6,}; m LIBRARY "’ J ‘ Michigan State University This is to certify that the thesis entitled CONSERVATION OF THE CBF LOW TEMPERATURE RESPONSE PATHWAY IN CEREALS presented by Keenan Loder Amundsen has been accepted towards fulfillment of the requirements for the MS. degree in Crop and Soil Sciences fiMLK‘ / Major Professor’s Signature / L/v/Aj / / Date MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 c:/CIRC/DateDue.p65-p.15 CONSERVATION OF THE CBF LOW TEMPERATURE RESPONSE PATHWAY IN CEREALS By Keenan Loder Amundsen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 2003 ABSTRACT CONSERVATION OF THE CBF LOW TEMPERATURE RESPONSE PATHWAY IN CEREALS By Keenan Loder Amundsen Freezing temperatures limit plant productivity which significantly affects grain and forage production. Many plants from temperate climates acquire improved fieezing tolerance afier exposure to low non-freezing temperatures; a process known as cold acclimation. In Arabidopsis, members of the CBF gene family regulate the low temperature expression of a family of highly expressed COR genes by binding to a conserved nucleotide sequence (CRT/DRE) in the promoter of the COR genes and inducing transcription of the COR genes. The cold acclimation response and conservation of COR genes in wheat, rye and barley has been previously demonstrated. Regions of the WCSIZO promoter involved in low temperature regulation contain CRT/DRE sequences (Vazquez-Tello et al., 1998), suggesting the possible role of a CBF- like system in the regulation of cereal COR genes. Novel members of the cereal CBF gene family can be identified fiom cereal sequence databases by conserved CBF protein signature sequences flanking the APZ/EREBP DNA binding domain. Expression of seven candidate CBF genes in Arabidopsis revealed that HvCBF 3 and HvCBF 6 induce . expression of the Arabidopsis COR genes and the remaining five candidate CBF genes do not. Gel retardation experiments and expression of domain swap constructs in transgenic Arabidopsis further supported these findings. The seven cereal CBF proteins are separated into two groups, based on a phylogenetic comparison of the protein sequences, which is consistent with the experimental findings. ACKNOWLEDGEMENTS I would like to thank my advisor, Mike Thomashow, for having an incredible amount of patience and offering encouragement while I was learning the skills of molecular biology. Additionally, I would like to thank Rick Ward for participating on my graduate committee and for giving me my first taste of lab research as an undergraduate. I would also like to thank Rebecca Grumet for participating on my committee and giving me advice during the review of my research. Special thanks go to Dan Zarka, Sarah Gilmour and Eric Stockinger who helped me throughout the years with seemingly silly and not so silly questions about methods, techniques, the literature and life in general. I appreciate the countless ways the other members of the lab have contributed in to my success as a graduate student: Dan Cook, Colleen Doherty, Sarah Fowler, Xin Zhang, Lahong Sheng, Huanying Qin, Jonathan Vogel, Heather VanBuskirk, Carri Duncan, Dona Canella, Ritu Shanna, Katie Diller, Ria Crawford, and Ryan Akema. I would like to give credit to James Zhang and Volker Haake at Mendel Biotechnology for supplying me DNA clones of Rye22, Rye24, and Rye31 and seed stocks of transgenic Arabidopsis expressing Rye31. Gratitude is also due Tony Chen, Patrick Hayes, and Jeff Skinner for providing me with clones of HvCBF3, HvCBF4, HvCBF6, and HvCBF9. iii PREFACE The previously unreported candidate CBF genes from barley HvCBF 6 and HvCBF9 were generously supplied along with HvCBF 4 and HvCBF 3 by Jeffrey Skinner with the consent of Tony Chen and Patrick Hayes (Oregon State University). All experiments presented in Chapter 2 were conducted by the author of this thesis with the exception of the whole plant freeze test which was graciously carried out by Jonathan Vo gel. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ........................................................................................................ ix PREFACE ....................................................................................................................... iv TABLE OF CONTENTS .................................................................................................. v CHAPTER 1: THE COLD ACCLHVIATION RESPONSE IN CEREALS INTRODUCTION ............................................................................................................ l PLANT RESPONSES TO FREEZING TEMPERATURES ........................................... 1 COLD ACCLIMATION ................................................................................................... 2 CHANGES IN GENE EXPRESSION AND COR GENE INDUCTION ........................ 3 LOW TEMPERATURE REGULATION OF THE COR GENES ................................... 4 THE LOW TEMPERATURE RESPONSE PATHWAY IN CEREALS ......................... 5 COMPONENTS OF THE CBF RESPONSE PATHWAY IN CEREALS ...................... 6 OBJECTIVES ................................................................................................................... 9 REFERENCES ............................................................................................................... l 0 CHAPTER 2: CONSERVED FUNCTION OF CEREAL CBF GENES INTRODUCTION .......................................................................................................... l 5 MATERIALS AND METHODS .................................................................................... 16 AMINO ACID SEQUENCE ALIGNMENT OF CBF PROTEINS ....................... 16 CONSTRUCTION OF CBF pGEM-T EASY PLASMIDS ................................... l7 CLONING OF TRANSLATIONAL FUSION CONSTRUCT S ............................. 20 PLANT GROWTH ............................................................................................... 23 RNA ISOLATION, NORTHERN TRANSFERS AND H YBRIDIZA T IONS .......... 24 CONSTRUCTION OF PLANT EXPRESSION VECTORS ................................. 25 DEVELOPMENT OF T RANSGENIC ARABIDOPSIS EURESSING CBF GENES ............................................................................................................................. 25 WHOLE PLANT FREEZE TEST ........................................................................ 27 H YDROPHOBIC CLUSTER ANALYSIS ............................................................ 27 RECOMBINAN T PROTEIN EXPRESSION AND EXT RA C T ION ...................... 28 WESTERN BLOT ANALYSIS .............................................................................. 28 ELECT ROPHORETIC MOBILITY SHIFT ASSA YS ........................................... 28 RESULTS ....................................................................................................................... 29 S UBFAMILY OF SIGNATURE SEQUENCE DOMAIN CONTAINING APZ/EREBP PROTEINS ..................................................................................... 29 CBF AND COR GENE TRANSCRIPT S ACC UMULA T E IN WHEA T AND RYE IN RESPONSE T 0 LOW TEMPERATURE ................................................ 32 ARABIDOPSIS COR GENE EXPRESSION IS INDUCED BY HvCBF 3 AND HvCBF 6 IN T RANSGENIC PLANTS .................................................................. 36 WHOLE PLANT FREEZE TEST ........................................................................ 39 CEREAL CBF GENE FAMILY IS COMPOSED OF TWO GROUPS ................ 39 EHRESSION OF CEREAL CBF DNA BINDING DOMAIN F USED T O ARABIDOPSIS AC T I VA T ION DOMAIN INDUCES EXPRESSION OF COR GENES ................................................................................................................ 41 vi RE C OMBINAN T HvCBF 3 AND HvCBF 6 PROTEINS BIND T 0 THE ARABIDOPSIS CR T /DRE ELEMENT ................................................................ 51 DISCUSSION ................................................................................................................. 55 REFERENCES ............................................................................................................... 62 APPENDIX 1 RECOMBINANT CEREAL CBF PROTEIN EXPRESSION CONSTRUCTS ............................................................................................................... 65 vii LIST OF TABLES Table 2.1 Primer pairs used to amplify CBF genes. ....................................................... 19 Table 2.2 Primer pairs used to amplify domain swap fragments. ................................... 22 Table 2.3 Oligonucleotides used in EMSA experiments. ............................................... 30 Table A.l Constructs to express recombinant CBF proteins. ................................... 66-67 viii LIST OF FIGURES Figure 2.1 Construction of CBF Translational Fusion Products .................................... 21 Figure 2.2 Map of Binary Vector pGA643. .................................................................... 26 Figure 2.3 “Signature sequence” containing proteins form a distinct subfamily within the APZ/EREBP gene family. .............................................................................. 