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Isolation and Characterization of Components of Low Temperature-Induced Signal Transduction Pathways in Poncirus trifoliata (L.) Raf. and Citrus paradisi Macf.

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Title:
Isolation and Characterization of Components of Low Temperature-Induced Signal Transduction Pathways in Poncirus trifoliata (L.) Raf. and Citrus paradisi Macf.
Creator:
CHAMP, KAREN IRISA ( Author, Primary )
Copyright Date:
2008

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Acclimatization ( jstor )
Cold tolerance ( jstor )
Complementary DNA ( jstor )
Freezing ( jstor )
Gels ( jstor )
Genes ( jstor )
Low temperature ( jstor )
Polymerase chain reaction ( jstor )
RNA ( jstor )
Species ( jstor )

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University of Florida
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University of Florida
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Copyright Karen Irisa Champ. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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4/30/2005
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436097554 ( OCLC )

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ISOLATION AND CHARACTERIZATION OF COMPONENTS OF LOW TEMPERATURE-INDUCED SIGNAL TRANSDUCTION PATHWAYS IN Poncirus trifoliata (L.) Raf. AND Citrus paradisi Macf. By KAREN IRISA CHAMP A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Karen Irisa Champ

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This document is dedicated to my husband, fa mily, friends and loved ones who have been so supportive of my aspirations

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ACKNOWLEDGMENTS The completion of this work is the cumulative result of the actions of many people. First and foremost, I am appreciative of the opportunity to work and conduct research under the direction of Dr. Gloria Moore. Her laboratory provided me much more than a facility, but also a supportive and encouraging environment. She encouraged critical, independent thinking and enabled me the opportunity to try many different techniques. Because of all this, I am better equipped as a scientist. I would like to express my gratitude to my committee members, Dr. Fred Davies, Dr. Charles Guy, and Dr. Andrew Hanson, for their time, advice and guidance. I thank Dr. Vicente Febres for his time, advice and technical support from the day I began my graduate studies to the completion of this work. I thank Dr. Ken Cline, Dr. Kevin Folta, and Dr. Manjunath Keremane for their assistance and use of their labs. I am also grateful to all the members of the Moore Lab for their assistance and friendship throughout my studies. Finally, I thank all my family and friends for their support and encouragement. I thank my parents, Hale and Rebecca, for their belief in me and unconditional support of my goals. I am especially grateful to my husband Chad for his undying love, support and confidence in me. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT ....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................4 Introduction...................................................................................................................4 Chilling Stress vs. Freezing Stress................................................................................4 Freezing Injury..............................................................................................................5 Cold-Responsive Genes................................................................................................7 Regulatory Networks in Cold Stress Responses.........................................................10 ABA Independent Regulatory Pathways.............................................................10 The CBF pathway.........................................................................................10 An eskimo1 pathway ....................................................................................14 A vernalization pathway...............................................................................14 ABA Dependent Regulatory Pathways ...............................................................14 ABRE-mediated pathways ...........................................................................14 MYB and MYC-mediated pathways............................................................16 Engineering Cold Tolerance.......................................................................................16 3 INVESTIGATING A POTENTIAL C-REPEAT BINDING FACTOR (CBF) PATHWAY IN Citrus USING TRANSGENIC PLANTS ........................................19 Introduction.................................................................................................................19 Results.........................................................................................................................22 Overexpression of Arabidopsis CBF1.................................................................22 Analysis of the Arabidopsis COR78 Promoter....................................................24 Discussion...................................................................................................................27 Materials and Methods ...............................................................................................29 Vector Construction.............................................................................................29 pCAMBIA 2201/FMV-CBF1 ......................................................................29 pCAMBIA2201/COR78Pr-CBF1 ................................................................32 v

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pCAMBIA2201/COR78Pr-GFP ..................................................................33 Particle Bombardment..................................................................................34 Transformation to Agrobacterium tumefaciens strain Agl1.........................35 Citrus Transformation .........................................................................................35 PCR Analysis of Shoots ......................................................................................37 Southern Blot.......................................................................................................38 Fluorescence Microscopy....................................................................................39 Relative Quantitative RT-PCR............................................................................40 4 ISOLATION AND CHARACTERIZATION OF COLD REGULATED (COR) GENES IN Poncirus trifoliata (L.) Raf. AND Citrus paradisi Macf. .......................42 Introduction.................................................................................................................42 Results.........................................................................................................................44 cDNA Sequence Isolation and Analysis..............................................................44 Characterization of COR Gene Expression .........................................................48 Discussion...................................................................................................................50 Materials and Methods ...............................................................................................55 Rapid Amplification of cDNA Ends (RACE).....................................................55 Northern Blots .....................................................................................................57 5 ISOLATION AND CHARACTERIZATION OF LOW TEMPERATURERESPONSIVE PROMOTERS IN Poncirus trifoliata (L.) Raf. ................................61 Introduction.................................................................................................................61 Results.........................................................................................................................65 Nuclear Run-On Assay........................................................................................65 COR Promoter Isolation and Characterization....................................................66 Discussion...................................................................................................................75 Materials and Methods ...............................................................................................78 Nuclear Run-On Assay........................................................................................78 Isolation of Promoters .........................................................................................80 Analysis of Promoter Sequences.........................................................................82 6 ISOLATION AND CHARACTERIZATION OF C-REPEAT BINDING FACTOR (CBF) HOMOLOGS IN Poncirus trifoliata (L.) Raf. AND Citrus spp. ...................83 Introduction.................................................................................................................83 Results.........................................................................................................................86 Discussion...................................................................................................................93 Materials and Methods .............................................................................................100 Cloning of PtCBF and CpCBF..........................................................................100 Isolation of PtCBF 5’ Regulatory Sequence .....................................................103 Southern Blots ...................................................................................................104 Northern Blots ...................................................................................................107 vi

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7 CONCLUSIONS ......................................................................................................110 LIST OF REFERENCES.................................................................................................113 BIOGRAPHICAL SKETCH ...........................................................................................125 vii

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LIST OF TABLES Table page 3-1. Agrobacterium-mediated transformation results.......................................................24 5-1. Poncirus trifoliata COR promoter motifs predicted by MEME analysis..................73 5-2. Summary of predicted cis-acting regulatory elements present in Poncirus trifoliata COR promoters.........................................................................................................74 5-3. Restriction enzymes, primers and annealing temperatures used for the isolation of COR promoters.........................................................................................................81 6-1. PtCBF identity and similarity to CBF homologs in other species a ...........................90 6-2. PtCBF promoter motifs predicted by MEME analysis. ............................................96 6-3. Summary of predicted cis-acting regulatory elements present in the PtCBF promoter. ..................................................................................................................97 viii

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LIST OF FIGURES Figure page 3-1. Transformation constructs. A) Transformation “cassettes” constructed in pUC118. B) Map of pCAMBIA2201 .....................................................................................23 3-2. Southern blot analysis of COR78Pr-GFP transgenic plants .....................................25 3-3. GFP expression in COR78/RD29A-GFP transgenic plants kept at 28C .................26 3-4. Relative Quantitative RT-PCR results of COR78Pr-GFP transgenic C. paradisi cv. Duncan .....................................................................................................................26 4-1. CORc102 sequence analysis and protein alignment. A.) CORc102 cDNA sequence. B.) CORc102 amino acid alignment with Cu/Zn superoxide dismutases...............46 4-2. CORc410 cDNA sequence ........................................................................................47 4-3. CORc410 amino acid alignment with the closest matching peroxidases ..................48 4-4. CORc510 cDNA sequence ........................................................................................49 4-5. Northern blot analysis of CORc410 and CORc115 during cold acclimation............50 4-6. Active oxygen scavenging reactions .........................................................................52 5-1. Nuclear run-on assay of CORc115............................................................................66 5-2. Illustration of inverse PCR. A) Diagramatic representation. B) Example of PCR results using different template concentrations........................................................67 5-3. CORc115 Promoter Sequence ...................................................................................68 5-4. CORc119 Promoter Sequence ...................................................................................69 5-5. CORc102 Promoter Sequence ...................................................................................70 5-6. CORc410 Promoter Sequence ...................................................................................71 5-7. CORc510 Promoter Sequence ...................................................................................72 ix

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6-1. Southern blot of genomic DNA from P. trifolata, C. paradisi, C. grandis and A. thaliana using 32 P-labeled CBF1 (Arabidopsis) as a probe. ....................................86 6-2. Alignment of CBF coding sequences........................................................................87 6-3. Alignment of CBF proteins. ......................................................................................89 6-4. Northern blot analysis of PtCBF expression levels during cold acclimation............91 6-5. Southern blot of genomic DNA from P. trifoliata and C. paradisi using DIG-labeled PtCBF as a probe......................................................................................................91 6-6. Alignment of PtCBF and CpCBF sequences. A.) Nucleotide alignment of the coding sequences. B.) Protein alignment. ................................................................93 6-7. Comparison of the expression patterns of PtCBF and CpCBF during cold acclimation. ..............................................................................................................94 6-8. PtCBF Promoter Sequence........................................................................................95 6-9. Alignment of the PtCBF promoter with the Arabidopsis CBF promoters................98 x

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ISOLATION AND CHARACTERIZATION OF COMPONENTS OF LOW TEMPERATURE-INDUCED SIGNAL TRANSDUCTION PATHWAYS IN Poncirus trifoliata (L.) Raf. AND Citrus paradisi Macf. By Karen Irisa Champ May 2004 Chair: Gloria A. Moore Major Department: Plant Molecular and Cellular Biology Citrus species are damaged by temperatures just below freezing, yet Poncirus trifoliata, an interfertile Citrus relative, is extremely cold tolerant and can survive freezes at -20C if fully cold acclimated. However, the molecular basis of this difference in cold tolerance is poorly understood. The CRT/DRE Binding Factor (CBF) pathway plays a major role in low temperature-regulated gene expression in many plants. The primary objective of this research was to better characterize the molecular basis of cold acclimation and investigate the presence of a CBF pathway in Poncirus trifoliata and Citrus paradisi. To accomplish this, we used several approaches: overexpression of heterologous CBF genes in Citrus, isolation and characterization of P. trifoliata cold regulated genes (COR) and their promoters and lastly, isolation and characterization of CBF homologs from P. trifoliata and C. paradisi. xi

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P. trifoliata and Citrus do contain a CBF pathway for cold-induced gene expression, although we suggest these pathways may possess unique characteristics or components. Characterization of COR promoters identified the presence of motifs conserved in the promoters of COR genes of other species. Notably, the 5' sequences of four COR promoters contained elements resembling the CRT/DRE core motif CCGAC. Additional motifs previously implicated in low temperature signaling were also present including ABREs, and MYB and MYC recognition sites. CBF-like cDNAs were isolated from P. trifoliata and C. paradisi, and similar to other species, P. trifoliata and Citrus appear to contain small CBF families. Although the nucleotide and amino acid sequences of PtCBF and CpCBF are highly similiar, their expression patterns during low temperature treatment differ. PtCBF mRNA accumulates earlier and to higher levels than CpCBF mRNA, perhaps contributing to the differences in cold tolerance. As this difference may reside in the promoter regions, PtCBF 5’ regulatory sequence was isolated. This sequence contains multiple MYC recognition sites, similar to that of the Arabidopsis CBF promoters. However, in contrast to these promoters, the PtCBF promoter contains one copy of the CRT/DRE core element. xii

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CHAPTER 1 INTRODUCTION Plants inhabit almost all climatic regions of the world, from the tropics to the desert to the tundra. Plants that grow in a specific habitat are adapted (or have evolved) to local conditions and are able to adjust to the typical environmental changes that occur. Some of those environmental changes might be unfavorable for their growth and development. However, there is great genetic variation in the ability of different plants to survive different abiotic stresses (Bohnert et al. 1995). That is, plants differ genetically in their ability to both sense stress and activate genes to cope with the consequences of stress. These differences have led to drastic variation in the freezing tolerance of economically important species. Many plants can increase their tolerance to low temperatures through a process known as cold acclimation. This process, induced by specific environmental stimuli, enables a plant to withstand temperatures which previously would have been lethal. While cold acclimation leads to many biochemical and physiological changes, the process is controlled by the coordinated expression of many genes. Current knowledge indicates that the molecular basis of cold acclimation is conserved, even between species which vary widely in cold tolerance. Citrus species are generally considered moderately cold sensitive. Even so, the most widely grown Citrus species, C. sinensis, leaves can withstand -6.7C if fully acclimated (Yelenosky 1985). The closely related trifoliate orange, Poncirus trifoliata, can withstand -20C if fully acclimated. Despite this extensive variation in cold 1

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2 tolerance, the underlying molecular basis is unknown. Durham et al. (1991) showed that there are changes in RNA species following cold acclimation in both P. trifoliata and C. grandis, and specific cDNAs representing unique cold-induced sequences have been cloned from P. trifoliata (Cai et al. 1995). Molecular maps indicate that cold hardiness in citrus does segregate. Quantitative trait loci (QTL) mapping of a Citrus grandis x Poncirus trifoliata F1 population indicated there is one major QTL or group of QTLs located in Poncirus that has a large effect on the level of cold tolerance (Weber et al. 2003), yet the gene or genes responsible for this QTL remain unknown. Citrus is commercially produced in more than one hundred countries and is a multi-billion dollar industry world-wide. United States citrus production alone totaled 15.2 million metric tons during the 2002-03 season, with a production value of 2.3 billion dollars (USDA 2003). Citrus cultivation is primarily limited to the region between 40N and 40S latitudes, yet freezing temperatures continue to be a major factor affecting production. A series of severe freezes in Florida, the greatest producer of citrus in the US, have significantly reshaped the industry and limited production to primarily the southern portions of the state. Additionally, periodic freezes cause economic losses and threaten citrus production in other countries including Japan and Spain. Even modest improvement of the freezing tolerance of citrus cultivars could reduce tree and crop losses worldwide. Arabidopsis research has demonstrated that molecular approaches can be used to increase the freezing tolerance of plants (Jaglo-Ottosen et al. 1998; Gilmour et al. 2000). An increased understanding of the molecular basis of cold acclimation in citrus should facilitate the improvement of citrus freeze tolerance and thus reduce fruit and tree losses

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3 in traditional growing regions. The primary objective of this research was to better characterize the molecular basis of cold acclimation in P. trifoliata and Citrus. Specifically, transformation experiments were used to investigate the presence of a CBF pathway in Citrus; previously identified P. trifoliata COR genes were isolated and characterized; COR promoter sequences were isolated and analyzed for low temperature-responsive elements; and lastly, CBF homologs were isolated from P. trifoliata and C. paradisi and characterized.

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CHAPTER 2 LITERATURE REVIEW Introduction Cold acclimation, the process by which plants achieve maximum freezing tolerance in response to specific environmental stimuli, involves adjustments in biochemical and physiological processes. The cellular and metabolic changes include alterations in lipid composition, increases in sugars and soluble proteins, and the appearance of new protein isoforms (Levitt 1980). In 1970, Weiser proposed that changes associated with cold acclimation involve modifications in gene expression, and in 1985, Guy et al. demonstrated that cold acclimation in spinach resulted in altered gene transcripts. Although much time has passed since these early revelations, the intricate complexities of cold acclimation are only recently becoming evident. Chilling Stress vs. Freezing Stress Temperature influences all aspects of plant metabolism and physiology, and plants differ significantly in their ability to withstand low temperatures (Guy 1999). Some tender species are damaged by exposure to “chilling” temperatures. These plants, known as chilling sensitive, are damaged when temperatures decrease below 12C. They include economically important species such as maize, rice and tomato. Chill-tolerant plants can endure chilling temperatures but are susceptible to freezing. Lastly, freeze tolerant plants can withstand temperatures below 0C (Pearce 1999). While plants from tropical regions may suffer injury at temperatures well above freezing, many woody species from temperate regions routinely survive -30C. Notably, 4

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5 these plants can increase their freezing tolerance. In autumn, hardy temperate plants begin to cold acclimate in response to specific environmental stimuli, including a decrease in temperature and day length. By winter, the plant is fully acclimated and can withstand maximum freezing temperatures (Kaye and Guy 1995). Arabidopsis thaliana, a freezing tolerant plant, has been established as a model for cold acclimation research. Arabidopsis cold acclimates rapidly, with a substantial increase in freezing tolerance apparent after only 24 hours of low temperature exposure. Freezing Injury Current knowledge suggests that susceptibility to chilling is a direct effect of low temperatures on cells, whereas freezing acts indirectly by damaging cells via dehydration and ice formation (Pearce 1999). Plant cells freeze when they cannot avoid the formation of ice nuclei and prevent the growth of ice. One strategy for freezing tolerance is supercooling or decreasing the temperature of ice formation. However, prevention of ice formation is usually only a successful strategy at relatively high freezing temperatures. A contrasting strategy is to allow extracellular ice formation and tolerate the resulting cellular dehydration and ice masses. While the accumulation of extracellular ice masses can potentially result in physical damage including the separation of cell layers and the formation of cavities (Pearce 2001), the formation of intercellular ice is considered a lethal event. Therefore, the limitation of ice formation to extracellular spaces is one mechanism of freezing tolerance. Ice crystals have a lower chemical potential than liquid water of the same temperature. As a result, the growth of ice crystals draws water from the cells, resulting in cellular dehydration. The solute concentration of the intracellular fluid and the freezing temperature determine the extent of dehydration. As the temperature decreases,

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6 the water potential of ice also decreases. Thus, cellular dehydration progressively worsens as temperature decreases (Gusta et al. 1975). It is this freezing-induced cellular dehydration that is believed to cause the most severe injury (Levitt 1980; Steponkus 1984). Although the injury caused by freezing-induced cellular dehydration includes many “solution effects” (Levitt 1980; Guy 1990), the primary site of injury is widely thought to be the plasma membrane (Steponkus 1984). Membrane destabilization may result either by the direct interaction of solutes with the membrane or by removal of water from the plasma membrane surface. The freezing temperature and the severity of cellular dehydration determine the specific type of membrane damage. “Expansion-induced lysis” is the predominant injury at relatively high freezing temperatures (-2C to -4C), due to the cycle of osmotic contraction and expansion that occurs with freezing and thawing. As temperatures decrease to -4C to -10C, membrane damage results from local lamellar-to-hexagonal-II phase transitions and the cells become osmotically unresponsive. Below -10C, membrane damage results from “fracture jump lesions” (Steponkus et al. 1993; Uemura and Steponkus 1997). Membranes are a central site of freezing injury. Therefore, multiple mechanisms of freezing tolerance contribute to increasing the cryostability of membranes. Changes in membrane lipid composition, including increased levels of fatty acid desaturation, seem to contribute to membrane stabilization (Anchordoguy et al. 1987; Steponkus et al. 1988). Lipid unsaturation reduces the temperature at which membrane phase transition occurs, while large hydrophobic groups and small head groups favor non-lamellar phases (Williams 1990). Therefore, these changes can contribute to freezing tolerance

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7 (Steponkus et al. 1988), and cold acclimated plants do show changes in lipid composition (Lynch and Steponkus 1987; Uemura et al. 1995). Solute accumulation can also have stabilizing effects on membranes. While the accumulation of solutes in the cytosol reduces the amount of water lost during freezing-induced dehydration, solutes can also stabilize macromolecules and membranes directly by interaction with membrane surfaces, or indirectly by strong interactions with the surrounding water (Crowe et al. 1992; Close 1996). Moreover, solutes do accumulate during cold acclimation, including sugars, osmoprotectants and hydrophilic proteins such as dehydrins or late embryogenesis abundant proteins (LEAs) (Pearce 1999; Pearce 2001). Cold-Responsive Genes The ability to cold acclimate has long been considered a quantitative trait, as indicated by early genetic studies. While early research focused on identifying quantitative trait loci (QTLs) involved in low temperature responses (Hughes and Dunn 1996), the realization that cold acclimation results in altered gene expression changed the focus of cold acclimation research (Guy et al. 1985). Since this discovery, a central goal of cold acclimation research has been to identify cold-responsive (COR) genes, genes which are upregulated in response to low temperatures. To date, COR genes, also designated LTI (low temperature induced), KIN (cold inducible), RD (responsive to desiccation), and ERD (early dehydration inducible), have been identified in both cold tolerant and cold sensitive species, although the types of cold-induced genes are very diverse. Many COR genes encode enzymes with roles relating to primary metabolism, while others likely perform protective functions. Still others probably function in gene regulation. However, most COR genes encode proteins

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8 of unknown function (Hughes and Dunn 1996; Pearce 1999; Smallwood and Bowles 2002). In addition, most genes induced by low temperatures are not unique to cold. Rather, they are induced in response to many types of stress. COR genes are often members of small multigene families. In Arabidopsis, four gene families, consisting of two genes each, are physically linked in tandem array (Thomashow 1998). Within these gene pairs, individual genes are distinctly regulated. Due to promoter divergence, the genes are expressed differently in response to specific stress conditions (Wilhelm and Thomashow 1993; Yamaguchi-Shinozaki and Shinozaki 1993). Dehydrins within Hordeum vulgare are differently regulated. Of three cold-induced dehydrins, only two are also induced by drought (Close 1996). H. vulgare non-specific lipid transfer proteins are also distinctly regulated (White et al. 1994). Low temperatures induce the expression of numerous genes with specific roles in stress responses. Extreme temperatures, both high and low, can affect protein stability, and many cold-induced genes are proposed to play roles in stabilization. Heat shock proteins (HSPs) are not only induced by heat, but also by cold (Anderson et al. 1994). Some HSPs are essential for protein folding and assembly, whereas others function in prevention of protein denaturation. An abundance of COR genes are extremely hydrophilic, have simple amino acid compositions, have repeated amino acid motifs and remain soluble upon boiling. These genes encode novel polypeptides or homologs of late embryogenesis abundant proteins known as LEAs or dehydrins. LEAs accumulate to high levels during dehydrative stresses (Bray 1993; Close 1997). Although the precise function is largely unknown, enzymatic activity is unlikely due to the simple amino acid composition and structure. However, a role in membrane and protein stabilization has

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9 been proposed. COR15A, an Arabidopsis LEA gene, stabilizes membranes against freeze-induced injury in overexpressing plants and increases freezing tolerance (Artus et al. 1996). Furthermore, overexpression of LE25, a Lycopersicon esculentum LEA gene, confers freezing tolerance to yeast cells (Imai et al. 1996). Other cold-induced genes possess antifreeze activities. These proteins, known as antifreeze proteins (AFPs), have the ability to decrease the temperature at which ice is formed by inhibiting ice crystal growth. AFPs accumulate in the apoplast of many winter cereals, and accumulation of AFPs has been correlated with freezing tolerance in winter rye and wheat (Marentes et al. 1993). Microarray technology has enabled the analysis of genome-wide gene expression (Schena et al. 1995), and the vast quantities of genes involved in cold acclimation are now becoming apparent. Microarrays have been used by numerous groups to identify cold-inducible genes in Arabidopsis. Using a microarray containing approximately 1300 full-length cDNAs, Seki et al. (2001) identified 19 cold inducible cDNAs, 10 of which were novel stress-induced genes not reported previously. A larger microarray facilitated the identification of 53 cold-induced genes (Seki et al. 2002). Fowler and Thomashow expanded this number to 306 cold-responsive genes, including 218 up-regulated genes and 88 down-regulated genes. If these numbers are representative of the entire genome, then as much as 4% of the genome may be altered during low temperature exposure (Fowler and Thomashow 2002). Other genomics approaches have also proved useful for studying cold acclimation. Transcriptome profiling using Serial Analysis of Gene Expression (SAGE) identified 272 cold-responsive genes in Arabidopsis leaves (Jung et al. 2003). Proteome studies

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10 identified 38-54 proteins that were upor downregulated by cold stress (Bae et al. 2003; Kawamura and Uemura 2003). Regulatory Networks in Cold Stress Responses Cold acclimation is the end result of genome-wide changes in gene expression, with both increases and decreases in transcripts. To achieve maximum freezing tolerance, plants must sense inductive conditions and activate mechanisms leading to the altered expression of hundreds of genes. Plants control the coordinated expression of these genes by complex regulatory networks. These signaling pathways that ultimately lead to cold acclimation involve multiple signaling molecules, transcription factors and cis-acting regulatory elements (CAREs). CAREs determine the spatial and temporal pattern of gene expression and they are found in the promoter regions of genes regulated by a common stimulus. Transcription factors recognize and bind specifically to CAREs and ultimately result in altering the rate of transcription. The coordinated expression of many genes during cold acclimation involves specific CAREs located in the regulatory regions of COR genes. Furthermore, gene expression during cold acclimation is controlled by both ABA independent and ABA dependent mechanisms (Thomashow et al. 2001; Viswanathan and Zhu 2002). ABA Independent Regulatory Pathways The CBF pathway The first low-temperature-induced regulatory element (LTRE) was identified by analyzing the promoter regions of coldand dehydration-induced genes (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1994). These groups, working independently on cold and dehydration stress, studied the expression of COR15A and RD29A (also known as COR78 and LTI78), respectively. Both groups isolated 5’ regulatory sequences

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11 and characterized the ability of the promoters to facilitate low temperature-induced expression of a reporter gene. By fusing progressive deletions of the promoters to -glucuronidase (GUS), the promoter regions conferring low temperature-induced gene expression were identified. The cold-responsive region of COR15A was designated the C-repeat (CRT) (Baker et al. 1994). The RD29A promoter contained a similar region which was designated the dehydration-responsive element (DRE) (Yamaguchi-Shinozaki and Shinozaki 1994). The CRT/DRE, which is characterized by the core sequence CCGAC, has been found in the promoters of other Arabidopsis COR genes, including, but not limited to, COR6.6, COR47, KIN1 and ERD10 (Thomashow et al. 2001). Additional CRT/DRE-controlled genes have been identified by genomic-based approaches. Microarray techniques have identified 45 CRT/DRE-controlled genes, 37 upregulated by cold and 8 downregulated by cold (Seki et al. 2001) (Fowler and Thomashow 2002). Arabidopsis genomic estimates place the number of CRT/DRE controlled genes around 100, which includes at least 12% of known cold-responsive genes (Fowler and Thomashow 2002). In addition to Arabidopsis, similar elements have been identified in the promoters of Brassica napus BN115 (Jiang et al. 1996), Triticum aestivum WCS120 (Ouellet et al. 1998) and WCOR15 (Takumi et al. 2003), and Hordeum vulgare BLT4.9 (Dunn et al. 1998) and multiple dehydrins (Choi et al. 1999). The presence of the CRT/DRE in many Arabidopsis genes and in diverged taxa, including both dicots and monocots, indicates that this LTRE is conserved and plays an important role in cold-induced gene expression. The CRT/DRE is specifically recognized by a small family of transcription factors known as CRT/DRE Binding Factors (CBFs) (Stockinger et al. 1997; Gilmour et al.

