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V 1 III.“ 90H3Uu3§vfi~¢z Isl (41:11. $.35 [iiia’llt . f :vrlloi‘il‘ ”fvrr... :53; . dun? xii... 1!. 5 {dilignnehwfiug . z . , 5 r I rdnfifiuz... "Swim? I r1235 5.. . ‘ Y'r”f—ll’ .I ..Il:fv'!ff 4300...}..21152.‘ «I (ll. ll!!!(¢¢flli tryiz .! . . Ill» ‘rn . cut... I.) . V v II «(.1 Irv....‘11. 27.x ;o.,IY.t.V:I.'l .01. :3 . . a!...-.x _ ., 3.21.21... .1 ‘ IIIIII IIIIIIIIIIIIIIII III IIIIIIIIIIIIIIIII II This is to certify that the \ dissertation entitled DEVELOPMENTAL AND SALICYLIC ACID REGULATED EXPRESSION OF THE ALTERNATIVE OXIDASE OF HIGHER PLANTS presented by David Michael Rhoads has been accepted towards fulfillment of the requirements for Ph.D. degree in Biochemistry Date 9éf/72 MSU Lt an Affirmative Action/Equal Opportunity Institution 0-12771 'OM 11.25252531' Hichigan State I University \__ “‘1 fl PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or bdora date duo. r———————_—__——__—__———-1I—_—_——_———— DATE DUE DATE DUE DATE DUE W II I " I—’__I ——II ‘TTTTTI MSU In An Affirmative Action/Equal Opportunity Institution cmmn' DEVEIDPMENTAL AND SALICYLIC ACID REGULATED EXPRESSION OF THE ALTERNATIVE OXIDASE OF HIGHER PLANTS By David Michael Rhoads A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1992 ABSTRACT DEVHDPMENTAL AND SAUCYLIC ACID REGULATED EXPRESSION OF THE ALTERNATIVE OXIDASE OF HIGHER PLANTS BY David Michael Rhoads Alternative respiration was described sixty years ago as respiration that is not sensitive to cyanide. But, to this day, the exact nature and function of alternative respiration remains a mystery. Research in just the last 10 years has provided a compelling argument that a terminal oxidase protein is involved. The flow of electrons through the alternative pathway is much less energy conserving than the electron flow through the cytochrome pathway. The purpose for such a potentially "wasteful" pathway is a source of great controversy, which will only be settled by future experimentation. Molecular-genetic analyses may provide new ways to study the function of the alternative pathway in plant cell metabolism. Chapter 2 of this thesis describes the first isolation and characterization of a cDNA clone encoding an alternative oxidase protein. The sequence of this cDNA has provided insight into the possrble structure of the protein that it encodes. It has also provided a molecular probe with which to isolate genes from other Species and investigate the regulation of expression of alternative oxidase genes. The results presented in Chapter 3 show that the developmental and salicylic-acid- directed increases in alternative pathway capacity are accompanied by the accumulation of alternative oxidase proteins and transcripts in the appendix tissue of Sauromatum guttatum Schott (an aroid plant that uses the heat generated by the "lost" energy of alternative respiration to volatilize putrid compounds that attract insect pollinators). Chapter 4 describes the isolation and analysis of a genomic clone that corresponds to the cDNA and, therefore, encodes the putative alternative oxidase precursor protein of S. guttatum. A highly conserved region of the gene, which encodes a domain of the protein that is predicted to embed the mature protein in the inner mitochondrial membrane, is contained in one of the four exons. These data suggest that this region of the protein is functionally important and was established early in its evolution. The promoter region of this gene is of particular interest since the promoter region of other salicylic acid- "responsive" genes have been analyzed. Chapter 5 shows the changes in alternative oxidase expression and alternative pathway and cytochrome pathway capacities in suspension cultured tobacco cells at various stages after subculturing and following the addition of salicylic acid. To Neera and My Parents, for their love and support. AWOWLEDGEMENTS I thank my mentor, Dr. Lee McIntosh, for the support and guidance he has given me over the laSt five years. Although I did not always understand his ways, I believe he taught me more about being a top scientist and running a lab than most others could have. He will always have my respect and deep gratitude. I greatly appreciate the help and guidance I received from each of the individuals who served on my advisory committee: Dr. Andrew Hanson; Dr. Zachery Burton; Dr. Mike Thomashow; Dr. John Wilson; and Dr. N. Edward Tolbert. I thank those I consider to be my honorary guidance committee members because of all the support and guidance that each has given me: Dr. David McConnell; Dr. Hans Kende; and, especially, Dr. Thomas Elthon. I thank all the past and present members of the Plant Research Laboratory and the Biochemistry Department who have helped me with their assistance in administrative and personal matters. I thank my colleagues who provided technical advice and friendship: especially Dr. John Sherwood, Dr. Thea Wilkins, Dr. Mike Mansfield, Dr. David Hondred, Dr. John Scott-Craig, Dr. John Shanklin, Dr. Thomas Newman; Dr. Ellen Johnson, Dr. Timothy Casper, Dr. Thomas Johnson, Dr. Arun Goyal, John Jacobs, Crispin Taylor, Todd Black, Frank van de Loo, Sharon Thoma, Dr. Robert Meeley, Dr. Christoph Benning and Susanne Hoffmann, Therese Best, Kristen Rorer, Dr. Linda Briggs, Lydia Herdies, Dr. Greg Vanlerberghe, Dr. Idah Sithole, Dr. Neil Bowlby, Dr. Carrie Hiser, and Roxy Nickels. I am especially thankful to Dr. John Fitchen, Vladimir Orbovic, Dr. Ellen Kearns, Dr. Ursula Hect, Dr. Beate Drechsler-Thielmann, Dr. Shawn Anderson and Denny Gerber, Larry Smart and Chris Durban, Dr. David Lerner, Harley Seeley, and Kurt Stepnitz for their close friendship. TABLE OF (”NTEN'I‘S PAGE LIST OF FIGURES ............................................ xi LIST OF ABBREVIATIONS .................................... xiii CHAPTER 1: INTRODUCTION .................................. 1 Characterization of the alternative respiratory pathway .............. 1 Partitioning of electrons between pathways ....................... 3 Alternative pathway in aroids ................................. 7 Alternative pathways in other organisms ......................... 9 Salicylic acid in aroids ..................................... 12 Salicylic acid in other organisms .............................. 13 References ............................................. 15 CHAPTER 2: ISOLATION AND CHARACTERIZATION OF A cDNA CLONE ENCODING AN ALTERNATIVE OXIDASE PROTEIN OF SAUROMATW GUT'I’ATUM (SCI-101T) . . 25 Abstract ............................................... 26 Introduction ............................................. 27 Materials and Methods .................................... 29 Plant material ...................................... 29 Isolation of mitochondria .............................. 29 Antisera .......................................... 29 Gel electrophoresis, immunoblotting and fluorography .................................... 30 Protein sequencing .................................. 30 Poly A+ RNA isolation ............................... 30 In vitro translation of poly A+ RNA and immunoprecipitation .............................. 31 Isolation of cDNA clones .............................. 31 Hybrid selection .................................... 32 vi Expression of phagemid inserts in E. coli .................. 32 DNA sequencing and analysis .......................... 33 Results ................................................ 33 In vitro translation of total poly A+ RNA and immunoprecipitation .............................. 33 Isolation of cDNA clones .............................. 34 In vitro translation and immunoprecipitation of hybrid selected RNA ............................... 34 Expression of plasmid inserts in E. coli ................... 37 DNA sequence analysis ............................... 40 Discussion .............................................. 41 Acknowledgements ....................................... 49 References ............................................. 49 CHAPTER 3: SALICYLIC ACID REGULATION OF RESPIRATION IN HIGHER PLANTS: ALTERNATIVE OXIDASE EXPRESSION .................................... 53 Abstract ............................................... 54 Introduction ............................................. 55 Materials and Methods .................................... 57 Plant material ...................................... 57 SA and inhibitor treatment of tissue sections ............... 58 Isolation of mitochondria and respiration assays ............. 59 Gel electrophoresis, immunoblotting and antisera ............ 60 Plasmid insert isolation and radiolabeling .................. 60 RNA isolation and blots .............................. 6O Isolation of nuclei and in vitro transcription ................ 61 Isolation of in vitro synthesized RNA ..................... 62 Hybridization of in vitro synthesized RNA to DNA probes ..... 63 Results ................................................ 64 Developmental expression of the alternative oxidase gene of voodoo lily .................................. 64 vii Time course of salicylic acid induced alternative oxidase expression .......................... 67 Inhibition of the effects of applied salicylic acid ............. 70 Nuclear in vitro transcription ........................... 73 Discussion .............................................. 78 Acknowledgements ....................................... 83 References ........... b .................................. 83 CHAPTER 4 ISOLATION AND CHARACTERIZATION OF A GENOMIC CLONE CONTAINING THE SAUROMATW GUTI‘ATUM ALTERNATIVE OXIDASE GENE AOXI ..... 87 Footnotes .............................................. 88 Abstract ............................................... 89 Introduction ............................................. 90 Materials and Methods .................................... 93 Plant material and SA treatment ........................ 93 Isolation of mitochondria and respiration assays ............. 93 Gel electrophoresis, immunobloting, and antisera ............ 94 Plasmid insert isolation and radiolabeling .................. 94 RNA isolation, blots and autoradiography ................. 95 Genomic DNA isolation and southern blot analysis .......... 95 Isolation of genomic clones and subcloning ................. 95 DNA sequencing and analysis .......................... 96 Synthesis of radiolabeled transcripts and mapping of aox1 mRNA with RNase .................. 96 Primer extension mapping of aox] mRNA ................. 97 Results ................................................ 98 Salicylic acid regulated expression of the alternative oxidase .............................. 98 Southern blot analysis to determine gene copy number ........ 98 Sequence of aox1 ................................... 99 Mapping of sex] mRNA using RNase ................... 109 Primer extension mapping of 30x1 mRNA ................ 109 viii Discussion ............................................. 114 References ............................................ 117 CHAPTER 5: CYTOCHROME AND ALTERNATIVE PATHWAY RESPIRATION IN TOBACXX): EFFECIS OF SALICYLIC ACID .................... 122 Footnotes ............................................. 123 Abstract .............................................. 124 Introduction ............................................ 125 Materials and Methods ................................... 127 Tobacco cultures ................................... 127 Salicylic acid and inhibitor treatment of suspension culture cells ............................ 127 Isolation of mitochondria and respiration assays ............ 128 Gel electrophoresis, immunoblotting and antisera ........... 129 Results ............................................... 129 Growth of suspension culture tobacco cells ................ 129 Respiratory pathway capacities during growth of suspension culture cells ...................... 129 Developmental regulation of the alternative oxidase expression in suspension cultured cells ................... 132 Time course of salicylic acid induced changes in respiratory pathway capacities ................. 137 Salicylic acid induced accumulation of alternative oxidase ..................... 137 Inhibition of salicylic acid induced accumulation of alternative oxidase ..................... 142 Salicylic acid concentration required for increase in alternative oxidase and capacity ............... 142 Discussion ............................................. 147 Acknowledgements ...................................... 151 References ............ 151 SUMMARY OF THESIS ....................................... 154 CHAPTER 2 ........................................... 154 The results ....................................... 154 The future ........................................ 155 CHAPTER 3 ........................................... 155 The results ....................................... 155 The future ........................................ 156 CHAPTER 4 ........................................... 157 The results ....................................... 157 The future ........................................ 158 Chapter 5 ............................................. 158 The results ....................................... 158 The future ........................................ 159 LIST OF FIGURES FIGURE PAGE CHAPTER 2 2-1. Products from in vitro translation of hybrid-selected , poly A+ RNA which could also be immunoprecipitated with M9 polyclonal antibodies ............................... 36 2-2. Western blot of the 48 kDa alternative oxidase/ fl-galactosidase fusion protein expressed in E coli ................. 39 2-3. Nucleotide and deduced amino acid sequence of the insert of pAOSG81-119 ............................... 43 2-4. Secondary structure prediction for the protein deduced from the nucleotide sequence of the insert of pAOSG81-119 ................ 45 CHAPTER 3 3-1. Developmental expression of the alternative oxidase proteins and transcript in voodoo lily appendix tissue ..................... 66 3-2. Time course graph of alternative pathway capacity in response to applied salicylic acid ..................... 69 3-3. Time course of accumulation of alternative oxidase proteins and transcript after salicylic acid application ..................... 72 3-4. Inhibition of salicylic acid induced expression of alternative oxidase proteins and transcript .................... 75 3-5. Nuclear in vitro transcription assays ........................... 77 CHAPTER4 4-1. Immunoblot and northern blot analysis of SA induction of alternative oxidase expression ............................. 101 4-2. Southern blot analysis of genomic DNA from S. guttatum .......... 103 4-3. Schematic diagram of genomic clone AGAOSGll ................ 106 4-4. Nucleotide and deduced amino acid sequence of the 30x1 gene of S. guttatum ............................. 108 4-5. RNase protection mapping of the 5’ end of the 30x1 transcript ...... 111 4-6. Primer extension mapping of the 5’ end of the 30x1 transcript ...... 113 CHAPTER 5 5-1. Growth curve for suspension cultured NT1 tobacco cells ........... 131 5-2. Respiratory pathway capacities of suspension cultured NTl tobacco cells ................................. 134 5-3. Immunoblot showing expression of tobacco alternative oxidase at various times after subculturing ........................... 136 5-4. Time course of SA effects on alternative and cytochrome pathway capacities .......................... 139 5-5. Immunoblot showing the time course of salicylic acid induction of increased alternative oxidase accumulation ........... 141 5-6. Immunoblot showing inhibition of salicylic acid induced increase in alternative oxidase expression ...................... 144 5-7. Immunoblot showing alternative oxidase accumulation caused by various concentrations of salicylic acid ................ 146 xii ADP ATP BSA cDN A DEP DNA EDTA EPR FCCP GRP HEPES IPTG kb kD/kDa MOPS mRN A NADH nt PAGE PR PV PVPP RBCS RNA rRNA SA SAR SDS SHAM Tris tRNA Tween-20 LIST OF ABBREVIATIONS adenosine diphosphate adenosine triphosphate basepairs (of DNA) bovine serum albumin complementary DNA (reverse-transcribed from RNA) cultivar diethylpyrocarbonate deoxyribonucleic acid ethylenediaminetetraacetate, disodium salt electron paramagnetic resonance spectroscopy p-trifluoromethoxycarbonyl cyanide glycine rich protein N-hydroxyethylpiperazine-N’-2-ethanesulfonic acid isopropyl fl-D-thiogalactopyranoside kilobase pairs (of DNA) kilodaltons 3-(n-morpholino)propanesulfonic acid messenger RNA reduced nicotinamide adenine dinucleotide nucleotides polyacrylamide gel electrophoresis pathogenesis related pathovar polyvinylpolypyrrolidone ribulose bisphosphate carboxylase small subunit ribonucleic acid ribosomal RNA salicylic acid systemic acquired resistance sodium dodecyl sulfate (sodium lauryl sulfate) salicylhydroxamic acid tris (hydroxymethyl) aminomethane transfer RNA polyoxyethylenesorbitan monolaurate xiii CHAPTER 1: INTRODUCTION CHARACTERIZATION OF THE ALTERNATIVE RESPIRATORY PATHWAY Plant thermogenesis was reported as early as 1778 when Lamarck demonstrated that heat was produced during the flowering of some aroid plants (referenced in 57). Garreau later linked this heat production to an increase in the rate of oxygen consumption (referenced in 5 7). In the 1930’s van Herk determined that this elevated rate of respiration associated with heat production during flowering of the aroid species 53 aroma tum guttatum Schott (voodoo lily) was not inhibited by cyanide (referenced in 45,57). These early studies using aroid plants and similar studies by Genevois using sweet pea (referenced in 49) initiated scientific interest in cyanide-resistant respiration, which is now commonly referred to as alternative respiration. Alternative respiration is insensitive to cyanide, azide, carbon monoxide, and antimycin A. Azide, carbon monoxide, and cyanide inhibit cytochrome pathway respiration by interfering with complex IV (see references 16,49 for reviews of cytochrome pathway components and inhibitors). Antimycin A inhibits Cytochrome pathway respiration by binding to a protein in complex III (16). 2 These inhibitors have been valuable in establishing that the alternative pathway diverges from the cytochrome pathway after the ubiquinone pool (4,77,88). Some evidence suggests that the cytochrome and alternative pathways interact with different pools of ubiquinone in the mitochondrial membranes (2,32,62,75,80,83). The alternative pathway is inhibited by iron-chelating compounds (49), disulfuram (27) and propyl gallate (68,85). The most widely used inhibitors are the hydroxamic acid derivatives, especially SHAM (salicylhydroxamic acid)(82). SHAM inhibits the alternative pathway respiration and lipoxygenase activity completely at a concentration of 1 mM, but does not inhibit cytochrome pathway respiration below 2 mM (12). The general nature of alternative respiration has, until recently, been a source of great controversy (49). Cyanide-resistant respiration has been proposed to be due to: 1) free radicals formed in mitochondrial membranes (49,78); 2) lipoxygenase (49,68); 3) a protonmotive ubiquinone cycle (49,77) cytochrome b-, (49); and 5) a discrete terminal oxidase (49,77). Several lines of evidence now provide a compelling argument in favor of cyanide-resistant respiration occurring via a discrete terminal oxidase (7), which is called the alternative oxidase. The alternative oxidase has also been localized to the matrix surface of the inner mitochondrial membrane (74). There have been numerous proposals (49,83,89) concerning the composition of the alternative oxidase. Flavin has been proposed as a cofactor (83); however, this seems unlikely since the affinity of mitochondrial flavoproteins for 02 is too low and the kinetics of reoxidation ' are too slow (4,83). , Also, auto-oxidizable flavoproteins produce H202 (83,89), whereas the alternative 3 pathway produces H20 (49,83,89). Nonheme iron (4,49,83) is an unlikely cofactor since no clear electron paramagnetic resonance (EPR) signal from a nonheme iron has been correlated with the presence of the alternative pathway (49). Copper (83,89), is likewise an unlikely candidate because of the lack of a clear EPR signal (63). Nevertheless, iron and copper cannot yet be ruled out completely since it is possible that an EPR signal from one or the other is masked (49,89) as in the case of coupled binuclear copper sites, designated as "type 3" copper (33). The determination of the exact composition of the alternative oxidase will, obviously, require a great deal more research. PARTITIONING OF ELECI'RONS BETWEEN RESPIRATORY PATHWAYS The partitioning of electrons between the alternative pathway and cytochrome pathway must be regulated because this has significant consequences for the physiology of the organism. The mechanisms to control the partitioning of electrons between the two pathways can be divided into two general categories: those that control the capacity of each pathway and those that control the degree of engagement of each pathway. The capacity of each pathway defined as the maximum number of electrons that can flow through the pathway. The capacity of the cytochrome pathway (Vm) is the rate of oxygen uptake sensitive to cyanide in the presence of SHAM using an exogenously added substrate. The capacity of the alternative pathway (V,,,) is measured as the rate of oxygen uptake sensitive to SHAM in the presence of 4 cyanide using an exogenously added substrate. The necessity for SHAM to be present in determining the capacity of the cytochrome pathway is debatable and based on the argument of whether or not inhibition of the cytochrome pathway results in increased electron flow through the alternative pathway (49,60,62). Although it is generally accepted that inhibition of the alternative pathway by SHAM does not cause an increase in electron flow to the cytochrome pathway (and will not cause erroneous determination of alternative pathway capacity), this assumption may not always be valid (60,62,99). The factors that could influence the capacity of each pathway include: 1) the amount of each component that is present in the mitochondria, which is determined by transcription rates, RNA stability, and translation rates for protein components and synthesis of cofactors associated with the components; 2) targeting of components to their proper locations in the mitochondria; 3) assembly of protein complexes; 4) interaction of the pathway components with the proper electron donors and acceptors; and 5) the presence or absence of pathway regulators. The activity of the alternative pathway (v,.,) is the rate of 02 reduction that is occurring via the alternative oxidase in vivo. The activity of the alternative pathway is related to the capacity by the following relationship: v... = pV,,,. The engagement of the pathway, p, is the portion of the alternative pathway capacity that is being utilized and has values of 0 (no engagement) to 1 (full engagement). Methods for measuring alternative pathway activity include: 1) the simple addition of SHAM (to whole cells or tissues) and determination of the difference in the 5 rate of 0; consumption before SHAM addition and the rate after SHAM addition (60), assuming that inhibition of the alternative pathway does not divert electrons to the cytochrome pathway; 2) the Bahr and Bonner method, which utilizes titration with SHAM (1,12) and assumes that inhibition of the alternative pathway does not result in an increase in electron flow to the cytochrome pathway (see above), and that the cytochrome pathway is always engaged to its maximum capacity (which has also been questioned [84,91]); and 3) the Lambowitz et a1. method, in which the ATP/O ratios are determined for a given substrate in the presence and absence of inhibitors of the alternative and cytochrome pathways and used to calculate the fraction of electrons flowing through each pathway using equations that take into account the different amounts of ATP produced by electrons from different substrates flowing through each pathway (47,49). Recently, a non-invasive method for measuring the engagement of the alternative pathway was introduced (28). This method relies on the observation that the alternative oxidase discriminates against 180 to a much greater extent than does cytochrome oxidase (28). The factors that could influence the in viva activity (or engagement) of the alternative pathway include the presence or absence of inhibitors and activators of the pathway, the identity of the substrates being oxidized (13,49,62), the physical arrangement of the components of each pathway, and the reduction level of the ubiquinone pool (2,11,19). Succinate (even in the presence of malonate, an inhibitor of succinate dehydrogenase) and malate (even in the presence of oxaloacetate, which inhibits malate oxidation) stimulate the rate of NADH 6 oxidation via the alternative pathway above the levels predicted by simple addition of the rates for the single substrates in Petunia bybn'da callus mitochondria and potato tuber callus mitochondria (96). However, in mitochondria that poorly oxidize NADH via the alternative pathway, this stimulation was not observed (96) and succinate inhibits NADH oxidation via the alternative pathway in soybean cotyledon mitochondria (13). In addition, malonate, which inhibits succinate dehydrogenase because it is structurally similar to succinate, is not capable of stimulating NADH oxidation via the alternative pathway (96). These observations argue against the hypothesis that succinate and/or malate affect respiration via the alternative pathway by interacting with the alternative oxidase (96). Electrons from succinate may flow through the alternative pathway more readily than electrons from NADH in the mitochondria from some sources, such as wounded potato tuber slices (49,96); while electrons from succinate seem to have little access to the alternative pathway in mitochondria from other sources, such as corn and fresh potato tuber slices (49). These studies illustrate the complex and poorly understood partitioning of electrons from different substrates to the alternative and cytochrome pathways in various tissues of assorted plant species. At least four hypotheses have been proposed to explain the observations: 1) there is a longer diffusion path between external NADH dehydrogenase and the alternative oxidase than between succinate dehydrogenase and the alternative oxidase (13,62); 2) there is a direct interaction between succinate dehydrogenase and the alternative oxidase (13,62); 3) there is compartmentation of distinct alternative oxidase pools (13,96); and 4) there is compartmentation of distinct 7 quinone pools associated with different dehydrogenases and/or oxidases (2,11,13,49,62,80,91). The actual