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Molecular and physiological studies of nitrate and sulfate uptake in roots of barley Vidmar, Joseph John 1999

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Molecular and physiological studies of nitrate and sulfate uptake in roots of barley by Joseph John Vidmar B.Sc, Concordia University, 1990 M.Sc, Concordia University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1999 © Joseph John Vidmar, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Ct>\tX*f\ The University of British Columbia Vancouver, Canada Date 15 Sr-rVcW 133*1 DE-6 (2/88) Abstract Molecular and physiological approaches were employed to characterize nitrate and sulfate transporters from roots of Hordeum vulgare cv. Klondike. A cDNA, hvstl (accession no. U52867), by heterologous complementation in E. coli. This cDNA encodes a high-affinity sulfate transporter, that is 2442 bp in length and encodes a protein of 660 amino acids. Under steady-state conditions of sulfate supply, ranging from 2.5 to 250 pM, sulfate influx (measured at 100 uM external sulfate concentration) and hvstl transcript levels were inversely correlated with sulfate concentrations in the culture solution. A time-course study, designed to investigate effects of S-withdrawal on the abundance of hvstl transcript, showed a 5-fold increase of the latter within the first two hours after removing external sulfate, followed by a further slight increase during the period up to 48 h. These changes were accompanied by a parallel increase in sulfate influx and a decrease of root glutathione concentrations. When plants that had been deprived of sulfate for 24 h were exposed to 1 mM L-cysteine, or glutathione, for a period of 3 h, glutathione was the more effective down regulator of hvstl transcript levels, reducing the latter to a level that was below that of unstarved controls. Both hvstl transcript abundance and sulfate influx increased as a function of N-supply to N-starved plants. Two new cDNAs, bch3 and bch4 (homologous with an Aspergillus nidulans gene encoding a nitrate transporter, crnA) were isolated from barley roots by R A C E PCR. Bch3 and bch4 are 1822 and 1705 bp, encode putative polypeptides of 507 amino acids, with a predicted m.w. of 54.6 kDa. Predicted BCH3 and BCH4 proteins are members of a nitrate/nitrite transporter subfamily of the major facilitator superfamily. Northern blot analysis, revealed that supplying NO3" to N-deprived plants increased both the abundance of bch transcripts and NO3" influx. All four bch genes (bchl, bchl, bch3, bch4) are co-ordinately up-regulated in response to NO3" treatment. Plants provided with 50 pM NO3" showed the highest bch transcript abundance and , J N0 3 " influx. The effects of exogenous provision of various amino acids on bch transcript levels was investigated, when plants were co-supplied with nitrate. Asparagine, aspartate, glutamate and glutamine decreased transcript levels by >60% and 13NC»3 influx by 50-80%. Analysis of amino acid concentrations of roots showed that the decrease of bch transcript was correlated with increased glutamine levels. ii T A B L E OF CONTENTS Abstract ii Table of Contents iii List of Tables viii List of Figures ix List of Abbreviations xi Acknowledgments xii CHAPTER I Introduction 1 1.1.1.1 Ion transport in plants 2 1.2.1.1 Importance of sulfate 4 1.2.2.1 Metabolism and assimilation of sulfur 5 1.2.2.2 Roles of sulfur and main sulfur containing compounds 5 1.2.2.3 Sulfur assimilation 7 1.2.3.1 Absorption and activation of sulfate 11 1.2.3.2 Absorption of sulfate 11 1.2.4.1 Sulfate transport systems 14 1.2.4.2 Sulfate transport in prokaryotes 14 1.2.4.3 Sulfate permeases in fungi 15 1.2.4.4 Sulfate transporters in plants 16 1.2.4.5 Sulfate transporters: Na+ and H+ dependent in animals .17 1.2.5.1 Glutathione 18 1.2.5.2 Synthesis and localization 18 1.2.5.3 Role of glutathione in plants 19 1.2.5.4 Regulation of uptake of sulfate by glutathione 20 1.3.1.1 Importance of nitrogen 22 1.3.2.1 Nitrate assimilation 22 1.3.2.2 Nitrate reductase 23 1.3.2.3 Nitrite reductase 24 1.3.2.4 GS/GOGAT cycle 26 1.3.2.5 Glutamine synthetase 27 1.3.2.6 Glutamate synthase 29 1.3.3.1 Physiology of nitrate uptake 30 1.3.3.2 Molecular biology of nitrate uptake 32 1.3.3.3 Bacterial and cynobacterial nitrate transporters 32 1.3.3.4 Fungal and lower plants nitrate transporters 33 1.3.3.5 Higher plants nitrate transporters 34 1.3.4.1 Regulation of nitrate uptake 36 1.4.1.1 Aims 37 CHAPTER II Isolation and characterization of hvstl cDNA encoding high-affinity sulfate transporter 39 2.1.1.1 Introduction 40 2.2.1.1 Materials and Methods 41 2.2.1.2 Bacterial strains, media and growth conditionns 41 2.2.1.3 cDNA libraries and genomic libraries 41 2.2.1.4 Preparation and analysis of DNA and RNA 42 2.2.1.5 Isolation and sequencing, of promoter region of hvstl 43 2.2.1.6 Plant material 44 2.2.1.7 Influx measurements 45 2.2.1.8 Compartmental and thiol analysis 45 2.3.1.1 Results 46 2.3.1.2 Cloning and identification of hvstl cDNA encoding sulfate transporter. . . 46 2.3.1.3 Predicted structure of protein encoded by hvstl, phylogenetic analysis and comparison with other plant sulfate transporters 47 2.3.1.4 Isolation of the promoter region of hvstl 49 2.3.1.5 Effect of S status on sulfate influx, thiol levels and hvstl mRNA abundance 51 2.3.1.6 Efflux analysis 60 2.3.1.7 Interaction between N and S nutrition, effect on SO42" influx and hvstl transcript abundance 62 2.4.1.1 Discussion • 66 CHAPTER UJ Isolation and characterization of bch3 and bch4 cDNAs encoding inducible high-affinity nitrate transporters 72 3.1.1.1 introduction 73 3.2.1.1 Materials and methods 76 3.2.1.2 Plant material 76 3.2.1.3 RNA and DNA isolation 77 3.2.1.4 cDNA and genomic library 78 3.2.1.5 Northern blot analysis 78 3.2.1.6 Isolation and screening of bch3 and bch4 cDNAs by RACE-PCR 79 3.2.1.7 Isolation of promoter region of bchl, bch2 and bch3 80 3.2.1.8 Nitrate influx 81 3.3.1.1 Results 82 3.3.1.2 Isolation ofbch3 and bch4cTMAs 82 3.3.1.3 Protein structure, genetic analysis, comparison of the nucleotide and protein sequences 83 3.3.1.4 Isolation, analysis and comparison of promoter regions of bchl, bch2 and bch3 86 3.3.1.5 Time profile of NO3" induction of bch multigene family mRNA accumulation and NO3" influx 87 3.3.16 Effect of various external NO3" concentrations on bch transcript levels and N0 3" influx 93 3.3.1.7 The effect of NCY and N H 4 + on transcript levels of bch multi-gene family 93 3.4.1.1 Discussion 98 3.4.1.2 Characterization of bch cDNAs and promoter regions 98 3.4.1.3 Time profile of bch transcript levels and nitrate transport 99 3.4.1.4 Effect of N O 2 " - or NFlY" on NO3" influx and bch transcript accumulation. 101 3.5.1.1 Conclusion 102 CHAPTER IV Regulation of bch multigene family 104 4.1.1.1 Introduction 105 4.2.1.1 Materials and methods 108 4.2.1.2 Plant material 108 4.2.1.3 Nitrate influx 109 4.2.1.4 RNA isolation and Northern blot analysis 110 4.2.1.5Nitrate analysis I l l 4.2.1.6 Amino acid and ammonium analysis I l l 4.3.1.1 Results 112 4.3.1.2 Time course study of the effect of 10 mM NO3" on 13NC»3~ influx and bch transcript accumulation 112 1 "X 4.3.1.3 The effect of external application of amino acids on NO3" influx and... bch transcript accumulation 115 4.3.1.4 The effect of N-assimilation inhibitors on NO3" influx and bch transcript accumulation 115 4.3.1.5 The effect of N H / on 13NC>3~ influx and bch transcript accumulation.. . 122 4.4.1.1 Discussion 128 4.4.1.2 The effect of NO3" on the regulation of nitrate influx and the accumulation of bch transcript 130 4.4.1.3 Amino acid regulation of nitrate influx and the accumulation of bch transcript 131 4.4.1.4 The effect of N H / on the regulation of nitrate influx and the accumulation of bch transcript 134 4.4.1.5 Model for the regulation of bch transcript and nitrate uptake 135 CHAPTER V Conclusion and Recommendations for Further Work 138 CHAPTER VI Bibliography 142 List of Tables 1. Efflux analysis of sulfate at 10 and 100 nM 63 2. Effect of exogenously supplied amino acids on organic and inorganic nitrogen levels in plant roots 119 3. Effect of N-assimilation inhibitors on organic and inorganic nitrogen levels in plant roots. 126 4. Effect of ammonium and MSO on bch transcript abundance 127 5. Regulation of IHATS, hypothesis and observations 136 viii List of Figures 1. Sulfate assimilation in plants 8 2. Pathway for N assimilation 25 3. Phylogeny of plant sulfate transporters 48 4. hvstl promoter sequence and alignments 50 5. Effect of external sulfate supply on transcript abundance of hvstl 52 6. Effect of external sulfate supply on sulfate influx 54 7. Effect of external sulfate supply on GSH content in roots 55 8. Effect of removal of external sulfate supply on hvstl transcript abundance in barleyroots. . . .56 9. Effect of removal of external sulfate supply on sulfate influx 57 10. Effect of removal of external sulfate supply on GSH content in roots 58 11. Effect of re-supply of external sulfate to S starved plants on hvstl transcript abundance in barley roots 59 12. Regulation of hvstl transcript abundance as functions of treatments with various S-metabolites 61 13. Effect of N treatment on hvstl transcript abundance and sulfate influx 64 14. Effect of N treatment on sulfate influx 65 15. Alignment of predicted amino acid sequence of bch3 and bch4 with other high affinity nitrate transporters 84 16. Phylogeny of predicted amino acid sequences of inducible high affinity nitrate transporters.85 17. Time course of 1 mM nitrate treatment on bch mRNA accumulation 89 18. Time course of 10 mM nitrate treatment on bch mRNA accumulation 90 19. Time course of nitrate treatment on bch mRNA accumulation 91 ix 20. Time course of nitrate treatment on nitrate influx 92 21. Northern blot analysis, of known members of the bch family of genes 94 22. Effect of external nitrate concentration on bch mRNA accumulation 95 23. Effect of external nitrate concentration on nitrate influx 96 24. Effect of nitrite, and ammonium and nitrate co-supply on bch transcript abundance 97 25. Time course of changes of root nitrate and ammonium concentration 113 26. Effect of 10 mM nitrate pretreatment on amino acid concentration in barley roots 114 27. Effect of exogenously applied amino acids on nitrate influx 117 28. Effect of exogenously applied amino acids bch transcript accumulation in roots 118 29. The enzymatic steps involved in nitrate assimilation, and the respective points of inhibition. . 120 30. The effect of N assimilation inhibitors on nitrate influx 121 31. The effect of N assimilation inhibitors bch transcript accumulation in roots 123 32. Time course effect of tungstate and MSO on bch transcript accumulation 124 33. Time course effect of tungstate and MSO in the presence of Glu on bch transcript accumulation 125 34. Proposed model for regulation of nitrate transporter M A T S and bch expression 137 L i s t o f A b b r e v i a t i o n s A O A : Amino oxy-acetate APS : Adenosine 5'-phosposulfate ATP : Adenosine triphosphate A Z A : Azaserine Asn : Asparagine Asp : Aspartate BSO : Butyl sulfoximine B C H : //vNRT2 Bch : HvNrt2 cDNA : copy DNA CHATS : Constitutive high-affinity transport system Cys : Cysteine DEPC : Diethylpurocarbonate FAD : flavin adenine dinucleotide (oxidized form) GSH : Glutathione (reduced) GSSG : Glutathione (oxidized) GOGAT : Glutamate synthase GS : Glutamine synthetase Glu : Glutamate Gin : Glutamine JHATS : Inducible high-affinity transport system LATS : Low-affinity transport system MSO : Methylsulfoximine mRNA : messenger ribonucleic acid N : Nitrogen NAD : Nicotinamide adenine dinucleotide (oxidized form) N A D H : Nicotinamide adenine dinucleotide (reduced form) NADP : Nicotinamide adenine dinucleotide phosphate (oxidized form) NADPH : Nicotinamide adenine dinucleotide phosphate (reduced form) NiR : Nitrite reductase NR : Nitrate reductase O-AS : O-acetylserine PAPS 3'-phopho 5'-phoshosulfate R A C E : Rapid amplification of cDNA ends RT : Reverse transcription S : Sulfur Ooc : flux outside to cytoplasm Ocv : flux cytoplasm to vacoule Ox : flux to xylem Oco : flux cytoplasm to outside Ovc : flux vacoule to outside Acknowledgments I would like to start this off by thanking my wife Claudine whoses love, caring, support and sacrifice over the last seven years has made this thesis possible. Especially for her constant persistence in pushing me over last two years to get the damn thing finished. My supervisor Dr.Glass, for his mentoring, my committee members, Drs. Louise Glass, Carl Douglas and Doug Kilborn for their support and direction during the thesis. I would like to also mention Drs. Jan Schjoerring, Yaeeshi Siddiqi. Herbert Kronzucker and Bruno Touraine for the many wonderful scientific outings to the Pendulum, where science was always discussed. I would like to thank the many friends I have made at UBC Adlane, Dave, Dev, Diana, Garth, Sven, Mark, Tony, and Richard, and the ones outside Eddy, Maurice, Frasier, Pat, Mercedes and Anamaria for making life in Vancouver an enjoyable and interesting experience. xii CHAPTER 1 INTRODUCTION 1.1.1.1 Ion transport in plants One of the main characteristics of plants lies in their ability to assimilate elements taken from the external environment. Plants provide to animals a source of organic substances, which they are unable to synthesize. Apart from carbon (from atmospheric CO2), hydrogen and oxygen (from ground water), plants absorb the majority of the mineral elements they need to grow and to develop from the soil, in ionic form. These ions are often found in very low concentrations, all the more since absorption depletes resources at the root surface, in spite of the increase of the area of soil exploited by the root system during its growth. To ensure their mineral content, plants have developed absorption systems for ions that are quite efficient (affinity constants often in the uM range; Wyn Jones, 1975; Glass, 1975; Lass and Ullrich-Eberius, 1984; Glass, 1990). Another characteristic of the nutrition source for roots lies in the frequency of fluctuations of ionic concentrations in the soil environment. To deal with these variations, plants have evolved mechanisms that control root absorption. The operation of these processes, the intake of mineral elements by the plant does not depend on their concentrations at the surface of the roots, or on the chemical characteristics of rhizosphere, but rather varies according to the organism's needs to sustain current growth rate and development stage. The existence of regulation systems driven by the nutritional demand of the plant, has been recognized for most of the major ions, including potassium (Glass, 1975; Petterson and Jensen, 1979; Drew and Saker; 1984; De la Guardia et al, 1985; Siddiqi and Glass, 1986, 1987), phosphate (Clarkson and Scattergood, 1982; Drew and Saker, 1984; Lee, 1993), nitrate (Lee and Rudge, 1986; Bowman et al., 1983) and sulfate (Hart and Filner 1969; Smith 1975, 1980; Hawkesford and Belcher, 1991; Clarkson et al., 1983). Therefore, this is a general feature in higher plants. Physiological studies, however, show that a specific regulation of absorption 2 occurs for the specific ions in question. For example, the culture of a plant in an environment deficient of one element stimulates the absorption of the corresponding ion, but not of the other ones (Glass, 1975; Datko and Mudd, 1984; Lee and Rudge, 1986; Clarkson and Saker, 1989; Hawkesford and Belcher, 1991; Lee, 1993; Lappartient and Touraine, 1996). Another major characteristic of this type of regulation is that it operates as an integrated mechanism, where the root absorption is under the control of the needs of the whole plant. If we consider the quantitative importance of the shoots compared to the roots this control should involve some kind of interaction between both organs. The demand-driven regulation of root uptake has been described in its broad lines, but the underlying mechanisms remain largely unknown. The major obstacle in their study is the lack of knowledge about the molecular identity of the systems responsible for ion absorption. Indeed, at the start of this study, only a few genes encoding proteins which play a role in ion transport in roots had been cloned (potassium; Anderson et al., 1992; Sentenac et al., 1992; nitrate: Tsay et al., 1993), and none of the proteins themselves had been isolated. The initial goal of this work was to isolate a high-affinity nitrate transporter from barley (Hordeum vulgare L.). Barley plants, and particularly roots of barley plants have been used as model plants for the study of ion transport since the pioneering studies of Hoagland and Broyer (1936) and Epstein (1956) to present day. This has provided a wealth of information about ion transport in this species. By contrast relatively few studies have been reported on ion transport in Arabidopsis, perhaps because of the tiny root system and the difficulties encountered growing this species hydroponically. For these reasons, the experiments described in this thesis used barley (Hordeum vulgare L. cv Klondike) as the experimental system for parallel molecular and physiological studies. However, I have serendipitously isolated a high-affinity sulfate transporter and then characterized its regulation (Chapter 2). I next isolated two inducible high-affinity 3 nitrate transporters from barley (Chapter 3). Finally, the expression of the genes encoding the nitrate transporter, in response to nutritional treatments and to putative signals that would control their functions, has been characterized (Chapter 4). 1.2.1.1 Importance of sulfur One of the effects of excessive sulfur is a reduction of plant growth and a retardation of flowering. In 1984, it was estimated that excess sulfate costs agriculture industries of the 11 OECD countries 500 million dollars (Rennenberg, 1984). This problem was noticed in the early 1970s, as a consequence of the progressive increase in sulfur levels in the atmosphere in certain regions of the world (industrialized countries), essentially in the form of sulfur dioxide. This gas has two origins: one natural (volcanic activity and oceanic), the other from human activity (emissions of sulfur dioxide by the chemical industry and the combustion of fossil fuels). At the beginning of the 1980s it was estimated that 20% of sulfur released into the atmosphere originated from Western Europe, a territory which makes up only 1% of the earth's surface (Rennenberg, 1984). Also in the European Union, an area of more than a million hectares has potentially been exposed to excess sulfur levels (Rennenberg, 1984). Conversely, sulfur deficiency in soils is also damaging to agriculture. For example, in canola, this results in frail stems, yellowish marbling of leaves, and a decrease in the laminar size. These deficiency symptoms are accompanied by production of anthocyanins. Sulfur starvation causes the canola to be more susceptible to pathogen attack. One of the reasons for this is that there is a decrease in the levels of sulfur containing compounds, such as glucosinolates, which are thought to play a role in the mechanisms of defense. Sulfur deficiencies have also been implicated in a decrease in pollen production, and a decrease in the 4 seed number (Haneklaus and Schnug, 1992). Moreover there is a decrease in the utilization of nitrogen by plants, which in turn decreases protein synthesis. The resulting effect is that plants store nitrogen in the form of nitrate, which diminishes their use as consumable food. This situation has particularly developed in crop soils of Northern Europe. The origin of this problem is the removal of increasing amounts of sulfur by intensive farming while sulfate inputs to the soil diminished because of the modification in chemical composition of N and P fertilizers (Haneklaus and Schnug, 1992). The requirements of sulfur for crops have been to date underestimated. For example although sulfate made up approximately 11% of superphosphate fertilizers, it has been thought of only as a contaminant, not as a required nutrient. Since 1965, the replacement of ammonium sulfate by liquid ammonia and the utilization of triple phosphate with low levels of sulfur (2% of the fertilizer) (Jolivet, 1993) led to decreased sulfur input. Also, the regulation of pollution emissions by industrialized nations resulted in a 30% decrease in sulfur dioxide in the atmosphere. The amount of sulfur dioxide released into the soils due to acid rain has played an important role in the availability of sulfur for agriculture in the recent past, and this has now changed. Thus, in developed countries, intensive agricultural programs have increased the removal of sulfur from the soil whilst this removal has not been accompanied by replacement of sulfur by fertilizer use and uncontrolled input of sulfur has been stopped. Therefore, problems of sulfur starvation are now occurring. 1.2.2.1 M e t a b o l i s m a n d a s s i m i l a t i o n o f s u l f u r 1.2.2.2 R o l e s o f s u l f u r a n d m a i n s u l f u r c o n t a i n i n g c o m p o u n d s The products of assimilation of sulfur play an important role in the growth and metabolism of plants. Seventy percent of the compounds containing organic sulfur are found in proteins, in two amino acid forms: cysteine and methionine (Anderson, 1980). Twenty nine percent are associated with soluble amino acids or peptides, for example glutathione. One-percent or less is found in the form of sulfolipids. Cysteine plays an important role in the structure of proteins, by the formation of di-sulfur bridges, reactions of oxygen reduction, and the fixation of metal groups (e.g. Fe-S groups) and the process of energy transfer (thioesters present in active sites). Plant proteins rich in sulfur must contain high levels of cysteine and methionine, which are localized in leaves and seeds. These proteins are capable of inhibiting the activity of certain enzymes and play a role in the resistance of predators and pathogens (Bohlman, 1993). Also, the major role of these proteins in the fixation of metals allows the plant to protect itself when levels of these metals become excessive. Phytochelatins and metallothioneines, for example, which have a high level of the amino acid cysteine, are able to chelate cadmium and inhibit its irreversible binding to - S H functional groups of enzymes and coenzymes. They thus play a role in detoxification (Marschner, 1986). Cysteine is one of three amino acids present in glutathione, which makes up 90% of the thiols present in soluble non-proteins. This tripeptide (y-glutamyl-cyteinyl-glycine) is implicated in the mechanism of detoxification and in the defense of plants against oxidative and environmental stresses. It is also the major storage and long distance transport form of sulfur in the plants (Bagmen and Rennenberg, 1993; Stulen and De Kok, 1993). The sulfur containing amino acids play an important role in the nutrition of animal species, which are incapable of synthesizing sulfur containing amino acids. Sulfur is also present in sulfolipids, which are the greatest in abundance in lipids in the biosphere, and may play a regulatory role in membrane transport (Heinz, 1993). Sulfur also is important in one of the two electron chain transport systems, in binding iron in the heme molecule. Sulfur is also found in reduced organic form (glucosinolates) or in the oxidized form, sulfate, which contrary to nitrate can be incorporated into organic structures. 1.2.2.3 Sulfur assimilation The main functioning aspects of sulfur assimilation in plants have been proposed by analogy with the mechanism known in microorganisms; The pathway have been elucidated by biochemical studies in plants. The results of the latter were sometimes contradictory because of the instability of certain enzymes and of the properties of thiols (cf. Leustek, 1996). The availability of mutant microorganisms, missing in one or in the other enzymes of the pathway, recently enabled the isolation of plant genes encoding these different proteins using heterologous complementation of mutants. Hence, a study of the regulation of the different enzymes involved in S assimilation in plants has become possible. 9 . The assimilation path from the ion SO4 to the synthesis of cysteine (Figure 1) can be divided into four steps: (1) the absorption of sulfate by root cells; (2) the activation of sulfate; (3) the reduction of "activated" sulfate to sulfide; (4) the incorporation of sulfide into cysteine. It appears that steps (2) and (3) may follow two distinct reaction pathways, involving different metabolic intermediates (Leustek, 1996). One of the paths is identical to the one known in bacteria, whereas the other one would be typical of the assimilation of sulfate in all photosynthetic eukaryotes and some photosynthetic prokaryotes (Brunold, 1990; Schiff et al., 1993). The debate on the role and importance of one or the other path in plants is still open (Leustek, 1996). The assimilation of sulfate is an expensive process in energy terms. The energy is provided by ATP and reductants (ferredoxin, thioredoxin) produced by photosynthesis. The 7 Absorpt ion sulfate (ext) (D Activation sulfate (int) (2) ATP PPi APS (3) GS H AMP (5) ATP ADP PAPS (6) ^ — Thi ored Thioox + PAP Reduction thiosulfonate 6Fdred 6Fdox (4) ^ — c sulfite (7) ^— 6F dred 6Fdox Incorporation thiosulfur (9) C sulfur O-acetyl-serine (8) acetate CoA serine acetylCoA cysteine Figure 1: Sulfate assimilation in plants. It is divided in four steps: absorption, activation, reduction and incorporation. Two alternative pathways are proposed for the reduction step. However, the pathway on left is preferred. However, there are results supporting the right pathway (similar that observed in bacteria) which may exist in plants. The enzymes or proteins involved are: (1) sulfate transporter, (2) ATP sulfurylase, (3) APS sulfotransferase, (4) thiosulfonate reductase, (5) APS kinase, (6) PAPS sulfotransferase, (7) sulfite reductase, (8) serine acetyltransferase, (9) 0-acetylserine(thiol)lyase. Taken from Leustek (1996). assimilation of sulfate occurs essentially in the leaves, at the plastid level. However, some enzymes are not only present in the plastid, but also in the cytoplasm, particularly in the roots. Therefore, these organs have the potential machinery for the assimilation of sulfate. Consequently, excised roots are able to grow by receiving only sulfate as a sole source of sulfur (Leustek, 1996). Respiration would then provide the energy source needed for the functioning of the pathway. According to Leustek (1996), this root assimilation of sulfate could play a key role under conditions of high demand for sulfur. In plants, the APS synthesized by ATP sulfurylase could be used as a substrate for APS sulfotransferase and then transformed, with glutathione, hence creating the thiosulfonate. It could also be activated by phosphorylation as for micro-organisms such as E. coli (Kredich, 1987) or S. cervisiae (Cherest and Surdin-Kerjan, 1992), to produce the adenosine 3'-phospho 5'-phosphosulfate (PAPS) during a reaction catalyzed by the APS kinase. The PAPS can be used following the assimilation path of sulfate or take part in the formation of sulfate esters and sulfolipids (Marschner, 1986; Anderson, 1980). The role of the APS kinase in plant tissues is unknown so far since this protein, which is in low abundance, has not been purified and no mutant as been characterized. However, cDNA clones encoding this protein (Arz et al., 1994; Jain and Leustek, 1994) have been isolated recently, and it is now possible to determine its function in higher plants. Recent results have demonstrated that the same enzyme has both APS kinase and APS sulfotransferase activity. The APS sulfotransferase activity appears when alterations of the tetrameric structure of APS kinase occur during interactions with glutathione (Schiffmann and Schwenn, 1994). The APS sulfotransferase is regulated positively by sulfate deficiency and by environmental conditions, which increase the need for reduced sulfur in the plant. In contrast, its activity is inhibited in planta by external application of cysteine (Brunold 9 and Schmidt, 1978; Wyss and Brunold, 1979). This regulation does not seem to be allosteric since cysteine has no in vitro effect on the activity of the protein (Schmidt, 1975). Therefore the APS kinase could also be regulated by the nutritional status of the plant. Results of Chen and Leustek (cf Leustek, 1996) support this hypothesis. These authors have shown that a deficiency of sulfate increases the accumulation of mRNA of the APS kinase. The activities of PAPS sulfotransferase, sulfite reductase and thiosulfonate reductase were measured in the plant, and a cDNA of sulfite reductase was recently cloned (Leustek, 1996). Hence both reduction paths of sulfate appear to co-exist in plants. During the last step of the assimilation path, cysteine synthase catalyses the synthesis of cysteine from sulfide, serine and acetyl Coenzyme A. The cysteine synthase is an enzymatic complex which associates the enzymes O-acetylserine(thiol)lyase and serine acetyltransferase (Ruffet et al., 1994). The presence of this complex was demonstrated in chloroplasts, in the cytosol and the mitochondria (Bumold and Sutter, 1989; Lunn et al., 1990; Leustek, 1996). The cDNA encoding these two enzymes were cloned in spinach (Saito et al, 1992, 1993, 1994; Hell et al., 1994; Bogdanova et al., 1995; Murillo et al., 1995). The expression of acetyl serine transferase does not seem to be modulated by sulfate deficiency (Bogdanova et al., 1995; Murillo et al., 1995). In tobacco, over-expression of the second enzyme of the cysteine synthase complex, i.e 0-acetylserine(thiol)lyase, does not increase the cysteine level. In contrast, the incubation of isolated chloroplasts of plants that do not overexpress the 0-acetylserine(thiol)lyase, with only O-acetylserine or with O-acetylserine and sulfate, enables an increased synthesis of cysteine; this phenomenon disappears when sulfate is added alone. Therefore the acetyl serine transferase and not the (9-acetylserine(thiol)lyase would be the limiting enzyme of the cysteine synthase complex (Saito et al., 1994). The assimilation path of sulfate seems to be regulated at various levels. The first two steps, the absorption of sulfate and its activation catalyzed by ATP sulfurylase, are similarly regulated 10 by sulfate deficiency. The study of the regulation of the assimilation path of sulfate by the nutritional status of the leaves could hence occur by a study of these first two steps. 