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High-Affinity NH₄⁺ transport in rice (Oryza sativa L.) : physiology, biochemistry and molecular biology Kumar, Anshuman 2003

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High-Affinity N H Transport in Rice (Oryza sativa L.): Physiology, Biochemistry and Molecular Biology. 4  by  Anshuman Kumar  M. Sc., University o f Delhi, 1994 M. Phil., University o f Delhi, 1995  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E REQUIREMENTS FOR T H E DEGREE OF  DOCTOR OF PHILOSOPHY In T H E F A C U L T Y OF G R A D U A T E STUDIES (Department o f Botany) We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A June 2003 © Anshuman Kumar, 2003  In presenting  this  degree at the  thesis  in  University of  freely available for reference copying  of  department  this or  publication of  partial fulfilment  of  British Columbia,  I agree  and study.  this  his  or  her  Department of  ^  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  that the  representatives.  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  thesis for scholarly purposes by  the  is  that  an  advanced  Library shall make it  permission for extensive  granted  by the  understood be  for  allowed  that without  head  of  my  copying  or  my written  Abstract  In plants, N H  influx occurs via a saturable high-affinity, and a low-affinity transport  + 4  system. Regulation of  1 3  NH  influx was studied by altering N H  + 4  pretreatments with amino acids. Diurnal variations in N H Increased N pools caused N H NH  + 4  + 4  supply and  influx were also studied.  influx to decrease, whereas reduced N pools increased  + 4  influx. When rice plants were transferred from 10 p M to 10 m M N H , H A T S  +  +  4  4  influx of N H 1 3  + 4  was rapidly down-regulated to < 20% within 72 h. Conversely,  1 3  NH  + 4  influx exhibited a steep increase following transfer from 10 m M to 10 p M N H . Upon +  4  pretreatments with amino acids, N H NH  influx when N H  + 4  + 4  + 4  influx was reduced. There was no major decline in  assimilation was blocked by M S X treatment; however when  either Gin or Asn was added together with M S X , N H concentrations of N H regulation of N H  + 4  + 4  + 4  influx declined rapidly. Root  and all 4 amino acids increased substantially during down-  influx and were reduced during the up-regulation. Pretreatments with  Gin, Asn, Glu, or Asp led to increased root concentrations of these amino acids as well as that of N H . When treated with M S X , root [NH ] increased several fold, while root +  +  4  4  [Gin] declined accompanied with increased root [Asn], [Glu], and [Asp]. Transcript levels of OsAMTl.l  in roots decreased several fold within 48 hours of  transfer from 10 p M to 10 m M N H . Likewise when plants acclimated in 10 mM N H +  4  + 4  were transferred to 10 u M N H , there was an equally rapid up-regulation of OsAMTl.l +  4  and  1 3  NH  + 4  influx in the roots. Changes in transcript abundance following these  treatments were in order OsAMTl.l  > OsAMTl.l  > OsAMTl.3.  Amino acid  ii  pretreatments also reduced OsAMTl transcripts. M S X pretreatment slightly reduced OsAMTl.l transcripts in 12 h. However, additions of Gin or Asn with M S X caused a much greater reduction. OsAMTl.3 expression and NH4 influx increased approximately 3-fold late in 15  the photoperiod, while OsAMTl.l  +  and OsAMTl.2 exhibited only modest changes.  Sucrose application during the dark period increased root sucrose concentration and OsAMTl.3 expression. Growth analysis and N H / influx of transgenic lines, over-expressing OsAMTl.l in two cultivars were also undertaken. One over-expression line, 75-4, showed increased influx.  iii  Table of Contents Abstract  ii  Table of Contents  iv  List of Tables and Figures  vii  List of Abbreviations.  xii  Acknowledgements  xiii  Dedication  x  Chapter 1. General Introduction  v  1  1.1 Earlier research  2  1.2 Energetics of NH Influx  3  1.3 Kinetics of N H  5  +  4  + 4  Influx  1.4 Regulation of N H / Influx 1.5 Molecular Biology N H  + 4  •  Influx  8  Chapter 2. Nitrogen Effects  2.1 Physiology and Biochemistry of N H  6  10  + 4  Transport  10  2.1.1 Introduction  10  2.1.2 Materials and Methods  13  2.1.3 Results  20  2.1.4 Discussion  62  iv  2.2 Molecular Biology of N H  Transport  + 4  69  2.2.1 Introduction  69  2.2.2 Materials and Method  70  2.2.3 Results  73  2.2.4 Discussion  104  Chapter 3. Diurnal Studies  108  3.1 Physiology and Biochemistry of N H  + 4  Transport  108  3.1.1 Introduction  108  3.1.2 Materials and Method  109  3.1.3 Results  110  3.1.4 Discussion  115  3.2 Molecular Biology of N H  + 4  Transport  118  3.2.1 Introduction  118  3.2.2 Materials and Method  119  3.2.3 Results  119  3.2.4 Discussion  127  Chapter 4. Transgenic Studies  4.1.1 Introduction  130  -130  4.1.2 Materials and Method  132  4.1.3 Results  137  v  4.1.4 Discussion  151  Chapter 5. Summary, Conclusions and Future Prospects  158  References  164  vi  List of Figures and Tables  Figure 2.1.1. Down-regulation of N H 1 3  + 4  influx in rice roots with differentN sources... 22  Figure 2.1.2.Down-regulation and up-regulation of N H 1 3  Figure 2.1.3. Down-regulation of N H  4  Figure 2.1.4. Down-regulation of N H  4  1 3  1 3  Figure 2.1.5. Changes in N H  4  Figure 2.1.6. Changes in N H  4  Figure 2.1.7. Changes in N H  4  1 3  1 3  1 3  + 4  influx in rice roots  23  +  influx in rice roots with Gin, and Asn  26  +  influx in rice roots with Glu, and Asp  27  +  influx in rice roots, M S X treatment  28  +  influx in rice roots, M S X and Gin treatment  29  +  influx in rice roots, M S X and Asn treatment  30  Figure 2.1.8. Changes in Gin and Asn concentrations in rice seedling roots  33  Figure 2.1.9. Changes in Glu and Asp concentrations in rice seedling roots  34  Figure 2.1.10. Changes in N H  35  + 4  concentrations in rice seedling roots.  Figure 2.1.11. Changes in Gin and Asp concentrations in roots during up-regulation.... 36 Figure 2.1.12. Changes in Glu and Asp concentrations in roots during up-regulation.... 37 Figure 2.1.13. Changes in N H  + 4  concentrations in rice roots during up-regulation  38  Figure 2.1.14. Gin and Asn concentrations in rice roots, with Gin treatment  41  Figure 2.1.15. Glu and Asp concentrations in rice roots, with Gin treatment  42  Figure 2.1.16. Changes in N H  concentrations in roots, with Gin treatment  43  Figure 2.1.17. Gin and Asn concentrations in rice roots, with Asn treatment  44  Figure 2.1.18. Glu and Asp concentrations in rice roots, with Asn treatment  45  Figure 2.1.19. Changes in N H  concentrations in roots, with Asn treatment  46  Figure 2.1.20. Gin and Asn concentrations in rice roots, with Glu treatment  47  Figure 2.1.21. Glu and Asp concentrations in rice roots, with Glu treatment  48  Figure 2.1.22. Changes in N H  49  + 4  + 4  + 4  concentrations in roots, with Asn treatment  vii  Figure 2.1.23. Gin and Asn concentrations in rice roots, with Asp treatment  50  Figure 2.1.24. Glu and Asp concentrations in rice roots, with Asp treatment  51  Figure 2.1.25. Changes in N H  4  52  Figure 2.1.26. Changes in N H  4  +  concentrations in roots, with Asn treatment  +  concentrations in roots, with M S X , M S X and Gin, and  M S X and Asn treatments  55  Figure 2.1.27. Gin and Asn concentrations in rice roots, with M S X treatment  56  Figure 2.1.28. Glu and Asp concentrations in rice roots, with M S X treatment  57  Figure 2.1.29. Gin and Asn concentrations in roots, with M S X and Gin treatment  58  Figure 2.1.30. Glu and Asp concentrations in roots, with M S X and Gin treatment  59  Figure 2.1.31. Gin and Asn concentrations in roots, with M S X and Asn treatment  60  Figure 2.1.32. Glu and Asp concentrations in roots, with M S X and Asn treatment  61  Figure 2.1.33. N H  assimilation enzymes  65  Figure 2.2.1. Sequence alignments of OsAMTl family members  74  Figure 2.2.2. Phylogenetic tree of ammonium transporter proteins  75  Figure 2.2.3. Expression patterns of OsAMTl.l in roots during down-regulation  77  Figure 2.2.4. Expression patterns of OsAMTl.2 in roots during down-regulation  78  Figure 2.2.5. Expression patterns of OsAMTl.3 in roots during down-regulation  79  Figure 2.2.6. Expression patterns of OsAMTl.l in rice roots during up-regulation  80  Figure 2.2.7. Expression patterns of OsAMTl.2 in rice roots during up-regulation  81  Figure 2.2.8. Expression patterns of OsAMTl.3 in rice roots during up-regulation  82  Figure 2.2.9. Expression patterns of OsAMTl.l in roots during Gin treatment  85  Figure 2.2.10. Expression patterns of OsAMTl.2 in roots during Gin treatment  86  Figure 2.2.11. Expression patterns of OsAMTl.3 in roots during Gin treatment  87  + 4  Figure 2.2.12. Expression patterns of OsAMTl.l  in roots during Asn treatment  88  Figure 2.2.13. Expression patterns of OsAMTl.l  in roots during Asn treatment  89  Figure 2.2.14. Expression patterns of OsAMT1.3 in roots during Asn treatment  90  Figure 2.2.15. Expression patterns of OsAMTl.l  in roots during Glu treatment  91  Figure 2.2.16. Expression patterns of OsAMTl.l  in roots during Glu treatment  92  Figure 2.2.17. Expression patterns of OsAMTl.3 in roots during Glu treatment  93  Figure 2.2.18. Expression patterns of OsAMTl.l  in roots during Asp treatment  94  Figure 2.2.19. Expression patterns of OsAMTl.l  in roots during Asp treatment  95  Figure 2.2.20. Expression patterns of OsAMT1.3 in roots during Asp treatment  96  Figure 2.2.21. Expression patterns of OsAMTl.l  in roots during MSX, or MSX and Gin  treatment  98  Figure 2.2.22. Expression patterns of OsAMTl.l  in roots during MSX, or MSX and Gin  treatment  99  Figure 2.2.23. Expression patterns of OsAMT1.3 in roots during MSX, or MSX and Gin treatment  100  Figure 2.2.24. Expression patterns of OsAMTl.l  in roots during MSX, or MSX and Asn  treatment  101  Figure 2.2.25. Expression patterns of OsAMTl.l  in roots during MSX, or MSX and Asn  treatment  102  Figure 2.2.26. Expression patterns of OsAMTl.3 in roots during MSX, or MSX and Asn treatment  103  Figure 3.1.1. NFL; influx in rice roots during a complete diurnal cycle 15  +  112  ix  Figure 3.1.2. N H 1 5  influx in rice roots with and without sucrose treatment  + 4  113  Figure 3.1.3. Internal sucrose concentrations in rice roots with and without exogenous sucrose treatment  114  Figure 3.2.1. Expression patterns of OsAMTl.l  in rice roots during a complete diurnal  cycle  122  Figure 3.2.2. Expression patterns of OsAMTl.2  in rice roots during a complete diurnal  cycle  123  Figure 3.2.3. Expression patterns of OsAMTl.3  in rice roots during a complete diurnal  cycle  124  Figure 3.2.4. Expression patterns of OsAMTl.l,  OsAMTl.2,  and OsAMTl.3  in roots  during the daytime with and without sucrose Figure 3.2.5. Expression patterns of OsAMTl.l,  125 OsAMT1.2, and OsAMTl.3  in roots  during the night time with and without sucrose  126  Figure 4.1. OsAMTl.l  135  Figure 4.2. N H B  + 4  influx in the transgenic lines and the two wild type rice cultivars  grown in 1 0 p M N H Figure 4.3.  1 3  NH  + 4  gene construct for rice reformation  + 4  138  influx in the transgenic lines and the two wild type rice cultivars  grown in 2 mM N H  + 4  139  Figure 4.4. Biomasses of the transgenic lines and the wild type cultivar Jarrah grown in 10pMNH  + 4  144  Figure 4.5. Biomasses of the transgenic lines and the wild type cultivar Taipei grown in 10pMNH  + 4  145  x  Figure 4.6. Biomasses o f the transgenic lines and the w i l d type cultivar Jarrah grown in 2mMNH  146  + 4  Figure 4.7. Biomasses of the transgenic lines and the w i l d type cultivar Taipei grown in 2mMNH  147  + 4  Figure 4.8. N H  4  Figure 4.9. N H  4  +  Efflux from roots o f wild-type rice plants  149  +  Efflux from roots o f Jarrah cultivar 75-4 rice plants  150  Table 4.1. List o f transgenic lines used  136  xi  List of Abbreviations  A A T : Aspartate amino transferase A S : Asparagine synthetase Asn: Asparagine Asp: Aspartate C: Carbon D W : D r y weight F W : Fresh weight G D H : glutamate dehydrogenase Gin: Glutamine Glu: Glutamate G O G A T : Glutamate synthase GS: Glutamine synthetase H A T S : High-affinity transport system L A T S : Low-affinity transport system M S X : Methionine sulfoximine m R N A : messenger ribonucleic acid N : Nitrogen NH4 : Ammonium +  N0 ": Nitrate 3  N A D : Nicotinamide adinine dinucleotide (oxidized form) N A D H : Nicotinamide adinine dinucleotide (reduced form) N A D P : Nicotinamide adinine dinucleotide phosphate (oxidized form) N A D P H : Nicotinamide adinine dinucleotide phosphate (reduced form) P A R : photosynthetically active radiation R T - P C R : Reverse transcription-polymerase chain reaction  Acknowledgements  First of all I would like to thank my supervisor professor Anthony D. M . Glass for extending his challenging ideas to me, besides providing the intellectual and financial supports. He made me think like the scientist and philosopher like him. Besides he has always shown the human side of a scientist during time of need throughout my research. Also, I will always remember his ever smiling face that hides a thoughtful mind and a caring soul. I wish to thank my committee members, professors Anthony J. F. Griffiths, Paul J. Harrison and X i n L i for their helpful discussions and suggestions. I can never ever thank enough my professor and pro vice-chancellor at the University of Delhi, C. R. Babu whose countless helps established foundations of a scientist inside me, during my masters' degree and beyond. I am also grateful to Professors M . R. Vijayaraghavan and K. R. Sharma of University of Delhi for helping me to reach my goal of undertaking Ph.D. research. M y special thanks go to Dr. Salim Silim, who when working as a research associate of Prof. Glass, was like an executive supervisor for me during all his stay in our lab. He stood by me during all my good and bad times against all odds. The generous helps extended by past and present Glass lab members Brent Kaiser, Mamoru Okamoto, John Vidmar, Stefanie Wienkoop, Aniko Varga, and Yaeesh Siddiqi supported my work and made research enjoyable. My thanks also go to them. I am also thankful to Prof. Tom Rufty of North Carolina State University for the collaboration in performing sugar and amino acid analysis.  xiii  I have no word in my vocabulary that would state my true gratefulness to my mother Mrs. Jayanti Devi Mishra, father Mr. Aditya N. Mishra, and my elder brother Arvind K. Mishra, without whose persistent help and encouragements I would have ever aspired or achieved my objectives. Actually, it was my mother's gold medal that she earned as best graduating student in her university in her master's degree inspired me to achieve something similar by earning first class first position in the university, when I earned my B.Sc. honors degree, which served as the foundation stone o f my higher studies and research. Therefore, as I did with my master's thesis, I have dedicated this work and the Ph.D. thesis to my respected mother.  xiv  Dedicated to my Respected Mother  1 General introduction  Rice (Oryza sativa L.) is one o f the most important crops in the world with an annual production o f approximately 595.26 million tonnes in 2001 ( F A O , 2001). It is consumed as a staple food by over 4 0 % o f the world's population (De Datta, 1981), and is the energy source for over half o f the world's population (Buresh and De Datta, 1991). Nitrogen is commonly the single most important element that limits crop yield, and both the yield and protein content o f rice increases with proper application o f N (Gomez and De Datta, 1975; Allen and Terman, 1978). Among the nitrogen sources, ammonium is the preferred species taken up b y rice plants (Sasakawa and Yamomoto, 1978; Goyal and Huffaker, 1984). The energetics and kinetics o f N H 4 (Wang et al.  +  fluxes into rice roots have been worked out  1993a; Wang et al. 1993b; Wang et al. 1994a; Wang 1994b), but the  biochemical and molecular mechanism o f control is virtually unknown. Moreover, until now there has been no success in developing functional rice Rhizobium (or functional  1  rice-N2 fixing bacteria) symbioses. Therefore, in order to meet the demand o f an evergrowing population we need rice plants that are more efficient i n nitrogen uptake, translocation and assimilation. A n understanding o f the biochemical and molecular mechanism o f N H / acquisition and transport particularly via the High-Affinity Transport System (HATS) w i l l enable plant breeders and molecular biologists to develop and select varieties o f rice that are more efficient i n N H / uptake, translocation and assimilation. Hence, the initial aim o f my research was to contribute towards a better understanding o f biochemical and molecular control mechanisms o f these physiological processes.  1.1  Earlier research  The earliest published work on N H / uptake by plant roots was by Becking (1956) in which he did extensive experiments on the relationships between rates o f ammonium ion uptake by maize roots and external ammonium ion concentrations. Later, Ullrich et al. (1984) measured tissue [ N H / ] and electrical potential differences in  Lemna  fronds and concluded that below 67 p M ammonium  uptake was active, while at higher [ N H / ] , uptake was passive. Other work especially by Kosola and Bloom (1994) on tomato; Fentem et al. (1983) on Hordeum; and Jackson (1988) on wheat and oat; Dubois and Grenson (1979) on cerevisiae;  Morgan  Saccharomyces  Wang et al. ( 1993a, b, 1994b) on rice; Kronzucker et al. (1995 a, b, 1996)  on spruce have concluded that at relatively low external [ N H / ] the ammonium uptake is by ( H A T S ) , while at high external [ N H / ] ( >1 m M ) the uptake is via a low affinity transport system (LATS).  