Molecular Biochemical Characterization of Rubber Biosynthetic Machinery in Hevea and Dandelion, and Evaluation of Rabbitbrush As a Potential Domestic Rubber Crop

Home , Hevea brasiliensis, Parthenium argentatum, Phytochemical

University of Nevada, Reno

Molecular biochemical characterization of rubber biosynthetic machinery in Hevea and Dandelion, and evaluation of Rabbitbrush as a potential domestic rubber crop

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology

Upul Indrajith Hathwaik

Dr. David K. Shintani/ Dissertation Advisor

December, 2012

THE GRADUATE SCHOOL

We recommend that the dissertation prepared under our supervision by

UPUL INDRAJITH HATHWAIK

entitled

Molecular Biochemical Characterization Of Rubber Biosynthetic Machinery In Hevea And Dandelion And Evaluation Of Rabbitbrush As A Potential Domestic Rubber Crop

be accepted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

David K. Shintani, Ph.D., Advisor

Gary Blomquist, Ph.D., Committee Member

John C. Cushman, Ph.D., Committee Member

Jeffrey F. Harper, Ph.D., Committee Member

Patricia M. Berninsone, Ph.D., Graduate School Representative

Marsha H. Read, Ph. D., Dean, Graduate School

December, 2012

ABSTRACT

Natural rubber is an irreplaceable material that is used in manufacturing over

40,000 products including tires and numerous medical devices. Hevea brasiliensis is the sole producer of world natural rubber and more than 90% of the Hevea plantations are located in less than five South Asian countries. In 2011, the US spent 4.4 billion dollars to import natural rubber. When considering the unpredictability and high cost of NR imports, the limited growing conditions of Hevea plants, the increasing allergic reactions caused by Hevea rubber, and the irreplaceability by synthetics, it is clear that domestic sources of natural rubber are needed. Furthermore, the rubber transferase that is responsible for producing rubber is currently unknown, and understanding rubber biosynthetic pathway will help to improve domestic rubber crops. In this study several experiments were done to identify the rubber transferase. The rubber transferase is responsible for the polymerization of isopentenyl pyrophosphate (IPP) monomers into high molecular weight cis-1,4-polyisoprene polymer with the help of farnesyl pyrophosphate (FPP) primer. In all rubber producing plants rubber is made in cytosolic compartments know as rubber particles. The analyses of H. brasiliensis rubber particle proteins with liquid chromatography mass spectrometry (LC/MS) and western blot analysis using cis-prenyltransferase (CPT) antibodies confirmed the CPT localization in washed rubber particles (WRPs). Direct evidence of CPT involvement in rubber polymerization was established by in vitro cross-linking studies done with two different

FPP analogues. The in vivo role of CPT was identified by CPT under-expressing transgenic Taraxacum kok-saghyz (Russian Dandelion) plants.

Additionally, this research was focused in identifying a domestic rubber crop.

Ericameria nauseosa (Rabbitbrush) is one of the very few domestic rubber producers.

Wild Rabbitbrush populations were analyzed for their rubber amount and molecular weight. Patterns of rubber accumulation and molecular weight in shoots with different diameter tissue were established over time. Rabbitbrush collected from Austin, NV had high quality rubber in high yield. The extracted rubber had several physical properties that were analogous to existing commercial natural rubber producers. The resins and the biomass of Rabbitbrush were also analyzed. Both resin and leftover biomass residue had promising characteristics. Overall, Rabbitbrush collected from Austin, NV has the potential of becoming a domestic natural rubber, resin, and biomass crop.

iii

DEDICATION

To my parents Nobert N. Hathwaik and Nimali Wijeweera who always believed in me. Your unconditional love, sacrifices, and words of encouragement have led me to accomplish this work. Thank you.

To my brother Prabath, who was there for me whenever I needed him.

To my loving daughter Lyra, who gave me a boost of energy every time you smile and call me Daddy in many different ways.

To my yet to be born Baby Hathwaik, who gave me an extra motivation to finish my Ph.D.

To my wife and best friend, Leyla Trinidy Hathwaik, who supported me every step of the way to my dissertation and loving me for who I am. This would not be possible without you.

I love you all

Thank you from the bottom of my heart.

ACKNOWLEGEMENTS I would like to thank my advisor David Shintani for his guidance. Dave you have taught me so much during the time I worked with you. Thank you for taking me into your lab when I was just a young undergrad, and pointing me in the right direction. One of the very first things I remember is taking time out of your busy schedule to teach me how to design primers. Since then you have spent countless hours teaching me not only numerous lab skills, but also to become a better scientist and a leader. You are a brilliant scientist, but you always made everybody else feel special and value their opinions. Your support, encouragement and enthusiasm have made me a better person. I have never met a professor or a scientist, who care about their students academically and personally as you care about your students. I am grateful to you for helping and caring about me throughout the years, and I know I can always count on you. I consider you in many ways as my academic father figure. No matter where I end up, one day I hope to make you proud. I would like to thank my committee members, Drs. Gary Blomquist, John Cushman, Jeffrey Harper, and Patricia Berninsone, who were more than generous with their expertise and precious time. Thank you for all the advisements, criticisms, and support you have given me throughout the years. You have made me a better scientist. I am thankful to Drs. Colleen McMahan and Wenshuang Xie, our collaborators in USDA- ARS Albany, CA, for numerous productive conversations that we had. I am grateful to Dr. Ron Pardini, who took me into his lab and gave me my first research project which ignited my scientific career. Thank you to Dr. Christie Howard for helping me with seminar preparations and advisements. I would like to express my thanks to Dr. Kathy Schegg, who helped me with many projects. No matter how bad of a day I had, I always felt better after I talked to you. Thank you. I thank you to the great staff in biochemistry department, Rebecca Hess, Marianne Davis, Ronald Robards, Jerry McGraw, Michael Jackson, and previous staff for your considerable support. I am thankful to the past and present Shintani lab members. I thank Connie Ribas for showing me the way around the lab. Special thanks to Drs. Yoseph Tsegaye, Imad Ajjawii, Meral Tunc, Huma Taban, and Jillian Collins-Silva. Since I was an undergrad, all of you have helped me more than I can say. I know all of you will achieve great things in your lives. Mohammad Yazdani, you have helped me in many ways last two years that I cannot tell you how much I appreciate you. There are many undergraduate students who helped me throughout the years. Some of you worked directly with me on related projects; Amanda Skaggs, Stephania Cheng, Hong Vong, Jourfika Gestoso, Ryan Wong, Mel Bersaba, Justin Lee, Thivanka Muthumalage, Evan Villaluz, Janice Cho, Mitch Hegedus, Arthur Lewis, Kacey Durant, and Leyla Hathwaik. I thank you for your hard work. Other members including Travis, Michelle, Regina, Tony, Sam, Liz, Jake, Kristine, Tatiana, Katie, Andrea, Severin, Nate, Michael, Scott and Brent have helped me in many ways that made my lab life much easier. I thank to past and present Shintani lab members and apologized for any of you who I did not mentioned. I am truly grateful to know each and every one of you. I could not have done my Ph.D. without your help. Thanks JR (Richard), Steve, Gadi, Evan, Mark, Imad, Kristine, Severin for all the fun we had together. Thank you to Becky, Bahay, Mariam, Sangho, Matt, Jeremiah, Liz, Ryan,

Danny, Ruby, Paul, and Mike for being wonderful lab neighbors and sharing your expertise with me. I want to thank my friends Todd, Cynthia, and Juan. You have being very supportive and I know you will always be good friends. I also would like to thank my life-long friends, Udaya, Dilupa, Rodney, and Chaminda, who I know since elementary school. No matter where we lived, we have managed to keep in contact and take care of each other. You have heard me complaining and celebrating, many many times. Thank you for being there for me and I cannot wait to see you guys. Thank you for your support. I would like to thank my family. Mom and Dad, this work is a direct result of your sacrifices and dedication to make a better life for us. I cannot begin to understand how hard it must have been for you to leave my brother and I, in order to go to foreign countries for work. Because you were strong enough to sacrifice your lives, we were able to have a better education and life in the US. Without you none of this would have been possible. You are my role models. I hope one day I would be as good of a parent as you are to me. Words cannot express how lucky I am to have a younger brother like Prabath. Even though you were bigger than me since fifth grade, you always treated me as your big brother. I know if you wanted, you could have beaten me up many times when we were kids, but you never did. Thank you for being the greatest brother anybody can ask for. Especially, during past several months taking care of Lyra and everything else at home, so Leyla and I can finish our work. I would always be grateful to my grandparents, David and Missinona Wijeweera who helped to raise my brother and me. My Aunt Kamali Wijeweera and her family, and Uncle Tuition Wijeweera (Pody Mama), who dedicated their lives to be with us. My Cousins Chandi and her family, Rohana and Thushara, who were like sister and brothers to me. You all have protected us and guided us throughout our childhood. I will always be indebted to you. Thank you to my in-laws, Juan Jose Hernandez and Carolina Gomes who have helped us taking care of our baby, Lyra, this past year and for raising a wonderful daughter. Also thank you to Juan, for being such a wonderful brother-in law. I want to thank you to Lyra, for bringing such joy to our lives. Things you have done during this past year have made me realized doing a Ph.D. might not be such a hard thing. You have learned to walk by nine months and are trying to learn three languages at the same time. Most of all, I love your ability to make everybody around you feel special. I also want to thank our unborn baby for giving me the extra motivation to finish this work. I am looking forward to see you and to the great life ahead of us. Finally, I want to thank Leyla Hathwaik, the love of my life. I would not be here without your support. You are a brilliant scientist, even though you do not recognize that, a terrific daughter and a sister, a wonderful mother, and a perfect wife. I really, do not know how you managed to be all of that. We got married just before I started my grad school and we managed to finish our dissertation at the same time. I know I have not being the easiest person to deal with this past several months while we were writing, but you have managed to helped me whenever I needed, finish your dissertation, and take care of Lyra while you are expecting a another child. You are truly the perfect wife. I love you.

TABLE OF CONTENT

LIST OF FIGURES AND TABLES…………………………………………………...ix

CHAPTER ONE- INTRODUCTORY CHAPTER – THE IMPORTANCE OF NATURAL RUBBER…………....……………………...... 1 The ancient and modern uses ………………….……………………………...…..1 History and the present-day of natural rubber..……………………………..….....1 World natural rubber production and demand.……………………………..…...... 2 Reasons to develop alternative rubber sources and the need of domestic natural rubber crops………….……………………………..…………………………...... 3 Natural rubber and synthetic rubber………………………………………….……5 Alternative rubber crops……………...... ………………………………….5 Guayule………………….………...…………………………………...... 6 Dandelion………...……………………………………...... 7 Rabbitbrush……………………….…………………………………….....8 Rubber biosynthesis….…………………………………………..……………...... 9 The rubber biosynthetic pathway...………...……………………...... 9 Rubber particles…..…………………...………………………….……....10 Rubber particle associated proteins……………………………………………....11 Rubber elongation factor (REF).………………….……………………...11 Small rubber particle protein (SRPP)…………………………………….12 Cis-prenyltransferase (CPT)………………………………………..…….13 References…………..…………………………………….…………………….. 16

CHAPTER TWO: Multiple lines of evidence for the association of cis-1,4- polyisoprene synthase with active rubber particles from Hevea brasiliensis….….....23 Abstract……………………………………………………………………...... 24 Introduction…………………………………………………………………...... 25 Material and methods………………………………………………………….....28 Purification of Hevea brasiliensis washed rubber particles…...……...... 28 SDS-PAGE analysis of H. brasiliensis WRP proteins and liquid chromatography with tandem mass spectrometry (LC/MS/MS)...... 28 Expression of recombinant Hevea CPT, antibody production and Purification……..…………………………………………...... 30 Western blot analyses of Hevea brasiliensis latex, C-serum, and WRPs proteins………………………………….....……..………...... 32 In vitro rubber transferase assay....…………………...... 33 Synthesis of benzophenone-containing analogues, A and B ...... 33 Photolabeling reactions and identification of photolabeled prot...... 34 Results and Discussion..………………………………………………………….35 Hevea brasiliensis CPT identified by liquid chromatography mass spectrometry………………………………………………...... 35 Immunological and enzymatic evidence for CPT association with

vii

H. brasiliensis rubber particles……...…………………………………...36 Photolabeling of washed rubber particle associated proteins with radiolabeled FPP analogues………………………………...... 37 Acknowledgments……………………………………………………………...... 40 References…………..…………………………………….……………………...42

CHAPTER THREE: The role of cis-prenyltransferase in determining the yield and the molecular weight of rubber in Taraxacum kok-saghyz………………………...... 56 Abstract……………………………………………………………………...... 56 Introduction…………………………………………………………………...... 57 Materials and methods…..…………………………………………………….....59 Taraxacum koksaghyz plant material…..……………………………...... 59 Assembling TkCPT under-expressing RNAi constructs.…...... 60 Development of transgenic Taraxacum kok-saghyz…....……………...... 60 Rubber extraction and analysis of Taraxacum kok-saghyz roots…...... 61 RNA extraction and quantitative real-time PCR (qRT-PCR) of Taraxacum kok-saghyz plants…………………...….……...... 62 Protein extraction from Taraxacum kok-saghyz plants and western blot analyses…..………………………………...... 63 Statistical analyses…..…………………..……...... 64 Results and Discussion..………………………………………………………….64 Analysis of TkCPT temporal gene expression profile and rubber accumulation in Taraxacum koksaghyz…...………………...... 64 The suppression of TkCPT2 expression in T. koksaghyz plants...... 65 Acknowledgments……………………………………………………………...... 69 References…………..…………………………………….……………………...70

CHAPTER FOUR: Ericameria nauseosa (Rabbitbrush): A potential candidate for renewable source of natural rubber……...…...... …………………………….....76 Abstract…………………………………………………………………………..76 Introduction………………………………………………………………………77 Material and methods………………………………………………………….....78 Rabbitbrush plant material…………………………………………….....78 Soil pH measurements...…………………………………...………….....78 Rubber extraction……………...…………...………………………….....78 Statistical analysis…………….....…………………………………….....80 Results and discussion…………………………………………………………...80 The amount and the molecular weight of Rabbitbrush collected from Nevada and California………………………………………...…………80 Seasonal patterns of rubber production in young Rabbitbrush tissue collected in 2006……………………………………………...…….……81 The amount and the molecular weight of whole Rabbitbrush plants collected in 2006...……………………………………………………….83

viii

Seasonal patterns of rubber production in whole Rabbitbrush plants collected from EV in 2007………………………………..…………...... 84 Conclusions………………………………………………………………………85 Acknowledgments………………………………………………………………..86 References…...………………………………………………………………..….87

CHAPTER FIVE: Ericameria nauseosa subsp. consimilis (Rabbitbrush): A renewable source of natural rubber, resin, and bioenergy feedstock.…………...... 94 Abstract……………………………………………………………………….….95 Introduction………………………………………………………………………96 Material and methods………………………………………………………….....98 Rabbitbrush plant material…………………………………………….....98 Rubber extraction from different branch diameters………………..…….98 Purification of Rabbitbrush Washed Rubber Particles and particle size determination ….…………………………….………………..…….99 Purification of Rabbitbrush Washed Rubber Particles proteins….…….100 Nuclear Magnetic Resonance (NMR) of Rabbit brush rubber…………100 Rubber and resin extraction using a commercial Soxhlet extractor….…………………………………………………….100 Rubber molecular weight determination………………………………..101 Determination of rubber gel…………………………………………….102 Bulk Viscosity determined by Advanced Polymer Analyzer…………..102 Acetone extraction of rubber, the percent extractables…………………102 Determination of Plasticity Retention Index (PRI)…………………….103 Hydrothermal Carbonization (HTC) of Rabbitbrush tissue…………….103 Analysis of HTC products…………………………………………...…104 Results and discussion………………………………………………………….105 Rabbitbrush rubber particles………………………….…………...……105 Deposition of rubber in Rabbitbrush shrubs……………………………107 The physical properties of bulk solid Rabbitbrush rubber………...……108 The components of Rabbitbrush resin………………………………….110 Analysis of lignocellulosic biomass residue……………………………111 Acknowledgments………………………………………………………………113 References…...…………………………………………………………….……114

CHAPTER SIX: Concluding remarks….…………………...…………………...…. 127 References…...………………………………………………………………….133

LIST OF FIGURES AND TABLES

CHAPTER ONE

Table 1. Alternative sources of natural rubber…...……………………………..21

Figure 1. Rubber biosynthetic pathway…..………..…………………………....22

CHAPTER TWO

Figure 1. Hevea brasiliensis washed rubber particle proteins……...... 45

Table 1. CPT associated with Hevea brasiliensis WRP proteins, identified by mass spectrometry……………………………………………. .………….….46

Figure 2. Western blot analysis of Hevea brasiliensis latex, C-serum and WRP proteins with Hevea CPT antibody. ……………………………………….…….47

Figure 3. The rubber transferase activity of Hevea brasiliensis latex and WRPs. ……………………………………………………………………….….48

Figure 4. Structures of FPP and benzophenone-modified FPP analogues….…...49

Figure 5. Phosphorimage of H. brasiliensis washed rubber particle proteins identified by 32P FPP analogues A and B…...…………………………………..50

Table 2. Mass spectrometry of the H. brasiliensis washed rubber particle proteins identified by 32P labeled FPP analogues………...………………...…...51

Supplemental Table 1. Peptide sequences identified by mass spectrometry of the H. brasiliensis WRP proteins with analogue A……………………….…..52

Supplemental Table 2. Peptide sequences identified by mass spectrometry of the H. brasiliensis WRP proteins with analogue B.………………….……….54

CHAPTER THREE

Figure 1. The micro-propagation of dandelion plants……………...... 72

Figure 2. The CPT gene expression and the rubber production in developing dandelion roots... .…………………………………………………….……….….73

Figure 3. The analyses of TkCPT RNAi transgenic dandelion plants.……….….74

Figure 4. TkCPT transcript abundance and protein levels in transgenic dandelion plants……………..….…………………………………………………………...75

CHAPTER FOUR

Figure 1. . The amount and the molecular weight of Rabbitbrush rubber collected from various locations in Nevada and California, along with the soil pH of each location……………………………………..……….. …………………………..88

Figure 2. The amount and the molecular weight of rubber from young Rabbitbrush tissue collected on year 2006………………...... 89

Figure 3. The amount and the molecular weight of rubber from Rabbitbrush roots and shoots collected on year 2006…………………...…….………………90

Figure 4. The amount and the molecular weight of rubber from whole Rabbitbrush samples collected on year 2007...……………………………….…91

Supplemental Figure 1. The amount and the molecular weight of rubber from individual Rabbitbrush samples collected form EV and G on year 2006...... 92

Supplemental Figure 2. The amount and the molecular weight of rubber from individual Rabbitbrush samples collected from GP and SR on year 2006. ..……………………………....………………………………….….…….93

CHAPTER FIVE

Figure 1. 13C Nuclear Magnetic Resonance (NMR) spectra of Rabbitbrush rubber………………………………………...…………………….…………..117

Figure 2. The amount of bulk rubber and molecular weight of Rabbitbrush shoots……………………………………………………………. …..………..118

Figure 3. Whole Rabbitbrush plant rubber and resin extracted by Accelerated Solvent Extractor (ASE). . …………..….……………………………..………119

Table 1. The properties of bulk solid Rabbitbrush rubber compared to other rubber producing species…………………………………………………….…120

Table 2. The washed rubber particle diameter and the protein content of Rabbitbrush and other rubber producing species…………….……………...…121

Table 3. The analysis of gases produced during Hydrothermal Carbonization (HTC) of Rabbitbrush residue…………………………………………………122

Table 4: The mass balance of Hydrothermal Carbonization (HTC) char…….123

Table 5. Energy densification of Hydrothermal Carbonization (HTC) char….124

Table 6. Pellet Abrasion test of raw and Hydrothermal Carbonized Rabbitbrush…………………………………………………………………….125

Table 7. Pellet characteristics after water immersion test of raw and Hydrothermal Carbonized Rabbitbrush tissue…………………………………126

Chapter I

The importance of natural rubber

The ancient and modern uses

Natural rubber (NR) was used as early as 1600 B.C. by ancient Mesoamerican peoples (Hosler et al. 1999). The latex harvested from Castilla elastic, was processed using Ipomiea alba (a species of morning glory vine) extract to strengthen the rubber and was used in producing solid rubber balls, solid and hollow human figurines, wide rubber bands and other items (Hosler et al. 1999). The solid rubber balls were used in the

Mesoamerican ball games, an important central ritual component in all ancient

Mesoamerican civilizations.

More than 2,500 plants species have been identified as natural rubber producers.

Hevea brasiliensis (Brazilian rubber tree) is the only commercial source of NR in the world. Harvested latex, a milky sap, from H. brasiliensis (Hevea) is subjected to curing process called vulcanization, before use in many industrial applications. At present, NR is used in the manufacture of over 40,000 products, including tires, surgical gloves, more than 400 medical devices, numerous engineering and consumer products.

History and the present-day of natural rubber

The Brazilian rubber tree is the nearly exclusive natural rubber source in the world. It is indigenous to Amazon rainforest. When Christopher Columbus discovered the New World in 1495-96, he reported to have seen American Indians playing with balls that bounced. However, it was Charles de la Condamine, a French scientist, who made the earliest attempts to industrialized natural rubber. After his visit to Ecuador in 1736 he presented a paper by Francois Fresneau to the Academie Royale des Sciences of France

2 about their travel to South America and the uses of Hevea brasiliensis latex. Francois

Fresneau was later referred to as the father of the modern rubber industry (Schurer 1957).

Until late 19th century, South America remained the main source of natural rubber. In

1876, a large amount of Hevea seeds were collected from Brazil by Henry Wickham and transported to England. The Hevea seedlings produced were sent to several British colonies at the time; Ceylon (Sri Lanka), Indonesia, Singapore and Malaysia

(International Rubber Study Group, IRSG). Later, Hevea plantations have spread to other Asian and African countries. In year 2010, the top five NR producers were

Thailand, Indonesia, Malaysia, India, and Viet Nam (Food and Agriculture Organization of the United Nations, FAOSTAT data, 2010).

World natural rubber production and demand

More than 90% of the Hevea plantations are located in South and Southeast Asian countries, particularly Thailand, Indonesia, and Malaysia. Hevea plantations are limited to a very small region of the world, due to their specific growing conditions. NR is an indispensable raw material that is used in thousands of civilian and military industrial applications in US and the rest of the world. Almost 60% of the world’s natural rubber is used for the manufacture of tires for the agricultural, automotive and aviation industries

(International Rubber Study Group, IRSG). World NR production in year 2010 was over

10 million metric tons, and US spent 3.3 billion dollars on natural rubber imports

(International Rubber Study Group, IRSG). In 2011, the US spent 4.4 billion dollars to import NR while, it is forecasted to increase to $4.5 and $4.8 billion dollars in years 2012 and 2013, respectively (USDA 2012). Between 2000- 2011, the world’s natural rubber consumption was similar or in most cases, higher than the world’s rubber production.

Natural rubber production clearly needs to be increased to meet world NR demand. The

US is currently investigating alternative natural rubber sources such as Parthenium argentatum (Guayule) and Taraxacum kok-saghyz (Russian Dandelion) which will be discussed in a later section.

Reasons to develop alternative rubber sources and the need of domestic natural rubber crops

Hevea plants can only be grown in regions where the environmental conditions are similar to those found in the Amazon rain forest. These tropical conditions make

Hevea plantation growing areas restricted to regions 15-20 degrees latitude north or south of the equator (International Rubber Study Group, IRSG). Latex from rubber trees could be collected after five-eight year of growth and continue for another 20-30 years. The projected NR production is inadequate to meet future demand (Davis 1997; Cornish

2001; van Beilen et al. 2007). Natural rubber production needs to be increased by either improving latex production in Hevea plants, increasing the number of Hevea plantations and by finding alternative rubber producers that have a broad range of growing conditions. To make matters worse, current rubber plantations are being replaced by more profitable palm plantations to produce palm oil for the purpose of biofuel (Belcher et al. 2004). Because the current rubber plantations are concentrated in a very few countries, the world rubber supply is at risk due to natural disasters and political instability. For example, during WWII the US and its allies lost their rubber supplies due to Japanese control over the rubber producing countries (Davis 1997). During this time research of Hevea rubber alternatives such as synthetic rubber took place. In addition,

4 rubber producing alternative crops were also developed. For example, the US was successful in making army vehicle tires using dandelion rubber (Mooibroek et al. 2000).

Other than natural and political reasons, there are also biological threats to the world’s Hevea rubber supply. The main reason that Hevea production is not doing well in Brazil, even though Hevea is native to Brazil, is due to a disease called South

American Leaf Blight (SALB) cause by Microcyclus ulei. The SALB is a fungal disease that almost wiped-out the Brazilian rubber industry in the early 1900s. Considering the narrow genetic variability of rubber trees, SALB could have a similar devastating effect if it were to spread to Asia (Davis 1997). In spite of the extensive research done to date, scientists have been unable to obtain SALB resistant rubber trees (Guen et al. 2007). To prevent such a disaster, the Asian rubber producing countries have taken serious precautions of intensive control of international air traffic and freight shipments from

South America to other tropical countries (Lieberei 2007). A disease such as SALB is another key reason to develop alternative rubber crops.