1.2.3.1 Absorption and activation of sulfate 1.2.3.2 Absorption of sulfate The absorption of sulfate is a net flux, a balance of two unidirectional fluxes through the plasma membrane of root cells, namely influx and efflux. In Lemna, Thoiron et al. (1981) calculated by compartmental analysis that the sulfate which penetrates into the cell, is partitioned as follows: 1/3 is reduced, 1/3 is stored in the vacuole and 1/3 leaves the cell by efflux across the plasma membrane. To measure the influx of sulfate, the cells or the roots must be allowed to incorporate radioactive sulfate for only a few minutes because longer exposures would result in a determination of net flux due to the increasing contribution of efflux. This labeling phase must be followed by short washes in order to remove tracer from extracellular spaces. This conclusion is reinforced by the results of Bell et al. (1994) who determined that the half-lives of sulfate in the cytoplasm and the apoplasm were respectively, 16 and 3 minutes. Efflux was also measured for carrot slices (Cram, 1983) and for Macroptilium atropurpureum (Bell et al., 1995). Low values for efflux compared to influx were reported in Lemna paucicostata (Datko and Mudd, 1984a). Analysis of the curves where the influx of sulfate is plotted against external concentration of sulfate shows two phases. One of them, which is non-saturable, varies linearly 2-with the external concentration of sulfate and becomes predominant only for SO4 concentrations in excess of 10 mM (Datko and Mudd, 1984a). The other, saturable component 11 shows high affinity for sulfate and has a K m range inferior to 100 uM for the different species analyzed (Lass and Ullrich-Eberius, 1984; Deane-Drummond, 1987; Hawkesford et al., 1993). Sulfate transport to the cytoplasm occurs against both an electrical gradient and a concentration gradient (Bell et al., 1994). Energy is therefore necessary to allow transport. The use of inhibitors of metabolism such as 2,4-dinitrophenol (Jensen and Konig, 1982) causes an inhibition of sulfate uptake. By contrast, fusicoccin (an activator of the plasma membrane type ATPase) stimulates this absorption (Lass and Ullrich-Eberius, 1984). These results are consistent with the hypothesis of the energy coupling of sulfate transport with the H+-ATPase. Sulfate uptake is generally accompanied by a depolarization of the membranes, showing that a net transport of positive "charges" across the plasma membrane has occurred, thus Lass and Ulrich-Eberius (1984) proposed the hypothesis of a 3H +: ISO42" "symport". The environment affects the active transport of sulfate. The absorption of sulfate is thought to be derepressed, when a sulfur deficiency occurs, whether it be in cultures of tobacco cells (Hart and Filner, 1969; Smith 1980; Harrington and Smith, 1980), isolated roots of tomato (Hawkesford and Belcher, 1991) or roots from Macroptilium atropurpureum (Clarkson et al., 1983). This regulation seems to originate from leaves, and but not from roots nourished by a nutritive solution with no sulfate. In fact, the deficiency of one part of the root system creates an increase in the absorption of sulfate in the other part of the root system, even if the latter receives a normal nourishment (Clarkson et al., 1983). Considering the vascular links between the organs in plants, this type of regulation by nutritional demand necessarily implies the circulation of a signal(s) translocated from leaves to roots via the phloem sap (Touraine et al., 1994; Imsande and Touraine, 1994; Lappartient et al., 1999). Several molecules linked with the nutritional status of the plant could play this role of regulator for the active transport of sulfate. Sulfate is thought to regulate its own uptake as 12 reported by several authors (Smith, 1975, 1980; Jensen and Konog, 1982; Datko and Mudd, 1984a; Lass and Ulrich-Eberius, 1984; Bell et al., 1995). This is based on the negative correlation between the absorption of sulfate and the concentration of sulfate in the roots. 2- 2-Products of SO4 assimilation have also been shown to have an effect on SO4 uptake. Examples of this include L-cysteine or L-methionine, L-homocysteine, thiosulfate, sulfite, and glutathione (Hart and Filner 1969; Brunold and Schmidt, 1978, Maggioni and Renosto, 1977; Clarkson et al., 1983; Smith, 1975, 1980; Harrington and Smith, 1980; Herschbach and Rennenberg, 1991, 1994; Rennenberg et al, 1988, 1989). However, the plants can quickly oxidize cysteine to form sulfate. Therefore it was (Smith, 1975; Harrington and Smith, 1980) proposed that the sulfate produced by this reaction is responsible for the inhibitory effect of cysteine on the absorption of sulfate. The inhibiting action of glutathione could then be explained by the release of cysteine during the degradation of this tripeptide. However, Herschbach and Rennenberg (1991) have shown that cysteine is unable to inhibit sulfate uptake in the presence of BSO, an inhibitor of the synthesis of glutathione, which contradicts the previous hypothesis. Conversely, the effect of cysteine on sulfate transport is likely exerted via the synthesis of glutathione. Hart and Filner (1969) gave four hypotheses to explain the regulation of sulfate transport by the various sulfur assimilation intermediates: (1) simply, the effect of the mass action law, chemical equilibria being changed one after the other due to the supply of one intermediary of sulfur metabolism; (2) the occurrence of competition for the sites of sulfate transport; (3) a direct allosteric regulation; (4) an indirect regulating effect due to the inhibition of the synthesis of the transporter. In tobacco cells, the absorption of sulfate is decreased by glutathione (GSH), and the transfer of these cells into a medium lacking GSH restores the absorption of sulfate to the same 13 level as that of the control (Rennenberg et al., 1989). This restoration of sulfate uptake does not occur in the presence of cycloheximide or puromycin (inhibitors of the protein synthesis). The repressor effect of GSH on the absorption of sulfate would therefore be due to the inhibition of the synthesis of the sulfate transporter. Finally, the results of Hawkesford and Belcher (1991) showed an increase in the abundance of a 36kDa polypeptide in the plasma membrane in response to S starvation, indicating the occurrence of a regulation at a transcriptional or translational level. 1.2.4.1 Sulfate transport systems 1.2.4.2 Sulfate transport in prokaryotes For the enterobacteria such as E.coli and Salmonella typhimurium, as well as for the cyanobacteria Synechoccus PCC7942, the sulfate transport system is made of five polypeptides (Hryniewicz et al., 1990; Sirko et al., 1990; Laudenbach and Grossman, 1991; Sirko et al., 1995). The genes cysT, cysWand cysA, encoding three of the proteins, which are associated with the plasma membrane. The protein CYSA has a binding site for ATP, and the proteins CYST and CYSW would associate to enable the transport of sulfate through the plasm membrane (Laudenbach and Grossman, 1991). The other polypeptides, encoded by the genes sbp and cysP, are periplasmic proteins, involved respectively in the binding of sulfate and thiosulfate. The ion SO4 is fixed on the SBP (sulfate binding protein) by the formation of seven hydrogen bonds, by the association of three alpha helices, which extend out of the tertiary structure of the protein, at a ratio of one ion per protein (Pflugrath and Quiocho, 1985). The hydrolysis of one ATP molecule allows the movement of sulfate into the cytoplasm (Ritchie, 1996). 14 1.2.4.3 S u l f a t e p e r m e a s e s i n f u n g i In yeast, the transport of sulfate is mediated by at least two types of sulfate permeases, one responsible for high-affinity and another for low-affinity transport (Breton and Surdin-Kerjan, 1997). The transport of sulfate across the plasma membrane requires the co-transport of 3H + and the exit of 1K + (Roomans et al., 1979). Two genes encoding high-affinity transporters, sull and sul2, have been cloned and characterized (Smith et al., 1995a; Cherest et al., 1997). At present no low-affinity transporter has been cloned. In the filamentous fungus Neurospora crassa, sulfate transport is mediated by sulfate permease I and II, respectively, low- and high-affinity for sulfate (Marzluf, 1970; Hillenga et al., 1996). Sulfate permease II is predominantly expressed in mycelium, whereas sulfate permease I is expressed in conidia. These two systems function almost identically to those in yeast. 1.2.4.4. S u l f a t e t r a n s p o r t e r s i n p l a n t s In canola and in the unicellular alga Chlamydomonas reinhardtii, the transport of sulfate through the plasma membrane is accomplished by co-transport with H + , which uses the gradient of protons generated by the H+/ATPase (Hawkesford et al., 1993; Yildiz et al., 1994). Sulfate is accumulated predominately in the vacuole of plant cells when it is not reduced. It is transported through the tonoplast against a gradient of protons and via an active transport system whose 15 energy comes from the tonoplastic H+/ATPase (Martinoia et al., 1986; Mornet et al., 1997). When plants are removed from a S-sufficient to a S-deprived environment, sulfate is re-mobilized from the vacuole to the cytpoplasm by passive transport (Cram, 1983), which causes a decrease in sulfate concentration of the plant tissue. A similar distribution of sulfate would also exist in filamentous mushrooms (Hunter and Segel, 1985). Three cDNA's encoding transporters of sulfate have been isolated in the tropical plant Stylosanthes hamata (Smith et al., 1995b). Two among them, shstl and shst2 encode proton-dependent high-affinity sulfate transporters, and are expressed in the roots. The third cDNA isolated, shst3, codes a transporter with low affinity mainly expressed in the leaves. The transporters SHST1 and SHST2 could enable the acquisition of sulfate present in the soil whereas SHST3 would be involved in the transport of sulfate between the different cellular and subscellular compartments of the plant. cDNA's encoding transporters homologous to those of the group SHST have been isolated in H. vulgare (Smith et al., 1997) Sporolobus stapfianus (Ng et al., 1996) and Arabidopsis thaiiana (Takahashi et al., 1996; 1998; Yamaguchi et al., 1997). The gene of the noduline gmakl70 that is expressed during the formation of the symbiotic nodules of soybean encodes a sulfate transporter responsible of the transport of sulfate into the nodule (Sandal and Marcker, 1994). Plant cells are made of four major compartments in which sulfate can be transported: the cytoplasm, the vacuole, the mitochondria and the chloroplasts. No transport system for sulfate has so far been identified in the mitochondria, the chloroplasts or tonoplast. However, in the chloroplastic genome of Marchantia polymorpha, there are two genes, mbpX and mbpY, coding proteins strongly homologous to the proteins encoded by cys A and cysT, respectively, of bacteria (Laundenbach and Grossman, 1991). These two genes had also been identified in the chloroplastic genome of the unicellular alga Chlorella vulgaris (Wakazugi et al., 1997). No gene 16 coding proteins homologous to that of procaryote type sulfate transporters has been identified in the chloroplastic genome of higher plants. It has been proposed that chloroplasts arose from an endosymbiosis between a cyanobacterium and a primary eukaryote (Raven et al., 1986), and the transporter of chloroplastic sulfate could therefore have a cyanobacterial origin. Most of the genes necessary for the expression of this transporter would have been transferred into the nuclear genome during evolution, particularly in higher plants. 1.2.4.5 S u l f a t e t r a n s p o r t e r s : N a + a n d H + d e p e n d e n t i n a n i m a l s In animals, sulfate transport is mediated by at least two different types of permeases. In the rat, a Na+/SC>42"co-transporter is expressed in the cortical cells of the kidney (gene nasi) (Busch et al., 1994; Markowitch et al., 1994). It is located in the membrane of the brushed edge of these cells, and enables the co-transport of a sulfate ion, selenate or thiosulfate, with three sodium ions. Genes coding sulfate transporters strongly homologous to the H+/S04 2" co-transporters of plants and mushrooms have been isolated in animals. In the rat, the gene satl codes an active sulfate transporter in the basolateral membrane of heptocytes (Bissig et al., 1994). A sulfate transporter homologous to the transporter satl is also expressed in the kidney cells of the rat, is located in the basolateral membrane of the cortical cells of this organ (Bissig et al, 1994; Markowitch et al., 1994). In humans, mutation of the gene DTD, which codes a sulfate transporter, affects sulfation of the polysaccharides of the matrix of cartilage, leading to serious congenital malformations of the skeleton (Hastbacka et al., 1994). In the mouse, the gene dra codes for a sulfate transporter expressed specifically in the intestine, whose expression is repressed in tumor cells (Silberg et al., 1995). 17 The H+/S04 2" sulfate transporters have an identical structure in all the eucaryotes (Sandal and Marcker, 1994). They have 12 potential membrane spanning domains and numerous basic amino acids distributed on both sides of the membrane. The ultra-conserved sequence "YGLY" at the beginning of the second transmembrane domain is characteristic of these transporters. The Na+/SC»42~ transporter of the kidney of the rat also has 12 potential transmembrane domains but does not have any similar sequence with the Na+/S042" transporters, which indicates that these two transport systems have no common origin in animals. 1.2.5.1 Glutathione 1.2.5.2 Synthesis and localization Glutathione (glutamine-cysteine-glycine) is a thiol with low molecular mass that is most abundant in animals and plants. It can be replaced by homoglutathione (glutamine-cysteine-alanine) in the Fabaceae (Price, 1957) or by hydroxymethylglutathione (glutamine-cysteine-serine) in the Poaceae (Klapheck et al., 1992). Microorganisms only accumulate glutathione at low concentrations despite high contents in thiols (Rennenberg and Lamoureux, 1990). Two steps are involved in the synthesis of this tripeptide. The y-glutamylcysteine synthetase catalyses the synthesis of the y-glutamylcysteine from cysteine and glutamic acid. Then, the glutathione synthesase fixes a glycine in C-terminal position on this dipeptide. Available data on the regulation of internal content of glutathione are scarce. However, the activity of y-glutamylcysteine synthetase is inhibited by glutathione (Rennenberg and Lamoureux, 1990). Glutathione synthesis occurs essentially in the leaves; it is then exported towards the roots in which it takes part in protein synthesis (Rennenberg et al., 1979). Vast 18 quantities of glutathione have also been shown in seeds. However, contrary to what has been observed in leaves and roots, most of the glutathione present in seeds is in the oxidized form, GSSG (Rennenberg and Lamoureux, 1990; Lappartient and Touraine, 1997). In cells, glutathione seems to be equally distributed between the cytosol and the chloroplasts (Smith, 1985; Klapheck et al, 1988), but the presence of a significant part of glutathione (17%) has also been observed in vacuoles. Cells can take up glutathione from an external solution. An active transport of glutathione has been identified in tobacco cells (Schneider et al., 1992). Metabolic inhibitors reduce this transport. In the concentration corresponding to the high-affinity transport systems, the transport of glutathione is reduced in the presence of L-cysteine; sulfate ions have a similar effect in the concentration range of the low-affinity transport system. This competition with L-cysteine and sulfate has only been identified in heterotrophic cell cultures. Finally, GSH transport is pH-dependent, with the highest uptake rates at pH values ranging between 5.5 and 6.5. 1.2.5.3 Role of glutathione in plants In its reduced form, glutathione (GSH) is a storage form of reduced sulfur (Rennenberg, 1982) as well as the main form of sulfur transport within the plant (Bonas et al., 1982). The increase in the content of glutathione in spinach leaves (De Kok et al., 1986), or in homoglutathione in bean and clover leaves (Buwalda et al., 1993), when plants are exposed to H2S, illustrates the reduced S storage function of glutathione and homologous compounds. In order to follow the distribution of the organic S compounds in tobacco, Rennenberg et al. 35 2-(1979), have supplied roots or leaves with SO4 . The organic sulfur compounds were then extracted from parts of the stem and separated by thin layer chromatography. In both cases of 19 feeding, the distribution of S in the organic sulfur compounds ranged between 67-70% in glutathione, 27 and 30% in methionine, and 3 and 8% in cysteine. Therefore, the authors concluded that GSH is the major type of reduced S transported in the phloem sap. 1.2.5.4 Regulation of the uptake of sulfate by glutathione As presented above, the intake of sulfate is controlled by the concentration of S compounds within the plant. Among these molecules, glutathione is seemingly the best candidate for the role of regulator of sulfate absorption. The work by Rennenberg and collaborators has shown that external application of glutathione resulted in an inhibition of sulfate transport in tobacco cells (Rennenberg et al., 1988), excised roots (Herschbach and Rennenberg, 1991) and intact roots of tobacco (Herschbach and Rennenberg, 1994). Lappartient and Touraine (1996) have shown that glutathione, which was directly supplied to roots with the nutrient solution, down-regulated sulfate influx and ATPsulfurylase in roots. Glutathione also inhibits the secretion of sulfate into the xylem sap in excised roots of tobacco (Herschbach and Rennenberg, 1991). In contrast, no effect of this compound on sulfate transport to the leaves was detected in intact tobacco plants. Cysteine also inhibits the absorption of sulfate and its movement in the xylem. However, this effect disappears in the presence of BSO, an inhibitor of the enzyme y-glutamylcysteine synthetase, indicating that the cysteine effect was mediated by glutathione (Herschbach and Rennenberg, 1991, 1994; Lapportient and Touraine, 1996). Because the time needed for complete inhibition of sulfate transport by GSH in a tobacco cell culture was about 14 h (Rennenberg et al., 1988), GSH might not have a direct action on the transporter. Rennenberg et al. (1988) are in favor of the hypothesis that GSH causes an inhibition of de novo synthesis of 20 the sulfate transporter. The inhibition of sulfate absorption by GSH is reversible. However, cycloheximide inhibits this reversion (Rennenberg et al., 1989), supporting the protein synthesis hypothesis. Glutathione, produced by the assimilation of sulfate, thus seemingly plays a key role in the regulation of plant sulfur nutrition by acting on the first step of the pathway. The assumed mechanism for this regulation is as follows. When the plant absorbs sulfate in excess, higher rates of sulfate enter the leaves, where a surplus of organic S-compounds are synthesized, accumulated mainly as GSH. This compound is then transported in the phloem to the roots, where it inhibits sulfate uptake through effects on the synthesis of sulfate transporter. However, the concentrations of glutathione and cysteine in tobacco leaves and roots did not increase during a 1-hour exposure to 0.1 or 1 mM GSH (Herschbach and Rennenberg, 1994). To reconcile the previous hypothesis with this observation, it can be imagined that the regulation of sulfate transport results from changes of small metabolic pools of glutathione and not by the entire stored glutathione. Furthermore, there is nothing to demonstrate that this thiol is the primary regulator of SO42" transport. Rather, GSH might be one of the links of a complex transduction chain. The oxidized form of gluthathione (GSSG), y-glutamylcysteine and ophthalmic acid mimic the inhibiting effect of the GSH on the absorption of sulfate (Gunz et al., 1992). The latter is a glutathione analog without the thiol group, which therefore does not seem to be necessary to obtain the inhibiting effect of glutathione. Amino acids such as alanine or aspartate or else glutamate also inhibit the absorption of sulfate, suggesting the occurrence of cross-regulation between the assimilation pathway of sulfate and nitrate (Gunz et al., 1992). 1.3.1.1 Importance of nitrogen 21 Nitrogen is the fourth most abundant element in living organisms after carbon, oxygen, and hydrogen and this element represents about 2-6% of plant dry matter. It is the principal factor limiting plant growth (Olson and Kurtz, 1982). It is an important element in that it is present in amino acids, proteins, a number of heterocyclic compounds (purines and pyrimidines), coenzymes, plant pigment (chlorophyll), and a number of secondary metabolites. Nitrogen thus plays an essential role in living organisms. With the advent of industrialization at the turn of the century, and the exodus of population from rural to urban areas, coupled to population growth, great pressure was imposed upon agriculture to keep up with the growing demand for food and other products. To meet with this demand, farmers intensified cultivation and agricultural practice. The current high crop yield is now dependent on the large scale use of fertilizers, the prevention of crop loses due to insects and plant pathogens, and the use of cultivars which partition greater amounts of plant biomass to the harvestable matter. For example in 1989, in the U.S. alone 3 billion dollars were spent on fertilizer. The nitrogen required for plant growth, is withdrawn by roots from the environment in the form of NO3", NH4+, amino acids. The assimilation of inorganic and organic nitrogen can be divided into three categories nitrate, ammonium, and amino acids. In most agricultural soils nitrate is the most abundant form of nitrogen, although in acidic soils ammonium predominates because nitrification is largely inhibited. The assimilation of amino acids seems to play a minor role in agricultural soils. 1.3.2.1 Nitrate assimilation Nitrate assimilation is the process by which inorganic nitrogen (in the form of NO3 ) is 22 reduced and incorporated into amino acids. This pathway involves a number of steps. The first one is the transport of NO3" from the external environment into the plant cell; membrane transporters mediate this process. After being absorbed, nitrate ions can be assimilated within the roots, transported to the shoots, or stored in the vacuole of root cells (Sivasankar and Oaks, 1996). The transport of nitrogen from the roots to the shoots occurs essentially in the xylem vessels. The relative proportion of nitrate and organic nitrogen that is transported to the shoots depends on the extent of nitrate reduction in the roots vs. reduction in the shoots. This varies from one species to another (Andrews, 1986; Pate, 1972), and also according to the concentration of available NO3 in the external medium. The enzymatic steps from NO3" to amino acids are shown in Figure 2. 1.3.2.2 Nitrate reductase Once nitrate has entered the cell it is reduced by nitrate reductase to nitrite. Nitrate reductase (EC 1.6.6.1 and EC1.6.6.2) is substrate inducible, has a high turnover rate, and is present in the cytoplasm (Oaks et al, 1979). There are two different isoforms, the NADH-dependent NR, which uses N A D H as a source of electrons, and the bispecific NAD(P)H-dependent NR that can use either N A D H or NADPH as sources of reducing power. Both have been shown to be functional in the root and shoot, and depending on the age and growth conditions of the plant (Guerrero et al., 1981). The NR enzyme in higher plants has been shown to be a homodimer of 105-kDa to 120-kDa subunits. The subunit consists of three prosthetic groups, namely flavin, heme and a molybdenum-pterin cofactor (Campbell and Smarelli, 1986). The NR expression is controlled by many different factors, both internal and external, including 23 Caboche and Rouze, 1990). The isolation of mutants has been important for understanding the role of nitrate reductase. Using chlorate as a toxic analog of nitrate, Oostinder-Braaksna and Feenstra (1973) isolated 10 chlorate resistant Arabidopsis plants. These mutations were organized into seven different loci. Chl2 and Chl3 have been demonstrated to be the location of NR genes. At present, two different cDNAs have been cloned in Arabidopsis nial and nia2, which encode two nitrate reductase enzymes (Cheng et al., 1988; Crawford et al , 1988). Both proteins encode NADH-specific NR, but no NAD(P)H-bispecific NR has been found in Arabidopsis. Nia2 gene has been mapped to Chl3 locus (Wilkinson and Crawford, 1991). In barley, two loci, which contain the NR genes narl and narl have been isolated (Warner and Keinhoff, 1986). These loci were found by analyzing the NO3 content of shoot tissue of barley seedlings. The narl gene encodes a N A D H specific NR. This gene is expressed in both shoot and root tissues. In the Narl barley mutant, there is a 90% decrease in NR activity, resulting in higher levels of NO3" accumulation. Nar7 gene encodes the NAD(P)H-bispecific NR enzyme, which is expressed in roots of wild type barley plants, but not in shoots. In Narl mutant narl is expressed both in roots and shoots. 