i LIIRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE mih51°442004T MSU ie An Affirmative ActiorVEquel Opportunity institution chHi A MOLECULAR GENETIC STUDY OF COLD ACCLIMATION IN ARABHDOPSIS THALIANA By Chentao Lin A DISSERTATION Submitted to Michigan State Universi _ in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Program in Genetics 1992 0/73- 3/769 ABSTRACT A MOLECULAR GENETIC STUDY OF COLD ACCLIMATION IN ARABEOPSIS THALIANA BY Chentao Lin Changes in gene expression have been suggested to have roles in plant cold acclimation. The functions of cor (gold-regulated) genes, however, remains to be determined. Plant cor genes and their products were studied using Arabidopsis as a model system. Cold acclimation of Arabidopsis was found to be associated with the accumulation of cor gene transcripts encoding polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Genes encoding three ”boiling-stable” polypeptides, COR160, COR47, and COR15 were shown to be represented by three cor cDNA clones pill-128, pill-17.2, and pLCI’lO, respectively. The polypeptide CORIS and cDNA clones encoding it were further characterized. It was found that CORIS has potent cryoprotective activity in vitro: it was 106 fold more effective than sucrose, and 102-103 fold more effective than other proteins tested in protecting lactate dehydrogenase against freeze inactivation. DNA sequence analysis of the cDNA clone pLCTlO indicated that the N-terminal amino acid sequence of CORIS resembles chloroplast transit peptides. Immunological studies further demonstrate that CORIS is indeed targeted to the chloroplast and processed to a mature form of approximately 9 kDa. Attempts to create and analyze transgenic Arabidopsis plants that overexpress or underexpress car15 are discussed. ACKNOWLEDGEMENTS I would like to thank my adviser, Professor Michael Thomashow, for his insightful guidance, continuing support and encouragment throughout this research project. I also greatly appreciate the help and advice kindly provided by the members of ‘ my guidance committee, Dr. John Ohlrogge, Dr. Chris Somerville, and Dr. Steven Triezenberg. I wish to express my appreciation to Dr. Sarah Gilmour, for all her advice and help during my research as well as for her critical reviews of this manuscript. I would also like to express my gratefulness to my colleagues, Todd Cotter, David Horvath, Weiwen Guo, Guowei Fang, Thanda Wai, Rom Bada, Sue Hammar, Dr. Julia Bell, Dr. Ravindra Hajela, Dr. Stokes Baker, Dr. Nancy Artus, Dr. Steve Krebs, and all the members in Dr. Thomashow's laboratory and Dr. Rebecca Grumet's laboratory. Finally, I would like thank my wife Hongyun Yang, for her support and understanding, and for her technical help in some of my experiments. iii TABLE OF CONTENT LIST OF FIGURES LIST OF TABLES CHAPTER 1 literature Review Introduction Freezing Injury Biochemistry of Cold Acclimation Genetics of Cold Acclimation Literarue Cited CHAPTER 2 Arabidopsis cor Genes Encoding Boiling-Stable COR Polypeptides Summary Introduction Materials and Methods Results Discussion Literature Cited iv 10 16 23 29 30 3 1 36 48 52 CHAPTER 3 Arabidopsis Gene cor15 Encodes a Polypeptide Having Potent Cryoprotective Activity Summary 55 Introduction 55 Materials and Methods 57 Results 59 Discussion 66 Literature Cited 68 CHAPTER 4 Characterization of Arabidopsis cor15 Gene Product Summary 70 Introduction 71 Materials and Methods 72 Results 77 Discussion 109 Literature Cited 113 APPENDIX Creation and Analysis of Arabidopsis Mutants Overexpressing or Underexpressing car 15 I. Plasmid 117 II. Arabidopsis Transformation 117 III. Analysis of Transgenic Plants 120 IV. Conclusion 150 V. References 150 SUMMARY AND PERSPECTIVES 152 LIST OF TABLES Table 3.1 CPSO values of various agents. Table 4.1 Amino acid compositions of CORIS and COR15m. Table A.l Analysis of kanamycin resistance of T2 progeny for some of the transgenic plants vi Page 61 80 121 LIST OF FIGURES Figure 2.1. Accumulation of transcripts encoding boiling-stable polypeptides in cold acclimated Arabidopsis. Figure 2.2 Analysis of boiling-stable polypeptides synthesized by total RNAs isolated from nonacchmated (NA) and cold-acclimated (AC) spinach (spin.), and Solanum species Figure 2.3. Hybrid-arrest in vitro translation experiments indicating that Arabidopsis cDNA clone pHH28, pHH7 .2 and pLCTlOA (indicated as pLCTlO) represent cor genes encoding boiling -stable COR polypeptides. Figure 2.4 cDNA clone pLCTlOB encodes a 15 kDa boiling-stable polypeptide comigrating with COR15 in SDS-PAGE. Figure 2.5. Accumulation of transcripts encoding COR polypeptides and other boiling-stable polypeptides in drought stressed Arabidopsis. Figure 3.1 In vitro synthesis of COR15. Figure 3.2 Cryoprotection of LDH. Figure 4.1. Nucleic acid sequence of the cDNA insert in pLCl‘lOA and the deduced amino acid sequence of COR15. Figure 4.2. Hydropathy profile of COR15. Figure 4.3. The predicted secondary structure of COR15m according to Garnier et al (Garnier et al. , 1978). Figure 4.4. Nucleic acid sequence of the cDNA insert in pHH71.l. Figure 4.5. Antibody prepared against the protein A-CORlS fusion polypeptide recognizes COR15. Figure 4.6. Evidence that the COR15 polypeptide is processed in planta. Figure 4.7. Comparison of the chloroplast targeting transit peptide (cTP) and the putative cTP of COR15. Figure 4.8. Detection of COR15 m in leaves and chloroplasts isolated from cold acclimated plants. Figure 4.9. Detection of earbonic anhydrase, COR160, and glycolate oxidase in protein fractions isolated from nonacclimated and cold vii Page 37 39 42 46 62 78 81 83 86 88 91 96 98 acclimated plants. Figure 4.10. Comigration of chloroplast COR15m with recombinant COR15m. Figure 4.11. Migration of CORIS on a native polyacrylamide gel. Figure 4.12. Immunoblot showing the formation of multimers of recombinant COR15m. Figure 4.13. A helical wheel diagram of the utative COR15m containing 88 amino acids residues (amino acids m 51 to 139 of COR15). Figure A. 1. Nothem blot analysis showing the cor15 transcript in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD), and "sense” transgenic lines 71-1-3 (71-1), 71-2-23 (71-2), and 71—3-2 (71-3). Figure A.2. Nothem blot analysis showing the antisense RNA of cor15 in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD) and ”antisense" transgenic lines 72-2A, 72-2B, and 72-20. Figure A.3. Nothem blot analysis showing the cor15 transcript in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD) and antisense transgenic lines 72-2A, 72-28, and 72-20. Figure A.4. Immunoblot analysis showing the accumulation of COR15m in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD) and sense transgenic lines 71-1-3, and 71-2-23. Figure A.5a. Oxygen evolution of leaf discs prepared from nonacclimated wild RLD plants (broken line) and the sense transgenic line 71-2- 3 (solid line). Figure A.5b. Same as Figure A.5a using a different batch of plants. Figure A.6a. Oxygen evolution analysis of leaf discs prepared from cold-acclimated plants of wild type RLD (broken hne) and the sense transgenic line 71-2-23 (solid line). Figure A.6b. Same as Figure A.6a using a different batch of plants. Figure A.7. Photopho horylation assay showing the response of chloroplasts to a ze/ thaw cycle. Figure A. 8a. Photophosphorylation assay showing the response of chloroplast to different concentrations of NaCl. Figure A.8b. Same as Figure A.8a using a different batch of plants. viii 101 103 105 107 123 126 128 130 134 136 138 140 144 146 148 Chapter 1 LITERATURE REVIEW I. INTRODUCTION Freezing is a stress of widespread occurrence to plants; for about 50% of the earth's land, the average minimum temperature is below -10°C (see Sakai and Larcher 1987). Freezing stress represents the most important environmental constraint limiting the distribution of plants (Parker, 1963). Together with drought and salt stress, freezing also represents one of the major limiting factors in productivity of agriculture crops. For example, if winter wheat and rye could be made 2°C more cold hardy, these new cultivars could replace much of the spring wheat and rye grown in North America and territories of the former Sovite Union to increase the crop yields by 25 to 40 percent (See Salisbury and Ross 1985). Through evolution, plants have developed mechanisms to allow them to survive different environmental stresses occuring in the habitat in wild they originated. Most plant species from temperate and alpine regions can increase their resistance to freezing stress via a process triggered by exposure to low but non-freezing temperature (see Levitt, 1980; Sakai and Larcher, 1987; Thomashow, 1990). This process is termed cold acclimation or cold hardening. Plant cold acclimation ean be of such manifestation that some acclimated woody plants like dogwood can readily survive 2 liquid nitrogen temperature of 496°C. These same plants, when actively growing and nonacclimated, are severely injured or killed by only -3°C (Weiser, 1970). Herbaceous plants can also attain a certain level of cold hardiness. Non-acclimated wheats are killed upon freezing to about -5°C while acclimated ones can survive the temperature down to -20°C. Non-acclimated Arabidopsis thaliana are killed at about -3°C but they can survive a freezing temperature of -10°C after cold acclimation F (Gilmour er al 1988). For decades, the study of plant cold acclimation has been focused on two primary questions: the mechanism(s) of freezing injury, and the biochemieal and physiological changes that occur during cold acclimation. Enormous amounts of information have been generated which have improved our understanding of plant cold acclimation. However, our knowledge about the mechanism(s) of cold acclimation and freezing resistance is still far from satisfactory either for our scientific curiosity to understand its nature or for the practicality of achieving crop improvment. More recently, the development of molecular biology has brought a new approach to the study of cold acclimation (see Thomashow, 1990). This approach is based on an early idea that variation in hardiness among plant species and seasonal variation within the tissues of a single species have a genetic basis (Weiser 1970). By determination and characterization of the genes whose expression are altered by low temperatures, this new approach may provide insight into which biochemical and physiological changes in cold acclimation are directly responsible for altered freezing resistance in plants. 3 II. FREEZING INJURY Freezing injury may occur in all plants, in contrast to chilling injury that only occurs in tropical and subtropical plants. Among the more commonly recognized types of freezing injury are spring and autumn frost damage to tender annuals, flowers and fruits, crown kill of winter cereals and herbaceous perennials, die-back in citrus, sun seald of tree trunks, and frost cankers and burls in conifers and hardwoods. Upon freezing-injury, the plant tissues appear decolorized or bleached, and the tissues shrink or dieback. When injuries occur to meristematic and incompletely differentiated tissue, the organs that subsequently develop would exhibit distortions, stunting or fragmentation. Freezing damage can also delay the onset of various stages of development. By definition, freezing is a process in which the crystallization of ice occurs. Depending on the rate of cooling, freezing can happen in two markedly distinct loeations within the plant tissues: extracellular or intracellular. If the cooling rate is slow, as most frequently occurs in nature (< 1-2°C/hr), freezing is most likely to occur extracellularly (Mazur, 1969; Steffen et al 1989). It has been suggested that ice normally crystallizes first in the large vessels, probably because of their large diameter which does not favor supercooling, and their dilute sap which also contributes to the higher freezing point (Olien, 1967). In almost every case, ice crystals will spread from the initial site throughout the extracellular region of the tissue and continue to grow if the duration of subzero temperature is extended. At a slow cooling rate, ice crystals are barred from entering the cell by the plasma membrane surrounding each cell, where intracellular ice nucleation is usually not favored. Consequently, ice from vessels only spread through the intercellular spaces. On the other hand, if the cooling rate is moderately rapid (5-20°C/hr), freezing may occur intracellularly. At temperatures that 4 occur in nature, intracellular freezing is considered to be universally and instantaneously lethal to plant cells (Burke et al. , 1976; Sakai and Larcher, 1987). It should be noted that the rapid cooling rate causing intracellular freezing is seldom found in nature (Levitt, 1980). Since it is hard to know whether a cell surving a freeze-thaw cycle has been ”injured”, research into the processes involved in freezing injury in plants has always been directed primarily towards the elucidation of causes of cell death. Of the many hypotheses and theories that have been proposed over the years, none have proved entirely satisfactory (see Sakai and Larcher, 1987). The main reason for this is probably lies in the diversity of aspects depending upon species, state of acclimation, and the condition of freezing, so that one general mechanism can not be singled out to be responsible for cell death or survival. It is, however, generally accepted that intracellular ice formation always eauses direct injury leading to the death of the frozen cells, and extracellular freezing results in indirect physical injury to the protoplasm. Indirect injury may have different manifestations such as mechanical damage, air expulsion, freeze smothering, and freeze dehydration (Levitt, 1980). Among these, freeze-induced dehydration may be the most well documented. Freeze dehydration may also be the most important mechanism of freezing injury to plant cells because of the fact that intracellular freezing is rarely observed in nature (Levitt, 1980). As mentioned before, the plasma membrane is an effective barrier for ice crystals to invade the protoplast. When water in the intercellular spaces freezes gradually, the extracellular water potential declines rapidly compared to the intracellular water potential. First, the absolute value for osmotic potential of the extracellular spaces decreases (i.e. it becomes more negative) as a result of the freeze 5 -induced concentration of salt. Secondly,as the temperature drops, pressure potential (or vapor pressure), another component of water potential, of the ice outside of the cell decreases faster than the vapor pressure of liquid water inside the cell. A water potential gradient is thus established, with liquid water moving down the gradient to the extracellular spaces. It has been calculated that the decline in the water potential of ice with decreasing temperature can be as large as -1.16 MPa/0C (see Guy, 1990). This decline in water potential outside of the cell can create a very steep gradient across the plasma membrane, considering the osmotic potential for most plant saps lie between -0.4 and -2.0 MPa (Salisbury and Ross, 1985). Consequently, protoplasts at equilibrium with extracellular ice will dehydrate in a strict temperature-dependent relationship. One of the extreme consequences resulting from extracellular freezing is "frost plasmolysis. " Cells killed by extracellular freeze characteristically show frost plasmolysis, a contraction of the dead protoplast leaving a large space between it and its cell wall (see Levitt 1980). Frost plasmolysis is due the osmotic contraction of the cell during extracellular ice formation and the inability of the dead protoplast to reabsorb the water formed in the intercellular spaces on thawing of extracellular ice. As a result, the cell wall expands back to nearly its original shape, while the dead protoplast remains contracted. This would seem to indicate that the injury had occurred during the freezing and the cells were nonfunctional by the time the ice began to thaw. Although it is not clear exactly how freeze dehydration ean cause cell death, various kinds of deteriorating effects of freeze—dehydration have been proposed (see Levitt, 1980, Steponkus, 1990). As mentioned above, the plasma membrane plays a central role in cellular behavior during freezing. As early as 1912 Maximov suggested that disruption of cellular membrane, particularly the plasma membrane, was the primary form of 6 freezing injury in plants, and the subsequent studies have supported this view (see Levitt, 1980; Steponkus, 1990). It has been proposed that freeze-induced dehydration results in two different forms of injury depending on the temperature and the extent of cell dehydration (Steponkus et a1 1982; Steponkus and Lynch 1989; Steponkus 1990). The first type of freezing injury in the plasma membrane is "expansion-induced lysis", which occurs at a relatively high (-3 to -7°C) temperature. In contrast, when cells are cooled to a lower temperature (e. g. -10°C) and subjected to a greater extent of cell dehydration (e.g. about 90% of the osmotically active water is removed), the protoplasts are osmotically unresponsive during drawing. This second form of injury is referred to as ”loss of osmotic responsiveness.“ Although both forms of injury occur as a result of cell dehydration, the mechanisms of injury are vastly different. It was suggested that the elastic expansion/contraction of the plasma membrane is limited to changes in area of about 2-3% (Klosson and Krause, 1981). For larger changes in the surface area of the plasma membrane, as usually occurs during cell dehydration, membrane material is either deleted from the membrane during osmotic contraction or incorporated into the membrane during osmotic expansion. The deletion has been observed as endocytotic vesicles in nonacclimated rye protoplast by computer enhanced microscopy (Dowgert and Steponkus, 1984) and electron microscopy (Gordon-Kamm and Steponkus, 1984a; Jonhson-Flanagan and Singh, 1986). The deleted membrane materials formed during osmotic contraction do not re-enter the membrane during subsequent osmotic expansion. As a result, during cell rehydration, the irreversible deletion of the membrane leads to an intolerable osmotic pressure and the protoplast bursts. In contrast, when protoplast isolated from cold acclimated plants were subjected to similar osmotic contraction, the plasma membrane formed exocytotic extrusions instead of endocytotic vesicules. The important difference between exocytotic extrusion and endocytotic vesiculation is that the membrane material of 7 exocytotic extrusions remains associated with the plasma membrane and is reincorporated during rehydration. As a result, the protoplasts can swell back to their original size rather than lyse as is the case with endocytotic vesiculation. The second type of plasma membrane injury has been shown to occur at -10°C and below. At these lower temperatures, the dehydration experienced by the cell is more severe than freezing at -5°C, and results in the ”loss of osmotic responsiveness” because the plasma membranes lose their semipermeability during osmotic contraction. A conceivable consequence of this type of injury is the frost plasmolysis described before. The loss of osmotic responsiveness is associated with several changes in ultrastructure of the plasma membrane, including the appearance of lateral phase separations and lamellar-to—hexagonal II phase transitions (Gordon-Kamm and Steponkus, 1984a,b; Pearce and Willison, 1985; Johnson-Flanagan and Singh, 1986; Singh et al., 1987). Again, the second type of injury and the ultrastructural changes associated with it are greatly reduced in cold acclimated cells. The membrane ultrastructural changes and the loss of osmotic responsiveness are suggested to be the consequences of freeze-induced dehydration rather than the exposure to subzero temperature per se. This is indicated by the fact that the same injury can be induced by subjecting protoplasts to equivalent osmotic-dehydration (5.4 osm sorbitol) in the absence of ice formation. In addition, protoplast suspensions can be supercooled to temperatures of -5°C to -15°C without deliterious effects (Gordon-Karnm and Steponkus, 1984b; Dowgert er al., 1987). It has been suggested that dehydration increases the membrane liquid-crystalline to solid-gel phase transition temperature (see Steponkus and Lynch, 1989). One consequence