An additional incentive to search for alternative rubber sources is increase in allergenic reactions to Hevea-derived latex products. Furthermore, about the 6% of general population and up to 17 % of healthcare workers are at risk of developing life- threatening latex allergies due to continuous use of natural rubber products made from H. brasiliensis latex (Bousquet et al. 2006). Proteins associated with Hevea rubber are known to be responsible for these latex allergies. Alternative natural rubber crops consisting of less rubber associated proteins would likely resolve these latex allergy problems. Researchers have found that Parthenium argentatum (guayule), as an alternative hypoallergenic rubber producing plant.

Natural rubber and synthetic rubber

A possible replacement for natural rubber is petroleum-based synthetic polymers.

However, at present, synthetic polymers cannot compete with natural rubber (cis-1,4- polyisoprene) due to NR’s outstanding physical properties, such as heat dispersion, abrasion resistance, elasticity, resiliency, and malleability at cold temperatures (Carrier et al. 1999; Cornish 2001). These exceptional physical properties are believed to be due to interactions between non-rubber components and the rubber polymer. Commercial

Hevea rubber contain about 2-6% non-rubber components, which include 2% proteins, 2-

3% lipids, 0.4% carbohydrates and 0.2% metal ions (Eng et al. 1993; Toki et al. 2009).

Additionally, none of the synthetic rubber alternatives contain the amount of cis-bond,

99.5%, that natural rubber possesses (Hayashi 2009). Industrial research done for six decades on creating synthetic alternatives such as butyl rubber, butadiene rubber, chloroprene and polyisoprene was unsuccessful in replacing natural rubber (van Beilen et al. 2007). Although synthetic rubbers are used in creating certain products, the manufacture of many strategically important products, such as aircraft tires, requires

100% natural rubber (Carrier et al. 1999). Thus, the need for development of alternative natural rubber crops remains a high priority.

Alternative rubber crops

The high cost and the unpredictability of NR imports, the limited growing conditions of Hevea plants, the increasing allergic reactions caused by Hevea rubber, and the irreplaceability by synthetics all suggests that domestic alternative sources of natural rubber are needed. Consequently, many plants have been investigated throughout the years as potential rubber crops (Bowers 1990). Eight botanical families, 300 genera, and

2500 species have been identified to contain natural rubber in their latex, but only a few of them were able to produce large amount of high quality rubber (Mooibroek et al. 2000;

Bushman et al. 2006). There are many candidate rubber alternatives including Guayule

(Parthenium argentatum), dandelion (Taraxacum kok-saghyz), and Rabbitbrush

(Ericameria nauseosa), Goldenrod (Solidago virgaureaminuta), Sunflower (Heliantus sp.), Fig tree (Ficus carica, Ficus bengalensis, Ficus elastic), Lettuce (Lactuca serriola).

The best candidates, Guayule, Dandelion, and Rabbitbrush are discussed below.

Guayule

Guayule (Parthenium argentatum), a desert Woody shrub is native to semi-arid regions in northern Mexico and Texan Chihauhaun desert (Lloyd 1911). Commercial exploitation of guayule rubber was started in the early 20th century when there were shortages of imported natural rubber from South America. In 1910, ten thousand metric tons of natural rubber were harvested from wild guayule stands per year (Ray et al.

2005). Guayule produces high molecular weight rubber of 1,280 kDa, similar to Hevea, and in relatively high amounts (20% dwt) (Table 1). In the 1980s, successful investigations were done with guayule rubber made tires on army trucks and airplanes

(Schloman Jr 2005). In addition, guayule rubber contains very low protein content compared to Hevea rubber, which is associated with allergic reactions. In fact, guayule natural rubber latex has recently been explored by Yulex Corporation to manufacture medical products that might be safer for people with Type I IgE-mediated Hevea latex allergies to use (Cornish et al. 2006).

However, guayule has several disadvantages. Guayule is a partially domesticated crop resulted from breeding efforts done during 20th century. Guayule does not tolerate

7 low temperatures found in most of Europe and the US. Most of the rubber is produced during the winter months, so it is currently used as a biannual or multiannual crop

(Cornish et al. 2003). In current commercial cultivations, guayule branches are harvested several times, instead of harvesting whole plants after 2-5 years (Foster et al. 2005). At present, rubber from guayule is purified either as latex or solid rubber. Rubber extracted using solid rubber extraction methods usually resulted in co-purifying lower molecular weight rubber and compounds such as resins and terpene (Schloman Jr 2005).

Even though, current rubber production from guayule is not cost effective, the higher cost might be justified for the manufacture of hypoallergenic medical-related products such as tubing and catheters (van Beilen et al. 2007). The bagasse after extracting resin and rubber can be used as a biomass energy source. The resin, low molecular weight rubber, and wax can be utilized to produce coatings, adhesives, organic pesticides, wood preservatives, and specialty chemicals. These by-products and co- products might increase the economic feasibility of guayule as a rubber crop (van Beilen et al. 2007).

Dandelion

Russian dandelion (Taraxacum kok-saghyz or TKS), is native to Kazakhstan and

Uzbekistan. During 1931-32, TKS was discovered as the top candidate out of almost

1,100 indigenous plant species through a strategic program to identify domestic natural rubber crops in the USSR (van Beilen et al. 2007). Extensive Russian dandelion studies were conducted in the US during the Emergency Rubber Project. For example, dandelion test plots were planted in more than 41 states by 1942 (Whaley et al. 1947). Tires made from dandelion rubber were similar to those made from Hevea rubber (Whaley et al.

1947). Russian dandelion produces high molecular weight rubber with an average MW of 2,180 kDa (Table 1) in their root laticifer cells with yields as high as 5-20% root dwt

(Whaley et al. 1947; Hallahan et al. 2004; McMahan 2009).

However, TKS as a rubber crop have several disadvantages. One of the major problems is that dandelion rubber contains more proteins than Hevea, so it might be more problematic due to potential risk of allergic reactions (Cornish et al. 2005). This could be avoided by using dandelion rubber for manufacturing non-medical products such as tires.

TKS also needs to be bred for more favorable agronomic properties. Because dandelion has a short life span, breeding and transgenic methods can be easily implemented to further improve it as a rubber crop.

Dandelion is responsive to both transformation and tissue culture and rubber phenotypes can be obtained in as little as 6 months (Collins-Silva et al. 2012). An additional benefit of TKS is that it is much smaller than Hevea plants. For these reasons dandelion can be used to understand rubber biosynthesis in greenhouse studies.

Rabbitbrush

Rabbitbrush (Ericameria nauseosa), a desert Woody shrub is endemic to western

North America and it consist of 16 species (McArthur et al. 1987). Rabbitbrush was also considered as an emergency source of natural rubber during WWII, and it was estimated to produce more than 300 million metric tons of rubber from wild Rabbitbrush stands

(Doten 1942). Previous studies have shown that Rabbitbrush was capable of producing

1.5 % - 6.5 % rubber in shoots (Hall et al. 1919; Ostler et al. 1984; Yeang et al. 1995).

Similar to rubber produced by Hevea, Rabbitbrush was also able to produce high quality rubber with a molecular weight greater than 1,300 kDa in shoots (Table 1).

However, Rabbitbrush has not undergone breeding to improve it as a rubber crop.

Given its highly diverse genetics due to large wild populations, and a diploid genome, genetic improvements are highly possible by modern breeding programs. Similar to guayule, rubber extracted from Rabbitbrush also contain lower molecular weight rubber and compounds such as resins (Hegerhorst et al. 1987).

Rabbitbrush shoots contain up to 36 % dwt oleoresins. The majority of the over

60 chemicals identified in the oleoresin fraction belong to the terpene class of chemicals, which can be converted to high quality biodiesel (Hegerhorst et al. 1987). The bagasse after extracting resin and rubber can be used as a biomass energy source. Rabbitbrush can grow in areas where temperatures range from -20 °F - 110 °F with less than 2 inches of annual precipitation (Doten 1942). Additionally, Rabbitbrush can be grown on marginal, alkaline lands under drought conditions without irrigation, making it an ideal crop for arid environments (Ostler et al. 1984). More details of Rabbitbrush as potential rubber, resin and biomass crop are presented in Chapter V.

Rubber Biosynthesis

The rubber biosynthetic pathway

The essential steps of rubber biosynthesis are indicated in Figure 1. Cytosolic acetyl-CoA is the primary building blocks of the mevalonate (MVA) pathway and the synthesis of rubber. The substrate of natural rubber, isopentenyl pyrophosphate (IPP) is made using acetyl-CoA through a series of condensation steps. Several allylic pyrophosphates (APPs), namely dimethylallyl-PP (DMAPP) geranyl-PP (GPP) and trans,trans-farnesyl-PP (FPP) were synthesized by the interaction of IPP and DMAPP.

The majority of the IPP used in the rubber production is derived from the cytosolic MVA

10 pathway (Chow et al. 2007). However, a small amount of IPP can originate from the plastid localized 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (Ko et al. 2003).

The APP primer used in rubber polymerization can be varied, including FPP, GPP,

DMAPP and more. However, kinetic analysis of rubber transferase and the structural analysis of NR have identified FPP as the in vivo APP primer (Archer et al. 1987; Tanaka

1989; Cornish et al. 1995; Tanaka et al. 1996; Tanaka 2001; Xie et al. 2008).

The rubber transferase enzyme initiates the synthesis of a new rubber molecule with the binding of FPP primer, followed by progressive addition of varying number of

IPP monomers producing cis-1,4-polyisoprene (Archer et al. 1987; Tanaka 2001).

Depending on the number of polymerized IPP units, the molecular weight vary from 10 -

10,000 kDa, of which >1,000 kDa represents a high quality rubber (Schmidt et al. 2010).

Rubber particles

Rubber is synthesized in monolayer cytosolic vesicles know as rubber particles.

These particles can be stored in specialized cells, such as the specialized laticifers found in H. brasiliensis (d'Auzac et al. 1989) and T. kok-saghyz (Polhamus 1962) or generalized cells, as in bark parenchyma cells of P. argentatum (Backhaus 1985). The average size of the particles in different plant species ranges from 0.2 - 4.0 µm (Siler et al. 1997). The general structure of the rubber particles is similar in that they contain a homogeneous rubber core surrounded by an intact monolayer membrane but, they are different in that they contain a highly species-specific protein and lipids composition. The surface of the particles are hydrophilic due to the hydrophilic head groups of the phospholipids and the sugar groups attached to the particle bound proteins (Cornish 2001).

The amount of particle bound proteins differs greatly among species, ranging from 0.1% in P. argentatum to 4.8% in Euphobia lactiflua (Siler et al. 1997). Also, the number of different proteins in the particles can vary significantly from more than 80 in

H. brasiliensis to less than 10 in Ficus elastic and P. argentatum (Cornish et al. 1993).

Importantly, rubber can be produced in vitro when active washed rubber particles

(WRPs) were provided with IPP substrates, APP primers (favor FPP in vivo), and magnesium ion cofactors (Archer et al. 1967; Light et al. 1989; Madhavan et al. 1989).

In summary, rubber particles contain the necessary proteins/machinery for rubber biosynthesis and many scientists are involved in identifying and understanding the WRP components and rubber biosynthesis.

Rubber particle associated proteins

Rubber elongation factor (REF)

In an effort to identify rubber transferase enzyme, the WRP proteins were analyzed for the past two decades. A 14.6 kDa protein, which was tightly associated with

H. brasiliensis particles, was identified as a possible rubber transferase in 1989 and was named Rubber Elongation Factor (REF) (Dennis et al. 1989) but, later found not to be true. REF was also found as a homotetramer of about 58 kDa and comprised about 60% of the total latex proteins (Goyvaerts et al. 1991; Czuppon et al. 1993). REF, also known as Hev b1, has been identified as a major latex allergens in Hevea latex (Czuppon et al.

1993).

A transcriptome analysis of Hevea latex study with 10,040 expressed sequence tags (ESTs) found REF was represented by the highest EST frequencies and was classified as a rubber biosynthetic and/or stress-related responses protein (Chow et al.

2007). In Hevea, there are two distinct subsets of rubber particles, small rubber particles

(SRPs) with 0.2 µm diameter and large rubber particles (LRPs) with 1 µm diameter, wherein SRPs have a much higher in vitro rubber transferase activity (Archer et al. 1963;

Xiang et al. 2012). A two-dimensional difference in-gel electrophoresis (2D-DIGE) combined with Matrix-assisted laser desorption/ionization time of flight/ time of flight

(MALDI-TOF/TOF) analyses done with SRP and LRP proteome identified REF as one of the seven most highly expressed proteins in LRPs (Xiang et al. 2012).

Perhaps the most important finding of REF was the identification of REF protein in Hevea WRP proteins by photoaffinity labeling using FPP analogues, showing possible interaction between REF and rubber transferase during rubber production (Degraw et al.

2007). Further studies conducted in the Shintani lab with FPP analogues were able to identify REF proteins in Hevea WRP proteins and these experiments are described in

Chapter II.

Small rubber particle protein (SRPP)

Small rubber particle protein (SRPP) is a 22 kDa protein found in high abundance in WRPs. Similar to REF, SRPP is also a latex allergen known as Hev b3 (Wagner et al.

1999). SRPP and REF belong to the same REF/SRPP/SRP (stress related protein) Pfam domain.

Several SRPPs have been identified in Hevea, guayule, and dandelion, all of which are known to produce high quality rubber. A transcriptome analysis of Hevea latex with 10,040 ESTs found that SRPP to have the second highest EST frequency, behind

REF, and it was classified as a rubber biosynthetic and/or stress-related responses protein similar to REF (Chow et al. 2007). The 2D-DIGE combined with MALDI-TOF/TOF

13 analyses done with Hevea SRP and LRP proteome, discussed in the previous section, also identified SRPP as one of fifteen highly expressed proteins in the SRPs. This is interesting because SRPs are more active than the LRPs and it might be due to SRPP.

Recently, Hevea SRPP was described as a glycoprotein that was also involved in in planta latex coagulation, suggesting its involvement in rubber biosynthesis and regulation

(Wititsuwannakul et al. 2008; Wititsuwannakul et al. 2008; Wititsuwannakul et al. 2008).

A guayule SRPP was identified in 2004, and the corresponding recombinant protein was able to stimulate the IPP incorporation in WRPs in vitro (Kim et al. 2004).

Recently, three SRPPs (TkSRPP1-3) were identified in T. kok-saghyz, dandelion,

WRP proteins, and the most abundant SRPP, TkSRPP3, was shown to have temporal and spatial patterns of gene expression correlated with the rubber accumulation (Collins-Silva et al. 2012). In addition, transgenic TkSRPP RNAi lines of T. kok-saghyz, had substantially reduced rubber content and significantly lower molecular weight (Collins-

Silva et al. 2012). Studies conducted with the FPP analogues and Hevea WRPs to identify rubber transferase and other related proteins have identified SRPP in close proximity to rubber transferase. These FPP analogue studies are discussed in Chapter II.

Cis-prenyltransferase (CPT)

Natural rubber is produced by polymerizing of isopentenyl pyrophosphate monomers in cis-configuration with the help of farnesyl pyrophosphate initiator molecules. The chemical structure of the natural rubber polymer is cis-1,4-polyisoprene.

In Hevea, rubber molecular weight varies from about 100,000 to 4,000,000 Da with mostly of 1,000,000 Da rubber (Dennis et al. 1989). Given the chemical structure of rubber, rubber transferase (RT) is most likely a cis-prenyltransferase. Research has

14 shown that RT is a particle bound enzyme or enzyme complex, not a soluble enzyme that temporarily associates with rubber particles when producing rubber (Madhavan et al.

1989; Cornish et al. 1990; Cornish 1993; Cornish et al. 1996).

In microbes and mammals, CPTs are ubiquitous enzymes that are able to produce short-chain (C15), medium-chain (C50-55), and long-chain (C70-120) products (Kharel et al. 2006). In bacteria, undecaprenyl diphosphate synthase is a CPT that synthesizes the

C55 medium-chain lipid (Koyama 1999). In yeast, Dedol-PP synthase (RER2) is a CPT that synthesizes the C65-C105 long-chain dehydrodolichol diphosphate (Sato et al. 1999).

In plants, the first CPT was identified and cloned from Arabidopsis thaliana. This CPT is predicted to be membrane associated, and it produced long-chain (C120) dolichols, but it did not make very long chain polymers such as rubber (Oh et al. 2000).

Recently, three CPTs from T. kok-saghyz, TkCPT1-3, have been cloned, characterized, and expressed (Schmidt et al. 2010). The structural conservation between the TkCPTs and the Hevea CPTs, along with their latex specific expression, suggests that they have a vital role in rubber biosynthesis. Transgenic Taraxacum brevicorniculatum made with depleted TbCPT1-3 (similar to TkCPT1-3) by laticifer-specific RNA interference (RNAi) showed a significant reduction in rubber biosynthesis, and a corresponding 50% increase of competing compounds triterpenes and storage carbohydrate, inulin (Post et al. 2012). The studies done in Shintani lab with T. kok- saghyz transgenic dandelion plants showed similar results of very low amounts of rubber and significantly reduced rubber molecular weight. These studies are discussed in

Chapter III.

Two CPTs from H. brasiliensis, Hevea rubber transferases (HRT1 and HRT2) were identified and characterized (Asawatreratanakul et al. 2003). The in vitro rubber transferase assay done with recombinant HRT1 and HRT2 proteins made with

Escherichia coli, were able to produce long chain polyprenyl products of 2,000 - 10,000

Da, with HRT2, but not with HRT1 (Asawatreratanakul et al. 2003). However, HRT2 was not able to create polymers with lengths similar to rubber or did not exhibit significant activity independently. In a recent study, Saccharomyces cerevisiae and A. thaliana T87 cultured cells were used to express recombinant HRT1 and HRT2 proteins

(Takahashi et al. 2012). A distinct CPT activity was observed, producing C80-C100 polymers, but as before, either HRT1 or HRT2 was not able to produce long chain polymers similar to rubber (Takahashi et al. 2012). In order to produce high molecular weight rubber, Hevea CPTs might require certain activation factors or co-factors that resides in the rubber particles (Asawatreratanakul et al. 2003; Takahashi et al. 2012).

As previously described, FPP is closely associated with rubber transferase during rubber production. The studies conducted in Shintani lab with the FPP analogues and

Hevea WRPs to identify rubber transferase and other related proteins have resulted in identifying CPTs, making this the first direct identification of a CPT involved in rubber production. These FPP analogue studies identifying CPT are discussed in Chapter II.

REFENCES

Archer, B., B. Audley, E. Cockbain and G. McSweeny (1963). "The biosynthesis of rubber - incorporation of mevalonate and isopentenyl pyrophosphate into rubber by Hevea brasiliensis -latex fractions." Biochem J 89: 565 - 574. Archer, B. L. and B. G. Audley (1967). Biosynthesis of Rubber. 29: 221-257. Archer, B. L. and B. G. Audley (1987). "New Aspects of rubber biosynthesis." Bot. J. Linn. Soc. 94: 181 - 196. Asawatreratanakul, K., Y. W. Zhang, D. Wititsuwannakul, R. Wititsuwannakul, S. Takahashi, A. Rattanapittayaporn and T. Koyama (2003). "Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis." Eur J Biochem 270: 4671-4680. Backhaus, R. A. (1985). "Rubber Formation in Plants - A Mini-Review." Isreal Journal of Botany 34: 283-293. Belcher, B., Rujehan, N. Imang and R. Achdiawan (2004). "Rattan, rubber, or oil palm: cultural and financial considerations for farmers in Kalimantan." Economic Botany 58: S77-S87. Bousquet, J., A. Flahault, O. Vandenplas, J. Ameille, J.-J. Duron, C. Pecquet, K. Chevrie and I. Annesi-Maesano (2006). "Natural rubber latex allergy among health care workers: A systematic review of the evidence." Journal of Allergy and Clinical Immunology 118: 447-454. Bowers, J. E., Ed. (1990). Natural rubber-producing plants for the United States. Beltsville, Maryland, National Agricultural Library. Bushman, B. S., A. A. Scholte, K. Cornish, D. J. Scott, J. L. Brichta, J. C. Vederas, O. Ochoa, R. W. Michelmore, D. K. Shintani and S. J. Knapp (2006). "Identification and comparison of natural rubber from two Lactuca species." Phytochemistry 67: 2590-2596. Carrier, T. A. and J. D. Keasling (1999). "Investigating autocatalytic gene expression systems through mechanistic modeling." J Theor Biol 201: 25-36. Chow, K. S., K. L. Wan, M. N. Isa, A. Bahari, S. H. Tan, K. Harikrishna and H. Y. Yeang (2007). "Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex." J Exp Bot. 58: 2429-2440. Epub 2007 Jun 2421. Collins-Silva, J., A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish and D. Shintani (2012). "Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism." Phytochemistry 79: 46-56. Cornish, K. (1993). "The separate roles of plant cis and trans prenyl transferases in cis- 1,4-polyisoprene biosynthesis." Eur J Biochem 218: 267-271. Cornish, K. (2001). "Similarities and differences in rubber biochemistry among plant species." Phytochemistry 57: 1123-1134. Cornish, K. and R. Backhaus (1990). "Rubber transferase activity in rubber particles of guayule." Phytochemistry 29: 3809 - 3813.

Cornish, K. and R. A. Backhaus (2003). "Induction of rubber transferase activity in guayule (Parthenium argentatum Gray) by low temperatures." Industrial Crops and Products 17: 83-92. Cornish, K., C. McMahan, C. Pearson, D. Ray and D. Shintani (2005). BIOTECHNOLOGICAL DEVELOPMENT OF DOMESTIC RUBBER- PRODUCING CROPS. Spring 167 th Technical Meeting of the American Chemical Society, Rubber Division. Cornish, K., C. McMahan, W. Xie, J. Williams, K. Nguyen, D. Kostyal and D. Marsh (2006). ASTM D1076 Category 4 Latex and Quantifying Guayule (NRG) and Hevea (NR) Latex Protein. Proceedings of the International Latex Conference. Cornish, K., D. Siler, O. Grosjean and N. Godman (1993). "Fundamental similarities in rubber particle architecture and function in three evolutionarily divergent plant species." J Nat Rubber Res 8: 275 - 285. Cornish, K. and D. J. Siler (1995). "Effect of different allylic diphosphates on the initiation of new rubber molecules and on cic- 1, 4 - polyisoprene biosynthesis in guayule (Parthenium argentatum Gray)." J. Plant Physiology 147: 301 - 305. Cornish, K. and D. J. Siler (1996). "Characterization of cis-prenyl transferase activity localised in a buoyant fraction of rubber particles from Ficus elastica latex." Plant Physiology 34: 377-384. Czuppon, A. B., Z. Chen, S. Rennert, T. Engelke, H. E. Meyer, M. Heber and X. Baur (1993). "The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex." J Allergy Clin Immunol 92: 690-697. d'Auzac, J., J. L. Jacob and H. Chrestin (1989). Physiology of rubber tree latex. The laticiferous cell and latex-a model of cytoplasm, CRC Press Inc. Davis, W. (1997). The rubber industry's biological nightmare. Fortune: 86 - 95. Degraw, A. J., Z. Zhao, C. L. Strickland, A. H. Taban, J. Hsieh, M. Jefferies, W. Xie, D. K. Shintani, C. M. McMahan, K. Cornish and M. D. Distefano (2007). "A Photoactive Isoprenoid Diphosphate Analogue Containing a Stable Phosphonate Linkage: Synthesis and Biochemical Studies with Prenyltransferases." J Org Chem. Dennis, M. S. and D. R. Light (1989). "Rubber Elongation Factor from Hevea brasiliensis." Journal of Biological Chemistry 264: 18608-18617. Doten, S. B. (1942). "Rubber from rabbit brush (Chrystothamnus nauseosus)." Univ. Nevada Agr. Expt. Sta. Bull. 157: 22. Eng, A. H. and Y. Tanaka (1993). Trends in Polymer Science. 3: 297. Foster, M. and T. Coffelt (2005). "Guayule agronomics: establishment, irrigated production, and weed control." Industrial Crops and Products 22: 27-40. Goyvaerts, E., M. Dennis, D. Light and N. H. Chua (1991). "Cloning and Sequencing of the cDNA Encoding the Rubber Elongation Factor of Hevea brasiliensis." Plant Physiol 97: 317-321. Guen, V. L., D. Garcia, C. R. R. Mattos, F. Doaré, D. Lespinasse and M. Seguin (2007). "Bypassing of a Polygenic Microcyclus ulei Resistance in Rubber Tree, Analyzed by QTL Detection." New Phytologist 173: 335-345. Hall, H. M. and T. H. Goodspeed (1919). "Chrysil, A New Rubber from Chrysothamnus nausuosus." University of California Publications in Botany 7: 183-264.

