoA and acrylyl-CoA and that these compounds might increase to toxic levels when isobutyrate, propionate, or valine are presented in excess (21, 44). Because the end-point assays presented above suggest that the metabolism of exogenous isobutyrate occurred primarily in peroxisomes, and was dependant on the function of CHY1, we compared the sensitivity of wild-type and chy1-3 seedlings to isobutyrate, propionate, and valine. The chy1-3 seedlings displayed an increased sensitivity to both isobutyrate and propionate, as compared to wild-type seedlings (Fig. 2.9). In contrast, the chy1-3 mutant exhibited the same level of sensitivity to valine as wild-type seedlings. These data suggest that the toxicity of isobutyrate and propionate may be related to the peroxisomal production of methylacrylyl-CoA and acrylyl-CoA, respectively, and that the disposal of these compounds depends on CHY1. Furthermore, peroxisomal CHY1 appears to play no role in the toxicity of valine, consistent with the theory that valine metabolism occurs primarily in mitochondria.

Figure 2.7 Pathway for the metabolism of valine to leucine. Pathway shows appropriate carbon shifts and labeled carbons according to the Human Metabolome Database (http://www.hmdb.ca/).

20.6* O Valine 31.8 63.3 176.9 * 19.3 * * * OH

NH2

* O Ketoisovalerate * * * * OH O

* O 2-Isopropylmalate * * * * OH HO

O OH

* O 3-Isopropylmalate * * * * OH OH HO O

2-Isopropyl- * O 3-oxosuccinate * * * * OH OH O O * *CO2 2-Ketoisocaproate * * * OH O O 23.6 * Leucine 26.8 * 42.6 24.7* * OH H2N O

Figure 2.8 13C NMR analysis of A. thaliana seedlings with U-13C-valine. Four-day-old, wild-type seedlings were incubated with 2 mM U-13C-valine for 24 hr in liquid MS medium and analyzed by 13C NMR as described in Experimental Procedures. A, Spectrum from wild-type seedlings incubated with U-13C-valine. B, Control spectrum from wild-type seedlings with no labeled valine. Spectrum was calibrated according to DSS.

A Valine C4/C5

Valine C3

Leucine C3 Valine C2 Leucine C4 Leucine C5/C6

65 60 55 50 45 40 35 30 25 20 15 ppm B

65 60 55 50 45 40 35 30 25 20 15 ppm

Figure 2.9 Effect of exogenous valine, isobutyrate and propionate on seedling growth. Wild-type and chy1-3 seedlings were grown on agar plates for 7 days with MS media and either valine, isobutyrate, or propionate. For A-D, wild-type seedlings are on the left, and chy1-3 seedlings are on the right. A, Control (untreated seedlings), B, 0.25 mM valine, C, 0.25 mM isobutyrate, and D, 0.25 mM propionate. E-F, Effects of varying concentrations of valine, isobutyrate and propionate on root length. The percent root elongation of seedlings treated versus control seedlings is shown. Gray bars indicate wild-type seedlings. White bars indicate chy1-3 seedlings. Error bars indicate the standard error of the mean. A B C D

100% 100% 100% rol)

80% 80% 80% vs. cont vs.

60% 60% 60% ion ( ion

40% 40% 40%

longat E

20% 20% 20%

Root % 0% 0% 0% 0.1mM 0.25mM 0.5mM 0.1mM 0.25m M 0.5mM 0.1mM 0.25mM 0.5mM

Valine Isobutyrate Propionate

2.5 Discussion The availability of a complete and well-annotated genome database for A. thaliana allows for the identification of genes with putative function in various metabolic pathways (27). This information also provides clues regarding the likely sub-cellular location of the corresponding enzymes. With regard to propionate metabolism, this capability is valuable considering there are pathways for the production of propionyl-CoA in both the mitochondria and peroxisomes. Examination of the A. thaliana genome shows that all of the enzymes required for conversion of valine to propionyl-CoA may reside in the mitochondria. The problem that arises is the subsequent fate of the propionyl-CoA produced in mitochondria. Although propionyl-CoA could possibly be converted to acetyl-CoA by a similar pathway as to that of isobutyryl-CoA, this may not be the case for mitochondrial propionyl-CoA. A primary obstacle for this pathway is the apparent lack of a mitochondrial acyl-CoA dehydrogenase functional for oxidation of propionyl-CoA (27). An isovaleryl-CoA dehydrogenase (IVD) has been characterized from several plant species including A. thaliana (28, 47, 48). However, this enzyme has been shown to be highly specific for branched-chain acyl groups and was inactive with short-chain acyl-CoA substrates like butyryl-CoA and propionyl-CoA (28). A mitochondrial enoyl-CoA hydratase specifically capable of producing β-hydroxypropionyl-CoA has also not been clearly identified, however mitochondrial enoyl-CoA hydratase activity has been reported (49). Putative mitochondrial enzymes exist that could potentially catalyze subsequent steps including hydroxyacyl-CoA hydrolases, hydroxyacid dehydrogenases, and a methylmalonate/malonate semialdehyde dehydrogenase (Table 2.1). However, the substrate specificities and kinetic parameters for these enzymes have not yet been determined. The enzymes listed in Table 2.1 may support the complete metabolism of valine to propionyl-CoA in mitochondria, but not necessarily the conversion of propionyl-CoA to acetyl-CoA as shown in Figure 2.1. Alternatively, it is also possible that propionyl-CoA could exit the mitochondrion by conversion to propionyl-carnitine and enter other pathways for further degradation. Acyl-carnitine carrier proteins have recently been identified in A. thaliana (50). There are also, clearly, genes coding for a specific subset of this pathway in the peroxisomes (Fig. 2.1 and Table 2.1). Identification of gene products with likely peroxisomal localization has been greatly advanced through the recognition of type 1 and type 2 peroxisomal targeting sequences (51). There are multiple isoforms of acyl-CoA oxidase (ACX1 through

ACX6) with clear peroxisomal targeting sequences. The expression and activities of these enzymes have been partially characterized (34-38). Multiple homologues also exist for enoyl- CoA hydratases and hydroxyacyl-CoA hydrolases (Fig.1; step 5) with clear peroxisomal targeting sequences (21, 27, 39, 40). However, a β-hydroxyacid dehydrogenase (step 6) homolog is not present in the A. thaliana genome that contains a clear type 1 or type 2 peroxisomal targeting signal (Fig. 1; step 6). There also appears to be no peroxisomal homologue of methylmalonate semialdehyde dehydrogenase (step 7), the enzyme believed to convert malonate semialdehyde to acetyl-CoA (41). It is plausible that β-hydroxypropionate could be oxidized by an as yet unidentified peroxisomal dehydrogenase. However, in the absence of a functional malonate semialdehyde dehydrogenase, peroxisomal oxidation of β- hydroxypropionate to malonate semialdehyde should be deleterious without any other means of disposal of this potentially toxic compound. Therefore, it appears that peroxisomal catabolism of propionate could be expected to produce β-hydroxypropionate as an end product rather than acetyl-CoA. The observed accumulation of 2-13C-hydroxypropionate from 2-13C-propionate is consistent with the genetic features described above. No evidence was found for the production of acetate from 2-13C-propionate as was previously reported for lima bean leaves and stems (13). This may be a difference between the plant materials studied- whole A. thaliana seedlings vs. lima bean leaves and stems. It may also be attributed to the metabolism of acetyl-CoA being too rapid to see any further accumulation of metabolites beyond β-hydroxypropionate. The time course that was examined does however show that the production of β-hydroxypropionate increases from 2 hours to 10 hours and then decreases. Whether this decrease represents further disposal of the β-hydroxypropionate or just a diminishment of the labeled propionate is not clear. Accumulation of β-hydroxyisobutyrate from exogenous isobutyrate is also consistent with this metabolic model. That the production of β-hydroxyisobutyrate was abolished in the chy1 mutant conclusively shows that this pathway occurs in peroxisomes. The data presented in this study suggest that there might be multiple metabolic sources of isobutyryl-CoA in plants. Isobutyryl-CoA is expected to be an early product of valine catabolism and, as such, should be produced in the mitochondria. However, this study also provides evidence for a role of the peroxisomal enzyme CHY1 in metabolism of isobutyryl-CoA. Possible sources of both isobutyryl-CoA and propionyl-CoA in peroxisomes may include branched-chain fatty acids such

as phytanic acid. There is evidence for peroxisomal metabolism of phytanic acid in mammalian systems but little is known of this pathway in plants. Although there have been preliminary reports of branched-chain amino acid degradation in plant peroxisomes (2, 13), this still remains to be conclusively shown. In light of this, it is particularly noteworthy that the chy1-3 mutant displayed increased sensitivity to isobutyrate but not valine, suggesting that this enzyme may not have a central role in valine degradation. However, the best evidence for a role in peroxisomes 14 14 in valine catabolism is the observation of reduced production of CO2 from U- C-valine in a chy1 mutant A. thaliana (44). This observation remains difficult to explain in light of the probable mitochondrial localization of key enzymes such as BCAT and BCKDH that produce isobutyryl-CoA from valine. Nevertheless, certain enzyme activities of the valine catabolic pathway may be present in both mitochondria and peroxisomes for metabolism of isobutyryl- CoA derived from compounds other than valine. No evidence for the production of β-hydroxypropionate from valine was observed in this study. Surprisingly, the major product observed from the metabolism of U-13C-valine was leucine. The pattern of incorporation of labeled carbon into leucine was exactly as one would expect for the conversion of U-13C-valine to β-ketoisovalerate and entry into the known pathway for leucine biosynthesis (Fig. 2.8). This may be the result of gene expression during seedling growth favoring amino acid biosynthesis rather than degradation. β-Ketoisovalerate is an intermediate shared by the expected pathways for valine degradation and for valine and leucine biosynthesis. If the enzymes for valine and leucine biosynthesis were highly expressed during seedling growth, then exogenous valine may produce α-ketoisovalerate and enter into this biosynthetic pathway. It is also possible that other pathways, such as a β-oxidation, may be fully functional, but may have occurred too rapidly to observe the accumulation of metabolic intermediates. Therefore, one cannot conclude that leucine was the only product produced from U-13C-valine. Nevertheless, from this study it is clear that leucine is a significant product of valine metabolism in A. thaliana seedlings. Therefore, whether propionyl-CoA is produced from valine in mitochondria of plants still remains a major question. The increased sensitivity of the chy1 mutant to excess propionate and isobutyrate represents new insight into the previously reported phenotypes of this mutant. Mutations in this gene are known to produce defects in peroxisomal β-oxidation and in responses to indole butyric acid (IBA) and 2,4-dichlorophenoxybutyric acid (21, 44). These effects are believed to result

from increased production of methylacrylyl-CoA, a toxic metabolite of valine. Altered responses to IBA are also believed to be the result of defects in β-oxidation caused by increased production of methylacrylyl-CoA (21, 44). IBA is converted to the active hormone, indole acetic acid, by the peroxisomal β-oxidation enzymes (52), and this process is presumably inhibited by the increased production of methylacrylyl-CoA. Conclusive data have been provided for a specific effect of methylacrylyl-CoA on β-ketoacyl-CoA thiolase, an enzyme important to β-oxidation (44). The increased sensitivity of the chy1 mutant to propionate and isobutyrate is also likely due to the increased production of acrylyl-CoA and methylacrylyl-CoA, respectively.

2.6 Acknowledgements We thank Dr. Bonnie Bartel for chy1–3 seedlings and Dr. Meghan Holdorf for endless assistance with growing plants and maintaining cell cultures. We greatly appreciate Dr. Chris Makaroff for the critical comments on the manuscript.

2.7 References

1. Halarnkar, P. P., and Blomquist, G. J. (1989) Comparative Aspects of Propionate Metabolism. Comp. Biochem. Physiol. B. 92, 227-231.

2. Gerbling, H., and Gerhardt, B. (1989) Propionyl-CoA Generation and Catabolism in Higher Plant Peroxisomes, in Biological Role of Plant Lipids (P. A. Basics, K. Gruiz, and T. Kremmer, Eds.) pp 21-26, Plenum Publishing, New York.

3. Dieuaide-Noubhani, M., Asselberghs, S., Mannaerts, G. P., and Van Veldhoven, P. P. (1997) Evidence that Multifunctional Protein 2, and Not Multifunctional Protein 1, is Involved in the Peroxisomal Beta-Oxidation of Pristanic Acid. Biochem. J. 325 ( Pt 2), 367-373.

4. Snell, K. D., and Peoples, O. P. (2002) Polyhydroxyalkanoate Polymers and their Production in Transgenic Plants. Metab. Eng. 4, 29-40.

5. Rezzonico, E., Moire, L., and Poirier, Y. (2002) Polymers of 3-Hydroxyacids in Plants. Phytochemistry Rev. 1, 87-92.

6. Horswill, A. R., and Escalante-Semerena, J. C. (1999) Salmonella typhimurium LT2 Catabolizes Propionate Via the 2-Methylcitric Acid Cycle. J. Bacteriol. 181, 5615-5623.

7. Beck, W. S., Flavin, M., and Ochoa, S. (1957) Metabolism of Propionic Acid in Animal Tissues. III. Formation of Succinate. J. Biol. Chem. 229, 997-1010.

8. Flavin, M., and Ochoa, S. (1957) Metabolism of Propionic Acid in Animal Tissues. I. Enzymatic Conversion of Propionate to Succinate. J. Biol. Chem. 229, 965-979.

9. Wurtele, E. S., and Nikolau, B. J. (1990) Plants Contain Multiple Biotin Enzymes: Discovery of 3-Methylcrotonyl-CoA Carboxylase, Propionyl-CoA Carboxylase and Pyruvate Carboxylase in the Plant Kingdom. Arch. Biochem. Biophys. 278, 179-186.

10. Yanai, Y., Kawasaki, T., Shimada, H., Wurtele, E. S., Nikolau, B. J., and Ichikawa, N. (1995) Genomic Organization of 251 kDa Acetyl-CoA Carboxylase Genes in Arabidopsis:

Tandem Gene Duplication has made Two Differentially Expressed Isozymes. Plant Cell Physiol. 36, 779-787.

11. Giovanelli, J., and Stumpf, P. K. (1958) Fat Metabolism in Higher Plants. X. Modified Beta Oxidation of Propionate by Peanut Mitochondria. J. Biol. Chem. 231, 411-426.

12. Hatch, M. D., and Stumpf, P. K. (1962) Fat Metabolism in Higher Plants. XVIII. Propionate Metabolism by Plant Tissues. Arch. Biochem. Biophys. 96, 193-198.

13. Halarnkar, P. P., Wakayama, E. J., and Blomquist, G. J. (1988) Metabolism of Propionate to 3-Hydroxypropionate and Acetate in the Lima Bean Phaseolus limensis. Phytochemistry. 27, 997-999.

14. Poston, J. M. (1978) Coenzyme B12-Dependent Enzymes in Potatoes: Leucine 2,3- Aminomutase and Methylmalonyl-CoA Mutase. Phytochemistry. 17, 401-402.

15. Poston, J. M. (1977) Leucine 2,3-Aminomutase: A Cobalamin-Dependent Enzyme Present in Bean Seedlings. Science. 195, 301-302.

16. Reeves, H. C., and Ajl, S. J. (1962) Alpha-Hydroxyglutaric Acid Synthetase. J. Bacteriol. 84, 186-187.

17. Buckel, W., and Barker, H. A. (1974) Two Pathways of Glutamate Fermentation by Anaerobic Bacteria. J. Bacteriol. 117, 1248-1260.

18. Hawes, J. W., Jaskiewicz, J., Shimomura, Y., Huang, B., Bunting, J., Harper, E. T., and Harris, R. A. (1996) Primary Structure and Tissue-Specific Expression of Human Beta- Hydroxyisobutyryl-Coenzyme A Hydrolase. J. Biol. Chem. 271, 26430-26434.

19. Shimomura, Y., Murakami, T., Fujitsuka, N., Nakai, N., Sato, Y., Sugiyama, S., Shimomura, N., Irwin, J., Hawes, J. W., and Harris, R. A. (1994) Purification and Partial Characterization of 3-Hydroxyisobutyryl-Coenzyme A Hydrolase of Rat Liver. J. Biol. Chem. 269, 14248- 14253.

20. Shimomura, Y., Murakami, T., Nakai, N., Huang, B., Hawes, J. W., and Harris, R. A. (2000) 3-Hydroxyisobutyryl-CoA Hydrolase. Methods Enzymol. 324, 229-240.

21. Zolman, B. K., Monroe-Augustus, M., Thompson, B., Hawes, J. W., Krukenberg, K. A., Matsuda, S. P., and Bartel, B. (2001) Chy1, an Arabidopsis Mutant with Impaired Beta- Oxidation, is Defective in a Peroxisomal Beta-Hydroxyisobutyryl-CoA Hydrolase. J. Biol. Chem. 276, 31037-31046.

22. Herter, S., Farfsing, J., Gad'On, N., Rieder, C., Eisenreich, W., Bacher, A., and Fuchs, G.

(2001) Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation Via the 3-Hydroxypropionate Cycle. J. Bacteriol. 183, 4305- 4316.

23. Rougraff, P. M., Paxton, R., Goodwin, G. W., Gibson, R. G., and Harris, R. A. (1990) Spectrophotometric Enzymatic Assay for S-3-Hydroxyisobutyrate. Anal. Biochem. 184, 317- 320.

24. Menon, G. K., Stern, J. R., Kupiecki, F. P., and Coon, M. J. (1960) Enzymic Synthesis and Reduction of Malonyl Semialdehyde-Coenzyme A. Biochim. Biophys. Acta. 44, 602-604.

25. Gamborg, O. L., Miller, R. A., and Ojima, K. (1968) Nutrient Requirements of Suspension Cultures of Soybean Root Cells. Exp. Cell Res. 50, 151-158.

26. Murashige, T., and Skoog, F. (1962) A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Physiol Plant. 15, 473-497.

27. Graham, I. A., and Eastmond, P. J. (2002) Pathways of Straight and Branched Chain Fatty Acid Catabolism in Higher Plants. Prog. Lipid Res. 41, 156-181.

28. Daschner, K., Couee, I., and Binder, S. (2001) The Mitochondrial Isovaleryl-Coenzyme a Dehydrogenase of Arabidopsis Oxidizes Intermediates of Leucine and Valine Catabolism. Plant Physiol. 126, 601-612.

29. Reumann, S., Ma, C., Lemke, S., and Babujee, L. (2004) AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes. Plant Physiol. 136, 2587-2608.

30. Schuster, J., and Binder, S. (2005) The Mitochondrial Branched-Chain Aminotransferase (AtBCAT-1) is Capable to Initiate Degradation of Leucine, Isoleucine and Valine in almost all Tissues in Arabidopsis thaliana. Plant Mol. Biol. 57, 241-254.

31. Diebold, R., Schuster, J., Daschner, K., and Binder, S. (2002) The Branched-Chain Amino Acid Transaminase Gene Family in Arabidopsis Encodes Plastid and Mitochondrial Proteins. Plant Physiol. 129, 540-550.

32. Fujiki, Y., Sato, T., Ito, M., and Watanabe, A. (2000) Isolation and Characterization of cDNA Clones for the e1beta and E2 Subunits of the Branched-Chain Alpha-Ketoacid Dehydrogenase Complex in Arabidopsis. J. Biol. Chem. 275, 6007-6013.