situation in each plant mitochondrion may be a specific combination of these various possibilities suited to the metabolism of the cell in which the mitochondrion exists ( 13). The partitioning of electrons to the alternative pathway may depend upon the reduction level of the ubiquinone pool (2,11,19,91). It has been postulated that the ubiquinone pool must be completely reduced before electrons will be diverted to the alternative pathway (2). A second model postulates that the partitioning of electrons is based upon the relative rate constants for the reactions between reduced ubiquinone and the cytochrome and alternative oxidase (11,91). In soybean cotyledon mitochondria, a quinone reduction level of 35-40% was sufficient to allow engagement of the alternative pathway (19), which argues against the first model. It must also be kept in mind that the reduction level of the quinone pool itself depends on the relative rates of electron flow into and out of the quinone pool, which depends on the substrate and the ADP level (18,19). ALTERNATIVE PATHWAY 1N AROIDS Since electron flow through the alternative pathway does not result in proton translocation at complex III or complex IV (4,61,90), much of the potential energy of the system is not conserved as chemical energy and is lost as heat (90). Specific floral tissues of some aroid plants develop such a high level of the alternative pathway that they become thermogenic (12,5 7,70). The day the 8 inflorescence of the aroid species S. guttatum blooms (D-day), the capacity of the alternative pathway in the appendix tissue is 300-600 natoms O/min/mg protein and the temperature of the tissue increases about 9-12°C above ambient temperature (23,70,72). In S. guttatum, the heat produced during blooming is used to volatilize foul smelling compounds, such as indoles, which attract insect pollinators (56). Other aroids have also been good sources of mitochondria with which to study the alternative respiratory pathway. Mitochondria from Arum maculatum and Symplocarpus foetidus (skunk cabbage) have been used extensively for biophysical experiments (1,33,35,36,76,90) and in attempts to purify the alternative oxidase (6,34,38). Three mitochondrial proteins with apparent molecular masses 35-, 36-, and 37 kilodaltons (kD) strongly correlate with the alternative pathway in S. guttatum (21). Several lines of evidence suggest that the alternative oxidase is comprised of at least one of these proteins. The initial identification was made by solubilizing the active mitochondrial proteins (20), followed by CM-Sepharose and phenyl-Sepharose column chromatography to attain a 166-fold purified preparation of the alternative oxidase activity (21). Mouse polyclonal antibodies raised to this preparation inhibited alternative oxidase activity and immunoprecipitated the 35-, 36- and 37 kD proteins which copurify with the activity. Three distinct monoclonal antibodies were raised to the 36 kD protein: the ADA monoclonal antibodies recognize all three proteins; the AOU antibodies recognize primarily the 37 kD protein, but do recognize the 35- and 36 kD proteins to a lesser extent; and the AOL antibodies recognize the 35- and 36 kD 9 proteins primarily and the 37 kD protein to a lesser extent (22). The observation that the three proteins are immunologically related is consistent with the hypothesis that these three proteins are posttranslationally modified versions of a single gene product. Secondly, the levels of the 35-, and 36 kD proteins correspond to changes in the level of alternative oxidase activity at various stages of development of the appendix tissue. The protein levels also increase after application of salicylic acid, which is known to cause an increase in alternative pathway activity and has been shown to be an endogenous "trigger" of thermogenesis in appendix tissue (70, see below). In addition, the ADA monoclonal antibodies recognize proteins in other tissues known to have high levels of alternative pathway activity (21). At least two mitochondrial proteins in specific tissues of all aroid plants investigated are also recognized by the ADA monoclonal antibodies (21,22). Finally, the ADA monoclonal antibodies recognize a protein that is correlated with alternative oxidase activity in Neurospora crassa mitochondria (46). These data provide strong evidence that the 35-, 36- and 37 kD proteins are components of the alternative oxidase of S. guttatum. ALTERNATIVE PATHWAY IN OTHER ORGANISMS The alternative pathway has been studied in a variety of plants other than aroids (3,4,84) as well as in algae (26,98), fungi (29,59,81,91,102), and "lower" animals including a Paramecium and some of the brucei group of African trypanosomes (8,83). The genetic studies using the fungus Neurospora have 10 contributed to the identification of the branch point of the alternative pathway (95 ), the identification of the alternative oxidase, and early hypotheses on the location of the genes. A great deal of biochemical, biophysical, and physiological work has been done with corn, mung bean, pea, potato, and soybean mitochondria (14,31,39,49,65,84,86,_87,99). A single alternative oxidase protein that is recognized by the AOA monoclonal antibodies has been identified in mitochondrial proteins from specific tissues of potato (22), mung bean (22), tobacco callus (22), soybean (39), and siratro (39). These results suggest that only one protein is required for alternative oxidase activity. Also, cDNA clones encoding alternative oxidase proteins have been isolated from potato (30), rice (R. Nickels and L. McIntosh, personal communication), Arabidopsrls (D. 3611, personal communication), and yeast (81). These data will provide information on the evolutionary conservation of the alternative oxidase. The protein produced from the Arabidopsis cDNA clone allows CN-resistant, SHAM sensitive respiration in E. 0011', indicating that a single alternative oxidase protein is sufficient for alternative respiration (D. Still, personal communication). Hansenula anomala may be a rich source of the alternative oxidase (81) to be used for biophysical analysis. The role of the alternative pathway in non aroid plants is unknown (45,84). Over the years there have been many theories to explain the existence of the (presumably) wasteful alternative pathway in the mitochondria of non-aroid higher plants (45,reviewed in reference 84). It has been proposed that the pathway 4 developed as a protection against periodic bursts of cyanide in cyanogenic plants (43,84), but this is generally disputed (48,84). Increased electron flow through the 11 alternative pathway is implicated in response to environmental stresses such as: wounding (15); pathogen attack, based on the observations of increased salicylic acid levels in plants during pathogen attack (see below); and low temperatures (10,45,54,55,86,87,94,101). While no direct evidence yet exists to support the idea that thermogenesis is an adaptive response to low temperatures, many of these studies support the hypothesis that respiration, at least in part through the alternative pathway, may be involved with plant responses to lowered temperature. Since leaf mitochondria preferentially oxidize glycine during photorespiration, the alternative oxidase may remove excess N ADH during this process (24). Reoxidation of excess NADH generated during glycine oxidation in isolated soybean mitochondria from greening cotyledons was mainly via the alternative pathway (24). However, separate studies using pea or spinach leaf mitochondria did not show a correlation between glycine oxidation and the alternative pathway (18,25). A second association between alternative pathway respiration and substrate oxidation has been proposed for malate (49). In this scheme, excess reducing power can be removed without control by the phosphate potential or the energy charge by malate oxidation via malic enzyme and the alternative respiratory pathway (49). Such an association has been observed (79,80), although the interpretation of these data has been disputed (50). The "energy overflow" hypothesis asserts that alternative pathway respiration provides increased respiratory capacity when the cytochrome pathway is saturated, allowing oxidation of cellular substrates in excess of those needed for 12 growth, osmoregulation, storage as carbohydrate, or for ATP production (44,45 ). The arguments in favor of and against this hypothesis will not be discussed since they have been extensively reviewed elsewhere (17,44,45,84). Finally, another hypothesis for the function of the alternative oxidase asserts that the pathway allows a high rate of flux through the tricarboxylic acid cycle for the continued production of carbon skeletons required for growth and maintenance when the cytochrome pathway is inhibited by a high energy charge (45,92) or by a lack of cytochrome pathway components (42,45). This hypothesis implies that the cytochrome pathway must be saturated before the alternative pathway is engaged. However, one study indicated a non-linear relationship of quinone pool reduction with electron flow through the alternative pathway and demonstrated that a quinone pool reduction of 35-40% was sufficient for the engagement of the alternative pathway (19). SALICYLIC ACID IN AROIDS Salicylic acid has been shown to be an endogenous "trigger" of thermogenesis in S. guttatum appendix tissue (70). Salicylic acid is produced in the male floral region of the inflorescence and moves into the appendix beginning early on D-1 and "triggers" thermogenesis at about noon of D-day (70,72). The concentration of salicylic acid in the appendix increases from below 100 ng/g fresh weight as late as D-2 to over 1.0 ug/g fresh weight on D-day (72). Salicylic acid has also been shown to dramatically increase both the alternative pathway activity 13 and the levels of the 35-, 36-, and 37 kD alternative oxidase proteins when it is applied to S. guttatum appendix tissue sections (23). Prior to the application of salicylic acid, the 37 kD protein is expressed at a moderate level in immature appendix tissue (23). All three alternative oxidase proteins are present in great abundance in the tissue sections following incubation in a phosphate-buffered solution containing 1.0 mM salicylic acid (23). In addition, Raskin et a1. (72) showed that light is required for salicylic acid to cause thermogenesis in appendix tissue sections. SALICYLIC ACID IN OTHER ORGANISMS Application of salicylic acid (and some of its derivatives) induces flowering in many species of the Lemnaceae family (9,37,40,41), in Impatiens balsamina (64), and Pistia stratioles L. (69). However, the concentration and function of endogenous salicylic acid in these organisms is unknown. The endogenous level of salicylic acid is high in the heat-producing tissues of many plants during thermogenesis and in the non-thermogenic flowers of Passiflora caerulea L. during anthesis (71). The highest recorded concentration of endogenous salicylic acid is found in the sporophst of male cones of Dioon eduIe, where it approaches 0.1 mg/g fi’esh weight during heat production (71). The highest level of salicylic acid in a non-thermogenic plant is found in the leaves of Oryza sativa (rice), where it ranges between 24 and 68 ug/g fresh weight depending on the cultivar (71). Again, the function of the endogenous salicylic acid in these organisms has not 14 been established. Addition of salicylic acid to high COz-grown Chlamydomonas cultures in the log phase of growth also caused an increase in alternative pathway capacity, but it is not known if salicylic acid functions as a regulator in vivo (26). Salicylic acid may also be a messenger in systemic acquired resistance (SAR) in tobacco plants infected with tobacco mosaic virus (TMV)(51,100) and in cucumber plants infected with: 1) tobacco necrosis virus (TNV)(58); 2) the fungal pathogen Colletotn’cbum lagenan'um (5 8); or 3) Pseudomonas syringae pv synngae, a bacterial pathogen (73). TMV infection induces the accumulation of pathogenesis related proteins (PR proteins)(66,100) and the transcripts that encode them (5,51,52,53,97,100) in leaves of resistant tobacco plants. The level of salicylic acid increases in the infected leaves and the upper, non-infected leaves of a resistant tobacco plant that has been inoculated with TMV (51,100). Likewise, the level of salicylic acid increases in the phloem of a cucumber plant following an inoculation with C. Iagenarium, to which the plant has become resistant by initial infection with TNV or C Iagenan'um (5 8). Furthermore, application of salicylic acid to the leaves of TNV-resistant tobacco plants induces the accumulation of the some of the acidic PR1 proteins (5,52,53,66,97,100), including PRla, and a glycine rich protein (GRP8) and their corresponding transcripts (5,51,97,100). . Accumulation of the transcripts corresponding to other "SAR genes" has also been observed in primary and secondary leaves following inoculation with TMV or salicylic acid (97). Therefore, salicylic acid appears to act in disease resistance by regulating gene expression since application of salicylic acid has been shown to cause the accumulation of pathogenesis related (PR) proteins, and their 15 corresponding transcripts (51,66,97). The promoter regions of the genes encoding GRP8 and PRla have been analyzed in order to identify salicylic acid responsive, oils-acting sequence elements (67,93). Although some of the data on the region of the PRla promoter that conveys salicylic acid responsiveness is contradictory, a sequence motif found in both the PRla promoter and the GRP8 promoter has been identified (67,93). In summary, these data provide evidence that salicylic acid is involved in SAR, at least in tobacco resistance to TMV. Therefore, identification of the mechanism(s) by which salicylic acid regulates gene expression will be valuable in understanding the role(s) of salicylic acid in various plant tissues, the physiology of plant responses to pathogens, and the physiology of thermogenic tissues. REFERENCES 1. Baht JT & Bonner WD Jr (1973) Cyanide-insensitive respiration. I. The steady states of skunk cabbage spadix and bean hypocotyl mitochondria. J Biol Chem 248: 3441-3445. 2. Baht JT & Bonner WD Jr (1973) Cyanide-insensitive respiration. 11. Control of the alternate pathway. J Biol Chem 248: 3446-3450. 3. Bendall DS (1958) Cytochromes and some respiratory enzymes in mitochondria from the spadix of Arum maculatum. Biochem J 70: 381-390. 4. Bendall DS & Bonner WD Jr (1971) Cyanide-insensitive respiration in plant mitochondria. Plant Physiol 47: 236-245. 5. Bo] JF, Ijnthont HJM & Cornelislen BJC (1990) Plant pathogenesis- related proteins induced by virus infection. Ann. Rev. Phytopathol. 28: 113- 138. 10. 11. 12. 13. 14. 15. 16. 16 Bonmr Jr WD, Clarke SD & Rich PR (1986) Partial purification and characterization of the quinol oxidase activity of Arum maculatum mitochondria. Plant Physiol 80: 838-842. Chauverm M, Dizengrernel P & lance C (1978) Thermolability of the alternative electron transport pathway in higher plant mitochondria. Physiol Plant 42: 214-220. Clarhon AB Jr, Bienen EJ, Pollakis G & Grady RW (1989) Respiration of bloodstream forms of the parasite trypanosoma brucei brucei is dependent on a plant-like alternative oxidase. J Biol Chem 264: 17770-17776. (Eland CF (1974) The influence of salicylic acid on flowering and grth in the long-day plant Lemna y'bba G3. In RL Bieleski, AR Ferguson & MM Creswell (eds) Mechanisms of Regulation of Plant Growth. The Royal Society of New Zealand, Wellington, pp 553-557. Cook ND & Camack R (1985) Effects of temperature on electron transport in arum maculatum mitochondria. Plant Physiol 79: 332-335. Cottingharn IR & Moore AL (1983) Ubiquinone pool behavior in plant mitochondria. Biochim Biophys Acta 724: 191-200. Day DA, Arron GP & Iaties GG (1980) Nature and control of respiratory pathways in plants: The interaction of cyanide-resistant respiration with the cyanide-sensitive pathway. In: DD Davies (ed) The Biochemistry of Plants, Vol 2, Metabolism and Respiration. Academic Press, New York. pp. 197-241. Day DA, Dry IB, Soole KL, W‘nkich JT & Moore AL (1991) Regulation of alternative pathway activity in plant mitochondria. Deviations from Q-pool behavior during oxidation of NADH and quinols. Plant Physiol 95: 948-953. Day DA, Moore AI, Dry IB, Wiskich JT & Anon-Bieto 1(1988) Regulation of nonphosphorylating electron transport pathways in soybean. cotyledon mitochondria and its implications for fat metabolism. Plant Physiol 86: 1199-1204. Dizengrernel P & Lance C (1976) Control of changes in mitochondrial activities during aging of potato slices. Plant Physiol 58: 147-151. Douce R, Brouquiue R & Journet E-P (1987) Electron transfer and oxidative phosphorylation in plant mitochondria. In: Davies DD (ed) The Biochemistry of Plants, Vol 11, Biochemistry of Metabolism. Academic Press, New York pp.177-211. 17. 18. 19. 20. 21. 22. 23. 24. 26. 27. 17 Dance R & Neuburger M (1989) The uniqueness of plant mitochondria. Ann Rev Plant Physiol Plant Mol Biol 40: 371-414. Dry IB, Bryce JH & Wiskich JT (1987) Regulation of mitochondrial respiration. In: DD Davies (ed) The Biochemistry of Plants, Vol 1], Biochemistry of Metabolism. Academic Press, New York. pp. 213-251. Dry IB, Moore AL, Day DA & Wiskich JT (1989) Regulation of alternative pathway activity in plant mitochondria: Nonlinear relationship between electron flux and the redox poise of the quinone pool. Arch Biochem Biophys 273: 148-157. Ethan TE & McIntosh L (1986) Characterization and solubilization of the alternative oxidase of Sauromatum guttatum mitochondria. Plant Physiol 82: 1-6. Ethan TE & McIntosh L (1987) Identification of the alternative terminal oxidase of higher plant mitochondria. Proc Nat Acad Sci USA 84: 8399-8403. Ethan TE, Nickels RL & McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89: 1311-1317. Ethan TE, Nickels RL & McIntosh L (1989) Mitochondrial events during development of thermogenesis in Sauromatum guttatum (Schott). Planta 180: 82-89. Gerard J & Diungrernel P (1988) Properties of mitochondria isolated from greening soybean and lupin tissues. Plant Sci 56: 1-7. Goya] A, Hirer C, Talbert NE & McIntosh L (1991) Progress no.9 cultivar of pea has alternative respiratory capacity and normal glycine metabolism. Plant Cell Physiol 32: 90-96. Gayal A & Talbert NE (1989) Variations in the alternative oxidase in Chlamydomonas grown in air or high C02. Plant Physiol 89: 958-962. Graver SD & Iatiel GG (1978) Characterization of the binding properties of disulfiram, an inhibitor of cyanide-resistant respiration. In: G Ducet, C Lance (eds) Plant mitochondria. Elsevier/North Holland Biomedical Press, Amsterdam. pp. 259-266. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 18 Guy RD, Berry JA, Fopl ML & Hoering TC (1989) Differential fractionation of oxygen isotopes by cyanide-resistant and cyanide-sensitive respiration in plants. Planta 177: 483-491. Henry MF & Nyrn E1 (1975) Cyanide-insensitive respiration. An alternative mitochondrial pathway. Sub-Cell Biochem 4: 1-65. H'uer CB (1991) Molecular and developmental aspects of respiratory complexes in higher plant mitochondria. Doctoral Dissertation, Michigan State University, Biochemistry Department. Hirer C & McIntosh L (1990) Alternative oxidase of potato is an integral membrane protein synthesized de novo during aging of tuber slices. Plant Physiol 93: 312-318. Huq S & Pahner JM (1978) The involvement and possible role of quinone in cyanide resistant respiration. In: G Ducet, C Lance (eds) Plant Mitochondria. Elsevier/North-Holland Biomedical Press, New York, pp. 225-232. Huq S & Palmer JM (1978) Isolation of a cyanide-resistant duroquinol oxidase from Arum maculatum mitochondria. FEBS Lett 95: 217-220. Huq S & Pahner JM (1978) Superoxide and hydrogen peroxide production in cyanide resistant Arum maculatum mitochondria. Plant Sci Lett 11: 351-358. James WO & Beavers H (1950) The respiration of Arum spadix. A rapid respiration, resistant to cyanide. New Phytol 49: 353-374. James WO & Eliot DC (1955) Cyanide-resistant mitochondria from the spadix of an Arum. Nature 175: 89. Kailmra S, Watanabe K & Takirnoto A (1981) Flower-inducing effect of benzoic and salicylic acids in various strains of Lemna paucicostata and ‘L. minor. Plant & Cell Physiol 22: 819-825. Kay CJ & Palnnr JM (1985) Solubilization of the alternative oxidase of cuckoo-pint (Arum maculatum) mitochondria. Stimulation by high concentrations of ions and effects of inhibitors. Biochem J 228: 309-318. Kearnr A, Whelan J, Young S, Ethan TE & Day DA (1992) Tissue- specific expression of the alternative oxidase in soybean and siratro. Plant Physiol 99: 712-717. 41. 42. 43. 45. 47. 49. 50. 19 Khurana JP & Maheshwari SC (1980) Some effects of salicylic acid on growth and flowering in Spirodela polyrrhiza SP”. Plant Cell Physiol 21: 923-927. Humane JP & Maheshwari SC (1983) Flora] insuction in Wolfiia microscopica by salicylic acid and related compounds under non-inductive long days. Plant Cell Physiol 24: 907-912. Berk-Kiebert De YM, Kncppcrr TJA & Plar van der LHW (1982) Influence of chloramphenicol on grth and respiration of soybean (Glycine max I...) suspension cultures. Physiol Plant 55: 98-102. Larnberr H (1980) The physiological significance of cyanide-resistant respiration in higher plants. Plant Cell Environ 3: 293-302. Lambert H (1982) Cyanide-resistant respiration: A non-phosphorylating electron transport pathway acting as an energy overflow. Physiol Plant 55: 478-485. Imhera H (1985) Respiration in intact plants and tissues: Its regulation and dependence on environmental factors, metabolism and invaded organisms. In: R Douce & DA Day (eds) Encyclopedia of Plant Physiology (New Series), Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York, pp.418-473. ImbawitzAI,SabaminJR,BertrandI-I,NickehR&McIntoshL(1989) Immunological identification of the alternative oxidase of Neurospora crassa mitochondria. Mol Cell Biol 9: 1362-1364. Iarnbawitz AM, Smith EW & Slayrnan CW (1972) Oxidative phosphorylation in Neurospora mitochondria. Studies on wild-type, poky and chloramphenicol induced wild type. J Biol Chem 247: 4859-4865. Lance C (1981) Cyanide-insensitive respiration in fruits and vegetables. In: J Friend & MJC Rhodes (eds) Recent Advances in the Biochemistry of Fruits and Vegetables. Academic Press, New York, pp.63-87. Lance C, Chauveau M & Dinngremel P (1985) The cyanide-resistant pathway of plant mitochondria. In: R Douce, DA Day (eds) Encyclopedia of Plant Physiology (New Series), Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York. pp. 202-247. Latier GG ( 1982) The cyanide-resistant, alternative path in higher plant respiration. Ann Rev Plant Physiol 33: 519-555. 51. 52. 53. 54. 55. 56. 57. 58. 59. 61. 20 Malamy J, Carr JP, Kleuig DF & Raskin I (1990) Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250: 1002-1004. Matuaka M, Alan S & Ohaahi Y (1988) Regulation mechanisms of the synthesis of pathogenesis-related proteins in tobacco leaves. Plant Cell Physiol 29: 1185-1192. Matsuoka M & Ohashi Y (1986) Induction of pathogenesis-related proteins in tobacco leaves. Plant Physiol 80: 505-510. McCaig TN & Hill RD (1977) Cyanide-insensitive respiration in wheat: cultivar differences and effects of temperature, carbon dioxide, and oxygen. Can J Bot 55: 549-555. McNulty AK & Cumminr WR (1987) The relationship between respiration and temperature in leaves of the arctic plant Sara'fi'aga cernua. Plant Cell Environ 10: 319-325. Meeure BJD (1966) The voodoo lily. Sci Am 215: 80-88. Meeuae BJD (1975) Thermogenic respiration in aroids. Ann Rev Plant Physiol 26: 117-126. M6trauxJP,SignerI-I,RyalsJ,WardFeWyIl-BenzM, GaudinJ, Raschdorf K, Schmid E Blum W & Inverardi B (1990) Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250: 1004-1006. MinagawaN,SakajoS,KomiyarnaT&YoshimotoA(1990)A36kDa ‘ mitochondrial protein is responsible for cyanide-resistant respiration in Hansenula anomala. FEBS Lett 264: 149-152. ManerMBercziAVanDerPlaIIHW&IamberrH(l988) Measurement of the activity and capacity of the alternative pathway in intact plant tissues: Identification of problems and possible solutions. Physiol Plant 72: 642-649. Moore AL & Bonner Jr WD (1982) Measurements of membrane potentials in plant mitochondria with the safranine method. Plant Physiol 70: 1271-1276. 62. 63. 65. 67. 69. 70. 71. 72. 73. 21 Moore AL & Rich PR (1985) Organization of the respiratory chain and oxidative phosphorylation. In: R Douce & DA Day, (eds) Encyclopedia of Plant Physiology (New Series), Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York, pp.134-172. Moore AI. Rich PR, Bonner WD Jr & Igeldew WJ (1976) A complex EPR signal in mung bean mitochondria and its possible relation to the alternative pathway. Biochem Biophys Res Commun 72: 1099-1107. Nanda KK, Kumar S & Sood V (1976) Effect of gibberellic acid and some phenols on flowering of Impatiens balsamina, a qualitative short-day plant. Physiol Plant 38: 53-56. Obenland D, Hirer C, McIntosh L Shibles R & Stewart CR (1988) Occurrence of alternative respiratory capacity in soybean and pea. Plant Physiol 88: 528-531. Ohashi Y & Matuoka M (1986) Induction of pathogenesis-related proteins in tobacco. Plant Physiol 80: 505-510. OhshimaM,ItohI-I,MatsuokaMMurakamiT&OhashiY(1990) Analysis of stress-induced or salicylic acid-induced expression of the pathogenesis-related la protein gene in transgenic tobacco. Plant Cell 2: 95-106. Parrish DJ & leapald AC (1978) Confounding of alternate respiration by lipoxygenase activity. Plant Physiol 62: 470-472. Pieterse AH (1978) Experimental control of flowering in Pistia stratiotes 1... Plant & Cell Physiol 19: 1091-1093. Raskin I, Ehmann A, Melander WR & Meeuse BJD (1987) Salicylic acid: A natural inducer of heat production in Arum lilies. Science 237: 1601-1602. Raskin I, Skubatz H, Tang W & Meeuse BJD (1990) Salicylic acid levels in thermogenic and non-thermogenic plants. Annals of Botany 66:369-373. Raskin I, Turner IV & Melander WR (1989) Regulation of heat production in the infloresences of an Arum lily by endogenous salicylic acid. Proc Natl Acad Sci 86: 2214-2218. Rasmussen JB, Hammerschmidt R & Zoak MN (1991) Systemic induction of salicylic acid accumulation in cucumber after inoculation with pseudomonas syringae pv syringae. Plant Physiol 97: 1342-1347. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 22 Rasmusson AG, Moller IM & Palmer JM (1989) Component of the alternative oxidase localized to the matrix surface of the inner membrane of plant mitochondria. FEBS Lett 259: 311-314. Rich PR (1981) A generalized model for the equilibration of quinone pools with their biological donors and acceptors in membrane-bound electron transfer chains. FEBS Lett 130: 173-178. Rich PR & Bonner Jr WD (1978) Paramagnetic centers associated with higher plant and poky Neurospora crassa mitochondria. In: G Ducet & C Lance (eds) Plant Mitochondria. Elsevier/North-Holland Biomedical Press, New York, pp.61-68. Rich PR & Moore AL (1976) The involvement of the proton motive ubiquinone cycle in the respiratory chain of higher plants and its relation to the branchpoint of the alternative pathway. FEBS Letters 65: 339-344. Rustin P, Dupont J & Lance C (1983) Oxidative interactions between fatty acid peroxy radicals and quinones: Possrble involvement in cyanide-resistant electron transport in plant mitochondria. Arch Biochem Biophys 225: 630-639. Rustin P & Moreau F (1979) Malic enzyme activity and cyanide-insensitive electron transport in plant mitochondria. Biochem Biophys Res Comm 88: 1125-1131. Rmtin P, Moreau F & lance C (1980) Malate oxidation in plant mitochondria via malic enzyme and the cyanide-insensitive electron transport pathway. Plant Physiol 66: 457-462. Sakajo S, Minagawa N, Komiyama T &. Yoshimoto A (1991) Molecular cloning of cDNA for antirnycin A-inducible mRNA and its role in cyanide- resistant respiration in Hansenula anomala. Biochim Biophys Acta 1090: 102-118. Schanbaum GR, Bonner WD, Storey BT & Bahr JT (1971) Specific inhibition of the cyanide-insensitive respiratory pathway in plant mitochondria by hydroxamic acids. Plant Physiol 47: 124-128. S'mdaw JN (1982) The nature of the cyanide-resistant pathway in plant , mitochondia. Rec Adv in Phytochem 16: 47-84. Siedaw JN & Berthald DA (1986) The alternative oxidase: A cyanide- resistant respiratory pathway in higher plants. Physiol Plant 66: 569-5 73. 87. 89. 91. 93. 94. 95. 23 Siedaw JN & Girvin ME (1980) Alternative respiratory pathway. Its role in seed respiration and its inhibition by propyl gallate. Plant Physiol 65: 669-674. Stewart CR, Martin BA, Redling L & Cerwick S (1990) Respiration and alternative oxidase in corn seedling tissues during germination at different temperatures. Plant Physiol 92: 755-760. Stewart CR, Martin BA, Redh'ng L & Cerwick S (1990) Seedling growth, mitochondrial characteristics, and alternative respiratory capacity of corn genotypes differing in cold tolerance. Plant Physiol 92: 761-766. Storey BT ( 1976) Respiratory chain of plant mitochondria. XVIII. Point of interaction of the alternative oxidase with the respiratory chain. Plant Physiol 58: 521-525. Storey BT (1980) Electron transport and energy coupling in plant mitochondria. In: DD Davies (ed) The Biochemistry of Plants, Vol.2. Academic Press, New York, pp.125-195. Storey BT & Bahr JT (1969) The respiratory chain of plant mitochondria. 11. Oxidative phosphorylation in skunk cabage mitochondria. Plant Physiology 44: 126-134. deTroastembergh J-C & Nyns E-J (1978) Kinetics of the respiration of cyanide-insensitive mitochondria from the yeast Saccbaromycopsis Iipolytica. Eur J Biochem 85: 423-432. Turner JF & Turner DH ( 1980) The regulation of glycolysis and the pentose phosphate pathway. In: DD Davies (ed) The Biochemistry of Plants, Vol.2. Academic Press, New York. pp. 279-316. VandeRheeMD,VanKanJAI, Ganzalez-JaenMT&BolJF(1990) Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection. Plant Cell 2: 357-366. van de Venter HA (1985) Cyanide-resistant respiration and cold resistance in seedlings of maize (Zea mays L.). Ann Bot 56: 561-563. van Jagaw G & Bohrer C (1975) Inhibition of electron transfer from cyt b to ubiquinone, cyt c, and duroquinone by antimycin. Biochim Biophys Acta 387: 409-424. 97. 98. 100. 101. 102. 24 WagnerAM,KraakMI-IS,vanEmmerikWAM&vanderPlasIHW (1989) Respiration of plant mitochondria with various substrates: alternative pathway with NADH and TCA cycle derived substrates. Plant Physiol Biochem 27: 837-845. Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhald DW, Alexander DC, Ahl-Gay P, Metraux J-P & Ryals JA (1991) Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell 3: 1085-1094. Weger HG, Guy RD & Turpin DH (1990) Cytochrome and alternative pathway respiration in green algae. Measurements using inhibitors and 1302 discrimination. Plant Physiol 93: 356-360. Wilson SB ( 1988) The switching of electron flux from the cyanide- insensitive oxidase to the cytochrome pathway in mung-bean (Phaseolus aureus L.) mitochondria. Biochem J 249: 301-303. Yalpani N, Sflverman P, Wilson TMA, Kleier DA & Raskin I (1991) Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco. The Plant Cell 3: 809-818. Yoshida S & Tagawa F (1979) Alteration of the respiratory function in chill-sensitive callus due to low temperature stress. I. Involvement of the alternative pathway. Plant Cell Physiol 20: 1243-1250. Yoshirnoto A, Sakajo S, Minagawa N & Komiyama T (1989) Possible role of a 36 kDa protein induced by respiratory inhibitors in cyanide-resistant respiration in Hansenula anomala. J Biochem 105: 864—866. CHAPTERZ ISOLATION AND CHARACTERIZATION OF A cDNA CLONE ENCODING AN ALTERNATIVE OXIDASE PROTEIN OF Saummatum gummm (Schott) DavidMRhoadsandLeeMcIntosh MSU-DOE Plant Research Laboratory and Biochemistry Department Michigan State University East Lansing, MI 48824 Published in Proc. Natl. Acad. Sci. USA 88: 2122-2126 (1991). 26 ABSTRACT Polyclonal and monoclonal antibodies, which recognize the 35, 36, and 37 kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 in E. coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63 amino acid transit peptide, and a nine amino acid match to authentic N-terminal sequence of the 36 kDa alternative oxidase protein. Analyses of the deduced amino acid sequence indicate: 1) that the transit peptide is predicted to form amphiphilic helices; and 2) that three regions of the processed protein are likely to form transmembrane alpha-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum. 27 INTRODUCTION All higher plants that have been investigated, as well as many fungi, algae, and some protists ( 1), contain two respiratory pathways: the cytochrome pathway and the alternative pathway (2). The alternative pathway diverges from the cytochrome pathway after the ubiquinone pool (3,4,5). Since electron flow through the alternative pathway does not result in the formation of a proton gradient at the cytochrome b-c, complex or at the cytochrome a-a, complex (3,6,7), much of the potential energy of the system is not conserved as chemical energy and is lost as heat. Some plant tissues that express a high level of the alternative pathway are thermogenic (8). The role of thermogenesis in the appendix tissue of the Aroid species Sauromatum guttatum Schott (voodoo lily) is to volatilize foul-smelling compounds which attract insect pollinators (9). However, the role of the alternative respiratory pathway in non-thermogenic plants, and most other organisms, is problematic. Alternative pathway respiration may function to increase respiration when the cytochrome pathway is "restricted", allowing a high rate of flux through the tricarboxylic acid cycle, thus producing carbon skeletons required for growth (10,11). This hypothesis implies that the cytochrome pathway must be saturated before the alternative pathway is engaged. However, a recent study indicated a non-linear relationship of quinone pool reduction with electron flow through the alternative pathway and demonstrated that a quinone pool reduction of 35-40% was sufficient for the engagement of the alternative pathway (12). Increased 28 electron flow through the alternative pathway has also been implicated in response to low temperatures (13). While no direct evidence yet exists to support the idea that alternative pathway thermogenesis is an adaptive response to low temperatures, it has been proposed that increased respiration in response to low temperatures may be required; possibly to prevent accumulation of torn'c metabolites (14). Three mitochondrial proteins with apparent molecular masses, as determined on denaturing polyacrylamide gels, of 35-, 36-, and 37 kilodaltons (kDa) strongly correlate with alternative oxidase activity in S. guttaturn (15). Previous results also showed that a single monoclonal antibody recognized all three of these polypeptides and was capable of inhibiting alternative oxidase activity in vitro (16). Taken together, these data strongly indicate that the three alternative oxidase proteins are closely related, possibly as post-translationally modified versions of a single gene product. The same monoclonal antibody raised to the S. guttatum alternative oxidase also recognizes this oxidase from Neurospora crassa and has been employed to characterize mutants of the oxidase in this organism (17). The structural component of the N. crassa alternative oxidase is probably encoded by a single nuclear gene (18,19), thus lending support to the hypothesis that the three proteins correlated with alternative oxidase in S. guttatum are the products of a single gene. Salicylic acid has been shown to increase alternative oxidase activity in isolated S. guttatum appendix sections dramatically (20). The 37 kDa protein is present in small amounts prior to the increase and all three polypeptides are 29 present in great abundance in the tissue sections following a 20 hour incubation in a solution of salicylic acid (21). These results suggest that de novo synthesis of alternative oxidase is required for the increased activity in S. guttatum appendix tissue, and that posttranslational modifications of the alternative oxidase may also be needed (ie. for the appearance of the 35 kDa and 36 kDa proteins). MATERIALS AND METHODS Plant Material. Sa uromatum guttatum Schott (voodoo lily) plants were maintained in a glasshouse at 27°C : 4°C under long-day conditions, as previously described (22). The day the appendix region of the spadix heats is referred to as D-day (see ref. 9 for anatomy of S. guttatum inflorescence). Other developmental stages of the plant are indicated as the number of days before or after D-day (D-l being the day before and D+1 the day after D-day). Isolation of Mitochondria. Mitochondria were isolated by a modification of the procedure of Schwitzguebel and Siegenthaler (23). After the first centrifugation at 19600 x g for 10 minutes the mitochondrial pellet was resuspended directly in reaction medium (250 mM sucrose, 30 mM MOPS, pH 6.8). Antisera. Antibodies used were as follows: M13 polyclonal antibodies, which recognize approximately 13 proteins on Western blots of total S. guttatum mitochondrial proteins (14); M9 polyclonal antibodies, which only recognize the 30 35-, 36-, and 37 kDa proteins (15); and the AOA, AOU, and AOL monoclonal antibodies (15). The AOA antibodies recognize all three polypeptides, AOU antibodies recognize primarily the 37 kDa polypeptide, and AOL antibodies recognize primarily the 35- and 36 kDa polypeptides (15). Gel Electrophoresis, Immrmablotting, and Fluoragraphy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein samples was performed using the buffer system of Laemmli (24) as previously described (14). Immunoblotting was done according to the procedure of Blake et a1. (25) except that antibody incubations were for 1 hour at room temperature. After SDS- PAGE, the gels were subjected to sodium salicylate fluorography using the procedure of Chamberlin (26). Protein Sequencing. The 36 kDa alternative oxidase protein was electroeluted from polyacrylamide gel slices and SDS was extracted from the samples by the procedure of Konigsberg and Henderson (27). The N-terminus of the protein was sequenced following the procedure of Vandekerckhoue et a]. (28). However, the first three amino acids were not identified. Poly (A)+ RNA Isolation. Total RNA was isolated from frozen (in liquid N2) S. guttatum appendices by the procedure of McIntosh and Cattolico (29), except that the tissue was homogenized with a mortar and pestle, 0.015 M EDTA was used in the extraction buffer, and the RNA was extracted three times with phenol, 31 three times with phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol), and three times with chloroform. The poly (A)+ fraction was isolated using a poly U Sepharose (Sigma, St. Louis, MO) column by the method of Cashmore (30). In Vitro Translation of Poly (A)+ RNA and Immunoprecipitation. Poly (A)+ RNA and hybrid-selected RNA transcripts were translated in-vitro using a rabbit reticulocyte lysate system (Promega, Madison, WI) with [35S]-methionine (Amersham, Arlington Heights, IL) as the radioactive label. The protocol of the manufacturer (Promega, Madison, WI) was used, except that 165 mM potassium acetate and 1.05 mM magnesium acetate (final concentrations) were used in a 25 ul final reaction volume and incubations were done at 37°C. Specific in vitro translation products were immunoprecipitated by the addition of M9 polyclonal antibodies or preimmune antiserum and formalin-fixed Staphylococcus aureus cells using a modification of the procedure of Anderson and Blobel (31) as described in Hondred et a1. (32). Isolation of cDNA (lanes. A S. guttatum cDNA expression library was constructed in the EcoRI sites of lambda ZAPII (33) by Stratagene (La Jolla, CA). Poly (A)+ RNA from the appendix of a D-day plant and a mixture of oligo dT and random primers were used to synthesize the cDNA. Both M13 polyclonal sera and a mixture of AOA, AOU, and AOL monoclonal antisera were employed to screen the cDNA expression h’brary following Stratagene’s picoBlue Immunodetection protocol. Inserts from phage isolates which produced proteins 32 that were recognized by both polyclonal and monoclonal antisera were subcloned by Stratagene’s excision procedure as described by Short et a]. (33). Small and large scale phagemid isolation and subcloning into pUC119 were done using standard procedures (34). Hybrid Selection. Phagemids pAOSG81, pAOSG83, and pBLUESCRIPT (see Results for descriptions) were used to select homologous RNA transcripts from total poly (A)+ RNA by the procedure given in Maniatis et a1. (34). The selected RNA was precipitated by addition of MgClz to 10 mM, KAc to 100 mM, and cold ethanol to 67% (v/v) and incubation at -20°C. kpression of Phagemid Inserts in E. coli. Liquid cultures of E. coli strain T61 cells containing phagemids pAOSG81-119, pAOSG81i-119, or pUC119 (see Results for descriptions) were used for expression experiments. The insert of the phagemid pAOSG81-119 is in-frame with the lacZ gene in pUC119 so that a fusion protein was produced after addition of isopropyl-B-D-thiogalactopyranoside (IPTG) to the growth media. After one hour of growth at 37°C in LB medium (see 34 for description) containing 25-50 pg ampicillin/ml, IPTG was added to experimental cultures to a final concentration of 25 ug/ml; no IPT G was added to negative control cultures. Growth was continued for 8 hours, at which time the cells were pelleted and resuspended in 30 ul of SDS-PAGE sample buffer (24). 33 DNA Sequencing and Analysis. Single-stranded deletions of pAOSG81-119 and pAOSG81i-119 were made using the procedure of Dale (35). Sequencing of the phagemid inserts was done by the dideoxy method of Sanger et a1. (36) using Sequenase® T7 DNA Polymerase (U.S. Biochemical Corporation, Cleveland, OH). Protein sequence was deduced from the nucleotide sequence using the Editbase DNA sequence analysis program. Helical wheel projections of the deduced amino acid sequence were made according to von Heijne (37). The structure prediction plot of the deduced amino acid sequences was made using the Seqanal Predictor program (38), a modified version of the Chou-Fasman protein secondary structure predictor program which is adapted for membrane proteins. RESULTS In Vitm Translation of Total Poly (A)+ RNA and Immunoprecipitatian. An in vitro translation product with an apparent molecular mass of 42 kDa was readily immunoprecipitated from total in vin’o translation products made from poly (A)+ RNA with the M9 polyclonal antibodies (Fig. 2-1A & 2-1B, lane 1), but not with preirnmune antiserum (data not shown). The alternative oxidase proteins previously identified (15) have apparent molecular masses of 35-, 36-, and 37 kDa, suggesting that the 42 kDa protein is a nuclear-encoded precursor of one or more of the alternative oxidase proteins. 34 Isolation of cDNA Clones. After two rounds of screening, five plaques which produced proteins that were recognized by both polyclonal and monoclonal antisera were selected for further analysis. The inserts from these clones were "rescued" as Bluescript phagemids. Phagemids from two of these putative alternative oxidase clones were designated pAOSG81 and pAOSG83 and contained inserts of 1400 base pairs and 1100 base pairs in length, respectively. The inserts of pAOSG81 and pAOSG83 were subcloned into pUC119 and the resulting vectors were designated pAOSG81-119, pAOSG83-119, and pAOSG81i- 119 (pUC119 containing the insert of pAOSG81 in an inverted orientation). In VitmTranslatianandImmrmoprecipitatiananybrid Selected RNA. Phagemids pAOSG81, pAOSG83, and pBLUESCRIPT were used to select homologous transcripts from total poly (A)+ RNA isolated from D-day appendix tissue. Upon in vitro translation of the selected RNA, SDS-PAGE, and fluorography, several diffuse bands representing proteins of widely distributed molecular masses were observed (Fig. 2-1A). These bands probably represent proteins translated from poly (A)+ RNA that bound nonspecifically to the nitrocellulose paper since they are present in all lanes. However, in the lanes corresponding to pAOSG81 and pAOSG83 a very abundant protein with an apparent molecular mass of 42 kDa was observed (Fig. 2-1A, lanes 2 & 3). This protein was not observed among the in vitro translation products from RNA selected by vector DNA alone (Fig. 2-1A, lane 4) and it comigrated with the protein made from total poly (A)+ RNA that was also immunoprecipitated with 35 Figure 2-1. Products from in vitro translation of hybrid-selected poly (A)+ RNA (Panel A) which could also be immunoprecipitated with M9 polyclonal antibodies (Panel B). Panel A: total poly (A)+ RNA and hybrid-selected poly (A)+ RNA transcripts were translated in vitro in the presence of [355]-methionine. The products were seperated by SDS-PAGE and visualized by fluorography. Lane 1; total poly (A)+ RNA translated in vitro and immunoprecipitated (see Materials and Methods) to indicate the position of the 42 kDa protein. The remaining lanes contained only products translated in vitro from RNA selected by the following phagemid samples: Lane 2, pAOSG81; Lane 3, pAOSG83; and Lane 4, pBLUESCRIPT. Panel B: total poly (A)+ RNA and hybrid selected poly (A)+ RNA transcripts were translated in vitro, immunoprecipitated, and visualized by fluorography (see Materials and Methods). The in vitro translation products were made from the following RNA samples proir to immunoprecipitation: Lane 1; total poly (A)+ RNA, Lane 2; pAOSG81 selected poly (A)+ RNA, Lane 3; pAOSG83 selected poly (A)+ RNA, and Lane 4; pBLUESCRIPT selected poly (A)+ RNA. kDa 66.2- 45.0- 42+ 31.0- 21.5- 1 36 234 kDa 66.2- 45.0- 42+ 31 .0- 1 8.4- 12.3- 37 M9 polyclonal antisera (Fig. 2-1A, lane 1). Fluorography of an SDS-polyacrylamide gel demonstrated that the 42 kDa protein translated from poly (A)+ RNA selected by phagemids pAOSG81 and pAOSG83 could also be immunoprecipitated by the M9 polyclonal antibodies (Fig. 2-1B, lanes 2 & 3). These data indicate that clones pAOSG81 and pAOSG83 encode a 42 kDa protein that is immunologically related to the 35-, 36-, and 37 kDa alternative oxidase proteins. Expression of Plasmid Inserts in E coli. Fig. 2-2, lane 2 shows that the AOA monoclonal antibodies recognize the protein encoded-by pAOSG81-119 when it is expressed in E. coli The protein has an apparent molecular mass of about 48 kDa, and was not expressed in uninduced cells (Fig. 2-2, lane 3), in cells that contained only pUC119 (Fig. 2-2, lane 4), or in cells that contained pAOSG81i- 119, which has the insert from pAOSG81-119 inverted relative to the lacZ gene (data not shown). The increased size of the polypeptide arose as a result of cloning. A portion of the cDNA for 3011:], a region which does not encode protein in the isolated gene, was inserted in such a manner so that it is now "in frame" and recognized as coding sequence behind the lacZ gene. DNA sequence analysis later showed that pAOSGSl-119 is predicted to express a fusion protein of 44,794 Da and consist of 21 amino acids of B-galactosidase, 24 amino acids now encoded by the 5’ non-coding region of the insert, and the 349 amino acids of the precursor alternative oxidase protein. 38 Figure 2-2. Western blot of the 48 kDa alternative oxidase/B-galactosidase fusion protein expressed in E. coli. Cell cultures containing phagemids were grown in the presence or absence of IPTG. An equal number of cells from each culture were pelleted, solubilized, and seperated by SDS-PAGE. The proteins were transferred onto nitrocellulose paper and detected using AOA monoclonal antibodies and alkaline-phosphatase conjugated goat anti-mouse IgG. Lane 1; total mitochondrial proteins from S. guttatum appendix tissue. The remaining lanes contained proteins from E. coli cultures grown in the presence (Lanes 2 & 4) or absence (Lane 3) of IPTG and which contained the following phagemids: Lane 2; pAOSG81-119, Lane 3; pAOSG81-119, Lane 4; pUC119. 39 kDa 1 2 3 4 48+ -— 37 36-\- ~_.__._..---—-- 40 DNA Sequence Analysis. The insert of pAOSG81-119 contains an open reading frame of 1047 base pairs which encodes a deduced polypeptide sequence of 349 amino acids (Fig. 2-3) with a calculated molecular mass of 38,931 Da. There is also a nine amino acid match between the deduced amino acid sequence of pAOSG81-119 and N -terminal amino acid sequence data that had been previously obtained using gel-purified 36 kDa alternative oxidase protein. Because of some ambiguity in protein sequencing, it was not possible to determine the specific N- terminal amino acid. However, the sequencing data do indicate that Ala-64 is the most likely N-terminal amino acid, and our analyses are based upon this assumption. The nine amino acid match begins at Leu-67 of the protein deduced from pAOSG81, as indicated in Fig. 2-3. If the first 63 amino acids of the deduced protein were removed during import into the mitochondria, then the mature protein would contain 286 amino acids and have a calculated molecular mass of 32,200 Da and a calculated isoelectric point of about 6.6, which is in relative agreement with the observed isoelectric point of 7.2-7.3 (39). Helical wheel projections of the putative transit peptide of the protein deduced from the sequence of pAOSG81 indicate that this region of the protein is capable of forming segments of amphiphilic helices (data not shown). Three adjacent regions of the deduced protein are predicted to be in alpha helical conformations and are likely to be membrane spanning as determined by the statistical Rao-Argos protein structure analysis program (Fig. 2-4). These three regions are as follows: a region from amino acids 171 to 202 (numbering based on the unprocessed Protein), a region from amino acids 207 to 228, and a region from amino acids 41 233 to 262. Furthermore, between these regions of predicted helical conformation there are regions which are predicted to form turns in the protein backbone (40). DISCUSSION We have isolated a cDNA clone, pAOSG81, representing a S. guttatum nuclear gene, which we have called aoxI, encoding a precursor alternative oxidase protein with a calculated molecular mass of 38.9 kDa and an apparent molecular mass of 42 kDa. The fusion protein expressed from pAOSGSl in E. coli has an apparent molecular mass of 48 kDa and was recognized by monoclonal antibodies to the alternative oxidase proteins. The observation that the 42 kDa precursor protein is nuclear-encoded is consistent with the results of Bertrand et a1. (41), which indicate that a single nuclear gene encodes the alternative oxidase in Neurospora crassa. Our results do not rule out the possibility that there is more than one gene encoding the alternative oxidase in S. guttatum. However, this is unlikely since 1) it appears that a single 42 kDa protein was immunoprecipated from the products made in vitro from total poly (A)+ RNA (Fig. 2-1A & 2-1B, lane 1); 2) transcripts of a single size were homologous to the insert of pAOSGSl on Northern blots of total RNA from S. guttatum appendix tissue (data not shown); and 3) Southern blots of genomic DNA probed with pAOSG81 also indicate that the nuclear gene, am], is present in a single copy (data not shown). It is also possible that one of the alternative oxidase proteins is encoded by the mitochondrial genome. However, this is likewise unlikely since cycloheximide (an 42 Figure 2-3. Nucleotide and deduced amino acid sequence of the insert of pAOSG81-119. Both strands of the insert of pAOSGSl-119 were sequenced by making unidirectional, single-stranded deletions of pAOSGSl-119 and pAOSG81i- 119 and sequencing by the dideoxy chain termination method. The triangle indicates the assumed start codon and first amino acid of the protein. The open box indicates the region of the deduced amino acid sequence which matches the amino acid sequence previously obtained by chemical sequencing. The first in- frame stop codon is indicated by the star. GAATTCCGAGCCAGTGCAGILLLLiLLLLILLILrrLLACCACCCATTTCTGTTCTGCCACCCGCCGGAGATC CAG CTC Q L GCG AGC 500 CCC ATG TGG ATC H I GTG CTG V L GAG GCC E A 980 CTG CCG L P TAC CAG Y Q 20 4 120 AGT CAC GTC S H V 220 CAG AGA GCG Q R A GCA GGA ACA A G T AAA GAG GAC K E D ACC ACC ATT T T I 600 ATG CTG GAG H L E 700 AGG GCC CTC R A GCG GTG CAG A V O ATC CAC TCC I H S CAG GGC TCC Q G S 1080 GAT CTT GAG D L E 120 I CTCGAAGACATTAGTATAACTATAAGATATTTCTATTCGAGTACTATTAACGATCGATCG 1320 ACC T CTG 800 GGG TAC ACC CTG L 0 GTA CCT CAG V P Q CTC TGG CCG L H P GGG AAG GTG G K V 420 TCC GAG TGG S E H 520 GAC AAG CTG D K L GTC GCG GCG A GAG GAG GCC E E A GTC TTC TTC V F F 900 ACC GAG TTC T E F 1000 CTG CGC GAC D AAG ACG ACG K T T 1340 140 TAC Y CCC CCG CGC GCC 620 GTG GAG E GTC V 1100 CCC P 122 43 0 TTG CCT GCC L P A 240 AGC TGG TTC S H F 340 CCG GGT GAG P G E TGG ACC TGC H T C TTG CGC ACC L R CCG GGC ATC P G H 720 AAC GAG CGG N 820 GCC TAC TTC A Y F AAG GAC ATC K D I GTC V ACC GTC T V GCG CCG CTC A P L 0 CTC TCG GAC D 440 TTC GTC V GTG V ATG H CTG 920 GAC GTC V 666 G 1360 160 CGT CCC ACC R P T CCC CCC CGC P P R GGC GGC GCC G A AGG CCA TGG R P H 540 AAG GCC CTC K A L 640 GGC GGC GTA G G V CAC CTG ATG H L H 666 TAC CTG G Y L AGT GGG GCC 5 A 1020 CGC GCA GAC R A D 1120 TAC cxc roa v u 't 24 GCG 260 CAC GAG GAG E CGG R ATC GAG E ATG ATG H GAC ACG GCG A GCG AGC ACG A S T 360 AAG GAG GCG K E A 460 ACG TAC CAG T Y Q TGG CCC ACC H T CTC CAC CTC L H L TTC ATG GAG F H E 840 TCC CCC AAG S P K 940 CAG GAC TGC O D C GCA A CAC CAC H H 1380 80 ACC 5 ACC S TCG CGT CTG S R L 180 TCA CTC CTG S L GTC V 100 ACC GCT T A GGC G GGA TGT TCA G C S CTC TGC L C AGG R 200 GCG GCG GCG A A A 300 CTG L 280 TCA GCT CCC S A P CAG GAC GGA Q D G GGG G AAG GAG K E GTG V GCG A 560 GAC AAG GTC V TTC CCC P GTG AGC TAC V S Y GAC CTC TCC D L S ATC TTC TTC I F F 660 TCC CTC CGC S L R 760 GCG CAG CCG A Q P GCC CAC CGG A H R GCC CCG GCC A P A 1040 CGC R GAC GTC AAC D V H 1140 TGCGCCGCCGGCCCGTTAATTGTTGCCGATCGACAGACGCCAAGATGGTCGATCGAAGT 0 1260 ATATATATGATCTGTTAAAAATTAAGAATTAACAG 1400 CTCCGCTGATTTCTTAAIIILUULIIIDULIILLITLLAIblILIAGGGTCCTCCTATATATATATGAACCACTGATTTCGGAATTCATCAATTC 128 AAACCATCTCCATATATATGTAGGTGGGAGGT 860 GTT ATC CAC H 0 CCC GTG CCG A V P 480 GAC CTG CAC L H 580 CGG CGG TAC R R Y TTC GAG CAC F E H TGG TAC GAG H Y E GTG GGC TAC V G Y 960 GCC CTG GAC A L D 1060 TTC GCC TCC F A S 1160 400 CCG TCC AAG P S K CAC CAC H H TGC CGG C R 080 AGC GGC GGG G G 780 GCG CTG A L C66 880 GAG GAG E E TGG CGG H R GAC GTC CAT D V H 1180 1300 Figure 24. Secondary structure prediction for the protein deduced from the nucleotide sequence of the insert of pAOSGSl-119. The solid line represents the values obtained using the Seqanal Predictor program. The dashed line represents the values obtained using the Chou-Fasman program. Abscissa indicates amino acid position starting from the first amino acid of the putative precursor protein. Ordinate indicates P, with a window of 15 amino acids (52). 