Molecular studies of dicamba-resistant Kochia scoparia L. by Anthony John Kern A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Sciences Montana State University © Copyright by Anthony John Kern (2002) Abstract: Extensive use of the auxinic herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in Montana grain production systems has selected for biotypes of Kochia scoparia that are insensitive to the herbicide. Dicamba is thought to induce the same physiological responses as the natural phytohormone auxin (indole-3-acetic acid), including changes in gene expression that may be involved in growth and developmental responses. Since the mechanism of dicamba resistance in K. scoparia is currently unknown, differential mRNA display techniques were conducted to compare patterns of dicamba-induced gene expression in resistant (R) and susceptible (S) biotypes. Examination of >80,000 mRNA fragments showed that changes in mRNA abundance occurred within minutes after dicamba treatment, and most changes were similar in R and S plants. From 106 cDNAs isolated, sequenced, and used as probes on northern blots, 14 represented mRNAs whose abundance changed after dicamba treatment. Of these 14 cDNAs, four were repressed similarly in R and S, two were similarly induced, and eight responded differentially to dicamba treatment in R and S. Eight cDNAs were assigned putative functions based on DNA or deduced amino acid sequence similarities to known genes, and included enzymes involved in basic carbon metabolism, cellular chloride uptake, photosynthesis, initiation of protein synthesis, synthesis and degradation of cell wall material, and a protein with a chaperone function. A partial cDNA encoding choline monooxygenase (CMO), an enzyme involved in the biosynthesis of the osmoprotectant glycine betaine (GB), was chosen for more detailed study. Characterization of expression patterns indicated that levels of CMO mRNA were increased by osmotic stress but rapidly declined after dicamba treatment. Levels of the CMO enzyme and GB were similarly reduced in R and S plants following dicamba treatment. However, R and S plants prestressed with NaCI showed differential CMO response after dicamba treatment, likely indicating a fundamental difference in dicamba translocation, perception, or signal transduction between the two biotypes. This research demonstrates that differential display is a useful technique for discovering changes in gene expression that may be initially involved in basic plant responses.  Molecular Studies of Dicamba-Resistant Kochia scoparia L. by Anthony John Kern A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Sciences MONTANA STATE UNIVERSITY Bozeman, Montana April 2002 APPROVAL ib of a dissertation submitted by Anthony John Kern This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies. William E. Dyer Y W o 2 'Date' Approved for the Department of Plant Sciences and Plant Pathology /hiPLUL ■//>4 ‘ signature Date Norman F. Weeden Approved for the College of Graduate Studies iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as described in the U S. Copyright Law. . Requests for extensive copying or reproduction of this dissertation should be referred to Bell & Howell Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non-exclusive rightto reproduce and distribute my abstract in any format in whole or in part.” Signature Date - P O - TABLE OF CONTENTS LIST OF TABLES.................................................................................................. vi LIST OF FIGURES.... .................................................... vii ABSTRACT.......................:........ ............i........................... ..................................x 1. INTRODUCTION............................................................................................... 1 Mechanisms of Herbicide Resistance The Auxinic Herbicides...................... Resistance to the Auxinic Herbicides. Arabidopsis Auxin-Related Mutants................... 10 Genes Responsive to Auxin and Auxinic Herbicides....................................... 15 Water Deficit in Plants,.....,.................................................................... *....... 25 2. ISOLATION OF DIFFERENTIALLY EXPRESSED GENES IN RESPONSE TO DICAMBA TREATMENT IN DICAMBA-RESISTANT AND SUSCEPTIBLE K.scoparia.........................37 I ntrod uction....................................................................................... . ■ ........ -37 Methods and Materials.....................................................................................41 Plant Material...... .......................................................................................41 RNA Extraction................................................................... 43 Differential Display RT-PCR................................................................. 44 Northern Blot Analysis............................................................................... 48 Sequencing...............................................................;............................... 50 Sequence Alignments................................................................................50 DNA Extraction...........................................................................................50 Cloning a Gene Fragment of 1-AminocycIopropane- 1 -carboxylate Synthase................................................. 51 Results and Discussion.............. 54 Differential Display Using D i-and Mono-Nucleotide-Specific Anchored Primers............................................................... -54 mRNAs Down-Regulated By Dicamba Treatment..................................... 57 C01R120.......................... 57 D416S0.............. 57 A05S0............... 60 C03S0.............................................................................................60 mRNAs Up-Regulated By Dicamba Treatment.........................................65 C07S120.........................................................................................65 G14S120....................................... 69 I iv CN CO O ) VTABLE OF CONTENTS-CONTINUED mRNAs Differentially Expressed Between R and S K. scoparia after Dicamba Treatment.................................. 69 A02S120........................................................................................ .69 C07S60.............................................. :........................................... 73 G07R0.............................................................................................76 C08R60...........................................................................................81 C08R60-2........................................................................................84 B16R120.......................... 87 A13S60................ :......................................................................... 87 C04R60............. 90 Cloning a Fragment of the 1 -AminocycIopropane- 1-carboxylate Synthase Gene From K. scoparia............................92 3. EXPRESSION OF K. SCOPARIA CHOLINE MONOOXYGENASE IN RESPONSE TO SALT STRESS AND DICAMBA TREATMENT............................................ 96 Introduction...... ............................... ................................ ..........:....................96 Methods and Materials................................... 98 3' RACE.......... .................................................... 98 NaCIT reatment............................................................ 100 Dicamba Treatment of NaCI-Treated Plants........... ............... 100 Northern Blot Analysis................ 101 Protein Extraction...... .............................. 101 Western Blot Analysis.......... ................................................................ ..,102 Glycine Betaine Extraction.... .................................................................. 103 1H NMR..,.......... .................................. 103 Results and Discussion................ 105 Analysis of the CMO 3' RACE Product.................................................... 105 CMO Expression in Response to NaCI Stress........................................ 108 CMO Expression in Response to Dicamba Treatment in R and BK. scoparia..................................................................................113 CMO Expression in Response to Dicamba Treatment in Salt-Stressed K. scoparia........................................................ 117 4. CONCLUSIONS AND FUTURE WORK....................................................... 123 Screening for Genes Responsive to Dicamba Treatment.......................123 Analysis of Choline Monooxygenase Expression.................................... 128 LITERATURE CITED:................................................................................. 131 vi LIST OF TABLES Table Page 1. Representative auxin induced genes............. ................................. ....18 2. Representative auxin repressed genes....:....................... 23 3. Primer sequences used for differential display.................................. ...45 r' vii LIST OF FIGURES Figure Page 1. Structures of auxin (indole-3-acetic acid) and representative auxinic herbicides............... 7 2. Biosynthetic pathways leading to GB production.............................. ..30 3. A representative northern blot containing RNA isolated 0,60, and 120 minutes after dicamba treatment................ ......... 49 4. mRNA amplification and differential display using dinucleotide-specific anchored primers.................... 55 5. Expression of C01R120 after dicamba treatment..... .........................58 6. Expression of D416S0 after dicamba treatment.................................59 7. Expression of A05S0 after dicamba treatment................................... 61 8. Expression of C03S0 after dicamba treatment................................... 62 9. C03S0 is mostly 3' untranslated region...............................................63 10. Expression of C07S120 after dicamba treatment............................ .66 11. C07S120 is mostly 3' untranslated region................................. ........67 12. Expression of G14S120 after dicamba treatment.............................70 13. Expression of A02S120 after dicamba treatment.......... ....................71 14. A02S120 is mostly 3' untranslated region..................... ....................72 15. Expression of C07S60 after dicamba treatment......... .......................74 LIST OF FIGURES-CONTINUED Figure Page 16. C07S60 has high sequence similarity to SUM translation initiation factors........ ............ 75 17. Expression of G07R0 after dicamba treatment............. .....................77 18. G07R0 open reading frame.................................................................78 19. PILEUP comparisons of G07R0 and similar amino acid , sequences as recovered by BLAST.......... .......... 79 20. Expression of C08R60 after dicamba treatment................................82 21. Translated amino acid sequence of the C08R60 cDNA......................83 22. Expression of C08R60-2 after dicamba treatment.............................85 23. C08R60-2 is similar to hypothetical proteins in several photosynthetic organisms.................................................. 86 24. Expression of B16R120 after dicamba treatment........................... ...88 25. Expression of A13S60 after dicamba treatment........... .................... .89 26. Expression of C04R60 after dicamba treatment................................91 27. PILEUP analysis of the deduced TACC1-6 amino acid sequence and ACC synthase sequences from other species........ 93 28. ACC synthase gene expression is induced by dicamba treatment......................... 94 29. WCMO 3' RACE product..................................... 106 30. PILEUP comparisons of WCMO and similar amino acid sequences as recovered by BLAST.................... 107 viii LIST OF FIGURES-CONTINUED Figure Page 31. Expression of CMO mRNA in response to salt stress.... ..................109 32. Accumulation of CMO protein and glycine betaine in response to salt stress.................................................................. 110 33: Expression of CMO mRNA after dicamba treatment of unstressed plants.................. 114 34. Reduction of CMO protein and glycine betaine levels in unstressed plants after dicamba treatment.................................. 115 35. Expression of CMO mRNA after dicamba treatment in salt-stressed plants...... ........................................... 118 36. Levels of CMO protein and glycine betaine in salt-stressed plants after dicamba treatment................................120 ix ABSTRACT Extensive use of the auxinic herbicide dicamba (3,6-dichloro-2- methoxybenzoic acid) in Montana grain production systems has selected for biotypes of Kochia scoparia that are insensitive to the herbicide. Dicamba is thought to induce the same physiological responses as the natural phytohormone auxin (indole-3-acetic acid), including changes in gene expression that may be involved in growth and developmental responses. Since the mechanism of dicamba resistance in K. scoparia is currently unknown, differential mRNA display techniques were conducted to compare patterns of dicamba-induced gene expression in resistant (R) and susceptible (S) biotypes. Examination of >80,000 mRNA fragments showed that changes in mRNA abundance occurred within minutes after dicamba treatment, and most changes were similar in R and S plants. From 106 cDNAs isolated, sequenced, and used as probes on northern blots, 14 represented mRNAs whose abundance changed after dicamba treatment. Of these 14 cDNAs, four were repressed similarly in R and S, two were similarly induced, and eight responded differentially to dicamba treatment in R and S. Eight cDNAs were assigned putative functions based on DNA or deduced amino acid sequence similarities to known genes, and included enzymes involved in basic carbon metabolism, cellular chloride uptake, photosynthesis, initiation of protein synthesis, synthesis and degradation of cell wall material, and a protein with a chaperone function. A partial cDNA encoding choline monooxygenase (CMO), an enzyme involved in the biosynthesis of the osmoprotectant glycine betaine (GB), was chosen for more detailed study. Characterization of expression patterns indicated that levels of CMO mRNA were increased by osmotic stress but rapidly declined after dicamba treatment. Levels of the CMO enzyme and GB were similarly reduced in R and S plants following dicamba treatment. However, R and S plants prestressed with NaCI showed differential CMO response after dicamba treatment, likely indicating a fundamental difference in dicamba translocation, perception, or signal transduction between the two biotypes. This research demonstrates that differential display is a useful technique for discovering changes in gene expression that may be initially involved in basic plant responses. 1CHAPTER 1 INTRODUCTION Since the introduction of 2,4-D (2,4-dichlorophenoxyacetic acid) in 1952, selective herbicides have been an integral part of modern agriculture, and are used in essentially all non-organic cropping systems to help ameliorate the impacts of weedy plants. While these herbicides have been generally very effective at reducing competition from weeds, the evolution of weed populations resistant to specific herbicides poses a serious threat to global agricultural production. In 1970, Ryan reported the first instance of herbicide resistance in Senecio vulgaris (Ryan, 1970), and by 2001, the International Survey of Herbicide Resistant Weeds documented over 250 populations of weeds resistant to different herbicides, with the numbers expected to continue to increase as herbicide use and selection continues (Heap 2001). Despite the problems herbicide-resistant weeds pose to agricultural production, these mutant phenotypes can provide useful tools to dissect important genetic and metabolic pathways. Weedy species are problematic in agricultural systems largely because of vigorous growth and reproduction, phenotypic plasticity, and the ability to grow under conditions of diverse stress. Some of these characteristics could be useful in an agronomic system if introduced into crop species (Snow et al., 1998), and a great deal of basic and applied research has focused on one weedy plant in particular (Arabidopsis thaliana] hereafter referred to as 2Arabidopsis). Mutants in this model plant have been the focus of an unusual amount of research, especially related to efforts to elucidate genetic control over plant hormone physiology and response to environmental stress. It is likely that other weedy species may provide useful tools to address questions related to both basic plant biology and potential agronomic applications. This report addresses changes in expression patterns mediated by a mimic of the natural phytohormone auxin in a mutant of the weedy plant K. scoparia, which has evolved resistance to the synthetic auxin dicamba (3,6-dichl0ro-2- methoxybenzoic acid). This mutant may provide a unique tool to gain insight into the genetics of auxin physiology. Additionally, this report characterizes a gene from K. scoparia that has received considerable attention as a potential target for transgenic applications that aim to improve drought tolerance in crops. Mechanisms of Herbicide Resistance Mechanisms of herbicide resistance vary greatly across plant species, but all stem from changes in the physiological fate of the herbicide: how it is absorbed, transported within the plant, metabolized, or exerts its toxic effects In most cases, herbicides exhibit their phytotoxic activities by binding to and inhibiting the normal biochemical activity of a single enzyme. Mutations in the genes encoding such enzymes that lower or abolish herbicide binding result in plants that are no longer sensitive to the herbicide. This so-called target site resistance is most thoroughly documented for the triazine and phenylurea herbicides, which normally inhibit photosynthesis by blocking electron transport 3from plastoquinone Qa (bound to the D2 protein in photosystem II) to plastoquinone Q6 (bound to the neighboring D1 protein); this blockage is probably mediated by displacement of Qb with the herbicide (Vermass et al., 1983). In resistant plants, one or more mutations in the amino acid sequence of D1 severely reduce the binding affinity of the enzyme for the herbicide, thereby conferring resistance. Plants that are herbicide resistant because of target-site enzyme mutations (such as the D1 protein, acetolactate synthase, and acetyl- coenzyme A carboxylase) account for well over half of the documented cases to date (Heap, 2001). Altered herbicide movement has also been shown to confer the resistance trait in several plant species. Slow absorption of herbicides through the cuticle is thought to confer resistance to triasulfuron (2-(2-chloroethoxy)-n-[[(4-methoxy-6- methyl-1,3,5-triazin-2-yl) amino]carbonyl] benzene sulfonamide) in Triticum aestivum (Meyer and Muller, 1989), and several species of Equisetum exhibit natural tolerance to the herbicide glyphosate (A/-phosophonomethyl glycine) due to similar mechanisms (Marshall et al., 1987). In Erigeron philadelphicus and E. canadensis (Tanaka et al., 1986), Hordeum glaucum (Bishop et al., 1987), Conyza bonariensis (Fuerst et al., 1985), and Lolium perenne (Faulkner, 1976), resistance to the herbicides paraquat (1:1-dimethyl-4,4'-bipyridinium dichloride) and diquat (6,7-dihydroipyridol[1,2-a:2T-c]pyrazidinium dibromide) is due to reduced translocation of the herbicide within the plant, even though rates of absorption were similar to susceptible biotypes. Sequestration of herbicides into 4vacuoles has been documented for 2,4-D resistance in soybean (Schmitt and Sandermann, 1982) and is also thought to confer resistance to diclofop-methyl (methyl-2-[4-(2,4-dichlorophenoxy) phenoxyl] propanoate) in Avena fatua populations (Bourgeois et al„ 1997). Herbicide sequestration into extracellular cell wall matrices is associated with resistance to difenzoquat (1,2-dimethyl-3,5- diphenyl-1 H-pyrazolium) in Avena fatua (Kern and Dyer, 1998a), and has been hypothesized as a mechanism of resistance to paraquat (1,T-dimethyl-4,4'- bipyridinium ion) in other species as well (Norman and Fuerst, 1997). Herbicide resistance can be caused by alterations in herbicide metabolic pathways. In most cases, these metabolism-based cases of resistance stem from increased rates of herbicide catabolism or conjugation of herbicides with endogenous cellular components. Cytochrome P450-mediated hydroxylation, sulfoxidation, and deesterification reactions are responsible for metabolic detoxification of herbicides in several species (Brown, 1991). Conjugation of herbicides with reduced glutathione (Dean et al., 1991), sugars (Gronneau et al., 1988) , or other compounds (Gronwald, 1994) can quickly mitigate herbicidal activity. The natural levels of tolerance exhibited to atrazine in Zea mays and certain biotypes of Abutilon theophrasti (Timmerman, 1989, Gronwald et al., 1989) are thought to be mediated by high levels of glutathione-S-transferase activity. While most metabolism-based mechanisms of herbicide resistance are due to increased rates of herbicide catabolism, other work has shown that decreased metabolism of certain herbicides like triallate (S-(2,3,3-trichloro-2- propenyl) bis(l-methylethyl)carbamothioate) that require an in vivo activation 5reaction also confer resistance (Kern et al., 1998b). Under conditions of extensive herbicide selection pressure, the evolution and increase of herbicide-resistant plants is highly likely (Maxwell and Mortimer, 1994). In most cases, inheritance of resistance can be explained by the action of a single dominant gene, although recessive (loss of function) alleles have been documented in Setaria viridis and Avena fatua for resistance to diclofop and triallate, respectively (Jasieniuk et al., 1994; Kern et al., 2002). Herbicide- resistant weed populations typically become resistant to other members of the herbicide class and mode of action to which they were repeatedly exposed; this characteristic is termed cross-resistance. Populations of Kochia scoparia that developed resistance to chlorsulfuron (2-chloro-A/-[[(4-methoxy-6-methyl-1,3,5 triazin-2-yl)amino]carbonyl]benzenesulfonamide) were also resistant to several other sulfonylurea herbicides (Sivakumaran, 1992), and trifluralin (1,6-dinitro- N,N-diprppy!