of such crystalline-to-gel phase transition is the occurrence of laterally separated regions with clusters of intramembrane particles enriched in some areas and smooth, particle-depleted surfaces in other areas of the membrane, a characteristic 8 feature of the lateral phase transition observed in freeze-injured membranes. A dehydration-induced crystalline-to-gel phase transition also results in demixing of the lipid mixture and localized enrichment of nonbilayer-forming lipids such as phosphatidylethanolamine which, upon further dehydration, would undergo lamellar-to- hexaganol 11 phase transition. The hexaganol 11 phase is not semipemeable and not responsive to osmotic changes. Besides the direct injury to plasma membranes, both freem-induced dehydration and freezing temperature also impose deleterious effects to other organelles and macromolecules of the cell. For example, the chloroplast has been regarded as another major site of injury by freezing (Levitt, 1980; Hincha and Schmitt, 1991). When leaves are frozen to lethal temperatures, photosynthesis is irreversibly damaged (Krause et al., 1988; Steflen et al., 1989). It has been shown that both freezing and severe dehydration can eause rupture of thylakoid membranes as indicated by the release of the lumen protein plastocyanin (Hincha et al. , 1987; Hincha et al 1989). When spinach leaves were frozen at 6°C and thawed slowly, more than 90% of the plastocyanin was released from the thylakoid lumen. Cold acclimation ean prevent freeze-induced or dehydration-induced leakage of the plastocyanin. It is not clear what mechanism is responsible for the freeze-induced rupture of chloroplast membranes. Neither is it known how cold acclimation can prevent this type of injury. Another form of freezing injury is the direct damage to protein molecules as many proteins are freeze labile (Levitt, 1980). Many enzymes lose their activity after freezing and thawing, such as lactate dehydrogenase, eatalase, phosphofructokinase, malate dehydrogenase, pyruvate kinase, and ATPase (Carpenter and Crowe, 1988). Protein structures may undergo a number of changes at low temperature or in the dehydrated environment. First, the quaternary structure may be altered leading to dissociation of a large protein complex into monomeric subunits (Markert, 1965). For 9 example, all of the microtubules in the green alga (Losterium ehrenbergir) depolymerize after only 5 min at 0°C (l-Iogetu, 1986), and the coupling factor CFl is released from the chloroplast ATPase complex by either freezing or hypertonic conditions (Santarius, 1984, Hincha and Schmitt, 1985). Secondly, the tertiary structure of some proteins may be changed during freezing or dehydration (Brandts, 1967), and the unfolded molecules may aggregate by forming physical bonds (salt I bridge, H bond, hydrophobic interactions, S-S bonds, etc) between the newly exposed active chemical groups that were previously buried within the folded molecules. Freeze-induced protein aggregation or precipitation was noticed more than 80 years ago by Gorke (Gorke, 1906), when he found that proteins in the sap expressed from nonacclimated cereal leaves and other plants precipitated upon a freeze-thaw cycle. The more resistant the plant to freezing injury, the lower was the temperature required to precipitate the proteins. Gorke explained freezing injury as a precipitation of the protoplasmic proteins due to the increased concentration of the cell salts that occurs during freezing. Finally, the freezing or dehydration damage to cellular membrane systems has profound effects on the protein molecules previously associated with these membranes or compartmented within organelles. This has been clearly shown by observation on one of the ultrastructural changes in the plasma membrane described previously, lateral phase separation, where some part of the membranes is depleted of membrane protein particles upon freeze-induced dehydration. On the other hand, it has been shown that the leakage of stroma or thylakoid lumen proteins such as coupling factor CPI and plastocyanin is associated with the inactivation of photophosphorylation and the irreversible damage to photosynthesis during freezing (Hincha et al 1987). It can be concluded that freezing injury to plant cells can be the consequence of either intracellular freezing which is always lethal, or the extracellular freezing that is pontentially tolerable. Extracellular freezing of plant tissues leads to impairment of the 10 structure and function of plasma membranes, chloroplasts, and other cellular components. Both freezing temperatures and freeze-induced dehydration result in changes in and damage to cellular structures and enzymes. Therefore, it was suggested that when plants become cold acclimated they must have developed both intracellular -freeze avoidance and extracellular-freeze tolerance mechanisms to survive the subsequent freezing environment (Levitt, 1980). Although the above notion is now generally accepted, the detaiils of the mechanisms involved are still controvertial (see Levitt, 1980; Sakai and Larch, 1987; Steponkus, 1990; Guy, 1990). II. BIOCHEMISTRY OF COLD ACCLMATION Research on the biochemistry of cold acclimation has been informative in terms of our understanding about mechanism(s) responsible for the increased freezing tolerance of acclimated plants. However, the majority of these studies have been restricted to either correlative studies of gross changes in certain types of molecules during cold acclimation or comparative studies of these changes in species or varieties of different hardiness (Levitt, 1980). Unfortunately, these studies have not significantly improved our understanding of how cold acclimation leads to increased freezing tolerance in plants. More recently, biochemieal studies have been directed more toward direct cause and effect analysis and most progress has been made in membrane lipid studies (see Stenpokus, 1990). A. Membrane lipid changes during cold acclimation Early studies on membrane lipid change during cold acclimation were restricted to lipid analysis of whole tissue extracts or crude membrane fractions rather than 11 plasma membrane per se. The improvement of techniques for isolating purified plasma membrane fractions made possible the more direct studies of the alteration in the lipid composition of the plasma membrane during cold acclimation. By comprehensive analysis of more than 50 lipid species of the plasma membrane from cold acclimated and nonacclimated rye leaves, it has been found that cold acclimation alters the proportion of virtually every lipid component; although there are no lipid species that are unique to cold acclimated or nonacclimated plasma membranes (see Lynch and Steponkus, 1987). Following cold acclimation, free sterols increase about 25 % , while the steryl glycosides and acylated steryl glycoside decrease about 30% and 25 % respectively. The phospholipid content of the plasma membrane also increases for about 25 % following cold acclimation. Two major phospholipids, PC (phosphatidylcholine) and PE (phosphatidylethanolamine) increase from 15 to 20 mol % and 11 to 16 mol % , respectively. In addition, the levels of diunsaturated fatty acid species (18:2/ 18:2, 18:2/18z3, 18:3/ 18:3) of PC and PE double in cold acclimated cells. As described in the previous section, the cryobehavior of the plasma membrane from cold acclimated and nonacclimated rye cells is different. Freeze-induced osmotic contraction result in endocytotic vesiculation and exocytotic extrusions in the plasma membranes of nonacclimated and cold acclimated cells, respectively. In addition, the dehydration-induced lamella to hexagonal 11 phase transition occurs only in plasma membranes of nonacclimated cells but not in plasma membrane of cold acclimated cells (Gordon-Kamm and Steponkus, 1984b). The eausal relationship between the changes in membrane lipid druing cold acclimation and the cryobehavior of plasma membranes has been demonstrated by Steponkus and colleagues in an elegant series of experiments (Steponkus et al., 1988; Steponkus and Lynch, 1989). The cryobehavior of liposomes prepared from plasma membrane lipids extracted from cold acclimated and 12 nonacclimated rye leaves during freeze-induced osmotic contraction was analyzed and it was shown that the differential cryostability of the plasma membrane of cold acclimated and nonacclimated plant cells was indeed determined by alterations in the composition of the lipid bilayer. In liposomes made from plasma membranes of nonacclimated cells, numerous vesicles were subdued from the liposome bilayer and sequestered into the liposome interior during freeze-induced osmotic contraction. These liposomes lysed during subsequent osmotic expansion upon thawing. Under similar conditions, liposomes prepared from membrane lipids of cold acclimated cells did not exhibit such behavior. Instead, osmotic contraction resulted in the formation of either exocytotic extrusions or vesicles that remained contiguous with the parent bilayer. During subsequent thawing and osmotic expansion, the extruded vesicles were drawn into the plane of the parent liposome bilayer and lysis was avoided. The specific lipid composition responsible for the cryostability of cold acclimated plant cells has been studied using pH-induced protoplast x liposome fusion techniques (Steponkus et a1. , 1988). It was found that the freezing tolerance of nonacclimated protoplasts at temperatures ranging from 0°C to -5°C could be dramatically increased (from 50% survival to 100% survival) by fusing nonacclimated protoplasts with liposomes composed of mono-unsaturated or diunsaturated molecules of phosphotidylcholine. At temperature over the range of 0°C to -5°C, the principal form of injury in nonacclimated protoplasts was expansion-induced lysis, which was probably the consequence of endocytotic vesiculation in the plasma membrane during osmotic contraction. Remarkably, when the nonacclimated protoplasts were fused with mono— or diunsaturated species of phosphotidylcholine, they formed exocytotic extrusions during freeze-induced osmotic contraction and the incidence of osmotic expansion induced lysis diminished. However, it did not appear that mono— or 13 diunsaturated phosphotidylcholine were responsible for the functional and morphological behavior of cold acclimated protoplasts at temperatures of around -10°C. Nevertheless, the changes in plasma membrane lipid composition appear to be important for one facet of the cold acclimation process. B. Protein Changes Associated with Cold Acclimation Two lines of evidence suggest that alteration in protein composition of plants may contribute to the freezing tolerance developed during cold acclimation. The evidence consists of repeated observations that a number of enzymes show shifts in isozyme composition during cold acclimation, and numerous studies have shown both quantitative and qualitative differences in the protein composition between nonacclimated and cold acclimated plant tissues (see Levitt, 1980; Thomashow, 1990; Guy, 1990). Studies of a number of enzymes from cold-acclimated plants have demonstrated changes in activity, stability, and isozyme variation when compared to nonacclimated ones. One of the best-characterized enzyme relative to cold acclimation is ribulose bisphosphate caboxylase/oxygenase (Rubisco) from winter rye, one of the most freezing-tolerant cereals. It was found that cold acclimated and nonacclimated rye have different Rubisco isozymes (Huner and Macdowall, 1976, 1978, 1979). The enzyme isolated from cold acclimated plants was not only more stable to denaturants and freezing storage, but also had a lower Km for C02 at temperatures below 5°C. Similar studies using the enzyme isolated from freezing-sensitive and tolerant potato species also demonstrated structural differences that paralleled the variation in freezing tolerance much in the same way the enzyme did from acclimated and nonacclimated rye (Huner et al., 1981). When the Rubisco isozymes from cold acclimated and nonacclimated cabbage were isolated (Shomer-Ilan and Waisel, 1975), their amino acid 14 compositions were found to be significantly different, which implied an involvement of differential gene expression, as described later. A number of other examples of cold-acclimation-associated alteration in isozyme profiles has been documented. These include peroxidase (McCown er al. , 1969), invertase (Roberts, 1974), ATPase, esterase, acid phosphatase, leucine aminopeptidase (Krasnuk er al. , 1976), and a number of dehydrogenase associated with the respiratory pathway (Hall et al., 1970). It has been suggested in many eases that the "acclimated" isozymes might be better "suited” to a low temperature environment. For example, a study of glutathione reductase from spinach has demonstrated that the isozyme isolated from acclimated plants was less sensitive to freeze/thaw inactivation than its nonacclimated counterpart (Guy and Carter, 1984). An indication that de novo protein synthesis is required for the induction of freezing resistance comes from studies using the protein synthesis inhibitor cycloheximide. It has been found in a number of plants, including winter wheat, winter rape, and potato species that application of cycloheximide could inhibit the development of freezing tolerance by cold acclimation (Trunova and Zvereva, 1977; Kacperska-Palacz er al., 1977; Chen et al., 1983). In addition, a general observation has been made that the soluble protein content of plants increases during cold acclimation (see Levitt, 1980; Sakai and Larcher, 1987). Examples include studies as long as 40 years ago in the black locust tree (Siminovitch and Briggs, 1949) where the protein content was found to increase during cold acclimation. In a more recent study, the content of protein of acclimated wheat was estimated to be 300% greater than that of the nonacclimated plants (Trunova, 1982). Alterations have also been reported in membrane protein composition during cold acclimation in orchard grass and winter rye (Yoshida and Uemura 1984, Uemura and Yoshida, 1984), where the plasma membrane 15 protein profiles were found to be different between cold-acclimated and nonacclimated plants. Moreover, the polypeptide composition of tissues from a variety of cold ~acclimated and nonacclimated plants have been shown to be different when examined by electrophoresis and in vivo radiolabeling (see Thomashow 1990; Guy, 1990). Studies of cold-acclimation associated changes in polypeptide composition and protein from a variety of plant species can be summarized as follows: (1) Cold acclimation is associated with both the appearance of new polypepetides and increase and/or decreases in others. (2) Although a number of changes occur, the overall patterns of protein synthesis in cold-acclimated and nonacclimated plants appears to be quite similar, which is in contrast with other stress responses like that of heat shock or anaerobiosis. Another difference between the response of protein synthesis to cold stress and other stresses is that the response is relatively slower to cold in comparison to other stresses. (3) The newly synthesized proteins, also referred to as COR (mid-regulated) proteins (Thomashow, 1990), range from large (200 kDa) to small (< 10 kDa) and from basic (pl > 8) to acidic (p1 < 4). They can be soluble or membrane-bound proteins. There is not a simple pattern which is universal between species. (4) The appearance of most, if not all, of the COR polypeptides generally coincides with the onset of freezing tolerance; their synthesis continues as long as the plants are kept at cold-acclimating temperatures, and their synthesis declines quickly during deacclimation. In most cases, the functional significance of changes in isozyme or other protein composition during cold acclimation is not known. Some of these changes may contribute indirectly to the overall fitness of the plant for low temperame survival, which in turn could increase plant freeze tolerance. For example, new isozymes may be necessary to accomplish the necessity to shift metabolic pathways at low 16 temperature, or replace cold labile isozymes so that the plant can continue to operate the pathway at low temperature. Other changes may contribute directly to the freezing tolerance of acclimated plants. For example, Volger and Heber have reported that some proteins (10-20 kDa) which accumulate in acclimated spinach leaves have cryoprotective activity in an in vitra assay (Volger and Heber, 1975), as do sucrose, trehalose, proline and other small molecules (Crowe and Crowe, 1986; Carpenter and Crowe, 1988; Caffrey et al., 1988). The activity of these cryoprotective proteins, measured as the ability to protect photophosphorylation of the thylakoid membrane against freezing damage, was found to be much higher (more than 10,000 times higher on a molar basis) than that of small molecular weight cryoprotectants. Similar cryoprotective proteins have also been reported in cold acclimated cabbage (Hincha et al. , 1989; Hincha and Schmitt, 1990). The cabbage proteins enriched of a polypeptide of about 28 kDa, was found to be 20,000 to 40,000 times as effective as sucrose in preventing freeze-thaw induced rupture of isolated thylakoid membranes. Both the spinach and cabbage proteins are very hydrophilic and stable to conditions that are usually detrimental to proteins, such as extreme pH, and extremely high temperature (95°C to boiling). However, these "cryoprotective” proteins have not been isolated to purity and whether these proteins have important cryoprotective functions in viva and contribute significantly to freezing tolerance of the plants also remain to be determined. III. Genetics of Cold Acclimation A. Inheritance of freezing tolerance The inheritance of freezing tolerance was studied first by Nilsson-Ehle (1912) who crossed two winter wheat varieties intermediate in freezing tolerance and found 17 transgressive segregation (progeny segregate outside the parental boundaries) for the trait of freezing tolerance. He concluded that freezing tolerance was a quantitatively inherited trait controlled by multiple genes. Subsequent studies have confirmed this conclusion (Hayes and Admodt, 1927; Rudolph and Nienstaedt, 1962; Norell et al. , 1986; Limin and Fowler, 1988). In wheat, barley and oat, several studies have reported evidence that freezing tolerance is controlled by partially dominant and/or recessive genes (Rhode and Pulham, 1960; Jenkins, 1969; Gullord er al., 1975; Sutka, 1981; Orlyuk, 1985). The complexity of genetics for freezing tolerance in plants is further revealed by the detailed cytogenetic studies of wheat using monosomic and substitution analysis. Eleven out of 21 chromosomes of hexaploid wheat have been suggested to be involved in freezing tolerance although chromosome 5A and 5D appear to earry the major effects (see Roberts, 1986; Thomashow, 1990). The repeated verification of the quantitative nature and the complexity of the genetics of plant freezing tolerance is not unexpected given the fact that morphology, physiology, developmental processes, and environmental interactions all influence the ability of a plant to survive in winter. B. Changes in gene expression during cold acclimation Over the last two deeades, it has become apparent that plants respond to adverse environmental stresses (anacrobiosis, heat shock, water deficit, salt stress, etc.) through alterations in gene expression (Key and Kosuye, 1984; Sachs and Ho, 1986). In general, when the environmental conditions become unfavorable for optimal growth and development of the plant, new stress-related genes are induced and their products accumulate. Although the functional identity for most stress-related genes remains undetermined, it is certain that at least some of them function to enhance the plant's survival (Sachs and Ho, 1986). Prior to advances in the understanding of gene 18 expression and regulation in anaerobic and heat shock stresses, the two most extensively studied cases, a role for changes in gene expression in cold acclimation and freezing tolerance was suggested. As early as 1970 Weiser proposed that altered gene expression and the synthesis of new proteins was necessary for the induction of maximum freezing tolerance in temperate perennials (Weiser, 1970). As previously described, studies in protein biochemistry have been generally supportive of Weiser's hypothesis, but the