33. Lutziger, I., and Oliver, D. J. (2001) Characterization of Two cDNAs Encoding Mitochondrial Lipoamide Dehydrogenase from Arabidopsis. Plant Physiol. 127, 615-623.

34. Hooks, M. A., Kellas, F., and Graham, I. A. (1999) Long-Chain Acyl-CoA Oxidases of Arabidopsis. Plant J. 20, 1-13.

35. Adham, A. R., Zolman, B. K., Millius, A., and Bartel, B. (2005) Mutations in Arabidopsis Acyl-CoA Oxidase Genes Reveal Distinct and Overlapping Roles in Beta-Oxidation. Plant J. 41, 859-874.

36. Eastmond, P. J., Hooks, M. A., Williams, D., Lange, P., Bechtold, N., Sarrobert, C., Nussaume, L., and Graham, I. A. (2000) Promoter Trapping of a Novel Medium-Chain Acyl- CoA Oxidase, which is Induced Transcriptionally during Arabidopsis Seed Germination. J. Biol. Chem. 275, 34375-34381.

37. Froman, B. E., Edwards, P. C., Bursch, A. G., and Dehesh, K. (2000) ACX3, a Novel Medium-Chain Acyl-Coenzyme A Oxidase from Arabidopsis. Plant Physiol. 123, 733-742.

38. Hayashi, H., De Bellis, L., Ciurli, A., Kondo, M., Hayashi, M., and Nishimura, M. (1999) A Novel Acyl-CoA Oxidase that can Oxidize Short-Chain Acyl-CoA in Plant Peroxisomes. J. Biol. Chem. 274, 12715-12721.

39. Richmond, T. A., and Bleecker, A. B. (1999) A Defect in Beta-Oxidation Causes Abnormal Inflorescence Development in Arabidopsis. Plant Cell. 11, 1911-1924.

40. Goepfert, S., Vidoudez, C., Rezzonico, E., Hiltunen, J. K., and Poirier, Y. (2005) Molecular Identification and Characterization of the Arabidopsis Delta(3,5),Delta(2,4)-Dienoyl- Coenzyme A Isomerase, a Peroxisomal Enzyme Participating in the Beta-Oxidation Cycle of Unsaturated Fatty Acids. Plant Physiol. 138, 1947-1956.

41. Kirch, H. H., Bartels, D., Wei, Y., Schnable, P. S., and Wood, A. J. (2004) The ALDH Gene Superfamily of Arabidopsis. Trends Plant Sci. 9, 371-377.

42. Small, I., Peeters, N., Legeai, F., and Lurin, C. (2004) Predotar: A Tool for Rapidly Screening Proteomes for N-Terminal Targeting Sequences. Proteomics. 4, 1581-1590.

43. Emanuelsson, O., Nielsen, H., Brunak, S., and von Heijne, G. (2000) Predicting Subcellular Localization of Proteins Based on their N-Terminal Amino Acid Sequence. J. Mol. Biol. 300, 1005-1016.

44. Lange, P. R., Eastmond, P. J., Madagan, K., and Graham, I. A. (2004) An Arabidopsis Mutant Disrupted in Valine Catabolism is also compromised in Peroxisomal Fatty Acid Beta- Oxidation. FEBS Lett. 571, 147-153.

45. Dunford, R., Kirk, D., and Ap Rees, T. (1990) Respiration of Valine by Higher Plants. Phytochemistry. 29, 41-43.

46. Wu, K., Mourad, G., and King, J. (1994) A Valine-Resistant Mutant of Arabidopsis thaliana Displays an Acetolactate Synthase with Altered Feedback Control. Planta. 192, 249-255.

47. Daschner, K., Thalheim, C., Guha, C., Brennicke, A., and Binder, S. (1999) In Plants a Putative Isovaleryl-CoA-Dehydrogenase is Located in Mitochondria. Plant Mol. Biol. 39, 1275-1282.

48. Faivre-Nitschke, S. E., Couee, I., Vermel, M., Grienenberger, J. M., and Gualberto, J. M. (2001) Purification, Characterization and Cloning of Isovaleryl-CoA Dehydrogenase from Higher Plant Mitochondria. Eur. J. Biochem. 268, 1332-1339.

49. Miernyk, J. A., Thomas, D. R., and Wood, C. (1991) Partial Purification and Characterization of the Mitochondrial and Peroxisomal Isozymes of Enoyl-Coenzyme A Hydratase from Germinating Pea Seedlings. Plant Physiol. 95, 564-569.

50. Lawand, S., Dorne, A. J., Long, D., Coupland, G., Mache, R., and Carol, P. (2002) Arabidopsis A BOUT DE SOUFFLE, which is Homologous with Mammalian Carnitine Acyl Carrier, is Required for Postembryonic Growth in the Light. Plant Cell. 14, 2161-2173.

51. Reumann, S. (2004) Specification of the Peroxisome Targeting Signals Type 1 and Type 2 of Plant Peroxisomes by Bioinformatics Analyses. Plant Physiol. 135, 783-800.

52. Ludwig-Muller, J. (2000) Indole-3-Butyric Acid in Plant Growth and Developement. Plant Growth Regul. 32, 219-230.

Chapter 3: Functional Diversity of Mitochondrial β-Hydroxyacyl-CoA Hydrolases in A. thaliana

Kerry A. Lucas, Zhenlian Ke and John W. Hawes*

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056

*Corresponding author: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056; Tel.: 513-529-8072; fax: 513-529-5715. Email:[email protected]

Author contributions: ZK determined parameters for coupled assay and purified HHYD, all other experiments were performed by KAL.

The data in this chapter will be submitted to Plant Physiology and Biochemistry

3.1 Summary In plants, the classical pathway for valine catabolism likely has multiple metabolic functions for disposal of valine and of isobutyryl-CoA from diverse sources such as valine, other amino acids, branched-chain fatty acids, and phytanic acid derived from chlorophyll. These functions appear to be matched by the presence of diverse β-hydroxyacyl-CoA hydrolase isoforms. Eight distinct β-hydroxyacyl-CoA hydrolase homologs are present in the Arabidopsis thaliana genome. Three of these genes are reported to code for peroxisomal hydrolases, and three are reported to code for mitochondrial hydrolases. Only one of these genes, CHY1, encoding a peroxisomal hydroxyacyl-CoA hydrolase protein, has been functionally examined to date. The functional importance of the three putative mitochondrial β-hydroxyacyl-CoA hydrolase genes of A. thaliana have been examined by characterization of sequence homology, T-DNA insertion mutations, enzyme activity, and mRNA expression profiles during growth and development. The isoforms are designated as CHY4 (At4g31810), CHY5 (At3g60510) and CHY6 (At3g24360). CHY4 and CHY5 display a higher level of amino acid sequence homology and display very characteristic mitochondrial targeting sequences compared to CHY6. The genes coding for these three proteins differ with respect to both expression profiles and effects of T-DNA insertion mutations. Mutations in CHY4 appear to be lethal suggesting critical and non- redundant functions, while mutations in CHY5 result in resistance to inhibitory levels of valine.

3.2 Introduction β-Hydroxyisobutyryl-CoA hydrolase (3-hydroxy-2-methyl-propionyl-CoA hydrolase, EC 3.1.2.4) catalyzes the hydrolysis of β-S-hydroxyisobutyryl-CoA (HIBYL-CoA), an intermediate in the valine catabolic pathway, producing free coenzyme A and β-hydroxyisobutyrate (1, 2) (Fig. 3.1). This aspect of the catabolism of valine differs from that of other branched-chain amino acids that are metabolized solely as CoA esters. In some mammalian tissues, the β- hydroxyisobutyrate produced by this reaction is oxidized to methylmalonate semialdehyde, where in others it enters the blood stream and serves as a substrate for hepatic gluconeogenesis (3). The enzymes catalyzing these reactions in mammalian systems are strictly mitochondrial (4). In plants, the same reactions are utilized for valine catabolism but likely have multiple metabolic functions for disposal of isobutyryl-CoA in both mitochondria and peroxisomes (5, 6). In contrast to the mammalian genome, the plant genome codes for multiple homologs of HIBYL-CoA hydrolase. The A. thaliana genome codes for eight distinct homologs of this enzyme (7) and a similar number of homologous genes appear in the genomes of a wide variety of plant species including the completed rice and poplar genomes. Three of the A. thaliana homologs were proposed to have mitochondrial leader sequences, whereas three of the other homologs contain peroxisomal targeting sequences (7). Only one of these enzymes, CHY1, has been examined experimentally and was shown to be an authentic HIBYL-CoA hydrolase with peroxisomal localization (7). HIBYL-CoA hydrolase catalyzes a reaction unique to the catabolism of valine (as compared to other branched-chain amino acids) and may also be important for the catabolism of isobutyryl-CoA derived from branched-chain fatty acids. Therefore it is important to note that -oxidation of fatty acids in plants occurs exclusively in peroxisomes (8-11), and peroxisomal HIBYL-CoA hydrolases may have metabolic functions aside from that associated with valine catabolism (6). The existence of this enzyme was also postulated as a mechanism to protect cells from the effects of the toxic metabolite, methacrylyl- CoA (2). Evidence for this hypothesis first came from metabolic observations of a human patient with a genetic defect in the HIBYL-CoA hydrolase gene (12) and later from phenotypic observations of mutant lines of A. thaliana (7, 13). Previous data also suggest that peroxisomal methylacrylyl-CoA may react with β-ketoacyl-CoA thiolase in A. thaliana peroxisomes affecting peroxisomal β-oxidation (14).

Figure 3.1 Valine catabolic pathway. BCAT, Branched-Chain Aminotransferase; BCKDH, Branched-Chain α-Ketoacid Dehydrogenase; HIBADH, Hydroxyisobutyrate Dehydrogenase; MMSDH, Methylmalonate Semialdehyde Dehydrogenase.

CO2- Valine

NH3+

BCAT α-ketoglutarate glutamate

α-Keto- CO2- isovalerate O

+ BCKDH NAD , CoA-SH NADH, CO2

SCoA Isobutyryl-CoA

FAD+ Acyl-CoA FADH2 Dehydrogenase

SCoA Methacrylyl-CoA

O Enoyl-CoA H2O Hydratase HO Hydroxy- SCoA isobutyryl-CoA O HIBYL-CoA Hydrolase H2O HO Co-ASH

O Hydroxy- isobutyrate O

NAD+ HIBADH + O NADH, H

O Methylmalonate semialdehyde O MMSDH NAD+, Co-ASH NADH, CO2

SCoA Propionyl-CoA

O 62

The toxicity of methylacrylyl-CoA is well established and is believed to result from Michael addition reactions with important cellular thiols like glutathione, reduced coenzyme A, and protein cysteine residues, providing a possible functional link to oxidative stress responses (12). Though the existence of multiple homologous genes coding for HIBYL-CoA hydrolases in plants is well established, the functional importance of these separate isoforms, both mitochondrial and peroxisomal, has not been established. We have examined the functional importance of the three putative mitochondrial -hydroxyacyl-CoA hydrolases of A. thaliana by characterization of amino acid sequence homology, T-DNA insertion mutations, enzyme activity analysis, and mRNA expression profiles during growth and development. Data presented in this study are consistent with critical and non-redundant metabolic functions for separate mitochondrial forms of HIBYL-CoA hydrolase in A. thaliana.

3.3 Experimental Procedures

3.3.1 Materials All chemicals were purchased from Sigma-Aldrich unless otherwise noted.

3.3.2 Plant Materials and Growth Conditions Wild type A. thaliana ecotype Columbia (Col-0) was used for all studies. Mutant lines, derived from Col-0, were obtained from The Arabidopsis Resource Center (ABRC, Columbus, OH). Seedlings were surface sterilized and planted on soil (Sun Gro Metro-Mix 360), placed in 4 C for two days, then transferred to growth chambers. Plants were grown under 16-h-light/8-h- dark photoperiods at 21 23 C. Seeds were also surface sterilized and grown on plates using ½- strength Murashige and Skoog (MS (15)) medium and 1% (w/v) sucrose solidified with 0.8% (w/v) agar (Teknova Inc.) under the same growth conditions described above. For root growth measurements, primary roots were measured under the conditions described. Landsberg erecta green cell cultures were maintained using Gamborg basal salts ((16) Research Products International Corp.), supplemented with 2, 4-dichlorophenoxyacetic acid, and subdivided weekly under conditions described above.

3.3.3 T-DNA Insertion Mutations Seeds containing specific T-DNA insertion mutations were purchased from ABRC: CHY4, SALK_002356; CHY5, SALK_100127; CHY6, SALK_032288. After confirming the presence of heterozygous mutant plants, seeds were isolated and plants screened by PCR using primers that flank the T-DNA insertion site and the left border primer on the T-DNA insert from second and third generation heterozygous plants. The primers used are as follows: MC1.1 5’- GTGGAAGAGCCTAGATTTGC-3’, MC1.2 5’-TGAAGCTGCTAATTCGTGCA-3’, MC2.1 5’- GGTGCTGCATCCTTGTCTAT-3’, MC2.2 5’-GAAATGTTGGCATGTGGCCT-3’, MC3.1 5’-GCATATCAGGCTATACTCAG-3’, MC3.2 5’-CTCAACGCCATGAATCTAG-3’, and LBa1 5’-ATGGTTCACGTAGTAGTGGGCCATC-3’. DNA sequence analysis confirmed insertion site as seen by PCR.

3.3.4 Bioinformatic Analysis Putative amino acid sequences and DNA sequences were obtained from TAIR (www.arabidopsis.org) and sequences were compared using BLAST. Predictions for localization of leader sequences were preformed using TargetP (http://www.cbs.dtu.dk/services/TargetP/) and Predotar (http://urgi.infobiogen.fr/predotar/predotar.html).

3.3.5 Recombinant Hydrolases Total RNA from six-day-old suspension cell cultures was isolated using RNeasy mini-kit (Qiagen) according to the manufacturer’s instructions except for the following modifications: frozen, powdered material was resuspended in lysis buffer, vortexed, homogenized with a glass homogenizer and then centrifuged at top speed in a micorcentrifuge for two minutes at room temperature. The supernatant was then used according to the instructions. Full-length cDNAs, excluding the leader sequences, were synthesized using the iScript cDNA synthesis kit (Bio-Rad) and PCR amplified using the following primers: CHY4rec1 5’-ATGTTCGAAGACCAGGTTC- 3’, CHY4rec2 5’-TCAAAATAAGGCTCTCGTT-3, CHY5rec1 5’- ATGCTTGATTACCAGGTTC-3’, CHY5rec2 5’-CTAAATGCTTCTCTGAGTT-3’. The cDNA was PCR amplified (iCycler, Bio-Rad) at 95 C for 3 min, 45 cycles of 95 C for 15 sec, 50 C for 15 sec and 72 C for 2 min, followed by one cycle at 72 C for 7 min to complete

extension and then held at 4 C. Within 24 hours the PCR product was used for cloning using the pTrcHis TOPO TA cloning kit (Invitrogen) according to manufacturer’s instructions. The resulting constructs were transformed into DH10B competent cells, incubated on TY agar plates containing ampicillin (50 μg/mL). Positive colonies were confirmed by colony PCR using the following primers: pTrcHis Forward (5’-GAGGTATATATATTAATGTATCG-3’) and the 3’ primer for each hydrolase gene for proper orientation. Positive colonies were made competent (17) for co-transformation with pGroESL (18) as above. Positive colonies were selected with ampicillin (50 μg/mL) and chloramphenicol (50 μg/mL). For enzyme purification, cells were grown in TY media with ampicillin (50μg/mL) and chloramphenicol (50 μg/mL) in a 37 °C shaking incubator to OD600 of approximately 0.60 and induced with 1-thio-β-D- galactopyranoside overnight (0.5 mM). Recombinant proteins were purified from 2 L cultures using Ni-CamTM HC Resin (Sigma) according to manufacturer’s instructions except lysis, wash and elution buffers each contained 10 mM β-mercaptoethanol. Purified protein was analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining, trypsin digestion, and MALDI-TOF mass spectrometry.

3.3.6 Enzyme Activity Kinetic assays of recombinant proteins were carried out on Varian Cary UV-Vis Spectrophotometer. A coupled assay measuring the production of NADH by recombinant hydroxyisobutyrate dehydrogenase (HIBADH, (19)) at 340 nm, 37 °C, was used to measure the specific activity of the hydrolase proteins. Final concentrations of assay components included 100 mM Tris-HCl (pH 8.0), 100 mM β-NAD+ (made fresh), HIBADH (108 μg), CHY4 (21 µg), or CHY5 (6 µg), or human HIBYL-CoA hydrolase (28 μg), and saturating amounts of HIBYL- CoA to a total volume of 1 mL. Each reaction was started by the addition of HIBYL-CoA, after measuring a baseline rate for several minutes. HIBYL-CoA was synthesized according to (20). Recombinant human HIBYL-CoA hydrolase was purified as according to (1).

3.3.7 Measuring mRNA Levels Seedlings grown on plates were treated with valine, isobutyrate, and menadione where indicated. Total RNA was isolated as described above. cDNA was synthesized using cDNA synthesis kit from Bioline (Taunton, MA). The cDNA was PCR amplified using the following

primers: CHY4.RU 5’-AAGGCTCTCGTTGAGCTGTT-3’, CHY4.RD 5’- CTCTCAATGTGTTTCTGCG-3’, CHY5.RU 5’-ATCAATCGAGCTCGTACTCC-3’, CHY5.RD 5’-ATGCACTCACCACTCACATG-3’, CHY6.RU 5’- CATCTACTTCGTCTAAGCTC-3’, CHY6.RD 5’-CTCATTACTCTTGATCGACC-3’, CHY1.RU 5’- TTCTGCTCTGCTGCTTTGG-3’, CHY1.RD 5’- CAAGGAGAACAGTCGAAGA-3’, E1Beta.1 5’-GAGCTAAAGGGTAACGTCGA-3’, E1Beta.2 5’-GCCACAAACTCTGCTTACAG-3’, 968-Actin 5’- GGCAGGATTAGCAGGAGAAGATGC-3’ and 969-Actin 5’- CCTGATATCCACTCACACTTCAT-3’ . PCR products were visualized on 0.7% (w/v) agarose gel stained with ethidium bromide.

3.3.8 Complementation Plasmid containing full-length cDNA for CHY4 (U10305) was obtained from ABRC. The full-length cDNA (including mitochondrial leader sequence) was PCR amplified using primers containing restriction sites for XhoI and XbaI. Following restriction digestion and gel purification (Qiagen, Qiaquik Gel Purification), the gene was ligated directly into pFGC5941 (GenBank Accession no. AY310901, obtained from Dr. Chris Makaroff, Miami University) at 16 °C, 16 hrs. Transformants were selected with kanamycin (50 μg/mL) and positive colonies were confirmed by PCR and restriction digests of purified plasmid. Constructs were transformed into GV3103/PMP90 Agrobacterium cells and transferred into heterozygous chy4 plants according to (21). BASTA (Farnam Companies, Inc) resistant transformants were selected and confirmed by PCR screening, restriction digests and evidence for rescue of phenotype.

3.3.9 Microscopy For seed analysis, siliques from heterozygous chy4 plants, complementation and wild- type plants were dissected according to (22) and examined using an Olympus SZX-12 Stereoscope and pictures taken with a Nikon D200. Confocal microscopy was performed on an Olympus FV500 Laser Scanning Confocal System using Fluoview software. Fixation and staining of seeds were performed according to (23) and cleared and mounted with methyl salicylate.