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12 1998) or DRE Binding Factors (DREBs) (Liu et al. 1998). CBFs bind specifically to DNA through a conserved AP2/EREBP DNA-binding domain. The AP2/EREBP domain, originally identified in Arabidopsis APETALA2 (Jofuku et al. 1994) and tobacco EREBP1 (Ohme-Takagi and Shinshi 1995), is present in more than 140 Arabidopsis transcription factors (Riechmann et al. 2000). In Arabidopsis, three CBF family members (CBF1, CBF2 and CBF3) are located in tandem on Arabidopsis chromosome 4 (Gilmour et al. 1998; Shinwari et al. 1998; Medina et al. 1999). CBFs transcripts are rapidly induced by low temperatures followed by the induction of CRT/DRE-containing COR genes (Gilmour et al. 1998). Overexpression studies have confirmed the roles of CBFs as transcription regulators of cold-induced gene expression in Arabidopsis. CBF-overexpressing transgenic plants demonstrate increased COR gene expression without a low-temperature stimulus. Moreover, the transgenic plants are more freezing tolerant than wild type plants without acclimation (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000). All these data together indicate that CBF genes control the expression of a cold-induced “regulon” of CRT/DRE-controlled genes. Cold acclimation results in many biochemical and physiological changes, and CBF transcription factors are proposed to integrate the activation of numerous components of the cold acclimation response. CBF3-overexpressing plants mimic multiple biochemical changes that occur during cold acclimation (Gilmour et al. 2000), all of which are believed to contribute to enhanced freezing tolerance. Specifically, CBF3 overexpression results in the accumulation of COR transcripts and polypeptides, the accumulation of proline, and the accumulation of soluble sugars.

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13 In addition to the conservation of the CRT/DRE motif, CBFs are also present in highly diverse taxa, including both dicots and monocots. CBF homologs have been identified in B. napus (Jaglo et al. 2001; Gao et al. 2002), H. vulgare (Choi et al. 2002; Xue 2002; Xue 2003), T. aestivum (Jaglo et al. 2001; Shen et al. 2003a; Vagujfalvi et al. 2003), Lycopersicon esculentum (Jaglo et al. 2001), Oryza sativa (Dubouzet et al. 2003), Secale cereale (Jaglo et al. 2001) and Atriplex hortensis (Shen et al. 2003b). The AP2 domain of CBF proteins is highly conserved. While the AP2/EREBP protein family of transcription factors is rather large (Riechmann et al. 2000), short conserved signature sequences distinguish the CBF family from other AP2/EREBP domain proteins (Jaglo et al. 2001). These signature sequences, PKK/RPAGRxKFxETRHP and DSAWR, flank the AP2 domain. Many of the identified CBF genes are induced by low temperature. Moreover, in species where target genes have been identified, cold-induced accumulation of CBFs is followed by increases in target gene transcripts (Gilmour et al. 1998; Jaglo et al. 2001). Expression of CBFs in heterologous systems demonstrates that CBF function is also conserved. Overexpression of AtCBF1 in B. napus and L. esculentum increases cold tolerance without a low temperature stimulus (Jaglo et al. 2001; Hsieh et al. 2002; Lee et al. 2003). Similar results have been obtained using CBFs from other species. TaDREB1 has been overexpressed in O. sativa and Arabidopsis (Shen et al. 2003a) and AhDREB1 in tobacco (Shen et al. 2003b). Likewise, TaDREB1, AhDREB1 and OsDREB1A were able to activate CRT/DRE-controlled genes (Dubouzet et al. 2003; Shen et al. 2003a; Shen et al. 2003b). Therefore, the CBF pathway is conserved in structure and function.

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14 An eskimo1 pathway Among ABA-independent cold-regulated pathways, the CRT/DRE is the most studied and best understood. However, evidence indicates that additional pathways do exist. Eskimo1 (ESK1), a “constitutively” freezing tolerant mutant, is more freezing tolerant than wild type plants without cold acclimation (Xin and Browse 1998). ESK1 does not express known members of the CBF regulon and affects only a subset of the mechanisms controlled by CBF genes. One hypothesis is that ESK1 controls a separate acclimation pathway. A vernalization pathway Distinct from cold acclimation, many plants require exposure to low, non-freezing temperatures for competence to flower. This process, called vernalization, requires the activation and repression of specific genes, although the cold signaling components are unknown. Recent studies indicate that vernalization is not mediated by the CBF pathway (Liu et al. 2002). Arabidopsis CBF1 overexpression does not affect flowering time or the expression of genes known to be involved in the vernalization response, pointing to a distinct cold-induced vernalization pathway. Transcriptome analysis during cold acclimation revealed the long-term cold-induced presence of eight known or putative transcription factors in addition to the CBFs (Fowler and Thomashow 2002). These include ZAT12, RAV1, AtMYB73, ATHB-12, H-protein binding factor 2a, RAP2.1, a zinc finger protein (At4g38960) and RAP2.7. ABA Dependent Regulatory Pathways ABRE-mediated pathways Abscisic acid (ABA) was suggested to play a role in cold acclimation as early as 1968 (Irving and Lanphear 1968) and levels of ABA increase during cold acclimation

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15 and drought. Moreover, ABA treatment and drought can trigger cold acclimation and increases in cold tolerance in many species (Chen et al. 1983; Chandler and Robertson 1994). The significance of ABA signaling is evidenced by the fact that the expression of many cold-induced genes is modulated by ABA, and many mutants affected in ABA biosynthesis and ABA signaling are also impaired in cold-induced gene expression (Gilmour and Thomashow 1991; Nordin et al. 1991). These observations indicated that, in addition to ABA-independent pathways, cold acclimation must also include pathways that are dependent on ABA. ABA regulates the transcription of many COR genes, indicating the presence of common promoter elements. The functional dissection of ABA-responsive promoters has identified several types of CAREs involved in mediating ABA-induced gene expression. ABA-response elements (ABREs) are defined as a sequence of 8-10bp characterized by a G-box core motif, ACGT (Busk and Pages 1998; Leung and Giraudat 1998; Hattori et al. 2002). This sequence has been identified in the promoters of many ABA-responsive COR genes, including COR15A (Baker et al. 1994) and RD29A (Yamaguchi-Shinozaki and Shinozaki 1994), genes which are also controlled by the CRT/DRE. The ABRE motif is highly conserved and has been found in the ABA-responsive promoters of many species, including but not limited to A. thaliana, O. sativa (Skriver et al. 1991), T. aestivum (Guiltinan et al. 1990) and H. vulgare (Shen and Ho 1995). The ABRE interacts specifically with many basic leucine zipper (bZIP) transcription factors, and ABRE binding factors (ABFs) have not only been cloned (Guiltinan et al. 1990; Uno et al. 2000; Kang et al. 2002), but some are themselves induced by cold (Choi et al. 2000).

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16 In general, single ABRE motifs are not responsive to ABA (Skriver et al. 1991). A second ABRE or “coupling element (CE)” is required for optimal ABA responsiveness. ABA-induction of HV22 and HVA1 require CEs. Analysis of these promoters defined a minimal ABA-responsive complex, consisting of a CE and an ABRE. CE sequences are different but contain a high content of cytosines and guanines (Shen and Ho 1995; Shen et al. 1996). Interestingly, the CRT/DRE has been implicated as a CE in Arabidopsis (Narusaka et al. 2003) and maize (Kizis and Pages 2002). The ABA-responsiveness of the RD29A promoter, which contains a single ABRE, is enhanced by the CRT/DRE. Similarly, the ABA-induction of the maize RAB17 promoter is dependent on two DRE-like elements (Busk et al. 1997). These elements are recognized specifically by DBF1 and DBF2, AP2/EREBP transcription factors (Kizis and Pages 2002). MYB and MYC-mediated pathways ABRE-directed gene expression, which does not usually require new protein synthesis, represents one pathway for ABA-dependent cold-induced gene expression. ABA-induced gene expression requiring new protein synthesis can be directed through MYB and MYC recognition sites (Iwasaki et al. 1995; Abe et al. 1997). The ABA-induced expression of RD22, which requires new protein synthesis, is modulated by MYB and MYC recognition sites (Abe et al. 1997). These sites are recognized and bound specifically by AtMYC2 and AtMYB2, which function as transcriptional activators of ABA-inducible gene expression (Abe et al. 2003). ABA-induction of AtADH1 is regulated by a single MYC binding site (de Bruxelles et al. 1996). Engineering Cold Tolerance Low temperatures limit the geographic distribution of plants and can result in plant damage and crop losses. The application of molecular research towards the production of

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17 plants more tolerant to low temperatures has met with limited success. Most molecular approaches have attempted to improve tolerance by the introduction of a single gene, and plants have been transformed with many different genes in attempts to confer low temperature tolerance. These have included genes involved in the metabolism of membrane lipids, active oxygen species and compatible solutes (Pearce 1999; Iba 2002; Sung et al. 2003). Although in many cases the plants were more freezing tolerant, the improvement was modest. Because freezing tolerance is a quantitative trait, the modification of numerous genes may be required to significantly improve freezing tolerance. Many genes are transcriptionally induced during cold stress. Thus, one strategy for cold tolerance improvement involves modification of transcription factor expression. The Arabidopsis CBF family of proteins was the first group of transcriptional activators shown to be involved in the induction of COR genes during acclimation (Thomashow et al. 2001), thus offering a new approach for stress engineering. The overexpression of the CBF genes in Arabidopsis and other species results in the overexpression of many COR genes, and the freezing tolerance of the plants is significantly improved (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Gilmour et al. 2000; Jaglo et al. 2001). Overexpression of other transcription factors has also resulted in significant improvements in cold tolerance. SCOF1, a coldand ABA-inducible C2H2-type zinc finger protein, resulted in constitutive expression of stress-responsive genes and increased freezing tolerance (Kim et al. 2001). Enhanced drought tolerance was achieved by constitutive expression of ABF3 and ABF4 (Kang et al. 2002). Furthermore,

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18 overexpression of AtMYC2 and AtMYB2 resulted in the constitutive expression of several ABA-induced genes and increased tolerance to osmotic stress (Abe et al. 2003). These results demonstrate the effectiveness of engineering regulons. Quantitative trait engineering is likely to require adjustments in the expression of many genes. Transcription factor engineering is one mechanism for altering the expression of many genes. An improved understanding of cold acclimation signal transduction pathways will reveal new possibilities for engineering tolerant plants.

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CHAPTER 3 INVESTIGATING A POTENTIAL C-REPEAT BINDING FACTOR (CBF) PATHWAY IN Citrus USING TRANSGENIC PLANTS Introduction The maximum freezing tolerance of a plant is not constitutive; rather it is induced in response to specific inductive conditions including exposure to low, non-freezing temperatures. This phenomenon is a product of a physiological process, known as cold acclimation, which has been defined as the expression of the plant’s genetic potential under inductive conditions (Pearce 1999). Cold acclimation results in numerous changes in plant cells, including increases in membrane unsaturation, soluble sugars, osmoprotectants and organic acids as well as altered gene expression (Pearce 1999; Iba 2002). Manipulation of cold acclimation offers immense potential for many crop species. As a result, identification and characterization of these changes continues to be an area of intense research. Studies have identified many genes differentially expressed upon exposure to low temperatures. These cold-responsive (COR) genes, also designated LTI (low temperature induced), KIN (cold-inducible), RD (responsive to desiccation), and ERD (early dehydration-inducible), have been identified in numerous plant species of varying degrees of cold tolerance (Hughes and Dunn 1996). Genomic approaches have begun to hint at the complexity of cold acclimation. For example, transcriptional profiling of Arabidopsis thaliana during cold acclimation identified 308 cold-responsive genes 19

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20 (Fowler and Thomashow 2002). The expression of 218 genes increased while 88 genes decreased. The application of molecular research towards the production of more cold tolerant plants, however, has met with limited success. Plants have been transformed with many different genes in attempts to confer cold tolerance, and most molecular approaches have attempted to improve tolerance by the introduction of a single gene (Pearce 1999; Iba 2002; Sung et al. 2003). Although in many cases the plants were more freezing tolerant, the improvement was modest. Cold acclimation is accomplished by the coordinated expression of many COR genes. Thus, freezing tolerance is considered a quantitative trait, i.e. controlled by many genes. Therefore, expression of the entire array of COR genes would be expected to have a greater impact on freezing tolerance than ex pression of a single ge ne. This principle was first demonstrated in Arabidopsis (Jaglo-Ottosen et al. 1998), thus illustrating that molecular approaches can be used to significantly increase the freezing tolerance of plants. The Arabidopsis CBF [CRT (C-repeat)/DRE (dehydration-responsive element) binding factor] family of proteins is the first group of transcriptional activators shown to be involved in the induction of COR genes during cold acclimation (Stockinger et al. 1997; Gilmour et al. 1998; Liu et al. 1998; Medina et al. 1999), thus offering a new approach for stress engineering. Upon exposure to a low temperature stimulus, CBF transcription factors bind specifically to CRT/DRE elements located in COR gene promoters and activate COR gene transcription. In fact, Arabidopsis microarray studies indicate that at least 12% of cold induced genes are regulated by the CBF regulon

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21 (Fowler and Thomashow 2002). CBF overexpression in Arabidopsis results not only in the overexpression of many COR genes, but also results in multiple biochemical changes associated with cold acclimation including increases in compatible solutes (Gilmour et al. 2000). Furthermore, the freezing tolerance of the overexpressing plants is substantially improved in both non-acclimated and cold acclimated plants (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000). Cold-induction of genes encoding CBF-like transcriptional activators appears to be a common feature of cold acclimation. CBF-like transcripts have been demonstrated to accumulate rapidly in response to low temperature in Arabidopsis (Gilmour et al. 1998; Liu et al. 1998; Medina et al. 1999), Brassica napus (Jaglo et al. 2001; Gao et al. 2002), Hordeum vulgare (Choi et al. 2002; Xue 2002; Xue 2003), Oryza sativa (Dubouzet et al. 2003), Lycopersicon esculentum (Jaglo et al. 2001), Triticum aestivum (Jaglo et al. 2001; Shen et al. 2003a), Secale cereale (Jaglo et al. 2001) and Atriplex hortensis (Shen et al. 2003b). Durham et al. (1991) showed that there are changes in RNA species following cold acclimation in Citrus, and specific cDNAs representing unique cold-induced sequences have been cloned from Poncirus trifoliata, an extremely cold-tolerant interfertile Citrus relative (Cai et al. 1995). In addition, quantitative trait loci (QTL) mapping of a Citrus grandis x Poncirus trifoliata F1 population indicated there is one major QTL or group of QTLs located in P. trifoliata that has a large effect on the level of cold tolerance (Weber et al. 2003). It is, however, unknown whether COR gene expression in Citrus is regulated by a CBF homolog. Severe freezes in Florida have significantly limited commercial citrus production in the northern growing region, and they continue to be a threat for the remaining industry.

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22 Modest improvement of the freezing tolerance of commercial citrus cultivars would potentially save growers millions of dollars in tree and crop losses. The purpose of this research was to investigate whether a CBF pathway for low temperature gene regulation may be present in citrus. We hypothesized that if a conserved CBF pathway exists in Citrus species, then overexpression of Arabidopsis CBF family members should induce Citrus COR gene expression and increase tolerance to low temperatures, as has been shown in Arabidopsis (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000), Brassica napus (Jaglo et al. 2001), and Lycopersicon esculentum (Hsieh et al. 2002). We also speculated that if Citrus contains a conserved CBF family, then cold acclimating conditions should induce expression of a reporter gene under the control of an Arabidopsis CRT/DRE-containing promoter. Results Overexpression of Arabidopsis CBF1 In an effort to obtain Citrus plants with an increased capacity to withstand freezing temperatures, Citrus paradisi cv. Duncan was transformed with constructs expressing Arabidopsis CBF1 (Figure 3-1). Histochemical GUS staining of regenerated shoots identified 104 plants transformed with the blank pCAMBIA2201 plasmid (Table 3-1). However, only one COR78Pr-CBF1 shoot showed GUS staining, and no FMVCBF1 transgenic plants were identified via GUS staining. The proper functioning of the GUS reporter gene was verified by transient expression in cauliflower epidermal cells. To confirm the absence of the transgene, regenerated shoots were analyzed by PCR. Many shoots containing coding regions of both the CBF1 and GUS transgenes and numerous shoots containing only the CBF1 or GUS transgene were identified. All shoots were rooted, and no phenotypic differences were observed between the wildtype and

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23 transformed plants. However, when approximately 3-4 months old and upon transfer to ex vitro conditions, all CBF1 transformed plants died. AB AB Figure 3-1. Transformation constructs. A) Transformation “cassettes” constructed in pUC118. B) Map of pCAMBIA2201. All cassettes were excised from pUC118 with PstI and inserted into the PstI site of pCAMBIA2201.

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24 Table 3-1. Agrobacterium-mediated transformation results. 0.26% 1 389 13865 COR78Pr-CBF1 pCAMBIA2201 18847 5440 3126 Total Explants 15.15% 0.00% 57.8% % Shoots GUS + 70 463 COR78Pr-GFP pCAMBIA2201 0 188 FMV-CBF1 pCAMBIA2201 104 188 Blank pCAMBIA2201 Total Shoots GUS + Total Shoots Produced Construct Plasmid 0.26% 1 389 13865 COR78Pr-CBF1 pCAMBIA2201 18847 5440 3126 Total Explants 15.15% 0.00% 57.8% % Shoots GUS + 70 463 COR78Pr-GFP pCAMBIA2201 0 188 FMV-CBF1 pCAMBIA2201 104 188 Blank pCAMBIA2201 Total Shoots GUS + Total Shoots Produced Construct Plasmid Analysis of the Arabidopsis COR78 Promoter To determine whether the Arabidopsis CRT/DRE could induce low temperature gene expression in Citrus, we also transformed Duncan grapefruit with a construct containing S65T GFP (green fluorescent protein) driven by the COR78/RD29A promoter, which contains the CRT/DRE (Figure 3-1). Seventy COR78Pr-GFP transformed shoots were obtained as demonstrated by histochemical GUS staining (Table 3-1). Southern blot analysis was used both to confirm the presence of the transgene and estimate the copy number (Figure 3-2). Transgene copy number ranged from single to multiple copies. The growth and development of the transgenic plants was indistinguishable from wildtype. The COR78/RD29A promoter contains one copy of the CRT/DRE (Yamaguchi-Shinozaki and Shinozaki 1994), and in Arabidopsis, COR78/RD29A is induced within 3 hours of transfer to 4C (Yamaguchi-Shinozaki and Shinozaki 1993). To analyze the ability of the CRT/DRE to activate transcription at low temperatures, COR78/RD29A-controlled GFP expression was analyzed in transgenic plants. Leaves collected from both non-acclimated and cold-acclimated transgenic plants were analyzed by

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25 fluorescence microscopy (Figure 3-3). Differences in GFP fluorescence between non-acclimated and cold-acclimated plants were not observed. Instead, GFP observations Figure 3-2. Southern blot analysis of COR78Pr-GFP transgenic plants. Genomic DNA was digested with EcoRI. Plant identification numbers are listed across the top. Lane 1 contains the DIG-labeled MW Ladder VII (Roche Molecular Biochemicals). revealed the COR78/RD29A promoter has a significant basal level of expression in Citrus. Moreover, while GFP protein was seen throughout the leaves, several plants were observed to have a higher concentration of GFP in the stomatal guard cells (Figure 3-3, plant 25). Relative quantitative reverse transcription PCR (RT-PCR) was used as a more sensitive assay of the GFP expression levels during cold acclimation (Figure 3-4). The Arabidopsis COR78/RD29A promoter induced very slight, if any, changes in GFP levels during cold acclimation. Of the plants analyzed by RT-PCR, GFP levels appeared unchanged during cold acclimation in half of the plants (Figure 3-4; plants 13, 10 and 12). In contrast, very slight changes in GFP transcript levels during cold acclimation were observed in the remaining plants (Figure 3-4; plants 4, 16 and 25). While these plants were observed to have a basal level of GFP expression, small fluctuations in GFP transcript levels were observed following transfer to 4C.

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26 Figure 3-3. GFP expression in COR78/RD29A-GFP transgenic plants kept at 28C. Photos show the adaxial surface of a leaf as viewed through a stereomicroscope (60X magnification). Numbers represent individual transgenic plants. Figure 3-4. Relative Quantitative RT-PCR results of COR78Pr-GFP transgenic C. paradisi cv. Duncan. Seedlings were either not treated (NA) or treated with cold (4C) for the indicated durations. (7d-RT=negative control without reverse transcription of 7 day 4C RNA sample). Plant identification numbers are listed along the left side. The first and last lanes contain the Invitrogen 1Kb ladder. In each panel, the top band represents GFP mRNA levels and the bottom band represents the 18S rRNA levels.