31 Figure 2.4 Conservation of “ Signature Sequences” within the CBF family of AP2/EREBP proteins. ..................................................................................................... 33 Figure 2.5 Northern hybridization of T riticum aestivum L. vars Manitou and Norstar Exposed to 4 degrees C. .................................................................................................. 34 Figure 2.6 Northern hybridization of Secale cereale L. vars Florida401 and Puma grown at 4 degrees C. ................................................................................................................. 35 Figure 2.7 Northern hybridization of non-acclimated transgenic Arabidopsis plants expressing candidate cereal CBF genes. ................................................................... 37-38 Figure 2.8 Whole plant freeze test of HvCBF3-overexpressing Arabidopsis plants. ..... 40 Figure 2.9 Phylogeny of members of the CBF gene family. .......................................... 42 Figure 2.10 Amino Acid Sequence Comparison of the COOH-terminal end of CBF gene Family Members. ...................................................................................................... 43-44 Figure 2.11 Conservation of hydrophobic clusters within the C-terminus of members of the CBF gene family. ................................................................................................ 45-46 Figure 2.12 Northern hybridizations of non-acclimated transgenic Arabidopsis expressing CBF gene fusion products. ........................................................................................ 48-49 Figure 2.13 Expression of recombinant CBF proteins in E. coli. .................................... 50 Figure 2.14 Expression of recombinant AtCBFl and HvCBF 3 in E. coli ....................... 52 Figure 2.15 Electromobility shifi assay of an Arabidopsis CRT/DRE DNA element....53 Figure 2.16 Electromobility shift assay of recombinant HvCBF6 Binds an Arabidopsis CRT/DRE Element From COR15a ................................................................................. 54 ix CHAPTER 1: THE COLD ACCLIMATION RESPONSE 1N CEREALS INTRODUCTION Agronomically valuable crops are predominantly grown in conditions that are not optimal for plant growth or productivity. Abiotic stresses such as drought, excess water, salinity, and high and low temperatures are factors that limit plant production. As a result of growing in these suboptimal conditions the productivity of a plant does not reach its full potential 030yer, 1982). Low temperature (LT) damage contributes significantly to crop loss on an annual basis (Levitt, 1980). Traditional plant breeding techniques have not proven successful in improving the freezing tolerance of many crops due in part to the compromise between improved freezing tolerance and desirable growth and yield characteristics (Prasad, 2001). For example, the freezing tolerance of wheat varieties used in today’s agriculture is not much improved over those used a century ago despite considerable breeding efforts (Thomashow, 1990). Improvements in the understanding of how plants sense and respond to LT could give rise to new strategies to improve productivity in LT growth conditions. PLANT RESPONSES TO FREEZING TEMPERATURES Plants that have evolved in temperate regions are generally resistant to chilling stress, but are susceptible to freezing stress; freezing stress occurs at temperatures below zero degrees C (Levitt, 1980). When plants undergo freezing stress the water in the extracellular space begins to freeze. Since the osmotic potential of ice is less than that of water an osmotic potential gradient is generated, resulting in the movement of most of the osmotically active water out of the cell. As this water fi'eezes, more water is drawn from the cell leading to cellular dehydration (Sakai and Larcher, 1987; Steponkus et al., 1993). Levitt (1980) suggests that 70 to 80 % of the liquid water in the plant tissue is frozen as ice in the extracellular space. The primary site of freeze induced injury is the cellular membrane. Cellular membrane damage results in electrolyte leakage from the cells (Steponkus, 1984; Steponkus, 1993). Improved stability of the membrane at freezing temperatures has been associated with increased accumulation of certain cryoprotectants such as the soluble sugars raffinose and sucrose (Anchordoguy et a1., 1987; Crowe et al., 1988). These simple sugars have also been implicated in improved freezing tolerance of cold . acclimated plants (Guy et a1., 1992; Koster and Lynch, 1992; Wanner and J unttila, 1999). Accumulation of specific amino acids such as proline may also contribute to fi‘eezing tolerance (N anj 0 et a1., 1999) by providing membrane and protein stability against freeze induced damage (Rudolph and Crowe, 1985; Carpenter and Crowe, 1988). Additionally, alterations in the lipid composition at the site of cellular membranes are also associated with improved cryostability (Steponkus et a1., 1990; Steponkus et a1., 1993). COLD ACCLIMATION A number of plant species from temperate climates are able to cold acclimate; a process whereby a plant gains the ability to withstand freezing temperatures following exposure to low non-freezing temperatures (Hughes and Dunn, 1996; Thomashow, 1999). For example, non-acclimated Arabidopsis thaliana L. (Heyn) cvs. Columbia and Landsberg erecta are able to survive being frozen to ~3° C whereas these ecotypes are able to survive being frozen to approximately -10° C, after exposure to 4° C for 9 days based on the temperature at which 50% of the plants are killed (Gilrnour et al., 1988). Accumulation of the previously mentioned cryoprotectants is associated with the process of cold acclimation. CHANGES IN GENE EXPRESSION AND COR GENE INDUCTION In 1985, Guy et al. (1985) established that there is an increase in mRNA accumulation during cold acclimation. Understanding the changes in gene expression which occur during the process of cold acclimation could open the doors to exploring new methods to improve LT tolerance through marker assisted selection, altering management practices that influence gene expression or developing transgenics. The initial approach in studying changes in gene expression associated with cold acclimation was to identify those genes that are expressed to high levels during exposure to LT and determine if they are involved in freezing tolerance. To this end, a number of LT induced genes have been identified including a number of genes encoding LEA (late embryogenesis abundant) or novel polypeptides (Thomashow, 1998). In Arabidopsis the family of COR (cold regulated) genes are some of the most highly expressed LT induced genes; examples include COR6. 6, CORISa, COR4 7, COR 78. The COR genes are small hydrophilic, boiling stable polypeptides and increased expression of the COR genes is closely correlated with improved freezing tolerance in plants. Constitutive expression of COR15a in transgenic Arabidopsis does not significantly improve the fieezing tolerance of the transgenics (J aglo-Ottosen et al., 1998). However, expression of CORl 5a has been shown to increase the freezing tolerance of protoplasts by 1 to 2° C (Artus et a1., 1996; Steponkus et al., 1998). Steponkus et al. (1998) demonstrated that the increased freezing tolerance is a result of a decreased incidence in the number of freeze-induced lamellar-to-hexagonal H phase transitions, 3 specific type of freeze-induced damage affecting the plasma membrane. It was then speculated that simultaneous expression of a number of COR genes might lead to improved freezing tolerance at the whole plant level. LOW TEMPERATURE REGULATION OF THE COR GENES Promoter deletion studies on COR genes revealed LT responsive cis-acting elements, CRT (C-repeat; Baker et al., 1994)/DRE (dehydration responsive element; Yamaguchi-Shinozaki and Shinozaki, 1994). These cis-acting elements contain the core nucleotide sequence CCGAC. Stockinger et al. (1997), using a yeast one-hybrid assay, identified the first transcription factor AtCBFl (A. thaliana C-repeat binding factor 1) to bind to the CRT/DRE. Stockinger et al. demonstrated that AtCBFl binds specifically to the CCGAC nucleotide sequence by electrophoretic mobility shift assay and by expressing AtCBFl and a lacZ reporter under control of the COR15a promoter in yeast. Arabidopsis AtCBF 1 is a member of the CBF family of transcription factors including AtCBFl, AtCBF2 and AtCBF3 (Gihnour et al., 1998), also known as DREBlb, c, and a, respectively (Liu et al., 1998; Shinwari et al., 1998). These transcription factors mediate the LT induction of CRT/DRE regulated genes. Constitutive expression of AtCBFl in non-acclimated transgenic Arabidopsis results in an increase in COR gene expression and improved freezing tolerance (J aglo-Ottosen et al., 1998). Transgenic Arabidopsis expressing AtCBF3 have increased proline and sucrose levels consistent with plants exhibiting enhanced freezing tolerance (Gilrnour et al., 2000). The CBF family of transcription factors is a subset of the AP2/EREBP family of DNA binding protein (Stockinger et al., 1997). The AP2/EREBP DNA binding domain is thus far unique to plants; however there are over 600 entries of AP2/EREBP DNA binding domain proteins in the NCBI database (Riechmann