3.4 Results and Discussion

3.4.1 Sequence Identity of Putative Mitochondrial Hydrolases Examination of the A. thaliana genome previously showed that this organism codes for eight separate genes homologous to mammalian -hydroxyisobutyryl-CoA hydrolase (GenBank Accession no. AAC52114) (7). Three of these homologs clearly contain Type I peroxisomal targeting sequences, whereas three others contain N-terminal leader sequences proposed as mitochondrial targeting sequences (7, 24). Each of these proteins display sequence identity in specific domains including conserved Glu and Asp residues previously shown to be critical for catalysis of β-hydroxyisobutyryl-CoA hydrolysis (7). Two of the proposed mitochondrial homologs coded for proteins with a higher level of sequence identity compared to the other homologs, including the third proposed mitochondrial homolog. We have designated these two isoforms as CHY4 and CHY5. Figure 3.2A shows an amino acid sequence alignment of the mammalian hydrolase, the three putative mitochondrial CHY proteins of A. thaliana and the established peroxisomal hydrolase, CHY1. CHY4 and CHY5 display 52% identity to each other compared to 30% identity to the human enzyme and 45% identity to the peroxisomal CHY1 protein. The third isoform, which we have tentatively designated CHY6 differs from CHY4 and CHY5 in several respects. CHY6 displays a similar level of sequence identity with the mitochondrial homologs as well as the other homologs including CHY1. However, the N- terminal leader sequence of CHY6 shows structural features characteristic of targeting sequences for both mitochondrial and chloroplast proteins. Helices with a predominance of basic residues on one face and hydrophobic residues on the opposite side are characteristic of mitochondrial targeting structures. In contrast, chloroplast-targeting sequences display fewer basic residues and a predominance of serine and threonine residues (25). Cursory examination of the CHY6 leader sequence shows several basic amino acids; however, these residues are not evenly distributed and do not position well on a single face of a helical wheel. CHY6 also contains a higher number of serine residues (Fig. 3.2B).

Figure 3.2 Sequence comparison of A. thaliana CHY proteins with human HIBYL-CoA hydrolase. A, The three putative mitochondrial CHY amino acid sequences were aligned with the known peroxisomal CHY1 amino acid sequence and the known human hydrolase, HHYD. CHY4 and CHY5 show higher levels of sequence identity, whereas CHY6 shows significantly less identity. Sequences were aligned using BLAST. Shaded areas indicate where at least three residues are identical. Basic residues in the N-terminal leader sequences are highlighted in gray and serine residues in CHY6 are starred. B, Predicted helical wheels showing the distribution of the first 30 N-terminal amino acids of each protein, highlighting Ser, Arg, and Lys residues. CHY4 and CHY5 have similar leader sequences, matching well with typical mitochondrial leader sequences.

CHY4 CHY5 CHY6

The leader sequences of CHY4 and CHY5 show a greater number of basic residues, which is in agreement with predicted mitochondrial localization. Bioinformatic analysis of these sequences using algorithms for prediction of targeting sequences also predicts a strong probability of mitochondrial localization for CHY4 and CHY5 but not for CHY6. TargetP predicts a 0.430 probability for localization of CHY6 to chloroplasts and a 0.283 probability of mitochondrial localization. The entry for this protein in The Arabidopsis Information Resource (TAIR) currently lists this protein as localized to chloroplasts based on these bioinformatic predictions. However this is based on weak prediction scores and CHY6 may still function as a mitochondrial protein, considering there are no other enzymes in the valine degradation pathway localized to the chloroplast.

3.4.2 Activity of Recombinant Hydrolases CHY4 and CHY5 were co-expressed with pGroESL to aid in protein folding (26-30). Column elution resulted in co-purification of GroEL (60 kDa) as seen in other labs (31). CHY4 and CHY5 were purified to 53.7% and 52.9%, respectively, with the only other contaminating band being that of GroEL (data not shown). Initial attempts to measure hydrolysis of HIBYL-CoA by purified CHY4 and CHY5, using the conventional DTNB (5,5′-dithiobis(2-nitrobenzoic acid)) assay, showed only slight activity and a high degree of variability. Examination of the number of Cys residues in these CHY proteins compared to those previously studied shows that the A. thaliana mitochondrial hydrolases are much more Cys rich, providing a likely explanation for difficulty with the DTNB assay method. DTNB is known to modify the free sulfhydryl groups within proteins, potentially inhibiting enzyme activity (32, 33). CHY4 and CHY5 have ten and eleven Cys residues, respectively, which are evenly distributed throughout their amino acid sequences. Some of these lie very near amino acid residues of known catalytic importance (7). Therefore, to measure activity of these proteins, we employed a coupled assay based on the oxidation of the β- hydroxyisobutyrate released from the CoA ester using purified recombinant β- hydroxyisobutyrate dehydrogenase. This enzyme is highly specific for oxidation of hydroxyisobutyrate and has been well characterized (34). Using this assay method, hydrolysis of HIBYL-CoA was consistently measured with CHY4 and CHY5 as well as the human enzyme.

As shown in Table 3.1, CHY4 has reduced activity compared to CHY5 and the human hydrolase (HHYD). These data show that CHY4 and CHY5 are in fact HIBYL-CoA hydrolases with activity similar to that of the well characterized human HIBYL-CoA hydrolase.

3.4.3 Mitochondrial CHY mRNA Levels of Seedling Growth and the Effects of Sucrose Gene expression of BCAA degradation is known to be high during germination and seedling establishment due to the degradation of protein and lipid storage reserves (35-37). Total RNA was isolated, and mRNA levels were measured from seedlings grown on MS plates over 10 days after imbibition (DAI) from whole seedlings. CHY4 and CHY5 showed higher message levels than CHY6; however, they all exhibited consistent levels similar to (13) (Fig. 3.3A). This is similar to gene expression data published by the expression analysis program, Genevestigator (38) (Fig 3.3B). Under conditions where carbon starvation is induced (namely, during extended periods of darkness or shading), plants have evolved to use alternative sources such as lipids and amino acids from protein breakdown to fuel metabolism (37). Previous data have shown that gene expression for branched-chain α-ketoacid dehydrogenase (39, 40), isovaleryl-CoA dehydrogenase (41), and CHY1 (13) are repressed in the presence of exogenous sucrose. It has yet to be examined whether mitochondrial proteins in the later half of the valine catabolic pathway respond similarly during carbon starvation. Wild-type seedlings were grown for five days on ½- strength MS agar plates with or without 1% sucrose under a long-day, light cycle. These conditions are similar to those previously used to measure CHY1 expression (13). There does not appear to be a similar response for CHY4 or CHY5 as seen for CHY1 (Fig. 3.4). In contrast to CHY1 expression, CHY6 appears to have lower message levels in the absence of sucrose, demonstrating that it may have a distinct functional role, different from the other mitochondrial hydrolases. The functional role of CHY6 may be to aid in the metabolism of HIBYL-CoA from sources other than valine under normal growing conditions (6) but become less necessary during times of carbon starvation.

Table 3.1 Specific activity of recombinant hydrolases. Specific activity was measured using a spectrophotometric, coupled assay based on the oxidation of β-hydroxyisobutyrate. Each reaction was initiated by the addition of the substrate, HIBYL- CoA, and activity was measured according to the reduction of NAD+ at 340 nm and 37 °C over the course of several minutes. Activity is expressed as μmoles of NADH per minute per mg of purified protein. Hydrolase protein ranging from 5-30 μg was used in a 1 mL assay volume. Specific activities were averaged for triplicate runs.

HHYD CHY4 CHY5 Specific Activity 109 (±38) 21 (±2) 69 (±7) (μmol/min/mg)

Figure 3.3 Mitochondrial CHY mRNA levels during seedling growth. Two-step RT-PCR of mRNA levels during seedling growth is shown. Total RNA was isolated from seedlings grown on ½-strength MS plates and cDNA synthesized and PCR amplified according to Experimental Methods. A, Seedlings were grown for the number of days indicated after imbibition at 21-23 °C and 16-hr-light/8-hr-dark photoperiod. B, Graph shows microarray data as depicted by the expression analysis program, Genevestigator, as a correlation between the average of absolute signal intensity values using MAS 5.0 normalization factor TGT set to 1000 and the age of the plant.

DAI 4 6 8 10 A

CHY4

CHY5

CHY6

ACT2

2500 CHY4 CHY5 CHY6 2000

1500

1000

500

0 Avg. of Absolute signal intensity values intensity signal of Avg.Absolute 1.0-5.9 6.0-13.9 14.0-17.9 18.0-20.9 21.0-24.9 25.0-28.9 29.0-35.9 36.0-44.9 45-50 Age (days)

Figure 3.4 Effects of sucrose on mitochondrial CHY mRNA levels. Two-step RT-PCR of mRNA levels in the presence and absence of sucrose is shown. Total RNA was isolated from seedlings grown on ½-strength MS plates for 5 days and cDNA synthesized and PCR amplified according to Experimental Procedures. E1β codes for a subunit of BCKDH known to be repressed by the presence of sucrose. ACT2 served as the loading control. 1% Sucrose + -

CHY4

CHY5

CHY6

E1β

ACT2

3.4.4 Effects of Metabolites and Oxidative Stress on Mitochondrial CHY mRNA Levels Analysis of microarray data using the program Genevestigator (38) revealed that these mitochondrial hydrolases show little change in message levels under common stress conditions. To take a more detailed look at conditions specific for these hydrolase genes, seedlings were grown on plates for 7 to 8 days with various concentrations of valine, isobutyrate, or menadione. For valine and isobutyrate mRNA expression profiles, a range of concentrations were used to see the effects with increasing concentrations. Using two-step RT- PCR, CHY4, CHY5, and CHY6 showed comparable mRNA levels when treated with exogenous valine or isobutyrate, with no effect seen under either stress (Fig. 3.5A, B). It has been previously shown that the production of β-hydroxyisobutyrate in wild-type seedlings treated with exogenous isobutyrate was greatly depleted in the peroxisomal, chy1 mutants (6). No change in message levels for these mitochondrial hydrolase genes under similar conditions demonstrates that exogenous isobutyrate is mainly metabolized in peroxisomes and not mitochondria, previously reported by Lucas et al (6). It has also been shown that some exogenous valine can be taken up by the chloroplast and converted to leucine (6). Plants may do this in order to avoid excess accumulation of methacrylyl-CoA, resulting in an unaltered response to exogenous valine by the mitochondrial hydrolases. The valine pathway intermediate, methylacrylyl-CoA, is reactive with reduced glutathione and other cellular thiols. This high reactivity forces the reaction towards the production of more methylacrylyl-CoA, resulting in a greater depletion of cellular thiols and creating an oxidative environment (12). Menadione is known to lead to production of oxidants such as superoxide ions and peroxides (42) and to deplete cellular thiols causing oxidative damage (43). Previous studies using menadione as an oxidative stress inducer with A. thaliana showed that morphological changes in the mitochondria do occur during the early stages of cell death (42). To test the effects of oxidative stress on mRNA levels, seedlings were grown on plates with 50 μM menadione. CHY4 showed an increase in message levels whereas CHY5 and CHY6 showed very little change (Fig. 3.5C). This may be an important part of the biological role of CHY4 - to prevent accumulation of methylacrylyl-CoA, thereby avoiding further damage of key cellular thiols during times of oxidative stress.

Figure 3.5 Effect of metabolites and oxidative stress on mitochondrial CHY mRNA levels. Two-step RT-PCR of mRNA levels is shown. Total RNA was isolated from seedlings grown on ½-strength MS plates supplemented with valine, isobutyrate and menadione for 6 days and cDNA synthesized and PCR amplified according to Experimental Procedures. Seedlings were treated with A, 1 mM valine; B, 1 mM isobutyrate; C, 50 μM menadione.

A B C

CHY4

CHY5

CHY6

ACT2 - + - + - + Valine Isobutyrate Menadione

The highly active metabolic state of germinating seedlings has been associated with increased oxidative stress. Increased production of reactive oxygen species at the onset of germination has been demonstrated in Glycine max (44), but similar studies have not been performed with A. thaliana, leaving the mechanism for the CHY4 response to oxidative stress unclear.

3.4.5 Effects of T-DNA Insertion Mutations Reverse genetics, through T-DNA insertion mutants for example, can provide phenotypic information that may be helpful for determining the functions of particular proteins. Heterozygous T-DNA insertion seeds for each mitochondrial CoA hydrolase gene were obtained from ABRC and screened by PCR for viable homozygous mutants. Figure 3.6 shows the location of the T-DNA insert for each gene as confirmed by PCR and DNA sequencing. CHY4 heterozygous seeds produced no viable homozygous plants (approximately 500 screened). Failure to obtain homozygous mutant plants suggests homozygous lethality for this gene. Further investigation of the seed within mature siliques of CHY4 heterozygotes, showed that 24% (of 378) of the seeds were phenotypically different (Fig. 3.7A,C), suggesting a deficiency in endosperm or embryo development (22). Further analysis by confocal microscopy revealed that 21% (of 518) of the embryos arrested at heart stage (Fig. 3.8A). This stage is the end of the first phase of embryogenesis, with subsequent stages occurring more rapidly until a mature embryo is developed and thus is a common stage for mutations to be visible (45). It is possible that CHY4 is expressed during the earliest stages of embryo development and when the gene is knocked-out, toxic levels of methylacrylyl-CoA accumulate causing inhibitory effects on embryogenesis. Complementation of heterozygous plants with the full-length CHY4 gene rescued the white seeds found in heterozygous siliques (Fig. 3.7B) and the arrest in embryo development (data not shown). Seed from heterozygous chy5 plants produced viable homozygous mutant plants. Homozygous seedlings grown on 1% sucrose, ½-strength MS plates with inhibitory amounts of valine showed resistance compared to wild-type (Fig. 3.9A-C). Even at levels of valine when the roots of chy5 mutants begin to respond similarly to wild-type, cotyledons and newly emerged rosette leaves still display resistance to valine compared to wild-type (Fig. 3.9B). Valine resistance is a well-known feature of mutations in acetohydroxy acid synthase (AHAS, EC 2.2.1.6), a gene found in the branched-chain amino acid biosynthesis pathway (46).

Figure 3.6 Location of T-DNA insertion sites for each mitochondrial CHY gene. Blocks represent exons, while lines represent introns and untranslated regions for each genomic DNA sequence. Arrows depict direction of T-DNA insert. A, CHY4; B, CHY5; C, CHY6.

A SALK_002356

5’ 3’ I II III IV V VI VII VIII IX X XI XII XIII XIV

B SALK_100127

5’ 3’ I II III IV V VI VII VIII IX X XI XII XIII XIV

SALK_032288 C 5’ 3’ I II III IV V VI VII VIII IX X

1kb

Figure 3.7 Light microscopy of A. thaliana siliques. All siliques were collected at similar times after flowering and captured at the same magnification. A, Heterozygous chy4 silique; B, chy4 complement silique; C, Wild-type Col-0 silique.

A B C

Figure 3.8 Confocal microscopy of chy4 seeds from a single heterozygous silique. Seeds were fixed and stained according to that found in Experimental Procedures. A, Stages of embryo development; B, chy4 homozygous embryo arrested at heart stage, bar = 50 μM; C, CHY4 heterozygous or wild-type embryo at bent torpedo stage, bar = 100 μM.

A B

Figure 3.9 Valine resistance of chy5 mutant seedlings. chy5 mutant and Col-0 wild-type seedlings were grown on ½-strength MS plates at 21-23 °C during 16-hr-light/8-hr-dark days. A, Seedlings were grown for 6 days with 0.5 mM valine; B, Seedlings were grown for 12 days with 1.5 mM valine; C, Primary roots were measured at 12 DAI and are shown as a percentage of control plate (no valine). Solid line, chy5 knockout; dashed line, wild-type.

WT CHY5 B

WT CHY5

C 90.0% 80.0% 70.0% 60.0% 50.0% 40.0%

% of Control of % 30.0% 20.0% 10.0% 0.0% 0.25 0.5 1 1.5 Valine Concentration (mM)

By adding valine exogenously to AHAS mutants, the plant was able to restore branched-chain amino acid metabolism and subsequently, protein synthesis. AHAS mutants show resistance to valine at much higher concentrations than seen with chy5 mutants (46). Under normal growing conditions, valine turnover is low with little possibility of the accumulation of toxic, downstream intermediates, primarily because of repressed expression of BCKDH subunits due to adequate carbon availability. When seedlings are subsequently stressed with exogenous valine, flux through the degradation pathway increases in order to use valine as a potential carbon source. However, this could cause the accumulation of toxic intermediates that would inhibit growth. chy5 mutants, on the other hand, may have reduced flux through this pathway and thus avoid accumulation of downstream intermediates, such as methylmalonate semialdehyde, resulting in growth resistance to inhibitory levels of valine (Fig. 3.9A-C). CHY6 heterozygous seeds also produced viable homozygous mutants with no visible phenotype along with no altered responses to valine, isobutyrate, or indole-3-butyric acid compared to wild-type. Subtle, undetected phenotypic differences may still exist, and further examination of this gene by metabolomic or DNA array methods may be required to determine if it has specific metabolic functions.

3.5 Conclusion Multiple mitochondrial and peroxisomal forms of β-hydroxyacyl-CoA hydrolase proteins are expressed in A. thaliana. Although they are homologous in sequence, these enzymes appear to have critical, and non-redundant, biological functions. From the data presented in this chapter, it is evident that mitochondrial β-hydroxyacyl-CoA hydrolases in A. thaliana show functional diversity. Their separate roles illustrate that CHY4 is necessary for embryogenesis and is responsive to oxidative stress, whereas chy5 knock-out mutants show resistance to inhibitory levels of valine in response to the apparent alleviation of methylmalonate semialdehyde. chy6 exhibits the greatest sequence diversity but showed no obvious phenotypic differences when mutated. It does however, appear to exhibit altered mRNA levels under carbon starvation, differing from the message levels of CHY4 and CHY5. From the data presented in this chapter, it is clear that mitochondrial β-hydroxyacyl-CoA hydrolase proteins have diverse functions, most likely including the detoxification of methylacrylyl-CoA and methylmalonate semialdehyde and the disposal of valine mobilized as a result of breakdown of storage proteins during germination.

3.6 Acknowledgements This work was supported in part by Miami University and a grant from the Ohio Plant Biotechnology Consortium. We would like to thank Dr. Meghan M. Holdorf and Dr. Chris A. Makaroff for advice and assistance with Arabidopsis plant and suspension cell culture methods.

3.7 References

1. Hawes, J. W., Jaskiewicz, J., Shimomura, Y., Huang, B., Bunting, J., Harper, E. T., and Harris, R. A. (1996) Primary Structure and Tissue-Specific Expression of Human Beta- Hydroxyisobutyryl-Coenzyme A Hydrolase. J. Biol. Chem. 271, 26430-26434.

2. Shimomura, Y., Murakami, T., Fujitsuka, N., Nakai, N., Sato, Y., Sugiyama, S., Shimomura, N., Irwin, J., Hawes, J. W., and Harris, R. A. (1994) Purification and Partial Characterization of 3-Hydroxyisobutyryl-Coenzyme A Hydrolase of Rat Liver. J. Biol. Chem. 269, 14248- 14253.