1.3.2.3 Nitrite Reductase Nitrite reductase (NiR) (E.C.I.7.7.1) mediates the reduction of nitrite to ammonium (Beevers and Hageman, 1980). This enzyme has been localized in plastids, but the gene encoding NiR is localized in the nuclear genome. NiR is synthesized with a transit peptide designated for the chloroplast (Sander et al., 1995; Wray et al., 1993). NiR was localized in chloroplasts of leaf tissue and in proplastids of roots (Kleinhofs and Warner, 1990; Miflin, 24 NO, NO, N A D H , H N A D + ) Nitrate Reductase T NO, Fd Nitrite Reductase A T P . NHS Glutamine synthetase GOGAT -A , Glutamate Glutamate Amino-acyl transferases Amino Acids gure 2. The pathway for Nitrogen assimilation 1974). NiR is a metalloprotein with a molecular mass of 60-64 kDa, that contains two prosthetic groups (siroheme) which interact with NO2", and a 4Fe/4S core, which is probably used as the primary electron acceptor. The affinity of NiR is high ( K m of 230 uM) which decreases the chance of accumulation of this toxic ion in cells. initially nitrite reducyase was thought not to function in roots due to the fact that its source of electrons is reduced ferredoxin (not present in roots). Ferredoxin-like proteins, however, have been isolated from proplastids of roots (Suzuki et al., 1984). A ferredoxin-like protein is coupled with pyrimidine nucleotide reductase in root tissue, which can transfer electrons from N A D H or NADPH to root "ferredoxin" (Suzuki et al. 1984). The first isolation of genes encoding NiR was from spinach and com (Bach et al, 1988; Lahners et al , 1988), and the isolation of a partial cDNA clone in barley (Ward et al., 1993). At present in higher plants only one mutant of NiR (Nirl) has been isolated by screening azide mutagenized seeds of barley (Duncanson et al., 1993 ; Ward et al., 1995). The use of the Nirl mutant and plants transformed with sense and antisense NiR has helped in the understanding of the function of NiR (Vaucheret et al., 1992) 1.3.2.4 G S / G O G A T Cycle In 1974, the discovery of glutamate synthase (GOGAT) in higher plants (Lea and Miflin, 1974) underlined the major role of the enzymes glutamine synthetase (GS) and GOGAT in the assimilation of ammonium. It is estimated that 95% of the assimilation of NH4 + is performed by the GS/GOGAT cycle (Rhodes et al., 1989). GS is responsible for the incorporation of ammonium into glutamate to form glutamine. The amide group of glutamine is transferred by GOGAT to a-ketoglutarate. The result of both reactions is the formation of two molecules of glutamate, where one is used in recycling for the formation of glutamine (Figure 2). 26 1.3.2.5 Glutamine Synthetase (GS) Glutamine synthetase plays a central role in nitrogen metabolism since it catalyzes the transfer of inorganic nitrogen into an organic form. Plant glutamine synthetase (GS) has a molecular weight of 320-380 kDa. GS is made up of eight subunits in the form of two tetramers. GS isoforms can be divided into two types: GS1 present in the cytosol and GS2 present in the plastid. A comparative analysis demonstrates the differences between these two enzymes in thermal stability, pH optimum and affinities for glutamate and ATP (Hirel et al., 1993). The optimum pH of GS1 is generally around 7-7.5, whereas for GS2 a value of 8 is usually obtained (Hirel and Gadal, 1980; McNally and Hirel, 1983). The K m of GS for ammonium is in the 10-20 pM range, denoting a high affinity of the enzyme for its substrate. On the other hand, the K m of GS2 for the glutamate is 2-13 mM, which is 10 times higher than that ofGSl . It has been established for several species, that GS is encoded by a multi-gene family and that the heterogeneity of isoforms has a genetic origin. Only one gene encoding for the GS1 from alfalfa was completely sequenced. The translated portion of the gene is 3820 bp in length. The gene contains 12 exons and 11 introns rich in A T nucleotides whose size varies from 90 to 715 bp. The majority of plants presently studied, contain only one gene per haploid genome coding for the plastidial GS and several genes coding for the cytosolic GS. The genes that encode for the cytosolic form account for at least three loci in Arabidopsis thaiiana (Peterman and Goodman, 1991), pea (Tingey et al., 1988; Tingey et al., 1987), bean (Cullimore et al, 1984; Gebhardt et al., 1986 Lightfoot et al., 1988) and soybean (Miao et al, 1991; Roche et al, 1993) and five for corn (Li et al., 1993). The exception to the rule is Sinapis alba which contains a 27 single gene for GS, corresponding to the plastidial form (Hopfher et al., 1991). In tobacco, an amphidiploid species, there are two genes coding for the plastidial GS (of which only one is identified at present) (Becker et al., 1992) and at least two genes coding for the cytosolic GS (Dubois etal., 1996). Comparisons between the different nucleotidic and amino acid sequences show the existence of a high homology between the different groups of GS genes and proteins (Forde and Cullimore, 1989). For instance, in bean, at the nucleotide level, the sequence homology reaches 85% for the coding regions between the three cytosolic GS genes. However, there is a greater divergence between the chloroplastic and the cytosolic GS, with an homology of 70 % (Forde and Cullimore, 1989). At the protein level, the different polypeptides representing the GS enzymes in higher plants have a similarity of 54% with the GS of mammals (Hayward et al., 1986; Tisher et al., 1986), whereas with most of the GS enzymes of procaryote origin, it only reaches 15 to 25% compared to plants (Sanangelantoni et al., 1990). However, for bacterial GS (GSH) of Bradyrhizobium japonicum, the homology with the plant enzymes reaches 47%. This result implies that GS from plants and bacteria evolved from a common ancestral gene, before the divergence of the prokaryotes and the eucaryotes (Carlson et al., 1987; Forde and Cullimore, 1989; Kumada et al., Tateno, 1994). 1.3.2.6 Glutamate Synthase (GOGAT) GOGAT catalyses the second reaction involved in the assimilation of ammonium that leads to the production of two glutamate molecules. In higher plants, two types of activities were identified, one is dependent on ferredoxin (Fd-GOGAT: E.C. 1.4.7.1) and the other on pyridine nucleotides (NADH-GOGAT: E.C. 1.4.1.14 and NADPH-GOGAT: E.C. 1.4.1.13). 28 Glutamate synthase was identified for the first time in the bacterium Aerobacter aerogenes (Tempest et al., 1970). Then the NADH- dependent glutamate synthase was identified in bacteria (including cyanobacteria), fungi (S. cerevisiae and Neurospora crassa), green algae and higher plants. Ferridoxin-dependent glutamate synthase was identified in cyanobacteria, red and green algae, and higher plants. The two GOGAT enzyme activities correspond to distinct proteins, with different molecular masses and different charges without common antigen determinants (Susuki and Gadal, 1984). It is unknown whether in higher plants the N A D H and NADPH-dependent activities reflect the existence of two proteins or of a single NADH-dependent protein that uses dephosphorylated NADPH. The Fd-GOGAT is a flavo protein (FMN) with Fe/S monomer with a molecular weight ranging from 130 to 180 kDa. The enzyme has a pH optimum of 6.9-7.5 and apparent K m values, for ferredoxin, glutamine and a-ketoglutarate ranging between 2 and 6 uM, 100 and 1000 uM, and 7 and 70 uM respectively (Lea et al., 1990a). The high affinity of the Fd-GOGAT for ferredoxin has allowed the purification of the enzyme by chromatography on a ferredoxin-Sepharose column (Suzuki and Gadal, 1984). In higher plants, NADH-GOGAT is also a flavoprotein (FMN and/or FAD with Fe/S) monomer with a molecular weight of 225 to 230 kDa. This enzyme has a pH optimum ranging between 7.5 and 8.5 and apparent K m for NADH, glutamate and a-ketoglutarate ranging between 4 and 13 pM, 400 and 1000 uM and 39 and 960 uM respectively (Lea et al., 1990a). The Fd-GOGAT proteins of com and Arabidopsis thaliana as well as the NADH-GOGAT proteins of rice and alfalfa contain a sequence with characteristics similar to the peptides localized within the plastid. Surprisingly, these presequences were identified in all the GOGAT enzymes from eukaryotes, including yeast and nematode (Temple et al., 1997). Three inhibitors of glutamate synthase are generally used in metabolic studies of this 29 enzyme: azaserine (0-diazoacetylserin), 6-diazo-5-oxonorleucin (DON) and albizziin. These substances are competitive inhibitors among a great number of enzymes using glutamine as a substrate to transfer the amide group to other molecules (Oaks and Hirel, 1985). When they are used in order to inhibit GOGAT activity, they create an accumulation of glutamine and ammonium in plant tissues (Lea et al., 1992). 1.3.3.1 Physiology of nitrate uptake The movement of nitrate into the plant is thermodynamically uphill (Glass et al., 1992), due to the negative electrical potential difference across the cell membrane (Thibaud and Grignon, 1981). Transport of N O 3 " across the plasma membrane causes a rapid depolarization (the electric potential difference becomes less negative; Glass et al., 1992), which has been considered as indicative of the operation of an electrogenic proton / nitrate symporter, which drive a net flux of positive charges inside the cell. This process is, therefore, likely to have a stoichiometry of at least 2 protons for 1 N O 3 " entering the cell (Ullrich and Novacky, 1981; McClure et al , 1990; Glass et al., 1992). Using CIO3" as a tracing analog of N O 3 " , or using 13 15 the short half-life (9.9 min) radioisotope N or the stable isotope N, it has been shown that N O 3 " uptake is a net flux, that is the balance between two opposite unidirectional fluxes, influx and efflux (Minotti et al., 1969). This behavior is not specific to N O 3 " , since the occurrence of both influx and efflux through the plasma membrane has been recognized for most of the ions absorbed by root cells, but it is of particular significance for N O 3 " because of the relative importance of efflux. Also, efflux values can be attributed to the presence or absence of external 30 NC»3~ prior to uptake measurements and of the concentration of nitrate supplied during these measurements. Most authors concluded that influx and efflux are not of the same order of magnitude (e.g. Morgan et al., 1973; Jackson et al., 1976; Deane-Drummond and Glass, 1982; Lee and Clarkson, 1986; Oscarson et al., 1987; Lee, 1993; Muller et al., 1995). These two fluxes are likely to be regulated separately (e.g. Oscarson et al., 1987; Weineke, 1994), so that NO3 uptake rate may be regulated by changes in one or the other. The individual responses of influx and efflux to treatments which affect net NO3" uptake rate have been analyzed in plants subject to N starvation (Hole et al., 1990, and Siddiqi et al., 1990) and amino acids, (Muller et al., 1995). The general opinion is that the control of NO3" uptake is mainly exerted on the influx component. It has been proposed that up to three inward nitrate transport systems co-exist in the plasma membrane of root cells (Siddiqi et al., 1990; Aslam et al , 1992). These are the constitutive high-affinity system (CHATS), the inducible high-affinity system (MATS) and the low affinity transport system (LATS). CHATS has been shown to operate at low concentrations of nitrate (200 pM concentration range) and displays saturation kinetics (Siddiqi et al., 1990). It functions in the roots of plants that were never supplied with NO3 prior to uptake measurements, which is the reason why it is referred to as "constitutive". H A T S is induced by nitrate or nitrite (Siddiqi et al., 1990); the magnitude of this induction is seemingly dependent on the concentration of nitrate provided. IHATS also displays saturation kinetics (Siddiqi et al., 1990), and has a K m value of 20 to 140 pM varying from one plant species to another. Studies have shown that LATS displays a linear (non-saturating) concentration-dependence (Glass, 1988; Siddiqi et al., 1990), and long-term studies indicate that the system is subject to negative feedback (Clement et al., 1978). All three transport systems require energy for the transport of 31 NC>3~ across the plasma membrane. 1.3.3.2 M o l e c u l a r b i o l o g y o f n i t r a t e u p t a k e At present, nitrate transporters have been isolated from bacteria, fungi, algae and higher plants. These transporters can be classified into three distinct groups: the A B C superfamily (ATP-binding cassette) in bacteria, the POT (proton-dependent oligopeptide transporters) and the MFS (major facilitator superfamily). 1.3.3.3 B a c t e r i a l a n d c y a n o b a c t e r i a l n i t r a t e t r a n s p o r t e r In the family Enterobacteriaceae, some species have the ability to use NO3" or NO2" as the sole nitrogen sources for aerobic growth and as a terminal electron exceptor in anaerobic respiration (e.g. Klebsiella pneumonia). On the other hand, other species have the ability to use nitrate only for anaerobic respiration (e.g. Esherichia coli, Salmonella typhimurium). In K. pneumonia there are two nitrate transport systems with K m values of 4.9 pM and 4.2 mM, respectively for the high- and low-affinity systems. NH4+ repressed both high- and low-affinity systems (Thayer and Huffaker, 1982). The genetic organization of the genes partially responsible for nitrate transport and its reduction to ammonium are organized in the nasFEDCBA operon (Lin et al, 1995). The structural gene nasB codes for NASB protein, which is homologous with NADH-dependent nitrite reductase of both prokaryotic and eukaryotic origin (Lin et al, 1993). The structural gene nasA, which codes for NASA protein, has homology to molybdopterin guanine dinucleotide-containing subunits of prokaryotic 32 molybdoproteins (Lin et al., 1993). The NASC protein encoded by the nasC gene mediates the transfer of electrons from N A D H to the NASA protein (Lin et al, 1993). The genes encoding proteins involved in nitrate transport are nasF, nasE, and nasD. These genes have homology to a gene cluster (nrtABCD) in Synechococcus sp. PCC7942 (Omata, 1991; Omata et al., 1993). The predicted sequence of NASF, NASE and NASD proteins are homologous to NRTA, NRTB, and NRTD, respectively. The nrtA gene codes for a 45 kDa cytoplasmic membrane protein, involved in active nitrate transport. NrtB gene codes for a hydrophobic protein having similarity to integral membrane components of bacterial transport systems, which are dependent on periplasmic-substrate binding proteins. NrtC and nrtD genes encode proteins, which resemble an ATP-binding component of binding protein-dependent transport systems (Omata et al., 1993). The nrtABCD and nasFED gene products both form A B C type transporters. In Esherichia coii, a bacterium which can only use nitrate as an electron acceptor during anaerobic respiration, one gene (narK) which encodes a nitrite transporter (responsible for NO2" efflux) has been isolated (Rowe et al., 1994). N A R K polypeptide is a transmembrane protein of 463 amino acids with a molecular weight of 49 kDa (Noji et al., 1989). 1.3.3.4 F u n g a l a n d l o w e r p l a n t s n i t r a t e t r a n s p o r t e r s Chlamydomonas reinhardtii and Aspergillus nidulans, have been used as model systems for genetic and molecular analysis of nitrate transport. In A.nidulans the use of chlorate as a toxic analog of nitrate resulted in the isolation of a crnA' strain, defective in nitrate transport in young mycelial stages (7-8 hours) (Browlee and Arst, 1983). The crnA gene (Johnstone et al., 1990; Unkles et al., 1991) encodes the protein responsible for nitrate transport. The crnA gene is clustered with the niiA and niaD genes. NiaD encodes the protein nitrate reductase, whereas niiA 33 encodes the protein nitrite reductase. The crnA gene encodes a protein product of 507 amino acids (54.9 kDa) containing 12 membrane spanning regions (Unkles et al., 1991). The expression of the crnA is induced by nitrate (and nitrite), and repressed by nitrogen metabolites (Unkles et al., 1991). In the unicellular green algae Chlamydomonas reinhardtii, the genes narl, nrtl.lCr and nrtl.lCr have been identified as high-affinity nitrate/nitrite transporters (Quesada et al., 1993; Quesada et al., 1994). nrtl.lCr and nrtl.lCr code for putative plasma membrane proteins. NRT2.1Cr protein is 527 amino acids in size and contains 12 membrane spanning regions. In analysis of a C. reinhardtii strain deleted in the nitrate assimilation cluster (nrtl.lCr-narl), transformed with combinations of NT genes, it was shown that narl/nrtl.lCr was responsible for high-affinity nitrate and nitrite transport, while narl/nrt2.1Cr restored only high-affinity nitrate transport. To date the high-affinity nitrate transporters isolated in eukaryotes have all been related to the CRNA type from A. nidulans. They include nrtl.lCr and nrtl.lCr from C. reinhardtii (Quesada et al., 1994), yntl from Hansenula polymorpha (Perez et al., 1997), nrtl.lNp from N. plumbaginifolia (Quesada et al., 1997), nrtlGm from Glycine max (Amarashinge et al., 1998), and nrtl.lAt and nrtl.lAt from A. thaiiana (Zhuo et al., 1999; Filleur and Daniel-Vedele, 1999). 1.3.3.5. H i g h e r p l a n t s n i t r a t e t r a n s p o r t e r s 36 Chlorate and CIO3 have been used as analogs for NO3 uptake, though the validity of these ions as tracers of nitrate has been debated (Deane-Drummond and Glass, 1982; Guy et al., 1988; Touraine and Glass, 1997). Chlorate is a herbicide and defoliant because the chlorite 34 produced by NR is toxic and is not assimilated (Humburg, 1989; Stimmann and Ferguson, 1990). Chlorate has been used as a selective agent for the isolation of mutants defective in nitrate uptake and assimilation. It was used in the isolation of a series of Arabidopsis thaliana mutants which are chlorate resistant (Doddema and Telkamp, 1979). Of these seven groups, one designated as the B-l (Chll) mutant had decreased NO3" transport capacity at high NO3 concentrations (>1 mM). It also was impaired in the transport of CIO3", Cl", and K + (Scholten and Feenstra, 1986). The first plant gene coding for a nitrate transporter (chll (renamed nrtl)) was cloned in Arabidopsis (Tsay et al., 1993) by screening for mutants resistance to chlorate. The sequence of amino acids corresponding to the gene nrtl shows no homology with proteins involved in nitrate transport from bacteria or fungi. The protein has two potential transmembrane domains divided in 2 groups of 6 segments by a central hydrophilic domain (Tsay et al., 1993). The nrtl gene is expressed in the roots and transcript levels increase due to nitrate treatment (Tsay et al., 1993). The NRT1 protein is a member of the POT (peptide oligopeptide transporters) family of transporters. Two homologs of nrtl (nrtl.lLe and nrtl.2Le) were identified recently from roots of tomato. The mRNA transcripts corresponding to nrtl.lLe can only be detected in root hairs and their amount increases in the presence of nitrate (Lauter et al., 1996). The use of chlorate resistance allowed for the isolation a putative Arabidopsis thaliana mutant deficient in constitutive high affinity transport (CHAT) (Wang and Crawford, 1996). This mutant, designated as Nrt2 (Chl8), displayed no nitrate transport at low concentration (<0.1 mM) of NO3" in uninduced plants, while normal NO3" uptake was demonstrated in NO3-induced plants. This would indicate that the mutation has no effect on the IHATS. At present the gene affected by this mutation has not been isolated. 35 The use of the crnA gene and its membership in the major facilitator superfamily, has allowed for the isolation of genes involved in IHATS. Trueman et al. (1996), used conserved motifs of the MFS to design primers, which allowed for the isolation of a 130 bp DNA fragment with high homology to the crnA gene sequence. This fragment, BCRNA, was then used in screening a cDNA library. The screening resulted in the isolation of two cDNAs which code for nitrate transporters, bchl and bch2. Using the same approach Quesada et al. (1997) isolated nrt2.1Np from Nicotiana plumbaginifolia by PCR. Messenger RNA transcript for nrt2.1Np decreased with the addition of glutamine and NH.4+ to NO3" -grown plants. The pattern of mRNA accumulation, due to nitrate treatment (induction) and the addition of ammonium or glutamine (repression) are consistent with the possibility that these genes encode nitrate transporters (Krapp et al., 1998). 1.3.4.1 Regulation of nitrate uptake The capacity to regulate NO3" uptake enables the plant to satisfy the requirement for N, even under unfavorable conditions (i.e. low N availability). Experimentally, this is shown by apparent constancy of NO3" uptake under conditions in which the concentration of NO3 in the nutrient solution is varied. For instance, when Lolium plants were grown on nutrient solutions with N03~concentration differing from 14 pM to 14 mM, neither the amount of nitrogen taken up by the plants nor their growth rates were affected (Clement et al., 1978). Conversely, changing the light intensity supplied to soybean plants resulted in changes of the shoot growth rate, and correlatively in changes of the NO3" uptake rate in roots, while the concentration of 36 this ion in nutrient solution was maintained constant (Touraine et al., 1994). This regulation of NO3" uptake is responsible for the increased intake of NO3" when plants starved of N for various periods of time are resupplied with NO3". This demand-driven regulation, that resembles features observed for other nutrients, has two important characteristics: (1) it is specific for N (i.e. NO3" uptake is not enhanced in plants starved of S or P; Lee, 1993). (2) It is a remote control that involves the transport of signaling molecules from the shoot to transduce the N demand information to the roots (hnsande and Touraine, 1994). Although the nature of these signal molecules is not unequivocally known, it is likely that by-products of NO3" assimilation, especially some amino acids, are involved in this process. The translocated signal (from shoots to roots), would then repress expression of the NO3" transport system. In support of this hypothesis, amino acids supplied to roots, either directly in the external solution (Doddema and Otten, 1979; Breteler and Siegerist, 1984; Breteler and Arnozis, 1985) or via the phloem sap (Muller et al., 1992) inhibited net NO3" uptake rate. 1.4.1.1 Aims An investigation of the molecular biology of nitrate uptake was undertaken. At the start of this research project the initial aim was to isolate the gene(s) involved in inducible high-affinity transport of nitrate and characterize the signals involved in regulation of these genes. Various approaches were used in attempt to reach this goal, as follows: differential display of PCR-products, subtractive libraries, heterologous expression in yeast using negative screen (screening using high concentrations of nitrite), heterologous expression in E. coli positive 37 screen (growth on nitrate), and PCR using degenerative oligonucleotide and/or RACE-PCR. Using heterologous expression in E. coli, I have isolated a cDNA, hvstl, encoding a high-affinity sulfate transporter from Hordeum vulgare. I have also characterized the signals involved in regulation of hvstl transcript levels, vis-a-vis physiological responses (sulfate influx). I used a combination of PCR using degenerative oligonucleotide and RACE-PCR to isolate two cDNAs, bch3 and bch4, which encode inducible high-affinity nitrate transporters. These cDNA belong to a multigene family in H. vulgare. I have also characterized the expression of the family of genes, looking at both positive (nitrate) and negative (amino acids) effectors, in combination with physiological responses (nitrate influx). 38 C H A P T E R 2 T H E I S O L A T I O N A N D C H A R A C T E R I Z A T I O N O F HVST1, c D N A E N C O D I N G H I G H -A F F I N I T Y S U L F A T E T R A N S P O R T E R 39 2.1.1.1 Introduction In higher plants the transport and assimilation of sulfate, an essential inorganic nutrient, is highly regulated. Sulfate transport into plant roots is mediated by at least two transport systems, namely high-affinity and a low-affinity transport system, (Datko and Mudd, 1984; Lass and Ullrich-Eberius, 1984; Hawkesford et al., 1993). The transport of sulfate across the plasmalemma of root cells is thought to be an active process driven by proton motive force, and thought to occur through a F f 7 S O 4 2 " symporter (Lass and Ullrich-Eberius, 1984; Clarkson et al., 1993). The withdrawal of sulfate from the external environment causes sulfate transport to be up-regulated (Clarkson et al., 1983, Hawkesford and Belcher 1991). This regulation is thought to be mediated by negative feedback control, in which sulfate or a product of sulfate assimilation: L-cysteine, L-methionine or glutathione (Herschbach and Rennenberg, 1991, 1994) serves as the effector molecule. It has also been shown that O-acetyl-L-serine may play a role as a positive acting compound by increasing sulfate uptake (Smith et al., 1997). hi the last four years a plethora of cDNAs encoding sulfate transporters has been isolated and characterized: shstl, shstl, and shst3 from Stylosanthes hamata (Smith et al., 1995), hvstl from Hordeum vulgare (Smith et al., 1997), and ast56 and ast6S from Arabidopsis thaiiana (Takahashi et al., 1996; 1997). The cDNAs, shstl and shstl, encode proteins which function as high-affinity sulfate transporters with reported K m values <10 pM in the yeast heterologous system. Of the other three reported sulfate transporter genes a K m (<100 pM) has been demonstrated only for shst3 in yeast heterologous expression system. Nevertheless, ast68 has been shown to restore growth of the YSUL1 sulfate transport mutant in low sulfate media. In this chapter, I describe the cloning of a high-affinity sulfate transporter (hvstl) by a novel bacterial heterologous expression system and examine its regulation with respect to 40 external sulfate supply and internal metabolites of sulfate. Also I have monitored the effect of N on hvstl transcript accumulation and sulfate uptake. I have isolated a genomic DNA by PCR comprising the promoter sequence of the gene consisting of 1350 bp from the start codon and compared this to the promoter sequences of other sulfate transporters, which are derepressed following withdrawal of sulfate. I have also characterized SO42" levels in planta using compartmental analysis. 