2  1.2  Energetics of  NH Influx +  4  The electrochemical potential difference o f a charged solute is determined using the following equation: A Uo-i = RT  In ( C o / C j ) - z F (E  1  -E°)  where, A  Lio_i  = electrochemical potential difference  R = Gas constant (8.3 J m o l " , d e g " ' ) 1  T = Absolute temperature (273 + C ) 0  In  (Co/Q)  = log base e o f the concentration o f solute outside/ solute inside  z = Charge on the solute (+1 for N F f / ) F = Faraday constant (96.5 J mol" . m V ' ) 1  1  (E -E°) = E m = Electrical potential difference in mV. 1  Thus we must estimate the electrical potential difference (Em) across the plasmalemma and cytoplasmic [ N H / ] ( C i ) to calculate the electrochemical potential difference (A p .;) across the membrane for NH4 . Only then can the thermodynamics +  0  o f NH4 influx be evaluated definitively to determine, whether N H  +  +  4  is actively or  passively transported across the plasma membrane.  Higinbotham et al. (1964) reported a marked depolarization effect o f [ N H V ] on the electrical potential difference ( E m ) in coleoptile cells in oats. Later Ullrich et al. (1984) reported the electrochemical potential difference (Em) in Lemna to  3  be between -200  and  -280 m V . They also calculated the approximate diffusion  potential ( E D ) , (with the Nernst Equation) which varied between -80 and -125 mV. Using these two values they concluded that an inwardly directed driving force acts at external concentrations down to 67 p M , and energy is required only at a lower external concentration. Wang et al. (1993c) reported the electrical potential difference (Em) in rice roots to be in the range o f -120 to -140 m V in the absence o f nitrogen and at steady state E m was -112 m V for plants grown on 2 p M [ N H ] and -89 m V for plants grown +  4  at 100 p M [ N H ] . Earlier Wang et al. (1993a) undertook a compartmental analysis +  4  using  1 3  NH  + 4  and estimated [ N H ] +  4  c y t  to be from 3.7 m M , in plants grown in 2 p M  [ N H ] , to 38 m M in plants grown in I m M [ N H ] . They concluded that below 42 p M +  +  4  4  for plants grown at 2 p M [ N H ] and below 655 p M for plants grown at 100 p M +  4  [ N H ] , ammonium influx was an active process and that above these concentrations +  4  ammonium uptake was passive in these intact roots o f rice plants. Later, Kronzucker et al. (1995a) estimated [ N H ]  in roots o f spruce to be 13 m M in plants grown in 100  +  4  c y t  p M [ N H ] to 34.7 m M in plants grown at 1.5 m M [ N H ] . The values obtained by +  +  4  4  other workers range from 78 m M in excised roots segments o f Allium cepa (Macklon et al.  1990) down to < 1 5 p M , in roots of maize (Roberts and Pang, 1992). A l l these  reports demonstrate that in rice and other plants, thermodynamically active transport occurs at low external [ N H ] and at higher external [ N H ] transport is passive. Also +  +  4  4  the break-point for active and passive influx is dependent upon the [ N H ] in the +  4  growth solution, and  the switching-point from active transport to passive transport  increases with increasing [ N H ] +  4  which in turn is increased by high [ N H ] . +  c y t  4  0  4  1.3  Kinetics of N H  + 4  Influx  Becking (1956) first described net uptake o f N H  as a hyperbolic function o f  + 4  external [ N H ] . The uptake studies in different plants e.g. wheat (Tromp, 1962; Cox +  4  and Reisensuer, 1973), ryegrass (Lycklama, 1963), rice (Fried et al. 1965, Youngdahl et al.  1982), maize (McNaughton and Presland, 1983; Presland and McNaughton,  1986), and tomatoes (Smart and Bloom, 1988) demonstrate that N H concentration  dependent  and follows  Michaelis-Menten  kinetics.  uptake is  + 4  Most  o f the  experiments were done using net depletion method.  Using that N H  + 4  1 3  NH  + 4  to measure unidirectional influx Wang et al. (1993b) demonstrated  influx in rice is saturable with K  m  values o f 32 p M in plants grown in 2 p M  [ N H ] and 188 p M in plants grown in 1 m M [ N H ] , while at higher [ N H ] a second +  +  4  +  4  transport system became apparent, indicating that N H  4  influxes across the plasma  + 4  membrane into rice roots follows a biphasic pattern. Thus, in the low range (< 1 m M external [ N H ] ) , influx occurred via a saturable high-affinity transport system, while +  4  from 1 m M to 40 m M external [ N H ] , a nonsaturable low-affinity transport system +  4  operates. Kronzucker (1996) determined that in spruce, K  m  values varied in the range  from 20 to 40 p M and concluded that in N-deprived plants, a Michaelis-Menten type high-affinity transport system operates in 2.5 to 350 p M external [ N H ] and from 500 +  4  p M to 50 m M external [ N H ] , a linear low-affinity transport system operates. This +  4  biphasic pattern o f uptake was earlier reported by Ullrich et al. (1984) in Lemna for NH  + 4  uptake; by Kochian and Lucas (1982) in corn roots for K uptake and by Siddiqi +  et al. (1990) in barley roots for NO3" uptake.  5  The H A T S for N H / may be a proton co-transporter (Glass, 1988; Wang et al. 1994a). The L A T S may be an electrogenic uniport system. Recently, however, Shelden et al. (2001) reported that heterologous expression o f AtAMT1.2, one o f the several genes coding for H A T S  in Arabidopsis  methylammonium uptake with K  m  thaliana in yeast resulted in biphasic  values o f 36 p M and 3.0 m M , respectively,  suggesting that a single protein might mediate both H A T S and L A T S fluxes.  1.4  Regulation of N H  + 4  Influx  Feedback inhibition o f N H / uptake by either N H / or other nitrogenous molecules has been suggested in lower plants (Flores et al. 1980; Wiame et al. 1985, Bagchi et al.  1985; Thomas and Harrison, 1985); Saccharomyces (Dubois and  Grenson, 1979); Synechococcus (Suzuki et al. 1993) and higher plants (Ullrich et al. 1984; Lee and Rudge, 1986; Morgan and Jackson, 1988).  Lee and Rudge (1986) showed that withholding external N from barley plants previously growing on ammonium caused a 2.4 fold increase in their subsequent capacity to absorb N H / . Although they also indicated that these responses to mild N stress may depend upon an N fraction other than ammonium or nitrate. Morgan and Jackson (1988) showed that net ammonium uptake by wheat and oat seedlings were increased 5 to 10 fold when the seedlings were deprived o f nitrate. They concluded that this finding was the result of: (a) the operation o f two ammonium influx systems, (b) the interplay o f tissue ammonium and a product o f its assimilation respectively acting  6  as positive and negative effectors o f a single influx system, and (c) variations in energy supply from the shoots. Lee et al. (1992) concluded that high intracellular levels o f glutamine or aspargine reduced the influx and net uptake o f N H / as well as that o f NO3", while reduced levels o f glutamine and/or aspargine had the opposite effect. Other studies on barley (Lee and Ayling, 1993) and maize (Jackson et al. Ayling 1993; Feng et al.  1993; Lee and  1994) also indicate the involvement o f glutamine or some  other N-assimilation product as potential regulators o f N uptake. However, Bagchi et al. (1985) after experimenting with glutamine auxotrophs o f Anabaena indicated that both glutamine and N H / might participate i n the regulation o f NO3" uptake. Wang et al. (1993a) demonstrated that N H / uptake was down-regulated with an increase in external [ N H / ] from 2 p M to 1000 p M , while N H / efflux from seedling roots was increased as the external [ N H / ] increased from 2 p M to 1000 p M . They interpreted this as a result o f increased cytoplasmic [ N H / ] . Further experiments by Glass et al. (1997) using methionine sulfoximine ( M S X ) to block N H / assimilation indicated that in low-N roots, glutamine was important i n regulating N H / influx, while in high-N roots, N H / itself appeared to participate in down-regulating influx. Some researchers have demonstrated N-cycling from root to shoot and vice-versa. (Cooper and Clarkson, 1989; Larsson et al. 1991) as a possible way o f information transfer on the nitrogen status o f the shoot back to roots, i n order to regulate N absorption according to the demand imposed by growth o f the plant as a whole.  In summary, although there is evidence for an important role o f glutamine or some other reduced N compounds in regulation o f N H / uptake, the role o f N H / itself can not be ruled out. Actually the identity o f the signal molecule responsible for  7  regulating H A T S expression is far from clear and that for regulation o f L A T S is even more uncertain. Particularly in rice, much research is required before concluding about the biochemical and/or molecular regulatory mechanism o f N H  + 4  uptake via H A T S .  In the present study, I explored the regulation o f H A T S and three OsAMTl genes {OsAMTl.l,  OsAMTl.2, and OsAMTl.3) putatively encoding a H A T S for N H  while simultaneously measuring N H  + 4  influx,  influx into rice roots by means o f time-course  + 4  treatments together with additions o f high or low concentrations o f N H , amino acids +  4  and amides plus or minus metabolic inhibitors. I n addition, I have investigated diurnal patterns o f influx with  1 5  N H , while simultaneously monitoring the expression patterns +  4  o f all three genes in seedling roots under the same conditions o f N status. I have measured  1 3  NH  + 4  influx and efflux in transgenic rice plants in which OsAMTl.l, the  first isolated member o f the AMT1 gene family i n rice, was over-expressed. For all the experiments except the diurnal and transgenic studies, detailed amino acid and tissue ammonium analysis was also performed for all the treatments. Also I have explored the physiology o f transgenic rice plants, over-expressing OsAMTl.l  1.5  Molecular Biology of N H  The first N H (Marini et al.  + 4  + 4  in various lines.  Influx  transporter {AMI) genes were cloned from yeast and Arabidopsis  1994; Ninnemann, Jauniaux & Frommer 1994). AMT1 was by  complementation o f the yeast double mutant, meplmep2, which is resistant to methylamine, a toxic analog o f ammonium. The meplmepl double mutant is defective in both ammonium transport systems and can grow only at an external [ N H ] o f about +  4  8  20 m M (Dubois and Grenson, 1979). The yeast meplmep2 double mutant expressing an Arabidopsis AMT1 gene takes up [ C ] methylamine in a saturable concentration 1 4  dependent manner consistent with a carrier mediated uptake, with a K which is in the same as that range reported for N H  + 4  m  o f 65 p M ,  influx by H A T S in rice and spruce  roots.  During the last few years, a number o f researchers have cloned additional N H  + 4  transporter genes (AMTs) from various higher plants and other organisms, e.g. tomato (Lauter et al. 1996; von Wiren et al. 2000), Arabidopsis (Gazzarrini et al. 1999), Lotus (Salvemini et al. 2001), Brassica napus (Pearson, Finnemann & Schjoerring 2002), rice (accession numbers: AF289477, AF289478 and AF289479), and from Homo sapiens and mouse ( L i u et al. 2000; Marini et al. 2000). Recently a total o f ten AMI  genes have been reported in rice (Suenaga et al. 2003). Interestingly in most o f  these plants, NO3" rather than N H  + 4  has been considered to be the predominant form in  which N is absorbed from the external environment. This conclusion is largely based upon the greater concentrations o f NO3" than N H 4 found in most agricultural soils (See +  e.g. Wo It, 1994). Moreover, despite a wealth o f physiological information regarding nitrogen, and particularly N H , uptake by rice and other cereals (Lee & Rudge 1986; +  4  Morgan & Jackson 1988; Wang et al. 1993a, b ) , there is to date no published work dealing with molecular regulation o f genes encoding N H  + 4  transporters i n any cereals.  The cloning o f the AMT genes from rice now makes it possible to investigate their role(s) in high-affinity N H  + 4  transport by means o f correlations between patterns o f  gene expression and high-affinity N H  influx determined by use o f N H 1 3  4  + 4  and  1 5  NH , +  4  and internal concentration o f N H , amino acids and amides. +  4  9  2 Nitrogen Effects 2.1 Physiology and Biochemistry of N H Transport +  4  2.1.1 Introduction As described in chapter 1, nitrogen is the single most important element limiting crop yield, and both the yield and protein content o f rice increase with proper application o f N (Gomez & De Datta 1975; Allen & Terman 1978). Under field conditions, ammonium ( N H / ) represents the major source o f N for rice plants (Sasakawa & Yamamoto 1978; Goyal & Huffaker 1984). Although under laboratory conditions, nitrate ( N O 3 ) may also be effectively absorbed (Kronzucker et al. 2000) when both N species are present, NO3" uptake is severely reduced by the presence o f N H / (Youngdahl et al. 1982). The earliest kinetic analysis o f N H / uptake was by Becking (1956), who undertook extensive experiments on the relationships between rates o f N H / ion uptake and N H / ion concentrations by maize roots. Later, Ullrich et al. (1984) measured tissue [ N H / ] and electrical potential differences in Lemna fronds  10  and concluded that below 67 U.M ammonium uptake was active, while at higher [NH4 ], uptake was passive. Other works especially by Kosola & Bloom (1994) in tomato, Fentem, Lea & Stewart (1983) i n Hordeum, Morgan & Jackson (1988) in wheat and oat, Dubois & Grenson (1979) in Saccharomyces cerevisiae, Wang et al. (1993a, b) i n rice, and Kronzucker, Siddiqi & Glass (1995a, b, 1996) i n spruce have concluded that at relatively l o w external [NH4 ], ammonium uptake is by a high-affinity transport +  system ( H A T S ) , while at high external [ N H ] (>1 m M ) uptake is via a low-affinity +  4  transport system (LATS). Recently, however, Shelden et al. (2001) reported that heterologous expression o f AtAMT1.2 in yeast resulted in biphasic methylammonium uptake with K m values o f 36 p M and 3.0 m M , respectively, suggesting that a single protein might mediate both H A T S and L A T S fluxes. Feedback inhibition o f N H molecules  has been  implied  uptake by either N H  + 4  in Saccharomyces  + 4  (Dubois  or other nitrogenous &  Grenson  1979),  Synechococcus (Suzuki, Sugiyama & Omata 1993), lower plants (Flores, Guerrero & Losada 1980; Ullrich et al. 1984; Wiame, Grenson & A r s t 1985; Bagchi et al. 1985; Thomas & Harrison 1985) and higher plants (Lee & Rudge 1986; Morgan & Jackson 1988; Wang et al. 1993a, b; Glass & Siddiqi 1995). Lee & Rudge (1986) showed that withholding external N from barley plants previously growing on N H  + 4  caused a 2.4  fold increase in subsequent capacity to absorb NH4 . They concluded that these +  responses to mild N stress probably depended upon a N fraction other than NH4 or +  NO3", their main argument being that feedback from a down-stream product o f NO3" or NH4  +  assimilation would best integrate the uptake o f various N forms. Morgan &  Jackson (1988) showed that net N H  + 4  uptake by wheat and oat seedlings was increased  11  5- to 10-fold when the seedlings were deprived o f N. They suggested that this increase was a result o f (a) the operation o f two ammonium influx systems, (b) the interplay o f either tissue N H  + 4  or a product o f its assimilation, respectively, acting as positive and  negative effectors o f a single influx system, and/or (c) variations in energy supply from the shoots. Other studies with barley (Lee & Ayling 1993), maize (Jackson et al. 1993; Feng, V o l k & Jackson 1994), sorghum (Feng et al. 1994) and rice (Glass et al. 1997) indicate the involvement o f glutamine or other amino acids as potential regulators o f N uptake. However, Bagchi et al. (1985), after experimenting with glutamine auxotrophs o f Anabaena, suggested that both glutamine and N H  + 4  might participate in the  regulation o f NO3" uptake. Wang et al. (1993a) demonstrated that  1 3  NH  + 4  influx into  rice roots measured at 100 p M , was down-regulated when external [ N H ] was +  4  maintained at 1000 p M rather than at 2 p M . They also showed that under steady state conditions, N H  + 4  efflux was 20-fold higher at 1000 p M than at 2 p M . One source o f  feedback for the down-regulation o f high-affinity N H  + 4  influx suggested by Wang et al.  (1993a) was increased cytoplasmic [ N H ] , a result that was borne out in their estimates +  4  o f cytoplasmic  [NH ] +  4  by compartmental analysis. Some researchers have also  suggested that the cycling o f amino acids from root to shoot and recycling back to the root (Cooper &  Clarkson 1989; Larsson et al. 1991) may present a means o f  information transfer regarding plant nitrogen status from the shoot back to roots, in order to regulate N-absorption according to the demand imposed by growth o f the plant as a whole (for recent reviews see Glass et al. 2001,2002).  