3. Letto, J., Brosnan, M. E., and Brosnan, J. T. (1986) Valine Metabolism. Gluconeogenesis from 3-Hydroxyisobutyrate. Biochem. J. 240, 909-912.

4. Harper, A. E. (1984) Branched-Chain Amino Acid Metabolism. Annu. Rev. Nutr. 4, 409.

5. Dunford, R., Kirk, D., and Ap Rees, T. (1990) Respiration of Valine by Higher Plants. Phytochemistry. 29, 41-43.

6. Lucas, K. A., Filley, J. R., Erb, J. M., Graybill, E. R., and Hawes, J. W. (2007) Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants. J. Biol. Chem. 282, 24980.

7. Zolman, B. K., Monroe-Augustus, M., Thompson, B., Hawes, J. W., Krukenberg, K. A., Matsuda, S. P., and Bartel, B. (2001) Chy1, an Arabidopsis Mutant with Impaired Beta- Oxidation, is Defective in a Peroxisomal Beta-Hydroxyisobutyryl-CoA Hydrolase. J. Biol. Chem. 276, 31037-31046.

8. Gerhardt, B. (1992) Fatty Acid Degradation in Plants. Prog. Lipid Res. 31, 417-446.

9. Kindl, H. (1993) Fatty Acid Degradation in Plant Peroxisomes: Function and Biosynthesis of the Enzymes Involved. Biochimie. 75, 225-230.

10. Lazarow, P. B. (1993) Genetic Approaches to Studying Peroxisome Biogenesis. Trends Cell Biol. 3, 89-93.

11. Olsen, L. J. (1998) The Surprising Complexity of Peroxisome Biogenesis. Plant Mol. Biol.

38, 163-189.

12. Brown, G. K., Hunt, S. M., Scholem, R., Fowler, K., Grimes, A., Mercer, J. F., Truscott, R. M., Cotton, R. G., Rogers, J. G., and Danks, D. M. (1982) Beta-Hydroxyisobutyryl Coenzyme A Deacylase Deficiency: A Defect in Valine Metabolism Associated with Physical Malformations. Pediatrics. 70, 532-538.

13. Lange, P. R., Eastmond, P. J., Madagan, K., and Graham, I. A. (2004) An Arabidopsis Mutant Disrupted in Valine Catabolism is also Compromised in Peroxisomal Fatty Acid Beta-Oxidation. FEBS Lett. 571, 147-153.

14. Hayashi, M., Toriyama, K., Kondo, M., and Nishimura, M. (1998) 2,4- Dichlorophenoxybutyric Acid-Resistant Mutants of Arabidopsis have Defects in Glyoxysomal Fatty Acid Beta-Oxidation. Plant Cell. 10, 183-195.

15. Murashige, T., and Skoog, F. (1962) A Revised Medium for Rapids Growth and Bioassays with Tobacco Tissue Cultures. Physiol Plant. 15, 473-497.

16. Gamborg, O. L., Miller, R. A., and Ojima, K. (1968) Nutrient Requirements of Suspension Cultures of Soybean Root Cells. Exp. Cell Res. 50, 151-158.

17. Dagert, M., and Ehrlich, S. D. (1979) Prolonged Incubation in Calcium Chloride Improves the Competence of Escherichia coli Cells. Gene. 6, 23.

18. Zhao, Y., Hawes, J., Popov, K., Jaskiewicz, J., Shimomura, Y., Crabb, D., and Harris, R. (1994) Site-Directed Mutagenesis of Phosphorylation Sites of the Branched Chain Alpha- Ketoacid Dehydrogenase Complex. J. Biol. Chem. 269, 18583-18587.

19. Njau, R. K., Herndon, C. A., and Hawes, J. W. (2000) Novel Beta -Hydroxyacid Dehydrogenases in Escherichia coli and Haemophilus influenzae. J. Biol. Chem. 275, 38780- 38786.

20. Hawes, J. W., and Harper, E. T. (2000) Synthesis of Methacrylyl-CoA and (R)- and (S)-3- Hydroxyisobutyryl-CoA. Methods Enzymol. 324, 73-79.

21. Chung, M., Chen, M., and Pan, S. (2000) Floral Spray Transformation can Efficiently Generate Arabidopsis Transgenic Plants. Transgenic Res. 9, 471.

22. Meinke, D. W. (1994) Seed Development in Arabidopsis, in Arabidopsis thaliana (E. M. Meyerowitz, and C. R. Somerville, Eds.) pp 253-295, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

23. Braselton, J. P., Wilkinson, M. J., and Clulow, S. A. (1996) Feulgen Staining of Intact Plant Tissues for Confocal Microscopy. Biotech. Histochem. 71, 84.

24. Reumann, S., Ma, C., Lemke, S., and Babujee, L. (2004) AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes. Plant Physiol. 136, 2587-2608.

25. von Heijne, G. (1986) Mitochondrial Targeting Sequences may Form Amphiphilic Helices. EMBO J. 5, 1335-1342.

26. Bochkareva, E. S., Lissin, N. M., and Girshovich, A. S. (1988) Transient Association of Newly Synthesized Unfolded Proteins with the Heat-Shock GroEL Protein. Nature. 336, 254.

27. Ostermann, J., Horwich, A. L., Neupert, W., and Hartl, F. U. (1989) Protein Folding in Mitochondria Requires Complex Formation with hsp60 and ATP Hydrolysis. Nature. 341, 125.

28. Rothman, J. E. (1989) Polypeptide Chain Binding Proteins: Catalysts of Protein Folding and Related Processes in Cells. Cell. 59, 591.

29. Ellis, R. J. (1990) The Molecular Chaperone Concept. Semin. Cell Biol. 1, 1.

30. Mendoza, J., Lorimer, G., and Horowitz, P. (1991) Intermediates in the Chaperonin-Assisted Refolding of Rhodanese are Trapped at Low Temperature and show a Small Stoichiometry. J. Biol. Chem. 266, 16973-16976.

31. Kedishvili, N. Y. (2000) Mammalian Methylmalonate-Semialdehyde Dehydrogenase. Meth. Enzymol. 324, 207.

32. Kato, H., Tanaka, T., Nishioka, T., Kimura, A., and Oda, J. (1988) Role of Cysteine Residues

in Glutathione Synthetase from Escherichia coli B. Chemical Modification and Oligonucleotide Site-Directed Mutagenesis. J. Biol. Chem. 263, 11646-11651.

33. Nakanishi, Y., Isohashi, F., Ebisuno, S., and Sakamoto, Y. (1989) Sulfhydryl Groups of an Extramitochondrial Acetyl-CoA Hydrolase from Rat Liver. Biochim. Biophys. Acta. 996, 209.

34. Rougraff, P. M., Paxton, R., Goodwin, G. W., Gibson, R. G., and Harris, R. A. (1990) Spectrophotometric Enzymatic Assay for S-3-Hydroxyisobutyrate. Anal. Biochem. 184, 317- 320.

35. Anderson, M. D., Che, P., Song, J., Nikolau, B. J., and Wurtele, E. S. (1998) 3- Methylcrotonyl-Coenzyme A Carboxylase is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants. Plant Physiology. 118, 1127-1138.

36. Sodek, L., and Wilson, C. M. (1973) Metabolism of Lysine and Leucine Derived from Storage Protein during the Germination of Maize. Biochim. Biophys. Acta. 304, 353.

37. Aubert, S., Alban, C., Bligny, R., and Douce, R. (1996) Induction of β-Methylcrotonyl- Coenzyme A Carboxylase in Higher Plant Cells during Carbohydrate Starvation: Evidence for a Role of MCCase in Leucine Catabolism. FEBS Lett. 383, 175-180.

38. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox. Plant Physiol. 136, 2621-2632.

39. Fujiki, Y., Sato, T., Ito, M., and Watanabe, A. (2000) Isolation and Characterization of cDNA Clones for the E1β and E2 Subunits of the Branched-Chain Alpha-Ketoacid Dehydrogenase Complex in Arabidopsis. J. Biol. Chem. 275, 6007-6013.

40. Fujiki, Y., Ito, M., Itoh, T., Nishida, I., and Watanabe, A. (2002) Activation of the Promoters of Arabidopsis Genes for the Branched-Chain α-Keto Acid Dehydrogenase Complex in Transgenic Tobacco BY-2 Cells Under Sugar Starvation. Plant Cell Physiol. 43, 275.

41. Daschner, K., Couee, I., and Binder, S. (2001) The Mitochondrial Isovaleryl-Coenzyme a

Dehydrogenase of Arabidopsis Oxidizes Intermediates of Leucine and Valine Catabolism. Plant Physiol. 126, 601-612.

42. Yoshinaga, K., Arimura, S., Niwa, Y., Tsutsumi, N., Uchimiya, H., and Kawai-Yamada, M. (2005) Mitochondrial Behaviour in the Early Stages of ROS Stress Leading to Cell Death in Arabidopsis thaliana. Ann. Bot. (Lond). 96, 337-342.

43. Tzeng, W. F., Chiou, T. J., Wang, C. P., Lee, J. L., and Chen, Y. H. (1994) Cellular Thiols as a Determinant of Responsiveness to Menadione in Cardiomyocytes. J. Mol. Cell. Cardiol. 26, 889-897.

44. Puntarulo, S. (1994) Effect of Oxidative Stress during Imbibition of Soybean Embryonic Axes. Proc Roy Soc Edinb B Biol. 102B, 279-286.

45. Bard, J. (1994) Embryos : Color Atlas of Development. Wolfe, London.

46. Wu, K., Mourad, G., and King, J. (1994) A Valine-Resistant Mutant of Arabidopsis thaliana Displays an Acetolactate Synthase with Altered Feedback Control. Planta. 192, 249-255.

Chapter 4: Evidence for Propionate and Isobutyrate Metabolism involving Mitochondrial Methylmalonate Semialdehyde Dehydrogenase in A. thaliana Mitochondria

Kerry A. Lucas and John W. Hawes*

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056

*Corresponding author: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056; Tel.: 513-529-8072; fax: 513-529-5715. Email:[email protected]

The data in this chapter will be submitted to Plant Physiology and Biochemistry

4.1 Summary The localization of valine degradation and propionyl-CoA metabolism in plants has been of interest for many years. It has been shown that valine and propionyl-CoA are metabolized, at least partially, in both the mitochondria and peroxisomes. This study is the first to show a direct relationship between mitochondria and peroxisomes in the metabolism of both valine and propionyl-CoA through experiments with plants containing a knockout methylmalonate semialdehyde dehydrogenase (MMSDH) in Arabidopsis thaliana. MMSDH knockout plants are viable but are sensitive to valine, which is metabolized in mitochondria, as well as isobutyrate and propionate, which have been shown to be metabolized in peroxisomes. Mutant seeds become wrinkled when desiccated, have delayed germination, and exhibit inverted cotyledons. AtMMSDH also showed very different message levels compared to those previously published for rice, showing that A. thaliana MMSDH is not responsive to gibberellin or auxin treatment. Also, unlike some enzymes in the valine degradation pathway, AtMMSDH mRNA levels are not repressed by sucrose. These data suggest AtMMSDH is critical during early seedling growth, most likely to metabolize both mitochondrial and peroxisomal intermediates of valine and propionyl-CoA metabolism.

4.2 Introduction Methylmalonate semialdehyde dehydrogenase (MMSDH, acylating CoA, E.C. 1.2.1.27) catalyzes the oxidative decarboxylation of methylmalonate semialdehyde and malonate semialdehyde to propionyl-CoA and acetyl-CoA, respectively. MMSDH is responsible for the last step in the valine degradation pathway and is involved in propionate and thymine metabolism (1). It belongs to the aldehyde dehydrogenase (ALDH) superfamily and is CoA dependent, which is unique among the ALDH family proteins (2). Earlier work on genetic diseases in humans showed HsMMSDH deficiencies resulted in an increased accumulation of S- and R-β-hydroxyisobutyrate, S- and R-aminoisobutyrate, and methylmalonate semialdehyde (3, 4). Likewise, a study demonstrated these genetic defects also result in the decreased production 14 of CO2 from labeled valine (5, 6). Clinically, patients exhibiting HsMMSDH deficiency experience developmental delay, but otherwise mature normally (6). This is in stark contrast to deficiencies in β-hydroxyisobutyryl-CoA hydrolase, an enzyme which acts upstream from MMSDH in the valine degradation pathway. Defects in β-hydroxyisobutyryl-CoA hydrolase cause severe birth defects ultimately leading to death shortly after birth (7). This is attributed to an accumulation of the highly reactive, toxic intermediate, methylacrylyl-CoA. Expression of MMSDH has been linked to many stress responses in mammalian systems, often times separate from its role in valine degradation or propionyl-CoA metabolism. For example, MMSDH serves as a biomarker for depression (8) and as a cardiac protein in aging rats (9). The same seems to hold true in higher plants as MMSDH appears to have several roles ranging from phytohormone responses affecting plant growth and development to metabolic responses like oxidative stress (10-12). A proteomics study in which Oryza sativa suspension cell cultures were treated with the synthetic auxin, 2, 4-dichlorophenoxyacetic acid (2, 4-D), found that protein levels of OsMMSDH were significantly induced, remaining high over 6 days (10). Auxins are a phytohormone involved in numerous processes including cell division, cell expansion, and cell differentiation (13-16). Interestingly, levels of auxin have also been directly linked to zinc availability (17). Due to this direct relationship of auxin and zinc availability, OsMMSDH expression was investigated in the presence of a combination of zinc and auxin, showing both enhanced protein and gene expression over auxin alone (17). Additionally, it was noted that OsMMSDH protein levels in the constitutive gibberellin (GA) response mutant, slr1, were

increased over two-fold in comparison to wild-type plants (10). Similar to auxin, GA is also a phytohormone shown to initiate germination and promote cell elongation and division (18). This led to the suggestion that OsMMSDH levels are regulated by the phytohormones, auxin, and GA, and therefore OsMMSDH may play a role in both plant growth and development (10). Recently, a more detailed study on OsMMSDH and GA signaling in rice was reported (11). Proteomics confirmed OsMMSDH increased close to seven-fold in slr1 suspension cell cultures compared to wild-type (11). MMSDH mRNA expression is also higher in roots and leaf sheaths of slr1 mutants, correlating to areas where GA signal transduction would be highest. Antisense, transgenic rice plants with reduced OsMMSDH expression had thinner seminal roots, an increased number of root hairs, and produced slightly shorter plants compared to the control. These data suggest that OsMMSDH does in fact play a role in root development and possibly stem elongation in rice (11). In a separate study in A. thaliana, Sweetlove et al found that AtMMSDH protein levels significantly decreased when cell cultures were treated with various oxidants, including peroxide and menadione. This is similar to the decrease observed in some TCA cycle proteins, a GABA transaminase, and ATP synthase (19) and signifies a possible role for AtMMSDH as a stress response marker. It is evident that MMSDH plays a significant role in rice development separate from its role in metabolism. However, there is a lot yet to be learned about the degradation of valine and the metabolism of propionyl-CoA, which can be accomplished through message response and knockout studies of MMSDH in A. thaliana. The valine degradation pathway is well characterized in mammalian systems and occurs solely in the mitochondria. In plants, this pathway is known to occur in multiple organelles (20- 28). Three enzymes in the pathway have isoforms in both the mitochondria and the peroxisomes. They include acyl-CoA dehydrogenase/oxidase, enoyl-CoA hydratase, and β- hydroxyisobutyryl-CoA hydrolase and these enzymes are responsible for the reactions in the middle of the pathway (Fig. 4.1, steps 3, 4, and 5). Interestingly, the enzymes in the pathway that do not have multiple isoforms are the initial steps, branched-chain aminotransferase (Fig. 4.1, step 1) and branched-chain α-ketoacid dehydrogenase (Fig. 4.1, step 2), and the final, AtMMSDH (Fig. 3.1, step 7), reported to be mitochondrial (12, 23, 29).

Figure 4.1 Valine degradation pathway in mitochondria and peroxisomes. 1, branched-chain aminotransferase; 2, branched-chain α-ketoacid dehydrogenase; 3, acyl-CoA dehydrogenase/oxidase; 4, enoyl-CoA hydratase; 5, hydroxyacyl-CoA hydrolase; 6, β- hydroxyacid dehydrogenase; 7, MMSDH. Intermediate abbreviations: KIV, ketoisovalerate; MA-CoA, methacrylyl-CoA; HIBYL-CoA, hydroxyisobutyryl-CoA; HPA-CoA, hydroxypropionyl-CoA; HIBA, hydroxyisobutyrate; HPA, hydroxypropionate; MMS, methylmalonate semialdehyde; MS, malonate semialdehyde.

Valine 1 KIV Propionate / Isobutyrate 2 Propionyl-CoA Isobutyryl-CoA Propionyl-CoA / Isobutyryl-CoA 3 3 MA-CoA Acrylyl-CoA / MA-CoA

4 4 HPA-CoA / HIBYL-CoA HIBYL-CoA 5 5 HPA / HIBA HIBA HPA Peroxisomes 6 6

MMS MS 7 7 Acetyl-CoA

Mitochondria

Recently, it was shown that both isobutyrate and propionate are metabolized in the peroxisomes (Fig. 4.1), leading to the accumulation of β-hydroxyisobutyrate and β- hydroxypropionate, respectively, but the metabolic fate of these hydroxyacids was speculative, as there are no peroxisomal enzymes to metabolize these end products further (28). The reverse genetics, the data presented here demonstrates further metabolism of these products by AtMMSDH in mitochondria. AtMMSDH knockout mutants produced wrinkled seeds that exhibited delayed germination, and inverted cotyledons compared to wild-type seedlings. The AtMMSDH mutant displays dramatically increased sensitivity to propionate and isobutyrate, as well as valine. This suggests that AtMMSDH may have multiple roles in plant metabolism and development, separate from that previously seen in rice.

4.3 Experimental Procedures

4.3.1 Plant Materials and Growth Conditions Wild-type A. thaliana ecotype Columbia (Col-0) was used for mRNA expression studies where indicated. Seedlings were surface sterilized (30 sec 70% EtOH, 30 min 10% bleach, rinse several times with dH2O) and placed on ½-strength Murashige and Skoog medium (MS (30)) and 1% (w/v) sucrose solidified with 0.8% (w/v) agar (Teknova Inc.). For expression studies with phytohormones, seeds were placed on MS plates containing 10 μM GA3, and 0.45 μM 2, 4- D. Plates were kept in the dark at 4 °C for 2 days and then allowed to germinate under 16-hour- light/8-hour-dark photoperiods at 21-23 °C for the number of days indicated. The T-DNA mutant line, WiscDsLox24A12 derived from Col-0, was obtained from The Arabidopsis Resource Center (ABRC, Columbus, OH). MMSDH heterozygous plants were screened using left border primer of the T-DNA insert and the AtMMSDH gene specific primer 5’-GCCAACATAGCACGTGAATTC-3’. Homozygous plants containing T-DNA were confirmed by DNA sequencing. Maintenance of the mutant line was conducted by surface sterilization and growth of the seeds on MS plates. Seeds were allowed to germinate for 6-10 days before being transferred to well hydrated soil (Sun Gro Metro-Mix 360) and pots were covered with plastic wrap to ensure high humidity during root re-establishment. Once seedlings began growth, the plastic wrap was removed.