specific changes in gene expression responsible for freezing tolerance is still unknown. It is now clear that changes in gene expression occur during cold acclimation at both the transcriptional and post transcriptional levels (Thomashow, 1990). The first evidence along this these lines came from the work of Guy and colleagues (Guy at al. , 1985). By comparing in vitra translation products of poly(A+) RNA isolated from control and cold acclimated spinach plants, these authors found that mRN As encoding 180 and 82 kDa polypeptides accumulated in cold acclimated plants. Upon a longer period of cold acclimation, additional RNA species encoding different polypeptides accumulated in acclimated plants. It was also clear from this work that the overall RNA populations in nonacclimated and cold acclimated plants did not differ dramatically. This result is in agreement with the results from the studies of protein synthesis studies discussed before. Cold acclimation associated changes in mRN A populations have since been reported in a number of plant species. The general observation is similar to the one reported by Guy et al., although the specific RNAs altered by cold temperature are different from species to species. For example, in cold acclimated rapeseed (Brassica napus), RNAs encoding nine polypeptides ranging from 25 to 80 kDa accumulated (Meza-Basso er al. , 1986). Translatable RNA for seven polypeptides (11 to 95 kDa) in alfalfa (Medicaga falcara cv Anik) were found to increase during cold acclimation (Mohapatra et al. , 1987). Levels of RNA for 160 and 19 47 kDa polypeptides were found to be elevated in cold acclimated Arabidapsis thaliana (Gilmour et al., 1988). Since the late 19803, cDNA clones representing car (mld regulated) genes have been isolated from several plant species including alfalfa, barley, wheat, rapeseed, and Arabidapsis (Mohapatra et al., 1988, 1989; Kurkela and Franck, 1990; Hajela er al., 1990; Cattivelli and Bartels, 1990; Houde et al., 1991; Singh et al., 1991). In all these eases, the car cDNA clones were isolated using the technique of differential screening which has been almost a standard approach in the isolation of stress -responsive genes (Key and Kosuge, 1984). Many car genes have been suggested to be members of small gene families (Mohapatra et al., 1989; Kurkela and Franck, 1990; Houde et al. , 1991). Northern analysis of car gene expression showed that the steady state level of car gene transcripts accumulated as quickly as 12 hours after cold induction in Arabidapsis (Hajela et al. , 1990) or as slowly as 2 to 3 days in alfalfa (Mohapatra et al., 1988). The level of these transcripts usually increased to 10 fold or higher in cold acclimated plants compared to the nonacclimated level, and returned to to lower level in a few hours upon deacclimation. Nuclear run-on transcription analysis indieated that of four Arabidapsis car genes, three were regulated primarily at the post-transcriptional level while the other one was controlled largely at the transcriptional level (Hajela et al. , 1990). C. Characterization of car genes and products No functional identity of any car gene has been determined. However, a correlation between the plant freezing tolerance and the car gene expression has been reported in alfalfa (Mohapatra et al. , 1989). Alfalfa cultivars Anik (Medicag falcata), Iroquois (M. saliva), Algonquin (M. media), and Trek (M. sativa) have different degrees of freezing tolerance: when cold acclimated, they have LT50 (the temperature 20 resulting in 50% mortality) values of -14.6, -11.8, -11.5, and -9.7°C, respectively. The relative level of expression of three cor genes, represented by cDNA clones pSM7844, pSM2358, and pSM2201 were evaluated in the alfalfa cultivars by Northern blot analysis. A positive correlation (with correlation coefficients larger than 0.9) was observed between the RNA levels for the genes represented by the three cDN A clones and the degree of freezing-tolerance in four alfalfa cultivars. It is not clear whether this type of correlation exists in other plant species. Many studies on the characterization of car genes have been directed at determining whether their expression is regulated by the plant hormone abscisic acid (ABA) and by drought stress. The reason that ABA is of interest is that exogenous application of ABA has been shown to increase freezing tolerance in whole plants and plant cell cultures in the absence of cold treatment (Irving, 1969; Rikin er al., 1975; Chen and Gusta, 1983; Chen et al., 1983; Orr et al., 1986; Reaney and Gusta, 1987; Mohapatra et al., 1988; Lang et al., 1989). Moreover, those species that increase in freezing tolerance in response to cold (eg. Medicaga sativa, Brassica inermis, Daucus carota, and Mticum aestivum) also become more freezing tolerant in response to ABA while those species that do not frost harden in response to cold (eg. Datum innoxia, Catharanthus raseus, Glycine max, Vicia hajastana, and cell lines of Triticle) do not accliarnte in response to ABA. Therefore, an important question raised by the ABA experiments is whether the expression of car genes is also modulated by ABA. This seems to be the case for some car genes but not for others. For example, all of the Arabidapsis car genes whose cDNA clones have been isolated are responsive to ABA (Hajela er al., 1990; Kurkela and Frank, 1990); on other hand, of four alfalfa car cDNA clones (Mohapatra et al., 1988, 1989), only one, (pSMl409, the relationship of its expression with the degree of freezing tolerance is not known) was found to be responsive to ABA (Mohapatra er al. , 1988) but only in the relatively frost-resistant 21 cultivars; other alfalfa car genes whose expression was strongly correlated with the freezing tolerance of the alfalfa cultivars described previously, however, were not responsive to ABA (Mohapatra er a1 . , 1989). Cold-regulation and ABA-regulation of the car genes have been suggested to be mediated through independent control mechanisms by the fact that ABA-regulated expression of Arabidapsis car genes were dramatically impaired in an ABA-insensitive Arabidapsis mutant, abiI , while the cold -regulated expression of these genes was unaffected in the obi] plants (Gilmour and Thomashow, 1992). To determine Whether car gene expression is affected by water deficit is of interest for at least three reasons. First, it is well established that drought stress results in the accumulation of ABA (see Levitt, 1980), and the increased ABA content of plant during cold acclimation has also been reported (Chen et al., 1983). Therefore, car genes which are responsive to ABA might as well be responsive to drought stress. Second, several studies have found that drought stress could induce freezing tolerance at normal growth temperature for some plants, such as cabbage, wheat, and rye (Cox and Levitt, 1976; Cloutier and Siminovitch, 1982; Siminovitch and Cloutier, 1982,1983). Finally, severe dehydration of the plant cell is one of the consequences resulting from extracellular freezing. As a consequence of the similarity between freezing and drought stress at the cellular level, plants may have developed similar responses to both forms of the stress, that involve common adaptive mechanisms. Thus, it is reasonable to hypothesize that genes with roles in drought stress might also have functions in freezing tolerance, and that a set of genes might be regulated by both kinds of stress. There have been limited studies in alfalfa and Arabidapsis on the expression of car genes in response to drought stress. In alfalfa, it was found that the expression of one car gene (pSMl409; the one responsive to ABA), was indeed increased 10 fold in water stressed plants (Mohapatra et al., 1988). In contrast, three 22 other alfalfa car genes which were not responsive to ABA were apparently not regulated by water stress (Mohapatra et al. , 1989). On the other hand, all Arabidapsis cor genes whose cDNA clones had been isolated are stimulated by water stress (Hajela er al. , 1990). Although it is clear that reproducible changes in gene expression occur during cold acclimation, the roles of these genes in cold acclimation are unknown. There is no report on the detailed characterization of any COR polypeptide, nor is their cellular localization known. Most importantly, there is no direct evidence for or against the hypothesis that COR polypeptides have significant roles in freezing tolerance as was first proposed about two decades ago (Weiser, 1970). Detailed studies on car genes and COR polypeptides should provide answers to the above questions. I 2 3 Literature Cited Brandts El (1967) Thermobiology. Academic Press, New York, pp 25-73 Burke MJ, Gust LV, Quamme HA, Weiser CJ, and Li PH (1976) Freezing and injury in plants. Annu Rev Plant Physiol 27, 507-528 Caffrey M, Fonseca V, and Leopold AC (1988) Lipid-sugar interactions, relevance to anhydrous biology. Plant Physiol 86, 754-758 Carpenter IF, and Crowe JH (1988a) The mechanism of cryoprotection of proteinsby solutes. Cryobiology 25 , 244-255 Carpenter IF, and Crowe JH (1988b) Models of stabilization of protein by organic solutes during desiccation. Cryobiology 25, 459-470 Cattivelli L, and Bartels D (1990) Molecular cloning and characterization of cold -regulated genes in barley. Plant Physiol 93, 1504-1510 Chen H-H, and Gusta LV (1983) Abscisic acid-induced freezing resistance in cultured plant cells. 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Science 169, 1269-1278 Yoshida S, and Uemura M (1984) Protein and lipid composition of isolated plasma membrane from orchard grass (Dactylis glamerara L.) and changes during cold acclimation. Plant Physiol 75, 31-37 Chapter 2 ARABIDOPSIS car GENES ENCODING BOILING-STABLE POLYPEPTIDES SUMMARY Changes in gene expression during cold acclimation of Arabidapsis were studied by analyzing the in vitra translation products of RNAs isolated from control and cold -acclimated plants. Transcripts encoding polypeptides with molecular weights of approximately 160, 47, 24, and 15 kDa were found to accumulate in cold-acclimated plants. These polypeptides, referred to as COR160, COR47, COR24, and COR15, respectively, share the unusual biochemical property of remaining soluble upon boiling in aqueous solution. The accumulation of transcripts encoding ”boiling-stable" polypeptides was also detected in cold-treated spinach and potato. The Arabidapsis genes encoding COR160, COR47, and COR15 were shown to be represented by cDNA clones pHH28, pHH7 .2, and LCT10, respectively. Drought-stressed Arabidapsis plants were found to accumulate transcripts for COR160, COR47, COR24, and COR15 as well as a number of drought-specific transcripts that also encode boiling-stable polypeptides. 29 30 - INTRODUCTION Cold acclimation is a complex response involving a variety of physical and biochemical changes (Levitt, 1980; Sakai and Larcher, 1987). One of the hallmarks of cold acclimation is increased freezing tolerance; in many species of plants, a period of exposure to low nonfreezing temperatures results in an increased tolerance to freezing temperatures (Levitt, 1980; Sakai and Larcher, 1987). Biochemical changes that have been associated with cold acclimation include alterations in lipid composition, increased sugar and soluble protein content, and the appearance of new isozymes (Levitt, 1980; Sakai and Larcher, 1987; Steponkus and Lynch, 1989). In most cases, the precise role that a given biochemical change has in the cold acclimation process is uncertain. Presumably, some contribute to the overall fitness of the plant for low temperature survival while others have specific roles in bringing about increased freezing tolerance. Indeed, it has been demonstrated that changes in membrane lipid composition can contribute directly to the freezing tolerance of plant cells (Steponkus and Lynch, 1989; Steponkus et al., 1988). In addition, there is evidence that proline and many simple sugars (Carpenter and Crowe, 1988; Santarius, 1973; Strauss and Hauser, 1986), as well as certain soluble polypeptides from spinach (V olger and Heber, 1975), have cryoprotective effects in vitra. Whether these molecules contribute signifieantly to freezing tolerance in viva, however, remains to be determined. The physical and biochemical changes that occur in plants during cold acclimation could be brought about by preexisting structures and enzymes that undergo alterations in their properties at low temperature. It is also possible, as first proposed by Weiser (Weiser, 1970), that cold acclimation involves changes in gene expression. Indeed, recent studies with a variety of plant species including Arabidapsis have 31 demonstrated that changes in gene expression occur during cold acclimation (see Guy, 1990; and Thomashow, 1990). Very little is known, however, about the nature of the car (cold-regulated) genes. Are any of the polypeptides encoded by car genes related at the structural or functional level? Are car genes involved in freezing tolerance or other aspects of low temperature survival? Are car genes responsive to environmental signals other than low temperature? To address these issues, I have initiated a characterization of car genes and their products in Arabidapsis thaliana. I have chosen Arabidapsis thaliana as a model system as it has the ability to cold acclimate (Gilmour er al. , 1988) and it is well suited for genetic and molecular genetic studies (Meyerowitz and Pruit, 1985; Estelle and Somerville, 1986). MATERIALS AND METHODS Plant Material Arabidapsis thaliana L.(Heyn) ecotype Landsberg or Columbia was grown in controlled environment growth chambers at 21°C under constant illumination from cool white fluorescent lights. Light intensity was approximately 150 uE m"zs'1 near soil level, and the relative humidity was about 80% . Arabidapsis plants (approximately two weeks old) were cold acclimated by transferring them to chambers set at 5°C for 3 days with continuous light at an intensity of approximately 50 uE m’2 s'1 . Drought stress was applied to plants grown at 21°C by withholding water until they became visibly wilted (approximately 8 days). Seedlings of spinach (Spinacia aleracea L.) and potato species (Salomon tuberasum, S. acaule, S. cananersanir) were grown under similar conditions to that of Arabidopsis, and were cold acclimated at 4°C for 4 days and 7 days, respectively. 32 For RNA extraction, Arabidopsis rosette leaves, and spinach and potato leaves, were excised, ground in liquid N2 with a mortar and pestle and either used directly or stored at -80°C prior to use. Total and Poly(A+) RNA Isolation Total RNA and poly(A+) RNA were prepared from plants essentially as described (Gilmour et al. , 1988). Frozen pulverized plant material was collected and extracted in a buffer (4 ml per gram tissue) containing 100 mM Tris-HCl (pH 7.6), 50 mM EGTA, 100 mM NaCl, 1% (w/v) SDS, 10 mM D'l'l‘, 6% (w/v) p-aminosalicylic acid (sodium salt) (Kodak), and 1% (w/v) tri-isopropylnaphthalene-sulfonic acid (sodium salt) (Sigma), and an equal volume of buffer-saturated phenol/chloroform/isoamyl alcohol (25/24/1, v/v/v). After centrifugation (10,000 g, 10 min) to separate the phases, the aqueous phase was extracted again with phenol/chlorofomr/isaomyl alcohol, and the nucleic acids were ethanol precipitated. All the solutions used after this point were made free of RNase by DEPC treatment (Sambrook et al. , 1989). The pellets from ethanol precipitation were resuspended in water, then precipitated with 2 M LiCl, followed by ethanol precipitation. The pellets were dissolved in water and stored at -80°C. Poly(A +) RNA was further isolated from total RNA using poly(U) Sepharose chromatography according to Cashmore (Cashmore, 1982)., In Vitra Translation Poly(A+) RNA or total RNA was translated in vitro using a rabbit reticulocyte lysate system (Promega) with [358] methionine (103 Ci/mmole) as a radiolabel according to the manufacturer's instruction. Typically, 1 ug of poly(A+) RNA or 5 to 33 10 ug of total RNA was translated in a 50 ul reaction containing 35ul lysate and about so uCi [35S]methionine at 30°C for 1 h. Polyacrylamide Gel Electrophoresis SDS-PAGE gels were run according to the method of Laemmli (Laemmli, 1970). For most of the experiments, equal volumes of the in vitra translation reaction were analyzed, generally 2 to 4 ul for SDS-PAGE. Gels were stained with Coomassie brilliant blue to visualize the molecular weight standards, dried, and autoradiographed for up to 1 week. In some experiments, the gel was soaked in 1 M sodium salicylate prior to drying, then fluorographed for 1 to 3 days. Kodak X-Omat AR5 x-ray film was used for both the autoradiography and fluorography. Detection of Boiling-Stable COR Polypeptides Poly(A +) RNA or total RNA was translated in vitra. The translation reactions were diluted with 5 volumes of 50 mM Tris-HCl (pH 7.0), the samples placed in a boiling water bath for 10 min, and the insoluble material removed by centrifugation in an Eppendorf microcentrifuge (15 min). Polypeptides that remained soluble were precipitated with 5 volumes of acetone and collected by centrifugation in a microcentrifuge (15 min). The pelleted material was analyzed by SDS-PAGE gels as described above. cDN A Clones of car Genes cDNA clones pill-128, pHH7.2 and pI-IH67 were previously described (Haj ela, et al., 1990). pLCTlOA and pLCTlOB are cDNA clones homologous to pI-IH67, but were isolated from a different cDNA library prepared as follows. Double-stranded cDNA was synthesized from poly(A +) RNA isolated from 3-day-cold-acclimated 34 Arabidapsis (Columbia) essentially as described by Gubler and Hoffman (Gubler and Hoffman, 1983). EcaRI linkers were added to the cDNAs and the fragments inserted into the EcoRI site of lambda ZAP (Stratagene). Recombinant phage were packaged in vitra using Packagene (Promega) and transduced into Escherichia cali BB4 (Stratagene). Approximately 5x106 primary recombinants were obtained. The library was amplified once and stored at -70°C in SM buffer [SM buffer is 100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH 7.5, 0.01% (w/v) gelatin] containing 7% (v/v) DMSO (Sambrook et al., 1989). The cDNA library was screened to isolate clones homologous to car cDN A clone pill-171.1, a homologue of pill-I67 (Hajela et al. , 1990). Plaque lifts were prepared on N ytran membranes (Schleicher and Schuell) using standard methodologies (Ausubel er al., 1987; Sambrook et al., 1989). The cDNA insert from pI-IH71.1 was 32P—labeled by random priming (Feinberg and Vogelstein, 1983). Hybridization was at 60°C in buffer containing 6 x ssc (1 x ssc is 0.15 M NaCl, 0.015 M sodium citrate), 0.5% (w/v) SDS and 0.25% (w/v) nonfat dry milk. Washes were done at moderate stringency: 2 X SSC, 0.5% (w/v) SDS at 60°C. Phage displaying strong homology to the pl-IH7 1.1 probe were plaque purified and the cDNA inserts ”subcloned" in pBluescript SK(-) by a temperature induced 'phagemid rescue” procedure according to the supplier's instructions (Stratagene). The resulting plasmids was analyzed by Southern hybridization using the pHH71 .1 probe. One clone chosen for further analysis was pLCTlOA. The orientation of the EcoRI insert in pLCTlOA was reversed using standard recombinant DNA methods (Sambrook et al. , 1989) to give pLCTlOB. 35 Hybrid-Arrest in Vitra Translation Hybrid-arrest translations were done as described (Jagus, 1987) using poly(A+) RNA and single-stranded DNA. Single-stranded DNA antisense to the mRNAs were prepared from cDNA clones pI-IHZ8, pHH7.2, pLCTlOA, and the cloning vector pBluescript SK" by phage rescue method as described by Vieira and Messing (V ieira and Messing, 1987). To initiate a hybrid-arrest translation reaction, 1 ug poly(A+) RNA isolated from 3-day-cold-acclimated plants, 0.5 ug single-stranded DNA, and 10 ug tRNA were precipitated together with ethanol. The pellet was resuspended in 3 ul water, boiled in a water bath for 30 seconds, snap—frozen in liquid N2, thawed on ice, and mixed with 23 ul of hybridization solution (hybrid solution is 80% deionized formamide, 400 mM NaCl, 10 mM Pipes-NaOH pH 6.4, 8 mM EDTA). The reaction was incubated at 37°C for 10 hours, the nucleic acids precipitated with ethanol, the pellet resuspended, translated in vitra, and the translation products analyzed by SDS -PAGE. In Vitra Transcription/Translation pLCTlOA and pLCTlOB were linearized by digestion with BamI-II and the inserts transcribed in vitna with T7 RNA polymerase (Promega) using the T7 promoter carried on the pBluescript vector. The resulting transcripts were extracted with phenol:chloroform:isomayl alcohol (25:24: 1), and precipitated with ethanol. Transcription products were translated in vitra using the rabbit reticulocyte lysate system (Promega) containing [35S]methionine as described. Boiling-stable polypeptides were prepared, and the radioactive polypeptides were fractionated by SDS-PAGE and visualized by autoradiography. 