45 46 inhibitor of cytoplasmic translation but not of mitochondrial translation) blocks expression of the alternative oxidase proteins in S. guttatum appendix tissue sections which were treated with salicylic acid (Rhoads and McIntosh, in preparation). This raises the intriguing possrbility that the 35-, 36-, and 37 kDa proteins are all posttranslationally-modified products of the 42 kDa protein. From the nucleotide sequence of pAOSGSl and the amino acid sequence of the 36 kDa alternative oxidase protein we determined that the N-terminus of the 36 kDa protein is internal to the amino acid sequence of the 38.9 kDa protein deduced from the nucleotide sequence, though the exact site of processing is not clear since the exact N-terminal amino acid of the 36 kDa protein was not determined from the sequencing. The putative transit peptide possesses many properties which are common to previously described mitochondrial transit peptides (42). It has a high content of amino acids considered to be either alpha helix formers (a total of 28) or neutral (a total of 22) relative to the number of helix breakers (a total of 13). The transit peptide also has regions that are likely to form amphiphilic helices and it has a net positive charge. Similar to many other transit peptides of mitochondrial proteins, there is a high abundance of the amino acids Ala, Leu, Arg, and Ser and with low abundance of Asp, Glu, Ile, and Lys (42). These data are consistent with the hypothesis that the 42 kDa protein is a precursor of one or more of the alternative oxidase proteins with a 63 amino acid transit peptide which is removed as the protein is transported into the mitochondria. 47 It has been shown that the alternative oxidase of another member of the Aroid family, Arum maculatum, is associated with the inner surface of the inner mitochondrial membrane (43). Analysis of our sequence data indicates that the region of the 42 kDa alternative oxidase precursor protein that is most likely associated with the mitochondrial membranes of S. guttatum is between amino acids 108 to 199. This region contains three stretches of amino acids which are predicted to form transmembrane alpha helices. There have been numerous proposals (44,45,46) concerning the identity of the higher plant alternative oxidase. Flavin has been proposed as a cofactor (46); however, this seems unlikely since the affinity of mitochondrial flavoproteins for 02 is too low and the kinetics of reoxidation are too slow (3,46). Also, auto- oxidizable flavoproteins produce H202 (45,46), whereas the alternative pathway produces H20 (44,45,46). Nonheme iron (3,44,46) is an unlikely cofactor since no clear electron paramagnetic resonance (EPR) signal from a nonheme iron has been correlated with the presence of the alternative pathway (47). Copper (44,45), is likewise an unlikely candidate because of the lack of a clear EPR signal (48). Nevertheless, iron and copper cannot yet be ruled out completely since it is possrble that an EPR signal from one or the other is masked (44,46) as in the case of coupled binuclear copper sites, designated as "type 3" copper (49). ' Since there is little precise structural data on the active sites of enzymes containing these cofactors, the deduced amino acid sequence of the alternative oxidase of S. guttatum does not allow us to draw many conclusions about possible cofactors of this enzyme. However, we can conclude from the sequence that this protein 48 alone does not contain consensus iron-sulfur protein sequence motifs which form Cys ligands in the 4Fe-48 ferredoxins and the 2Fe-28 type of ferredoxins (50,51). It has also been proposed (45) that the alternative oxidase itself may transfer electrons directly from ubiquinone to oxygen and that it may, therefore, contain a ubiquinone binding site. Since the structures of quinone binding sites are still unknown, we cannot determine, on the basis of deduced protein sequence alone, if the alternative oxidase protein encoded by pAOSG81 contains such a site. The isolation of an alternative oxidase cDNA clone is a new step toward answering questions concerning the role of the oxidase in higher plants and other organisms. While the mechanism and function of the alternative oxidase cannot be deduced in the absence of more biochemical and physiological data, it is now possible to use the gene to investigate the developmental regulation of alternative pathway appearance in S. guttatum and other higher plants. We intend to explore the role of the alternative oxidase in non-thermogenic plants through genetic manipulations that produce plants modified in their expression of the alternative oxidase. 49 AWOWLEDGEMENTS We would like to thank the following: Dr. B. J. D. Meeuse for providing the S. guttatum plants; Dr. Thomas E. Elthon and Roxy Nickels for isolating the 36 kDa protein and for helpful advice; Dr. J. F. Vandekerckhoue for sequencing the 36 kDa protein; Dr. David Hondred for providing the S. aureus cells and for helpful advice; Dr. John Fitchen for helpful advice; Dr. Niels Nielson for the use of the Editbase program; and Dr. A. Crafts for the use of the Seqanal Package. This work was supported in part by U. S. Department of Energy contract DE- AC02-76ERO-1338 and National Science Foundation grant DCB-89045 18. 10. 11. 12. 13. REFERENCES Henry, M. F. & Nyns, E. J. (1975) Subcell. Biochem. 4, 1-65. Bendall, D. S. (1958) Biochem. J. 70, 381-390. Bendall, D. S. & Bonner, W. D. Jr. (1971) Plant Physiol. 47, 236-245. Rich, P. R. & Moore, A. L. (1976) FEBS Letters 65, 339-344. Storey, B. T. (1976) Plant Physiol 58, 521-525. Storey, B. T. & Bahr, J. T. (1969) Plant Physiol. 44, 126-134. Moore, A. L. & Bonner, W. D. Jr. (1982) Plant Physiol. 70, 1271- 1276. Meeuse, B. J. D. (1975) Ann. Rev. Plant Physiol. 26, 117-126. Meeuse, B. J. D. (1966) Sci. Am. 215, 80-88. Lambers, H. (1985) in Encyclopedia of Plant Physiology: Higher Plant Cell Respiration, eds. Douce, R. & Day, D. A. (Springer, Berlin), pp. 418-473. Turner, J. F. & Turner, D. H. (1980) in The Biochemistry of Plants: A Comprehensive Treatise, Vol. 2, Metabolism and Respiration, ed. Davies, D. D. (Academic Press, New York), pp. 279-316. Dry, I.B., Moore, A.L., Day, D.A., & Wiskich, J .T. (1989) Archiv. Biochem. Biophys. 273, 148-157. McNulty, A.K. & Cumming, W.R. (1987) Plant Cell Environ. 10, 319-325. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 26. 27. 50 Stewart, C.R., Martin, B.A., Reding, 1.. & Cerwick, S. (1990) Plant Physiol. 97, 755-760. Elthon, T. E. & McIntosh, L. (1987) Proc. Natl. Acad. Sci. USA 84, 8399-8403. Elthon, T. E., Nickels, R. I... & McIntosh, L. (1989) Plant Physiol. 89, 1311-1317. Lambowitz, A., Sabourin, J. R., Bertrand, H., Nickels, R. & McIntosh, L. (1989) M01. and Cell Biol. 9, 1362-1364. Lambowitz, A. M. & Slayman, C. W. (1971) J. Bacterial 108, 1087- 1096. Bertrand, I-L, Argan, C. A. & Szakacs, N. A. (1983) in Mitochondria 1983, ed. Schweyen, R. J., Wolf, K. & Kaudewitz, F. (Walter de Gruyter & Co., Berlin), pp.495-507. Raskin, I., Ehmann, A., Melander, W. R. & Meeuse, B. J. D. (1987) Science 737, 1601-1602. Elthon, T. E, Nickles, R. L & McIntosh, L. (1989) Planta 180, 82- 89. Elthon, T. E. & McIntosh, L. (1986) Plant Physiol. 82, 1-6. Schwitzguebel, J. P. & Siegenthaler, P. A. (1984) Plant Physiol. 75, 670-674. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Blake, N. S., Johnston, K. H., Russel-Jones, G. J. & Gotschlich, E. C. (1984) Anal. Biochem. 136, 175-179. Chamberlin, J. P. ( 1979) Anal. Biochem. 98, 132-135. Konigsberg, W. M. & Henderson, 1.. (1983) Methods Enzymal. 91, 254-259. Vandekerckhoue, J. F., Bauw, G., van Damme, J ., Puype, M. & van Montagu, M. (1987) in Methods in Protein Sequence Analysis, ed. Walsh, K. (Humana Press, Clifton, New Jersey), pp. 261-276. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 51 McIntosh, L. & Cattolico, R. A. (1978) Analytical Biochem. 91, 600- 612. Cashmore, A. R. (1982) in Methods of Chloroplast Molecular Biology, eds. Edelman, M., Hallick R. B. & Chua, N.-H. (Elsevier Biomedical Press, Amsterdam), pp. 387-392. Anderson, D. J. & Blobel, G. (1983) Methods in Enzymal. 96, 111- 120. Hondred, D., Wadle, D. M., Titus, D. E. & Becker, W. M. ( 1987) Plant Mal. Biol. 9, 259-275. Short, J. M., Fernandez, J. N., Sarge, J. A. & Huse, W. D. (1988) Nucleic Acids Res. 16, 7583-7600. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Dale, R. M., McClure, B. A. & Houchins, J. P. (1985) Plasmid 13, 31.41. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977) Proc. Natl. Acad. Sci. USA 74, 5463-5 467. von Heijne, G. (1987) in Sequence Analysis in Molecular Biology, (Academic Press, New York), pp.81-118. Seqanal Package, Copyright © 1987, A. Crofts, Univ. of 111., Urbana, Il 61801. Hiser, C. & McIntosh, L. (1990) Plant Physiol. 93, 312-318. Cohen, F. E., Abarbanel, R. M., Kuntz, I. D. & Fletterick, R. J. (1983) Biochemistry, 22, 4894-4904. Bertrand, H., Argan, C. A. & Szakaco, N. A. (1983) in Mitochondria 1983, eds. Schwegen, R. J. & Kandewitz, F. (Walter de Gruyter & Co., Berlin), pp. 495-507. von Heijne, G., Steppuhn, J. & Herrmann, R. G. (1989) Eur. J. Biochem. 180, 533-545. 43. 44. 45. 47. 49. 50. 51. 52. 52 Rasmussom, A. G., Moller, I. M. & Palmer, J. M. (1990) FEBS Letters 259, 311-314. Storey, B. T. (1980) in The Biochemistry of Plants: A Comprehensive Treatise, Vol. 2, Metabolism and Respiration, ed. Davies, D. D. (Academic Press, New York), pp. 125-195. Siedow, J. N. (1982) Recent Adv. in Phytochem. 16, 47-84. lance, C., Chauveau, M. & Dizengremel, P. (1985) in Encyclopedia of Plant Physiol: Higher Plant Cell Respiration, eds. Douce, R. & Day, D. A. (Springer, Berlin), pp. 202-247. Moore, A. L., Rich, P. R., Bonner, W. D. Jr. & Igeldew, W. J. (1976) Biochem. Biophys. Res. Commun. 72, 1099-1 107. Huq, s. & Pahner, J. M. (1978) FEBS Letters 95, 217-220. Solomon, E. I. (1981) in Copper Proteins, ed. Spiro, T. G. (Wiley- Interscience, New York), pp. 41-108. Yasunobu, K. T. & Tanaka, M. (1980) Methods in Enzyrnal. 69, 228- 238. Stout, C D. (1982) in Iran-Sulpher Proteins, ed. Spiro, T. G. (Wiley- Interscience, New York), pp. 97-146. Chou, P. Y. & Fasman, G. D. ( 1974) Biochemistry 13, 211-222. m3 SALICYLIC ACID REGULATION OF RESPIRATION IN HIGHER PLANTS: ALTERNATIVE OXIDASE EIPRESSION DavidMRhoadsandLeeMcIntosh Department of Biochemistry and Department of Energy Plant Research Laboratory Michigan State University East Lansing, MI 48824 In Press in The Plant Cell. 53 54 ABSTRACT Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (four days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid in the appendix is very low early on D-l, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in salicylic acid by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of salicylic acid to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to salicylic acid was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of 30x]. 55 INTRODUCIION All higher plants examined contain two pathways for mitochondrial electron flow: the cytochrome respiratory pathway and the alternative respiratory pathway (Bendall, 1958; Bendall and Bonner, 1971). The alternative pathway diverges from the cytochrome pathway after the ubiquinone pool (Bendall and Bonner, 1971; Rich and Moore, 1976; Storey, 1976). Therefore, electron flow through the alternative pathway is not coupled to ATP synthesis at the two sites of proton gradient formation (complex III and complex IV) that are downstream of the ubiquinone pool (Storey and Bahr, 1969; Moore and Bonner, 1982). The energy of electron flow through the alternative pathway is not conserved as chemical energy, but is lost as heat (Storey and Bahr, 1969). Specific floral tissues of some aroid plants develop such a high level of the alternative pathway that they become thermogenic (Meeuse, 1975; Day et al., 1980; Raskin et al., 1987). The day the inflorescence of the aroid species voodoo lily blooms (D-day), the capacity of the alternative pathway in the appendix tissue is 300-1000 natoms O/min/mg protein and the temperature of the tissue increases about 9-12°C above ambient temperature (Raskin et al., 1987). Immature appendix tissue (8 to 2 days prior to blooming, D-8 through D-2) has a relatively low alternative pathway capacity, 50- 100 natoms O/min/mg protein. In voodoo lilies, the heat produced during blooming is used to volatilize foul smelling compounds, such as indoles, which attract insect pollinators (Meeuse, 1966). The role of the alternative pathway in non aroid plants is unknown (Lambers, 1985). 56 The appearance of three antigenically related mitochondrial proteins with apparent molecular masses of 35-, 36-, and 37 kD strongly correlates with the activity of the alternative oxidase, the terminal oxidase of the alternative respiratory pathway, in voodoo lily appendix tissue (Elthon and McIntosh, 1987). A 42 kD protein that is a putative precursor of all three of these alternative oxidase proteins has been identified (Rhoads and McIntosh, 1991). Since only the 42 kD protein was immunoprecipitated from products made by in vitro translation of total voodoo lily RNA, it is likely that the 35-, 36-, and 37 kD proteins are past- translationally modified products of the 42 kD protein. A cDNA clone, pAOSGSl, corresponding to the nuclear gene, aaxI, encoding the 42 kD protein has been isolated and characterized (Rhoads and McIntosh, 1991). Salicylic acid has been shown to be an endogenous "trigger" of thermogenesis in voodoo lily appendix tissue (Raskin et al., 1987). Salicylic acid is produced in the male floral region of the inflorescence and moves into the appendix beginning early on D-1 and "triggers" thermogenesis at about noon of D- day (Raskin et al., 1987, 1989). The level of salicylic acid in the appendix increases from below 100 ng/g fresh weight as late as D-2 to more than 1.0 ug/g fi'esh weight in the evening of D-1 (Raskin et al., 1989). Salicylic acid has also been shown to dramatically increase both the alternative pathway activity and the levels of the 35-, 36-, and 37 kD alternative oxidase proteins when it is applied to voodoo lily appendix tissue sections (Elthon et al., 1989b). Prior to the application of salicylic acid, the 37 kD protein is constitutively expressed and is present at a moderate level in immature appendix tissue. All three alternative oxidase proteins 57 are present in great abundance in the tissue sections following incubation in a phosphate-buffered solution containing salicylic acid. In addition, Raskin et al. (1989) showed that light is required for salicylic acid to cause thermogenesis in appendix tissue sections. Salicylic acid may be involved in flowering in many plant species (Raskin, 1992) and in systemic acquired resistance (SAR) in others (Malamy et al., 1990; Métraux et al., 1990; Rasmussen et al., 1991). Furthermore, and the endogenous level of salicylic acid is very high in a few species of higher plants, including rice (Raskin et al. 1990). However, the pathway by which salicylic acid is produced and the mechanisms by which salicylic acid acts remain unknown. Therefore, identification of the mechanism(s) by which salicylic acid regulates gene expression in voodoo lilies will be valuable in understanding the role(s) of salicylic acid in various plant tissues, the physiology of plant responses to pathogens, and the physiology of thermogenic tissues. MATERIALS AND METHODS Plant Material Voodoo lily (Sauromatum guttatum) plants were maintained as previously described (Elthon and McIntosh, 1987). The day the inflorescence blooms and the appendix tissue of the spadix heats is referred to as D-day (for anatomy see Meeuse, 1966). Other developmental stages of the plant are indicated as the number of days before or after D-day (D-l being the day before and D+1 the day after D—day). Between the time the inflorescence begins to 58 develop and the end of D-2, the inflorescence is referred to as "immature." SaliqlicAcidandInhibitorTreatmentafT’nsueSections. At8AM (time 0) of D-6, D5, or D-4 the appendix tissue of a voodoo lily plant was cut into 1.0-1.5 cm sections. Sections were placed into 10 mL beakers containing 1.0 mL of phosphate buffer (10 mM KHZPO, and 50 ug/mL streptomycin) and a drop of buffer was placed on top of each section. For the time course experiments, one set of sections at each time point was incubated in phosphate buffer alone and one set in phosphate buffer containing 1.0 mM salicylic acid. The portion of the appendix remaining on the plant until the inflorescence bloomed is referred to as the D-day tissue sample. Application of calorigen served as a positive control for the induction of the accumulation of the alternative oxidase proteins (Raskin et al., 1987). Calorigen, a crude extract of the male floral region of the inflorescence, was prepared as described by Raskin et a1. (1987). For the inhibitor experiments, actinomycin D (60 ug/mL final concentration; Sigma), an inhibitor of transcription, was added to one of four sets of sections and cycloheximide (15 ug/mL final concentration; Sigma), an inhibitor of translation by 808 ribosomes, was added to a second set. After 2 hr of incubation at room temperature, salicylic acid was added (1.0 mM final concentration) to the two experimental sets of sections and the positive control set. The negative control set was incubated in phosphate buffer for the duration . of the experiment. The beakers were covered and incubated at room temperature 59 for an additional 22 hr. For the nuclear, in vitro transcription experiments, six to eight sections from all along the length of an appendix were incubated in phosphate buffer for 24 hr while other sections from the same appendix were incubated in phosphate buffer containing 1.0 mM salicylic acid. Isolation of Mitochondria and Respiration Assays. Mitochondria were isolated by a modification of the procedure of Schwitzguebel and Siegenthaler (1984) as described previously (Rhoads and McIntosh, 1991). The oxygen content of air- saturated water was estimated according to Estabrook (1987). Respiration rates were measured as oxygen uptake using a Rank Brothers (Cambridge, UK) oxygen electrode. The capacity of the alternative pathway was measured in 1.0 mL of reaction medium (250 mM sucrose, 30 mM 3-(N-morpholino)propanesulfonic acid (Mops), pH 6.8) at 25 °C with 1 mM NADH as the substrate. Carbonylcyanide p- trifluoro-methoxyphenylhydrazone (FCCP) was added to 0.5 uM after NADH in order to diminish the electrochemical gradient prior to measurement of the pathway capacity (Elthon et al., 1986). The capacity of the alternative pathway was taken as the rate of oxygen uptake sensitive to 1 mM salicylhydroxamic acid in the presence of 1 mM KCN (Elthon et al., 1987). The capacities were standardized by dividing each value by the total amount of protein in the reaction (which is directly proportional to the number of mitochondria in the assay). The amount of total protein in each sample was determined by the procedure of Larson et al. (1986). 60 Gel Electrophoresis, Immunoblotting, and Antisera. SDS-PAGE and immunoblotting were performed as described previously (Elthon et al., 1987). Antibodies used were the AOA monoclonal antibodies raised to the 36 kD alternative oxidase protein of voodoo lily, and which recognize all three alternative oxidase proteins of voodoo lily (Elthon et al., 1989a). Since the same amount of total mitochondrial protein was loaded onto each lane of each polyacrylamide gel, the protein detected on protein blots represents the proportion of total mitochondrial proteins that constitutes the alternative oxidase proteins. Plasmid Insert Isolation and Radiolabeh'ng. The 1400-hp EcoRI insert of pAOSG81 (Rhoads and McIntosh, 1991) was purified by as described in Maniatis et al. (1989) except that the elution buffer was 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.05% SDS, and 0.3 M LiCl and the insert was electrophoresed through two successive polyacrylamide gels. The purified fragment was resuspended to a concentration of 60 ng/ul in Tris-EDTA. The purified inserts were used to make DNA radiolabeled with a-nP-dATP (Amersham Corporation) by the random primer method (Feinberg and Vogelstein, 1983). Specific activity of the DNA probes was routinely about 107 cpm/ug template, as determined by the sodium phosphate wash method (Maniatis et al., 1989). RNA Isolation and Blots. Total RNA was isolated from frozen (in liquid N2) voodoo lily appendix tissue by the procedure of McIntosh and Cattolico (1978) as described by Rhoads and McIntosh (1991. RNA was separated on agarose gels 61 containing formaldehyde as described by Ausubel et al. (1987), and transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) as described by Maniatis et al. (1989). All RNA blots were probed with radiolabeled DNA made using the purified insert of pAOSG81 as described above. Hybridization was done under high-stringency hybridization conditions as described by Maniatis et al. (1989). Autoradiography using Kodak diagnostic film XAR-S was used to view the hybridization results as described by Ausubel at al. (1987). The amount of radiolabeled probe that hybridized to each sample was determined using a Phospholmager (Molecular Dynamics, Sunnyvale, CA). Since each lane of each RNA blot contained the same amount of total RNA, the amount of hybridized probe detected represents the proportion of total RNA that constitutes the alternative oxidase transcript. Isolation of Nuclei and In Vitro Transcription. Nuclei were isolated following method 11 of Luthe and Quatrano (1980a) except that the discontinuous gradients contained 25, 50, and 80% (v/v) Percoll layers, all on top of a 2.0 M sucrose cushion and the centrifugation for the washesiwas done at 1100g. Most of the nuclei were found at the interface between the 80% Percoll and 2.0 M sucrose layers as determined by fluorescence microscopy of a 50/50 (v/v) mixture of nuclei and stain [20 mg/mL 4’,6-diamidino-2-phenylindole (DAPI), 0.1X phosphate- buffered saline, 0.05% (w/v) sodium azide, 90% (v/v) glycerol]. Nuclei were isolated from the appendix tissue of voodoo lily plants on D-6, D-5, D4, or on D- day. In a separate experiment, tissue sections from a D-6 plant were incubated 62 for 24 hr in phosphate buffer or in phosphate buffer containing 1.0 mM salicylic acid. Nuclei were then isolated from the appendix tissue sections. In vitro RNA synthesis was performed essentially as described by Luthe and Quatrano (1980b). The final concentrations of ATP and GTP were both 0.5 mM, 3-5 uL of a-32P- UTP (20 mCi/mL, 800 Ci/mmol) was used in each synthesis, and the final volume wasZOO uL Isolation of In Vitro Synthesized RNA. After the in vitro synthesis, RNase-free DNase I was added to a final concentration of 0.5 ug/uL and the mixture was incubated at 26°C for 5 min. An equal volume of proteinase K mix (200 ug/mL proteinase K, 2% SDS, 10 mM EDTA, 20 mM Tris-HCl pH 7.4, and 200 ug/mL yeast tRNA) was added to each sample and the samples were incubated at 37°C for 30 min. Each sample was extracted twice with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1[v/v]). The RNA was precipitated twice using ammonium acetate and ethanol as described by Maniatis et al. (1989) and resuspended in 50 uL of STE (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA). NaOH was added to a final concentration of 0.2 N, and the RNA was incubated on ice for 10 min, followed immediately by the addition of 1 M Hepes to a final concentration of 0.25 M. The RNA was ethanol precipitated and resuspended in 50 uL of Tris-EDTA. The total number of counts in each RNA sample was determined by the sodium phosphate wash method (Maniatis et al., 1989). To compare gene expression at different developmental stages, we used the same number of total counts per minute of the RNA made from immature 63 appendix tissue nuclei as the number of total counts per minute of the RNA made from the D-day appendix tissue nuclei. To compare gene expression in salicylic acid treated and untreated tissue, we used the same number of total counts per minute of the RNA made from untreated appendix tissue nuclei as the number of total counts per minute of the RNA made from the nuclei from the salicylic acid- treated appendix tissue. Hybridintian of In Vitro Synthesimd RNA to DNA Probes. The unlabeled DNA samples used to determine the levels of the specific transcripts produced by in vitro transcription in isolated nuclei are as follows: 1) the single-stranded form of the phagemid pAOSG81, corresponding to the antisense of the 1.6-kb alternative oxidase transcript; 2) a single-stranded form of phagemid p25SSGlO; and 3) denatured pSAc3 DNA (a generous gift of Dr. R. B. Meagher). Phagemid pZSSSGIO is a cDNA clone that has been partially sequenced and corresponds to a voodoo lily 258 nbosomal RNA gene based on the observation that it has >90% sequence similarity to bases 846 to 1982 of the rice 25S rRNA gene (Takaiwa at al., 1985). Plasmid pSAc3 is a genomic clone that represents a soybean actin gene (Shah at al., 1982). The DNA samples were denatured and applied to a nitrocellulose membrane as described by Ausubel et al. (1987). Prehybridization, hybridization, and washing conditions were essentially as described by Ausubel et al. (1987), except that 50 mM sodium phosphate was used instead of potassium phosphate, 25 ug/mL of salmon sperm DNA, and 1X Denhardt’s solution (1X Denhardt’s solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA) 64 were used in the prehybridization and hybridization solutions, and 12.5% dextran sulfate was used in the hybridization solution. Hybridization was performed at 42°C for 72 hr and the filters were washed 4 times in 2X SSC (1X SSC is 0.15 M NaCl, 0.015 sodium citrate) containing 0.1% (v/v) SDS for 5 min at room temperature and once in 0.1X SSC containing 0.1% (v/v) SDS for 5 min at 50°C. The amount of radiolabeled RNA that hybridized to each probe was visualized by autoradiography (Maniatis at al., 1989). RESULTS DevelopmentalEpressionaftheAlternativeridaseGeneafVoodoaLily. Figure 3-1A shows that the 37 kD alternative oxidase protein was constitutively expressed in the appendix tissue and was present at about the same level in the appendix on D-5 and D-4 (lanes 1 and 2). The level increased significantly by 10 AM of D-3 (lane 3) and reached a peak on D-day (lane 6). The levels of the 35- and 36 kD proteins were very low in the appendix tissue at D-5 and D-4 (lanes 1 and 2) and increased dramatically by 10 AM of D-3 (lane 3). The levels continued to increase steadily on D-2 (lane 4) and D] (lane 5), and were the highest on D-day (lane 6). It is interesting to note that the levels of the alternative oxidase proteins were already fairly high by D-3, even though thermogenesis does not occur until about noon on D-day. Figure 3-lB shows that the 1.6-kb alternative oxidase transcript accumulated to a very high level in the appendix tissue of the voodoo lily 65 Figure 3-1. Developmental Expression of the Alternative Oxidase Proteins and Transcript in Voodoo Lily Appendix Tissue. Panel A: Immunoblot of total mitochondrial proteins from a single voodoo lily appendix at different days during inflorescence development: D-5, lane 1; D-4, lane 2; D-3, lane 3; D-2, lane 4; D-l, lane 5; D-day, lane 6. Mitochondria were isolated at 10 AM each day and assayed for alternative pathway capacity. The mitochondrial proteins (30 pg per lane) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the AOA monoclonal antibodies. Alternative pathway capacity in natoms O/min/mg protein of each mitochondrial sample is indicated at the bottom. Apparent molecular masses in kilodaltons are indicated to the left. Panel B: RNA blot of total RNA from a second appendix. All RNA was isolated from the appendix tissue at 10 AM of each day during inflorescence development: D-2, lane 1; D-l, lane 2; D-day, lane 3. The RNA (10 pg per lane) was separated by formaldehyde- agarose gel electrophoresis and transferred to nitrocellulose. The radiolabeled probe was made from the purified insert of pAOSG81. Transcript lengths in kilobases are indicated to the left. A B kD 1 2 3 4 5 6 kb 9.5 - 7.5 - 97.4 - 4_4 _ 66.2 ‘ 42.7- 2-4- 37.. 