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27 Discussion Cold acclimation results from the coordinated expression of many genes. CBFs, which are very early induced, function in the activation of many downstream COR genes. CBF overexpression in Arabidopsis results in the overexpression of many COR genes, and the freezing tolerance of the plants is significantly improved (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000). To investigate whether expression of a heterologous CBF gene in Citrus would result in activation of the cold acclimation pathway, we attempted to transform Duncan grapefruit with Arabidopsis CBF1. Expression of CBF1 from both a constitutive (Figwort mosaic virus 34S) and stress-inducible (COR78/RD29A) promoter was unsuccessful. These results were unexpected as CBFs have been overexpressed in Arabidopsis (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000; Dubouzet et al. 2003), B. napus (Jaglo et al. 2001), L. esculentum (Hsieh et al. 2002), O. sativa (Shen et al. 2003a) and N. tobacum (Shen et al. 2003b) without difficulty. Although CBFs have been overexpressed in many species, transformed plants have consistently shown a dwarf phenotype (Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000; Jaglo et al. 2001; Hsieh et al. 2002; Dubouzet et al. 2003; Shen et al. 2003a). Moreover, the extent of growth retardation has been correlated with transgene expression levels (Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000). As CBF expression increased, plant growth decreased. In addition to growth retardation, CBF1 overexpressing L. esculentum plants exhibited a reduction in fruit set and seed number per fruit (Hsieh et al. 2002). Taken together, these results indicate that CBF overexpression can have adverse effects on plant growth. Although some transgenic C. paradisi plants were obtained, no plants survived transfer to ex vitro conditions. It is

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28 hypothesized that CBF1 expression in Citrus also has negative effects on plant growth, and in our case, CBF1 overexpression was lethal. To reduce the negative effects on plant growth, Kasuga et al. expressed CBF3 from the stress-inducible COR78/RD29A promoter. Expression of CBF3 with the COR78/RD29A promoter in Arabidopsis minimized adverse effects on growth (Kasuga et al. 1999) while continuing to provide an appreciable increase in cold hardiness. Additionally, transgenic L. esculentum plants expressing AtCBF1 from the stress inducible HVA22 promoter were similar to wildtype in both growth and yield, yet more tolerant to chilling temperatures (Lee et al. 2003). Experiments utilizing the COR78/RD29A promoter in Citrus indicate that this promoter does not function as in Arabidopsis. Instead, the COR78/RD29A promoter has a significant basal expression level in Citrus. Therefore, inducible expression of CBF genes in Citrus will require identification of a more suitable promoter. Upon exposure of Arabidopsis to low temperatures, CBF family members activate expression of genes containing CRT/DRE promoter motifs (Thomashow et al. 2001). The COR78/RD29A promoter contains the conserved CRT/DRE, and expression of COR78/RD29A increases following exposure to cold (Yamaguchi-Shinozaki and Shinozaki 1994). In Arabidopsis, COR78/RD29A expression levels increase substantially within 3 hours of transfer to 4C and remain elevated for at least 24 hours (Yamaguchi-Shinozaki and Shinozaki 1993). If Citrus species contain a CBF family with a conserved binding specificity, then a reporter gene driven by the COR78/RD29A promoter should be induced by low temperatures. In this study, GFP expression in Duncan grapefruit plants transformed with COR78Pr-GFP was not substantially induced in response to low

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29 temperatures. Moreover, the transgenic plants maintained a constitutive level of GFP even under non-stress conditions. Although both Arabidopsis and Citrus can cold acclimate, these results indicate differences among the Arabidopsis and Citrus low temperature pathways. Although Citrus may contain a CRT/DRE pathway complete with CBF homologs, the low temperature-responsive promoter elements and binding specificities of the Citrus CBFs may be slightly different. As a result, Arabidopsis homologs and promoter elements may be functionally unable to substitute in the Citrus pathway. Furthermore, Arabidopsis transcriptome profiling indicates that multiple regulatory pathways, in addition to the CBF pathway, are activated during cold acclimation (Fowler and Thomashow 2002). It is unknown what role, if any, the CBF regulon plays in Citrus cold acclimation. A clearer understanding of this pathway in Citrus will be necessary to enable its use for the molecular improvement of Citrus. Materials and Methods Vector Construction pCAMBIA 2201/FMV-CBF1 The high homology of the coding sequence of the Arabidopsis CBF family members made specific amplification of CBF1 (GenBank Accession: NM 118681) from genomic DNA difficult. Therefore, a two-step strategy was used to amplify the CBF1 coding sequence for expression in citrus. In the first step, primers CBF1 Extended-FWD (5’CAAGACAGATATACTATCTTTTATTAATCC 3’) and CBF1 Extended-RVS (5’TAGAAAACGATTTGTTTTGTATTGAATTAT 3’) were designed to recognize unique sequences in regions flanking the CBF1 coding sequence. Pfu Turbo Polymerase (Stratagene) was used for amplification in a 100 l PCR reaction containing 1X Cloned

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30 Pfu Buffer [20 mM Tris-HCl pH 8.8, 2 mM MgSO 4 , 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100, and 100 ng/ml BSA], 200 M each dNTP, 250 nM each primer, and 2.5 units enzyme. The PCR consisted of 30 cycles (94C 1 minute, 55C 1 minute, 72C 1 minute) with an initial denaturation of 94C for 3 minutes and a final extension at 72C for 10 minutes. Using Arabidopsis thaliana ecotype Wassileweskija (WS) DNA as template, a CBF1 band was amplified that extended from -104 to +783 with reference to the translation start site. The extended CBF1 PCR product was diluted 1:10 and 1l was used as a template for specific amplification of the CBF1 coding sequence. Primers CBF1 CDS-FWD (5’TTATAGGGCCCACCATGAACTCATTTTCAGCTTTTTC 3’) and CBF1 CDS-RVS (5’ATTAATACTCGAGTTAGTAACTCCAAAGCGACACGT 3’) were designed to amplify the coding sequence of CBF1 (642 bp) and incorporate ApaI and XhoI sites at the 5’ and 3’ ends, respectively. PCR reactions were performed, as described previously, using Pfu Turbo Polymerase (Stratagene) and the following program: 94C 3 minutes, 30 cycles of 94C 1 minute/55C 1 minute/72C 1 minute, and 72C for 10 minutes. Ten microliters of purified CBF1 was digested with 10 units ApaI (Promega) at 37C for 2.5 hours and purified. The CBF1 (ApaI) band was then digested with 10 units XhoI (Gibco BRL) at 37C for 2.5 hours and purified. pUC118/FMV P-T (Vicente J. Febres) was used as an intermediate cloning vector, as this vector contained an appropriate promoter and termination signal for expression in plants. Approximately 1 g pUC118/FMV P-T plasmid DNA was digested with ApaI and XhoI and purified in the same manner as the CBF1. Eleven microliters of CBF1 (ApaI, XhoI) and 5 l of pUC118/FMV P-T (ApaI, XhoI) were ligated with 1 unit T4 DNA Ligase (Promega) at 14C overnight. One hundred microliters of chemically

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31 competent E. coli DH5 were transformed with 10 l of the ligation reaction using the heat shock method. Transformed colonies were selected by plating on 2XYT (16 g/L tryptone, 10 g/L yeast extract, 5 g NaCl, 15 g/L agar) medium containing ampicillin (100 mg/L). All cloned plasmids were identified by PCR. The cloned plasmid was designated pUC118/FMV-CBF1. To release the FMV-CBF1-Term cassette from pUC118/FMV-CBF1, approximately 1 g of plasmid DNA was digested with 10 units PstI (Gibco BRL) at 37C for 2 hours. The reaction was separated on a 1 % agarose gel and the 1225 bp band containing the FMV-CBF1-Term (PstI) cassette was excised. The DNA was purified using the Gel Extraction Kit (Qiagen). Approximately 1 g pCAMBIA2201 DNA was digested with 10 units PstI (Gibco BRL) in a 30 l reaction volume. After 2 hours of incubation at 37C, 4 l of 10X Dephosphorylation Buffer and 6 l Calf Intestinal Alkaline Phosphatase (CIAP) (Gibco BRL) were added. The reaction proceeded at 37C for an additional 1 hour followed by 15 minutes at 56C. The plasmid DNA was then purified. Eleven microliters of FMV-CBF1-Term (PstI) and 5 l of pCAMBIA2201 (PstI, CIAP) were ligated with 1 unit T4 DNA Ligase (Promega) at 14C overnight. E. coli DH5 cells were transformed and plated on 2XYT medium containing chloramphenical (25 mg/L) and positive colonies were identified by PCR. The cloned plasmid was designated pCAMBIA2201/FMV-CBF1. The orientation of the insert was checked by restriction digestion with XhoI and HindIII (Gibco BRL). The PCR Purification Kit (Qiagen) was used for purification of all enzymatic reactions, and the Plasmid Miniprep Kit (Qiagen) was used for all plasmid minipreps. The sequences of all of the transformation plasmids were confirmed by DNA sequencing performed by the

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32 University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core Facility. pCAMBIA2201/COR78Pr-CBF1 A pUC118 plasmid containing the Arabidopsis COR78/RD29A (GenBank Accession: L22568) promoter and the 35S termination signal was constructed to facilitate cloning of genes under the control of this Arabidopsis promoter. This was accomplished by replacing the 35S promoter in pUC118/35S P-T(-SmaI)2-1 (Vicente J. Febres) with the COR78/RD29A promoter. KS1 (5’ATTATCTAGACTGCAGGATCTCAAAGTTTGAAAGAAAATTTAT3’) and KS2 (5’TTAAGGGCCCTTTGTGAGTAAAACAGAGGAGGG3’) were designed to amplify the COR78/RD29A promoter (-808 to +5, with reference to the transcription start site) and incorporate a XbaI and PstI site on the 5’ end and an ApaI site on the 3’ end. The COR78/RD29A promoter was amplified from WS Arabidopsis DNA with Pfu Turbo (Stratagene) using the following cycles: 94C 1 minute, 30 cycles of 94C 1 minute/55C 1 minute/72C 1 minute, and 72C 10 minutes. Reaction conditions were as previously described. The PCR product was purified and 10 l of the COR78/RD29A promoter was sequentially digested with ApaI (Promega) and XbaI (Gibco BRL) as described previously. Approximately 1 g pUC118/35S P-T(-SmaI)2-1 plasmid DNA was digested similarly with ApaI and XbaI to release the 35S promoter. The reaction was separated on a 1% agarose gel and the band containing the plasmid (minus the 35S promoter) was excised. The band was purified using the Gel Extraction Kit (Qiagen). Eleven microliters of the COR78/RD29A promoter (ApaI, XbaI) and 5 l of plasmid were ligated with 1 unit T4 DNA Ligase (Promega) at 14C overnight. E. coli DH5 were

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33 transformed and plated on 2XYT medium containing ampicillin (100 mg/L). The new plasmid was designated pUC118/COR78Pr. Approximately 1 g of pUC118/COR78Pr DNA was digested with ApaI (Promega) and XhoI (Gibco BRL) and purified as described for pUC118/FMV P-T previously. Eleven microliters of CBF1 (ApaI, XhoI) and 5 l of pUC118/COR78Pr (ApaI, XhoI) were ligated with 1 unit T4 DNA Ligase (Promega) at 14C overnight and transformed into chemically competent E. coli DH5 cells. Cells were plated on 2XYT medium containing ampicillin (100 mg/L). The plasmid was designated pUC118/COR78Pr+CBF1. To move the COR78Pr-CBF1-Term cassette into pCAMBIA2201, approximately 1 g pUC118/COR78Pr+CBF1 DNA was digested with 10 units PstI (Gibco BRL). The reaction was separated on a 1 % agarose gel and the released cassette was excised from the gel. The DNA was purified using the Gel Extraction Kit (Qiagen). Eleven microliters of COR78Pr-CBF1-Term (PstI) and 5 l of pCAMBIA2201 (PstI, CIAP) were ligated with 1 unit T4 DNA Ligase (Promega) at 14C overnight and transformed into E. coli DH5. Cells were plated on 2XYT medium containing chloramphenical (25mg/L). This plasmid was designated pCAMBIA2201/COR78Pr+CBF1. The orientation of the insert was checked by restriction digestion with XhoI and HindIII (Gibco BRL). pCAMBIA2201/COR78Pr-GFP A standard miniprep of pCAMBIA 2202 was diluted 1:20 in water and 1 l was used as a template for PCR. VF26 (5’ATAATAGGGCCCATGGTGAGCAAGGGCGAGGAG3’) and KC17 (5’ATTAATACTCGAGTTACTTGTACAGCTCGTCCCATGC3’) were designed to

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34 amplify S65T GFP (720 bp) and add ApaI and XhoI sites on the 5’ and 3’ ends of GFP, respectively. GFP was amplified with Pfu Turbo Polymerase (Stratagene) using the following cycling parameters: 94C 3 minutes, 30 cycles of 94C 1 minute/55C 1 minute/72C 1 minute, and 72C for 10 minutes. Cloning was performed as previously described. Briefly, purified GFP was digested sequentially with ApaI (Promega) and XhoI (Gibco BRL). Eleven microliters of GFP (ApaI, XhoI) and 5 l of pUC118/COR78Pr (ApaI, XhoI) were ligated with 1 unit T4 DNA Ligase (Promega) at 14C overnight. E. coli DH5 were transformed and plated on 2XYT medium containing ampicillin (100 mg/L). This plasmid was designated pUC118/COR78Pr+GFP. To move COR78Pr-GFP-Term into pCAMBIA2201, approximately 1 g pUC118/COR78Pr+GFP DNA was digested with PstI (Gibco BRL). The reaction was separated on a 1 % agarose gel and an approximately 1774 bp band containing COR78Pr-GFP-Term (PstI) was excised. The band was purified using the Gel Extraction Kit (Qiagen). Eleven microliters of COR78Pr-GFP-Term (PstI) and 5 l of pCAMBIA2201 (PstI, CIAP) were ligated and transformed into E. coli DH5. This plasmid was designated pCAMBIA2201/COR78Pr+GFP. The orientation of the insert was checked by restriction digestion with XhoI and HindIII (Gibco BRL). Particle Bombardment Plasmids were transiently expressed in cauliflower epidermal cells to verify reporter gene activity. For each plasmid, 3 mg of microcarriers (prepared in 50 % glycerol) were combined with 5 l DNA (1 g/l), 50 l 2.5 M CaCl 2 , and 20 l 0.1 M spermidine. The microcarriers were vortexed for 5 minutes, allowed to settle for 1 minute, and pelleted by spinning in a microfuge for 2 seconds. The supernatant was discarded and the microcarriers were washed sequentially with 140 l 70 % ethanol and

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35 140 l 100 % ethanol. The microcarriers were gently resuspended in 48 l 100 % ethanol. Cauliflower epidermal tissue (1 cm 2 and 2-4 mm thick) was bombarded with 8 l of coated microcarriers using 1100 dpi rupture discs from a distance of 6 cm. The bombarded tissue was incubated in the dark for 2 days and assayed for reporter gene activity. Transformation to Agrobacterium tumefaciens strain Agl1 Two microliters of a standard miniprep was used to transform 100 l of freshly prepared chemically competent Agrobacterium tumefaciens strain Agl1 using the freeze-thaw method. Cells were plated on YEP (10 g/L peptone, 10 g/L yeast extract, 5 g/L NaCl, 15 g agar) medium containing chloramphenical (100 mg/L), carbenicillin (50 mg/L) and rifampicin (50 mg/L). Plates were incubated at 28C and colonies appeared after 2-3 days. To confirm positive clones, colonies were added to 5 l Lyse-N-Go PCR Reagent (Pierce). The tubes were placed in a thermocycler and incubated at 95C for 2 minutes, followed by 99C for 1 minute. The Lyse-N-Go mixture was then used as template in a 50 l PCR reaction with appropriate primers. Citrus Transformation Citrus transformation was performed as described (Luth and Moore 1999). Briefly, seeds of C. paradisi cv. Duncan (grapefruit) were surfaced sterilized by washing in 70 % ethanol for 2 minutes, 0.525 % hypochlorite solution and 0.05 % Tween 20 for 10 minutes, and sterile water three times for 1 minute each. Seeds were placed in culture tubes containing MS medium with 0.7 % agar. Seeds were germinated in the dark at 27C to produce etiolated seedlings. Approximately 4-6 week old seedlings were used in all experiments. On the day of the experiment, the seedlings were cut into epicotyl

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36 segments of approximately 2 cm in length and placed in petri plates containing sterile water until inoculation. Fifty milliliters of liquid YEP media containing chloramphenical (100 mg/L), carbenicillin (50 mg/L) and rifampicin (50 mg/L) was inoculated from a frozen glycerol stock with the appropriate culture. The culture was grown overnight at 28C with shaking at 280 rpm until it reached an OD 620 between 0.6 and 1.0. The culture was transferred to a 50 ml conical tube and centrifuged at 3500 g for 10 minutes at 4C. The supernatant was discarded, and the pellet was resuspended in liquid MS medium supplemented with 100 M acetosyringone to an OD 620 =1.0. Prepared epicotyl segments were soaked in the bacteria suspension for approximately 1 minute, blotted dry on sterile paper towels, and co-cultivated by plating on solid MS media supplemented with 100 M acetosyringone. During the two day co-cultivation period, plates were kept in the dark at 25C. On the third day after the inoculation, segments were transferred to selection media (MS media containing 50 mg/L kanamycin, 500 mg/L claforan, and 0.5 mg/L benzyladenine) and maintained in a growth chamber at 28C with a 16 hour photoperiod. Every 4-6 weeks, segments were transferred to fresh selection media. Regenerated shoots were tested for GUS expression by cutting a piece of tissue from the shoot base (Moore et al. 1992). The tissue was placed in microtiter plates containing 30 l stain (1 mg/ml 5-bromo-4-chloro-3-indolyl--D-glucuronide in 0.1 M NaPO 4 pH 7.0 with 10 mM Na 2 EDTA) per well and incubated at 37C overnight. The following day, a 3:1 solution of ethanol: glacial acetic acid was added to the plate to bleach the chlorophyll. GUS expression was evaluated by analyzing the tissue under a

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37 dissecting microscope. Shoots testing positive for GUS were transferred to rooting media ( MS media supplemented with 0.5 mg/L naphthalene acetic acid) until roots developed. When roots were visible, the shoots were transferred to soil. PCR Analysis of Shoots Shoots obtained from experiments using pCAMBIA2201/FMV-CBF1 and pCAMBIA2201/COR78Pr-CBF1 were subjected to PCR analysis. To extract DNA, tissue from 6 shoots was combined, frozen and ground to a fine powder in a 1.5 ml microfuge tube. Seven hundred microliters of extraction buffer (0.1 M Tris-HCl pH 8.0, 50 mM EDTA, 0.5 M NaCl, 3 % SDS and 11 nM -mercaptoethanol) was added to each tube and the samples were incubated at 65C. After 10 minutes, 350 l each of phenol and 24:1 chloroform: isoamyl alcohol was added. Samples were centrifuged at 13,000 g for 5 minutes, and the supernatant was removed to a new tube. Three hundred microliters each of phenol and 24:1 chloroform: isoamyl alcohol was added to the supernatant and the tube was centrifuged at 13,000 g for 5 minutes. One additional chloroform extraction was performed and the supernatant was precipitated with 10 l Na acetate and 300 l isopropanol. Tubes were centrifuged at 13,000 g for 10 minutes. The pellet was washed with 500 l of 70 % ethanol, dried, and resuspended in 30 l TE buffer. DNA was also extracted from individual samples in the same manner. Five microliters of extracted DNA was used as templated in 50 l PCR reactions containing 200 M each dNTP, 0.2 M each CBF1-CDS FWD and CBF1-CDS RVS or GUS FWD (5’CAACGAACTGAACTGGCAG3’) and GUS RVS (5’CATCACCACGCTTGGGTG3’), 2.5 mM MgCl 2 , and 1 unit Taq Polymerase (Promega) in 1X PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X-100). Cycling was as follows: 94C 3 minutes, 22 cycles of 94C 1 minute/55C 1

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38 minute/72C 1 minute, and 72C 10 minutes. Ten microliters of each reaction was analyzed by electrophoresis in a 1% agarose TAE (40 mM Tris-acetate pH 7.6, 1 mM Na 2 EDTA) gel. Southern Blot Southern blot experiments were conducted using GUS-positive grapefruit seedlings. Leaf tissue was collected, ground in the presence of liquid nitrogen and stored at -80C. Genomic DNA was extracted from approximately 250 mg of ground tissue with 800 l DNAzol ES (Molecular Research Center, Inc.). The DNA pellet was resuspended in 30 l TE buffer. To quantify the amount of DNA in the samples, 2 l of DNA was diluted in 100 l water and the absorbance at 260 nm was measured. To digest the DNA, 10 g DNA was combined with 20 units EcoRI (Invitrogen) and 2 l RNase A (1g/ml, Ambion). The reactions were incubated at 37C for 6 hours. The samples were electrophoresed in a 0.8 % agarose TAE gel for approximately 18 hours. Following electrophoresis, the digested DNA was stained by soaking the gel in 1X TAE buffer containing 0.25 g/ml ethidium bromide for 10 minutes. The DNA samples were visualized by illumination with a UV transilluminator. Prior to transfer, the DNA was depurinated, denatured and neutralized by gentle shaking in the appropriate solution. Between steps, the gel was rinsed with water. The DNA was depurinated by incubation in 0.25 M HCl for 10 minutes. The gel was incubated twice (15 minutes each) in denaturation solution (0.5 N NaOH, 1.5 M NaCl) and twice (15 minutes each) in neutralization solution (0.5 M Tris-HCl pH 7.5, 3 M NaCl). 20X SSC was used to transfer the DNA from the gel to a positively-charged nylon membrane by capillary transfer. The transfer was allowed to proceed for approximately 20 hours. The DNA was crosslinked to the membrane using a UV Stratalinker 1800 (Stratagene).

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39 The probe was prepared by PCR amplification of GFP in the presence of Digoxigenin (DIG)-11-dUTP. GFP was amplified with 1 unit Taq Polymerase (Promega) using pCAMBIA2201/COR78Pr-GFP as template. Reaction conditions were as follows: 1X PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X-100), 2.5 mM MgCl 2 , 200 M dATP, 200 M dGTP, 200 M dCTP, 130 M dTTP, 70 M DIG-11-dUTP, and 200 nM each KC17 and VF26. Sufficient labeling was confirmed by electrophoresis, as incorporation of DIG-11-dUTP results in a PCR product with a reduced migration rate. Reconstituted DIG Easy-Hyb granules (Roche Molecular Biochemicals) were used for both prehybridization and hybridization. Prehybridization was performed at 42C for 1 hour using 15ml DIG Easy-Hyb. During prehybridization, 50 l of water was added to 20 l of probe. The probe was denatured by incubation at 100C for 5 minutes, followed by immediate chilling on ice. The denatured probe was added to 10 ml of pre-warmed DIG Easy-Hyb. Hybridization proceeded at 42C for approximately 16 hours. The membrane was washed twice at 25C for 5 minutes under low stringency conditions (2X SSC containing 0.1 % SDS), and twice at 65C for 15 minutes under high stringency conditions (0.5X SSC containing 0.1 % SDS). The detection procedure was performed according to the manufacturer’s protocol (Roche Molecular Biochemicals), and disodium 3-{4-metho xyspiro[1,2-dioxetane-3,2-(5-chloro) tricyclo (3.3.1.1 3 ,7)decan]-4-yl}phenyl phosphate (CSPD) was the substrate used for all detections. Following addition of CSPD, the membrane was incubated at 37C for 15 minutes and detected by incubation with X-ray film. Fluorescence Microscopy GFP expression was analyzed in plants testing positive for GUS expression. Leaves were collected and analyzed by fluorescence microscopy using a Zeiss

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40 stereomicroscope SV11 equipped with an Atto Arc HBO 100W lamp and emission filter FITC 535 nm. Relative Quantitative RT-PCR Relative quantitative reverse-transcription PCR was used to monitor GFP expression levels in the pCAMBIA2201/COR78Pr-GFP transgenic grapefruit plants. Approximately 1 year old seedlings were placed in a growth chamber at 4C with a 16 hour photoperiod. Leaves were collected at specific time points (0, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 1 day, 3 days, 5 days, and 7 days) and immediately frozen in liquid nitrogen. Frozen leaf tissue was ground in 1.5 ml microfuge tubes in the presence of liquid nitrogen using sterile tips. RNA was extracted from approximately 200 mg ground tissue using 1 ml TRIzol reagent (Invitrogen). The RNA pellet was dissolved in 30 l water. Contaminating DNA was removed by addition of 3 l 10X DNase I buffer (100 mM Tris-HCl pH 7.5, 25 mM MgCl 2 , and 1 mM CaCl 2 ) and 2 units DNase I (Ambion). Tubes were incubated at 37C for 30 minutes, followed by the addition of 5 l of DNase Inactivation Reagent. The reaction was mixed by gently flicking the tube and incubating at 25C for 2 minutes. Tubes were centrifuged at 10,000 g for 1 minute to pellet the DNase Inactivation Reagent, and the supernatant was carefully removed to a new tube. Two microliters of RNA was diluted in 100 l water and quantified by measuring the absorbance at 260 nm. cDNA was synthesized using the First Strand Synthesis Kit for RT-PCR (Ambion). Two micrograms of total RNA were combined with 100 mol random decamers in 12 l water. The samples were denatured by incubation at 85C for 3 minutes and immediately transferred to ice. Two microliters of 10X RT buffer (500 mM Tris-HCl pH 8.3, 750 mM KCl, 30 mM MgCl 2 , and 50 mM DTT), 4 l 2.5 mM dNTPs, 10 units RNase Inhibitor,

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41 and 100 units MMLV Reverse Transcriptase were all added to the tube. The tube was incubated at 42C for 1 hour, followed by 92C for 10 minutes. The QuantumRNA 18S Internal Standards (Ambion) were used for relative quantitative RT-PCR. The number of PCR cycles and the ratio of 18S primer: competimer were optimized using 100 ng cDNA prepared from two transgenic plants (#12 and #16) incubated at 4C for 3 days. Twenty-two PCR cycles and an 18S primer: competimer ratio of 2:8 was determined to be optimum. Fifty microliter PCR reactions were composed of 100 ng cDNA, 1 unit Taq Polymerase (Promega), 0.2 M each KC52 (5’ TTATATTCCATGGTGAGCAAGGGCGAGGAG3’) and KC53 (5’ TTATATTCTCGAGCTTGTACAGCTCGTCCATGCC3’), 400 nM 2 (18S primers): 8 (competimers), 200 M each dNTP, 2.5 mM MgCl 2 , and 1X PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X-100). Cycling was as follows: 94C 3 minutes, 22 cycles of 94C 1 minute/55C 1 minute/72C 1 minute, and 72C 10 minutes. Ten microliters of each reaction was analyzed by electrophoresis in a 1 % agarose TAE gel.