36 RESULTS Cold Acclimation is Associated with Accumulation of Transcripts Encoding Boiling-Stable Polypeptides. Cold acclimation of Arabidapsis results in the accumulation of transcripts encoding polypeptides of 160 kDa, 47 kDa, 24 kDa and 15 kDa (Gilmour et al., 1989; Lin unpublished); these polypeptides are referred to as COR160, COR47, COR47, and COR15, respectively. As a first step towards determinng the functions of these COR polypeptides, I initiated a characterization of their physical properties. I found that all four major COR polypeptides share an unusual biochemical property: they remain soluble upon boiling in aqueous solution. This was shown as follows. Poly(A+) RNA isolated from both cold acclimated and nonacclimated plants was translated in vitra and the polypeptide products were fractionated either directly by SDS-PAGE or were first boiled, centrifuged to remove the boiling-insoluble material, and then fractionated by SDS-PAGE gels (Figure 2.1). As anticipated, the boiling treatment resulted in precipitation of the majority of the polypeptides translated from the RNA isolated from either the acclimated or nonacclimated plants. However, all four of the major COR polypeptides remained soluble. In addition, transcripts encoding boiling-stable polypeptides of 18, 20, and 21 kDa appeared to increase in the acclimated plants. Accumulation of transcripts encoding boiling-stable polypeptides at low temperatures is not unique to Arabidapsis; it also occurs in at least spinach and potato. In particular, boiling-stable polypeptides with the apparent molecular weight of approximately 24 and 12 kDa synthesized by RNAs isolated from cold-acclimated but not nonacclimated spinach plants (Figure 2.2). Similarly, cold-treatment resulted in the accumulation of RNA species encoding boiling-stable polypeptides in three Salanum species (Figure 2.2). For example, the boiling-stable polypeptides specific to cold _ _. 14“.... 37 Figure 2.1. Accumulation of transcripts encoding boiling-stable polypeptides in cold acclimated Arabidapsis. Poly(A) RNA isolated from cold acclimated (AC) and nonacclimated (NA) Arabidapsis was translated in vitra and the polypeptide products were either fractionated directly by SDS-PAGE (TOTAL) or were first boiled and treated as described in Materials and Methods and then fractionated (BOILED). The TOTAL and BOILED samples represent approximately 5 and 25 p1 of the in vitra translation products, respectively. Film exposures were for approximately 3 days. 38 $31—15 ' .0 Figure 2.1 39 Figure 2.2 Analysis of boiling-stable polypeptides synthesized by total RNAs isolated from nonacclimated (NA) and cold-acclimated (AC) spinach (spin.), and Salomon species: S. acaule (S.a.), S. cammersanii (S.c.), and S. tuberasum (S.t.). Asterisks indicate the boiling-stable COR polypeptides. MW indicates the molecular weight (x10' ) of the standards. arms-rams 40 w—w Figure 2.2 41 -treated plants include those of approximately 20 and 12.2 kDa in S. acaule, 20 and 11.8 kDa in S. cammersanii, and 20.2 kDa in S. tuberasum (Figure 2.2). Identification of cDN A Clones Encoding Ambidapsis Boiling-Stable Polypeptides. A previous study resulted in the isolation of four Arabidapsis car genes (Hajela et al. , 1990). It was of interest to determine whether any of these genes encoded a boiling-stable COR polypeptide. Hybrid-arrest in vitra translation experiments indicated that at least three of them did. In particular, pHH28, pHH7.2, and pLCl‘10A inhibited the translation of COR160, COR47, and COR15, respectively (Figure 2.3). That the pLCTlOA insert encoded COR15 was confirmed by an in vitra transcription/translation experiment (Figure 2.4). When pLCTlOB, which has the same insert as pCTLlOA but in the opposite orientation, was transcribed in vitra using T7 polymerase (the vector carries a T7 promoter) and translated in vitra, a boiling -stab1e polypeptide of 15 kDa was synthesized. This polypeptide was not synthesized in the in vitra transcription/translation reactions using pLCTlOA. The gene encoding COR15 was designated car15. Drought Stress in Ambidapsis is Associated with Accumulation of mRN As Encoding Boiling-Stable Polypeptides Freezing stress, to a large extent, is a dehydration stress. Therefore, mechanisms enabling plants to tolerate freezing stress might have components in common with those for dehydration tolerance. Specifically, similar changes in gene expression might occur in both low temperature and drought stresses. This was demonstrated to be the case by analyzing the in vitra translation products of mRN As isolated from control, cold-acclimated, and drought stressed plants (Figure 2.5). It is clear that mRNAs encoding COR160, COR47, COR24, and COR15 accumulated in 42 Figure 2.3. Hybrid-arrest in vitra translation experiments indicating that Arabidapsis cDNA clone pHH28, pHH7.2 and pLCTlOA (indicated as pLCTlO) represent car genes encoding boiling-stable COR polypeptides. Poly(A+) RNA isolated from nonacclimated (NA) and cold acclimated (AC) plants was either translated in vitra without hybrid-arrest (NA and AC), or after hybrid-arrest with single stranded DNA of pHH28, pHH7.2, pLCT 10A, and cloning vector pBluescript (p88). The boiling-stable products of the translation were fractionated with a 15% SDS-PAGE, and the autoradiograph shown. 43 <- CORIOO are - <- COR47 <- COR24 <- COR15 , , wise”... . S a and... dug, . , anti“; ., . anaemia: i s .« “N in. is is. an; ., . m. ...w .1 .,.v r..... mw m .. ...:Lfler .....Jw . ... .7. .. Figure 2.3 44 Figure 2.4 cDN A clone pLCTlOB encodes a 15 kDa boiling-stable polypeptide comigrating with COR15 in SDS-PAGE. Lanes 1 and 2 show the boiling-stable polypeptides synthesized by in vitra translation of poly(A ) RNAs isolated from nonacclimated (lane 1) and cold-acclimated (lane 2) plants. Lanes 3 and 4 show the in vitra transcription/translation products of :pLCT 103 (lane 3) and pLCTlOA (lane 4). MW indicats the molecular weight (x 10' ) of the major COR polepeptides. 45 mw 160 47 Figure 2.4 46 Figure 2.5. Accumulation of transcripts encoding COR polypeptides and other boiling-stable polypeptides in drought stressed Arabidapsis. lanes 1 and 2 show the total in vitra translation products synthesized from poly(A+) RNA isolated from control plants (lane 1), and drought—treated plants (lane 2). Lanes 3 and 4 show the boiling soluble fractions of the in vitra translation products synthesized from poly(A+) RNA isolated from drought-treated plants (lane 3), and cold-treated plants (lanes 4). The asterisks indicate the boiling-stable COR polypeptides, and the arrow heads indicate the boiling-stable polypeptides specific to drought. 47 kDa —180 mm _ _ is 6 6 3 2 _ _ .— '— em .«m.fifl ”in: 1.31%; fl , ., r If ..m . as s a. s ,. N 813.... a; ”a sung” .. ...s.... ._ , H ... afiewfi meg... I new V 4 1 O 2 _ ; m. we , 3 , a . . a , mm». C I .: ,_. 5i; .. a: ..fi Jain... . E‘s-fin,” ”as,“ a E Z“ w bbbb 1234 Figure 2.5 48 both cold-treated and drought-stressed plants (Figure 2.5, lanes 4 and 3), although the level of these car transcripts seemed to be lower in drought-stressed plants than in the cold-treated plants. Besides the accumulation of the car gene products, drought -stress resulted in accumulation of transcripts encoding an additional set of boiling -stable polypeptides of approximately 22, 20, 19 and 18 kDa (Figure 2.5, lane 3), which were not found in either control or cold-treated plants (Figure 2.5, lane 1, and 4). DISCUSSION The data presented indicate that several of the car transcripts from Arabidapsis encode polypeptides share the unusual biochemieal property of remaining soluble upon boiling in aqueous solution. Four Arabidapsis car genes have been isolated in a previous study by differential screenings of a cDNA libaray (Hajela et al. , 1990). Three of these car genes, represented by cDNA clones pHH28, pHH7.2, and pLCTlOA, are shown here to encode the boiling-stable COR polypeptides COR160, COR47, and COR15, respectively; a fourth cDNA, pHH29, has recently been shown to also encode a boiling-stable polypepetide (6.6 kDa) (Gilmour and Lin , unpublished). Since all car cDNA clones derived from the differential screenings encode boiling-stable polypeptides, it appears that the transcripts encoding boiling -stab1e polypeptides are the most abundant car gene products in Arabidopsis. The accumulation of transcripts encoding boiling-stable polypeptides is not unique to Arabidapsis; it also occurs in cold-treated spinach and three Solarium species. It is possible that the induction of car genes encoding boiling-stable polypeptides will be proven to be a common low temperature response in plants. Indeed, studies have 49 indicated that this response also occurs in winter wheat, and one of the wheat car genes, encoding a boiling-stable polypeptide of 39 kDa, was found to be a homolog of Arabidapsis car4 7 (Lin et al. , 1990; Guo and Thomashow, 1992). Given the evolutionary distance between Arabidapsis and wheat, it would seem likely that related car genes encoding boiling-stable polypeptide will be found in additional plant species. Future studies will determine whether this is true and whether any of the other ’ Arabidapsis car genes have counterparts in other plant species. The finding that certain car genes encode boiling-stable polypeptides is significant in terms of their potential function. First, it strongly suggests that the expression of these genes in response to low temperature is not fortuitous. Otherwise, it would be hard to explain why so many would encode polypeptides sharing the same unusual property, boiling-stability, and that such a large proportion of the boiling-stable polypeptides would be cold-regulated. Further, it would seem unlikely that the accumulation of car transcripts encoding the boiling-stable polypeptides would occur in a variety of plant species including wheat, and one of the wheat car genes is actually homolog to the Arabidopsis can! 7 gene (Lin et al. , 1990; Guo and Thomashow, 1992). The more likely situation might be that the car genes encoding boiling-stable polypeptides have a fundamental role in plants acclimating to cold temperatures and that the boiling-stable nature of the polypeptides is reflective of their function. What function(s) might the boiling-stable COR polypeptides have? One possibility is suggested by the work of Volger and Heber (Volger and Heber, 1975). These investigators have reported that cold acclimated spinach synthesizes polypeptides that, on a molar basis, are greater than 10,000 times more effective in protecting thylakoid membranes against freezing damage (in vitra) than are known low molecular weight cryoprotectants such as sucrose and glycerol. Intriguingly, these polypeptides appear to have distinctive features in common with the Arabidapsis COR polypeptides 50 described here: notably cold-regulation (the polypeptides were detected in cold acclimated plants but not in nonacclimated plants) and heat-stability (they were not irreversibly denatured by high temperatures). In addition, the spinach polypeptides were found to be very hydrophilic. Likewise, DNA sequence analysis of the Arabidapsis car cDN A clones indicates that Arabidapsis COR polypeptides COR160, COR47, COR15, and COR6.6 are all hydrophilic (Lin and Thomashow, 1992; Gilmour et al. , 1992; Gilmour, McLamey, and Horvath, unpublished). Given these similarities, the possibility is raised that the Arabidopsis COR polypeptides are analogs of the spinach cryoprotective polypeptides. Whether the Arabidapsis COR polypeptides have cryoprotective properties and whether they contribute significantly to the freezing tolerance of cold-acclimated plants remain to be tested. A second observation presented in this chapter is that the accumulation of transcripts encoding boiling-stable polypeptides is also a response of plants to drought stress. The drought-stressed Arabidapsis plants accumulated transcripts encoding all four boiling-stable COR polypeptides, in addition to a number of boiling-stable polypeptides that were not found in cold-treated plants. This observation leads to intriguing speculation that plants may have common genetic mechanisms for drought and freezing tolerance. A potential connection between cold acclimation and tolerance to desiccation and drought lies in the fact that plant cells become severely dehydrated during a freeze-thaw cycle (Levitt, 1980; Sakai and Larcher, 1987; Steponkus and Lynch, 1989). Freezing tolerance must, therefore, include tolerance to dehydration. In support to this notion is the fact that drought stress has been observed to increase the freezing tolerance of wheat, rye (Siminovich and Cloutier, 1983), and cabbage (Cox and Levitt, 1976). Given this, it is reasonable to hypothesize that drought and freezing tolerance might involve related genetic mechanisms and gene products. In this regard, it is of interest to compare COR polypepetides with another family of heat-stable 51 polypeptides in plants, the LEA (late—embryogenesis-abundant) proteins (Dure er al. , 1989). LEA proteins are normally produced late in embryogenesis just prior to seed desiccation (Dure et al. , 1983) and in certain eases, they have been shown to be synthesized in plant seedlings in response to water stress (Close et al., 1989; Mundy and Chua, 1988). It has been suggested that the LEA proteins might have a role in desiccation and drought tolerance (Baker et al., 1988; Close et al., 1989). Like the boiling-stable COR polypeptides, LEA proteins remain soluble upon boiling (Close er al., 1989; Jocobsen and Shaw, 1989;) and are very hydrophilic (Close er al., 1989; Dure et al., 1989; Jacobsen and Shaw, 1989; Mundy and Chua, 1988). The structure similarities of COR polypeptides and LEA proteins are further demonstrated by the fact that Arabidopsis COR47 (Gilmour er al. , 1992) and its wheat counterpart (Guo and Thomashow, 1992) both had the lysine rich repeat that is characteristic to the Group II LEA proteins (Dure er al. , 1989). Taken together, these results suggest the possibility that freezing and drought tolerance involve related genetic mechanisms that include the action of car and lea genes. Future studies will be needed to test the validity of this hypothesis. A final point regarding the COR polypeptides described above is that all the data presented here only demonstrate the accumulation of car transcripts in cold -acclimated plants. To be functionally relevant, the accumulation of COR polypeptides needs to be studied in cold-acclimated plants. It is known that COR160, COR47, as well as COR24 accumulate in such plants (Gilmour, er al., 1988; Gilmour and Hajela, unpublished). It is not known, however, if the accumulation of the COR15 polypeptide occurs in the plants. Neither is the subcellular compartmentalization known for any of the COR polypeptides identified in Arabidapsis and other plants (Thomashow, 1990). These basic questions will be addressed in the following chapters. 52 LITERATURE CITED Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmon JG, Smith JA, Struhl K (1987) Current Protocols in Molecular Biology. Greene Publishing Associates, Wiley Interscience, New York Baker J, Steele C, Dure III L (1988) Sequence and characterization of 6 Leo proteins and their genes from cotton. Plant Mol Biol 11: 277-291 Carpenter JF, Crowe JH (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25: 244-255 Close TJ, Kortt AA, Chandler PM (1989) A cDNA-based comparison of dehydration -induced proteins (dehydrins) in barley and corn. Plant Mol Biol 13: 95-108 Cox W, Levitt J (1976) Interrelation between environmental factors and freezing resistance of cabbage leaves. Plant Physiol 57: 553-555 Dure III L, Crouch M, Harada J, Ho T-HD, Mundy J, Quatrano R, Thomas T, Sung ZR (1989) Common amino acid seguence domains among the LEA proteins of higher plants. Plant Mol Biol 12: 475-48 Dure 111 L, Galau GA, Chlan CA, Pyle J (1983) Developmentally regulated gene sets in cotton embryogenesis. In RB Goldberg, ed, Plant Molecular Biology. Alan R Liss, New York, pp 331-342 Estelle MA, and Somerville CR (1986) The mutants in Arabidapsis. Trends in Genetics 2, 89-93 Fienberg AP, Vogelstein B ( 1983) A technique for radiolabeling restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6-13 Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol 87: 745-750 Gilmour SJ, Artus A, and Thomashow FI‘ (1992) cDNA sequence analysis and expression of two cold-regulated genes of Arabidapsis thalinan. Plant Mol. Biol. 18,13-21 Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263-269 Guo WW, and Thomashow (1992) Expression of a cold-regulated wheat gene encoding a novel LEA-like polypeptide. Plant Physiol, submitted Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41: 187-223 Hajela RK, Horvath DP, Gilmour SJ, Thomashow MF (1990) Molecular cloning. and expression of car (geld-regulated) genes in Arabidopsis thaliana. Plant Physrol, in press 53 Hincha, D.K., Heber, U., and Schmitt, J.M (1989) Freezing ruptures thylakoid membranes in leaves, and rupture can be prevented in vitra by cryoprotective proteins. Plant Physiol. Biochem. 27: 795 -801 . Hincha, D.K., Herber, U., and Schmitt, J.M. (1990) Protiens from frost-hardy leaves protect thylakoids against freeze-thaw damage in vitra. Planta 180, 416-419. Li PH, Palta JP, and Chen H (1979) Freezing stress in potato. In Lyons JM, Graham D, and Raison JK eds, Low temperature stress in crop plants, the role of the membrane. Academic Press, New York, pp291-303 Lin C, Guo WW, Everson E, and Thomashow MF (1990)Cold acclimation in Arabidopsis and wheat. A response associated with expression of related genes encoding 'boiling-stable' polypeptides. Plant Physiol. 94: 1078-1083 Lin C, and Thomashow MF (1992) DNA sequence analysis of a cDNA for cold -regulated Arabidapsis gene cor15 and characterization of the COR15 polypeptide. Plant Physiol, in press Jacobson JV, Shaw DC (1989) Heat-stable proteins and abscisic acid action in barley aleurone cells. Plant Physiol 91: 1520-1526 Jagus R (1987) Hybrid selection of mRNA and hybrid arrest of translation. Meth Enzymol 152: 567-572 Kurkela S, Franck M, Heino P, Lang V, Palva ET (1988) Cold induced gene expression in Arabidapsis thaliana L. Plant Cell Reports 7: 495-498 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 : 680-685 Levitt J. (1980) Responses of Plants to Environmental Stress. Chilling, Freezing, and High Temperature Stresses, Ed 2. Academic Press, New York Meyerowitz EM, and Pruitt RE (1985) Arabidapsis thaliana and plant molecular genetics. Science 229, 1214-1218 Mundy J, Chua N-H (1988) Abscisic acid and water stress induce the expression of a novel rice gene. EMBO J 7: 2249-2286 Sakai A, Larcher W (1987) Frost Survival of Plants. Responses and Adaptations to Freezing Stress. Springer-Verlag, Berlin Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Santarius KA (1973) The protective effect of sugars on chloroplast membranes during temperature and water stress and its relationship to frost, desiccation and heat resistance. Planta 113: 105-114 Siminovitch D, Cloutier Y (1983) Drought and freezing tolerance and adaptation in plants: some evidence of near equivalences Cryobiology 20: 487-503 54 Steponkus PL (1990) Cold acclimation and freezing injury from a perspective of the plasma membrane. In Katterrnan E ed. Environmental rnj ury to plants. Academic Press, New York, ppl- -16 Steponkus PL, Lynch DV (1989) Freeze/thaw-induced destabilization of the plasma rznembrane and the effects of cold acclimation. J Bioenergetics and Biomembranes 21: 1-41 Steponkus PL, Uemura M, Balsamo RA, Arvinte T, Lynch DV (1988) Transformation of the cryobehavior of rye protoplasts by modification of the plasma membrane lipid composition. Proc Natl Acad Sci USA 85: 9026-9030 Strauss G, Hauser H (1986) Stabilization of lipid bilayer vesicles by sucrose during freezing. Proc Natl Acad Sci USA 83. 