Hallahan, D. L. and N. M. Keiper-Hrynko (2004). Cis-prenyltransferases from the Rubber-Producing Plants Russian Dandelion (Taraxacum Kok-saghyz) and Sunflower (Helianthus Annus) US Patent 2004/044173. Hayashi, Y. (2009). "Production of natural rubber from Para rubber tree." Plant Biotechnology 26: 67-70. Hegerhorst, D. F., D. J. Weber, E. D. McArthur and A. J. Khan (1987). "Chemical analysis and comparison of subspecies of Chyrsothamnus nauseosus and other related species." Biochemical Systematics and Ecology 15: 201-208. Hosler, D., S. L. Burkett and M. J. Tarkanian (1999). "Prehistoric Polymers: Rubber Processing in Ancient Mesoamerica." Science 284: 1988-1991. Kharel, Y., S. Takahashi, S. Yamashita and T. Koyama (2006). "Manipulation of prenyl chain length determination mechanism of cis-prenyltransferases." FEBS Journal 273: 647-657. Kim, I. J., S. B. Ryu, Y. S. Kwak and H. Kang (2004). "A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro." J Exp Bot 55: 377-385. Ko, J. H., K. S. Chow and K. H. Han (2003). "Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree)." Plant Mol Biol 53: 479-492. Koyama, T. (1999). "Molecular analysis of prenyl chain elongating enzymes." Bioscience, biotechnology, and biochemistry 63: 1671-1676. Lieberei, R. (2007). "South American leaf blight of the rubber tree (Hevea spp.): new steps in plant domestication using physiological features and molecular markers." Annals of Botany 100: 1125-1142. Light, D. and M. Dennis (1989). "Purification of a prenyltransferase that elongates cis- polyisoprene rubber from the latex of Hevea brasiliensis." J Biol Chem 264: 18589 - 18597. Lloyd, F. E. (1911). Guayule (Parthenium argentatum Gray), a rubber-plant of the Chihuahuan Desert. Washington, D.C.,, Carnegie Institution of Washington. Madhavan, S., Greenblatt, M. A. Foster and C. R. Benedict (1989). "Stimulation of isopentenyl pyrophosphate incorporation into polyisoprene in extracts from guayule plants (Parthenium argentatum Gray) by low temperature and 2-(3, 4 - dichlor-phenoxy) triethylamine." Plant Physiol. 89: 506 - 511. Madhavan, S., G. A. Greenblatt, M. A. Foster and C. R. Benedict (1989). "Stimulation of Isopentenyl Pyrophosphate Incorporation into Polyisoprene in Extracts from Guayule Plants (Parthenium argentatum Gray) by Low Temperature and 2-(3,4- Dichlorophenoxy) Triethylamine." Plant Physiol. 89: 506-511. McArthur, E. and S. Meyer (1987). A review of the taxonomy and distribution of Chrysothamnus. Proc. Fourth Utah Shrub Ecology Workshop. College of Natural Resources, Utah State University, Logan. McMahan, C. (2009). Natural Rubber from Domestic Crops. Meeting Abstract, American Chemical Society (ACS) Rubber Division Meeting, Pittsburgh, PA. Mooibroek, H. and K. Cornish (2000). "Alternative sources of natural rubber." Appl Microbiol Biotechnol 53: 355-365.

Oh, S., K. Han, S. Ryu and H. Kang (2000). "Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana." J Biol Chem 275: 18482 - 18488. Ostler, W. K., C. M. McKell and S. White (1984). Chrysothamnus nauseosus: A potential source of natural rubber. Symposium: Biology of Artemisia and Chrysothamnus, USDA Forset Service, General Technical Report INT-200. Polhamus, L. G. (1962). Rubber Botany, Production and Utilization, Leonard Hill Ltd.: 106-413. Post, J., N. van Deenen, J. Fricke, N. Kowalski, D. Wurbs, H. Schaller, W. Eisenreich, C. Huber, R. M. Twyman, D. Prüfer and C. S. Gronover (2012). "Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum." Plant Physiology 158: 1406-1417. Ray, D. T., T. A. Coffelt and D. A. Dierig (2005). "Breeding guayule for commercial production." Industrial Crops and Products 22: 15-25. Sato, M., K. Sato, S. Nishikawa, A. Hirata, J. Kato and A. Nakano (1999). "The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis." Mol Cell Biol 19: 471 - 483. Schloman Jr, W. W. (2005). "Processing guayule for latex and bulk rubber." Industrial Crops and Products 22: 41-47. Schmidt, T., A. Hillebrand, D. Wurbs, D. Wahler, M. Lenders, C. Schulze Gronover and D. Prüfer (2010). "Molecular cloning and characterization of rubber biosynthetic genes from Taraxacum koksaghyz." Plant molecular biology reporter 28: 277-284. Schmidt, T., M. Lenders, A. Hillebrand, N. van Deenen, O. Munt, R. Reichelt, W. Eisenreich, R. Fischer, D. Prufer and C. Gronover (2010). "Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz." BMC Biochemistry 11: 11. Schurer, H. (1957). "Rubber, a magic substance of ancient America." Rubber J 132: 543- 549. Siler, D., M. Goodrich-Tanrikulu, K. Cornish, A. Stafford and T. McKeon (1997). "Composition of rubber particles of Hevea brasiliensis, Parthenium argentatum, Ficus elastica, and Euphorbia lactiflua indicates unconventional surface structure." Plant Physiol Biochem 35: 881 - 889. Takahashi, S., H. J. Lee, S. Yamashita and T. Koyama (2012). "Characterization of cis- prenyltransferases from the rubber producing plant Hevea brasiliensis heterologously expressed in yeast and plant cells." Plant Biotechnology 29: 411- 417. Tanaka, Y. (1989). "Structure and biosynthesis mechanism of natural polyisoprene." Progress in Polymer Science 14: 339-371. Tanaka, Y. (2001). "Structural Characterization of Natural Polyisoprenes: Solve the Mystery of Natural Rubber Based on Structural Study." Rubber Chemistry and Technology 74: 355-375. Tanaka, Y., Aik-Hwee, E. , N. Ohya, N. Nishiyama, J. Tangpakdee, S. Kawahara and R. Witisuwannakul (1996). "Initiation of rubber biosynthesis in Hevea brasiliensis:

characterizationof initiating species by structural analysis." Phytochemistry 41: 1501 - 1505. Toki, S., B. S. Hsiao, S. Amnuaypornsri and J. Sakdapipanich (2009). "New insights into the relationship between network structure and strain-induced crystallization in un-vulcanized and vulcanized natural rubber by synchrotron X-ray diffraction." Polymer 50: 2142-2148. USDA (2012). Outlook for U.S. Agricultural Trade. van Beilen, J. and Y. Poirier (2007). "Establishment of new crops for the production of natural rubber." Trends Biotechnol 25: 522 - 529. van Beilen, J. B. and Y. Poirier (2007). "Guayule and Russian Dandelion as Alternative Sources of Natural Rubber." Critical Reviews in Biotechnology 27: 217-231. Wagner, B., M. Krebitz, D. Buck, B. Niggemann, H. Y. Yeang, K. H. Han, O. Scheiner and H. Breiteneder (1999). "Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida." J Allergy Clin Immunol 104: 1084-1092. Whaley, W. G. and J. S. Bowen (1947). "Russian Dandelion (Kok-saghyz): An emergency source of natural rubber." USDA Micellaneous Publication 618: 1- 212. Wititsuwannakul, R., P. Pasitkul, P. Jewtragoon and D. Wititsuwannakul (2008). "Hevea latex lectin binding protein in C-serum as an anti-latex coagulating factor and its role in a proposed new model for latex coagulation." Phytochemistry 69: 656-662. Wititsuwannakul, R., P. Pasitkul, K. Kanokwiroon and D. Wititsuwannakul (2008). "A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation." Phytochemistry 69: 339-347. Wititsuwannakul, R., K. Rukseree, K. Kanokwiroon and D. Wititsuwannakul (2008). "A rubber particle protein specific for Hevea latex lectin binding involved in latex coagulation." Phytochemistry 69: 1111-1118. Xiang, Q., K. Xia, L. Dai, G. Kang, Y. Li, Z. Nie, C. Duan and R. Zeng (2012). "Proteome Analysis of the Large and the Small Rubber Particles of Hevea brasiliensis Using 2D-DIGE." Plant Physiology and Biochemistry. Xie, W. S., C. M. McMahan, A. J. DeGraw, M. D. Distefano, K. Cornish, M. C. Whalen and D. K. Shintani (2008). "Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species." Phytochemistry 69: 2539-2545. Yeang, H. Y., F. Yusof and L. Abdullah (1995). "Precipitation of Hevea brasiliensis Latex Proteins with Trichloracetic Acid and Phosphotungstic Acid in Preparation for the Lowry Protein Assay." Analytical Biochemistry 226: 35-43.

FIGURES AND TABLES

Table 1. Alternative sources of natural rubber.

Figure 1. Rubber biosynthetic pathway.

Chapter II

Multiple lines of evidence for the association of cis-1,4-polyisoprene synthase with

active rubber particles from Hevea brasiliensis

Upul I. Hathwaik1, Wenshuang Xie3, Olivier Henry2, Huma N. Taban1, Jillian Collins-

Silva1, Maureen Whalen3, Colleen McMahan3, Mark Distefano2, David K. Shintani1*

1Department of Biochemistry, University of Nevada, Reno, 89557, USA

2Department of Chemistry, University of Minnesota, 55455, USA

3USDA, Western Region Research Center, CA, 94710, USA

*Corresponding Author,

David Shintani

Tel: 1 (775) 784-1095, Fax: 1 (775) 784-4227

Email: [email protected]

ABSTRACT

The rubber transferase is responsible for the polymerization of isopentenyl pyrophosphate (IPP) monomers into high molecular weight cis-1,4-polyisoprene polymer. It was hypothesized that cis-prenyltransferase (CPT) is the rubber transferase and that it was localized to the rubber particles, the known site of rubber synthesis and sequestration. The studies conducted revealed strong evidence of CPT being the rubber transferase. Liquid chromatography mass spectrometry, western blot analysis with CPT antibodies, and rubber transferase enzyme assays revealed that CPT was localized and active in the rubber particles. In vitro cross-linking studies done with two FPP analogues were able to cross-link and identify CPT from proteins purified from WRPs, strongly suggesting the direct role of CPT in rubber biosynthesis. These results provide significant evidence to support the idea that CPT as rubber transferase in the plant rubber biosynthesis.

INTRODUCTION

Natural rubber (NR), cis-1,4-polyisoprene, is a plant derived material essential to the US economy. NR is used in thousands of civilian and military industrial applications and is a significant US import ($4.4 billion in 2011 International Rubber Study Group,

IRSG). Unfortunately, a constant supply of natural rubber is at risk due to economic, political, environmental and biological factors. Currently, the majority of the world’s commercial supply of natural rubber is derived from one plant species, Hevea brasiliensis

(Brazilian rubber tree). Because all commercial Hevea varieties are derived from highly inbred clonal varieties, the global rubber crop is highly susceptible to disease and environmental calamities. Due to the economic expansion occurring in India and China, the demand for natural rubber is outpacing supplies. The growing conditions of Hevea are limited to a very small region of the world. Some of the alternative options are either increase the capacity of Hevea rubber production and the development of alternative rubber crops. Unfortunately, one of the major limiting factors in the genetic manipulation of Hevea and other alternative NR crops are a lack of understanding of the mechanism of rubber production.

Rubber is known to be synthesized from an allylic pyrophosphate (APP) primer to which isopentenyl pyrophosphate (IPP) monomers are sequentially attached to form cis-

1,4-polyisoprene polymers (Archer et al. 1987; Tanaka 2001). Molecular weights can vary from 10 -10,000 kDa, with polymers >1,000 kDa representing high quality rubber

(Schmidt et al. 2010). Cytosolic IPP used in rubber biosynthesis is primarily derived from the mevalonate (MVA) pathway. However, IPP derived from plastid localized 2-C- methyl-D-erythritol 4-phosphate (MEP) pathway might also contribute to rubber

26 generation (Ko et al. 2003; Chow et al. 2007). While studies have shown that various

APP primers can be used in rubber polymerization, including farnesyl pyrophosphate

(FPP), geranyl pyrophosphate (GPP), dimethylallyl pyrophosphate (DMAPP), kinetic analysis of rubber transferase and structural analysis of NR have identified FPP as the in vivo APP primer (Archer et al. 1987; Tanaka 1989; Cornish et al. 1995; Tanaka et al.

1996; Tanaka 2001; Xie et al. 2008).

Rubber is produced on cytosolic vesicles known as rubber particles that consist of a proteinaceous phospholipid monolayer surrounding a hydrophobic core of sequestered rubber. Rubber particles are alone sufficient of producing cis-1,4-polyisoprene, when provided with IPP, an APP primer, and magnesium ion cofactors (Archer et al. 1967;

Light et al. 1989; Madhavan et al. 1989). Surprisingly, after several decades of research, the identification of the proteins associated with rubber transferase activity are still largely unknown. This is primarily because rubber transferase activity is labile and lost when WRP integrity was disturbed. Therefore, attempts to identify the rubber transferase through classical biochemical approaches have been unsuccessful.

Several proteins have been hypothesized to function in rubber transferase activity, including the rubber elongation factor (REF), the small rubber particle protein (SRPP), and cis-prenyltransferase (CPT). The rationale for assigning REF and SRPP to rubber transferase activity was first based on their high abundance in purified rubber particles

(Dennis et al. 1989). However, photoaffinity labeling studies with benzophenone analogues of FPP, the APP primer for rubber transferase, have identified REF as a protein closely associated with the rubber transferase substrate binding site (DeGraw et al. 2007).

Recently, altered gene expression of SRPP in transgenic rubber producing dandelion has

27 been shown to alter rubber yields, molecular weight and rubber particle morphology

(Collins-Silva et al. 2012; Hillebrand et al. 2012). Therefore, spatial and functional evidence exists to support the hypothetical role of REF and SRPP in rubber transferase activity.

However, the role of CPT in rubber biosynthesis is less clear. CPT was first identified in Hevea based on its high degree of shared protein sequence similarity with prokaryotic and eukaryotic undecaprenyl pyrophosphate synthases. These enzymes produce short chain cis-1,4-polyisoprene membrane anchors for oligosaccharide carriers involved protein glycosylation in eukaryotes and lipopolysaccharide formation in bacteria. While recombinant CPTs were able to synthesize cis-1,4-polyisoprene in vitro, the molecular weight of the polymers produced were much shorter than commercial natural rubber (Asawatreratanakul et al. 2003). Recently, transgenic studies performed in a rubber producing dandelion species showed that by decreasing CPT gene expression, rubber yields and molecular weights were significantly decreased (Post et al. 2012).

While this result suggest CPT is involved in rubber biosynthesis, evidence has not been presented that this enzyme is a component of the rubber transferase or that it is localized to the rubber particle, the known location of rubber biosynthesis. An alternative hypothesis to the possible function of CPT in rubber transferase function is that CPT functions to produce short chain cis-1,4-polyisoprene primers that are elongated by a rubber particle associated rubber transferase enzyme (Takahashi et al. 2012).

In this study, a series of experiments were performed to provide evidence for the physical association of CPT with the rubber particle and the rubber transferase.

MATERIALS AND METHODS

Purification of Hevea brasiliensis WRPs

Enzymatically active washed rubber particles were purified from H. brasiliensis

(RRIM600) latex as described previously by Siler and Cornish, 1993 with some modifications. Hevea latex was washed four times with wash buffer containing 100 mM

Tris-HCI (pH 7.5), 5 mM Dithiothritol (DTT) and 0.1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). Each wash was done with increasing g force from 1000 xg to 7000 xg at 4 C, while collecting floated rubber particles after each spin. Collected washed rubber particles were stabilized with 10% glycerol, frozen drop-wise into liquid nitrogen and stored in -80 C (Siler et al. 1993; Cornish et al. 1997).

SDS-PAGE analysis of H. brasiliensis WRP proteins and liquid chromatography with tandem mass spectrometry (LC/MS/MS)

Total protein from H. brasiliensis WRPs were extracted using Bio-Rad®

(Hercules, CA), ReadyPrep Sequential Extraction kit reagent 3; 5 M Urea, 2 M thiourea,

2% CHAPS, 2% SB 3-10, 40 mM Tris, 0.2% Bio-Lyte 3/10 ampholyte (Bio-Rad®) .

Four volumes of Extraction buffer 3 (Extraction buffer A) was added to Hevea WRPs and mixed for 1 h in room temperature. Rubber was coagulated-out from the solution by centrifuging at 16,000 x g at room temperature for 15 min. The supernatant was filtered with an Acrodisc® 25 mm Syringe filter with a 0.45 μm Supor® Membrane (PALL Life

Sciences, East Hills, NY). Extracted protein was concentrated by precipitating with four volumes of 100% cold acetone, followed by 2 washes with four volumes of 80% v/v cold acetone, dried under nitrogen, and resuspended with BioRad® ReadyPrep Sequential

Extraction Kit Reagent 3 (Collins-Silva et al. 2012). The extracted proteins were

29 quantified using EZQ® Protein Quantitation Kit (Molecular Probes Inc., Grand Island,

NY) according to the manufactures instruction.

The purified WRP proteins, 20 µg, were ran on a 12 % SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250 (BioRad®, Hercules, CA). Nevada

Proteomics Center analyzed selected bands by trypsin digestion and LC/MS/MS analysis.

Bands were reduced and alkylated using 10 mM dithiothreitol and 100 mM iodoacetamide respectively and were digested overnight at 37 °C with 75 ng trypsin in 25 mM ammonium bicarbonate. Peptides were separated by Michrom Paradigm Multi-

Dimensional Liquid Chromatography (MDLC) instrument using a Magic C18AQ 3µ

200Å (0.2 x 50 mm) column (Michrom BioResources Inc., Auburn, CA) with an Agilent

ZORBAX 300SB-C18 5µ (5 x 0.3 mm) trap (Agilent Technologies, Santa Clara, CA).

The gradient was as follows: 0.1% formic acid in water (pump A) and 0.1% formic acid in Acetonitrile (Pump B), Time (mins) , Flow (µl/min), Pump B (%): (0.0, 4.00, 5.00),

(5.0, 4.0, 5.00), (35.0, 4.0, 45.00), (35.10, 4.0, 80.00), (36.1, 4.0, 80.00), (36.2, 4.0, 5.00),

(41.0, 4.0, 5.00). Eluted peptides were analyzed using a Thermo Finnigan (Ringoes, NJ)

LTQ-Orbitrap using Xcalibur v 2.0.7. MS spectra (m/z 300-1800) were acquired in the positive ion mode with resolution of 60,000 in profile mode. The top 4 data-dependent signals were analyzed by MS/MS with CID activation, minimum signal of 2,000, isolation width of 3.0, and normalized collision energy of 35.0. The reject mass list included: 355.0700, 356.0690, 371.1010, 372.1010, 374.0970, 420.6591, 445.1200,

516.3020, 572.5, 590.3040, 597.3110, 633.3210, 650.7670, 679.3, 691.3260, 692.3490,

695.8410, 717.8880, 738.3, 752.6860, 854.3870, 858.9320, 883.3690, 1288.2555, and

1523.3550. Dynamic exclusion settings were used with a repeat count of 2, repeat duration of 10 seconds, exclusion list size of 500 and duration of 30 seconds.

Database search was performed using Sequest (Thermo Fisher Scientific, San

Jose, CA, version v.27, rev. 11). Tandem mass spectra were extracted; charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest. Sequest was set up to search the NCBI non-redundant database (2010,

9,694,989 entries) assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10 ppm. A fixed modification of iodoacetamide derivative of cysteines and variable modification of oxidation of methionine was set. Scaffold (V3.6.1, Proteome Software Inc., Portland,

OR) was used to validate MS/MS based peptide and protein identifications. Peptide and protein identifications were accepted if they could be established at greater than 95.0% probability and greater than 95.0% probability and contained at least 2 identified peptides, respectively, as specified by the Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

Expression of recombinant Hevea CPT, antibody production and purification

Hevea HRT1 was amplified using a cDNA clone with following primers: sense

5′-GAATTCATGGAATTATACAACGG-3′, antisense primer 5′-

CTCGAGTTTTAAGTATTCCTTATGTTTCTCC-3′. The primers were designed to incorporate Eco RI and XhoI sites into the 5′ and 3′ ends of the PCR fragment respectively. Resulted PCR fragment was sub-cloned into pCR™-Blunt II-TOPO® vector

(Invitrogen, Grand Island, NY) and then cloned into pGEX-6P-1(Amersham Biosciences,

Piscataway, NJ), which resulted a glutathione S-transferase (GST) fused HRT1 protein.

Protein expression was facilitated by transformation of pGEX-6P-1-HRT1 vector into

Rosetta 2(DE3) pLysS cells. Protein expression and purification was done according to manufacturer’s instructions with minor modifications (GE Healthcare, Piscataway, NJ).

After cell culture reached A600 between 0.5–2, they were induced with 0.1 M isopropyl β-

D thiogalactoside (IPTG) and incubated for 7.5 h at 28 °C. Cells were harvested by centrifugation at 7700 x g for 10 min at 4 °C. A modified PBS cell lysis buffer: 140 mM

NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 supplemented with 1 mM EDTA, 10 mM DTT, 1 mM PMSF (phenylmethylsulfonyl fluoride) was used to lyse the cells. After the cell lysis GST-HRT1 fusion protein was purified using Glutathion

Sepharose 4B (GE Healthcare). PreScission™ Protease (GE Healthcare) was used to cleave the fusion protein and purify HRT1 protein according to the manufacturer’s instructions (Collins-Silva et al. 2012). After confirming the purification of HRT1 protein by SDS-PAGE gel, about 1 mg of proteins was sent to Cocalico Biologicals, Inc

TM (Reamstown, PA) for antibody production.

To purify the antibodies a Glutathion Sepharose 4B (GE Healthcare) column was made with GST-HRT1 as follows. Glutathion Sepharose beads were resuspended 1:1 with NETN (0.5 % NP-40, Sigma, 20 mM Tris (pH 8.0), 100 mM NaCl, and 1 mM

EDTA) buffer and incubated overnight at 4 °C. Beads were washed several times with

0.1 M borate buffer (pH 8) and incubated with 40 mM dimethylpimelimidate for 1 h at 4

°C. After washing beads twice with 0.1 mM borate buffer (pH 8) beads were incubated with 40 mM ethanolamine in 0.1 M borate buffer for 45 min at 4 °C. After washing the column three times with 0.2 M glycine-HCl (pH 2.5) and 1 M K2HPO4, the beads were

32 ready for antibody binding. The GST-HRT1 column was washed with PBS containing

0.02 % Tween20, and incubated the column overnight at 4 °C after adding the serum containing PBS (1:1). After washing the column several times with PBS, antibody was eluted with 0.2 M glycine-HCl (pH 2.5), and collected into tubes containing 250 µl of 1M

K2HPO4. A total of 10 fractions were collected and the extracted proteins were quantified using EZQ® Protein Quantitation Kit (Molecular Probes Inc., Grand Island,

NY.) according to the manufacture’s instruction. The identified peak protein fractions were pooled and solvent exchanged with PBS and stored with 1 % BSA at -80 °C.

Western blot analyses of Hevea brasiliensis latex, C-serum, and WRPs proteins.

Total protein from H. brasiliensis latex, C-serum, and WRPs were extracted using

NuPAGE LDS sample buffer: 10% glycerol, 247 mM Tris-HCI, pH 8.5, 2% lithium dodecyl sulphate, 0.51 mM EDTA, 0.22 mM SERVA Blue 250, 0.175 mM Phenol Red

(Invitrogen, Grand Island, NY). Samples were mixed with NuPAGE LDS sample buffer for 1 h and rubber was coagulated-out from the solution by centrifuging at 16,000 x g at room temperature for 3 min. Supernatant was vortexed and centrifuged several times until all the rubber was gone. The extracted proteins were quantified using EZQ® Protein

Quantitation Kit (Molecular Probes Inc., Grand Island, NY) according to the manufactures instruction. After the proteins were denatured for 5 min at 100 °C, proteins were fractionated by 12 % SDS-PAGE and transferred to nitrocellulose according to manufacturer’s instructions (Bio-Rad, Hercules, CA). Membrane was stained with

SYPRO® Ruby Protein blot stain (Molecular Probes Inc., Grand Island, NY). Affinity purified Hevea CPT antibody was used at 1:200 dilution as primary antibody and a peroxidase-conjugated Affinitypure Goat Anti-Mouse IgG (Jackson Immunoresearch,

West Grove, PA) was used at 1:10,000 dilution as secondary antibody. Proteins were detected by chemiluminescence using ECL Plus kit according to manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL).

In vitro rubber transferase assay

The activity of rubber particles were measured using a modified version of a previously described method (Mau et al. 2000). A typical 40 µl volume reaction contained 100 mM Tris-HCl, pH 7.5, 1.25 mM MgSO4, 5 mM DTT, 15 µM FPP, 0.9 nmol [14C] IPP (American Radiolabeled Chemicals, Inc., St. Louis, MO), 1 mM unlabeled IPP, 0.5 mg WRPs using a multiscreen 96-well filter plate (Millipore

Corporation, Billerica, MA). The transferase assay was done for 4 h at 25 °C and the reaction was stopped by adding 40 mM EDTA (Mau et al. 2000; Xie et al. 2008). The reaction was filtered and the plate was washed twice with 150 µl water and 95 % ethanol, dried at 37 °C for 30 min and the filters were put on to a vial containing 1.5 ml of

ScintVerse BD Cocktail (Fisher Science). The [14C] IPP incorporation was measured by scintillation counting (Beckman Coulter, Fullerton, CA) and was converted to IPP incorporation to a per gram dry rubber basis.