Despite a wealth o f physiological information regarding nitrogen, and particularly N H , uptake by rice and other cereals (Lee & Rudge 1986; Morgan & Jackson 1988; +  4  12  Wang et al. 1993a,b), there is to date no published work dealing with expression patterns and/or regulation of genes encoding N H  + 4  transporters in any cereals. The  cloning of the AMT genes from rice now makes it possible to investigate their role(s) in high-affinity N H / transport by means of correlations between patterns of gene expression and high-affinity NH4 influx determined by use of N H / a n d 13  15  NH . +  4  In this chapter I set out to investigate the regulation of N H / influx by various N derivatives other than N H / itself. Clearly, when plants are deprived of N H / several N derivatives such as the amino acids involved in N H / assimilation (Gin, Asn, Glu, and Asp) may also decline in concentration. The opposite situation applies when N deprived plants are supplied with high concentrations of N H / . To investigate the role of N H / and/or various amino acids in regulating N H / influx the N status of rice seedlings was altered by transfer from low-to high-N conditions and from high to lowN conditions. N H / influx and root N H / , Gin, Asn, Glu, and Asp concentrations as well as AMT gene expressions were monitored during these perturbations. In addition various amino acids were supplied to the rice roots exogenously and in some cases N assimilation blocker was supplied simultaneously. Again, N H / influx and root N H / , Gin, Asn, Glu, and Asp concentrations as well as AMT gene expressions were monitored.  2.1.2 Materials and Methods  2.1.2.1 Plant Material and Growth Conditions  13  For all experiments, hydroponically grown rice plants (Oryza sativa I R 72 H D 137) were used. First the rice seeds were surface sterilized with 1 % NaOCl, then allowed to imbibe for 24 h in aerated de-ionized water at 38°C in the dark. Seeds were then transferred to plastic meshes fixed near the bottom o f Plexiglas cups (1.5 inches in diameter and 6 inches long) and covered with vermiculite. These cups were transferred to Plexiglas tanks (40 L capacity) containing modified Johnson's nutrient solution (MJNS) and were allowed to germinate i n the dark for 3-4 days, before providing light. The outer walls as well as the bottom o f the Plexiglas tanks were painted black to prevent light from entering inside the tanks (to prevent algal growth). The composition o f MJNS was as follows:  N a and H P 0 " , 300 p M ; M g , 2 m M ; S 0 \ 2.5 m M ; C a , 1 m M ; Cl", 2 m M ; +  2 +  2  2  4  K , 1 m M ; Fe-EDTA, 20 p M ; M n , 9 p M ; M o 0 +  5pM; Z n  2+  4  2 +  2 +  and C u , 1.5 p M . N H 2 +  + 4  2 4  \ 35 p M ; B 0  was added as ( N H ) S 0 4  2  4  3 3  \ 20 p M ; S i 0 \ 2  3  according to the  requirement o f the experiment (10, 100 p M etc.). Required concentrations o f nutrients were maintained by continuously supplying appropriate stock solution using a peristaltic pump (Technicon Proportioning Pump I I , Technicon Inst. Corp.). The concentration o f the stock solution and/or pump speed were adjusted after determining the concentration o f N H  + 4  b y chemical analysis o f the media (Solorzano, 1969) in the  Plexiglas tanks twice daily. The nutrient solution in the tanks was continuously aerated and mixed with a circulating pump (Circular Model IC-2, Brinkmann Inst., Inc.). The p H o f the medium was monitored daily and maintained at 6 ± 0.2 by addition o f excess CaCC>3. The tanks were maintained in a walk-in growth room with  day/night  temperature o f 25/20 ± 2°C and 12/12 h o f light/dark conditions. The irradiance during  14  the light period was provided at 400 pmol m" s" (plant level) by fluorescent tubes 2  1  (Vita Lite, Duro-Test).  2.1.2.2 N H 4 Production 13  +  The radioactive isotope  1 3  N (t ./ = 9.98 min) was produced as 2  13  NG*3"  at the T r i -  University Meson Facility ( T R I U M F ) , University o f British Columbia Campus, by 20 M e V proton irradiation o f H2O using an A C E L CP42 cyclotron. Details o f the protocol are described in Siddiqi et al. (1989). The reduction o f  13  to N H was achieved 1 3  NC»3"  3  using Devarda's alloy (Cu/Al/Zn), saturated with N a O H at 70°C in a water bath as described in Vaalburg et al. (1975) and Meeks et al. (1978). The ammonia thus formed was then distilled and trapped i n acid solution to generate  NH . 4  2.1.2.3 NH4 Influx Measurements 13  +  The standard procedures for NH4 influx measurements were: (a) Pre-wash: 13  +  prior to exposure to the isotope, roots were pre-washed for 5 min with an unlabeled MJNS, identical to the loading solution in all other respects, (b) Loading: rice roots were loaded in NH -labeled MJNS for 10 min to measure the unidirectional 13  +  4  1 3  NH  + 4  influx, (c) Post-wash: isotope loading was terminated by transferring the roots to unlabeled MJNS for 3 min in order to desorb the isotope from the free space. These times were based upon earlier measurements o f influx and efflux o f N H 1 3  + 4  by Wang et  al. 1993a. Immediately after the post-wash period, plants were cut into roots and shoots  15  and the surface liquid adhering to the roots was removed by a 30 sec spin in a slow speed table centrifuge (International Chemical Equipment, Boston). Fresh weights o f the roots and shoots were recorded, after which the roots and shoots were introduced into separate scintillation vials and immediately counted in a gamma counter ( M I N A X I y- 5000, Packard, Canberra-Packard, Mississauga, Ontario, Canada). A l l the  I 3  NH  + 4  influx measurements were carried out between 11:00 A M and 12:00 noon unless otherwise specified.  2.1.2.4 Analysis of Up-regulation and Down-regulation of N H 4 Influx 13  +  For down-regulation smdies, rice seedlings were grown in solutions maintained at 10 p M [NH4 ] for 21 days and then transferred to solutions containing various N forms, +  namely 1 m M K N 0 , 0.5 m M N H N 0 , or 0.5 m M ( N H ) S 0 for periods up to 72 h. 3  4  3  4  2  4  In subsequent experiments (NH4)2S0 was used to avoid confusing altered tissue [ N 0 " 4  3  ] and [ N H ] . In one set o f experiments plants grown in 10 p M N H +  4  transferred to media containing 10 p M N H  + 4  + 4  solution were  supplemented with 10 m M o f either Gin,  Asn, Glu, or Asp. In addition in order to block N H  + 4  assimilation via GS, the inhibitor  M S X was used. Further, 10 m M G i n , or A s n was supplied along with M S X to selectively modulate the tissue N pools. This allowed the distinction between N H  + 4  itself and a product o f its assimilation, as the source o f the regulatory effect. After the periods o f pretreatment in media with altered N H and inhibitor treatments,  1 3  NH  + 4  + 4  concentration, amino acids, amides  influxes were measured in 100 p M [ N H ] in MJNS. +  4  Root tissue samples were frozen for subsequent amino acid and tissue ammonium  16  analysis. Staggered transfers were made so that all the influx measurements were obtained at the same time and root tissue samples saved at the same time o f day to avoid diurnal effects. This was also necessary as the  1 3  N tracer could be delivered only  once a day and because o f its very short half-life (9.98 minutes). The present flux data, expressed on a root fresh weight basis, represent means o f at least four replicates. A l l experiments were repeated three or more times and statistical differences evaluated by means o f t tests. A l l the  1 3  NH  + 4  influx measurements were carried out between 11:00  A M and 12:00 noon unless otherwise specified.  17  2.1.2.5 Analysis of Internal N H  + 4  and its Assimilation Products by HPLC  To achieve the objective o f finding out which molecule(s) might control(s) H A T S activity and the members o f OsAMTl gene family, it was necessary to determine tissue concentrations o f amino acids and amides, particularly those o f the key ones in the NH  + 4  assimilation pathways, namely Gin, Asn, G l u , Asp, and tissue [ N H ] itself. +  4  Keeping this in mind and to develop a comprehensive picture, analysis o f quantities o f various amino acids, amides, and ammonium present in the roots o f plants grown in low external [ N H ] and plants grown in high external [ N H ] as well as many other +  +  4  4  treatments were undertaken. A l l experiments were repeated using four replicates and were repeated at least twice. 10 p M [ N H ] steady state-grown plants were transferred +  4  to 10 m M [ N H ] for studying the chemical changes in the root tissues that might be +  4  correlated with the observed down-regulation  of N H  + 4  influx. Similarly plants  previously grown in 10 m M external ammonium were transferred to MJNS containing 10 p M N H NH  + 4  + 4  for monitoring the internal changes in the key biochemical species in the  assimilation pathways. Plants grown under steady state conditions in 10 p M N H  were transferred to 10 p M N H  + 4  + 4  plus either 10 m M Gin, Asn, Glu, or Asp. In separate  experiments the GS inhibitor M S X was used to block N H  + 4  assimilation in the presence  or absence o f external Gin or Asn. A l l the root and shoot tissues were saved by freezing them in liquid N immediately after harvesting. Then they were stored in minus 80°C 2  freezers and analyzed at later dates. Statistical analyses o f differences among means were evaluated by t tests.  18  Before the amino acid analyses, in collaboration with Dr. Salim Silim, I developed a very simple method o f sample preparation. In this method l O m M sodium acetate at p H 6.42 was used as the buffer for sample preparation rather than the freezedrying method. The advantage of this method was that I could also quantitate the tissue ammonium concentrations using appropriate standards. This eliminated a separate step that would otherwise be required in order to quantitate the tissue ammonium. For all these analysis HPLC supplied by Waters Corporation (24, Maple Street, Milfords, M A ) which included: Waters 717 plus Autosampler, Waters 600 Controller, and Waters 474 Scanning Fluorescent Detector was used. The columns used were: Waters AccQ.TAG Cig.  All  samples were derivatized using  6-aminoquinolyl-N-hydroxysuccinimidyl  Carbamate AQCFlour reagent (Waters Corporation). For the separation o f amino acids and [ N H ] , six methods were developed and eventually one was optimized for use in +  4  all the analysis. A l l the methods developed were based on the methods developed by van Wandelen and Cohen (1997) and Cohen and van Wandelen (1997). A l l the treatments were carried out between 11:00 A M and 12:00 noon unless otherwise specified.  19  2.1.3 Results  2.1.3.1  1 3  NH  Influx Rates During Down-regulation and Up-regulation by  + 4  Manipulating External NH4 Availability +  Pretreatments which increased plant N status caused N H  influx to decrease,  + 4  whereas treatments that reduced plant N status resulted in increased values o f N H influx. The down-regulation o f N H previously grown at 10 p M N H  + 4  + 4  + 4  influx was most pronounced when the plants  were transferred to 0.5 m M ( N H ) S 0 4  2  4 ;  when  compared with that caused by 1 m M NH4NO3 and 0.5 m M N H N 0 3 . The influx rate 4  measured at 100 p M N H  + 4  fell from 8.11 ± 0.45 (SE) pmol g" h" F W to 3.98 ± 0.22 1  1  (SE) pmol g" h" FW, due to KNO3 treatment, whereas the influx fell to 1.39 ± 0.08 1  1  (SE) pmoles g" h" F W and 1.29 ± 0.09 (SE) pmoles g" h" F W as a result o f N H N 0 1  1  1  1  4  3 )  and ( N H ) 2 S 0 treatments respectively, i n 72 h (Fig. 2.1.1). After determining that the 4  4  reduction o f N H  + 4  influx was most effectively achieved by using ( N H ) 2 S 0 all other 4  4;  ammonium treatments were performed using ammonium sulphate as the source o f N H . The H A T S influx o f +  4  1 3  N H , into rice roots was rapidly down-regulated when  rice plants grown at 10 p M N H  +  4  + 4  were transferred to 10 m M N H  for periods up to 72  + 4  h. The influx declined from 8.01 ± 0.48 (SE) pmol g" h" F W at zero time to 1.24 ± 1  0.09 (SE) pmol g" h" F W after 72 h o f the high N H 1  1 3  NH  + 4  1  + 4  1  treatment (Fig. 2.1.2).  influx into roots o f plants grown at 10 m M N H  steep increase following transfer to 10 p M N H  + 4  + 4  MJNS exhibited a  MJNS growth media, the highest  influx rate being recorded 48 to 72 h after transfer (Fig. 2.1.2). Influx increased from  20  1.19 ± 0.11 (SE) pmol g" h" F W to a maximum o f 7.92 ± 0.51 (SE) pmol g" h" F W (a 1  1  1  1  6.7 fold increase) after 72 h o f exposure to 10 p M N H / .  21  Figure 2.1.1: Down-regulation o f  1 3  in MJNS containing 10 p M N H containing 1 m M K N 0 , 0.5 m M 3  NH + 4  + 4  influx in rice roots. The rice plants were grown  for three weeks and then transferred to media  NH4NO3,  or 0.5 m M ( N H ) S 0 as nitrogen sources for 4  2  4  up to 72 hours. The values shown are means o f four replicates. Bars represent SE.  22  Figure 2.1.2: Down-regulation and up-regulation o f NEL; influx in rice roots. For the 13  +  down-regulation o f influx measurements, the rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 m M N H  + 4  as  nitrogen source for up to 72 hours. For the up-regulation o f influx measurements, the rice plants were grown in MJNS containing 10 m M N H / for three weeks and then transferred to media containing 10 p M N H / as nitrogen source for up to 72 hours. The values shown are means o f four replicates. Bars represent SE.  23  2.1.3.2 Changes i n  1 3  NH  I n f l u x Rates Following Pretreatment w i t h Various  + 4  A m i n o Acids and Amides and/or M S X  Pretreatments with four amino acids (Gin, Asn, Glu, or Asp) reduced NH4 influx rates. The M S X treatment failed to show major decline in N H when M S X was accompanied with either Gin or Asn, N H When N H  + 4  +  influx, however  + 4  influx declined rapidly.  assimilation was blocked via GS by using M S X , influx remained relatively  + 4  high. When GS is blocked the assimilation o f N H  + 4  to glutamine stops resulting in  lowering o f internal Gin concentration and resultant increase in internal N H . When +  4  the rice seedlings previously grown in 10 p M N H transferred to MJNS containing 10 p M N H  + 4  media at steady state were  + 4  and 10 m M Gin, the N H  influx fell  + 4  rapidly from 8.01 ± 0.48 (SE) pmol g" h" F W to 1.02 ± 0.09 (SE) pmol g h" F W in 1  1  1  1  24 h (Fig. 2.1.3). Similarly the influx rate fell to 1.12 ± 0.08 (SE) pmol g" h" F W in 24 1  1  h when the plants were transferred to media containing Asn in place o f Gin (Fig. 2.1.3). The decrease in the influx rates were similar when the rice seedlings were preheated with 10 m M Glu or Asp, with the N H  + 4  influx values falling to 1.07 ± 0.09 (SE) pmol  g" h" FW, and 1.54 ± 0.11 (SE) pmol g" h" F W within 24 h (Fig. 2.1.4). 1  1  1  1  When the rice plants acclimated in 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  medium at steady state were  and 1 m M M S X , the N H  + 4  influx rate did  not change significantly after 6 h o f treatment, but showed a small decrease to a value o f 6.82 ± 0.36 (SE) pmol g" h" F W compared to the (-MSX) value o f 8.78 ± 0.51 (SE) 1  1  pmol g" h" F W after 12 h o f treatment (Fig. 2.1.5). However when the plants grown in 1  10 p M N H  1  + 4  were transferred to media containing 10 m M G i n , in addition to 10 p M  24  NH  + 4  and 1 m M M S X , the influx rate fell rapidly to 2.52 ± 0 . 1 2 (SE) pmol g" h" FW 1  within 12 h o f the transfer (Fig. 2.1.6). The N H  + 4  1  influx rate was reduced to 2.93 ± 0.21  (SE) pmol g" h" F W when the transfer was made to media containing 10 m M Asn 1  1  along with 10 p M N H  + 4  and 1 m M M S X (Fig. 2.1.7).  25  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.3: Down-regulation o f NFLi influx in rice roots. The rice plants were grown 13  in MJNS containing 10 p M N H  +  + 4  for three weeks and then transferred to media  containing 10 p M NH.4 and 10 m M glutamine or 10 m M asparagine for up to 24 hours. +  The values shown are means o f four replicates. Bars represent SE.  26  Duration of Pretreatment (h)  Figure 2.1.4: Down-regulation o f NH4 influx in rice roots. The rice plants were grown 13  in MJNS containing 10 p M N H containing 10 p M N H  + 4  +  + 4  for three weeks and then transferred to media  and 10 m M glutamate or 10 m M aspartate for up to 24 hours.  The values shown are means o f four replicates. Bars represent SE.  27  0  6  12  Duration of Pretreatment (h)  Figure 2.1.5:  Changes in NFLt influx in rice roots. The rice plants were grown in 13  MJNS containing 10 p M N H 10 p M N H  + 4  +  + 4  for three weeks and then transferred to media containing  with or without 1 m M M S X for up to 12 hours. The values shown are means  o f four replicates. Bars represent SE.  28  Duration of Pretreatment (h)  Figure 2.1.6:  Changes in  1 3  MJNS containing 10 p M N H 10 p M N H  + 4  NH + 4  + 4  influx in rice roots. The rice plants were grown in  for three weeks and then transferred to media containing  with 1 m M M S X and with 1 m M M S X and 10 m M glutamine for up to 12  hours. The values shown are means o f four replicates. Bars represent SE.  29  0  6  12  Duration of Pretreatment (h)  Figure 2.1.7:  Changes in  1 3  MJNS containing 10 p M N H 10 p M N H  + 4  NH + 4  + 4  influx in rice roots. The rice plants were grown in  for three weeks and then transferred to media containing  with 1 m M M S X and with 1 m M M S X and 10 m M asparagine for up to 12  hours. The values shown are means o f four replicates. Bars represent SE.  30  2.1.3.3 Changes in I n t e r n a l N H  and K e y A m i n o A c i d Concentrations D u r i n g  + 4  Down-regulation and Up-regulation of N H  Influx  + 4  Root [ N H ] and the root concentrations o f all four amino acids increased +  4  during down-regulation o f N H  influx and were reduced substantially during the up-  + 4  regulation. During the study o f down-regulation o f N H transferred from MJNS containing 10 p M N H m M N H . Root N H +  4  + 4  + 4  + 4  influx, the rice seedlings were  at steady state to media containing 10  concentration increased from 1.09 ± 0.09 (SE) pmol g" FW to 1  13.67 ± 0.92 (SE) pmol g" F W (Fig. 2.1.8) by 72 h. B y 24 h root [ N H ] had already 1  +  4  increased to 8.89 ± 0.43 pmol g" FW. Root Gin, and Asn concentrations also increased 1  rapidly to several times the initial values within 24 h and climbed to 15.91 ± 0.68 (SE) pmol g" F W and 11.18 ± 0.43 (SE) pmol g" FW, respectively, from initial value o f 1  1  1.31 ± 0.09 (SE) pmol g" FW, and 1.12 ± 0.08 (SE) pmol g" F W respectively (Fig. 1  1  2.1.9). The increases in the concentrations o f Glu and Asp were from 0.61 ±0.06 (SE) pmol g  F W and 0.54 ± 0.04 (SE) pmol g  1  1  F W to 5.54 ± 0.43 (SE), and 4.87 ± 0.35  pmol g" F W respectively (Fig. 2.1.10). 