2.2.1.1 M a t e r i a l s a n d m e t h o d s 2.2.1.2 B a c t e r i a l s t r a i n s , m e d i a a n d g r o w t h c o n d i t i o n s The Escherichia coii strains used were DH5cc, XLl-Blue MRF' , SOLR and JM109. DH5a was used for transformations and manipulation of plasmid DNA. XLl-Blue MRF' and SOLR strains were used for cDNA packaging into X phage and in vivo excision of cDNA library. JM109 strain was used for heterologous complementation assay. Plasmids pVSJ1344 and pVSJ1358 were gifts from Dr. V. Stewart. Plasmid pVSJ1344 contains the nasFEDCBA operon, while pVSJK1358 contains only the nasCBA genes under the control of the nas promoter. JM109 (containing plasmid pVSJ1358) was used for heterologous complementation on modified M9 minimal medium without NH 4C1, replaced with 2 mM K N O 3 supplemented with 1 uM thiamine and 0.5 mM IPTG. 2.2.1.3 c D N A l i b r a r i e s a n d g e n o m i c l i b r a r y A Hordeum vulgare cv. Klondike cDNA library was constructed in Lambda ZAP (Stratagene, La 41 Jolla, Ca) from mRNA isolated from roots of 7 day-old seedlings which were grown on 1/10 strength (N-free) Modified Johnson's solution for 4 days then treated with 10 mM K N O 3 for 6 h. The plasmid library for heterologous complementation in E. coli was derived by mass excision of the X phage library. Marathon cDNA library (Clontech, Palo Alto, Ca) was obtained by use of reverse transcriptase with mRNA isolated from 6 h nitrate-induced barley seedlings as template. Construction of PromoterFinder DNA walking libraries (Clontech, Palo Alto, Ca) used genomic DNA isolated from leaves of Hordeum vulgare cv. Klondike. 2.2.1 A Preparation and analysis of DNA and RNA Plant genomic DNA was isolated as described by Dellaporta et al. (1983). Total RNA was isolated using TriZol Reagent (Life Technologies, Gaithersburg, Md), according to the protocol provided by the manufacturer. Messenger RNA was isolated using the FASTTRACT mRNA isolation kit (Invitrogen, Carlsbad, Ca) according to the manufacturer's instructions. In all experiments 20 pg of Total RNA were fractionated on denaturing 1.2% agarose gel containing 1.1 M formaldehyde and 50 mM MOPS pH 7.0, and transferred to nylon membranes (Hybond N+, Amersham, Piscataway, NJ). Blots were hybridized for 12 to 16 h at 42°C. Hybridization was performed in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 20 pg ml"1 sonicated herring sperm DNA. Blots were washed twice for 10 min at 22-25°C with 2X SSC and 0.1% SDS, followed by 15 min wash at 42°C in 1XSSC and 0.1% SDS, then a 15 min wash at 42°C in 0.1X SSC and 0.1% SDS. A 1.4 Kb EcoW-XhoI fragment from initial clone pSB201 (3' end of hvstl) was radioactively labeled with 3 2 P y dCTP (Amersham, Piscataway, NJ) using Prime-A-Gene kit (Promega, Madison, Wi) and used for hybridization experiments. For control levels of total 42 RNA, the Xhol fragment of plasmid pV25S, which contains a 25S ribosomal gene was used as a probe. 2.2.4.1 I s o l a t i o n a n d s e q u e n c i n g , o f the p r o m o t e r r e g i o n o f hvstl Sequences on both strands were determined by the dideoxy chain termination method (Sanger et al., 1973) using Applied Biosystems 373A automated sequencer. After detailed restriction analysis of resulting plasmids, sequencing was pursued on overlapping subclones using Ml3 reverse and forward primers. Oligonucleotides were designed for sequencing hvstl and the promoter of hvstl. RACE-PCR was employed for the isolation of the 5' coding region of hvstl by use of a Marathon cDNA amplification kit (Clontech, Palo Alto, Ca). Copy DNA was obtained by reverse transcription from mRNA isolated from barley seedlings induced for 6 h with 10 mM nitrate as template. Expand high-fidelity PCR system (Boehringer Mannheim, Montreal, PQ) was used for polymerase chain reactions with gene specific oligonucleotides HS1: 5' GGC A A C C A T T C C G G A T A C T A C A C C , HS2: 5 ' C C A A A C G C A A G T A T G G G C C A G A C C , API and AP2 were supplied by the manufacturer. Oligonucleotides HS1 and API were used for the first round of PCR, with the following conditions of 30 cycles at 94° C for 45 s, 60°C for 30 s and 68°C for 3 min. The resulting amplification (1/50) was then re-amplified using the above cycle with oligonucleotides HS2 and AP2. A specific PCR product of 1.05 kb was isolated and cloned into pCR2.1 (Invitrogen, Carlsbad, Ca). To ensure the sequence integrity of the PCR product, 3 independent sets of PCR were conducted. The resulting PCR products were then sequenced. The 5' non-coding region of hvstl was isolated using PromoterFinder DNA Walking Kit (Clontech, Palo Alto, Ca). The genomic library was based on complete digestion of DNA from Hordeum vulgare cv. Klondike using oligonucleotides specific for the 5' UTR of hvstl 43 HSG1: 5' G C A C A A G T A T G A G T G T A T G A C T A C T G G HSG2: 5' T C T T G C G G A C G T T G C G T G C G C C T A T G A , API and AP2 (supplied by manufacturer). The Expand Long PCR system (Boehringer Mannheim, Montreal, PQ) was used for DNA amplifications. The first round of amplification used primers HSG1 and API, with the reaction conditions of 92°C for 30 s, 62°C for 45 s, and 72°C for 15 min, for 30 cycles. The amplicon was diluted 1/100 then re-amplified with oligonucleotides HSG2 and AP2 under the same conditions as above. This amplification resulted in a specific PCR product (from PvuTL library HS33, and Seal library HS42) which was subcloned into pCR2.1 (Invitrogen, Carlsbad, Ca) resulting in the plasmids pHS33 and pHS42. 2.2.1.6 Plant material Plants of H. vulgare cv. Klondike were used as the source of RNA and DNA. Seeds were surface sterilized with 1% commercial bleach, and rinsed with de-ionized H 2 0 . Seeds were imbibed for 6 h in de-ionized H2O, then placed on plastic mesh fitted into Plexiglas discs (8 seeds). The discs containing the seeds were transferred to moist sand for germination in the dark at 20°C for 3 d. Discs holding the seedlings were then transferred to Plexiglas hydroponic tanks (40 L) containing 1/10 Johnson's solution (100 pM SO42") for 4 days (Epstein, 1973). In S-minus treatments MgS04 was replaced with MgC^. For nitrogen treatments plants were grown on 1/10 Modified Johnson's solution (-N) with sulfate provided at 250 pM, as follows: 200 pM K H 2 P 0 4 , 150 pM MgS0 4 , 50 pM K2SO4, 50 uM CaS0 4 , and micronutrients and Fe (as Fe-EDTA) pM: Cl 50, B 25, Mn 2, Cu 0.5, Fe 20, Zn 5. Nitrogen was supplied at 2 mM concentration in the form of K N O 3 or NH4CI. The plants were grown in walk-in growth rooms with a 16h light-8 h dark cycle at a temperature of 20±2°C, with 70% relative humidity. The light source was provided by 44 fluorescent tubes having a spectral composition similar to sunlight, with a photon flux density of 300 umol m"2 s"1 at plant level. 2.2.1.7 Influx measurements In all experiments, influx was measured from 0.1 pM SO42" labeled with 35SC>42"; all other nutrients were provided as in the respective growth medium, with a pH of 6.2 maintained by the addition of excess calcium carbonate. The volume of influx solution (mL): root weight (g) ratio was approximately 50:1 so that depletion of SO42" during the 5 min influx period was 5% or less. Influx was measured into intact roots that were pre-washed for 5 min in fresh non-radioactive 35 2 1 35 2 solution before transfer to the uptake solution, which contained SO4 "(10 MBq mol" SO4 " specific activity (Amersham, Piscataway, NJ). After five min, uptake was terminated by transferring roots to 250 mL of desorption solution for 3 min. Roots and shoots were excised and separated, then placed into glass scintillation vials in which they were extracted with 10 mL of 10 N HC1 per gram of tissue (FW) for one hour. Eight mL of scintillation cocktail (Ultima Gold, Packard-Bell) were added to 2 mL of the extraction solution and counted in a Beckman LS6001C scintillation counter. 2.2.1.7 Compartmental analysis and thiol analysis Six day-old seedlings of Hordeum vulgare cv. Klondike were grown in 1/10 Johnson's solution containing 10 uM or 100 uM sulfate. For the last 16 h, seedlings were transferred to 1 L vessels containing the above growth solution labeled with SO4 " (loading solution). After labeling, the seedlings were introduced to the efflux funnel where the roots were eluted with 20 mL of sample 45 solution for specific durations, of 1x5 s, 1x10 s, 6x15 s, 4x1 min, 1x2 min, 2x3 min, 2 x 4 min, 1 X 5 min, 1 x 8 min, 1 x 10, 1 x 15 min, 2 x 30 min, and 10 x 1 h. The elution solutions were then dried at 80°C and re-suspended in 2 mL 0.1 N HCI. Eight mL of scintillation cocktail (Ultima Gold, Packard-Bell) was added to resuspended elution solution, and counted in a Beckman LS6001C scintillation counter. The radioactivities released from the barley roots during the 8 h efflux period, were converted to efflux rates, and plotted verses time in semi-log plots. The analysis of the log of rate of 35S042" release over time, were plotted, as described by Lee and Clarkson (1986) for 13N03~ by using a computer analysis program (Siddiqi et al., 1991). Thiol analysis was conducted as reported by Lappartient and Touraine (1996) 2.3.1.1 R e s u l t s 2.3.1.2 C l o n i n g a n d i d e n t i f i c a t i o n o f the hvstl gene e n c o d i n g su l fa te t r a n s p o r t e r A partial clone, SB201, was isolated using a heterologous bacterial expression system, designed for the isolation of nitrate transporters from higher plants. This heterologous bacterial expression system is based on the nas operon from K. pneumonia. The genes nasFEDCBA are responsible for the uptake of nitrate and the reduction of nitrate to ammonium (Lin et al., 1994). The nas operon can be expressed in E.coli, and allows growth on nitrate, as the sole nitrogen source. The proteins encoded by nasFED genes form a transporter complex, while nasCBA genes encode nitrate reductases, nitrite reductases and a protein involved in the provision of elections required in the reduction process. A plasmid cDNA library derived from root-rnRNA of nitrate-treated H. vulgare was transformed into an E.coli strain (VJSK1090) containing plasmid pVSJK1358 (containing the nasCBA genes but lacking the nasFED genes responsible for nitrate 46 transport), and screened on modified M9 minimal media containing 2 mM NO3". Six complemented clones able to grow on nitrate were isolated and re-transformed into VSJK1090 strain containing plasmid pVSJK1358, to ensure that growth on nitrate was indeed due to the cDNA clone and not a mutation in the bacterial strain. One of the positive partial clones, SB201, contained a cDNA fragment of 1.4 kb, which was sequenced and found to have high homology to shstl, shst2 and shst3, sulfate transporters from Stylosanthes hamata. The SB201 clone nucleotide sequence was inframe with respect to the LacZ a portion to form a fusion protein. pSB201 encodes the 3' end of the hvstl gene, and the 5' end of the gene was isolated by R A C E -PCR. A fusion of the SB201 cDNA fragment and 5'RACE-PCR product resulted in the full length gene, hvstl, 2442 bp in length. 2.3. 1.3 P r e d i c t e d s t r u c t u r e o f p r o t e i n e n c o d e d b y hvstl, p h y l o g e n e t i c ana lys i s a n d c o m p a r i s o n w i t h o t h e r p l a n t sul fate t r a n s p o r t systems The deduced amino acid sequence of the protein encoded by hvstl contains 660 amino acids, with a predicted molecular weight of 72500 and a pl of 9.04 using Compute pI/Mw program (Wilkins et al., 1998) at Expasy Server. Using SOSUI program (Hirokawa et al., 1998) at Expasy Server, the polypeptide was predicted to contain twelve membrane spanning domains. Figure 3 presents the result of phylogenetic analysis using TreeGen program (Gonnet, 1994) at the Darwin server (http://cbrg.inf.ethz.ch) obtained by comparing amino acid sequence of the known (HVST1, SHST1, SHST2, AST56, AST68) and putative (SSST1, HSTl . lAt , ATST1, AST1, AST91 and ASTC1) plant S0 4 2" transporter. Four clusters, named A, B, C, and 47 Figure 3. Phylogeny of predicted amino acid sequences of plant sulfate transporters. The phylogeny was obtained by using the GenTree program (Darwin, ETHZ Switzerland) with a) HVST1 (acc. no. U52867), b) SHST1 (acc. no. X82255), c) SHST2 (acc. no. X82256), d) HSTl . lAt (acc. no. AJ30018695),e) AST1 (acc.no. AB004060), f) AST68 (acc. no. AB003591), g) ASTC1 (acc. no. AB008782), h) SHST3 (acc. no. X82454), i) ATST1 (acc. no. D89631.), j) AST56 (acc. no. D85416) and k) SSST1 (acc. no. X96761). 48 D, have thus been identified. Members of cluster A are HVST1, SHST1, SHST2 and HSTl. lAt, while cluster B is composed of SHST3, AST56 and AST68. Very little is known about the members of cluster C, except that their transcripts accumulate in above ground portions of the plant, while cluster D has only one member ASTC1 (AB008782) whose predicted protein contains a chloroplastid signal peptide (probability of 0.942); all other sulfate transporters were destined for insertion in the plasma membrane PSORT program (Nakai and Kanehisa, 1992). Using PROPSEARCH program (Hobohm and Sander, 1995), I found that there may be a structural relationship between HVST1 and NRT1 protein (reliability of > 99.6) from Arabidopsis thaiiana (Tsay et al., 1993), which is a low affinity nitrate transporter. 2.3.1.4 Isolation of the 5' upstream region of hvstl I isolated a genomic DNA fragment 5' upstream of the hvstl by PCR. This fragment is 1356 bp in length. The sequence of this fragment is presented in Figure 4a. Using the NNPP program (Reese et al., 1996) the putative transcription start site was determined (Figure 4a), with the T A T A box at -32 to -22 from this site. Using TfSEARCH program (Heinemeyer et al, 1998), cis-acting DNA elements implicated in the expression of genes involved in sulfate assimilation in yeast and N. crassa (reviewed by Marzluf, 1997) were not present in the hvstl promoter. I compared the hvstl promoter sequence with known cis-acting elements in plants, using the PLACE program (Ugawa et al., 1998). The resulting analysis found three putative cis-acting elements, box 1 from chsl5 from bean (Lawton et al, 1991); PE1, a positive acting element from the phya3 promoter from oat (Bruce and Quail, 1990); and L box element from rbcs promoter from tobacco (Giuliano et al., 1988). The boxl element was also present in hstl.lAt, ATP sulfurylase (aps2, acc. no. U59737), and APS kinase (apk acc. no. U59759) promoter sequences 49 HVST1 -583 T A A A A G T T A A A A -572 AST88 -479 T A A A A A T T A A A A -467 APS2 -1118 T A A A A G T T A G A T -1130 APSK -1389 C A A A A G T C A A A A -1391 consensus Y A A A A R T Y A R A A CHS15 BOX T A A A A G T T A A A A BOX I HVST1 -932 A A T T C G G C T T A C T A T A AST88 -1075 A A T T C G G T T T A C T A T A consensus A A T T C G G Y T T A C T A T A BOX II HVST1 -710 A T C G G C A G A C c G C A T AST88 -245 A T C T T C A C A C c G C A T consensus A T C N N C A N A C c G C A T BOX V HVST1 -264 T G T C A C C T G T c G C T G A A AST88 -143 T G T T G G C T T T c G C T G A A concensus T G T Y R N C T N T c G C T G A A Figure 4. a) Sequence of hvstl promoter and location of putative cis-acting elements, putative transcription start site (Capitalized) and TATA box (underlined), b) Alignment of putative cis-acting element Box IV in the 5' upstream region of hvstl, hstl.lAt (ast88), aps2 and apsk c) comparison of 5' upstream regions of hvstl and hstl.lAt (ast88). 50 with the consensus sequence (T/C)AAAA(G/A)T(T/C)A(A/G)A(A/T). Figure 4b and 4c show the alignment of this consensus, present in the above promoters and location. I compared the promoter sequences of hvstl with two sulfate transporters induced by SO42" starvation in root: ast68 (Takahashi et al , 1997) and hstl.lAt (Vidmar, unpublished data). Using MEME2.2 program (Bailey and Gribskov, 1998), I found no high scoring sequence similarities between the promoters of hvstl and ast68. The comparison between hvstl and hstl.lAt, demonstrated the presence of four elements containing a high degree of sequence similarities between the two promoters (Figure 4b and 4c). 2.3.1.5 Effect of S status on sulfate influx, thiol levels and hvstl mRNA abundance In order to investigate the effect of external SO42" concentrations on hvstl mRNA levels in roots of barley grown under steady state conditions, plants were grown on <2.5, 10, 50, 100, or 250 uM SO42" for 4 days after seedlings were transferred from sand to hydroponic tanks. To ensure that the concentrations of SO42" in hydroponic media remained constant, media was exchanged every 12 h. Shoots and roots were harvested by blade-cutting ca. 4 mm above and under the seed, respectively, in order to avoid contamination of different tissue types. Total RNA was separately extracted for Northern blots. The membranes probed with hvstl showed a single band at2.4 kb in all the lanes loaded with root extracts, while no hybridization signal was detected in leaf extracts (Figs. 5a and 5b). In roots, the level of hvstl hybridizing transcripts was inversely correlated with sulfate supply, the highest level being observed in roots of plants grown on <2.5 uM (Fig. 5a). The pattern of 3 5 S04 2 " influx in roots of intact barley plants corresponded in pattern with the 2 2 Northern blots. When SO4 " influx was plotted against external SO4 " concentration in steady-state experiments, influx decreased markedly from the lowest concentration used (< 2.5 uM) to 50 pM, then tended to decrease more slowly with increasing external 51 A) 7 [SO ' (uM) 2.5 10 50 100 250 4 %XT Root HVST1 25S Shoot HVST1 25S ifltttift J H ^ ^ Jltoik W t * ^mwmW ""iSP^ ^ ^ ^ ^ ^ ^ ^ ^ R^|P^  B) 2.5 10 50 100 250 External S04 2" [pM] Figure 5. Effect o f external sulfate supply on transcript abundance of sulfate transporter gene hvstl. Roots and shoots were sampled from barley plants grown on <2.5, 10, 50, 100, 250 u M SO4 ". A ) Northern blot analysis of expression of hvstl in barley roots. Northern blot analysis of hvstl transcript levels in barley roots, 20 pg of total R N A was introduced into each lane. Northern blots were probed with the hvstl probe (full length c D N A ) and 25 S ribosomal subunit was to ensure equal loading of R N A . Blots were washed at high stringency. B) Quantification of hvstl transcript levels in roots using densitometry. Transcript levels were normalized to the 2.5 u M sulfate treatment. 52 concentration up to 250 p M (Fig. 6). The thiol analyses of root extracts by H P L C revealed four identifiable peaks, corresponding to cysteine, y-glutamyl-cysteine, glutathione (y-glutamyl-cysteinyl-glycine) and hydroxymethyl-glutathione (y-glutamyl-cysteinyl-serine), a homologue of glutathione that is found in plants belonging to the Poaceae (Klapheck et al., 1992). Among these four components, glutathione appeared to exceed any other thiol in concentration by ca. two orders of magnitude in all the barley root extracts examined. Growing plants on increasing SO42" concentration led to increasing internal levels of thiols, especially glutathione, in root tissues. The plot of glutathione (GSH) concentration vs. external SO42" concentration (Fig. 7) revealed a strong increase of G S H for the lower S0 4 2 ~ concentrations tested, followed by further smaller increases at higher S042"concentrations. To investigate the response patterns of hvstl transcript levels, S 0 4 2 " influx and thiol pools in roots to S status, a time course study of the effects of S 0 4 2 " withdrawal from the nutrient solution was performed. Barley plants previously grown under conditions of sufficient S supply (100 p M S0 4 2 ") were transferred to a S-free solution for time periods, varying from 1 to 48 h. The duration of culture on 100 p M SO42" varied so that all plants were 7 day-old at harvest time. After transfer to S-free solution, hvstl transcript levels increased 5-fold within two hours, and remained elevated for longer duration of S starvation (Fig. 8). Similarly, S starvation caused rapid changes in SO42" influx and glutathione levels: S 0 4 2 " influx increased by 65% within the first two hours, from 0.075 to 0.124 pmol g _ 1 F W h"1, and then by 60% within the following 46 h, up to 0.2 pmol g _ 1 F W h"1 at 48 h (Fig. 9); glutathione levels declined from 140 to 120 nmol g"1 F W within the first two hours, and then to 75 nmol g"1 F W at 48 h (Fig. 10). When plants starved of S for 24 h were re-supplied with 100 p M S 0 4 ", the level of hvstl transcript in roots returned to the level measured in roots of SO4 "-grown plants withm 15 mm (Fig. 11). In order to investigate the specific effects of the major intermediates of S assimilation, 53 0.25-0.201 0.05i O.OfjH • 1 1 1 1 r-0 50 100 150 200 250 [UM] External Sulfate Figure 6. Effect of external sulfate supply on sulfate influx. Barley plants grown on <2.5, 10, 50, 100, 250 p M S 0 4 2 " and measured at 100 p M 3 5S-sulfate for 5 min (4 replicates for each concentration point with error bars = ± 1 S.D.). 54 200 -| 180H 40 H 20-0-\ 1 1 ' 1 1 1 • 1 1 r 0 50 100 150 200 250 [UM] External Sulfate Figure 7. Effect of external sulfate supply on G S H content in roots. Barley plants were grown on <2.5, 10, 50, 100, 250 p M SO4 " (4 replicates for each concentration point with error bars = ± 1 S.D.). 55 A) ' -S (h) 0 1 2 3 6 9 12 24 48 _ t | p „ a | | | M | ^ | | 25S — g f f | M ( f A f , f f H p m p ^ I P ' Figure 8. Effect of removal of external sulfate supply on hvstl transcript in barley roots. Roots were sampled from barley plants grown on 100 p M sulfate then deprived of SO42" for 0, 1,2, 3, 6, 9, 12, 24 or 48 hours. A ) Northern blot analysis of hvstl transcript levels in barley roots, 20 pg of total R N A was introduced into each lane. Northern blots were probed with the hvstl (full length fragment) and 25S ribosomal subunit was to ensure equal loading of R N A . Blots were washed at high stringency. B) Quantification of transcript levels using densitometry transcript level normalized to the 100 p M sulfate grown treatment (average of two independent R N A isolations and Northern blots). 56 0.12n 0.02-j . • . 1 • • • , , 0 10 20 30 40 50 time (h) sulfate starvation Figure 9. Effect of removal of external sulfate supply on sulfate influx in barley seedlings. Plants were sampled from barley plants grown on 100 p M sulfate then deprived of SO42" for 0, 2, 6, 12, 24 or 48 hours. Sulfate influx was determined as a function of as a function of sulfate withdrawal during pretreatment prior to influx determination at 100 p M SO42" (4 replicates for each time point with error bars = ± 1 S.D.). 57 Figure 10. Effect of removal of external sulfate supply on G S H concentrations in barley roots. Roots were sampled from barley plants grown on 100 p M sulfate then deprived of SO4 " for 0, 1, 2, 3, 6, 9, 12, 24 or 48 hours. G S H concentrations of root tissue were determined as a function of sulfate withdrawal during pretreatment (4 replicates for each time point with error bars = ± 1 S.D.). 58 A) B ) D. g 'E 1 § Time Figure 11. Effect of re-supply of external sulfate to S starved plants on hvstl transcript abundance, in barley plants previously deprived of S for 24 h. Plants re-supplied with 100 p M sulfate for 0, 15, 30, 60 or 180 min. A ) Northern blot analysis of expression of hvstl in barley roots, 20 pg of total R N A was introduced into each lane. Northern blots were probed with the hvstl (full length fragment) and 25S ribosomal subunit to ensure equal loading of R N A . Blots were washed at high stringency. B) Quantification of transcript levels using densitometry transcript level normalized to S deprived plant treatment. 59 2 24 h S-starved plants were exposed to the following treatments for 3 h: (a) 100 u M SO4 ", (b) 1 m M Cys, (c) 1 m M G S H , (d) 100 u M S 0 4 2 " plus 1 m M B S O , and 1 m M Cys plus 1 m M B S O (an inhibitor of y-glutamyl-cysteine synthetase, the first enzyme involved in the synthesis of G S H from Cys). The experiment presented in Figure 12, showing the results of re-supplymg SO4 " to S-starved plants for 3 h, established that this treatment (lane 3) resulted in a decrease of hvstl m R N A abundance in roots almost down to the level observed in the roots of plants continuously grown with S 0 4 2 " (Lane 2, Fig . 12). Figure 12 also shows that Cys had a similar effect to that of SO42" re-supply, while G S H treatment induced a more substantial decline in hvstl transcript accumulation, and B S O partially prevented the effects of both SO42" and Cys. 2.3.1.6 Eff lux analysis Compartmental analysis of SO42" was undertaken in order to get insights into the internal distribution of SO42" within root cells and to estimate the fluxes between the different compartments. This type of study requires that plants be at a quasi-steady state. It cannot be used to look for short-term responses to changes in solution composition, as in the case of SO4 " withdrawal or re-supply, or in the case of Cys and G S H treatments, but only to compare SO4 " patterns in plants grown under steady-state conditions of different SO4 " concentrations. The lowest concentration of S 0 4 2 " that I could maintain at a constant level for several hours was 10 u M . Thus to compare roots of plants grown on a low to a high level of SO42" concentrations we grew plants on nutrient solution containing either 10 p M or 100 p M SO4 ". According to the SO42" concentrations used, we observed different values for SO42" influx, internal G S H concentration, and hvstl transcript abundance in roots (see above). Efflux analysis experiments 60 A ) 1 2 3 4 5 6 7 HVST1 • "* • • * Figure 12. Regulation of hvstl transcript abundance as functions of treatment with various S-metabolites. A ) lane 1: sulfate-starved for 24 h; lane 2: 100 pM-grown plants; lane 3: sulfate-starved for 24 h and then re-supplied with 100 p M sulfate for 3 h; lane 4: sulfate-starved for 24 h and then supplied with 1 m M cysteine for 3h; Lane 5: sulfate-starved for 24 h and then supplied with 100 u M sulfate and 1 m M B S O ; lane 6: sulfate-starved for 24 h and then supplied with 1 m M cysteine and 1 m M B S O ; lane 7: sulfate-starved for 24 h and then supplied with 1 m M glutathione for 3 h. Northern blots were probed with the hvstl (full length fragment) and 25S ribosomal subunit to ensure equal loading of R N A . Blots were washed at high stringency. B) Quantification of transcript levels using densitometry transcript level normalized to S deprived plant treatment. 61 were performed on 7-day-old plants grown with either 10 u M or 100 u M SO4 " and labeled with 35SC>42" for the last 24 h. The logarithm of rates of tracer release from roots were plotted against time. The same general pattern was obtained with plants grown on 10 and 100 u M SO4 " (data not shown). A s classically described for various ions, within various plant species, these plots reveal distinct phases, characterized by different half-lives of exchange, that are identified as the surface film of solution, apparent free space, cytoplasm and vacuoles (Kronzucker et al., 1995). The results of the compartmental analysis are given in Table 1. The net flux (rate of SO42" uptake), increased ~4-fold as the external concentration of SO42" increased from 10 to 100 u M , which was the result of more dramatic, but concomitant, -14 and -17 increases, respectively, in both influx and efflux. Consequently the efflux/influx ratio remained essentially unchanged. 2 2 Every other S 0 4 " flux estimated by compartmental analysis increased with external S 0 4 " concentration, though not to the same extent. Both the vacuolar and cytoplasmic concentrations of SO4 " o f root cells increased by 4 fold 2 2 when plants were grown on 100 u M SO4 " compared to those grown on 10 u M SO4 ". The half-life of S O 4 2 " turnover (to.5) for the cytoplasmic phase varied with external S O 4 2 " concentration (12 and 5 min respectively for roots of 10 and 100 u M S O 4 2 " grown plants), while the vacuolar t0.5 was not significantly affected by the external concentration of SO4 ". 