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CHAPTER 4 ISOLATION AND CHARACTERIZATION OF COLD REGULATED (COR) GENES IN Poncirus trifoliata (L.) Raf. AND Citrus paradisi Macf. Introduction Low temperatures are a major limiting factor in the productivity and distribution of many commercial crops. Citrus, a billion dollar industry worldwide, is vulnerable to freeze injury at C and below (Yelenosky 1985). However, commercial citrus continues to be grown in many freeze-prone locations, including Japan, Spain, and regions of the United States. The citrus industry of Florida, the largest citrus producer in the United States, has been dramatically reshaped due to severe freezes. While commercial citrus once grew in northern regions of Florida, freezes have reduced the growing regions to include primarily the southern portions. Although no citrus species can withstand lengthy hard freezes, Citrus species do vary somewhat in their susceptibility to freezing temperatures. In general, mandarins (C. reticulata Blanco) are the most cold hardy and limes (C. aurantifolia Christm. Swingle) are the most cold sensitive commercial species. While Citrus grandis, the pummelo, is prone to injury just below 0C, C. sinensis (L.) Osb. (sweet orange), the most widely distributed commercial citrus species, is capable of withstanding about -7C leaf temperatures for limited periods of time (Yelenosky 1985). Thus, there is limited diversity for freezing tolerance traits within the Citrus genus, which might allow limited improvement in the cold hardiness of sensitive citrus types. In contrast with Citrus, Poncirus trifoliata (L.) Raf., an interfertile Citrus relative, is extremely cold tolerant and 42

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43 can survive freezing at -20C if fully cold acclimated (Yelenosky 1985). The fundamental differences that create these variances in cold tolerance, however, are largely unknown. Citrus, similar to numerous other species, achieves maximum freezing tolerance through exposure to specific inductive conditions including low, non-freezing temperatures. This process is known as cold acclimation and is characterized by specific physiological and biochemical changes (Pearce 1999; Iba 2002). As early as 1970 Weiser proposed that changes associated with cold acclimation involve modifications in gene expression. In 1985 Guy et al. demonstrated that cold acclimation in spinach does result in altered gene expression. In fact, it is now known that cold acclimation is accomplished by the expression of many cold-regulated genes (Thomashow 1999), designated cold-responsive (COR), low temperature induced (LTI), cold-inducible (KIN), responsive to desiccation (RD), and early dehydration-inducible (ERD). The function of identified genes varies from enzymatic activities that potentially contribute to freezing tolerance to genes with potential roles in signal transduction. Most identified genes, however, do not have known physiological functions (Hughes and Dunn 1996; Pearce 1999; Thomashow 1999). The significant freezing tolerance of P. trifoliata makes it a desirable germplasm source for improved cold tolerance in citrus species. Molecular maps indicate that cold hardiness in citrus does segregate. Quantitative trait loci (QTL) mapping of a C. grandis x P. trifoliata F1 population indicated there is one major QTL or group of QTLs located in Poncirus that has a large effect on the level of cold tolerance (Weber et al. 2003). Additionally, Durham et al. (1991) showed that there are changes in RNA species

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44 following cold acclimation in both P. trifoliata and C. grandis, and specific cDNAs representing unique cold-induced sequences have been cloned from P. trifoliata (Cai et al. 1995). Cai et al. isolated six P. trifoliata cDNAs representing unique cold-induced sequences (Cai et al. 1995). Full-length sequence was obtained for two of these cDNAs, and they were shown to be members of the conserved group II LEA (late embryogenesis abundant) family. Expression of LEA genes has been demonstrated to increase during low temperature stress in many species (Thomashow 1999). LEA genes encode boiling-soluble, hydrophilic proteins that may serve a stabilizing function during low-temperature-associated dehydration (Artus et al. 1996). The differential expression of the remaining clones following exposure to low temperatures was confirmed via Northern hybridization, although full-length cDNAs were not isolated (Cai et al. 1995). The main goal of this research was to better understand the cold acclimation process in P. trifoliata and Citrus. More specifically, this work focused on the isolation and characterization of previously identified P. trifoliata COR genes (Cai et al. 1995). Identification of the full-length COR genes and characterization of their expression patterns in both P. trifoliata and C. paradisi should facilitate a better understanding of cold acclimation similarities and differences in these species. An improved understanding may assist in the production of improved citrus cultivars. Results cDNA Sequence Isolation and Analysis To identify the previously isolated COR genes, the full cDNA sequences were isolated using 5’ Rapid Amplification of cDNA Ends (RACE). 5’ RACE using primers specific to CORc102 amplified a 466 bp band having a 138 bp overlap with the published

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45 sequence (GenBank Accession L39003.1). The amplified cDNA was roughly 663 bases and contained a 61 nucleotide (nt) 5’ UTR and a 281 nt 3’ UTR. An open reading frame extended from nt 119 to 435 (Figure 4-1, A). The cDNA encoded a protein of 106 amino acids with a molecular weight of 12 kDa and a pI of 11.2. tBLASTx and BLASTp searches of NCBI GenBank revealed significant homology with copper/zinc superoxide dismutases (E value 1e-08). An NCBI conserved domain search identified a copper/zinc superoxide dismutase domain (E value 1e-06), although only 36.4% of the domain aligned with CORc102 (Figure 4-1, B). CORc102 was predicted to be localized in the mitochondria (probability 0.707) as determined by TargetP. The CORc410 specific primer amplified a 1063 bp band having a 137 bp overlap with the published sequence (GenBank Accession L39006.1). Assembly of the fragments resulted in a cDNA of roughly 1403 bases. The 5’ UTR was 41 nt and the 3’ UTR was 353 nt. An open reading frame extended from nt 42 to 1046 (Figure 4-2). The cDNA encoded a 335 amino acid protein with a molecular weight of 37.8 kDa and a pI of 9.03. Homology searches using tBLASTx and BLASTp identified extensive homology with plant peroxidases (Figure 4-3). An NCBI conserved domain search identified a secretory peroxidase domain (E value 3e-100), and TargetP predicted CORc410 to be targeted to the secretory pathway (probability 0.977). A series of 5’ RACE reactions using CORc510 specific primers amplified bands ranging in size from 350 to 450 bp. All amplified bands represented homologous sequences of varying lengths. Assembly of the amplified fragments and published sequence (GenBank Accession L39007.1) into a single contig resulted in a cDNA of 847

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46 Figure 4-1. CORc102 sequences analysis and protein alignment. A.) CORc102 cDNA sequence. The open reading frame is shown in blue. The amino acid sequence is indicated above the corresponding nucleotide sequence. The black arrow indicates the location of the primer used for 5’ RACE. B.) CORc102 amino acid alignment with Cu/Zn superoxide dismutases. T10450, Citrus sinensis; NP197311, Arabidopsis thaliana; Q02610, Pisum sativum; Q07796, Ipomoea batatas; AAD05576, Raphanus sativus; O65768, Carica papaya; O65174, Zantedeschia aethiopica; and AAQ14591, Citrus limon. The red line indicates the portion of the conserved Cu/Zn superoxide dismutase domain present in CORc102. Solid circles () indicate residues important for the binding of Cu 2+ and Zn 2+ ; open circles () indicate residues important for maintaining active site geometry; crosses (+) indicate residues important for -barrel stability (Fridovich et al. 1986; Bordo et al. 1994).

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47 M G T K A V F L L L A L L 1 CAAGGTTGGT CACCTTTAGT GAGTGAAGAG TAGAAGAGAA AATGGGTACGAAAGCTGTCT TCTTGCTTTT AGCTTTGCTTS F S A V S L R S A L A E N E E D P G L V M N F Y K D81 TCCTTCTCAG CTGTATCTCT GAGGTCTGCT TTGGCAGAAA ATGAAGAGGA CCCAGGTCTT GTTATGAATT TTTACAAGGAT C P Q A E D I I R E Q V K L L YK R H K N T A F S W161 TACATGCCCT CAGGCCGAGG ACATTATCAG GGAACAAGTT AAGCTTCTGT ACAAGCGCCA CAAGAACACT GCATTTTCTTL R N I F H D C A V Q S C D A S L L L D S T R K T L 241 GGCTTAGAAA CATTTTCCAT GACTGTGCTG TCCAGTCTTG TGATGCTTCA CTGCTTCTGG ACTCGACAAG AAAGACCTTGS E K E M D R S F G M R N F R Y I E N I K E A V E R E321 TCTGAGAAGG AGATGGACAG GAGCTTTGGT ATGAGGAACT TCAGGTACAT TGAGAACATC AAAGAAGCTG TTGAAAGAGAC P G V V S C A D I L V L S G R DG I V A L G G P H I401 GTGCCCTGGT GTTGTTTCCT GTGCTGATAT TCTTGTCCTG TCCGGTAGAG ATGGCATTGT TGCGCTTGGAGGCCCTCACA P L K T G R R D G R K S R A E I L E Q Y L P D H N D 481 TTCCTCTCAA GACAGGAAGA AGAGATGGTA GAAAAAGCAG AGCAGAGATA CTTGAGCAGT ATCTCCCAGA TCACAATGACS M S V V L E R F A A I G I D A P G L V A L L G S H S561 AGCATGTCTG TTGTTCTTGA GAGGTTTGCA GCCATTGGCA TTGACGCCCC TGGACTTGTT GCTCTGCTAG GATCTCACAGV G R T H C V K L V H R L Y P E VD P A L N P D H V P641 TGTTGGCAGA ACTCATTGTG TGAAGCTGGT GCACCGTCTG TACCCAGAAG TTGACCCTGC ACTGAACCCT GACCATGTTCH M L H K C P D A I P D P K A V Q Y V R N D R G T P 721 CGCATATGCT CCATAAGTGT CCTGATGCAA TCCCAGACCC CAAGGCTGTT CAGTATGTGA GGAATGACCG TGGCACACCCM V L D N N Y Y R N I L D N K G L M M V D H Q L A T D801 ATGGTGCTGG ACAACAACTA CTATAGGAAC ATATTGGACA ACAAGGGCTT GATGATGGTT GATCATCAGC TAGCCACCGAK R T R P Y V K K M A K S Q D Y FF K E F S R A I T I881 CAAGAGGACA AGACCTTATG TTAAGAAGAT GGCCAAGAGT CAAGACTACT TCTTCAAGGA ATTTTCAAGA GCCATTACTAL S E N N P L T G T K G E I R K V C N F A N K L H D 961 TCCTTTCTGA GAACAACCCT CTCACCGGTA CAAAGGGTGA GATCAGAAAG GTTTGCAATT TTGCCAACAA GCTCCACGACK S * 1041 AAGTCCTAGC TAGCTAATAG CTGTATCTCC TGCAACAAGT TAATAGCTCC AAATTTTCTT CCCTTGTTTC TCCTATGAGG 1121 AAGAAAAGAG TGTGAGATGA GCTCCCAATA AGATGGTTTT CTTAGATGGGTTTGTTTCCT ATAAGAGGTT CGTGTTACTA 1201 CTACTATGTC CCTTAGATGT ACCGTCTAAT GTTTAAGCCT AGGCTTTCCTTGTCCTCGAT GGTGTGAGCA TGATGTTATT 1281 AGTATTTAGT AATGGCAATG TAGATGTGGG ATGTATGTAT GTATGCATGGATGGTGGTGA TGGTGTATGG TGCATGGATT 1361 TATACAATGA TGATAATTGA TTATTCATGC CAATGGCAAT AAA M G T K A V F L L L A L L 1 CAAGGTTGGT CACCTTTAGT GAGTGAAGAG TAGAAGAGAA AATGGGTACGAAAGCTGTCT TCTTGCTTTT AGCTTTGCTTS F S A V S L R S A L A E N E E D P G L V M N F Y K D81 TCCTTCTCAG CTGTATCTCT GAGGTCTGCT TTGGCAGAAA ATGAAGAGGA CCCAGGTCTT GTTATGAATT TTTACAAGGAT C P Q A E D I I R E Q V K L L YK R H K N T A F S W161 TACATGCCCT CAGGCCGAGG ACATTATCAG GGAACAAGTT AAGCTTCTGT ACAAGCGCCA CAAGAACACT GCATTTTCTTL R N I F H D C A V Q S C D A S L L L D S T R K T L 241 GGCTTAGAAA CATTTTCCAT GACTGTGCTG TCCAGTCTTG TGATGCTTCA CTGCTTCTGG ACTCGACAAG AAAGACCTTGS E K E M D R S F G M R N F R Y I E N I K E A V E R E321 TCTGAGAAGG AGATGGACAG GAGCTTTGGT ATGAGGAACT TCAGGTACAT TGAGAACATC AAAGAAGCTG TTGAAAGAGAC P G V V S C A D I L V L S G R DG I V A L G G P H I401 GTGCCCTGGT GTTGTTTCCT GTGCTGATAT TCTTGTCCTG TCCGGTAGAG ATGGCATTGT TGCGCTTGGAGGCCCTCACA P L K T G R R D G R K S R A E I L E Q Y L P D H N D 481 TTCCTCTCAA GACAGGAAGA AGAGATGGTA GAAAAAGCAG AGCAGAGATA CTTGAGCAGT ATCTCCCAGA TCACAATGACS M S V V L E R F A A I G I D A P G L V A L L G S H S561 AGCATGTCTG TTGTTCTTGA GAGGTTTGCA GCCATTGGCA TTGACGCCCC TGGACTTGTT GCTCTGCTAG GATCTCACAGV G R T H C V K L V H R L Y P E VD P A L N P D H V P641 TGTTGGCAGA ACTCATTGTG TGAAGCTGGT GCACCGTCTG TACCCAGAAG TTGACCCTGC ACTGAACCCT GACCATGTTCH M L H K C P D A I P D P K A V Q Y V R N D R G T P 721 CGCATATGCT CCATAAGTGT CCTGATGCAA TCCCAGACCC CAAGGCTGTT CAGTATGTGA GGAATGACCG TGGCACACCCM V L D N N Y Y R N I L D N K G L M M V D H Q L A T D801 ATGGTGCTGG ACAACAACTA CTATAGGAAC ATATTGGACA ACAAGGGCTT GATGATGGTT GATCATCAGC TAGCCACCGAK R T R P Y V K K M A K S Q D Y FF K E F S R A I T I881 CAAGAGGACA AGACCTTATG TTAAGAAGAT GGCCAAGAGT CAAGACTACT TCTTCAAGGA ATTTTCAAGA GCCATTACTAL S E N N P L T G T K G E I R K V C N F A N K L H D 961 TCCTTTCTGA GAACAACCCT CTCACCGGTA CAAAGGGTGA GATCAGAAAG GTTTGCAATT TTGCCAACAA GCTCCACGACK S * 1041 AAGTCCTAGC TAGCTAATAG CTGTATCTCC TGCAACAAGT TAATAGCTCC AAATTTTCTT CCCTTGTTTC TCCTATGAGG 1121 AAGAAAAGAG TGTGAGATGA GCTCCCAATA AGATGGTTTT CTTAGATGGGTTTGTTTCCT ATAAGAGGTT CGTGTTACTA 1201 CTACTATGTC CCTTAGATGT ACCGTCTAAT GTTTAAGCCT AGGCTTTCCTTGTCCTCGAT GGTGTGAGCA TGATGTTATT 1281 AGTATTTAGT AATGGCAATG TAGATGTGGG ATGTATGTAT GTATGCATGGATGGTGGTGA TGGTGTATGG TGCATGGATT 1361 TATACAATGA TGATAATTGA TTATTCATGC CAATGGCAAT AAA Figure 4-2. CORc410 cDNA sequence. The open reading frame is shown in blue. The amino acid sequence is indicated above the corresponding nucleotide sequence. The black arrow shows the location of the primer used for 5’ RACE. bases (Figure 4-4). Sequence analysis revealed a 92 nt 5’ UTR, a 491 nt 3’ UTR, and an open reading frame extending from nt 93 to 356. The cDNA encoded a 9.9 kDa protein of 88 amino acids with a pI of 9.87. BLASTn, tBLASTx, BLASTp, and conserved domain homology searches did not identify any proteins of close homology.

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48 Figure 4-3. CORc410 amino acid alignment with the closest matching peroxidases. The red line indicates the conserved secretory peroxidase domain. T10790, Gossypium hirsutum; AF149251_1, Nicotiana tabacum; T06227 and AF145348_1, Glycine max. While the amino acid sequence did not possess unique patterns or repeats, there was a high amount of leucine residues (18 total, or 20.45%). TargetP predicted CORc510 to be targeted to the mitochondria (probability 0.851). Characterization of COR Gene Expression The COR cDNAs were originally isolated as being differentially expressed during cold acclimation (Cai et al. 1995). Furthermore, previous RNA blots revealed differences in the cold-induced expression of CORc410 and CORc510 between P. trifoliata and C.

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49 1 ACGCGGGGAT AATGGGTCAT CTAAGGCTGA TAGTGGCAAG CGTGGGGACTCTGACAGCTC ATCTATCTTA AGATCTGCTG M M M S V W V R F Q R P P G Q C H K S L L L L81 AGGAAGAACT AGATGATGATGAGTGTTTGG GTCAGGTTCC AGAGGCCTCC TGGGCAGTGC CACAAATCCC TTCTCCTCCTQ H L G S T G Q K I L R T N M R AL L C L T Y T T I L161 ACAGCATCTG GGCTCTACTG GCCAAAAAAT CCTCAGAACC AATATGAGAG CGCTTTTGTG CCTGACATAT ACCACCATTCS L I A L P Q E G G C T K P L L D V S C L Q Y H L F 241 TCAGCTTAAT CGCGCTGCCA CAAGAAGGTG GTTGCACTAA ACCTTTATTG GATGTCTCNT GTCTTCAATA TCACCTTTTCR F A S T Q L S P L V C * 321 AGGTTTGCTT CTACTCAATT ATCTCCTCTGGTTTGCTAAA ACTAAAATTC GATGATCTAT CTGCATGGGA TTTGACCGGC 401 GCTTTCCCTG GTGCCTGCTC TTGATACAGT ATGGGTTTTC GAAGATGAAAGCATAAATAA GAAGTGTGTT TGGAGAGCGG 481 CTTCTTTGCC TTAGTTGTGG AAGTTATAGT TATACTGTTA GCTGTAGTAGTCAATAGTCC AAGTTAAACA AAGTTAGTAT 561 TTGTGTGGTT GAACCATTCT AATTGACTAA ATATCTTGAC CAAATCAATGTATTTCTCTA TTCTTTAGAA TTGGAAACAA 641 GGGTATTTCG ATATCTGCAA ATAAATGGTA AGGACATCCC AAATGGTGAGAACAATGTTC TAGTGCCTGG ATTCTTTTGC 721 ATGTCTTCTT GTTACTTATT GCATGCATTT GGGATTTCCT GGATTACATTTCTTGGCATT TGATTTGCTT GTTGTGAGTA 801 CTAACTCTCC TTAATTCTTT TATTATTCCT TTCCATTGGA TTTGAAA KC44KC261 ACGCGGGGAT AATGGGTCAT CTAAGGCTGA TAGTGGCAAG CGTGGGGACTCTGACAGCTC ATCTATCTTA AGATCTGCTG M M M S V W V R F Q R P P G Q C H K S L L L L81 AGGAAGAACT AGATGATGATGAGTGTTTGG GTCAGGTTCC AGAGGCCTCC TGGGCAGTGC CACAAATCCC TTCTCCTCCTQ H L G S T G Q K I L R T N M R AL L C L T Y T T I L161 ACAGCATCTG GGCTCTACTG GCCAAAAAAT CCTCAGAACC AATATGAGAG CGCTTTTGTG CCTGACATAT ACCACCATTCS L I A L P Q E G G C T K P L L D V S C L Q Y H L F 241 TCAGCTTAAT CGCGCTGCCA CAAGAAGGTG GTTGCACTAA ACCTTTATTG GATGTCTCNT GTCTTCAATA TCACCTTTTCR F A S T Q L S P L V C * 321 AGGTTTGCTT CTACTCAATT ATCTCCTCTGGTTTGCTAAA ACTAAAATTC GATGATCTAT CTGCATGGGA TTTGACCGGC 401 GCTTTCCCTG GTGCCTGCTC TTGATACAGT ATGGGTTTTC GAAGATGAAAGCATAAATAA GAAGTGTGTT TGGAGAGCGG 481 CTTCTTTGCC TTAGTTGTGG AAGTTATAGT TATACTGTTA GCTGTAGTAGTCAATAGTCC AAGTTAAACA AAGTTAGTAT 561 TTGTGTGGTT GAACCATTCT AATTGACTAA ATATCTTGAC CAAATCAATGTATTTCTCTA TTCTTTAGAA TTGGAAACAA 641 GGGTATTTCG ATATCTGCAA ATAAATGGTA AGGACATCCC AAATGGTGAGAACAATGTTC TAGTGCCTGG ATTCTTTTGC 721 ATGTCTTCTT GTTACTTATT GCATGCATTT GGGATTTCCT GGATTACATTTCTTGGCATT TGATTTGCTT GTTGTGAGTA 801 CTAACTCTCC TTAATTCTTT TATTATTCCT TTCCATTGGA TTTGAAA 1 ACGCGGGGAT AATGGGTCAT CTAAGGCTGA TAGTGGCAAG CGTGGGGACTCTGACAGCTC ATCTATCTTA AGATCTGCTG M M M S V W V R F Q R P P G Q C H K S L L L L81 AGGAAGAACT AGATGATGATGAGTGTTTGG GTCAGGTTCC AGAGGCCTCC TGGGCAGTGC CACAAATCCC TTCTCCTCCTQ H L G S T G Q K I L R T N M R AL L C L T Y T T I L161 ACAGCATCTG GGCTCTACTG GCCAAAAAAT CCTCAGAACC AATATGAGAG CGCTTTTGTG CCTGACATAT ACCACCATTCS L I A L P Q E G G C T K P L L D V S C L Q Y H L F 241 TCAGCTTAAT CGCGCTGCCA CAAGAAGGTG GTTGCACTAA ACCTTTATTG GATGTCTCNT GTCTTCAATA TCACCTTTTCR F A S T Q L S P L V C * 321 AGGTTTGCTT CTACTCAATT ATCTCCTCTGGTTTGCTAAA ACTAAAATTC GATGATCTAT CTGCATGGGA TTTGACCGGC 401 GCTTTCCCTG GTGCCTGCTC TTGATACAGT ATGGGTTTTC GAAGATGAAAGCATAAATAA GAAGTGTGTT TGGAGAGCGG 481 CTTCTTTGCC TTAGTTGTGG AAGTTATAGT TATACTGTTA GCTGTAGTAGTCAATAGTCC AAGTTAAACA AAGTTAGTAT 561 TTGTGTGGTT GAACCATTCT AATTGACTAA ATATCTTGAC CAAATCAATGTATTTCTCTA TTCTTTAGAA TTGGAAACAA 641 GGGTATTTCG ATATCTGCAA ATAAATGGTA AGGACATCCC AAATGGTGAGAACAATGTTC TAGTGCCTGG ATTCTTTTGC 721 ATGTCTTCTT GTTACTTATT GCATGCATTT GGGATTTCCT GGATTACATTTCTTGGCATT TGATTTGCTT GTTGTGAGTA 801 CTAACTCTCC TTAATTCTTT TATTATTCCT TTCCATTGGA TTTGAAA KC44KC26 Figure 4-4. CORc510 cDNA sequence. The open reading frame is shown in blue. The amino acid sequence is indicated above the corresponding nucleotide sequence. The black arrows show the location of the primers used for 5’ RACE. grandis. To gain a better understanding of citrus cold acclimation and the roles of these COR genes, we characterized their expression profiles following exposure to low temperatures. Northern blots were used to monitor changes in RNA levels during cold acclimation of P. trifoliata and C. paradisi. The expression pattern of CORc410 mRNA during cold acclimation was similar in both P. trifoliata and C. paradisi (Figure 4-5). In both species, an increase in expression levels could be detected as early as 15 minutes following transfer to 4C. However, the CORc410 RNA levels began decreasing as early as 8 hours and the expression continued to decrease until levels were lower than those under non-acclimating conditions. CORc115 mRNA levels increased during cold acclimation in both P. trifoliata and C. paradisi (Figure 4-5). While P. trifoliata CORc115 expression began increasing approximately 4 hours following transfer to 4C, C. paradisi CORc115 mRNA levels