Synthesis of benzophenone-containing analogues, A and B

Benzophenone-containing analogues were synthesized as previously described: analogue A (Marecak et al. 1997), and analogue B (Henry et al. 2009). In brief, GPP- based analogue A was prepared from a protected form of geraniol (Gaon et al. 1996;

Gaon et al. 1996) followed by O-alkylation (Turek et al. 2001) to install the benzophenone unit. Analogue B was prepared from phosphonic acid as described in

1 Henry et al. (2009). All compounds were characterized by H NMR (500 MHz, CDCl3),

31 P NMR (202 MHz, CDCl3), HRMS (ESI), and UV spectrometry to confirm their structures and reverse-phase HPLC have confirmed the purity to be greater than 90% for each compound.

Photolabeling reactions and identification of photolabeled proteins

All photolysis reactions were conducted at 4 °C in a UV Rayonet mini-reactor

(The Southern New England Ultraviolet Company Inc., Branford, CT), as described in

DeGraw et al. (2007). Briefly, all reactions (~ 100 µl) were performed in salinized quartz test tubes (10 x 75 mm) and contained 0.1 M Tris-HCl (pH 7.5), 1.25 mM MgSO4, 5 mM

DTT, 10 µM 32P analogue, and 30 µl of WRPs (4.51µg protein/ µl). Reaction tubes were photolyzed for 6 h in the mini reactor. Loading buffer ( 4 % SDS, 12 % glycerol, 50 mM

Tris-HCl, 2 % 2-mercaptoethanol, 0.01% bromophenolblue) was added to each reaction and samples were heated to 100 °C for 5 min. After photolysis of WRPs with analogues

A and B, 20 µg of purified WRP proteins were fractionated on 4-16 % and 12% SDS-

PAGE respectively. Gels were stained with Coomassie Brilliant Blue R-250 and radioactivity was detected by phosphorimaging using a Cyclone Storage phosphor scanner (PerkinElmer, Waltham, MA). The radioactive bands identified from the gel were analyzed by mass spectrometry. Nevada Proteomics Center analyzed selected proteins by trypsin digestion and MALDI TOF/TOF analysis as described by Collins-

Silva et al. (2012). The data was extracted from the Oracle database and a peak list was created by GPS Explorer software v 3.6 (Applied Biosystems) from the raw data generated from the ABI 4700. The filtered data were searched by Mascot v 1.9.05

(Matrix Science) using NCBI nr database (NCBI 20070908), containing 5,454,477 sequences.

RESULTS AND DISCUSSION

Hevea brasiliensis CPT identified by liquid chromatography mass spectrometry

(LC-MS)

A proteomics-based approach was used to determine if Hevea rubber particles harbored a CPT. For this study, rubber particles isolated from Hevea latex were subjected to an extensive purification process to remove contaminating cytosolic proteins and assayed prior to use to ensure the presence of rubber transferase activity. The total rubber particle protein fraction was treated with a detergent containing buffer to ensure the complete extraction of soluble and membrane associated proteins. This protein extract was then fractionated by 12% SDS PAGE after which the region of the gel lane corresponding to proteins with ~35 kDa molecular weights was excised from the gel. The gel slice was subjected to trypsin digestion and subsequent liquid chromatography-mass spectrometry (LC-MS) protein sequencing analysis (Figure 1). The results of this analysis revealed that the excised band contained two CPT isoforms and one REF with

29.7%, 17.2%, and 19.6% sequence coverage, respectively (Table 1). Seven and four tryptic fragments corresponded to two known Hevea CPTs HRT1 and HRT2 (accession numbers AB061234 and AB064661) respectively (Asawatreratanakul et al. 2003). The first seven unique peptides, identified as gi|20563020, were 100% identical to HRT1. On the other hand, three of the peptides were 100% identical and four of the peptides were at least 86% identical to HRT2. In the case of the gi|22213589, the peptides were at least

64% identical to HRT1 and 50% identical to HRT2 (Table 1). Therefore, the identified

Hevea WRP proteins were likely to be HRT1 and HRT2. In addition to the two Hevea

CPTs, tryptic fragments corresponding to REF were also identified in the excised 35 kDa

36 protein band. This result is consistent with previous photoaffinity labeling studies performed on Hevea rubber particles using benzophenone analogues of FPP (DeGraw et al. 2007).

Immunological and enzymatic evidence for CPT association with H. brasiliensis rubber particles

Immunolocalization studies in conjunction with rubber transferase assays were performed to confirm the rubber particle association of CPT. Affinity purified polyclonal antibodies generated to a recombinant Hevea HRT1 protein were used to detect CPT in protein extracts derived from rubber particles. This analysis was performed on rubber particles subjected to a sequential series of increasing washes to eliminate contaminating cytosolic CPT and to determine the relative affinity of CPT to the rubber particle surface.

Crude latex and C-serum (i.e. the latex fraction devoid of rubber particles) protein extracts were used as positive and negative controls, respectively. Positive signals corresponding to a 35 kDa band were identified in each lane containing a rubber particle protein extracts. As expected, the enriched latex fraction contained relatively lower amounts of CPT compared to C-serum and the C-serum was essentially devoid of CPT.

The highest CPT level was found in the proteins from one time washed WRPs and the amount of CPT decreased with each successive wash and there were no visible CPT band in the nine time washed WRPs (Figure 2). The fact that CPT was highly visible on washes 1-4 and somewhat visible on washes 5-8, indicated that CPT was present in the

WRP surface, but rigorous washing can reduce the amount or inactivate the rubber transferase. However, the sensitivity of the western blot analysis might have hindered the

37 visibility of CPT in 5-9 washes, because the rubber transferase activity of the same 5-9 washes expressed a different story; nine-time-washed particles still had activity.

To verify the rubber synthetic capacity of the rubber particles used in this study, in vitro rubber transferase assays were performed on the Hevea latex and 1-9-time- washed Hevea washed rubber particles. In each case [14C] IPP incorporation was measured in the rubber fraction using a scintillation counter (Figure 3). The rubber transferase activity correlated with the CPT protein levels observed in the immunoblot analysis (Figure 2). While the rubber transferase activity of the one- and two-time- washed particles were essentially the same, activity decreased with each successive purification in a manner consistent with the amount of CPT detected. The nine-times- washed rubber particles had ~55 % of the rubber transferase activity associated with the highly active two times washed particles. Therefore, CPT protein appears to be tightly bound to WRPs and its associated activity remained despite of rigorous washing.

Photolabeling of washed rubber particle associated proteins with radiolabeled FPP analogues

A series of experiments was conducted to identify proteins that work in close proximity with rubber transferase enzyme during natural rubber production. A group of benzophenone (Bz)-containing photoactive FPP analogues have been developed to identify enzymes that synthesize FPP and employ FPP as substrates (Dorman et al. 1994;

Yokoyama et al. 1995; Gaon et al. 1996; Turek et al. 1997; Zhang et al. 1998; Turek et al.

2001; DeGraw et al. 2007). Bz-containing photoaffinity analogues undergo stable covalent C-H bond insertion reactions when excited with 350 nm light as presented in

Xie et al., (2008). Moreover, recent studies have utilized Bz-containing analogues of

38 rubber biosynthetic initiator, FPP, to identify rubber transferase and its activity (DeGraw et al. 2007; Xie et al. 2008). This approach allows the tracking and identification of rubber transferase and other enzymes/proteins that are directly associated in the rubber production machinery. Rubber elongation factor (REF) protein was one of the rubber transferase related proteins identified using FPP analogues (DeGraw et al. 2007).

Previous studies have shown that analogue A (Xie et al. 2008) and analogue B (Henry et al. 2009) were able to function as APP primers in in vitro rubber transferase assays

(Figure 4). According to Xie et al. (2008), rubber transferase had a higher affinity for analogue A and is, therefore, an ideal photoaffinity substrate to identify the rubber transferase.

Analogue B consists of unmodified FPP group connected to a benzophenone moiety with a pyrophosphate group (Figure 4). Structurally, analogue B is identical to

FPP with an addition of a benzophenone moiety to the pyrophosphate group. Because the Bz moiety of analogue B is not within the binding site, it might cross link with other proteins in close proximity. Furthermore, analogue B was a competitive inhibitor of

Saccharomyces cerevisiae protein farnesyl transferase (ScPFTase) with respect to FPP, making analogue B a better candidate to identify rubber transferase (Henry et al. 2009).

Analogues A and B were incubated with active Hevea rubber particles and photolyzed, resulting the formation of stable cross-links between the benzophenone moiety and protein subunits of the rubber transferase (Figure 4). The phosphate groups of both analogues were 32P-labeled. Therefore, cross-linked proteins were detected by phosphorimaging followed by mass spectrometry identification.

Analogue A was able to successfully cross link with Hevea particle proteins as shown by the phosphorimage (Figure 5). The radioactive bands identified from Hevea particle proteins were at 10 kDa, 15 kDa, 18 kDa, 25 kDa, and 35 kDa (arrows to the right side of each gel image). The bands were excised from SDS-PAGE gel, alkylated, enzymatically digested with trypsin, peptides were analyzed by MALDI TOF/TOF mass spectrometry (Table 2). The 35 kDa band contained cis-prenyltransferase (CPT), small rubber particle protein (SRPP), and rubber elongation factor (REF). Five peptides were identified for CPT, six peptides for SRPP, and five peptides for REF. SRPP and REF was identified in 25 kDa and 18 kDa bands, whereas only REF was identified in 15 kDa and 10 kDa bands. The peptide sequences were shown in supplemental Table 1.

The results of analogue A were further supported by the results from the reaction between Hevea particles and the analogue B (Figure 5). The radioactive bands identified from the phosphorimage of Hevea WRP proteins were analyzed by MALDI TOF/TOF mass spectrometry as before. CPT and REF proteins were identified from the 35 kDa band. SRPP and REF were identified from the 25 kDa band, whereas only REF was identified in the 20 kDa band (Table 2). The 35 kDa band consists of seven peptides corresponding to CPT and six peptides related to REF. The analysis of 25 kDa band resulted in eight SRPP peptides and five REF peptides, whereas 20 kDa band contained eight REF peptides (Table 2). The peptide sequences for each peptide are shown on supplemental Table 2.

Two analogues were able to cross link to proteins, shown by the radiolabeled bands in the phosphorimage (Figure 5). Analogue A was able to mimic FPP and closely interact with rubber transferase when cross-linking was initiated, analogue A was able to

40 covalently bind to CPT. Analogue B, which consists of a unmodified FPP connected to a benzophenone moiety with a pyrophosphate group (Figure 4), was able to covalently bind to CPT. All the CPT peptide sequences identified by both analogues had 100% identity to

HRT1 and HRT2 (Suppl. Table 1 and 2). These results suggest that CPT is the rubber transferase. Although, in vitro studies with recombinant Hevea HRT1 and HRT2 synthesized cis-1,4-polyisoprene polymer, the molecular weight was significantly lower than of natural rubber from Hevea (Asawatreratanakul et al. 2003). In order to produce high molecular weight rubber, rubber transferase enzyme might be required to be associated with the rubber particles and to work together with other rubber particle associated proteins. In this study, several SRPP and REF proteins were also identified by both analogues (Figure 5 and Table 2). SRPP and REF proteins were found in abundance in Hevea latex and WRPs (Chow et al. 2007; Collins-Silva et al. 2012). During rubber production, FPP closely interacted with rubber transferase and might also interact with other proteins in the complex (Tanaka et al. 1996; Tanaka 2001). In order to be cross- linked to analogues, proteins have to be in very close proximity. Therefore, the identified

SRPP and REF are either a part of the rubber transferase complex or aid rubber transferase in producing high molecular weight natural rubber.

ACKNOWLEDGEMENTS

This work was supported by funding from The National Science Foundation Plant

Genome Research Program (DBI-03211690), the Nevada Agricultural Experimental

Station and the NIH IDeA Network of Biomedical Research Excellence (INBRE, RR-03-

008). The Nevada Proteomics Center is supported by NIH Grant Number P20 RR-

016464 from the INBRE Program of the National Center for Research Resources. This project's contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. Also was supported by DOA-ARS-USDA grant

REFERENCES

Archer, B. L. and B. G. Audley (1967). Biosynthesis of Rubber. 29: 221-257. Archer, B. L. and B. G. Audley (1987). "New Aspects of rubber biosynthesis." Bot. J. Linn. Soc. 94: 181 - 196. Asawatreratanakul, K., Y. W. Zhang, D. Wititsuwannakul, R. Wititsuwannakul, S. Takahashi, A. Rattanapittayaporn and T. Koyama (2003). "Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis." Eur J Biochem 270: 4671-4680. Chow, K. S., K. L. Wan, M. N. Isa, A. Bahari, S. H. Tan, K. Harikrishna and H. Y. Yeang (2007). "Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex." J Exp Bot. 58: 2429-2440. Epub 2007 Jun 2421. Collins-Silva, J., A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish and D. Shintani (2012). "Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism." Phytochemistry 79: 46-56. Cornish, K. and D. L. Bartlett (1997). "Stabilisation of Particle Integrity and Particle Bound cis-Prenyl Transferase Activity in Stored, Purified Rubber Particles." Phytochemical Analysis 8: 130-134. Cornish, K. and D. J. Siler (1995). "Effect of different allylic diphosphates on the initiation of new rubber molecules and on cic- 1, 4 - polyisoprene biosynthesis in guayule (Parthenium argentatum Gray)." J. Plant Physiology 147: 301 - 305. DeGraw, A. J., Z. B. Zhao, C. L. Strickland, A. H. Taban, J. Hsieh, M. Jefferies, W. S. Xie, D. K. Shintani, C. M. McMahan, K. Cornish and M. D. Distefano (2007). "A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: Synthesis and biochemical studies with prenyltransferases." Journal of Organic Chemistry 72: 4587-4595. Dennis, M. S. and D. R. Light (1989). "Rubber Elongation Factor from Hevea brasiliensis." Journal of Biological Chemistry 264: 18608-18617. Dorman, G. and G. D. Prestwich (1994). "Benzophenone Photophores in Biochemistry." Biochemistry 33: 5661-5673. Gaon, I., T. C. Turek and M. D. Distefano (1996). Tetrahedron Lett. 37: 8833. Gaon, I., T. C. Turek, V. A. Weller, R. L. Edelstein, S. K. Singh and M. D. Distefano (1996). "Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase." The Journal of Organic Chemistry 61: 7738- 7745. Henry, O., F. Lopez-Gallego, S. A. Agger, C. Schmidt-Dannert, S. Sen, D. Shintani, K. Cornish and M. D. Distefano (2009). "A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes." Bioorganic & Medicinal Chemistry 17: 4797-4805. Hillebrand, A., J. J. Post, D. Wurbs, D. Wahler, M. Lenders, V. Krzyzanek, D. Prüfer and C. S. Gronover (2012). "Down-Regulation of Small Rubber Particle Protein

Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum." PLOS ONE 7: e41874. Ko, J. H., K. S. Chow and K. H. Han (2003). "Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree)." Plant Mol Biol 53: 479-492. Light, D. and M. Dennis (1989). "Purification of a prenyltransferase that elongates cis- polyisoprene rubber from the latex of Hevea brasiliensis." J Biol Chem 264: 18589 - 18597. Madhavan, S., G. A. Greenblatt, M. A. Foster and C. R. Benedict (1989). "Stimulation of Isopentenyl Pyrophosphate Incorporation into Polyisoprene in Extracts from Guayule Plants (Parthenium argentatum Gray) by Low Temperature and 2-(3,4- Dichlorophenoxy) Triethylamine." Plant Physiol. 89: 506-511. Marecak, D. M., Y. Horiuchi, H. Arai, M. Shimonaga, Y. Maki, T. Koyama, K. Ogura and G. D. Prestwich (1997). Bioorg. Med. Chem. Lett. 7: 1973. Mau, C. J. D., D. J. Scott and K. Cornish (2000). "Multiwell filtration system results in rapid, high-throughput rubber transferase microassay." Phytochemical Analysis 11: 356-361. Post, J., N. van Deenen, J. Fricke, N. Kowalski, D. Wurbs, H. Schaller, W. Eisenreich, C. Huber, R. M. Twyman, D. Prüfer and C. S. Gronover (2012). "Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum." Plant Physiology 158: 1406-1417. Schmidt, T., M. Lenders, A. Hillebrand, N. van Deenen, O. Munt, R. Reichelt, W. Eisenreich, R. Fischer, D. Prufer and C. Gronover (2010). "Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz." BMC Biochemistry 11: 11. Siler, D. J. and K. Cornish (1993). "A protein from Ficus elastica rubber particles is related to proteins from Hevea brasiliensis and Parthenium argentatum." Phytochemistry 32: 1097-1102. Takahashi, S., H. J. Lee, S. Yamashita and T. Koyama (2012). "Characterization of cis- prenyltransferases from the rubber producing plant Hevea brasiliensis heterologously expressed in yeast and plant cells." Plant Biotechnology 29: 411- 417. Tanaka, Y. (1989). "Structure and biosynthesis mechanism of natural polyisoprene." Progress in Polymer Science 14: 339-371. Tanaka, Y. (2001). "Structural Characterization of Natural Polyisoprenes: Solve the Mystery of Natural Rubber Based on Structural Study." Rubber Chemistry and Technology 74: 355-375. Tanaka, Y., Aik-Hwee, E. , N. Ohya, N. Nishiyama, J. Tangpakdee, S. Kawahara and R. Witisuwannakul (1996). "Initiation of rubber biosynthesis in Hevea brasiliensis: characterizationof initiating species by structural analysis." Phytochemistry 41: 1501 - 1505. Turek, T. C., I. Gaon, M. D. Distefano and C. L. Strickland (2001). "Synthesis of Farnesyl Diphosphate Analogues Containing Ether-Linked Photoactive Benzophenones and Their Application in Studies of Protein Prenyltransferases." The Journal of Organic Chemistry 66: 3253-3264.

Turek, T. C., I. Gaon, D. Gamache and M. D. Distefano (1997). "Synthesis and evaluation of benzophenone-based photoaffinity labeling analogs of prenyl pyrophosphates containing stable amide linkages." Bioorganic & Medicinal Chemistry Letters 7: 2125-2130. Xie, W. S., C. M. McMahan, A. J. DeGraw, M. D. Distefano, K. Cornish, M. C. Whalen and D. K. Shintani (2008). "Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species." Phytochemistry 69: 2539-2545. Yokoyama, K., P. McGeady and M. H. Gelb (1995). "Mammalian Protein Geranylgeranyltransferase-I: Substrate Specificity, Kinetic Mechanism, Metal Requirements, and Affinity Labeling." Biochemistry 34: 1344-1354. Zhang, Y. W., T. Koyama, D. M. Marecak, G. D. Prestwich, Y. Maki and K. Ogura (1998). Biochemistry 37: 13411.

FIGURES AND TABLES

Figure 1. Hevea brasiliensis washed rubber particle proteins. H. brasiliensis WRP proteins (Twenty micrograms) were fractionated by 12 % SDS PAGE and stained with Coomassie Brilliant Blue R-250. Lane 1: protein ladder (10-250 kDa, New England Biolabs); Lane 2: rubber particle protein purified using Sequential Extraction kit reagent 3. The band corresponding to about 35 kDa (Black box) was cut and subjected to trypsin digestion followed by liquid chromatography mass spectrometry (LC /MS).

Table 1. CPT associated with Hevea brasiliensis WRP proteins, identified by mass spectrometry.

Figure 2. Western blot analysis of Hevea brasiliensis latex, C-serum and WRP proteins with Hevea CPT antibody. Proteins from H. brasiliensis, latex, C-serum, and 1-9 times washed WRPs (1x-9x) were purified using NuPAGE LDS sample buffer. Proteins were fractionated by 12 % SDS-PAGE and transferred to nitrocellulose. Cis-prenyltransferase was detected using affinity purified Hevea CPT antibody.

Figure 3. The rubber transferase activity of Hevea brasiliensis latex and WRPs. The rubber transferase assay was performed with Hevea latex and 1-9 times washed (1x-9x) Hevea WRPs shown in x-axis. The [14C] IPP incorporation as IPP incorporation to a per gram dry rubber basis is shown on y-axis.

Figure 4. Structures of FPP and benzophenone-modified FPP analogues.

Figure 5. Phosphorimage of H. brasiliensis washed rubber particle proteins identified by 32P FPP analogues A and B. Five bands were cut from the analogue A sample while only three bands were analyzed from analogue B (bands 1-5 and 1-3, respectively are shown to the right of each lane).

Table 2. Mass spectrometry of the H. brasiliensis washed rubber particle proteins identified by 32P labeled FPP analogues.

SUPPLEMENTAL TABLES

Supplemental Table 1. Peptide sequences identified by mass spectrometry of the H. brasiliensis WRP proteins with analogue A.

Supplemental Table 2. Peptide sequences identified by mass spectrometry of the H. brasiliensis WRP proteins with analogue B.

Chapter III

The role of cis-prenyltransferase in determining the yield and the molecular weight

of rubber in Taraxacum kok-saghyz

ABSTRACT

Despite the importance of natural rubber, scientists know very little about the key enzymes involved in rubber biosynthetic pathway. The rubber transferase enzyme is responsible for the polymerization of isopentenyl pyrophosphate (IPP) monomer into cis-

1,4-polyisoprene polymer, natural rubber, that contains about 16000 IPP units. We hypothesize that cis-prenyltransferase (CPT) is a component of the rubber transferase enzyme complex and it is localized to the rubber particles, the known site of rubber synthesis and sequestration. As support for its role in rubber biosynthesis, the CPT gene from the rubber producing species Taraxacum kok-saghyz (TKS), showed patterns of temporal expression that strongly correlated with patterns of rubber accumulation in this species. To confirm the in vivo role of CPT in rubber biosynthesis, CPT under- expressing transgenic TKS plants were studied. The rubber yield, rubber molecular weight, CPT gene expression, and CPT proteins levels of transgenic plants were measured. The CPT under-expressing transgenic lines showed a reduced rubber yield and considerably lower molecular weight. These results provide evidence for the involvement of CPT as rubber transferase in plant rubber biosynthesis.

INTRODUCTION

Natural rubber (NR) is essential to US economy in regards to manufacturing of many industrial and medical products. In 2011, the US spent $4.4 billion to import NR while, it is forecasted to increase to $4.5 and $4.8 billion dollars in years 2012 and 2013 respectively (USDA 2012). Hevea brasiliensis (Brazilian rubber tree) is the primary producer of NR in the world. Hevea NR is currently obtained from economically and politically unstable Southeast Asian countries. Furthermore, H. brasiliensis varieties are vulnerable to pathogen attack and abiotic stress due to inbreeding. Unfortunately, the lack of understanding of the rubber production mechanism and the enzymes involved, mainly the rubber transferase, has prevented the enhancement of rubber production by genetic manipulation of Hevea and other alternative NR crops.

Eight botanical families, 300 genera, and 2500 species have been identified to contain natural rubber in their latex, but only a few of them were able to produce large amount of high-quality rubber (Mooibroek et al. 2000; Bushman et al. 2006). Russian dandelion (Taraxacum kok-saghyz) is one of the top alternative rubber producing candidates. Russian dandelion produce high molecular weight rubber with 2,180 kDa in their root laticifer cells and the rubber yield can be as high as 20% of root dry weight

(Whaley et al. 1947; Hallahan et al. 2004; McMahan 2009). Dandelion plants respond to both tissue culture and transformation. In addition, T. kok-saghyz plants are much smaller than Hevea plants and rubber phenotypes can be obtained as early as in 6 months

(Collins-Silva et al. 2012). For these reasons dandelion plants are being used as a model system for rubber producing plants to understand rubber biosynthesis.

Rubber is synthesized in monolayer vesicles known as rubber particles. Rubber transferase enzyme, located in particle surface, initiates the synthesis of a new rubber molecule with the binding of farnesyl pyrophosphate (FPP) primer, followed by progressive addition of isopentenyl pyrophosphate (IPP) monomers into producing cis-

1,4-polyisoprene polymer (Archer et al. 1987; Tanaka 2001). Depending upon the number of polymerized IPP units, the molecular weight vary from 10 -10,000 kDa with

>1000 kDa representing the high quality rubber (Schmidt et al. 2010).

Currently the rubber transferase enzyme is unknown. However, several candidate proteins have been identified due to their rubber particle localization (Dennis et al. 1989;

Light et al. 1989; Collins-Silva et al. 2012) and by correlation of gene expression patterns with their rubber production (Kush et al. 1990; Chow et al. 2007; Collins-Silva et al.

2012). Rubber elongation factor (REF), and small rubber particle protein (SRPP) were among the top candidates. However, REF or SRPP are unlikely to be the rubber transferase (RT) given that the rubber structure contain cis-1,4-polyisoprene. Notably,

REF and/or SRPP likely closely interact with the RT when producing rubber (Collins-

Silva et al. 2012).

Cis-prenyltransferase (CPT) has been the number one RT candidate. The first

CPT identified in plants was from Arabidopsis thaliana. This CPT is predicted to be membrane associated and to produce long-chain (C120) dolichols, but was unable to produce very long chain polymers such as rubber (Oh et al. 2000). Asawatreratanakul et al. (2003) have identified and characterized two CPTs from H. brasiliensis: Hevea rubber transferases 1 and 2 (HRT1 and HRT2). The in vitro rubber transferase assays performed with recombinant HRT1 and HRT2 proteins made with Escherichia coli showed that

HRT2 but not HRT1 was able to produce long-chain polyprenyl products of 2-10 kDa

(Asawatreratanakul et al. 2003). However, HRT2 was not able to create polymers with lengths similar to rubber or did not exhibit significant activity independently of WRPs.