2422-2426 Thomashow MF (1990) Molecular genetics of cold acclimation in higher plants. Adv Genet, 28: 99-131 Thomashow MF, Gilmour SJ, Hajela R, Horvath D, Lin C, Guo W (1990) Studies on cold acclimation in Arabidapsis thaliana. In AB Bennett, SD O'Neill eds, Horticultural Biotechnology. Wiley-Liss, New York, pp 305-314 Vieira J, Messing J (1987) Production of single-stranded plasmid DNA. Meth Enzymol 153: 3-11 XflgjeigHG, Heber U (1975) Cryoprotective leaf proteins. Biochim Biophys Acta 412: 5- Weiser CJ (1970) Cold resistance and injury in woody plants. Science 169: 1269-1278 Chapter 3 ARABIDOPSIS GENE car15 ENCODES A POLYPEPTIDE HAVING POTENT CRYOPROTECTIVE ACTIVITY SUMMARY The cold-regulated gene of Arabidapsis thaliana, car15, encodes a 15 kDa polypeptide designated COR15. In this chapter, I show that COR15 has potent cryoprotective activity in a standard in vitra cryoprotection assay. Specifieally, COR15 was very effective in protecting the freezing-labile enzyme lactate dehydrogenase against freeze-inactivation; on a concentration basis, it was about 10°°times more effective than sucrose and 102-103 times more effective than other proteins including bovine serum albumin. The possible role of car15 in cold acclimation is discussed. INTRODUCTION In many species of higher plants, a period of exposure to low nonfreezing temperature results in an increased level of freezing tolerance (Levitt, 1980; Thomashow, 1990). Considerable effort has been directed at understanding the molecular basis of this cold acclimation response, yet the mechanism remains poorly 55 56 understood. In 1970, Weiser (Weiser, 1970) suggested that cold acclimation might involve changes in gene expression. Since then, it has been clearly established that changes in gene expression occur during cold acclimation and cold-regulated genes have been isolated from a number of plant species (Thomashow, 1990). The roles that these genes have in the cold acclimation process, however, remain to be determined. Heber and colleagues (Volger and Heber, 1975; Hincha er al., 1989; Hincha er al. , 1990) have found that polypeptide fractions prepared from the leaves of cold -acclimated spinach and eabbage can prevent freeze-induced rupture of isolated 1‘qu thylakoid membranes. They have suggested that these polypeptides might have an important role in the enhancement of freezing tolerance that occurs with cold acclimation. The spinach and cabbage polypeptides, which appear to be cold-regulated (they could only be isolated from cold-acclimated plants), have molecular masses in the range of 10-30 kDa, are hydrophilic and remain soluble upon treatment with high heat. None of them, however, have been isolated to purity nor have the genes which encode them been isolated. Three Arabidapsis cDNA clones representing car (cold-regulated) genes have been identified to encode COR polypeptides that remains soluble upon boiling in aqueous solution (see Chapter 2). DNA sequence analysis of these cDN A clones indicated that the polypeptides they encode are hydrophilic (Lin and Thomashow, 1992; and Gilmour et al. , 1992). Thus, each of these cor genes encodes a polypeptide that has at least three characteristics in common with the cold-regulated polypeptides described by Heber and colleagues (Volger and Heber, 1975; Hincha er al., 1989; Hincha er al. , 1990): cold-regulated expression, hydrophilicity, and ”heat-stability. " In addition, it has been found that one of these polypeptides, COR47, is related to certain LEA (late embryogenesis abundant) proteins" (Gilmour er al. , 1992), a group of hydrophilic polypeptides (also boiling-stable) that have been hypothesized to have roles 57 in water-stress tolerance (Baker et al., 1988; Close et al., 1989; Jacobsen and Shaw, 1989). The possible relevance of this to cold acclimation is that the cellular damage that results from a freeze-thaw cycle is due in large part to the dehydration that occurs during freezing (Levitt, 1980; Steponkus and Lynch, 1989). These and other considerations have led to the speculation that the boiling-stable Arabidapsis COR polypeptides might be analogues of the polypeptides described by Heber and colleagues (Volger and Heber, 1975; Hincha er al., 1989; Hincha er al., 1990) and that they might act as cryoprotectants by helping plant cells withstand the dehydration stress associated with freezing (Lin et al., 1990). HereI show that COR15, the cold -regulated polypeptide encoded by Arabidapsis gene car15, does indeed have potent cryoprotective activity in a standard in vitra assay. MATERIALS AND METHODS Preparation of COR15 cDNA clone pLCTlOA and pLCTlOB are described in the previous chapter. Since repeated attempts to express COR15 in E. coli with a number of expression vectors were unsuccessful (unpublished results), the COR15 polypeptide was prepared by in vitra transcription/ translation. pLCTlOB was linearized by digestion with HindIII and the insert was transcribed in vitra with T7 RNA polymerase (Stratagene) using the T7 promoter carried on the pBluescript vector. The resulting transcripts were extracted with phenol/chloroform/isoamyl alcohol (25 :24: 1), precipitated with ethanol and in certain cases ”capped" in vitra using the mCAPTM eapping kit (Stratagene). The resulting transcript was then translated in vitro using a rabbit reticulocyte lysate system with [35 S] methionine (Promega, Madison, WI). All these experiments were 58 carried out according to the manufacturer' s instructions. The resulting translation mix containing [3SS]COR15 was boiled for 10 min and centrifuged (15 min in an Eppendorf centrifuge) to remove most of the proteins present in the in vitra translation mix. The boiling-soluble fraction, which contained the [35S]COR15 polypeptide, was fractionated by SDS-PAGE (Laemmli, 1970) using 15 % (w/v) polyacrylamide gels, and the COR15 polypeptide was located using a Betagen 603 Blot Analer (Betagen Corp.). The region of the gel containing the COR15 polypeptide was excised with a razor blade and the polypeptide recovered by electroelution as suggested by the manufacturer (CBS Science Inc. , Del Mar, CA). The sample was then electrodialyzed against 20 mM Tris-HCl (pH 8.9) for 24 hours at 150 V, and precipitated with 3x volumes of acetone. The amount of COR15 obtained was estimated from the total dpm of the preparation, the specific activity of the [358]methionine used in the in vitra translation reaction, and the number of methionine residues per polypeptide as determined by DNA sequencing (see Chapter 4). The yields of COR15 in three experiments ranged from 0.4 pg to 1.5 pg. As a control for the cryoprotection assays, mock preparations of COR15 were made using transcripts synthesized from the control plasmid pLCTlOA. In this case, the region of the SDS-PAGE gel that corresponded to the position of the [35S]COR15 produced in the test reactions was excised and treated the same way as the bona-fide COR15 samples. Cryoprotection Assay The cryoprotective activities of COR15 , sucrose and various proteins were assayed essentially as described by Tamiya er al. (Tamiya et al. , 1985). A solution of the freeze-labile enzyme L-lactate dehydrogenase (LDH) (EC 1.1.1.27, rabbit muscle lactate dehydrogenase-5N4] isoenzyme type V-S; obtained from Sigma [St. Louis, 59 Mo]) was prepared (2.5 rig/ml) in 10 mM potassium phosphate buffer pH7.5. 50 1110f this solution was placed in a plastic microcentrifuge tube and 50 ltl of the test compound suspended in 10 mM potassium phosphate buffer pH7 .5 was added. The LDH/cryoprotectant mixtures were frozen at -20°C for 24 h, thawed at room temperature for 5-10 min and assayed for enzyme activity as described by Tamiya et al. (Tamiya et al., 1985). Briefly, 20 ul of sample was added to 1 ml of assay mix (at room temperature) which contained 80 mM Tris-HCl pH7 .5, 100 mM KCl, 2mM pyruvic acid and 0.3 mM NADH. The absorbance decrease at 340 nm was measured (at room temperature) using a RESPONSE recording scanning spectrophotometer (Gilford, Oberlin, Ohio). The rate of decrease in absorbance during the first 3 min of the reaction was used to calculate activity (rates were linear over this time interval). All samples were assayed in triplicate. The CP50 (50% cryoprotection) value was defined as the concentration of the additive required to give 50% residual LDH activity after the freeze-thaw cycle, and the average value of the means were compared. The proteins B-galactosidase (B-Gal; from E. colt), ovalbumin (from chicken egg), bovine serum albumin (BSA; Cohn fraction V, from bovine), and RNase A (type I-AS, from bovine pancreas) were purchased from Sigma. RESULTS The cryoprotective activity of COR15 was examined using the LDH freezing -inactivation assay, a well characterized model system commonly used in the studies of cryoprotectants (Greiff and Kelly, 1966; Soliman and Van Den Berg, 1970; Tamiya, et al., 1985; Carpenter and Crowe, 1988). COR15 and mock preparations of it were synthesized by in vitra translation of the in vitra transcription products of LCTIOB and pLCTlOA, respectively (Figure 3.1), and were purified by SDS-PAGE (see Materials 60 and Methods). The cryoprotective activity of the samples was then assessed by determining whether they could protect LDH against freeze-inactivation. Without addition of a cryoprotectant, a freeze/thaw cycle resulted in the LDH solution losing more than 90% of its enzymatic activity (not shown). Addition of the mock preparations of COR15 had no detectable effect; LDH still lost more than 90% of its enzymatic activity after a freeze! thaw cycle (not shown). However, addition of COR15 at a concentration of approximately 1.0 ug/ ml resulted in almost complete protection of LDH against freeze-inactivation (Figure 3.2). The results of the LDH cryoprotective assay are shown in Figure 3.2 and summarized in Table 3.1. Figure 3.2 represents a single experiment that shows the cryoprotective effects of different agents at varying concentrations, while Table 3.1 summarizes the CP50 values of the different agents from the number of experiments indicated. A comparison of the protective effect of COR15 with that of other agents indicated that COR15 has relatively potent cryoprotective activity. Whereas sucrose, a compound that is commonly regarded as an effective cryoprotectant and protein stabilizer (Lee and Timasheff, 1981; Carpenter and Crowe, 1988), had a CP50 of greater than 100 mg/ml (8x108 nM), the cpso of COR15 was about 0.1 rig/m1 (5.6 nM) (Figure. 3.2, Table 3.1). Thus, on a concentration basis, COR15 was approximately 106 times more effective than sucrose in protecting lactate dehydrogenase from freeze-inactivation. COR15 was also more effective in protecting LDH against freeze-inactivation than were a number of other proteins. For example, BSA, a protein which has been shown to be effective in protecting LDH against freeze -inactivation (Tamiya et al., 1985; Greiff and Kelly, 1966), had a CP50 of 28 ug/ml 61 Table 3.1: CP50 values of various agentsa Agents MW n CP50(ug/m1) CP50(nM) Sucrose 340 4 270,000 800,000,000 Ovalbumin 45,000 4 84 1,900 B-Gal. 116,000 4 160 1,400 RNase A 13,700 3 16 1,200 BSA 66,000 1 28 400 COR15 15,000 4 0.083 5.6 a The CP50 values were determined as described in the Materials and Methods. “2 Standard errors (the standard deviation of a mean which was calculated as (s.d.)/(n ) for the CP50 shown are: Sucrose :i; 9.3%; Ovalbumin j; 15%; B-Gal j; 13%; RNase A j; 4.5% and COR15 :1: 6.0%. n = the number of the experiments performed. 62 Figure 3.1 In vitra synthesis of COR15. The boiling-stable fractions of the in vitra transcription/translation products of pLCTlOB (lanes 1-4) and pLCTlOA (lanes 5-8) were fractionated on a 15% SDS-polyacrylamide gel, the gel was sealed in a plastic bag, and scanned for 50 min using a Betagen blot analyzer. The RNAs used in the in vitra translation were 0.05 pg (lanes 1 and 5), 0.1 pg (lanes 2 and 6), and 1 pg (lanes 3, 4 and 7, 8), and they were either translated directely (lanes 1-3 and 4-7) or capped in vitra before translation (lanes 4 and 8). 63 Figure 3.1 64 Figure 3.2 Cryoprotection of LDH. The curve shows the percentage LDH activity remaining after a freeze/thaw cycle in the presence of different concentrations of COR15 (open circles), BSA (solid circles), ovalbumin (solid squares), and sucrose (open squares). 65 ~09 m . n when?“ :5an 8.. _ o _.I C) in Y I T—fi -O S (KilAleD96) H01 66 (4x102 nM) (Figure 3.2, Table 3.1), a value that was about lOO-fold higher than that of COR15. Other proteins, picked either randomly or because they were known to be hydrophilic like COR15 (i.e., RNase A) gave CP50 values ranging from approximately 300 to 2000-fold higher than COR15 (Table 3.1). Thus, on a concentration basis, COR15 was some 102 to 103 times more effective than BSA and other proteins in protecting LDH against freeze-inactivation. DISCUSSION It has been hypothesized that at least some of the boiling-stable polypeptides encoded by Arabidapsis car genes might act as cryoprotectants in cold acclimated plants (Thomashow, 1990). The results presented here are consistent with this notion. Specifically, the data indicate that COR15, the boiling-stable polypeptide encoded by Arabidapsis car15, has potent cryoprotective activity in a standard in vitra cryoprotection assay. The fact that COR15 demonstrated activity in the assay is not unexpected as it is generally observed that increased protein concentration stabilizes proteins against denaturation by many factors (Greiff and Kelly, 1966; Shikama and Yamazaki, 1966). However, what is significant is that COR15 appears to have particularly effective cryoprotective activity; on a concentration basis, it was about 102 times better in protecting LDH against freeze-inactivation than was BSA, a protein that is recognized as being an effective cryoprotectant and protein stabilizer (Tamiya et al. , 1985; Greiff and Kelly, 1966). While the data presented here encourage the notion that certain of the boiling -stable polypeptides encoded by Arabidapsis car genes act as cryoprotectants, the hypothesis is, of course, far from proven. First of all, the relevance of the 67 cryoprotective feature of COR15 in vitra to its function in viva is uncertain. Indeed, there is no direct evidence that these polypeptides contribute significantly to freezing tolerance in cold-acclimated plants. In addition, as described in the following chapter, the COR15 polypeptide is processed and targeted to the chloroplasts. Whether the mature polypeptide, COR15m, has greater, lesser or the same activity as COR15 in the LDH cryoprotection assay remains to be determined. More importantly, it remains to be established whether COR15 m has a role in protecting chloroplasts against freeze -induced damage. 68 LITERATURE CITED Baker J, Steele C, Dure III L (1988) Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant Mol Biol 11, 277-291 Carpenter JF, and Crowe JH (1988) The mechanism of cryoprotection of protein by solutes. Cryobiology 25, 244-255. Close TJ, Kortt AA, Chandler PM (1989) A cDNA-based comparison of dehydration -induced proteins (dehydrins) m barley and corn. Plant Mol Biol 13, 95- 108 Gilmour SJ, Artus A, and Thomashow PT (1992) cDNA sequence analysis and eréprgssiion of two cold-regulated genes of Arabidapsis thalinan. Plant Mol. Biol. 1 1 - 1 Greiff D, and Kelly RT (1966) Cryobiology of enzymes. Cryobiology 2, 335-341 . Hincha DK, Heber U, and Schmitt JM (1989) Freezing ruptures thylakoid membranes in leaves, and rupture can be prevented in vitra by cryoprotective proteins. Plant Physiol. Biochem. 27, 795- 801. Hincha DK, Heber U, and Schmitt JM (1990) Proteins from frost-hardy leaves protect thylakoids against freeze-thaw damage in vitra. Planta 180, 416-419. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lee JC, and Timasheff SN (1981) The stabilization of proteins by sucrose. J. Biol. Chem. 256, 7193-7201. Levitt J. (1980) Responses of Plants to Environmental Stress. Chilling, Freezing, and High Temperature Stresses, Ed 2. Academic Press, New York Lin C, Guo WW, Everson E, and Thomashow MF (1990) Cold acclimation in Arabidopsis and wheat. A response associated with expression of related genes encoding 'boiling-stable' polypeptides. Plant Physiol. 94, 1078-1083 Lin C, and Thomashow MF (1992) DNA sequence analysis of a cDN A for cold- regulated Arabidapsis gene car15 and characterization of the COR15 polypeptide. Plant Physiol., in press Jacobsen JV, Shaw DC (1989) Heat-stable proteins and abscisic acid action in barley aleurone cells. Plant Physiol 91, 1520-1526 Sarnbrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning. A laboratory manual, Ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Shikama K, and Yamazaki I (1961) Denaturation of catalase by freezing and thawing. Nature 190, 83-84. 69 Soliman FS, and Van Den Berg L (1970) Factors affecting damage of lactate dehydrogenase. Cryobiology 8, 73-78. Steponkus PL, Lynch DV (1989) Freezeltllaw-induced destabilization of the plasma membrane and the effects of cold acclimation. J Bioenergetics and Biomembranes 21, 21-41 Tamiya T, Okahashi N, Sakuma R, Aoyarna T, Akahane T, and Matsarnoto JJ (1985) Frsegze denaturation of enzymes and prevention with additives. Cryobiology 22, 446 -4 . Thomashow MF (1990) Molecular genetics of cold acclimation in higher plants. Adv Genet, 28, 99-131 Volger HG, and Heber U (1975) Cryoprotective leaf proteins. Biochim. Biophys. Acta. 412, 335-349. Weiser CJ (1970) Cold resistance and injury in woody plants. Science 169, 1269-1278 Chapter 4 CHARACTERIZATION OF THE ARABIDOPSIS car15 GENE PRODUCT SUMMARY In the previous chapter I showed that car15 encodes a boiling-stable polypeptide, COR15 , which has potent cryoprotective activity in an in vitra assay. In this chapter, cDNAs for car15 were characterized by DNA sequence analysis. The data indicated that car15 encodes a 14.6 kDa hydrophilic polypeptide. The N-terminal amino acid sequence of this polypeptide closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunoblot analysis indicated that COR15 is processed in planta and that the mature polypeptide, COR15m, is present in the soluble fraction of chloroplasts. The biochemieal activity of COR15 m is not known, however, a strong intermolecular interaction of COR15 m with itself is suggested by the observation that COR15m can form multimers even in the presence of SDS. 70 71 INTRODUCTION It is now clear that changes in gene expression occur during cold acclimation in a wide range of plant species (Weiser, 1970; Guy, 1990; Thomashow, 1990). It is not yet known, however, whether cold-regulated genes have critical roles in freezing tolerance. To address this issue, investigators have begun to isolate and characterize genes that are induced during cold acclimation. While no functions have yet been determined for these genes, results have been intriguing. Mohapatra er al. (Mohapatra et al. , 1989), for example, have found that the levels of expression of three cold- regulated genes from alfalfa correlate positively with the freezing tolerances of four different alfalfa cultivars. Further characterization of these cDNA clones, however, has not been reported. Another approach to study the function of the cold-regulated genes in plant cold acclimation is to analyze the amino acid sequence of COR polypeptides. For example, one of the Arabidapsis cold-regulated genes, kin] , has been reported to encode an alanine-rich polypeptide that has amino acid sequence similarities with certain fish antifreeze or thermal hysteresis proteins (Davies and Hew, 1990). Whether KINl has antifreeze properties in vitra or in planta is not known. In the previous chapters, it has been established that in Arabidapsis and some other plants, cold acclimation results in the accumulation of transcripts encoding polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Arabidapsis genes encoding the boiling-stable COR160, COR47, and COR15 polypeptides have been identified to be represented by the cDNA clones pill-128, pHH7 .2, and pLCTlO, respectively. Moreover, one of the COR polypeptides, COR15 , has been found to have potent cryoprotective activity in an in vitra assay. In this chapter, COR15 is further characterized by DNA sequence analysis of cDNAs for 72 car15. The accumulation of the car15 gene product in cold-acclimated Arabidapsis is also investigated by immunological analysis. MATERIALS AND METHODS Plant Material Arabidopsis thaliana ecotype RLD was grown in clay pots in planting soil (Baccto Co., TX) with a photoperiod of 14 h or 18 h. The photoperiod was 14 h if the vegatative tissue was needed, and it was 18 h if the plants were used for seed production. For cold acclimation, 2 to 3 week old plants were transferred to a cold room with a temperature of 4°C at soil level for 3 days. Other conditions were the same as described in Chapter 2. DNA Sequencing DNA sequences of the cDNA inserts of clones pHH71. 