4.3.2 Measuring mRNA Levels Total RNA was isolated using RNeasy mini-kit (Qiagen) according to the manufacturer’s instructions except for the following modifications: frozen, powdered plant material was resuspended in lysis buffer, vortexed, homogenized with a glass homogenizer and then centrifuged at top speed in a microcentrifuge for two minutes at room temperature. The supernatant was then used according to the instructions. AtMMSDH cDNA was synthesized using the cDNA synthesis kit (Bioline, Taunton, MA) and PCR amplified using the following gene specific primers used for germination and sucrose mRNA response studies: MMSDH.R5 5’-AATGCCTCCGAGAGTACCAA-3’, MMSDH.R3 5’-CTCCTGCTTTGCCATAGAAG-3’, E1Beta.1 5’-GAGCTAAAGGGTAACGTCGA-3’, E1Beta.2 5’- GCCACAAACTCTGCTTACAG-3’, ACT2.5 5’- GGCAGGATTAGCAGGAGAAGATGC-3’ and ACT2.3 5’- CCTGATATCCACTCACACTTCAT-3’. To confirm absence of expression in AtMMSDH homozygous knockout mutants, total RNA was isolated and cDNA synthesized as above from mature knockout and wild-type leaves as above. RT-PCR was performed using MMSDH.R5 and MMSDH.R3 primers shown above.

4.4 Results

4.4.1 AtMMSDH mRNA levels during germination Studies have shown that the enzymes in the branched-chain amino acid (BCAA) degradation pathway have increased gene expression during both germination and seedling establishment (31-33). To confirm presence of AtMMSDH in young seedlings, total RNA was isolated from seedlings and mRNA levels measured by RT-PCR over the first ten days of growth. Expression was found to be consistent throughout seedling growth, similar to other genes in the degradation pathway (21) (Fig. 4.2).

Figure 4.2 mRNA levels of MMSDH during germination and seedling establishment. Two-step RT-PCR of mRNA levels during the first 10 days of growth is shown. Total RNA was isolated from seedlings grown on ½-strength MS plates and cDNA synthesized and PCR amplified as described in Experimental Procedures. Seedlings were grown for the number of days indicated at 21-23 °C and 16-hr-light/8-hr-dark photoperiod.

DAI 2 4 6 8 10

MMSDH

ACT2

Valine degradation in plants is also associated with carbon depletion as plants are known to use alternative carbon sources during extended periods of darkness (21, 23, 34, 35). Gene expression studies for valine degradation have shown that subunits of branched-chain α-ketoacid dehydrogenase (BCKDH) are repressed by the presence of exogenous sucrose (23, 34), also seen for CHY1, a peroxisomal β-hydroxyisobutyryl-CoA hydrolase (21) and AtIVD, a mitochondrial acyl-CoA dehydrogenase/oxidase (35). Total RNA was isolated from 5 day old seedlings grown on MS plates with or without 1% sucrose. Unlike BCKDH subunits, CHY1 and AtIVD, AtMMSDH did not show a decrease in mRNA levels in the presence of sucrose, relative to BCKDH E1β, which was used as the control (Fig. 4.3). This suggests AtMMSDH may only play a minor role in promoting valine degradation when carbon sources are depleted. The mRNA levels of AtMMSDH also differed from results with rice seedlings that showed a significant increase in OsMMSDH expression in the presence of auxin and GA (10, 11). Message levels of AtMMSDH only showed a slight increase after 5 days with auxin and GA compared to hormone free control plates (Fig 4.4).

4.4.2 AtMMSDH Knockout Mutant Affects Seed Viability OsMMSDH antisense rice plants were previously shown to have reduced gene expression and demonstrated phenotypic differences, including thinner seminal roots, increased root hairs, and truncated plants in comparison to wild-type (11). To date, there have been no reported knockout studies in any plant species. Therefore, to provide further insight into the role that MMSDH has in plants, seeds heterozygous for a T-DNA insertion mutant of A. thaliana MMSDH were obtained from the ABRC. MMSDH heterozygous plants were screened by PCR and plants homozygous for the mutation were confirmed by the presence of a T-DNA insert by DNA sequencing (Fig. 4.5A). RT-PCR studies of mmsdh confirmed complete loss of AtMMSDH expression. Therefore the results presented below represent the phenotypic result of a true MMSDH knockout (Fig. 4.5B).

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Figure 4.3 MMSDH mRNA levels in the presence and absence of sucrose. Total RNA was isolated from seedlings grown on ½-strength MS plates in the presence or absence of 1% sucrose for 5 days at 21-23 °C and 16-hr-light/8-hr-dark photoperiods. cDNA was synthesized and PCR amplified as described in Experimental Procedures. E1β codes for a subunit of BCKDH known to be repressed by the presence of sucrose and used here as a control.

1% Sucrose + -

MMSDH

E1β

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Figure 4.4 MMSDH mRNA levels with GA and 2,4-D. Total RNA was isolated from seedlings grown on ½-strength MS plates supplemented with 10 μM GA and 0.45 μM 2, 4-D for 5 days at 21-23 °C and 16-hr-light/8-hr-dark photoperiods. cDNA was synthesized and PCR amplified according to Experimental Procedures.

10μM 0.45μM control GA3 2, 4-D MMSDH

Act2

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Figure 4.5 Location of T-DNA insertion and confirmation of homozygous knockout. A, T-DNA insert is located on intron 7 of chromosome 2. Squares represent exons, line represents introns and untranslated region. B, Total RNA was isolated from rosette leaves of fully developed knockout and wild-type plants. cDNA was then synthesized and PCR amplified according to Experimental Procedures.

A WISCDSLOX242A12

5’ 3’ I II III IV V VI VII VIII IX X XI XII XIII XIV XVI XVIII XIX XV XVII

1kb

WT Atmmsdh B AtMMSDH

Act2

103

Surprisingly, even though homozygous mmsdh knockout plants could be obtained from a MMSDH heterozygous population, the resulting mmsdh seeds exhibited low germination rates on soil. To investigate this result further, we studied seed and growth characteristics of the mmsdh plants. Desiccated seeds from mmsdh plants exhibit a wrinkled appearance in comparison to wild-type (Fig. 4.6A); however, imbibed mmsdh seeds (8-12 hrs) plated on MS plates supplemented with sucrose show no difference from those of wild-type (Fig. 4.6B). Young plantlets do exhibit characteristic differences though. First, seedlings appear to have delayed germination (approximately 12 hrs) compared to wild-type and then develop inverted cotyledons that persist throughout germination and establishment (Fig. 4.6C, D). This could be due to the inability to properly metabolize storage reserves and/or the accumulation of reactive intermediates such as methylacrylyl-CoA or (methyl)malonate semialdehyde. In contrast, after approximately 20-25 days on soil, mmsdh plants show no obvious differences in growth from wild-type (data not shown). This result is contrary to the observations that OsMMSDH RNA knockdown plants are shorter than wild-type (11).

4.4.3 Knockout Mutants Show Sensitivity to Peroxisomal Metabolites Given that AtMMSDH is the only dehydrogenase known to metabolize the last step in valine degradation in A. thaliana, it was predicted that knockout mutants treated with exogenous valine would have enhanced sensitivity to valine. This sensitivity could be attributed to an accumulation of reactive intermediates within valine and propionyl-CoA metabolism, like methylmalonate semialdehyde and malonate semialdehyde. When grown on MS plates containing inhibitory concentrations of valine, mmsdh seedlings do in fact show greater sensitivity to valine compared to wild-type as evidenced by shorter primary roots and defects in leaf development (Fig. 4.7B). Exogenous isobutyrate and propionate are metabolized in the peroxisomes (28). Exogenous propionate leads to the accumulation of β-hydroxypropionate, which appears to be further consumed over a 30 hr period (28), but the exact enzymes and sub-cellular localization for this metabolism are not known. Interestingly, Atmmsdh seedlings exhibited significantly increased sensitivity to inhibitory levels of exogenous isobutyrate and propionate compared to wild-type (Fig. 4.7C, D).

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Figure 4.6 Growth development of mmsdh knockout seedlings. Knockout and wild-type seedlings were grown on ½-strength MS plates for the number of days indicated at 21-23 °C under 16-hr-light/8-hr-dark photoperiods. A, Desiccated seeds stored at room temperature, bar = 0.32 cm. B, Seeds imbibed for 8-12 hrs at room temperature in nano- pure water, bar = 0.32 cm. C, 2 day old seedlings, bar = 0.1 cm. D, 4 day old seedlings, bar = 0.1 cm.

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Figure 4.7 mmsdh Knockout seedlings grown with valine, isobutyrate, and propionate. Knockout and wild-type seedlings were grown MS plates for 5 days. A, control seedlings, others were supplemented with B, 0.25 mM valine; C, 0.25 mM isobutyrate; D, 0.25 mM propionate. Bar = 0.1 cm.

WT Atmmsdh

Valine WT Atmmsdh A

Isobutyrate

Propionate

Control D

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4.4.4 Sequence Homology across Species MMSDH is a member of the ALDH superfamily and has high sequence identity across species (2). Failure to amplify the full-length cDNA (TAIR accession: 1007965274) with primers at the annotated start and stop codons resulted in a closer examination of the translated amino acid sequence. Sequence alignment with other published MMSDH homologs (human, rat, and rice) revealed high sequence similarity (74% with OsMMSDH (AAC03055), and 55% with RnMMSDH (NP_112319) and HsMMSDH (NP_005580)). It also revealed that AtMMSDH best aligned with the other sequences beginning at Met74 and not the annotated start codon (Fig. 4.8). Most Expressed Sequence Tags (ESTs) for AtMMSDH thus far appear to start at Met74 as well. These ESTs are also more similar to all other known MMSDH cDNAs. Primers located at the annotated start codon and 64 bases downstream from the annotated start codon would not amplify cDNA in multiple reactions (data not shown). Furthermore, when a primer was placed at the downstream ATG (corresponding to Met74), PCR resulted in amplification of a cDNA for AtMMSDH. Also of note, if Met74 were in fact the actual start codon for AtMMSDH, it would have a much improved mitochondrial leader sequence than that currently predicted (Table 4.1). This is supported by Sweetlove et al who showed MMSDH protein expression in mitochondrial extracts from A. thaliana suspension cell cultures (19).

4.5 Discussion In mammalian systems MMSDH appears to be highly responsive to various stress conditions and in some cases is thought to serve as a stress marker (44). Until recently, however, little had been known about MMSDH in plants. It seems that MMSDH serves a similar role in plants as it does in mammalian systems, which may be beneficial in predicting metabolic or developmental responses in plants under certain stress conditions. Expression of genes for BCAA degradation tends to be highest in fast growing tissues during germination where there is a large need for protein turnover. This results in degradation of storage proteins and the synthesis of new ones to support this rapid growth. In contrast to some genes involved in the valine degradation pathway that demonstrated increasing expression during germination (21, 45, 46), AtMMSDH mRNA levels did not change during the early days of seedling growth and maintained expression well into maturity (Fig. 4.2).

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Figure 4.8 Sequence alignment of MMSDH from A. thaliana, rice, human, and rat. Protein sequences were aligned using ClustalW2 (36). AtMMSDH (NP_001077888), OsMMSDH (AAC03055), HsMMSDH (NP_005580), RnMMSDH (NP_112319). Black highlights represent identity among all four species; grey highlights represent identity between OsMMSDH and AtMMSDH. Underline for AtMMSDH likely represents the inframe, 5’untranslated region. Underline for human and rat sequences are the known mitochondrial leader sequences (37).

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Table 4.1 Mitochondrial localization prediction scores and potential cleavage sites for AtMMSDH. MitoProt II 1.0a4a TargetP 1.1b Predator v.1.03c MITOPREDd Long form 0.646 25 0.04 32 0.78 N/A NMe N/A (607aa) Short form 0.7168 NPf 0.8969 86 0.910 N/A M N/A (534aa) a (38), b (39, 40), c (41), d (42, 43), eNM, Non-mitochondraial; M, Mitochondrial, f Not Predictable

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If AtMMSDH is metabolizing intermediates from both mitochondria and peroxisomes or from multiple precursors, then it may be necessary for it to sustain constant gene and protein expression throughout development and growth. Also, unlike some enzymes in the valine degradation pathway, AtMMSDH did not exhibit decreased message levels in the presence of sucrose (Fig. 4.3). The largest response to sucrose is gene expression for subunits of BCDKH, which have been shown to be repressed by the presence of sucrose. The repression of BCKDH gene expression suggests that utilizing valine as an alternative carbon source during carbon starvation may be largely controlled by BCKDH. The difference between Oryza and A. thaliana MMSDH is significant. It has been shown that MMSDH protein and RNA levels are induced by GA and 2, 4-D (10, 11). However, in A. thaliana the RNA levels were not increased with either GA or 2,4-D (Fig. 4.4). AtMMSDH gene expression data analyzed by the program Genevestigator, also did not show an increase with GA or 2,4-D (48). In support of this, Graham and Eastmond commented that in cereals, GA is known to be involved in the regulation of β-oxidation of protein and lipid storage reserves during germination (47). However this does not seem to be the case in A. thaliana, suggesting that genes involved in this metabolism are not regulated by the same mechanism in dicotyledons (47). This study reports a true mmsdh knockout mutant in plants, with phenotypes that differ from Osmmsdh knockdown mutants (11). Atmmsdh plants produce seeds with wrinkled seed coats that exhibited a delay in germination. In addition, seedlings display inverted cotyledons (Fig. 4.6) suggesting AtMMSDH has roles in both seed development and establishment. Amino acid sequence alignments showed AtMMSDH appears to be structurally similar to other MMSDH proteins, however further studies are needed to confirm the actual N-terminal sequences of the processed and unprocessed protein, as well as the 5’ untranslated region. Of greater significance are the effects of exogenous valine, isobutyrate, and propionate on this mitochondrial knockout protein as it has been previously shown that isobutyrate and propionate are metabolized in the peroxisomes (28). Mutant seedlings show increased sensitivity towards valine, isobutyrate, and propionate in comparison to wild-type (Fig. 4.7). Interestingly, the toxic effect of the mmsdh mutant is not as drastic in valine treated seedlings as it is in isobutyrate and propionate. This is probably due to valine being taken up and metabolized in both mitochondria and chloroplasts (28), whereas isobutyrate and propionate are initially

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metabolized in the peroxisomes and then likely transported to mitochondria for disposal of their respective β-hydroxyacids. In support of this theory, we have previously shown that 2-13C-β- hydroxypropionate accumulates and then is consumed over time in A. thaliana suspension cell cultures (28). Also in support of this model, is a report by Lange et al regarding the depletion of 14 14 CO2 from C-valine in young A. thaliana seedlings when a peroxisomal β-hydroxyacyl-CoA hydrolase is knocked out (Fig. 4.1, step 5) (21). 14 14 Previous reports have shown that isolated peroxisomes produced CO2 and C-acetyl- CoA from 14C-propionate (27, 49, 50), however there is no evidence for the presence of a peroxisomal β-hydroxyacid dehydrogenase or MMSDH which would be needed to convert propionate to CO2 or acetyl-CoA. The findings in this report are the first to show the effects of a true MMSDH knockout and a refined model of valine degradation and propionyl-CoA metabolism in the mitochondria and peroxisomes of A. thaliana.

4.6 Acknowledgements The authors would like to thank Dr. Meghan Holdorf for her comments on the manuscript.

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4.7 References

1. Goodwin, G., Rougraff, P., Davis, E., and Harris, R. (1989) Purification and Characterization of Methylmalonate-Semialdehyde Dehydrogenase from Rat Liver. Identity to Malonate- Semialdehyde Dehydrogenase. J. Biol. Chem. 264, 14965-14971.

2. Kirch, H. H., Bartels, D., Wei, Y., Schnable, P. S., and Wood, A. J. (2004) The ALDH Gene Superfamily of Arabidopsis. Trends Plant Sci. 9, 371-377.

3. Roe, C. R., Struys, E., Kok, R. M., Roe, D. S., Harris, R. A., and Jakobs, C. (1998) Methylmalonic Semialdehyde Dehydrogenase Deficiency: Psychomotor Delay and Methylmalonic Aciduria without Metabolic Decompensation. Mol. Genet. Metab. 65, 35.

4. Chambliss, K. L., Gray, R. G. F., Rylance, G., Pollitt, R. J., and Gibson, K. M. (2000) Molecular Characterization of Methylmalonate Semialdehyde Dehydrogenase Deficiency. J. Inherit. Metab. Dis. 23, 497.

5. Gray, R. G. F., Pollitt, R. J., and Webley, J. (1987) Methylmalonic Semialdehyde Dehydrogenase Deficiency: Demonstration of Defective Valine and β-Alanine Metabolism and Reduced Malonic Semialdehyde Dehydrogenase Activity in Cultured Fibroblasts. Biochem. Med. Metab. Biol. 38, 121.

6. Gibson, K. M., Lee, C. F., Bennett, M. J., Holmes, B., and Nyhan, W. L. (1993) Combined Malonic, Methylmalonic and Ethylmalonic Acid Semialdehyde Dehydrogenase Deficiencies: An Inborn Error of Beta-Alanine, L-Valine and L-Alloisoleucine Metabolism? J. Inherit. Metab. Dis. 16, 563.

7. Brown, G. K., Hunt, S. M., Scholem, R., Fowler, K., Grimes, A., Mercer, J. F., Truscott, R. M., Cotton, R. G., Rogers, J. G., and Danks, D. M. (1982) Beta-Hydroxyisobutyryl Coenzyme A Deacylase Deficiency: A Defect in Valine Metabolism Associated with Physical Malformations. Pediatrics. 70, 532-538.

8. Mu, J., Xie, P., Yang, Z., Yang, D., Lv, F., Luo, T., and Li, Y. (2007) Neurogenesis and Major Depression: Implications from Proteomic Analyses of Hippocampal Proteins in a Rat Depression Model. Neurosci. Lett. 416, 252. 112

9. Kanski, J., Behring, A., Pelling, J., and Schoeneich, C. (2005) Proteomic Identification of 3- Nitrotyrosine-Containing Rat Cardiac Proteins: Effects of Biological Aging. Am. J. Physiol. Heart Circ. Physiol. 288, H371.

10. Oguchi, K., Tanaka, N., Komatsu, S., and Akao, S. (2004) Methylmalonate-Semialdehyde Dehydrogenase is Induced in Auxin-Stimulated and Zinc-Stimulated Root Formation in Rice. Plant Cell Rep. 22, 848.

11. Tanaka, N., Takahashi, H., Kitano, H., Matsuoka, M., Akao, S., Uchimiya, H., and Komatsu, S. (2005) Proteome Approach to Characterize the Methylmalonate-Semialdehyde Dehydrogenase that is Regulated by Gibberellin. J. Proteome Res. 4, 1575-1582.

12. Millar, A. H., Sweetlove, L. J., Giege, P., and Leaver, C. J. (2001) Analysis of the Arabidopsis Mitochondrial Proteome. Plant Physiol. 127, 1711-1727.

13. Casimiro, I., Marchant, A., Bhalerao, R. P., Beeckman, T., Dhooge, S., Swarup, R., Graham, N., Inze, D., Sandberg, G., Casero, P. J., and Bennett, M. (2001) Auxin Transport Promotes Arabidopsis Lateral Root Initiation. Plant Cell. 13, 843-852.