1.6 ~> “—3“ 31 .o- 1-4‘ 21 .5- 67 inflorescence by 10 AM of D-l (lane 2). Comparatively little of the 1.6-kb transcript was present at 10 AM of D-2 (lane 1). The level of the 1.6—kb alternative oxidase transcript, relative to the total RNA of the appendix tissue, was significantly higher at 10 AM of D-day (lane 3) than it was at 10 AM of D-1 (lanes 2). Time Course ofSalicylicAcidInducedAlternative OxidaseEtprenion. Figure 3-2 shows that the capacity of the alternative pathway in immature appendix tissue sections treated with salicylic acid reached a peak by about 16 hr after salicylic acid application. The capacity was slightly lower by 24 hr after application. Quantitatively the capacity at the 16 hr time point was almost three higher than the capacity at time 0, and the capacity decreased about 20% by the 24 hr time point. Figure 3-3A shows that the levels of the alternative oxidase proteins, particularly the 35- and 36 kD proteins, at each time point corresponded to the alternative pathway capacity (compare to Figure 3-2). A slight increase in the levels of the 35- and 36 kD proteins, compared to a time 0 and negative control, was observed as early as 5 hr after salicylic acid application (D. M. Rhoads and L. McIntosh, unpublished results). The peak in the accumulation of the proteins always occurred by 16 hr after salicylic acid application (lanes 16- and 16+), as did the peak in the capacity. Qualitatively, the levels of the proteins remained constant between the 16 and the 24 hr time points (lanes 24— and 24+), although the capacity usually decreased slightly (Figure 3-2). 68 Figure 3—2. Time Course Graph of Alternative Pathway Capacity in Response to Applied salicylic acid. Voodoo lily appendix tissue sections were incubated in phosphate buffer (triangles and broken line) or buffer plus 1.0 mM salicylic acid (circles and solid line). Mitochondria were isolated at each time point, and the capacity was determined using a Rank Brothers oxygen electrode. Results are averages of three experiments. . a A o. .. x \ \ \ \\ \ \ \ ‘ \i)\.”0) ‘7 69 #- H_ fit .1 -a 1 w. c\ .n A .. o 1 .,. .... 18 mmmmm .u 3.805 9:326 2885 9.8% 536 A‘. A A. A A " ‘ I\\ \. 4 \~. \\ \\ Figure 3-2. Time Course Graph of Alternative Pathway Capacity in Response to Applied salicylic acid. Voodoo lily appendix tissue sections were incubated in phosphate buffer (triangles and broken line) or buffer plus 1.0 mM salicylic acid (circles and solid line). Mitochondria were isolated at each time point, and the capacity was determined using 3 Rank Brothers oxygen electrode. Results are averages of three experiments. 69 +84 “upon”..- ‘ s s ‘s “‘1 IN s s ‘s 16 24 "ME (m7 70 Figure 3-3B shows that the accumulation of the 1.6-kb alternative oxidase transcript corresponded precisely to the accumulation of the alternative oxidase proteins. Transcript accumulation above the control level was apparent by 4 hr after the application of salicylic acid (lanes 4- and 4+). The accumulation was increased by the 8 hr time point (lanes 8- and 8+). The highest level of the transcript was reached by 16 hr after salicylic acid application (lanes 16- and 16+), and the peak level was maintained until at least 24 hr after the application (lanes 24- and 24+). Lanes D5 and D0 of Figure 3-3 show the level of the alternative oxidase transcript that was present at D-5 and D-day, respectively. It is interesting to note that the amount of the 1.6-kb transcript was slightly higher at time 0 (lane D5) than in the negative control at the 4 hr time point (lane 4»). Furthermore, the level of the alternative oxidase transcript increased steadily over the course of the experiment rather than rapidly at a specific time point. Inhibition of the Efiectl of Applied Salicylic Acid. Figure 3-4A shows that application of salicylic acid to immature (D-4) appendix tissue sections for 24 hr induced the accumulation of the alternative oxidase proteins, particularly the 35- and 36 kD proteins (lane 2), relative to the amount present in untreated tissue sections (lane 1). The accumulation of the proteins was significantly blocked by incubating the tissue sections for 2 hr in a cycloheximide solution (lane 4) or for 2 hr in an actinomycin D solution (lane 3) prior to the addition of salicylic acid for 22 hr. Slightly more of the 35- and 36 kD proteins was present in the sample treated with actinomycin D than in the sample treated with cycloheximide. This 71 Figure 3-3. Time Course of Accumulation of Alternative Oxidase Proteins and Transcript after Salicylic Acid Application. Panel A: Immunoblot showing the levels of the alternative oxidase proteins at specific time points after salicylic acid application. Voodoo lily appendix tissue sections were incubated in phosphate buffer (-) or buffer plus 1.0 mM salicylic acid (+) or buffer plus calorigen (lane C). Mitochondria were isolated from the tissue sections from a D-4 plant at time 0 (lane D4) or at 8 hr (lanes 8- and 8+), 16 hr (lanes 16- and 16+), 24 hr (lanes C, 24-, and 24+), or 48 hr (lanes 48- and 48+) after the application of salicylic acid. The capacity of the alternative pathway was determined. The mitochondrial proteins from each sample (30 pg per lane) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the AOA monoclonal antibodies. Panel B: RNA blot showing the level of the 1.6-kb alternative oxidase transcript at specific time points after salicylic acid application. Tissue sections from a D-S plant were frozen in liquid N; at time 0 (lane D5); 4 hr (lanes 4- and 4+), 8 hr (lanes 8- and 8+), 16 hr (lanes 16- and 16+), or 24 hr (lanes 24- and 24+) after salicylic acid application; and at 10 AM of D-day (lane DO). Total RNA was then isolated, separated by formaldehyde-agarose gel electrophoresis (10 pg per lane) and transferred to nitrocellulose. The radiolabeled probe was made from the purified insert of pAOSG81. 72 A D408 8161624244848 -+ —+-+-+ 37kD +—-.——-.--—-"' r? 3m +4- IO +09 I8 +3 '5‘: +23 1.6kb+-‘- '0 . . . 73 would be expected if actinomycin D did not completely inhibit transcription. Figure 348 shows that application of salicylic acid to immature (D-4) appendix tissue sections for 24 hr induced the accumulation of the 1.6-kb alternative oxidase transcript (lane 2) relative to the level of the transcript found in untreated tissue sections (lane 1). Again, the accumulation of the 1.6-kb transcript was blocked by preincubation in either cycloheximide (lane 4) or actinomycin D (lane 3). Nuclear, In Vitro Transcription. Figure 3-5A shows that nuclear, in vitro transcription of the voodoo lily rRNA gene(s) occurred in nuclei isolated from D-6 appendix tissue (slot 11), but in vitro synthesis of the alternative oxidase transcript was very low in the nuclei isolated from appendix tissue at this stage of development (slot 12). Figure 3-SB shows that in vitro transcription of rRNA gene(s) also occurred in nuclei isolated from D-day (mature) appendix tissue (slot M1), but in vitro synthesis of the alternative oxidase transcript was very low in these nuclei as well (slot M2). Figure 3-5C shows that in vitro transcription of rRNA gene(s) (slot C1) and the actin gene(s) (slot C2) occurred in nuclei isolated from control D-4 tissue, but in vitro synthesis of the alternative oxidase transcript was very low in these nuclei (slot C3). Finally, Figure 3-5D shows that in vitro transcription of rRNA gene(s) (slot D1) and the actin gene(s) (slot D2) also occurred in nuclei isolated from D-4 tissue treated with salicylic acid, but that in vitro synthesis of the alternative oxidase transcript was very low in these nuclei as well (slot D3). 74 Figure 3-4. Inhibition of Salicylic Acid Induced Expression of Alternative Oxidase Proteins and Transcript. Panel A: Immunoblot of the salicylic acid induced expression of the alternative oxidase and inhibition of expression. Tissue sections from a single voodoo lily appendix from a D-4 plant were incubated for 24 hr as follows: in phosphate buffer, lane 1; in buffer containing 1.0 mM salicylic acid, lane 2; in buffer plus actinomycin D for 2 hr followed by the addition of salicylic acid for 22 hr, lane 3; in buffer plus cycloheximide for 2 hr followed by the addition of salicylic acid for 22 hr, lane 4. Mitochondria were isolated after the 24 hr incubations and the capacity of the alternative pathway was determined for each sample. For the protein analysis, the mitochondrial proteins from each sample (30 ug per lane) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the AOA monoclonal antibodies. Alternative pathway capacity in natoms O/min/mg protein of each mitochondrial sample is indicated at the bottom. The results are representative of four experiments. Panel B: RNA blot of the salicylic acid induced expression of the alternative oxidase and inhibition of expression. The tissue sections from a separate appendix from a D-S plant were treated as follows for 24 hr: in phosphate buffer, lane 1; in buffer containing 1.0 mM salicylic acid, lane 2; in buffer plus actinomycin D for 2 hr followed by the addition of salicylic acid for 22 hr, lane 3; in buffer plus cycloheximide for 2 hr followed by the addition of salicylic acid for 22 hr, lane 4. Total RNA from each sample (10 pg per lane) was separated by formaldehyde- agarose gel electrophoresis and transferred to nitrocellulose. The radiolabeled probe was made from the purified insert of pAOSGSl. Results are representative of two experiments. 75 1234 M ._.._...-——--- as e 2 1 234 76 Figure 3-5. Nuclear, in vitro Transcription Assays. Panel A: Radiolabeled RNA made from D-6 nuclei was hybridized to nitrocellulose-bound, single-stranded DNA probes corresponding to the antisense strand of pZSSSGlo, slot 11; or pAOSG81, slot 12. Panel B: Radiolabeled RNA made from D-Day nuclei was hybridized to nitrocellulose-bound, single-stranded DNA probes corresponding to the antisense strand of pZSSSGIO, slot M1; or pAOSG81, slot M2. Panel C Radiolabeled RNA made from nuclei from untreated D-4 appendix tissue was hybridized to nitrocellulose-bound, single-stranded DNA probes corresponding to the antisense strand of pZSSSGlo, slot C1; pAOSG81, slot C2; or a double- stranded, denatured I-IindIII-MspI fragment of pSAc3, slot C3. Panel D: Radiolabeled RNA made from nuclei from salicylic acid treated D-4 appendix tissue was hybridized to nitrocellulose-bound, single-stranded DNA probes corresponding to the antisense strand of pZSSSGIO, slot SAl; pAOSG81, slot 8A2; or a double-stranded, denatured HindIII-Mspl fragment of pSAc3, slot 8A3. Nuclei isolated from the appendix tissue of a single voodoo lily plant on D-6 and on D-day were used for the experiment yielding the results shown in Panel A and Panel B. Nuclei isolated from appendix tissue sections of a single D-4 voodoo lily plant were used for the experiment yielding the results shown in Panel C and Panel D. The sections for Panel C were incubated in phosphate buffer for 24 hr, while the sections for Panel D were incubated in buffer plus 1 mM salicylic acid for 24 hr. 77 A D-6 B D-daY I2 _ M2 C -SA D +SA 78 DISCUSSION In the appendix tissue of the voodoo lily inflorescence, salicylic acid is an endogenous signal that "triggers" an increase in the capacity of alternative respiratory pathway, which results in thermogenesis (Meeuse, 1966; Raskin et al., 1987). The levels of the alternative oxidase proteins, particularly the 35- and 36 kD proteins, present at each day (from D-5 until D-day) in the development of the inflorescence corresponded to the level of the alternative pathway capacity. The level of the 1.6-kb transcript, which encodes the 42 kD alternative oxidase precursor protein, increased from a relatively low level at 10 AM on D-2 to a high level on D-1. The level of the 1.6-kb transcript remained high until at least 10 AM of D-day, when the level of each of the alternative oxidase proteins was highest and when thermogenesis of the appendix tissue is approaching a peak (Raskin et al., 1987, 1989). However, Raskin et al. (1989) have demonstrated that salicylic acid begins to appear in the appendix tissue early on D—l, but does not reach a peak until late in D-1 or during theudark period between D-1 and D-day. Taken together these data indicate that the capacity of the alternative pathway and the levels of the alternative oxidase proteins in the appendix tissue begin to rise relatively early in the development of the inflorescence (D-3), when the level of salicylic acid is, presumably, quite low. In contrast, the amount of the 1.6-kb transcript is relatively low until at least 10 AM of D-1, at which time there is a dramatic increase in the level. This increase may correspond to the appearance of salicylic acid in the appendix tissue. We have not determined the 79 level of salicylic acid in the appendix tissue used for this these experiments, but the peak in the salicylic acid level usually occurs late on D-1 (Raskin et al., 1989). The basal level (Fig. 3-3, lane D5) of the transcript probably accounts for the constitutive expression of the 37 kD protein and allows for the steady rise in the accumulation of the 35- and 36 kD proteins. We have not determined if the level of the 1.6-kb transcript changes between D-5 and D-2. Because the accumulation of the alternative oxidase proteins in developing appendix tissue seems to precede the dramatic rise in the salicylic acid levels observed by Raskin et al. (1989), it is possible that the developmental regulation of expression of the alternative oxidase gene, aoxI, of voodoo lily appendix tissue is controlled by one or more additional regulators, not by salicylic acid alone. salicylic acid may then act as a light-induced (Raskin et al., 1987, 1989) "booster" of the transcript and protein levels at the critical time of D-1 through D-day so that thermogenesis occurs at precisely the proper time. The developmental experiments and the time course studies indicate that the accumulation of the alternative oxidase transcript is the first step in the dramatic burst of alternative pathway capacity between D-1 and D-day, which results in thermogenesis. Therefore, it is likely that salicylic acid functions at the level of transcript accumulation. Applied salicylic acid causes immature appendix tissue sections to become thermogenic between 16 and 24 hr after application. We have demonstrated that thermogenesis is accompanied by an increase in the capacity of the alternative pathway, which peaks at 16 hr after application. The accumulation of the 80 alternative oxidase proteins, which began as early as 5 hr after the application of salicylic acid, also peaked at 16 hr and remained essentially constant until 24 hr after application. The level of the 1.6-kb alternative oxidase transcript began to rise by about 4 hr after the application of salicylic acid, continued to rise to a peak at 16 hr after the application, and remained essentially constant until 24 hr. Thus, the time course for increase in alternative pathway capacity, the time course for the accumulation of the alternative oxidase proteins, and the time course for the accumulation of the transcript coincided precisely. Furthermore, these results agree with a developmental time course in which salicylic acid, released from the male floral region on D-l, "boosts" the levels of the alternative oxidase transcript and proteins, resulting in thermogenesis at about noon on D-day. In addition, these results demonstrate that salicylic acid alone (in lighted conditions) was sufficient to cause these changes. It is difficult to derive a clear picture of how salicylic acid regulates the accumulation of the alternative oxidase proteins in the mitochondria of the appendix tissue from the inhibitor data presented here. It is possible that the effects of cycloheximide are due to the inhibitor simply blocking the synthesis of a protein(s) involved in general translation or a protein(s) involved in general transcription. Likewise, it is possible that actinomycin D blocks the transcription of a gene(s) whose product(s) is involved in general translation or transcription. Although we do not know the components of the salicylic acid signal transduction pathway that are eliminated by cycloheximide and/or actinomycin D, it is clear that both de novo transcription and de novo translation are required for the 81 accumulation of the alternative oxidase transcript and proteins. If the inhibitors are not having general effects, then it appears that salicylic acid is affecting the accumulation of the 1.6-kb alternative oxidase transcript because actinomycin D does block the accumulation of the transcript when salicylic acid is present. The nuclear, in vitro transcription data presented indicate that the rate of transcription of 30x1 was the same at D-6 and D-day. Likewise, nuclear, in vitro transcription assays using nuclei isolated from untreated, immature (D-5) appendix tissue and sections from the same appendix treated with salicylic acid for 24 hr indicate that the rate of transcription of am] was the same in the treated tissue and the untreated tissue. It is possible that the level of detection of the nuclear, in vitro transcription assays was not sensitive enough to detect the levels of the alternative oxidase transcript produced in these tissues. However, this possibility seems unlikely because the amount of the transcript is extremely high in the appendix tissue at D-day and after treatment with salicylic acid. Furthermore, the rate of transcription of 30x1 may increase at a specific time in the development of the appendix and at a specific time after the application of salicylic acid, and we may not have isolated nuclei from the appendix tissue at one of these specific times. Based on the data presented, there are several possible mechanisms by which salicylic acid is regulating the expression of the alternative oxidase proteins and the capacity of the pathway. Because salicylic acid does induce an accumulation of the 1.6-kb alternative oxidase transcript, we assume that this is the ultimate purpose of salicylic acid in regulating the alternative pathway in the 82 appendix tissue. However, this does not eliminate the possibility that salicylic acid also regulates the accumulation of the alternative oxidase proteins in the mitochondria by increasing the production of an unidentified protein(s) involved in transport of the precursor protein to the inner mitochondrial membrane. Alternatively, salicylic acid may cause a stabilization of the 1.6-kb transcript by binding to a protein that stabilizes the 1.6-kb 30x1 transcript or initiating a complex series of events that eventually results in a stabilization of the aox] transcript. Any of these mechanisms would be inhibited by both actinomycin D and cycloheximide if the protein(s) involved must be made by de novo transcription and translation. A putative, soluble salicylic acid-binding protein in tobacco has recently been identified (Chen at al., 1991). It is possible that this protein is involved in some pathway leading to a response to salicylic acid in tobacco. Finally, it is likely that a complex mechanism accounts for the salicylic acid regulation of the alternative respiratory pathway. However, once the mechanism is elucidated, it will be very interesting to determine if salicylic acid regulates the expression of other proteins in other higher plants, such as the PR and SAR proteins in tobacco and cucumber, via the same mechanism. 83 ACKNOWLEDGEMENTS This study was supported in part by the US. Department of Energy contract DE-AC02-76ERO-1338 (L.M.) and the National Science Foundation DCB-8904518 (L.M.). We thank the following: Dr. Bastiaan J. D. Meeuse for providing the S. guttatum plants; Dr. Richard B. Meagher for providing the pSAc3 DNA; Dr. Ilya Raskin, Dr. Thomas E. Elthon, and Dr. Michael A. Mansfield for helpful advice and discussions. REFERENCES Ausubel F.M., Brent R., Kingston RE, Moore D.D., Siedman J.G., Smith LA, and Struhl K. (1987). Current Protocols in Molecular Biology (New York Greene Publishing Associates and Wiley-Interscience). Bendall, D.S. (1958). Cytochromes and some respiratory enzymes in mitochondria from the spadix of Arum maculatum. Biochem. J. 70, 381-390. Bendall D.S., and Bonner W.D., Jr. (1971). Cyanide-insensitive respiration in plant mitochondria. Plant Physiol. 47, 236-245. Chen Z., and Kleuig DP. (1991). Identification of a soluble salicylic acid- binding protein that may function in signal transduction in the plant disease-resistance response. Proc. Natl. Acad. Sci. USA 88, 8179-8183. Day D.A., Arron G.P., and Iaties G.G. (1980). Nature and control of respiratory pathways in plants: the interaction of cyanide-resistant respiration with the cyanide-sensitive pathway. In The Biochemistry of Plants: A Comprehensive Treatise, Vol 2, DD. Davies, ed (New York: Academic Press), pp. 197-241. ElthonT.E,StewartC.R.,McCoyC.A.,andBonnerW.D.,Jr. (1986). Alternative respiratory path capacity in plant mitochondria: effect of grth temperature, the electrochemical gradient, and assay pH. Plant Physiol. 80, 378-383. Elthon T.E., and McIntosh L (1987). Identification of the alternative terminal oxidase of higher plant mitochondria. Proc. Natl. Acad. Sci USA 84, 8399-8403. Elthon TE, Nickels RL, and McIntosh L (1989a). Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol. 89, 1311-1317. 84 Elthon TE, Nickeh KL, and McIntosh L (1989b). Mitochondrial events during the development of thermogenesis in Sauromatum guttatum (Schott). Planta 180, 82-89. Estabrook RW. (1987). Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10, 41-47. Feinberg AR, and Vogelstein B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 13?. 6-13. Imbers, H. (1985). Respiration in intact plant tissues: Its regulation and dependence on environmental factors, metabolism and invaded organisms. In The Encyclopedia of Plant Physiology: Higher Plant Cell Respiration, R. Douce, and DA. Day, eds (Berlin: Springer), pp. 418-473. [arson E, Howlett B., and Jagendorf A. (1986). Artificial reductant enhancement of the Lowry method for protein determination. Anal. Biochem. 155, 243-248. Luthe D.S., and Quatrano RS. (1980). Transcription in isolated wheat nuclei. 1. Isolation of nuclei and elimination of endogenous ribonuclease activity. Plant Physiol. 65, 305-308. Luthe D.S., and Quatrano RS. (1980). Transcription in isolated wheat nuclei. II. Characterization of RNA synthesized in vitro. Plant Physiol. 65, 309-313. Malamy J., Carr J.P., Klessig DE, and Raskin I. (1990). Salicylic acid: A likely endogenous signal in the resistance response of tobacco to viral infection. Science 250, 1002-1004. McIntosh L, and Cattolico RA. (1978). Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation. Anal. Biochem. 91, 600-612. Meeuse BJD. (1966). The voodoo lily. Sci. Am. 215, 80-88. Meeuse B.J.D. (1975). Thermogenic respiration in aroids. Ann Rev Plant Physiol. 26, 117-126. 85 Metraux J.P., Signer H., Ryals J., Ward R., Wyn-Benz M., Gaudin J., Ranchdorf K, Schmid E, Blum W., and Inverardi B. (1990). Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250, 1004-1006. Moore AL, and Bonner W.D., Jr. (1982). Measurements of membrane potentials in plant mitochondria with the safranine method. Plant Physiol. 70, 1271-1276. Raskin 1., Ehmann A., Melander W.R., and Meeuse B.J.D. (1987). Salicylic acid: a natural inducer of heat production in Arum lilies. Science 237, 1601-1602. Raskin L, Turner LV., and Melander W.R. (1989). Regulation of heat production in the infloresences of an Arum lily by endogenous salicylic acid. Proc. Natl. Acad. Sci. USA 86, 2214-2218. Raskin I., Skubatz H., Tang W., and Meeuse HID. (1990). Salicylic acid levels in thermogenic and non-thermogenic plants. Ann. Bot. 66, 369-373. Rasmussen J.B., Hammerschmidt R., and look MN. (1991). Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syn'ngae pv syn'ngae. Plant Physiol. 97, 1342-1347. Rhoads DM, and McIntosh L (1991). Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott). Proc. Natl. Acad. Sci. USA 88, 2122-2126. Rich P.R., and Moore AL (1976). The involvement of the protonmotive ubiquinone cycle in the respiratory chain of higher plants and its relation to the branchpoint of the alternate pathway. FEBS Lett. 65, 339-344. Sambrook J., Fritsch ER, and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed, (Cold Spring Harbor, New York: Cold Spring Harbor laboratory Press). Schwitzguebel LE, and Siegenthaler P.A. ( 1984). Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation. Plant Physiol. 75, 670-674. Shah D.P., Hightower RC, and Meagher RB. (1982). Complete nucleotide sequence of a soybean actin gene. Proc. Natl. Acad. Sci. USA 79, 1022- 1026. 86 Storey B.T. (1976). Respiratory chain of plant mitochondria. XVIII. Point of interaction of the alternate oxidase with the respiratory chain. Plant Physiol. 58, 521-525. Storey B.T., and Bahr J.T. (1969). The respiratory chain of plant mitochondria. 11. Oxidative phosphorylation in skunk cabbage mitochondria. Plant Physiol. 44, 126-134. Tahiwa F., 00110 K., Iida Y., and Sagiura M. (1985). The complete nucleotide sequence of a rice 258 rRNA gene. Gene 37, 255-259. CHAPTER4 ISOLATION AND CHARACTERIZATION OF A GENOMIC CLONE comma THE SAUROIWATUM GUITATW ALTERNATIVE OXIDASE GENE AOXI DavidMRhoadsandIeeMcIntoch Biochemistry Department and Department of Energy Plant Research Laboratory Michigan State University East Lansing, MI 48824 87 FOOTNOTES 1Supported in part by US. Department of Energy contract DE-AC02-76ERO-1338 (LM.) and the National Science Foundation DCB-8904518 (LM.). 2Abbreviations: PR, pathogenesis related; GRP, glycine rich protein; SAR, systemic acquired resistance; RBCS, ribulose bisphosphate carboxylase small subunit; Mops, 3-(N-morpholino)propanesulfonic acid; FCCP, carbonylcyanide p- trifluoro-methoxyphenylhydrazone; SHAM, salicylhydroxamic acid 89 ABSTRACT We have isolated and characterized a genomic clone, lAOSGl 1, corresponding to aoxI, which encodes the 42 kD alternative oxidase precursor protein of Sauromatum guttatum Schott. The sequence of AAOSGll revealed that aox] consists of four exons separated by three short introns. Exon three contains the region of 801:] that: 1) is highly conserved in the corresponding genes of potato, rice, and yeast; and 2) encodes a region of the deduced protein that is predicted to form two transmembrane a-helices, which are predicted to embed the mature protein in the inner mitochondrial membrane. Southern blot analysis of restriction endonuclease digested genomic DNA, indicated that 30x1 is a single, nuclear- encoded gene in S. guttatum. We have determined the transcriptional start site of am] using nuclease protection and primer extension experiments. Comparison of the putative promoter region of aox1 to promoters of PR la and GRP8 revealed some sequence similarity. 90 INTRODUCTION The alternative and cytochrome respiratory pathways exist together in the mitochondria of higher plants, fungi, algae, and protista (10,40). If electrons flow through the cytochrome pathway, a proton gradient is established at three sites along the pathway (3,41,24). The potential energy of the proton gradient is used to produce chemical energy in the form of ATP (25). If electrons flow through the alternative pathway, a proton gradient is formed from, at most, one site (3,35,41). Therefore, the partitioning of electrons between the two pathways must be regulated because it may have significant consequences on cellular energy metabolism (18,40). The energy of electron flow through the alternative pathway that is not coupled to proton gradient formation is released as heat (22). Some plant tissues that express a high level of the alternative pathway are thermogenic (29). The role of thermogenesis in the appendix tissue of the Aroid species Sauromatum guttatum Schott (voodoo lily) is to volatilize foul-smelling compounds which attract insect pollinators (21). The appearance of three mitochondrial proteins with apparent molecular masses of 35-, 36-, and 37 kD correlates with the activity of the alternative oxidase, the terminal oxidase of the alternative respiratory pathway, in S. guttatum appendix tissue (4). A 42 kD protein that is a putative precursor of all three of these alternative oxidase proteins has been identified and a cDNA clone, pAOSG81, corresponding to the nuclear gene, aox1, encoding the 42 kD protein has been isolated and characterized (33). One of the factors that may 91 determine the alternative pathway activity (the actual rate of 02 reduction that is occurring via the alternative pathway in vivo) is the amount of the alternative oxidase present in the mitochondria. The amount of alternative oxidase in the mitochondria may be determined, in part, by the level of expression of the corresponding gene(s). We demonstrated that the level of the alternative pathway capacity (the maximum rate of 02 reduction of the pathway in isolated mitochondria) in developing S. guttatum appendix tissue correlates well with the levels of the 35- and 36 kD alternative oxidase proteins (6,34) and the level of the 1.6 kb alternative oxidase transcript (34). The accumulation of the transcript may be due to an increase in the rate of transcription of aox] or an increase in the stability of the transcript (34). Previous work failed to show an increased rate of transcription of 301:] (34). However, since these results are not direct proof that RNA stability is the mechanism by which the transcript accumulates, it is possible that an increase in the rate of transcription of 30x1 does play a role in the transcript accumulation. Salicylic acid is an endogenous "trigger" of thermogenesis in voodoo lily appendix tissue (29). Salicylic acid is produced in the male floral region of the inflorescence and moves into the appendix beginning early on D-1 and "triggers" thermogenesis at about noon of D-day (29,30). Raskin et al. (30) showed that light is required for salicylic acid to induce thermogenesis in appendix tissue sections. Salicylic acid induces an increase in alternative pathway capacity in S. guttatum appendix tissue by causing an accumulation of the alternative oxidase transcript, resulting in an accumulation of the alternative oxidase proteins (34). 92 Salicylic acid may also be a messenger in systemic acquired resistance (SAR) in tobacco and cucumber (19,20,31,46). Salicylic acid may act in disease resistance by regulating gene expression since application of salicylic acid caused the accumulation of pathogenesis related (PR) proteins, including PR1a and a glycine rich protein (GRP) called GRP8, and their corresponding transcripts in tobacco leaves, which have increased levels of salicylic acid following innoculation of certain pathogens (19,26,45). The promoter regions of the genes encoding GRP8 and PR1a have been analyzed in order to identify salicylic acid responsive, cis-acting sequence elements (27,43). One study indicated that only the first 300 basepairs (bp) of the 5’-upstream region of PR1a gene were sufficient to allow the increased expression of a reporter gene (B-glucuronidase gene) in transgenic tobacco plants following infection with tobacco mosaic virus (TMV) or application of salicylic acid (27). A separate study indicated that the first 643 bp were required for an increase in expression of the same reporter gene in transgenic tobacco (43). However, a sequence motif found in both the PR1a promoter and the GRP8 promoter has been identified (42). Salicylic acid may participate in thermogenesis in aroids and the response to pathogen attack in tobacco and cucumber by regulating the expression of (apparently) unrelated genes, am] and the PR genes. Therefore, identification of the mechanism(s) by which salicylic acid regulates gene expression will be valuable in understanding the role of salicylic acid in various plant processes. In this paper we report on the identification and characterization of am], the salicylic acid "responsive" gene encoding the alternative oxidase of S. guttatum. 93 MATERIAIS AND METHODS Plant Material and Salicylic Acid Treatment. Sauroma tum gutta tum Schott (voodoo lily) plants were maintained in a glasshouse at 27°C : 4°C under long- day conditions, as previously described (4). D-day is the day the inflorescence blooms and the appendix tissue of the spadix heats (see reference 21 for description). Other developmental stages of the plant are indicated as the number of days before or after D-day (D-1 being the day before and D+ 1 the day after D-day). Between the time the inflorescence begins to develop and the end of day D-2, the inflorescence is referred to as "immature". For the salicylic acid treatment experiments, appendix tissue of a S. guttatum plant was cut into 1.0-1.5 cm sections. Sections were placed into each of two 10 ml beakers containing 1.0 ml of phosphate buffer (10 mM KHzPO4 and 50 ug/ml streptomycin). Salicylic acid was added to a final concentration of 1.0 mM to one beaker and a drop of buffer or buffer plus salicylic acid was placed on top of each section. Isolation of Mitochondria and Respiration Assays. Mitochondria were isolated as described previously (4). Respiration rates were measured as oxygen uptake using a Rank Brothers (Cambridge, UK) oxygen electrode. The oxygen content of air- saturated water was estimated according to Estabrook (7). The capacity of the alternative pathway was measured in 1.0 mL of reaction medium (250 mM sucrose, 30 mM 3-(N-morpholino)propanesulfonic acid [Mops], pH 6.8) at 25 °C with 1 mM NADH as the substrate. Carbonylcyanide p-trifluoro- 94 methoxyphenylhydrazone (FCCP) was added to 0.5 uM after NADH in order to diminish the electrochemical gradient prior to measurement of the pathway capacity (4). The capacity of the alternative pathway was taken as the rate of oxygen uptake sensitive to 1 mM salicylhydroxamic acid (SHAM) in the presence of 1 mM KCN (4). The amount of total protein in each sample was determined by as described previously (4). Gel Eectrophorecia, Immunoblotting, and Antisera. Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and immunoblotting were performed as described previously (4). The antibody used was the AOA monoclonal antibody raised to the 36 kD alternative oxidase protein of voodoo lily, and which recognize all three alternative oxidase proteins of voodoo lily (5 ). Since the same amount of total mitochondrial protein was loaded onto each lane of each polyacrylamide gel, the protein detected on protein blots represents the proportion of total mitochondrial proteins that constitutes the alternative oxidase proteins. Plasmid Insert Isolation and Radiolabeling. The 1400-bp EcoRI insert of pAOSG81 (33) was purified by as described in Sambrook et al. (37) except that the elution buffer was 10 mM Tris-H01, pH 7.5, 0.1 mM EDTA, 0.05% SDS, and 0.3 M LiCl and the insert was electrophoresed through two successive I polyacrylamide gels. The purified fragment was resuspended to a concentration of 60 ugly] in Tris-EDTA and was used as a template to make DNA radiolabeled 95 with a-nP-dATP (Amersham Corporation) by the random primer method (8). Specific activity of the DNA probes was routinely about 107 chug template, as determined by the sodium phosphate wash method (37). RNA Isolation, Blots, and Autoradiography. Total RNA was isolated from frozen (in liquid N2) S. guttatum appendix tissue as described previously (33). RNA was separated on agarose gels containing formaldehyde as described (1) and transferred to nitrocellulose (Schleicher and Schuell, Keene, NH) as described in Sambrook et a1. (37). RNA blots were probed with radiolabeled DNA made using the purified insert of pAOSG81. Hybridization was done under high stringency hybridization conditions (37). Autoradiography using Kodak diagnostic film XAR- 5 (Eastman Kodak Company, Rochester, NY) was used to view the hybridization results (1). Genomic DNA Isolation and Southern Blot Analyst. S. guttatum plants were grown in the dark for 24 h prior to genomic DNA isolation. Genomic DNA was isolated from S. guttatum leaves by the procedure of Ausubel et a1. (1). Southern blotting was performed according to the procedure of Sambrook et a1. (37) using 10 ug per lane of restriction endonuclease digested genomic DNA and radiolabeled DNA probes made using the insert of pAOSG81, as described above. Isolation of Genomic (Jones and Subcloning. A library of the genomic DNA partially digested with Sau3A was constructed in the Barn HI site of lambda 96 EMBL3 vector by Stratagene (LaJolla, CA). Library screening and all subcloning were performed according to the procedures of Sambrook et al. (37). The 2.2 kbp Nco I fragment (see Figure 4—3 for location of sites) and the 2.9 kbp Bam HI/Sal I fragment were subcloned from lGAOSGll into pUC119 to form pGAOSG-N22 and pGAOSG-B829, respectively. The 2.3 kbp Sma I/Sac I fragment from lGAOSGll was subcloned into pBluescript (Stratagene, LaJolla, CA) to form pGAOSG-S823. DNA Sequencing and Analysis. Sequencing of the inserts of phagemids pGAOSG-N22 and pGAOSG-B829 was done by the dideoxy method of Sanger et al. (38) using Sequenase® T7 DNA Polymerase version 2.0 (US. Biochemical Corporation, Cleveland, OH). Primers for sequencing and primer extension experiments were made in the laboratory of Dr. C. S. Sommerville or the Michigan State University Macromolecular Facility. All sequence analysis was performed using the EDITBASE DNA sequence analysis program. SynthesisofRadiolabeledTramuiptsandMappingofaaxImRNAwithRNase. Radiolabeled transcripts used to map the 5’ end of the mRNA encoding the 42 kD alternative oxidase protein were synthesized in vitro by the procedure of Sambrook et al. (37). The template used for synthesis was an I digested pGAOSG-SSZ3 (see Fig. 4-3 for location of sites). T7 RNA polymerase was used to synthesize a 544 nucleotide transcript that contained 10 bases of pBluescript sequence, 173 bases of sequence of pGAOSG-SS23 that is the same as the 5’ end 97 of the insert of pAOSGSl, and 361 bases of pGAOSG-SS23 preceding the first base that corresponds to the 5’ end of the insert of pAOSG81. The radiolabeled transcripts were hybridized to 10 ug of total S. guttatum RNA isolated from D-day appendix tissue and digested with RNase T1 (BRL, Gaithersburg, MD) and RNase A (Sigma Chemical Co., St. Louis, MO) following the procedure of Sambrook et al. (37). Radiolabeled RNA protected from digestion was analyzed by electrophoresis through a 7% (w/v) polyacrylamide sequencing gel and autoradiography. The products of the sequencing reactions using primer FDRS 2- 4, which corresponds to bases 1349 to 1366 of the insert of AGAOSGl 1, were loaded into adjacent lanes as markers. Each of these products (DNA) migrates through the sequencing gel at the same rate as an RNA molecule that is 5%-10% shorter (37). Primer Etension Mapping of 8:21 mRNA. Primer extension mapping of the aoxI transcript was done by the procedure of Sambrook et a1. (37) using a 25 base oligonucleotide, called PE-25, of the sequence 5’-GGTACGGGGACGTGACI‘GA GCI‘GCC-3’, corresponding to the complementary strand of bases 118 to 142 of AGAOSGll and, therefore, complementary to the 30x1 transcript. Products of the primer extension were analyzed by electrophoresis through a 7% (w/V) polyacrylamide sequencing gel and autoradiography. REULTS Salicylic Acid-Regulated Expression of the Alternative Oxidase. The alternative pathway capacity and the amounts of the 35- and 36 kD alternative oxidase proteins are increased by 5 h following the application of salicylic acid to day D-5 S. guttatum appendix tissue sections (Fig. 4-1A, lane 2) as compared to the levels before salicylic acid application (lane 1). The capacity and amount of each of the three alternative oxidase proteins are greatly increased by 24 h following salicylic acid addition (lane 3). The amount of the 1.6 kb alternative oxidase transcript increased by 4 h following salicylic acid addition to day D-S appendix tissue (Fig. 4-1B, lane 2) as compared to the amount present before salicylic acid application (lane 1). The amount of the transcript is greatly increased by 24 h following salicylic acid (lane 3). Southern Blot Analyfi to Determine Gene Copy Number. The copy number of alternative oxidase genes was determined by Southern blot analysis of S. guttatum genomic DNA digested with various restriction endonucleases. Figure 4-2 shows that single DNA fragments were detected when genomic DNA digested with 5 either Barn HI (lane 1), Hind III (lane 3), Nco I (lane 4), Sal I (lane 7) or Xba I (lane 8) was probed with radiolabeled DNA made using the insert of pAOSG81. Two bands were detected when the genomic DNA was digested with either Eco RI (lane 2), Pst I (lane 5), or Sac I (lane 6). Based on the sequence of AGAOSGl 1, there should not be two bands recognized by the probe when 99 genomic DNA is digested with EcoRI. We can only explain this as an error in fidelity in the production of clone lGAOSGll. All other bands agree with the sequence data. Southern blot data obtained using genomic DNA digested with Cla I, Hinc II, Kpn I, Nde I, and Sa] I also agreed with the sequence data (data not shown). Sequence of amI. Figure 4-3 shows a schematic diagram of the exon/intron structure of 30x1 deduced from the sequence of AGAOSGll and the locations of key restriction endonuclease recognition sites. The sequence of the phagemid subclones, pGAOSG-N22 and pGAOSG-BS29, derived from genomic clone AGAOSGI 1, revealed that 30x1 is organized as four exons separated by three short introns (Fig. 4-4). Exon 1, exon 2, exon 3, and exon 4 are 452 bp, 133 bp, 493 bp and approximately 570 bp in length, respectively. They are separated by intron 1, intron 2, and intron 3, which are 117 bp, 79 bp, and 119 bp in length, respectively. Exon 3 contains the entire region of 30x1 that is highly conserved among the alternative oxidase cDNA clones from potato (11), Hansenula anomala (36), and rice (Dr. L McIntosh and R. Nickels, personal communication). This region of 30):] encodes a region of the deduced protein that is predicted to form two transmembrane a-helices (33). The putative TATA box with the sequence TATAAA is located at bases -32 to -27. Two putative CAAT boxes are at bases - 83 to -79 (CCCAT) and at bases -67 to ~62 (CAAAAT). Many cis-acting transcriptional elements (see 12,14, and 15 for descriptions) were identified in the promoter of am], but only the potential zinc finger (GATA) and GT-l/GT-2 100 Figure 4-1. Immunoblot and RNA blot analysis of salicylic acid induction of alternative oxidase expression. Panel A: Total mitochondrial proteins were separated by SDS-PAGE and transferred to nitrocellulose. The immunoblot was probed with the AOA monoclonal antibody. The mitochondrial proteins were from D-5 S. guttatum appendix tissue sections before application of salicylic acid (lane 1), following incubation in phosphate buffer containing 1.0 mM salicylic acid for 5 h (lane 2), and 24 h following salicylic acid application (lane 3). Alternative pathway capacity in natoms O/min/mg protein is indicated at the bottom of each lane. Protein relative molecular masses in kD are indicated at the side. Panel B: Total RNA (10 pg per lane) was electrophoresed through 1.2% (w/v) agarose- formaldehyde gels and transferred to nitrocellulose. The blot was probed with radiolabeled DNA made using the insert of pAOSG81 by the random primer method and hybridization that occurred at high stringency was detected by autoradiography. The RNA samples were from appendix tissue from a D-5 S. guttatum appendix tissue sections before application of salicylic acid (lane 1), following incubation in phosphate buffer containing 1.0 mM salicylic acid for 4 h (lane 2), and 24 h following salicylic acid application (lane 3). Transcript length in kilobases is indicated at the side. 101 123 37\ 337:: 1.6-v 123 102 Figure 4-2. Southern blot analysis of genomic DNA. S. guttatum genomic DNA (10 pg) was digested with individual restriction endonucleases, electrophoresed through a 0.7% (w/v) agarose gel, and transferred to nitrocellulose. The blot was probed with radiolabeled DNA made using the insert of pAOSG81 by the random primer method. Hybridization at high stringency was detected by autoradiography. The DNA samples were digested with the following restriction endonucleases: Barn HI (lane 1), Eco RI (lane 2), Hind III (lane 3), Nco I (lane 4), Pst I (lane 5), Sac I (lane 6), Sal I (lane 7), and Xba I (lane 8). DNA marker sizes in base pairs are indicated to the side. 103 123 45 67 8 23.1 - 9.41 - 6.56 - 4.37- - clingy-C. 2.32 - 2.03 - 104 (GTGG) target sequences were identical to the consensus sequence of each (Fig. 4-3). Regions with sequence similarity to the following classes of promoter elements were identified: bZIP (TGACG, GACGTG, CACGTG, GACGTG), at bases -695 to -689 (matched 5/6 bases), bases -195 to -190 (5/6), bases -152 to -147 (5/6), and bases -57 to -51 (5/6); HLH (CAGGTGC), -1281 to -1275 (6/7); heat shock (GAAnnTTC), at bases -1446 to -1439 (7/8), bases -1183 to -1174 (7/8), bases -1064 to -1057 (7/8), bases -1043 to -1036 (7/8), bases -860 to -853 (7/8), bases -597 to -590 (7/8), bases -265 to -258 (7/8), and bases -161 to 154 (7/8); and SV40 (GTGGA/I‘ M M G), at bases -606 to -599 (7/8) and bases -139 to -132 (7/8). Sequences similar to each of the "boxes" identified in the promoter of the gene encoding the small subunit of ribulose bisphosphate carboxylase (RBCS) were also detected: Box I at bases -1458 to -1446 (10/13), bases -903 to -897 (6/7), bases -759 to -753 (6/7), bases -366 to -360 (6/7), bases -278 to -271 (6/7), and bases -117 to -111 (6/7); Box 11 at -138 to -128 (10/14); Box 11* at bases -234 to - 223 (9/12); Box 111 at bases -378 to -369 (8/10); Box IV at bases -806 to -797 (8/10), bases -784 to -775 (8/10), bases -634 to -624 (8/10), and bases -99 to -90 (8/10); and Box V at bases -611 to -601 (8/11) and bases -109 to -99 (8/11). A region of the am] promoter, from base -524 to base -468 can be aligned with the -50 to +1 regions the PR1a and GRP8 promoters (42) as follows: “1 '526 GTlTl‘ltTl-Tm'AT-'01Mt1’AT-mtm -tTABTMATC-MTITC-Tc-A 468 =1=1== 11111 1=11111= =111====111111=111=111 an -54 rr-urecnrrm-Aru-u-cnrm- mac-cc- 1m mm-mnmc-A +1 1 1 =1111111 1 111111 1111 111111 111111111 n1. ~56 cr-mrmummcrc-curm ----- rac-ccrreeucrmrcrmrr-rrcu +1 in which capital letters in the 30x1 sequence dots between the sequences indicate . bases that are present in either the GRP8 sequence or the PR1a sequence and 105 Figure 4-3. Schematic diagram of genomic clone AGAOSGll. Exons are shown as open boxes, introns as filled boxes, the untranscribed regions as lines, and the arms of the lambda phage as hatched boxes. The positions of recognition sites of the following restriction endonucleases are shown: Bam HI, indicated as B; Nco I, N; Sac I, S; Sal I, Sa; Sma I, Sm; and an I, X. 106 I3 11 I2 Ikh 107 Figure 4-4. Nucleotide and deduced amino acid sequence of the aox] gene of S. guttatum. The sequence of cDNA clone pAOSG81 is shown in capital letters. The transcription start site (+1) is marked by a bent arrow. The putative TATA box, potential CAAT boxes, and other potential cis-acting transcriptional elements are underlined or overlined and discussed in the text. 108 A v 4541 4‘51 W 4“] 4171 4181 40'! Jul A A v v AAA AA J” as: _ ~73] AA A AA -“l 451 ‘ ~48! ‘ A AAAA AAA .-_—_-_ A _-.. J" A‘—f— .- 481 ~10] cu Ar-anH'Pffflffffl’ff‘lfnmnlflflKIWI A A A A A -ll sm rm to su in um um pm pm in .m I“ .m pm pm an LG on an am am an um um :w am pm pm '1 AI in um 1a in ow am J; ..m rm xm on am ow 4m in am 1.... u on :u um sm 1... 8 .m sm .w a V" n- .u I VK 9! :3 U..— n O. O. 1“ cw xm um um um um u ‘3 I! u Vt 1. 1m an I. pm LT cu 9‘ L7 95 in ya in um .m .u .m .m In .m L»... Pb nu .u 4m ya rm lm Aim .- n C 8 um um um um vm cm I“ L" :u an ..m (m cu ..m (u I." 'K C ol an um um 1m um um as 1... it um um cu m m 1‘ C18 CW 1 m .u pm cu .w a In .I in ca um um .I on 1m ..m sm in in 'un& su 'I um ‘0 V V Y 0 V V W 61C OTC ”C "C ITC S Y L I ICCECCTSC‘C I I L P 0 6 IUCCC‘I’ICCOCMN V K I A A A AA A A A V 11 ' IKCKIM L I cream I Y 1' A, ummccsocsccs I. II I. I "INC TC Y Q 0 YEW“ V D 1390 OK 811: CI! Iln-“‘r‘l IWIIMIIIII—-uu-—--- 1880 I!“ .YfT-u I‘ll! “)0 MICICCNIMTTCYTMIT TMV I186C11661112111811“WittTKIA‘IMAIAIAIWIMTYYCIC'H 1720 1810 A A AA I,” 1990 109 lines between sequences indicate nucleotides that are present in all three sequences. In addition, the 30x1 promoter has three regions that have sequence similarity to the -689 to -643 region of the PR1a promoter and the promoter of GRP8. These regions are bases -146 to -134 (10/13), bases -139 to -128 (10/11), and bases -442 to -457 (11/16). Mapping of 80:1 mRNA Using RNase. A large amount of the 544 nucleotide, radiolabeled transcript was made by the in vitro system (Figure 4-5, lane 6). The largest fragment of the 544 nucleotide transcript that is protected from RNase digestion by hybridization to the aox] transcript was about 200 nucleotides in length (lane 5). The transcriptional start site that corresponds to a fragment of this size is base +1 of lGAOSGl 1, which is marked with a bent arrow in Figure 4-4. The other bands present in lane 5 correspond to RNA molecules of about 199, 198, 185, 173, 172, 171, 169 and 129 nucleotides. Primer Eaten Mapping of 8:21 mRNA. A large amount of primer PE-25 was end labeled with a-32P and that there may have been some contamination with oligonucleotides of about 50 nucleotides in length (Figure 4-6, lane 6). The most abundant product made by primer extension was 142 nucleotides (lane 5) and corresponds to a transcription start site at base + 1 of lGAOSGll, which is marked with a bent arrow in Figure 44. Bands corresponding to products of about 141, 138, 91, 90, and 89 nucleotides were also detected. 110 Figure 4-5. RNase protection mapping of the 5’ end of the 30x1 transcript. An in vitro synthesized, 544 nucleotide transcript was hybridized to 10 pg of total RNA from D-day appendix tissue and digested with RNase T1 and RNase A. Radiolabeled RNA samples were analyzed by electrophoresis through a 7% (w/v) polyacrylamide sequencing gel and autoradiography. A protected fragment of about 200 nucleotides (lane 5) is indicated by an arrow. The undigested, radiolabeled probe transcript (lane 6) is also indicated by an arrow. Sequencing products made using the single stranded form of phagemid pGAOSG-BSZ9 and primer FDR52-4 (lanes 1-4) were used as size markers. The positions of some of the sequencing products are indicated in DNA base pairs at the side. Each of these products (DNA) migrates through the sequencing gel at the same rate as an RNA molecule that is 5%-10% shorter (37). 111 123456 a $1? _ 5 7 1 225 200 a... we}... _ O 5 1 125— .: L ,1 F 112 Figure 4—6. Primer extension mapping of the 5’ end of the 30x1 transcript. Primer extension was performed using oligonucleotide RDR22-7, which is complementary to the 30x1 transcript and corresponds to bases 1661 to 1685 of lGAOSGll. The products of primer extension (lane 5), the unextended oligonucleotide primer (lane 6), and products of sequencing reactions using the same primer and the single stranded form of phagemid pGAOSG-SSZ3 were analyzed by electrophoresis on a 7% (w/v) polyacrylamide gel and autoradiography. DNA molecule sizes in base pairs are indicated at the side. The most abundant primer extension product (142 nucleotides) is indicated by an arrow. 