-4-(trifluoromethyl) benzeneamine)-resistant populations of Eleusine indica and Setaria viridis were also shown to be cross-resistant to numerous other dinitroaniline herbicides (Mudge et al., 1984). In some cases, however, the evolution of resistance to one chemical class of herbicides may also confer resistance to herbicides with different mechanisms of action. Avena fatua populations resistant to the thiocarbamate herbicides triallate and diallate (S- (2,3-dichloro-2-propenyl) bis(l-methylethyl) carbamothioate) were shown to be cross-resistant to the unrelated bipyridiliUm herbicide difenzoquat (Kern et al., 1996, Blackshaw et al., 1996). Lolium rigidum populations from Australia were shown to be resistant to over 20 different herbicides from nine different chemical 6classes (Hall et al., 1994). However, multiple resistance mechanisms, including target-site mutations and increased metabolism-based herbicide detoxification, were present in resistant populations. The auxinic herbicides Auxinic herbicides have long played a key role in controlling dicot weed species in grass crops. Although the precise mode of action of the individual auxinic herbicides is unknown, it is widely thought that they act as synthetic versions of the phytohormone auxin (indole-3-acetic acid, IAA; Sterling and Hall, 1997). In low doses, the auxinic herbicides 2,4-D and dicamba are commonly used to replace auxin in plant cell tissue culture, supporting the idea that these herbicides act as synthetic auxin mimics. In higher doses, auxinic herbicides cause cell and plant death through uncontrolled growth, vascular tissue proliferation, and cell membrane destruction (Devine, 1993). Despite this evidence, individual auxinic herbicides have chemical structures very different from auxin, although all have a carboxylic acid moiety (Figure 1). The phenoxycarboxylic acids include MCPA (4-chloro-2- methylphenoxy acetic acid), 2,4-D, 2,4-DB (4-(2,4-dichlorophenoxy butanoic acid), and mecoprop (2-(4-chloro-2-methylphenoxy) propanoic acid); dicamba and chloramben (3-amino-2,5-dichloro benzoic acid) are derivatives of benzoic acid; picloram (4-amino-3,5,6-trichloro 2-pyridinecarboxylic acid) and triclopyr ([(3,5,6-trichloro-2-pyridinyl] oxy)acetic acid) are pyridine carboxylic acids, and quinclorac (3,7-dichloro-8-quinolinecarboxylic acid) and quinmerac (7-chloro-3- 7/ S ^ r ---^-CH2 COOH U L / indole-3-acetic acid Phenoxycarboxylic acids CH3 mecoprop Benzoic acid derivatives Pyridine carboxylic acids H2N Cl Cl— ^ ^ — COOH>N picloram O -C H 2-CH2-C h2-COOH 2,4-DB COOH chloramben Quinoline carboxylic acids COOH quinmerac COOH quinclorac Figure 1. Structures of auxin (indole-3-acetic acid) and representative auxinic herbicides. Revised from Sterling and Hall, 1997. 8methyl 8-quinolinecarboxylic acid) belong to the quinoline carboxylic acid class of compounds. Recent reviews (Grossmann, 2000; Sterling and Hall, 1997; Hansen and Grossmann, 2000) suggest that multiple modes of action, coupled with different perturbations in plant hormone homeostasis, exist for different auxihic herbicides. Soon after application of all auxinic herbicides, the rate of ethylene biosynthesis increases dramatically (Sterling and Hall, 1997). This ,is primarily thought to be the effect of rapid induction of 1 -aminocyclopropane-1 -carboxylic acid (ACC) synthase (ACS), the enzyme that catalyzes the rate-limiting step in ethylene biosynthesis (conversion of S-adenosyl methionine to ACC). In some plants, induction of ethylene biosynthesis is thought to be the primary mechanism by which the auxinic herbicides cause leaf epinasty, stem curvature, and leaf abscission (Wei et al., 2000). Additionally, Arabidopsis mutants defective in ethylene perception and tomato plants containing an overexpressed antisense version of the ACS gene were not sensitive to auxin (Sitrit and Bennett, 1998), whereas ethylene applications caused symptoms normally associated with auxinic herbicide application. Other evidence suggests that ethylene overproduction is not the causative agent of phytotoxicity after treatment with auxin: one byproduct of ethylene biosynthesis is cyanide, which is thought to play a role in plant death after treatment with quinclorac (Grossman, 1998). As with IAA, the auxinic herbicides have been shown to increase proton pumping into the tonpplast within minutes of application, subsequently activating cell wall degrading enzymes in support of the acid growth hypothesis (Salisbury 9and Ross, 1992). Activation of these enzymes may cause irreversible degradation of plant cell wall materials, leading to necrosis and cell death (Sterling and Hall, 1997). Resistance to the auxinic herbicides Despite extensive use for over 50 years, there are relatively few examples of plant resistance to the auxinic herbicides. The few biotypes that have developed resistance generally have lower levels of fitness and survivability, making them less likely to become a widespread threat to agronomic systems utilizing these herbicides. In 1957, Commelina diffusa plants were isolated in Hawaii that were not controlled by field rates of 2,4-D (Hilton, 1957), but have not been documented since. Populations of wild carrot (Caucus carota) that were resistant to 2,4-D were a similarly minor problem (Whitehead and Switzer, 1963). Other examples include 2,4-D-resistant Carduus species (Bonner et al., 1998), Centaurea solstitialis (Sabba et al., 1998) resistant to picloram, MCPA-resistant populations of Cirsium arvense (Fogelfors, 1979; Solymosi et al., 1987), populations of K. scoparia resistant to dicamba, MCPA, butyrac, bicloram, and 2,4-D (Miller et al., 1997), as well as a few other cases (for review, see http://www.weedscience.org). With the exception of 2,4-D-resistant Carduus spp. and K. scoparia, most instances of auxinic-herbicide resistant weeds have resulted in relatively few agricultural problems. Little is known about the mechanism(s) of resistance to the auxinic herbicides, although there typically are severe morphological effects associated 10 with the resistance trait. R biotypes of Sinapis arvensis tend to be shorter, more branched, darker green, have poorly-branched roots, and had aberrations in cytokinin and other hormone levels (Hall and Romano, 1995). R S. arvensis biotypes showed decreased binding of 3H-IAA to unidentified microsomal proteins; however, it is unclear if such binding assays represent physiologically relevant binding activities (Webb and Hall, 1995). Studies on mecoprop resistance in Steilaria media (Coupland et al., 1991), picloram resistance in Sinapis arvensis (Peniuk et al., 1993), picloram resistance in Centaurea solstitialis (Fuerst et al., 1996) and dicamba resistance in K. scoparia (Cranston et al., 2001) indicated that resistance to the auxinic herbicides was not due to reduced herbicide uptake or translocation, or increased metabolism. Arabidoosis auxin-related mutants Insights into the mechanism of resistance to the auxinic herbicides may be gained by understanding the biochemical and genetic differences between wild-type plants and plants with altered auxin responsiveness; Numerous mutant phenotypes have been isolated from Arabidopsis and provide the opportunity to study auxin physiology at many different levels, and in some cases these studies have identified specific genes that result in altered auxin biosynthesis, metabolism, transport, or cellular perception/signal transduction. Auxin is produced in plant meristems by transamination or decarboxylation of the amino acid tryptophan (Salisbury and Ross, 1992), and is often subsequently conjugated to free amino acids, short polypeptides, and 11 sugars. Alterations in auxin biosynthesis and metabolism have been shown to cause several auxin mutant phenotypes, including the Arabidopsis trp1 mutant, which is deficient in tryptophan production and exhibits symptoms similar to auxin deficiency (Last et al., 1991). In contrast, other auxin-production mutants are overproducers: the sur1 (superrootl) mutant exhibits morphology suggestive of severe auxin overproduction, including elongated hypocotyls, increased adventitious root formation, and epinasty (Boerjan et al., 1995). The st/pe/roof2 mutant sur2, which also overproduces auxin and has a similar morphology to sur1, was generated using transposon mutagenesis. Tagging studies indicated that SUR2 encodes a cytochrome P450 gene, and the gene is upregulated in both sur1 and sur2 populations (Delarue et al., 1998). These mutants, along with the trp2 and trp3 populations (Normanly et al., 1993), are thought to overproduce auxin via upregulation of cytochrome P450-mediated oxidation of indole-3-acetonitrile (IAN), suggesting the presence of a tryptophan-independent pathway for auxin biosynthesis. Indeed, two P450 genes have been isolated from Arabidopsis that may mediate this pathway (Hull et al., 2000), and other auxin-insensitive Arabidopsis mutants are defective in the NIT1 gene, which is also thought to catalyze the formation of auxin from IAN (Normanly et al., 1997). The sur2 mutants reverted to wild-type morphology when treated with exogenously-applied auxin or when grown on an acidic growth medium; concomitant decreases in both indole-3-acetonitrile and auxin content were noted (Barlier et al., 2000). Aside from auxin production, other auxin mutants appear to have altered \auxin transport characteristics. It is thought that auxin transport is mediated by two important cellular proteins: auxin efflux carriers, which are located at the basal ends of cells and are responsible for basipetal flow of auxin in stems (Palme and Galweiler, 1999), and auxin influx proteins, which are thought to mediate cellular auxin uptake by a proton symport system (Lomax et al., 1995). The latter system suggests a plasmalemma-bound auxin receptor (an auxin binding protein) responsible for initiation of cellular secondary messenger cascades. The auxin binding protein ABP1 has been purified from maize (Lobler and Klambt, 1985) and shows features characteristic of a protein located in the lumen of the endoplasmic reticulum (ER), as it contains a carboxy-terminal KDEL motif (Tillman et al., 1989). However, an auxin receptor has been shown to be located on the surface of the plasma membrane (Napier and Venus, 1995), and it is thought that the ABP protein is stored in the ER and secreted to the cell surface at levels which depend on the physiological state of the cell (Shimomura etal., 1999). Perhaps the most-studied of these influx or efflux mutants is the auxl mutant from Arabidopsis. Initially created by mutagenesis (Maher and Martindale 1980), the auxl phenotype is characterized by root agravatropism and reduced lateral root formation (Pickett et al., 1990). Auxl is resistant to auxin and 2,4-D treatment (Yamamoto and Yamamoto 1998); but not to 1- napthaleneacetic acid (NAA), which crosses membranes by simple diffusion, suggesting a defect in cellular auxin uptake. Bennett et al. (1996) recapitulated the auxl mutant from wild-type plants by transposon mutagenesis and showed 12 13 AUX1 has high sequence homology to amino acid permeases, which are also proton symporters. Promoter-GUS fusions showed XhatAUXI is expressed in root tips ahd in emerging lateral roots, but not in shoots, further supporting the hypothesis that the AUX1 protein is involved in root auxin uptake (Marchant et al., 1999). Auxin efflux carriers are thought to be the driving force of auxin transport, and physiological studies have shown that one of these proteins, pin1, exists on the basal ends of vascular parenchyma cells (Galweiler et al., 1998). Mutation in the PIN1 gene results in altered inflorescence and vascular development consistent with permutations in auxin transport. Additionally, treatment of wild- type plants with auxin transport inhibitors yields phenotypes similar to pin1 mutants. These inhibitors (2,3,5-triiodobenzoic acid and AM -napthylphtalamic acid; NPA) act as direct inhibitors of auxin efflux proteins, as in the tir3 auxin transport mutant, which has fewer sites that bind NPA in microsomal preparations than do wild-type plants (Ruegger et al., 1997). Other research suggests that reduced NPA binding is associated with reduced auxin polar transport activity. The interfasicularfiberless/revoluta (ifU/rev) mutant is characterized by a pin-like inflorescence and lack of normal interfascicular fiber differentiation (Zhong et al., 1997), altered xylem differentiation (Zhong and Ye, 1999), and dark green leaves with delayed senescence. The ifU/rev mutant shows a 40% reduction in NPA binding to plasma membranes compared to the wild-type, and expression of P/A/3 and other putative auxin efflux carrier genes is reduced in ifU/rev mutants (Zhong and Ye, 2001). The IFL1 gene encodes a 14 homeodomain leucine-zipper, and it is thought that mutations in this gene are responsible for decreased expression of the PINauxin efflux carriers) and resultant phenotypes (Ratcliffe et al., 2000). A second putative auxin efflux carrier termed AGR (Utsuno et al., 1998) is allelic to EIR1 (Luschnig et al., 1998), has been shown to be involved in root gravitropism, and encodes a protein with likely membrane-spanning domains that also has sequence homology to toxin efflux systems in E. coll (Luschnig et al., 1998). Other mutations in auxin efflux carrier-associated proteins appear to have similar effects as mutations in the efflux proteins themselves. The rent mutant is deficient in protein phosphatase 2A activity and exhibits defects in root curling and other phenotypes requiring differential cell elongation (Rashotte et al., 2001). While auxin influx and efflux mutants have been fairly well characterized, less is known about mutants that have altered auxin perception or signal transduction, likely because these pathways have yet to be elucidated. Mutants of these types carry a variety of genetic lesions. The axrl mutant is one of only a few that show reduced auxin sensitivity in all tissues, reduced induction of auxin-responsive genes such as the SAUR genes (see below), and do not have altered endogenous auxin concentrations (Lincoln et al., 1990). Although the AXRi gene has been cloned and has sequence similarity with the ubiquitin- activating enzyme E1 (Leyser et al., 1993), it is not clear what role this gene plays in reduced auxin sensitivity (Leyser, 1997). The arg1 mutant was generated through transposon mutagenesis and has been proposed to be deficient in signal transduction pathways after auxin treatment (Sedbrook et al., 15 1999). The ARG1 gene was positionally cloned using bulked segregant analysis (interestingly, ARG1 was not transposon tagged, despite being generated using transposon mutagenesis; this may be due to a transposon excision event, leaving a footprint responsible for the mutant phenotype) and shown to encode a DnaJ-Iike protein, suggesting it may modify protein kinase or calmodulin activity by acting as a chaperone protein (Sedbrpok et a!., 1999). Certainly, auxin-response mutants in Arabidopsis have given insight into auxin physiology and have provided strong evidence for an active transport system which includes auxin efflux and influx proteins. Additionally, studies investigating auxin autotrophs and auxotrophs show alternative metabolic pathways for auxin production, and clearly indicate severe morphological effects when in vivo auxin concentrations are perturbed. Despite this, these mutants provide limited information on the ultimate mechanism of auxin action (changes in cell physiology that may be mediated by alterations in gene expression). It is likely that genetic studies investigating changes in gene expression as affected by auxin will provide considerable insight into auxin action. Some of the experiments reported here investigate such changes in an auxin-mimic mutant of K. scoparia. Genes responsive to auxin and auxinic herbicides While it is not entirely clear if the auxinic herbicides affect plants in ways that are identical to IAA1 numerous bioassays indicate both have similar effects on various aspects of plant growth such as cell elongation, tissue differentiation, 16 turgor pressure, and cell polarity (Abel and Theologis, 1996; Sitbon and Perrot- Rechenmann1 1997). Although molecular mechanisms (incontrovertible verification of an auxin receptor, secondary messenger cascades, and changes in gene expression that are ultimately responsible for alterations to plant growth) by which auxin exerts its effects have not been identified, considerable research has concentrated on the roles that auxin and auxin mimics have on changes in gene expression. It is important to note that while auxin and the auxinic herbicides (in particular, 2,4-D) are often used in these studies interchangeably, in some cases the two have different effects on gene expression (IVIito and Bennett, 1995). Most systems to date have used subtractive hybridization methodologies in auxin-starved cell suspension cultures or auxin-treated elongating tissues to isolate differentially-expressed genes. While dozens of these auxin-responsive genes have been isolated, considerable effort has focused on the expression events that occur within minutes after auxin application (Abel and Theologis, 1996); these are termed primary-response genes. It is thought that changes in the expression patterns of these primary-response genes occur independently of de novo protein synthesis, indicating that the cellular components necessary to change expression patterns are preexisting in the cell. It follows that these genes may be some of the most important in initiating the cascade of transcriptional events ultimately leading to the various physiological responses to auxin, and may offer the most insight into auxin action. Many of the genes isolated to date are members of large gene families, 17 and homology-based PCR cloning has enabled researchers to isolate and identify multiple members of these families. The majority of auxin-responsive genes isolated to date are induced, or up-regulated, by auxin treatment and loosely fall into one of eight categories (Table I ). Although auxin has been only loosely implicated in the initiation of cell division (Kitamiya et al., 2000), several cell cycle-associated cDNAs have been isolated that are responsive to auxin treatment. Homologs of the protein kinase p34 (cdc2) have been isolated from Arabidopsis (Martinez et al., 1990), Medicago (Hirt et al., 1993), Nicotiana (John et al., 1993), Pisum (Hemerly et al., 1993), and Glycine protoplasts (Miao et al., 1993). However, since cytokinin