and Meyerowitz, 1998). J aglo et al. (2001) reported conserved anrino acid regions PKK/RPAGRXKFXETRHP and DSAWR are conserved in CBF genes. These conserved sequences, which flank the AP2/EREBP domain, are called the CBF family signature sequence and are found in CBF proteins from different species including wheat and rye (Jaglo et al., 2001). THE LOW TEMPERATURE RESPONSE PATHWAY IN CEREALS Wheat, rye and barley, are examples of cereal crops that have, like Arabidopsis, adapted a cold acclimation response to tolerate freezing temperatures. For instance, non- acclimated winter wheat and rye plants have an LTso (the temperature at which 50% of the population is killed) value of approximately -5° C, whereas cold acclimated winter wheat and rye plants have minimum LT50 values near —20° and -25° C, respectively (Fowler et a1., 1996). Genes influencing frost resistance have been mapped to linkage group 5 and chromosomes 4B, 4D and 7A in wheat. The location of these genes was carried out by mapping in monosomic and substitution lines (Sutka, 1981; Roberts, 1986; Galiba and Sutka, 1988). There is a great deal of synteny among wheat, rye and barley (Herrrnan et a1., 1996; Karn-Morgan et al., 1989). For example, Sh2 on chromosome 5H of barley is consistent with the location of VrnI on chromosome 5A of wheat; both genes contribute significantly to winter hardiness (Laurie et al., 1995). Additional genes affecting cold hardiness have been mapped to barley chromosome 5H in accordance with the similar location of these genes on chromosome 5A of wheat (Hayes et al., 1993). A number of these LT responsive genes are reviewed by Thomashow (1999). COMPONENTS OF THE CBF RESPONSE PATHWAY IN CEREALS Following the identification of the CBF cold response pathway in Arabidopsis, it was of interest to determine whether a similar system is conserved in the cereals. There are three sequence regions containing the core CRT/DRE nucleotide sequence (CCGAC) at the nucleotide positions 167, 353, and 430 upstream of the translational start site of the cold responsive barley gene DHN5 (AF 1 8 1455). Similarly the LT responsive H VA] promoter (AF 343067) has two CRT/DRE sequences at nucleotide positions eight and 214. The promoter region of WCS120, a member of the LT responsive WCS120 gene family, was characterized by Vazquez-Tello et al. (1998). Within the promoter region of WCSI 20, there are 10 nucleotide regions that contain the Arabidopsis CRT/DRE core sequence. A number of these were shown to be in regions of the promoter that are regulated by LT. Additionally, experiments were conducted to demonstrate that the promoter of WCS120 is cold inducible in both monocots and dicots (Oullet et al., 1998). Since CRT/DRE sequences were found in the promoters of LT induced genes in cereals, it was of interest to determine whether the key regulators of these genes in Arabidopsis, the CBF genes, are also conserved in cereals. Indeed a few AP2/EREBP domain- containing proteins have been identified in cereals that do contain the CBF family signature sequences, suggesting that these species have CBF-like genes. Some of these signature sequence containing APZ/EREBP genes are quickly responsive to LT. J aglo et al. (2001) were the first to demonstrate that CBF-like genes are present in T riticum aestivum and Secale cereal. It was shown that these cereal CBF genes, T aCBF 1 (AF 376136), Rye22, Rye24, and Rye31, have the CBF family signature sequence. Also their mRNA expression is responsive to LT, and the expression of the CBF genes precedes the expression of a target COR gene, COR39. The temporal expression of the CBF genes followed by COR gene expression is similar to that of the cold responsive CBF genes in Arabidopsis. Similar to T aCBF I, Rye22, Rye24, and Rye31 identified by Jaglo et al., a second candidate wheat CBF gene, T aDREBI (AF 30337 6), has been shown to be rapidly induced by LT (Shen et al., 2003). The T aDREBI protein was shown by Shen et al. to bind specifically to a DRE element and induce expression of a LacZ reporter gene in a yeast one hybrid system. They showed that T aDREBI induces expression of the target gene Rd29a, using non-acclimated transgenic rice plants constitutively expressing TaDREBI . ’ Additional CBF genes have been identified from Hordeum vulgare, BCBFI and BCBF 3 [HvCBF4 (AAK01088) and HvCBF 3 (AF 239616) respectively; Choi et a1, 2002], HvCBF] (AAL84170; Xue, 2002a) and HvCBF 2 (AAM13419; Xue, 2003). Choi et a1. (2002) demonstrated that HvCBF3 mRNA is rapidly induced by LT and precedes induction of Dhn8. HvCBF 3 was mapped between markers WG364b and saflp58 on chromosome 5H of barley and may coincide with the chI locus on wheat chromosome 5A. Vaguj falvi et a1. (2000) identified two loci, chI and ch2 (regulator for COR genes), with additive effects controlling the expression of c0r14b in wheat. Experiments by Xue (2002a, 2002b) demonstrated that transient expression of HvCBF] in barley leaves induces expression of a GFP reporter under control of the LT responsive HvaI promoter. Additionally it was shown that HvCBF 1 binds to a G/aC/tCGAC motif closely matching the CRT/DRE Arabidopsis element. Northern blot experiments demonstrated that HvCBF 2 is constitutively expressed while an assay of protein activity revealed that the DNA binding activity of HvCBF2 protein was enhanced as the temperature decreased (Xue, 2003). This is different fi'om Arabidopsis CBF proteins, which function at normal grth temperatures as demonstrated by transgenic Arabidopsis plants constitutively expressing these CBF genes (J aglo-Ottosen et al., 1998; Gilrnour et al., 2000). In recent years efforts have increased to demonstrate the conservation of the CBF cold responsive system in agronomically important crops (J aglo et al., 2001; Choi et a1., 2002; Shen et al., 2003; Xue, 2002). Even though candidate CBF genes have been identified in cereal crops, the demonstration of the functional homology of these genes to their Arabidopsis counterparts is lacking. It is clear that the CBF system in cereals is more complicated than that previously noted in Arabidopsis and additional studies are needed in order to understand the level of functional conservation of these genes in cereals. OBJECTIVES This study was designed to investigate whether there was an efficient way to identify candidate CBF genes fiom cereal species and survey the fimctional characteristics of a larger group of cereal CBF genes. It was anticipated that this investigation of a larger group of cereal CBF genes will offer insight into the extent of firnctional homology between these cereal CBF genes and the Arabidopsis CBF genes and will lead to further elucidation of the LT response pathway in cereals. These CBF genes were compared to the Arabidopsis CBF genes to determine whether they were functionally homologous, i.e., whether the candidate cereal CBF proteins bind to the CRT/DRE element and induce expression of the Arabidopsis COR genes in vivo. The results of this study will lead to a better understanding of the CBF LT responsive system in cereals and will offer insights into approaches for developing more LT tolerant varieties. REFERENCES Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH. (1987) Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology. 24(4):324- 3 1 . Artus NN, Uemura M, Steponkus PL, Gilrnour SJ, Lin C, Thomashow MF. (1996) Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc Natl Acad Sci U S A. 93(23):]3404-13409. Baker SS, Wilhelm KS, Thomashow MF. (1994) The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Biol. 24(5):701-13. Boyer, J S. 1982. Plant productivity and enviromnent. Science. 218:443-448. Carpenter JF, Crowe J H (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25: 244—255 Choi, DW, Rodriquez, EM, and Close, TJ. 2002. Barley CBF 3 gene identification, expression pattern, and map location. Plant Physiol. 129(4): 1 781-17 87. Crowe J H, Crowe LM, Carpenter JF, Rudolph AS, Wistrom CA, Spargo BJ, Anchordoguy TJ. (1988) Interactions of sugars with membranes. Biochim Biophys Acta. 947(2):367-84. Dubouzet, JG, Sakuma, Y, Ito, Y, Kasuga, M, Dubouzet, EG, Miura, S, Seki, M, Shinozaki, K, and Yamaguchi-Shinozaki, K. 2003. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, hi gh-salt-, and cold- responsive gene expression. Plant J. 33(4):751-763. Fowler, DB and Gusta, LV. 1979. Selection of winterhardiness in wheat. 1. Identification of genotypic variability. Crop Sci. 19:769-772. Fowler, DB, Limin, AE, Wang, S-Y, and Ward, RW. 1996. Relationship between low- temperature tolerance and vemalization response in wheat and rye. Can. J. Plant Sci. 76:37-42. Fowler S, Thomashow MF. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell. 14(8): 167 5-90. Galiba, G and Sutka, J. (1998) A genetic study of frost resistance in wheat callus culture. Plant Breeding 101:132-136. 