2.3.1.7 I n t e r a c t i o n b e t w e e n N a n d S n u t r i t i o n : effect o n SO42" i n f l u x a n d hvstl t r a n s c r i p t a b u n d a n c e The roots of seedlings grown in the absence of any N source displayed a low abundance of hvstl. However, the amount of hvstl transcripts in roots increased 3 fold upon supply of NO3" or 62 Table 1. Efflux analysis of sulfate at 10 u M and 100 u M . A l l values are derived from the average of three individual experiments. Treatment 1 0MM 100 LIM Onet (Mmol g"1 FW h"1) 0.010 0 083 O o c (Limol g 1 FW h"1) 0.028 0.387 O c o (pmol g-1 FW h"1) 0.018 0.304 O c v (Mmol g 1 FW h"1) 0.011 0.064 O v c (Mmol g"1 FW h'1) 0.002 0.029 O r e d (Mmol g"1 FW h"1) 0.008 0.021 O x (Mmol g 1 FW h"1) 0.002 0.015 k c 3.41 8 - 8 6 k v (h_1) 0.169 1.63 ti/2Cyto (min) 12.3 4 8 ti/2vac (min) 256 201 [S04 2 -] cyto (MM) 18 8 0 [S042"]Vac (MM) 86 335 63 A ) +NO3- (h) 0 2 6 12 24 48 HVST1 2 5 S nitrate 2 m M llllll preteatment (h) Figure 13. Effect of N treatment on the hvstl transcript abundance. Plants were exposed to 2 m M NO3" for 0, 2, 6, 12, 24 or 48 h. A ) Northern blot analysis of hvstl expression in barley roots; 20 pg of total R N A was loaded into each lane. Northern blots were probed with the hvstl (full length fragment) and 25S ribosomal subunit to ensure equal loading. Blots were washed at high stringency. B) Quantification of transcript levels using densitometry transcript level normalized to N starved plant treatment. 64 Figure 14. Effect of N treatment on sulfate influx. Plants were exposed to 2 m M N G y for 0, 2, 6, 12, 24 or 48 h. Sulfate influx as a function of treatment with 2 m M NO3" (4 replicates for each time point with error bars = ± 1 S.D.). 65 N H 4 + for 6 h and remained elevated for 24 (Fig. 13). In parallel, SO42" influx increased by 2 fold (from to 0.034 ± 0.004 to 0.074 ± 0.008 pmol g _ 1 F W h"1) within 6 h (Fig. 14). 2.4.1.1 Discussion The first plant sulfate transporter c D N A s were isolated from Stylosanthes hamata (Smith et al., 1995) by functional complementation of a yeast mutant disrupted in the yeast high-affinity sulfate transport system, encoded by the sull gene. A n Arabidopsis thaiiana sulfate transporter c D N A has been identified by homology with SO42" transporters of S. hamata, S. cerevisiae and N. crassa (Takahashi et al., 1996). Using the yeast mutant complementation approach, Smith et al. (1997) isolated a high-affinity SO42" transporter c D N A , designated hvstl. Their c D N A was isolated from a c D N A library constructed from m R N A of barley roots which were S-starved (Smith et al., 1997). Here I report the independent cloning of the same c D N A hvstl using the complementation of a bacterial strain as an alternative strategy for isolating plant ion transporter genes. In fact, my initial aim was to isolate genes responsible for NO3" transport. Therefore I specifically used an E. coli strain, which contained the nasCBA genes from K. pneumoniae (Lin et al., 1995) that code for the enzymes nitrate reductase and nitrite reductase. This strain would allow the transformants to grow on a nitrate medium provided that NO3" can be transported across the plasmalemma into the bacteria. Screening the transformants after complementation with a barley roots c D N A library led to the isolation of a D N A fragment with high homology to shstl and shst2. The full length gene, that was cloned and sequenced, is identical to the sequence published for hvstl (Smith et a l , 1997). This result was entirely unexpected and may indicate that under the conditions imposed, the SO42" transporter can function as a NO3" transporter to 66 some extent. For example, the H V S T 1 protein may have structural homologies to nitrate transporters. To investigate this hypothesis, we used the P R O P S E A R C H program (Hobohm and Sander, 1995) which neglects the order of amino acid residues in a sequence, but analyzes the amino acid composition, molecular weight, content of bulky residues, content of small residues, average hydrophobicity, average charge and the content of selected amino acid residue doublets. This search revealed that N R T 1 (originally named C H L 1 ) protein, which is a low-affinity nitrate transporter from Arabidopsis thaliana (Tsay et al., 1993), scored highest after the sulfate transporter family (shstl, shst2 and shst3), with a reliability of greater than 99.6 %. These results suggest that in this heterologous complementation assay, the partial sulfate transporter may be sufficiently lacking in selectivity that it allows for the transport of N O V into the bacteria. The analysis o f phylogenetic relationships between all known and putative plant SO4 " transporters showed that they fall into four clusters that would have diverged before the separation of dicots and monocots. Cluster A consists in high-affinity SO4 " transporters (SHST1, SHST2, H V S T 1 and H S T l . l A t ) , and m R N A is detected exclusively in roots. These transporters SHST1, SHST2, H V S T 1 have apparent K m values in the 10 u M range when measured in the yeast expression system, and transcript levels increase under conditions of S-starvation for shstl, shst2, hvstl and hstl.lAt (shstl and shst2, Smith et al., 1995; hvstl, Smith et al., 1997, this work; hstl.lAt, Vidmar unpublished data). Cluster B contains low-affinity SO42" transporter proteins, which can be expressed in both roots and shoots but predominately in shoots. Within this group, only shst3 has been characterized in yeast ( K m , 100 u M ; Smith et. al., 1995), while the Arabidopsis c D N A ast68 can restore growth of S. cerevisiae the Y S U L 1 mutant at 100 u M SO4 " (Takahashi et al., 1997). The m R N A levels o f both genes in roots are up-regulated by S-starvation (shst3, Smith et al., 1995; ast68, Takahashi et al., 1997). Due to the lack of any functional data, little is known about the function of cluster C genes, except that they are 67 expressed in shoot tissue. Cluster D contains only one member A S T C 1 (accession no. AB008782). This out-lier is presently the only protein which does not fall into any of the clusters, so it may have a function which is distinct from the SO4 " transporters; using P S O R T program (Nakai and Kanehisa, 1992) we found that it has a 0.934 probability of being localized to the chloroplast. Promoter analysis of hvstl indicated the presence of putative-cis acting elements related to PE1 positive acting element from oat and Box 1 from chsl5 o f bean, which are elements that interact with transcription factors SBF1 and GT-1 , respectively. The presence of this element with a consensus sequence of T A A A A ( G / A ) T ( T / C ) A ( G / A ) A ( A / T ) in hvstl, hstl.lAt, aps2 and apk indicates that transcription factors related to GT-1/SBF1 (Lawton et al., 1991) may be involved in the regulation of high-affinity sulfate transport and S-assimilation. A s has been shown for the other major nutrients absorbed by roots, SO4 " uptake rate is governed by the nutritional status of the plant. Thus, SO42" influx in roots is enhanced when S-sufficient plants are deprived of S, for 1 to 3 days, as shown by experiments performed with Macroptilium atropurpureum (Clarkson et al., 1983), canola (Lappartient and Touraine, 1996) and barley (Clarkson et al., 1992). Conversely, a few hours after the restoration of the SO4 " supply to these plants, SO42" uptake capacity declined back to rates that are typical for plants continuously supplied with adequate concentrations of SO4 ". Consistent with the results reported by Smith et al. (1997), withdrawing S from the nutrient solution and re-supplying barley plants with SO42" resulted in concomitant changes (increase and decrease, respectively) in the root uptake capacity and the level o f root hvstl transcripts (Figs. 8, 9 and 11). Though the general pattern of the response to S deprivation was identical to that described by Smith et al. (1997), the levels of hvstl m R N A increased more rapidly in our own experiments: a 5 fold increase occurred within the first two hours following the transfer of plants to S-free nutrient solution and the level 68 of expression plateaued for the duration of the experiment (48 h). B y contrast, in the report by Smith et al. (1997), the amount of hvstl transcripts increased steadily for up to 7 days. These differences in the time-course of the SO42" transporter gene expression during S deprivation may be due to differences in the concentration of SO42" in culture solution (250 u M for Smith et al. (1997) compared to 100 u M in our conditions), to differences in the age of seedlings (15 days in the Smith study compared to 7 days), differences in cultivar and/ or culture conditions (e.g. light, temperature or medium composition). The increase/decrease in the rate of ion uptake in roots is thought to be a response to increased/decreased nutritional demand of the plant. It has been proposed that root ion transporters are normally down-regulated, and that the actual uptake rate depends upon the extent of repression rather than the potentiality of the root for ion uptake (Clarkson and Liittge, 1991; Touraine et al., 1994). Feeding plants with a S-free solution as in the experiments discussed above created a high demand for S, whereas restoring SO42" supply to roots of plants previously deprived of S resulted in decreased level of S demand. Another way to experimentally diminish S demand consists in growing plants on a solution lacking any N source. Indeed, it has been observed in many species that the S/N ratio is remarkably constant, reflecting the proportion of S-amino acids in proteins (Dijkshoom and Van Wijk, 1967). Consistent with this pattern, the roots of plants deprived of N for a few days exhibited lower S 0 4 2 " influx, while, for example, the influx of H2PO4" was only slightly affected or not at all (Lee, 1993; Lappartient and Touraine, 1996). The results obtained in the course of the present study support this coordination of S and N acquisition by plant roots: supplying NO3" or N H 4 + to N-deficient plants rapidly enhanced SO42" influx. This change in the activity of the SO42" transport system in roots was accompanied by parallel changes in the amounts of hvstl transcripts level, suggesting again that a regulation of the expression of this transporter gene is, at least partly responsible for the control of S 0 4 " 69 uptake by the high-affinity system. Experiments performed in canola indicated that the signaling process responsible for the demand-driven regulation of SO42" uptake involved phloem-translocated glutathione (Lappartient and Touraine, 1996), probably in its reduced form, G S H (Lappartient and Touraine, 1997). In the Poaceae (e.g. barley), a priori the situation is more complex than in the Cruciferae due to the presence of a glutathione homologue, namely hydroxymethyl-glutathione, that has been identified in several plant species of this family (Bergmann and Rennenberg, 1993). Hydroxymethyl-glutathione was detected in roots of our barley plants, but glutathione was the predominant thiol. The concentration of glutathione in the root tissues decreased when plants were supplied with S-free nutrient solution (Fig. 10), as already observed by Smith et al. (1997). These authors reported that glutathione concentrations increased when plants previously deprived of sulfur were re-supplied with SO42". In summary, in both situations, hvstl transcripts levels and SO42" influx were negatively correlated with glutathione concentrations in the root tissues. In order to investigate the relationships between the expression of the hvstl SO4 " transporter gene and the nutritional status of plants while growing under steady state conditions • 2 of S supply, sets of barley seedlings were continuously fed with concentrations of SO4 ranging from <2.5 p M to 250 p M . The higher the concentration of SO42" the lower was the transcript level of hvstl gene (Fig. 5). The negative effect of increased external concentration of SO4 " was specially marked at values lower than 50 p M , i.e. within the same order of magnitude as the K m value for SO42" absorption by the high-affinity transport system measured in barley roots (10 p M , (Leggett and Epstein, 1956)) or when hvstl was measured in yeast (6.9 u M , (Smith et al., 1997)). The level o f transcript o f hvstl correlated positively with SO4 " influx from an uptake solution containing 100 p M SO42" (Fig. 5 and 6), a concentration chosen to maximize expression of the high-affinity system and to minimize expression of the low-affinity system. Again, as in the S-70 deprivation experiments, we observed a negative correlation between hvstl expression levels and SO42" influx on the one hand, and the concentration of glutathione in root tissue on the other hand, this latter increasing with external SO42" concentration, especially for lower values of external SO42" (Fig. 7). Thus glutathione is a likely candidate for the role of the regulatory signal that would act by repressing gene expression according to its cellular concentration. The addition of glutathione in the nutrient solution dramatically inhibited the expression of the hvstl gene, to a greater extent than did high external SO42" concentrations or additions of cysteine. Furthermore, the down-regulation of hvstl expression by cysteine partially depended on the synthesis of glutathione from cysteine, since the decrease in the amount of transcripts was partially prevented by B S O , an inhibitor of y-glutamyl-cysteine synthetase (Fig. 12). Although not excluding the possibility that other process(es), such as the positive regulation by O-acetylserine postulated in Escherichia coii (Kredich, 1993), is (are) operating in barley as proposed by Smith et al. (1997), my results clearly implicate glutathione as a major 2 • 2 regulatory molecule involved in SO4 " uptake via repression of the expression of the hvstl SO4 " transporter gene. 71 C H A P T E R 3 T H E I S O L A T I O N A N D C H A R A C T E R I Z A T I O N O F c D N A s BCH3 A N D BCH4 E N C O D I N G P U T A T I V E I N D U C I B L E H I G H - A F F I N I T Y N I T R A T E T R A N S P O R T E R S 72 3.1.1.1 Introduction The absorption of nitrate (NO3") by root cells is driven by at least three, kinetically distinct, thermodynamically active transport systems which coexist in the plasma membranes of root cells. These three systems were characterized on the basis of their differential responses to external NO3" concentrations and by their differential NO3" inducibility (reviewed by Glass and Sidiqqi, 1995; Crawford and Glass 1998). The constitutive high-affinity transport system ( C H A T S ) is a low-capacity, high-affinity transporter which is expressed without the necessity of prior exposure to N 0 3 " (Behl et a l , 1988; Siddiqi et a l , 1990; As lam et al., 1992). This transporter represents the main pathway for NO3" entry into roots from low external NO3" on first exposure to NO3". This entry of NO3" leads to the induction of a high-capacity, high-affinity inducible transport system (IHATS) which can be induced by both NO3" and by NO2" (Siddiqi et a l , 1992; Aslam et al., 1996). A notable feature of the time-course of this induction is that it is typically followed by down-regulation of NO3" influx to a much lower steady-state level. While it is very evident that the induction of the I H A T S is mediated by NO3", the subsequent down-regulation has been attributed by different authors to NO3", N H 4 + , and /or various amino acids (Ingemarsson et al., 1987; Siddiqi et al., 1990; Muller and Touraine, 1992). A t high external NO3" concentration (>200 m M ) a low-affinity transport system ( L A T S ) becomes apparent. This system, like the C H A T S , is expressed in barley plants grown in the complete absence of NO3" and showed no evidence of saturation even at NO3" concentrations as high as 50 m M (Siddiqi et al., 1989). The I H A T S and L A T S transporters are thought to function as proton cotransport systems, with a H + : NO3" stoichiometry equal to or greater than 2 (Ullr ich and Novacky, 1981; McClure et al., 1990; Glass et al, 1992), consistent with the rapid depolarization of the plasma membrane upon exposure of roots to nitrate. 73 A t present, the only genetic information on the C H A T S comes from the isolation of a chlorate-resistant mutant Nrt2 (Chl8) from Arabidopsis thaiiana (Wang and Crawford, 1996). Physiology studies of this mutant showed that plants grown in submerged cultures without NO3" appear to lack expression of the C H A T S . B y contrast, the I H A T S and L A T S activities were normal. Genes that are thought to encode the L A T S and I H A T S transporters have been isolated from various higher plants. In the case of L A T S , this was accomplished by screening A. thaiiana mutants with chlorate (a toxic analog of NO3"). This resulted in the isolation and characterization of the chlorate resistant mutants (Doddema and Telkamp, 1979). O f these mutants, B-l, was affected in nitrate transport (the other loci were involved in M0C0 and N R synthesis). Using the same screening method, (Tsay et al., 1993) isolated the T - D N A tagged mutant that mapped to the same loci as B-l. This was followed by the isolation of the T - D N A tagged chll gene, and the wild-type homolog. The expression of chll cDNA in Xenopus oocytes resulted in the accumulation of nitrate, and depolarization of the oocyte membrane upon exposure to nitrate. These results indicate that the protein encoded by the chll gene mediates NO3" transport (Tsay et a l , 1993). Physiological analysis of the Chll (Chll-5) deletion mutant by (Touraine and Glass, 1997) and A. thaiiana transformed with chll under the control of the 35 S promoter (Huang et al., 1996) was interpreted to indicate the existence of two low-affinity nitrate transport systems in A. thaiiana. These transport systems and/or their respective genes are possibly differential regulated by ammonium. Currently there is a debate involving the role of chll gene in high-affinity transport of N 0 3 " (inducible, L i u et al., 1999; constitutive, Wang et al., 1998). The first inducible high-affinity nitrate transporter in eukaryotes was cloned from Aspergillus nidulans (Unkles et al., 1991), The crnA mutant (Tomsett and Cove, 1979) was defective in NO3" uptake in conidiospores and young mycelia (Brownlee and Arst, 1983). The 74 crnA gene, which was able to restore NO3" uptake in this mutant (Unkles et al., 1991), encodes a protein that is 507 amino acids, contains 12 putative membrane-spanning regions and belonging to major facilitator superfamily (MFS) (Trueman et al., 1996). The M F S is a superfamily of membrane proteins, which contains two conserved amino acid motif of ( D / N ) R X G R ( R / K ) and IX2RX3GX3G between membrane spanning domains 2 and 3, (Henderson, 1991). A number of genes that are homologous with crnA has been cloned from other eukaryotes. These include yntl from Hansenula polymorpha (Perez et al., 1997), nrt2.1Cr and nrt2.2Cr from Chlamydomonas reinhardtii (Quesada et al., 1994), bchl and bch2 from Hordeum vulgare (Trueman et al., 1996), nrt2Np from Nicotiana plumbaginifolia (Quesada et al., 1997), nrt2Gm from Glycine max (Amarashinghe et al., 1998) and nrt2.1At and nrt2.2At from A. thaliana (Zhuo et al., 1999; Filleur and Daniel-Vedele, 1999). A l l of the above are thought to encode high-affinity nitrate transporters and belong to the M F S . In barley the isolation of a D N A fragment (bcrna) by P C R using oligonucleotides directed at a conserved M F S motif led to the isolation of the first bchl and bchl genes, encoding putative N03 _-inducible high-affinity transporters in higher plants (Trueman et al., 1996). Northern blot analysis using roots of nitrogen-starved barley plants showed that bch transcript accumulated rapidly in response to NO3" provision (Trueman et al., 1996). This is in agreement with physiological data which has shown that NO3" influx can increase up to -30- fold in the high-affinity range upon NO3" treatment (Siddiqi et al., 1989). Subsequently, in both N. plumbaginifolia and A. thaliana, levels of respective transcripts (nrt2.1Np and nrt2.1At), decreased when the nitrate supply was maintained beyond the period of peak induction (Krapp et al., 1998; Zhuo et al., 1999). This pattern of expression correlates with the overshoot of high-affinity NO3" transport and subsequent decline to a lower steady-state level (Siddiqi et al., 1990). Reduced N-forms, for example N H 4 + or glutamine, which are known to diminish nitrate uptake 75 when applied in the presence of NO3", decreased nrt2. INp and nrt2. lAt transcript levels in roots of N. plumbaginifolia and A. thaiiana, respectively (Quesada et al., 1997; Krapp et al., 1998; Zhuo et al., 1999). B y use of metabolic inhibitors, particularly methionine sulfoximine ( M S X ) and azaserine ( A Z A ) , which block the enzymes glutamine synthetase and G O G A T , respectively, it was established that both N H 4 + and glutamine were active in the down-regulation of nrt2.1At expression (Zhuo et al., 1999). In barley, the genome organization may allow for the presence of multiple members of the bch gene (7-10 possible members, (Trueman et al., 1996)). Due to the considerable physiological data available in this species, barley is an important model system in which to investigate the mechanism of transcriptional regulation of NO3" transport. In this chapter we describe the isolation of two new c D N A s , bch3 and bch4 that are closely related to bchl and bch2, and the isolation of the 5' upstream region of bchl, bch2 and bch3. We have also characterized the expression pattern of the bch family of genes in response to the provision of various N sources and dissected the involvement of the intermediates of NO3" assimilation by use of specific inhibitors of key enzymes of this pathway. In order to integrate this molecular information with patterns of ion transport, we have measured NO3" influx, using NO3"', in parallel experiments. 3.2.1.1 M a t e r i a l s a n d m e t h o d s 3.2.1.2 P l a n t m a t e r i a l Seven-day-old seedlings of Hordeum vulgare cv. Klondike were used in all experiments. Seeds were surface sterilized with 1% commercial bleach solution and rinsed with de-ionized H 2 O . The seeds were placed on to a nylon mesh (pore size, 4 mm) which was fixed onto 20 mm (8 seeds) 76 or 60 mm (25 seeds) plexi-glass discs, depending on the experiment. The discs were placed in moist sand, the seeds being covered with 10 mm of moist sand, in the dark. After 3 d the seedlings were transferred to 40 L hydroponic tanks, and grown in N-free 1/10 strength modified Johnson's solution (Siddiqi et al., 1991). According to the experiment, N was supplied in the form of NO3*, NO2", or N H / . Potassium concentrations were monitored daily and K + and other nutrients resupplied to restore their concentrations. The p H of the solution was maintained at 6.2 ± 0.3 by the addition of excess CaC03 powder. Plants were maintained in a controlled environmental chamber with a 16 h/8 h light dark cycle at 20 ± 2°C and 70% relative humidity. Light (photon flux density at plant level of 300 pmol m" 2 s"1) was provided by fluorescent tubes with a spectral composition similar to sunlight. 3.2.1.3 R N A and D N A isolation. Total R N A was isolated using Tr iZol Reagent (Life Technologies, Gaithersburg, M D ) , with two modifications. Firstly, after the tissue was ground in a mortar and Trizol reagent was added at a ratio of 0.2 g tissue/ 1 m l Trizol , the homogenate was centrifuged at 8000 xg for 30 min to remove cellular debris. Secondly, after the total R N A was isolated, it was again extracted with phenol:chloroform:iso-amyl alcohol (25:24:1), and precipitated with 0.3 M sodium acetate (final concentration), and two volumes of ethanol. FastTrack m R N A isolation K i t (Invitrogen, Carlsbad, Ca) was used in the isolation of m R N A , as per manufacturer's instructions. Genomic D N A was isolated as described by Asubel et al. (1995). 77 3.2.1.4 cDNA and genomic library Messenger R N A isolated from roots of 7-day old barley seedlings, treated for 2 and 6 h with 10 m M K N O 3 , were used as template for c D N A synthesis. A Marathon c D N A synthesis kit (Clontech, Palo Al to , Ca) was used for the construction of a c D N A library. A PromoterFinder D N A Walking kit (Clontech, Palo Alto , Ca) was used for the construction of a barley genomic D N A library. 3.2.1.5 Northern blot analysis Total R N A was separated on a 1.2% agarose gel containing I X M O P S with 2.2 M formaldehyde, at 60 V for 3.5 h, then washed twice in H2O, then R N A was transferred by capillary action to an N+ nylon membrane (Amersham, Piscataway, NJ) . The membrane was baked for 2 h at 80°C to fix the R N A , then it was placed in prehybridization solution for 1 h or 4 h (random labeled probe or oligonucleotide probe, respectively). Membranes were then placed in hybrization solution with 3 2 P labeled probe for 12-16 h. For random labeled probes prehybridization solution and hybridization solution were 6 X SSC, 5 X Denhardt's solution, 0.5% SDS and 20 pg ml" 1 sonicated herring sperm D N A , respectively. Random labeled probes were made with Prime-A-Gene kit (Promega, Madison, WI) using internal fragment from bch3 gene from plasmid p B C F B by digestion with EcoRV and AfllR. Control levels o f total R N A were probed with a fragment of the 25S gene, on plasmid pV25S, by digestion with Xhol. Membranes were washed according to manufacturer's instructions with 0.25 S S C and 0.1 % SDS at 42°C for 15 min for the final wash. Oligonucleotide probing, prehybridization solution and hybridization solution consisted of 50% formamide, 6 X SSC, 0.01% SDS and 0.05 mg/ml sonicated herring 78 sperm D N A . Prehybridization was done at 37°C for 4 h, while hybridization was for 12-16 h. The washing of the membrane consisted of 2 X 15 min washes at R T with 2 X SSC and 0.05% SDS. The oligonucleotides used as probes were D X 46: 5' C T G T A G T T C A G T A C T T G T A C A T A G G for bchHl gene, D X 4 8 : 5 ' C A C T G T A C G T G T A C A C A G G T A A A G for bchl; B C H 3 : 5 , G G T C C A A A T G G A G G T G G A G G for bch3; and B C H 4 : 5' C A A A A T T T G A A A C T T A T A C G T G T A G G for bch4. End-labeling of oligonucleotides used T4 D N A kinase (Life Technologies, Gaithersburg, M D ) and 3 2Poc A T P (Amersham, Piscataway, NJ) . G-25 spin columns (Pharamacia, Montreal, Que) were used for separation of unincorporated P a A T P from the reaction mixture. 3.2.1.6 Isolation and screening of bch3 and bch4 c D N A s by R A C E P C R The isolation of bch3 was by 5' and 3' R A C E P C R . Oligonucleotide bcnra-7: 5 ' G T A T G G G T G T G C C T T C C T was used for the 3' prime race. For 5' R A C E P C R and isolation of a full length c D N A bch3, 5 ' T G C C T T A T A C C T G C T G C T G G G G T G was used. The c D N A template was fabricated using the marathon c D N A Ki t . 