1  During the study o f the up-regulation o f N H were transferred from 10 m M N H  + 4  + 4  influx rate, the rice seedlings  steady state media to 10 p M NH -containing +  4  media and this brought about a rapid decline in root [ N H ] and the concentrations o f +  4  key amino acids and amides. The concentrations o f Gin fell from 15.01 ± 0.91 (SE) pmol g" F W to 1.52 ± 0.11 (SE) pmol g" F W and that o f Asn fell from 11.21 ± 0.64 1  1  (SE) pmol g" F W to 1.25 ± 0.11 pmol g" F W within 72 h of the transfers (Fig. 2.1.12). 1  1  The decreases in Glu and Asp concentrations were from 7.23 ± 0.37 (SE) pmol g" F W 1  31  and 6.12 ± 0.46 (SE) pmol g F W to 0.67 ± 0.04 (SE) pmol g" F W and 0.61 ± 0.05 1  1  (SE) pmol g" FW, respectively, during the same treatment (Fig. 2.1.13). The decline in 1  internal N H  + 4  concentration was equally rapid, from 14.02 ± 0.81 (SE) pmol g" F W to 1  1.11 ± 0.04 (SE) pmol g" F W in the 72 h o f transfer (Fig. 2.1.11). 1  32  O)  o  E  3. c o  "•C  2 c  0)  o c o o  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.1.8: Changes in N H  + 4  concentrations in rice seedling roots. The rice plants were  grown in MJNS containing 10 p M NH4 for three weeks and then transferred to media +  containing 10 m M N r l / for up to 72 hours. The values shown are means o f four replicates. Bars represent SE.  33  18  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.1.9: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 m M N H  + 4  + 4  for three weeks and then  for up to 72 hours. The values shown are  means o f four replicates. Bars represent SE.  34  l  1  1  1  r  Duration of Pretreatment (h)  Figure 2.1.10: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 m M N H / for up to 72 hours. The values shown are means o f four replicates. Bars represent SE.  35  Ui  o  E  3. c o  ^5 (0  c u c o o  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.1.11:  Changes in N H / concentrations in rice seedling roots. The rice plants  were grown in MJNS containing 10 m M N H / for three weeks and then transferred to media containing 10 p M N H / for up to 72 hours. The values shown are means o f four replicates. Bars represent SE.  36  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.1.12: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 m M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  for up to 72 hours. The values shown are  means o f four replicates. Bars represent SE.  37  8  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.1.13: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing l O m M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  for up to 72 hours. The values shown are  means o f four replicates. Bars represent SE.  38  2.1.3.4 Changes in Internal N H  + 4  and Key Amino Acid Concentrations Rates  Following Pretreatment with Various Amino Acids  Pretreatments with any one o f the four amino acids (Gin, Asn, Glu, or Asp) increased root N H  and concentrations o f all four amino acids. When GS was blocked  + 4  by M S X , root [ N H ] increased substantially, while root [Gin] declined and root [Asn], +  4  [Glu], and [Asp] increased. When the rice seedlings grown in MJNS containing 10 p M NH  + 4  at steady state were transferred to media containing 10 m M Gin in addition to 10  pM NH  in order to study the down-regulation o f N H  + 4  + 4  influx rate, rapid increases in  root [NF£ ] and concentrations o f key amino acids were observed. Root [Gin] increased +  4  from 1.26 ± 0.08 (SE) pmol g" F W to 16.57 ± 0.51 (SE) pmol g" F W in 24 h 1  1  treatment. Similarly the Asn concentration increased from 1.04 ± 0.07 (SE) pmol g"  1  F W to 11.48 ± 0.44 (SE) pmol g" F W during the same period (Fig. 2.1.15). Increases 1  in the Glu and Asp concentration were from 0.63 ± 0.05 (SE) pmol g" F W to 5.83 ± 1  0.41 (SE) pmol g" F W and from 0.58 ±0.06 (SE) pmol g" F W to 4.12 ± 0.38 (SE) 1  1  pmol g" F W respectively during same 24 h o f Gin treatment (Fig. 2.1.16). During the 1  same period o f time the root [ N H ] increased from 1.05 ± 0.09 (SE) pmol g" F W to +  1  4  12.62 ± 0.76 (SE) pmol g F W ( F i g . 2.1.14). 1  Increases in the root [ N H ] and N H +  4  + 4  assimilation products were also recorded  when the seedlings previously maintained in 10 p M N H containing 10 p M N H  + 4  + 4  were transferred to media  plus 10 m M Asn. Root concentrations o f Gin, and A s n  increased to 11.38 ± 0.97 (SE) pmol g" F W and 12.98 ± 0.63 (SE) pmol g" F W , 1  respectively, in 24 h from the untreated values (Fig. 2.1.18).  1  The Glu and Asp  39  concentrations increased to 4.39 ± 0.39 (SE) umol g" F W and 5.25 ± 0.34 (SE) umol 1  g" F W (Fig. 2.1.19). Root [ N H ] went up to 12.45 ± 0.63 (SE) pmol g" F W in the 24 1  +  1  4  h o f the treatment (Fig. 2.1.17). The root concentrations o f Gin and Asn increased to 13.83 ± 0.65 (SE) pmol g" 1  F W and 9.42 ± 0.41 (SE) pmol g" FW when media containing 10 p M N H 1  + 4  were  supplemented with 10 m M Glu (Fig. 2.1.21) after 24 h. During the same period the Glu, Asp, and N H  + 4  concentrations increased to 5.14 ± 0.52 (SE), 4.12 ± 0.33 (SE), and  11.06 ± 0.51 (SE) pmol g F W respectively (Figs. 2.1.22 and 2.1.20). 1  When the rice seedlings grown in 10 p M N H containing 10 p M N H  + 4  + 4  were transferred to media  plus 10 m M Asp, root concentrations o f N H , Gin, Asn, Glu, +  4  and Asp increased to 10.78 ± 0.57 (SE) pmol g" FW(Fig. 2.1.23), 8.79 ± 0.42 (SE), 1  11.23 ± 0.66 (SE) (Fig. 2.1.24), and 4.42 ± 0.32 (SE), 4.68 ± 0.31 (SE) (Fig. 2 . 1 . 25), respectively, within 24 h of the transfers.  40  Ui  o E C  o s c o> o c o o  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.14:  Changes in N H  + 4  concentrations in rice seedling roots. The rice plants  were grown in MJNS containing 10 p M N H media containing 10 p M N H  + 4  + 4  for three weeks and then transferred to  and 10 m M glutamine for up to 24 hours. The values  shown are means o f four replicates. Bars represent SE.  41  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.15: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 10 m M glutamine for up to 24 hours.  The values shown are means o f four replicates. Bars represent SE.  42  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.16: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 10 m M glutamine for up to 24 hours.  The values shown are means o f four replicates. Bars represent SE.  43  Ui  o E e o  2  *•>  c u c o o  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.17: Changes in N H  + 4  concentrations in rice seedling roots. The rice plants  were grown in MJNS containing 10 p M NH4 for three weeks and then transferred to +  media containing 10 p M NH4 and 10 m M asparagine for up to 24 hours. The values +  shown are means of four replicates. Bars represent SE.  44  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.18: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 10 m M asparagine for up to 24 hours.  The values shown are means of four replicates. Bars represent SE.  45  Duration of Pretreatment (h)  Figure 2.1.19: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H  + 4  and 10 m M asparagine for up to 24 hours.  The values shown are means of four replicates. Bars represent SE.  46  Ul  o  E ^. c o  2 C  o o c o o  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.20: Changes in N H  + 4  concentrations in rice seedling roots. The rice plants  were grown in MJNS containing 10 p M N H media containing 10 p M N H  + 4  + 4  for three weeks and then transferred to  and 10 m M glutamate for up to 24 hours. The values  shown are means of four replicates. Bars represent SE.  47  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.21: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 10 m M glutamate for up to 24 hours.  The values shown are means of four replicates. Bars represent SE.  48  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.22: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 10 m M glutamate for up to 24 hours.  The values shown are means of four replicates. Bars represent SE.  49  LL.  O) O  E O  (0  o o c o o  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.23: Changes in N H  + 4  concentrations in rice seedling roots. The rice plants  were grown in MJNS containing 10 p M N H media containing 10 p M N H  + 4  + 4  for three weeks and then transferred to  and 10 m M aspartate for up to 24 hours. The values  shown are means of four replicates. Bars represent SE.  50  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.24: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H / and 10 m M aspartate for up to 24 hours. The values shown are means o f four replicates. Bars represent SE.  51  0  6  12  24  Duration of Pretreatment (h)  Figure 2.1.2.5: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 10 m M aspartate for up to 24 hours. The  values shown are means o f four replicates. Bars represent SE.  52  2.1.3.5 Changes in I n t e r n a l N H  + 4  and K e y A m i n o A c i d Concentrations Following  Pretreatments w i t h M S X plus o r minus G i n o r Asn  The effect o f M S X on internal N H / concentration was rapid and pronounced. When the seedlings grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  at steady state were  + 4  plus 1 m M M S X , the root ammonium  concentration increased several fold within 6 h and in 12 h was 10.81 ± 0.77 (SE) pmol g" F W as compared with the - M S X value o f 1.09 ± 0.11 (SE) pmol g" FW. The N H 1  1  + 4  concentrations were even higher when the plants were treated with 10 m M o f Gin or Asn together with 1 m M M S X and reached 12.98 ± 086 (SE), and 12.79 ± 0.85 (SE) pmol g" FW, respectively (Fig. 2.1.26). Gin concentration in the root tissue decreased 1  from 1.35 ± 0.11 (SE) pmol g" F W to 1.02 ± 0.08 (SE) pmol g 1  1  F W in 12 h o f  treatment with M S X , however Asn concentration increased from 1.09 ± 0.09 (SE) to 1.79 ± 0.13 (SE) pmol g" F W during the same period o f time (Fig. 2.1.27). The 1  concentrations o f Glu and Asp increased several fold from 0.61 ± 0.04 (SE) and 0.57 ± 0.04 (SE) pmol g" F W to 3.78 ± 0.16 (SE) and 2.87 ± 0.15 (SE) pmol g" FW, 1  1  respectively, during the 12 h o f M S X treatment (Fig. 2.1.28). When the rice seedlings grown at 10 p M N H  + 4  were treated with 10 m M Gin in the presence o f I m M M S X , the  root Gin concentration increased to 7.88 ± 0.39 (SE) pmol g" F W and root [Asn] 1  increased to 5.04 ± 0.43 (SE) pmol g" F W (Fig. 2.1.29) in 12 h. The same treatment 1  increased the root Glu and Asp concentrations to 3.71 ± 0.21 (SE) and 2.72 ± 0.12 (SE) pmol g" FW, respectively, (Fig. 2.1.30). When plants were transferred to media 1  containing 10 m M Asn along with 1 m M M S X , the root concentrations o f Gin, Asn,  53  Glu and Asp increased to 4.21 ± 0.41 (SE), 6.49 ± 0.48 (SE), 3.68 ± 0.23 (SE), and 2.96 ± 0.18 (SE) pmol g" FW, respectively, in the 12 h o f treatment (Fig. 2.1.31, and 1  2.1.32).  54  15  Duration of Pretreatment (h)  Figure 2.1.26:  Changes in N H  concentrations in rice seedling roots. The rice plants  + 4  were grown in MJNS containing 10 p M N H media containing 10 p M N H  + 4  + 4  for three weeks and then transferred to  with 1 m M M S X , or 10 p M N H  10 m M glutamine or 10 p M N H  + 4  + 4  with 1 m M M S X and  with 1 m M M S X and 10 m M asparagine for up to 12  hours. The values shown are means o f four replicates. Bars represent SE.  55  Figure 2.1.27: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  with 1 m M M S X for up to 12 hours. The  values shown are means o f four replicates. Bars represent SE.  56  T  1  r  0  6  12  Duration of Pretreatment (h)  Figure 2.1.28: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  with 1 m M M S X for up to 12 hours. The  values shown are means o f four replicates. Bars represent SE.  57  0  6  12  Duration of Pretreatment (h)  Figure 2.1.29: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  with 1 m M M S X and 10 m M glutamine for  up to 12 hours. The values shown are means o f four replicates. Bars represent SE.  58  Figure 2.1.30: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H ^ for three weeks and then transferred to media containing 10 p M NH4 " with 1 m M M S X and 10 m M glutamine for 4  up to 12 hours. The values shown are means o f four replicates. Bars represent SE.  59  Figure 2.1.31: Changes in glutamine and asparagine concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  with 1 m M M S X and 10 m M asparagine for  up to 12 hours. The values shown are means o f four replicates. Bars represent SE.  60  Figure 2.1.32: Changes in glutamate and aspartate concentrations in rice seedling roots. The rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H  + 4  with 1 m M M S X and 10 m M asparagine for  up to 12 hours. The values shown are means o f four replicates. Bars represent SE.  61  2.1.4 Discussion  Initially the down-regulation and up-regulation o f NH4 influx via H A T S appeared to be regulated by external N H / provision that in turn increased the root ammonium concentration. Root [ N H / ] and  1 3  N H / influx rates are inversely correlated  (Fig. 2.1.2). When the N H / influx rate was reduced to < 15% o f the initial value (Fig. 2.1.2) following transfer o f the plants from medium containing 10 p M N H / to 10 m M N H / , the root  [ N H / ] increased by over 12-fold during the same time period (Fig.  2.1.8). Likewise, when the plants were transferred from media containing 10 m M N H / to media containing 10 p M N H / the influx increased rapidly within the first 24 h o f transfer to > 6.5 times its initial value (Fig. 2.1.2). This was accompanied by a huge decrease o f root [ N H / ] to < 10% o f the original value (Fig. 2.1.11). Similar downregulation and up-regulation o f N H / influx into rice roots was also reported by Wang et al. (1993a, 1994). Lee and Rudge (1986) reported similar results, using different cereal species, and suggested that the observed response might be due to N derivatives rather than N H / itself. In the present study, when the plants were transferred from 10 p M N H / to media containing 10 p M N H / plus 10 m M Gin, Asn, Glu, or Asp, the N H / influx rates were sharply reduced to around 12.5%, 14%, 13%, and 19% o f the respective initial values (Fig. 2.1.3, 2.1.4) in 24 h, accompanied by increased root [ N H / ] to 12-fold (Fig. 2.1.14), 11.8-fold (Fig. 2.1.17), 10.5-fold (Fig. 2.1.20), and 10fold (Fig. 2.1.23), respectively. Thus the influx o f N H / appeared to be  inversely  correlated with root [ N H / ] . However, when the plants were treated with 1 m M M S X in addition to the 10 p M N H / , the influx did not fall in the first 6 h o f treatment, although it was reduced by  62  22% after 12 h o f this treatment (Fig. 2.1.5). Despite the fact that root [ N H ] was +  4  increased to values comparable to those observed in the absence o f M S X , the conditions that reduced N H  to < 12% o f initial values, i n the presence o f M S X the  + 4  influx value remained near the control levels. The only difference between these two treatments was that the conversion o f N H  + 4  to Gin was blocked by the M S X treatment.  This absence o f comparable reduction o f the N H  + 4  influx rate when the internal [ N H ]  went as high as in the case o f 10 m M external N H  +  4  + 4  treatment rules out significant  direct effect o f internal ammonium concentration on the N H  + 4  influx via H A T S , and  suggests that the other N assimilates may be responsible for regulating the N H  + 4  influx  via H A T S . Similar observations were made i n Arabidopsis by Rawat et al. (1999). However, in studies o f rice, it was concluded that when M S X was used as inhibitor o f GS, i n the roots o f low nitrogen grown plants Gin was important i n down-regulating NH  + 4  influx, while N H  + 4  itself appeared to prevent up-regulation o f N H  + 4  influx when  high-N plants were transferred to low-N conditions (Glass et al., 1997). So what actually regulates the N H when the internal N H  + 4  + 4  influx via HATS? During the present study  concentration was ruled out, the focus shifted to its assimilation  products, Gin, Asn, Glu, and Asp. The main enzymes involved i n the N H  + 4  assimilation  pathway, GS, G O G A T , A A T , and A S result in the biosynthesis o f the above amino acids and amides. A l l the N H Fig. 2.1.33. Regulation o f N H  + 4  + 4  assimilation pathways and enzymes are presented in uptake by feedback inhibition by N H  + 4  and/or other N -  containing molecules had been reported earlier from Saccharomyces (Dubois and Grenson, 1979), and Synechococcus (Suzuki et al. 1993). Similar observations have been made in some higher plants by Lee and Rudge (1986), and Morgan and Jackson  63  (1988). It has also been suggested that in maize Gin or some other N assimilation products are potential regulators o f N uptake (Jackson et al. 1993, Lee and Ayling 1993, Feng etal. 1994).  64  GDH 1.  2- Oxaloglutarate (a Ketoglutarate) + N H  + 4  + NAD(P)H ^  ^ Glutamate + N A D ( P )  MSX GS 2.  NH  4  + A T P + Glutamate  •  Glutamine + A D P + Pj GOGAT  3.  (i) Glutamine + a Ketoglutarate + F D  •  r  2 Glutamate + F D  0 X  GOGAT 3. (ii) Glutamine + a Ketoglutarate + N A D ( P ) H  • 2 Glutamate + N A D ( P )  AAT 4. Oxaloacetate + Glutamate  •  Aspartate + a Ketoglutarate  AS 5. Aspartate + Glutamine ( N H ) + A T P +  4  Figure  2.1.33: N H  + 4  •  Asparagine + Glutamate + A D P + P;  assimilation enzymes. G D H , glutamate  dehydrogenase; GS,  glutamine synthase; G O G A T , Glutamate synthase; A A T , aspartate amino transferase; A S , asparagine synthase.  65  When the plants were transferred from MJNS with 10 p M N H containing 10 p M N H  + 4  and 10 m M Gin, Asn, Glu, or Asp, the N H  + 4  + 4  to media  influx rates were  sharply reduced to around 12.5, 14, 13, and 19% o f the respective initial values (Fig. 2.1.3, 2.1.4). These treatments also increased the root concentrations o f the four key NH  + 4  assimilation products. The root [Gin], [Asn], [Glu], and [Asp] rapidly increased  13, 1 1 , 9 and 7-fold, respectively (Fig. 2.1.15, 2.1.16), when the plants were subjected to 24 h o f 10 m M Gin treatment. When the plants were treated with the same external concentration o f Asn, the root concentrations o f Gin, Asn, Glu, and Asp also increased 9, 12.5, 7 and 9-fold in 24 h (Fig. 2.1.18, 2.1.19). The high external Glu treatment also increased the root [Gin], [Asn], [Glu], and [Asp] to 11, 9, 8 and 7-time the values in the untreated rice seedling roots (Fig. 2.1.21, 2.1.22). External Asp treatments increased root [Gin], [Asn], [Glu], and [Asp], 7, 1 1 , 7, and 8-fold, respectively compared to the initial values (Fig. 2.1.23, 2.1.24). Consistently, it appeared that all amino acids and amides had similar effects on N H  + 4  influx via the H A T S , as the root concentrations o f  the four chemical species were inversely correlated with N H  + 4  influx (data not shown).  Interestingly, the external addition o f any one o f these four amino acids increased root concentrations o f all four amino acids in the rice root tissues to varying degrees. However, when the plants were treated with M S X , as discussed above, the N H  + 4  influx rates did not fall significantly in the first 6 h and the reduction was modest even after 12 h o f treatment (Fig. 2.1.5). Interestingly, the same treatment resulted in considerably higher root concentrations o f Glu, and Asp. The internal [Glu] rapidly increased by over 6-fold above the control, and [Asp] increased by over 5-fold by 12 h o f M S X treatment (Fig 2.1.28). This was to be anticipated, because M S X blocks GS,  66  and since Glu is also a substrate o f this GS reaction (Fig. 2.1.33), this results in increased root [Glu], and in turn increased root [Asp] through the involvement o f the enzyme A A T . N o w , i f the internal concentrations o f Glu, and Asp were regulating the N H / influx rates via H A T S , the high recorded internal [Glu], and [Asp] should have resulted in a similar down-regulating effect on the N H / influx rates as were apparent when these two amino acids were supplied externally. As observed with M S X treatment, the reduction o f N H / influx was modest when compared with the massive increases in the internal concentrations o f Glu, and Asp. This clearly indicates that Glu, and Asp are probably not exerting any major effect on the rate o f N H / influx and hence are not key regulators o f N H / influx via H A T S in rice seedling roots. Therefore, the evidence strongly suggests that the N H / influx is regulated by internal [Gin], and/or [Asn]. This was further substantiated by the results o f the Gin, or Asn addition together with M S X . When the plants were treated with 10 m M Gin, in the presence o f M S X , the N H / influx rate fell sharply to < 33% o f the initial influx rate (Fig. 2.1.6), and this was accompanied by 6-fold increased root [Gin] (Fig. 2.1.29). Similarly the Asn treatment in presence of M S X reduced the N H / influx to 36 % o f the initial value in 12 h (Fig. 2.1.7). This was also accompanied by a 6-fold increased root [Asn] (Fig. 2.1.31). The potential down-regulatory effect o f Asn was always less than that o f Gin, the latter being the primary product of N H / assimilation in plants. These treatments also increased the root [Glu], and [Asp] (Fig. 2.1.30, 2.1.32), but as discussed earlier it is unlikely that Glu or Asp are important in regulating N H / influx. Lee et al. (1992) also concluded that when intracellular levels o f Gin and Asn were higher net uptake o f N H / and NO3" were high and the low intracellular levels o f Gin  67  and Asn were always accompanied by low N H  + 4  and NO3" uptake rates. However,  Bagchi et al. (1985), while studying glutamine auxotrophs o f Anabaena, had concluded that there was participation o f N H  + 4  as well as Gin in regulation o f NO3" uptake.  Similar conclusions were arrived at by Vidmar et al. (2000) with respect to the regulation o f NO3" influx in barley. It is likely that a model describing the regulation o f N (NO3" or N H ) influx might operate on a common basis, facilitating the regulation o f +  4  both NO3" and N H  + 4  influx by the same N derivatives. Some others have suggested that  the cycling o f the N derivatives from root to shoot and in the other direction could be the way o f achieving the information transfer (Copper and Clarkson, 1989; and Larsson et al. 1991). Their conclusions were based on the observations that the root concentrations o f N H  + 4  and all four key assimilation products changed with transfer o f  plants to different N sources.  68  2.2 Molecular Biology of N H Transport +  4  2.2.1  Introduction As described in section 2.1.1, after studies regarding the physiology and  biochemistry, and the cloning o f various OsAMTl genes, it was logical to seek answers concerning the regulation o f N H / influx via H A T S at the transcript levels for the three genes that have been, cloned, and which are thought to encode the high-  OsAMTl  affinity N H  + 4  transporters in rice roots. In particular, it is possible to determine the  extent o f changes in transcript abundances o f these genes under the conditions described in the previous section (2.1) namely, conditions that perturbed plant N status and N H  + 4  influx.  Prior to the present study, there was no published data on the  regulation o f AMT genes that putatively encode the H A T S proteins for ammonium influx in rice seedling roots. Therefore, with this topic in mind, the present research work was undertaken.  This section deals with the changes o f transcript abundances o f three OsAMTl genes (OsAMTl.l,  OsAMTl.2,  and OsAMTl.3)  in rice roots by simultaneously  monitoring the expression patterns o f all three genes in rice seedling roots under steady state conditions o f nitrogen supply, following transfers from low-to-high and high-tolow nitrogen provisions, and exogenous applications o f Gin, A s n , G l u , or Asp. I n addition the expression patterns o f OsAMTl  family members were also quantified  following treatment with M S X in the presence or absence o f Gin or Asn and in order to  69  gain insights into which N derivative might be responsible f o r regulation o f these genes.  2.2.2 Materials and Methods  2.2.2.1 Plant Material and Growth Conditions  The growth conditions and the plant materials for all the expression analysis discussed i n this sub-chapter were described in section 2.1.2.  2.2.2.2 RNA Extraction, RT-PCR Analysis and Verification of PCR Products.  For all m R N A expression analyses, root tissue samples were frozen i n liquid nitrogen for all the treatments corresponding to up-regulation and down-regulation o f influx, or time points for the diurnal studies. The frozen root tissue samples were ground with mortar-and-pestle and total-RNA samples were isolated using a plant R N A isolation k i t (Qiagen Inc., Mississauga, O N ) following the protocol provided. Then 50100 ng o f different total-RNA samples were used for semi-quantitative RT-PCR analysis. A l l RT-PCR reactions were undertaken using One-Step RT-PCR K i t (Qiagen Inc. Mississauga, O N ) , following the manufacturer's protocol. The product c D N A sizes for OsAMTl.l,  OsAMTl.2 and OsAMTl.3 were 769 bp, 561 bp, and 394 bp  respectively. The specific primers f o r the genes were as follows: f o r OsAMTl.l Forward:  5 'GTCGTTCACCACCATCCTCAAGACGTA3'  5' TCCTTCGCTGTG A C G T C G T T C G T T C 3 ' ;  for  and  OsAMTl.2:  Reverse: Forward  70  5 'GATCTACGGCGAGTCGGGCACGAT3' 5 TTCCATCTCTGTCGAGGTCGAGACG3'; 5 'TCAAATCCTACGGCCCGCCCGGTAG3'  and and  for  OsAMTl.3: and  Reverse: Forward Reverse:  5 ' G C C G A A G A T C T G G T C C A C G T A C T C C T T 3'. A l l three specific RT-PCR products were cloned, sequenced and compared with the sequences o f the respective AMT genes to confirm the specificity o f the RT-PCR products. For each o f the three genes, the optimum cycle number used in the quantitative RT-PCR was individually determined. After amplifying the respective product c D N A for 3 to 37 cycles, equal amounts o f the product D N A were electrophoresed through 1.4% agarose gel, stained with S Y B R Gold (Molecular Probes Inc., Eugene, OR) for 30 min. Then the signals o f the gene products were captured and analyzed densitometrically using an Alphalmager™ 1200 (Alpha Innotech Corp., San Leandro, CA). For each gene the number o f cycles for RT-PCR was optimized by plotting the I D V (Integrated Density Value) on a log scale and the number o f cycles used for the quantitative RT-PCR for each o f the gene was picked from the middle o f the log-linear part o f the graph, representing the linear phase of the amplification o f the c D N A . For this, after the RT step, aliquots were taken every two cycles in the PCR step. After this number o f cycle standardization, different experimental RT-PCRs were run for 23 or 25 cycles. For all the RT-PCR reactions, QuantumRNA Universal 18S internal standard (Ambion Inc., Austin, T X ) was used to show equal initial use and loading o f the R N A samples. The amounts o f 18S r R N A internal standard primers were determined according to the manufacturer's protocol to amplify both the target gene and the 18S at an appropriate level. A l l experiments were  71  undertaken at least three times and differences among means were evaluated by means o f t tests.  2.2.2.3 Bioinformatics Analyses  A l l the sequence homology searches were performed on the B L A S T server (http://www.ncbi.nlm.nih.gov/blast).  All  mRNA,  DNA/cDNA,  and amino  acid  sequences were obtained from online databases and/or gene-bank. Sequence alignment analysis  and  phylogenetic  tree  analysis  were  done  using  ClustalW  (http://www2.ebi.ac.uk/clustalw/) (Thompson et al. 1994) and ClustalX (Thompson et al. 1997) as well as Treemap (http://taxonomy.zoology.gla.ac.ulc/rod/heemap.html) and PAUP (http://paup.csit.fsu.edu). Transmembrane helices were predicted using different methods: T M A P version 46 (Persson & Argos 1994), D A S (Cserzo et al. 1997) and T M H M M (Sonnhammer et al. 1998). Other programs like GeneDoc (Nicholas and Nicholas 1997) and Bioedit (Hall 1999) were also used.  72  2.2.3 Results  2.2.3.1 The OsAMTl Gene Family and the AMT Proteins The three members o f the OsAMTl analyses after cloning. OsAMTl.l,  gene family were subject to various  OsAMTl.2, and OsAMTl.3 encode 56.8-kD, 53.4-  k D , and 52.5-kD polypeptides o f 532, 497, and 495 amino acid residues respectively (Fig. 2.2.1). Furthermore, OsAMTl.l  is closely related to OsAMTl.3  with 8 3 %  similarity at the amino acid level, and is more distantly related to OsAMTl.2 with 7 3 % similarity, while OsAMTl.2 showed a 6 8 % similarity with OsAMTl.3. More detailed phylogenetic relationship among several known members o f different AMT families is shown in Figure 2.2.2. Sequence alignments and phylogenetic relationships were analyzed using ClustalW, ClustalX as well as Treemap and PAUP. Using T M A P version 46 (Persson & Argos 1994), D A S (Cserzo et al. 1997) and T M H M M (Sonnhammer et al. 1998), 9 to 11 transmembrane helices were predicted.  73  j f i A l j ANATDYLCNRFADTTSAVDATYLLFSAYLVFAMQLGFAMLCAGSVRAKNT & \ S : iBBABBiYHCNRFRDTBSAVDATYLLFSAYLVFAMOLGFAMLCAGSVRAKNE jAAJ|ANATDYLCMgFADTTSAVDBTYLLFSAYLVFAMQLGFAMLCAGSVRAKNT  OsAMTl.1 OsAMTl.2 OsAMTl.3 OsAMTl.1 OsAMTl.2 OsAMTl.3  MNIMLTNVLDAAAGALFYYLFGFAFAFGTPSMGFIGKQFFGLKHMPQTGFDYDFFLFQWAFAIAAAGI MNIMLTNVI3DAAAGALFYYLFGFAB35BTPSI5GFIGKOFFGLKHMPOTGBDYDFFLFOWAFAIAAAGI MNIMLTNVLDAAAGALFYYLFGFAFAFGBPSNGFIGKJEFFGLK^PQBGFDY^FFLFQWAFAIAAAGI  OsAMTl.1 OsAMTl.2 OsAMTl.3  TSGSIAERTQFVAYLIYSAFLTGFVYPWSHWIWSADGWASASRTSGPLLFGSGVIDFAGSGWHMVG TSGSIAERTSFGAYLIYSAFLTGFWPWSHV^WSBDGWASAGRBBGPLLFIGSGVIDFAGSGVVHJLVG TSGSIAERTQFVAYLIYSAFLTGFVYPWSHWIWSADGWASASRTSGGLLFGSGVIDFAGSGWHMVG  OsAMTl.1 OsAMTl.2 OsAMTl.3  'IEGPRIGRFDHAGRSVALKGHSASLWLGTFLLWFGWYGFNPGSFTTILKHYGPBG llEG PRIGRFD0AGR@VA^IKGHSASLVVLGTFLLIWFGV^GFNPGSFTTI@KJJYG^BG ACR-TLffiRP^GPl3!8iiiRFDHAGRSVALigGHSASLVVLGBFLLWFGWYGFNPGSFilTILK@YGPBG  OsAMTl.1 OsAMTl.2 OsAMTl.3  GQWSEVGRTAVTTTLAGSVAALTTLFGKRLQTGHWNVBDVCNGLLGGFAAITAGCSVVDPWAAIICGF  GQWSAVGRTAVTT@LAGSVAAL!5ESE^3!SSGS33S33I^^S3^3AITAGCSWDPWAS5ICGF GQWSAVGRTAVTTTLAGSHAALTTLFGKRLQTGHV>JNVBDVCNGLL,GGFAAITAGCSWDPWAAIICGF  338 339 337  OsAMTl.1 OsAMTl.2 OsAMTl.3  VSAWLIGLNALAARLKFDDPLEAAQLHGGCGAWGIJSFTALFARSAYVEAIYGHAGRPYGLFMGGGG VSAWVLIGBNELAEE!LKFDDPLEABQLHGGCGAWGIIFTALFARKEYVEIIYGBPGRPYGLFMGGGGF VSAWVLIGLNALAARLKFDDPLEAAQLHGGCGAWGEIFTALFARKEYVSIIBGBPGRPYGLFMGGGG""  405 407 405  iWiajablEHDHs- -G : 471  OsAMTl.1 OsAMTl.2 OsAMTl.3  iDEpy^GORRVRAKB : 47 5 [gASGBPDRg : 473  JCLQQQQPSVTNPERTTSQRRKKSRVSLPLRSRSSRHKFDPHI OsAMTl.l : V ^ 3 S 3 R ^ Q - B R f f l E P g OsAMTl.2 : AAETARVEPRKSPEQf" EIM OsAMTl. 3 : FWdatiilKHBlHGHoSBABlE^  532 497 495  Figure 2.2.1: Alignments o f OsAMTl  family members. Amino acid sequence alignments o f  and OsAMTl.3  were initially obtained using ClustalW and then adjusted  OsAMTl.l,  OsAMTl.2,  using GeneDoc (Nicholas & Nicholas 1997). Amino acids in the black background indicate identical residues. The accession numbers are: AF289477 (OsAMTl.l), AF289479  AF289478  (OsAMTl.2),  (OsAMTl.3).  74  and  AsAMTB OsAMTl. 1 OsAMTl .3 OsAMTl .2 AtAMTI .1 LJAMT2 AtAMT2 RhCG PDRC2 •  MvMEPa  jj— MEP1  1 — MEP3 |— NaAMTI — I SsAMTA AtAMTI .2 UMEP1  H:;  AtAMt1.3 BnAMT1.2 LeAMT2 LJAMT1.1 AbAMTB SmAMTB AtAMTB CrAMTH 2 LeAMTI .3 PsAMTB2  HI •  MEP2 MtAMTI HcAMTI  l r - AaAMTB '— EcAMTB ^ r - CrAMTI SsAMTI MjAMTB Ma AMT CaNRGA AfAMT3  Figure 2.2.2. Phylogenetic tree o f ammonium transporter proteins. Amino acid sequence alignment and phylogenetic tree analysis were obtained using ClustalX and PAUP. The tree includes members o f the OsAMTl family and some other AMTs, and does not include all known AMTs.  75  2.2.3.2 Changes in OsAMTl  Transcript Abundances in Roots During Up-  regulation and Down-regulation of N H / Influx  When the rice seedlings previously grown in MJNS containing 10 p M N H / at steady state were transferred to media containing 10 m M N H / , the transcript abundance o f OsAMTl.l  decreased rapidly b y several fold within 24 h and was only  14% o f the initial value at 72 h after transfer (Fig. 2.2.3). OsAMTl.l  transcript  abundance also decreased and at the end o f 72 h treatment it was only 2 5 % o f the original value (Fig. 2.2.4). The reduction in the OsAMTl.3 was least and at 72 h after the transfer it remained at 4 7 % o f the initial value (Fig. 2.2.5.). When the plants grown in MJNS containing 10 m M N H / at steady state were transferred to media containing 10 p M N H / for 72 h, the transcript abundances o f OsAMTl.l,  OsAMTl.2, and OsAMTl.3 increased 7-times (Fig. 2.2.6) for  OsAMTl.l,  almost 4-times for OsAMTl.2 (Fig. 2.2.7), and approximately 2.5 times for OsAMTl.3 (Fig. 2.2.8) as compared to their respective zero time values.  76  l_l  0-1—I 0  l_l 6  l_l 12  l_l 24  L_J 48  I I 72  Duration of Pretreatment (h)  Figure 2.2.3: RT-PCR analysis o f the expression patterns o f OsAMTl.l  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 m M N H / for up to 72 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  77  OsAMT1.2  IMP  mm  18S  mm  mm-  mm  1mm  •  f*<*» I  120  o o  c  (0 T3 C 3 X!  (0  O  c ro  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.2.4: RT-PCR analysis o f the expression patterns o f OsAMTl.2 in rice roots during down-regulation. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 m M N H / for up to 72 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  78  12  OsAMT1.3 18S  1 W ""1 fHH  mm  24  48  72  H mmm 'mm.  -4^01$  120  a> o c  90  (0 TJ §  60  «j  a •c  o w  30-  2  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.2.5: RT-PCR analysis o f the expression patterns o f OsAMTl.3  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 m M N H / for up to 72 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  79  120  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.2.6: RT-PCR analysis o f the expression patterns o f OsAMTl.l  in rice roots  during up-regulation. Rice plants were grown in MJNS containing 10 m M N H / for three weeks and then transferred to media containing 10 p M N H / for up to 72 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values based upon the 72 h value as 100%.  80  Sox&fafbp. ' ^ W W  ifel&titffi  W^&$IIF'  WlWiilA WPBBH?  <£ti&&£5ttfc '^|l$HpP  120  a) u c  03  "O  c 3 X I OB  O (A C CO  0  6  12  24  48  72  Duration of Pretreatment (h)  Figure 2.2.7: RT-PCR analysis o f the expression patterns o f OsAMTl.2  in rice roots  during up-regulation. Rice plants were grown in MJNS containing 10 m M N H / for three weeks and then transferred to media containing 10 p M N H  + 4  for up to 72 hours. Total  R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values based upon the 72 h value as 100%.  81  12  24  48  72  OsAMTl.3 18S 120  0  6  12  24  48  Duration of Pretreatment  72 (h)  Figure 2.2.8: RT-PCR analysis o f the expression patterns o f OsAMTl.3 in rice roots during up-regulation. Rice plants were grown in MJNS containing 10 m M N H weeks and then transferred to media containing 10 p M N H  + 4  + 4  for three  for up to 72 hours. Total  R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values based upon the 72 h value as 100%.  