10 Gilrnour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF. (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J. 16(4):433-42. Gilrnour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF. (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124(4):1854-65. Guy CL, Niemi KJ, Brambl R. (1985) Altered gene expression during cold acclimation of spinach. Proc Natl Acad Sci U S A. 82(11):3673-7. Guy CL, Huber J LA, Huber SC (1992) Sucrose phosphate synthase and sucrose accumulation at low temperature. Plant Physiol 100: 502—508. Hayes, PM, Blaket, T, Chen, THH, Tragoonrung, S, Chen, S, Pan, A, Lin, B. (1993) Quantitative trait loci on barley (Hordeum vulgare L.) chromosome 7 associated with compnents of winterhardiness. Genome 36:66-71. Hen'mann, RG, Martin, R, Busch, W, Wanner, G, Hohmann, U. (1996) Physical and topographical mapping among Triticeae chromosomes. Symp Soc Exp Biol. 50:25-30. Hughes MA, Dunn MA (1996) The molecular biology of plant acclimation to low temperature. J Exp Bot 47: 291-305. J aglo, KR, Kleff, S, Amundsen, KL, Zhang, X, Haake, V, Zhang, JZ, Deits, T, and Thomashow, MF. 2001. Components of the Arabidopsis c-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol. 127(3):910-917. J aglo-Ottosen KR, Gilrnour SJ, Zarka DG, Schabenberger O, Thomashow MF. (1998) Arabidopsis CBFl overexpression induces COR genes and enhances freezing tolerance. Science. 280(5360): 104-6. Kam-Morgan, LNW, Gill, BS, Muthukrishnan, S. (1989) DNA restriction fragment polymorphism: a strategy for genetic mapping of D genome of wheat. Genome 32:724- 732. Koster KK, Lynch DV (1992) Solute accumulation and compartrnentation during the cold acclimation of puma rye. Plant Physiol 98: 108—113. Laurie, DA, Pratchett, N, Bezant, J, Snape, JW. (1995) RF LP mapping of five major genes and eight quantitative trait loci controlling flowering time in a winter x spring barley (Hordeum vulgare L.) cross. Genome 38:575-585. 11 Levitt, J. 1980. In Responses of plants to environmental stresses. Vol. 1: Chilling, freezing, and high temperature stresses. 2nd ed, Academic Press, New York. Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two transcription factors, DREBl and DREBZ, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low- temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10: 1 391 - 1406. Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (1999) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461: 205—210. Ouellet F, Vazquez-Tello A, Sarhan F. (1998) The wheat wcs120 promoter is cold- inducible in both monocotyledonous and dicotyledonous species. FEBS Lett. 423(3):324- 8. Prasad, TK. (2001) Mechanisms of Chilling Injury and Tolerance. 2001. in Basra, AS., ed Crop responses and adaptations to temperature stress. Food Products Press, New York. pp 1-52. Riechmann, JL and Meyerowitz, EM. (1998) The AP2/EREBP family of plant transcription factors. Biol Chem. 379(6):633-46. Roberts, DWA (1986) Chromosome in ‘Cadet’ and ‘Rescue’ wheats carrying loci for cold hardiness and vemalization response. Can J Genet Cytol 28:991-997. Rudolph AS, Crowe JH (1985) Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline. Cryobiology 22: 367—3 77 Sakai, A. and Larcher, W. 1987. In Frost survival of plants. Springer Verlag, Berlin. Shen, YG, Zhang, WK, He, SJ, Zhang, J S, Liu, Q, and Chen, SY. 2003. An EREBP/AP2- type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress. Theor. Appl. Genet. 106(5):923-930. Shinwari ZK, Nakashima K, Miura S, Kasuga M, Seki M, Yamaguchi-Shinozaki K, Shinozaki K (1998) An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochem Biophys Res Commun 250: 1 61 -1 7O Steponkus, PL. (1984) Role of the plasma-membrane in freezing-injury and cold- acclimation. Annu. Rev. Plant. Physiol. 35:543-585. 12 Steponkus, PL, Lynch, DV, Uemura, M. (1990) The influence of cold acclimation on the lipid composition and cryobehaviour of the plasma membrane of isolated rye protoplasts. Philos Trans R Soc Lond B 326:571-583. Steponkus, PL, Uemura, M, Webb, MS. (1993) A contrast of the cryostabiliy of the plasma membrane of winter rye and spring oat-two species that widely differ in their freezing tolerance and plasma membrane lipid composition. in Steponkus, PL. Advances in Low-temperature Biology. Vol 2. J AI Press, London, pp 211-312. Stockinger, EJ, Gilrnour, SJ, Thomashow, MF. (1997) Arabidopsis thaliana CBF] encodes an AP2 domain-containing transcriptional activator that binds to the C- repeat/DRE, a cis-acting DNA regulatory elemtrr that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. 94: 1035-1040. Sutka, J. (1981) Genetic studies of frost resistance in wheat. Theor Appl Genet 59:145- 52. Thomashow, MF. (1990) Molecular genetics of cold acclimation in higher plants. Adv Genet. 28:99-131. Thomashow, MF. (1999) Plant Cold Acclirnation: Freezing Tolerance Genes and Regulatory Mechanisms. Annu. Rev. Plant Physiol. 50:571-599. USDA-NASS, Agricultural Statistics 2002. 1 March 2003. US. Department of Agriculture, National Agricultural Statistics Service.http://www.usda.gov/nass/pubs/ agrO2/acroO2.htrn. Vaguj falvi A, Crosatti C, Galiba G, Dubcovsky J, Cattivelli L (2000) Two loci on wheat chromosome 5A regulate the differential cold-dependent expression of the cor14b gene in frost tolerant and sensitive genotypes. Mol Gen Genet. 263: 194-200 Vazquez-Tello A, Ouellet F, Sarhan F. (1998) Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheat. Mol Gen Genet. 257(2):157-66. Wanner LA, J unttila O (1999) Cold-induced freezing tolerance in Arabidopsis. Plant Physiol 120: 391—400 Xue, GP. 2002a. An AP2 domain transcription factor HvCBFl activates expression of cold-responsive genes in barley through interaction with a (G/a)(C/t)CGAC motif. Biochim. Biophys. Acta. 1577(1):63-72. Xue, GP. 2002b. Characterisation of the DNA-binding profile of barley HvCBFl using an enzymatic method for rapid, quantitative and hi gh-throughput analysis of the DNA- binding activity. Nucleic Acid Res. 30(15):e77. l3 Xue, GP. 2003. The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperature. Plant J. 33(2):373-383. Zhu B, Choi DW, Fenton RD, Close TJ. (2000) Expression of the barley dehydrin multigene family and the development of freezing tolerance. Mo] Gen Genet 264: 145- 1 53 14 CHAPTER 2 CONSERVED FUNCTION OF CEREAL CBF GENES INTRODUCTION Cold acclimation is a dynamic process in which plants increase their ability to withstand freezing temperatures to different degrees depending upon their conditioning prior to the freeze. Usually a period of treatment with low non-fi'eezing temperatures, a cold acclimation, will increase the freezing tolerance significantly in freezing tolerant species. Although several cereals are able to cold acclimate, a better understanding of the causes of low temperature (LT) damage and the events that occur during cold acclimation in wheat and other cereal crops would offer potential benefits to combat damage and yield loss due to LT. Over the past century, wheat breeders have had limited success improving the LT tolerance of wheat (Fowler and Gusta, 1979). In fact, the LT tolerance of cultivated wheat is not much improved over the land races. A better understanding of how wheat and other cereal plants sense and respond to LT could result in methods to improve plant productivity and tolerance. The significant loss of wheat and other cereal crops as a result of LT could be reduced through improved LT tolerance of cultivated varieties. In Arabidopsis, members of the CBF gene family encode low temperature responsive transcription factors that are involved in the cold acclimation response (Stockinger et al., 1997; Gilrnour et al., 1998; Liu et al., 1998; Shinwari et al., 1998). Plants exposed to low temperatures exhibit increased expression of cold regulated CBF gene family members. The CBF proteins bind to conserved cis-acting sequences 15 (CRT/DRE) in the promoters of the COR (Qld regulated) genes and activate transcription of the COR genes (Stockinger et al., 1996; Jaglo-Ottosen et al., 1998). Increased expression of the COR genes is associated with improved freezing tolerance in plants. In wheat, the WCS120 gene family is composed of a number COR genes whose expression is induced upon exposure to low temperature (Limin et al., 1997). Both transcript and protein levels of WCSlZO are elevated when the plants are grown at low temperature. The promoter of this gene has cis-acting elements that are important for the cold responsive gene expression (Vazquez-Tello et al., 1998). Promoter deletion analysis, conducted by Vazquez-Tello et a1. revealed a number of conserved elements similar to the Arabidopsis CRT/DRE elements. Similarly, sequence information fi‘om the barley LT responsive H VAI (AF 343067) and DHN5 (AF 1 81455) genes revealed a number of CRT/DRE elements in these promoters. This suggests that a CBF relative may be involved in the regulation of cereal COR genes. The current study was undertaken with two primary goals: to identify candidate cereal CBF genes and to determine whether they were functionally homologous to the Arabidopsis CBF genes. Sequence analysis and database searches were employed to identify candidate CBF genes from wheat, rye and barley. Protein expression studies of a subset of the identified cereal CBF genes were conducted to determine functional homology to the known CBF genes. MATERIALS AND METHODS AMINO ACID SEQUENCE ALIGNMENT OF CBF PROTEINS l6 The amino acid sequence of the AP2/EREBP DNA binding domain of Arabidopsis thaliana AtCBFl (AAC49662) and Nicotiana tabacum ERFl (BAAO7321) were queried against the NCBI database using Blast vs. 2.0 (Altschul and