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50 Figure 4-5. Northern blot analysis of CORc410 and CORc115 during cold acclimation. The lengths of the time of treatment at 4C are indicated above the blot. NA=not acclimated began increasing approximately 24 hours after transfer to 4C. In both species, levels of CORc115 mRNA continued to increase throughout the remainder of the experiment. Northern blots using CORc102 and CORc510 probes produced unclear results. Although some changes in RNA levels were observed throughout the cold treatment, the results were not consistent among independently conducted experiments. Furthermore, we conclude that under our experimental conditions, these genes are not cold-regulated. Discussion Previous studies identified cold-regulated (COR) genes in P. trifoliata. These COR genes not only increased in expression in response to low temperatures, but differences in expression between the cold-hardy P. trifoliata and the cold-sensitive C. grandis were also observed (Cai et al. 1995). Further analysis of these genes should assist in understanding the roles they may play in cold acclimation. Towards this goal, we isolated the derived full-length sequence of these clones and analyzed their expression in both cold-hardy and cold-sensitive species. Two of the genes isolated in this study encode enzymes involved in antioxidation. Environmental stresses such as low temperatures, drought and high light intensity cause an increase in active oxygen species (AOS). AOS are generated by the reduction of

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51 molecular oxygen (O 2 ) and hydroxyl radicals (OH) and include both superoxide anions (O 2 ) and hydrogen peroxide (H 2 O 2 ). AOS production causes damage to cellular components including membranes and macromolecules. As a result, plants have developed several detoxification strategies to scavenge these reactive compounds. AOS scavengers include enzymes such as catalase, superoxide dismutase (SOD), peroxidase and glutathione reductase, as well as small molecules such as ascorbate, glutathione, carotenoids and anthocyanins (Mittler 2002). Genes encoding enzymes involved in scavenging AOS have been shown previously to be upregulated in plants subjected to low temperatures (Saruyama and Tanida 1995; O'Kane et al. 1996). In fact, transcriptome studies have shown that antioxidant metabolism and oxidative stress are a major component of temperature stress. In one such study, cold stress was shown to increase the expression of three known or putative glutathione S-transferases and nine known or putative peroxidases. In addition, the expression of seven hydrogen peroxide-inducible genes was also increased (Fowler and Thomashow 2002). Other RNA profiling studies have also reported increases in the expression of oxygen scavenging genes including catalase and SOD (Seki et al. 2001; Nogueira et al. 2003). Not only have studies identified scavenging enzymes as being upregulated in response to low temperatures, but attempts to overexpress these in numerous plant species have resulted in slight improvements in chilling and freezing tolerance (Iba 2002; Sung et al. 2003). SODs, which catalyze the conversion of superoxide radicals to hydrogen peroxide and molecular oxygen (Figure 4-6), can be divided into three classes based on the catalytic center (Fe 2+ , Mn 2+ , or Cu 2+ and Zn 2+ ). All three classes are found in plants.

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52 FeSODs are typically plastidial, MnSODs are mitochondrial, and Cu/ZnSODs are either plastidial or cytosolic (Bowler et al. 1992). Conserved structural and functional Figure 4-6. Active oxygen scavenging reactions. Superoxide dismutase (EC 1.15.1.1) converts superoxide radicals (O 2 ) to hydrogen peroxide (H 2 O 2 ), while peroxidase (EC 1.11.1.7) further metabolizes H 2 O 2 to H 2 O. features of Cu/ZnSODs have identified specific amino acid residues that play key roles in determining the active dimeric SOD molecule. In particular, seven residues coordinate the copper (H45, H47, H62, H119) (numbering based upon Q02610; Figure 4-1, B) and zinc (H62, H70, H79, D82) molecules (Fridovich et al. 1986). G43, G60, P65, G81, G137 and G140 are important for maintaining active site geometry; and G16, L37, F44, L105 and G146 are important for the stability of the -barrel fold (Bordo et al. 1994). CORc102 encodes a partial Cu/ZnSOD domain (36.4% aligned; Figure 4-1, B). However, the partial CORc102 SOD domain does not include many highly conserved amino acids thought to be responsible for coordination of catalytic metals, maintainance of active site geometry and stability of the -barrel fold (Figure 4-1, B). Without these key residues, it seems unlikely that CORc102 encodes a functional SOD. CORc102 shares the highest similarity with a putative Cu/ZnSOD from C. sinensis (T10450). T10450 also lacks many of the conserved residues and likely SOD function. Further characterization of CORc102 and T10450, including assays of enzymatic activity, will assist in uncovering the functions of these genes.

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53 While SOD scavenges superoxide species, catalase and peroxidase detoxify hydrogen peroxide (Figure 4-6). Based upon sequence homology, CORc410 encodes a class III/secretory peroxidase. Class III peroxidases are a large family of plant specific oxidoreductases, with 73 members in Arabidopsis thaliana (Duroux and Welinder 2003). Class III peroxidases participate in diverse plant processes, including lignification, suberization, auxin catabolism, pathogen defense, salt tolerance and senescence (Hiraga et al. 2001). Expression analysis of 21 class III peroxidases in rice demonstrated that peroxidase genes are unique in their expression patterns. These results support the involvement of individual peroxidases in diverse biological processes and suggest that peroxidases may function differently or cooperatively in the same physiological processes (Hiraga et al. 2000). Cold acclimation led to an increase in CORc410 and CORc115 expression. Upon exposure to 4C, CORc410 expression increased rapidly in both P. trifoliata and C. paradisi and was followed by a rapid decline (Figure 4-5). Low temperatures in the light can cause the accumulation of AOS due to an increase in photosystem II excitation pressure (Huner et al. 1998). Furthermore, transient increases during cold acclimation in enzymes involved in AOS metabolism, including peroxidases, have been previously reported (Fowler and Thomashow 2002). CORc410 likely functions in helping the plant adjust to fluctuating environmental conditions, rather than the direct effects of low temperature. LEA-type proteins accumulate in plant species in response to dehydration resulting from drought, osmotic stress and temperature extremes. These proteins are believed to stabilize macromolecules during freeze-induced dehydration (Close 1996). The overall

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54 expression pattern of CORc115 was similar for both P. trifoliata and C. paradisi. Expression of CORc115 increased dramatically during cold acclimation and continued to increase for up to 10 days (Figure 4-5). If CORc115 performs a stabilizing function, then the expression would be expected to remain high during low temperatures in order to prevent damage from dehydration. While P. trifoliata CORc115 expression increased within 4 hours of transfer to 4C, C. paradisi expression did not noticeably increase until after 24 hours. The delay in CORc115 expression is likely just one small example of the genome-wide differences that are expected between P. trifoliata and C. paradisi during cold acclimation. In contrast to previous results (Cai et al. 1995), CORc102 and CORc510 were not induced by low temperatures in this study. This inconsistency could be attributed to potential differences in plant materials, environmental conditions, sampling procedures or laboratory techniques. In conclusion, dehydrins, peroxidases and genes involved in antioxidant metabolism are common stress-response genes. CORc410 and CORc115 may play important roles in cold acclimation, yet it is not likely that the expression of these genes alone can account for the dramatic differences in the cold tolerance of Poncirus and Citrus. In addition to low temperature stress, CORc115, CORc102, CORc410 and CORc510 may play significant roles in other stress responses. The identification and full characterization of genes with potential roles in multiple stress pathways should assist in identifying mechanisms of crosstalk between these pathways. Furthermore, as components of multiple stress responses, these genes present potential targets for plant improvement.

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55 Materials and Methods Rapid Amplification of cDNA Ends (RACE) Poncirus trifoliata (L.) Raf. seedlings were grown at 4C for 14 days prior to tissue collection. Leaves were collected, immediately frozen in liquid nitrogen, and stored at -80C. Leaf tissue was ground to a fine powder with a mortar and pestle in the presence of liquid nitrogen. TRIzol reagent (Invitrogen) was used to extract total RNA. Briefly, 1.5 ml TRIzol reagent was added to approximately 250 mg ground tissue. Samples were centrifuged for 10 minutes at 12,000 g at 4C to remove particulate matter. Three hundred microliters of chloroform was added to the supernatant, mixed well by shaking, and incubated at 25C for 3 minutes. Samples were centrifuged for 15 minutes at 12,000 g at 4C to separate the phases. The upper phase was removed to a new tube and 750 l isopropanol was added to precipitate the RNA. Samples were incubated at 25C for 10 minutes, followed by centrifugation for 10 minutes at 12,000 g at 4C. The pellet was washed with 75 % ethanol and resuspended in 30 l TE buffer. The SMART RACE cDNA Amplification Kit (Clontech) was used to obtain the 5’ cDNA ends. According to the protocol, cDNA was prepared from 1 g total RNA extracted from plants treated at 4C for 14 days. The 5’ RACE-ready cDNA was diluted with 100 l Tricine-EDTA buffer (10 mM Tricine-KOH pH 8.5, 1 mM EDTA). All RACE PCR was performed according to the manufacturer’s protocol using 2.5 l prepared cDNA. CORc102 was amplified using 0.2 M KC24 (5’GGCATCGGTCACGATGAATGAAACGGCTTCTCTCTGG3’) and the following cycles: 5 cycles 94C 5 seconds/ 72C 2 minutes, 5 cycles 94C 5 seconds/70C 10 seconds/72C 2 minutes, and 25 cycles 94C 5 seconds/68C 10 seconds/72C 2 minutes. The 5’ end of CORc410 was amplified using 0.2 M KC25

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56 (5’CAGCTATTAGCTAGCTAGGACTTGTCGTGGAGC3’) and the following cycles: 5 cycles 94C 5 seconds/ 72C 2 minutes and 38 cycles 94C 5 seconds/70C 10 seconds/72C 2 minutes. The 5’ end of CORc510 was amplified through a series of RACE reactions using different sequence specific primers. CORc510 RACE reactions were performed with 0.2 M KC26 (5’CCAGGCACTAGAACATTGTTCTCACCATTTGGGATGTCC3’) and KC44 (5’ GAGCAGGCACCAGGGAAAGCGCCG3’). The reaction conditions were 5 cycles 94C 5 seconds/ 72C 2 minutes, 5 cycles 94C 5 seconds/70C 10 seconds/72C 2 minutes, and 25-35 cycles 94C 5 seconds/68C 10 seconds/72C 2 minutes. All reactions were visualized by electrophoresis of 5 l in a 1 % agarose TAE (40 mM Tris-acetate pH 7.6, 1 mM Na 2 EDTA) gel. To clone RACE products, the remaining PCR reaction was run in a 1 % agarose TAE gel. The band was excised and purified using the Gel Extraction Kit (Qiagen). Seven microliters of purified RACE product was combined with 50 ng pGEM-T Easy vector (Promega) and ligated overnight by incubation at 4C with 3 units T4 DNA Ligase (Promega). Ten microliters of ligation reaction was transformed into 100 l chemically competent E.coli DH5 cells. Transformants were selected by plating on 2XYT (16 g/L tryptone, 10 g/L yeast extract, 5 g NaCl, 15 g/L agar) media supplemented with 100 mg/L ampicillin and 20 mg/L X-gal (5-bromo-4-chloro-3-indolyl--D-galactoside). Minipreps of positive clones were prepared and sequenced. All DNA sequencing was performed by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core Facility.

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57 Nucleotide and protein sequences were analyzed using Vector NTI Suite 8 (Invitrogen). Sequence homology was analyzed using BLAST similarity searches at the National Center for Biotechnology Information ( www.ncbi.nlh.nih.gov ) (Altschul et al. 1990; Altschul et al. 1997). Alignments were made using CLUSTAL (Thompson et al. 1997). Protein targeting was analyzed by TargetP (Emmanuelsson et al 2000). Northern Blots Northern blot experiments were conducted using two-year old P. trifoliata (L.) Raf. and C. paradisi Macf. (cv. Duncan) seedlings with new growth flushes. The plants were treated at 4C with a 16 hour photoperiod. Following transfer to 4C, leaves were collected at specific time points, frozen immediately in liquid nitrogen, and stored at -80C. Total RNA was extracted using TRIzol reagent (Invitrogen) as described previously. Three to ten micrograms of total RNA was used for all blots. RNA samples were combined with two volumes of freshly made Loading Buffer [50 % deionized formamide, 6 % formaldehyde, 1X MOPS (20 mM morpholineopropanesulfonic acid, 5 mM Na acetate, 2 mM EDTA), 0.0005 % bromophenol blue]. RNA samples were denatured prior to electrophoresis by incubation at 65C for 10 minutes. Samples were loaded onto a 1.5 % agarose gel (2 % formaldehyde, 1X MOPS) and run in 1X MOPS buffer until the bromophenol blue dye had migrated two-thirds through the gel. To estimate RNA quantity and quality, the gel was stained by incubating in 0.25 g/ml ethidium bromide for 10 minutes and visualized by exposure to UV light. To remove formaldehyde from the gel before transfer, the gel was soaked twice (2 x 15 minutes) in 20X SSC. 20X SSC was used to transfer the DNA from the gel to a positively-charged nylon membrane by capillary transfer. The transfer was allowed to proceed for

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58 approximately 20 hours. The DNA was crosslinked to the membrane using a UV Stratalinker 1800 (Stratagene). Antisense RNA probes were used for all northern blots. cDNA was prepared from RNA extracted from P. trifoliata seedlings treated for 14 days at 4C using TRIzol reagent (Invitrogen). cDNA was synthesized using the First Strand Synthesis Kit for RT-PCR (Ambion). Two micrograms of total RNA was combined with 100 mol random decamers in 12 l water. The samples were denatured by incubation at 85C for 3 minutes and immediately transferred to ice. Two microliters of 10X RT buffer (500 mM Tris-HCl pH 8.3, 750 mM KCl, 30 mM MgCl 2 , and 50 mM DTT), 4 l 2.5 mM dNTPs, 10 units RNase Inhibitor, and 100 units MMLV Reverse Transcriptase were all added to the tube. The tube was incubated at 42C for 1 hour, followed by 92C for 10 minutes. To prepare the transcription template, RT-PCR primers were designed complementary to the sequences to be transcribed and the sequence of the T7 RNA polymerase promoter. Primers used for the probe preparation were: CORc410, KC81 (5’TAATACGACTCACTATAGGGGGACTTGTCGTGGAGCTTG3’) and KC82 (5’GTGAAGAGTAGAAGAGAAAATGG3’); CORc115, KC91 (5’GATCGGAGAAGCCCTTCACG3’) and KC92 (5’TAATACGACTCACTATAGGGGCAAATTACACACCGACTGGG3’); CORc102, KC93 (5’AACAAAATGCTGAAAGCAGTTGC3’) and KC94 (5’TAATACGACTCACTATAGGGACAGTGCATAGAACTCCTTGAC3’); and CORc510, KC83 (5’TAATACGACTCACTATAGGGGGATGTCCTTACCATTTATTTGC3’) and KC84 (5’GGATAATGGGTCATCTAAGGC3’). One unit of Taq DNA Polymerase (Promega)

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59 was used to amplify coding sequence from 200 ng cDNA in a reaction containing 0.2 M each gene-specific primer, 200 M each dNTP, 2.5 mM MgCl 2 , and 1X PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X-100). Cycling was as follows: 94C 3 minutes, 30 cycles of 94C 1 minute/55C 1 minute/72C 1 minute, and 72C 10 minutes. Ten microliters of each reaction was analyzed by electrophoresis in a 1 % agarose TAE gel. The probe was labeled by in vitro transcription using the DIG RNA Labeling Kit (Roche Molecular Biochemicals). Four microliters of the RT-PCR reaction was combined with 2 l 10X Transcription Buffer (0.4 M Tris-HCl pH 8.0, 60 mM MgCl 2 , 100 mM dithiothreitol, 20 mM spermidine, and 100 mM NaCl), 1 mM CTP, 1 mM GTP, 1 mM ATP, 0.65 mM TTP, 0.35 mM DIG-11-UTP, 20 units RNase Inhibitor and 40 units T7 RNA Polymerase in a 20 l reaction volume. The reaction was incubated at 37C for 2 hours. To terminate the reaction, 2 l 0.2 M EDTA was added. All northern blot hybridization and detection was performed according to the manufacturer’s protocol (Roche Molecular Biochemicals). Membranes were prehybridized at 68C for 30 minutes in reconstituted DIG Easy Hyb (Roche Molecular Biochemicals). Two microliters of labeled probe was combined with 50 l water, denatured at 100C for 5 minutes, and chilled on ice. The denatured probe was added to 10ml prewarmed (68C) DIG Easy Hyb. The prehybridization solution was poured off the membrane and the hybridization solution was added. All hybridizations were performed at 68C overnight. Stringency washes and detection were performed according to the standard protocol. Following detection, the probes were stripped from the membranes by washing (2 X 60 minutes) at 80C with a solution of 50 % deionized

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60 formamide/5 % SDS/50 mM Tris-HCl pH 7.5. The stripped membranes were re-probed with a DIG-labeled 18S RNA probe.

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CHAPTER 5 ISOLATION AND CHARACTERIZATION OF LOW TEMPERATURE-RESPONSIVE PROMOTERS IN Poncirus trifoliata (L.) Raf. Introduction Severe freezes in Florida and elsewhere cause millions of dollars of damage to citrus fruit and trees. In fact, freeze injury is likely the most important factor limiting citrus production in Florida, an area susceptible to some of the most historically damaging freezes to the world citrus industry. Although Citrus species are damaged by temperatures just below freezing, they do vary in their susceptibility to low temperatures. While C. grandis is subject to injury at temperatures just below 0C, C. sinensis, the most widely grown species, can withstand leaf temperatures of -7C for limited periods of time (Yelenosky 1985). Poncirus trifoliata, an interfertile citrus relative, can withstand -20C when fully acclimated. Although the molecular basis of these differences is unknown, an increased understanding should help to facilitate the improvement of citrus freeze tolerance and thus reduce fruit and tree losses in traditional growing regions. Cold acclimation, the accumulation of maximum freezing tolerance in response to specific stimuli, results in numerous biochemical and physiological changes in a plant cell. Studies in the 1980s indicated that changes in gene expression accompany cold acclimation (Guy et al. 1985) though the specific genes and mechanisms were unclear. A growing number of genes have since been identified, although many have known physiological functions (Hughes and Dunn 1996; Pearce 1999). Microarray studies have begun to shed light on the vast quantities of genes involved in coping with low 61

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62 temperature extremes. One such study has indicated that as much as 4 % of the Arabidopsis genome may be affected by low temperature exposure (Fowler and Thomashow 2002). The cold acclimation response is accepted to be a cascade of interactions that occur following the perception of a low temperature stimulus. Understanding the molecular basis of cold acclimation requires knowledge of the signal transduction pathways responsible for producing the vast transcriptome changes. Transcription is regulated by the interaction of transcription factors with precise cis-acting regulatory elements (CAREs). CAREs, short conserved motifs present in a gene’s 5’ regulatory sequence or promoter, recruit specific proteins including basal transcriptional machinery, as well as transcriptional activators and repressors. More than 5 % of the Arabidopsis genome is believed to be involved in transcription, and over 1500 genes are expected to encode transcription factors (Arabidopsis Genome Initiative 2000). Although 48 cold-responsive genes encoding known or putative transcription factors have been identified by microarray analysis (Fowler and Thomashow 2002), knowledge of their specific promoter binding sites is limited. The identification of specific cold-induced genes and their promoters facilitated the identification of the first low temperature-responsive element (LTRE). The cis-acting C-repeat (CRT)/dehydration-responsive element (DRE) was identified independently by two different research groups (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1994). These groups, working separately on dehydrationand low temperature-induced gene expression in Arabidopsis, studied the expression of the RD29A (also known as COR78 and LTI78) and COR15A genes, respectively. Using similar approaches, the

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63 promoters of the respective genes were isolated and analyzed for their ability to facilitate low temperature-induced expression of a reporter gene. By analyzing promoter deletions, these experiments revealed which promoter regions retained the capacity for low temperature-induced gene expression. The CRT/DRE is characterized by a core of five conserved bases, CCGAC. This element has been found in the promoters of other Arabidopsis COR genes, including, but not limited to, COR6.6, COR47, KIN1 and ERD10 (Thomashow et al. 2001). These Arabidopsis genes are coordinately stimulated by low temperature, and genomic approaches using microarrays have identified 45 CRT/DRE-regulated genes in Arabidopsis (Seki et al. 2001; Fowler and Thomashow 2002). Furthermore, the CRT/DRE-regulated genes include both transiently induced and long-term induced genes. In addition to Arabidopsis, CRT/DRE-like elements also regulate expression of low temperature-induced genes in other species. Specifically, the CRT/DRE core is present in the promoters of Brassica napus BN115 (Jiang et al. 1996), Triticum aestivum WSC120 (Ouellet et al. 1998) and WCOR15 (Takumi et al. 2003), Hordeum vulgare BLT4.9 (Dunn et al. 1998) and a number of H. vulgare dehydrins (Choi et al. 1999). CRT/DRE binding factors (CBFs) are a small family of AP2 domain-containing proteins that activate transcription through specific interactions with the CRT/DRE. Transcript levels of the CBF genes can increase within 15 minutes of exposure to low temperature, and this is followed by the induction of CRT/DRE-containing COR genes (Gilmour et al. 1998). CBFs have been proposed to integrate the activation of multiple components of the cold acclimation response. CBF3-overexpressing plants mimic multiple biochemical changes that occur in plants during cold acclimation (Gilmour et al.

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64 2000). CBF3 overexpression results in the accumulation of COR transcripts and polypeptides, accumulation of proline, and the accumulation of soluble sugars. All of these changes are believed to contribute to enhanced freezing tolerance. However, not all cold-inducible genes contain the CRT/DRE element in their promoters. In addition to the CBF regulon, Arabidopsis contains other pathways that contribute to cold tolerance (Fowler and Thomashow 2002). The expression of many COR genes is increased by drought or abscisic acid (ABA) treatment. Moreover, drought and ABA treatment can result in an increase in cold tolerance. These observations indicated that cold acclimation must also include pathways that are dependent on ABA. Careful analysis of many ABA-responsive genes has identified CAREs involved in mediating ABA-regulated gene expression (ABA-response elements, ABREs). ABREs have been identified in the promoters of many ABA-responsive COR genes, including COR15A (Baker et al. 1994) and RD29A (Yamaguchi-Shinozaki and Shinozaki 1994), genes which are also controlled by the CRT/DRE. Additionally, ABRE binding factors (ABFs) have not only been cloned (Guiltinan et al. 1990; Uno et al. 2000; Kang et al. 2002), but some are themselves induced by cold (Choi et al. 2000). Moreover, MYB and MYC recognition sites are also involved in the cold-induced transcription of some genes (Iwasaki et al. 1995; Abe et al. 1997). To date, COR genes are known to be regulated by CRT/DRE, ABRE, MYB and MYC cis elements by means of specific transcription factors. However, genomic profiling experiments indicate that several regulatory networks are involved in adaptation to low temperatures (Fowler and Thomashow 2002). Therefore, more CAREs and their corresponding transcription factors remain to be identified.