is also required for their induction, and induction requires several hours, they are probably not primary-response genes, In contrast, Xie et al. (2000) suggests that the rapidly activated TIR1 (Gray et al., 1999) and AXR1 (Leyser et al., 1993) genes, both of which encode proteins involved in the pathways of protein ubiquitination, allow pericycle cells (normally arrested in the G2 phase of the cell cycle) to re-enter the cell cycle and initiate lateral root formation. Most genes induced by auxin treatment appear to be directly involved in biochemical processes mediated by IAA. Because of the broad array of gene transcription events associated with auxin treatment, it is not surprising that transcription of several putative DNA-binding proteins is induced by auxin treatment. The histone genes DBP and HSC-IIwere induced within 4 hours after auxin treatment in Arabidopsis and Medicago (Alliotte et al., 1989; Kapros et al., 1992), suggesting that expression of histones may be involved in auxin-mediated Table 1. Representative auxin induced genes. Modified and updated from Sitbon and Perrot-Rechenmann (1997) Gene Function Experimental system Effective auxins Response time Reference Genes encodina DNA bindind proteins PS/IAA4/5 transcription factor Pisum hypocotyls 20 pM IAA1 NAA, 2,4-D 10 min Theologis et al., 1985 DBP histone Arabidopsis peduncles 1 pM NAA <4 hr Aiiotteetal., 1989 H3C-11 Histone Medicago callus 100 pM 2,4-D <1 hr Kapros et al., 1992 AT-IAA1 transcription factor Arabidopsis seedlings 20 pM IAA1 NAA, 2,4-D 4 min Abel et al., 1995 SGBF-2 G-box factor Glycine hypocotyls 23 pM 2,4-D <2 hr Hong etal., 1995 ATHB-8 homeobox protein Arabidopsis leaves 10 pM IAA, NAA, 2,4-D <1 hr Baima et al., 1996 Genes encodina calcium-modulated oroteins PCM-1 calmodulin Fragaria fruit 1 mM NAA 12 hr Jena et al., 1989 MBCAM-1 calmodulin Vicia leaves 500 pM IAA <3 hr Botella and Ateca, 1994 TCH3 calcium-binding protein Arabidopsis leaves 1 pM IAA <30 min Antosiewicz et al., 1995 ARCAM calmodulin Vicia hypocotyls 1 pM IAA <1 hr Okamotoetal., 1995 VR-CDPK1 Ca-dependent kinase Vicia seedlings 500 pM IAA 3 hr. Botella etal., 1996 Genes encodina cell cvcle-associated oroteins CDC2 CDC2 protein kinase Arabidopsis roots 0.1 pM IAA <2 d Martinez et al., 1990 CDC2MS CDC2 protein kinase Medicago cell culture 100 pM 2,4-D (pulse) >8 hr Hirtetal., 1993 P34CDC2 CDC2 protein kinase Pisum roots 50 pM IAA <1 hr John etal., 1993 CDC2-S5, CDC2-S6 CDC2 protein kinase Glycine roots 0.5 mM NAA <24 hr Miao etal., 1993 Genes encodina cell wall-associated orotiens and hvdrolvtic enzymes - EU P-glycanase Hordeum alurorie 5 pM IAA <20 hr Slakeski and Fincher, 1992 DCPRP I proline-rich protein Caucus roots 10 pM IAA, NAA, 2,4-D 3-24 hr Ebeneretal., 1993 SBPRP2 proline-rich protein Glycine roots 10 pM IAA, NAA, 2,4-D <20 hr Suzuki etal., 1993 HGRPNT3 hydroxyproline-rich GP Nicotiana roots 10 pM IAA 1-2 hr Vera et al., 1994 TCH4 xylogiucan transglycosylase Arabidopsis seedlings I pM IAA <10 min Xu etal., 1995 EXT xyloglucan transglycosylase Arabidopsis seedlings 1 pM IAA 10-30 min Xuetal., 1996 EG L I P-1,4 glucanase Pisum hypocotyls 5 pM 2,4-D <5 hr Wu etal., 1996 DD21.4-1 a-expansin Pinus hypocotyls 10 p_M IBA <24 hr Hutchison etal., 1999 Table 1 (continued). Representative auxin induced genes. Modified and updated from Sitbon and Perrot-Rechenmann (1997) Gene Function Experimental system Effective auxins Response time Reference Genes encodina hvdrolitic enzvmes ATSEH epoxide hydrolase Arabidopsis plants . 100 pM 2,4-D, IAA <1 hr Kiyosue et al., 1994 VR-ACS6 ACC synthase Vigna hypocotyls 500 pM IAA <4 hr Yoon et al., 1997 AT-ACS4 ACC synthase Arabidopsis seedlings 20 pM IAA1 NAA1 2,4-D 20 min Abel et al., 1995 ADC arginine decarboxylase Pisum ovaries 45 pM 2,4-D <12 hr Perez-Armador et al., 1995 Genes encodina oxidative enzvmes A0P1 ascorbate oxidase Cucurbita fruit 4.5 pM 2,4-D 24 hr Esakaetal., 1992 AAO ascorbate oxidase Zea roots 5 pM 2,4-D <48 hr Kerk and Feldman, 1995 PS-ACO ACC oxidase Pisum seedlings 100 pM IAA <4 hr Peck and Kende, 1995 GERMIN oxalate oxidase Hordeum seedlings 10 pM IAA <4 hr Hurkman and Tanaka, 1996 Genes encodina GSTs GH2/4 GST Glycine hypocotyls 2.2 pM 2,4-D, NAA, IAA 15 min van derZaal et al., 1991 CNT103 GST Nicotiana cell culture 2.2 pM 2,4-D, NAA, IAA 15 min van derZaal et al., 1987 PARA GST Nicotiana protoplasts 4.5 pM 2,4-D, NAA, IAA 10 min Takahashi et al., 1991 PARB GST Nicotiana protoplasts 4.5 pM 2,4-D <20 min Takahashi and Nagata11992 HMGST-1 GST Hyoscyamum culture 10 pM 2,4-D, 2,4,5-T <24 hr Bilang and Sturm, 1995 Genes encodina oroteins of unknown function SAUR Glycine hypocotyls 100 pM 2,4-D, IAA 3 min McClure and Guilfoyle, 1987 GH3 Glycine hypocotyls 100 pM 2,4-D, IAA <15 min Hagen et al., 1985 ARCA Nicotiana cell culture 0.9 pM 2,4-D, NAA 2 hr Ishidaetal., 1993,1996 SAR1, SAR2 Fragaria fruit 1 mM NAA 2 hr Reddyetal., 1990 MU-3 Vicia leaves 500 pM IAa <2 hr Chen etal., 1996 G015-13 Nicotiana seedlings 1 pM NAA <2 hr Rouxetal., 1998 20 DNA replication. Other DNA-binding proteins with homologies to transcription factors have been isolated, and at least one (Ps-iaa4/5) has DNA binding domains characteristic of repressor proteins (Abel et al., 1994). Not surprisingly, genes encoding proteins thought to be involved in secondary-messenger cascades have been shown to be up-regulated after exposure to auxin. Several studies have shown that treatment with auxin increases calcium levels within the cytosol (Irving et al., 1992; Gehring et al., 1990), suggesting that calcium is involved in these signal transduction pathways. In support of this idea, the calmodulin-homologs PCM-1 from Fragana (Jena et al., 1989), MBCAM-1 and ARCAM from Vicia faba (Botella and Arteca, 1994), and TCH3 from Arabidopsis (Okamoto et al., 1995) are induced by auxin treatment, although the time required for induction ranges from one to 24 hours (Antosiewicz et al., 1995). Similarly, the calcium-dependent protein kinase gene VR-CDPK1 from Vicia was induced after auxin treatment, mechanical stimuli, or NaCI treatment (Botella et al., 1996). The role of these putative secondary messenger proteins in auxin perception or signal transduction has yet to be elucidated. Glutathione-S-transferases (GSTs) are enzymes involved in the detoxification of numerous herbicides and various other electrophilic substances (Dean et al., 1991; Gronwald et al., 1989), and have been shown to be induced , ■ ■ . by auxin, ethylene, and many stress treatments. PARA and PARB from Nicotiana (Takahashi and Nagata, 1992; Takahashi et al., 1991) are induced within 20 minutes of auxin treatment, as are other GST-like proteins in Nicotiana 21 and Glycine (Table 1). The role of GSTs in the auxin response is unclear, but they may represent a general response to cell stress rather than a primary- response gene (Abel and Theologis, 1996). Cell wall-associated proteins such as hydroxyproline-rich glycoproteins, glycoproteins, and glycine- and proline-rich glycoproteins are known to play a role in pathogenesis and wounding by increasing the barrier to pathogen infection and spread (Somssich and Hahlbrock, 1998). After auxin treatment, expression levels of DCPRP1 in Daucus (Ebener et al., 1993), SBPRP2 in Glycine (Suzuki et al., 1993), and HGRPNT2 from Nicotiana (Vera et al., 1994) were increased, suggesting that auxin may be involved in stress responses. Activities of cell wall hydrolytic enzymes have independently been shown to be elevated by auxin treatment, possibly associated with concomitant acidification of the apoplast in preparation for cell elongation (Rayle and Cleland 1992). In Arabidopsis, TCH4 and EXT(Xu et al., 1995, 1996), both encoding xyloglucan transglycosylases, were induced within 30 minutes after auxin treatment, although tch4 is well known to be responsive to mechanical stimuli as well. A (3- 1,4-endoglucanase (EGL1) from Pisum increased almost 10-fold after treatment with 2,4-D (Wu et al., 1996). Similarly, the P-glucanase EU from Hordeum vulgare is up-regulated after treatment with IAA (Slakeski and Fincher, 1992). Increases in ethylene production have been documented within minutes of auxin treatment (Devine et al., 1993). Peck and Kende (1995) indicated that the Pisum sativum PS-ACO gene, encoding ACC oxidase (the rate-limiting step in ethylene biosynthesis), was induced within 4 hours after treatment with 100 uM 22 IAA. Also, the Arabidopsis AT-ACS4 gene encoding ACC synthase was induced within 20 minutes after auxin treatment. While the majority of auxin-responsive genes investigated thus far are induced by auxin treatment, there are several examples of auxin down-regulated genes (Table 2). The most dramatically attenuated genes are the ADR family from Glycine (Datta et al., 1993), with transcript levels dropping over 100-fold within 24 hours after whole-plant treatment with 2,4-D. While this family of auxin down-regulated genes has no known function, genes encoding enzymes of common metabolism like tryptophan decarboxylase (Goddijn et al., 1992), chalcone synthase and phenylalanine ammonia lyase (Ozeki et al., 1990), and strictodine synthase (Pasquali et al., 1992) were down-regulated within 6 hours of auxin or 2,4-D treatment. While it is unknown why these aspects of plant metabolism are inhibited by auxin and auxin mimics, the attenuation of other genes involved in plant defense may provide more insight. An endo-p-1,3- glucanase from Nicotiana (GL43) is down-regulated by trace amounts of NAA (IVIohnen et al., 1985), and both the mRNA and enzymatic activity of a class of chitinases (exemplified by CHN50) are reduced by NAA (Shinshi et al., 1987). Transgenic expression of the proteinase PIN2 promoter indicated that this promoter region is responsive to auxin down-regulation within 18 hours after treatment (Kernan and Thornburg, 1989), and similar work suggests that another endo-P-1,3-glucanase from tobacco (GLB1) is down-regulated by auxin treatment (Vogelie-Lange et al., 1994). It is not clear what role the attenuation of these genes may have in response to auxin treatment, but these results are Table 2. Representative auxin repressed genes. Modified and updated from Sitbon and Perrot-Rechenmann (1997) Gene Function Experimental system Effective auxins Response time Reference Genes encodina oathoaenesis-related oroteins GL43 (3-1,3-glucanase Nicotiana pith culture 11 pM NAA <7 d Mohnen etal., 1985 CHN50 chitinase Nicotiana pith culture 11 pM NAA <7 d Shinshi etal., 1987 CA125 unknown PR protein Capsicum leaves \AA, Xanthomonas <24 hr Jung and Hwang, 2000 Genes encodina enzvmes in secondary metabolism TDC tryptophan decarboxylase Catharanthus culture 0.1 pM NAA, 2,4-D, IAA <2 hr Goddijn etal., 1992 . SSS strictodine synthase Catharanthus culture 10 pM NAA <6 hr Pasquali etal., 1992 CHS chalcone synthase Caucus culture 0.5 pM 2,4-D <3 hr Ozeki etal., 1990 PAL phenylalanine ammonia lyase Daucus culture 0.5 pM 2,4-D <3 hr Ozeki etal., 1990 CGS cystathione-y-synthase Fragaria fruit 1 pM NAA (spray) <24 hr Marty et al., 2000 Genes encodina miscellaneous oroteins SAR5 unknown function Fragaria fruit 1 mM NAA (spray) <2 hr Reddy and Poovaiah. 1990 SAM46 superoxide dismutase Glycine culture 10 pM NAA <4 hr Crowell and Amasino, 1991 GB8 ribosomal protein Pisum shoots 10pM NAA <24 hr Stafstrom and Sussex, 1992 ADR1-12 unknown function Glycine seedlings 2.5 mM 2,4-D (spray) <24 hr Dattaetal., 1993 VSP vegetative storage protein Glycine leaf petioles I pM NAA <6 hr DeWaId etal., 1994 N I-POX peroxidase/GUS fusion Nicotiana protoplasts 30 pM IAA1 NAA <30 min Klotz and Lagrimini, 1996 24 consistent with the finding that wounding leads to reduced auxin levels (Thornburt and Li, 1990; others), and causes increased expression of pathogenesis-related genes. These findings, coupled with the observation that the activities of other stress-related enzymes (superoxide dismutase and peroxidase) are also reduced upon treatment with 2,4-D (Crowell and Amasino, 1991; Klotz and Lagrimini, 1996), suggest that auxin perception may act as an “all-clear” signal, indicating that limited stresses are being placed upon the plant. Other evidence that auxin may counteract stress-related signals in plants can be found in studies of the physiological and genetic changes mediated by the plant hormone abscisic acid (ABA), which can be reversed by auxin treatment. Heat, drought, salt, and cold stress on plants are all thought to cause a decrease in cellular turgor pressure (Tamminen et al., 2001), and this decrease in cellular water content causes significant responses in many plants. A well- , known rapid effect of drought stress is an increase in ABA levels which in turn lead to stomatal closure (for review, see Jensen et al., 1996). Long-term growth under drought conditions or after treatment with ABA results in smaller stomata in Tradescantia virginiana (Franks and Farquhar, 2001), while treatment with auxin rapidly overcomes these effects, possibly mediated by increases in ethylene production (Merritt et al., 2001). Several ABA-induced stress genes are down-regulated by auxin treatment. For example, induction of GAD1 and GAD2 mRNAs, encoding wound-inducible proteinase inhibitors, is inhibited by auxin pretreatment (Jacobsen and Olszewski, 1996). The dessication-related LEA (late embryogenesis-abundant) protein EDP31 mRNA is also induced by ABA, 25 while transfer of developing embryos to auxin-free growth media also induces expression, suggesting a repressor role for auxin in the expression of this gene (Kiyosue et al., 1992). As a corollary, the auxin-induced expression of various genes encoding ACC synthases (Yoon et al., 1999; Botella et al., 1992; Kim et al., 1992) are suppressed by ABA treatment. Despite these examples of the reciprocal control of physiological and genetic responses between auxin and ABA, there are numerous reports that confound our understanding of plant hormone interactions. As mentioned above, many auxin effects are thought to be mediated by induction of ethylene production, and auxin treatment has been shown to increase in in vivo ABA levels (Grossman, 1998). Certainly, additional research is needed to help elucidate the roles of both hormones in controlling plant response to stress. Water deficit in plants Water deficit is one of the most common environmental limitations of plant productivity (Boyer, 1982), and can be caused by drought, cold temperatures, or excessively saline soils. In all cases, plants must respond to decreased water potential in the soil, which limits water uptake into tissues. Of special interest are the stresses imposed on a plant as a result of saline soils, which not only decrease soil water potential, but also contain potentially toxic concentrations of sodium, carbonate, and chloride ions. At high concentrations, these ions (especially sodium) exert their toxic effects primarily by the denaturation of proteins and cell membranes (Cheesman, 1988), and plants have evolved 26 specialized strategies to overcome these effects. Some halophytes (plants that grow naturally in saline environments) have developed succulence to dilute sodium concentrations; as sodium content in the plant increases, the plant maintains turgor pressure and osmotic potential by absorbing more water (Salisbury and Ross, 1992). Other halophytes (such as Atriplex and members of the Chloridoideae) exude excess salt on the surface of leaves, and have developed specialized salt glands to actively pump sodium out of the cytoplasm (Troughton and Donaldson, 1972; Marcum, 1999). Since charged molecules do not typically move through a lipid bilayer by diffusion (Schachtman and Liu, 1999), plants must use active transporter systems to absorb micro- and macronutrients. Salt tolerance in some species can be attributed to the species’ ability to exclude sodium while still absorbing essential nutrients, in particular potassium (Mumms, 1993). It has been shown that sodium ions compete with the active uptake of potassium, and potassium is typically present in the soil at much lower concentrations than sodium. Some halophytes such as mangrove exclude nearly 100% of the sodium in the soil (or brackish water), limiting cellular exposure to sodium (Ball, 1988). Genetic variation in salt tolerance in Triticum has been attributed to differing affinity of cation uptake channels, with tolerant cultivars excluding more sodium than susceptible cultivars while still maintaining potassium uptake (Davenport and Tester, 2000), and other cereals have been shown to accumulate sodium in old leaves and actively transport potassium to young leaves (Wolf et al., 1991, Colmer et al., 1995). Certain plants also actively pump sodium out of the cytoplasm and into the vacuole, probably powered by 27 the pH gradient across the tonoplast, utilizing Na+/H+ antiporters (Darley et al., 2000) . The increased salt tolerance observed in wild Lycopersicon over that of cultivated varieties has been attributed to variability in these antiporters (Taha et al., 2000). Additional molecular evidence for the importance of vacuolar sequestration of sodium was found in transgenic maize suspension cells, where overexpression of a Na+/H+ antiporter significantly increased NaCI tolerance (Gruwel et al., 2001). Similar results have been noted in transgenic Arabidopsis overexpressing the AVP1-1 and AVP1-2 vacuolar proton pumps (Gaxiola et al., 2001) . A more common way for plants (especially nonhalophytes) to avoid sodium toxicity and associated dessication is by the production and accumulation of certain organic solutes, which maintain a more negative osmotic potential in tissues than in the soil and thus increase the ability of cells to retain water. These compounds, termed compatible solutes, can exist in cells at high concentrations without denaturing enzymes and cell membranes (Yancey et al., 1982). The sugar-alcohols glycerol, pinitol, mannitol, and arabitol have been identified as compatible solutes in yeast (Blomberg, 2000) and in the algae Dunaliella (Gimmler, 1989), the plant Mesembryanthemum crystallinum (Vera-Estrella et al., 1999), and other prokaryotic and fungal species. The amino acid proline has long been known to act as a compatible solute in plants, and dozens of plant species accumulate proline to high levels in response to drought or salt stress (for review, see Yoshiba et al., 1995; Csonka and Hanson, 1991). Not surprisingly, genes encoding proline biosynthesis in Arabidopsis are induced 28 in response to drought and water stress, and this response appears to be dependent on ABA perception (Strizhov et al., 1997). There is considerable interest in genetic modification of crop plants to increase proline production in response to drought or salinity stress; however, the unavailability of appropriate genes is currently a major constraint in this effort (Jiban-Mitra and Mitra, 2001). To date, genetic modification to overproduce proline has met with limited success, except in the case of overexpression of the defense- and drought- related polypeptide osmotin (Barthakur et al., 2001). Another compatible solute common in a wide variety of plant species is the quaternary ammonium compound glycine betaine (GB; for review, see Rhodes and Hanson, 1993). GB is zwitterionic but electrically neutral at physiological pH and appears to be a particularly effective compatible solute (LeRuduIier et al., 1984). In addition to acting as an osmoregulant, GB acts as an osmoprotectant in stabilizing the structure of cell membranes in vitro as well as the tertiary and quaternary structures of enzymes like ribulose bisphosphate carboxylase and components of photosystem Il (Papageorgiou and Mutata, 1995) against salt stress (Gorham, 1995). GB may protect the cell against damage induced by free radicals (Jolivet et al., 1982), and Holmstrom et al. (2000) noted that GB protects photosynthetic machinery against light-induced damage in tobacco. Sakamoto and Murata (2001) suggested that enhanced tolerance to temperature stress in transgenic GB-producing Arabidopsis is due to a similar protection of membrane integrity. Bourot et al. (2000) showed that GB behaves like a chaperonin, and suggested that GB may stabilize the transcription 29 and translation machinery under conditions of stress. In this light, Allard et al. (1998) showed that exogenous GB activates different cold-inducible genes. Additionally, GB lowers the melting temperature of double-stranded DNA, which may facilitate transcription and DNA replication in high-salt environments (Rajendrakumar et al., 1997). In all cases studied to date, GB is synthesized from choline utilizing one or two enzymes to carry out the oxidation of choline (Figure 2), and three distinct pathways are known. In Arthrobacter globifoijmis and Arthrobacterpascens, the oxidation reaction and synthesis of GB are carried out by the flavoenzyme choline oxidase (COD), releasing hydrogen peroxide (lkuta et al., 1977). In mammalian cells and in E. coli, choline undergoes a dehydrogenation reaction mediated by the oxygen-dependent enzyme choline dehydrogenase (CDH), producing the toxic intermediate betaine aldehyde, which is then converted to GB by the NAD+-dependent enzyme betaine aldehyde dehydrogenase (BADH; Landfald and Strom, 1986). In plants, choline is oxidized by the enzyme choline monooxygenase (CMO), a ferredoxin-dependent soluble protein which is localized primarily in the chloroplast stroma (Rathinasabapathi et al., 1997), to produce betaine aldehyde. As in mammalian cells, the toxic betaine aldehyde intermediate is then converted to GB via the activity of BADH, which is also localized to the chloroplast stroma in members of the Chenopodiaceae. GB accumulates almost exclusively in the chloroplast of the chenopods, and likely serves to protect the photosynthetic machinery (McNeil et al., 1999). In contrast, BADH appears to be localized to the peroxisomes in members of the 30 1. Plants CH2OH CH2| H3C— Ni -C H 3 CH3 CMO CHO I CH2 H3C — Ni - CH3 CH3 BADH COO" I CH2 H3C — N^— CHg CH3 choline betaine aldehyde glycine betaine 2. Escherichia coli CH2OH CH2 H3C— N^-CH 3 CDH CH3 choline CHO I CH2I H3C— N ^-CH 3 CH3 betaine aldehyde BADH COO" I CH2 H3C — Ni - CH3 CH3 glycine betaine 3. Arthrobacter globiformis CH2OH CH2 H3C— N-^-CH3 COD CH3 choline COO" I H3C CH2 - N ^ C H 3 CH3 glycine betaine Figure 2. Biosynthetic pathways leading to GB production. Choline is oxidized by two enzymes in plants and E. coli and by a single enzyme in Arthrobacter globiformis. Abbreviations: CMO1 choline monooxygenase; BADH, betaine aldehyde dehydrogenase; CDH, choline dehydrogenase; COD, choline oxidase. Revised from Sakamoto and Murata1 2000. 