5' and 3' R A C E P C R conditions were 7 min at 94°C then 35 cycles of 94°C for 45 s, 62°C for 45 s, with a 5 min extension period at 72°C. The isolation of bch4 was by 5' and 3' R A C E P C R . The oligonucleotide DZ44: 5' G G A C T A G C A G C G G G T was used in the initial 3' R A C E - P C R . R A C E - P C R conditions were 94° for 7 min, then 35 cycles of 94°C for 45 s, 50°C for 45 s, and 72°C for 4 min. The P C R reaction products were purified, then separated on a 1.2% agarose gel. The digested D N A was then transferred to nylon membrane (N+ hybond, Amersham, Piscataway, NJ) for Southern analysis. Positive P C R products were cloned into pCR2.1 (Invitrogen, Carlsbad, Ca) and 79 sequenced. Oligonucleotide bch4: 5' C A A A A T T T G A A A C T T A T A C G T G T A G G , was used for the isolation of 5' R A C E P C R product. Ful l length clones for bch3 and bch4 were generated by P C R using the Expand high-fidelity P C R system (Boehringer Mannheim, Montreal, Que). For bch.3, oligonucleotides used were bch3-5, 5 ' G G T C C A A A T G G A G G T G G A G G and bch3, while for bch4, oligonucleotides bch4-5, 5' C T C A G T A G A T A T G G A G G T G A G G C and bch4 were used. The full length P C R products were subcloned into pCR2.1 (Invitrogen, Carlsbad, Ca). After restriction endonuclease analysis o f resulting c D N A s , resulting overlapping fragments were subcloned into pBlueScript I KS+ (Stratagene, L a Jolla, Ca). Sequences were determined on both strands using M l 3 forward and reverse primers. For regions where subclones could not be generated specific oligonucleotides were designed for sequencing. 3.2.1.7 Isolation of promoter sequences of bchl, bch2 and bch3 PromoterFinder D N A walking kit (Clonetech, Palo Alto , Ca) was used for the isolation of the upstream region of bchl, bch2 and bcKh. Specific oligonucleotides were designed to hybridize with the 5' U T R . For bchl the oligonucleotides were G B 1 : 5 ' C A A C A A C T A G A A G C A G C T A A T G G T G G C and GB1-2: 5 ' G T T G C A G C T C T T G A G C T T G G C T T G C A A ; for bch2 G B 2 : 5 ' T C G A G C T A G C T A G C T T A G T C G C A C T G G and GB2-2: 5 ' G T G T G T C T T T A A T G G T G G T T G C T G C T G ; for bch3 G B 3 : 5 ' G G A C C T T G C T T G A T C G A G C T A G T C T C C and GB3-2: 5' G G A G C T A G C T T G C T T G A T C A G C T G C A G . A l l P C R reactions used the Expand Long P C R system (Boehringer Mannheim, Montreal, Que). For the first round of P C R the oligonucleotide A P I was used (PromoterFinder D N A walking kit) with G B 1 , G B 2 or G B 3 , at 92°C for 3 min, then 30 cycles of 92°C for 25 s, 65°C for 30 s, and 68°C 80 for 10 min. The amplicon was then diluted 1/10 then a second round of P C R (nested) was conducted using A P 2 (PromoterFinder D N A walking kit) and GB1-2 , GB2-2 or GB3-3 . The resulting P C R products were purified using Gene Clean K i t ( B I O L A B S , Mississauga, Ont), and cloned into pCR2.1 (hivitrogen, Carlsbad, Ca. 3.2.1.8 Nitrate influx Nitrate influx experiments were carried out essentially as described by Siddiqi et al. (1989). Barley plants were grown on sand for 3 days than transferred to hydroponic tanks for 4 days, and treated according to experimental design. Plants were then transferred to 0.5 L vessels containing unlabeled uptake solution for 5 min so as to bring the apoplasm to the same NO3" concentration as was used for the influx determination. After this period they were transferred to 0.5 L vessels containing 50 u M NO3" influx solution labeled with 13N03~ for a period of 5 min. Thereafter, plants were transferred back to the 0.5 L vessel of unlabelled nutrient solution for 3 min to remove tracer from the cell wall . Roots and shoots were harvested separately, and placed into 20 m L scintillation vials for counting in a Packard y-counter (Minaxi 8, Auto-y 5000 series). Production of 13N03~ was as described by Kronzucker et al. (1995). 81 3.3.1.1. Results 3.3.1.2 Isolation of bch3 and bchA c D N A s The isolation of bch3 (acc. no. AF091115) was accomplished by the use of R A C E - P C R . Oligonucleotides directed to the bcrna fragment (Trueman et al., 1995) were designed and subsequently used for 5' and 3 ' R A C E P C R . The sequencing of the 3' R A C E P C R product indicated a new member of the bch family of genes in barley, which was designated as the 3BCH3 fragment. The 5' R A C E - P C R product was a contaminant of bchl, therefore, a new oligonucleotide was designed based on sequence data from 3' U T R of the 3 B C H 3 fragment. 5' R A C E P C R resulted in the isolation of a full length c D N A . The c D N A sequence revealed that it was a new member of the bch family of genes in barley. This c D N A , which is 1822 bp in length, was designated as bch3. A different strategy was used in the isolation of the bch4 (acc. no. AF091116) c D N A . I designed an oligonucleotide encoding protein consensus sequences found in the nrtl.lCr, nrt2.2Cr, bchl and bch2 c D N A s . This consensus sequence represents amino acid position 166 to 174 of the B C H 1 protein. This amino acid motif has the sequence G L A A G W G N M , which is conserved among the nitrate/nitrite subgroup of the M F S (Trueman et al., 1995). The c D N A library used for 3 ' R A C E - P C R was digested with a number of restriction endonucleases that digest within bchl and bch2 c D N A s . This was done in order to remove the possibility that bchl and bch2 c D N A s would be amplified. The 3 ' R A C E - P C R products were transferred to nylon membrane and probed with bchl c D N A at medium stringency. The resulting R A C E - P C R products that hybridized with the bchl probe were cloned and sequenced. The sequencing data indicated that one of the R A C E - P C R products was another member of the bch gene family in 82 barley. This fragment is designated 3 ' B C H 4 . A n oligonucleotide specific to the 3' U T R of this gene was designed and synthesized. 5' R A C E P C R was carried out resulting in the isolation and cloning of bch4 c D N A . This c D N A is 1705 bp in length. 3.3.1.3 P r o t e i n s t r u c t u r e , genet ic ana lys i s , a n d c o m p a r i s o n s o f the n u c l e o t i d e a n d p r o t e i n sequences The predicted B C H 3 and B C H 4 proteins are 507 amino acids in length with a predicted m.w. of 54.6 kDal using Compute pI /Mw program at Expasy Server (Wilkins et al., 1998). The predicted p l of the B C H 3 and B C H 4 proteins are 8.21 and 8.54, respectively (Compute pI /Mw program). Using SOSUI program (Hirokawa et al., 1998) both proteins contain 12 putative membrane spanning regions, and have the M F S conserved sequence of ( D / N ) R X G R ( R / K ) and IX2RX3GX3G (Henderson, 1991; Marger and Saier, 1993). We analyzed the predicted protein sequences of B C H proteins with P R O S I T E program (Bairoch et al., 1997), and found possible sites for post-translation modifications (Figure 15). B C H 3 and B C H 4 proteins have three possible protein kinase C phosphorylation sites (Woodgett et al., 1986; Kikkawa et al., 1988) at position 28 to 30, 381 to 383, and 484 to 486, with the residues composition of SFR, SRR, and SER. Also B C H 3 and B C H 4 proteins have three casein kinase II sites (Pinna, 1990) at positions 453 to 456, 463 to 466, and 482 to 485, with the residues composition of T E E E , S E E E , and SRSE. The phosphorylation sites are all on the predicted cytoplasmic face of the B C H proteins. These phosphorylation sites were also present in B C H L The predicted localization of B C H 3 and 83 BCH3 BCH1 BCH4 BCH2 NRT2.INp NRT2.lAt NRT2Gm consensus BCH3 BCH1 BCH4 BCH2 NRT2.INp NRT2 . lAt NRT2Gm consensus 138 138 13S 14 0 161 161 161 161 FVSCQYWMSTMFNSK FVSCQYWMSTMFNSK FVSCQYWMSTMFNSK FVSCQYWMSTMFNSK 'FVSCQYWMSTMFNSK „ GFglATFVSCQYWMSTMFNSgllGgVNGjlAAGWGNMGGGQTQLlM IGFSLATFVSCQYWMSTMFNSKIIGBSNGBAAGWGNMGGGATQLIM: GWGNM GATQLIMPL 5 E BCH3 316 BCH1 i i i BCH4 218 BCH2 220 NRT2 . INp 241 NRT2.lAt 241 NRT2GH1 241 consensus 241 BCH3 296 BCH1 2 9 S BCH4 298 BCH2 300 NRT2.INp 321 NRT2.lAt 321 NRT2Gm 321 consensus 321 BCH3 377 BCH1 376 BCH4 378 BCH2 360 NRT2.INp 401 NRT2.lAt 401 NRT2Gm 401 consensus 401 BCH3 454 BCH1 455 BCH4 455 BCH2 457 NRT2.INp 4 8 1 NRT2.lAt 481 NRT2Gm 4B1 consensus 48 1 LVLTMGQDLPDGNLASLQKRGDMAKDKFSKVLWGAVTNYRTWI LVLTMGQDLPDGNLASLQKBGDMAKDKFSKVLWGAVTNYRTWI LVLTMGQDLPDGNLASLQKMGDMAKDKFSKVLWGAVTNYRTWI LVLTMGQDLPDGNIASLQKBGGWKDKFSKVGWGAVTNYRTWI LVLTjGQDLPDGNJffl^QKJGgvgKDKF2Ji;LWBAgTNYRTWI LVLiffiGQDLPDGN^JljKfflG|KKDKF|KlLW^VTNYRTWI I.VLTEGQDLPDGK I ediBm '^VAKDKFSKVLWRAITNYRTWI FVLLYGYCMGVELTTDNVIAEYYFDHFHLDLRBAGTI FVLLYGYCMGVELTTDNVIAEYYFDHFHLDLRHAGTI FVLLYGYCMGVELTTGNVIAEYYFDHFHLGLR^GTI FVLLYGYCMGVELTTDNVIAEYYFDHFHLDLR^SGTI FVLLYGYgMGVELgTDNVIAEVFFDRFiJl.g: R«A -H FVLLYGY^GVEL|[TDNVIAEY^DHFHLELKAGBI FSLLYGYHMGVELTTDNV I AE YFYDR FISI S i .HBACQ I PFVSRRSLGIISGLTGAGG PFVSRRSLGIISGLTGAGG PFVSRRSLGIISGLTGAGG PFVSRRSLGIISGL[GAGG PF|SRRSLGIISG§TGAGG PFVSRR[LGI I SGLTGAGG PFLSRRSLGIISGLTGAGG Figure 15. Predicted amino acid sequences of BCH3 and BCH4 with alignment of 8 full length sequences representing other inducible high-affinity nitrate transporters BCH1, BCH2, NRT2.1Np, NRT2.1At, NRT2Gm, CRNA, YNT1 and NRT2.1Cr. The alignment was made using Clustal W1.7 with Blosum 62 weight matrix (Thompson et al., 1994) at BCM launcher server (Baylor College of Medicine, Texas, USA). 84 Figure 16. Phylogeny of predicted amino acid sequences of inducible high-affinity nitrate transporters. The phylogeny was obtained by using the GenTree program (Gonnet, 1994) with a) B C H 1 (acc. no. U34198), b) B C H 2 (acc. no. U34290), c) B C H 3 (acc. no. AF091115), d) B C H 4 (acc. no. AF009116), e) NRT2 .1At (acc.no. Z97058), f) NRT2 .1Np (acc. no. Y08210), g) N R T 2 G m (acc. no. AF047718 ), h) C R N A (acc. no. U34382) i) Y N T 1 (acc. no. Z69783) and j) NRT2.1Cr (acc. no. Z25438) 85 B C H 4 proteins, using the P S O R T program (Nakai and Kanehisa, 1992), was the plasma membrane. I analyzed and compared the protein and nucleotide sequences of the four known members of the bch multi-gene family, in comparison to each other and to other nitrate transporters. Figure 15 shows the alignment of the predicted protein sequences, while Figure 16 shows the phylogenetic relationship of the four predicted B C H proteins in comparison to the other nitrate transporters N R T 2 N p , NRT2.1At , NRT2.1Cr , N R T 2 G m , Y N T 1 and C R N A . The amino acid sequences of the predicted proteins show considerable identity (>87%) among the different barley members, with B C H 2 being the most divergent. The highest level of nucleotide sequence divergence was observed at 5' and 3' U T R s of all four c D N A s . In comparing the predicted protein sequence of bch genes from barley with the other plant nrt2 genes, we found a stretch of 21 amino acids near the amino terminus of the N R T 2 proteins (encoded by the c D N A s nrt2Gm, nrt2.1At and nrt2.1Np) which was not present in the B C H proteins. Analysis o f this protein sequence predicts a possible protein kinase C phosphorylation site (TGR), and/or a casein kinase H phosphorylation site (TGRE) (Woodgett et al., 1986; Kikkawa et al., 1988; Pinna, 1990). 3.3.1.4 Isolation, analysis and comparison of promoter regions for bchl, bch2 and bch3 The promoter region of bchl, bch2 and bch3 ( D N A fragments of 535, 635 and 1436 bp in length, respectively) was isolated by the use of a Genome Walk kit (Clontech, Palo Alto , Ca). The T A T A boxes were located at -48, -37 and -45 for bchl, bch2 and bch3, respectively of the translation start site. In comparing the promoter sequences of bchl, bch2 and bch3,1 found that bchl has 65% homology with bch3, and 58% homology with to bch2. In comparison, bch2 had 86 53.2% homology with bch3. One stretch of D N A present in the promoter sequences of bchl, bch2 and bch3, was found to be highly homologous, (domain I) 16/19 (84.2% identity) with the consensus sequence T G A T T C C G T N N G N T G C A A T . If the areas adjacent to domain I of bchl and bch3 are compared, this domain increased both in D N A size and homology 29/33 (87.8% identity). Another stretch of D N A , domain U , with 62/69 identical nucleotides (89.8% identity) was also identified. Analysis of the sequence for the presence of a core sequence of a putative cis-acting element involved in nitrate induction of genes encoding nitrate reductase in Arabidopsis thaiiana (Hwang et al., 1997), revealed that this AT- r i ch sequence, preceding the A ( G / C ) T C A core sequence, was not present in the promoter sequences of the bch promoters, but the core sequence of this element was present in all bch promoter sequences, bchl has one copy of the core sequence at -430; bch2 has 2 copies at -120 and -299; bch3 has four copies at -684, -764, -994 and -1267 from the transcription start site. 3.3.1.5 T i m e p r o f i l e o f NO3" i n d u c t i o n o f bch m u l t i - g e n e f a m i l y m R N A a c c u m u l a t i o n a n d N 0 3 " i n f l u x The effects of two NO3" concentrations, (1 m M and 10 m M ) on the expression of the bch multi-gene family and NO3" influx were investigated for various pretreatment times (0 to 48 h). Northern blot analysis, using an internal fragment of bch3 (recognizing all members of the bch family), showed that 1 m M nitrate induced the accumulation of bch transcript in roots to their highest level within 3 hours of treatment; thereafter, transcript levels steadily decreased to undetectable levels by 24 h (Figure 17). The same overall pattern was observed for the 10 m M nitrate treatment bch m R N A accumulation peaked at 6 h, and then decreased steadily to 87 undetectable levels by 24 h (Figure 18). In short-term experiments, in which plants were supplied with 10 m M NO3", bch transcript accumulation was observed within 30 min of the onset of NO3" treatment (Figure 19). However, transcript was not apparent in shoots. In parallel, influx measurements, using 50 u M external NO3" to measure high-affinity NO3" influx (Figure 20), a similar pattern: was observed, whereby influx increased 20 fold from the onset of 1 m M NO3" pretreatment to a maximum value at 9 h, then slowly decreased until the end of the experiment (48 h). Nevertheless, even at 48 h, influx remained relatively high despite the fact that transcript abundance had decreased to undetectable levels. This may indicate the participation of other transport systems to the measured influx. The pattern of response to pretreatment with 10 m M NO3" was essentially similar to the 1 m M pretreatment (Figure 20). To specifically investigate the expression of each of the known bch c D N A s , oligonucleotide probes directed to the 3' U T R were designed and used in Northern blot analysis. Figure 21 shows the overall accumulation of specific bch m R N A in N-starved plants fed with 1 m M NO3" for 0, 3, 6, 9, 12, 24, and 48 h: with accumulation of the specific transcripts in roots peaking at 3 h, then a diminishing to undetectable levels by 12-24 h for bchl, bch2 and bchS. B y contrast, bch4 transcript levels, which increased within 3 h of NO3" feeding, remained at elevated levels for the duration of the experiment (48 h). 88 Nitrate pretreatment (h) 0 3 6 9 12 24 48 t t ••••••• B) Pretreatment nitrate (h) Figure 17. Time course of NO3" treatment on bch m R N A accumulation. 7 day-old seedlings of barley grown in N-free Johnson's solution then supplied with 1 m M NO3" for: 0, 3, 6, 9, 12, 24, and 48 h. 20 pg of total R N A was introduced into each lane. Northern blots were probed with the bch3 probe (EcoRV and AflDl fragment) and 25S ribosomal subunit was to ensure equal loading of R N A . B) Quantification of bch transcript levels in roots using densitometry. Transcript levels were normalized to 3h nitrate treatment. 89 Nitrate pretreatment (h) 0 2 6 12 24 48 BCH Figure 18. Time course of NO3" treatment on bch m R N A accumulation. 7 day old seedlings of barley grown in N-free Johnson's solution then supplied with 10 m M NO3" for: 0, 2, 6, 12, 24 and 48 h. 20 pg of total R N A was introduced into each lane. Northern blots were probed with the bch3 probe (EcoRV and AflHl fragment) and 25S ribosomal subunit was to ensure equal loading of R N A . 90 A) 1 2 3 4 5 6 7 8 9 BC" l I f f I 25S | ^ ljp ^ | fgf N 0 3 - t i m e ( h ) 0 0.5 1 2 4 6 0 2 6 root (R) or shoot (S) R R R R R R S S S treatments Figure 19. Time course of NO3" treatment on bch m R N A accumulation. 7 day-old seedlings of barley grown in 1/10 strength N-free Modified Johnson's solution then supplied with 10 m M NO3": Lanes 1-6 root for 0, 0.5, 1, 2, 4, and 6 h; Lanes 7-9 shoots 0, 2 and 6 h. 20 pg of total R N A was introduced into each lane. Northern blots were probed with the bch3 probe (EcoRV and Afllll fragment) and 25S ribosomal subunit was to ensure equal loading of R N A . B) Quantification of bch transcript levels in roots and shoots using densitometry. Transcript levels were normalized to 2 h nitrate root treatment. 91 Figure 20. Time course of NO3" treatment on NO3" influx. 7day-old seedlings of barley grown in 1/10 strength N-free Modified Johnson's solution then supplied with 1 m M (•) or 10 m M NO3" ( • ) . Influx were measured at 50 p M for 5 min (4 replicates each time point with error bars = ± 1 S.D). 3.3.1.6 E f f e c t o f v a r i o u s e x t e r n a l NO3" c o n c e n t r a t i o n s o n bch t r a n s c r i p t levels a n d NO3" i n f l u x In order to investigate the effect of external NO3" concentrations on bch transcript levels and N 0 3 " influx under steady-state culture conditions, barley plants were grown in hydroponic solutions containing 0, 10, 50, 100 and 500 p M NO3" for 4 d. Both bch transcript levels (Northern blot analysis Figure 22) and 13NC>3" influx from 50 p M NO3" (Figure 23) were subsequently monitored. Nitrate influx varied from 0.54 pmol g"1 F W h"1 in plants maintained at 0 external NO3" to its maximum rate (5.32 pmol g"1 F W h"1) for 50 p M NO3" plants. A t higher NO3" concentrations influx decreased to 3.15 pmol g"1 F W h"1 in plants maintained at the 500 p M level. The levels of bch transcript in the roots followed the same pattern. 3.3.1.7 T h e effect o f N 0 2 " a n d N H 4 + o n t r a n s c r i p t levels o f bch m u l t i - g e n e f a m i l y Figure 24 shows the effect of 10 m M N H 4 + on the accumulation of bch transcript by 10 m M NCV-treated barley seedlings. B y 2 h after providing N H 4 + and NO3" together, no effect on bch transcript accumulation was observed, compared to plants supplied NO3" only. However, by 13 4 and 6 h respectively, little transcript was detectable. In parallel, we measured NO3" influx at 50 p M NO3" in plants co-supplied with NO3" and NH4 for 6h. NO3" influx in these plants was ~2-fold higher than N-starved plants (uninduced) levels (0.97 ± 0 . 1 3 compared to 0.48 + 0.12 pmol g _ 1 F W h"1). Plants pretreated only with 10 m M N H 4 + as a displayed no increase in bch transcript levels compared to N-starved control, with influx varying from levels (0.42 ± 0 . 1 3 compared to 0.58 ± 0.12 pmol g _ 1 F W h"1 for the duration of the experiment). 93 Nitrate pretreatment (h) 0 3 6 9 12 24 48 B C H f i t * 25S H P fjlji life A f t A f t BCH2 BCH3 BCH4 Figure 21. Northern blot analysis, o f known members of the bch family of genes. Time course profile of 1 m M N 0 3 " pretreatment on N-starved plants for 0, 3, 6, 9, 12, 24, and 48 h. 20 pg of total R N A was introduced into each lane, and probed with bch3 internal fragment (EcoRI - AflUT) then washed with medium stringency, or 25S to show equal loading of R N A , or probed with specific oligonucleotides to bchl, bchl, bch3, and bch4 transcripts. 25S and oligonucleotide probes were washed at high stringency. 94 A) External [N03"] 0 10 50 100 500 ••••••• -„*->-3±- jMsfMm&i 25S B ) 0 10 50 100 500 External NO," mi Figure 22 . Effect of external N03~concentration on bch mRNA accumulation. 7 day-old seedlings of barley grown for 4 d in 1/10 strength N-free Johnson's solution containing 0, 10, 50, 100, or 500 uM KN03. 20 ug of total RNA was introduced into each lane. A) Northern blots were probed with the bch3 probe (EcoRW and Afllll fragment) and 25S ribosomal subunit was to ensure equal loading of RNA. B) Quantification of bch transcript levels in roots using densitometry. Transcript levels were normalized to 50 uM nitrate treatment. 95 6 I External N0 3 " [uM] Figure 23. Effect of external MLVconcentration on NO3" influx. 7 day-old seedlings of barley grown for 4 d in 1/10 strength N-free Modified Johnson's solution containing 0, 10, 50, 100, or 500 u M KNO3". Influx was measured at 50 u M for 5 min (4 replicates each concentration point with error bars = ± 1 S.D). 1 2 3 4 5 6 7 9 10 BCH 25S N C y N H 4 + N 0 2 " duration • m i t i i i t J t t J j £ j g k + + + + + + + + + + + 2 4 6 2 4 6 4 6 B ) 100 rt S 60 Cd 40 -| 1 2 3 4 5 6 7 8 9 10 treatments Figure 24. Effect of N02" alone or providing N H 4 + together with N03" on bch transcript abundance. Total RNA from barley roots, lane 1: plants grown in 1/10 strength N-free Modified Johnson's solution; lanes 2, 3 and 4: 10 mM N03" -supplied plants for 2, 4, and 6 h, respectively; lanes 5, 6, and 7: 10 mM N H 4 + and 10 mM N03" co-supplied plants for 2, 4, and 6 h, respectively; and lanes 8, 9, and 10: 10 mM N02"-supplied plants for 2, 4, and 6 h, respectively. 20 ug of total RNA was introduced into each lane, and probed with bch3 internal fragment (EcoRI-Afllll) washed with medium stringency. Northern blot was probed with the 25S ribosomal subunit was to ensure equal loading of RNA. B) Quantification of bch transcript levels in roots using densitometry. Transcript levels were normalized to 2 h nitrate treatment. 97 3.4.1.1 D i s c u s s i o n 3.4.1.2 C h a r a c t e r i z a t i o n o f bch c D N A s a n d p r o m o t e r r e g i o n s In this study R T - P C R and R A C E - P C R were employed for the isolation of two c D N A s (bch3 and bch4) which encode new putative high-affinity nitrate transporters from barley roots. The coding regions of these c D N A s are highly conserved in comparison to each other and to bchl and bch2 (Trueman et al., 1996), with greater than 87% identity at the protein level. B C H 3 and B C H 4 both have the conserved motif of the major facilitator superfamily, and possibly protein kinase C and casein kinase II phosphorylation sites. The location and number of sites are constant for all the known B C H proteins, and the sites appear to be located on the cytoplasmic face of the protein. The major difference between the B C H proteins and the N R T 2 proteins from Nicotiana plumbaginifolia, Arabidopsis thaliana, and Glycine max, is a deletion of 21 amino acids in the B C H proteins at the amino terminus. In this deletion, are located proteins kinase C and casein kinase II phosphorylation sites. This may indicate that there are differences in post-translation modification of B C H proteins compared to the other N R T 2 proteins. The role of these phosphorylation sites was not assessed in this study, but it is interesting that protein kinase C and casein kinase II sites were present in other known B C H proteins, and also present in the N -terminus (21 amino acid sequence) of other N R T 2 proteins. In barley, Southern blot analysis demonstrated that there are 7 to 10 homologues (Trueman et al., 1996), while in Nicotiana plumbaginifolia, A. Thaliana and in soybean, there are only two copies of the gene (Quesada et a l , 1997; Zhuo et al., 1999; Amarashinghe et al., 1998). Why barley should possess 7 to 10 copies of this gene family is unknown. 98 Analysis of the 5' upstream region of bchl, bchl, and bchl, revealed that these sequences are less conserved (>53% homology), than the coding sequence (89% homology), One region of D N A (domain I) was conserved in the promoter sequences of bchl, bch2 and bch3. Also present was domain II, a region of D N A comprising 69 Bp with a homology of 89%. The roles of these sequences are unknown, but they may function in the regulation of the bch genes. Analysis of the sequence for the presence of an N I E element, found to be implicated in nitrate induction of the N R genes in Arabidopsis thaliana (Hwang et al., 1997), established that the core sequence of the element, without the preceding AT-r ich region, was present in multiple copies in the promoter sequence of bch genes. In narl and nar7 genes, which encode N R in barley, only the core sequence is present (Hwang et al., 1997). If this element is functional in barley, it is interesting to note the loss of the A T rich region, given that barley has a genome which is CG-r ich compared to the AT- r i ch Arabidopsis. 3.4.1.3 T i m e p r o f i l e of bch t r a n s c r i p t levels a n d n i t r a t e t r a n s p o r t The substantial increase of high-affinity NO3" uptake following first exposure to NO3" has been referred to as nitrate induction (Jackson et al., 1973; Goyal and Huffaker, 1986). This process typically increases rates of net NO3" uptake by 5 fold (Warner and Huffaker, 1989). Using the Klondike variety of barley, and 1 3 N03~ to measure plasma membrane influx (Siddiqi et al., 1989) demonstrated a -30 fold increase. It is evident that the extent of increase is to a large extent governed by the constitutive value of C H A T S influx and the induced flux. In Steptoe barley (King et al., 1993), was much higher than in Klondike. In the present experiments, using 1 m M or 10 m M NO3" treatments, bch transcript levels increased with the same time-dependence pattern. This involved increases of transcript during the first 2-12 h, followed by declines to a uii-99 13 detectable levels. NO3" Influx on plants treated in a similar fashion increased during the first 6-12 h then plateaued to a level o f 75- 80% of the maximum influx value. This indicates that other nitrate transport system are involved in the high-affinity uptake. Plants pretreated at 10 m M NO3" had lower values of influx than those treated with 1 m M NO3" and this probably reflects greater down-regulation by various internal N pools associated with larger internal N pools in the former treatment (Siddiqi et al., 1990). This decline of bch transcript has been shown for other c D N A s encoding putative nitrate transporters when plants were treated to long exposure to NO3" >12 h as in Nicotiana (Quesada et al., 1997) and Arabidopsis (Zhuo et al., 1999). The investigation of effects of NO3" treatment on individual members of the bch family of genes revealed that these genes are coordinately induced in root tissue in approximately the same pattern as observed in using a bch probe, which recognized all members. However, this result does not preclude maybe differential expression vis-a-vis different cell types o f the root (e.g. endodermis, stele, root hairs etc.). In tomato, the nrtl.lLe c D N A which encodes a putative low-affinity nitrate transporter, transcript is predominately localized in the root cylinder, while nrtl.2Le is predominately localized in root hairs (Lauter et al., 1996). Also , nrt2Np has been shown to be highly expressed in epidermal and endodermal cells at the root tip, and in lateral root primordia and epidermis of mature roots (Krapp et al., 1998). Clearly, a significant question remains as to the functional roles of the multiple representatives of the nrt2/bch family of genes. The long-term effect of external NO3" concentration (0, 10, 50, 100, or 500 p M ) revealed that both bch transcript levels and influx were greatest in plants grown with 50 p M NO3". The reported K m of I H A T S , 25 to 100 p M , varies with genotype and as a function of nitrate pretreatment (Siddiqi et al., 1990). The present correlation between bch transcript levels and influx rates confirms earlier observations to this effect in Arabidopsis (Zhuo et al., 1999) and 100 provides further evidence that bch genes encode a component of the inducible high-affmity transport systems for NO3" influx. 