1, pLCTlOA and part of pI-IH67 were determined on either single or double stranded DNA templates by the dideoxy chain termination method (Sanger et al. , 1977) using SequenaseTM (U. S. Biochemieal) according to the manufacturer's instructions. Single stranded plasmid DNA was prepared from E. cali strand MV1190 using the helper phage M13K07 (V icira and Messing, 1987). Double stranded sequencing was performed according to the method of Zhang et al (Zhang et al., 1988). Deletions of the cDNA insert in pBluescript SK(-) were generated by digestion with exonuclease III and mung bean nuclease (Henikoff, 1987). The complete sequence of each strand of the inserts in pHI-17 l .1 and pLCTlOA was determined. Nucleotide and amino acid sequence analysis was performed using the DNAsis and PROsis programs of Hitachi (Hitachi Engineering 73 Co. , Ltd) and the CGC (University of Wisconsin Genetics Computer Group) programs (version 6.0) of the University of Wisconsin Biotechnology Center (Madison, WI). Antiserum Antiserum that recognized COR15 was raised by immunizing rabbits with a protein A-COR15 fusion protein. A gene encoding the hybrid protein was created by ligating the EcoRI cDNA insert of pLCTlOB into the EcoRI site of pRIT2T, a protein A fusion vector (Nilsson er al. , 1985) (Pharmacia). The recombinant plasmid was transformed into E. cali N4830-l (Pharmacia) and the fusion protein expressed as recommended by the supplier. Cells were disrupted using a French Press (Aminco, Urbana, IL) at 16,000 psi and the extract centrifuged at 10,000g for 15 min. The supernatant was collected and the protein A-COR15 fusion was enriched by affinity chromatography using a column of IgG Sepharose (Pharmacia) according to manufacturer's instructions. The fusion protein was further purified by preparative SDS-PAGE. Gel slices containing the 41 kD fusion protein were homogenized in a buffer consisting of 0.1 M Tris-HCl (pH 8.0), 0.1% (w/v) SDS using a mortar and pestle and the suspension stirred at room temperature for 5 h. The material was centrifuged at 10,000g for 15 min and the fusion protein in the supernatant was concentrated using a CentriconTM 10 filter (Amicon). Analogous procedures utilizing the unmodified pRIT2T vector were employed to obtain the protein A polypeptide (26 kDa). New Zealand white rabbits were immunized with the protein preparations by subcutaneous injection of 80 pg protein and boosted once after 4 weeks with the same amount of protein. Antiserum recognizing COR160, an Arabidapsis cold-regulated boiling-stable polypeptide, was obtained from Sarah Gilmour (S Gilmour, M Thomashow, unpublished). Antisera to carbonic anhydrase (Fawcett et al. , 1990) and glycolate 74 oxidase (V olokita and Somerville, 1987) of spinach were kindly provided by Chris Somerville. Chloroplast Isolation Chloroplasts were isolated by modification (8 Hugly, C Somerville, unpublished) of a previously described procedure (Somerville at al. , 1981). Leaves (10 g) harvested from 2-3 week old plants (rosette stage) were immersed in ice water for 5 min, blotted dry, placed in 150 ml cold grinding buffer [20 mM Tris-HCl (pH 8.4), 1% (w/v) BSA, 1.25% (w/v) Ficoll-400, 2.5% (w/v) Dextran-40, 0.45 M sorbitol, 10 mM EDTA, 1 mM DTT], out into small pieces with scissors, and ground for 10 sec in a Tissumizer (Tekmar, Cincinnati, OH) at maximum speed. The homogenates were passed through Miracloth (Calbiochem) and centrifuged at 1400g for l min. Pelleted material was gently resuspended with a camel hair paint brush in 2 ml resuspension buffer [lOOmM Tris (pH7.9), 300 mM glycerol, 1 mM MgC12, 1 mM DT'I'], layered onto a discontinuous percoll gradient [1 ml 60% (v/v) percoll, 10 ml 25 % (v/v) percoll in resuspension buffer] and centrifuged in a swinging bucket rotor (Sorval I-IB-4) at 6000 rpm for 3 min. Chloroplasts banding at the interface between the percoll layers were collected, suspended in 5x volume of resuspension buffer and collected by centrifugation at 1300g for l min. Preparation of Protein from Ambidapsis Total soluble protein was prepared by grinding plant tissue in liquid N2 followed by grinding in extraction buffer [50 mM Tris (pH 8.0), 5 mM EDTA, 2.5% (w/v) polyvinylpolypyrrolidone] (4 ml buffer/ gram tissue). The material was then centrifuged (10,000g, 10 min), the pellet discarded, and the proteins in the supernatant 75 collected either by ammonium sulfate precipitation or by addition of 3 vol acetone followed by centrifugation (14000g for 10 min). Chloroplast proteins were separated into soluble and membrane fractions by suspending isolated chloroplasts in 5x volume of lysis buffer [100 mM Tris (pH 7.9), 5 mM EDTA, 1 mM PMSF], freezing the suspension at -20°C for 30 min, thawing it at 37°C for 5 min, mixing it using a vortex (about 1 min), and centrifuging it in a microfuge at approximately 14,000g for 15 min. Proteins in the supernatant and pellet were collected by adding 2 vol acetone followed by centrifugation (14,000g for 10 min) and were designated the soluble and membrane fractions, respectively. Gel Electrophoresis of Proteins There were three types of the polyacrylamide gel electrophoresis used for the experiments described in this chapter: SDS-PAGE, native PAGE, and tricine-SDS - -PAGE. SDS-PAGE was as described (Laemmli, 1972). Native PAGE was prepared and performed basieally the same as with Laemmli gels except that no SDS was included in any solutions. Tricine-SDS-PAGE was essentially according to the method of Schagger and Van J agow (Schagger and Van J agow, 1987), where tricine was used as the trailing ion in replace of the glycine that is used in the Laemmli gel, and breifly described as follows. The separation gel (10% acrylamide, 0.6% bisacrylamide, 1 M Tris-HCl pH 8.5, 0.1% SDS) was overlaided with a layer of stacking gel (3.84% acrylamide, 0.23% bisacrylamide, 1 M Trise-HCl pH 8.5, 0.1 % SDS); protein samples were disolved in the same SDS sample buffer for SDS-PAGE (Laemmli, 1972), and electrophoresis was conducted with the anode buffer (0.2 M Tris-HCl pH 8.9) and the cathode buffer (0.1 M tricine, 0.1 M Tris base, 0.1% SDS) at a constant current. 76 Immunoblot Analysis Proteins were fiactionated by SDS-PAGE and transferred to nitrocellulose (Schleicher and Schuell) as described (Towbin et a1 . , 1979). Immunoblots were then treated with antisera and bound antibody detected using protein A-conjugated alkaline phosphatase (Sigma) as described (Blake et al., 1984). Preparation of A Putative COR15m Recombinant Polypeptide The putative COR15m is a polypeptide consisting of the amino acids 51 to 139 of COR15 (see Results). A pair of primers was synthesized in the Macromolecular Structural Facility in the Biochemistry Department of Michigan State University. Primer I, (5 ' TC’I‘QCAIEGCTAAAGGTGA CGGC 3'), corresponds to nucleotides 209 -231 of the cDNA insert of pLCTlO with 4 mismatches (underlined). The mismatches in Primer I result in the generation of the restriction site NcaI and a methionine codon at the 5' end of the primer. Primer II (3' ACGGTGTITCATCCCTALEETGG 5'), corresponds to nucleotides 474-495 with 4 mismatches (underlined). The mismatches in Primer II result in the generation of the restriction site BamHI at the 5' end of the primer. The DNA fragment corresponding to nucleotides 209-495 of the pLCTlO insert was synthesized using the primers and standard PCR protocols (Innis et al. , 1990), then end-repared, and cloned into the Smal site of pBluescript by standard recombinant DNA techniques (Sambrook et al. , 1989). This DNA fragment has an open reading frame encoding the putative COR15m plus a methionine at the N -terminus; this polypeptide is referred to as recombinant COR15m. The NcaI/BamHI fragment of the resulting plasmid (pLCTlOm) was isolated and cloned into the expression vector pET9d (Novagen, Madison, WI) carrying the bacteriophage T7 RNA polymerase promoter (Rosenberg et al. , 1987). The resulting plasmid (pLCT103) was transformed into E. cali strain BL21 (DE3, plysS), the expression of the protein 77 induced by IPTG, and cells harvested and lysed according to the supplier's instructions. The lysates were boiled, the insoluble material removed by centrifugation, and the recombinant COR15m polypeptide was purified using preparative tricine-SDS-PAGE. Antiserum was raised against COR15m as described for the protein A-COR15 fussion protein. The relative migration of COR15m and the recombinant COR15m were analyzed by tricine-SDS-PAGE. RESULTS DNA Sequence Analysis of cDNAs for COR15 The DNA sequences of three cDNA inserts representing COR15 were determined. The data for the cDNA insert in pLCTlOA is shown in Figure 4.1. The data indieate that the insert has an open reading frame that could encode a 139 amino acid long polypeptide with a predicted molecular weight of 14,604 daltons, a value consistent with the in vitro transcription/ translation experiment presented in Chapter 2. The gene encoding this polypeptide has been designated car15. The deduced polypeptide, designated COR15, had a high alanine (17.9 mol%), lysine (14.3 mol%), and glutamic acid (9.3 mol %) content and was devoid of cysteine, tryptophan and proline residues (Table 4.1). The hydropathy profile of the polypeptide indicated that the N-terminal third of COR15 had both hydrophobic and hydrophilic regions, but that the latter two thirds of the polypeptide was primarily hydrophilic (Figure 4.2). Analysis of the potential secondary structure of the polypeptide using the algorithm of Robson (Garnier et al. , 1978) indicated that the latter two thirds of the polypeptide (from residue 56 to 131) was likely to assume an til-helical configuration (Figure 4.3). Computer searches of the Genbank (release 68, June 1991), EMBL (release 27, May 1991), SwissProt (release 18.0, May 1991) and PIR Prat (release 28.0, May 1991) data 78 Figure 4.1. Nucleic acid sequence of the cDNA insert in pLCTlOA and the deduced amino acid sequence of COR15. The loosely defined consensus cleavage site for chloroplast transit peptides (Gavel and Heijne, 1990) is double-underlined and the predicted site of cleavage is indicated by an asterisk (see text). The predicted amino acid sequence for COR15m following the alanine residue is underlined. The two nucleotides underlined indicates the location of the 306 bp intron in the insert of pHH71. l. 79 008 who «we 08m mmm and omv NNH mnv 00H can om can oh man an com me mad 0N ova OH mm we .n <<< <<< 008 888 08< <88 8<< <08 0<< 0<0 8<0 8<< 0<8 80< 80< 8<< och 008 o¢< (88 0:0 mmHIoH¢ mac mug was <0< 000 8<0 0<< 808 <08 888 H.e ousmflm 8<0 88< 08< <08 <88 088 800 <<8 000 <80 080 <08 888 808 88< 0<0 <<8 008 <<0 <00 088 <8< <0< 8<8 008 880 8<< 008 80< <08 <8< 0<< 0<< <8< Och 088 :04 080 0<0 Hm> 880 new 00¢ GH< 800 8<8 080 8<0 08< 008 0<< 008 >8 you <88 0<< 08< n80 mNo 88m mam and Hmv MNH nmc bad man Hm saw me man mm acm nv an 8N mQH HA 8m mv 80 Table 4.1 Amino acid composition of COR15 and COR15m Amino Acid Mol% (COR15) Mol% (COR15m) Glycine (G) 8.57 7.78 Alanine (A) 17.86 21.11 Valine (V) 7.14 4.44 Leucine (L) 4.29 4.44 Isoleucine (I) 1.43 1.11 Serine (S) 7.86 2.22 Threonine (T) 5.76 6.67 Cysteine (C) 0.00 0.00 Methionine (M) 2.14 0.00 Aspartic acid (D) 6.43 10.00 Asparagine (N) 2.86 4.44 Glutamic acid (E) 9.29 14.44 Glutamine (Q) 2.86 0.00 Arginine (R) 1.43 0.00 Lysine (K) 14.29 17.78 Histidine (H) 0.71 0.00 Phenylalanine (F) 4.29 2.22 Tyrosine (Y) 2.14 2.22 Tryptophan (W) 0.00 0.00 Praline (P) 0.00 0.00 Molecular weight 14604.51 9352 66 Isoelectric point 7.52 4.56 81 Figure 4.2. Hydropathy profile of COR15. Plots are according to Kyte and Doolittle (Kyte and Doolittle, 1982) using a window of 9 residues. Negative values indicate hydrophilic regions. Numbers on the abscissa indicate amino acid residues. m . e 382 ON« 00H co 00 av ON .Ouduzmomnhz . . 82 Omnozhoza>z O.Ni m.di o.di . m.Oi m.o O.d m.« 83 Figure 4.3. The predicted secondary structure of COR15m according to Garnier et a1 (Garnier et al. , 1978). 84 n.e apnoea AHOU sz8 8mmmw m33mmmmmmmmmmwxmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm: xHAmm nm8><<>NDMh oea OMH ONH OHH OOH om ow 0 0 000 0000000 00 AHOU 88 8888 888 88 8 ZMD8 mmmmmm mmmmm mm 8mmmm mmmmmmmmmmmmmmm mmmmwxm I XHAHE QmHAmMMMOm>>>hO8MO0>m><0mmm0M<0mmhmm<208A>92 m . e 382 III "' nemem~_ nemem~_ 98 Figure 4.9. Detection of carbonic anhydrase, COR160, and glycolate oxidase in protein fractions isolated from nonacchmated and cold acclimated plants. Immunoblots were prepared from protein preparations fractionated by SDS-PAGE on 10% (w/v) gels. The transfers were then treated with antisera to either carbonic anhydrase (A), COR160 (B), or glycolate oxidase (C) and developed as described in Materials and Methods. The samples are: lanes 1 and 2, total protein prepared from chloroplasts isolated from nonacclimated (lane 1) and acclimated plants (lane 2), respectively; lanes 3 and 4, total soluble protein prepared from leaves of acclimated (lane 3) and nonacclimated (lane 4) plants, respectively. 99 Figure 4.9 100 COR15m Comigrates on SDS-PAGE with Recombinant COR15m Expressed in E. cali A gene that encodes the putative COR15 m with the addition of a methionine residue at the N-terminal end was created using the polymerase chain reaction (see Materials and Methods). The gene was then cloned into the expression vector pET9d, expressed in E. cali, and the recombinant COR15m polypeptide purified (see Materials and Methods). The relative migration of the purified recombinant COR15 m was compared with that of the authentic COR15 m of Arabidapsis chloroplast on high resolution tricine-SDS-PAGE. This system has superior resolution to conventional glycine-SDS-PAGE (Laemmli, 1970) for proteins in the range of 5 to 25 kDa (Schagger and Van Jagow, 1987). An immunoblot analysis indicated that the recombinant COR15m migrated to the same position as the COR15m prepared from cold acclimated Arabidapsis (Figure 4.10). Thus, the data are consistant with COR15 being processed at the putative cleavage site of the chloroplast transit peptide (Figure 4.1 and Figure 4.7). Intermolecular Interactions of Polypeptides COR15 and Recombinant COR15m Preparations of COR15 polypeptide synthesized by in vitra transcription ltlanslation did not migrate as a single band in the native PAGE (Figure 4.11A) as it did in the SDS-PAGE (Figure 4.11B). Instead, it migrated as multiple bands at much higher molecular weight positions, and the majority of the proteins barely migrated into the gel (Figure 4.11A). This could have resulted from COR15 binding to other unlabelled proteins in the translation system (rabbit reticulocyte lysate) or from it forming multimers itself. The abnormal migration of COR15 was not effected by the addition of nonionic detergents (Figure 4.11A). More interestingly, when the recombinant COR15m was purified as a single band from tricine-SDS-PAGE gels (see 101 Figure 4.10. Comigration of the chloroplast COR15m with recombinant COR15m. The gene encoding the putative COR15m with an additional methionine residue at the N-terminal was created, expressed in E. cali, and the recombinant COR15m purified as described in the Materials and Methods. (A). The boiling-stable proteins of the E. cali cell hosting vector plasmid pET9d (lane 4) or plasmid pLCT103 (lane 3), purified COR15m (lane 2) and the mock preparation (lane 1) were fractionated by tricin-SDS -PAGE on a 10% gel, and stained with Coomassie brilliant blue. (B). Soluble proteins of the chloroplast prepared from nonacclimated (lane 1) and 3-day-cold-acclimated (lane 2) Arabidapsis, and the recombinant COR15m (0.01 pg) (lane 3.3) were fractionated by tricine-SDS-PAGE on a 10% gel, the bottom half of the gel blotted to nitrocellulose, and the immunoblot was developed with antiserum against COR15m (1:50 dilution). The arrow head indicates COR15m. 102 kDa 20 l7 14 10 Figure 4.10 2 3 kDa 103 Figure 4.11. Migration of COR15 on a native polyacrylamide gel. [358]methionine -1abelled COR15 was prepared by in vitra transcription/translation of pLCTlOB as described in the Materials and Methods. Translation products were fractionated either by native PAGE on a 15% gel (A) or by SDS-PAGE on a 15% gel (B). Proteins were either fractionated directely (lanes A.l, A.6, 8.1 and B.2), or incubated in the sample buffer containing nonionic detergent NP-40 of 0.1% (lane A2) and 2% (lane A.3), or Triton X-100 of 0.1% (lane A.4) and 2% (lane A5) at 65°C for 15 min. Lane A.6 and B2 show the transcription/translation of pLCTlOA. The positions of the molecular weight markers are indicated for the panel A. The positions of 160, 47, 24, and 15 kDa COR polypeptides are indicated in panel B. 104 ,2 kDa 1234551433 _'60 > "*** —116 —84 —47 > —58 —4s —36 D —24 —24 D *9- —15 A 8 Figure 4.11 105 Figure 4.12. Immunoblot showing the formation of multimers of recombinant COR15m. The recombinant COR15m was purified as a single band from SDS-PAGE (see Materials and Methods), fractionated on a 11% tricine-SDS-PAGE, and the immunoblot prepared with antiserum against the recombinant COR15m (1:50 dilution) as described in the Materials and Methods. The arrow indicates the recombinant COR15m, and arrow heads indicate multimers. 106 kDa Figure 4.12 107 Figure 4.13. A helical wheel diagram of the putative COR15m containing 88 amino acids residues (amino acids from 51 to 139 of COR15). Each tit-helix contains 3.6 residues, so the adjacent residues are separated by 100° of arc on the wheel and each wheel contains 5 oz-helices. The chemical properties of the amino acid side chains are shown as: +, positively charged (lys, arg); -, negatively charged (asp, glu); blank, polar (asn, gln, ser, thr); and shaded, apolar (leu, ile, val, phe, tyr, trp, ala,). 