Lipman, 1990) to identify AP2/EREBP domain containing proteins. Clustal X vs. 1.8 (Thompson et al., 1997) was used to align the full-length amino acid sequences and generate a phylogenetic tree of 245 unique members of this AP2/EREBP family. CONSTRUCTION OF CBF pGEM-T EASY PLASMIDS PCR was used to amplify the DNA regions coding for the presumed open reading frames of Arabidopsis AtCBF] (Stockinger et al., 1997;AAC49662), Rye22 (J aglo et al., 2001; AF370730), Rye24 (Jaglo et a1., 2001; AF370729), Rye31 (Jaglo et al., 2001; AF 370728), wheat T aCBFI (J aglo et a1., 2001; AF376136), barley HvCBF 4 (HvCBFI ; Choi et a1., 2002; AAK01088), HvCBF 3 (Choi et al., 2002; AAK01089), HvCBF 6 (Dr. Patrick M. Hayes, Oregon State University, Unpublished), and HvCBF 9 (Dr. Patrick M. Hayes, Oregon State University, Unpublished). The PCR primers used to amplify each candidate CBF gene are presented in Table 2.1. Arabidopsis AtCBF] was amplified from pACT-Bgl+ (Stockinger et al., 1997). Rye22, Rye24 and Rye 31 were sub cloned into the SalI and NotI restriction sites of pBluescriptSK- and amplified. The candidate rye CBF genes were generously supplied in proprietary vectors harboring the full-length rye clones by James Zhang (Mendel Biotechnology, Hayward, CA). T aCBF 1 was amplified from KLA21; a fill] length T aCBF 1 cloned into the EcoRI restriction site of pBluescriptSK-. Full length HvCBF 4, HvCBF 6 and HvCBF 9 cloned into the EcoRI and 17 XhoI restriction sites of pBluescriptSK- and HvCBF 3 cloned into the pGEM-Teasy vector (Promega, Madison, WI) were supplied by Tony Chen (Oregon State University, Corvallis, OR). Candidate genes were amplified separately using Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) with the addition of 0.5% dirnethyl sulfoxide to each 50 ul PCR reaction. A touchdown PCR protocol was used on an MJ Research DNA Engine PTC-200 therrnocycler with the following conditions: 94° C for 4 min; 10 cycles of 94° C for 1 min, 65° C for 1 min (-1° C per cycle), 72° C for 1 min; 25 cycles of 94° C for 1 min, 54° C for 1 min, 72° C for l min; 72° C for 5 min. The PCR products were fractionated on a 1% agarose 1 x TBE gel run at 100 volts for 1 hr. The amplified products were gel purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) as described by the manufacturer. The purified DNA was then ligated to pGEM-T easy; the ligation reaction was performed as described in the pGEM-Teasy protocol supplied by Promega. The ligation products were then transformed into Escherichia coli strain DHSOL via electroporation using the BioRad Bacterial Electroporator (Bio-Rad Laboratories, Hercules, CA). Transformations were conducted as recommended by the electroporator manufacturer. Transformed E. coli were selected based on their ability to grow in Luria-Bertani grth medium (LB) containing ampicillin (100 ug/ml). Selected colonies underwent a colony PCR screen to determine the presence of the candidate CBF genes. The PCR conditions and primers were the same as those described above. A —80° C glycerol stock was prepared fi'om each E. coli colony harboring the appropriate plasmid. Plasmid DNA was extracted from the afore mentioned cultures using the Wizard Plus Minipreps DNA Purification System (Promega). The pGEM-Teasy plasmids harboring the candidate CBF genes were 18 Table 2.1 Primers pairs used to amplify CBF genes. Gene nt Name Sequence Linker AtCBF] 23 KLA34 tctagaATGAACTCATTI‘TCAGC Fwd XbaI 37 KLA19 agatctattccatggTGTAACTCCAAAGCGACACGTC Rev Ncolnglll HvCBF3 24 KLA16 tctagaATGTCTCCCACACTCTCG Fwd XbaI 36 KLA17 agatctattccatggACATATCGACACT ATAGCT CC Rev NcoI:BglII HvCBF4 24 KLA14 tctagaATGGACGTCGCCGACATC FWD XbaI 36 KLA15 agatctattccatggTGCAGTCGAACAAATAGCT CC REV NcolnglIl-l HvCBF6 24 KLA20 tctagaAAGATGTGTCAGATCAAG Fwd XbaI 36 KLA21 agatctattccatggTGCT CT GGTAGCT CCAGAGTG Rev NcoI:BglII HvCBF 9 25 KLA24 tctagaAGCATGTCGAATCCGATTC fwd XbaI 37 KLA25 agatctattccatggTGTTGAACAAGCAGCTCCATAG Rev NcolnglII Rye22 24 KLA14 tctagaATGGACGTCGCCGACATC FWD XbaI 35 KLA26 agatctattccatggTAATTAGTCGAACAAACAGC Rev NcolnglII Rye24 24 KLA27 tctagaACTCGGATGGACGTCGCC Fwd XbaI 37 KLA28 agatctattccatggTGTCGAACAAGCAGCTCCATAG Rev NcolnglII Rye31 24 KLA29 tctagaACGCTGCAACTGATGGAC FWD XbaI 37 KLA30 agatctattccatggTGT'I‘CCAAAGCGGCGTGTGGAC Rev NcoI:BglII TaCBF 1 24 KLA29 tctagaACGCTGCAACTGATGGAC Fwd XbaI 37 KLA31 agatctattccatggTGTTCCAAAGCGGCGTGTAGAC Rev NcolnglII Primer pairs used to amplify AtCBF], HvCBF 4, HvCBF 3, HvCBF 6, HvCBF 9, Rye22, Rye24, Rye31 and T aCBF 1 . Linker shows the relative orientation of the primer (fwd, forward; rev, reverse) and the restriction site linker DNA (XbaI, NcoI, BglII). The XbaI, BglII and N001 linker fragments are indicated with the lowercase fonts. 19 sequenced by the Genomics Technology Support Facility (East Lansing, MI), using M13 forward and reverse primers on an Applied Biosystems ABI Prism 3100 Genetic Analyzer. After DNA sequencing to verify the presence of the correct insert, the —80° C glycerol stocks were given a KLA number (AtCBFlpGEM-Teasy, KLA152; HvCBF4pGEM-Teasy, KLA129; HvCBF3pGEM-Teasy, KLA130; HvCBF6pGEM- Teasy, KLA131; HvCBF9pGEM-Teasy, KLA157; Rye22pGEM-Teasy, KLA133; Rye24pGEM-Teasy, KLA134; Rye3lpGEM-Teasy, KLA135; TaCBFlpGEM-Teasy, KLA136). CLONING OF TRANSLATIONAL FUSION CONSTRUCTS Fragments of AtCBF 1, HvCBF 4, HvCBF 3, HvCBF6 or HvCBF 9 were amplified using the primers indicated in Table 2.2. PCR was conducted as described above using PfuTurbo DNA Polymerase (Stratagene, La J olla, CA) in place of Taq Polymerase, resulting in blunt ended DNA fragments. The PCR product was then phosphorylated using T4 DNA Kinase (New England Biolabs, Beverly, MA) as described by the enzyme supplier. Fractionation of amplified DNA products and isolation of DNA was performed as described above. The N-terminal CBF fragment was amplified to include the upstream amino acids through the NF amino acids at the COOH-tenninal end of the AP2/EREBP DNA binding domain or the COOH-terminus was amplified to include the ADS amino acids just downstream of the AP2/EREBP domain to the COOH-terminus of the CBF gene. The N-terminal AtCBFl fragment (N -AtCBF1) was ligated to the COOH- terminal 20 AtCBFl —216 HvCBF4 I 1228 HvCBF 3 l 1296 HvCBF6 L J 247 109 HvCBF9 _ 294 104 105 N-HvCBF4zC-AtCBF1 l:—216 152 105 N-HvCBF32C-AtCBF1 :— 216 123 105 N-HvCBF6zC-AtCBFl 216 109 105 N-HvCBF9:C-AtCBFl 216 105 104 N-AtCBFl :C-HVCBF4 —Y:: 229 105 152 N-AtCBFl:C-HvCBF3 _::2 249 105 123 N-AtCBFlzC-HVCBF6 d: 229 105 109 N-AtCBFl:C-HvCBF9 h 290 Figure 2.1. Construction of CBF Translational Fusion Products Cartoon of CBF translational fusion constructs. The construct names are on the left. The length of the peptides is indicated at the right. Internal numbers of the wild type CBF proteins indicates the point of truncation. The under bar indicates the predicted AP2/EREBP DNA binding domain. This image is presented in color. 21 Table 2.2 Primer pairs used to amplify dormin swap fi‘agments. Primer 1 Primer 2 Table 2.1 DNA Fragment Name Name Sequence 5 ’ to 3 ’ N-AtCBFl KLA34 MFT674 GAAGTTGAGACATGCTGATCGGC C-AtCBF l KLA19 KLA39 GCTGAC TCGGC TTGGCGGCTACG N-HvCBF4 KLA14 KLA4O GAAGTTGAGGCAGGC GGCGC GGC C-HvCBF4 KLA15 KLA41 GCCGACTCCGC GTGGCGGATGCG N-HvCBF 3 KLA16 KLA36 GAAGTTGAGGCAC GCGGC GGAGC C-HvCBF3 KLA17 KLA35 GCCGACTCCGC GTGGCTGCTCGC N-HvCBF6 KLA20 KLA42 GAAGTTGAGGCAGCCCGCGCCCC C-HvCBF 6 KLA21 KLA43 GCCGACTCCGCCGAGC TAC TCGC N-HvCBF9 KLA24 KLA44 GAAGTTGAGGCAGGC GGCGC GGC C-HvCBF9 KLA25 KLA45 GCCGACTCCGCC TGGCGGATGC Primer combinations used to amplify domain swap fragments of the indicated CBF genes. 22 fi'agment of HvCBF4 (C-HvCBF4), HvCBF 3 (C-HvCBF3), HvCBF6 (C-HvCBF6), and HvCBF9 (C-HvCBF9) resulting in the fusion constructs N-AtCBFl :C-HvCBF4, N- AtCBFl :HvCBF3, N-AtCBFl :HvCBF 6, and N-AtCBFl :HvCBF9 respectively. Similarly the N-terrninal portion of HvCBF 4 (N-HvCBF4), HvCBF 3 (N-HvCBF3), HvCBF6 (N -HvCBF6), and HvCBF 9 (N -HvCBF 9) were ligated to the COOH-tenninal end of AtCBFl (C-AtCBF 1) resulting in the constructs N-HvCBF4zC-AtCBF1, N- HvCBF3:C-AtCBF1, N-HvCBF6:C-AtCBF1, N-HvCBF9:C-AtCBF1 . These fusion constructs were amplified using Invitrogen HiFi Taq DNA Polymerase as described previously using the appropriate primers from Table 2.1. The amplified DNA products were isolated and ligated to pGEM-Teasy. Isolation and sequencing of fusion constructs in pGEM-Teasy was conducted as described above. PLANT GROWTH Wheat, T riticum aestivum L. vars ‘Norstar’ and ‘Manitou’ (seeds generously supplied by Dr. D.B. Fowler, University of Saskatchewan), and Rye, Secale cereale L. vars Puma and F lorida401 (contributed by Harold Bockelman, curator at the USDA- ARS, National Small Grains Research Facility, Aberdeen, ID), seeds were genninated and grown in Bacto High Porosity Professional Planting Mix (Michigan Peat, Houston, TX) in 8 x 8 cm pots. The seeds were germinated under controlled growth conditions in continuous fluorescent illumination of 150-200 uEm'zs'1 light intensity and 20-22° C. Pots containing 23-26 day old plants were moved to a chamber with controlled growth conditions of 4° C and continuous fluorescent illumination of 50 uEm'zs'l. At the times 23 indicated in the time course experiments (Figs 2.5, 2.6), leaf tissue was harvested and frozen