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65 Previous research indicates that the mechanism of cold acclimation in Arabidopsis and Citrus is conserved. Citrus species can cold acclimate, and gene expression changes during cold acclimation have been documented in both Citrus species and Poncirus trifoliata (Durham et al. 1991; Cai et al. 1995). QTL studies have shown that cold hardiness in citrus does segregate (Weber et al. 2003), and specific cDNAs representing unique cold-induced sequences have been cloned from P. trifoliata (Cai et al. 1995). The mechanisms of cold induced gene activation in woody perennial plants, including P. trifoliata and Citrus species, have not been extensively studied, nor have low-temperature response elements (LTREs) have not been identified in these species. Woody perennials and herbaceous plants are expected to contain both common and unique mechanisms for withstanding low temperatures. If the mechanism of cold acclimation is conserved, Poncirus COR promoters are expected to contain LTREs responsible for their low-temperature activation. The goal of this study is to isolate and characterize stress-responsive promoters from P. trifoliata with the ultimate goal of identifying LTREs. Results Nuclear Run-On Assay P. trifoliata CORc115 expression increases during cold acclimation (Cai et al. 1995). However, in some cases, an increase in transcription is not the primary cause of the accumulation of mRNA during cold acclimation. Rather, mRNA accumulation is regulated post-transcriptionally. To distinguish between these two mechanisms, nuclear run-on assays were performed (Figure 5-1). CORc115 transcription increased relative to 18S rRNA transcription within 4 hours of cold treatment, indicating that CORc115 is indeed transcriptionally induced.

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66 Figure 5-1. Nuclear run-on assay of CORc115. Nuclei were extracted from leaves treated at 4C for the times indicated above the membrane. NA=not acclimated. COR Promoter Isolation and Characterization To better understand the mechanism of gene expression during cold acclimation, inverse PCR (IPCR) was used to isolate 5’ regulatory sequence of Poncirus COR genes (Ochman et al. 1988; Triglia et al. 1988; Cai et al. 1995) (Figure 5-2). Promoter sequence was successfully isolated for Poncirus CORc115, CORc119, CORc102, CORc410 and CORc510. Following the isolation of 5’ regulatory sequence, the promoters were analyzed using a variety of databases (Higo et al. 1999; Rombauts et al. 1999; Wingender et al. 2000) and programs (Bailey and Elkan 1994). All promoters contained common promoter features including TATA boxes and CAAT boxes. The isolated CORc115 promoter includes 1248 bp of sequence upstream of the putative start of translation (Figure 5-3). Statistically significant motifs were predicted by MEME (Table 5-1) including 5 imperfect copies of a 15 bp motif (E-value 7.9e+002), 4 imperfect copies of a 31 bp motif (E-value 2.7e+003), 2 perfect copies of a 7 bp motif (GATGGCG) (E-value 7.4e+003), 2 imperfect copies of a 6 bp motif (E-value 3.8e+003) and 4 imperfect copies of a 28 bp motif (E-value 7.4e+003). In addition, searches for regulatory elements identified many potential CAREs including 1 CRT/DRE, 4 ABREs, 9 MYB sites, 3 MYC sites and 5 TCA elements (Table 5-2).

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67 Figure 5-2. Illustration of inverse PCR. A) Pictorial representation of inverse PCR. Black lines represent genomic DNA with specific restriction sites indicated by green triangles; orange boxes represent known sequence; arrows represent the locations of PCR primers. B) Example of PCR results using different template concentrations. The isolated CORc119 promoter includes 687 bp of sequence upstream of the putative translation start codon (Figure 5-4). MEME predicted six significant motifs including 2 imperfect copies of a 42 bp motif (E-value 8.3e+002), 2 imperfect copies of an 8 bp motif (E-value 2.0e+003), 2 perfect copies of a 9 bp motif (E-value 3.4e+003), 2 near perfect copies of a 15 bp motif (E-value 4.3e+003) and 2 imperfect copies of an 11 bp motif (E-value 5.2e+003)(Table 5-1). Regulatory element searches identified many potential CAREs including 1 CRT/DRE, 3 ABREs, 4 MYB sites, 2 MYC sites and 5 TCA elements (Table 5-2). The isolated CORc102 promoter includes 1399 bp of sequence upstream of the predicted translation start codon (Figure 5-5). MEME sequence analysis identified an imperfect 15 bp motif present in five copies (E-value 1.2e+001) and two different 6 bp motifs, CACCGG and GGCACG, present in two copies each (both E-value 8.1e+003)

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68 (Table 5-1). CARE predictions are summarized in Table 5-2. Specifically, 1 CRT/DRE, 4 ABREs, 13 MYB sites, 3 MYC sites and 3 TCA elements were identified. Figure 5-3. CORc115 Promoter Sequence. Solid double lines indicate the TATA box; asterisks indicate CRT/DREs; dashed double lines indicate ABREs; solid single line indicates MYB sites; dotted line indicates MYC sites; crosses indicate TCA sites; black arrow indicates the start of translation; boxes represent selected motifs (motif 1, blue; motif 2, red; motif 3, green; motif 4, orange; and motif 5, purple). All numbering is relative to the putative start of translation.

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69 The isolated CORc410 promoter includes 1767 bp of sequence upstream of the putative start of translation (Figure 5-6). MEME predicted an 8 bp motif GCTGCCCG present in two direct repeats (E-value 2.5e+003) (Table 5-1). Table 5-2 summarizes the CAREs predicted. Notably, the CORc410 promoter contains 3 CRT/DREs, 2 ABREs, 10 MYB sites, 7 MYC sites and 7 TCA elements. The isolated CORc510 promoter includes 1495 bp of sequence upstream of the putative start of translation (Figure 5-7). MEME predicted an imperfect 28 bp motif (E-value 4.1e+003) with five sites. Additionally, a 7 bp motif (E-value 8.4e+003), consensus GGGAGCC, was present in two copies (Table 5-1). Table 5-2 summarizes the predicted CAREs, including 2 ABREs, 12 MYB sites, 8 MYC sites and 4 TCA elements. Figure 5-4. CORc119 Promoter Sequence. Solid double lines indicate the TATA box; asterisks indicate CRT/DREs; dashed double lines indicate ABREs; solid single line indicates MYB sites; dotted line indicates MYC sites; crosses indicate TCA sites; black arrow indicates the start of translation; boxes represent selected motifs (motif 1, blue; motif 2, red; motif 3, green; motif 4, orange; and motif 5, purple). All numbering is relative to the putative start of translation.

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70 Figure 5-5. CORc102 Promoter Sequence. Solid double lines indicate the TATA box; asterisks indicate CRT/DREs; dashed double lines indicate ABREs; solid single line indicates MYB sites; dotted line indicates MYC sites; crosses indicate TCA sites; black arrow indicates the start of translation; boxes represent predicted motifs (motif 1, blue; motif 2, red). All numbering is relative to the putative start of translation.

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71 Figure 5-6. CORc410 Promoter Sequence. Solid double lines indicate the TATA box; asterisks indicate CRT/DREs; dashed double lines indicate ABREs; solid single line indicates MYB sites; dotted line indicates MYC sites; crosses indicate TCA sites; black arrow indicates the start of translation; boxes represent predicted motifs (motif 1, red). All numbering is relative to the putative start of translation.

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72 Figure 5-7. CORc510 Promoter Sequence. Solid double lines indicate the TATA box; dashed double lines indicate ABREs; solid single line indicates MYB sites; dotted line indicates MYC sites; crosses indicate TCA sites; black arrow indicates the start of translation; boxes represent predicted motifs (motif 1, blue; motif 2, red). All numbering is relative to the putative start of translation.

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73 Table 5-1. Poncirus trifoliata COR promoter motifs predicted by MEME analysis. Predicted MotifsllrE-valueSequenceLocationStrandCORc115 Motif 1747.9e+002 tcGGCGGCGGCG AGGGAAGAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAGAGAGAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAGAGAAAAAGGAAACCAAAAAGAAAAGAAAAGAAAAGGAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAGGAGAGAAGAAGAGAGAAGGAAGAAAAAAGGAAAAAAAAGAAAAAAAAGAGAAAAGAAAAAAAAGAAAAGAAAG Cacagaagata-1245+gattacaaagCTGGCGGTTCGGCttttgatggc-1200-attacgtgtaCGCGTCTTCCTGGCtttttgggta-279-aatctatctaCGTGTCGGCTTTCaaactacgat-1169+gatgatgcaaCGTGTCGTTTGGCttggcagtat-322+CORc115 Motif 21072.7e+003aaaataatctTTCTTCCTTTCTCCCCTCCTTTtggagcctat-165+tgtcgatattCTCCTTCTTTCGTCGTCTCCCTCTctatattttc-623+ccctttacccTTCGTCCTTTGTTCCGCTCGCTTTtgcacatttt-1075+cttgtttgctTTTTCTCTTCTTCTTCTTGTTCTTGTTGTgaatatcgta-92-CORc115 Motif 3237.4e+003aacgatatttGTGGCGaaattaatac-695-tcggacttttGTGGCGaataaatgat-1211-CORc115 Motif 4193.8e+003agacgaaaatGGCGGGagttattcac+3+tacacgtaatGGGGGCaaaagagtaa-254+CORc115 Motif 5967.4e+003aaatttatttCCCTCCCTTCTTTCctgaatcaaa-875-tggacaactaCTCCCCGCTCCTGTGTGCaacgtgtcga-352+aagattaagaTCTGTTCTGGCCCTTCTTTCGattaacaaat-534+agactgttaaCCGGCTGCTTTCCTCTGTTtttagtttcc-778+CORc115 Motif 6538.5e+003cattgtctttCTTTTCTGGGCaacaataaat-1022-actaacaaaaCTTTCTTGGCcctttaatta-944+aatatcgtaaGGTGTCTTGGCttggtattta-115-aacggataatCTTTTCTTCCCaaaatcagaa-215+CORc119 Motif 1978.3e+002cccatgaagaCGCGTCCGTTGGGGGCTTCgaaaggataa-264+agtagaaaatCGCCTCTTTCGTTGCCtttcttttta-58+CORc119 Motif 2242.0e+003ttgtgaataaCTCCCGCCattctctctt+3-cctaattctaGTCCCTCCttttagagcc-142+CORc119 Motif 3253.4e+003ctaggatttcTGGCCTacatacagca-349+aatatcgttaTGGCCTgattctcaac-665+CORc119 Motif 4384.3e+003agttgtccatCGTTTTGTGGcgaaa-681-attgtcaactCGTTTTGTTGtctgacaggt-645+CORc119 Motif 5285.2e+003acatacagcaCCGCTCCTgatgatgcaa-330+aaatactgttCTGGCCCTtctattcgat-512+CORc119 Motif 6225.4e+003aacaaatataTGCCGTataattaaat-480+tttgattgtcTGCTgtcgatatta-628+CORc102 Motif 1791.2e+001agttccagttTTCCCTCCCTtactgatttc-752+ctcttataaaTTTGTCCCCCCTaaaccaatct-929+aatctgtaacTTCCCTCCCaaaccatatt-798-tacaagcattGTTGTCCCCGTctatgttgca-992-ccatcatttaTTGGCCGCCCGTgttattgcct-1185+CORc102 Motif 2208.1e+003gtaacacttcCCCGGcatccgatgt+31-gaactgacttCCCGGtgtgcttgaa-1113+CORc102 Motif 3208.1e+003gcaaggctaaGGCCGaactttaaac-269-acacctgtttGGCCGcgactttggt-359+CORc410 Motif 1272.5e+003gctgcccgctGCTGCCCGaaatcttaat-467+atgttcactcGCTGCCCGctgctgcccg-477+CORc510 Motif 11154.1e+003cctagtcttcTGTCTCCTTTTTTCTCTCctaattggtt-839+tacagtctttTGGGTCCTTCTTTCCCTCctagtcttct-876+atagatgagcTGTCTCTCCCGCTTGCCCTTCagcctttaag-63-atgtcactacTGGCCCCGTTGGCTCCTCCTCcaacaaggga-933-actttgttcaTTGCTCGCCCTCCCCCTtgcaggtaaa-1379-CORc510 Motif 2228.4e+003agccttttctGGGCCagtacttaat-678+gtactggttgGGG A G CCtctatctttt-1174+

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74 Table 5-2. Summary of predicted cis-acting regulatory elements present in Poncirus trifoliata COR promoters. PromoterSite NameFunctionCopiesCORc115ABREAbscisic acid responsiveness4EREEthylene responsiveness3HSEHeat stress responsiveness12LTRELow-temperature responsiveness1MYB siteDrought-inducibility9MYC siteDrought-inducibility3P-boxGibberellin-responsive element4TCA elementSalicylic acid responsiveness/cold responsive5WUN-motifWound-responsive element8Multiple sitesLight responsivenessmanyCORc119ABREAbscisic acid responsiveness3EREEthylene responsiveness1HSEHeat stress responsiveness2LTRELow-temperature responsiveness1MYB siteDrought-inducibility4MYC siteDrought-inducibility2P-boxGibberellin-responsive element4TCA elementSalicylic acid responsiveness/cold responsive5WUN-motifWound-responsive element4Multiple sitesLight responsivenessmanyCORc102ABREAbscisic acid responsiveness4GC motifAnoxic specific inducibility4HSEHeat stress responsiveness10LTRELow-temperature responsiveness1MYB siteDrought-inducibility13MYC siteDrought-inducibility3P-boxGibberellin-responsive element2TATC-boxGibberellin-responsive element3TCA elementSalicylic acid responsiveness/cold responsive3TGACG-motifMeJA-responsiveness1WUN-motifWound-responsive element12Multiple sitesLight responsivenessmanyCORc410ABREAbscisic acid responsiveness2Aux-RRAuxin responsiveness2GC motifAnoxic specific inducibility1EREEthylene-responsive element2HSEHeat stress responsiveness16LTRELow-temperature responsiveness3MYB siteDrought-inducibility10MYC siteDrought-inducibility7P-boxGibberellin-responsive element3TCA elementSalicylic acid responsiveness/cold responsive7TGACG-motifMeJA-responsiveness1WUN-motifWound-responsive element19Multiple sitesLight responsivenessmanyCORc510ABREAbscisic acid responsiveness2GC motifAnoxic specific inducibility2EREEthylene-responsive element1HSEHeat stress responsiveness8MYB siteDrought-inducibility12MYC siteDrought-inducibility8P-boxGibberellin-responsive element2TATC-boxGibberellin-responsive element1TCA elementSalicylic acid responsiveness/cold responsive4WUN-motifWound-responsive element9Multiple sitesLight responsivenessman y

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75 Discussion The differential expression of genes depends on the interaction of specific transcription factors with specific CAREs. As a result, eukaryotic promoters contain numerous CAREs specifying the timing and pattern of gene expression. Identification of promoter sequences and characterization of these regulatory elements is of utmost importance in unraveling the mechanisms of gene induction in response to a vast number of specific stimuli, including environmental stresses. Not all increases in transcripts, however, are due to increases in transcription. Post-transcriptional regulation is responsible for low temperature-induced increases in transcript abundance in many species. For example, H. vulgare BLT14.0 transcripts are believed to be stabilized by a protein factor (Phillips et al. 1997). In addition, glycine-rich RRM (RNA-recognition motif)-containing proteins, proteins assumed to play a role in transcript stability, are cold-induced in many species, including A. thaliana (Carpenter et al. 1994) and H. vulgare (Dunn et al. 1996). Transcripts of several P. trifoliata genes have been shown to accumulate during cold acclimation (Cai et al. 1995). This study demonstrates that the transcript accumulation of at least one of these genes, CORc115, is due to an increase in transcription (Figure 5-1). Transcriptional induction during cold acclimation is controlled by specific promoter elements. As a result, CORc115 is a good candidate for the identification of potential CAREs responsible for low temperature-induction in Poncirus. For this purpose, 5’ regulatory sequence was isolated for CORc115 and several other identified Poncirus COR genes. Analysis of the COR promoter sequences identified many potential CAREs (Table 5-2), some of which have been demonstrated previously to be involved in low

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76 temperature-induced gene expression. While MEME predicted multiple motifs within each COR promoter, none of these motifs resembled previously identified LTREs (Table 5-1). In contrast, putative LTREs were predicted by PLACE, PlantCARE and Transfac. Of the five promoter sequences analyzed, all but one (CORc510) show homology to previously identified LTREs. CORc115 and CORc410 each contain one copy of the Arabidopsis COR15A LTRE, CCGAC (Baker et al. 1994). The COR15A LTRE, which forms the core of the Arabidopsis CRT/DRE, is present in the promoters of many low temperature-induced Arabidopsis genes (Thomashow et al. 2001). In addition to Arabidopsis, this LTRE has been identified in cold-inducible promoters of both dicots and monocots, including B. napus BN115 (White et al. 1994), and T. aestivum WSC120 (Ouellet et al. 1998) and WCOR15 (Takumi et al. 2003). The promoter of H. vulgare BLT4.9, a cold-inducible non-specific lipid transfer protein, contains a similar LTRE, CCGAAA (Dunn et al. 1998). CORc119 and CORc102 each contain one copy of the H. vulgare LTRE, while CORc410 contains two copies. The presence of these LTRE core sequences in multiple cold-induced Poncirus promoters suggests similar LTREs may function in both herbaceous and woody perennial plants. Signaling during cold acclimation involves multiple mechanisms, including both ABA-independent and ABA dependent pathways (Gilmour and Thomashow 1991) (Kurkela and Franck 1990; Nordin et al. 1991). The CRT/DRE pathway represents an ABA-independent pathway (Yamaguchi-Shinozaki and Shinozaki 1994). However, many Arabidopsis CRT/DRE-regulated genes contain additional CAREs which may also regulate gene expression at low temperatures. RD29A, KIN1, COR6.6 and COR47 contain CRT/DRE and ABRE motifs (Thomashow 1999; Seki et al. 2001). In addition,

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77 ABREs have been found in cold-inducible promoters that are not part of the CBF regulon (Nordin et al. 1993; Welin et al. 1994; Yamaguchi-Shinozaki and Shinozaki 1994). Endogenous ABA levels increase transiently during cold acclimation, and these increased ABA levels regulate gene expression through ABREs. In general, promoters containing multiple ABRE motifs are responsive to ABA, while promoters containing a single ABRE motif are not ABA-responsive (Leung and Giraudat 1998). All of the promoters isolated in this study contain multiple ABRE motifs (Table 5-2), indicating that they may also be ABA-inducible. However, these promoters have not been tested for ABA-inducibility. It is also important to note that the ABRE is only a subset of elements defined by an ACGT core (Busk and Pages 1998). Functional analysis of these elements will be needed to confirm an ABA-responsive function. Numerous copies of MYB and MYC binding sites were present in all of the promoters. MYB and MYC recognition sites function cooperatively in the ABA and dehydration-induced expression of Arabidopsis RD22 (Abe et al. 1997), while a single MYC binding site is necessary for the ABA-induction of AtADH1 (de Bruxelles et al. 1996). ICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, recognizes multiple MYC sites present in the AtCBF3 promoter leading to increased AtCBF3 expression in the cold (Chinnusamy et al. 2003). Additionally, a cold-responsive region of the Arabidopsis CBF2 promoter, ICE r1, contains a MYC recognition site (Zarka et al. 2003). Multiple TCA elements, originally defined as enhancer-like elements present in the promoters of salicylic acid (SA)-inducible genes (Goldsbrough et al. 1993), were found in all of the isolated promoters (Table 5-2). A novel role of TCA-like elements in cold

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78 induction was detailed in studies of the H. vulgare BLT101.1 promoter (Brown et al. 2001). A single TCA-like element, AAGAAGATCG, functions in the low temperature-induction of BLT101.1. While low temperature signaling has been most studied in Arabidopsis, accumulating research indicates that the molecular mechanisms of cold acclimation are highly conserved among plants. These results lend support that cold acclimation pathways in woody perennials parallel those in herbaceous plants. However, it is expected that the process of cold acclimation in woody perennials will also contain unique characteristics. Functional experiments will be needed to confirm the identified promoter elements. In addition, these functional studies may also uncover new LTREs unique to woody perennial plants. Materials and Methods Nuclear Run-On Assay Transcriptionally active nuclei were isolated as described by Kanazawa et al. (2000). Leaves collected from P. trifoliata seedlings were treated at 4C (0, 30 minutes, 4 hours) and frozen immediately in liquid nitrogen. Three milliliters of extraction buffer (1.14 M sucrose, 10 mM Tris-HCl pH 7.6, 5 mM MgCl 2 , 0.1 mM phenylmethylsulfonylfluoride, 0.1 mM 1,10-phenanthroline and 0.1 % thiodiglycol) was added to approximately 1 gram finely-ground leaf tissue and mixed thoroughly by inversion. The suspension was filtered through two layers of Miracloth and carefully transferred onto a 2 ml sucrose cushion (2 M sucrose, 10 mM Tris-HCl pH 7.6 and 5 mM MgCl 2 ). The tube was centrifuged for 5 minutes at 1800 g. The layer/pellet above the cushion was transferred to new tube. Two milliliters of extraction buffer containing 0.15 % Triton X-100 was added to the pellet and mixed thoroughly using a wide bore pipet.