109 Materials and Methods), fractionated again on a tricine-SDS-PAGE gel and subjected to immunoblot analysis, multiple bands were detected at positions higher than 9.4 kDa in addition to a 9.4 kDa band (Figure 4.12). In this case, it was clear that the higher molecular weight bands could only result from the formation of multimers of the recombinant COR15m because it was the only protein in the sample. The relative migration positions of the COR15m multimers indicated that they might be dimers, and trimers (Figure 4.12). DISCUSSION The results of DNA sequence and immunoblot analyses indicate that the car15 gene of Arabidapsis encodes a 14.7 kDa cold-regulated polypeptide, COR15 , that is processed in planta to a polypeptide of about 9 kDa. The mature protein, designated COR15m, is soluble, hydrophilic, and is predicted to form an a-helix. The predicted amino acid sequence of COR15m indicated that it was rich in alanine (21.1 mol%), lysine (17.8 mol%), and glutamic acid (14.4 mol%), but devoid of cysteine, proline, tryptophan, histidine, and methionine (Table 4.1). The amino acid sequence of COR15 failed to show any significant sequence homology with that of other proteins including the fish antifreeze proteins (Davies and Hew, 1990) and the plant LEA proteins (Baker et al., 1988; Close et al., 1989), which have been suggested to have roles in the freezing tolerance of fish and dehydration tolerance of plants, respectively. However, an analysis of the secondary structure (Schiffer and Edmunson, 1967) of COR15m revealed that it could form an amphiphilic a-helix (Figure 4.13). This amphiphilic a -helical structure has been predicted for some LEA proteins (Dure et al. , 1988), and has been suggested to allow for the formation of tertiary filamentous polymers by 110 intramolecular or intermolecular interactions (Dure et a1 . , 1988). The relationship between the suggested tertiary structure of LEA proteins and their speculated function in dehydration protection is not clear. It appears that COR15 m can form intermolecular multimers (Figure 4.12B). The formation of intermolecular disulfide bound is excluded as COR15 m has no cysteine residues (Figure 4.2). Whatever interaction enables the COR15m to aggregate is resistant to the detergent SDS (Figure 4.123). Whether the formation of multimers of COR15m results in a filamentous polymer suggested for LEA proteins remains to be determined. Immunoblot analysis indicated that COR15m is localized in chloroplasts. Specifically, COR15m can be detected in soluble protein extracts prepared from chloroplasts purified on percoll gradients (Figure 4.8). In addition, amino acid sequence comparisons indicate that the N-terminal sequence of COR15 has a number of characteristics in common with chloroplast transit peptides, including sequences that match the loose consensus stromal-targeting cleavage site (Gavel er al. , 1990; see Figure 4.7). The size of COR15m is consistent with COR15 being processed at the putative consensus cleavage site; the apparent molecular weight of COR15m is about 9 kDa (Figure 4.6), while the predicted molecular weight of COR15m, assuming processing at residue 50 of COR15 (the putative cleavage site) is 9.4 kDa (Table 4.1). Attempts to determine directly the N-terminal sequence of COR15m, and thus confirm the site of processing, have failed, apparently due to chemical blockage of the N -terminal amino acid (unpublished result). However, when the putative COR15m sequence (amino acids 51-139) was fused to a methionine codon and expressed in E. cali, the recombinant COR15m (also boiling-stable) polypeptide comigrated on tricine -SDS-PAGE with authentic COR15m prepared from chloroplasts (Figure 4.10). Taken together, these data indieate that at least some of the cellular COR15 is transported into chloroplasts and proteolytically processed to COR15m of approximately 9 kDa. 111 Confirmation of this conclusion and a determination of the suborganeller location of COR15m will be accomplished by future immunolocalization studies. Based on the fact that COR15m is present in the soluble fraction of the chloroplasts and that the putative transit peptide of COR15 resembles the stroma targeting signals, the suborganeller localization of COR15m is speculated to be in the chloroplast stroma. Finally, it should be noted that the data presented here do not rule out the possibility that COR15 is targeted to more than one cellular compartment. ‘1‘“ ‘5 A major challenge now is to determine the function(s) of COR15m. One 1" intriguing possibility is suggested by the work of Heber, Hincha, Schmitt and colleagues (Voger and Heber, 1975; Hincha er al., 1989; Hincha er al., 1990). These investigators have found that cold acclimated spinach and cabbage, but not nonacclimated plants, synthesize proteins that ean protect isolated thylakoid membranes against freeze damage in vitra. The cryoprotective activity of these proteins is high; on a molar basis, they are over 10,000 times more effective than sucrose in protecting thylakoids against damage caused by a freeze-thaw cycle. To date, none of these cryoprotective proteins have been isolated to purity. However, initial studies (V oger and Heber, 1975; Hincha et al., 1989; Hincha et al., 1990) indicate that they have a number of properties in common with COR15m: they are cold-regulated, hydrophilic, they remain soluble upon boiling in aqueous solution, they are small (about 10 to 30 kD) and at least some of them appear to be present in chloroplasts (see Discussion of Voger and Herber, 1975). Results presented in the previous chapter indicate that COR15 does indeed have cryoprotective activity in an in vitra assay. The obvious question raised is whether COR15 m also has cryoprotective properties. The expression of COR15m in E. cali should enable the purification of COR15m in large amount and its cryoprotective activity be tested directly. The key question, of course, is whether 112 COR15m and the spinach and cabbage proteins have cryoprotective roles in planta. In particular, do they have specific functions in protecting chloroplasts against freeze -induced damage? The construction of transgenic Arabidapsis plants that either overexpress or underexpress car15, should help in answering these basic questions . “a... f” 113 LITERATURE CITED Baker J, Steele C, Dure In L ( 1988) Sequence and characterization of 6 lea proteins and their genes from cotton. Plant Mol Biol 11, 277-291 Blake MS, Johnston KH, Russell-Jones GT, Gotschlich EC (1984) A rapid, sensitive method for detection of alkaline phosphtase-conj ugated anti-antibody on western blots. Anal Biochem 136, 175-179 Close TJ, Kortt AA, Chandler PM (1989) A cDNA-based comparison of dehydration -induced proteins (dehydrins) in barley and corn. Plant Mol Biol 13, 95-108 Crowe JH, Carpenter JF, Crowe LM, and Anchordoguy JT (1990) Are Freezing and dehydration similar stree vectors? a comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 27 , 219-231 Dagises PL, Hew CL, (1990) Biochemistry of fish antifreeze proteins. FASEB 4, 2460 -24 Dure 111 L, Crouch M, Harada J, Ho T-H D, Mundy J, Quatrano R, Thomas T, Sung ZR (1989) Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol Biol 1, 475-486 Fawcett TW, Browse JA, Volokita M, Bartlett SG (1990) Spinach carbonic anhydrase primary structure deduced from the sequence of a cDNA clone. J Biol Chem 265, 5414-5417 Garnier J, Osguthorpe DJ, Robson B (1978) Analysis of the accuracy and implications of sim (l)e glethggs for predicting the secondary structure of globular proteins. J Mol Biol 1 , 7- Gavel Y, van Heij ne G (1990) A conserved cleavage-site motif in chloroplast transit peptides. FEBS Letters 261, 455-458 Gilmour SJ, Artus NN, Thomashow MF (1992) cDNA sequence analysis and expression of two cold-regulated genes of Arabidapsis thaliana. Plant Mol Biol 18, 13-21 Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold acclimation in Arabidapsis thaliana. Plant Physiol 87, 745-750 Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann Rev Plant Physiol Plant Mol Biol 41, 187-223 Hajela RK, Horvath DP, Gilmour SJ, Thomashow MF (1990) Molecular cloning and expression of car (mid-regulated) genes in Arabidapsis thaliana. Plant Physiol 93, 1246-1252 Henikoff S (1987) Unidirectional digestion with exonuclease III in DNA sequence analysis. Meth Enzymol 155, 156-165 114 Hincha DK, Heber U, and Schmitt JM (1989) Freezing ruptures thylakoid membranes in leaves, and rupture can be prevented in vitra by cryoprotective proteins. Plant Physiol. Biochem. 27, 795-801. Hincha DK, Heber U, and Schmitt JM (1990) Proteins from frost-hardy leaves protect thylakoids against freeze-thaw damage in vitra. Planta 180, 416-419. Kurkela S, Franck M (1990) Cloning and characterization of a cold- and ABA -inducible Arabidapsis gene. Plant Mol Biol 15, 137-144 K J, Doolittle RF (1982) A simple method for displaying the hydropathic character a aprotein. J Mol Biol 157, 105-132 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Levitt J (1980) Responses of Plants to Environmental Stress. Chilling, Freezing, and High Temperature Stresses, Ed 2. Academic Press, New York Lin C, Guo WW, Everson E, Thomashow MF (1990) Cold acclimation in Arabidapsis and wheat. A response associated with expression of related genes encoding 'boiling -stable' polypeptides. Plant Physiol 94, 1078-1083 Mohapatra SS, Wolfraim L, Poole RJ, Dhindsa RS (1989) Molecular cloning and relation to freezing tolerance of cold-acclimation-specific genes of alfalfa. Plant Physiol 89, 375-380 Nilsson B, Abrahamsen L, and Uhlen M (1985) Immobilization and purification of enzymes with staphylcoccal protein A gene vectors. EMBO J 4, 1075-1080 Sarnbrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467 Schiffer M, Edmundson AB (1967) Use of helical wheels to represent the structure of proteins and identify segments with helieal potentials. Biophys J 7, 121-135 Shagger H, and Van Jagow (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379 Siminovitch D, Cloutier Y (1983) Drought and freezing tolerance and adaptation in plants: some evidence of near equivalences. Cryobiology 20, 487-503 Somerville CR, Somerville SC, Ogren WL (1981) Isolation of photosynthetically active protoplasts and chloroplasts from Arabidapsis thaliana. Plant Sci Lett 21, 89-96 Thomashow MF (1990) Molecular genetics of cold acclimation in higher plants. Adv Genet 28, 99-131 115 Towbin H, Staehlelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354 Vieira J, Messing J (1987) Production of single-stranded plasmid DNA. Meth Enzymol 153, 3-11 Volger HG, and Heber U (1975) Cryoprotective leaf proteins. Biochim. Biophys. Acta. 412, 335-349. Vololn'ta M, Somerville CR (1987) The primary structure of spinach glycolate oxidase deduced from the DNA sequence of a cDNA clone. J Biol Chem 262, 15825-15829 Weiser CJ (1970) Cold resistance and injury in woody plants. Science 169, 1269-1278 Zhang H, Scholl R, Browse J, and Somerville C (1988) Double stranded DNA sequencing as a choice for DNA sequencing. Nucl Acids Res 16, 1220 APPENDIX CREATION AND ANALYSIS OF ARABIDOPSIS MUTANTS OVEREXPRESSING OR UN DEREXPRESSING car15 I have initiated a study to create and analyze Arabidapsis mutants that overexpress or underexpress car15, in an attempt to determine if the gene has an important role in Arabidapsis cold acclimation. Transgenic Arabidapsis plants that constitutively express car15 under the control of the CaMV 35 S promoter were obtained by Agrabacterium-mediated transformation. The transgenic plants were analyzed to determine if constitutive accumulation of COR15m resulted in increased freezing tolerance of whole plant tissues or isolated chloroplasts. The results were encouraging but inconclusive. Attempts to create transgenic plants that underexpress car15 were made by transforming plants with a plasmid containing an antisense car15 gene under the control of the CaMV 358 promoter. More than 130 independent transformants were obtained. The immunoblot analysis of the accumulation of COR15 m for approximately 100 transformants indicated that none of them dramatically underexpressed car15. 116 117 I. Plasmids A ScaI DNA fragment containing the whole ORF (open reading frame) for COR15 and part of the sequence from pBluescript was isolated from pLCTlOA, and BamHI linkers were added to the ends of the fragment that had been repaired by a standard E. cali DNA polymerase I reaction (Sambrook et al., 1989). The DNA was then digested with BamHI, and cloned into the BamI-II site downstream of the CaMV 35 S promoter in the plasmid vector pCIB710 (Rothstein et al., 1987), and introduced into E. cali strain DH5a (Barnsbrook et al. , 1989). Clones containing recombinant plasmids with the COR15 ORF oriented in sense or antisense direction with respect to the CaMV 35S promoter in pCIB710 were isolated, and referred to as pLCT61 and 62, respectively. The WUKpnI DNA fragment of the plasmid pLCT61 (containing the sense 35S-cor15 gene), and pLCT62 (containing the antisense 358-car15 gene) were isolated, cloned into the corresponding sites of the binary vector pCIBlO that provides the T-DNA borders and the kanamycin-resistant gene nptII (Rothstein et al. , 1987), and referred to as pLCT71 and pLCT72, respectively. The plasmids pLCT71 and pLCI‘72 were then introduced into Agrabacterium tumefaciens strain LBA4404 (Hoekema et al. , 1983) by triparental mating with pRK2073 providing the mobilization functions. H. Arabidapsis Transformation Arabidapsis thaliana ecotype RLD was transformed by the root explant cocultivation and regeneration method either exactly following the procedure of Valvekens (Valvekens er al. , 1988) or with the slight modifications as described bellow. 118 A. Preparation of Arabidapsis root explants and cocultivation Arabidapsis seeds were surface sterilized by incubating seeds (about 100 mg) in 1.2 ml of LDTM (Alcide Co. , Norwalk, CT) disinfectant solution (waterzbasezactivator = 10:1:1) at 30°C for 30 min. The seeds were then washed 3 times with sterile water, suspended in sterile water, and spread as straight lines on 150 x 25 mm Petri dishes containing germination medium (GM) (Valvenkens er al. , 1988) supplemented with B5 vitamins (0.1 mg/l thiamine, 0.01 mg/l nicotinic acid, 0.01 mg/l pyridoxine) and 1 mg/l myo-inositol. About 1 mg md was spread as one line on the surface of one GM plate. The sterilizing effect of LDTM solution was found to be better than the standard ethanol/NaOCl method (V alvenkens er al. , 1988). The plates were sealed with two layers of parafilm (American National Can, CT), and placed at a 45° to 60° angle under white fluorescent lights so that the roots would only grow on the surface of the agar medium but not penetrate into the agar. The light intensity was about 100 pE s"1 m'2 , the photoperiod 16 h, and the temperatures ranged from 23°C to 27°C. After 3 weeks, plantlets were cut with a razor blade to remove the green parts, the roots were peeled off from surface of the agar medium with forceps and placed onto Petri dishes containing eallus inducing medium (CIM) (Valvenkens et al., 1988) supplemented with B5 vitamins. The intact roots were incubated on CIM for 3 days (never in excess of 4 days to avoid a dramatic decrease in the transformation efficiency), and then transferred to a clean Petri dish, cut to small pieces of 0.5 to 1 cm long, and incubated with Agrabacterium cells for 3 min. The Agrabcteriwn cells harboring the plasmids (pCIBlO, pLCT71, or pLCT72) were grown in LB (0.5% NaCl, 0.5% yeast extract, 1% Bacto-tryptone) supplemented with 50 mg/l rifampicin and 50 mg ll kanamycin, at 30°C for 24 to 48 h. The bacterial cells were washed twice with fresh LB containing no antibiotics, resuspended in LB, and mixed with the root explants. The root explants 119 were then transferred onto CIM plates, incubated for 2 to 3 days, the root explants were collected, washed at least 3 times by placing them in bottles containing more than 150 ml of sterile water, shaking vigorously for 1 to 2 min, changing the water and washing again until the water was clear after shaking. B. Shoot induction, root induction, and seed production The root explants were placed onto the shoot inducing medium (SIM) (Valvenkens et al. , 1988) supplemented with B5 vitamins, with a few pieces of roots clumped together. The antibiotics vancomycin (500 mg/l) or carbenicillin (500 mg/l) was used to inhibit the growth of Agrabacterium; carbenicillin was found to be much more effective than vancomycin for this purpose. Green calli appeared in about 10 days, and they developed into shoot rosettes in another 1 to 2 weeks. The calli or small shoots were transferred to fresh SIM plates every week until the shoots were big enough for root induction. When the shoots were big enough (1-2 weeks from calli), they were individually excised from the calli but with a small chunk of the callus attached to the bottom of the shoot, incubated about 2 to 5 min in a drop of root inducing hormone solution (1 mg/ml indole-3-butric acid [sodium salt], 2 mg/ ml 1 -naphtaleneacetamide in water) (Last et a1 . , 1991), and placed in a magenta box containing GM medium. A hole (about 2.5 cm in diameter) was made the in lids of the magenta boxes and the hole was plugged with a polyurethane foam plug (Apico Inc. , Baltimore, MD) to accelerate air exchange and prevent contamination. Roots appeared within one week. After about 2 to 3 weeks grown on GM medium, plantlets were pulled out from the medium, all agar surrounding the roots was removed by washing with water, the plantlets were transferred to soil, and covered with plastic wrap. These transgenic plants (referred to as T1 plants) were grown in growth chambers under the conditions described in Chapter 2, the plastic wrap covering the pots was cut to 120 decrease the humidity one week after transfer and removed another week later. Seeds were harvested from individual plants about 4 weeks after planting in soil, and referred to as T2 seeds. Seeds from individual T2 plants were harvested and referred to as T3 P1983“- III. Analysis of transgenic plants A. Kanamycin assay Transformed plants were evaluated initially as those able to grow normally on GM medium containing kanamycin. The seeds were surface sterilized as described previously, and germinated on Petri dishes containing GM medium supplemented with 75 mg/l kanamycin. The germinating transformants grew normally, but the untransformed plantlets became yellowish within a week, became bleached and died within 10 days. The surviving plantlets were transferred to soil after two weeks on GM plates, and the transplants were covered with plastic wrap. The plastic wrap was removed after 3 days, and the plants were allowed to grow and set seed. Segregation of kanamycin-resistant and sensitive individuals was scored for T2 plants, and summarized in Table A. 1. The transgenic lines CIB-l, 71-2, and 71-3 segregated in a 3:1 ratio for kanamycin resistant to sensitive individuals (Table A. l). B. Detection of car15 sense and antisense transcript in transgenic plants Total RNA was extracted from wild type RLD plants and transgenic plants as described in the Chapter 2. RNA (15 pg per lane) was resuspended in formaldehyde loading buffer containing EtBr (1 mg/ ml), and fractionated on denaturing formaldehyde agarose gels (Sambrook et al. , 1989). Northern blots were prepared on Nytran membranes 121 Table A.1 Analysis of kanamycin resistance of the T2 progeny for some of the transgenic plants Progeny Kant Kans Kant/Kans Plasmid transformed CIB-l 10 3 3.3:1 pCIBlO (vector) 71-1 5 0 5:0 * pLCT71 (sense car15) 71-2 15 8 2.5:] ,, 71-3 10 4 3.3:1 ,, 72-31 30 13 2.3:1 pLCT72 (antisense carI5’) 72-33 1 90 1:90 * ,, 72-34 75 14 5.3:1 ,, 72-35 19 7 3.7:1 ,, 72-36 107 12 8.9:1 "' ,, 72-37 130 37 2.8:1 ,, 72-38 0 37 0:37 * ,, 72-39 40 8 4.8:1 ,, 72-40 85 28 3.0:] ,, 72-41 86 27 3.2:1 ,, 72-42 78 6 13 :l * ,, 72-43 51 31 1.6:1 * ,, 72-44 15 26 0.5:] * ,, 72-46 25 5 5.0:1 ,, 72-47 4 2 2.0:1 ,, 72-48 56 17 3.3:1 ,, 72-49 36 6 6.0:1 ,, 72-52 46 16 2.9:] ,, 72-55 26 14 1.9:1 ,, 72-59 32 2 16:1 * ,, 72-60 2 2 1.0:] ,, 72-63 32 0 32 :0 * ,, 72-64 27 12 2.3:1 3’ * The asterisks indicate that the segregation differ sigmficantly from a 3:1 ratio [kanamycin resistant (kanr):kanamycin sensitive (Kan )] by a Chi-square test (P01)- 122 (Schleicher and Schuell) using 20 x SSPE (Sambrook et al. , 1989). The blotted RNA was visualized and photographed with UV irradiation, blots baked at 80°C for 3 h in a vacuum oven, and then washed in 2 x SSPE containing 0.5% SDS at 55°C for 3 h. Sense and antisense car15 RNA probes (5-7 x 105 cpm) were prepared by in vitro transcription of the plasmid pLCTlOB and pLCTlOA, respectively. The plasmid DNAs (0.2 pg) digested with Barn HI were transcribed in 20p] reactions (containing 40 mM Tris-HCl pH 7.5, 6 mM MgC12, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 unit RNAsein, 500 pM each of ATP, GTP, and CTP, 12 pM UTP, 250 pCi [32P]UTP, and 25 units of T7 RNA polymerase) at 37°C for 1 h. The reactions were then mixed with 10 unit of DNAse l, incubated at 37°C for 30 min, diluted with 100 pl of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA), heated at 65°C for 5 min, chilled in ice, and the RNAs purified using a G-75 Sephadex spun column (Sambrook et al. , 1989). Hybridization was carried out according to standard procedures (Sambrook et al., 1989) at 42°C for 16 h, the blots were then washed twice with 0.1 x SSPE containing 0.5% SDS, incubated (without shaking) in 0.1 x SSPE containing 0.5% SDS at 60°C for 48 h, wrapped in a plastic membrane, and exposed to X-ray films. car15 transcripts were detected in T3 plants (71-1-3, 71-2-23, 71-3-1) of all three transgenic lines transformed with the sense 35S-car15 gene (Figure A. l). The transcripts of the transformed gene was slightly larger than that of the native ones (Figure A. 1), and were expressed at lower levels in comparison to that of the native car15 transcripts (Figure A. 1). 71-1-3 and 71-2-23 are T3 progeny (of the lines 71-1 and 71-2, respectively) that were homozygous for kanamycin resistance, while 71-3-1 plants (T3 progeny of the line 71-3) segregated in a 3:1 ratio for kanamycin-resistant to sensitive individuals. 123 Figure A.1. Nothem blot analysis showing the car15 transcript in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD), and ”sense” transgenic lines 71-1-3 (71-1), 71-2-23 (71-2), and 71-3-2 (71-3). The blot was hybridized with an antisense RNA probe for car15 prepared by in vitra transcription. Exposure was overnight. 