82  2.2.3.3  Changes  in OsAMTl  Transcript  Abundances in Roots Following  Pretreatment with Various Amino Acids and Amides  The transfers o f rice seedlings to MJNS containing high external amino acids and amides also affected the expression patterns o f the three members o f the OsAMTl gene family. When the plants grown in 10 p M N H / were transferred to media containing 10 m M o f Gin, the OsAMTl.l  transcripts were reduced to 10% o f the  original value within 24 h o f the treatment (Fig. 2.2.9). The effect o f this treatment on OsAMTl.2  was also rapid and reduced the expression level to 15% o f the initial  expression i n 24 h (Fig. 2.2.10). The transcript abundance o f OsAMTl.3  was least  reduced and was at 2 9 % o f the initial value after 24 h o f the same treatment (Fig. 2.2.11). The treatment with 10 m M Asn also caused a significant reduction o f transcript abundance. The OsAMTl.l,  OsAMTl.2,  and OsAMTl.3  transcripts were reduced to  12% (Fig. 2.2.12), 2 0 % (Fig. 2.2.13), and 2 9 % (Fig. 2.2.14), respectively, after 24 h o f treatment.  When the rice seedlings previously acclimated in MJNS containing 10 p M N H / were transferred to media containing 10 p M N H  + 4  and 10 m M Glu, the transcript  abundances o f AMTs were also reduced. The largest reduction was i n followed b y that o f OsAMTl.2  and least in the case o f OsAMTl.3.  values at the end o f 24 h treatment were 14% f o r OsAMTl.l  OsAMTl.l,  The respective  (Fig. 2.2.15), 2 3 % f o r  OsAMTl.2 (Fig. 2.2.16), and 2 9 % for OsAMTl.3 (Fig. 2.2.17).  83  Similar effects on the transcripts levels were observed when the plants were transferred from low NHL/ steady state to low N H  + 4  plus 10 m M o f external Asp. The  effect o f Asp treatment was least when compared with that o f Gin, Asn, and G l u treatments. The transcript abundances o f OsAMTl.l,  OsAMTl.2, and OsAMTl.3 were  reduced to 29 % (Fig. 2.2.18), 34 % (Fig. 2.2.19), and 40 % (Fig. 2.2.20), respectively, after 24 h o f treatment.  84  Figure 2.2.9: RT-PCR analysis o f the expression patterns o f OsAMTl.l in rice roots during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M glutamine  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  85  Figure 2.2.10: RT-PCR analysis o f the expression patterns o f OsAMTl.2  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M glutamine  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  86  0  OsAMTI.3  6  jgjlgljij^ ^^^^^^^^^r  12 mm^m  ^^^^^^^^^W  ^^^^^^^^^r  *8S  24  nin  iiTiii-i  ^^^^w^^w  4MMMi  0  6  12  24  Duration of Pretreatment (h)  Figure 2.2.11: RT-PCR analysis o f the expression patterns o f OsAMTl.3  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M glutamine  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  87  Y8S  ^^^WBPHI^Ir  '^^^^^^HPf  4NNMfr  4MMNMfe  4MMMMt  4tHMMk  120  o c  TO TJ  c  3 XI TO  u (fl c TO  0  6  12  24  Duration of Pretreatment (h)  Figure 2.2.12: RT-PCR analysis o f the expression patterns o f OsAMTl.l  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H  + 4  and 10 m M  asparagine for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RTPCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  88  Figure 2.2.13: RT-PCR analysis o f the expression patterns o f OsAMTl.2 in rice roots during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M  asparagine for up to 24 hours. Total R N A was extracted f r o m roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RTPCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  89  Figure 2.2.14: RT-PCR analysis o f the expression patterns o f OsAMTl.3 in rice roots during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M  asparagine for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RTPCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  90  Figure 2.2.15: RT-PCR analysis o f the expression patterns o f OsAMTl.l  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M glutamate  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  91  Figure 2.2.16: RT-PCR analysis o f the expression patterns o f OsAMTl.2  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M glutamate  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  92  Figure 2.2.17: RT-PCR analysis o f the expression patterns o f OsAMTl.3  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M glutamate  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  93  Figure 2.2.18: RT-PCR analysis o f the expression patterns o f OsAMTl.l  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H three weeks and then transferred to media containing 10 p M N H  + 4  + 4  for  and 10 m M aspartate  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  94  Figure 2.2.19: RT-PCR analysis o f the expression patterns o f  OsAMT1.2  in rice roots  during down-regulation. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H  + 4  and 10 m M aspartate  for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates rRNA internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  95  Figure 2.2.20: RT-PCR analysis o f the expression patterns o f OsAMTl.3 in rice roots during down-regulation. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H / and 10 m M aspartate for up to 24 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  96  2.2.3.4 Changes  in OsAMTl  Transcript  Abundances in Roots Following  Pretreatment with MSX with or without Gin or Asn  When the rice seedlings grown i n MJNS with 10 p M N H media containing 10 p M N H OsAMTl.l,  + 4  + 4  were transferred to  with 1 m M M S X , the transcript abundance o f  OsAMTl.2, and OsAMTl.3 showed little response by 6 h after the transfer  but showed some reductions after 12 h o f treatment. The values for the transcript abundances for OsAMTl.l,  OsAMTl.2, and OsAMTl.3 after 12 h o f treatments were  79% (Fig. 2.2.21), 83% (Fig. 2.2.22), and 86% (Fig. 2.2.23) o f the initial values.  However, when 10 m M o f Gin was also added to the media along with M S X , the reductions in the expression levels o f the three genes were much more pronounced and rapid and were at 16% (Fig. 2.2.21), 2 0 % (Fig. 2.2.22), and 36% (Fig. 2.2.23) for OsAMTl.l,  OsAMT1.2, and OsAMTl.3 respectively.  The transfer o f the plants to media with 10 m M Asn and 1 m M M S X also reduced transcript abundances. After 12 h o f the treatment, the transcript abundances o f OsAMTl.l,  OsAMT1.2, and OsAMTl.3 were reduced to 2 3 % (Fig. 2.2.24), 28% (Fig.  2.2.25), and 39% (Fig. 2.2.26) o f the initial values in the untreated plants.  97  Figure 2.2.21: RT-PCR analysis o f the expression patterns o f OsAMTl.l Rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  in rice roots.  for three weeks and then  and 1 m M M S X or 10 p M N H  + 4  plus 1 m M  M S X and 10 m M glutamine for up to 12 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates rRNA internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  98  MSX  6 OsAMT1.2  mm  MSX + Gin  12  mm mm  0 6 m •: * 1 ^mmm afi. | i mmm  12 mjQmm  1  18S  MHMHM^  m)mninmt  mmmm  mmmm ^mm%%  120  0  6  12  Duration of Pretreatment (h)  Figure 2.2.22: RT-PCR analysis o f the expression patterns o f OsAMTl.2 in rice roots. Rice plants were grown in MJNS containing 10 p M N H / for three weeks and then transferred to media containing 10 p M N H / and 1 m M M S X or 10 p M N H / plus 1 m M M S X and 10 m M glutamine for up to 12 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  99  Figure 2.2.23: RT-PCR analysis o f the expression patterns o f OsAMTl.3 in rice roots. Rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 1 m M M S X or 10 p M N H  + 4  plus 1 m M  M S X and 10 m M glutamine for up to 12 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  100  MSX 0  —  OsAMTl.l  6  12  0  mm — *  —  MSX + Asn 6 12  18S  120 I I MSX 188583 MSX + Asn  90  0)  o c ro TJ  c 3 .a  60  ro o w  c  ro  30 A  0  6  12  Duration of Pretreatment (h)  Figure 2.2.24: RT-PCR analysis o f the expression patterns o f OsAMTl.l Rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  in rice roots.  for three weeks and then  and 1 m M M S X or 10 p M N H  + 4  plus 1 m M  M S X and 10 m M asparagine for up to 12 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  101  Figure 2.2.25: RT-PCR analysis o f the expression patterns o f OsAMTl.2 in rice roots. Rice plants were grown in MJNS containing 10 p M N H transferred to media containing 10 p M N H  + 4  + 4  for three weeks and then  and 1 m M M S X or 10 p M N H  + 4  plus 1 m M  M S X and 10 m M asparagine for up to 12 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates rRNA internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  102  Figure 2.2.26: RT-PCR analysis o f the expression patterns o f OsAMTl.3 in rice roots. Rice plants were grown in MJNS containing 10 p M N H  + 4  for three weeks and then  transferred to media containing 10 p M NFL* and 1 m M M S X or 10 p M N H  + 4  plus 1 m M  M S X and 10 m M asparagine for up to 12 hours. Total R N A was extracted from roots at the time intervals shown. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture and corresponding data has been shown. Transcript abundances shown in the graph represent percent values defining the 0 time value as 100%.  103  2.2.4 Discussion  Expression levels o f all three members, OsAMTl.l, were high in roots o f plants grown on 10 p M N H  + 4  OsAMTl.2, and OsAMTl.3  (Fig. 2.2.3-2.2.8) suggesting that  perhaps all three members o f the A M T 1 gene family play a role in the influx o f N H / . Nevertheless, OsAMTl.l  transcript abundance in roots showed the largest reduction in  response to increased ambient N H  + 4  concentration. Further, this reduction  correlated  13  quantitatively and temporally with the corresponding decreases o f root  "I" •  NH  4  influx,  while changes o f OsAMTl.2 were next in magnitude and changes o f OsAMTl.3 were least o f all. The reduction o f the transcript abundances o f these three genes due to transfer o f rice plants from media containing 10 p M N H NH  + 4  to media containing 10 m M  + 4  were accompanied by rapid and considerable increases in the root concentrations  o f N H , G i n , Asn, G l u , and Asp as discussed in 2.1.4. OsAMTl.l +  4  transcript  abundances were negatively correlated with the root [ N H ] , [Gin], [Asn], [Glu], and +  4  [Asp]. However this was also the case for OsAMTl.2, although the extent o f transcript changes was less. The least effect o f these perturbations in the root concentrations o f these N derivatives was on the OsAMTl.3 transcript abundances. Similar effects were observed when the root N status was perturbed b y exposures to solutions containing 10 m M o f either o f Gin (Fig. 2.2.9 - 2.2.11), Asn (Fig. 2.2.12 - 2.2.14), Glu (Fig. 2.2.15 2.2.17), or Asp (Fig. 2.2.18 - 2.2.20). A l l these treatments led to increased values o f root [ N H ] , [Gin], [Asn], [Glu], and [Asp]. Expression levels o f +  4  OsAMTl.2,  and to a lesser degree OsAMTl.3  OsAMTl.l,  were negatively correlated with root  concentrations o f the N compounds. Thus, without further information it would be  104  difficult to distinguish between N H , Gin, Asn, Glu, and Asp as potential regulators o f +  4  OsAMTl gene expressions. However, a different picture emerges when the expression analyses were undertaken in the roots o f the plants treated with 1 m M M S X in the presence or absence o f 10 m M Gin or Asn. The expression level o f OsAMTl.l  showed no significant  change in 6 h o f M S X treatment and was still 79% o f the untreated value at 12 h (Fig. 2.2.21). Levels o f OsAMTl.l,  and OsAMTl.3  followed similar patterns and were 83  and 86% o f untreated values at the end o f 12 h treatment (Figs. 2.2.22-2.2.23). Yet as described in section 2.1.4, the root [ N H ] , as well as that o f [Glu] and [Asp] increased +  4  several fold as a result o f this treatment. In the absence o f M S X , when root [ N H ] was +  4  increased to this extent, transcript levels o f OsAMTl genes were substantially reduced. Again the only difference between these treatments was that in the presence o f M S X , conversion o f N H  + 4  to Gin was blocked. This indicates that the root [ N H ] level as +  4  well as [Glu] and [Asp] have very little effect in regulating the expression levels o f these three genes. When the plants were treated with M S X together with 10 m M G i n , the expression levels o f OsAMTl.l,  OsAMTl.l,  and OsAMTl.3  fell sharply (Figs.  2.2.21 - 2.2.23). The effects o f M S X and M S X in the presence o f glutamine were also reported by Rawat et al. (1999), and they also concluded that the expression levels o f AtAMTl.l  were inversely correlated with the root [Gin] in Arabidopsis roots, however  all the experimental plants they used were also supplied with a high concentration o f sucrose. Similar effects were observed when the plants were transferred to solutions containing M S X together with 10 m M Asn. Again the effect on transcript abundance was highest in the case o f OsAMTl.l,  followed by OsAMTl.l,  and OsAMTl.3 (Fig.  105  2.2.24 - 2.2.26). These treatments, as discussed in section 2.1.4, also resulted in rapid increases in the internal [Gin] and [Asn]. Therefore, it might be concluded that the internal concentrations of Gin and to a lesser extent Asn regulate the expression levels of OsAMTl.l,  OsAMTl.l,  and to a lesser extent OsAMTl.3. However, Marini et al.  (1994, 1997) concluded that in Saccharomyces treatments with glutamate and glutamine led to repression of transcript abundances of Mepl, Mepl, and Mep3 encoding for HATS expression. Interestingly, Lauter et al. (1996) reported that the expression of LeAMTI was similar under all nitrogen conditions i.e. were constitutively expressed in tomato. Further, in the roots of Arabidopsis Gazzarrini et al. (1999) reported steep increases in the ATAMTI. 1 expression levels when N nutrition became limiting and that of ATAMTI.3 increased slightly in addition to ATAMTI. 1 being constitutively expressed. Shelden et al. (2001) reported similar expression patterns for ATAMTI.1.  Also in Arabidopsis, Zhou et al. (1999) suggested that the  AtNRTl.l  expression was down-regulated by N H / , Gin or other amino acids. Again, interestingly it was reported that in tomato LeAMTI.1 expression increased after N H / or NO3" supply in the roots of hydroponically grown plants, however LeAMTI. 1 was induced by N deficiency and coincided with low Gin concentrations (von Wiren et al. 2000). Recently, while studying the regulation of HATS in Brassica, Pearson et al. (2002) reported that mRNA expression of BnAMTl.l  and N H / uptake were stimulated when  detached leaves were supplied with increasing [ N H / ] , however when Gin or Glu was supplied to the leaves the BnAMTl.l  expression was the lowest. As the treatments with  high external [ N H / ] or [Gin] or [Glu] all lead to increase internal [Gin] as well as [Glu], the results reported by Pearson et al. (2002) become difficult to understand.  106  Therefore based on the results of my present study, I conclude that downregulation and up-regulation of OsAMTl.l  and OsAMTl.2,  and to a lesser extent  OsAMTl.3 transcript in roots of rice plants in response to root [Gin] and/or [Asn] are primarily responsible for the corresponding changes of NH4 influx and therefore 13  +  mediate changes of H A T S that appear to be regulated by the N status of the plants. The closer quantitative correspondence between changes of OsAMTl.l transcript abundances and N H  + 4  + 4  OsAMTl.2  influx rates than those OsAMTl.3 and N H  suggest that it is primarily through changes of OsAMTl.l expression that the N H  and  + 4  and OsAMTl.2  influx gene  influx via H A T S is regulated. However, this does not preclude  contributions from OsAMTl.3. The  1 3  NH  + 4  influx declined more rapidly than the decline of OsAMTl.l  and  OsAMTl.2 transcript abundances during down-regulation, suggesting the possibility of additional levels of control, namely, post-transcriptional control of HATS. It could be via a direct effect of root [NH ] on the AMT proteins and/or effects of combinations of +  4  N H , Gin, and/or Asn at the protein level. In lotus, Salvemini et al. (2001) also +  4  reported high expression of LjAMTl.l  under low nitrogen conditions. However in many  of these reports, extensive analysis of root concentrations of N H , Gin, Asn, Glu, and +  4  Asp were not undertaken. By contrast to the pattern of down-regulation, the increase of lagged behind that of the increase of OsAMTl.l  1 3  NH  + 4  influx  and OsAMTl.2 transcript level in roots  following transfer of plants from high to low N H , as would be expected of a system +  4  whose first level of control is at the transcript level.  107  3 Diurnal Studies 3.1 Physiology and Biochemistry of Diurnal N H  3.1.1  + 4  Transport  Introduction  As described in 2.1, the influx rate o f  1 3  N H / changes in response to different external  N pretreatments and various other treatments. I n addition, diurnal changes in NOV and N H / influx have been reported in a number o f species (Gazzarini et al, 1999; Matt et al, 2001). It was therefore anticipated that diurnal changes in N H / influx into roots o f rice might be observed, and in order to explore this phenomenon, N H / influx into rice roots was measured at four hour intervals throughout a complete 24 h period using 1 5  N H / . I n addition, roots were treated with sucrose supplied exogenously to determine i f  the influx which was reduced at night could be restored to its daytime value. This was accompanied by simultaneous monitoring o f the expression patterns o f all three genes in  108  seedling roots under the same conditions of N status, and other treatments as described in 3.2.1.  