3.4.1.4 E f f e c t s o f e x t e r n a l NO2" o r N H 4 + o n NO3" i n f l u x a n d bch t r a n s c r i p t a c c u m u l a t i o n Nitrogen signals other than nitrate that may be involved in the regulation of bch genes were investigated by supplying nitrite and/or ammonium during induction. Although NO2" is rarely present in soil solution, under laboratory conditions it is able to induce both NO2" and NO3" uptake (Siddiqi et a l , 1992; K i n g et al., 1993; Aslam et al., 1996). 10 m M N 0 2 " increased bch transcript levels within two hours of treatment. It may be possible that the slower response to NO2" is due to slower transduction of the NO2" signal or lower rates of NO2" uptake. In Arabidopsis thaliana NO2" was unable to increase nrt2.1At transcript levels when plants were pretreated with NO2" for 3 h (Zhuo et al., 1999). When N H 4 + was provided together with NO3", bch transcript accumulation was unaffected for the first 2 h, of the treatment, but by 4 h and 6 h there was a dramatic decrease in transcript levels. It is thought that N H 4 + may: (1) inhibit NO3" influx directly at the transport step, through effects at the extracellular or intracellular face of the membrane (King et al., 1993), and (2) function as a signal repressing transcription of I H A T S . In the experiment design, we used high levels of NO3" pretreatment (10 m M external) to ensure that NO3" would enter the root by both high-affinity and low-affinity transport systems, despite an inhibitory effect of N H 4 + on NO3" uptake. With the results presented in this chapter, it is not clear i f N H 4 + itself or an assimilate of N H 4 or both, acts as a signal involved in transcriptional regulation (down regulation), of bch transcripts. However Zhuo et al, (1999), demonstrated that ammonium is an important regulator of nrt2.1At in Arabidopsis. They found that co-supply of ammonium and nitrate, decreased transcript levels to 50% of nitrate induced plants levels within 101 3 h, this is in agreement with results in Figure 24, which demonstrates dramatic decrease in bch transcript levels by 4 h. Also , the report o f K i n g et al. (1993) demonstrated in barley that M S O failed to relieve the down regulation of NO3" influx. Therefore, in this chapter we are unable to distinguish between the effect of N H 4 + as a regulatory signal in transcription, or as a precursor to other products downstream of the N-assimilation pathway. The decrease of nrt2. INp transcript levels resulting from direct application of NFL;"1" or glutamine (Krapp et al., 1998) in N. plumbaginifolia, and also fro the addition amino acids and inhibitors of N-assimilation (Zhuo et al., 1999), are consistent with this conclusion. Earlier work, based on physiological treatments, are in disagreement over the role of N H 4 + (see Glass and Siddiqi, 1995) for discussion). Further investigation of the effect of NFf 4 + and amino acids w i l l be discussed in chapter 4. 3.6.1.1 Conclusion Transcripts of members of the bch gene family are induced by both NO3" and NO2". The presence of N H 4 + during induction appears not to effect bch transcript accumulation initially, suggesting that induction may not be sensitive to this form of N . Rather, it may be that N H 4 + and/or its assimilation products have a more pronounced effect on the down-regulation of bch transcript accumulation. Transcript levels of all known members of the bch c D N A s (bchl, bchl, bch3, and bch4) increased following provision of NO3". The promoter sequence, contains a core N I E domain, which has been implicated in the induction pathway of N R genes in Arabidopsis. This domain may be responsible for this pathway in barley, but that there are also evolutionary difference in these elements due the absence of the AT- r i ch region, which precedes the A ( G / C ) T C A core sequence in Arabidopsis ; this is also the case for the barley N R genes. Also 102 present in the promoter sequences of bchl, bch2 and bch3 genes was a common domain I whose function is presently unknown. 103 C H A P T E R 4 R E G U L A T I O N O F BCH c D N A s E F F E C T O F A M M O N I U M A N D A M I N O A C I D S O N T R A N S C R I P T A C C U M U L A T I O N A N D N I T R A T E I N F L U X 104 4.1.1.1 Introduction The uptake of nitrate in terrestrial plants is mediated by specific transporters. It has been shown that at least three transport systems coexist in the plasma membrane of root cells (see review by Glass and Siddiqi, 1995). These fall into two classes, referred to as low- and high-affinity transport systems ( L A T S and H A T S , respectively). A gene considered to encode the low-affinity transport system (nrtl.lAt, originally chll) was the first higher plant nitrate transporter gene to be cloned, from Arabidopsis thaiiana by Tsay et al. (1993). A t a physiological level this L A T S is involved in NO3" uptake at high external concentrations of NO3" (>0.5 m M ) . In physiological terms, it is constitutively expressed in barley roots, and is thought to be down-regulated by end products of N-metabolism (Clement et al., 1978; Siddiqi et al., 1993). However, in Arabidopsis roots, in contrast to the constitutive character of the L A T S in barley roots, the nrtl.lAt (chll) gene which is considered to be one of the genes responsible for L A T S (Huang et al., 1996; Touraine and Glass, 1997) is substrate (NO3") -inducible. The high-affinity transport systems can be further separated into constitutive and inducible systems. In plants, putative inducible high-affinity transporters ( IHATS) have been cloned from barley (Trueman et al., 1996; Chapter 3), A. thaiiana (Zhuo et al., 1999), Nicotiana plumbaginafolia (Quesada et al., 1997), and soybean (Amarasinge et al., 1998). In barley, putative I H A T S are encoded by a multigene family of 7-10 members (Trueman et al., 1996). To date, four members of this family, originally named the bch family in barley, have been isolated (Trueman et al., 1996; Chapter 3). The bch genes encode proteins belonging to the Major Facilitator Superfamily (MFS) , as are the other plant I H A T S . The B C H proteins have are composed of 507-509 amino acids, with a molecular weight of 54-55 kDa, including 12 hydrophobic (transmembrane) regions. It has been shown that the m R N A levels of these I H A T S 105 genes rapidly increase following the provision of nitrate, (a process referred to as 'induction') to N-deprived plants (Trueman et a l , 1996; Quesada et al., 1997; Amarashinge et a l , 1998; Zhuo et al., 1999). This increase in transcript levels is correlated at the physiological level with nitrate uptake, which is also first upregulated when NO3" is first supplied (Hole et al.,1990; Siddiqi et al.,1990 and Glass et al., 1990). In barley, the four bch transcripts studied are coordinately upregulated on the provision of NO3" (Chapter 3). Under quasi-steady state conditions of NO3" supply, the highest levels of I H A T S m R N A level and 1 3 N03~ influx were obtained when external NO3" concentration was 50 u M . In N plumbaginifolia, genes involved in N-acquisition and assimilation, namely, nrt2.1Np (bch homolog), nia (nitrate reductase) and nii (nitrite reductase), were coordinately expressed under conditions of NO3" induction and N repression (Krapp et al., 1998; Zhuo et al., 1999). Furthermore, in N. plumbaginifolia, nitrate reductase mutants showed elevated levels of nrt2.1Np (the bch homologue) transcript (Krapp et al., 1998), consistent with the proposal that nrt2. INp transcript abundance is regulated by feedback from reduced forms of N rather than NO3" itself. The down-regulation of I H A T S to a lower steady state level, a phenomenon which has been demonstrated to follow induction in several plants supplied with an ample source of N (Siddiqi et al., 1989; K i n g et al., 1993), has been argued to result from effects of accumulated NO3" or product(s) of its assimilation. Physiological data indicates that NO3" influx is likely down-regulated by NO3" itself (Ingermarsson et a l , 1987, K i n g et al., 1993; Doddema et al., 1978), by N H 4 + (Aslam et al., 1996) and/or by amino acids, (Doddema and Otten, 1979; Muller and Touraine, 1992; Mul ler et al., 1995). Some of the reports which support the hypothesis that internal NO3" down-regulates NO3" influx (e.g. Ingermarsson et al., 1987), were based upon the use of tungstate (an inhibitor of N R ) . Likewise, the results of experiments which made use of N R double mutants in barley (King et a l , 1993), or correlations between root [NO3"] and 1 3 N 0 3 _ 106 influx (Siddiqi et al., 1993) have been interpreted as evidence for a role of tissue NO3". The effect of N H 4 + on NO3" uptake is more complex, due to the possibility of affecting NO3" uptake at a number of levels (transcript abundance, protein levels or direct effects of N H 4 + on the NO3" transporter). This has resulted in a lack of consensus concerning the mechanism(s) of the ammonium effects on NO3" fluxes. Using NO2" as a tracer of NO3" As lam et al. (1994) suggested that N H 4 + resulted in enhanced efflux, while (by contrast) diminished influx was demonstrated by the use of 1 3 N 0 3 " (Glass et al., 1985; Lee and Drew, 1989; Kronzucker et al., 1999). A recent paper by Kronzucker et al. (1999) established that in barley roots, both a decrease of NO3" influx (King et al., 1993) and an increase in efflux resulted from the applications of N H 4 + in the external medium. Moreover, this effect occurs within minutes of supplying N H 4 + . Thus direct effects of N H 4 + are in addition to long term effects. On the other hand, Breteler and Siegerist (1984) showed that M S O , an inhibitor of GS , relieved the effect of N H 4 + on N 0 3 " uptake in dwarf bean. These authors concluded that N H 4 + was not acting per se, but v ia the products of its assimilation. K i n g et al. (1993) observed no relief of N H 4 + inhibition by M S O . B y contrast, de la Haba et al. (1990) suggested that N H 4 + and not its assimilation products were responsible for inhibiting NO3" uptake. Clearly, a part of the confusion in the literature has resulted from the aforementioned multiple levels at which N H 4 + is capable of inhibiting NO3" influx. There is every reason to expect that N H 4 + may have both direct effects on the transport system as well as effects at transcription via products of N H 4 + assimilation. Nevertheless, Zhuo et al. (1999), by use of inhibitors of N assimilation argued that elevated N H 4 + could affect NO3" uptake at a transcriptional level. Feeding plants with amino acids may mimic the shoot signals that control uptake by roots (Imsande and Touraine, 1994). It has been demonstrated that application o f amino acids in the nutrient solution can decrease NO3" uptake, in A. thaiiana (Doddema and Often, 1979), in 107 Phaseolus vulgaris (Breteler and Arnozis, 1985), in soybean (Muller and Touraine, 1992) and in wheat (Rodgers and Barneix, 1993). Repression was usually preceded by a lag period of 3 h or more. This indicates that the effect of amino acid on NO3" uptake is not direct or allosteric (Muller and Touraine, 1992). Again, it is necessary to interpret the results o f such experiments with caution, since the effects of particular amino acids may be influenced by the extent o f uptake, the extent of biochemical transformation as well as their effect upon the expression of the NO3" transport system. Furthermore, the demonstration of an effect due to exogenous application of amino acids must be carefully considered in light o f two questions. Is the amino acids normally present in the phloem sap as a likely signal of plant N status, and does this amino acid normally accumulate in roots (presumably as a result of transfer from shoots)? In this chapter I investigate the regulation of NO3" influx, bch transcript abundance and changes in NO3", N H 4 + and amino acid concentrations in roots during the down regulation of NO3" influx that follows the peak of induction. For this purpose barley seedlings were exposed to NO3" together with N H 4 + or amino acids (Asn, Asp, Gin , and Glu), or treated with inhibitors (tungstate, M S O , azaserine, and A O A ) of key enzymes of the N-assimilation pathway. 4.2.1.1 Materials and methods 4.2.1.2 Plant material Seven-day old seedlings of Hordeum vulgare cv. Klondike were used in all experiments. Seeds were surface sterilized with 1% commercial bleach solution and rinsed with de-ionized H 2 0 . The seeds were placed on a nylon mesh (pore size, 4 mm) which was fixed onto 20 mm (8 seeds) or 60 mm (25 seeds) Plexiglas discs, depending on the experiment. The discs were placed 108 on moist sand in the dark, and the seeds were covered with 10 mm of moist sand. After 3 d the seedlings were transferred to 40 L hydroponic tanks, and grown in N-free 1/10 strength Modified Johnson's solution (Siddiqi et al., 1991), then treated with nitrogen in the form of KNO3, KNO2, or NH4SO4. Potassium concentration of the growth medium was monitored daily and a concentrated nutrient solution was supplied to the tanks in the same ratio as in the original Modified Johnson's solution to restore nutrient concentrations. The p H of the solution was maintained at 6.2 ± 0.3 by the addition of excess CaCC»3 powder. Plants were grown in a controlled environmental chamber with a 16 h/8 h light dark cycle at 20 ± 2°C and 70% relative humidity. Light (photon flux density at plant level, 300 pmol ni" 2 s"1) was provided by fluorescent tubes with a spectral composition similar to sunlight. 4.2.1.3 Nitrate influx Nitrate influx experiments were carried out essentially as described by Siddiqi et al. (1989). Seven-day old barley plants grown in hydroponic tanks, and treated according to the particular experimental design, were transferred into 0.5 L vessels containing unlabeled uptake solution for 5 min, to equilibrate to the prevailing conditions to be employed for influx determination. After this period they were transferred to 0.5 L of uptake solution containing 50 u M NO3" labeled with 1 3 N C V for 5 min, and then transferred back into 0.5 L vessel of unlabelled solution for 3 min, to remove unabsorbed tracer residing in the cell wal l space. Roots and shoots were harvested separately, and placed into 20 m L scintillation vials for counting in a Packard y-counter (Minaxi 8, Auto-y 5000 series). Production of 13NC»3" was as described by Kronzucker et al. (1995). 109 4.2.1.4 RNA isolation and Northern blot analysis Total R N A was isolated using Trizol Reagent (Life Technologies), with two modifications. Firstly, after the tissue was ground in a mortar and Trizol reagent was added at ratio of 0.2 g tissue to 1 m L Trizol , the homogenate was centrifuged at 8000x g for 30 min to remove cellular debris. Secondly, after the total R N A was isolated, it was re-extracted with phenol: chloroform: iso-amyl alcohol (25:24:1), and precipitated with 0.3 M sodium acetate (final concentration), and two volumes of ethanol. Total R N A was separated on a 1.2% agarose gel containing I X M O P S with 2.2 M formaldehyde, at 60 V for 3.5 h, then washed twice in H2O, and R N A transferred by capillary action to N"1" nylon membranes (Amersham). The membranes were baked for 2 h at 80° C in order to fix the R N A , and then placed in prehybridization solution for 1 h, after which they were placed in hybrization solution with P labeled probe for 12-16 h. Prehybridization and hybridization solutions contained 6 X SSC, 5 X Denhardt's solution, 0.5% SDS and 20 pg/ml sonicated herring sperm D N A . Random prime probes were made with Prime-A-Gene kit (Promega, Madison, WI) using internal fragment from bch3 (plasmid p B C H 3 by digestion with AfUJi and EcoKV), which recognizes all known members of the bch family of c D N A s . Control levels of total R N A were probed using a fragment of the 25S gene, on plasmid pV25S, by digestion with Xhol. Membranes were washed as recommended by the manufacturer's instructions with 0.25 SSC and 0.1% SDS at 42° C for 15 min for the final wash. 110 4.2.1.5 Nitrate analysis Nitrate concentrations were determined from fresh tissues, by extracting with boiling water, at a ratio of l g root tissue to 10 m L H 2 O . The extracts were centrifuged at 8000 x g, and the supernatant was filtered through a 0.45 u M filter. Nitrate was analyzed using the cadmium-copper reduction method on a Technicon Autoanalyzer (Henricksen and Selmer-Olsen, 1970). 4.2.1.6 Amino acid and ammonium measurements Amino acids and ammonium were extracted from root material (ground to a fine powder using a morter and pestle) with a buffer containing 58% ethanol, 0.2 M formic acid, and 0.25 m M alpha-amino butyric acid as an internal standard. After centrifugation (21000 x g, 5 min) and filtration (0.45 pm P V D F micro-centrifuge tube filter, Whatman, England), amino acids were measured by use of Waters AccQ-Tag Amino A c i d Analysis on H P L C (two 626 H P L C pumps; 4 pm Nova-pak d g column, 3.9x150 mm, thermostatted at 39°C; using a 474 scanning fluorescence detector; 717 p i u s autosampler; 600S controller; all Waters components, Mill ipore, M A , U S A ) . Mobi le phase A consisted of 100 m M N a A c (Sigma, M o . , U S A ) , 5.4 m M triethylamine (Fluka, Germany), 3.5 p M E D T A (Sigma, M o . , U S A ) adjusted to p H 5.7 with phosphoric acid. Mobi le phase B had a composition similar to that of A except for the p H which was 6.7. Mobi le phase C was acetonitrile (JT Baker, Netherlands), and mobile phase D was M i l l i - Q water (resistance 18.2 M Q ) . A l l solutions were degassed before use. Gradient conditions were: 0.5 min with 90% A , 10% B ; 16.5 min with 89% A , 10 % B , 1% C; 9 min 80% A , 18% B , 2% C; 6 min 68% A , 27% B , 5% C; 1,5 min 63% A , 27% B , 10% C; 3.5 min with 87.5% B , 12.5% C; 11 min with 87% B , 13% C; 0.1 min with 85% B , 15% C, 2.90 min with 60% C, 40% i l l D and 9 min with 90%A, 10% B . Initial flow rate 1.0 m l min" 1 changing to 1.3 ml min" 1 after 33.8 min. Amino acid standard curves was made using standards (Sigma, M o . , U S A ) . 4.3.1.1 R e s u l t s 4.3.1.2 T i m e c o u r s e s t u d y o f the effect o f 10 m M NO3" o n 1 3NC>3" i n f l u x , a n d bch t r a n s c r i p t a c c u m u l a t i o n When seedlings, previously grown on N-free medium, were fed 10 m M N 0 3 " , 1 3 N 0 3 " influx 13 displayed a typical time-course response (Figure 20) with a 7-fold increase of NO3" influx measured at 50 u M (from 0.42 to 2.9 pmol g"1 F W h"1) within the first 12 h. This was followed by a steady decrease to 2.4 pmol g"1 F W h"1 (at 48 h). Parallel Northern blot analysis showed that bch transcript levels in root tissue increased dramatically within the first 6 h, and then steadily decreased to undetectable by after 24 h (Figure 18). Analysis of inorganic N (Figure 25), showed that NO3" concentration in root tissue increased steadily from 5.1 ± 2.7 to 46.2 ± 1 1 . 2 pmol g" *FW within 6 h of NO3" provision, and increased to 89.2 ± 8.5 pmol g _ 1 F W by 48h. In contrast, N H 4 + levels remained basically unchanged, varying between 3.5 and 4.5 pmol g _ 1 F W (Figure 25). The increase in root concentrations of the amino acids Gin , Glu , A s n and Asp in root are shown in Figure 26. This analysis revealed that the concentrations of Gin , G lu , Asp and Asn peaked by 10, 8.5, 13 and 4 -fold, respectively. A n interesting note is that G i n increased within the first 12 h after the onset of NO3" supply, then decreased with longer duration. 112 Duration of N 0 3 " pretreatment (h) Figure 25. Time course of changes of root NO3" ( • ) and N H 4 + ( • ) concentrations in seven day-old barley seedlings grown in 1/10 strength Modified (N-) Johnson's solution then supplied with 10 m M NO3" (5 replicates for each time point with error bars = ± 1 S.D.). 1400 4 > o o 1200 4 1000 4 Duration of N03" pretreatment (h) Figure 26. The effect of 10 m M NO3" pretreatment on amino acid concentration in roots of 7-day old seedlings of barley. Values are in % of N-starved control. A s n (•), Asp (•), G i n ( • ) and Glu (T). Each time point represents 5 replicates with error bars = ± 1 S.D. 114 4.3.1.3 The effect of external application of amino acids on NO3" influx and bch transcript accumulation The effect on 1 3 N 0 3 _ influx of pretreating plants with Gin , G lu , Asn , or Asp at concentrations of 1 m M for 6 h, during induction of NO3" influx with 10 m M NO3", was investigated in a subsequent influx measurement using 50 u M 1 3 N03~ to measure I H A T S activity. A l l the amino acids tested decreased 1 3 N03~ influx (Figure 27), the most effective being Glu (70%), then Asp (65%), Asn (30%) and G i n (25%). Northern blot analysis of R N A isolated from roots showed a similar pattern for the abundance of bch transcript (Figure 28) as for influx. Amino acid pretreatments affected NO3" concentrations in root tissue; Asp and A s n reduced [NO3"] by 43 and 32 % respectively, G in caused a 40% increase, while G l u add no effect (Table 2). The exogenous application of amino acids Glu , Gin , Asp and Asn , also increased amino acid pools in root tissue (Table 2). The changes of bch transcript levels were most strongly correlated with increases of G l u (r 2= 0.92) and G i n (r2= 0.68) concentrations, while Asp and Asn concentrations were poorly correlated (r 2 values of 0.4 and 0.22, respectively). 4.3.1.4 The effect of N-assimilation inhibitors on 13NC>3" influx, and bch transcript accumulation The effects of 0.5 m M tungstate, 1 m M methionine sulfoximine (MSO) , 0.25 m M azaserine ( A Z A ) and 0.5 m M amino oxyacetate acid ( A O A ) when co-supplied with 10 m M NO3" for 6 h to N-starved plants was also evaluated. A l l four of these compounds inhibit enzymes of nitrate assimilation at different steps of the pathway of NO3" assimilation as 115 shown in Figure 29. Tungstate inhibits the enzyme N R ; while M S O , G S ; A Z A , G O G A T and A O A ; amino transferases and glutamate decarboxylase, respectively. A priori, these treatments could be anticipated to increase or decrease root concentrations of NO3" assimilates by blocking the appropriate enzymes. B y examining the effects of these inhibitors on transcript abundance and NO3" influx, the role of these assimilates in regulating I H A T S influx might be assessed. A l l four treatments decreased 1 3 N 0 3 _ influx in comparison to the control treatment, which was the 6 h exposure to 10 m M NO3" (Figure 30). Tungstate increased bch transcript by 30% (Figure 31 and 32). Surprisingly, this treatment failed to significantly impact upon root NO3", N H 4 + Asp, or G l u concentrations but decreased both Asn and G i n concentrations (Table 3). When exogenous G l u was added to the tungstate treatment solution, bch transcript level was reduced by 70% (lane 5, Figure 33). M S O (Fig. 31 and 32), A O A and A Z A (Figure 33) decreased transcript abundance by 30, 60 and 95%, respectively. When 1 m M Glu was added to M S O treatment a further drop (63%) in bch transcript abundance was observed (Figure 33). N H 4 + levels increased 4.5- and 2-fold in roots of M S O and A Z A treated plants (Table 3), respectively, compared to the control treatment (plants with 10 m M N O 3 " , without inhibitor), but A O A treatment had virtually no effect on root [Ntf_4+]. As expected of an inhibitor of GS activity, M S O treatment decreased both G i n and Glu concentrations, but had little or no effect on Asn and Asp levels (Table 3). A Z A , on the other hand, increased A s n levels by 1.5-fold, slightly increased (25%) Gin , but dramatically decreased Glu and Asp. A O A increased Asn levels in root tissue by 2-fold, but this was accompanied by a decrease in Asp levels in the root tissue by 70%. A Z A , M S O and A O A treatments decreased the concentration o f NO3" in root tissue by 80, 25 and 30%, respectively, while tungstate was without effect (Table 3). 116 bo §1 Figure 27. The effect of exogenous pretreatment with amino acids on 1 3 N03~ influx. Treatment 1: N starved; treatments 2-6 plants were treated with 10 m M NO3" alone (treatment 2) or with amino acids (treatments 3 to 6) for 6 h. Treatment 3, 1 m M Asn; treatment 4: 1 m M Asp; treatment 5, 1 m M Gin ; and treatment 6, 1 m M Glu . (4 replicates for each treatment with error bars = ± 1 S.D.). 117 A) 1 2 3 4 5 6 B) -N NCy Asn Asp Gin Glu Treatments Figure 29. The effect of exogenous pretreatment with amino acids on bch transcript accumulation in roots. A ) R N A isolated from roots of N starved: lane 1; lane 2-6 contains R N A isolated from roots treated with 10 m M NO3" alone (lane 2) or with amino acids (lane 3 to 6) for 6 h. Lane 3, 1 m M Asn; lane 4: 1 m M Asp; lane 5, 1 m M Gin ; and lane 6, 1 m M Glu. B) Quantification of transcript levels by phosphoimager average of two experiments (standardized by 25S transcript) using S T O R M phosphoimager (Molecular Dynamics). Table 2. Effect of exogenously supplied amino acids on inorganic and organic nitrogen levels in plant roots. (Concentrations are in umol g"1 F W , and based upon four independent replicates with error bars = ± S.D.). Means followed by different letter are significantly different from other (within column) values at the P = 0.05. Treatments Amino Acids Inorganic N Asn Asp Gin Glu NCV -N 0.82 ± 0.41a 0.24 ± 0.02a 0.71 ± 0.06a 2.11 ±0.81a 5.1 ±2.7a 6h N03" 1.32 ± 0.25a 0.59 ± 0.14a 3.41 ± 0.21b 5.29 ± 1.07ab 46.2 ± 11.2b 6h N03"+ Asn 4.02 ± 2.01b 1.32 ± 0.22b 4.62+ l.Olcb 8.50±2.13bc 26.2 ± 12.7c 6h N03"+ Asp 2.58 ± 1.28ab 0.98 ± 0.16b 5.52 ± 0.42c 9.42±2.61bc 31.5±2.1bc 6h N03"+Gln 2.51 ± 1.02ab 0.62 ± 0.21a 5.02 ± 0.58c 8.51 ±2.61bc 64.8±9.4d 6h NCV+Glu 1.46 ± 0.68a 0.92 ± 0.11b 6.82 ± 0.51c 10.21 ± 1.94c 47.5 +4.1b 119 Figure 29. The enzymatic steps involved in NO3" assimilation, together with inhibitors, which block certain of these steps E N Z Y M E nitrate reductase nitrite reductase glutamine synthetase glutamate synthase aspartate amino transferase glutamate decarboxylase M E T A B O L I C STEP N O 3 - • N0 2 - • N H 4 + • glutamine f oxaloacetate > glutamate * I N H I B I T O R N0 2" Tungstate N H 4 + glutamine MSO glutamate AZA aspartate AOA aminobutyrate AOA Z.5-, 1 2 3 4 5 6 Figure 30. The effect of N assimilation inhibitors on 1 3 N C V influx. Treatment 1: N starved; treatments 2-6 plants were treated with 10 m M NO3" for 6 h; inhibitors were added to treatments 3 through 6. Treatments were: 3, 1 m M M S O ; 4, 0.5 m M tungstate; 5, 0.25 m M A Z A ; and 6, 0.5 m M A O A . Each treatment consists of 4 replicates with error bars = ± 1 S.D. 4.3.1.5 E f f e c t o f N H 4 + o n 13NC>3" i n f l u x a n d bch t r a n s c r i p t a c c u m u l a t i o n A s previously described, concurrently supplying 10 m M N H 4 + together with 10 m M NO3" for 2 h had no effect on bch transcript abundance in comparison to 10 m M NO3" feeding alone. However, treatments of longer durations e.g. 4 or 6 h resulted in a dramatic decrease in the abundance of bch (Chapter 3). This decrease of the abundance of bch transcript levels was accompanied by a marked decrease of 1 3 N03~ influx. The down-regulation of bch transcript accumulation may have been due to the NH4 + ion itself or a product of its assimilation. 