113 01 O 1:11 ‘- 1 mm Hillilllilillwl n :1“ ~1 11 llllllllll“h I11 llllllmu i 1 150— 15‘ _ =: —- <- 125— =E 12:5 100— E: ‘__ - -- —' ‘ ‘,_ I— 75— 2‘ "— _=-.- ‘— * -— _— a - - .fi- 50— - - .. ' -- - 9 - . - . - . ‘d' - ‘ — - - 1 I 1 1 4 DISCUSSION The amount of alternative oxidase present in plant mitochondria is one of the factors that will determine the partitioning of electrons between the alternative and cytochrome pathways. The data presented here indicates that alternative pathway capacity and the amounts of the 35- and 36 kD alternative oxidase proteins were increased by 5 h following salicylic acid application to immature S. guttatum appendix tissue. The amount of the 1.6 kb alternative oxidase transcript increased by 4 h following salicylic acid application. It is likely that the expression of the alternative oxidase in S. guttatum appendix tissue is affected by salicylic acid at the level of transcript accumulation (34). In order to better understand this regulation, we have isolated a genomic clone corresponding to am], which encodes the 42 kD alternative oxidase precursor protein. Three lines of evidence suggest that this gene is the only expressed alternative oxidase gene in S. guttatum appendix tissue and is present in a single copy in this organism: 1) single DNA fragments were detected on Southern blots when genomic DNA was digested with several different restriction endonucleases; 2) a single, 1.6 kb transcript is detected on RNA blots; and 3) a single, 42 kD protein is immunoprecipitated from products made by in vitro translation of poly (A)+ RNA from appendix tissue (33). The war] gene is organized as four exons that are separated by three short introns. One interesting feature of this gene structure is that exon 3 contains the entire region of ear] that: 1) is very highly conserved among several higher plant 115 species, including potato (11) and rice (R. Nickels and L McIntosh, unpublished results), and yeast (36); and 2) encodes the region of alternative oxidase protein that is predicted to embed the protein in the inner surface of the inner mitochondrial membrane (33). Each intron begins with GU and ends with AG, a pattern typical of eukaryotic introns (2). Each intron is AU-rich, a characteristic of dicots (9), and contains the YURAY sequence common to introns of higher plants (13). We have mapped the 5’ end of the am] transcript by RN ase protection and primer extension experiments. The results from each of these suggest that the first base of the 30x1 transcript corresponds to the adenine marked as base + 1 on Fig. 4-4, which is 14 bp upstream of the 5’ end of the start of the match with pAOSG81. The smaller RNA fragments that appear in the RNase protection experiment may be due to multiple transcription start sites that are not detected by RNA blot analysis due to the transcripts differing by a small number of nucleotides. Alternatively, the bands could have resulted from imperfect conditions during the RNase digestion, which could result in slight separation of the strands at the ends. The primer extension experiment also showed multiple products that are shorter than the main product. These products may have resulted from premature termination of reverse transcription. The first base of the proposed TATA box is 32 bases from the start site, which is the distance often observed for the TATA box of a higher plant gene transcribed by RNA polymerase II (44). The most likely CAAT site occurs at bases -67 to -62, which agrees with the position of the CAAT site of other plant genes (44). 116 Several sequence regions in the promoter of 30x] are similar to the plant transcription elements that have been identified (see references 12,14,15,17,28 for element descriptions), but only the potential zinc finger and GT-l/GT-Z target sequences were identical to the consensus sequence of each of these cis-acting regulatory elements. Regions with sequence similarity to the following classes of promoter elements are located in the promoter of am]: bZIP, HLH, heat shock, and SV40. Since light is required for applied salicylic acid to cause thermogenesis in immature S. guttaturn appendix tissue sections (30), it is interesting to note that several regions were identified that are similar to the "boxes" of the light-regulated RBCS promoter (16). A region of the am] promoter, from base -524 to base - 468 can be aligned with the -50 to +1 regions the PR1a and GRP8 promoters (42). In addition, the 80):] promoter has three regions that have sequence similarity to the -689 to -643 region of the PRla promoter and the promoter of GRP8. It is possible that one or more of these regions plays a role in the potential salicylic acid controlled regulation of these genes. The polyadenylation site of the am] transcript is not known and cannot be accurately deduced from the sequence of AGAOSGl 1. In summary, our results are consistent with the hypothesis that salicylic acid "triggers" thermogenesis in S. guttatum appendix tissue by causing, at least in part, an increase in the accumulation of the 1.6 kb 30x1 transcript, which results in increased accumulation of the 35- and 36 kD alternative oxidase proteins and increased capacity of the alternative pathway. Accumulation of the 1.6 kb transcript may be through an increase in transcription rate or an increase in RNA 117 stability. Salicylic acid may play a role in SAR by causing an accumulation in the transcripts encoding certain PR and SAR genes, although relative transcription rates have not been determined for these events. The promoters of PR1a and GRP8 have been analyzed to determine regions that are important for salicylic acid directed gene regulation. It may now be possible to use IlGAOSGll to: 1) identify any protein(s) that may interact with the am] promoter in nuclei of S. guttatum appendix tissue; and 2) determine the regions of the 30x] promoter that may be responding to salicylic acid using a transgenic system. Results from such experiments may be useful in determining the general mechanism by which salicylic acid regulates gene expression REFERENCES 1. AusubelFMBrentR,KingstonRE,MooreDD,SiedmanJG,SmithJA& Struhl K ( 1987) Current Protocols in Molecular Biology (New York Greene Publishing Associates and Wiley-Interscience). 2. Breathnach R & Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Ann Rev Bioch 50: 349-383. 3. Bendall DS & Bonner WD Jr (1971) Cyanide-insensitive respiration in plant mitochondria. Plant Physiol 47: 236-245. 4. Ethan TE & McIntosh L (1987) Identification of the alternative terminal oxidase of higher plant mitochondria. Proc Nat Acad Sci USA 84: 8399-8403. 5. Ethan ’TF, Nickels RL & McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89: 1311-1317. 10. 11. 12. 13. 14. 15. 16. 17. 118 Ethan TE, Nickels RL & McIntosh L (1989) Mitochondrial events during development of thermogenesis in Sauromatum guttatum (Schott). Planta 180: 82-89. Estabrook RW (1987) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol 10: 41-47. Feinberg AP & Vogelstein B ( 1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. ' Goodall GJ & Filipowiez W (1991) Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants. EMBO J 10: 2635-2644. Henry MF & Nyns E-J ( 1975) Cyanide-insensitive respiration. An alternative mitochondrial pathway. Sub-Cell Biochem 4: 1-65. I-Iiser CB ( 1991) Molecular and developmental aspects of respiratory complexes in higher plant mitochondria. Doctoral Dissertation, Michigan State University, Biochemistry Department. Jones NC, Rigby PWJ & Zifi (1988) Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev 2: 267-281. Joshi CP (1987) Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucl Acids Res 15: 9627-9640. Katagiri F & Chua N-H (1992) Plant transcription factors: present knowledge and future challenges. Trends in Genet 8: 22-27. Kuhlerneier C (1992) Transcriptional and post-transcriptional regulation of gene expression in plants. Plant Mol Biol 19: 1-14. Kuhlemeier C, Green PJ, & Chua N-H (1987) Regulation of gene expression in higher plants. Ann Rev Plant Physiol 38: 221-257. Lam F, Benfry PN, Gilmartin PM, Fang R-X & Chua N-H (1989) Site- specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc Natl Acad Sci USA 86: 7890- 7894. 18. 19. 20. 21. 22. 23. 24. 26. 27. 119 Lambers H (1985) Respiration in intact plants and tissues: Its regulation and dependence on environmental factors, metabolism and invaded organisms. In: R Douce & DA Day (eds) Encyclopedia of Plant Physiology, Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York, pp.418-473. Malamy J, Carr JP, Klessig DF & Raskin I (1990) Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250: 1002-1004. M6trmrxJP,SignerI-I,RyalsJ,WardEWyss-BenZMGaudinJ, Raschdorf K, Schmid E, Blurn W & Inverardi B (1990) Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250: 1004-1006. Meeuse BJD (1966) The voodoo lily. Sci Am 215: 80-88. Meeuse BJD (1975) Thermogenic respiration in aroids. Ann Rev Plant Physiol 26: 117-126. MinagawaN,SakajoS,KomiyamaT&YoshimotoA(l990)A36 kDa mitochondrial protein is responsible for cyanide-resistant respiration in Hansenula anomala. FEBS Lett 264: 149-152. Moore AL & Bonner Jr WD (1982) Measurements of membrane potentials in plant mitochondria with the safranine method. Plant Physiol 70: 1271-1276. Moore AL & Rich PR (1985) Organization of the respiratory chain and oxidative phosphorylation. In: R Douce & DA Day, (eds) Encyclopedia of Plant Physiology, Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York. pp. 134-172. Ohashi Y & Matsuoka M (1986) Induction of pathogenesis-related proteins in tobacco. Plant Physiol 80: 505-510. OhshimaM,ItohH,MatsuokaM,MurakamiT&OhashiY(l990) Analysis of stress-induced or salicylic acid-induced expression of the pathogenesis-related 1a protein gene in transgenic tobacco. The Plant Cell 2: 95-106. Pfitzner U, Pfitzner AJP & Goodman HM (1988) DNA sequence analysis of a PR-Ja gene from tobacco: Molecular relationship of heat shock and pathogen responses in plants. Mol Gen Genet 211: 290-295. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 120 Raskin I, Ehmann A, Melander WR & Meeuse BJD (1987) Salicylic acid: A natural inducer of heat production in Arum lilies. Science 237: 1601-1602. Raskin I, Turmr 1M & Melander WR (1989) Regulation of heat production in the inflorescean of an Arum lily by endogenous salicylic acid. Proc Natl Acad Sci USA 86: 2214-2218. Rasmussen JB, Harnmerschmidt R, & Zook MN (1991) Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv syn'ngae. Plant Physiol. 97: 1342-1347. Rasmusson AG, Moller IM & Pahner JM (1989) Component of the alternative oxidase localized to the matrix surface of the inner membrane of plant mitochondria. FEBS Lett 259: 311-314. Rhoads DM & McIntosh L (1991) Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott). Proc Nat Acad Sci USA 88: 2122-2126. Rhoatk DM & McIntosh L (1992) Salicylic acid regulation of respiration in higher plants: Alternative Oxidase Expression. The Plant Cell, in press. Rich PR & Moore AL (1976) The involvement of the protonmotive ubiquinone cycle in the respiratory cnain of higher plants and its relation to the branchpoint of the alternate pathway. FEBS Letters 65: 339-344. Sakaja S, Minagawa N, Komiyama T & Yoshimota A (1991) Molecular cloning of cDNA for antimycin A-inducible mRNA and its role in cyanide- resistant respiration in Hansenula anomala. Biochim Biophys Acta 1090: 102-118. Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Sanger F, Nicklen S & Coulsan AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467. Schwitzguebel JP & Siegenthaler PA (1984). Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation. Plant Physiol 75: 670-674. Siedow JN & Berthold DA (1986) The alternative oxidase: A cyanide- 41. 42. 43. 45. 47. 121 resistant respiratory pathway in higher plants. Physiol Plant 66: 569-573. Storey BT (1976) Respiratory chain of plant mitochondria. XVIII. Point of interaction of the alternate oxidase with the respiratory chain. Plant Physiol 58: 521-525. Van Kan JAL, Carnelissen BJC & Bol JF (1988) A virus-inducrble tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphospate carboxylase small subunit gene. Mol Plant- Microbe Interactions 1: 107-112. vandeRheeMD,VanKanJAL,Gonzalez-JaenMT&BolJF(l990) Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection. Plant Cell 2: 357-366. Waibel F & Filipowia (1990) RNA-polymerase specificity of transcription of Arabidopsis U snRNA genes determined by promoter element spacing. Nature 346: 119-202. Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DW, Alexander DC, Ahl-Gay P, Metraus J-P & Ryals JA (1991) Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell 3: 1085-1094. YalpaniN,SilvermanP,WilsonTMA,KbierDA&Raskinl(1991) Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco. The Plant Cell 3: 809-818. Yoshimoto A, Sakajo S, Minagawa N & Komiyama T (1989) Possible role of a 36 kDa protein induced by respiratory inhibitors in cyanide-resistant respiration in Hansenula anomala. J Biochem 105: 864—866. CHAPTERS CYTOCHROME AND ALTERNATIVE PATHWAY RESPIRATION IN TOBACG): EFFECTS OF SALICYLIC ACID DavidMRhoadsandLeeMantosh Biochemistry Department and Department Of Energy Plant Research Laboratory Michigan State University East Lansing, Michigan 48824 122 FOOTNOTES 1Supported in part by US. Department of Energy contract DE-AC02-76ERO-1338 (LM.) and the National Science Foundation DCB-8904518 (L.M.). 2Abbreviations: FCCP, p—trifluoromethoxycarbonylcyanide; SHAM, salicylhydroxamic acid 123 124 ABSTRACT In suspension cultures of NT 1 tobacco (Nicotiana tabacum L cv. bright yellow) cells the cytochrome pathway capacity increased between day 3 and day 4 following subculturing, reached the highest level observed on day 7, decreased significantly by day 10, and was at the same level on day 14. Both alternative pathway capacity and the amount of the 35 kD alternative oxidase protein increased significantly between day 5 and day 6, reached the highest point observed on day 7, remained constant until day 10, and decreased by day 14. The highest points of the alternative and cytochrome pathway capacities and the largest amount of the 35 kD protein were attained on the day cell cultures reached a stationary phase of growth. Addition of salicylic acid to cell cultures on day 4, caused a significant increase in alternative pathway capacity and a dramatic accumulation of the 35 kD protein by 12 hours. The capacity and the protein level each reached the highest point observed by 16 h after salicylic acid addition and the cytochrome pathway capacity was at about the same level at each time point. The accumulation of the 35 kD protein was significantly decreased by addition of actinomycin D one hour before salicylic acid and was blocked by addition of cycloheximide. These results indicate that de novo transcription and translation were necessary for salicylic acid to cause the maximum accumulation of the 35 kD protein. 125 INTRODUCTION The regulation of electron flow between the alternative and cytochrome respiratory pathways in higher plants may be important in determining the overall carbon balance of plant cells (8,23). If electrons flow through the cytochrome pathway, a proton gradient is established from three coupling sites (complex I, complex III, and complex IV) along the pathway (1,14,24). The potential energy of the proton gradient is used to produce chemical energy in the form of ATP (reviewed in reference 15). If electrons flow through the alternative pathway, only complex I can contribute to establishing a proton gradient, but this depends on the substrate being oxidized (1,20,25). The energy of electron flow that is not used to produce ATP in the alternative pathway is released as heat, which is used in the reproductive physiology of the aroid plants such as the species Sauromatum guttatum Schott (12,13). The role of the alternative pathway in non-aroid plants is not yet defined (8,23). The appearance of three mitochondrial proteins with apparent molecular masses of 35-, 36-, and 37 kD strongly correlates with the activity of the alternative oxidase, the terminal oxidase of the alternative pathway, in S. guttatum appendix tissue (2). These proteins are believed to be posttranslationally modified products from a single, nuclear-encoded precursor protein (18). A monoclonal antibody that recognize all three alternative oxidase proteins of S. guttatum was prepared and named AOA monoclonal antibody (3). This antibody also recognizes a single, putative alternative oxidase protein of 35 kD in tobacco and a single 126 mitochondrial protein from other non-aroid higher plants (2,3). A 42 kD protein that is a putative precursor of all three proteins has been identified (18). A cDNA clone, pAOSG81 corresponding to the gene, 30):], encoding the precursor protein has been isolated and characterized (18). Salicylic acid is an endogenous "trigger" of thermogenesis in S. guttatum appendix tissue (16). Raskin et al. (17) also showed that light is required for salicylic acid to "trigger" thermogenesis in appendix tissue sections. We previously used pAOSG81 and the AOA monoclonal antibody to study the developmental regulation of alternative oxidase expression and the expression of the alternative oxidase after application of salicylic acid in S. guttatum appendix tissue sections (19). Salicylic acid caused an increase in alternative pathway capacity in S. guttatum appendix tissue by causing an accumulation of the alternative oxidase transcript, which leads to an accumulation of the alternative oxidase proteins in the mitochondria ( 19). We are now establishing a system to study the regulation of the two respiratory pathways using suspension cultures of NTl tobacco cells. Alternative pathway capacity increases and a 35 kD alternative oxidase protein accumulates in these tobacco cells when they are transferred from the normal growth temperature of 30°C to 18°C (26) or when antimycin A, an inhibitor of the cytochrome pathway, is added to the culture medium (27). We report here on the developmental regulation of alternative oxidase expression and cytochrome and alternative pathway capacities in NT] cells and on the effects of exogenous salicylic acid on the capacities and expression of the alternative oxidase. 127 MATERIALS AND METHODS Tobacco Giltures. Suspension cultures of tobacco (Nicotiana tabacum L. cv.bright yellow) cells were grown in batch culture in LS medium (10) containing 0.2 pg/ml 2,4-D on a rotary shaker at 150 rpm and 30°C under heterotrophic conditions. In order to determine the cell density of a culture, a 5 mL aliquot was removed and the cells were pelleted at 1876:; in a Beckrnan RT6000B centrifuge. The supernatant was carefully removed, and the packed cells were weighed. The cell density was calculated as the grams of cells per 100 mL of grth medium and given as a percentage. The cell cultures were subcultured to a final density of 4% (w/v). The day following subculturing is referred to as day 1, the second day as day 2, and so on. Salicylic Acid and Inhibitor Treatment of Stupension Cultured Cells. For the time course experiments, salicylic acid was added to a final concentration of 1.0 mM to day 3 or day 4, and the cultures were then grown under normal conditions for 2 h, 4h,8h, 12h, 16h,20h,24h,and48h. For each inhibitor experiment, actinomycin D (50 pg/mL final concentration; Sigma, St. Louis, MO), an inhibitor of transcription, was added to one of four cultures and cycloheximide (100 pg/mL final concentration; Sigma, St. Louis, MO), an inhibitor of translation by 808 ribosomes, was added to a second culture. After one hour of incubation at 30°C, salicylic acid was added (1.0 mM final concentration) to the two cultures containing the inhibitors and the positive 128 control culture. The cultures were incubated at 30°C for an additional 15 h. The negative control culture was grown under normal conditions for 16 h and each experiment was started on day 3. For each concentration experiment, salicylic acid was added to final concentrations of 1.0 mM, 100 pM, 10 pM, or 1.0 pM to independent cultures. The cultures were incubated at 30°C for 16 h after addition of salicylic acid and experiments were started on day 3 or day 4. Isolation of Mitochondria and Respiration Assays. Suspension cultured cells were washed twice in fresh growth medium at room temperature. Mitochondria were isolated by a modification of the procedure of Schwitzguebel and Siegenthaler (22) as previously described ( 18). The cells were disrupted in a commercial blender by three bursts of 3 s each. Respiration rates were measured as oxygen uptake using a Rank Brothers oxygen electrode and 1.0 mL of reaction medium at 25 °C. The oxygen content of air-saturated water was estimated according to Estabrook (5). Capacities of the cytochrome and alternative pathways were determined as described by Elthon et al. (2). The capacity of the alternative pathway is the rate of oxygen uptake that is sensitive to SHAM in the presence of KCN. The capacity of the cytochrome pathway is the rate of oxygen uptake that is sensitive to KCN in the presence of SHAM. Total respiratory capacity is the sum of the alternative and cytochrome pathway capacities plus residual respiration (respiration in the presence of SHAM and cyanide), which was very low in the suspension cultured tobacco cells. The amount of total protein in each sample 129 was determined by the procedure of Larson et al. (9). Gel Ebctropharesis, Immrmoblotting, and Ant'uera. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as previously described (2). The antibody used was the AOA monoclonal antibody, which was raised to the 36 kD alternative oxidase protein of S. guttatum. This antibody recognizes all three alternative oxidase proteins of S. guttatum as well as putative alternative oxidase proteins from other higher plants, including tobacco (2,3). RESULTS Growth of Suspension Cultured Tobacco (blls. Figure 5-1 shows a grth curve for the suspension cultured NT] tobacco cells following subculture. The cell density increased by a factor of 1.5 each day from day 1, when the density was about’8%, until reaching a density of about 68% and a stationary phase at day 7. The cells remained in this stationary phase (at a density of about 70%) until at least day 14. Respiratory Pathway Capacities During Growth ofSuspension Culture Tobacco Cells. Changes that occurred in the capacities of the cytochrome and alternative pathways, measured in isolated mitochondria, during the grth of the suspension cultured tobacco cells are shown in panel A of Figure 5-2. The cytochrome 130 Figure 5-1. Growth curve for suspension cultured NTl tobacco cells. Cell density of each culture was determined by removing a 5 mL aliquot, pelleting the cells, removing the supernatant, and weighing the packed cells. The cell density, calculated as the grams of cells per 100 mL of growth medium, was plotted as a function of the number of days after subculturing. Results are averages from four experiments. 131 16 . - . p t p . O O 0 0 O 8 6 4 2 .523... ._E 8. 3:8 2 E23 =8 10 12 14 Culture Age [d] 132 pathway capacity increased rapidly between day 3 and day 4, and continued to increase until it reached the highest level observed on day 7. The capacity decreased between day 7 and day 10 and was at about the same level on day 14 as it was on day 10. The capacity of the alternative pathway was relatively high the day after the cells were subcultured, but decreased rapidly between day 1 and day 3. The capacity then increased rapidly between day 5 and day 6, reached the highest observed level on day 7, was at the same level on day 10 as it was on day 7, and decreased by day 14. Panel B of Figure 5-2 shows that the net result was that the percentage of the total respiratory capacity (see Materials and Methods for definition) of the cells that was attributable to the alternative pathway increased from 12% on day 5 to 36% on day 7. DevelopmentalRegtdationofAltemativeridaseExpressioninSmpension Cultured Tobacco Cells. Figure 5-3 shows that the amount of the 35 kD alternative oxidase protein in the mitochondria used for the capacity measurements (Fig. 5-2) increased dramatically between day 6 (lane 5) and day 7 (lane 6), increased slightly between day 7 and day 10 (lane 7), and decreased substantially by day 14 (lane 8). This pattern parallels the changes that occurred in the capacity of the alternative pathway during the grth of the cells (Fig. 5-2) and the dramatic increase occurred at the same time that the cells were approaching the stationary phase of growth. 133 Figure 5-2. Respiratory pathway capacities of suspension cultured NT1 tobacco cells. Mitochondria were isolated at each time point, and the capacities were determined using a Rank Brothers oxygen electrode. Panel A: The capacity of the alternative pathway (solid circles) and the cytochrome pathway (solid triangles) were plotted as functions of the number of days after subculturing. Panel B: The percentage of the total respiration that was attributable to the alternative pathway was plotted as a function of the number of days after subculturing. Results are averages of three experiments. 134 16 14 P D D D P D D P D D D D D D D D L D O O 0 O O O O m w W m m 06 5 4 3 2 1 En... .23 .o 8. 3.22.8 5a.. .2 329... 95555 2:22.. 2.330 due: 10 12 Culture Age [d] 135 Figure 5-3. Immunoblot showing accumulation of tobacco alternative oxidase at various times following subculturing. Mitochondria were isolated from suspension cultured NT1 tobacco cells at various times after subculturing and the respiratory pathway capacities were determined (see Fig. 5-2). The mitochondrial proteins (100 pg) were separated by SDS-PAGE, transferred to nitrocellulose, and the alternative oxidase proteins were identified using the AOA monoclonal antibody (3) and alkaline phosphatase linked to a secondary antibody. Lane 1: S. guttatum mitochondrial proteins; lane 2: day 3 NT 1 tobacco mitochondrial proteins; lane 3: day 4; lane 4: day 5; lane 5: day 6; lane 6: day 7; lane 7: day 10; lane 8: day 14. Mitochondria were isolated from S. guttatum appendix tissue as previously described (18) and used as a positive control (lane 1). Protein molecular masses in kilodaltons are indicated at the side. Results are representative of four experiments. 