124 RLD 7i-i 71-2 71-3 W C W C W C WC 0.7 kb Figure A.l 125 Northern blot analysis demonstrated the presence of the antisense-car15 RNA in T2 plant of all three randomly chosen transgenic lines that were transformed with the antisense 35S-car15 gene (Figure A.2). The levels of RNA of the native car15 gene in these lines, however, was not dramatically affected by the presence of the antisense RNA (Figure A.3). This is not necessarily surprising as the line producing the most antisense, 72-2, only produced only about 1/2 to 1/4 the amount of RNA (car15 antisense) as was produced by the endogenous car15 gene. C. Detection of COR15m in the transgenic plants Total soluble protein was isolated from wild type RLD and transgenic plants, proteins were fractionated on SDS-PAGE gels, and immunoblots analyzed with antibodies against COR15 as described in Chapter 4. Immunoblot analysis indicated that the sense transgenic plant accumulated COR15m constitutively. The levels of accumulation of COR15m in nonacclimated T3 plants 71-2-23 and 71-3-1 (sense transgenic lines 71-2 and 71-3, respectively) were found to be comparable to the level in the cold-acclimated wild type plants (Figure A.4). The levels of COR15m in cold-acclimated 71-2-23 and 71-3-1 plants appeared to be a bit higher than that the cold-acclimated wild type plants (Figure A.4). The COR15m synthesized in the nonacclimated transgenic plants was targeted to the chloroplasts (not shown). However, the immunoblot analysis of the antisense transgenic lines (kanamycin resistanth plants) indicated that none of these lines dramatically decreased its COR15 m level (not shown). D. COz-dependent oxygen evolution assay The effect of constitutive accumulation of COR15m on the freezing tolerance of plants was assessed using oxygen evolution assays. Light-dependent oxygen evolution 126 Figure A.2. Nothem blot analysis showing the antisense RNA of car15 in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD) and ”antisense" transgenic lines 72-2A, 72-2B, and 72-20. The blot was hybridized with a sense RNA probe for car15 prepared by in vitra transcription. Exposure was two days. 127 WCWCWCWC - 0.7 kb l__—JI._J L—J l__i RLD 72—2A 72-2e 72-20 Figure A.2 128 Figure A.3. Nothem blot analysis showing the car15 transcript in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD) and antisense transgenic lines 72-2A, 72-2B, and 72-20. The blot was hybridized with the antisense RNA probe for car15 prepared by in vitra transcription.Eexposure was overnight. 129 wCWCWCVVC ‘ ‘J'fiC‘Wf—"lfiwbl " ‘ . l :1 “Ti l——J “—4 RLD 72-2A 72"" 72"” Figure A.3 130 Figure A.4. Immunoblot analysis showing the accumulation of COR15m in nonacclimated (W) and cold-acclimated (C) plants of wild type (RLD) and sense transgenic lines 71-1-3, and 71-2-23. Total soluble protein was fractionated by SDS- PAGE on a 15% gel, transferred to a nitrocellulose, and the blot developed with antiserum (1:25 dilution) against COR15-protein A fussion protein (see Chapter 4). - uISIIIOZ) D 131 Figure A.4 132 activity reflects the integrity of the chloroplast electron transport apparatus. Arabidapsis RLD and T3 transgenic plants 71-2-23 were grown with or without cold acclimation under the conditions described in Chapter 4. 02 evolution assays were conducted with leaf discs according to Walker (Walker, 1985), using a Clark type oxygen electrode unit (Runk Brothers, Cambridge, England). leaf discs (approximately 1 cm in diameter) were obtained using a paper hole puncher. Each sample, consisting of 10 leaf discs, was equilibrated at -2°C in glass test tubes for 30 min in a low temperature bath (Model 2095 , Forma Sci. , Ohio). Freezing was then initiated by the addition of a drop of fine ice onto the leaf discs, and the temperature was decreased at a rate of 1°C/30min. After being frozen at appropriate temperatures for 30 min, tubes were transferred to ice, and kept on ice in the dark overnight to allow the leaf discs to thaw slowly. leaf discs were cut into 4 strips each with a razor blade, immersed in the assay solution (containing 100 mM tricine-KOH, pH8, 50 mM KCl, 5 mM NaHCO3) that was saturated with N2 and maintained at 21°C with a circulating water bath, and equilibrated in the dark for 2 min. The oxygen evolution reaction was started by illumination with white light of approximately 300 pE ln'2 s'1 , and the rate of oxygen evolution measured for 5 to 10 min. The plant material was recovered, blotted dry on filter paper, incubated in 1 ml acetone in an eppendorf microtube at room temperature for 1 h, and the content of chlorophyll measured at OD652 (pg chlorophyll = 2.78 OD652). The rate of oxygen evolution for freezing-treated samples was expressed as a percentage of the rate of untreated samples (approximately 50 to 100 11M 02 mg chl'1 h'l). Figure A.5 and A.6 demonstrate that freeze/thaw treatment severely damaged the ability of plant tissues to evolve oxygen for both nonacclimated wild type plants and 71-2-23 plants (Figure A.5). Cold acclimation increased the resistance of the photosynthetic apparatus in both kinds of plants to freeze inactivation (Figure A.6). 133 Freezing leaves at -7°C almost completely abolished the oxygen evolution activity of both nonacclimated wild type and 71-2-23 plants (Figure A.5). Freezing at this temperature, however, had little effect on the cold-acclimated plants (Figure A.6). In one experiment, leaves from 71-2-23 plants were more resistant to freezing damage than those from the wild type plants (Figure A.5a), but this increased freezing tolerance of 71-2-23 plants was not observed in another experiment (Figure A.5b) where a different batch of wild type and 71-2-23 plants were used. Similar inconsistencies in the experimental results were found when cold acclimated plants were used (Figure A.6). The reason for these inconsistencies is unknown. It may result from deviations derived from experimental conditions (e.g. time to prepare leaf discs, time to thaw leaf discs, etc) that were not precisely controlled, or it could beeause of some subtle differences in the growth conditions of the plants (e. g. temperature, light intensity, humidity, time the plant materials harvested, etc). E. Photophosphorylation The effect of constitutive accumulation of COR15m on the cryobehavior of the chloroplasts was analyzed directly by measurement of the photophosphorylation activities of the chloroplasts after a freeze/ thaw cycle or after an electrolyte (NaCl) treatment. The effect of freeze/thaw cycle and high concentration of NaCl on the photophosphorylation activity for chloroplasts isolated from the sense transgenic line 71-3-5 (homozygous for kanamycin resistance, and constitutively accumulates COR15m), and a quasi wild type line 71-3-3 (homozygous for kanamycin sensitivity, and does not accumulate COR15 m until cold acclimated), were compared. Chloroplasts were isolated essentially as described in Chapter 4 but without the step of Percoll gradient centrifugation; a different grinding buffer (0.45 M sorbitol, 20 mM Hepes-KOH pH 7.8, and 5 mM EDTA), and a slightly different resuspension 134 Figure A.5a. Oxygen evolution of leaf discs prepared from nonacclimated wild type RLD plants (broken line) and the sense transgenic line 71-2-23 (solid line). Leaf discs were treated with a freeze/ thaw cycle at the temperatures indicated proir to the assay. The oxygen evolution activity of each sample was expressed as percentage of that of the untreated sample that was kept on ice. 135 Figure A.5a (96) NOlin'lO/ia to 136 Figure A.5b. Same as Figure A.5a using a different batch of plants. 137 50's 1%) MorlmoAa to 10- Figure A.5b 138 Figure A.6a. Oxygen evolution analysis of leaf discs prepared from cold-acclimated plants of wild type RLD (broken line) and the sense transgenic line 71-2-23 (solid line). Leaf discs were treated with a freeze/thaw cycle at the temperatures indicated proir to the assay. The oxygen evolution activity of each sample was expressed as percentage of that of the untreated sample that was kept on ice. 139 mm.< ousowm te\eO— .mrQZ Moun'loaa to 140 Figure A.6b. Same as Figure A.6a using a different batch of plants. 141 I O in (96) Morlmoaa to 10‘ -H Figure A.6b 142 buffer (0.33 M sorbitol, 20 mM Hepes-KOH pH 7.8, and 5 mM MgC12) were used. Chloroplasts obtained from the first centrifugation were resuspended in theresuspension buffer to 2 pg chl/pl and stored on ice in the dark. The intactness of the chloroplasts was checked by microscopy, and by ferlicyanide-dependent oxygen evolution (Leegood and Malkin, 1986), and the chloroplasts were classified as type B (unbroken) according to the criteria described by Reeves and Hall (Reeves and Hall, 1980). For the freezing treatment, 100 pl of the chloroplast suspension (0.5 pg chl/pl) was added to a glass test tube, and incubated at -2°C for 10 min. Freezing was initiated by the addition of a few grains of sand cooled to -20°C, and the temperature decreased at a rate of 1°C/30 min. The samples were then thawed on ice for 2 to 3 h before the photophosphorylation assay. For the NaCl treatment, chloroplast suspensions were made of 0.5 pg chl/pl and 0.2 to 2 M NaCl in microcentrifuge tubes, and were incubated on ice for 3 h before the assay. Photophosphorylation of the chloroplasts was measured as described by Mills (Mills, 1986). Briefly, 60 pl of the chloroplast suspension (0.5 pg chi/pl) was mixed with 800 pl reaction solution (70 mM tricine-KOH pH 8, 50 mM glucose, 30 mM KCl, 12 mM MgC12, 1 mM NaHzPO4, 2 mM ADP, 0.1 mM Methylviologen, 2.5 pM diadenosinepentaphosphate, 4 unit/ml hexokinase, 5 pCi/ml ”PD in an microcentrifuge tube, allowed to equilibrate in the dark for 2 min, and illuminated with white light (approximately 200 pE m'2 s'l) for 3 min. The unincorporated 32Pi was extracted with molybdate (Mills, 1986), and the amount of 32m incorporated into ATP (approximately 105 cpm for the untreated controls) was measured in a liquid scintillation counter. The photophosphorylation reaction was completely inhibited by the addition of 20 mM DCMU (electron transport inhibitor), or 5 mM NH4C1 (uncoupler), or by omission of the light. The relative photophosphorylation activity of the freezing- or NaCl-treated samples was expressed as percentages of that of the untreated sample. 143 It is intriguing to find that the chloroplasts isolated from 71-3-5 plants that accumulated COR15m constitutively, had a slightly higher degree of freezing tolerance than those of the quasi wild type (71-3-3) plants (Figure A.7). Similar results were obtained when the chloroplasts isolated from a second batch of plants were analyzed (not shown). More experiments are needed, however, to comfirm these results. At temperatures occurring in nature, intracellular freezing is considered to be universally lethal to the plant cell, so freezing resistance can be more accurately defined as a tolerance to the consequences of extracellular freezing. One of the most detrimental consequences of extracellular freezing is dehydration of the protoplasm and concentration of the electrolytes to toxic levels. An analysis of the cryobehavior of isolated chloroplasts upon a direct freeze! thaw cycle, therefore, may not be the best way as chloroplasts may never freeze in plant cells surviving a freezing stress in nature. Thus, an alternative method to detect the effect of COR15m on chloroplast cryobehavior was employed by measuring the photophosphorylation activity of chloroplasts exposed to high concentrations of the electrolyte NaCl at low nonfreezing temperature (Figure A. 8). As shown in Figure A.8, photophosphorylation activity of chloroplast decreased dramatically when the concentration of NaCl increased from 0 to 1.5 M. It is interesting that chloroplasts isolated from the sense transgenic plants (71- 3-5) retained more than 90% of its photophosphorylation activity after incubating in 0.2 M NaCl while chloroplasts isolated from the quasi wild type plants (71-3-3) lost about 50% of its activity (Figure A.8a). However, this observation was not consistant under the experimental conditions used, as chloroplasts that were isolated from a different batch of plants of the lines 71-3-3 and 71-3-5, did not respond differentially to NaCl treatment (Figure A.8b). 144 Figure A.7. Photophosphorylation assay showing the response of chloroplasts to a freeze/thaw cycle. The chloroplasts were isolated from nonacclimated plants of the sense transgenic line 71-3-5 (solid line) or the quasi wild type line 71-3-3 (broken line). The photophosphorylation activity of the sample was expressed as a percentage of that of the untreated sample stored on ice. 145 9.¢ ouemflm 0.. :l 2.. or at T ct at or at o p _ p - p p P _ LT. rewo— / been (‘16) 'd OlOHd 146 Figure A.8a. Photophosphorylation assay showing the response of chloroplast to different concentrations of NaCl. The chloroplasts were isolated from nonacclimated plants of the sense transgenic line 71-3-5 (solid line) and the quasi wild type line 71-3-3 (broken line). The photophosphorylation activity of the sample was expressed as a percentage of that of the untreated sample stored on ice. 147 a.— om.¢ madman A3 .002 ed v.0 «.0 IO— Ian oo- (’/o ) 'd OlOHd 148 Figure A.8b. Same as Figure A.8a using a different batch of plants. 149 em.< oedema :5 .82 10— ton loc— (%)'dOLOHd 150 IV. Conclusion Attempts were made to study the effect of the constitutive accumulation of COR15m on the photosynthetic functions of the chloroplasts after a direct freeze! thaw cycle or after a treatment of a high concentration of NaCl. The studies, however, were inconclusive as to whether the accumulation of COR15m in nonacclimated plant cells or its accumulation at relatively higher levels in the cold-acclimated cells could increase the resistance of the chloroplasts to freeze! thaw cycle or to high concentrations of NaCl. The inconsistency in the experimental results may be as a result of the experimental conditions not being controlled precisely enough. On the other hand, accumulation of COR15m by itself, may not result any notable differences in the cryobehavior of plant tissue or chloroplasts, as COR15 m, even if necessary, may not be sufficient to protect chloroplasts from freeze inactivation. Therefore, construction of mutant plants that underexpress cor15 may be a better approach to assess the role of COR15m in cold acclimation. Attempts to create antisense mutants of car15 resulted in many transgenic plants. However, none of these transgenic lines tested show a dramatic decrease in the level of COR15m during cold acclimation. V. References Last RL, Bissinger PH, Mahoney DJ, Radwanski ER, and Fink GR (1991) Tryptophan mutant in Arabidapsis: the consequences of duplicated tryptophan synthase 8 genes. Plant Cell 3, 345-358 leegood RC, and Malkin R (1986) Isolation of sub-cellular photosynthetic systems. In Hipkins MF, and Baker NR, eds, Photosynthesis, energy transduction: a practical approach. IRL Press, Oxford, England Mills JD ( 1986) Photophosphorylation. In Hipkins MF, and Baker NR, eds, Photosynthesis, energy transduction: a practical approach. IRL Press, Oxford, England 151 Reeves SG, and Hall DO (1980) Higher plant chloroplast and grana: general preparative procedures (excludin n45 hrgh earbon dioxide fixation ability chloroplasts). Methods m Enzymology 69, 85 Rothstein SJ, Lahners KN, Lotstein RJ, Carozzi NB, Jayne SM, and Rice DA (1987) Promotgr cagsetttses, antibiotic-resistance genes, and vectors for plant transformation. Gene 5 15 -1 l Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Valvenkens D, Van Montagu M, and Van Lijsebettens M (1988) Agrabacterium- mediated transformation of Arabidapsis thaliana root explants by using kanamycin selection. Proc Natl Acad USA 85, 5536-5540 Walker DA (1985) Measurement of oxygen and chlorophyll fluorescence. In Coomos DH, Long SD, and Scurlock JMO eds, Techniques in bioproductivity and photosynthesis. Pergamon Press, New York SUMNIARY AND PERSPECTIVES The study described in this dissertation represents an attempt to understand possible function(s) of the cold regulated (car) gene in plant cold acclimation and freezing tolerance. It is demonstrated that the accumulation of transcripts encoding boiling-stable polypeptides is a common response to low temperature in different plant species. It is hypothesized that Arabidapsis cor genes encoding boiling-stable polypeptides have important function(s) in the plant cold acclimation and freezing tolerance. One possible role of the boiling-stable COR polypeptides is proposed to be cryoprotectant: a molecule which can protect cellular structures and macromolecules against freeze or conditions associated with freeze, such as dehydration, extreme pH, and extreme ionic strength. In support of this hypothesis, one of the boiling-stable COR polypeptides, COR15, is shown to be a potent cryoprotectant in an in vitra assay to protect LDH from freezing inactivation. It is not known, however, if COR15 has cryoprotective activity in viva. COR15 is found to be transported to chloroplast and processed to a mature form (COR15m) of 9.4 kDa. COR15m is also boiling-stable, and it is a hydrophilic polypeptide with an acidic p1 and unusual amino acid composition. More interestingly, COR15m is predicted to have an amphapathic at-helix structure, and it is found to form multimers even in the presence of SDS. A working hypothesis is that COR15m contributes to plant freezing tolerance by acting as a cryoprotectant in chloroplast, it protects chloroplast enzymes or membranes from freeze-induced dehydration damage. For example, COR15m may protect chloroplast enzymes from dehydration-induced 152 injuries. On the other hand, COR15m may prevent chloroplast membranes from the occurrence of lamellar-to-hexagonal 11 phase transition during freezing-induced dehydration, so that the osmotic responsive nature of the membrane can be protected. It is estimated that the concentration of COR15m in chloroplast is at pM range in comparison to the CP50 of COR15 that is only at nM range [the calculation of COR15m concentration is based on a measurement that the yield of COR15m extracted from 12 gram leave tissue is approximately 1 pg (not shown), and a speculative estimation that chloroplasts consist of approximately 10% of the tissue volume]. It . L‘.__.‘_‘_._._-._.___ may suggest that the concentration of COR15m is at a physiologically meaningful level as its hypothesized function is concerned. To test the working hypothesis, there are at least three approaches that can be employed. First, a gene expressing COR15m has been created and expressed in E. coli so that the purification of COR15m in large quantity is possible. The purified COR15m can be tested in vitra to see if it can protect the chloroplast enzyme from freeze- or dehydration-induced inactivation, or if it can protect the chloroplast membrane from dehydration-induced lamellelar-to hexagonal 11 phase transition. Secondly, the function of COR15m ean be tested in viva in the Arabidapsis mutants that constitutively express car15. This stratage is basically trying to answer the similar questions as does the first approach but with less artificial conditions used in the analysis. The analysis of such mutants has yield some interesting results (see Appendix), but more experiments are needed before a conclusion can be reached. Finally, the most direct approach to test the hypothesis is to creat and analyze the Arabidapsis mutants that do not express car15 gene. Initial attempts to creat such a mutant was not successful (see Appendix). This might be because of the following reasons. First, the promoter (CaMV 358) used to control the antisense car15 gene may not be capable of producing a sufficient amount of the antisense RNA to eliminate the 153 car15 RNA; employment of the promoter of car15 gene in the antisense gene construct should solve this possible problem. Secondly, the antisense RNA produced by the 358- antisense-car15 gene (see Appendix) may have secondary structures that interfer with its function; transgenic plants transformed with new antisense genes created by using the different part of the car15 sequences may be useful tests for this possibility. Finally, it is known that the expression of car15 is also regulated by dehydration stress and its expression may be necessary for plant dehydration tolerance; since the transgenic plants regenerated via the tissue culture method may need to survive dehydration stress at various stages of the regeneration, a null mutant of car15 may be selected against. Construction of a new antisense gene under the control of a regulated promoter (eg. substituted benzensulfonamide compound-induced promoter In 2-1 or In 2-2) may allow the recovery of the car15 deficient mutants. Alternatively, a "directe transformation” procedure without involvement of the tissue culture may overcome the same problem without change of the promoter. 154