14. Abel, S., and Theologis, A. (1996) Early Genes and Auxin Action. Plant Physiol. 111, 9.

15. Hagen, G., and Guilfoyle, T. (2002) Auxin-Responsive Gene Expression: Genes, Promoters and Regulatory Factors. Plant Mol. Biol. 49, 373.

16. Becker, D., and Hedrich, R. (2002) Channelling Auxin Action: Modulation of Ion Transport by Indole-3-Acetic Acid. Plant Mol. Biol. 49, 349.

17. Cakmak, I., Marschner, H., and Bangerth, F. (1989) Effect of Zinc Nutritional Status on Growth, Protein Metabolism and Levels of Indole-3-Acetic Acid and Other Phytohormones in Bean (Phaseolus vulgaris L.). J. Exp. Bot. 40, 405-412.

18. Kende, H., and Zeevaart, Jan A. D. (1997) The Five "Classical" Plant Hormones. Plant Cell. 9, 1197.

113

19. Sweetlove, L. J., Heazlewood, J. L., Herald, V., Holtzapffel, R., Day, D. A., Leaver, C. J., and Millar, A. H. (2002) The Impact of Oxidative Stress on Arabidopsis Mitochondria. Plant J. 32, 891-904.

20. Zolman, B. K., Monroe-Augustus, M., Thompson, B., Hawes, J. W., Krukenberg, K. A., Matsuda, S. P., and Bartel, B. (2001) Chy1, an Arabidopsis Mutant with Impaired Beta- Oxidation, is Defective in a Peroxisomal Beta-Hydroxyisobutyryl-CoA Hydrolase. J. Biol. Chem. 276, 31037-31046.

21. Lange, P. R., Eastmond, P. J., Madagan, K., and Graham, I. A. (2004) An Arabidopsis Mutant Disrupted in Valine Catabolism is also Compromised in Peroxisomal Fatty Acid Beta-Oxidation. FEBS Lett. 571, 147-153.

22. Daschner, K., Thalheim, C., Guha, C., Brennicke, A., and Binder, S. (1999) In Plants a Putative Isovaleryl-CoA-Dehydrogenase is Located in Mitochondria. Plant Mol. Biol. 39, 1275-1282.

23. Fujiki, Y., Sato, T., Ito, M., and Watanabe, A. (2000) Isolation and Characterization of cDNA Clones for the e1beta and E2 Subunits of the Branched-Chain Alpha-Ketoacid Dehydrogenase Complex in Arabidopsis. J. Biol. Chem. 275, 6007-6013.

24. Bode, K., Hooks, M. A., and Couée, I. (1999) Identification, Separation, and Characterization of Acyl-Coenzyme A Dehydrogenases Involved in Mitochondrial - Oxidation in Higher Plants. Plant Physiol. 119, 1305-1314.

25. Faivre-Nitschke, S. E., Couee, I., Vermel, M., Grienenberger, J. M., and Gualberto, J. M. (2001) Purification, Characterization and Cloning of Isovaleryl-CoA Dehydrogenase from Higher Plant Mitochondria. Eur. J. Biochem. 268, 1332-1339.

26. Gerbling, H., and Gerhardt, B. (1989) Peroxisomal Degradation of Branched-Chain 2-Oxo Acids. Plant Physiol. 91, 1387-1392.

27. Gerbling, H., and Gerhardt, B. (1989) Propionyl-CoA Generation and Catabolism in Higher Plant Peroxisomes, in Biological Role of Plant Lipids (P. A. Basics, K. Gruiz, and T. Kremmer, Eds.) pp 21-26, Plenum Publishing, New York. 114

28. Lucas, K. A., Filley, J. R., Erb, J. M., Graybill, E. R., and Hawes, J. W. (2007) Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants. J. Biol. Chem. 282, 24980.

29. Schuster, J., and Binder, S. (2005) The Mitochondrial Branched-Chain Aminotransferase (AtBCAT-1) is Capable to Initiate Degradation of Leucine, Isoleucine and Valine in almost all Tissues in Arabidopsis thaliana. Plant Mol. Biol. 57, 241-254.

30. Murashige, T., and Skoog, F. (1962) A Revised Medium for Rapids Growth and Bioassays with Tobacco Tissue Cultures. Physiol Plant. 15, 473-497.

31. Anderson, M. D., Che, P., Song, J., Nikolau, B. J., and Wurtele, E. S. (1998) 3- Methylcrotonyl-Coenzyme A Carboxylase is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants. Plant Physiology. 118, 1127-1138.

32. Sodek, L., and Wilson, C. M. (1973) Metabolism of Lysine and Leucine Derived from Storage Protein during the Germination of Maize. Biochim. Biophys. Acta. 304, 353.

33. Aubert, S., Alban, C., Bligny, R., and Douce, R. (1996) Induction of β-Methylcrotonyl- Coenzyme A Carboxylase in Higher Plant Cells during Carbohydrate Starvation: Evidence for a Role of MCCase in Leucine Catabolism. FEBS Lett. 383, 175-180.

34. Fujiki, Y., Ito, M., Itoh, T., Nishida, I., and Watanabe, A. (2002) Activation of the Promoters of Arabidopsis Genes for the Branched-Chain α-Keto Acid Dehydrogenase Complex in Transgenic Tobacco BY-2 Cells Under Sugar Starvation. Plant Cell Physiol. 43, 275.

35. Daschner, K., Couee, I., and Binder, S. (2001) The Mitochondrial Isovaleryl-Coenzyme a Dehydrogenase of Arabidopsis Oxidizes Intermediates of Leucine and Valine Catabolism. Plant Physiol. 126, 601-612.

36. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Clustal W and Clustal X Version 2.0. Bioinformatics. 23, 2947-2948.

37. Kedishvili, N., Popov, K., Rougraff, P., Zhao, Y., Crabb, D., and Harris, R. (1992) CoA- Dependent Methylmalonate-Semialdehyde Dehydrogenase, a Unique Member of the

115

Aldehyde Dehydrogenase Superfamily. cDNA Cloning, Evolutionary Relationships, and Tissue Distribution. J. Biol. Chem. 267, 19724-19729.

38. Claros, M. G., and Vincens, P. (1996) Computational Method to Predict Mitochondrially Imported Proteins and their Targeting Sequences. Eur. J. Biochem. 241, 779-786.

39. Emanuelsson, O., Nielsen, H., Brunak, S., and von Heijne, G. (2000) Predicting Subcellular Localization of Proteins Based on their N-Terminal Amino Acid Sequence. J. Mol. Biol. 300, 1005-1016.

40. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Identification of Prokaryotic and Eukaryotic Signal Peptides and Prediction of their Cleavage Sites. Protein Eng. 10, 1-6.

41. Small, I., Peeters, N., Legeai, F., and Lurin, C. (2004) Predotar: A Tool for Rapidly Screening Proteomes for N-Terminal Targeting Sequences. Proteomics. 4, 1581-1590.

42. Guda, C., Fahy, E., and Subramaniam, S. (2004) MITOPRED: A Genome-Scale Method for Prediction of Nucleus-Encoded Mitochondrial Proteins. Bioinformatics. 20, 1785-1794.

43. Guda, C., Guda, P., Fahy, E., and Subramaniam, S. (2004) MITOPRED: A Web Server for the Prediction of Mitochondrial Proteins. Nucl. Acids Res. 32, W372-374.

44. Tom, A., and Nair, K. S. (2006) Assessment of Branched-Chain Amino Acid Status and Potential for Biomarkers. J. Nutr. 136, 324S-330.

45. Eastmond, P. J., Hooks, M. A., Williams, D., Lange, P., Bechtold, N., Sarrobert, C., Nussaume, L., and Graham, I. A. (2000) Promoter Trapping of a Novel Medium-Chain Acyl- CoA Oxidase, which is Induced Transcriptionally during Arabidopsis Seed Germination. J. Biol. Chem. 275, 34375-34381.

46. Germain, V., Rylott, E. L., Larson, T. R., Sherson, S. M., Bechtold, N., Carde, J. P., Bryce, J. H., Graham, I. A., and Smith, S. M. (2001) Requirement for 3-Ketoacyl-CoA Thiolase-2 in Peroxisome Development, Fatty Acid Beta-Oxidation and Breakdown of Triacylglycerol in Lipid Bodies of Arabidopsis Seedlings. Plant J. 28, 1-12.

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47. Graham, I. A., and Eastmond, P. J. (2002) Pathways of Straight and Branched Chain Fatty Acid Catabolism in Higher Plants. Prog. Lipid Res. 41, 156-181.

48. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox. Plant Physiol. 136, 2621-2632.

49. Giovanelli, J., and Stumpf, P. K. (1958) Fat Metabolism in Higher Plants. X. Modified Beta Oxidation of Propionate by Peanut Mitochondria. J. Biol. Chem. 231, 411-426.

50. Hatch, M. D., and Stumpf, P. K. (1962) Fat Metabolism in Higher Plants. XVIII. Propionate Metabolism by Plant Tissues. Arch. Biochem. Biophys. 96, 193-198.

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Chapter 5: The Biosynthesis of Leucine and Its Effects on Protein Synthesis in A. thaliana

Kerry A. Lucas, Ellen C. Wetli, John W. Hawes*

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056

*Corresponding author: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056; Tel.: 513-529-8072; fax: 513-529-5715. Email:[email protected]

Author contributions: ECW isolated RNA, synthesized cDNA for expression studies, and performed all GFP plant studies and analysis. KAL performed all other experiments.

Some of the data presented in this chapter will be submitted to The FASEB Journal as a hypothesis article.

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5.1 Summary Branched-chain amino acids (BCAAs, valine, leucine, and isoleucine) have been extensively studied in plants, mammals, and bacteria as their metabolism has been shown to be related to both primary and secondary metabolic pathways. It is still difficult to know exactly what role each play in these systems or how important it is to maintain their regulation. Unlike many other organisms, plants have the ability to synthesize and degrade BCAAs. Interestingly, previous data have shown that leucine is produced from exogenous valine in the chloroplast instead of being degraded in the mitochondria as expected. This may be a mechanism to avoid production of toxic intermediates known to be produced in valine degradation as well as maintain levels of leucine in the cell. Expression data of genes in leucine biosynthesis indicate that synthesis and degradation pathways work together to maintain BCAA homeostasis under conditions where degradation is known to be most active and lend support for the importance of leucine in plant metabolism. Leucine is also known to stimulate protein synthesis in skeletal muscles in mammals through the mTOR signaling pathway by activation of several proteins associated with mTOR and translation in general. With the aid of the completed Arabidopsis thaliana genome, it has been determined that plants also possess a TOR signaling pathway that is involved in translation, plant growth, and stress resistance. It is likely then, that leucine has the ability to stimulate protein synthesis in plant systems as well. Preliminary data show increased expression of green fluorescent protein (GFP) under a constitutively expressed promoter in A. thaliana seedlings in the presence of leucine. This suggests that leucine does in fact stimulate translation and may have signaling functions in plants similar to that seen in mammalian systems.

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5.2 Introduction The BCAA, leucine, has received little attention in higher plants compared to the key role it has in mammalian systems. Leucine has been linked to several signaling and metabolic pathways in mammals, most recently discussed at the conference “Symposium of Branched- Chain Amino Acids” and highlighted by The Journal of Nutrition (2006)2. In plants, leucine biosynthesis appears to be tightly regulated through gene expression, substrate specificity, and by feedback inhibition (1). Like the other BCAAs, leucine biosynthesis is localized to the chloroplast, with gene expression said to be highest during germination and present in all areas of the plant (1). The synthesis pathway for leucine differs from other BCAAs in that it is initiated by a branch point in the valine synthesis pathway. The first step utilizes the valine synthesis intermediate, ketoisovalerate which condenses with acetyl-CoA by 2-isopropylmalate synthase (IPMS, EC 2.2.3.13, Fig. 5.1) to form 2-isopropylmalate (2-4). IPMS is the main regulatory protein in leucine synthesis and is regulated by feedback inhibition (3) (Fig. 5.1). Ketoisovalerate is also a regulatory intermediate, as it serves as a substrate for both IPMS, in leucine synthesis, and branched-chain aminotransferase (BCAT, EC 2.6.1.42) in valine synthesis (Fig. 5.1), where catalysis relies on substrate specificity (5-7). Also key to leucine synthesis is its ability to be synthesized from both pyruvate and valine (Fig. 5.1). All this combined, it may be a mechanism to conserve endogenous leucine levels by ensuring there are multiple pathways and regulatory points for its synthesis to occur.

2 Published in a supplement to The Journal of Nutrition. Presented at the conference "Symposium on Branched-Chain Amino Acids" held May 23–24, 2005, in Versailles, France. The conference was sponsored by Ajinomoto USA, Inc. The organizing committee for the symposium and guest editors for the supplement were Luc Cynober, Robert A. Harris, Dennis M. Bier, John O. Holloszy, Sidney M. Morris, Jr., and Yoshiharu Shimomura. Guest editor disclosure: L. Cynober, R. A. Harris, D. M. Bier, J. O. Holloszy, S. M. Morris, Y. Shimomura: expenses for travel to BCAA meeting paid by Ajinomoto USA; D. M. Bier: consults for Ajinomoto USA; S. M. Morris: received compensation from Ajinomoto USA for organizing BCAA conference. 120

Figure 5.1 BCAA biosynthesis pathway. TD, threonine deaminase; AHAS, acetohydroxyacid synthase; KARI, ketolacid reductoisomerase; DHAD, dihydroxyacid dehydratase; BCAT, branched-chain aminotransferase; IPMS, isopropylmalate synthase; IPMI, isopropylmalate isomerase; IPMDH, isopropylmalate dehydrogenase.

Threonine TD

2 x Pyruvate Ketobutyrate + Pyruvate AHAS 2-Acetolactate 2-Aceto-2-hydroxybutyrate

KARI 2,3-dihydroxy-3-isovalerate 2,3-dihydroxy-3-methylvalerate

DHAD IPMS 2-Isopropylmalate Ketoisovalerate Ketomethylvalerate

IPMI BCAT 3-Isopropylmalate Valine Isoleucine IPMDH Ketoisocaproate BCAT Leucine

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BCAA synthesis is most active in young tissues when plants need to synthesize proteins for support of fast growth (as reviewed in (1)). Likewise, BCAA degradation is high during this time as part of the breakdown of protein and lipid storage reserves that accumulated during seed development (8). BCAA degradation is also higher when plants need alternative carbon sources, such as during extended periods of darkness (9-12). Because BCAA degradation and synthesis occur in physically separate organelles, their balance must be maintained for BCAA homeostasis. In contrast to the little that is known about leucine in plant systems, leucine is known to play a key role in several signaling pathways in mammals. It has been associated with immune function (13) and neurotransmitter synthesis (14) but most notably, with stimulating protein synthesis in skeletal muscle (15-17). It was previously shown that leucine stimulates protein synthesis through the mammalian target of rapamycin (mTOR) (17, 18). mTOR is a serine/threonine protein kinase known to be involved in cell growth and proliferation, protein synthesis, and translation (19-23). Several studies since then have found that leucine’s relationship with mTOR is much more complex than previously thought (17, 24). Figure 5.2A shows a sketch of the mTOR pathway as characterized in mammalian systems and which proteins leucine is believed to stimulate. It is still not known exactly how leucine stimulates protein synthesis in mammalian systems but the same mechanism appears to be present in plants. It was only recently that A. thaliana TOR was identified and linked to mRNA translation as in mammals, as well as specifically to plant growth (25) (Fig. 5.2B). This invites the question of whether leucine stimulates protein synthesis in plants through a similar mechanism and also what implications this may have on plant science. In this chapter, preliminary data are shown to support a synergistic relationship between the synthesis of leucine, by way of valine, and subsequent degradation when carbon availability is low. Data are also presented that supports the hypothesis that leucine may stimulate protein translation in plants, similar to mammalian systems. The implications of these data would help in understanding BCAA metabolism in plants as well as highlight the importance of leucine in plant metabolism.

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Figure 5.2 TOR signaling pathway. A, mTOR pathway as understood in mammalian systems. B, Current knowledge of TOR pathway in A. thaliana. Both pathways are compiled from information obtained in (17, 26-28).

A Insulin B

IRS-1 PDK1 PI3K Stress

Akt ? ? TSC1 TSC2

[Leucine] [Leucine] RHEB

PDK1 TOR

mTOR RAPTOR GβL? eIF4G RAPTOR GβL PP2A

S6K1 PP2A

eIF4E-BP S6K1 S6

S6 eIF4E

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5.3 Experimental Procedures

5.3.1 Plant Materials and Growth Conditions Wild-type A. thaliana ecotype Columbia (Col-0) was used for measuring mRNA levels where indicated and as a control for protein stimulation studies. Seedlings were surface sterilized and placed on ½-strength Murashige and Skoog medium (MS (29)) and 1% (w/v) sucrose, where indicated, solidified with 0.8% (w/v) agar (Teknova Inc.). Plates were kept in the dark at 4 °C for 2 days and then allowed to germinate under 16-hour-light/8-hour-dark photoperiods at 21-23 °C for the number of days indicated. Green fluorescent protein (GFP) seeds were provided by Dr. Q. Quinn Li, Miami University. GFP is expressed by 35S promoter in pMDC83 (30) and grown on ½ -strength MS medium and 1% (w/v) sucrose solidified with 0.75% Gel-rite agar (Caisson Laboratories) with or without 0.5 mM leucine as indicated. Seedlings (GFP treated with leucine, GFP without leucine and wild-type) were removed from the plates, placed on a glass slide with dH2O and covered with a glass cover slip. Pictures were taken on a fluorescent microscope with a SPOT RT camera (Olympus BX51) under 10X magnification using a FITC filter for visualization.

5.3.2 NMR Sample Preparation and Procedure Samples were prepared and tested similar to (31). Approximately 300-400 wild type A. thaliana ecotype Columbia (Col-0) seedlings were surface sterilized and grown on plates using ½ -strength MS medium and 1% (w/v) sucrose solidified with 0.8% (w/v) agar (Teknova, Inc) as above. After 4 days, seedlings were removed from the plates and placed in 50 mL liquid, ½- strength MS medium for 24 h with or without U-13C-valine. Seedlings were then rinsed with sterile water, ground to a fine powder in liquid nitrogen, resuspended in 5 mL of 5% perchloric acid and stored at -80 °C prior to NMR analysis. All frozen samples were thawed to room temperature and neutralized with 10 M KOH. Samples were centrifuged at 1200 x g for 15-30 min at 4 °C. The supernatant was lyophilized and resuspended in 1-2 mL of 100% D2O. Samples were centrifuged at maximum speed for 10 min before placing in a 200 x 5-mm 535-PP NMR tube. 13C-spectra were acquired at 125.77 MHz with a deuterium lock on a Bruker AVANCE™-500 MHz with a 5 mm TXI probe. Spectra were obtained with a 30° RF pulse,

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relaxation time of 2 s, 1,024 scans, and the spectra were Fourier transformed using 1-Hz line broadening. Chemical shifts were calibrated to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS).