In a recent study Takahashi et al. (2012), have expressed recombinant HRT1 and HRT2 proteins in Saccharomyces cerevisiae and A. thaliana T87 cultured cells. A distinct CPT activity was observed, producing 1-1.4 kDa (C80-C100) polymers, but as before either

HRT1 or HRT2 was unable to produce long-chain polymers similar to rubber (Takahashi et al. 2012). In order to produce high molecular weight rubber, Hevea CPTs might require certain activation factors or co-factors that reside in the rubber particles

(Asawatreratanakul et al. 2003; Takahashi et al. 2012).

In this study Russian dandelion plants were used as a model plant system to investigate the effect of CPT on rubber polymer. One CPT gene was identified from a

TKS Expressed sequence tag (EST) collection and TkCPT RNAi lines were generated using stable Agrobacterium tumefacien transformation. TkCPT gene expression profiles and accumulated rubber were measured and compared in developing Taraxacum koksaghyz plants. The steady-state TkCPT transcript and protein abundances were measured and compared with the observed rubber yield and the molecular weight in transgenic RNAi plants.

MATERIALS AND METHODS

Taraxacum koksaghyz plant material

The plants used in this study were originated from a single T. koksaghyz accession

(NSL219131) from the USDA National Seed Storage Laboratory in Fort Collins,

Colorado. Plants were re-generated by micro-propagation and grown in greenhouse conditions (temperatures 72 °C and 68 °C with 16h and 8 h day and night, respectively, with 30 min watering for 3-times a week).

Assembling TkCPT under-expressing RNAi constructs

Russian dandelion cDNA clone TKN052F04 was used to PCR amplify the open reading frame of TkCPT. The following primers were used to amplify the PCR fragment: sense primer with CACC overhang 5ʹ- CACCGGATCCTGATATTTTAG-3 ʹ, antisense primer 5ʹ- CGTTCTTCTGCTCGTATAATGC-3 ʹ. The PCR fragments were first cloned into Invitrogen (Grand Island, NY) pENTR™/D-TOPO® entry vector according to manufacturer’s recommendations. The RNAi vector was constructed by performing a LR

Clonase reaction between the pENTR™/D-TOPO® vector and pK7GWIWG2(I) vector according to the manufacturer’s instructions (Karimi et al. 2002; Karimi et al. 2005). The binary vector carrying the TkCPT RNAi was transformed into Agrobacterium tumefaciens strain EHA 105 according to the standard methods.

Development of transgenic Taraxacum kok-saghyz plants

The transformation of Russian dandelion plants were done according to the established methods (Collins-Silva et al. 2012). Briefly, Agrobacterium cells carrying

TkCPT RNAi plant expression vectors and empty vectors were grown in 50 ml of YEP medium containing 100 µg/ml spectinomycin. Cells were harvested by centrifugation at

6000 x g for 10 min at 4 °C and resuspended in shooting medium. Surface sterilized dandelion leaves were cut into approximately 2 cm2 explants and incubated with shooting medium containing Agrobacterium (OD 600 between 1.5 and 1.65) for 10 min (Figure 1).

Dandelion explants were placed on plates containing solid shooting medium; 1 x

Murashige-Skoog with Vitamins (Caison Labs, North Logon, UT), 2% w/w Sucrose, pH

5.7, 0.7% Phytoagar, 1 mg/L 6-benzylaminopurine (BAP), 0.2 mg/L indole acetic acid

(IAA), and incubated in 22 °C incubator for 4 days. Explants were transferred to shooting medium containing 50 µg/ml kanamycin and 500 µg/ml amoxicillin. After 3-4 weeks, immerging small plantlets were transferred to shoot elongation medium (1 x

Murashige-Skoog with Vitamins (Caison Labs, North Logon, UT), 2% w/w Sucrose, pH

5.7, 0.7% Phytoagar, 0.5 mg/L kinetin, 0.1 mg/L IAA), and in another 3-4 weeks plantlets with shoots were transferred to rooting medium (0.5 x Murashige-Skoog with

Vitamins (Caison Labs, North Logon, UT), 2% w/w Sucrose, pH 5.7, 0.7% Phytoagar,

0.5% charcoal, 0.2 mg/L IAA). Once roots have established in about 3-4 weeks mature plantlets were transferred to small pots containing Pro-mix BX (Horticulture Source,

Vancouver, WA). Selected plants were transferred to 1 Gal pots after about two weeks and kept until harvest. Greenhouse conditions were as follows, temperatures 72 °C and 68 °C with 16 h and 8 h day and night, respectively, with 30 min watering for 3-times a week.

Rubber extraction and analysis of Taraxacum kok-saghyz roots

Mature dandelion plants that were about 3-4 months were harvested and root tissue was lyophilized and ground using a ball mill (Retsch mill MM301, Retsch, Inc.

Newtown, PA) at 4 °C for rubber analysis. Dionex Accelerated Solvent Extractor (ASE) model 200 (Sunnyvale, CA) was used to extract rubber as described in Collins et al.

(2012). Briefly, lyophilized root tissue from each RNAi line was extracted in triplicate.

Rubber extraction was performed using the following method: The 11 ml ASE cell was filled with n-hexane containing 2.5% ethanol for 1 min, heated to 80 °C and pressurized

62 to 1500 psi for 5 min, 3 cycles of: 15 min static extractions with 60 % flushing, and a final purge with nitrogen gas. The collected hexane/ethanol extract was dried under N2 and stored in -20 °C. Dried samples were resuspended in 2 ml of tetrahydrofuran (THF) overnight and analyzed using a HPLC-GPC system. The HPLC system (HP 1100 HPLC,

Agilent Technologies, Palo Alto, CA) contained one Phenogel guard column (50 x 7.80 mm) and two Phenogel 10 µ Linear columns (300 x7.80 mm) (Phenomenex, Torrance,

CA). The columns were connected in series with a Sedex Light Scattering Detector,

Model 75 (SEDERE, France). Mobile phase was 100% Toluene with a 1 ml/min flow rate at 70 °C. Data were analyzed using ChemStation software (LC Rev. A. 10.02, 1757,

Agilent technologies). Molecular weight of the rubber was calculated by a standard curve made using polystyrene standards (Polymer Laboratories, UK) with peak molecular weight ranging from 1,480 to 3,114,000 Da. The extracted rubber was quantified by an external standard curve made with serial dilutions of known amount of synthetic polyisoprene (Kraton Polymers IR-401, Houston, TX).

RNA extraction and quantitative real-time PCR (qRT-PCR) of Taraxacum kok- saghyz plants

Freshly ground dandelion root tissue, 100 mg, was used for extraction of total

RNA using the RNeasy Plant Mini Kit according to the manufacturer’s instructions

(Qiagen, Valencia, CA). The reverse transcription was done as follows: 2 µg of total

RNA, 250 ng of random primers, 0.5 mM of each dNTPs, 1x first Strand Buffer, 5 mM

DTT, and 100 units of Superscript III RT enzyme (Invitrogen, Grand Island, NY). PCR conditions were as described in Collins-Silva et al. (2012). The synthesized cDNA was diluted fifty times and 7.25 µl was used as template in the following: 1x TkCPT

Quantiprobe, 1x TkCPT primer pair, 1x elongation initiation factor 4a (EIF4a)

Quantiprobe, 1x EIF4a primer pair, 1x QuantiTech Multiplex PCR Master Mix (Qiagen),

0.25 units of uracil-N—glycosylase (Eurogentec, San Diego, CA), in a total reaction volume of 25 µl. Primers and probes were designed using the Qiagen QuantiTech

Custom Assay design. The probes/ primer sequences used as follows: TkCPT

Quantiprobe labeled with FAM 5ʹ- TGTTGCTTTGAGTATCTTCTGTTGCCATCCAT-

3ʹ, sense primer 5ʹ-TTTCTGTATCCCAAATACCAAATCAC-3ʹ, antisense primer 5ʹ-

GCCCTGCCCCATCGA-3ʹ; EIF4a Quantiprobe labeled with Yakima yellow 5ʹ-

ACCAACGCATCCTCTC-3ʹ, sense primer 5ʹ-GCCCAACAAATCGAAAAAG-3ʹ, antisense primer 5ʹ-TGATGTAGTCAGCGCGAAGT-3ʹ. ABI PRISM 7000 sequence detection system (Applied Biosystems) was used and reactions were carried out as previously performed (Collins-Silva et al. 2012). EIF4a was used as an internal control and the gene expression levels were normalized against it. Relative abundance and standard deviation were calculated using the 2-Δ ΔCT method (Livak et al. 2001).

Protein extraction from Taraxacum kok-saghyz plants and western blot analyses

Approximately 1 g of root tissue ground in liquid nitrogen was used to extract proteins using a modified phenol extraction method (Hurkman et al. 1986) and as described in Collins-Silva et al. (2012). The purified proteins were resuspended in Bio

Rad ReadyPrep Sequential Extraction Kit Reagent 3 (Bio-Rad, Hercules, CA) and quantitated using the EZQ Protein quantification kit (Molecular Probes Inc., Grand

Island, NY, Eugene, OR).

Purified proteins were denatured by heating for 5 min at 100 oC and proteins were fractionated by 12 % SDS-PAGE and transferred to a nitrocellulose membrane

64 according to manufactures instruction (Bio-Rad, Hercules, CA). Membranes were stained with SYPRO® Ruby Protein blot stain (Molecular Probe, Inc). Affinity purified

(using GST tag) Hevea CPT antibody (Cocalico Biologicals, Inc TM, Reamstown, PA) was used at 1:200 dilution as primary antibody and a peroxidase-conjugated Affinitypure

Goat Anti-Mouse IgG (Jackson Immunoresearch, West Grove, PA) was used at 1:10000 dilution as secondary antibody. ECL Plus kit was used to detect the proteins by chemiluminescence, according to manufacturer’s instructions (Thermo Fisher Scientific,

Rockford, IL).

Statistical analyses

Paired t-tests were done with two-tailed distribution using the Microsoft Excel student’s t-Test function.

RESULTS AND DISCUSSION

Analysis of TkCPT temporal gene expression profile and rubber accumulation in

Taraxacum koksaghyz

Experiments were performed to evaluate the correlation between the TkCPT transcript level and the amount of rubber accumulation in developing T. koksaghyz root tissue (Figure 2). To evaluate the TkCPT relative, steady-state transcript abundant, a real- time, quantitative RT-PCR approach was taken. Greenhouse grown wild type T. koksaghyz plants were collected several times during a 6.4 month period and the TkCPT transcript abundance was measured (Figure 2A). Relative TkCPT expression increased from 1 to 1.8 fold during first 2.1 months and thereafter, expression changed sporadically between 0.29 and 0.68 fold until the end of collections at 6.4 months. T. koksaghyz

65 plants showed increased amount of TkCPT transcript abundance during their early developmental stages.

To compare the TkCPT transcript levels to the rubber production, dandelion root tissue was harvested during the same time period and the rubber was extracted using ASE and quantitated using HPLC. The rubber was undetectable at 1.3 months, but increased gradually overtime (Figure 2B). The highest rate of rubber accumulation occurred from

2.1 to 2.6 months correlating to the high TkCPT transcript abundance occurred at 2.1 months (Figure 2A). Additionally, the rate of rubber accumulation decreased corresponding to the reduced TkCPT transcript levels after 2.1 months. The TkCPT transcript levels analyzed here represent the total root TkCPT. According to the Schmidt et al. (2010), there are 3 different TkCPTs (TkCPT1-3) (Schmidt et al. 2010). Although, at the time of the experiments, only one TkCPT (TkCPT2) was known and used to dedign

RT-PCR primers, they were able to identify the expression of all three CPTs due their high nt sequence identity. Schmidt et al. (2010), showed TkCPT1 and 3 were highly expressed during 4 -6 weeks and the highest expression of TkCPT2 was at 12 weeks.

But, TkCPT1 and TkCPT3 were expressed much higher than the TkCPT2. When considered the relative transcript abundance of all three CPTs, the data agree with the results found here. Overall, the TkCPT gene expression correlates with the temporal patterns of natural rubber production in T. koksaghyz root tissue.

The suppression of TkCPT2 expression in T. koksaghyz plants

To evaluate the role of TkCPT2, transgenic T. koksaghyz plants were generated using an RNAi approach. Dandelion leaf discs were transformed with Agrobacterium tumefaciens carrying a TkCPT2 RNAi vector (Figure 1). Positive transformants were

66 identified by antibiotic selection throughout the tissue culture process and by PCR of genomic DNA. Rubber content, and molecular weight (Figure 3), TkCPT gene expression and protein levels (Figure 4) were measured in mature transgenic dandelion plants. A total of fifteen RNAi lines were compared to four vector controls (controls) and four wild type plants (Figure 3).

Two of the RNAi transgenic lines had significantly lower amount of rubber in the root tissue (Figure 3A). Rubber content of RNAi 17a was 1.26 mg rubber/g dry weight, which was significantly lower than both the mean vector control (1.87 mg rubber/g dry weight) and the mean WT controls (2.52 mg rubber/g dry weight; p values <0.05). The

RNAi 31a had the lowest amount, 10-fold less, of rubber content among all the plants of

0.14 mg rubber /g dry weight (p value <0.0005). Four RNAi lines had lower molecular weight (Mw) rubber of 875 kDa, 827 kDa, 831 kDa, and 820 kDa with a p values of

<0.05. Transgenic RNAi31a line had significantly reduced Mw of 353 kDa with a p values of <0.005 whereas vector control molecular weights were near or higher than 1000 kDa (Figure 3B).

To determine if relative amounts of CPT gene expression correlated with the observed changes in rubber quality and quantity, TkCPT transcript and protein levels were measured in RNAi and control plants. The TkCPT transcript levels determined by qRT-PCR were sporadic among the transgenic lines (Figure 4A). The amount of rubber and molecular weight decreased in order; RNAi 7a > RNAi 17a > and 31a. The relative abundance of TkCPT also had the same pattern in RNAi 7a and RNAi 17a, however, the lowest rubber amount and the molecular weight occurred in line RNAi 31a, which had the highest relative TkCPT expression (Figure 4A). Western blot analysis using affinity

67 purified anti-Hevea- HRT1 CPT antibodies was also performed to determine CPT levels on selected plants (Figure 4B). The results showed significantly lower amount of TkCPT proteins in RNAi plants compared to the controls and wild type plants. The amount of proteins in RNAi lines were correlated to the TkCPT transcript levels in that RNAi 7a and

RNAi 17a were considerably lower than RNAi 31a. The transgenic plants used in the experiments were original RNAi plants. Because of the limited amount of available tissue, proteins were purified from whole root tissue, not specific tissue for rubber production such as laticifers. Therefore, the data presented in the western blot analysis

(Figure 4B) were actually representative of the TkCPT of the whole root tissue. An analysis of latex where rubber is produced actually would have been a better representation of the TkCPT expression correlating to rubber production.

The results found here showed that suppression of TkCPT had significant influence on dandelion rubber content and molecular weight as evidence by RNAi line

17a and 31a. Interestingly, the RNAi 7a line which had TkCPT expressions similar to controls also contained very similar rubber amounts and molecular weights to vector controls and wild type control plants (Figure 3 and 4). CPT is involved in many biosynthetic pathways responsible for producing numerous compounds. The CPT knock- down vectors consisted of a cauliflower mosaic virus (CaMV) 35S promoter, which expressed the TkCPT RNAi constitutively. Moreover, TkCPT was not targeted specifically to rubber production sites, resulting an overall reduction of the CPT gene throughout the plants. Therefore, the highly suppressed CPT transgenic lines might even not have survived. Although, the rubber content and the rubber quality of several transgenic RNAi lines correlated with the gene expression and the protein levels, for the

68 most part they were inconsistent. Further analyses of the transgenic lines need to be done to verify the transcript and protein amounts. Clones of those RNAi lines should be made and analyze for their transgene stability. Several clones of each original transgenic line should be pooled to purify washed rubber particles in order to perform [14C] IPP incorporation, which is directly related to the activity of the rubber particles.

Additionally, protein amounts should be measured and compared to CPT transcript expression. Specific qRT-PCR primers should be made so individual TkCPT gene expression can be measured.

Similar to transgenic plants with lower rubber and quality found here, Post et al.

(2012) also found CPT RNAi transgenic lines with significantly lower amount of rubber and lesser quality. The three RNAi lines generated contained a laticifer specific promoter and about 25%, 38%, and 84% less rubber content and significantly lower quality rubber compared to the wild type controls (Post et al. 2012). They have found that all three

RNAi lines had significantly lower [14C] IPP incorporation. Hevea CPT, HRT2 but not

HRT1, was also shown to have reduced in vitro [14C] IPP incorporation rates

(Asawatreratanakul et al. 2003). The transgenic TkCPT RNAi plant analyses done here, did not include [14C] IPP incorporation, but regarding the rubber amount, rubber molecular weight, CPT transcript abundance, and CPT protein amounts were consistent with other research data. Additionally, in Chapter 2, Hevea CPTs were identified as rubber transferase by multiple methods, including photoaffinity analysis of Hevea washed rubber particles with FPP analogues. The results found in transgenic TkCPT

RNAi plants strongly indicate that CPT influences both rubber amount and the molecular

69 weight in plants and the possibility of the dandelion CPT identified here as being the bone fide rubber transferase enzyme.

ACKNOWLEDGEMENTS

This work was supported by funding from The National Science Foundation Plant

Genome Research Program (DBI-03211690), the Nevada Agricultural Experimental

Station and the NIH IDeA Network of Biomedical Research Excellence (INBRE, RR-03-

008). Also was supported by DOA-ARS-USDA grant.

REFERENCES

Archer, B. L. and B. G. Audley (1987). "New Aspects of rubber biosynthesis." Bot. J. Linn. Soc. 94: 181 - 196. Asawatreratanakul, K., Y. W. Zhang, D. Wititsuwannakul, R. Wititsuwannakul, S. Takahashi, A. Rattanapittayaporn and T. Koyama (2003). "Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis." Eur J Biochem 270: 4671-4680. Bushman, B. S., A. A. Scholte, K. Cornish, D. J. Scott, J. L. Brichta, J. C. Vederas, O. Ochoa, R. W. Michelmore, D. K. Shintani and S. J. Knapp (2006). "Identification and comparison of natural rubber from two Lactuca species." Phytochemistry 67: 2590-2596. Chow, K. S., K. L. Wan, M. N. Isa, A. Bahari, S. H. Tan, K. Harikrishna and H. Y. Yeang (2007). "Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex." J Exp Bot. 58: 2429-2440. Epub 2007 Jun 2421. Collins-Silva, J., A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish and D. Shintani (2012). "Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism." Phytochemistry 79: 46-56. Dennis, M. S. and D. R. Light (1989). "Rubber Elongation Factor from Hevea brasiliensis." Journal of Biological Chemistry 264: 18608-18617. Hallahan, D. L. and N. M. Keiper-Hrynko (2004). Cis-prenyltransferases from the Rubber-Producing Plants Russian Dandelion (Taraxacum Kok-saghyz) and Sunflower (Helianthus Annus) US Patent 2004/044173. Hurkman, W. J. and C. K. Tanaka (1986). "Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis." Plant Physiology 81: 802-806. Karimi, M., B. De Meyer and P. Hilson (2005). "Modular cloning in plant cells." Trends in plant science 10: 103-105. Karimi, M., D. Inzé and A. Depicker (2002). "GATEWAY™ vectors for Agrobacterium- mediated plant transformation." Trends in plant science 7: 193-195. Kush, A., E. Goyvaerts, M.-L. Chye and N.-H. Chua (1990). "Laticifer-specific gene expression in Hevea brasiliensis (rubber tree)." Proceedings of the National Academy of Science 87: 1787-1790. Light, D. and M. Dennis (1989). "Purification of a prenyltransferase that elongates cis- polyisoprene rubber from the latex of Hevea brasiliensis." J Biol Chem 264: 18589 - 18597. Livak, K. J. and T. D. Schmittgen (2001). "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2− ΔΔCT Method." methods 25: 402- 408. McMahan, C. (2009). Natural Rubber from Domestic Crops. Meeting Abstract, American Chemical Society (ACS) Rubber Division Meeting, Pittsburgh, PA. Mooibroek, H. and K. Cornish (2000). "Alternative sources of natural rubber." Appl Microbiol Biotechnol 53: 355-365.

Oh, S., K. Han, S. Ryu and H. Kang (2000). "Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana." J Biol Chem 275: 18482 - 18488. Post, J., N. van Deenen, J. Fricke, N. Kowalski, D. Wurbs, H. Schaller, W. Eisenreich, C. Huber, R. M. Twyman, D. Prüfer and C. S. Gronover (2012). "Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum." Plant Physiology 158: 1406-1417. Schmidt, T., A. Hillebrand, D. Wurbs, D. Wahler, M. Lenders, C. Schulze Gronover and D. Prüfer (2010). "Molecular cloning and characterization of rubber biosynthetic genes from Taraxacum koksaghyz." Plant molecular biology reporter 28: 277-284. Schmidt, T., M. Lenders, A. Hillebrand, N. van Deenen, O. Munt, R. Reichelt, W. Eisenreich, R. Fischer, D. Prufer and C. Gronover (2010). "Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz." BMC Biochemistry 11: 11. Takahashi, S., H. J. Lee, S. Yamashita and T. Koyama (2012). "Characterization of cis- prenyltransferases from the rubber producing plant Hevea brasiliensis heterologously expressed in yeast and plant cells." Plant Biotechnology 29: 411- 417. Tanaka, Y. (2001). "Structural Characterization of Natural Polyisoprenes: Solve the Mystery of Natural Rubber Based on Structural Study." Rubber Chemistry and Technology 74: 355-375. USDA (2012). Outlook for U.S. Agricultural Trade. Whaley, W. G. and J. S. Bowen (1947). "Russian Dandelion (Kok-saghyz): An emergency source of natural rubber." USDA Micellaneous Publication 618: 1- 212.

FIGURES

Figure 1. The micro-propagation of dandelion plants. (A) Surface sterilized dandelion leaves that were cut into small explants. (B) and (C) Small explants growing in shooting medium. (D) Dandelion plants growing in Shoot elongation medium. (E) Dandelion plants growing in rooting medium. (F) Small dandelion plants growing in small pots. (G) and (H) Dandelion plants growing in greenhouse (Collins-Silva et al. 2012).

Figure 2. The CPT gene expression and the rubber production in developing dandelion roots. (A) A real time-PCR analysis of TkCPT expression during root development. (B) the amount of rubber in developing Taraxacum koksaghyz roots. Rubber was extracted from roots harvested between 1.3 and 6.4 months. Error bars represent the standard deviation of the mean of at least three biological replicates.

Figure 3. The analysis of TkCPT RNAi transgenic dandelion plants. (A) The amount of rubber in dandelion plants measured by ASE followed by HPLC. (B) The molecular weight of rubber in transgenic and control dandelion plants. *Student t-Test significance at p < 0.05, ** p < 0.005, and *** p < 0.0005 relative to the vector control. Colors Red, Blue, and Orange represent RNAi, Vector controls, and WT controls, respectively.

Figure 4. TkCPT transcript abundance and protein abundance in transgenic dandelion plants. (A) TkCPT transcript abundance. A qRT-PCR reaction was done using EIF 4a as the internal control. (B) Western blot analysis done with CPT antibodies. Proteins were fractionated by 12 % SDS-PAGE and transferred to a nitrocellulose membrane.

Chapter IV

Ericameria nauseosa (Rabbitbrush): A potential candidate for renewable source of

natural rubber

ABSTRACT

Ericameria nauseosa (Rabbitbrush) is able to produce more than 6% rubber by dry weight in its shoot tissue. Rabbitbrush is an ideal crop due to its ability of thriving in arid environments such as alkaline lands and drought conditions. The goal of this project is to investigate the potential of Rabbitbrush as a new domestic commercial source of natural rubber. In this study Rabbitbrush plants from various Nevada and California regions were surveyed for their rubber content and quality. Further studies were conducted in chosen locations on years 2006 and 2007. In 2006, shoots diameter < 3 mm were collected from Eagle Valley (EV), Gerlach (G), Gerlach Playa (GP), and Selenite

Range (SR) from June through November. In 2007, whole plants were collected from

EV, G, GP, and SR from May through October. The samples from both years were analyzed for their rubber accumulation and the quality. Rubber was extracted with hexane using Accelerated Solvent Extractor (ASE). The amount of rubber and the molecular weight was determined by HPLC-GPC. The results showed that there is a pattern between rubber accumulation and the time of the year. The highest amount of rubber was found in plants collected from EV on September. Furthermore, the wide range of locations of the plants collected, the quality and the quantity of the rubber, point toward a potential of developing wild Rabbitbrush as a natural rubber crop.

INTRODUCTION

Ericameria nauseosa (Rabbitbrush) has the remarkable ability to produce more than 6 % rubber in the bark tissue making it a potential crop for natural rubber (Hall et al.

1919; Ostler et al. 1984; Yeang et al. 1995). Rabbitbrush can be grown on marginal, alkaline lands without irrigation under drought conditions, currently considered unusable for agricultural uses, making it an ideal crop for arid environments (Ostler et al. 1984).