31 Graminaceae (Nakamura et al., 1997). GB is only produced by certain members of the Chenopodiaceae, Gramineae, and Amaranthaceae (McCue and Hanson, 1992) and GB levels increase on exposure to drought or salt stress. Levels of GB increase approximately 10-fold in salt-stressed leaves of Hordeum vulgare (Arakawa et al., 1992), and is preferentially produced in young leaves (Nakamura et al., . 1996). A similar induction was noted in sorghum, where GB levels increased up to.as high as 70 ^mol GB g"1 dry weight (Grieve and Maas, 1984). Other grasses that accumulate GB in response to drought or salt stress include Triticum aestivum, Secale cereale, and Zea mays, although there is considerable variation between cultivars. In corn, this variation is due to a nonsense mutation in the CMO gene in some maize genotypes, and the mutation is correlated with a decreased resistance to dry conditions (Lerma et al., 1990). Certain members of the Chenopodiaceae and Amaranthaceae appear to be the most vigorous producers of GB. For example, salt-stressed sugar beet and amaranth accumulate GB to >30 //mol GB g"1 fresh weight (Russell et al., 1998). Although numerous studies have documented increases in cellular GB concentration in response to water deficit (for review, see Rhodes and Hanson, 1993), only a few studies have investigated concomitant CMO and BADH expression in natural systems. The abundance of BADH mRNA was shown to increase under conditions of water stress in mangrove (Hibino et al., 2001), sugar beet (McCue and Hanson, 1992), rice (Kishitani et al., 2000), amaranth (Legaria et al., 1998), sorghum (Wod et al., 1996), spinach (Weretilnyk and 32 Hanson, 1990), and barley (Arakawa et al., 1992). Most of these studies reported a 2- to 10-fold increase in BADH mRNA under water stress conditions as compared to control plants. Immunological and in vitro assays for detecting BADH levels and activity have yielded similar results, although have only been performed in barley (Arakawa et al., 1992) and Amaranthus tricolor (Wang et al., 1999). Treatment with ABA also increased BADH mRNA levels in barley (lshitani et al., 1995). However, no other studies have looked at the effects of ABA or other plant hormones on altering expression of BADH or other enzymes involved in GB biosynthesis. To date, CMO has only been isolated and cloned from members of the Chenopodiaceae (Spinacia oleraceae, Beta vulgaris (Russell et al., 1998), and Atriplex hortensis (Shen and Chen, 2001)) and Amaranthaceae (Amaranthus tricolor (Wang et al., 1999), and its expression patterns seem to follow that of BADH. CMO mRNA levels increased 7-fold in salinized leaves of spinach, with CMO enzyme activity increasing approximately 4-fold (Rathinasabapathi et al., 1997). Similar results were seen in Beta vulgaris and Amaranthus caudatus (Russell et al., 1998), as well as in Amaranthus tricolor (Wang et al., 1999). As mentioned above, the chemical characteristics of GB make it an attractive target for use in the genetic modification of crops to increase their drought and salt tolerance (Sakamoto and Murata, 2000; Sakamoto and Murata, 2001), and attempts have been made using both bacterial COD genes and plant BADH and CMO genes. A spinach BADH controlled by the CaMV 35S promoter was introduced into tobacco and targeted to chloroplasts (Rasinasabapathi et al., 33 1994). BADH activity effectively conferred resistance to betaine aldehyde (the toxic intermediate in GB production), evidently by its conversion to GB. However, transgenic plants failed to accumulate GB in the absence of exogenously supplied betaine aldehyde, evidently because tobacco has no in vivo CMO activity and could not produce betaine aldehyde. Tobacco transformed with a barley BADH cDNA showed increased levels of BADH mRNA, in response to salt and ABA (lshitani et al. 1995). In a similar study, Kishitani et al. (2000) introduced a barley BADH cDNA into rice and noted that transgenic lines were able to convert exogenous betaine aldehyde to GB, and showed an increased tolerance to salt, cold, and heat stress over untransformed lines. To date, only one study has attempted to introduce CMO into a GB non­ accumulator. Nuccio et al. (1998) used a spinach CMC cDNA under the control of the CaMV 35S promoter to transform tobacco. Although high CMO expression levels were noted (nearly as high as CMO mRNA in salinized spinach leaves), the tobacco plants were only able to synthesize GB to -50 nmol g"1 FW, which is far below the levels seen in natural accumulators. Although the authors did not speculate what role the absence of a functional BADH enzyme played in GB production, they did note that CMO enzyme levels (as indicated by Western blot) increased 3- to 5-fold upon salinization of the transformants, leading to the conclusion that post-transcriptional events modify CMO expression. Indeed, the authors verified an unusually fast disappearance of the CMO protein upon release from salt stress, a finding noted by other researchers (Russell et al., 1998). 34 The Nuccio study (1998) also provides interesting evidence that the endogenous supply of choline (the precursor to GB) strongly influences GB production. In this study, supplying exogenous choline increased GB production by about 50-fold in the transformed plants, suggesting that the endogenous : choline supply limits GB production. Subsequent studies (McNeil et al., 2000; Huang et al., 2000) verified that choline production in tobacco must be increased before appreciable GB can be synthesized in this normally non-accumulating species. Indeed, in vivo choline production is increased in spinach plants undergoing salt stress (Summers and Weretilnyk, 1993; Weretilnyk et al., 1995), and the drought-induced enzymes involved in choline production have been recently characterized (Weretilnyk et al., 2001). Despite this potential limit, Sakamoto and Murata (2000) point out that tobacco contains only a small amount of choline, whereas rice and Arabidopsis have 10- to 30-fold higher concentrations of endogenous choline (Nuccio et al., 1998; Sakamoto et al., 1998; Hayashi et al., 1997), suggesting that some species are better targets for genetic modification to increase GB production. In addition to using plant-derived enzymes for the production of GB, other research has utilized the bacterial COD gene to transform plants. The major advantage of this approach is that only a single transformation with the relevant gene (codA) is needed to introduce the pathway to convert endogenous choline to GB. Hayashi et al. (1997) produced transgenic Arabidopsis that overexpressed the codA gene from Arthrobacter globiformis, and noted that GB accumulation was as high as 5 //mol g"1 fresh weight in salt-stressed plants. 35 Despite this relatively low production, the authors noted significantly enhanced tolerance to salt and cold stress, likely because the majority of GB was localized to chloroplasts, leading to physiologically significant GB concentrations in that organelle (Sakamoto et al., 2000). Subsequent work has shown that transformed plants had increased seed germination under high salt conditions (Hayashi et al., 1998), were less susceptible to photosynthetic inhibition under salt and cold stress (Sakamoto et al., 1998), were more tolerant of high temperatures (Alia et al., 1998a), had higher germination rates and better growth v under cold stress (Alia et al., 1998b), and showed decreased susceptibility to freezing temperatures (Sakamoto et al., 2000) than wild-type plants. Endogenous choline levels did not decrease in the transformed plants, although in vivo hydrogen peroxide levels doubled (Alia et al., 1999). Other studies utilizing the CODA gene have noted similar broad protection from stress in Brassica juncea (Prasad et al., 2000a, Prasad et al., 2000b) Diospyros khaki (Gao et al., 2000), and Nicotiana (Holstrom et al., 2000), even though GB production was nearly 1000-fold lower in these plants than in natural GB- accumulating plants. Clearly, cellular localization plays an important role in GB- mediated stress protection; targeting the cod A protein to the chloroplasts had a more significant protective effect against salt-induced photosynthesis deactivation in rice plants than targeting it to the cytosol (Sakamoto et al., 1998). While the one-enzyme codA system has been explored for transgenic applications to increase crop tolerance to drought stress, other researchers suggest that plant-derived systems (CMO and BADH) are a better choice for 36 manipulation for two reasons. First, CMO requires reduced ferredoxin, which links GB production to the light reactions of photosynthesis. This coupling would increase GB production during the day, when demand for osmotic adjustment is highest. Secondly, the CMO and BADH genes presumably have stress- responsive cis regulatory elements, which are needed to reproduce the natural pattern of drought-induced GB accumulation (Rathinasabapathi et al., 1997) and enable genetically modified plants to produce GB only when needed. The authors concluded that the isolation and characterization of CMO genes from new plant sources is an important step in bioengineering GB synthesis in crops. 37 CHAPTER 2 ISOLATION OF DIFFERENTIALLY EXPRESSED GENES IN RESPONSE TO DICAMBA TREATMENT IN DICAMBA-RESISTANT AND SUSCEPTIBLE K. SCOPARIA Introduction Auxin and other phytohormones mediate a diverse array of plant growth and developmental responses. Although numerous physiological studies have documented these effects, the molecular mechanisms of hormone action remain largely unknown. One strategy to investigate hormone-mediated signal transduction pathways is through the study of genes that respond rapidly to hormone treatments. Until recently, isolation of hormone-responsive genes relied on interpreting physiological responses and subsequently cloning genes likely involved in these responses. For instance, it has long been known that auxin treatment dramatically increases ethylene production (Bonner et al., 1980). Molecular evidence for this induction came from assaying and purifying the ACC synthase enzyme, determining its amino acid sequence, and designing degenerate PCR primers to isolate the corresponding cDNA (Nakagawa et al., 1991). Induction at the mRNA level was verified by northern blotting. Subtractive hybridization screening strategies of cell lines treated with auxin have also led to the discovery of several auxin-responsive genes, such as the Aux/IAA gene family (Ainley et al., 1988, Conner et al., 1990, Yamamoto et al., 1992), the 38 SAUR gene family (McClure et .al., 1989), and several genes encoding GSTs (Hagen et al., 1988, Takahashi and Nagata, 1992, Van derZaal et al., 1987; see Table 1, pp. 18-19). Although PCR-based cloning approaches have led to isolation of homologs of these genes from different species (Van der Straeten et al., 1992, Kim et al., 1992, Trebitsh et al., 1997, Abel et al., 1995; Chen and Singh, 1999; de Billy et al., 2001), few other hormone-responsive genes in plants ! had been isolated until fairly recently. . . In 1992, a new molecular technique was developed to isolate genes that are differentially expressed in response to specific treatments, or in different tissues in response to developmental cues (Liang and Pardee, 1992). Differential Display (DD) is a reverse transcription/PCR-based approach to amplify mRNAs and radiolabel the resulting partial cDNAs, which are then run in adjacent lanes on a denaturing polyacrylamide sequencing gel, followed by autoradiography. Comparisons of individual band intensities between treatments potentially identifies mRNA species whose abundance has changed as a result of the treatment in question, and their cDNAs can be isolated from the gel, subclohed, and used as probes on northern blots. Because it utilizes PCR, DD is thought to be an improvement over traditional subtractive hybridization methodologies because of a lack of discrimination against rare messages and its technical ease of use (Yoshida et al., 1994). DD relies on amplification of a subset of mRNAs primed at 1) the site of the polyadenylated 3' tail (the anchored primer), and 2) a random site on the first-strand cDNA upstream from the 3' tail (the arbitrary primer). Briefly, total 39 RNA samples are prepared from separate tissues undergoing the treatment of interest, and a subset of the mRNA is primed for reverse transcription of first- ! strand cDNA with an oligo-dT primer such as S1-T11CA-S'. In theory, this would only prime mRNAs ending with the sequence S1-TGAAAAAAAAAAA-3'. Each anchored primer would thus recognize one-twelfth of the total mRNA population (4x3 possibilities for the two 5' nucleotides, excluding thymidine from the penultimate position), permitting reverse transcription of only this subpopulation. After reverse transcription, partial second-strand synthesis is performed by annealing an arbitrary 10-mer, strand extension, and PCR amplification in the presence of 33P-dATP. Like most developing technologies, DD has seen many improvements since its introduction in 1992. A common problem with early DD screens was the generation of false positives (messages that appear to be differentially expressed by PCR amplification, but prove to be otherwise when examined by northern blotting). In 1997, Zegzouti et al. refined the technique by suggesting 1) the use of an anchored primer with only one selective base, such as T11C, and 2) that PCR reactions be split prior to amplification and subsequently run in adjacent lanes on the DD gel. These changes were thought to reduce the percentage of false positives from as high as 70% down to perhaps 25% by screening for, and avoiding, amplification patterns that are not reproducible. Since 1998, DD has seen significant utilization for the study of gene expression in response to plant hormone treatment, and this technique has more than doubled the number of hormone-responsive genes previously 40 characterized. More than 20 ethylene-responsive genes (Zegzouti et al., 1999; Hajouj et al., 2000), 14 ABA-responsive genes (Yoshida et al., 2001, Deleu et al., 1999, Karkthik et al., 2000), a gene induced by cytokinin treatment (Iwahara et al., 1998), and ten auxin-responsive genes have been isolated from various plant species. Interestingly, the recent DD-based studies show that significant numbers of such genes are down-regulated after hormone treatment, in contrast with earlier methodologies that isolated primarily hormone-induced genes. Of the auxin-responsive genes isolated using DD, six have homology to the Aux/IAA I gene family (Dargeviciute et al., 1998), one is an expansin involved in acid- induced loosening of cellulose and xyloglucan networks (Hutchison et al., 1999), one has sequence homology with the SAUR genes (Watillon et al., 1998), and two others show sequence homology to the auxin-induced aldo-keto reductases (Mazeyrat et al., 1998) and lignin degrading enzymes (Watillon et al., 1998). All ten of these genes isolated by DD are. induced by treatment with auxin or auxin analogs. The research reported here used DD to screen two inbred biotypes of K. scoparia that were either susceptible (S) or resistant (R) to the auxinic herbicide dicamba. Previous experiments in our lab showed that dicamba resistance was not due to altered herbicide uptake, translocation, or metabolism patterns (Cranston et al., 2001). Since auxin is known to have rapid effects on the expression of specific genes and subsequent plant developmental responses (Guilfoyle et al., 1998), we hypothesized that dicamba (an auxin-mimic herbicide) would have similar effects on gene expression patterns in TC. scoparia plants and that these changes may be characteristic of auxin treatment. Using DD as a screening tool, these studies investigated 1) if dicamba treatment has a rapid effect on gene expression patterns in K. scoparia; 2) which genes are induced or repressed shortly after treatment; and 3) whether there is a difference in gene expression patterns between S and R K. scoparia as a result of dicamba treatment that may provide insight into the mechanism of dicamba resistance. 41. Methods and Materials Plant material The R bibtype was derived from a population collected in a cultivated field near Fort Benton, MT, in 1992. The initial R population, designated as line R99, was derived from cuttings taken from 25 plants that survived field dicamba treatment. About 12% of the progeny from these cuttings showed no injury symptoms when treated with 70 g ai ha"1 of dicamba in the greenhouse. Eighteen of the uninjured plants were individually transplanted into 15-cm diameter 3.8 L pots containing greenhouse soil mix (1:1:1 Bozeman silt loam:washed sand:peat moss), fertilized weekly, and watered as needed. At maturity, seeds from these plants were bulked and planted 0.5 cm deep in 54 by 26 by 6 cm flats and grown for 4 weeks in the Montana State University Plant Growth Center greenhouses under natural light supplemented with mercury vapor lamps (14 hour daylength; 24/18°C day/night; 165 pmol m"2 s"1 PPF) and 42 fertilized and watered as above. After 4 weeks, 3- to 5-cm-tall plants were sprayed with 70 g ai ha"1 dicamba (formulated as the dimethylamine salt; Banvel® herbicide, BASF Corporation) using a greenhouse belt sprayer delivering a carrier volume of 94 L water ha"1. Plants were returned to the greenhouse and evaluated 21 days after treatment. Bulked seeds from 20 plants that showed no injury symptoms were designated as line R99 and used as the R phenotype in all experiments reported here. Even after two generations of selection, the R99 line contained about 10% S individuals. The S254 line was generated from /C scoparia plants that were susceptible to dicamba treatment and seeds were obtained by growing untreated plants under isolation conditions for two generations as described above. Both R and S lines were maintained as families rather than developed using single-seed descent in order to avoid symptoms of inbreeding depression, as previously observed in this highly outcrossing species (Mulugeta, 1991). Previous reports (Cranston et al., 2001) showed that R99 is about 5-fold more tolerant to dicamba treatment than S254. Interestingly, the R phenotype of K. scoparia does not exhibit significant morphological changes, as do dicamba-resistant Sinapis arvensis plants (Hall and Romano, 1995), which are shorter, more branched, darker green, and more tolerant to cold temperatures than S plants. Similarly, auxin- and 2,4-D-resistant Arabidopsis mutants are typically stunted, show reduced apical dominance, have reduced lateral root formation, or are agravitropic (Leyser, 1997). 