5.3.3 Measuring mRNA Levels Total RNA was isolated using RNeasy mini-kit (Qiagen) according to the manufacturer’s instructions except for the following modifications: frozen, powdered plant material was resuspended in lysis buffer, vortexed, homogenized with a glass homogenizer, and then centrifuged at top speed in a micorcentrifuge for two minutes at room temperature. The supernatant was then used according to the instructions. cDNA was synthesized from 1 μg RNA using cDNA synthesis kit (Bioline, Taunton, MA) and PCR amplified using the following primers: IPMS1.1 5’-AACTTGCTGACGCTGATGG-3’, IPMS1.2 5’- GAACCTAACTTCTGTCTGAC-3’, IPMS2.1 5’-TGCCTAGTGAGTTTGGTCAGT-3’, IPMS2.2 5’-TTAGGGCAAATGCCTTCAC-3’, BCAT2.1 5’-CTCTGGTTCTACCTCTTCA-3’, BCAT2.2 5’-TGTCTGGATACCTACGAGGA-3’, BCAT3.1 5’- CGACATAGATTGGGATACCG-3’, BCAT3.2 5’-GCAAAACCAAGCAGCCGAAT-3’, BCAT5.1 5’-CTCTTTGTCCTCCCTCAGAA-3’, BCAT5.2 5’- TTGAAGCATCAGAAGCATGG-3’, ACT2.5 5’- GGCAGGATTAGCAGGAGAAGATGC-3’ and ACT2.3 5’- CCTGATATCCACTCACACTTCAT-3’.

5.4 Results and Discussion

5.4.1 The Effects of Sucrose on Leucine Biosynthesis We previously showed that exogenous valine can be converted to leucine (31). When young seedlings were treated with U-13C-valine, a direct conversion to leucine was measured (31). Because the biosynthesis of leucine is known to occur in the chloroplast, these data became very intriguing as it was anticipated that valine would be taken up and degraded in the mitochondria to propionyl-CoA. This indicated that valine was taken up in the chloroplast and metabolized to leucine (31). It is possible that plants have found a way to utilize both organelles in order to dispose of valine, as its degradation produces highly reactive and toxic intermediates in plants (32, 33).

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To find alternative ways to measure valine degradation by 13C-NMR, we removed sucrose from the growth medium as it is well known that some enzymes in BCAA degradation are repressed by sucrose. However, what we saw was a decrease in the production of leucine (Fig. 5.3). This response may be a result of repressed expression of leucine biosynthesis genes, an increase in gene expression for valine degradation, or a combination of both. To test these hypotheses, we measured mRNA levels of two known IPMS isoforms, IPMS1 and IPMS2, and the aminotransferases in the chloroplast that are responsible for the last step of BCAA synthesis, in particular BCATs -2, -3, and -5. The chloroplast localized BCATs have not yet been characterized to know which BCAT is responsible for the synthesis of specific BCAAs. Seedlings were grown on MS plates with or without 1% sucrose for 5 days before RNA isolation and RT-PCR (Fig. 5.4). The data show a slight repression of the IPMS genes on plates containing no sucrose, as well as BCAT-3 and BCAT-5 (Fig. 5.4). BCAT-2, on the other hand, shows a slight increase in mRNA levels in response to sucrose availability (Fig. 5.4). The results support the first hypothesis, that leucine synthesis may be sucrose responsive in an equal but opposite manner to BCAA degradation under similar conditions. During periods when carbon availability is low, plants may restrict protein synthesis by down regulating BCAA synthesis and thus activating BCAA degradation to provide alternative carbon sources to maintain metabolism. Future research will need to be conducted on more genes in the biosynthesis pathway in addition to real-time RT-PCR to get a more accurate measure of gene expression in BCAA biosynthesis during carbon starvation.

5.4.2 Stimulation of Protein Synthesis by Leucine The intake of BCAAs, in particular leucine, during periods of intense exercise helps to promote protein synthesis, most likely through the signaling of the mTOR pathway (15-17). However, it is not known metabolically what effect the addition of BCAAs may have in plants. Plants contain all the machinery to have a similar effect of leucine stimulation on protein synthesis through the TOR signaling pathway (25). In order to get an idea of the potential role of leucine stimulation on protein synthesis, we utilized a constitutively-expressed GFP A. thaliana mutant as a representative of mRNA translation and thus protein synthesis.

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Figure 5.3 13C-NMR studies. Seedlings were grown on ½-strength MS plates with or without 1% sucrose for 5 days then treated with 1 mM U-13C-Valine for 24 hrs in liquid MS medium; extracts were prepared as written in Experimental Procedures. A, Originally published in (31), but shown here for comparison. B, No sucrose sample. DSS served as external standard.

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Figure 5.4 mRNA levels of genes expressed during leucine biosynthesis in response to sucrose. A, Total RNA was isolated from wild-type seedlings grown on ½-strength MS plates with or without sucrose for 5 days at 21-23 °C under 16-hr-light/8-hr-dark photoperiods. cDNA was synthesized and PCR amplified as described in Experimental Procedures. Act2 served as loading control.

1% Sucrose + -

IPMS1

IPMS2

AtBCAT-2

AtBCAT-3

AtBCAT-5

ACT2

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Wild-type and GFP expressing seeds were grown on MS plates with or without 0.5 mM leucine for 4 to 7 days. GFP expression was most visible in roots and found to have increased fluorescence on plates containing leucine, compared to GFP seedlings with no leucine and wild- type seedlings under both conditions (Fig. 5.5). These preliminary data support leucine stimulation of protein synthesis in plants. Currently it is not known specifically how leucine is causing the increase in GFP expression, but it could be due to leucine targeting parts of the TOR pathway in A. thaliana and thus stimulating mRNA translation for protein synthesis.

5.5 Conclusion BCAAs cannot be synthesized by mammals, making them essential amino acids and thus they must be obtained through dietary means. In recent years, reports have shown that BCAAs are utilized in several metabolic and signaling pathways (13, 14, 34, 35). When there is an imbalance or blockage in BCAA metabolism the consequences can be severe (33, 36, 37). This shows the importance of maintaining the metabolic homoeostasis for these amino acids. The same seems to be true for plant metabolism, where synthesis and degradation are known to occur in separate organelles (1, 3, 38). It is interesting to note that mutations in the biosynthesis pathway can result in herbicide resistant plants (1), whereas mutations in the degradation pathway result in an array of responses, from indole-3-butyric acid resistance (39, 40) to embryo lethality to (Lucas, submitted). Regardless, plants appear to have developed a system of pathways to maintain levels of BCAAs, in particular, leucine. NMR and mRNA expression studies illustrate how plants appear to have developed mechanisms to ensure synthesis of leucine and at the same time, seek alternate routes for valine degradation so as to avoid production of toxic intermediates. This lends support for the importance of leucine in plant metabolism and subsequent questions regarding what other roles leucine has in plant metabolism. Because leucine is known to play such a key role in mammalian systems, it is not surprising that leucine may have similar roles in plant systems.

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Figure 5.5 GFP response to leucine. Seedlings were grown on ½-strength MS plates containing 0.5 mM leucine for 4 days at 21-23 °C under 16-hr-light/8-hr-dark photoperiods. Seedlings were removed from the plates and rinsed in distilled water. GFP roots from seedlings grown on leucine plates (top), GFP roots of seedlings grown without leucine (middle) and wild-type seedlings (bottom). Fluorescence in wild-type seedlings was similar in the presence or absence of leucine.

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Preliminary data with constitutively expressing GFP plants show increased fluorescence when grown in the presence of leucine. This may represent an increase in mRNA translation, which is known to be linked to the mTOR pathway in mammalian systems, however more data is needed to support this hypothesis. If leucine is in fact able to stimulate protein synthesis in plants, then there are implications for the development of high protein vegetation, of interest for both food and agricultural industries.

5.6 Acknowledgements Support for this project was funded in part by Miami University through the DUOS grant to KAL and ECW. The authors would like to thank Dr. Q. Quinn Li for the GFP seeds and Dr. Xiaohui Yang for assistance with the microscopy.

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5.7 References

1. Singh, B. K. (1995) Biosynthesis of Branched-Chain Amino Acids: From Test Tube to Field. Plant Cell. 7, 935.

2. Hagelstein, P., and Schultz, G. (1993) Leucine Synthesis in Spinach Chloroplasts: Partial Characterization of 2-Isopropylmalate Synthase. Biol. Chem. Hoppe-Seyler. 374, 1105.

3. Singh, B. (1999) Biosynthesis of Valine, Leucine and Isoleucine. in Plant Amino Acids: Biochemistry and Biotechnology (B. K. Singh, Ed.) 1st ed., pp 227-247, Marcel Dekker, New York.

4. Oaks, A. (1965) The Synthesis of Leucine in Maize Embryos. Biochim. Biophys. Acta. 111, 79.

5. Sauer, A., and Heise, K. P. (1984) Regulation of Acetyl-Coenzyme A Carboxylase and Acetyl-Coenzyme A Synthetase in Spinach Chloroplasts. Zeitschrift fuer Naturforschung.Section B. 39, 268.

6. Hagelstein, P., Sieve, B., Klein, M., Jans, H., and Schultz, G. (1997) Leucine Synthesis in Chloroplasts. Leucine/isoleucine Aminotransferase and Valine Aminotransferase are Different Enzymes in Spinach Chloroplasts. J. Plant Physiol. 150, 23.

7. Schulze-Siebert, D., Heineke, D., Scharf, H., and Schultz, G. (1984) Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism. Plant Physiol. 76, 465-471.

8. Anderson, M. D., Che, P., Song, J., Nikolau, B. J., and Wurtele, E. S. (1998) 3- Methylcrotonyl-Coenzyme A Carboxylase is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants. Plant Physiology. 118, 1127-1138.

9. Fujiki, Y., Sato, T., Ito, M., and Watanabe, A. (2000) Isolation and Characterization of cDNA Clones for the e1beta and E2 Subunits of the Branched-Chain Alpha-Ketoacid Dehydrogenase Complex in Arabidopsis. J. Biol. Chem. 275, 6007-6013.

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10. Fujiki, Y., Ito, M., Itoh, T., Nishida, I., and Watanabe, A. (2002) Activation of the Promoters of Arabidopsis Genes for the Branched-Chain α-Keto Acid Dehydrogenase Complex in Transgenic Tobacco BY-2 Cells Under Sugar Starvation. Plant Cell Physiol. 43, 275.

11. Daschner, K., Couee, I., and Binder, S. (2001) The Mitochondrial Isovaleryl-Coenzyme a Dehydrogenase of Arabidopsis Oxidizes Intermediates of Leucine and Valine Catabolism. Plant Physiol. 126, 601-612.

12. Lange, P. R., Eastmond, P. J., Madagan, K., and Graham, I. A. (2004) An Arabidopsis Mutant Disrupted in Valine Catabolism is also Compromised in Peroxisomal Fatty Acid Beta-Oxidation. FEBS Lett. 571, 147-153.

13. Calder, P. C. (2006) Branched-Chain Amino Acids and Immunity. J. Nutr. 136, 288S-293.

14. Fernstrom, J. D. (2005) Branched-Chain Amino Acids and Brain Function. J. Nutr. 135, 1539S-1546.

15. Kimball, S. R., and Jefferson, L. S. (2001) Regulation of Protein Synthesis by Branched- Chain Amino Acids. Curr. Opin. Clin. Nutr. Metab. Care. 4, 39.

16. Anthony, J. C., Anthony, T. G., Kimball, S. R., Vary, T. C., and Jefferson, L. S. (2000) Orally Administered Leucine Stimulates Protein Synthesis in Skeletal Muscle of Postabsorptive Rats in Association with Increased eIF4F Formation. J. Nutr. 130, 139-145.

17. Norton, L. E., and Layman, D. K. (2006) Leucine Regulates Translation Initiation of Protein Synthesis in Skeletal Muscle After Exercise. J. Nutr. 136, 533S-537.

18. Anthony, J. C., Yoshizawa, F., Anthony, T. G., Vary, T. C., Jefferson, L. S., and Kimball, S. R. (2000) Leucine Stimulates Translation Initiation in Skeletal Muscle of Postabsorptive Rats Via a Rapamycin-Sensitive Pathway. J. Nutr. 130, 2413-2419.

19. Abraham, R. T. (2005) TOR Signaling: An Odyssey from Cellular Stress to the Cell Growth Machinery. Curr. Biol. 15, R139.

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20. Bjornsti, M., and Houghton, P. J. (2004) The TOR Pathway: A Target for Cancer Therapy. Nat Rev Cancer. 4, 335-348.

21. Dennis, P. B., Fumagalli, S., and Thomas, G. (1999) Target of Rapamycin (TOR): Balancing the Opposing Forces of Protein Synthesis and Degradation. Curr. Opin. Genet. Dev. 9, 49.

22. Rohde, J., Heitman, J., and Cardenas, M. E. (2001) The TOR Kinases Link Nutrient Sensing to Cell Growth. J. Biol. Chem. 276, 9583-9586.

23. Thomas, G., and Hall, M. N. (1997) TOR Signalling and Control of Cell Growth. Curr. Opin. Cell Biol. 9, 782.

24. Kimball, S. R., and Jefferson, L. S. (2006) New Functions for Amino Acids: Effects on Gene Transcription and Translation. Am J Clin Nutr. 83, 500S-507.

25. Deprost, D., Yao, L., Sormani, R., Moreau, M., Leterreux, G., Nicolai, M., Bedu, M., Robaglia, C., and Meyer, C. (2007) The Arabidopsis TOR Kinase Links Plant Growth, Yield, Stress Resistance and mRNA Translation. EMBO Rep. 8, 864.

26. Kimball, S. R., and Jefferson, L. S. (2006) Signaling Pathways and Molecular Mechanisms through which Branched-Chain Amino Acids Mediate Translational Control of Protein Synthesis. J. Nutr. 136, 227S-231.

27. Stipanuk, M. H. (2007) Leucine and Protein Synthesis: MTOR and Beyond. Nutr. Rev. 65, 122-129.

28. Mahfouz, M. M., Kim, S., Delauney, A. J., and Verma, D. P. S. (2006) Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals. Plant Cell. 18, 477-490.

29. Murashige, T., and Skoog, F. (1962) A Revised Medium for Rapids Growth and Bioassays with Tobacco Tissue Cultures. Physiol Plant. 15, 473-497.

30. Curtis, M. D., and Grossniklaus, U. (2003) A Gateway Cloning Vector Set for High- Throughput Functional Analysis of Genes in Planta. Plant Physiol. 133, 462-469.

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31. Lucas, K. A., Filley, J. R., Erb, J. M., Graybill, E. R., and Hawes, J. W. (2007) Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants. J. Biol. Chem. 282, 24980.

32. Shimomura, Y., Murakami, T., Fujitsuka, N., Nakai, N., Sato, Y., Sugiyama, S., Shimomura, N., Irwin, J., Hawes, J. W., and Harris, R. A. (1994) Purification and Partial Characterization of 3-Hydroxyisobutyryl-Coenzyme A Hydrolase of Rat Liver. J. Biol. Chem. 269, 14248- 14253.

33. Brown, G. K., Hunt, S. M., Scholem, R., Fowler, K., Grimes, A., Mercer, J. F., Truscott, R. M., Cotton, R. G., Rogers, J. G., and Danks, D. M. (1982) Beta-Hydroxyisobutyryl Coenzyme A Deacylase Deficiency: A Defect in Valine Metabolism Associated with Physical Malformations. Pediatrics. 70, 532-538.

34. Tom, A., and Nair, K. S. (2006) Assessment of Branched-Chain Amino Acid Status and Potential for Biomarkers. J. Nutr. 136, 324S-330.

35. Newsholme, E. A., and Blomstrand, E. (2006) Branched-Chain Amino Acids and Central Fatigue. J. Nutr. 136, 274S-276.

36. Chuang, D. T., Chuang, J. L., and Wynn, R. M. (2006) Lessons from Genetic Disorders of Branched-Chain Amino Acid Metabolism. J. Nutr. 136, 243S-249.

37. Gibson, K. M., Lee, C. F., Bennett, M. J., Holmes, B., and Nyhan, W. L. (1993) Combined Malonic, Methylmalonic and Ethylmalonic Acid Semialdehyde Dehydrogenase Deficiencies: An Inborn Error of Beta-Alanine, L-Valine and L-Alloisoleucine Metabolism? J. Inherit. Metab. Dis. 16, 563.

38. Binder, S., Knill, T., and Schuster, J. (2007) Branched-Chain Amino Acid Metabolism in Higher Plants. Physiol. Plantarum. 129, 68.

39. Zolman, B. K., Monroe-Augustus, M., Thompson, B., Hawes, J. W., Krukenberg, K. A., Matsuda, S. P., and Bartel, B. (2001) Chy1, an Arabidopsis Mutant with Impaired Beta- Oxidation, is Defective in a Peroxisomal Beta-Hydroxyisobutyryl-CoA Hydrolase. J. Biol. Chem. 276, 31037-31046.

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40. Zolman, B. K., Yoder, A., and Bartel, B. (2000) Genetic Analysis of Indole-3-Butyric Acid Responses in Arabidopsis thaliana Reveals Four Mutant Classes. Genetics. 156, 1323-1337.

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Chapter 6: Conclusion

6.1 Understanding Branched-Chain Amino Acids Through the years, research of branched-chain amino acids (BCAAs) has been studied in mammalian systems – in relation to genetic diseases, as second messengers and biomarkers (see references in Chapter 1). On the contrary, much less is known about BCAAs in plants. The major findings in plant research regarding BCAAs have thus far focused on the development of herbicide resistant plants (1). Other significant findings show the discovery of multiple isoforms for many of the biosynthesis and degradation enzymes (2). Interestingly, BCAA degradation is dependent on carbon availability (3) and despite great strides scientists have made in understanding BCAA metabolism, there still exists large gaps in knowledge regarding BCAA metabolism and whether plant BCAAs act similar to those in mammals. One of the goals of this dissertation was to piece together certain aspects of BCAA metabolism in plants in order to gain a more complete understanding of how these amino acids and their metabolism truly function in plants.

6.2 BCAA Metabolism Unlike mammals, plants have the ability to synthesize and degrade BCAAs and do so primarily during germination when the plants are consuming the last of the protein storage reserves as well as supporting the rapid growth plants experience during this time (4). The synthesis pathway is regulated by three enzymes (threonine dehydratase, acetohydroxyacid synthase and isopropylmalate synthase) through feedback inhibition (5-7). The degradation pathway seems to be controlled by a single enzyme-complex, branched-chain α-ketoacid dehydrogenase (8, 9). Several parts of each pathway have been studied, in some instances, at great lengths. The separation of the two pathways into separate organelles has allowed scientists the opportunity to study in depth, the degradation of BCAAs separate from their biosynthesis. Doing so has resulted in the production of herbicide resistant plants and the discovery of genes that have significant roles in fatty acid metabolism. However, an understanding of the whole picture, particularly BCAA degradation, has been lost in the details of this pathway. To date, no one has made an effort to fill in the gaps regarding the metabolism of valine and propionyl-CoA in

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plants. Knowing the localization and steps of this pathway will provide scientists with a model to explain or predict certain genetic or environmental responses that target BCAAs and their degradation.