immediately in liquid nitrogen and stored at -80°C until total RNA was extracted. A. thaliana ecotype Wassilewskija (WS-2) seeds were sterilized for 10 min in a bleach solution (10% Sodium Hypochlorite (v/v) and 0.01% Tween 20). The seeds were then rinsed twice with sterile H20. Approximately 100 seeds were plated to 10 cm petri plates containing 0.8% Phytagar (Invitrogen) and Gamborgs B-S medium with sucrose (Sigma, St. Louis, MO) and germinated in controlled growth chambers as described above. RNA ISOLATION, NORTHERN TRANSFERS AND HYBRIDIZATIONS Total RNA from Arabidopsis, wheat and rye plants was isolated using the Plant RNeasy Isolation Kit (Qiagen). Frozen tissue (200-300mg) was ground to a fine powder in liquid nitrogen then immediately transferred to a 1.5 ml eppendorf tube containing 1 ml RLT buffer (Qiagen). The rest of the RNA extraction procedure was completed as stated in the Qiagen Rneasy Mini Handbook. RNA samples were fractionated on a 1.5% agarose gel in the presence of formaldehyde at 100 volts for 90 min. Total RNA transfers were conducted as described by Stockinger et al. (1997). The EcoRI fragments from KLA152 (AtCBFI), KLA129 (HvCBF 4), KLA130 (HvCBF3), KLA131 (HvCBF 6), KLA157 (HvCBF9), KLA133 (Rye22), KLA134 (Rye24), KLA135 (Rye31) and KLA136 (TaCBF!) were isolated and 32p radiolabeled using the Random Primers DNA Labeling System (Invitrogen). The DNA probes for eIF4A, COR6. 6, COR15a, COR4 7, COR 78 and COR39 were described previously (J aglo- 24 Ottosen et al., 1998; Guo et al., 1992). Hybridizations and wash conditions were conducted as described in Jaglo et al. (2001). To prepare membranes for hybridization with a second probe, the initial probe was stripped from the membranes by washing the membranes in boiling 0.5% SDS solution until all measurable radioactivity was removed from the membrane. CONSTRUCTION OF PLANT EXPRESSION VECTORS The plasmid DNA ofKLA152, KLA129, KLA130, KLA131, KLA157, KLA133, KLA134, KLA135 and KLA136 and the translational fusion constructs described above was digested with the restriction enzymes XbaI and Bng (Roche, Basel, Switzerland). The fragment DNA was isolated and ligated to the XbaI and Bng sites of pGA643 (Fig 2.1; An, 1995) as previously described. The ligated products were transformed into E. coli DH5a as described previously. E. coli transformants were screened for the ability to grow on LB containing kanamycin (50ug/ml). The presence of the candidate CBF genes was verified by sequencing. These constructs were then transformed into Agrobacterium tumefaciens strain GV3101pMP90 via electroporation as described previously for E. coli transformations. A.tumefaciens harboring the pGA643 constructs were selected based on their ability to grow on LB containing kanamycin (50ug/ml) and gentamycin (50ug/ml). DEVELOPMENT OF TRAN SGENIC ARABIDOPSIS EXPRESSING CBF GENES 25 Hind III Xba I Bgl II BR nos-npt P358 3 BL Figure 2.2. Map of Binary Vector pGA643 Cartoon of the binary vector used for Agrobacterium mediated transformations of Arabidopsis. XbaI and BglII restriction sites were used for cloning. 26 A. thaliana ecotype WS-2 was transformed with the floral dip method as described by Clough and Bent (1998). Approximately 2000 seeds were screened from each construct for their ability to germinate and grow on Gamborgs-BS medium containing kanamycin (SOug/ml). Arabidopsis plants that grew in the presence of kanamycin were transplanted to Bacto soil and grown to maturity. Seeds were harvested from each line separately. Approximately 100 second generation seeds were then screened again with kanamycin to determine segregation of kanamycin resistance. Seed from transgenic Arabidopsis lines segregating 3:1 were plated to Gamborg-BS 0.8% phytagar in 10 cm petri plates and grown for 13 to 15 days. The plant tissue was then harvested and frozen in liquid nitrogen until RNA was extracted as described previously. WHOLE PLANT FREEZE TEST The whole plant freeze tests were generously performed by Jonathan Vogel (Michigan State University, East Lansing, Michigan). The freeze test was conducted, as described by Vlachonasios et al. (2003), on acclimated and non-acclimated wild type Arabidopsis plants and non-acclimated HvCBF3 expressing plants. HYDROPHOBIC CLUSTER ANALYSIS Hydrophobic cluster information was generated using the drawhca program (http://smi.snv.jussieu.fr/hca/hca-seq.html) on the amino acid sequences of AtCBFl, AtCBF2, AtCBF 3, HvCBF4, HvCBF3, HvCBF6 and HvCBF9. 27 RECOMBINANT PROTEIN EXPRESSION AND EXTRACTION The XbaI and Bng HvCBF 3 and HvCBF6 (from KLA130 and KLA131 respectively) fragments were cloned into the NheI and BamHI restriction sites of pET28a (+) expression vector (N ovagen, Madison, WI). This resulted in a translational fusion of a histidine tag to the N-terminus of HvCBF3 and HvCBF 6 under control of the T7 phage promoter. The construct was transformed into the E. coli host BL21 (DE3) codon+ (Stratagene) and expressed as recommended by the pET-28a vector supplier (Novagen). Total protein extracts were prepared as described by Stockinger et a1. (1997) and the protein samples were quantified using the Bradford assay. WESTERN BLOT ANALYSIS Total soluble protein extracts were fractionated on a 10% tricine SDS polyacrylamide gel (Schiigger and von Jagow, 1987) and transferred to a 0.2 micron nitrocellulose membrane as described by Towbin et al. (1979). The His Tag Monoclonal Antibody (N ovagen) directed against the histidine tag sequence was used as the primary antibody to detect recombinant CBF protein. The ECL+Plus Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ) were used to visualize expression of the recombinant protein as recommended by Amersharn Biosciences. ELECTROPHORETIC MOBILITY SHIFT ASSAYS 28 Electrophoretic mobility shift assays were conducted as described by Stockinger et al. (1997) with the following modifications. Complementary single stranded Oligonucleotides of the 23bp DNA fragment fiom the Arababidopsis COR15a promoter containing a single core CRT/DRE sequence and mutated versions of this sequence (Table 2.3) were annealed using a step down PCR protocol [94° C for 30 seconds, -0.5° C for each cycle. Temperature was reduced until a final temperature of 20° C was reached, at which time the temperature was rapidly reduced to 4° C]. The annealed wild type 23bp fragment was 32P labeled using T4 Polynucleotide Kinase (New England Biolabs) as described by Sarnbrook et al. (1989). RESULTS SUBFAMILY OF SIGNATURE SEQUENCE DOMAIN CONTAINING AP2/EREBP PROTEINS Amino acid sequences containing the CBF signature sequence form a distinct subfamily within the AP2/EREBP superfamily. An unrooted phylogenetic tree of 245 AP2/EREBP domain-containing proteins was generated using Clustal vs. 1.8 (Fig 2.3). The AP2/EREBP proteins that contain the CBF family signature sequence are indicated with the bold branch lines. The signature sequence containing proteins can be traced to a single node and they group together within this family of AP2/EREBP proteins; the subgroup is labeled CBF Family. 29 Table 2.3 Oligonucleotides used in EMSA experiments Oligonucleotide CRT/DRE Seflence MTSO COR15a gat CATTTCATGGCCGACCTGCTTTTT MTSZ M1 COR15a CACAATTTCAaGaat t caCTGCTTTTT‘I‘ MT80 M2CORI5a gat cATTTCATGGt atgt CTGCTTTTT MT 1 25 M3 COR 1 5a 9 at CATTTCATGGaat caCTGCTTTTT Oligonucleotides encoding wild-type (COR15a) or mutant (M1COR15a, M2CORI5a, M3COR15a) CRT/DRE sequences. Nucleotides present in wild-type CRT/DRE sequences are in uppercase letters. The core CRT/DRE sequence is in bold type. Lowercase letters at the beginning of a sequence indicate bases added to facilitate cloning. The underlined lowercase letters indicate the nucleotides mutated in MI -3 COR15a. 30 0.1 CBF Family Figure 2.3. “Signature sequence” containing proteins form a distinct subfamily within the AP2/EREBP gene family. Unrooted neighbor-joining tree of 244 unique AP2/EREBP domain proteins. Branch lines representing sequences containing the CBF Family “signature sequences”, PKK/RPAGRXKFXETRHP and DSAWR, are indicated by the label CBF Family and bold branch lines. 31 Amino acid sequence comparisons were made between Arabidopsis AtCBFl and HvCBF4, HvCBF3, HvCBF6, HvCBF 9, Rye22, Rye24, Rye31 or TaCBFl. The percent identity ranged from 32 to 40 for the full length sequences whereas in the AP2 domain the identity ranged from 67 to 77 percent (data not shown). An alignment of the AP2/EREBP DNA binding domain sequence region including the flanking signature sequences demonstrates a high degree of similarity within this sequence region across plant species (see fig 2.4). CBF AND COR GENE TRANSCRIPTS ACCUMULATE IN WHEAT AND RYE IN RESPONSE TO LOW TEMPERATURE In T riticum aestivum L vars. Manitou and Norstar, induction of COR39 transcripts in response to low temperature followed an expression pattern similar to that previously seen by Fowler et al. (1996). COR39 transcripts accumulated when the wheat plants were placed at 4° C for 12 hrs (see fig 2.5). A maximum level of COR39 transcript was reached between 1 and 7 days. In the winter variety Norstar the increased level of transcript was maintained for at least 49 days in plants kept at 4° C. Similarly in the spring variety Manitou an increase in COR39 mRNA accumulation was noted within 12 hours after the plants were placed at low temperature. However, after 7 days at 4° C the level of COR39 transcript was reduced to near the levels at the 0 hour time point. In both wheat varieties the candidate CBF genes, Rye22 and T aCBF I, were induced within 12 hours after exposure to 4° C. The Rye22 probe is most probably cross hybridizing with a related wheat CBF gene. The expression patterns of Rye22 and T aCBF I are different 32 .Ezsamca Exoutk .3. ”$8.86 33.8% .om ”name: GREEN :5 25833 93:06.2 JZ €552: flamohficxw J< SEE. EDD .mO SSMB; E3638: $3 360% :83 ofio>€ Ho confine a Sow gain can mahmx§é0<§m .moocoscom 2:3:me big mmo maniac EH Ems—ow mEHEE <75 mmmMmCNm/w 05 mo Eofiqwzm 85:68 Eon oEE< 6.589:— mmmmnénnz we 3:3.