136 12345678 1 8.4— 137 TimeComseafSalicylicAcidInduceanngesinRespiratoryPathway Capacities. Panel A of Figure 5-4 shows that the capacity of the alternative pathway, measured in isolated mitochondria, remained relatively constant through 8 h after the addition of salicylic acid to a day 3 culture. The capacity then increased dramatically between 8 h and 12 h after salicylic acid addition, continued to increase until it reached the highest observed level at 16 h, decreased between 16 h and 20 h, and was at the same level at the 48 h time point as it was at the 20 h time point. During this experiment, the capacity of the cytochrome pathway did not increase as it did in the cultures that did not contain salicylic acid (Fig. 5-2, Panel A). Panel B of Figure 5-4 shows that the net result of the salicylic acid- induced changes in the capacities was that the percentage of total respiratory capacity (see Materials and Methods for definition) of the cells that was attributable to the alternative pathway increased from 22% before salicylic acid was added to a maximum of 53% at the 16 h time point. Salicylic Acid Induced Accumulation of Alternative Oxidase. The increase in the accumulation of the 35 kD alternative oxidase protein in the mitochondria used to determine the respiratory pathway capacities (Fig. 5-4) is shown in Figure 5-5. The amount of the protein remained relatively low until about 8 h (lane 6) after addition of salicylic acid to 1.0 mM. The amount of the protein was dramatically higher at 12 h than at 8 h (lane 7), was at a slightly higher level by 16 h (lane 8), was at a decreased level by 20 h (lane 9), was at about the We level at 24 h (lane 10), and was at a lower level at 48 h (lane 11). 138 Figure 5-4. Time course of salicylic acid effects on alternative and cytochrome pathway capacities. Salicylic acid was added to 1.0 mM to cell cultures on day 3. Panel A: The capacities of the alternative (solid circles) and the cytochrome pathways (solid triangles) were plotted as functions of the number of hours after salicylic acid addition. The solid circle surrounded by a circle and the solid circle surrounded by a square represent alternative pathway capacity measured in uninduced cells at the 24 h time point and the 48 h time point, respectively. The solid triangle surrounded by a circle and the solid triangle surrounded by square represent cytochrome pathway capacity in untreated cells at the 24 h time point and the 48 h time point, respectively. Panel B: The percentage of the total respiration that was attributable to the alternative pathway was plotted as a function of the number of days after subculturing. The solid circle surrounded by a circle and the solid circle surrounded by a square represent the percentage of total respiration attributed to the alternative pathway at 24 h and 48 h time points, respectively. Results are averages of three experiments. 139 7 4 3 2 4| .58... 9:356 2.22.. .3038 .83. but 7] 24 If 43 52 20 “me with Sallcyllc Acld [h] 12 16 b P b n b n b o 0 0 0 0 0 5 4 3 2 1 2.8.. .28 S a. >680 5a.. .2 140 Figure 5-5. Immunoblot showing the time course of salicylic acid induction of increased alternative oxidase accumulation. Salicylic acid was added to 1.0 mM to cell cultures on day 3. Mitochondria were isolated at each time point, and the respiratory pathway capacities were determined (see Fig. 5-4). The mitochondrial proteins (100 pg) were separated by SDS-PAGE, transferred to nitrocellulose, and the alternative oxidase proteins were identified using the AOA monoclonal antibody (3) and alkaline phosphatase linked to a secondary antibody. Mitochondrial proteins were from the following sources; lane 1: day 3 suspension cultured tobacco cells as time zero control; lane 2: day 4 as control; lane 3: day 5 as end point control; lane 4: cells treated for 2 h; lane 5: cells treated for 4 h; lane 6: cells treated for 8 h; lane 7: cells treated for 12 h; lane 8: cells treated for 16 h; lane 9: cells treated for 20 h; lane 10: cells treated for 24 h; lane 11: cells treated for 48 h. Protein molecular mass in kilodaltons is indicated at the side. Results are representative of three experiments. 141 1234567891011 142 thibitionofSalicylicAcidInducedAccmnulationofAlternafive Oxidase. Figure 5-6 shows that the accumulation of the alternative oxidase protein between the time when salicylic acid was added (lane 2) and 16 h after addition (lane 4) was decreased by the addition of actinomycin D (lane 5) 1 h prior to salicylic acid and blocked by addition of cycloheximide (lane 6) 1 h prior to salicylic acid. SahcylicacidConcenu'afionReqlmedforIncreaseinAltemafiveridaseand Capacity. Addition of salicylic acid to 1.0 mM (lane 3) for 16 h caused an increase in the capacity and accumulation of the 35 kD alternative oxidase protein (Figure 5-7). Addition of salicylic acid to 100 pM (lane 4) also caused a slight increase in alternative pathway capacity (data not shown) and a slight increase in the accumulation of the 35 kD alternative oxidase protein above the control level (lane 4). However, addition of salicylic acid to 10 pM (lane 5) or 1 pM (lane 6) for 16 h did not result in a significant increase in alternative pathway capacity (data not shown) or in an accumulation of the protein that was greater than that of the negative control (lane 2). 143 Figure 5-6. Immunoblot showing inhibition of salicylic acid induced increase in alternative oxidase expression. Actinomycin D (lane 5) or cycloheximide (lane 6) was added to day 3 suspension cultured cells. One hour later salicylic acid was added to 1.0 mM to the cultures, which were then incubated under normal growth conditions for 16 h. For positive control samples salicylic acid alone was added. Mitochondria were isolated after each treatment and the respiratory pathway capacities were determined. The mitochondrial proteins (100 pg) were separated by SDS-PAGE, transferred to nitrocellulose, and the alternative oxidase proteins were identified using the AOA monoclonal antibody (3) and alkaline phosphatase linked to a secondary antibody. Mitochondrial proteins were from the following sources; lane 1: S. guttatum appendix tissue; lane 2: untreated day 3 cells as a time zero control; lane 3: untreated day 4 cells; lane 4: salicylic acid treated cells; lane 5: actinomycin D and salicylic acid treated cell culture; lane 6: cycloheximide and salicylic acid treated cell culture. Mitochondria were isolated from S. guttatum appendix tissue as previously described (18) and used as a positive control (lane 1). Protein molecular masses in kilodaltons are indicated at the side. Results are representative of two experiments. 8183 \II 144 123456 145 Figure 5-7. Immunoblot showing alternative oxidase accumulation caused by various concentrations of salicylic acid. Salicylic acid was added to the cells to a final concentration of 1.0 mM (lane 3), 100 pM (lane 4), 10 pM (lane 5), and 1.0 pM (lane 6) and the cells were grown under normal conditions for 16 h. Mitochondria were isolated after each treatment and the respiratory pathway capacities were determined. The mitochondrial proteins (100 pg) were separated by SDS-PAGE, transferred to nitrocellulose, and the alternative oxidase proteins were identified using the AOA monoclonal antibody (3) and alkaline phosphatase linked to a secondary antibody. Mitochondrial proteins were from the following sources; lane 1: untreated day 3 cell culture as a time zero control; lane 2: untreated day 4 cells; lane 3: 1.0 mM salicylic acid treated cells; lane 4: 100 pM salicylic acid treated cells; lane 5: 10 pM salicylic acid treated cells; lane 6: 1.0 pM treated cells. Protein molecular mass in kilodaltons is indicated at the side. Results are representative of two experiments. 146 12 3 4 5 6 147 DISCUSSION The data presented here demonstrate that the capacities of both the alternative and the cytochrome pathway, measured in isolated mitochondria, varied greatly over the growth cycle of suspension cultured cells of Nicotiana tobaccum L cv bright yellow. Specifically, the cytochrome pathway capacity increased dramatically between day 3 and day 4 after subculturing, while alternative pathway capacity increased dramatically between day 5 and day 6. The capacities of both pathways their highest observed levels on day 7, which is the day the cell cultures reached a stationary phase of growth. However, the net result of the changes in the capacities of the pathways was that the percentage of the total respiratory pathway that was attributed to the alternative pathway increased significantly as the cells approached and reached the stationary phase of growth. In contrast, another study, using the N. glutinosa L cells, showed that the level of the alternative pathway was greatest during the lag and log phases of growth (6). The capacity measurements presented by Horn and Mertz used whole cells and measured alternative pathway capacity as O; uptake that was sensitive to SHAM only; not in the presence of cyanide (6). The difference between the results presented here and the results of Horn and Mertz (6) may be due to the use of different lines of suspension cultured tobacco cells. Our protein immunoblot data indicated that the level of the 35 kD alternative oxidase protein of the NT1 tobacco cells coincided with the pathway capacity throughout the growth cycle. Each increased dramatically between day 5 and day 6 and reached the highest level observed on day 7. 148 We have not measured the amount of sucrose at the various stages of the grth of the culture cells, but we assume that the amount was much lower at the stationary phase than it was in the early stages. Based on this assumption, the data presented here are contradictory to the overflow hypothesis (8), which postulates that the alternative pathway is most active when there is a supply of carbohydrate that exceeds the demand. Indeed, it seems illogical that the culture cells would partition electrons to the alternative pathway, with the inherent decrease in energy conservation, when the carbon supply is becoming limited. In this case, it may be a way for the cells to slow their growth during periods of decreased carbohydrate supply. When the alternative pathway was increased by the addition of salicylic acid, the density of the cells after 24 h of growth was 23 i 4%, compared to 33 z 2% for the control cultures. We do not know if the slight decrease in grth was caused by an increase in alternative pathway respiration or the presence of salicylic acid in the medium. Salicylic acid may play a role in systemic acquired resistance (SAR) in tobacco (11,30). Chemicals that mimic the effects of salicylic acid are being actively pursued as possible agents for crop protection (28,29). Studies to determine the efiect of salicylic acid upon 'the regulation of expression of the alternative oxidase will add to our understanding of the consequences of increased levels of salicylic acid and salicylic acid-mimicking chemicals in plant cells. In the experiments presented here, 1.0 mM salicylic acid caused a dramatic accumulation of the 35 kD alternative oxidase protein in suspension cultures of tobacco cells. The amount of the protein increased dramatically between 8 h and 12 h following 149 the addition of salicylic acid to the growth medium. The greatest accumulation occurred 16 h after the addition of salicylic acid. It is interesting to note that a very similar time course was observed for the response of S. guttatum appendix tissue to salicylic acid application (19). As in the experiments using S. guttatum appendix tissue, the increased protein accumulation in the tobacco cells coincided with a striking increase in the capacity of the alternative pathway. The capacity of the cytochrome pathway in these tobacco cells remained unchanged. Therefore, the percent of the total respiratory pathway that was attributed to the alternative pathway increased greatly. Since cytochrome pathway capacity increased in control culture cells during the same time, it seems that salicylic acid caused an inhibition of the normal, developmental increase in cytochrome pathway capacity. Again, a similar phenomenon occurs during the development of the appendix tissue of S. guttatum (4). While the capacity of the alternative pathway increases dramatically as the appendix tissue becomes thermogenic, the capacity of the cytochrome pathway is greatly decreased (4). The connection between the presence of salicylic acid in the observed effect on cytochrome pathway capacity in these systems remains unknown. In an attempt to dissect the mechanism of regulation of the expression of alternative oxidase in suspension cultured tobacco cells, we have demonstrated that actinomycin D significantly decreased the salicylic acid-directed accumulation of the 35 kD protein in the mitochondria of these cells. In addition, cycloheximide completely inhibited the accumulation of the protein. These data indicate that de, nova transcription and translation were necessary for salicylic acid to cause an 150 increase in the accumulation of the 35 kD alternative protein in suspension cultured tobacco cells. The observation that actinomycin D greatly inhibited the accumulation of the protein is consistent with the theory that salicylic acid regulates alternative oxidase expression in tobacco cells at the level of transcription. We have also demonstrated that a concentration of 100 pM salicylic acid caused a slight increase in alternative pathway capacity (data not shown) and a slight increase in the accumulation of the 35 kD alternative oxidase protein, but that concentrations of 10 pM or less did not cause any significant increases. By comparison, Kapulnik et al. (7) reported that a salicylic acid concentration of only 20 pM was sufficient to increase alternative pathway capacity in whole cells of Nicatiana tobaccum cv. Xanthi-nc. Since we do not know the efficiency with which salicylic acid is taken up by the suspension cultured tobacco cells and directed to its place of action, it is difficult for us to draw any conclusions from our results. Furthermore, all our cultures were grown in the dark, leaving the possiblity that a light component may be required for salicylic acid to have a full effect, as is the case in S. guttatum appendix tissue (17). This is the first thorough investigation of the developmental and salicylic acid induced changes in the respiratory pathway capacities and alternative oxidase expression in isolated mitochondria of suspension cultured tobacco cells. Future experiments will focus on identifying the changes in metabolism and cell growth resulting from increased and decreased alternative oxidase expression in order to learn more about the function of the alternative oxidase in higher plant cells. 15 1 ACKNOWLEDGEMENTS The authors thank Dr. Gynheung An for the suspension cultured tobacco cells and Dr. Thomas E. Elthon, Dr. Beate Drechsler-Thielmann and Dr. Greg Vanlerberghe for helpful advice and discussions. REFERENCES 1. Bendall DS & Bonner WD Jr (1971) Cyanide-insensitive respiration in plant mitochondria. Plant Physiol 47: 236-245. 2. Ethan TE & McIntosh L (1987) Identification of the alternative terminal oxidase of higher plant mitochondria. Proc Nat Acad Sci USA 84: 8399-8403. 3. Ethan TE Nickels RL & McIntosh L (1989) Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol 89: 1311-1317. 4. Ethan TE Nickels RL & McIntosh L (1989) Mitochondrial events during development of thermogenesis in Sauromatum guttatum (Schott). Planta 180: 82-89. 5. Estabrook RW (1987). Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47. 6. Horn ME & Mertz D (1982) Cyanide-resistant respiration in suspension cultured cells of Nicatr'ana glutinasa L. Plant Physiol 69: 1439-1443. 7. Kapulnik Y, Linser R, Yalpani N & Raskin I (1992) Salicylic acid induces cyanide-resistant respiration in tobacco cell suspension culture. Suppl Plant Phys 99: 112. 8. Lambers H (1985) Respiration in intact plants and tissues: Its regulation and dependence on environmental factors, metabolism and invaded organisms. In: R Douce & DA Day (eds) Encyclopedia of Plant Physiology, Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York, pp. 418-473. 9. Larson E, Hewlett B & Jagendorf A (1986). Artificial reductant enhancement of the Lowry method for protein determination. Anal Biochem 155: 243-248. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 152 Limaier EM & Skaog F (1965) Organic growthfactor requirement of tobacco tissue cultures. Physiol Plant 18: 100-127. Malamy J, Carr JP, Klessig DF & Raskin I (1990) Salicylic acid: a likely endogenous signal in the resistance response of tabacco to viral infection. Science 250: 1002-1004. Meeuse BJD (1975) Thermogenic respiration in aroids. Ann Rev Plant Physiol 26: 117-126. Meeuse BJD (1966) The voodoo lily. Sci Am 215: 80-88. Moore AL & Bonner Jr WD (1982) Measurements of membrane potentials in plant mitochondria with the safranine method. Plant Physiol 70: 1271-1276. Moore AL & Rich PR (1985) Organization of the respiratory chain and oxidative phosphorylation. In: R Douce & DA Day, (eds) Encyclopedia of Plant Physiology, Vol 18, Higher Plant Cell Respiration. Springer-Verlag, New York. pp. 134-172. Raskin I, Ehmann A, Melander WR & Meeuse BJD (1987) Salicylic acid: A natural inducer of heat production in Arum lilies. Science 237: 1601-1602. Raskin I, Turner IM & Melander WR (1989) Regulation of heat production in the inflorescean of an Arum lily by endogenous salicylic acid. Proc Natl Acad Sci USA 86: 2214-2218. Rhoads DM & McIntosh L (1991) Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott). Proc Nat Acad Sci USA 88: 2122-2126. Rhoads DM & McIntosh L (1992) Salicylic acid regulation of respiration in higher plants: Alternative Oxidase Expression. The Plant Cell, in press. Rich PR & Moore AL (1976) The involvement of the protonmotive ubiquinone cycle in the respiratory cnain of higher plants and its relation to the branchpoint of the alternate pathway. FEBS Letters 65: 339-344. Sambrook J, Frillch EF & Maniatis T (1989). Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. 22. 24. 26. 27. 29. 30. 153 Schwitzguebel JP & Siegenthaler PA (1984). Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation. Plant Physiol. 75: 670-674. Siedaw JN & Berthold DA (1986) The alternative oxidase: A cyanide- resistant respiratory pathway in higher plants. Physiol Plant 66: 569-573. Storey BT & Bahr JT (1969) The respiratory chain of plant mitochondria. 11. Oxidative phosphorylation in skunk cabbage mitochondria. Plant Physiol 44: 126-134. Storey BT (1976) Respiratory chain of plant mitochondria. XVIII. Point of interaction of the alternate oxidase with the respiratory chain. Plant Physiol 58: 521-525. Vanlerberghe GC & McIntosh L (1992) Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco. Plant Physiol, in press. Vanbrberghe GC & McIntosh L (1992) Coordinate regulation of cytochrome and alternative pathway respiration in tobacco. Plant Physiol, submitted. UknesS,Mauch-ManiB,Moych,PotterS,WilliamsS,DincherS, Chandler D, Slusarenko A, Ward E & Ryals J (1992) Acquired resistance in Arabidopsis. The Plant Cell 4: 645-656. WardER,UknesSJ,WilliamsSC,DincherSS,WiederhaldDW,Alexander DC, Ahl-Goy P, Metraus J-P, Ryah JA (1991) Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell 3: 1085-1094. YalpaniN,SilvermanP,WilsonTMA,KleierDA&Rash'nI (1991) Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco. Plant Cell 3: 809-818. SUMMARY OFTHESIS The major goals of my research project were to isolate cDNA and genomic clones encoding the alternative oxidase of Sauromatum guttatum and to study the developmental and salicylic acid induced expression of the alternative oxidase in S. guttatum. The research expanded to encompass the developmental and salicylic acid induced expression of alternative oxidase in suspension cultured tobacco cells. CHAPTER 2: ISOLATION OF A cDNA CLONE ENmDING THE ALTERNATIVE OXIDASE OF SAUROIHATUIK GUTTATW TheResults The first major achievement from my thesis research was the isolation of a cDNA clone encoding an alternative oxidase protein of S. guttatum. This work benefitted greatly from p the prior isolation of polyclonal and monoclonal antibodies that recognize only the 35-, 36-, and 37 kD alternative oxidase proteins on immunoblots of total mitochondrial proteins from S. guttatum. This cDN A clone has already been used to: 1) study the developmental and salicylic acid induced expression of the alternative oxidase in S. guttatum (CHAPTER 3); 2) isolate a 154 155 genomic clone of the alternative oxidase gene, aaxI, of S. guttatum (CHAPTER 4); and 3) isolate alternative oxidase cDNA clones from several other higher plants. The identification of a single, 42 kD product of in vitro translation that is immunoprecipitated by polyclonal antibodies, supports our hypothesis that the 35-, 36-, and 37 kD alternative oxidase proteins are post-translationally modified products of a single, nuclear-encoded protein. The isolation of the cDNA clone encoding an alternative oxidase protein of S. guttatum has provided a tool for the isolation of alternative oxidase cDNA clones from other organisms. The comparison of the sequence of these various cDNA clones will provide information about the evolution of the alternative oxidase gene. GIAPTER 3: DEVEDPWTAL AND SALICYLIC ACID REGULATION OF ALTERNATIVE OXIDASE WON IN SAUROMQTUM GUTTATUM TheResults Exploring the salicylic acid regulation of the alternative respiratory pathway, and, to some extent, the cytochrome pathway was the most interesting aspect of 156 my thesis research. This research provided evidence that salicylic acid regulates the expression of the alternative oxidase in S. guttatum by causing an increase in the accumulation of the 1.6 kb transcript. However, the mechanism resulting in this accumulation remains a mystery and an exciting area of research. Research on pathogenesis related proteins indicates that the transcripts for many of these proteins accumulate following salicylic acid application. Analysis of transgenic plants containing constructs with the promoters of each of these genes fused to reporter genes is consistent with the hypothesis that salicylic acid acts at the level of transcription. Thus, it is attractive to hypothesize that salicylic acid functions at the level of transcription to regulate alternative oxidase expression. However, the research in this thesis provides no direct evidence to support this hypothesis. TheFuture One way of attempting to prove this hypothesis would be to create transgenic tobacco plants containing a construct with the promoter region of the S. guttatum alternative oxidase gene, 30):], fused to a reporter gene. These transgenic plants could be sprayed with salicylic acid and analyzed to determine if there is increased accumulation of the transcripts of the reporter gene. Alternatively, the existence of a protein that binds to the promoter region may be confirmed by using nuclear extracts mixed with a DNA corresponding to the promoter region, followed by gel retardation assays. The binding of a protein in the presence of salicylic acid, but not the absence, would support this hypothesis. 157 Another hypothesis to explain the accumulation of the alternative oxidase transcript is that salicylic acid causes an increase in the stability of the alternative oxidase transcript. In order to investigate this hypothesis it would be helpful to: 1) establish an in vitro assay system to analyze the rate of transcript degradation; and 2) construct individual vectors to produce transcripts consisting of individual regions of the 1.6 kb alternative oxidase transcript fused to transcripts that are stable in the in vitro system. CHAPTER 4: ISOLATION AND CHARACTERIZATION OF A GENOMIC CLONE CONTAINING THE SAUROMATUM GUTTATW ALTERNATIVE OXIDASE GENE AOXI TheResults The S. guttatum alternative oxidase gene, 30x1, is a single copy gene that consists of four exons separated by three short introns. Exon 3 contains the region of the deduced precursor protein that is predicted to form two transmembrane helices. It is likely that this arrangement has some evolutionary significance in terms of protein structural and/or functional domains. The promoter of aaxl has sequences that resemble previously identified promoter elements that are associated with transcriptional regulation resulting from various stimuli. More importantly, the promoter contains sites with homology to sites of other promoters that convey salicylic acid responsiveness to their respective genes. 158 The Future The significance of the putative promoter elements present in the aaxl promoter may be determined directly by "dissection" of the promoter and analyses using transgenic tobacco plants. Isolation of genomic clones encoding the alternative oxidase of other organisms may assist in the determination of the evolutionary significance of the structure of am]. CHAPTER 5: CYTOCHROME AND ALTERNATIVE PATHWAY RESPIRATION IN TOBACG): EFFECTS OF SALICYLIC ACID TbResults The capacity of the alternative pathway at various time points during the growth of suspension cultures tobacco cells was determined. The results indicate that both the cytochrome pathway capacity and the alternative pathway capacity reach the highest level observed as the cells enter the stationary phase of growth. These results are inconsistent with the "overflow" hypothesis, which postulates that the alternative pathway is most active when there is a supply of carbohydrate that exceeds the demand. For these cells it may be a way for the cells to slow their growth during periods of decreased carbohydrate supply. Salicylic acid caused an increase ' in the alternative pathway capacity and increased expression of the 35 kD alternative oxidase protein in these cells. Even if salicylic acid is not a normal, in 159 viva regulator of the pathway in these cells, this result is important since the level of salicylic acid rises in tobacco leaf cells following inoculation of some pathogens. This will be useful information for researchers who are considering the use of salicylic acid mimicking chemicals as agents to increase the resistance of plants to pathogens. The Future The suspension cultured tobacco cells may prove to be a useful model system for studying the function of the alternative respiratory pathway in higher plants. Comparison of cell cultures with high alternative oxidase expression (by fusion of an alternative oxidase cDNA clone to an "inducible" promoter) to cell cultures with lowered expression (by production of anti-sense alternative oxidase transcripts) and cell cultures with normal expression may provide information regarding the contribution by alternative pathway respiration to whole cell. Furthermore, the suspension cultured tobacco cells may be a used as a model system to study the mechanism of salicylic acid regulation of alternative oxidase expression, alternative pathway capacity, and cytochrome pathway capacity in higher plants. 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