3.1.2 Materials and Methods  3.1.2.1 Plant Material and Growth Conditions  Rice seedlings were grown in a greenhouse in MJNS maintained at 100 p M [NH ]. +  4  The ambient air and nutrient solution temperatures along with irradiance values were continuously measured with two temperature probes and a light probe and recorded in a computer using software from Strawberry Tree Inc (Sunnyvale, California). A l l sampling for  1 5  NH  + 4  influx and freezing of corresponding tissue samples was undertaken at  intervals of 4 h for a 24 h cycle normally starting at 6 A M and ending at 6 A M the following day. A l l the experiments were repeated at least three times. A t all time points fresh weights of the root and shoot tissues were recorded and samples were dried for 3 days at 75°C. The root and shoot dry weights were recorded and the dried tissue samples were kept in desiccators to avoid the absorption of atmospheric moisture. The dried root and shoot samples were ground by pestle-and-mortar and approximately 5 mg samples were packed individually in small tin capsules and shipped for  1 5  N analysis to the  IS  University of California Isotope Facility at Davis. The choice of N rather than  1 "\  N was  made because the short-lived isotope could not be procured as required for the diurnal analysis and because of other logistic problems associated with the use of this radioisotope. To investigate effects of exogenous sucrose, plants were transferred from 109  MJNS media to media o f the same inorganic composition with or without 1 m M sucrose. Plants were exposed to these treatments for 4 h at times corresponding to the highest and lowest recorded influxes, i.e. at 2:00 a.m. and 6 p.m. Similar procedures were used for 1 5  NH  + 4  influx analysis as were described before for  1 3  NH  + 4  influx. Additional root and  shoot samples were quickly frozen in liquid nitrogen and stored at - 8 0 ° C for sugar analysis. The samples for sugar analysis were then ground using a mortar and pestle, freeze-dried and shipped to the University o f North Carolina for analysis.  A l l other aspects o f the Materials and Methods were the same as described in section 2.1.2.  3.1.3 Results  Root  1 5  NH  + 4  influx varied considerably during the 24 h cycles, with values  in the light period at 2:00 P M and 6:00 P M being 3.2 and 3.4 times higher than those at 2:00 A M in the dark period (Fig. 3.1.1). Influx showed a sharp increase following the onset o f daylight and an equally sharp decrease corresponding to the beginning o f the dark period. The highest and lowest influx values were 9.91 and 2.95 pmol g" h" , 1  1  respectively. The highest irradiance on the day o f the experiment shown in Fig. 3.1.1 was 0  1050 pmol m  1  1  9  s", whereas values as high as 1600 pmol m" s" (data not shown) were  recorded during the prior three weeks o f growth. N H  + 4  influx into roots of the seedlings  treated with sucrose at a time corresponding to the highest influx rate in the absence o f exogenous sucrose was increased by 13.5% from 9.69 pmol g" h" to 11 pmol g" h" (Fig. 1  1  1  1  3.1.2). The increase o f influx resulting from the sucrose addition in the dark was over 110  95% when compared with that without sucrose treatment, from 3.02 pmol g" h" to 5.97 1  1  pmol g" h" (Fig. 3.1.2). Internal sucrose concentrations of the roots and shoots of the rice 1  1  seedlings showed no significant change as a result of the exogenous provision of sucrose during the light period. In the dark period however, there was a statistically significant increase of internal sucrose concentration (p<0.05). It increased from 20 pg  g  _1  D W to  26.7 pg g" D W in the roots of the rice seedlings (Fig. 3.1.3). Interestingly, the levels of 1  internal sucrose concentrations in the seedling roots were not significantly different during the light period and the dark period in the untreated plants (Fig. 3.1.3). Changes of internal concentrations of glucose and fructose in the root tissue showed no reproducible pattern (data not shown).  Ill  Figure 3.1.1: NH.4 influx in rice roots during a complete diurnal cycle. The rice plants 15  +  were grown in 100 p M NH4 containing MJNS for three weeks. The values shown are +  means of four replicates. Bars represent SE. Photon flux values are based upon one representative data set.  112  6 PM  2 AM  Time of the Day  Figure 3.1.2:  1 5  NH  + 4  influx in rice roots with and without sucrose treatment. The rice  plants were grown in 100 p M N H  + 4  containing MJNS for three weeks and treated with  (or without) 1 m M sucrose for 4 hours during the light and the dark period. The values shown are means of four replicates. Bars represent SE.  113  6 P M  2 A M  Time of the Day  Figure 3.1.3: Internal sucrose concentrations in rice roots with and without exogenous sucrose treatment. The rice plants were grown in 100 p M N H  + 4  containing MJNS for three weeks and  treated with (or without) 1 m M sucrose for 4 hours during the light and the dark period. The values shown are means o f four replicates. Bars represent SE.  114  3.1.4 Discussion  3.1.4.1 NH 15  + 4  Influx and Light Availability  The changes in N H  + 4  influx in the diurnal cycle corresponded with changes in the  availability o f photosynthetically active radiation (PAR), except that the influx was still significant during the dark period, although highly reduced even when the recorded irradiance was zero. This suggests that the diurnal variation in N H  + 4  influx in the rice  seedling roots is largely determined by light and/or carbon availability. This is true not only for rice, but it has been reported for other plants (see Introduction). Clearly, the sucrose generated by the photosynthetic machinery represents both the primary carbon source for assimilation o f ammonium; and also the source o f A T P required ammonium assimilation, since N H  + 4  for  absorbed by plant roots is typically assimilated in  situ rather than being transferred to shoots for assimilation as is generally the case for NO3". Despite the conversion o f chloroplast starch to sucrose and its mobilization to roots via phloem, the results o f the flux measurements and the effects o f exogenous sucrose suggest that in darkness carbon and energy are available to the seedling roots in reduced amounts and thus limit the N H substantial increase in N H  + 4  + 4  influx rate. Kubik-Dobosz et al. (2000) reported  uptake during daytime and decrease during nighttime, they  also recorded uptake o f NO3" increased in day and decreased in the night in tobacco roots. Recently, Matt et al. (2001) reported that the N 0 " uptake was about 4 0 % higher during 3  the day as compared to night. Moreover, the photoperiod also regulates the internal biological clock and the circadian rhythm o f the plants. Thus the irradiance could have a  115  direct effect on the rate o f N H / influx and/or an indirect effect via the product o f photosynthesis (sucrose) as discussed later.  3.1.4.2 N H I n f l u x and Carbon Availability 15  +  4  The diurnal variation in  15  NH4  influx was considerable. B y contrast, the  +  concentrations o f internal sucrose in the roots o f the rice plant seedlings during the light and dark periods were relatively constant under control conditions (i.e. without exogenous sucrose, Fig. 3.1.1), and failed to show changes that corresponded with the recorded influx values. One explanation for this observation is that the higher rates o f sucrose transfer to roots during daylight hours cause increased rates o f NH4 influx and +  corresponding increases in the rates o f NH4 assimilation. Both o f these processes depend +  upon a source o f energy and a source o f carbon skeletons. These increased rates o f carbon utilization in turn may deplete the sucrose pool so that there is little difference between daytime and night time values. Similarly, reduced sucrose transfer to roots at night, may cause reduced NH4  +  influx  and NH4  +  assimilation, resulting in lower  rates o f  consumption o f sucrose. B y these interactive processes root sucrose concentrations remain similar during day and night. However, sucrose may also be acting as a signaling molecule in addition to its metabolic effects. This issue remains unresolved. Another point that has to be acknowledged is that measured values o f root sucrose concentration represent combined cytoplasmic and vacuolar values. The effects upon NH4 influx reported here and elsewhere must almost certainly be determined by the +  cytoplasmic sucrose pool(s).  Thus i f values o f cytoplasmic sucrose increased during  116  daylight hours, they might show a strong correlation with N H  + 4  influx and assimilation  rates. In summary it is unclear whether the regulation o f diurnal N H  + 4  influx is achieved  through the effect o f a single limiting factor or through a complex interplay o f (i) PAR, (ii) C availability, and (iii) C/N ratio. The evidence for the C/N ratio or interactions and availability o f C affecting diurnal influx have been demonstrated in the regulation of high-affinity nitrate transport in roots o f Arabidopsis thaliana (Lejay et al., 1999), and in roots o f tobacco (Matt et al., 2001). Kubik-Dobosz et al. (2000) also reported the treatments with sucrose or glucose resulted in substantial increase in N H  + 4  uptake.  Availability o f such carbohydrates is essential for the rapid assimilation o f potentially toxic N H . +  4  The increase in N H  + 4  influx during the daytime after treatment with sucrose was  small but statistically significant, suggesting that carbon availability may limit N H  + 4  influx even during daylight hours. Nevertheless, there was no significant change in root sucrose concentration following exposure o f roots to exogenous sucrose (see above). The increase o f root sucrose concentration was significant during the dark period after the external treatment with sucrose. This might have increased the availability o f the carbon skeleton needed for the assimilation o f N H , hence resulting in a very significant +  4  increase o f N H  + 4  influx (Fig. 3.1.2). However the details o f the mechanisms whereby  these processes are regulated by carbohydrate and/or other diurnal signals is presently unknown.  117  3.2 Molecular Biology of Diurnal N H Transport +  4  3.2.1 Introduction  As described in 2.2.1, after the cloning o f various OsAMTl genes, it was logical to seek answers concerning the documented diurnal pattern o f N H / influx at the transcript levels for the three known OsAMTl genes and the extent to which changes o f transcript abundances o f these genes might be responsible for the observed diurnal variations in ammonium fluxes. Prior to the present study, there were no published data on the diurnal regulation o f genes encoding the H A T S for ammonium influx in rice seedling roots. Therefore, with this topic in mind, the present research work was undertaken.  This chapter deals with the contribution(s) o f three OsAMTl  genes  (OsAMTl.l,  OsAMTl.2, and OsAMTl.3) to N H / influx into rice roots by simultaneously monitoring the expression patterns o f all three genes in rice seedling roots under steady state conditions o f nitrogen supply, together with diurnal influx measurements using  I 5  NH/  and exogenous applications o f sucrose at the time when maximum and minimum influx rates were recorded in order to investigate the extent to which carbon supply to roots might influence transcript levels o f OsAMTl genes and N H / influx.  118  3.2.2 Materials and Methods  Rice seedlings were grown i n a greenhouse in 100 p M [ N H ] in MJNS medium. 4  Concentrations o f N H w e r e maintained at this concentration (± 10%) by daily sampling +  4  o f the medium and continuous addition o f concentrated MJNS into the growth tanks. Delivery rates o f the concentrated MJNS were adjusted to maintain the medium at 100 p M [ N H ] . A l l sampling for N H +  4  1 5  + 4  influx and freezing o f corresponding tissue samples  was undertaken at 4 h intervals for a 24 h cycle normally starting at 6 A M and ending at 6 A M the following day as described in 3.1.2. Sucrose treatments were given only at the times corresponding to the highest and lowest observed influxes and were given for a period o f four hours. A l l the experiments were repeated at least three times. A t all the corresponding time points root samples were quickly frozen in liquid nitrogen and stored at -80°C for m R N A analysis. A l l other conditions were the same as described in 3.1.2.  3.2.3 Results  As described in Section 3.1.3, root  1 5  NH  + 4  influx varied considerably during the 24  h cycles, with influx in the light period at 2:00 P M and 6:00 P M being 3.2 times and 3.4 times higher than influx at 2:00 A M in the dark period (Fig. 3.1.1). Root transcript levels o f OsAMTl.3 also increased approximately three times from the value at 2:00 A M to that at 6:00 P M (Fig. 3.2.3). OsAMTl.l  and OsAMTl.2 transcript levels also increased but  only modestly compared to the changes noted for OsAMTl.3.  (1.3 and 1.4 times,  respectively) compared to their respective minimum values (Fig. 3.2.1 and 3.2.2). A l l o f  119  these increases (day time over night time) were statistically significant (p<0.05). Only the changes o f OsAMTl.3 transcript abundance corresponded quantitatively with the changes in N H  + 4  influx. It is noteworthy that the decline o f N H  + 4  influx and expression levels o f  OsAMTl.3 lagged behind the diurnal decline o f irradiance. When the seedling roots were treated externally with sucrose during the light period, as reported in 3 . 1 , the increase in  1 5  NH  + 4  influx was small and statistically  insignificant (p<0.05). The increase o f transcript abundance o f OsAMTl.l  and OsAMTl.2  were small and statistically insignificant (p<0.05) (Fig. 3.2.4). However, the increase in OsAMTl.3  transcript abundance was significant (p<0.05) and the increase was 18% as  compared to the untreated roots (Fig. 3.2.4). When the external sucrose treatment was provided to the rice seedlings in the dark period,  1 5  NH  + 4  influx increased significantly (p<0.05). The increase was 9 5 % when  compared to the influx in the untreated roots (Fig. 3.1.2). The transcript abundances o f all the three OsAMTl  genes were also significantly higher (p<0.05). The increases were 26  and 25% for OsAMTl.l case o f OsAMTl.3  and OsAMTl.2 transcripts, respectively, while the increase in the  was over 95% o f the expression level in the untreated roots (Fig.  3.2.5).  As mentioned in 3.1.1, as a result o f the exogenous addition o f sucrose during the light period, internal sucrose concentrations o f the roots and shoots o f the rice seedlings showed no significant change. However, there was a small but statistically significant increase o f internal sucrose concentration (p<0.05) in the dark period when external sucrose was added in the MJNS growth medium. It increased from 20 to 26.7 p g g" D W 1  in the roots o f the rice seedlings (Fig. 3.1.3). Again as previously mentioned, the levels o f  120  internal sucrose concentrations in the seedling roots during the light period and the dark period were not significantly different in the untreated plants (Fig. 3.1.3) while changes of internal concentrations of glucose and fructose in the root tissue showed no clear pattern.  121  6:00  10:00  14:00  18:00 22:00  2:00  6:00  160  06:00 10:0014:00 18:00 22:00 02:00 06:00 Time of the day  Figure 3.2.1: RT-PCR analysis o f the expression patterns o f OsAMTl.l in rice roots during a complete diurnal cycle. Total R N A was extracted f r o m roots o f rice plants grown in 100 p M NHLt containing MJNS for three weeks at intervals o f 4 h during a 24+  h day/night cycle. 18S indicates rRNA internal control for loading. One representative set o f RT-PCR gel picture is shown. Transcript abundances shown represent percent values where the 2:00 A M value was set as 100%.  122  2  6:00 10:00 14:00 18:00 22:00 2:00 6:00 M M H I • I IJMIllll.ll•»«•  ^ilNliNiM^  ^IMWlM^  ^m^Ktm^  ^jttpi(t  ^liHlfc ^i^tmW^  ^m^m^ffc  ^m^Rtfe  f&tkWIt  %mWmm%  06:00 10:00 14:0018:00 22:00 02:00 06:00  Time of the day  Figure 3.2.2: RT-PCR analysis o f the expression patterns o f OsAMTl.2  in rice roots  during a complete diurnal cycle. Total R N A was extracted f r o m roots o f rice plants grown in 100 p M N H  + 4  containing MJNS for three weeks at intervals o f 4 hour during a  24-hour day/night period. 18S indicates r R N A  internal control f o r loading. One  representative set o f RT-PCR gel picture has been shown. Transcript abundances shown represent percent values where the 2:00 A M value was set as 100%.  123  6:00  OSAMT1.3  10:00  14:00  18:00  22:00  2.-00  f"' ^ " '""'W ' " W' mm mm mm mm nm mm m  ll  l  ! ll  IB  :  6:00  mm.  350  06:00 10:0014:0018:00 22:00 02:00 06:00  Time of the day  Figure 3.2.3: RT-PCR analysis o f the expression patterns o f OsAMTl.3  in rice roots  during a complete diurnal cycle. Total R N A was extracted from roots o f rice plants grown in 100 p M N H  + 4  containing MJNS for three weeks at intervals o f 4 hour during a  24-hour day/night period. 18S indicates r R N A  internal control f o r loading. One  representative set o f RT-PCR gel picture has been shown. Transcript abundances shown represent percent values where the 2:00 A M value was set as 100%.  