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79 The mixture was incubated on ice for 30 minutes, and the suspension was gently transferred onto a 2 ml sucrose cushion. The tube was centrifuged for 5 minutes at 1800 g. The layer/pellet above the cushion was transferred to 2 ml extraction buffer containing 0.15 % Triton X-100. The solution was mixed carefully with a wide bore pipet. The nuclei were pelleted by centrifugation at 1000 g for 5 minutes. The supernatant was discarded and the pellet was resuspended in a minimal volume of nuclear storage buffer (50 mM Tris-HCl pH 7.8, 10 mM 2-mercaptoethanol, 20 % glycerol, 5 mM MgCl 2 and 0.44 M sucrose). Nuclei were stored at -80C. The nuclear run-on assay was performed according to Folta and Kaufman (2000). Fifty microliters of isolated nuclei were incubated with 20 units of RNasin (Ambion) at 30C for 60 minutes. Fifty-five microliters of pre-warmed (30C) transcription assay reaction mixture [21 l 5X transcription assay buffer (250 mM Tris-HCl pH 7.8, 375mM NH 4 Cl, 50 mM MgCl 2 and 50 % glycerol), 10 l 100 mM CTP, 10 l 100 mM UTP, 10 l 100 mM ATP and 5 l 32 P GTP (3000 Ci/mmol)] was added to the nuclei. The reaction proceeded at 30C for 30 minutes. Ten units of DNase I (Ambion) was added and the tube was incubated at 30C for 10 additional minutes. To terminate the reaction, 200 l of termination buffer (7.5 M urea, 0.5 % SDS, 20 mM EDTA pH 7.5 and 100 mM LiCl) was added. Following transcription, a brief RNA extraction was performed. Three hundred microliters of phenol: chloroform was added and the reaction was mixed by vortexing. The phases were separated by centrifuging at 13,000 g for 5 minutes. The aqueous phase was mixed with 100 l 4 M ammonium acetate containing 200 g/ml yeast tRNA. Two volumes of 100 % ethanol were added, and the RNA was precipitated by incubation in a dry ice/ethanol bath for 10 minutes. The RNA was pelleted by

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80 centrifuging at 13,000 g for 10 minutes. The pellet was dried and resuspended in 500 l hybridization buffer (0.25 M NaH 2 PO 4 , 7 % SDS and 100 g/ml yeast tRNA). To determine the relative rate of transcription of specific nuclear genes, gene specific PCR bands were amplified, purified with the PCR Purification Kit (Qiagen), and quantified by measuring the absorbance at 260 nM. Three hundred nanograms of DNA was fixed to a positively-charged nylon membrane by crosslinking using a UV Stratalinker 1800 (Stratagene). Prehybridization (0.25 M NaH 2 PO 4 , 7 % SDS and 100 g/ml yeast tRNA) was performed at 42C for 24 hours. Membranes were hybridized with 32 P GTP-labeled RNA overnight at 42C in a rotary incubator. Two (2 x 15 minutes) low stringency washes (6X SSC, 0.1 % SDS) were performed at 25C, and two (2 x 15 minutes) high stringency washes (0.1X SSC, 0.1 % SDS) were performed at 55C. Signals were detected by exposure to autoradiography film. Isolation of Promoters Inverse PCR was used to isolate the 5’ regulatory regions of the P. trifoliata COR genes (Ochman et al. 1988; Triglia et al. 1988). Genomic DNA was isolated from the leaves of P. trifoliata seedlings using a CTAB extraction method (Dellaporta et al. 1983). The template was prepared by digesting 2 g of DNA with 10 units of restriction enzyme for 6 hours. Five hundred nanograms of digested DNA was self-ligated with 3 units T4 DNA Ligase (Promega) in a 100 l reaction volume at 14C overnight. Promoters were amplified in a 100 l inverse PCR reaction containing 1X Cloned Pfu Buffer [20 mM Tris-HCl pH 8.8, 2 mM MgSO 4 , 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1 % Triton X-100, and 100 ng/ml BSA], 200 M each dNTP, 250 nM each primer, 2.5 units Pfu Turbo (Stratagene) and 5 ng of prepared template. The PCR reactions were cycled at 94C 3 minutes; 35 cycles of 94C 1 minute, 56-65C 1 minute, and 72C 5 minutes; and 72C

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81 10 minutes. Table 5-3 lists the restriction enzymes, primers and annealing temperatures used to isolate each COR promoter sequence. CORc115, CORc119 and CORc410 promoter sequences were isolated in one-step, while two sequential inverse-PCR reactions were performed to isolate CORc102 and CORc510 promoter sequences. PCR reactions were analyzed Table 5-3. Restriction enzymes, primers and annealing temperatures used for the isolation of COR promoters. Promoter Restriction Enzyme Primers Tm CORc115 Cfo I KC2 (5’CGTGATACTGCTGCTGATGG3’) 65C KC12 (5’CTTGGTGGACAAGATCAAGC3’) CORc119 Hae III KC3 (5’CTCCTCAAGGTGGTTATTGC3’) 56C KC4 (5’GCTTGTCCTCCTCCTTTTGC3’) CORc102 Hinf I KC30 (5’GGAGACATAAGACTGGGTTGG3’) 60C KC31 (5’GGAAAAGGTAACACTTCCACCG3’) CORc102 Dra I KC41 (5’CCTCGTCTTGAGCTAAAACCG3’) 56C KC42 (5’CTTGTGGTGAAAAACTAAGCTGC3’) CORc410 Hind III KC28 (5’CAAGGATACATGCCCTCAGG3’) 60C KC29 (5’GGTCCTCTTCATTTTCTGCC3’) CORc510 Nsi I KC9 (5’GTGAGAACAATGTTCTAGTGC3’) 58C KC10 (5’GCAGATATCGAAATACCCTTG3’) CORc510 Taq I KC18 (5’GAAGGGATTTGTGGCACTGC3’) 60C KC19 (5’CCATTCTCAGCTTAATCGCG3’) by electrophoresis on a 1 % agarose TAE (40 mM Tris-acetate pH 7.6, 1 mM Na 2 EDTA) gel and amplified bands were purified using the Gel Extraction Kit (Qiagen). To increase the percentage of fragments containing dATP overhangs, an “A-Tailing” reaction containing 1X PCR Buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X

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82 100), 2.5 mM MgCl 2 , 250 M dATP, and 5 units Taq Polymerase (Promega) was incubated at 70C for 1 hour. The reactions were purified using the PCR Purification Kit (Qiagen) and eluted in 30 l EB buffer. Seven microliters of the purified reactions were ligated to 50 ng pGEM-T Easy (Promega) using 3 units T4 DNA Ligase (Promega) at 4C overnight. The ligated plasmids were transformed to E. coli DH5 competent cells and sequenced with M13 forward and reverse primers. All sequencing was performed by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) Sequencing Core. All promoter sequences were confirmed via PCR amplification using a primer located within the coding sequence and a primer at the extreme 5’ end of the promoter. Analysis of Promoter Sequences Sequences were analyzed using a variety of molecular biology programs and databases, including PlantCARE ( http://oberon.fvms.ugent.be:8080/PlantCARE/index.html ) (Rombauts et al. 1999), PLACE ( http://www.dna.affrc.go.jp/htdocs/PLACE/ ) (Higo et al. 1999) and TRANSFAC (Wingender et al. 2000). Motifs were predicted using MEME version 3.0 ( http://meme.sdsc.edu/meme/website/ ) (Bailey and Elkan 1994). The llr is the logarithm of the ratio of the probability of the occurrences of the motif given the motif model versus their probability given the background model. The statistical significance of a motif was based on its log likelihood ratio (llr), its width and number of occurrences, the background letter frequencies, and the size of the data set. The E-value is an estimate of the expected number of motifs with the given llr (or higher), and with the same width and number of occurrences, that one would expect in a similarly sized set of random sequences.

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CHAPTER 6 ISOLATION AND CHARACTERIZATION OF C-REPEAT BINDING FACTOR (CBF) HOMOLOGS IN Poncirus trifoliata (L.) Raf. AND Citrus spp. Introduction Plants possess complex molecular mechanisms for coping with adverse environmental conditions. One such mechanism, the process of cold acclimation, enables plants to increase their ability to withstand low temperatures. This process, which involves biochemical and physiological changes, results largely from global changes in gene expression, yet how the plant senses low temperatures and reacts with the expression of so many genes is largely unknown. Global changes in gene expression result from coordinated changes at the transcriptional level. In Arabidopsis, approximately 12% of the genome is involved in transcriptional control, with approximately 5% of the genome encoding transcription factors (Initiative 2000). As much as 4% of the genome may be altered during low temperature exposure, and transcriptional profiling during cold acclimation identified 48 putative transcription factors induced by low temperature (Fowler and Thomashow 2002). The changes in gene expression associated with cold acclimation result from the integration of multiple signal transduction pathways. The most studied low temperature regulatory pathway, the CBF (C-repeat binding factor) regulon, accounts for at least 12% of cold-induced genes (Fowler and Thomashow 2002). CBFs (also known as DREBs, dehydration responsive element binding proteins) encode a small family of 83

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84 transcriptional activators containing an AP2 DNA-binding domain which interacts specifically with the CRT (C-repeat)/DRE (dehydration-responsive element) (Stockinger et al. 1997; Gilmour et al. 1998; Liu et al. 1998). This element, originally characterized in the promoters of COR15A (Baker et al. 1994) and RD29A (Yamaguchi-Shinozaki and Shinozaki 1994), is present in the regulatory regions of many Arabidopsis coldand dehydration-inducible genes including, but not limited to, COR6.6, COR47, KIN1 and ERD10 (Thomashow et al. 2001). Additionally, CRT/DRE-like elements have been found in the promoters of Brassica napus BN115 (Jiang et al. 1996), Triticum aestivum WCS120 (Ouellet et al. 1998) and WCOR15 (Takumi et al. 2003), Hordeum vulgare BLT4.9 (Dunn et al. 1998) and a number of H. vulgare dehydrins (Choi et al. 1999). Upon exposure to low temperature, CBF genes are induced rapidly followed by the increased expression of their target genes (Dunn et al. 1998; Gilmour et al. 1998; Shinwari et al. 1998; Jaglo et al. 2001). Overexpression of CBF genes leads to an increase in the expression of downstream target genes without a low temperature stimulus and an increase in freezing tolerance (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000). Prior attempts to improve cold tolerance altered the expression of single genes (Iba 2002; Sung et al. 2003) and resulted in only slight improvements in tolerance. In contrast, these experiments, by overexpression of a transcription factor, resulted in the altered expression of many genes and a greater improvement in tolerance, thus providing new possibilities for engineering quantitative traits. The CBF family is highly conserved. In addition to Arabidopsis, CBF genes have been identified in B. napus (Jaglo et al. 2001; Gao et al. 2002), H. vulgare (Choi et al.

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85 2002; Xue 2002; Xue 2003), T. aestivum (Jaglo et al. 2001; Shen et al. 2003a; Vagujfalvi et al. 2003), Lycopersicon esculentum (Jaglo et al. 2001), Oryza sativa (Dubouzet et al. 2003), Secale cereale (Jaglo et al. 2001) and Atriplex hortensis (Shen et al. 2003b). Homologous CBF proteins share the highest conservation in the AP2 domain. Short highly conserved polypeptide sequences (signature sequences), PKK/RPAGRxKFxETRHP and DSAWR, flank the AP2 domain and distinguish the CBF family from other AP2/EREBP domain-containing proteins (Jaglo et al. 2001). In addition to sequence homology, CBF function is conserved. Overexpression of Arabidopsis CBF genes in B. napus and L. esculentum resulted in improved freezing tolerance and increased expression of B. napus BN115, a CBF target gene. Therefore, the CBF pathway is conserved in structure and function. To date, most cold acclimation studies have centered on Arabidopsis and other herbaceous plants. As a result, relatively little is known of the signaling pathways of perennial species. Current knowledge of Citrus cold acclimation parallels the Arabidopsis response. Citrus can acquire cold tolerance by cold acclimation (Yelenosky 1985), and cold acclimation in Citrus is associated with changes in RNA and protein accumulation (Guy and Haskell 1988; Durham et al. 1991). Mapping studies using Poncirus trifoliata (L.) Raf., a cold-hardy interfertile citrus relative, indicate that cold tolerance in Citrus does segregate (Weber et al. 2003), and multiple COR genes have now been isolated (Cai et al. 1995). The primary objective of this research is to isolate and characterize CBF homologs in P. trifoliata and Citrus species. P. trifoliata can withstand -20C, while some Citrus species are damaged by -2C. The molecular basis of this difference is largely unknown.

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86 As CBF genes play important roles in the cold acclimation and cold tolerance of other species, a comparison of CBF genes P. trifoliata and Citrus can potentially reveal differences that correlate with cold tolerance. While low temperatures are a major limiting factor in citrus production, the identification of transcription factors involved in cold acclimation will provide new opportunities for improvement of Citrus cold tolerance. Results In an initial effort to identify P. trifoliata and Citrus CBF homologs, a series of Southern blots were probed with AtCBF1 (Figure 6-1). Hybridizing bands were detected only in Arabidopsis DNA, indicating that the nucleotide sequences of P. trifoliata and Citrus CBF homologs do not have high identity with the Arabidopsis CBF sequences. However, the A. thaliana genome is estimated to contain 125Mb (Arabidopsis Genome Initiative 2000), while the P. trifoliata and Citrus spp. genomes are estimated to contain approximately 382Mb (Arumuganathan and Earle 1991). Therefore, the blot was not loaded with equivalent genome amounts. Figure 6-1. Southern blot of genomic DNA from P. trifolata, C. paradisi, C. grandis and A. thaliana using 32 P-labeled CBF1 (Arabidopsis) as a probe. 10g of genomic DNA from each species was digested with the enzymes listed above the respective lanes.

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87 Figure 6-2. Alignment of CBF coding sequences. The nucleotide sequences shown are for: Ca, Capsicum annuum CBF1B (accession no. AY368483); Le, Lycopersicon esculentum CBF1 (accession no. AY034473); At, Arabidopsis thaliana CBF1 (accession no. ATU77378); Gh, Gossypium hirsutum DREB1A (accession no. AY321150); Pa, Prunus avium DREB1-like protein (accession no. AB080965); Hv, Hordeum vulgare CBF1 (accession no. AF418204); Bn, Brassica napus CBF (accession no. AF370733); Sa, Schedonorus arundinaceus DREB1 (accession no. AY423713); Ta, Triticum aestivum CBF1 (accession no. AF376136); Sc, Secale cereale CBF (accession no. AF370730); and Os, Oryza sativa CBF (accession no. AF243384). The region encoding the AP2 domain is indicated by a dotted line.

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88 Figure 6-2. Continued.

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89 Figure 6-3. Alignment of CBF proteins. The amino acid sequences shown are for: Ca, Capsicum annuum CBF1B (accession no. AAQ88400); Le, Lycopersicon esculentum CBF1 (accession no. AAK57551); At, Arabidopsis thaliana CBF1 (accession no. AAC49662); Gh, Gossypium hirsutum DREB1A (accession no. AAP83936); Pa, Prunus avium DREB1-like protein (accession no. BAC20183); Hv, Hordeum vulgare CBF1 (accession no. AAL84170); Bn, Brassica napus CBF (accession no. AAL38242); Sa, Schedonorus arundinaceus DREB1 (accession no. AAQ98965); Ta, Triticum aestivum CBF1 (accession no. AAL37944); Sc, Secale cereale CBF (accession no. AAL35761); and Os, Oryza sativa CBF (accession no. AAG59619). The AP2 domain and signature sequences are indicated by a dotted line and red boxes, respectively.

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90 Table 6-1. PtCBF identity and similarity to CBF homologs in other species a CaLeAtGhPaHvBnSa TaScOsNT Identity58%57%57%61%57%42%49%37%37%33%32%AA Identity62%57%56%54%47%47%47%38%35%32%21%AA Similarity71%72%67%64%64%58%57%51%48%44%33% a Ca, Capsicum annuum CBF1B (accession no. AAQ88400); Le, Lycopersicon esculentum CBF1 (accession no. AAK57551); At, Arabidopsis thaliana CBF1 (accession no. AAC49662); Gh, Gossypium hirsutum DREB1A (accession no. AAP83936); Pa, Prunus avium DREB1-like protein (accession no. BAC20183); Hv, Hordeum vulgare CBF1 (accession no. AAL84170); Bn, Brassica napus CBF (accession no. AAL38242); Sa, Schedonorus arundinaceus DREB1 (accession no. AAQ98965); Ta, Triticum aestivum CBF1 (accession no. AAL37944); Sc, Secale cereale CBF (accession no. AAL35761); and Os, Oryza sativa CBF (accession no. AAG59619). A P. trifoliata CBF-like gene (PtCBF) was isolated using a PCR-based approach. PtCBF contains an open reading frame of 642 nucleotides and encodes a 214 amino acid protein with a molecular weight of 23.92 kDa and a pI of 5.67. PtCBF contains a conserved AP2 DNA binding domain and a putative nuclear localization signal. The PtCBF coding sequence is 57% identical to AtCBF1 at the nucleotide level (Figure 6-2, Table 6-1). At the protein level, PtCBF and AtCBF1 share 56% identity and 67% similarity. The highest homology is contained within the AP2 domain, although the sequences flanking the PtCBF AP2 domain vary slightly from the published CBF signature sequences (Jaglo et al. 2001) (Figure 6-3). PtCBF expression during cold acclimation was analyzed using northern blots (Figure 6-4). PtCBF transcripts became detectable within 2 hours of cold treatment. Expression levels reached a peak at 12 hours and began gradually decreasing. However, PtCBF transcripts were still detectable after one week of cold acclimation.

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91 Southern blot analysis using the PtCBF coding sequence as a probe indicated that P. trifoliata and C. paradisi contain multiple CBF-like genes (Figure 6-5). Despite differences in the cold tolerance of these species, the CBF copy numbers do not appear to NA15’30’1h2h4h8h12h1d2d4d7d PtCBFrRNA NA15’30’1h2h4h8h12h1d2d4d7d PtCBFrRNA 4C NA15’30’1h2h4h8h12h1d2d4d7d PtCBFrRNA NA15’30’1h2h4h8h12h1d2d4d7d PtCBFrRNA 4C Figure 6-4. Northern blot analysis of PtCBF expression levels during cold acclimation. The membrane was stained with methylene blue to verify equal RNA loading and transfer (lower panel). NA=not acclimated. Figure 6-5. Southern blot of genomic DNA from P. trifoliata and C. paradisi using DIG-labeled PtCBF as a probe. The enzymes used for the DNA digestion are listed above the respective lanes. Molecular weight ladder VII (Roche Molecular Biochemicals) was used to estimate molecular sizes (lane 1).

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92 vary considerably. In addition, comparison of the banding patterns of the BamHI-digested samples identified certain restriction fragments present in both species, indicating these species most likely share some of the same CBF genes. A highly homologous C. paradisi CBF-like gene (CpCBF) was isolated by PCR using primers complementary to PtCBF. The coding sequences of PtCBF and CpCBF are 97% identical at the nucleotide level and 95.8% identical at the amino acid level (Figure 6-6). Furthermore, the regions containing the signature sequences are identical. A comparison of the expression profiles of PtCBF and CpCBF during cold acclimation is shown in Figure 6-7. Similar to PtCBF, CpCBF was upregulated following exposure to 4C. However, transcripts of PtCBF accumulated earlier and in higher quantities than CpCBF. In an effort to identify potential cis-acting regulatory elements involved in the low temperature-induced expression of PtCBF, 5’ regulatory sequence was isolated. The isolated PtCBF promoter includes 1101 bp of sequence upstream of the putative start of translation (Figure 6-8). Statistically significant motifs were predicted by MEME (Table 6-2) including four copies of a 54 bp motif (motif 1, E-value 5.7e-003). Two copies of this motif formed larger, nearly perfect direct repeats of 86 bp. Searches for regulatory elements identified many potential CAREs including 1 CRT/DRE, 6 ABREs, 14 MYB sites and 7 MYC sites (Table 6-3). In addition, an alignment of the PtCBF promoter with the Arabidopsis CBF promoters is shown in Figure 6-9. Although extensive sequence homology is not evident, the PtCBF promoter does contain some regions conserved in the Arabidopsis CBF promoters.

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93 Discussion In many species, the mechanisms of low temperature-induced gene expression involve a CBF family. CBF proteins activate gene expression by binding to specific conserved sequences in the promoters of low temperature-induced genes. As temperatures decrease, CBF transcripts increase rapidly, followed by an increase in Figure 6-6. Alignment of PtCBF and CpCBF sequences. A.) Nucleotide alignment of the coding sequences. B.) Protein alignment. The red boxes indicate the CBF signature sequence.

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94 Figure 6-7. Comparison of the expression patterns of PtCBF and CpCBF during cold acclimation. The membrane was stained with methylene blue to verify equal RNA loading and transfer (lower panel). NA=not acclimated. the expression of downstream target genes. In Arabidopsis, the CBF regulon includes at least 12 % of all cold-responsive genes (Fowler and Thomashow 2002). The CBF family is conserved in a broad range of species, including both monocots and dicots (Jaglo et al. 2001; Choi et al. 2002; Gao et al. 2002; Xue 2002; Dubouzet et al. 2003; Vagujfalvi et al. 2003; Xue 2003). Moreover, CBF genes have been found in both freezing tolerant and chilling sensitive species (Jaglo et al. 2001), indicating that CBF genes are not limited to plants that are freezing tolerant. While CBF genes are widely present, most studies have been conducted in herbaceous plants. CBF homologs were identified in both P. trifoliata and C. paradisi. PtCBF and CpCBF encode a highly conserved AP2 domain, although the signature sequences, PKK/RPAGRxKFxETRHP and DSAWR, are slightly modified (Figure 6-3). In Arabidopsis, these signature sequences distinguish CBF proteins from other AP2/EREBP domain proteins, and these sequences are highly conserved in CBF homologs in other species (Jaglo et al. 2001). However, modified signature sequences have been reported in H. vulgare CBF1 and CBF2 (Xue 2002; Xue 2003) and O. sativa DREBs (Dubouzet et al.

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95 2003), and the ability of these modified CBF proteins to bind the CRT/DRE has been demonstrated. Within the AP2 domain, the 14 th valine and 19 th glutamic acid are conserved in the DREB subgroup of AP2/EREBP proteins (Sakuma et al. 2002) and are crucial to the binding activity of AtCBF3 (Cao et al. 2001). These amino acids are also conserved within the AP2 domains of PtCBF and CpCBF. Therefore, it is likely PtCBF Figure 6-8. PtCBF Promoter Sequence. Solid double lines indicate the TATA box; asterisks indicate the CRT/DRE; dashed double lines indicate ABREs; solid single lines indicate MYB sites; dotted lines indicate MYC sites; black arrow indicates the putative start of translation; boxes represent selected motifs (motif 1, red; motif 2, blue; motif 3, green; and motif 4, orange); single dashed lines represent a large repeated motif. All numbering is relative to the putative start of translation

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96 Table 6-2. PtCBF promoter motifs predicted by MEME analysis. Predicted MotifsllrE-valueSequenceLocationStrandMotif 11885.7e-003aatagctaaaCGGTTTTTCTGTTTCTCTCTCTGCCGCCGGGTtcattccctt-407+aacagctaaaCGTTTTTCTGTTTCGCTCTCTCGTCGGGTtcattccctt-517+ccaaccgcctCGCCTCTCCCCTGTGTTTTTCCCGCGGTTTCCTGGCGTggcaattcga-881ctaaCTTTCCTCTCCCGCGGTTGTGTTGGTTTGTTTTCCTTGTCGTTgtgatagctt-1096+Motif 2344.8e+003taggttccccGCCCCCGCCCcaaccgcctc-818-cgccacgtcaGCCTCCCCCCgactgttctt-721+Motif 3745.9e+003atctttatttGCCGTGTCCGGagcactcaca-299+cttaagatttCCGTGTCCTtctcataatt-446+gtgacgtcacCGCGTCCCCGGttgacagtta-788-tccaggaactCTCTTCTCTCaatccttccc-673+Motif 4538.2e+003tcagttacttCCCTGTCgagatacgac-968+cctgaagtgaCTCTGTCtgttaactgg-47-ggcggggaacCTCTGTCaacccgtgtt-799+ggattcatttCCCTGTCtccttatcat-192+aagctcaggtCCCCTTTCgaacatcgta-236+ AAAAAAAAGAGAGAAGAGAAAAGAAGAAAAAGAGAGAAGAGAGAAAAAAAAAAAAGGAGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAGAAAGAAAAAAAAAAAAGA Table 6-2. PtCBF p romoter motifs p redicted b y MEME anal y sis

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97 Table 6-3. Summary of predicted cis-acting regulatory elements present in the PtCBF promoter. Site NameFunctionCopiesABREAbscisic acid responsiveness6Aux-RRAuxin responsiveness1EREEthylene responsiveness3GC motifAnoxic specific inducibility7HSEHeat stress responsiveness7CRT/DRELow-temperature responsiveness1MYB siteDrought-inducibility14MYC siteDrought-inducibility7P-boxGibberellin-responsive element2TATC-boxGibberellin-responsive element1TGA Auxin responsiveness2TGACG-motifMeJA-responsiveness4WUN-motifWound-responsive element6Multiple sitesLight responsivenessman y and CpCBF do function in a CBF regulon. The promoter of CORc115, a P. trifoliata cold-induced Group II LEA gene, contains the CRT/DRE core, CCGAC (see chapter 5), and thus may be a potential target gene of PtCBF. Interestingly, PtCBF and CpCBF shared the highest protein similarity with Capsicum annuum CBF1B and Lycopersicon esculentum CBF1 (Table 6-1), two species which are chilling sensitive. Southern blot analysis indicated that both P. trifoliata and C. paradisi contain multiple CBF-like genes, constituting a small gene family (Figure 6-5). Restriction patterns indicate that these species share common CBF-like genes while also possessing unique CBF-like genes. Moreover, P. trifoliata and C. paradisi appear to possess a similar number of CBF-like genes. Therefore, it seems unlikely that the differences in cold tolerance of P. trifoliata and C. paradisi are due to differences in CBF numbers. A common feature of CBF genes is a transient increase in expression during cold treatment. In general, CBF transcripts increase within 1 hour of cold treatment, peak within 2-4 hours, and gradually decrease (Gilmour et al. 1998; Shinwari et al. 1998; Jaglo et al. 2001). Transcripts of PtCBF and CpCBF also increase during cold acclimation

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98 Figure 6-9. Alignment of the PtCBF promoter with the Arabidopsis CBF promoters. Red boxes represent conserved Arabidopsis CBF promoter motifs described by (Shinwari et al. 1998). (Figures 6-4, 6-7). Increases in the expression of PtCBF and CpCBF are detectable within 2-4 hours of transfer to 4C and reach a peak at 12-24 hours. Compared to herbaceous species, PtCBF and CpCBF accumulation is delayed. However, it would be