For example, Rabbitbrush grows in areas where temperature ranges from -20 °F to 110 °F with less than 2 inches of annual precipitation (Doten 1942). Furthermore, Rabbitbrush is endemic to western North America and is represented by 16 species (McArthur et al.

1987).

More than 2,500 plants species have identified as natural rubber (NR) producers, however, Hevea brasiliensis (Brazilian rubber tree) is the only commercial source of NR in the world. At present, NR is used in the manufacture of over 40,000 products, including tires, surgical gloves, more than 400 medical devices, numerous engineering and consumer products (Cornish 2001; Cornish 2001; van Beilen et al. 2007; van Beilen et al. 2007). More than 90% of the Hevea plantations are located in South and Southeast

Asian countries, particularly Thailand, Indonesia, and Malaysia. In 2011, the US spent

$4.4 billion to import NR while, it is forecasted to increase to $4.5 and $4.8 billion in years 2012 and 2013, respectively (USDA 2012). Rabbitbrush was considered as an emergency source of natural rubber during World War II with wild Rabbitbrush stands estimated to have the potential to produce more than 300 million tons of rubber (Doten

1942). Early Rabbitbrush seed collections, and exact locations were unavailable for

78 further studying, therefore, a survey of wild Rabbitbrush populations was needed in order to identify superior quality high rubber producing Rabbitbrush stands.

In this study, wild Rabbitbrush stands were collected from various regions of

Nevada and California. Eagle Valley (EV), Gerlach (G), Gerlach Playa (GP), and

Selenite Range (SR) were chosen for further analysis. Seasonal variation of rubber accumulation and rubber molecular weight was measured from plants collected in 2006.

Whole plants were collected and rubber was analyzed in different diameter shoot samples in 2007. The amount of rubber and molecular weight changes were described throughout

2006 and 2007 summer and fall months.

MATERIALS AND METHODS

Rabbitbrush plant material

The collected Rabbitbrush samples were stored and transported in paper bags and stored in room temperature until use.

Soil pH measurements

Soil was collected from fourteen different collection sites of the survey.

Collected soil was resuspended in water and pH was measured with Accumet AP61 pH meter according to the manufacture (Fisher scientific, Pittsburgh, PH).

Rubber extraction

Rabbitbrush branches from different diameters were finely ground using a ball mill (Retsch mill MM301, Retsch, Inc. Newtown, PA) at 4 °C. Ground tissue was lyophilized and all extractions were done in triplicates. Dionex Accelerated Solvent

Extractor (ASE) model 200 (Sunnyvale, CA) was used to extract resin and rubber

79 sequentially, as described in Collins et al. (2012), with modifications. Resin from

Rabbitbrush samples was extracted before extracting rubber. Resin extraction was done using the following method: an 11 ml ASE cell containing 0.3 g tissue was filled with acetone solvent for 1 min, heated and pressurized the cell to 40 °C and 1500 psi for 5 min, 3 cycles of: 15 min static extractions with 150 % flushing, and a final purge with nitrogen gas. The collected acetone was dried under N2 and stored in -20 °C. Rubber extraction was done using the following method: ASE cell was filled with n-hexane with

2.5 % ethanol for 1 min, heated and pressurized the cell to 140 °C and 1500 psi for 5 min,

5 cycles of: 20 min static extractions with 150 % flushing, and a final purge with nitrogen gas. The collected hexane/ethanol extract was dried under N2 and stored in -20 °C.

Dried samples were resuspended overnight in 2 ml of Tetrahydrofuran (THF) and analyzed by HPLC-GPC system. The HPLC system (HP 1100 HPLC, Agilent

Technologies, Palo Alto, CA) contained one Phenogel guard column (50 x 7.80 mm) and two Phenogel 10 µlinear columns (300 x7.80 mm) (Phenomenex, Torrance, CA). The columns were connected in series with a Sedex Light Scattering Detector, Model 75

(SEDERE, France). Mobile phase was 100% Toluene with a 1 ml/min flow rate at 70 °C.

Data was analyzed using ChemStation software (LC Rev. A. 10.02, 1757, Agilent technologies, Santa Clara, CA). Molecular weight of the rubber was calculated by a standard curve made using polystyrene standards (Polymer Laboratories, UK) with peak molecular weight ranging from 1,480 to 3,114,000 Da. The extracted rubber was quantified by an external standard curve made with serial dilutions of known amount of synthetic polyisoprene (Kraton Polymers IR-401, Houston, TX).

Statistical analyses

Paired t-tests were performed with two-tailed distribution using the Microsoft

Excel student’s t-test function. One-way ANOVA and Dunnet’s tests on JMP version 9 were performed to determine the significance of the amount of rubber and time.

RESULTS AND DISCUSSION

The amount and the molecular weight of Rabbitbrush collected from Nevada and

California

In order to find Rabbitbrush populations that contain high rubber yield with good quality rubber, wild Rabbitbrush samples were collected from fourteen different regions of Nevada and California (Figure 1). Rubber was extracted with hexane from the shoot tissue of < 3 mm in diameter using an Accelerated Solvent Extractor (ASE). The amount of rubber and the molecular weight was determined by HPLC-GPC. The rubber concentration of Rabbitbrush seemed to be higher under stress conditions including slope, elevation, soil electrical conductivity (Ostler et al. 1984). Therefore, as an additional measure, pH of the soil was also measured from each Rabbitbrush collection site. The lowest and the highest amount of rubber were found in samples collected from Southeast

Reno and Sand mountain, which contained 0.4 and 15.7 mg rubber/ g dry weight, respectively (Figure 1A). The second highest, 13.5 mg rubber/ g dry weight was found in

Eagle Valley Sand Dunes (EV), while the rest of the collection sites contained less than

5.8 mg rubber/ g dry weight. The lowest and the highest rubber molecular weight was found in samples collected from Ione and Southeast Reno (1.21 kDa and 50.82 kDa, respectively) (Figure 1B). The rest of the collection sites contained a molecular weight

81 of the extracted rubber between 24.90 and 50.82 kDa. The soil pH of each site ranged from 5.2 to 8.3 and the pH did not seem to correlate with either amount of rubber or the molecular weight. The samples collected were small branches with < 3 mm in diameter and were not expected to yield 60 mg rubber/ g dwt (6 %) as literature sited (Hall et al.

1919; Ostler et al. 1984; Yeang et al. 1995) due to its size. However the stem diameter should be an indicator of the total rubber production in the plants. The molecular weights of the samples collected were 20-fold or more, less than what considered as high quality rubber, which is about 1000 kDa. The molecular weight might be correlated to the branch diameter.

Seasonal patterns of rubber production in young Rabbitbrush tissue collected in

2006

In order to identify the seasonal patterns of rubber production, young Rabbitbrush tissue, < 3 mm in diameter, were collected from four different regions in Nevada from

June to November in 2006. The locations were decided using the data from the previous survey conducted at University of Nevada, Reno (UNR) (Figure 1), published data, and the proximity to the UNR (Ostler et al. 1984; Hegerhorst et al. 1987; Bhat et al. 1990).

EV region was chosen because the plants collected from there had the second highest amount of rubber (13.5 mg rubber/ g), the second highest molecular weight (31.15 kDa), and the close proximity to Reno, Nevada (Figure 1). Previous studies done with

Rabbitbrush grown in alkali flats near Gerlach, Nevada showed that the rubber contents ranged from 4.71% to 6.57 % (Hall et al. 1919; Yeang et al. 1995) . Even though,

Gerlach region was not part of the preliminary study, Gerlach, and in close proximity to

Gerlach, The Gerlach Playa and Selenite Range were selected for 2006 studies. Young

82 tissue was collected from the same plant approximately every two weeks for approximately 5 months. There were 7 plants from EV, 10 plants from G, 4 plants from

GP, and 7 plants from SR were collected. The mean amounts of rubber and molecular weights from each location are shown in Figure 2, and the individual plant rubber amounts and the molecular weights are shown in supplemental Figure 1 and 2.

The highest mean amount of rubber on each collection date was always found in tissue collected from EV except on the last collection date, November 9th (Figure 2A).

The lowest mean amount of rubber on each collection date was always found in tissue collected from SR except on the July 28th. The elevation of the EV collection site is about 1250 m compared to the SR collection site elevation of about 2000 m. The soil of

EV collection site had more of a dry sand consistency compared to the relatively moist clay consistency in SR. Rabbitbrush rubber production was shown to be positively influenced by the plant age, increase soil moisture and high air temperature, while it was uncertain with regard to elevation (Hall et al. 1919; Ostler et al. 1984; Adams et al.

1987). The high rubber amount in EV plants might be due to lower amount of soil moisture and elevation. The EV samples collected on September 7th, 23rd, and October

23rd and G samples collected on September 7th, October 23rd, and November 9th and GP samples collected from September 23rd were significantly higher than the first collection date of each region. The amount of rubber was increased from June to November and peaked around mid-September. The highest mean and the highest individual amount of rubber was found on September 7th in EV region of 15.97 and 28.12 mg/g dry weight respectively (Figure 2A and Supplemental Figure 1A). If the amount of rubber in young

Rabbitbrush shoots correlates with the total amount of rubber in the whole plants harvesting should be done during September and October.

On the other hand, a favorable mean molecular weight was not specific to any region (Figure 2B). The highest mean molecular weight was found in the first two collection dates in June 22nd and July 5th in EV and G. Unfortunately, Rabbitbrush samples from GP and SR were not collected until July 28th and 21st, respectively. The highest and lowest mean molecular weights were found in G site and were 43.7 kDa and

18.34 kDa, respectively. Gerlach also had the highest molecular weight individual plant with 72.77 kDa (Supplemental Figure 1D). EV had a high mean molecular weight of

41.58 kDa, which was very similar to the mean of G (Figure 2B). Both GP and SR plant populations had molecular weight between 20 to 50 kDa (Figure 2B and Supplemental

Figure 2). Similar to preliminary survey collections, Figure 1, the molecular weight was considerably lower than high quality rubber of 1,00kDa, presumably due to the small ste diameter of the collected samples.

The amount and the molecular weight of whole Rabbitbrush plants collected in 2006

In order to determine the localization of highest rubber amount and molecular weight, whole Rabbitbrush plants were collected from EV, G, GP, and SR sites. Each plant was fractionated to three groups; root (> 12 mm), lower shoots (>12 mm), and upper shoots (< 6 mm) in diameter (Figure 3). The rubber amounts were not consistent throughout the collection sites. In EV, roots had the highest mean rubber amount of 2.92 mg/g dry weight, whereas the highest amounts of rubber in G, GP, and SR were found in lower shoots, lower shoots, and roots, respectively (Figure 3A). Lower shoots on GP had

84 by far the highest amount of 19.3 mg/g dry weight rubber, which was more than three- fold higher than the second highest amount of GP upper shoots.

Unlike the amount of rubber, the molecular weight of rubber from whole plants were consistent in that roots, lower shoots, and upper shoots had decreasing molecular weights, respectively, except the lower shoots of GP (Figure 3B). Roots from all the areas had molecular weight ranging from 73 kDa to 132 kDa,whereas upper shoots had the lowest molecular weights ranging from 24 kDa to 43 kDa. GP lower shoots had the top molecular weight of 264 kDa.

The whole plants were collected during the early collection dates in 2006 from each site. Considering the upper shoots in whole plants were about 6 mm in diameter, which was twice the shoot diameter compared to the shoots collected for the seasonal variation study (Figure 2), the rubber amounts and the molecular weights were comparable (Figures 2 and 3) in each site. Even though GP shoots had higher rubber amount and molecular weight the Rabbitbrush population was small compared to the other sites. Therefore, EV was chosen for the seasonal whole plant study in 2007.

Seasonal patterns of rubber production in whole Rabbitbrush plants collected from

EV in 2007

In order to determine the plant fraction and the time of the year, where highest amount and molecular weight of rubber was produced, whole plants from EV were collected from May through October, 2007 (Figure 4). Three-four whole plants (only shoots) were collected on each collection day and fractionated into five groups; 1.5 mm,

3 mm, 6 mm, 12 mm, and 20 mm. Previous Rabbitbrush analyses showed that older stems which were closer to the ground level to contain more rubber (Hall et al. 1919;

Hegerhorst et al. 1987; Bhat et al. 1989; Bhat et al. 1990). As expected, the amount of rubber increased from small to bigger diameter fractions. Each fraction had similar pattern of amount of rubber over time. For example, all the fractions had a spike in amount of rubber on June 6th, 2007 (Figure 4A). The highest rubber amounts were found in 12 mm and 20 mm fractions on September 12th, 26th, and October 11th and was significantly higher compared to the first collection date of each fraction ( p-value <

0.05). The maximum mean amount of rubber in both 12 mm and 20 mm fractions were found on September 12th of 34.47 mg/ g and 40.76 mg/ g dry weight, respectively (Figure

4A).

In contrast to the amount of rubber, the mean molecular weights of each fraction was highest at the beginning of the season in most cases, where 6 mm fractions had the highest of 108.4 kDa on June 6th (Figure 4B). The only molecular weight data that was significantly higher than the first collection date was found in 3 mm fractions on July 5th

(p-value < 0.05). The molecular weights of the shoot diameter 6, 12, and 20 mm fractions were consistently higher than the smaller shoots. On this study even shoot diameters as high as 20 mm did not have high quality rubber of 1,000kDa at any time point. According to the amount of rubber whole plants should be harvested during mid-

September although the molecular weight would not be at its highest.

CONCLUSIONS

The studies conducted either using small shoot tissue from four different sites

(EV, G, GP, and SR) or the fractionated whole plants from EV showed a pattern of increasing amount of rubber with time, which peaks in September in both 2006 and 2007.

The highest amount (60.45 mg/ g dry weight) of rubber was found in an EV individual 20 mm fraction on September 12th. The highest rubber molecular weight of 211 kDa was found in an EV individual 6 mm fraction on July 5th. However, GP Lower shoots (>12 mm) had the top molecular weight of 264 kDa in one individual plant, but GP has a very small Rabbitbrush population. On average 20 mm Rabbitbrush fraction harvested in

September would have about 40 -50 mg rubber/ g dry weight with a molecular weight of about 50 kDa. Therefore, to identify high rubber yielding wild Rabbitbrush populations, bigger stems closer to the soil line should be harvested in September.

ACKNOWLEDGEMENTS

The authors would like to acknowledge funding support from the University of

Nevada Agricultural Experiment Station (NEV). DOE NSHE-DRI and Nevada

Renewable Energy Consortium (NVREC) grants.

REFERENCES

Adams, R. P. and S. Price (1987). "Seasonal variation in resource allocation of extractable compounds in Asclepias, Chrysothamnus and Grindelia." Biochemical Systematics and Ecology 15: 417-426. Bhat, R., D. Weber, D. Hegerhorst and E. McArthur (1990). "Rubber and resin content in natural and uniform-garden populations of Chrysothamnus nauseosus subspecies." Phyton, Buenos Aires 51: 35-42. Bhat, R. B., B. L. Welch, D. J. Weber and E. D. McArthur (1989). "Winter nutritive value of Chrysothamus nauseosus." J. of Range Manag. 43: 177-179. Collins-Silva, J., A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish and D. Shintani (2012). "Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism." Phytochemistry 79: 46-56. Cornish, K. (2001). "Biochemistry of natural rubber, a vital raw material, emphasizing biosynthetic rate, molecular weight and compartmentalization, in evolutionarily divergent plant species." Nat Prod Rep 18: 182-189. Cornish, K. (2001). "Similarities and differences in rubber biochemistry among plant species." Phytochemistry 57: 1123-1134. Doten, S. B. (1942). "Rubber from rabbit brush (Chrystothamnus nauseosus)." Univ. Nevada Agr. Expt. Sta. Bull. 157: 22. Hall, H. M. and T. H. Goodspeed (1919). "Chrysil, A New Rubber from Chrysothamnus nausuosus." University of California Publications in Botany 7: 183-264. Hall, H. M. and T. H. Goodspeed (1919). A rubber plant survey of western North America. Berkeley,, University of California press. Hegerhorst, D., D. W. Weber and E. D. McArthur (1987). "Resin and rubber content in Chrysothamnus." The Southwestern Naturalist: 475-482. McArthur, E. and S. Meyer (1987). A review of the taxonomy and distribution of Chrysothamnus. Proc. Fourth Utah Shrub Ecology Workshop. College of Natural Resources, Utah State University, Logan. Ostler, W. K., C. M. McKell and S. White (1984). Chrysothamnus nauseosus: A potential source of natural rubber. Symposium: Biology of Artemisia and Chrysothamnus, USDA Forset Service, General Technical Report INT-200. USDA (2012). Outlook for U.S. Agricultural Trade. van Beilen, J. and Y. Poirier (2007). "Establishment of new crops for the production of natural rubber." Trends Biotechnol 25: 522 - 529. van Beilen, J. B. and Y. Poirier (2007). "Guayule and Russian Dandelion as Alternative Sources of Natural Rubber." Critical Reviews in Biotechnology 27: 217-231. Yeang, H., E. Yip and S. Hamzah (1995). "Characterization of zone 1 and zone 2 rubber particles in Hevea brasiliensis latex." J Nat Rubber Res 10: 108 - 123. Yeang, H. Y., F. Yusof and L. Abdullah (1995). "Precipitation of Hevea brasiliensis Latex Proteins with Trichloracetic Acid and Phosphotungstic Acid in Preparation for the Lowry Protein Assay." Analytical Biochemistry 226: 35-43.

FIGURES

Figure 1. The amount and the molecular weight of Rabbitbrush rubber collected from various locations in Nevada and California, along with the soil pH of each location. (A) The mean amount of rubber and soil pH. (B) The mean molecular weight of rubber.

Figure 2. The amount and the molecular weight of rubber from young Rabbitbrush tissue collected on year 2006. (A) The mean amount of rubber. (B) The mean molecular weight of rubber. Rabbitbrush samples of about 3 mm in diameter were collected from Eagle Valley (EV), Gerlach (G), Gerlach Playa (GP), and Selenite Range (SR). The individual plant data for graph A and B are shown in supplemental figure 1 and 2. *Student T-test significance at p < 0.05 compared to the first collection date. n= 7 (EV), 10 (G), 4 (GP),and 7 (SR) plants. Individual plant data were shown in Supplemental figures 1 and 2.

Figure 3. The amount and the molecular weight of rubber from Rabbitbrush roots and shoots collected on year 2006. (A) The mean amount of rubber. (B) The mean molecular weight of rubber. Rabbitbrush samples of roots (> 12 mm), lower shoots (> 12 mm), and upper shoots (< 6 mm) of stated diameter were collected from Eagle Valley (EV), Gerlach (G), Gerlach Playa (GP), and Selenite Range (SR). n= 3-5 plants.

Figure 4. The amount and the molecular weight of rubber from whole Rabbitbrush samples collected on year 2007. (A) The amount of rubber. (B) The molecular weight of rubber. Rabbitbrush samples of 1.5 mm, 3 mm, 6 mm, 12 mm, and 20 mm in diameter were collected from Eagle Valley (EV). *Student T-test significance at p < 0.05 compared to the first collection date. n= 3 plants

SUPPLEMENTAL FIGURES

Supplemental Figure 1. The amount and the molecular weight of rubber from individual Rabbitbrush samples collected form EV and G on year 2006. (A) The amount of rubber on Eagle Valley (EV) samples. (B) The molecular weight of rubber on EV samples. (C) The amount of rubber on Gerlach (G) samples. (D) The molecular weight of rubber on G samples. Rabbitbrush samples of 3 mm in diameter were collected from EV and G.

Supplemental Figure 2. The amount and the molecular weight of rubber from individual Rabbitbrush samples collected from GP and SR on year 2006. (A) The amount of rubber on Gerlach Playa (GP) samples. (B) The molecular weight of rubber on GP samples. (C) The amount of rubber on Selenite Range (SR) samples. (D) The molecular weight of rubber on SR samples. Rabbitbrush samples of 3 mm in diameter were collected from GP and SR.

Chapter V

Ericameria nauseosa subsp. consimilis (Rabbitbrush): A renewable source of natural rubber, resin, and bioenergy feedstock

Upul Hathwaik1, Mohammad Yazdani1, Glenn Miller2, Curtis Robbins3, Colleen McMahan4, David Shintani1*

1Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557

2Department of Natural Resources and Environmental Sciences, University of Nevada, Reno, NV 89557

3Division of Atmospheric Sciences, Desert Research Institute, Reno, NV 89557

4Crop Improvement and Utilization Research, USDA-ARS, 800 Buchanan St, Albany, CA, 94710

*Corresponding Author,

David Shintani

Tel: 1 (775) 784-1095, Fax: 1 (775) 784-4227

Email: [email protected]

ABSTRACT

Ericameria nauseosa (Rabbitbrush) is a potential crop for natural rubber and biofuel production because it has the remarkable ability to produce more than 6% rubber,

36% of oleoresin and a large amount of lignocellulosic biomass. Rabbitbrush can be grown on marginal, alkaline lands under drought conditions, making it an ideal crop for arid environments, currently considered unusable for traditional agricultural applications.

In addition, genetic improvements should be possible considering its highly diverse genetics due to large wild populations and a diploid genome. The goal of this project is to investigate the potential of Rabbitbrush as a new domestic commercial source of natural rubber, resin and lignocellulosic biomass. In this study, Ericameria nauseosasubsp.consimilis collected from Austin, NV were analyzed. Rabbitbrush rubber was analyzed for its physical properties including molecular weight, insolubles, bulk viscosity, extractables, and thermal stability. The polymer molecular weight and some of the rubber physical properties were comparable to current commercial natural rubber producers, such as Hevea brasiliensis and Parthenium argentatum. The chemical components of the Rabbitbrush resin fraction were also examined. The evaluation of the lignocellulosic biomass showed promising characteristics in terms of hydrophobicity, abrasion resistance, and energy density similar to other biomass energy sources. Overall,

Rabbitbrush has the potential to be a renewable domestic crop for natural rubber, resin and bioenergy feedstock.

INTRODUCTION

Ericameria (Rabbitbrush) is endemic to western North America and it consist of

16 species (McArthur et al. 1987). It is a hearty desert shrub that can grow in areas where the temperature ranges from -20 °F to 110 °F with less than 2 inches of annual precipitation (Doten 1942). Rabbitbrush is of particular interest because it is a copious producer of a number of interesting and valuable chemical compounds including rubber, terpene resins and lignocellulosic biomass. As such, Rabbitbrush has the potential of being exploited as an alternative to petroleum for the production of industrial chemical feedstocks. Because Rabbitbrush can be grown on marginal without irrigation, it is an ideal industrial crop for arid lands that are currently considered unarable (Ostler et al.

1984).

Rabbitbrush has long been known as a rubber producing plant. The earliest record of rubber in Rabbitbrush was from the Great Basin Native Americans who used its bark as a type of chewing gum. Hall and Goodspeed (1919) later identified the elastic component of the bark as rubber and subsequent studies reported Rabbitbrush rubber contents ranging from 1.5 to 6.5% (Hall et al. 1919; Ostler et al. 1984; Yeang et al.

1995). Like the commercial rubber crop, Hevea brasiliensis, Rabbitbrush produces high molecular weight rubber, which can potentially be used in industrial applications (Weber and Fernandes, 1991). These findings have spurred investigations into Rabbitbrush as a domestic source for commercial rubber. In fact, during World War II, Rabbitbrush was considered an emergency source of natural rubber from which an estimated 300 million tons of rubber could be harvested from native stands (Doten 1942).

Rabbitbrush is in many ways similar to guayule, an emerging domestic source of natural rubber. Both species are desert shrubs belonging to the Asteraceae family and unlike other rubber producing plants, both produce rubber in specialized bark parenchyma cells. Furthermore, Rabbitbrush and guayule are known to synthesize rubber in response to environmental stress. However, whereas guayule rubber production is stimulated by cold temperatures, elevated temperatures are known to trigger rubber biosynthesis in Rabbitbrush (Ostler et al. 1984).

In addition to rubber, Rabbitbrush also produces high levels of energy rich compounds including various oleoresin compounds and lignocellulosic biomass.

Rabbitbrush has been reported to contain up to 36% oleoresin in shoot dry weight. Of the over 60 chemicals identified in the oleoresin fraction, the majority belonged to the terpene class of chemicals (Hegerhorst et al. 1987), from which a high quality biofuel can be produced through catalytic conversion. An estimated 12.5 barrels of crude oil equivalent can be generated from oleoresins harvested from one hectare of Rabbitbrush

(McLaughlin et al. 1982). Interestingly, the terpene resin content has been shown to be inversely proportional to the amount of rubber produced and was more responsive to environmental factors than rubber (Bhat et al. 1990). In addition to being used as a biofuel, specific Rabbitbrush terpene compounds have been reported to have medicinal properties. For example, chrysothol, a compound found in a closely related species,

Chrysothamnus viscidiflorus, has been shown to have anti-cancer activity against human breast cancer cells (Ahmed et al. 2006). Rabbitbrush also produces high amounts of lignocellulosic biomass with estimated yields ranging from 5 (McLaughlin et al. 1982) to

76 (Gordon et al. 1982) metric tons per hectare.

In this study, Ericameria nauseosa subsp. consimilis was analyzed for its potential as a domestic natural rubber, resin, and biomass crop. The molecular and physical properties of its natural rubber were analyzed and compared to other commercial rubber sources. The extracted Rabbitbrush resin fraction was analyzed for its chemical components and the remaining residue, after extracting rubber and resin, was analyzed for its biomass energy and evaluate the potential in biomass pellet industry.