43 RNA extraction Four-week-old R99 and S254 plants were sprayed with 70 g ai ha"1 dicamba as above and shoot tissue was harvested at 0 (untreated), 30, 60, 90, or 120 minutes after treatment and frozen in liquid nitrogen. Frozen tissue (0.5 g fresh weight) was pulverized under liquid nitrogen using a prechilled mortar and pestle and transferred to a 1.5-ml centrifuge tube. Total RNA was extracted using a phenol/chloroform extraction procedure modified from Ausubel et al. (1994). TLE (0.5 ml) extraction buffer (0.2 M Tris, pH 8.2, 0.1 M lithium chloride, 5 mM EDTA), 0.15 ml TLE-equilibrated phenol and 0.15 ml chloroform were added to the frozen powder, and the tube was vortexed and incubated at 50°C for 30 minutes. After centrifugation at 14,000g at room temperature to remove cell debris, the aqueous phase was transferred to a new tube containing 0.3 ml phenolichloroform (1:1), vortexed, and centrifuged as before. The aqueous layer was again removed and extracted in 1 volume of phenol:chloroform (1:1) two more times as above, and total RNA was precipitated by adding 0.33 volumes of 8 M LiCI and incubating for 16 hours at 4°C. After centrifugation for 30 min at 14,000g at 4°C, the RNA pellet was washed with 0.5 ml 2 M LiCI, resuspended in 100 pi sterile water, and ethanol precipitated. The RNA was again recovered by centrifugation as above and resuspended in 100 pi sterile water and stored at -80°C. RNA obtained by this method was judged to be intact by denaturing agarose gel electrophoresis and ethidium bromide staining. 44 Differential display RT-PCR Two approaches were utilized in the DD screens reported here. Initial efforts in 1997 and 1998 used the two-base selective anchored primer as described by Liang and Pardee (1992). After reports of reduced false positives by the Zegzouti group (1997) using the protocol changes mentioned above, a Single-base discriminatory anchored primer coupled with parallel PCR reactions became the method of choice in 1998 to 2000. All other procedures were identical between the two screens. Total RNA extracted as above was treated with RNase-free DNase I (10 units/25 pg RNA; Promega Corp.) in 100 mM Tris- HCI (pH 8.0), 100 mM KCI, and 15 mM MgCI2 for 60 min at 37°C. Samples were then extracted with an equal volume of water-saturated phenpkchloroform (1:1) and once with chloroform. RNA was ethanol precipitated, centrifuged 30 min at 14,000g at 4°C, washed with 70% ethanol, and resuspended in diethyl pyrocarbonate-treated water. Quantification of RNA samples was determined spectrophotometrically and by using denaturing agarose gel electrophoresis. . DNA-free RNA (2 pg) from the timepoints described above was incubated at 80°C in the presence of the anchored primer (2 pM; Table 3) for 5 min, cooled to 37°C over 20 min, and reverse-transcribed in a 20 pi reaction containing 50 mM Tris-Hcl (pH 8.0), 50 mM KCI, 5 mM MgCI2, 10 mM DTT, 100 pM each dNTP, and 200 units MMLV Superscript® reverse transcriptase (GIBCO-BRL) for 120 min at 37°C. Reactions were stopped by heating to 75°C for 10 min. cDNA (10 ng) from each treatment was amplified in a 10 pi PCR reaction containing 45 Arbitrary primers used with dinucleotide anchored primers Arbitrary primers used with mononucleotide anchored primers Name Seouence Name Sequence OPA01 CAGGCCCTTC H-APi AAGCTTGATTGCC OPA02 TGCCGAGCTG H-AP2 AAGCTTCGACTGT OPA03 AGTCAGCCAC H-AP3 AAGCTTTGGTCAG OPA04 AATCGGGCTG H-AP4 AAGCTT CT CAACG OPA05 AGGGGTCTTG H-AP5 AAGCTTAGTAGGC OPA06 GGTCCCTGAC H-AP6 AAGCTTGCACCAT OPA07 GAAACGGGTG H-AP7 AAGCTTAACGAGG OPA08 GTGACGTAGG H-AP8 AAGCTTTTACCGC OPA09 GGGTAACGCC H-AP9 AAGCTTCATTCCG OPA10 GTGATCGCAG H-AP10 AAGCTTCCACGTA OPA11 CAATCGCCGT H-AP11 AAGCTTCGGGTAA OPA12 TCGGCGATAG H-AP12 AAGCTTGAGTGCT OPA13 CAGCACCCAC H-AP13 AAGCTTCGGCATA OPA14 TCTGTGCTGG H-AP14 AAGCTTGGAGCTT OPA15 TTCCGAACCC H-AP15 AAGCTTTAGAGCG OPA16 OPA17 OPA18 OPA19 . AGCCAGCGAA GACCGCTTGT AGGTGACCGT CAAACGTCGG H-AP16 AAGCTTACGCAAC OPA20 GTTGCGATCC Table 3. Primer sequences used for differential display. (Left) Arbitrary 10-mer primers used in combination with dinucleotide-specific primers T11GC, T11CA, or T11GA for a total of 360 PCR reactions. (Right) Arbitrary 13-mer primers used in combination with mononucleotide-specific primers T11G, T11C1 or T11A for a total of 576 PCR reactions. 46 0.5 unit AmpIiTaq Gold® DNA polymerase (Perkin Elmer), 60 mM Tris-HCI (pH 8.9), 18 mM (NH4)2SO4, 2 mM MgCI2, 3 pM each dNTP, 2 pM anchored primer, 2 pM arbitrary primer, and 2 pCi 33P-dATP (DuPont-NEN). Cycling conditions included an initial 5-min denaturation step at 94°C, followed by 35 cycles of 94°C for 30 sec, 42°C for 60 sec, 72°C for 30 sec, and a final extension step at 72°C for 10 min in a Perkin Elmer 9600 thermocycler. When two parallel PCR reactions were used, the 20 pi reaction volume was split into two 10 pi reactions prior to thermocycling. After thermocycling, 3 pi from each reaction was heated for 5 min at 95°C with loading dye (95% formamide, 20 mM EDJA, 0.01% bromophenol blue, and 0.01% xylene cyanol FF) and separated by electrophoresis on a denaturing 6% polyacrylamide/7 M urea gel in Tris-borate-EDTA buffer at 500C until optimum separation of bands in the 200- to 500-bp range was achieved (about 5 hr at 1900 volts constant voltage). The gel was blotted to Whatman 3MM paper, fixed with 10% acetic acid/5% methanol for 10 min, dried at 60°C on a slab gel dryer (Hoefer Scientific), and exposed to Kodak X-OMAT AR film for 6 to 16 hours at room temperature. Differentially displayed cDNA bands were excised with a scalpel from the dried gel by aligning the exposed film over the gel (premarked with 0.001% 35S- dATP [DuPont-NEN] in India ink) and eluting the cDNA in 100 pi water at 37°C for 60 min followed by 15 min at 94°C. cDNAs were recovered by ethanol precipitation, redissolved in 20 pi nuclease-free water, and a 1 pi aliquot was 47 used for reamplification in a 50 pi PCR reaction using the above conditions, except that 33P-dATP was omitted and the dNTP concentration was increased to 200 pM. Ten pi of the PCR reaction was analyzed on a 1 % agarose gel with ' ethidium bromide staining to verify the expected fragment size and specificity of amplification. One pi of successful PCR reactions was then subcloned into the pCR 2.1/TOPO TA® cloning vector (Invitrogen Corp.) and used to transform competent cells (TOP10 E. coir, F mcrA A(mrr-hsdRMS-mcrBC) 80/acZAM15 A/acX74 recA1 deoR araD139 A(ara-/eu)7697 galU galK rpsL (Strp) endA1 nupG). After culture and selection on Luria-Bertani medium (10 g tryptone, 5 g yeast extract, and 10 g NaCI per L, pH 7.0) supplemented with 100 pg/ml ampicillin, master plates were made from 10 white colonies from each transformation, and the resulting insert sizes were determined by PCR as above, except that the M13 Universal Forward and Reverse primers (1 pi of 0.1 ng primer/pl) were used in a 20 pi PCR reaction. Anticipated insert sizes were verified by agarose gel electrophoresis/ethidium bromide staining as above, and corresponding single colonies were used to inoculate 3 ml cultures in Terrific Broth (0.017 M KH2PO*, 0.072 M K2HPO4, 12 g tryptone, 24 g yeast extract, and 4 ml glycerol per L) supplemented with 100 pg/ml ampicillin. Plasmid DNA purifications were performed using the SET/alkaline lysis buffer method (Sambrook et al., 1989), and plasmids were digested with EcoRI (New England Biolabs) under standard conditions. DNA inserts were isolated by electrophoresing the digestion mix on a 1.5% agarose gel, excising the insert 48 with a scalpel, and purifying the DNA from the gel matrix using the QIAQuick® Gel Extraction Kit (Qiagen Company). DNA inserts were resuspended in 50 pi sterile water and stored at -20°C. Northern blot analysis Analysis of differentially exposed bands was carried out by using the appropriate cDNA as a probe on northern blots. RNA (12 pg per lane) was denatured for 5 min at 65°C in 50% formamide/6% formaldehyde, electrophoresed at 50 volts constant voltage for 90 min in a 1% agarose gel containing 2% formaldehyde, stained with ethidium bromide to verify equal loading, transferred overnight to a positively charged nylon membrane (Osmonics Magna® nylon transfer membrane, 0.45 pm pore size), and bound to the membrane by UV-crosslinking. To make cDNA probes, 40 ng of purified insert was labeled with 50 pCi 32P-dCTP (specific activity 3000 Ci/mmol; DuPont/NEN) using a random hexamer priming kit (Ready-To-Go® labeling beads, Amersham Pharmacia) and unincorporated nucleotides were removed using G50 Sephadex columns (ProbeQuant® G50 micro columns, Amersham Pharmacia). After verification of labeling efficiency (> 50% incorporation of label using a Geiger-Mueller counter), the probe was denatured by boiling for 5 min and added to RNA blots preincubated for 4 hours at 57°C in 4 ml of aqueous hybridization buffer (Sambrook et al., 1989). Blots were hybridized overnight at 57°C, washed once for 15 min in 100 ml 2X SSC (0.3 M NaCI, 0.03 M Na3Citrate (dihydrate), pH 7.0) at room temperature, followed by three 20 min washes at 49 57°C in 100 ml 2X SSC1 1X SSC, and 0.5X SSC, respectively. Blots were sealed in plastic bags while wet and exposed to film as above for 10 min to 1 week, depending on signal intensity as detected by a Geiger-Mueller counter. For rare messages, film was exposed at -80°C in the presence of an intensifying screen (DuPont Lightning Plus®). Equal loading of lanes was determined by visual inspection of ethidium bromide-stained RNA on nothern blots (Figure 3). Figure 3. A representative northern blot containing RNA isolated 0, 60, or 120 minutes after dicamba treatment of susceptible (S) or dicamba-resistant (R) K. scoparia, showing intact RNA and uniform RNA loading of each lane. 50 Sequencing of isolated cDNA fragments in the pCR 2.1/TOPO TA plasmid was performed at the Washington State University DNA Sequencing Facility (Pullman, WA) using the BigDye dideoxy termination method. In all cases, primers used for sequencing were the M13 Forward (-40) and Reverse primers. Sequence alignments DNA sequences of selected cDNA fragments were compared to sequence databases using the National Center for Biotechnology Information’s BLASTN and BLASTX algorithms (Altschul et al., 1997) using default settings. For further analyses, sequences were compared to published sequences using the Genetics Computer Group (Madison, Wl) GCG® (v 10.0) PILEUP program. Sequencing DNA extraction Genomic DNA was isolated from S254 K. scoparia using a method modified from Sambrook et al. (1989). Tissue was harvested, frozen, and pulverized as above. Extraction buffer (0.5 ml; 100 mM Tris1 pH 8.0, 50 mM EDTA, 100 mM NaCI, 1% (w/v) sodium dodecyl sulfate, 10 mM (3- mercaptoethanol) was added and the sample was placed at 65°C for 30 min with occasional gentle mixing. Polysaccharides and protein were precipitated by adding 0.33 volume 5 M potassium acetate. The sample was incubated on ice for 60 minutes, centrifuged 20 min at 14,000g at room temperature, and the 51 supernatant removed and added to a new tube. DNA was precipitated by adding 1 volume ice-cold isopropanol, centrifuged as above, and washed with 0.5 m l, ice-cold 70% ethanol. The DNA pellet was redissolved in 100 pi sterile water and stored at 4°C. Quality of genomic DNA obtained by this method was determined by agarose gel electrophoresis and ethidium bromide staining. Cloning a gene fragment of 1 -aminocvcloprooane-1 -carboxvlate synthase Auxins are known.to stimulate ethylene production in a variety of plant tissues (Abeles, 1973), and numerous systems have shown that the mRNA for 1 -aminocyclopropane-1 -carboxylate synthase (ACC synthase), the enzyme mediating the rate-limiting step in ethylene production, is induced after auxin (Kim et al., 1992, Yoon et al., 1997, Arteca and Arteca, 1999) or 2,4-D (Jones and Woodson, 1999) treatment. Although dicamba is known to induce ethylene production in dicamba-susceptible Sinapis but not in R biotypes (Hall et al., 1993; Peniuk et al., 1993), the molecular basis for the dicamba-mediated , induction in ethylene production has not been examined. To address this question, efforts were undertaken to clone the K. scoparia ACC synthase gene and determine whether dicamba treatment causes accumulation of the ACC synthase mRNA. Such studies would address 1) what role ACC synthase mRNA accumulation plays in ethylene biosynthesis in R and S K. scoparia, and 2) whether dicamba is perceived similarly to auxin by K. scoparia (as measured by an induction of ACC synthase transcript accumulation). ACC synthase has been cloned from dozens of plant species and has 52 seven highly conserved regions, including the active site domain (Henskens et al., 1994). Because two of these conserved domains (amino acid sequences TNPSNPLGTT and MSSFGLV) do not span introns, PCR amplification using DNA as a template was successfully used to clone 290- and 328-bp fragments of ACC synthase genes from Malus and Vigna, respectively (Kim et al., 1992). Under my direct guidance, Mr. Thomas Emborg attempted a parallel study in K. scoparia while enrolled in BCHM 489 (Undergraduate Research Methods) at Montana State University. ACC synthase DNA and inferred amino acid sequences were aligned by hand, spanning the conserved domains listed above from Malus (Kim et al., 1992), three Vigna cDNAs (Kim et al., 1992, Botella et al., 1992, Yoon et al., 1997), Lycopersicon (Rottman et all, 1991; Olson et al., 1995), Dianthus (Henskens et al., 1994), Cucumis (Trebitsh et al., 1997), two Pelargonium cDNAs (Wang and Arteca, 1995), and Arabidopsis (Van Der Straeten et al., 1992). Two degenerate primers corresponding to the highly conserved amino acid sequences TNPSNP (5' AC(CGA)AA(TC)CC(ATG)TC(AGC)AA(TC)CC 3') and KMSSFG (5' TCC(AG)AA(GA)CTNGACAT(TC)TT 3') were designed to limit degeneracy but account for all wobble seen in each codon in the above species. Primers were synthesized by Integrated DNA Technologies (Coralville, IA) and used in PCR amplifications of K. scoparia DNA- A PCR master mix (240 pi) containing 100 ng genomic DNA, 1 pM each primer, 50 mM Tris (pH 8.6), 50 mM KCI, 1.5 mM MgCI2, 200 pM each dNTP, and 6 units AmpIiTaq Gold® DNA polymerase (Perkin Elmer) was split into12 aliquots and amplification was done 53 on a thermal gradient block in a RoboCycler® (Stratagene Company) thermocycler. Cycling conditions included an initial 5-min denaturation step at 94°C, followed by 30 cycles of 94°C for 30 sec, 409C to 62°C (gradient) for 30 sec, 72°C for 30 sec, and a final extension step at 72°C for 10 min. Amplification products were analyzed on a 1% agarose gel, and the 305 bp clone TACC1-6 from the 58°C annealing temperature was gel-purified, subcloned, sequenced, and used as a probe on northern blots as above. 54 Results and Discussion Differential Display using di- and mono-nucleotide-specific anchored primers Use of dinucleotide-specific anchored primers, coupled with individual 10- mer arbitrary primers, typically resulted in 20 to 70 radiolabeled amplification products per reaction in the 200-bp to 600-bp size range (Figure 4). A total of about 20,000 message fragments were evaluated using this experimental approach. A wide variety of message abundance and expression patterns was noted. Most messages (>90%) were not affected by dicamba treatment in R or S and therefore were not differentially expressed between the two biotypes (constitutive messages). About 1,000 (5%) of the messages were amplified in only R or S, and about 600 (3%) of the messages were induced or repressed equally in R and S up to 120 minutes after dicamba treatment. The relatively few messages that were differentially expressed between the two biotypes as a result of dicamba treatment were given priority for subcloning and verification of expression patterns by northern blotting. Of 44 cDNAs reamplified and used as probes on northern blots, 17 proved to be constitutively expressed (in contrast to the expression patterns noted on the DD film), 15 did not recognize a detectable message after 1 week of exposure, nine gave unusable northern blot results due to nonspecific hybridization (typically to ribosomal RNA), and three exhibited expression patterns consistent with the DD gel. Not including the cDNAs with an undetectable signal, these numbers indicate the occurrence of false positives to be nearly 90%. 55 2 O 60 120 0 60 120 C Figure 4. mRNA amplification and differential display using dinucleotide- specific anchored primers. DD was performed using the anchored primer T11GC and arbitrary primer OPA07 (group 1) and OPA08 (group 2). The six lanes in each group correspond to RNA from S (left three lanes in each group) and R (right three lanes in each group) K. scoparia isolated 0, 60, or 120 minutes after treatment with dicamba. In most cases, expression remained essentially unchanged as a result of dicamba treatment, as in “A”. In other cases, expression patterns changed as a result of treatment but were not different between R and S, as in the example shown by “B”. “C” and “D” denote cDNAs whose expression patterns differ between R and S and were changed as a result of treatment. In “C”, the message is more abundant in untreated S than in R, and is transiently induced in S and only slightly in R. In “D”, the message accumulates in S but only until 60 minutes after treatment, whereas it continues to increase in abundance in R up to 120 minutes after treatment. 