13 To further investigate this, I analyzed transcript abundance of bch, N 0 3 " influx, and N H 4 + and amino acid concentrations in roots grown either on N 0 3 " alone, with or without M S O , or on NO3" together with N H 4 + , with or without M S O . These treatments were designed to increase N H 4 + level in root tissue while monitoring its effect on 13N03~ influx (Table 4). While the treatment with NO3" plus M S O resulted in a 4-fold increase of ammonium level compared to the N 0 3 " treatment (Table 4). I found that [ N H 4 + ] increased by 10- fold in the roots of plants treated with N 0 3 " and N H 4 + or NO3", N H 4 + and M S O compared to roots treated with NO3" alone. Compared to control plants that were completely deprived of NO3", bch transcript levels increased ~8 fold after 6 h of NO3" treatment. When M S O was provided together with NO3" transcript, level was reduced by 15% (Table 4). Roots subjected to NO3" + N H 4 + , with or without M S O , displayed only 9 and 14% transcript abundance, respectively, compared to the NO3"-treated controls. The concentration of root G in increased by 2-fold when NO3" and N H 4 + were co-supplied. A l l the other treatments ( N 0 3 " alone or with M S O , or with both N H 4 + and M S O ) led to small changes of internal G in concentrations (Table 4). 122 A) B) bch 25 S 1 2 3 4 5 6 m mm m m m m * * * -N N03- MSO Tung AZA AOA treatments Figure 31. The effect of N assimilation inhibitors on bch transcript accumulation in roots. A ) Northern blot analysis of R N A isolated from roots of N starved plants: lane 1; lane 2-6 contains R N A isolated from roots treated with 10 m M NO3" for 6 h; lanes 3 to 6 contains R N A from plants treated with inhibitors. Lane 3, 1 m M M S O ; lane 4: 0.5 m M tungstate; lane 5, 0.25 m M A Z A ; and lane 6, 0.5 m M A O A . B) Quantification of transcript levels by phosphoimager average of two experiments (standardized by 25S transcript) using S T O R M phosphoimager (Molecular Dynamics). A) 1 2 3 4 5 6 7 8 9 10 - ffvtttftl - •••••••••• B) Treatments Figure 32. Time course effect of tungstate and M S O on bch transcript abundance. Lane LTota l R N A isolated from roots of 7-day old seedling grown on 1/10 strength N-free Modified Johnson's solution: (N-starved); Plants treated with 10 m M NO3" alone (lane 2) or with inhibitor, lane 5 tungstate, and lane 8 M S O for 2 h; Plants treated with 10 m M NO3" alone (lane 3) or with inhibitor, lane 6 tungstate, and lane 9 M S O for 4 h; Plants treated with 10 m M NO3" alone (lane 4) or with inhibitor, lane 7 tungstate, and lane 10 M S O for 6 h. Quantification of transcript levels by phosphoimager average of two experiments (standardized by 25S transcript) using S T O R M phosphoimager (Molecular Dynamics). Transcript abundance calculated as percent of 2 h NO3" treatment (as 100%). 124 1 2 3 4 5 6 Bch N 0 3 - + + + + + Glu + + Tung + + M S O + + 0 1 — m i l 1 2 3 4 5 6 Treatments Figure 33. The effect of N assimilation inhibitors tungstate and M S O in the presence of G lu on bch transcript accumulation in roots. Northern blot analysis of R N A isolated from roots of N starved plants: lane 1; lane 2-6 contains R N A isolated from roots treated with 10 m M NO3" for 6h (lane 2) or from roots of plants treated with 10 m M NO3" plus various inhibitors plus or minus G lu (lanes 3 to 6). Lane 3 and 5: 0.5 m M tungstate; Lane 4 and 6: 1 m M M S O ; lane 5 and 6: 1 m M Glu . Transcript abundance calculated as percent of 6 h NO3" treatment. Quantification of transcript levels by phosphoimager average of two experiments (standardized by 25S transcript) using S T O R M phosphoimager (Molecular Dynamics). Transcript abundance calculated as percent of 6 h N 0 3 " treatment (as 100%) 125 Table 3. Effect o f N-assimilation inhibitors on inorganic and organic nitrogen levels in plant roots (concentrations are in pmol g"1 F W , 4 independent replicates with error bars = ± 1 S.D.). Means followed by different letter are significantly different from other (within column) values at the P = 0.05. Treatments Amino Acids inorganic N Asn Asp Gin Glu N0 3" MI. ; -N 0.82 ±0.41 0.24 10.02 0.71 ± 0.06 2.11 ±0.81 5.1 ±2.7 4.2 ± 0.2 ab a a a a a 6hN03" 1.32 ±0.25 0.59 ±0.14 3.42 ±0.21 5.29 ± 1.07 46.2 ±11.2 4.5 ± 0.34 ab na be b b a 6h N03+Tungstate 0.61 ±0.11 0.57±0.21 0.65±0.28 4.80 ± 0.72 42.5 ± 15.6 3.6 ± 0.47 a a a b b a 6hN03"+MSO 0.71 ±0.41 0.54 ±0.12 2.12 ±0.81 2.90 ±0.62 34.2 ±7.4 18.5 ±1.4 a a ab a b b 6hN03" + AZA 1.78 ±0.87 0.27±0.12 4.88+ 1.47 0.54 ± 0.24 8.4 ±2.1 10.4 ±2.1 b a c c a c 6hN03" + AOA 1.4610.41 0.1910.20 3.21 1 1.41 4.63 10.17 31.415.2 4.1 1 1.3 a a be b b a 126 Table 4. Effect of N H 4 + and M S O on bch transcript abundance, N H 4 + concentrations in umol g"1 F W [Gin] concentrations in nmol g _ 1 F W , in plant roots (4 independent replicates with error bars = ± 1 S.D.). Plants were grown on 1/10 strength Modified (-N) Johnson's solution then treated for 6 h with the treatments shown above, using 10 m M N 0 3 \ 10 m M N H 4 , and 1 m M M S O . Transcript abundance was calculated using the ratio of bch I 25s hybridizing signal, quantified on Storm phosphoimager (Molecular Dynamics). Means followed by different letter are significantly different from other (within rows) values at the P = 0.05. Treatments -N 6h N03" 6hN03" + MSO 6hN03-+ NH 4 + . 6hN03" + NH 4 + + MSO Relative Transcript levels 0.12 1.00 0.85 0.14 0.09 N03" Influx umol g'FW h"1 0.48 ±0.12 a 3.20 ±0.25 b 1.74 ±0.21 c 0.97 ±0.13 d 0.84 ± 0.08 e [NH4+] 4.2 ±0.21 a 4.5 ±0.34 a 18.9 ± 1.4 b 47.2 ±3 .1 c 55.2 ±2 .8 d [Gin] 0.71 ±0.06 a 3.41 ±0.21 b 2.12 ±0.81 c 6.70 ± 0.54 d 4.54 ± 0.52 e 127 4.4.1.1 Discussion Nitrate uptake is a process that is subject to regulation, by induction and repression, the latter process being dependent on the plant's nitrogen requirements. NO3" uptake has been demonstrated to be mediated by at least three uptake systems. In this chapter factors responsible for regulating I H A T S were investigated. A t the physiological level it has been shown that the signal for the induction process of I H A T S is NO3" (Jackson et al., 1973; Goyal and Huffaker, 1983; Aslam et a l , 1996a), while NO2" can also serve as a signal for this process (Siddiqi et al., 1992; Aslam et al., 1996b). In higher plants, genes thought to be responsible for NO3" M A T S have been cloned. These include bch from barley (Trueman et al., 1996; Chapter 3), nrt2.1Np from Nicotinia plumbaginifolia (Quesada et a l , 1996), nrt2Gm from soybean (Amarashinge et al., 1998), and nrt2.1At and nrt2.2At from A. thaiiana (Zhuo et at., 1999; Filleur and Daniel-Vedele, 1999). Transcript levels for these genes increase dramatically in response to the provision of NO3" in the media, and the expression patterns have been correlated with nitrate influx, giving indirect evidence that they are responsible for high-affinity transport (Quesada et a l , 1997; Lejay et a l , 1999; Zhuo et al., 1999). Heterologous expression of the bch/nrt2 genes in an Hansenula polymorpha nitrate transport mutant, defective in the yntl gene, that is a member of the crnA family of nitrate transporters (Zhang et al., 1998), demonstrated that these genes encode functional nitrate transporters. However, in this heterologous system the K m for I H A T S was elevated in comparison to that measured in planta. Nitrate uptake has been hypothesized to be under negative feedback control (Glass 1988; Clarkson 1988). To investigate the factors responsible for negative feedback control of I H A T S and abundance of bch transcript, we designed a series of experiments, according to three criteria. First, plants were maintained on - N media. These plants would therefore contain low levels of 128 organic and inorganic N , so that major as well as minor changes of the nitrogen pools could be analyzed following the supply of N . Second, - N plants were challenged with high levels of nitrate (10 m M ) , so that NO3" could enter the root cells via low affinity NO3" transport systems even i f these treatments were inhibitory to high-affinity transport. Third, the treatments with amino acids and inhibitors of N-assimilatory enzymes were short-term (6 h) in order to reduce the possibility of secondary effects of the treatments. Several possible signals for the down-regulation of I H A T S have been proposed. These include root NO3", root N H 4 + , and/ or amino acids (Lee et al., 1992; Siddiqi et al., 1989; K i n g et a l , 1993; Muller and Touraine, 1992). In the present work we address the question of the transcriptional level of regulation by these signals, by several methods. A t a molecular level, Quesada et al. (1997) demonstrated that nrt2Np transcript levels in NCV-grown plants decreased due to the supply of N H 4 + or Gin . Unfortunately, these preliminary results failed to identify which signals ( N H 4 + itself or products of its assimilation) were responsible for the observed effects. The study by Zhuo et al. (1999) using inhibitors of NO3" assimilation, concluded that N H 4 + and glutamine were involved in the down regulation of the Arabidopsis nrt2. lAt gene. To investigate this question in barley roots, five parameters were monitored: NO3" influx, bch transcript levels, and NO3", N H 4 + and amino acid concentrations of root tissue. The typical time profile of NO3" influx upon providing NO3" to plants, previously starved of N , was observed. This pattern correlated well with bch transcript accumulation during the first 12 hours of NO3" 13 * provision, but transcript levels then decreased to very low levels. A t the same time NO3" influx remained relatively high. Two possibilities can account for this; 1) protein turnover of I H A T S is long, therefore even though the m R N A levels are very low, there is protein to maintain influx capacity, or 2) other transporters types ( C H A T S or L A T S ) are responsible for the influx. In the meantime, N H 4 + levels increased only slightly (10%). However the major observable difference 129 observed was in root NO3" concentrations, which increased 17-fold (Figure 27) following the same pattern as reported by Siddiqi et al. (1993). G in , Glu , A s n and Asp levels increased 4- to 13-fold. These results failed to reveal which effectors might be responsible for the down-regulation of I H A T S activity and bch transcript abundance, but indicated the possibility that NO3" and/or amino acids may participate in this down-regulation. 4.4.1.2 The effect of NO3" on nitrate influx and bch transcript accumulation. Tungstate has been shown to inhibit the activity of the enzyme nitrate reductase (Deng et al., 1993). The response of 13N03~ influx to tungstate treatment has been described by Ingemarsson et al. (1987) in Lemna: at high external NO3" concentration^ m M ) internal NO3" concentration increased and NO3" influx was inhibited. B y contrast at low external NO3" (10 pM) concentration NO3" influx was high. This experimental design provided a check against side effects of tungstate. These authors proposed that this provided evidence for regulation of nitrate influx by nitrate itself. In our experiments bch transcript levels increased (20%) in tungstate treated plant roots compared to control plants, while influx decreased by 50%. The blockage of N R with tungstate did not increase the concentration of NO3" in roots, but this does not negate the possibility of redistribution of NO3" between subcellular compartments. The tungstate treatment did decrease the level of amino acids, particularly G i n (which made up 30% of the amino acids concentration) by 80% (Table 3). These results imply that NO3" itself is not responsible for transcriptional down regulation, but it may effect NO3" influx at a post-transcriptional level. This is in agreement with physiological experiments using the narllnar7 N R double mutants of barley (King et al., 1993; Warner and Huffaker, 1989) which have 1-5% of wi ld type N R activity. These demonstrated that following the typical induction of I H A T S on supplying exogenous nitrate, 130 there was a decrease of NO3" influx and net NO3" uptake, which was correlated with increased nitrate levels in the plant tissue. These observations and our data suggest that NO3" may act as a signal for post-transcriptional regulation. When G l u (which decreased both bch transcript levels and NO3" influx) was supplied simultaneously with tungstate and NO3", bch transcript levels decreased dramatically, demonstrating that repression is acting independently of NO3" induction (Figure 35), since the effect of tungstate alone was to increase bch transcript levels. 4.4.1.3 A m i n o a c i d r e g u l a t i o n o f NO3" i n f l u x a n d bch t r a n s c r i p t a b u n d a n c e Both NO3" influx and abundance of bch transcript in root tissue declined in response to amino acid pretreatments. The effect of the four amino acids tested on NO3" influx were m the following order: Glu>Asp>Asn>Gln. Abundance of bch transcript displayed a similar pattern (Glu>Asp=Asn>Gln); the decrease was greater than 50% in all cases. The increased NO3" concentration in the roots of G i n treated plants, can be explained by the inhibition of N R by Gin (Vincentz et al., 1993; Sivasankar et al., 1997) or possibly a stimulation of L A T S . Exogenous application of amino acids has been shown to have varying effects on NO3" uptake/influx (see next paragraph), however, without examining the fate of exogenous applied amino acids due to uptake and/or metabolism of the amino acid it is impossible to determine the putative effectors of NO3" uptake/influx. A n example of this phenomena in maize was reported by Sivasankar et al. (1997), who found that feeding with either A s n or G i n resulted in increases of endogenous levels of both of the amides in the plant tissue. In our amino acid-treated barley plants, we found that the exogenous application of Gin , G lu , Asp, or A s n resulted in an increase of the levels of the applied amino acid but levels of the other amines and amides increase to the same extent as the 131 applied amino acid. In the above treatments, the increases in G i n and G l u concentrations in root tissue correlated with the decrease in the abundance of bch transcript. Breteler and Arnozis (1985) found that exogenously supplied Arg , Asp, Cys, and Glu inhibited NO3" uptake in N O 3 - induced plants; also Rodgers and Bameix (1993) demonstrated the same pattern of inhibition, while G i n had a minor effect. In the report by Muller and Touraine (1992), the authors fed the amino acid to root tissue either via the cotyledons, to mimic shoot signals (via the phloem) or by exogenous feeding. Whichever the mechanism used for supplying amino acids to soybean roots, these authors found that several amino acids had an inhibitory effect; G i n was one of these but not the most effective. However, since the changes in amino acids concentrations in the root tissue were not monitored the fate o f amino acids supplied in these treatments are unknown. For instance, the present results demonstrate that internal concentrations of Gin , G lu , A s n or Asp increased when amino acids are given to the root, whichever amino acid is supplied. Consequently, this does not discount the role of G in or any other amino acid as a regulator of NO3" influx. Moreover, Til lard et al. (1998) reported that transferring Ricinus communis grown on NO3" to N-free media resulted in transient increase in N 0 3 " influx, and that this increase correlated with a 40 % decrease in the amino acids concentration in the phloem sap (predominantly G i n and Ser). This evidence would support the hypothesis of a possible role of G i n in repression of NO3" influx. In summarizing the effects of exogenous amino acid application in the present experiments, although the effects of amino acids were in order Asp>Glu>Asn>Gln on influx and Glu>Asp=Asn>Gln on transcript abundance, the concentrations of all amino acids increased to the same extent (with notable exceptions) following exposure to single amino acid. Thus Asp feeding increased Asp (x2), A s n (x2), G i n (xl .6) and G l u (xl .8) , G lu increased Asp (xl .6), G i n (x2) and G l u (x2) but had no effect on Asn , G i n increased Asn (xl .9), G i n (xl.5) and G l u (xl .6) but had no effect on Asp, and A s n increased 132 Asp (x2.2), A s n (x3), G i n (xl .4) and Glu (xl .6). Since transcript decline in response to all four amino acids, it would seem that Asp and Asn are not important at the transcriptional level since their concentrations remained unchanged in the G i n and G l u treatments, respectively. This leaves G i n and G l u as the amino acids which consistently increased in concentrations together with decreases in bch transcript level. However, despite the fact that G l u appeared to be more effective than Gin , the amino acid analysis indicate that G l u and G i n increased by similar amounts in response to pretreatment with G lu or Gin . Thus the results of exogenous application of these amino acids must be interpreted with considerable caution. Inhibiting G O G A T or amino transferases activity (using A Z A and A O A respectively) in plants supplied with NO3" dramatically decreased NO3" influx and bch transcript levels in roots (Figure 30 and 31). A Z A treatment affected all the parameters: it decreased NO3" levels in the root tissue by 80%; it decreased influx to a lower level than that of uninduced plants; it increased N H i + levels by 2-fold; it decreased G l u levels and brought also an increase in G i n levels. If G lu were a major down regulator of bch transcript this treatment would have been expected to relieve the down regulation, leading to increased bch transcript abundance. The fact that transcript abundance declined seems to favor a major role for Gin . This treatment is possibly affecting the regulation of nitrate influx, by 1) decreasing the concentration of inducer (NO3"), 2) dramatically affecting the Gln /Glu ratio or possibly the ratio G i n to organic acid, and 3) increasing N H 4 + levels so that it can act as a repressor. 133 4.4.1.4 T h e effect o f N H / o n NO3" u p t a k e a n d bch t r a n s c r i p t a b u n d a n c e Previous reports in the literature suggest that N H 4 + has many effects on NO3" uptake. It is thought to affect influx and/or efflux specifically at the transporter level (Kronzucker et al., 1999). It has also been demonstrated that N H 4 + , in the presence of NO3", can down regulate influx (Aslam et al., 1996) and transcription of the I H A T (Quesada et a l , 1997). However, due to experimental design it was impossible to determine from these studies whether N H 4 + itself or a product of N H 4 + assimilation is responsible for the down regulation of bch m R N A levels. It has been shown that feeding plants with M S O (an inhibitor of GS) led to substantial increases in cytoplasmic N H 4 + concentrations (Lee et al., 1992). In our experiments, an increase of N H 4 + levels was observed in root tissue when NO3" and M S O were supplied together for 6 h, compared to NO3" treatment alone. However, we found only minor differences in bch transcript levels, with or without M S O . Considering that bch expression in control plants is under N-metabolite repression, this would indicate that the N H 4 + ion does affect the accumulation of bch transcript; otherwise, repression normally occurring in the absence of M S O would have been relieved by application of this inhibitor due to decreased inhibition by amino acids. Since influx decreased by 50%, while transcript levels slightly decreased, it is possible that a down-regulation of nitrate uptake at post-translational level occurs due to the N H 4 + accumulating in the root tissue; alternatively toxic effects of M S O on the plant may be responsible for this effect. Treatment of N-starved plants with NO3", M S O and N H 4 + increased N H 4 + concentration (10-fold) in root tissue, while bch transcript levels decreased dramatically, with a 2-fold increase in G i n concentrations. Zhou et al. (1999) demonstrated that root nrt2.1At transcript levels were lower in plants treated with N H 4 + + M S O than in those treated with M V + M S O . However, since their 134 plants were grown on N H 4 , internal concentrations of this ion and glutamine (which may amount to over 60% of the total amino acid concentration under these conditions, see Rawat et al., 1999) are high, and transcriptional down-regulation of nrt2.1At may occur. This provides further evidence that N H 4 + ion can act as a signal for transcriptional regulation. 4.4.1.5 Model for the regulation of bch transcription and nitrate uptake In conclusion, a model for the regulation of nitrate uptake is proposed which operates at multiple levels (Figure 34 and Table 5). 1. Nitrate may down regulate nitrate influx, presumably by a post-transcriptional modification, but not by down regulating bch transcript levels. 2. Ammonium can regulate nitrate uptake at a transcriptional and post-transcriptional level, but the effect at the transcriptional level only occurs when the N H 4 + level in roots is elevated, as for example when both nitrate and ammonium are provided in the root zone, or during treatments (e.g. M S O ) blocking GS activity. 3. Glutamine (also glutamate, aspartate and asparagine) has the ability to decrease NO3" uptake by decreasing bch transcript levels. However, the present study does not allow a distinction to be made between direct effects of Glu , Asp and A s n on transcriptional regulation and effects of these compounds mediated via conversion to Gin . Nevertheless, the evidence from the inhibitor study is consistent with the major role for Gin . This is based upon the A Z A experiment (Fig. 33) as well as the tungstate experiment (Table 3) as transcript levels increased. This increase of transcript was associated with a significant decline in [Gin] but virtually no change in [Glu]. 135 Table 5. Regulation of I H A T S : hypotheses and observations. Hypothesis observation Nitrate does not down regulate bch transcript levels. a) Decrease in nitrate influx (Fig. 30 tungstate treatment). b) sustained increase in bch transcript (Fig. 31, 32 and 33 tungstate treatment). Ammonium affects both at a transcriptional and post-transcriptional levels c) Decrease in bch transcript levels, when ammonium levels are high (Table 4). d) Ammonium affects I H A T S at a post-transcriptional level, decrease in influx (50%) with small affect in bch transcript levels (Figure 31 and 32, M S O treatment). Amino acid down regulation of bch transcript levels is mediated by G i n e) Ammonium treatment, dramatically decreases bch transcript level, when assimilation is not blocked (Figure 24). f) Addition of amino acids, decrease in bch transcript levels to varying degrees (Figure 27). g) Increase in G lu and G i n levels in root tissue correlated with decrease in bch transcript levels, (exogenous application of amino acids). h) Treatments with A Z A and A O A dramatically decreased bch transcript levels (Figure 31), and resulted in dramatic decrease in nitrate influx (Figure32). i) Correlation of levels of bch transcript with amino acid levels (including amino acid and N -inhibitor treatments) suggest G in as possible effector. 136 Figure 33. Proposed model for regulation of nitrate transporter IHATS and bch expression. 137 CONCLUSIONS 5.1.1.1 Conclusions Many different approaches have been used in the isolation of solute transporters. These include biochemical methods (e.g., the H + ATPase: Harper et a l , 1989), differential screening of c D N A library (e.g. the glucose transporter, Sauer and Tanner, 1989), functional complementation (e.g., the K + channel, Sentenac et al., 1992), screening for T - D N A tagged mutants (e.g., the low-affinity nitrate transporter nrtl (chll), Tsay et al., 1993). In this work, a c D N A encoding a high-affinity sulfate transporter has been isolated by a novel method involving heterologous complementation in bacteria. The mechanism(s) involved in controlling the transcriptional regulation of this gene were investigated by modifying the rate of sulfate supply to barley plants and through use of various metabolic inhibitors. The data presented indicate that derepression of the high-affinity sulfate transporter in response to withholding S, is the result of metabolite derepression. Evidence is provided that this metabolite is glutathione (Chapter 2). On the other hand, Smith et al. (1997) showed that O-AS may play a role as a positive acting signal capable of upregulating hvstl expression. In order to determine how these signals are transduced in the plants, the isolation of promoter regions (Chapter 2) and promoter analysis by deletions of promoter sequence, in vivo and in vitro D N A footprinting, and D N A mobility assays w i l l be of the utmost importance. There are many questions still to be addressed. For example, where is the high-affinity sulfate transporter localized? To answer this question it w i l l be necessary to undertake in situ hybridations, immuno-localizations and reporter gene studies. Are there other sulfate transporters in barley? A. thaiiana has seven putative sulfate transporters, while S. hamata has three. What are the roles of these other transporters? 139 Transcripts o f all members of the bch gene family (bchl, bch2, bch3, and bch4) increase (are induced) by provision of NO3". The presence of N H 4 + during the first hours of induction appears not to affect bch transcript accumulation, suggesting that the induction step of bch gene expression may not be the site of action of N H 4 + (chapter 3). Rather, it may be that N H 4 + and/or its assimilation products have a more pronounced effect on the down-regulation of bch transcript accumulation. The down-regulation of NO3" uptake, in contrast to its induction, appears to be exerted at multiple levels. Nitrate itself down regulates N03" influx at a post-transcriptional level, but does not seem to be involved in the transcriptional down-regulation. N H 4 + down-regulates both at a transcriptional and post-transcriptional level. In plants grown on NO3" for long durations, NO3" transport and bch transcript levels declined in root tissue, while no significant increase in N H 4 + concentrations occurred, but amino acid concentrations increase dramatically. Therefore under steady state conditions amino acids are likely key regulators involved in the adaptation of plants to various levels of NO3" availability. Plant concentrations of amino acids were altered by feeding amino acids exogenously or by inhibitor treatments. Such amino acid treatments decreased bch m R N A levels and NO3" influx to varying degrees, and an analysis of amino acid concentration in root tissue showed a negative correlation between G i n level and bch m R N A levels. Isolation of the promoter sequences of the bchl, bchl and bch3 genes, showed the presence of a core N I E domain, which has been implicated in the induction pathway of N R genes in Arabidopsis. This domain may be responsible for this pathway in barley, but that there are also evolutionary differences in these elements due the absence of the A T rich region, which precedes the A ( G / C ) T C A core sequence in Arabidopsis; this is also the case for the barley N R genes. Also present in the promoter sequences of bchl, bch2, and bch3 genes was domain I which at present has no known function. To further understand the role of these elements future research should 140 be directed to the analysis of their respective promoters. The type of analysis would include deletion analysis, in vitro and in vivo D N A footprinting, and D N A mobility assays. Once putative cis-acting elements have been characterized, the elements should be used as baits for one-hybrid screening and/or south-western screening of c D N A libraries, to isolate transcription factors. These types of approach should reveal the mechanisms of both induction and repression of nitrate transporter. In barley it has been demonstrated that 7-10 copies of the bch gene are present in the genome. At present 4 of these c D N A s have been isolated; What is the localization of these transporters? 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