MATERIALS AND METHODS

Rabbitbrush plant material

Wild Ericameria nauseosaplants collected from Austin, Nevada (Latitude: 39-

53'59'' N and Longitude: 116-35'08'' W) were transported to University of Nevada Reno for further experiments. Whole plants collected were stored with ice during harvesting and transported on the same day.

Rubber extraction from different branch diameters

Rabbitbrush branches from different plants were cut into five different diameters of 3, 6, 12, 20, and 25 mm. Each sample was finely ground using a ball mill (Retsch mill

MM301, Retsch, Inc. Newtown, PA) at 4 °C. Dionex Accelerated Solvent Extractor

(ASE) model 200 (Sunnyvale, CA) was used to extract resin and rubber as described in

Collins et al. (2012), with modifications. Lyophilized shoots from each of the five different fractions mentioned above was extracted in triplicate. Resin extraction was done as the following method: 11 ml ASE cell containing 0.3 g of plant tissue was filled with acetone solvent for 1 min, heated and pressurized the cell to 40 °C and 1500 psi for

5 min, 3 cycles of: 15 min static extractions with 150% flushing, and a final purge with

nitrogen gas. The collected acetone was dried under N2 and stored in -20 °C. Rubber extraction was done as the following method: ASE cell was filled with n-hexane with 2.5

% ethanol for 1 min, heated and pressurized the cell to 140 °C and 1500 psi for 5 min, 5 cycles of: 20 min static extractions with 150 % flushing, and a final purge with nitrogen gas. The collected hexane/ethanol extract was dried under N2 and stored in -20 °C.

Extracted rubber was measured gravimetrically.

Purification of Rabbitbrush Washed Rubber Particles and particle size determination

Enzymatically active washed rubber particles were purified from Rabbitbrush as described previously with some modifications (Cornish et al. 1990; Cornish et al. 1995).

Rabbitbrush bark was blend with extraction buffer: 100 mM Tris–HCl, 50 mM KF, 5 mM

MgSO4, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 1% ascorbic acid, 5 mM 2- mercaptoethanol, 0.05 mL/ml antifoam A emulsion, 20 % glycerol, and 0.07 g/ml polyvinylpolypyrrolidone (PVPP). Homogenate was filtered using cheese cloth and centrifuged in an increasing speed from 1000 x g to 7000 x g at 4 C, while collecting floated rubber particles after each spin. Collected rubber particles were washed in wash buffer: 100 mM Tris-HCI (pH 7.5), 5 mM Dithiothritol (DTT) and 0.1 mM 4-(2-

Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). As before second set of centrifugations were done in an increasing speed from 1000 x g to 7000 x g at 4 C, to collect the washed rubber particles to new wash buffer. Collected washed rubber particles were stabilized with 10% glycerol, frozen drop-wise into liquid nitrogen and stored in -80

C.

100

Latex particle size distribution was determined using Horiba LA-900 Laser Light

Scattering Particle Size Distribution Analyzer (HORIBA Scientific, Edison NJ) according to manufacturer’s instructions.

Purification of Rabbitbrush Washed Rubber Particles proteins

Total proteins from Rabbitbrush WRPs were extracted using extraction buffer; 7

M Urea, 2 M thiourea, and 2% ASB-14. Four volumes of extraction buffer was added to

Rabbitbrush WRPs and mixed for 1 h in room temperature. Rubber was coagulated-out from the solution by centrifuging at 16,000 x g at room temperature for 15 min. The supernatant was filtered with an Acrodisc® 25 mm Syringe filter with a 0.45 μm Supor®

Membrane (PALL Life Sciences, East Hills, NY). If needed, extracted proteins were concentrated by precipitating with four volumes of 100% cold acetone, followed by 2 washes with four volumes of 80% cold acetone, dried under nitrogen, and resuspended with the extraction buffer. The extracted proteins were quantified using EZQ® Protein

Quantitation Kit (Molecular Probes Inc., Grand Island, NY) according to the manufacture’s instruction.

Nuclear Magnetic Resonance (NMR) of Rabbit brush rubber

The rubber purified from WRPs were dissolved in 0.6 ml of dichloromethane and analyzed in a 400 MHz instrument. A cis-1,4-polyisoprene standard was also dissolved in 0.6 ml of dichloromethane and compared with Rabbitbrush rubber.

Rubber and resin extraction using a commercial Soxhlet extractor

Whole Rabbitbrush plants that were kept in cold room were ground with a commercial grade wood chipper (CH3 11HP, GXI international Clayton, NC). The partially ground plant material were further ground using a Hammer mill (AT Ferrell

101

Company Inc. Bluffton, IN). A commercial Soxhlet extractor (Eden Labs LLC,

Columbus, OH) was used to extract resin and rubber from 3 kg of finely ground plant material. Resin and low molecular weight compounds were extracted with 20 L of acetone at 70 °C temperature for 4 h. Extracted resin in acetone was filtered and concentrated using a BUCHI Rotavapor R-220 (BUCHI Corporation, New Castle, DE) rotary evaporator until all the volatiles were evaporated according to manufacturer’s instruction. Extracted resin was weighed and the volume was measured for future calculations. Sample was stored at room temperature until further analysis.

Rubber was also extracted using 3 kg of finely ground material with 25 L of pentane/acetone azeotrope (79: 21) by weight (Beinor et al. 1986).The sample was extracted at 50 °C temperature for 4 h. After extraction, azeotrope was collected and the total volume was measured. Azeotrope was concentrated using a Rotary Evaporator according to manufacturer’s instruction. An antioxidant was added and Rubber was precipitated using 1 volume of acetone (Schloman Jr 2005). After mixing for about 1 min the rubber precipitate was collected, flattened, and dried in a fume hood overnight.

Partially dried rubber was cut into small pieces and further dried for one more day and the weight was measured. Dried samples were stored at 4 °C until further analysis.

Rubber molecular weight determination

The molecular weight and their distributions were determined according to previously published methods (McMahan 2009). Briefly, extracted rubber was dissolved overnight in Tetrahydrofuran (THF). The solubilized rubber was filtered and injected into

2 PLgel MIXED B size-exclusion columns in series on a Hewlett-Packard 1100 series

HPLC, coupled to Dawn Heleos-II multiple angle light scattering (Wyatt Technologies,

102

Santa Barbara CA) and HP differential refractive index (RID) detectors. Molecular weights were determined from both chromatograms using ASTRA V4 software.

Determination of rubber gel

The gel content of extracted rubber was determined according to ASTM D3616.

About 0.4 g of rubber sample was cut into small pieces and dissolved in 100 ml of toluene for 20 h in the dark. The rubber that dissolved in toluene was measured by drying the toluene fraction. The rubber that did not dissolved was calculated as rubber gel.

Bulk Viscosity determined by Advanced Polymer Analyzer

The Advanced Polymer Analyzer 2000 (APA 2000, Alpha Technologies, Akron,

Ohio) measures dynamic mechanical properties of polymers as a function of temperature, frequency, and strain. The ASTM D 6204B was used to determine the bulk viscosity.

The test specimen was formed in situ by closing of two heated pressured dies at sufficient temperature to mold the test piece. Sheets of nylon were placed above and below the test specimen. One die then oscillates in shear while a torque transducer on the other die measures the resultant force.

Acetone extraction of rubber (the percent extractables)

About 0.5 g of rubber was extracted with acetone using a Accelerated Solvent

Extractor (ASE Dionex Corp., Sunnyvale, CA). Rubber extraction was done as the following method: ASE cell was filled with acetone for 1 min, pressurized to 1500 psi for

5 min, 3 cycles of: 20 min static extractions with 150% flushing, and a final purge with nitrogen gas. The collected acetone extract was dried and weighed.

103

Determination of Plasticity Retention Index (PRI)

PRI of the rubber was measured according to ASTM D3194. Three millimeter thick rubber film was cut into cylindrical pellet using a specimen cutter. Wallace instrument was used to measure the time to a given force. A comparison of room temperature pellets and pellets heated at 140 °C for 30 min were used in calculating the

PRI.

Hydrothermal Carbonization (HTC) of Rabbitbrush tissue

Ground whole Rabbitbrush plant tissue and tissue that were extracted with acetone/pentane azeotrope using Soxhlet extractor described above was used for these experiments. HTC experiments were carried out in a 2L pressurized Parr reactor system

(model 4522) developed by Desert Research Institute (DRI) (Hoekman et al. 2011). A known quantity of feedstock was added to water at a ratio of approximately 1:8. The Parr bomb was then sealed and purged with helium before heating begins (Figure 4). Electric heating coils on the outside of the bomb were used to increase the temperature inside to

255˚C as quickly as possible while maintaining a wall temperature less than 400˚C. The reactor was stirred throughout heating and the 30 min process hold time. Once the 30 min have expired, the bomb was removed from the Parr system and placed in an ice bath for rapid cooling. Upon completion of each experiment, there are three products to be analyzed: gases, liquids, and solids. During the HTC process, the high temperature and pressure produces gases. Once the bomb had cooled down, it was purged with helium to collect any produced gases. After purging, the contents of the reactor were removed; the solids and liquids were separated for analysis and drying.

104

Analysis of HTC products

The produced gases were collected to Tedlar bags (Figure 4) and analyzed by gas chromatograph (Hoekman et al. 2011). The collected liquid and HTC char was separated by vacuum filtration. The water balance and the non-volatiles in the liquids were measured as described by Hoekman et al, (2011). The produced HTC char was then placed in a laboratory oven at 104 ˚C for 24 h. The energy content of the HTC char was determined using a 6200 Isoperibol Calorimeter (Parr Instrument Company. Moline, IL) following manfacture’s instruction. Due to the composition and small particle size of the ground Rabbitbrush, it was necessary to transform the biomass into a material more suitable for transportation. Therefore, a manual, single-pellet press was used to produce pellets by using materials from with and without HTC process of Rabbitbrush tissue from before and after the rubber extraction. Two test methods, an abrasion test, and a water solubility test, were developed and performed to demonstrate the advantages of HTC pellets (Reza et al. 2012).

The abrasion test was done as follows. A small-scale abrasion test setup was first developed at the University of Nevada, Reno to simulate the ASTM E 873 standard abrasion test by tumbling 40 pellets in a modified gem tumbler with baffles. The standard requires that pellets lose less than 5% of their mass while tumbled at 50 rpm for

10 min. The developed test tumbles 40 pellets at 50 rpm for a total of 3000 revolutions, or 60 min. Because only enough HTC char was available to produce 3 pellets, the procedure was further modified such that only one pellet was used. An experimental matrix was tested using a varying number of pellets mixed with metal, plastic and wood ball bearings. Based on the results of this experiment, it was determined that wood ball

105 bearings were most representative. One pellet mixed with 39 wood ball bearings was determined to perform the same as 5 pellets with 35 wooden ball bearings.

A water solubility test was setup to display the hydrophobic properties of the

HTC pellets. Similar to abrasion test, this test does not follow any standard, but can be used to compare pellets of different feedstock. One pellet of each feedstock was placed in a vial of deionized water for 60 min. The pellets dimensions and weight are taken before and after the experiment to determine the amount of deformation. The pellets submerged in water during the test are shown in Figure 5. After the test, the pellets were removed from the vial, and dried in a laboratory oven at 80˚C for 24 h before measurements were taken.

RESULTS AND DISCUSSION

Rabbitbrush rubber particles

Rabbitbrush rubber can be harvested as a latex suspension or as bulk solid rubber.

Rabbitbrush latex is made up of a colloidal suspension of small rubber containing vesicles known as rubber particles (d'Auzac et al. 1989). Rubber particles are made up of a phospholipid monolayer surrounding a hydrophobic lipid core. Because rubber particles, like lipid bodies, are buoyant in aqueous buffers, they can be isolated as a floating pad upon centrifugation. Through a series of washes and recollections, these rubber particles can be purified from cellular contaminants. Because rubber particle sequestered rubber is protected from chemical and enzymatic degradation, analysis of this rubber will give the best representation of the native product produced in this plant species. However, when the rubber particle surface is disrupted, particles coagulate

106 forming an insoluble mass of solid rubber that is prone to oxidation induced polymer cleavage.

To determine Rabbitbrush’s native structure and polymer molecular weight, highly purified washed rubber particles (WRPs) were isolated from freshly harvested

Rabbitbrush shoot material. The rubber purified from these WRPs was analyzed by 13C nuclear magnetic resonance (NMR) at 400MHz as described in methods. The expected

135.19, 125.00, 32.20, and 26.38 ppm signals confirmed the presence of repeating cis-

1,4-carbon linkages commonly associated with polymers of natural rubber (Figure 1).

Surprisingly, NMR signals corresponding to trans carbon-carbon double bonds, consistent to the allylic pyrophosphate rubber polymer primer (Duch et al. 1970; Tanaka

2001), were not detected in the Rabbitbrush rubber. However, rubber isolated from a number of plant species including guayule have shown to lack the trans carbon-carbon double bond NMR signal (McMahan 2009). The reasons for this are not understood, but suggest a modification of the anterior end of the polymer (Tanaka 2001).

Size exclusion chromatography and multi-angle light scattering was used to determine the native molecular weight of the Rabbitbrush WRP rubber. This analysis showed that the Rabbitbrush WRPs had an average molecular weight of 800 kDa with molecular weight ranging from 60 to 2,500 kDa.

Both protein composition and rubber particle size have been shown to be species- specific for natural rubber-producing plants (Cornish et al. 1993; Siler et al. 1997).

Moreover, the protein composition and total protein content influence the immunogenic properties of the rubber. Approximately 20 million people in US suffers from life threatening Type I Hevea allergies, which include anaphylaxis to less severe allergic

107 reactions triggered by the proteins contained in Hevea WRPs (Tomazic et al. 1992;

Cornish 2001; Bousquet et al. 2006). Guayule latex have shown to be hypoallergenic due to its low protein content in WRPs resulting in an increase in demand of guayule rubber for the biomedical applications (Mooibroek et al. 2000). Rabbitbrush rubber also contains low protein content of up to 9.11 mg/g rubber in WRPs analogous to guayule

WRPs protein content of 4.30-9.04 mg/g rubber (Table 2), making it a possible candidate to produce hypoallergenic products. However, further studies need to be conducted to verify the immunogenic properties of Rabbitbrush rubber. Rabbitbrush latex particle diameter was 2 μm, slightly higher than that of Hevea, Guayule, and Dandelion (Table 2).

In Hevea, small rubber particles have higher protein content (Tarachiwin et al. 2005), enzyme activity (Dennis et al. 1989; Dennis et al. 1989; Ohya et al. 2000) and rubber molecular weight (Yeang et al. 1995; Tarachiwin et al. 2005). Further studies need to be conducted to determine the Rabbitbrush WRPs size relationship to enzyme activity and rubber molecular weight.

Deposition of rubber in Rabbitbrush shrubs

To determine the variation of the amount and the molecular weight of rubber produced in shoots, five different fractions of several Rabbitbrush shoots of each plant were analyzed as shown in Figure 2. The shoot diameter 3- 25 mm fractions had 5.4, 8.8,

33.3, 38.3, and 26.7 mg/g dry weight rubber respectively. The smallest amount of rubber was found to be in the 3 mm diameter shoot whereas the highest amount was found in the shoots with a diameter of 20 mm, but not of 25 mm (Figure 2 A). The reason 20 mm shoots to have more than 10 mg/g rubber compared to the 25 mm diameter shoots might due to the wood: bark ratio. The 25 mm diameter shoots might have more wood than

108 bark. The mean molecular weight in each shoot fraction increased with the diameter from 34 kDa to 268 kDa (Figure 2B). Even though the mean molecular weight was 268 kDa the molecular weight distribution was as high as 2000 kDa to a low of 5 kDa.

The physical properties of bulk solid Rabbitbrush rubber

The physical properties of alternative natural rubbers are important in determining its possible usages. Natural rubber was extracted from ground whole Rabbitbrush plants, using a pentane/acetone azeotrope method (Schloman Jr 2005). However, the current extraction methods need to be optimized as evident by the amounts of resin and rubber remained in the residue. About 20 mg resin/ g and 5 mg rubber/ g were not able to extract with the current extraction method (Figure 3). The extracted rubber was analyzed for its physical properties including molecular weight by Gel Permeation Chromatography

(GPC), insolubles by the percent gel test, bulk viscosity by Advanced Polymer Analyzer

(APA 2000), extractables by Accelerated Solvent Extractor (ASE), and thermal stability by Plasticity Retention Index (PRI) methods. The molecular weight of rubber is perhaps the most important factor that determines the quality of rubber. However, there are other components in Hevea rubber that contribute to its superior characteristics (Tanaka 2001).

Here we analyzed physical properties of Rabbitbrush rubber and compared them to rubber from other sources (Table 1). GPC analysis of Rabbitbrush revealed high molecular weight rubber of 995,800 Da, comparable to that of commercial Hevea (Table

1) strengthening the possibility of Rabbitbrush being an alternative rubber crop.

According to current theory, the outstanding physical properties of natural rubber are due to strain-induced crystallization on deformation, which is caused by formation of a "gel" network by polymer-protein and polymer-lipid linkages (Tanaka 2001). The extent of

109 formation of this network can be estimated by determination of insoluble gel. The percent insoluble gel, determined by ASTM D3616, of Rabbitbrush rubber was 2.75 %, significantly lower that than of other rubbers tested (Table 1). Lower gel percent suggests a lower level of protein-polymer interactions (vs. Hevea), as has also been observed for guayule. Nevertheless, a low percent gel value does not mean the performance of rubber is poor.

The bulk modulus, G’ or viscosity, η*, are properties related to both the structure of polymers and their processability. The bulk viscosity of raw polymers often reflects the molecular weight, but also includes the influence of secondary effects. For example, the insoluble gel in Hevea contributes to bulk viscosity, resulting in higher η* values than for fully soluble polyisoprene. Likewise, low molecular weight extractables in guayule can result in lower η* values than for low-extract polyisoprene (McMahan 2011). In this study, Rabbitbrush rubber had much lower bulk G' (8.1 kPa) and η*(2842 Pa.s) than

Hevea rubber (G' =34.5 kPa and η* =7981 Pa.s), but similar to guayule rubber (G'=12.3 kPa and η*=3886 Pa.s), another potential source of commercial natural rubber (Table 1).

The low bulk viscosity of Rabbitbrush might indicate the presence of components that melt below the testing temperature of 100 °C. In preliminary extractions the solvent extracted rubber was recovered by concentrating and air drying. When the dried rubber was analyzed, significant levels of a waxy material were observed during APA testing; material apparently co-extracted with the rubber, yielding bulk viscosities as low as 50 kPa/s. Rubber recovered by acetone precipitation in later extractions, effectively minimized the contaminant material, as evidenced by higher η* and no observed melted waxy material.

110

The low molecular weight components were quantified by ASE extraction of solid rubber with acetone and expressed as percent extractables (Table 1). Extractable low molecular weight components from the later Rabbitbrush rubber were quite low,

1.418 ± 0.01 % extractables which is very low compared to 6-7 % extractables in

Guayule rubber (McMahan, 2011). So while some contribution of wax material and residual resins might be responsible for the low η* in Rabbitbrush rubber, the nature of the components appears to be different from those found in guayule. Bulk plasticity, by

PRI was lower for Rabbitbrush rubber, in a agreement with the APA results. Thermal stability by PRI, however, showed plasticity retention index of over 70%, similar to synthetic polyisoprene (Table 1). Rabbitbrush rubber maintained 72.7% plasticity compared to 73.68 % of Natsyn synthetic polyisoprene and Hevea Technically specified rubber, TSR, specification of minimum 60%. The thermal stability of Hevea rubber has been attributed to its non-rubber constituents with anti-oxidative properties, especially proteins and amino acids (Tuampoemsab et al. 2007). Synthetic polyisoprene and guayule, which are both very low in protein, show consequently lower thermal stability than Hevea. In contrast, natural rubber from Rabbitbrush appears to have excellent thermal stability despite its low protein content. Other naturally-occurring non-protein constituents might contribute to thermal stability of Rabbitbrush rubber, a finding worthy of further study.

The components of Rabbitbrush resin

Gas chromatography mass spectrometry (GC/MS) of Rabbitbrush resin identified many components, including monoterpenes, diterpenes, and triterpenes in high concentration. Among them Geraniol, Coumarin (Esculin), (5-Amino-3H-imidazol-4-yl)

111 acetonitrile, and 2-Naphthalenemethanol were found in high concentrations.

Additionally, monoterpene: 1R-α-Pinene and Limonene, Diterpene: Geranylgeraniol, and triterpene: Squalene were also identified. Other resin component found included Indole,

1-Naphthalenol, 4-(2,2-Dimethyl-6-methylenecyclohexyl) butanal, Hexadeca-2,6,10,14- tetraene-1-ol, Eicosane, Tetratetracontane, Heptacosane, and Decanol. There were also numerous non-terpenes and unidentified chemicals that need to be properly identified.

More research needs to be done to identify and quantify Rabbitbrush resin components as well as conversion of resin to biofuel.

Analysis of lignocellulosic biomass residue

The analyses of Rabbitbrush lignocellulosic biomass residue resulted from resin and rubber extractions have shown promising results. In an effort to produce a biomass product with similar characteristics to coal, the lignocellulosic biomass residue after rubber and resin extractions were subjected to a process called Hydrothermal

Carbonization (HTC) (Figure 4). HTC performs energy densification by introducing biomass into a, “hot, pressurized, aqueous environment” (Hoekman et al. 2011). We analyzed the products of HTC process: gases, liquid, and solid. A gas chromatograph was used to determine the make-up of the gases that were purged out of the system. The results, shown in Table 3, demonstrate that the majority of produced gases contained small amounts of pollutant, including CO2 (18.41 %), CO (0.99 %) and H2 (trace amounts). The mass balance of the Rabbitbrush residue was very similar to unextracted biomass (Table 4). The mass balance of solid HTC Char showed a reduction as the amount of gases and liquids have been increased. The energy content of the dried HTC char was determined by calorimetry (Table 5). The HTC treated Rabbitbrush residue had

112 a 27.882 MJ/kg compared to 20.081 MJ/kg non-treated. The energy content of raw

Rabbitbrush was slightly higher in both cases. The energy content was increased by about 40 % in both raw Rabbitbrush and the Rabbitbrush residue. This increase was in- line with other feedstock that were processed, such as pine from the Lake Tahoe forest, and a mixture of Pinjon and Juniper collected in Nevada. The energy yield of

Rabbitbrush residue was increased by 62.45%.

The processed Rabbitbrush residue was consisted of small particles, whether or not it went through HTC. As a practical means of transportation it was necessary to produce pellets. Therefore, pellets were made from Rabbitbrush tissue, and analyzed for their quality. The abrasion test performed with pellets showed very low percent fines of

0.90% and 0.15% in both HTC treated Rabbitbrush and Rabbitbrush residue (Table 6).

Water solubility test was done to check the hydrophobic properties of the pellets (Figure

5). While pellets made from HTC treated tissue did not significantly changed 0 and 20%, pellets made from non-HTC tissue changed more than 300% in volume (Table 7).

The biomass pellet industry is attracting attention as governments from around the world are encouraging the use of renewable resources as a replacement to fossil-based resources. While using biomass as a solid fuel is not a new concept, making its use economical is difficult. In order to compete against coal, a biomass feedstock must maintain a comparable cost for the same output. The use of HTC, which has shown to increase the energy density of Rabbitbrush by a factor of 1.4, could be one solution to this problem. By increasing the energy content of Rabbitbrush residue to 27.88 MJ/kg, the

HTC char is comparable to High Vol. Bituminous coal of 27.87 MJ/kg (Narain et al.

2009). As a by-product from the production of resin and rubber, the residue is a readily-

113 available feedstock with no associated feedstock transportation costs. Furthermore, the

Rabbitbrush biomass pellets produced using HTC char had promising characteristics in terms of abrasion resistance and hydrophobicity. Based on these results, Rabbitbrush

HTC should be further researched as a feedstock for gasification, or co-firing with coal.

ACKNOWLEDGEMENTS

The authors would like to acknowledge funding support from the University of

Nevada Agricultural Experiment Station (NEV). DOE NSHE-DRI and Nevada

Renewable Energy Consortium (NVREC) grants.