56 DD using single-nucleotide discriminatory anchored primers and arbitrary 13-mers yielded similar patterns of expression, but this method proved to be more useful. As expected, the numbers of amplification products rose significantly, yielding about 80 to. 200 amplification products per PCR reaction. Occurrence of differentially expressed messages remained low, and performing parallel PCR reactions effectively removed about 50% of the bands from further consideration, as they failed to give repeatable amplification patterns. Approximately 60,000 message fragments were screened using this DD technique, and 62 were selected and successfully reamplified for use as probes in northern blots. Of these, 21 proved to be constitutively expressed, 24 did not recognize a detectable message after 1 week of exposure, six gave unusable northern blot results, and 11 exhibited differential expression patterns consistent with the DD gel. Not considering undetectable messages, the rate of false positives with this approach was about 70%. mRNAs that were detectable and not constitutively expressed after dicamba treatment could be assigned to one of three categories: 1) mRNAs that were similarly up-regulated in R and S, 2) mRNAs that were similarly down- regulated in R and S, or 3) mRNAs that were differentially expressed between R and S after dicamba treatment. The cDNAs for two constitutively expressed mRNAs were also sequenced because of unusually high expression levels and this writer’s interest. In all cases reported here, changes in gene expression were inferred from changes in steady-state mRNA levels as determined by northern 57 blotting. While other mechanisms may affect mRNA abundance, such as a change in the rate of mRNA degradation, the expression level of most genes is determined primarily by the rate of transcription (Alberts et al., 1989). I assumed that steady-state (“basal” levels of expression) mRNA abundance for each gene was exemplified in untreated tissue (i.e., 0 minutes after dicamba treatment). mRNAs down-reaulated bv dicamba treatment C01R120 The 287-bp cDNA C01R120 represents a 550-b message expressed at moderate levels (16-hour film exposure) that is down-regulated in R and S after dicamba treatment (Figure 5). It is attenuated about 8-fold in S and 5-fold in R, and this down-regulation occurs somewhat more quickly in S than in R. No significant sequence identities were detected at the nucleotide or translated amino acid levels. D416S0 cDNA D416S0 is a 245-bp fragment recognizing a 1.9-Kb message that is relatively abundant in untreated tissue (16-hour film exposure; Figure 6). By 120 minutes after dicamba treatment, the message is decreased about 7-fold in S and 10-fold in R. High sequence similarity was noted at the nucleotide (>80%) and translated amino acid levels (>77% identical; 88% similar) to the chloroplastic enzyme choline monooxygenase from Spinacia oleracea, Beta 58 A r S A_____\ r R A 550 bp 40000 Resistant ro ^ 30000 m ™ 20000 0)i t 70% amino acid identity to the Spinacia PSII-T protein and to the carboxyl terminus of the Gossypium psbT gene (Figure 14), as well as to the large polycistronic chloroplastic transcript encoding the psbB-psbT-psbH-petB-petD suite 70 A 1.5 Kb 30000 Susceptible j 20000 Resistant 5 10000 Time after treatment (min) C a t a a t t a a a a t g t g g t t a a g c a t a a t g t g t g t c t g c t t t g c t t a a t t g t g t a c t t g a g t g t a a g g t g c t a a t a a t g a c t a t t a t a a a t a a a c a c c a t g g c a c c c c c t t c c c c t a a a t a g t t t t c a a a t a t t t t g g g g g g t t a a g c g a a g a a t a g a t c g c t t a t g a a t g t t t t a t c t a g t c a t g t a t g g t t t t c a t t a g t t a a g t a c a t g a g g a a a a c a t t a t t g t g t g t t g a a t a c a g c a t a t t a a t a c a a c a t a t t a t c a g t t g t a g t a a a a a a a a a a a Figure 12. Expression of G14S120 after dicamba treatment. (A) RNA isolated from S and R K. scoparia 0, 30, 60, 90, or 120 minutes after treatment and probed with the radiolabeled cDNA. (B) Phosphorimager analysis of message abundance from blot (A). (C) Nucleotide sequence of the G14S120 cDNA. A 1.2 Kb 40000 Susceptible Co £ "2 c 30000 - Q ^ ro ra CU i= 20000 — ro, fV ^ 10000 Resistant Time after treatment (min) C a a g c a a a c g a g g g a c t g c a g a a g c t a a g a a g a a g t a t g c g c c c a t t t g c g t c a c t a t g c c a a c t g c c a g g a t c t g t t a c a a g t g a g g c a t c t c t t g a a a t t c c g a t a t g a a a a t c a g t a g c a a t a t c t a a t a a t g t t g a t a t t a t g t t t g t a a t g t t c t g t a a a a c t t g t a c t a c t t t a a t g c a t t a t g c g t c a t t t t t g c g a a a a a a a a a a a Figure 13. Expression of A02S120 after dicamba treatment. (A) RNA isolated from S and R K. scoparia 0, 60, or 120 minutes after treatment and probed with the radiolabeled cDNA. (B) Phosphorimager analysis of message abundance from blot (A). (C) Nucleotide sequence of the A02S120 cDNA. 72 A 2 agcaaacgagggactgcagaagctaagaagaagtatgcgcccatttgcgtcactatgcca S K R G T A E A K K K Y A P I C V T M P 62 actgccaggatctgttacaagtgaggcatctcttgaaattccgatatgaaaatcagtagc T A R I C Y K * 122 aatatctaataatgttgatattatgtttgtaatgttctgtaaaacttgtactactttaat 182 gcattatgcgtcatttttgcgaaaaaaaaaaa 213 B Kochia Arabidopsis (AY050320) Arabidopsis (AF339682) Gossypium Spinacia SKRGTAEAKK K Y A P ICVTMP TARICYK* PKRGTEAAKK KYAPVCVTMP T A K I CRN* PKRGTEAGKK KYAQVCVTMR T A K I CRY* PKRGSAEAKK AYAPVCVTMP T A R I CRN* PKRGTPEAKK KYAPVXVTMP SARIXYK* Figure 14. A02S120 is mostly 3' untranslated region. (A) DNA (lower case) and inferred amino acid sequence (upper case) of the A02S120 partial-length cDNA. The stop codon is indicated by an asterisk. (B) PILEUP comparisons of A02S120 and similar amino acid sequences as recovered by BLAST. Amino acid sequence identities between the cDNAs are in bold; the stop codon for each is indicated by an asterisk. of chloroplastic proteins from Arabidopsis (IVIeurer et al., 1996). Based on amino acid similarities, it thus appears that A02S120 encodes a fragment of a light- regulated photosystem Il component. The significance of the observed differential expression of this gene in R and S K. scoparia after dicamba treatment is unknown. 73 The 1.0 Kb mRNA recognized by the 434-bp C07S60 clone was present in S but not detectable in R after a 48-hour film exposure (Figure 15). C07S60 also hybridized at moderate levels to rRNA in both R and S samples. DNA sequence analysis indicated this clone has >80% sequence identity to the translation initiation factor elF-1 from Sporobolus stapfianus (Neale et al., 1999), and to homologs of the translation initiation factor SUM from Arabidopsis, Salix, Eucalyptus, and Pimpinella, and to the Zea translation initiation factor GOS2 (de Pater et al., 1992; Figure 16). At the deduced amino acid level, the sequence was >80% identical and 90% similar to translation initiation factors from a variety of organisms including Arabidopsis, Oryza, yeast, human, Mus, Schizosaccharomyces, and others. While most translation initiation factors are expressed at very low levels (Neale et al., 1999), the mRNA for the C07S60 appears to be relatively abundant and may be slightly induced by 90 minutes after treatment. The eiF-1 translation initiation factor is induced upon drought stress in Sporobolus, but it is not known whether dicamba treatment presents a similar challenge to S K. scoparia. Of the 106 clones generated by DD and used as probes on northern blots in the studies reported here, C07S60 is one of only two that hybridized to one K. scoparia biotype and not the other. One possible explanation is that the clone was originally generated from a cDNA from the S biotype, and allelic differences between R and S were significant enough to prevent hybridization to R RNA. C07S60 S A 74 R A C £ 15000 11 B 03 ^ 10000 0) 4= S e J5 to m ^ 5000 CK 0 C a a g c t t c g a c t g t t c a a g g t c t g a a g a a g g a t t t c a g c t a c g a g a a g a t a c t c a a a g a c c t c a a a a a g g a g t t c t g c t g c a a c g g c a a t g t t g t a c a g g a c a a g g a g c t a g g g a a g g t t a t c c a a c t g c a a g g c g a t c a g c g t a a g a a g g t t g c g a a t t t c c t g a c c c a a g c c g g t c t t g t g a a g a a g g a t c g g a t c a a a a t t c a t g g t t t t t a a g c t t t g a t t c g t g c t g t t g a g a t a c a a g a g c g a t t g a t t t g t t c t t g g a t a g t t t c t g t t t t c g t t t a g g g t t t g g t c g t t t g a t g t t g t t g g t t t c a g t t g c t t t c g t t t c t g t t g a g t g t t a a t g c a t c c c c a t g g a t g g c a c t g g t a g t c t g t t a t g t a t t g a g c t t t c g a a c t c g t t g c t a t t a a a t a a a a t c g g a t g c t t a c g t a a a a a a a a a a Susceptible y^x X / X / " / Resistant A--------- --------* ----- -------A------------------A----------------- * 0 30 60 90 120 Time after treatment (min) Figure 15. Expression of C07S60 after dicamba treatment. (A) RNA isolated from S and R K. scoparia 0, 30, 60, 90, or 120 minutes after treatment and probed with the radiolabeled cDNA. (B) Phosphorimager analysis of message abundance from blot (A). (C) Nucleotide sequence of the C07S60 cDNA. 75 A 3 gcttcgactgttcaaggtctgaagaaggatttcagctacgagaagatactcaaag.acctc A S T V Q ' G L ' K K D F S Y E K I L K D L 63 aaaaaggagttctgctgcaacggcaatgttgtacaggacaaggagctagggaaggttatc K K E F C C N G N V V Q D K E L G K V I 123 caactgcaaggcgatcagcgtaagaaggttgcgaatttcctgacccaagccggtcttgtg Q L Q G D Q R K K V A N F L T Q A G L V 183 aagaaggatcggatcaaaattcatggtttttaagctttgattcgtgctgttgagatacaa K K D R I K I H C F * 243 gagcgattgatttgttcttggatagtttctgttttcgtttagggtttggtcgtttgatgt 303 tgttggtttcagttgctttcgtttctgttgagtgttaatgcatccccatggatggcactg. 363 gtagtctgttatgtattgagctttcgaactcgttgctattaaataaaatcggatgcttac 423 gtaaaaaaaaaa 434. B Kochia Zea Phleum Sporobolus Arabidopsis Homo I 50 T V Q G L K K D F S Y E K I L K D L K K E F C C N G N W Q D K E L G K V I Q L Q G D Q R K K V A N T V Q G L K K E F S Y S K I L K D L K K E F C C N G T W Q D P E L G Q V IQ L Q G D Q R K N V S N T V Q G L K K E F S Y S K I L K D L K K E F C C N G T W Q D P E L G Q V IQ L Q G D Q R K N V S N T V Q G L K K E F S Y N K I L E C D L K K E F C C N G T W Q D P E L G Q V IQ L Q G D Q R K N V S N T V Q G L E C K E Y S Y E R IL iC D L E C K D F C C N G N W Q D K E L G K I I Q L Q G D Q R K K V S Q T V Q G I A D D Y D K K K L V K P F IC K K F A C N G T V IE H P E Y G E V IQ L Q G D Q R K N IC Q Kochia Zea Phleum : Sporobolus Arabidopsis Homo SI 6 8 F L T Q A G L V K K D R I K I H G F * F L V Q A G I V K K E H I K I H G F * F L V Q A G I V K K E H I K I H G F * F L V Q A G I V K K E H I K I H G F * F L V Q T G I A K K D Q I K I H G F * F L V E I G L A K D D Q L K V H G F * Figure 16. C07S60 has high sequence similarity to SUM translation initiation factors. (A) DNA (lower case) and translated amino acid sequence (upper case) of the C07S60 partial-length cDNA. The stop codon is indicated by an asterisk. (B) PILEUP comparisons of C07S60 and similar amino acid sequences as recovered by BLAST. Amino acid sequence identities between the cDNAs are in bold; the stop codon for each is indicated by an asterisk. Alternatively, this may be a member of a multiple gene family that is expressed in S but not R. Both these explanations seem somewhat unlikely, however, given the high similarity across diverse species seen in Figure 16B. 76 Figure 17 shows the expression patterns of the 1.2-Kb mRNA that hybridized to the 657-bp clone G07R0. The message appears to be differentially expressed in S but levels do not change in R. In S, the message is reduced by about half at 30 minutes after treatment, but increased to about 3 times basal levels by 60 minutes after treatment. DNA sequence analysis of the clone indicated 82-89% similarity to xyloglucan endotransglycosylases (XETs) from Arabidopsis, Carica, and Fagus. The predicted amino acid sequence (Figure 18) was about 65% identical or 74% similar to dozens of xyloglucan endotransglycosylases (also known as endoxyloglucan transferases) and xyloglucan endo-1,4-(3-D-glucanases from Arabidopsis and other dicots. Clone G07R0 has several characteristics that further suggest it is a xyloglucan endotransglycosylase, including the HDE(IAZ)D(F/I/M)EFLG motif (underlined in Figure 19) and the four invariant cysteine residues thought to be involved in disulfide bridge formation (Xu et. al., 1995a). Xyloglucan endo-1,4-P-D-glucanases have a cellulase activity and catalyze the internal hydrolysis of long xyloglycan chains, whereas XETs transfer a xyloglucan molecule onto another polysaccharide after the same internal cleavage (Arrowsmith and de Silva, 1995). Such enzymes appear to be critical in the creation, modification, and degradation of plant cell walls, which are composed of cellulose microfibrils embedded in a matrix of hemicellulose, proteins, and pectin (Xu et al., 1995b). In dicots, the primary hemicellulose is G07R0 A 1.2 Kb 20000 Susceptible / 5 2 ■o E 15000 10000 ■ > ■ 9 (D (0 Resistant 5000 Time after treatment (min) aacgaggttacactgctggagttataacagcattctatctttcgaacaaccaagttcatcccgggcaccacg atgaagtagacatggaattccttgggacaacatttggaaaaccatatgttttgcaaacaaatgtgtacataa ggggtagtggagatggcacaattattggaagagagatgaaatttcatctatggtttgatccaactaagggat ttcaccattatgccattttttggagtcccaaagaaatcatatttttggtagatgatattccaataagaagat atcctagaaagagtgcagccacatatcctctaaggccaatgtgggtgtatggctcaatatgggatgcatcat cttgggcaaccgaagatggaaaatacaaagcaaattataattaccaaccatttgttggtcaatacactaatt ttaaagctagtggttgctccgcctacgccccacgctcatgtcgcccggtgtccgtctcgccctaccggtccg ggggccttacccaaaagcaaacctacgtgatgaagtgggtacaaagacattacatgatttataactattgca aggaccgtaaaanggaccattcacataccccggaatgttggttacgtcgccgttaataattaaagttatata atggtgg Figure 17. Expression of G07R0 after dicamba treatment. (A) RNA isolated from S and R K. scoparia 0, 60, or 120 minutes after treatment and probed with the radiolabeled cDNA. (B) Phosphorimager analysis of message abundance from blot (A). (C) Nucleotide sequence of the G07R0 cDNA. 78 3 cgaggttacactgctggagttataacagcattctatctttcgaacaaccaagttcatccc R G Y T A G V I T A F Y L S N N Q V H P 63 gggcaccacgatgaagtagacatggaattccttgggacaacatttggaaaaccatatgtt G H H D E V D M E F L G T T F G K P Y V 123 ttgcaaacaaatgtgtacataaggggtagtggagatggcacaattattggaagagagatg L Q T N V Y I R G S G D G T I I G R E M 183 aaatttcatctatggtttgatccaactaagggatttcaccattatgccattttttggagt K F H L W F D P T K G F H H Y A I F W S 243 cccaaagaaatcatatttttggtagatgatattccaataagaagatatcctagaaagagt P K E I I F L V D D I P I R R Y P R K S 303 gcagccacatatcctctaaggccaatgtgggtgtatggctcaatatgggatgcatcatct A A T Y P L R P M W V Y G S I W D A S S 363 tgggcaaccgaagatggaaaatacaaagcaaattataattaccaaccatttgttggtcaa W A T E D G K Y K A N . Y N Y Q P F V G Q ' 423 tacactaattttaaagctagtggttgctccgcctacgccccacgctcatgtcgcccggtg Y T N F K A S G C S A Y A P R S C R P V 483 tccgtctcgccctaccggtccgggggccttacccaaaagcaaacctacgtgatgaagtgg S V S P Y R S G G L T Q K Q T Y V M K W 543 gtacaaagacattacatgatttataactattgcaaggaccgtaaaanggaccattcacat V Q R H Y M I Y N Y C K D R K X D H S H 603 accccggaatgttggttacgtcgccgttaataattaaagttatataatggtgg 655 T P E C W L R R R * * Figure 18. G07R0 open reading frame. DNA (lower case) and deduced amino acid sequence (upper case) of the G07R0 partial-length cDNA. Consecutive stop codons are indicated by asterisks. xyloglucan, which is structurally similar to cellulose but has numerous xylosyl side chains. Since xyloglucans form tight hydrogen bonds with cellulose, it has been proposed that modification of xyloglucans and subsequent turgor-mediated cell expansion is necessary for cell growth (Fry et al., 1992, McCann et al., 1992, others). Xyloglucan degradation and turnover is associated with auxin-mediated cell elongation in Pisum (Labavitch and Ray, 1974), Vigna (Nishitani and Masuda, 1982), and Oryza (Revilla and Zarra, 1987). 79 Kochia Arabidopsis Carica Tropaeolum Oryza Nicotiana I 50 R G Y T A G fV T T A F Y L S N fS fQ V H P G H H D E V D M E F L G T T F G K P Y V L Q T N V Y IR G S P G Y T A G V I T S L Y L S N N E A H P G F H D E V D IE F L G T T F G K P Y T L Q T N V Y IR G S P G Y T A G V I T S F Y L S N N E D Y P G N H D E I D I E F L G T t p g e p y t L Q T N V F IR G S S G Y T A G V I T S F Y L S N N Q D Y P G K H D E I D I E F L G T i p g k p y t L Q T N V F IE G S P G Y T A G V N T A F Y L S N T F O Y P G H H D E ID M E L L G T v p g e p y t L Q T N V Y V R G S G G D S A G V V T A F Y L S S N N . . . A E H D E I D F E F L G N r t g q p y i L Q T N V F T G G K .Kochia Arabidopsis arica Tropaeolum Oryza icotiana 51 G D G T I IG R E M G D G K I I G R E M G D R N IV G R E V G D Y N I I G R E M G D G N IV G R E M G D ............ R E Q K F H L H F D P T K K F R L W F D P T K K F H L W F D P T Q R IH L W F D P T Q R F H L W F D P T A R I Y L W F D P T K G F H H Y A IF W S D F H H Y A I L W S D F H N Y A I L W T D Y H N Y A IY W T G F H H Y A I L W N G Y H S Y S V L W N P K E I I F L V D D P R E I I F L V D D P S E I V F F V D D P S E I I F F V D D P D Q I L F L V D D T F Q I V I F V D D 100 I P I R R Y P R K S I P I R R Y P K K S V P IR R Y P R K S V P IR R Y P R K S V P IR R Y E K K V V P IR A F K N S K Kochia Arabidopsis Carica Tropaeolum Oryza Nicotiana 101 A . . A T Y P L . R A . . S T F P L . R D . . A T F P L . R D . . A T F P L . R E . . G T F P E . R D L G V K F P F N Q P M W V Y G S IW D P M W L Y G S IW D T M W V Y G S IW D P L W V Y G S V W D M W A Y G S IW D P M K IY S S L W D A S S W A T E D G K A S S W A T E D G K A S S W A T D D G K A S S W A T E N G K A S D W A T D G G R A D D W A T R G G L Y K A W Y N Y Q P F Y K A D Y K Y Q P F Y K A D Y Q Y Q P F Y K A D Y R Y Q P F Y R A D Y R Y Q P F E K T D W S N A P F 150 V G Q Y T N F K A S T A K Y T N F K A L V G R Y K N F K IA V G K Y E D F K L G V S R F A D L K V G T A S Y T S F H V D 151 Kochia G C S A Y A P R S . Arabidopsis. G C T A Y S S A R . Carica A C R A D G Q A S . Tropaeolum S C T V E A A S S . Oryza G C A T A A P P A . Nicotiana G C E A A T P Q E V 201 Kochia K D R K X D H S H T Arabidopsis K D Y K R D H S L T Carica L D P K R D H T P T Tropaeolum D D P T R D H T L T Oryza Q D Y S R D H T F Y Nicotiana T D R K R Y P T L P ..CRPVSVS. .P .YRSGGLT ..CYPLSAS. .P .YRSGGLT . .CRPPSVS. . .P .SGFGVLS . . CNPASVS . . P . . . YGQLS ..CSPVPAS. .SGGGSAALS QVCNTKGMRW WDQKAFQDLD 219 ' P E C W L R R R * P E C W R * P E C * P E C * P E C * P E C T K D R D * 200 Q K Q T Y V M K W V Q R H Y M IY N Y C R Q Q H Q A M R W V Q T H S M V Y N Y C P Q Q E S A M E W A Q R N S L V Y N Y C Q Q Q V A A M E W V Q K N Y M V Y N Y C P Q Q E A A M A W A Q R N A M V Y Y Y C A L Q Y R R L R W V R Q K Y T IY N Y C Figure 19. PILEUP comparisons of G07R0 and similar amino acid sequences as recovered by BLAST. Amino acid sequence identities among the cDNAs are in bold and stop codons are indicated by asterisks. Residues involved in the putative active site are underlined (Xu et at., 1995). Invariant cysteine residues are shown with a A. 80 The mRNAs for the XETs TCH4 and EXT were induced within 10 minutes in Arabidopsis seedlings treated with low concentrations of auxin (Xu et al., 1995, Xu et al., 1996). Similarly, RNAs for two endo-1,4-|3-D-glucanases from Hordeum (Slakeski and Fincher, 1992) and Pisum (IVIatsumoto et al., 1997) were induced after treatment with auxin and 2,4-D, respectively. In contrast, XET in S K. scoparia appears to be initially down-regulated by 30 minutes after treatment, followed by the induction noted by Xu et al. The difference in expression patterns between R and S K. scoparia is consistent with the resistance trait in that S plants become epinastic within a few hours after treatment. In some species, epinasty is thought to be the result of uncontrolled cell elongation (Sterling and Hall, 1997), and the induction of this XET in S but not R plants may partly account for this symptom. XETs and endo-1,4-|3-D-glucanases are large families of enzymes in Arabidopsis and other dicots, and different members appear to respond to various stimuli (including auxin treatment) differently. Expression of the CEL1, CEL5, and BAC1 genes, which encode endo-1,4-p-D-glucanases, is thought to be involved in the abscission process and these enzymes are induced by ethylene treatment. As this induction is strongly inhibited by auxin (Tucker et al., 1988, Wu et al., 1996), there appears to be complex regulation of these classes of enzymes. I 81 C08R60 The 522-bp clone C08R60 recognized a moderately-abundanf (24 hour exposure time) 1.4-Kb message in both R and S (Figure 20). Basal mRNA levels were somewhat higher in S than in R1 and levels fell slowly in S. However, mRNA levels declined rapidly in R by 30 minutes after treatment before returning to basal levels by 60 and 90 minutes after treatment. Sequence analysis of C08R60 indicated no nucleotide sequence similarities to any known genes, but the translated amino acid sequence was 71% identical and 83% similar to a hypothetical protein in Arabidopsis (Figure 21). Somewhat weaker similarities were noted between this clone and the human translation initiation factor HSPC182 (52% identical; 76% similar) as well as the human (50% identical; 68% similar) and yeast (40% identical; 59% similar) transcription initiation factor SSU72 (Pappas and Hampsey, 2000). Sequence similarities to these diverse genes prevents a putative identification of C08R60, but a more detailed analysis of the full-length clone could provide more information on the presence of functional domains common to translation or transcription initiation factors. If the apparently differential response between R and S can be confirmed on independent blots, this cDNA should be studied further. A 1.4 Kb \ Susceptible 15000 10000 Resistant Time after treatment (min) C gcttctcaacgctggcaagacaacgcacttgatggtgcttttgatgttgtgcttacatttgaagaaaaggtc tttgatatggttgttgaagatctataccaccgtgagcatgtacaaatgaagcctgttgtcatcatcaacctt gaggtaaaagacaatcacgaggaagcagctattggcggtcgccttacactagattttatgccaagagattga agagactgctgattcatgggaagacaagatcgatgagatcattgctgcttttgagagacaccataggcgcaa gattgtctacactatttccttttattgaacataagatattgttttgtaggcaatgaccaacaatttgtaaca gttctgttttacatgagcacagattcccgcatttcatcatattcatgcggtcacaaacgtgacatattgtat agttctagcctatgttactttgactcgtatacgagatgtccgactcggctaatttatgaaaattacctagat tttagccaaaaaaaaaaa Figure 20. Expression of C08R60 after dicamba treatment. (A) RNA isolated from S and R K. scoparia 0, 30, 60, 90, or 120 minutes after treatment and probed with the radiolabeled cDNA. (B) Phosphorimager analysis of message abundance from blot (A). (C) Nucleotide sequence of the C08R60 cDNA. 83 I gcttctcaacgctggcaagacaacgcacttgatggtgcttttgatgttgtgcttacattt A S Q R W Q D N A L D G A F D V V L T F 61 gaagaaaaggtctttgatatggttgttgaagatctataccaccgtgagcatgtacaaatg E E K V F D M V V E D L Y H R E H V Q M 121 aagcctgttgtcatcatcaaccttgaggtaaaagacaatcacgaggaagcagctattggc K P V V I I N L E V K D N H E E A A I G 181 ggtcgccttacactagattttatgccaagagattgaagagactgctgattcatgggaaga G R L T L D F M P R D * 241 caagatcgatgagatcattgctgcttttgagagacaccataggcgcaagattgtctacac 301 tatttccttttattgaacataagatattgttttgtaggcaatgaccaacaatttgtaaca 361 gttctgttttacatgagcacagattcccgcatttcatcatattcatgcggtcacaaacgt 421 gacatattgtatagttctagcctatgttactttgactcgtatacgagatgtccgactcgg 481 ctaatttatgaaaattacctagattttagccaaaaaaaaaaa 522 A B . 