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99 interesting to compare the expression patterns with CBF genes in other woody perennial species. Compared to CpCBF, PtCBF accumulates earlier and in greater quantities (Figure 6-7). As CBF proteins activate the expression of many cold-induced genes, the significance of this difference and how it relates to cold tolerance is of interest. Citrus species represent a wide range of cold-tolerance phenotypes (Yelenosky 1985), thus providing a germplasm source to investigate how CBF expression patterns correlate with cold tolerance.To examine the mechanism of PtCBF cold-induced expression, the PtCBF promoter was isolated. Database searches predicted many cis-acting regulatory elements with potential roles in cold acclimation (Table 6-3). In contrast with the Arabidopsis CBF promoters (Gilmour et al. 1998), the PtCBF promoter contains one copy of the CRT/DRE core sequence, CCGAC, creating the potential for autoregulation. ICE1, a positive regulator of AtCBF3, encodes a MYC-like bHLH transcription factor that specifically recognizes MYC recognition sites in the AtCBF3 promoter (Chinnusamy et al. 2003), and a cold responsive region of the AtCBF2 promoter also contains a MYC recognition site (Zarka et al. 2003). Although alignment of the PtCBF promoter with the Arabidopsis CBF promoters did not identify any regions of extensive homology (Figure 6-9), the PtCBF promoter contains seven potential MYC recognition sites, indicating the mechanism of PtCBF induction may be similar to Arabidopsis CBF induction. Furthermore, the PtCBF promoter contains two direct repeats of an 86bp sequence (Figure 6-8). These repeats contain one MYB recognition site, but are not conserved in the Arabidopsis promoters. In summary, the cold acclimation pathways in P. trifoliata and C. paradisi share conserved components with herbaceous plants. Despite their differences in cold

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100 tolerance, P. trifoliata and C. paradisi both contain small CBF families. The P. trifoliata and C. paradisi CBF pathways share characteristics in common with each other and CBF genes in other species, while also possessing unique differences. Further characterization of these genes among Citrus species of differing cold sensitivities should assist in the identification of strategies for the improvement of Citrus cold tolerance. Materials and Methods Cloning of PtCBF and CpCBF Block Maker ( http://blocks.fhcrc.org/blocks/make_blocks.html ) (Henikoff et al. 1995) identified two conserved amino acid stretches using a CLUSTAL (Thompson et al. 1997) alignment of the published CBF protein sequences, and the conserved blocks were used by CODEHOP (http://blocks.fhcrc.org/codehop.html) (Rose et al. 2003) to generate degenerate primers. Primers KC109 (5’CCAGCTGGCAGAACAAAGTTYMRNGARAC3’) and KC113A (5’GGTCTCGGGGATTGGCARNCKCCANGC3’) were expected to amplify a band of approximately 225 bp. PCR was performed using Advantage 2 Polymerase Mix (Clontech) with P. trifoliata (L.) Raf. genomic DNA as template. Cycling parameters were as follows: 94C 3 minutes, 40 cycles of 94C 30 seconds/60C 30 seconds/72C 30 seconds, and 72C 10 minutes. A band of approximately 250 bp was visualized on a 2 % agarose gel stained with ethidium bromide. The band was excised and purified using the Gel Extraction Kit (Qiagen). To increase the percentage of fragments containing dATP overhangs, an “A-Tailing” reaction containing 1X PCR Buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X-100), 2.5 mM MgCl 2 , 250 M dATP, and 5 units Taq Polymerase (Promega) was incubated at 70C for 1 hour. Seven microliters of the purified reaction was ligated to 50 ng pGEM-T Easy (Promega) using 3 units T4

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101 DNA Ligase (Promega) at 4C overnight. The ligated plasmid was transformed to E. coli DH5 competent cells and sequenced with M13 forward and reverse primers. All sequencing was performed by the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) Sequencing Core Facility. The degenerate band contained 247 bp of sequence highly homologous to other CBF family members. The remainder of the Poncirus CBF-like sequence was amplified using inverse PCR (Ochman et al. 1988; Triglia et al. 1988) and 5’ Rapid Amplification of cDNA Ends (RACE). Genomic DNA was isolated from the leaves of P. trifoliata seedlings using a CTAB extraction method (Dellaporta et al. 1983). The PCR template was prepared by digesting 2 g of DNA with 10 units Taq I (Promega) at 65C for 4 hours. Five hundred nanograms of digested DNA was self-ligated with 3 units T4 DNA Ligase (Promega) in a 100 l reaction volume at 14C overnight. Flanking sequence was isolated in a 100 l inverse PCR reaction containing 1X Cloned Pfu Buffer [20 mM Tris-HCl pH 8.8, 2 mM MgSO 4 , 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1 % Triton X-100, and 100 ng/ml BSA], 200 M each dNTP, 250 nM each primer (KC129, 5’CTGCCATTTCGGCGGTGGG3’ and KC130, 5’GCTTTGAGGGGGAGGTTAGC3’), 2.5 units Pfu Turbo (Stratagene) and 5 ng of prepared template. Cycling was as follows: 94C 3 minutes, 30 cycles of 94C 1 minute/62C 1 minute/72C 5 minutes, and 72C 10 minutes. A 750 bp band was purified using the Gel Extraction Kit (Qiagen). A-Tailing, ligation into pGEM-T Easy, transformation and sequencing were performed as described previously. The band, which was homologous to the previously isolated sequence, contained 651 bp of additional 3’ sequence.

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102 The SMART RACE cDNA Amplification Kit (Clontech) was used to obtain the 5’ cDNA end. According to the protocol, 5’ RACE-ready cDNA was prepared from 1 g total RNA extracted from plants treated at 4C for 14 days. RNA was extracted using TRIzol reagent (Invitrogen). Briefly, 1.5 ml TRIzol reagent was added to approximately 250 mg ground tissue. Samples were centrifuged for 10 minutes at 12,000 g at 4C to remove particulate matter. Three hundred microliters of chloroform was added to the supernatant, mixed well by shaking, and incubated at 25C for 3 minutes. Phases were separated by centrifuging for 15 minutes at 12,000 g at 4C. The upper phase was removed to new tube, and RNA was precipitated with 750 l isopropanol. Samples were incubated at 25C for 10 minutes, followed by centrifugation for 10 minutes at 12,000 g at 4C. The pellet was washed with 75 % ethanol and resuspended in 30 l TE buffer. The 5’ RACE-ready cDNA was diluted with 100 l Tricine-EDTA buffer (10 mM Tricine-KOH pH 8.5, 1 mM EDTA). All RACE PCR was performed according to the manufacturer’s protocol (Clontech) using 2.5 l prepared cDNA. A 700 bp band was obtained using primer KC133 (5’ CGATTACACTGAGGTGGTGGTGGTGGGGG 3’) and the following cycles: 5 cycles 94C 5 seconds/ 72C 2 minutes, 5 cycles 94C 5 seconds/70C 10 seconds/72C 2 minutes, and 25 cycles 94C 5 seconds/68C 10 seconds/72C 2 minutes. The band was purified using the Gel Extraction Kit (Qiagen), cloned into pGEM-T Easy, transformed into E. coli DH5, and sequenced as previously described. The amplified RACE band contained the entire 5’ coding sequence of the gene, and the gene was designated PtCBF. A PtCBF homolog was isolated from C. paradisi using 3’ RACE and primers complementary to the PtCBF sequence. RNA extraction and cDNA preparation were

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103 performed as described previously using leaves collected from C. paradisi Macf. (cv. Duncan) seedlings treated at 4C for 1 day. 3’ RACE-ready cDNA was diluted with 100 l Tricine-EDTA buffer (10 mM Tricine-KOH pH 8.5, 1 mM EDTA). PCR was performed according to the manufacturer’s protocol (Clontech) using 2.5 l prepared cDNA and primer KC134 (5’ CCCAGTTAACAGACAGTTAGTC 3’). A 700 bp band was obtained using the following cycles: 94C 3 minutes, 30 cycles 94C 30 seconds/58C 30 seconds/72C 1 minute, and 72C 10 minutes. The band was cloned and sequenced as described previously, and the isolated gene was designated CpCBF. Nucleotide and protein sequences were analyzed using Vector NTI Suite (Invitrogen). Sequence homology was analyzed using BLAST similarity searches at the National Center for Biotechnology Information ( www.ncbi.nlh.nih.gov ) (Altschul et al. 1990; Altschul et al. 1997). Protein targeting was analyzed by PSORT ( www.psort.nibb.ac.jp ) (Nakai and Kanehisa 1992). Alignments were performed using CLUSTAL. Isolation of PtCBF 5’ Regulatory Sequence Inverse PCR was used to isolate the PtCBF 5’ regulatory region (Ochman et al. 1988; Triglia et al. 1988). Genomic DNA was isolated from the leaves of P. trifoliata seedlings using a CTAB extraction method (Dellaporta et al. 1983). The template, which was prepared as described previously, was digested with 10 units of Sty I (Promega). The promoter was amplified in a 100 l inverse PCR reaction containing 1X Cloned Pfu Buffer [20 mM Tris-HCl pH 8.8, 2 mM MgSO 4 , 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1 % Triton X-100, and 100 ng/ml BSA], 200 M each dNTP, 250 nM each primer (KC136, 5’ CAACACCACGTCCTCGTCGG 3’ and KC137, 5’GAGGAGGAGGGATTCGGGC 3’), 2.5 units Pfu Turbo (Stratagene) and 5 ng of prepared template. The PCR reaction was

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104 cycled at 94C 3 minutes, 35 cycles of 94C 1 minute/62C 1 minute/72C 5 minutes, and 72C 10 minutes. The reaction, which was analyzed by electrophoresis, amplified a 1400 bp band. The band was cloned and sequenced as described previously. The promoter sequence was confirmed via PCR amplification using a primer located within the coding sequence (KC138, 5’ GGGGGCAACAGCATCCCTTGC 3’) and a primer near the 5’ end of the promoter (KC146, 5’ GTTTTGGACGTCGTGTGACG 3’). Sequences were analyzed using a variety of molecular biology programs and databases, including PlantCARE ( http://oberon.fvms.ugent.be:8080/PlantCARE/index.html ) (Rombauts et al. 1999), PLACE ( http://www.dna.affrc.go.jp/htdocs/PLACE/ ) (Higo et al. 1999) and TRANSFAC (Wingender et al. 2000). Motifs were predicted using MEME version 3.0 ( http://meme.sdsc.edu/meme/website/ ) (Bailey and Elkan 1994). The statistical significance of a motif was based on its log likelihood ratio (llr), its width and number of occurrences, the background letter frequencies, and the size of the data set. The llr is the logarithm of the ratio of the probability of the occurrences of the motif given the motif model versus their probability given the background model. The E-value is an estimate of the expected number of motifs with the given llr (or higher), and with the same width and number of occurrences, that one would expect in a similarly sized set of random sequences. Southern Blots Leaf tissue was collected, ground in the presence of liquid nitrogen and stored at -80C. Genomic DNA was extracted from approximately 250 mg of ground tissue with 800 l DNAzol ES (Molecular Research Center, Inc.). The DNA pellet was resuspended in 30 l TE buffer. The DNA was quantified by measuring the absorbance at 260 nm.

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105 To digest the DNA, 10 g DNA was combined with 20 units of restriction enzyme (Invitrogen, Figures 5-1 and 5-5) and 2 l RNase A (1 g/ml, Ambion). The reactions were incubated at 37C for 6 hours. The samples were electrophoresed in a 0.8 % agarose TAE (40 mM Tris-acetate pH 7.6, 1 mM Na 2 EDTA) gel for approximately 18 hours. Following electrophoresis, the digested DNA was stained by soaking the gel in 1X TAE buffer containing 0.25 g/ml ethidium bromide for 10 minutes. The DNA samples were visualized by illumination with a UV transilluminator. Prior to transfer, the DNA was depurinated, denatured and neutralized by gentle shaking in the appropriate solution. The DNA was depurinated by incubation in 0.25 M HCl for 10 minutes. The gel was incubated twice (15 minutes each) in denaturation solution (0.5 N NaOH, 1.5 M NaCl) and twice (15 minutes each) in neutralization solution (0.5 M Tris-HCl pH 7.5, 3 M NaCl). Between steps, the gel was rinsed with deionized water. 20X SSC was used to transfer the DNA from the gel to a positively-charged nylon membrane by capillary transfer. The transfer was allowed to proceed for approximately 20 hours. The DNA was crosslinked to the membrane using a UV Stratalinker 1800 (Stratagene). To prepare the Arabidopsis CBF1 probe, pUC118/COR78Pr+CBF1 was sequentially digested with ApaI (Promega) and XhoI (GibcoBRL) to release CBF1. One microgram of pUC118/COR78Pr+CBF1 was digested at 37C with 10 units ApaI (Promega) for two hours. The reaction was purified using the Qiagen PCR Purification Kit. The plasmid was then digested at 37C with 10 units of XhoI for 2 hours. Following separation on a 1 % agarose TAE gel, a 650 bp band containing CBF1 was excised and purified using the Qiagen Gel Purification Kit. CBF1 was quantified by measuring the absorbance at 260 nm. 50 ng of CBF1 in 40 l water was denatured by incubation at

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106 94C for 5 minutes and transferred immediately to ice. The denatured CBF1 was combined with RadPrime (Gibco-BRL) and mixed until the blue dye was evenly distributed. To label CBF1, 10 l of 32 P-dCTP was added. The reaction was incubated at 37C for 1 hour. The 32 P-CBF1 was purified in a G50 sephadex column equilibrated with 1X NTE (0.5 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) and denatured by addition of 1/10 volume of 1 M NaOH. The membrane was incubated with 7.5 ml of prehybridization solution for 1 hour. Following prehybridization, the denatured probe was added, and hybridization was conducted overnight at 60C. The membrane was washed (2 x 15 minutes) with 0.5X SSC, 0.1 % SDS at 60C. Signals were detected by incubation with X-ray film at -80C. The PtCBF probe was prepared by PCR amplification in the presence of Digoxigenin (DIG)-11-dUTP. PtCBF was amplified with 1 unit Taq Polymerase (Promega) from cDNA prepared from leaves treated at 4C for 2 hours. Reaction conditions were as follows: 1X PCR buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl and 0.1 % Triton X-100), 2.5 mM MgCl 2 , 200 M dATP, 200 M dGTP, 200 M dCTP, 130 M dTTP, 70 M DIG-11-dUTP, and 200 nM each KC134 and KC135 (5’ TAATACGACTCACTATAGGGGCAGAGCCCGCCTGATTACTGCTTC 3’). Sufficient labeling was confirmed by electrophoresis, as incorporation of DIG-11-dUTP results in a PCR product with a reduced migration rate. Reconstituted DIG Easy-Hyb granules (Roche Molecular Biochemicals) were used for both prehybridization and hybridization. Prehybridization was performed at 42C for 1 hour using 15 ml DIG Easy Hyb. During prehybridization, 50 l of water was added to 20 l of probe. The probe was denatured by incubation at 100C for 5 minutes, followed by immediate chilling on

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107 ice. The denatured probe was added to 10 ml of pre-warmed DIG Easy-Hyb. Hybridization proceeded at 42C for approximately 16 hours. The membrane was washed twice at 25C for 5 minutes under low stringency conditions (2X SSC, 0.1 % SDS), and twice at 65C for 15 minutes under high stringency conditions (0.5X SSC, 0.1 % SDS). The detection procedure was performed according to the manufacturer’s protocol (Roche Molecular Biochemicals), and disodium 3-{4-metho xyspiro[1,2-dioxetane-3,2-(5-chloro) tricyclo (3.3.1.1 3 ,7)decan]-4-yl}phenyl phosphate (CSPD) was used as substrate for all detections. Following addition of CSPD, the membrane was incubated at 37C for 15 minutes and detected by incubation with X-ray film. Northern Blots Northern blot experiments were conducted using two-year old P. trifoliata and C. paradisi Macf. (cv. Duncan) seedlings with new growth flushes. The plants were treated at 4C with a 16 hour photoperiod. Following transfer to 4C, leaves were collected at specific time points, frozen immediately in liquid nitrogen and stored at -80C. Total RNA was extracted using TRIzol reagent (Invitrogen) as described previously. Three to ten micrograms of total RNA was used for all blots. RNA samples were combined with two volumes of freshly made Loading Buffer [50 % deionized formamide, 6 % formaldehyde, 1X MOPS (20 mM morpholineopropanesulfonic acid, 5 mM Na acetate, 2 mM EDTA), 0.0005 % bromophenol blue]. RNA was denatured by incubating at 65C for 10 minutes. Samples were loaded onto a 1.5 % agarose gel (2 % formaldehyde, 1X MOPS) and run in 1X MOPS buffer until the bromophenol blue dye had migrated through two-thirds of the distance of the gel. To confirm the RNA quantity and quality, the gel was stained by incubating in 0.25 g/ml ethidium bromide for 10 minutes and visualized by exposure to UV light. To remove formaldehyde from the gel before

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108 transfer, the gel was soaked twice (2 x 15 minutes) in 20X SSC. 20X SSC was used to transfer the DNA from the gel to a positively-charged nylon membrane by capillary transfer. The transfer was allowed to proceed for approximately 20 hours. The DNA was crosslinked to the membrane using a UV Stratalinker 1800 (Stratagene). Antisense RNA probes were used for all northern blots. cDNA was prepared from RNA extracted from P. trifoliata seedlings treated for 14 days at 4C using TRIzol reagent (Invitrogen). cDNA was synthesized using the First Strand Synthesis Kit for RT-PCR (Ambion). Two micrograms of total RNA was combined with 100 mol random decamers in 12 l water. The samples were denatured by incubation at 85C for 3 minutes and immediately transferred to ice. Two microliters of 10X RT buffer (500 mM Tris-HCl pH 8.3, 750 mM KCl, 30 mM MgCl 2 , and 50 mM DTT), 4 l 2.5 mM dNTPs, 10 units RNase Inhibitor, and 100 units MMLV Reverse Transcriptase were added to the tube. The tube was incubated at 42C for 1 hour, followed by 92C for 10 minutes. To prepare the transcription template, RT-PCR primers were designed complementary to the probe sequence and the T7 RNA polymerase promoter. One unit of Taq DNA Polymerase (Promega) was used to amplify coding sequence from 200 ng cDNA in a reaction containing 0.2 M each gene-specific primer (KC134, 5’ CCCAGTTAACAGACAGTTAGTC 3’ and KC135, 5’ TAATACGACTCACTATAGGGGCAGAGCCCGCCTGATTACTGCTTC 3’), 200 M each dNTP, 2.5 mM MgCl 2 , and 1X PCR buffer (10 mM Tris-HCl pH9.0, 50 mM KCl and 0.1 % Triton X-100). Cycling was as follows: 94C 3 minutes, 30 cycles of 94C 1 minute/55C 1 minute/72C 1 minute, and 72C 10 minutes. Ten microliters of the reaction was analyzed by electrophoresis in a 1 % agarose TAE gel. The probe was

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109 labeled by in vitro transcription using the DIG RNA Labeling Kit (Roche Molecular Biochemicals). Four microliters of the RT-PCR reaction was combined with 2 l 10X Transcription Buffer (0.4 M Tris-HCl pH 8.0, 60 mM MgCl 2 , 100 mM dithiothreitol, 20 mM spermidine, and 100 mM NaCl), 1 mM CTP, 1 mM GTP, 1 mM ATP, 0.65 mM TTP, 0.35 mM DIG-11-UTP, 20 units RNase Inhibitor and 40 units T7 RNA Polymerase in a 20 l reaction volume. The reaction was incubated at 37C for 2 hour. To terminate the reaction, 2 l 0.2 M EDTA was added. All northern blot hybridization and detection was performed according to the manufacturer’s protocol (Roche Molecular Biochemicals). Membranes were prehybridized at 68C for 30 minutes in reconstituted DIG Easy Hyb (Roche Molecular Biochemicals). Two microliters of labeled probe was combined with 50 l water, denatured at 100C for 5 minutes, and chilled on ice. The denatured probe was added to 10 ml prewarmed (68C) DIG Easy Hyb. The prehybridization solution was discarded and the hybridization solution was added. All hybridizations were performed at 68C overnight. Stringency washes and detection were performed according to the standard protocol. Following detection, the probes were stripped from the membranes by washing (2 X 60 minutes) at 80C with a solution of 50 % deionized formamide/5 % SDS/50 mM Tris-HCl pH 7.5. To confirm equal loading, the membranes were either stained with methylene blue (Molecular Research Center, Inc.) or re-probed with a DIG-labeled 18S RNA probe.

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CHAPTER 7 CONCLUSIONS Transcriptome changes are required for cold acclimation. In Arabidopsis, the most widely studied model of plant cold acclimation, these changes are controlled by specific regulatory pathways. These include specific cis-acting regulatory elements which are recognized and bound specifically by transcription factors. The CBF pathway is the best understood pathway leading to cold acclimation. This pathway may control as many as 100 genes cold-regulated genes, making it an attractive intervention point for manipulating and controlling cold acclimation. Indeed, the CBF transcription factors have been exploited successfully in creating plants with enhanced cold tolerance. To date, little research has been directed towards understanding the mechanisms of cold acclimation in woody perennial species. We propose the use of Citrus, in addition to Arabidopsis, as a model organism for cold acclimation research. Citrus species represent a range of cold tolerance phenotypes and production is limited by freezing temperatures. Additionally, Citrus production is a multi-billion dollar industry worldwide, and modest improvement of the freezing tolerance of commercial citrus cultivars would not only save growers millions of dollars in tree and crop losses, but may also permit the re-establishment of previously productive northern growing regions. Therefore, the primary goal of this research was to establish P. trifoliata and Citrus as model organisms for cold acclimation research and investigate the molecular basis of cold acclimation in these species. 110

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111 The data presented indicate that the mechanisms of cold-induced gene expression in P. trifoliata and C. paradisi resemble those in Arabidopsis. This research has identified components of cold acclimation signaling at different levels, including cold-regulated genes, potential cis-acting regulatory elements and binding factors. The identified COR genes resemble cold-regulated genes in other species, as do the promoter elements and transcription factors. We report that P. trifoliata and Citrus contain small families of CBF-like genes. PtCBF and CpCBF, while highly homologous at the nucleotide and protein level, do vary in their cold-induced expression patterns. We speculate that the cold-induced expression of these genes may account for some of the difference in the cold tolerance between P. trifoliata and C. paradisi. Despite commonalities with Arabidopsis and other herbaceous plants, differences were also observed. CBF overexpression, despite producing cold tolerance in other plants, was unsuccessful in Citrus. The Arabidopsis CRT/DRE, within the context of the RD29A promoter, does not function in cold-induced gene expression in Citrus. If Citrus CBFs activate gene expression in response to low temperatures, they may function through a modification of this motif. The PtCBF and CpCBF signature sequences are modified, and the cold-induced expression patterns of CBFs are later and longer-lived in P. trifoliata and C. paradisi. Additionally, the PtCBF promoter contains one copy of the CRT/DRE core motif, a motif not present in the Arabidopsis CBF promoters. These results enhance our understanding of cold acclimation in P. trifoliata and C. paradisi, and provide a framework for future studies on stress-induced gene activation in these species. The identified CBF genes provide new targets for manipulation of cold

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112 tolerance in cold sensitive Citrus. Additionally, the data suggest one molecular explanation for the substantial differences in P. trifoliata and Citrus cold tolerance.

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BIOGRAPHICAL SKETCH Karen Irisa Champ was born on November 28, 1977, to Hale and Rebecca Stancil in Ocala, Florida. She was active in the Marion County 4H program where she discovered and nurtured an interest in agriculture. She graduated valedictorian of Vanguard High School in 1996 and enrolled at the University of Florida to study agriculture. She received a BS degree in horticultural sciences and a minor in plant molecular and cellular biology in 1999. Upon graduation, she was accepted into the Plant Molecular and Cellular Biology Program at the University of Florida. This manuscript details her research on the molecular basis of cold acclimation in Poncirus trifoliata and Citrus paradisi. She conducted her research under the supervision of Dr. Gloria Moore in the Horticultural Science Department. 125