6.3 Valine Degradation Since a review published by Gerhardt in 1992, it has been generally accepted that propionyl-CoA is metabolized solely in the peroxisomes through a series of steps that produce acetyl-CoA in plants (10). It was determined to be a CO2 independent pathway that produced β- hydroxypropionate as an intermediate through a pathway similar to β-oxidation (11, 12). This hypothesis was supported by Gerbling and Gerhardt who showed the production of acetyl-CoA from propionyl-CoA in isolated peroxisomes (13, 14). Studies conducted by other groups used whole-cell extracts from A. thaliana and other plants to prove this pathway existed (12, 13, 15). It is because of this data and that of other groups discussed in Chapter 2 that led us to propose a model for valine and propionate metabolism in higher plants (Fig 2.1). Through the use of 1H and 13C NMR spectroscopy, we were able to show that β- hydroxypropionate was produced from exogenous 2-13C propionate in both monocots and dicots (Fig. 2.6). Evidence for this pathway occurring in the peroxisomes was supported by the reduction of β-hydroxyisobutyrate in peroxisomal β-hydroxyisobutyryl-CoA hydrolase knockout mutants (Fig. 2.5). The most significant finding presented in Chapter 2 was in whole plant extracts used for NMR. No downstream intermediates were seen after the production of β- hydroxypropionate (Fig. 2.2). In addition, the peroxisomal β-hydroxyisobutyryl-CoA hydrolase knockout mutant was sensitive to only isobutyrate and propionate, and not valine, which is believed to be metabolized in mitochondria (Fig. 2.9). These findings allowed us to conclude that propionate is metabolized in the peroxisomes through a modified β-oxidation pathway, but that it can not be metabolized all the way to acetyl-CoA as predicted by Gerbling and Gerhardt (13, 14). This is because the enzymes necessary to catalyze the subsequent reactions do not appear to be present in plants. Therefore the intermediates must be further metabolized elsewhere. In an effort to refine our model of valine and propionyl-CoA metabolism, we further investigated two of the enzymes that catalyze steps in the later half of the valine degradation pathway. β-Hydroxyisobutyryl-CoA (HIBYL-CoA) hydrolase catalyzes a key step in an effort

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to avoid accumulation of the toxic intermediate, methacrylyl-CoA. Unlike in mammalian systems which only have one HIBYL-CoA hydrolase, there are eight different isoforms in A. thaliana (16). There are three each localized to the mitochondria and the peroxisomes and two, presumably in the cytosol (16). We believed that plants had multiple isoforms packed into different organelles for different functions. Our focus was the mitochondrial isoforms and we were able to show through reverse genetics and measurement of mRNA levels that these mitochondrial hydrolases are functionally distinct (Chapter 3). We discovered that CHY4 is necessary for embryogenesis, based on data from chy4 knockout mutants that arrested at an early stage in embryo development (Fig. 3.8). We were also able to show that valine is in fact degraded in the mitochondria, as evidence in chy5 mutant seedlings that are resistant to exogenous valine (Fig. 3.9). We hypothesize this is due to a reduction in methylmalonate semialdehyde, also known to be a downstream, reactive intermediate (17). CHY6 on the other hand was shown to be responsive to carbon availability, similar to other genes in valine degradation, but completely unlike CHY4 and CHY5, which are not responsive. The results in Chapter 3 were the first to show data regarding the mitochondrial hydrolases and their functional diversity. Data presented in Chapter 4 helped to finalize our model for valine and propionyl-CoA metabolism in plants. It is believed that there is only one methylmalonate semialdehyde dehydrogenase (MMSDH) in plants and it is known to be mitochondrial (18). However, the only data specific for MMSDH published thus far has been in rice, where it was shown to be associated with gibberellin and auxin signaling (19, 20). MMSDH RNAi plants exhibited abnormalities in root and leaf growth. We showed that mmsdh knockout plants exhibited delays in germination and deformities in cotyledon development, but was otherwise relatively normal (Fig. 4.6). MMSDH from A. thaliana did not appear to be related to gibberellin or auxin signaling based on experiments performed in our lab (Fig. 4.4) and shown in microarray data (21). Graham and Eastmond previously suggested that gibberellin and auxin may have little to do with lipid β-oxidation during germination in A. thaliana, contrary to that seen in cereals (22). However, the most significant information gained from our studies on the mmsdh mutant was its sensitivity to valine, isobutyrate, and propionate (Fig. 4.7). We had previously shown that valine is metabolized in the mitochondria, and that isobutyrate and propionate are both metabolized in the peroxisomes. This led us to refine our model for valine and propionyl-CoA

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metabolism in plants, resulting in a pathway that relies on the transport of propionyl-CoA to the peroxisomes and β-hydroxypropionate back to the mitochondria to be further metabolized to acetyl-CoA (Fig. 6.1). Evidence for an acyl-CoA transporter in plants was recently published (23). Also, a recent review highlighted the transport of metabolites out of the peroxisomes to other organelles, lending support for the transport of β-hydroxypropionate from the peroxisomes to mitochondria (24).

6.4 Valine Metabolism in the Chloroplast In an effort to measure valine degradation intermediates by 13C NMR spectroscopy, seedlings were treated with U-13C valine and unexpectedly produced leucine (Fig. 2.8). This meant some of the exogenous valine was taken up by the chloroplast and metabolized to leucine. We speculated this was a way to conserve BCAAs in plants, in particular leucine. Because it is well known that leucine plays multiple roles in mammalian systems, especially in stimulating protein synthesis, it became another focus of this dissertation (see references in Chapter 5). The original experiments showing the conversion of valine to leucine by NMR, were performed in the presence of sucrose (Fig. 2.8, (25)). It is well known that expression of genes in the degradation pathway are highest when carbon availability is low (3, 26-28). Therefore, in order to maintain BCAA homeostasis, we hypothesized that the conversion from valine to leucine would be responsive to carbon levels as well. As shown in Figure 5.3, the production of leucine from valine was drastically reduced when sucrose was removed from the media, suggesting valine was getting degraded in the mitochondria instead of getting converted to leucine in the chloroplast. We measured mRNA levels of the genes most responsible for the conversion to leucine, isopropylmalate synthase and branched-chain aminotransferase (Fig. 5.4). There was a slight decrease in RNA levels of almost all the genes tested, which would also support a decrease in the production of leucine from valine as seen with NMR spectroscopy.

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Figure 6.1 Final model for valine and propionyl-CoA metabolism in plants. 1, branched-chain aminotransferase; 2, branched-chain α-ketoacid dehydrogenase; 3, acyl-CoA dehydrogenase/oxidase; 4, enoyl-CoA hydratase; 5, hydroxyacyl-CoA hydrolase; 6, β- hydroxyacid dehydrogenase; 7, methylmalonate semialdehyde dehydrogenase. Intermediate abbreviations: KIV, ketoisovalerate; MA-CoA, methacrylyl-CoA; HIBYL-CoA, hydroxyisobutyryl-CoA; HPA-CoA, hydroxypropionyl-CoA; HIBA, hydroxyisobutyrate; HPA, hydroxypropionate; MMS, methylmalonate semialdehyde; MS, malonate semialdehyde.

Valine Mitochondria 1 KIV

2 Propionyl-CoA Peroxisomes Isobutyryl-CoA Propionyl-CoA 3 3 MA-CoA Acrylyl-CoA

4 4

HIBYL-CoA HPA-CoA

5 5

HIBA HPA HPA 6 6

MMS MS 7 7 Acetyl-CoA

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6.5 Leucine and Protein Synthesis Also of significance, was our results related to the stimulation of protein synthesis by leucine in plants. Recently, it was discovered that plants have a similar mechanism for regulating translation as mammals; however, it was not known if leucine plays a similar role in plants as in mammals (see references in Chapter 5). To test this hypothesis, we used plants that constitutively express green fluorescent protein (GFP) to probe mRNA translation. When seedlings were grown on plates containing low concentrations of leucine, an increase in fluorescence was seen. This suggests that leucine may in fact stimulate protein synthesis, although the data is still preliminary.

6.6 Future Directions Reverse genetics has provided us with the opportunity to refine our model for valine and propionyl-CoA metabolism in plants and hopefully resolve much of the ambiguity still present in this research area. We have been able to confirm several aspects of this pathway through various biochemical techniques and now, truly believe that the mitochondria and peroxisomes work together to metabolize propionyl-CoA. However, this hypothesis relies on several key theories that will need to be addressed in future studies.

6.6.1 Model for Valine and Propionyl-CoA Metabolism in Plants This model relies on initial reports regarding an acyl-carnitine carrier-like protein in order to transport propionyl-CoA from the mitochondria to the peroxisomes (23). Since a true acyl- CoA carnitine carrier protein has not yet been identified in A. thaliana, more studies are needed. The same is true for understanding transport of free acids out of the peroxisomes to be metabolized in the mitochondria. Initial reports point to the likely possibility of this transport system in A. thaliana, but again, this has yet to be confirmed (24) We have shown metabolic tracing by NMR as a useful tool for understanding metabolism in several different organisms (25). Another such technique that has been shown to detect metabolic intermediates and complementary to NMR is HPLC-MS (29). Although difficult, isolating pure mitochondria from young seedlings treated with exogenous valine would allow for the detection of CoA ester intermediates, such as propionyl-CoA or acetyl-CoA. By utilizing this technique, one would be able to confirm the steps taken to metabolize valine through the

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accumulation of propionyl-CoA and not acetyl-CoA. Similar studies, by NMR and/or HPLC- MS, could be performed with mmsdh knockout plants to confirm its ability to block propionyl- CoA production. These techniques could generate a lot of data that would help us to understand what is occurring metabolically, not just within valine and propionyl-CoA metabolism, but in plant metabolism as a whole. Further studies investigating the development of mmsdh knockout mutants would give answers to the importance of this gene in seedling development. It will be necessary to look at embryo and endosperm development as well as factors contributing to water content in seeds in order to understand why desiccated seeds are wrinkled and have difficulty germinating on soil. It will also be necessary to clone and over-express the MMSDH and check for activity with both methylmalonate semialdehyde and malonate semialdehyde to confirm our model for propionyl- CoA metabolism. These assays would be performed similar to those previously published with purified MMSDH from rats (30). It will also be necessary to confirm the activity of the peroxisomal HIBYL-CoA hydrolase, CHY1, with β-hydroxypropionyl-CoA. Lange et al 14 14 showed a decrease in CO2 production in chy1 mutants treated with C-valine giving support for β-hydroxypropionate as a substrate (31). It may also be necessary to clone and over-express the other two peroxisomal HIBYL-CoA hydrolases, as one may be more specific for HIBYL- CoA, while another specific for β-hydroxypropionyl-CoA.

6.6.2 Stimulation of Protein Synthesis We have shown an increase in GFP fluorescence when seedlings are treated with exogenous leucine and as noted, this suggests leucine may serve as a second messenger for protein stimulation in plants. After confirmation that message levels do not change with respect to the addition of leucine, it will be necessary to quantitate the increase in GFP protein expression. This can be done using a GFP anti-body (provided by Dr. Q. Quinn Li, Miami University) for western blot analysis. This will allow us to measure a true increase in cellular proteins with the treatment of leucine. We will also need to study effects leucine has on components of the TOR pathway in order to confirm that leucine stimulation is occurring through a similar mechanism as seen in mammals (discussed in Chapters 1 and 5). Initial studies would target the phosphorylation of S6 kinase, as it has been shown that leucine increases its phosphorylation in mammals (32, 33) and this has been shown to be present in plant systems as

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well (34, 35). There are other known leucine targets within the mammalian TOR pathway, but they have not yet been investigated in plants. Further studies with leucine may help researchers piece together the machinery needed for mRNA translation and protein synthesis for both organisms.

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6.7 References

1. Singh, B. K. (1995) Biosynthesis of Branched-Chain Amino Acids: From Test Tube to Field. Plant Cell. 7, 935.

2. Binder, S., Knill, T., and Schuster, J. (2007) Branched-Chain Amino Acid Metabolism in Higher Plants. Physiol. Plantarum. 129, 68.

3. Aubert, S., Alban, C., Bligny, R., and Douce, R. (1996) Induction of β-Methylcrotonyl- Coenzyme A Carboxylase in Higher Plant Cells during Carbohydrate Starvation: Evidence for a Role of MCCase in Leucine Catabolism. FEBS Lett. 383, 175-180.

4. Anderson, M. D., Che, P., Song, J., Nikolau, B. J., and Wurtele, E. S. (1998) 3- Methylcrotonyl-Coenzyme A Carboxylase is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants. Plant Physiol. 118, 1127-1138.

5. Sharma, R. K., and Mazumder, R. (1970) Purification, Properties, and Feedback Control of l- Threonine Dehydratase from Spinach. J. Biol. Chem. 245, 3008-3014.

6. Borstlap, A. C. (1972) Changes in the Free Amino Acids of Spirodela Polyrhiza during Growth Inhibition by L-Valine, L-Isoleucine, or L-Leucine. Gas Chromatographic Study. Acta Bot. Neerl. 21, 404.

7. Singh, B. (1999) Biosynthesis of Valine, Leucine and Isoleucine. in Plant Amino Acids : Biochemistry and Biotechnology (B. K. Singh, Ed.) 1st ed., pp 227-247, Marcel Dekker, New York.

8. Yeaman, S. J. (1989) The 2-Oxo Acid Dehydrogenase Complexes: Recent Advances. Biochem. J. 257, 625.

9. Shimomura, Y., Fujii, H., Suzuki, M., Murakami, T., Fujitsuka, N., and Nakai, N. (1995) Branched-Chain {Alpha}-Keto Acid Dehydrogenase Complex in Rat Skeletal Muscle: Regulation of the Activity and Gene Expression by Nutrition and Physical Exercise. J. Nutr. 125, 1762S-1765.

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10. Gerhardt, B. (1992) Fatty Acid Degradation in Plants. Prog. Lipid Res. 31, 417-446.

11. Giovanelli, J., and Stumpf, P. K. (1958) Fat Metabolism in Higher Plants. X. Modified Beta Oxidation of Propionate by Peanut Mitochondria. J. Biol. Chem. 231, 411-426.

12. Hatch, M. D., and Stumpf, P. K. (1962) Fat Metabolism in Higher Plants. XVIII. Propionate Metabolism by Plant Tissues. Arch. Biochem. Biophys. 96, 193-198.

13. Gerbling, H., and Gerhardt, B. (1989) Propionyl-CoA Generation and Catabolism in Higher Plant Peroxisomes, in Biological Role of Plant Lipids (P. A. Basics, K. Gruiz, and T. Kremmer, Eds.) pp 21-26, Plenum Publishing, New York.

14. Gerbling, H., and Gerhardt, B. (1989) Peroxisomal Degradation of Branched-Chain 2-Oxo Acids. Plant Physiol. 91, 1387-1392.

15. Halarnkar, P. P., Wakayama, E. J., and Blomquist, G. J. (1988) Metabolism of Propionate to 3-Hydroxypropionate and Acetate in the Lima Bean Phaseolus Limensis. Phytochemistry. 27, 997-999.

16. Zolman, B. K., Monroe-Augustus, M., Thompson, B., Hawes, J. W., Krukenberg, K. A., Matsuda, S. P., and Bartel, B. (2001) Chy1, an Arabidopsis Mutant with Impaired Beta- Oxidation, is Defective in a Peroxisomal Beta-Hydroxyisobutyryl-CoA Hydrolase. J. Biol. Chem. 276, 31037-31046.

17. Schauenstein, E., Esterbauer, H., Zollner, H., and Gore, P. H. (1977) Aldehydes in Biological Systems: Their Natural Occurrence and Biological Activities. Pion, London.

18. Sweetlove, L. J., Heazlewood, J. L., Herald, V., Holtzapffel, R., Day, D. A., Leaver, C. J., and Millar, A. H. (2002) The Impact of Oxidative Stress on Arabidopsis Mitochondria. Plant J. 32, 891-904.

19. Tanaka, N., Takahashi, H., Kitano, H., Matsuoka, M., Akao, S., Uchimiya, H., and Komatsu, S. (2005) Proteome Approach to Characterize the Methylmalonate-Semialdehyde Dehydrogenase that is Regulated by Gibberellin. J. Proteome Res. 4, 1575-1582.

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20. Oguchi, K., Tanaka, N., Komatsu, S., and Akao, S. (2004) Methylmalonate-Semialdehyde Dehydrogenase is Induced in Auxin-Stimulated and Zinc-Stimulated Root Formation in Rice. Plant Cell Rep. 22, 848.

21. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004) GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox. Plant Physiol. 136, 2621-2632.

22. Graham, I. A., and Eastmond, P. J. (2002) Pathways of Straight and Branched Chain Fatty Acid Catabolism in Higher Plants. Prog. Lipid Res. 41, 156-181.

23. Lawand, S., Dorne, A. J., Long, D., Coupland, G., Mache, R., and Carol, P. (2002) Arabidopsis A BOUT DE SOUFFLE, which is Homologous with Mammalian Carnitine Acyl Carrier, is Required for Postembryonic Growth in the Light. Plant Cell. 14, 2161-2173.

24. Visser, W. F., Van Roermund, Carlo W. T., Ijlst, L., Waterham, H. R., and Wanders, Ronald J. A. (2007) Metabolite Transport Across the Peroxisomal Membrane. Biochem. J. 401, 365- 375.

25. Lucas, K. A., Filley, J. R., Erb, J. M., Graybill, E. R., and Hawes, J. W. (2007) Peroxisomal Metabolism of Propionic Acid and Isobutyric Acid in Plants. J. Biol. Chem. 282, 24980.

26. Journet, E., Bligny, R., and Douce, R. (1986) Biochemical Changes during Sucrose Deprivation in Higher Plant Cells. J. Biol. Chem. 261, 3193-3199.

27. Dorne, A. J., Bligny, R., Rebeille, F., Roby, C., and Douce, R. (1987) Fatty Acid Disappearance and Phosphorylcholine Accumulation in Higher Plant Cells After a Long Period of Sucrose Deprivation. Plant Physiol. Biochem. 25, 589.

28. Brouquisse, R., James, F., Pradet, A., and Raymond, P. (1992) Asparagine Metabolism and Nitrogen Distribution during Protein Degradation in Sugar-Starved Maize Root Tips. Planta. 188, 384.

147

29. Hall, R., Beale, M., Fiehn, O., Hardy, N., Sumner, L., and Bino, R. (2002) Plant Metabolomics: The Missing Link in Functional Genomics Strategies. Plant Cell. 14, 1437- 1440.

30. Kedishvili, N. Y. (2000) Mammalian Methylmalonate-Semialdehyde Dehydrogenase. Meth. Enzymol. 324, 207.

31. Lange, P. R., Eastmond, P. J., Madagan, K., and Graham, I. A. (2004) An Arabidopsis Mutant Disrupted in Valine Catabolism is also Compromised in Peroxisomal Fatty Acid Beta-Oxidation. FEBS Lett. 571, 147-153.

32. Kimball, S. R., and Jefferson, L. S. (2006) Signaling Pathways and Molecular Mechanisms through which Branched-Chain Amino Acids Mediate Translational Control of Protein Synthesis. J. Nutr. 136, 227S-231.

33. Norton, L. E., and Layman, D. K. (2006) Leucine Regulates Translation Initiation of Protein Synthesis in Skeletal Muscle After Exercise. J. Nutr. 136, 533S-537.

34. Mahfouz, M. M., Kim, S., Delauney, A. J., and Verma, D. P. S. (2006) Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals. Plant Cell. 18, 477-490.

35. Deprost, D., Yao, L., Sormani, R., Moreau, M., Leterreux, G., Nicolai, M., Bedu, M., Robaglia, C., and Meyer, C. (2007) The Arabidopsis TOR Kinase Links Plant Growth, Yield, Stress Resistance and mRNA Translation. EMBO Rep. 8, 864.

148