114

REFERENCES

Ahmed, A. A., M.-E. F. Hegazy, N. M. Hassan, M. Wojcinska, J. Karchesy, P. W. Pare and T. J. Mabry (2006). "Constituents of Chrysothamnus viscidiflorus." Phytochemistry 67: 1547-1553. Beinor, R. T. and W. M. Cole (1986). Solvent fractionation of guayule rubber, Google Patents. Bhat, R., D. Weber, D. Hegerhorst and E. McArthur (1990). "Rubber and resin content in natural and uniform-garden populations of Chrysothamnus nauseosus subspecies." Phyton, Buenos Aires 51: 35-42. Bousquet, J., A. Flahault, O. Vandenplas, J. Ameille, J.-J. Duron, C. Pecquet, K. Chevrie and I. Annesi-Maesano (2006). "Natural rubber latex allergy among health care workers: A systematic review of the evidence." Journal of Allergy and Clinical Immunology 118: 447-454. Cornish, K. (2001). "Similarities and differences in rubber biochemistry among plant species." Phytochemistry 57: 1123-1134. Cornish, K. and R. Backhaus (1990). "Rubber transferase activity in rubber particles of guayule." Phytochemistry 29: 3809 - 3813. Cornish, K. and D. Siler (1995). "Effect of different allylic diphosphates on the initiation of new rubber molecules and on cis-1,4-polyisoprene biosynthesis in guayule (Parthenium argentatum Gray)." J Plant Physiol 147: 301 - 305. Cornish, K., D. J. Siler, O. K. Grosjean and N. Goodman (1993). "Fundamental similarities in rubber particle architecture and function in three evolutionarily divergent plant species." J. National Rubber Research 8: 275 - 285. d'Auzac, J., J. L. Jacob and H. Chrestin (1989). Physiology of rubber tree latex. The laticiferous cell and latex-a model of cytoplasm, CRC Press Inc. Dennis, M. S., W. J. Henzel, J. Bell, W. Kohr and D. R. Light (1989). "Amino Acid Sequence of Rubber Elongation Factor Protein Associated with Rubber Particles in Hevea Latex." Journal of Biological Chemistry 264: 18618-18626. Dennis, M. S. and D. R. Light (1989). "Rubber Elongation Factor from Hevea brasiliensis." Journal of Biological Chemistry 264: 18608-18617. Doten, S. B. (1942). "Rubber from rabbit brush (Chrystothamnus nauseosus)." Univ. Nevada Agr. Expt. Sta. Bull. 157: 22. Duch, M. and D. Grant (1970). "Carbon-13 chemical shift studies of the 1,4- polybutadienes and the 1,4-polyisoprenes." Macromolecules 3: 165 - 174. Gordon, A. V. E., J. R. Barker and C. M. McKell (1982). "Energy Biomass from Large Rangeland Shrubs of the Intermountain United States." Journal of Range Management 35: 22-25. Hall, H. M. and T. H. Goodspeed (1919). "Chrysil, A New Rubber from Chrysothamnus nausuosus." University of California Publications in Botany 7: 183-264. Hegerhorst, D. F., D. J. Weber, E. D. McArthur and A. J. Khan (1987). "Chemical analysis and comparison of subspecies of Chyrsothamnus nauseosus and other related species." Biochemical Systematics and Ecology 15: 201-208. Hoekman, S. K., A. Broch and C. Robbins (2011). "Hydrothermal Carbonization (HTC) of Lignocellulosic Biomass." Energy & Fuels 25: 1802-1810.

115

McArthur, E. and S. Meyer (1987). A review of the taxonomy and distribution of Chrysothamnus. Proc. Fourth Utah Shrub Ecology Workshop. College of Natural Resources, Utah State University, Logan. McLaughlin, S. P. and J. J. Hoffmann (1982). "Survey of biocrude -producing plants from the Southwest." Economic Botany 36: 323-339. McMahan, C. (2009). Natural Rubber from Domestic Crops. Meeting Abstract, American Chemical Society (ACS) Rubber Division Meeting, Pittsburgh, PA. Mooibroek, H. and K. Cornish (2000). "Alternative sources of natural rubber." Appl Microbiol Biotechnol 53: 355-365. Narain, M. and S. Watson (2009). "Coal Fact Sheet: Coal fuels 50% of global electricity demand and 25% of world primary energy needs." Massachusetts Institute of Technology Energy Club. Ohya, N., Y. Tanaka and T. Koyama (2000). "Activity of rubber transferase and rubber particle size in Hevea latex." Journal of Rubber Research 3: 214-221. Ostler, W. K., C. M. McKell and S. White (1984). Chrysothamnus nauseosus: A potential source of natural rubber. Symposium: Biology of Artemisia and Chrysothamnus, USDA Forset Service, General Technical Report INT-200. Reza, M. T., J. G. Lynam, V. R. Vasquez and C. J. Coronella (2012). "Pelletization of biochar from hydrothermally carbonized wood." Environmental Progress & Sustainable Energy 31: 225-234. Schloman Jr, W. W. (2005). "Processing guayule for latex and bulk rubber." Industrial Crops and Products 22: 41-47. Siler, D., M. Goodrich-Tanrikulu, K. Cornish, A. Stafford and T. McKeon (1997). "Composition of rubber particles of Hevea brasiliensis, Parthenium argentatum, Ficus elastica, and Euphorbia lactiflua indicates unconventional surface structure." Plant Physiol Biochem 35: 881 - 889. Tanaka, Y. (2001). "Structural Characterization of Natural Polyisoprenes: Solve the Mystery of Natural Rubber Based on Structural Study." Rubber Chemistry and Technology 74: 355-375. Tarachiwin, L., J. Sakdapipanich, K. Ute, T. Kitayama, T. Bamba, E. Fukusaki, A. Kobayashi and Y. Tanaka (2005). "Structural characterization of α-terminal group of natural rubber. 1. Decomposition of branch-points by lipase and phosphatase treatments." Biomacromolecules 6: 1851-1857. Tarachiwin, L., J. Sakdapipanich, K. Ute, T. Kitayama and Y. Tanaka (2005). "Structural characterization of α-terminal group of natural rubber. 2. Decomposition of branch-points by phospholipase and chemical treatments." Biomacromolecules 6: 1858-1863. Tomazic, V. J., T. J. Withrow, B. R. Fisher and S. F. Dillard (1992). "Latex-associated allergies and anaphylactic reactions." Clin Immunol Immunopathol 64: 89-97. Tuampoemsab, S. and J. Sakdapipanich (2007). "Role of Naturally Occuring Lipids and Proteins on Thermal Aging Behaviour of Purified Natural Rubber." KGK. Kautschuk, Gummi, Kunststoffe 60: 678-684. Yeang, H., E. Yip and S. Hamzah (1995). "Characterization of zone 1 and zone 2 rubber particles in Hevea brasiliensis latex." J Nat Rubber Res 10: 108 - 123.

116

Yeang, H. Y., F. Yusof and L. Abdullah (1995). "Precipitation of Hevea brasiliensis Latex Proteins with Trichloracetic Acid and Phosphotungstic Acid in Preparation for the Lowry Protein Assay." Analytical Biochemistry 226: 35-43.

117

FIGURES AND TABLES

Figure 1. 13C Nuclear Magnetic Resonance (NMR) spectra of Rabbitbrush rubber. Rubber purified from WRPs were compared to cis-1,4-polyisoprene standard. The cis- configuration of Rabbitbrush rubber was confirmed.

118

Figure 2. The amount of bulk rubber and molecular weight of Rabbitbrush shoots. Rabbitbrush shoots collected from Austin, NV were fractionated from four different plants according to their diameter from 3-25 mm. (A) The amount of rubber. The amount of rubber was shown as a function of stem diameter. (B) The molecular weight. The molecular weight of each sample was shown as a function of stem diameter. Error bars represent the standard error of at least three different plants.

119

Figure 3. Whole Rabbitbrush plant rubber and resin extracted by Accelerated Solvent Extractor (ASE). Tissue was obtained from before and after running Soxhlet extractor. (A) The amount of resin extracted using acetone. (B) The amount of rubber extracted using hexane/ethanol after the resin extraction. Error bars represent the standard error of three technical replicates.

120

Table 1. The properties of bulk solid Rabbitbrush rubber compared to other rubber producing species. The rubber samples Rabbitbrush, Natsyn Synthetic polyisoprene and Guayule were solid rubber extracted by solvent extraction. The rubber samples Hevea and Guayule dried latex were latex extracted by centrifugation. Mw refers to the molecular weight calculated using light scattering detector. The Gʹ and the ƞ* is an indicator of bulk viscosity. The P0 and the median PRI values represent the plasticity retention index. The percent extractables were contaminants that were extracted from rubber using ASE.

Sample Name Mw (LS only) Average % Gel G' (kPa) η* (Pa.s) Plasticity Retention Index Percent Dalton @ 1.0 Hz @ 1.0 Hz Po (mm) Median PRI Extractables

Rabbitbrush 995800 2.750 8.070 2842.500 11 72.727 1.42 ± 0.01 Natsyn Syn PI 1348500 25.200 18.830 5927.500 38 73.684 0.09 ± 0.06 Guayule 1313000 8.15 20.41 5834.00 33 77.1 1.06 ± 0.18

Hevea (dried latex) 1143000 87.90 34.547 7981.667 67.8 65.1 0.96 ± 0.07 Guayule (dried latex) 1192000 22.750 12.31 3886.33 31.3 21.6 8.67 ± 1.55

121

Table 2. The washed rubber particle diameter and the protein content of Rabbitbrush and other rubber producing species.

Sample Name Particle Protein content of Diameter (μm) WRP (mg/g rubber) Ericameria nauseosa (Rabbitbrush) 2.0 2.99 – 9.11 Hevea brasiliensis (Hevea) 1.0 6,732-19,846 Parthenium argentatum (Guayule) 1.4 4.30-9.04

Taraxacum kok-saghyz (Russian Dandelion) 1.1 2420

122

Table 3. The analysis of gases produced during Hydrothermal Carbonization (HTC) of Rabbitbrush residue. The produced gases during HTC process were collected to Tedlar bags and analyzed by gas chromatograph.

123

Table 4: The mass balance of Hydrothermal Carbonization (HTC) char.

Mass balance Rabbitbrush Rabbitbrush Lake Tahoe Pinyon/Juniper (percent) Residue Pine Mix Starting dry mass in 48.80% 44.98% 50.13% 50.58% solid Starting dry mass in 12.05% 12.95% 9.20% 12.15% non-volatile liquid Starting dry mass in 10.23% 14.84% 8.27% 10.03% gases Starting dry mass in 30.69% 25.80% 27.34% 5.00% water Starting dry mass -1.77% 1.43% 5.07% 16.32% unidentified

124

Table 5. Energy densification of Hydrothermal Carbonization (HTC) Char.

Biomass Energy content Mass yield Energy Energy (MJ/Kg) densification yield Raw Rabbitbrush 21.315 HTC Raw Rabbitbrush 29.902 48.80% 1.4 68.46% Rabbitbrush residue 20.081 HTC Rabbitbrush residue 27.882 44.98% 1.39 62.45%

Lake Tahoe Pine Mix 20.32 HTC Lake Tahoe Pine Mix 28.26 50.30% 1.39 70% Pinjon/Juniper 20.545 HTC Pinjon/Juniper 28.259 50.58% 1.38 69.56% Loblolly Pine 20.28 HTC Loblolly Pine 28.984 50.13% 1.4 70.18%

125

Table 6. Pellet Abrasion test of raw and Hydrothermal Carbonized Rabbitbrush. The pellets were tumbled in a modified gem tumbler and the material lost during tumbling was referred as %fines.

Feedstock % Fines Raw Rabbitbrush 1.8 HTC Raw Rabbitbrush 0.9 Rabbitbrush residue 1.5 HTC Rabbitbrush residue 0.15

Lake Tahoe Pine Mix 0.32

126

Table 7. Pellet characteristics after water immersion test of raw and Hydrothermal Carbonized Rabbitbrush tissue.

Biomass Mass Volume Pellet Density Loss (%) Increase (%) (kg/m3)

Raw Rabbitbrush* 6.31 300 255.90 Extracted Rabbitbrush 9.15 300 248.31 HTC Extracted Rabbitbrush 0.58 0 1240.30 HTC Rabbitbrush -0.33 20 1026.63 Pinyon/Juniper 1156.20 HTC Pinyon/Juniper** 1226.70 *Measured as several pieces that remained **HTC at 200°C

127

Chapter VI

Concluding Remarks

Natural rubber (NR) is an irreplaceable commodity that only a hand full of

Southeast Asian countries produce. Despite more than 2500 plants species that are able to produce NR, Hevea brasiliensis (Brazilian rubber tree) is the only commercial source of NR in the world (Mooibroek et al. 2000; Bushman et al. 2006). Interest in NR had increased many times in the past especially during WWI and WWII when the US could not import NR due to Japanese control over Asian countries (Davis 1997). Currently, NR is used in the manufacture of over 40,000 products, including tires, surgical gloves, more than 400 medical devices, numerous engineering and consumer products (Cornish 2001; van Beilen et al. 2007). World NR production in year 2010 was over 10 million metric tons, and Unites States spent $3.3 billion for natural rubber imports (International Rubber

Study Group, IRSG). In 2011, the US spent $4.4 billion to import NR while, it is forecasted to increase to $4.5 and $4.8 billion in years 2012 and 2013, respectively

(USDA 2012). When considering the high cost and the unpredictability of NR imports, the limited growing conditions of Hevea plants, the increasing allergic reactions caused by Hevea rubber, and the irreplaceability by synthetics, domestic alternative sources of natural rubber are clearly needed. Furthermore, the rubber biosynthetic pathway needs to be better understood, because currently the rubber transferase that synthesizes rubber is unknown. Better understanding of the pathway could lead to improved rubber production on the new alternative NR crops.

128

The rubber transferase (CPT) is responsible for the polymerization of isopentenyl pyrophosphate (IPP) monomers into high molecular weight cis-1,4-polyisoprene polymer. The studies conducted with H. brasiliensis washed rubber particles (WRP) have yielded multiple lines of evidence that cis-1,4-polyisoprene synthase is associated with the active WRPs (Chapter II). The analyses of H. brasiliensis WRP proteins with liquid chromatography mass spectrometry (LC/MS) and western blot analysis using CPT antibodies confirmed the CPT localization in WRPs. A direct evidence of CPT involvement in rubber polymerization was established by in vitro cross-linking studies done with two different farnesyl pyrophosphate (FPP) analogues. The FPP analogues were able to identify CPT in the WRPs (Chapter II). To confirm the in vivo role of CPT in rubber biosynthesis, CPT under-expressing transgenic Taraxacum kok-saghyz

(Dandelion) plants were studied (Chapter III). The mature transgenic plants were analyzed for the rubber yield, rubber molecular weight, CPT gene expression, and CPT proteins levels. The CPT under-expressing transgenic lines showed a 10-fold less rubber yield and 3-fold lower molecular weight, compared to the lowest transgene control

(Chapter III, Figure 3). The results provided multiple confirmations to strengthen the fact that CPT is rubber transferase. However, the studies conducted here were unable to identify the active sites of CPT. Further studies need to be conducted, perhaps using different FPP analogues, to confirm and reinforce the current evidence. For example, better FPP analogues have been used recently, in identifying FPP utilizing enzymes

(Henry et al. 2009). These new FPP analogues contain unmodified FPP groups with less bulky modifications. These modifications include Fluoride ions and 13C benzene rings so they are less bulky and eliminate the need to work with 32P phosphate groups. In vitro

129 studies conducted with recombinant Hevea rubber transferase 2 was only able to synthesized significantly lower molecular weight cis-1,4-polyisoprene polymer

(Asawatreratanakul et al. 2003). The current theory states that CPT requires other proteins in order to produce high molecular weight rubber (Tanaka et al. 1996; Tanaka

2001; Chow et al. 2007; Collins-Silva et al. 2012). Even though research done here with

FPP analogues identified Small Rubber Particle Protein (SRPP) and Rubber Elongation

Factor (REF), further studies need to be conducted to identify and confirm other crucial proteins that work with CPT and might be essential in the natural rubber production.

Further studies should be done with transgenic plants containing several of the genes mentioned above; CPT, SRPP, and REF. Furthermore, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) a known MVA pathway rate-limiting enzyme also should be used in these multi-gene transformations. Transformation systems are available for simultaneous transformation of 1-3 genes with reporter genes using

Gateway technology (Life Technologies, Grand Island, NY). These studies should be done in rubber producing plants as well as non-rubber producing plants. Taraxacum officinale (common dandelion) does not produce rubber but, is related to rubber- producing Russian dandelion. Common dandelion should be the perfect candidate to check the effect of the above mentioned candidate genes on rubber production and molecular weight. Nicotiana tabacum, Tobacco Bright Yellow 2 (TBY-2) cells is a good non-rubber producing model species to study rubber biosynthesis (Wentzinger et al.

2002; Hemmerlin et al. 2004). There are extensive studies done on MVA pathway in

TBY-2 cells. A major advantage of using the TBY-2 cells is their ability to readily

130 uptake specific inhibitors and stably- and/or radiolabeled precursors. Rubber is a product of a side branch of MVA pathway. The pool of available FPP for rubber production can be increased by introducing inhibitors such as squalstatin-1 and terbinafine, two specific squalene synthase and squalene epoxidase inhibitors (Wentzinger et al. 2002).

Ericameria nauseosa (Rabbitbrush) is one of the promising NR alternatives that was studied throughout the twentieth century (Hall et al. 1919; Ostler et al. 1984; Yeang et al. 1995). Early Rabbitbrush seed collections, and exact sites were unavailable for further studying, therefore, a survey of wild Rabbitbrush populations were needed in order to identify superior quality high rubber producing Rabbitbrush stands (Chapter IV).

A preliminary analysis was done with Rabbitbrush plants collected from fourteen different sites in California and Nevada. Extensive studies were conducted with small branches and whole Rabbitbrush plants collected from the Eagle Valley (EV), Gerlach

(G), Gerlach Playa (GP), and Selenite Range (SR) in 2006 and 2007. Seasonal variation of rubber accumulations and rubber molecular weights were measured on different diameter shoots. In both years, there was a pattern of rubber accumulation and time.

Additionally, a higher amount of rubber of about 6% was found in bigger diameter shoots, but the rubber molecular weight was inferior to that of high quality rubber (about

4-fold less). It was clear that Rabbitbrush needs to be harvested during September and

October in order to have the highest amount of rubber. When it comes to natural rubber, the molecular weight is a very important factor. Because the research here did not find any Rabbitbrush stands with superior quality rubber (1,000 kDa), further studies were conducted with Rabbitbrush collected from Austin, NV (Chapter V).

131

Preliminary studies done with wild Rabbitbrush plants collected from Austin, NV showed promising 800 kDa to 900 kDa high molecular weight rubber. Therefore,

Rabbitbrush from Austin was analyzed for its potential as a domestic natural rubber, resin, and biomass crop (Chapter V). The rubber from 20 mm diameter fractions contained on average 40 mg rubber /g dry weight. Precipitated rubber from whole plant had a very good quality molecular weight of 1,000 kDa. Several physical properties of rubber were analogous to existing commercial natural rubber producers, such as Hevea brasiliensis and Parthenium argentatum (McMahan 2009; McMahan 2011). Natural rubber from Rabbitbrush had excellent thermal stability despite its low protein content.

Other naturally-occurring non-protein constituents might contribute to thermal stability of

Rabbitbrush rubber. Industrial grade rubber extraction methods need to be developed and precautions should be taken to ensure the purity of the rubber and resin fraction because the quality of rubber and resin is very important in manufacturing many products. The chemical components of the Rabbitbrush resin fraction were also examined. Although several previously known resins were identified, further analysis need to be done to identify and quantify the total resins (Hegerhorst et al. 1987). Interestingly, the terpene resin content has been shown to be inversely proportional to the amount of rubber produced and was more responsive to environmental factors than rubber (Bhat et al.

1990). Further studies need to be conducted to manipulate rubber and the resin amounts depending on the consumer demand. The assessment of the lignocellulosic biomass showed encouraging characteristics in terms of hydrophobicity, abrasion resistance, and energy density similar to other biomass energy sources. Overall, Rabbitbrush found in

132

Austin, NV has the potential to be a renewable domestic crop for natural rubber, resin and bioenergy feedstock.

Future studies should focus on cultivating and breeding Rabbitbrush for rubber, resins, and biomass. Uniform-garden populations should be made in areas with diverse elevation, precipitation, and temperature conditions. Guayule, P. argentatum, is another rubber producing shrub that is native to Southwestern United States and Northern Mexico that grows with very little water. Guayule has been developed for rubber production over the past century. The agronomic practices developed for guayule including transplanting

(Foster et al. 2005; Coffelt et al. 2009; Coffelt et al. 2010), post-harvest handing of the shrubs (McMahan et al. 2006; Coffelt et al. 2009) should be useful in developing

Rabbitbrush as a crop. One of the best qualities of Rabbitbrush is its minimal water requirements. However, the impact of irrigation methods and water application on rubber, resin, and biomass production should be evaluated.

133

REFERENCES

Asawatreratanakul, K., Y. W. Zhang, D. Wititsuwannakul, R. Wititsuwannakul, S. Takahashi, A. Rattanapittayaporn and T. Koyama (2003). "Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis." Eur J Biochem 270: 4671-4680. Bhat, R., D. Weber, D. Hegerhorst and E. McArthur (1990). "Rubber and resin content in natural and uniform-garden populations of Chrysothamnus nauseosus subspecies." Phyton, Buenos Aires 51: 35-42. Bushman, B. S., A. A. Scholte, K. Cornish, D. J. Scott, J. L. Brichta, J. C. Vederas, O. Ochoa, R. W. Michelmore, D. K. Shintani and S. J. Knapp (2006). "Identification and comparison of natural rubber from two Lactuca species." Phytochemistry 67: 2590-2596. Chow, K. S., K. L. Wan, M. N. Isa, A. Bahari, S. H. Tan, K. Harikrishna and H. Y. Yeang (2007). "Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex." J Exp Bot. 58: 2429-2440. Epub 2007 Jun 2421. Coffelt, T., F. Nakayama, D. Ray, K. Cornish, C. McMahan and C. Williams (2009). "Plant population, planting date, and germplasm effects on guayule latex, rubber, and resin yields." Industrial Crops and Products 29: 255-260. Coffelt, T. and D. Ray (2010). "Cutting height effects on guayule latex, rubber, and resin yields." Industrial Crops and Products 32: 264-268. Coffelt, T. A., F. S. Nakayama, D. T. Ray, K. Cornish and C. M. McMahan (2009). "Post-harvest storage effects on guayule latex, rubber, and resin contents and yields." Industrial Crops and Products 29: 326-335. Collins-Silva, J., A. T. Nural, A. Skaggs, D. Scott, U. Hathwaik, R. Woolsey, K. Schegg, C. McMahan, M. Whalen, K. Cornish and D. Shintani (2012). "Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism." Phytochemistry 79: 46-56. Cornish, K. (2001). "Similarities and differences in rubber biochemistry among plant species." Phytochemistry 57: 1123-1134. Davis, W. (1997). The rubber industry's biological nightmare. Fortune: 86 - 95. Foster, M. and T. Coffelt (2005). "Guayule agronomics: establishment, irrigated production, and weed control." Industrial Crops and Products 22: 27-40. Hall, H. M. and T. H. Goodspeed (1919). "Chrysil, A New Rubber from Chrysothamnus nausuosus." University of California Publications in Botany 7: 183-264. Hegerhorst, D., D. W. Weber and E. D. McArthur (1987). "Resin and rubber content in Chrysothamnus." The Southwestern Naturalist: 475-482. Hemmerlin, A., E. Gerber, J. F. Feldtrauer, L. Wentzinger, M. A. Hartmann, D. Tritsch, J. F. Hoeffler, M. Rohmer and T. J. Bach (2004). "A review of tobacco BY-2 cells as an excellent system to study the synthesis and function of sterols and other isoprenoids." Lipids 39: 723-735.

134

Henry, O., F. Lopez-Gallego, S. A. Agger, C. Schmidt-Dannert, S. Sen, D. Shintani, K. Cornish and M. D. Distefano (2009). "A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes." Bioorganic & Medicinal Chemistry 17: 4797-4805. McMahan, C. (2009). Natural Rubber from Domestic Crops. Meeting Abstract, American Chemical Society (ACS) Rubber Division Meeting, Pittsburgh, PA. McMahan, C. (2011). Blends of guayule natural rubber latex with commercial latex polymers. Meeting Proceedings. McMahan, C. M., K. Cornish, T. A. Coffelt, F. S. Nakayama, R. G. McCoy, J. L. Brichta and D. T. Ray (2006). "Post-harvest storage effects on guayule latex quality from agronomic trials." Industrial Crops and Products 24: 321-328. Mooibroek, H. and K. Cornish (2000). "Alternative sources of natural rubber." Appl Microbiol Biotechnol 53: 355-365. Ostler, W. K., C. M. McKell and S. White (1984). Chrysothamnus nauseosus: A potential source of natural rubber. Symposium: Biology of Artemisia and Chrysothamnus, USDA Forset Service, General Technical Report INT-200. Tanaka, Y. (2001). "Structural Characterization of Natural Polyisoprenes: Solve the Mystery of Natural Rubber Based on Structural Study." Rubber Chemistry and Technology 74: 355-375. Tanaka, Y., Aik-Hwee, E. , N. Ohya, N. Nishiyama, J. Tangpakdee, S. Kawahara and R. Witisuwannakul (1996). "Initiation of rubber biosynthesis in Hevea brasiliensis: characterizationof initiating species by structural analysis." Phytochemistry 41: 1501 - 1505. USDA (2012). Outlook for U.S. Agricultural Trade. van Beilen, J. and Y. Poirier (2007). "Establishment of new crops for the production of natural rubber." Trends Biotechnol 25: 522 - 529. Wentzinger, L. F., T. J. Bach and M. A. Hartmann (2002). "Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3- hydroxy-3-methylglutaryl coenzyme A reductase." Plant Physiology 130: 334- 346. Yeang, H., E. Yip and S. Hamzah (1995). "Characterization of zone 1 and zone 2 rubber particles in Hevea brasiliensis latex." J Nat Rubber Res 10: 108 - 123.