1 50 Kochia A S Q R W Q D N A L D G A F D V V L T F E E K V F D M W E D L Y H R E H V Q M K P W I I N L E V Arabidopsis A P Q R W Q D N A G D G V F D W M T F E E K V F D S V L E D L N N R E Q S L T K T I L V M N L E V Homo K P R P E R F Q N C K D L F D L I L T C E E R V Y D Q W E D L N S R E Q E T C Q P V H W N V D I Saccharomyces A P E K W Q E S T - - K V F D F V F T C E E R C F D A V C E D L M N R G G K L N K I V H V I N V D I 51 ■ 79 Kochia K D N H E E A A IG G R L T L D F M P R D * Arabidopsis. K D N H E E A A IG G R L A L E L C Q E I * Homo Q D N H E E A T L G A F L I C E L C Q C IQ H T E D M E N Saccharomyces K D D D E N A K IG S K A I L E L A D M . L N D K IE Q C E Figure 21. Translated amino acid sequence of the C08R60 cDNA. (A) DNA (lower case) and translated amino acid sequence (upper case) of the entire C08R60 partial length cDNA, The stop codon is indicated by an asterisk. (B) PILEUP comparisons of C08R60 and similar amino acid sequences as recovered by BLAST. Amino acid sequence identities between the cDNAs are in bold; the stop codon for each is indicated by an asterisk. 84 Clone C08R60-2 recognized a moderately abundant (16 hour exposure) 1.2-Kb message in both R and S that was induced over 20-fold by 30 minutes after treatment in R (Figure 22). Message levels remained high at 60 minutes after treatment, but dropped to nearly basal levels by 120 minutes after treatment. In S, the message remained at very low levels throughout the time course. Sequence analysis showed that this 479-bp clone has 80% DNA sequence identity and 92% amino acid identity to an unknown, hypothetical protein in Arabidopsis (NCBI accession AF375454; Figure 23). Other amino acid similarities are to unknown, hypothetical proteins from Volvox (63% identical) and Synechocystis (64% identical). Hypothetical proteins are typically derived from large-scale genome sequencing efforts, and are identified by the presence of open reading frames recognized by sequence analysis software. These proteins have not been characterized biologically, so no putative function can be assigned to them. The dramatic increase in mRNA levels specifically in R plants after dicamba treatment suggests that this clone deserves further attention and may provide important insights into the physiological responses occurring in R plants. C08R60-2 85 A C SA____ R_____K.t O 30 60 120 0 30 60 120 1.2 Kb 20000 Resistant 3 15000 10000 Susceptible Time after treatment (min) ccgacccgggcgcagcccatgatgaagaatgttaatgaagggaaaggtatatttgctcctgtggtggttgtg actaggaacatcattggcaaaaagaggttcaatcaacttagaggcaaagccattgcccttcactcacaggtt ataacagagttttgcaagtcaataggagcagatgcaaagcaaagacaaggcttgattcgattggcaaagaag aatggagagaaattagggttccttgcataattaaatttgaacatttgtttgaatttgtcaaatgaaattgtt gagacttgtgtaaaaccttatgtatacatttcttgtgtagaagtaatctgtagaaatttctgttgcaattgc aattgcaaaattcatctaaatttggggattaaacttcaaattgatggtattattattttaggtttagtaaaa tcaaattagcaattttgtagttgttattgttattataaaaaaaaaaa Figure 22. Expression of C08R60-2 after dicamba treatment. (A) RNA isolated from S and R K. scoparia 0, 30, 60, or 120 minutes after treatment and probed with the radiolabeled cDNA. (B) Phosphorimager analysis of message abundance from blot (A). (C) Nucleotide sequence of the C08R60-2 cDNA. 86 I ccgacccgggcgcagcccatgatgaagaatgttaatgaagggaaaggtatatttgctcct P T R A Q P M M K N V N E G K G I F A P 61 gtggtggttgtgactaggaacatcattggcaaaaagaggttcaatcaacttagaggcaaa V V V V T R N I I G K K R F N Q L R G K 121 gccattgcccttcactcacaggttataacagagttttgcaagtcaataggagcagatgca A l A L H S Q V I T E F C K S I G A D A 181 aagcaaagacaaggcttgattcgattggcaaagaagaatggagagaaattagggttcctt K Q R Q G L I R L A K K N G E K L G F L 2 4 1■gcataattaaatttgaacatttgtttgaatttgtcaaatgaaattgttgagacttgtgta A * 301 aaaccttatgtatacatttcttgtgtagaagtaatctgtagaaatttctgttgcaattgc 361 aattgcaaaattcatctaaatttggggattaaacttcaaattgatggtattattatttta 421 ggtttagtaaaatcaaattagcaattttgtagttgttattgttattataaaaaaaaaaa 479 A P T R A Q P M M K N V N E G K G I F A P W W T R N I I G K K R F N Q L R G K A I A L H S Q V I T A IR A V P M M K N V N E G K G L F A P L W V T R N L V G K K R F N Q L R G K A I A L H S Q V I T _______________ M F A P I V I L V R Q Q L G K A K F N Q IR G K A I A L H C Q T I T P R R K P V T M M G N K A T T G P F A R L W V V R G A I G E K E F N Q F R G K A l S L H S Q V I K Si 81 Kochia E F C K S I G A D A K Q R Q G L I R L A K K N G E K L G F L A * Arabidopsis E F C K S IG A D A K Q R Q G L I R L A K K N G E R L G F L A * Synechocystis N F C N R V G ID A K Q R Q N L IR L A K S N G K T L G L L A * Volvox D F C K L L G V D N K Q V Q G V IR L A K K N G E K L G F L A * B Kochia Arabidopsis Synechocystis Volvox Figure 23. C08R60-2 is similar to hypothetical proteins in several photosynthetic organisms. (A) DNA (lower case) and translated amino acid sequence (upper case) of the C08R60-2 partial-length cDNA. The stop codon is indicated by an asterisk. (B) PILEUP comparisons of C08R60-2 and similar amino acid sequences as recovered by BLAST. Amino acid sequence similarities are in bold; the stop codon for each gene is indicated by an asterisk. 87 B16R120 Clone B16R120 recognized a moderately abundant (16 hour film exposure) 2.2-Kb message in both R and S Kochia (Figure 24). By 90 minutes after treatment there was about 8-fold more of the mRNA in R than in S K. scoparia. Expression was essentially constitutive in S plants after herbicide treatment, but increased about 3-fold in R. The 263-bp fragment showed no sequence similarity to known sequences at either the DNA or deduced amino acid level. A13S60 Expression levels of the moderately abundant (16 hour film exposure) A13S60 message increased in S and remained constitutive in R (Figure 25). The 231-bp fragment recognized a 1.9-Kb mRNA, which was induced about 5- fold in S by 60 minutes after treatment. Sequence analysis found no significant similarities to known sequences at the DNA or inferred amino acid level. 88 S R z----------A--------- \ z--------- A--------- \ O 30 60 90 120 0 30 60 90 120 4m # # # # # # - # » -------------2.2 Kb . J , ' Q i t A x . . J k . a— ^ , m e t ■' , Resistant<5 15000 -Q ^ ro (5 10000a> j= S e JD (D 80% identical, >90% similar) to choline monooxygenase sequences from Spinacia, Atriplex, Beta, and Amaranthus (Figure 30), and weaker identity (55%) to a hypothetical protein from Arabidopsis. The deduced amino acid sequence contains several motifs present in CMOs from other plants and related prokaryotic genes encoding proteins with oxygenase functions. First, the cysteine/histidine consensus sequence for a Rieske-type [2Fe-2S] cluster (Cys-X-His-X15„17-Cys-X2- His) is present in the K. scoparia CMO reported here, as it is in all CMOs isolated to date (Figure 30). Secondly, CMO cDNAs from Beta and Spinacia (Russell et al., 1998), Amaranthus, Kochia, and Atriplex (Figure 30), as well as numerous bacterial monooxygenases contain a proposed consensus sequence for coordination sites of mononuclear, nonheme iron (GluZAsp-X3^ -Asp-X2-His-Xw- His; Jiang et al., 1996). The same motif is present in plant CMOs except that the central asparagine and histidine residues are three residues apart instead of two. Further evidence that WCMO encodes a stromal enzyme could be gained by 106 2 tgccgagctggcgaagggaaagtgcatgcatttcacaacgtttgtactcatcgtgcttcg C R A G E G K V H A F H N V C T H R A S 62 attctcgcttgtggaagtggcaaaaagtcctgctttgtgtgcccttaccatggatgggtt I L A C G S G K K S C F V C P Y H G W V 122 tttggcatgaatggagatctcacaaaagcaacccaagctgaaactcaaacttttgatgct F G M N G D L T K A T Q A E T Q T F D A 182 aaggaacttgggctagtggccctaaaggttgcagtatggggaccattcgttctgatcagc K E L G L V A L K V A V W G P F V L I S 242 ttggacaaaactctccctgaaactgatgttggcactgaatggcttggcaaatctgctgaa L D K -T L P E T D V G ' T E W L G K S A E 302 gatgtcaaggcccatgcctttgatccctccctccaattcatccataggagtgaattcccc D V K A H A F D P S L Q F I H R S E F P 362 atggagtgcaattggaaggttttctgtgacaactatctggatagctcataccatgtccct M E C N W K V F C D N Y L D S S Y H V P 422 tatgcacacaaatactacgcaactgaactcgactttgacacatacgacactcaaatgatt Y A H K Y Y A T E L D F D T Y D T Q M I 482 gagaatgttgtgattcaaagggttggaggtaacaaaaacaagactgatgggatcgataga E N V V I . Q R V G G M K N K T D G I D R 542 cttggaaatcaagcattctatgcctttgcttatcccaactttgctattgaaaggtatggc L G N Q A F Y A F A Y P N F A I E R Y G 602 ccttggatgaccacaatgcatgttcaaccattgggactaaggaaatgcaagcttgttgtt P W M T T M H V Q P L G L R K C K L V V 662 gactattatattgaagactctaagttggaggacaaggactacatagagaaaggtatcgca D Y Y I E D S K L E D K D Y I E K G I A 722 atcaacgataacgtacagagtgaagataaggtgttatgtgaaagtgtccaaagggggtta I N D N V Q S E D K V L C E S V Q R G L 782 gagactccatcatacagcacagggagatatgtgatgccaattgagaaaggaatccaccac E T P S Y S T G R Y V M P I E K G I H H 842 ttccactgctggttgcaccaagtgttgcagtgattttcatagatctcttccatggatatc F H C. W L H Q V L Q * 902 aattacgacgcataattaaaatgtggttaagcataatgtgtgtctgctttgcttaattgt 962 gtacttgagtgtaaggtgctaataatgactattataaataaacaccatggcacccccttc 1022 ccctaaatagttttcaaatattttggggggttaagcgaagaatagatcgcttatgaatgt 1082 tttatctagtcatgtatggttttcattagttaagtacatgaggaaaacattattgtgtgt 1142 tgaatacagcatattaatacaacatattatcagttgtagtaaaaaaaaaaaaaaaagtac 1202 tagtcgacgcgtggccaa 1219 Figure 29. WCMO 3' RACE product. DNA (lower case) and deduced amino acid sequence (upper case) of the WCMO partial-length cDNA. The stop codon is indicated by an asterisk and the putative polyadenylation signal is underlined. 107 I Kochia C R A G E G K V H A Spinacia S R D G E G K V H A Atriplex C R D G E G K V H A Beta S R D G Q G E L H A Amaranthus C R D G Q G K V H A 5 1 Kochia T Q A . E T Q T F D Spinacia S K A K P E Q N L D Atriplex S K A T P E Q S L N Beta S K A T E T Q N L D Amaranthus T K . T E N Q V F D 1 0 1 Kochia S A E D V K A H A F Spinacia S A E D V K A H A F Atriplex C A E D V K A H A F Beta S A E D V K A H A F Amaranthus C A E E V K K H A F 1 5 1 Kochia T E L D F D T Y D T Spinacia T E L N F D T Y D T Atriplex T E L D F D T Y Q T Beta A E L D F D T Y N T Amaranthus A E L D F D T Y K T F H N V C T H R A S I L A C G S G K K S F H N V C T H R A S I L A C G S G K K S F H N V C T H R A S I L A C G S G K K S F H N V C T H R A S I L A C G S G K K S F H N V C T H R A S I L A C G T G K K S A K E L G L V A L K V A V W G P F V L I P K E L G L V P L K V A V W G P F V L I P D E L G L V P L K V A V W G P F I L I P K E L G L A P L K V A E W G P F I L I P K E L G L V T L K V A IW G P F V L I D P S L Q F IH R S E F P M E C N W K V D P S L Q F IH R S E F P M E S N W K I D P N L Q F IN R S E F P I E S N W K I D P N L K F T H R S E F P M E C N W K V D P S L Q F IN R S E F P M E S N W K V Q M I E N W I Q R V G G N K N K T D G Q M IE N V T IQ R V E G S S N K P D G D M V G N V T IQ R V A G T S N . .N G E M I E K C V I Q R V G S S S N K P D G D L L E K V V IQ R V A S S S N K P N G C F V C P Y H G W V F G M N G D L T K A C F V C P Y H G W V Y G M D G S L A K A C F V C P Y H G W V Y G M N G S L T K A C F V C P Y H G W V Y G L D G S L A K A C F V C P Y H G W V F G L D G S L M K A A A 100 S L D K T L P E . T . D V G T E W L G K S L D R S L E E . G G D V G T E W L G T S L D R S S R E . V G D V G S E W LG S S L D R S L D A . N A D V G T E W IG K S L D R S G S E G T E D V G K E W IG S 1 5 0 F C D N Y L D S S Y H V P Y A H K Y Y A F S D N Y L D S S Y H V P Y A H K Y Y A F S D N Y L D S S Y H V P Y A H K Y Y A F C D N Y L D S S Y H V P Y A H K Y Y A F C D N Y L D S A Y H V P Y A H K Y Y A ❖ ❖ ' ❖ ❖ 200 I D R L G N Q A F Y A F A Y P N F A I E F D R V G IQ A F Y A F A Y P N F A V E F N R L G T Q A F Y A F A Y P N F A V E F D R L G T E A F Y A F I Y P N F A V E F D R L G S E A F Y A F I Y P N F A V E 50 2 0 1 Kochia R Y G P W M T T M H Spinacia R Y G P W M T T M H Atriplex R Y G P W M T T M H Beta R Y G T M M T T M H Amaranthus R Y G P W M T T M H 2 5 1 Kochia E D K V L C E S V Q Spinacia E D W L C E S V Q Atriplex E D V V L C E S V Q Beta E D K V L C E S V Q Amaranthus E D W L C E S V Q V Q P L G L R K C K L W D Y Y I E D S I H P L G P R K C K L W D Y Y I E N S I V P L G P R K C K L W D Y Y I E K S W P M G Q R K C K L W D Y Y L E K A IG P L G P R K C K L W D Y Y L E N A R G L E T P S Y S T G R Y V M P IE K G R G L E T P A Y R S G R Y V M P IE K G K G L E T P A Y R S G R Y V M P IE K G R G L E T P A Y R S G R Y V M P IE K G R G L E T P A Y R S G R Y V M P IE K G 2 5 0 K L E D K D Y I E K G IA IN D N V Q S M L D D K D Y IE K G lA lN D N V Q R * K L D D K D Y I E K G I A I N D N V Q K M L D D K A Y ID K G I A I N D N V Q K M M N D K P Y IE K S IM IN D N V Q K 2 9 3 IH H F H C W L H Q V L Q * IH H F H C W L Q Q T L K * IH H F H C W L H Q V L K * IH H F H C W L H E T L Q * IH H F H C W L H Q T L N * Figure 30. PILEUP comparisons of WCMO and similar amino acid sequences as recovered by BLAST. Amino acid sequence identities among the cDNAs are in bold and the stop codons are indicated by asterisks. Conserved cysteine/histidine pairs involved in the putative Rieske-type cluster are indicated by a A. Conserved asparagine/histidine residues involved in mononuclear iron binding are underscored by a 0. 108. isolating the remainder of the cDNA and checking for chloroplast stroma targeting sequences that are poorly conserved but rich in polar amino acid residues (Cline and Henry, 1996). Experiments are underway to isolate the 5' end of the K. scoparia CMO cDNA. CMO expression in response to NaCI stress GB production in response to osmotic stress is widespread among members of the Chenopodiaceae (Russell et al., 1998), although the molecular details of induction are unknown. To date, induction of CMO mRNA or protein in response to osmotic stress has been documented in Spinacia (Rasinasabapathi et al., 1997), Beta (Russell et al., 1998), and Amaranthus (Wang et al., 1999). In K. scoparia, osmotic stress similarly induced expression of CMO mRNA, protein, ^ and GB (Figures 31 and 32). Basal CMO mRNA levels were moderately abundant as they could be detected with overnight film exposures of northern blots (Figure 31A). This finding is consistent with reports that some members of the Chenopodiaceae accumulate moderate levels of GB regardless of stress conditions (Weretilnyk and Hanson, 1990). The radiolabeled D416S0 probe recognized a 1.9-Kb message that was relatively abundant in unsallnized plants, and increased greatly (>20-fold in S) after plants had been treated with 500 mM NaCI for 2 days (Figure 31B). In R plants, the CMC mRNA was induced more slowly and mRNA levels increased by a maximum of about 12-fold. Mouse polyclonal antibodies raised against Spinacia CMC (Russell et al., ' -■ 1 109 30000 £ 25000 "O c .-ti 20000 Susceptible y ^ 15000 Resistant 5000 Time after salinization (d) Figure 31. Expression of CMO mRNA in response to salt stress. (A)RNA isolated 0, 2, 4, 6, and 8 days after salinization treatments began and probed with the radiolabeled D416S0 cDNA. (B) Phosphorimager analysis of message abundance from blot (A). 110 A B C 10000 Resistant 8000 6000 Susceptible 4000 2000 Time after salinization (d) Resistant ^ 40 Susceptible Time after salinization (d) Figure 32. Accumulation of CMO protein and glycine betaine in response to salt stress. (A) CMO protein as detected with mouse anti-CMO antibodies 0, 4, 6, and 8 days after salinization treatments began. (B) Densitometry analysis of protein bands in (A). (C) Corresponding GB levels in shoot tissues used in (A). 111 1998) recognized two protein bands of about 42 and 43 Kd in protein extracts from K. scoparia (Figure 32A). CMO enzyme levels followed the pattern of mRNA induction, and the enzyme accumulated more slowly as expected. By 4 days after treatment, S and R plants had about 8- and 9-fold more enzyme than untreated plants, respectively. After 8 days, CMO levels in R and S plants were about 15 times their original levels. CMO protein levels increased similarly in salt-stressed Amaranthus tricolor (Wang et al., 1999), Beta vulgaris, and Amaranthus caudatus (Russell et al., 1998) plants by 4 days after treatment with 300 mM NaCI. However, it is evident that K. scoparia has the ability to continue accumulating CMO protein under conditions of more severe osmotic stress (750 mM NaCI): Other studies using SDS-PAGE and western blotting have detected a single CMO protein of about 45 Kd in Beta vulgaris and Amaranfhus caudatus (Russell et al., 1998), as well as in Amaranthus tricolor (Wang et al., 1999). While it is possible that the presence of two CMO bands in K. scoparia is a degradation artifact, it is also reasonable to speculate that the two bands represent products of two different genes that may form a dimeric holoenzyme in vivo. Consistent with this idea, Southern blotting indicated that there are two to four CMO genes in the K. scoparia genome (data not shown). Glycine betaine accumulated in both biotypes in response to salt treatment (Figure 32C). Prior to salinization, GB was present at about 10 pmol g'1 fresh weight, a finding consistent with the idea that GB is a constitutive 112 chemical component of the Chenopodiaceae (Grieve and Maas, 1984). After 8 days of NaCI treatment; GB levels increased about 6-fold to more than 60 pmol g-1 fresh weight, and were apparently still increasing. Treatment of Amaranthus fr/co/or with 300 mM NaCI caused GB values to rise to about 30 pmol g"1 fresh weight (Wang et al., 1999), the highest value reported so far in the literature. The increases in CMO mRNA, protein, and GB content in response to salt stress are consistent with the natural drought tolerance of K. scoparia, which grows very well in xeric environments. In Montana, K. scoparia is often the only plant found growing in saline seeps, and the plant is especially well-adapted to the dry climate of the western U S. where it infests millions of hectares of cropland and rangeland (Eberlein and Fore, 1984). Even given the extreme NaCI stress in these experiments, neither K. scoparia biotype displayed any outward symptoms of drought stress (such as wilting), although growth essentially stopped. One cause of this stunting may due to the metabolic costs of GB production. At the end of these experiments, GB had accumulated to 2 to 3% of the total plant dry weight (data not shown), and thus likely represents a significant carbon and nitrogen sink. 113 CMO expression in response to dicamba treatment in R and S K. scooaria Initial differential display results indicated that the message for clone D416S0 was attenuated rapidly after dicamba treatment (page 59). To more thoroughly investigate the kinetics of this apparent down-regulation, CMO mRNA, protein, and GB levels were determined at several times after dicamba treatment of R and S K. scoparia. These experiments were designed to determine the effect of dicamba treatment on CMO expression in plants that had not been previously subjected to NaCI stress. As noted before, steady-state CMO mRNA levels were moderately high in untreated plants (Figure 33). Treatment of R and S plants with 70 g ai ha"1 of dicamba resulted in a rapid attenuation of the CMO message, with the apparent down-regulation occurring somewhat more rapidly in R than in S. By 90 min after treatment, both biotypes exhibited a >10-fold reduction in the CMO mRNA, which remained low in S plants through the course of this experiment, but recovered slightly in R by 6 and 24 hr after treatment. The difference in CMO mRNA levels at 24 hr after treatment may be associated with the different physiological responses to dicamba treatment in R and S plants. By this time, S plants began to show injury symptoms associated with dicamba treatment such as severe epinasty, whereas R plants did not. The failure of CMO mRNA levels to recover in S plants may thus be due to the eventual lethal effects of dicamba. Western analysis indicated that CMO enzyme levels closely followed the kinetics of mRNA abundance (Figure 34). Basal protein levels were low, and became nearly undetectable by 6 hr after treatment, even when 25 pg total S A 114 R A O 0.5 1 1.5 2 6 24 0 0.5 1 1.5 2 6 24 14000 o 12000 c ro ^ "g w 10000 3 'c m 13 8000 J \ Susceptible Cr -5 Resistantro 4000 2000 6.0 24.00.0 0 .5 1 .0 1 .5 2.0 Time after treatment (h) Figure 33. Expression of CMO mRNA after dicamba treatment of unstressed plants. (A) RNA isolated 0, 0.5, 1, 1.5, 2, 6, and 24 hours after dicamba treatment and probed with the radiolabeled D416S0 cDNA. (B) Phosphorimager analysis of message abundance from blot (A). S115 A RX_____\ 6 24 48 Susceptible 1500 1000 Resistant Time after treatment (h) Susceptible Resistant 6.0 24.00.0 0.51.01.5 2.0 Time after treatment (h) Figure 34. Reduction of CMO protein and glycine betaine levels in unstressed plants after dicamba treatment. (A) Western blots of CMO protein isolated 0, 1, 6, 24, and 48 hours after dicamba treatment. (B) Densitometry analysis of protein bands in (A). (C) GB levels in tissue used in (A). 