— mmU 05 5.5.: amass—com enigma: 2: he Staten—SO .v.~ 0.5“:— H «mom m3¢m0x x. _m>omw mmmemum mmmo->m m3¢moam2qo<.- .9 He . ommm<<__mmu swam m.m>wmw mxmewgmx Humo-m9 - -oaq. .momamae _mmo .oqm Lm>wmw mmmemflmuflm. cxHH mmo-om - -omqa. ..nm4mm¢1mmmmnmewq ImoxH m .o>m m.m>omw mmmem:mx ammo->m - -umqe ..oxm m.m>wm» mxmamlmx mm uxHH mmo-om - -omq. .xomamaaflm «Bream; imoxH oo>m m._ _m>wm> mmmemlmxamo. oxHH mmu-om - rogue .. omom» Amman. examo cxHH mmo-mo - -o.q« .. anam<<__mma.mauq H -Iexx -om_mmmHomHHmmmamm Humo-wq m- -ogqm .. omamamxmze.meoq H -Imxx -om.mo :>omemmme «mmo-u¢ m- -wLa.H.. omom»Hm:m9WW mmmo-u< mzmmoamzao.m- lugq. ..}nmem>memem mmmu-u< - -waq. .. omamm<_ .msoq H -Iexx -om_mo m>omemmmsm Humolua - -o.a. .. enema m¢e.mewq H -Imxx -omxm m>wm>Hmmmem cxHH mmo-em - to.q. .. exam mma.mewq H -Imxx -omxm m>omHHmmmem. oxHH www-cm - -o.q. .. enema m<9.meoa H -lmxx -omxm m>omemmmam memurem m-x-w;q.H.. ommmo-:m x «m94m2qo.m-m-o.H.H.. om<-uz x «momazqo.m-m-o;H.H.. ommmad:m.mmmaoq H llmox tom dHHH m-Hu\mu>¢-uz m-m-o.q. . omamdmc. a. eon -lmomHmmm>mo>z.-ooem. m>omw oxHH mmo-u¢ mzmo>3.-oo.mfimfiomw} mmmam.m Emo.. emu cxHH mmo-o< Hmmmoumzqma.-mmo.q a. H.. amam¢<:mme. Boa H I-mo .moo.x m>omw} mmmamamxvmo..gxm. oxHH m-uo\mu><-mo Mmmmommzqom.rm- o. .. ommmamxmmemmeoq H --oxm mmx>mo .-wm_m.m>w. Hmmu->m 8m 9m cacao 9.65m <20 ammmmanz 8533 932mm 33 Manitou Norstar 12 h 1d 2d 7d 14d 28d 49d 77d 112d 0 h 12 h 1d 2d 7d 14d 28d 49d 77d 1 12d TaCBF] .. l _—10h 1 Rye22 a... WCSIZO/COR39 Figure 2.5. Northern hybridization of T riticum aestivum L. vars Manitou and Norstar Exposed to 4° C Northern hybridization of spring wheat variety Manitou and winter wheat variety Norstar. Northern transfers of total RNA from wheat plants grown at 4° C for 0, 12 h, l, 2, 7, 14, 28, 49, 77, or 112 d. Membranes were hybridized with full length T aCBF I , Rye22 or COR39 as described in Materials and Methods. 34 Florida 401 Puma .: W“: 5.54 :qmsacnva OOO—NVOON— u-.-“ vi Rye22 CBF COR39 Rye22 CBF COR39 Figure 2.6. Northern hybridization of Secale cereale L. vars Florida401 and Puma grown at 4° C RNA was extracted from Secale cereale L. vars Florida401 and Puma grown at 4° C for 0, 0.25, 0.5, l, 2, 4, 8, 24 hrs and 1 wk. Northern transfers were as described. The membranes were hybridized with Rye22 or Cor39 as described in Materials and Methods. 35 suggesting that Rye22 and TaCBF] probes are hybridizing to different wheat CBF genes. Multiple bands are observed when COR39 is used as probe; this is presumably due to the COR39 probe cross hybridizing with other members of the wheat COR gene family. The COR39 probe cross hybridized with putative target COR transcripts from Secale cereale L. vars. Puma and F lorida401 (Fig 2.6) in a low temperature induced pattern that mimicked the induction of the COR genes in Arabidopsis (Gilrnour et a1., 1998). Both of the rye lines exhibited increased levels of COR39 transcript beginning 2 and 4 hours following exposure to 4° C, and transcript levels remained high in the plants through 1 week of exposure to 4° C. The candidate CBF gene, Rye22, was expressed in a similar pattern to that observed for the Arabidopsis low temperature responsive CBF genes (Gilrnour et a1., 1998). An increase in Rye22 transcript was observed within 15 minutes of exposure to 4° C and it reached a maximum level between 2 and 4 hrs after the cold treatment. ARABIDOPSIS COR GENE EXPRESSION IS INDUCED BY HvCBF3 AND HvCBF6 IN TRANSGENIC PLANTS Second generation transgenic Arabidopsis WS-2 plant lines constitutively expressing candidate cereal CBF genes were generated. In order to test if the cereal CBF genes have an affect on the endogenous COR genes in vivo, Northern blot hybridizations were performed on the non-acclimated transgenic plants testing expression levels of the candidate cereal CBF genes and Arabidopsis COR genes. Two transgenic plant lines expressing each candidate CBF gene were chosen for subsequent Northern hybridization 36 Probes COR 6.6 COR l5a COR 47 COR 78 CBF I HvCBF 4 HvCBF 3 HvCBF 6 Rye22 Rye24 Rye3 I 288 Transgenic Arabidopsis Lines U QEEEEE‘L’. v——~"‘"‘ r mmmmmmaaaanaaa 4: OOUOUQoooaxaoo v >>>>> >~>~>~mg¢gfiam _§ mmmmmmatrzmvmvt—r— 99- fl'I'TINIC":Nl‘rlmr"’IV’ITIHITI”’IV’I 08 cemmoooooo—n—v—v—NN P mmmmmmvvsrsrmtnvv >> v-fiv—II—tv—v—Iv—Iv—uv—flv—ivI—lv-flv—iv—iv-I . .‘ . " I. O - on .. ‘0 I. .-99 Figure 2.7. Northern hybridization of non-acclimated transgenic Arabidopsis plants expressing candidate cereal CBF genes Total RNA was isolated from transgenic Arabidopsis plants constitutively expressing HvCBF 4 (155_2, 155_9), HvCBF 3 (150_4, 150_7), HvCBF 6 (l38_2, 138_4), Rye22 (140_2, 140_5), Rye24 (l41_5, l41_l4), Rye31 (151_13, 151_l4) or TaCBF] (142_4, 142_5) candidate cereal CBF genes under control of the CaMV 35$ promoter. Northern 37 Figure 2.7 cont. hybridizations were conducted as described and the membranes were hybridized with COR6. 6, COR15a, COR4 7, COR 78, AtCBF], HvCBF 4, HvCBF 3, HvCBF 6, Rye22, Rye24 and Rye31. Rye3I and T aCBF 1 are both detected with the Rye3l probe as they are over 80% identical at the nucleotide level. The plasmid pGA643 was transformed into Arabidopsis as a control (vector). The ethidium bromide stained upper ribosomal band is labeled 288. 38 experiments. The RNA blot was hybridized with COR6. 6, COR15a, COR4 7, COR 78, AtCBF] , HvCBF4, HvCBF 3, HvCBF 6, Rye22, Rye24, and Rye31 (Fig 2.7). An increase in COR transcripts was observed in wild type WS-2 plants transformed with the pGA643 vector without a CBF gene insert grown at 4° C for 24 hrs and the transgenic lines expressing HvCBF3 and HvCBF6 (150_4, 150_7, 138_2, and 138_4 respectively) compared to non-acclimated WS-2 plants. Accumulation of the Arabidopsis COR gene transcripts was not observed in the transgenic Arabidopsis plants expressing HvCBF 4, Rye22, Rye24, Rye31, or T aCBF I. WHOLE PLANT FREEZE TEST A whole plant freeze test was conducted on two non-acclimated transgenic Arabidopsis lines expressing HvCBF 3 (KLA150_2 and KLA150_7) and on acclimated and non-acclimated wild type Arabidopsis plants (Fig 2.8). Wild type cold acclimated Arabidopsis plants survived freezing at -5° C, while non-acclimated Arabidopsis plants were killed as expected. Transgenic Arabidopsis lines expressing HvCBF 3 (KLA150_2 and 150_7) survived being frozen to -5° C, suggesting that the expression of HvCBF3 in Arabidopsis is able to activate cold acclimation pathways even in the absence of a cold stimulus. CEREAL CBF GENE FAMILY IS COMPOSED OF TWO GROUPS 39 Figure 2.8. Whole plant freeze test of HvCBF3-overexpressing Arabidopsis plants Whole plant freeze tests were conducted on wild type non-acclimated (WT), acclimated wild type (WT Acc) and HvCBF3 expressing (150_2 and 150_7) Arabidopsis plants. Survival was assessed after a recovery period from being frozen to -5° C. This image is presented in color. 40 An unrooted phylogenetic tree was generated by comparing the amino acid sequence of eight members of the CBF gene family (Fig 2.9). The four Arabidopsis CBF genes, AtCBF 1, AtCBF2, AtCBF 3 and AtCBF 4, formed one cluster. The candidate cereal CBF genes Rye22, Rye24, Rye31, T aCBF I, HvCBF 4, and HvCBF 9 formed a second cluster. HvCBF 3 and HvCBF 6 did not fall within either of these first two groups and therefore formed a third cluster. The amino acid sequences of AtCBF], AtCBF2, AtCBF3, AtCBF4, HvCBF3, HvCBF4, HvCBF6, HvCBF9, Rye22, Rye24, Rye3l and TaCBFl were aligned (Fig 2.10). There are 6 amino acids conserved in all 12 sequences compared as shown with the white font highlighted in black. A number of identical or similar amino acids (highlighted in grey) are conserved only within one group or the other (of CBF genes that induce expression of the Arabidopsis COR genes and those that do not) and not in both groups. The differences in the conservation of these amino acids are consistent with the divergence of HvCBF3 and HvCBF 6 from HvCBF 4, HvCBF 9, Rye22, Rye24, Rye3l, and TaCBFl (Fig 2.9). Hydrophobic cluster analysis revealed hydrophobic regions that were conserved within the activation domain of the Arabidopsis CBF proteins (Fig 2.11). These clusters are not present in any of the cereal CBF proteins. EXPRESSION OF CEREAL CBF DNA BINDING DOMAIN F USED TO ARABIDOPSIS ACTIVATION DOMAIN INDUCES EDRESSION OF COR GENES 41 HvCBF9 HvCBF 4 Rye24 TaCBFl Rye” Rye3 l AtCBF4 HvCBF6 AtCBF3 AtCBFl AtCBF2 0-1 HvCBF3 Figure 2.9. Phylogeny of members of the CBF gene family An unrooted tree was generated as described in Materials and Methods by comparing the amino acid sequences of AtCBFl, AtCBF 2, AtCBF 3, AtCBF4, TaCBF l, Rye22, Rye24, Rye31, HvCBF4, HvCBF3, HvCBF 6, and HvCBF 9. 