116 protein was used for SDS-PAGE as in Figure 34. Enzyme levels remained low in S plants up to 48 hours after treatment, whereas levels increased slightly in R plants by this timepoint. It is evident that levels of CMO protein decline rapidly in response to dicamba treatment in both R and S K. scoparia, although these studies do not address the molecular mechanism of attenuation. A similar decline in the CMO protein levels was reported in Beta after removal of drought stress (Russell et al„ 1998). However, it is unknown whether the mechanism of CMO mRNA and protein decline seen in response to dicamba treatment is similar to that caused by release from drought stress. If the value for R plants at 1 hr after treatment is disregarded, GB levels in unstressed tissues increased transiently following dicamba treatment, and then steadily declined to about one-half their initial levels after 24 hr. GB is metabolized very slowly, if at all, in members of the Gramineae that produce low levels (< 5 pmol g"1 fresh weight) in response to drought or salt stress (Grieve and Maas, 1984; Nakamura et al., 1996). However, some GB catabolism was noted in Beta vulgaris (Russell et al., 1998) and Sorghum b/co/or (Wood et al., 1996), but tissue GB levels remained higher than in unstressed plants, even several days after release from osmotic stress. These findings suggest that GB may also be catabolized slowly in K. scoparia. 117 CMQ expression in response to dicamba treatment in salt-stressed K. scoparia Since the auxinic herbicides dicamba and 2,4-D (data not shown) cause a rapid decline in CMO mRNA and protein levels and GB content in unstressed plants, experiments were conducted to determine whether these herbicides had similar effects on plants acclimated to severe osmotic stress. Herbicide efficacy commonly declines when target plants are stressed by Iow temperatures or water deficit, probably because of reduced herbicide uptake and translocation (Ashton and Crafts, 1981). When acclimated to 750 mM NaCI, K. scoparia plants showed no external symptoms of osmotic stress but essentially ceased growth. These experiments were conducted to determine if the attenuation caused by dicamba treatment would also be observed in plants under severe NaCI stress. For the results reported here, plants were treated with 750 mM NaCI for 4 days prior to dicamba treatment. CMO mRNA levels declined rapidly in salinized R K. scoparia plants by 1 hr after dicamba treatment, dropping about 7-fold (Figure 35). mRNA levels remained low until 2 hr after treatment, but then rapidly increased. By 24 hr after treatment, CMO mRNA levels had risen to levels higher than in unsprayed, NaCI- treated plants, possibly as a result of increased osmotic stress due to a concomitant decline in GB levels (Figure 36C). The transient nature of this attenuation is markedly similar to the decline and subsequent recovery of CMO mRNA seen in unstressed, dicamba-treated R plants on page 114, and likely represents a recovery from dicamba treatment. It is tempting to attribute the S A 118 A Z \ * O 0.5 1 1.5 2 6 24 Z * 0 0.5 1 R A ____ 1.5 2 6 24 30000 25000 "2 (/>c 20000 ^ Susceptible ^ 15000 10000 Resistant5000 6.0 24.00.0 0 .51 .01 .5 2.0 Time after treatment (h) Figure 35. Expression of CMO mRNA after dicamba treatment in salt-stressed plants. (A) RNA isolated 0, 0.5, 1, 1.5, 2, 6, and 24 hours after dicamba treatment and probed with the radiolabeled D416S0 cDNA. RNA isolated from unsprayed, unstressed S and R control plants is indicated with an asterisk. (B) Phosphorimager analysis of message abundance from blot (A). 119 recovery of CMO expression to a time-dependent metabolism of dicamba in vivo, but previous studies showed that dicamba remains essentially unmetabolized by 24 hr after treatment in both R and S K. scoparia (Cranston et al., 2001). In contrast to the mRNA attenuation seen in R plants, NaCI-stressed S plants showed less response to dicamba treatment, and CMO mRNA levels declined only about 2-fold by 24 hr after treatment. Consistent with visual observations that NaCI-stressed S plants did not show injury symptoms after dicamba treatment, these results may be explained by a reduction in herbicide absorption or translocation as a result of salt stress. However, the continued responsiveness to dicamba seen in NaCI-stressed R plants represents a fundamental difference between S and R plants and warrants further investigation. Changes in CMO protein levels were noted after dicamba treatment and closely paralled the changes in mRNA abundance. Protein abundance remained relatively uniform in S plants after dicamba treatment, declining only slightly (Figure 36). In contrast, the CMO protein declined rapidly in R plants, dropping about 3-fold before beginning to recover by 48 hr after dicamba treatment. GB levels declined slowly after herbicide treatment of both R and S salt-stressed plants (Figure 36C), consistent with other reports that GB is catabolized slowly (lshatani et al., 1995, Wood et al., 1996). S A 120 R A A r 0 1 6 111 24 48 - Resistant10000 8 0 0 0 6 0 0 0 Susceptible 4 0 0 0 2000 Time after treatment (h) Resistant O 4 0 Susceptible Time after treatment (h) Figure 36. Levels of CMO protein and glycine betaine in salt stressed plants after dicamba treatment. (A) Western blots of CMO protein levels isolated O1 1, 6, 24, and 48 hours after dicamba treatment. (B) Densitometry analysis of protein bands in (A). (C) GB levels in tissue used in (A). 121 With the exception of salt-stressed K. scoparia, the rapid decline of CMO mRNA and enzyme levels following dicamba treatment was similar in R and S plants. The most likely explanation for the relative unresponsiveness of CMO expression to dicamba treatment in NaCI-stressed S plants is a reduction in herbicide uptake and translocation, caused by osmotic stress. Even though R plants have slightly higher levels of CMO enzyme and GB than S plants in response to NaCI stress, the differences do not seem to be of sufficient magnitude to protect R plants from NaCI stress. Indeed, growth of both biotypes ceased during salinization. Little is known about the mechanism(s) of auxinic herbicide perception, but it is clear that there is a fundamental difference in the effects of osmotic stress on dicamba perception (as measured by CMO expression) between R and S K. scoparia. In light of this, logical follow-up experiments may study dicamba absorption and translocation in NaCI-stressed R and S plants. This is the first report documenting a rapid change in CMO expression in response to treatment with auxin or an auxin mimic. The few auxin down- regulated genes isolated to date generally encode proteins of unknown functions such as the Glycine ADR gene family (DaRa et al., 1993). Other down-regulated genes encode enzymes involved in plant defense (Ozeki et al., 1990; Pasquali et al., 1992) and are induced by ABA. Although the studies reported here did not investigate the effect of ABA treatment on CMO gene expression, it is possible that CMO provides another example of a gene induced by ABA and repressed by auxin. The ABA-induced expression of GAD1 and GAD2 mRNAs become 122 insensitive to ABA treatment after pretreatment with auxin (Jacobsen and Olszewski, 1996), and the gene for the dessication-related LEA protein EDP31 does not respond to ABA after auxin pretreatment (Kiyosue et al., 1992). One well-known effect of osmotic stresses similar to those reported here is an overall increase in ABA levels, leading to physiological responses consistent with reducing evaporative water loss (Jensen et al., 1996). Expression of CMO may thus exemplify the opposite effects of ABA and auxin. There is a considerable body of literature reporting the effects of auxin treatment on gene expression, yet there is no consensus on how these changes ultimately affect the physiological processes unique to plants. The findings of this study suggest that dicamba is perceived similarly to auxin at the level of gene expression, and provide verification of several genes previously reported to be responsive to auxin treatment. These studies also document other genes, some of which remain unidentified, that have not previously been shown to respond to auxin treatment. One of these genes encodes choline monooxygenase, an enzyme that has received attention as a target for genetic modification of crops to improve drought and salt tolerance. Genetic characterization of this enzyme is essential if it is to be pursued as such a target, and these studies suggest that its expression is regulated by a possibly complex interaction,of hormones and osmotic stress. 123 CHAPTER 4 CONCLUSIONS AND FUTURE WORK Screening for genes responsive to dicamba treatment One of the initial goals of this dissertation research was to isolate and characterize genes that may be involved in the mechanism of dicamba resistance in K. scoparia. Although none of the genes described here, with the possible exception of C08R60-2, appear to fulfill these criteria, several genes were isolated whose expression levels changed in response to dicamba treatment, and thus represent the first such genes to be reported. Several of these genes were previously reported to be responsive to auxin treatment, such as ACC synthase, xyloglucan endotransglycosylase, and a DnaJ protein. In addition to these, genes not previously shown to be responsive to auxin or auxinic herbicides are described here. Further, their expression patterns are consistent with auxin-mediated responses. For example, the apparent down- regulation of C03S0, putatively encoding 5,10-methylenetetrahydrofolate reductase, may increase the available pool of methyl group donors involved in thymidylate synthesis, probably in response to auxinic herbicide-mediated induction of mitotic cell division. Similarly, the induction of C07S120, putatively encoding a chloride channel protein, may serve to increase chloride ion uptake, 124 as previously noted after auxin treatment. The auxinic herbicides are thought to mimic auxin primarily because of their effects on plant physiological processes. Relatively few. studies have provided molecular evidence supporting this idea, and this research adds credibility to the notion that the auxinic herbicides affect gene expression in ways similar to the natural plant hormone. Still, several side projects not reported in this dissertation suggest that there may be important differences between auxin- and auxinic herbicide-mediated effects on gene expression. Several clones of known auxin induced genes from Arabidopsis were obtained from different research groups, and northern blot analysis indicated none of them were induced by dicamba treatment in K. scoparia. In addition, expression levels of several of the dicamba-responsive genes reported here were unaffected by treatment with 2,4-D or auxin. These disparities suggest that the auxinic herbicides may not be completely accurate mimics of auxin, which is a significant finding because many of the studies in the primary literature equate the auxinic herbicides with auxin. To Help understand these differences at the molecular level, our group is beginning studies to investigate the effects of different auxinic herbicides on gene expression. We plan to use many of the clones isolated in this research, coupled with known auxin-induced genes from other research groups, to gain a more complete understanding of the differences between auxin- and auxinic herbicide-mediated changes in gene expression in K. scoparia. One question that can be raised is whether differential display (DD) is the best technique to isolate genes responding to auxinic herbicide treatment. DD 125 has been extensively utlized for isolating genes differentially expressed in response to various treatments, and is the most widely used method for this purpose. Since its introduction in 1992, over two-thirds of published papers utilized DD as the method of choice, and more papers using DD have been published than all combined research using subtractive hybridization, serial analysis of gene expression, arbitrarily-primed RNA PCR, representational difference analysis, and DNA microarray analysis (see www.genhunter.com). However, despite its broad acceptance, DD is well known to be a somewhat inefficient method, largely due to the high occurrence of false positives, as was seen in this report. Another drawback of DD is the high percentage of 3' untranslated region in most clones, making it more difficult to assign putative functions based on nucleotide or inferred amino acid sequence. If these studies were to begin anew, I believe that microarray analysis would be faster, more useful, and a more rigorous method to isolate genes responsive to dicamba treatment. However, the studies could not be done in K. scoparia, but would rather have to be completed in a species with a large EST library such as Arabidopsis, and therefore could not address the mechanism of dicamba resistance. Essentially, there are three possible approaches utilizing array technology to address this research question. The first would be to obtain a large number (>1000) of random EST clones from the species of choice, amplifying the inserts using PCR, and then spotting the array. Next, the microarray would be simultaneously probed with differentially-labeled fluorescent RNAs isolated from two different treatments, such as dicamba-treated and ! 126 untreated Arabidopsis shoots. Differentially expressed clones are then identified by scanning the array and determining which clones hybridized preferentially to RNA from either treatment, potentially yielding both induced and down-regulated clones. The primary drawback of this approach is the cost of producing or purchasing EST clones and the time associated with PCR amplification and array preparation. A second approach to screening Arabidopsis EST libraries to isolate dicamba-responsive genes would be to use a commercially-available Arabidopsis microarray. Such arrays were offered to the public sector during 2001 through the Arabidopsis Functional Genome Consortium (http://afgc.stanford.edu/), and will be offered through the Genomic Arabidopsis Resource Network during the summer of 2002 (http://www.york.ac.uk/res/garnet/garnet.htm), but array availability is still quite limited and highly competitive. As of this writing, the only source for pre-made Arabidopsis microarrays is Affymetrix, a private company specializing in microarray technology. The Affymetrix systems require specialized array reading equipment which costs $220,000 (http://www.affymetrix.com). A third approach would be to create and screeen a DNA macroarray. Briefly, RNA is isolated from tissue, reverse transcribed, and used to make a cDNA library. Random cDNAs are isolated, amplified by PCR, and immobilized onto a nylon membrane. Macroarray analysis is conceptually similar to microarrays, but utilizes two identical arrays probed separately with radiolabeled cDNA reverse transcribed from mRNAs from two different treatments, such as 127 dicamba-treated and untreated K. scoparia shoot tissue. There are two major benefits to this approach. The first is that a researcher can preferentially find induced genes by making a cDNA library from dicamba-treated tissue and using this library to create the macroarray. This will skew the cDNA makup of the library, and thus the cDNAs immobilized onto the array towards dicamba-induced genes. A similar DNA macroarray approach was successfully used to isolate genes induced by pathogen infection in Capsicum (Jung and Hwang, 2000). Of 282 random cDNAs used as interrogation sequences on the array, 36 (13%) were differentially expressed between infected and uninfected leaves as a result ol Xanthomonas infection. Not surprisingly, all were induced and several were genes previously known to be induced by pathogens. A second major benefit that would be gained by utilizing macroarray technology is that K. scoparia RNA could be used, thus more directly addressing the issue of dicamba resistance. In addition, the use of radioactive cDNA populations prevents the need for specialized array reading equipment. The major drawback of using a random cDNA macroarray is that this approach severely discriminates against rare or down-regulated messages. While array technology probably holds more promise than DD for isolating genes whose expression patterns are affected by dicamba treatment, it cannot be used to elucidate the mechanism of dicamba resistance unless the resistance mechanism is correlated with changes in gene expression. Mutations in auxin binding proteins or proteins involved in herbicide transport, for instance, could theoretically confer resistance, and a better system to screen for such mutations 128 would be to isolate a dicamba-resistant Arabidopsis mutant. Because the Arabidopsis genome is saturated with molecular markers and has been sequenced, it would be relatively easy to use genetic tools to map the mutation. Once mapped, potential candidate genes can be identified and sequenced, allowing for putative identification of the genetic lesion responsible for the resistance trait. Such studies are now underway in the Dyer lab. The major drawback of using this model system is that a dicamba-resistant Arabidopsis mutant has not yet been isolated and may not have the same mutation as dicamba-resistant K. scoparia. Nonetheless, these studies could provide interesting insights into one possible mechanism of resistance to auxinic herbicides and could provide a valuable complement to my results. Analysis of choline monooxvaenase expression These studies provide the first evidence that choline monooxygenase (CMO) mRNA, protein, and product (glycine betaine, GB) are down-regulated in response to treatment with an auxinic herbicide, and suggest that in vivo auxin levels are an important indicator of plant stress. Because CMO has only been shown to be present in certain members of the Chenopodiaceae and Amaranthaceae, and because K. scoparia is highly tolerant to drought stress, this species provides an excellent model for characterizing this gene. Further, this approach may provide useful genetic information for future experiments using CMO to genetically modify crop plants to increase their ability to withstand water deficit. One such characterization that is still underway in our lab is to 129 identify c/s-acting promoter elements within the CMO gene that are involved in auxin-mediated downregulatidh of CMO mRNA. While there has been considerable work investigating promoter elements of auxin-induced genes (Guilfoyle et al., 1998; Hong etal., 1995; Sakaietal.,'1996.; Gil and Green, 1997; Plesch et al., 1997), only a few reports have characterized the promoter elements from auxin-repressible genes. Different regions of the Nicotiana (3-1,3- glucanase promoter have been shown to be essential for auxin-mediated mRNA attenuation, although it was noted that this promoter is most responsive to auxin- mediated increases in ethylene production and thus the changes are not attributable to auxin treatment perse (Vogeli-Lange et al., 1994). Even though auxin-responsive promoter elements probably also play a role in auxin-mediated gene attenuation, it is possible that other factors are involved in the reduction of cytoplasmic mRNA levels. Datta et al. (1993), using nuclear run-on transcription assays of the ADR genes, noted that the rapid reduction in ADR mRNA levels could not be attributed solely to a decrease in rates of transcription and suggested that post-transcriptional processes were more important in reducing mRNA levels in response to auxin treatment. The paucity of studies characterizing c/s-acting elements of auxin down- regulated genes, coupled with the excellent example of the auxin-repressible mRNA reported here, have prompted continued experiments to isolate and characterize the auxin-responsive CMO promoter elements from K. scoparia. A genomic library has been created and screened, and a 6.5-Kb clone has been isolated that likely contains regulatory regions. Future experiments will include 130 characterization of protein-binding regions in the CMO promoter using gel mobility shift assays, as well as performing run-on transcription assays to determine if translational control is the primary level at which the CMO message is attenuated in response to dicamba treatment. 131 i LITERATURE CITED Abel, S., M.D. Nguyen, W. Chow, and A. Theologis. 1995a. ASC4, a primary indoleacetic acid-responsive gene encoding 1 -aminocyclopropane-1 - carboxylate synthase InArabidopsis thaliana. d. Biol. Chem. 270:19093- 19099. 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