42 8:85 .wecnwznwfl 3% fine 28 “so.“ BE? a .23 82585 0.8 80538 5m “mac— 3 E 338:8 man 05:8 33:53 183 E BEE—Emma :8.“ 233 a 53, @8583 0.3 826395 2: mo NH =~ E 323:8 36a o£E< .voewzm 203 9303* can .cmmO>m .959: .359: .Emofi .3.on .3on .NNQHH .Emoé .252 .252. .382. Co as :08 2: 9:85 85:8” magma H=E£ mmO §-- reHea--ma- wxzreHH ..... a.mq mmmZmonH ...... owzommam-mwemmHomoaemmam-- vmmuua ----n m>- o anon-->nm._mz. o>m ..... a ma wqqrazmmz--m oomm-->om. .Hz .o>m ..... ammq QHHrmonzammaz----Hm4 ...... no 0 ...... rmHHHHHHHHmmza-- «amoua ..... m>--. oomm--unH .mz o>m-----n .mH QHHHmzomszmaz----Hm¢------omuo------ HHHH4m>zemmza-- Humou< --mo f.-> cam---¢. Hoeg..¢¢qm¢--a 4:. , -HazozanHQHmm ................. mm- 0 4m zo>mr -->.¢ o---oo omH mHHH>mommm rzamammaoemeemomommmmqm-- mmmo>m ..... z. J--HH>¢mH--mo.a_m.e.¢----mm m: . o ._m:-----o -----.m Ema---- .mme ----mwoemH--mo o m. 4----.m m: m mm-----o ----- .m HmH---- mam ----meo>o-- HmmHm onmqm.w _Hmem>wm---m m .mm m----ea_¢> o o.m--on ..... mHHm---m¢¢m ----m<-m¢-- ammo>m --HHm>m zaeommmmo .Hm, m----gm_¢>. o m.m--on ..... mszmmaqu<> HHH0000H¢>> mmmHm -oaHo. m--Ho>em---o mm a----ga.¢> e o m--aHm ..... mZHmmmaqoea HHHoxeoqa>> ammHm -zaqor. _--Ho>oe---o m mm..m----gm.<>. : o. o m--oqm . ..... r:.mZHmmmaqo¢>:HHHoomoq<>> mmmo>m .................................... deex-momeeem-------------------z-il . m.<.o.zgm---oeemiH ---- ammou< .................................... Homa-aeaoozm-------------------o . .e.o,ox_---oe m H ---- mamoua .................................... Hem _m> oH_mHmm¢< > ---- mmmu>m .................................... mo¢qo--mH -.-u mmmu>m ..................................... Hmoomzmmem>>mm.--------------- mH KH m mm .-4 Hr Hmmume ..................................... amenaemmem>mmms------------l-- m> K> m mm -< He HmmHm ...................................... m-mmau<,mHHHo -o-------------- > H.m em as. m ammu>m <><>m>mqooq¢>>¢><>m> ................ oxoxH¢>>¢.¢>m>o 00H<>>¢><>mHHox. H "MW a e on ;-4 H“ mmch ¢><>m>oqxoq<>><>e>m> ................ omooq¢>>¢.¢>m>o M0H¢>>¢>¢>HHH0H > a pm -< Hr emmHm ¢>¢>HHoqooqa>><>¢>m>ooooqam><><>m>oooxooqa>>¢.4>m>o H0H¢>>4>a>mHo¢m«mH. .> w on L-< -yHr mmmo>m 43 Figure 2.10 cont. amino acids are shown with a black font on a grey background. Gaps introduced to optimize the alignment of the sequences are shown as dashes. 44 AtCBF1 ml 010 A {ED A MEW— I D E? q t 2 F m A nv AtCBF3 HvCBF3 HvCBF6 HvCBF4 f hydrophobic clusters within the C-terminus of ion 0 ll Conservat Figure 2. members of the CBF gene family. 45 Figure 2.11 cont. Hydrophobic cluster analysis of the amino acid sequence regions downstream of the AP2 DNA binding domain starting with the conserved DSAWR CBF Family Signature sequence of AtCBF 1, AtCBF2, AtCBF3, HvCBF3, HvCBF 6, HvCBF9, and HvCBF4. The amino acid sequences are in duplicate in a downward diagonal and the amino acids are indicated by the single letter amino acid code with the exception that stars indicate proline, diamonds indicate glycine, squares indicate threonine and squares with dots indicate serine. Hydrophobic clusters are indicated by contour lines. The underbars indicate regions of conserved hydrophobic clusters between AtCBFl , AtCBF 2 and AtCBF3. 46 A. thaliana ecotype WS-2 plants were transformed with empty vector (pGA643) or pGA643 harboring AtCBF] , HvCBF 4, HvCBF 3, HvCBF 6, HvCBF 9, N-AtCBF1:C- HvCBF 4, N-AtCBFI :HvCBF3, N-A tCBFI :HvCBF 6, N-AtCBFI :HvCBF9, N-HvCBF 4 : C - AtCBFI, N—HvCBF 3: C—AtCBFI , N-HvCBF 6: C-AtCBFI , or N-HvCBF 9: C—AtCBFI . Northern blot hybridizations using COR15a or a pooled barley HvCBF probe (containing radiolabelled HvCBF 4, HvCBF 3, HvCBF 6, and HvCBF 9) were performed (Fig 2.12) to test if the domain swap constructs influenced the expression of Arabidopsis COR genes in vivo. The ethidium bromide stained ribosomal 288 band indicated that non-acclimated and cold acclimated wild type, AtCBF], and N-HvCBF9:C—AtCBFI lines were under loaded compared to the remaining samples. The HvCBF probe indicated expression of wild type HvCBF 4, HvCBF3, HvCBF 6, and HvCBF 9 and the domain swap fusion constructs N-AtCBFI :HvCBF3, N-AtCBF I :HvCBF 6, N-AtCBFI :HvCBF9, N-HvCBF 4 :C- AtCBF] , N-HvCBF3zC-AICBFI and N-HvCBF6zC-AtCBFI . Expression of N- HvCBF9zC-AtCBFI or N-AtCBF I :C—HvCBF 4 was not detected with the pooled HvCBF probe. Two exposures of COR15a hybridizations were presented and indicated by COR15a and COR15a *. Transcripts of COR15a were detected in cold acclimated wild type Arabidopsis plants. HvCBF 3 and HvCBF 6 transgenic Arabidopsis lines have increased COR15a transcript levels. COR15a mRNA was detected in the constructs N- HvCBF4zC-AtCBF1, N-HvCBF3:C-AtCBF1 and N—HvCBF 6 :C—AtCBF I . Elevated COR15a transcript levels were only observed in N-AtCBF I :C—HvCBF 3 or N-AtCBFI :C- HvCBF 6 lines. This experiment is evidence suggesting that the domains N-HvCBF 3, C- HvCBF 3, N-HvCBF 6, C-HvCBF 6, and N-HvCBF 6 are function in Arabidopsis. 47 Arabidopsis CBF DNA Binding Domain Fused to Barley CBF Activation Domain 3 L: Barley CBF DNA Fused to Arabidopsis CBF Activation Domain 93 8 Binding Domain Wild Type CBF é mum0>I- 0. EmOE-Z oumO>I- 0. EmOZ-Z - - a a $502.70“ Emma/$2 vum0>I- 0. 5502.. Z a [mug-0. mumO>I- Z EmOE-O. mumO>I- Z % EmO.<-Onm“_m0>I-Z é Emma/H70. vmm0>I- Z mmm0>I $9"!" #V” «(-mu. >I am. um >I l mumO>I Ho<-wn_mO>I Dm-cumngI mum0>I illL Em0>I aoz oméumoi ll Emoz. Ho<- [mug om- Emu? Proteins on u. m 0 > I 0000.00.00.00.- oafi'ns‘t. new §8> new 58> .oHoo> HoHom> . V o 28S RNA COR 15a COR15a * HvCBF Figure 2.12 Northern hybridizations of non-acclimated transgenic Arabidopsis 48 expressing CBF gene fusion products Figure 2.12 cont. Each lane represents independent total RNA isolated fi'om combined Arabidopsis plants that grew in the presence of kanamycin. Vector controls are plants transformed with pGA643 with no CBF insert. Vector 24h plants were acclimated at 4° C for 24 hrs. COR15a and a pooled HvCBF probe were hybridized to the membrane. CORISa“ represents a longer phosphorimager exposure of COR15a. HvCBF is a pooled probe of HvCBF 3, HvCBFf4, HvCBF 6 and HvCBF9. The ethidium bromide-stained upper ribosomal band is marked with 28S. CBF-BD represents the N-terminal portion of the CBF proteins to the end of the AP2/EREBP DNA binding domains. CBF-Act represents everything from the end of the AP2/EREBP DNA binding domains to the C-terminal end of the CBF proteins. This image is presented in color. 49 M M 2% 5225:8399 aeaeeaame 2 a “-12 2 E :E E "3 kDa .7 I ___, 95.5 55 43 36 29.2 18.4 12.5 Figure 2.13. Expression of recombinant CBF proteins in E.coli Total protein extracts isolated from BL21(DE3) codon+ Esherichia coli were fragmented on a 10% tricine SDS PAGE gel and stained with Coomassie brilliant blue. Total E. coli protein extracts were taken from E. coli harboring pET28a, or pET28a with AtCBF] , HvCBF 3 or antiHvCBF 3 inserts. 50 RECOMBINANT HvCBF 3 AND HvCBF6 PROTEINS BIND TO THE ARABIDOPSIS CRT/DRE ELEMENT Electrophoretic mobility shift assays were performed in order to determine if HvCBF3 and HvCBF 6 proteins are able to bind to a CRT/DRE sequence. Total protein extracts were prepared separately from E. coli host BL21 (DE3) codon+ harboring the expression cassettes AtCBFl, HvCBF3, or antiHvCBF3 (HvCBF3 cloned into the opposite orientation) in pET28a (+) or pET28a alone. Replicate protein extracts (100 ng) were fractionated in a 10% tricine SDS polyacrylamide gel. Gels of the replicate samples were either stained with coomassie blue (Fig 2.13) or transferred to a 0.2 micron nitrocellulose membrane. The His Tag Monoclonal Antibody (Novagen) directed against the histidine tag sequence was then used to detect the 6 His residues translationally fused to the amino terminus of HvCBF 3. The lanes containing protein extracts from AtCBFl and from HvCBF3 have a band present between the 29.2 and the 36 kD markers in both the coomassie stained gel and the western blot analysis (Fig 2.13 and 2.14). This band falls within the expected size range of the recombinant CBF genes and is not present in either the pET28a or the antiHvCBF 3 lanes (Figs 2.13 and 2.14). Similar results were obtained fi'om E. coli expressing HvCBF 6 recombinant protein in pET28a (data not shown). Attempts at expressing recombinant protein extracts of the remaining candidate cereal CBF genes failed and so no protein extracts were isolated to assess the binding activity of these additional candidate CBF genes (see Appendix 1). 51 MM as a£§§§§99 559999§§ kDa HH<3 HO >0 0 > 2 <> :I‘.> > I :12 {I} :1: Competitor - - - - - CRTMIMZ M3 Bound _ Probe Free _ Probe Figure 2.15. Electrophoretic mobility shift assay of HvCBF3 binding to an Arabidopsis CRT/DRE DNA element Total soluble proteins (40mg) isolated from E. coli expressing AtCBF 1 , HvCBF 3, antiHvCBF3 or empty vector were incubated with a P32 labelled CRT element (see Table 2.3). The protein samples were additionally incubated separately with or without the presence of the specific competitor DNAs, CRT, M1, M2 or M3 (see Table 2.3) as indicated. 53 _m~o~oo~o~o mmmmmmm . -ameaeeee Protein 99> >> >> >