124  0SAMT1.1 6PM-S 6PM+S  Os AMT 1.2 6PM-S 6PM+S  Os AMT 1.3 6PM-S 6PM+S  OsAMT Gene  Figure 3.2.4: RT-PCR analysis o f the expression patterns o f OsAMTl.l, OsAMTl.3  OsAMTl.2,  and  in roots during the daytime with and without sucrose. Total R N A was extracted  from roots o f rice plants grown in 100 p M N H / containing MJNS for three weeks and treated with (or without) 1 m M sucrose for 4 h during the daylight period. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture is shown. Transcript abundances shown represent percent values where the minus sucrose value was set as 100%.  125  OsAMT 1.1 2AM-^2AM+S  OsAMT 1.2 2AM-S  OsAMT 1.3  2AM+S  OsAMT 1 18S 250  1  T  I 200  I -sucrose +sucrose  J  o o  C CO TJ C 3 -Q < Q.  1504  100-|  u c  2  50H  1.1  1.3  1.2  OsAMTl  Gene  Figure 3.2.5: RT-PCR analysis o f the expression patterns o f OsAMTl.l,  OsAMTl.2,  and  OsAMTl.3 in roots during night-time with and without sucrose. Total R N A was extracted from roots o f rice plants grown in 100 p M N H / containing MJNS for three weeks and treated with (or without) 1 m M sucrose for 4 h during the dark period. 18S indicates r R N A internal control for loading. One representative set o f RT-PCR gel picture is shown. Transcript abundances shown represent percent values where the minus sucrose value was set as 100%.  126  3.2.4 Discussion  The changes in N H extent that o f OsAMTl.l  + 4  influx and the expressional level o f OsAMTl.3, and to a lesser  and OsAMTl.2, in the diurnal cycle corresponded with changes  in PAR. This indicates that the diurnal variation in the expression patterns o f OsAMTl.3 and to a lesser extent the expression levels o f OsAMTl.l  and OsAMTl.2  in the rice  seedling roots appear to be regulated by photon flux and/or availability o f carbon, and that in turn determines the level o f transporter proteins and thus N H  + 4  influx. However,  the influx as well as the expression levels o f all three genes were still significant (and not zero) during the dark period, although highly reduced even when recorded irradiance was zero. This suggests that ultimately it is the availability o f carbon that is impacting the gene expression and energy supply to support N H / influx and assimilation. This could be true not only for rice but for other plants, as the photosynthetic machinery that produces sucrose, the primary carbon source for assimilation o f ammonium, and the energy required ( A T P ) for assimilation o f ammonium taken in by the plant roots, are dependent on the availability o f light. Moreover, light also regulates the internal biological clock and circadian rhythms o f plants. Thus the irradiance could have a direct effect on the expression levels o f OsAMTl.3 and to a lower level to that o f OsAMTl.l and OsAMTl.2, thus regulating the rate o f N H  + 4  influx through the availability o f the  actual H A T S transporter proteins. As the availability o f carbon and energy declines during darkness, expression levels o f the gene(s) and hence the N H  + 4  influx rate decline  correspondingly.  127  The transcript abundance o f OsAMTl.3 in roots appeared least affected by changes in the external N H  status as mentioned in section 2.2, whereas it showed the highest  + 4  level o f change during the day/night cycle and these changes corresponded with diurnal changes o f  1 5  NH  + 4  influx and incident irradiance during the 24 h period (Fig. 3.2.3).  Similar results were reported for AtAMTI.3 in Arabidopsis (Gazzarrini et al. 1999) and LeAMTI.2 in tomato (von Wiren et al. 2000). B y contrast, levels o f root OsAMTl.l and OsAMTl.2  showed only modest changes during the 24 h period o f investigation.  Interestingly, von Wiren (2000) reported that LeAMTI.2  and LeAMTI.3  showed  reciprocal diurnal regulation with expression o f LeAMTI.3 transcripts being highest in the darkness, and that o f LeAMTI.2 after onset o f light. Therefore I hypothesize that the regulation o f the observed diurnal variation in the rice seedling root N H  + 4  influx was  achieved mainly via changes in the transcript abundance o f OsAMTl.3 and via transcript abundances o f OsAMTl.l OsAMTl.3 and N H  + 4  and OsAMTl.2 to a smaller extent. The expression level o f  influx were doubled in roots treated with sucrose during the dark  period. Also the expression levels o f OsAMTl.l  and OsAMT1.2 increased over 35%  when treated with external sucrose in the dark period, indicating that the regulation o f this diurnal response may result from C/N interactions or simply by making more carbon skeleton available for ammonium assimilation. The treatments with sucrose or glucose resulted in substantial increase in N H  + 4  uptake along with enhanced expression o f AMT1  in rutabaga (Brassica) roots also as reported by Kubik-Dobosz et al. (2000). This has also been postulated for the regulation o f AtNRT2.1, encoding high-affinity nitrate transport in roots o f Arabidopsis thaliana by Lejay et al. (1999), and NRT2 in roots o f tobacco by Matt et al. (2001). Observations dealing with changes in non-structural carbohydrates  128  during the diurnal cycle have also been demonstrated in roots o f soybean plants (Kerr, Rufty & Huber 1985). As in the present study, the authors reported that changes in the concentrations o f sucrose and other sugars were minimal and most o f the changes (over 80%) were due to changes in starch concentration that was mainly restricted to the shoot tissues. Thus even though an elevated supply o f such carbohydrates are essential for rapid assimilation o f potentially toxic NFL;" ", the elevated supply may not be visible as an 1  increased internal concentration o f sucrose as the higher supply could be rapidly utilized by higher N H  + 4  influx and rapid assimilation o f the ammonium. Thus, i f assimilation  keeps pace with supply, a steady state is maintained. Thus carbon supply rate to the roots may be responsible for the transcriptional control o f OsAMTl.3, OsAMTl.l  and to a lesser extent  and OsAMTl.2, expression. Indeed in similar studies o f the diurnal regulation  o f NOV influx, AtNRT2.1 transcript abundance and N G y influx were increased during 1 5  the dark period by exogenous provision o f sucrose (Lejay et al. 1999). Nevertheless, the details o f the mechanisms whereby these genes are regulated by carbohydrate and/or other diurnal signals are presently unclear.  129  4 Transgenic Studies  4.1 Introduction  Global fertilizer usage in the agricultural system, particularly that o f nitrogen and phosphorus, has increased  several times  in the past half century,  from  approximately 5 x 10 kg o f N and P fertilizer in 1950 to around 35 x 10 kg o f 9  9  phosphorus and over 90 x 10 k g o f nitrogen in 2000 (FAO 2000). A t current prices o f 9  approximately $0.5 kg" (U.S.) for nitrogen fertilizer, the total cost o f nitrogen addition 1  to crops is approximately $50 billion. O f this, roughly 30 to 4 0 % is recovered by crop plants, suggesting that losses o f the order o f 30 billion dollars (U.S.) are sustained every year (Raun and Johnson, 1999). This fertilizer loss could be either due to volatilization as N H accounting for < 45 kg o f N ha" , denitrification < 2 2 % o f applied N , and 1  3  leaching < 4 0 % o f applied N (Raun and Johnson, 1999). In summary, less than 50% o f the applied N is absorbed and used by the plants (Hauck et al., 1997; Raun and Johnson, 1999).  130  Nitrogen is the main limiting factor for rice yield, no matter whether up-land or low-land system is followed. Moreover, both the yield and protein content o f rice increase with proper application o f N (Gomez and De Datta, 1975; Allen and Terman, 1978). Among the nitrogen sources, ammonium is the preferred species taken up by rice plants (Sasakawa and Yamomoto, 1978; Goyal and Huffaker, 1984). One strategy to increase rice yield, might be to increase N influx. This would also serve to reduce the loss o f applied N from rice fields, and to protect the environment from the negative effects o f the volatilization, leaching and denitrification. Alternatively, farmers might make repeated and temporally well spaced planting and lower application rate o f N fertilizer. Such improved varieties would be better able to compete for N acquisition with soil microbes that include bacteria, fungi and algae. Under conditions wherein carbon availability is not limiting, the nitrogen acquisition and use efficiency might be increased by increasing the unidirectional influx o f ammonium by rice roots. As mentioned earlier, cloning o f rice AMT genes, particularly those that encode high-affinity ammonium transport systems (HATS) (accession numbers: AF289477, AF289478 and AF289479), has been done. Having completed the physiological, biochemical, and molecular studies (see chapters 2, and 3) dealing with the regulation o f expression o f these genes, it was interesting to determine i f over-expressing one or more o f these genes in two rice varieties would increase N H  + 4  influx in roots o f these  plants. Additionally it was important to investigate whether increased N H  + 4  influx  would adversely affect the biomass or other characteristics o f the over-expression lines. Because OsAMTl.l  appeared to be the most active and/or most N-responsive gene  responsible for H A T S activity, it was selected as the gene to be over-expressed in  131  transgenic lines to test the hypothesis. A t the same time that my focus was upon N H 4 influx, it was essential also to measure N H / efflux, since i f this parameter had increased by a value corresponding to any increase o f influx, then any potential advantage o f increased influx would be removed in the transgenic plants. Therefore this chapter describes the physiological characterization o f several over-expression lines o f two cultivars o f rice, Jarrah and Taipei.  4.2 Materials and Methods  4.2.1 Plant Material, Growth and Experimental Conditions  For all the transgenic studies, two cultivars, Taipei-309, and Jarrah were used. The transgenic lines (T2 generation) o f Taipei-309 cultivar were 40-2 and 38-1. The transgenic lines (T2 generation) o f Jarrah cultivar were 7 7 - 1 , 71-2, and 75-4. OsAMTl.l  was introduced into the genome o f Jarrah and Taipei cultivars using a T-  D N A construct and was driven by Ubil (I) promoter. This work was completed at the Australian National University by M . S. Haque (2001). Seeds o f transgenic lines were germinated in distilled water containing 80 mg L" hygromycin. Only surviving 1  seedlings resulting from this treatment were used as transgenic experimental plants. The w i l d type cultivar seeds were germinated in distilled water without hygromycin. For studies o f NH4 influx in transgenic lines, rice seedlings were grown in solutions 13  +  maintained at either 10 p M , or 2 m M [ N H ] , for 21 days. +  4  13  NH4  +  influxes were  measured in 100 p M [ N H ] in MJNS. A l l other growth conditions and experimental +  4  procedures used in influx studies were as described in Chapter 2. For biomass analyses,  132  root and shoot fresh weights o f the plants used for influx studies were recorded on the basis o f mass per unit plant.  4.2.2 N H 4 E f f l u x Measurements 15  +  For the efflux analysis, roots o f rice plants were immersed in loading solution containing  1 5  NH  + 4  and a specific concentration o f unlabelled N H  + 4  in MJNS for 45  minutes. The loaded plants' roots were then introduced into aerated MJNS medium contained in plastic funnels for elution with non-labelled MJNS medium. This was identical in all respects to that used for growth and to the solution used for loading, except that  1 5  NH  + 4  was absent and replaced by  1 4  N H . Roots were eluted with 100/200 +  4  mis o f the same MJNS medium by removing the medium in the funnel by means o f a valve at the base o f the funnel and immediately replacing the medium with fresh MJNS according to standard protocols in this laboratory (Siddiqi et al., 1991). After various intervals o f time for the exchange, the labelled efflux solution, resulting from efflux o f 1 5  N from roots was collected in appropriate flasks. The time intervals for successive  washes were 2 x 5 s, 2 x 10 s, 5 x 15 s, 4 x 30 s, 5 x 1 min, 7 x 2 min, 1 x 8 min, 10 x 15 min. After completion o f the elution for 3 h, all eluate media and root and shoot samples were evaporated to dryness at 75 °C. The root and shoot dry weights were recorded and the dried tissue samples were kept in desiccators to avoid the absorption o f atmospheric moisture. The dried root and shoot samples were ground using a pestle and mortar and approximately 5 mg samples were packed individually in small tin capsules. The wash solutions, after drying were reconstituted in 1 ml o f distilled water.  133  Finally all the samples were analysed for  1 5  N as described in chapter 2. A l l the data  were subject to regression analysis and were plotted as described in Siddiqi et al. (1991).  134  Figure 4 . 1 : OsAMTl.l  gene construction. Binary vector construct pPIMP161 containing  the Expression cassette from pPIMP56, iscoRI-endfilled/Mrtf/III fragment, inserted into the binary vector pWBvec8, Xbal-Qn&f\llQ&lHindlll  digest, which contains CaMV35S  promoter driven, intron- interrupted hygromycin resistance gene (hph) as selectable marker. The construct contains a 2.04 kb OsAMTl.l  c D N A inserted in sense orientation.  135  Table 4.1: List of the transgenic lines used  Line  Generation  Copy  Cultivar  77-1  T2  1  Jarrah  71-2  T2  1  Jarrah  75-4  T2  4-6  Jarrah  wild type  NA  NA  Jarrah  40-2  T2  1  Taipei-309  38-1  T2  3  Taipei-309  wild type  NA  NA  Taipei-309  136  4.3 R e s u l t s  4.3.1  1 3  NH  + 4  I n f l u x Analysis  When measured at 100 u M [ N H ] , the H A T S influx o f +  1 3  4  w i l d type, and into roots  N H , into roots o f the +  4  o f the transgenic rice lines were significantly different  (p<0.05). For plants grown in media containing 10 p M external [ N H ] containing +  4  MJNS, influx values were higher than those o f plants grown in 2 m M containing media. N H  + 4  [NH ] +  4  influx in the roots o f Jarrah cultivar (wild type) previously  grown in 10 p M was 4.5 ± 0.23 (SE) pmol g" h" , while in the transgenic 75-4 line 1  1  influx was 6.2 ± 0.30 (SE) pmol g" h" and was significantly higher (p<0.05), showing 1  1  an increased influx o f 37 percent as compared to the w i l d type cultivar. The influx recorded for 71-2, and 77-1 were 5.2 ± 0.15 (SE) and 5.9 ± 0.30 (SE) pmol  g" i f and 1  1  were significantly higher (p<0.05) showing increases o f 16 and 33 %, respectively, when compared with the influx rate in the w i l d type (Fig. 4.2).  1 3  NH  + 4  influx rates in  roots o f Taipei seedlings grown under the same conditions were 5 ± 0.18 (SE) pmol g"  1  hf . The transgenic over-expression lines 40-2 and 38-1 showed influx rates o f 4 ± 0.19 1  (SE) and 5 ± 0.17 (SE) pmol g^h" respectively (Fig. 4.2). Actually the influx in the 1  roots o f 40-2 was reduced by 20 percent as compared to the influx in the w i l d type and was significantly lower (p<0.05). The other transgenic line 38-1 showed no statistically significant (p<0.05) difference in the rate o f N H  + 4  influx (Fig. 4.2).  137  1—  8-i  1  r  C  2-1  T *  77-1 71-2 75-4 Jarrah 38-1 40-2 Taipei Transgenic Lines  Figure 4.2: Rates o f  1 3  NH  + 4  influx at lOOpM [ N H ] in the transgenic over-expression +  4  lines and the two w i l d type rice cultivars. A l l plants were grown in MJNS medium containing 10 p M N H  + 4  at steady state for three weeks. Each datum point represents the  average o f four replicates and vertical bars represent standard errors (SE).  138  o E  x 3  77-1 71-2 75-4 Jarrah 38-1 40-2 Taipei  Transgenic Lines  Figure 4.3: Rates of  1 3  NH  + 4  influx at lOOpM [NFL, "] in the transgenic over-expression 4  lines and the two w i l d type rice cultivars. A l l plants were grown in MJNS medium containing 2 m M N H  + 4  at steady state for three weeks. Each datum point represents the  average o f four replicates and vertical bars represent standard errors (SE).  139  For the rice seedlings grown in MJNS media containing 2 m M N H / ,  1 3  NH/  influx rates in roots o f the w i l d type Jarrah cultivar were measured to be 2 ± 0.08 (SE) pmol g" h" (Fig. 4.3). Influx in roots o f line 75-4 was 2.7 ± 0.25 (SE) pmol .g" h" 1  1  1  showing a significant increase o f 34 % percent (p<0.05).  1 3  1  N H / influx into roots o f  lines 71-2 and 77-1 were 2 ± 0.09 (SE) and 2.3 ± 0.12 (SE) pmol g" h" , Line 71-2 1  1  showed no statistically significant change in the rate o f influx (p<0.05), whereas line 77-1 showed an increase o f 13 % when compared with the influx rate o f the respective w i l d type plants (Fig. 4.3). It was not, however, statistically significant (p<0.05). The 1 3  NH  + 4  influx rate in the w i l d type Taipei cultivar was 2.4 ± 0 . 1 2 (SE) pmol g" h" . The 1  1  over-expression lines o f this cultivar, 40-2 and 38-1 showed influx rates o f 1.6 ± 0.06 (SE) and 2 ± 0.14 (SE) pmol g' h" respectively. Surprisingly, both o f the two over1  1  expression lines o f Taipei actually showed reduced influx rates o f 32 and 19 % respectively, when compared with that o f the wild type and were significantly lower (p<0.05) (Fig. 4.3).  140  4.3.2 Biomass Analysis  The biomasses o f the two w i l d type cultivars and those o f the overexpressing lines showed considerable variations. The total biomass o f the Jarrah rice seedlings was 1.5 ± 0.10 (SE) g per plant for plants grown in MJNS containing 10 p M NH  + 4  (Fig. 4.4). The same seedlings had an average root fresh weight o f 0.81 ± 0.05 (SE)  g per plant and shoot fresh weight o f 0.64 ± 0.04 (SE) g per plant. The total biomass o f line 75-4 was 1.4 ± 0.10 (SE) g per plant, not significantly different (p<0.05) from that o f the w i l d type. In contrast, line 71-2 showed a reduction o f 8% in total biomass at 1.3 ± 0.06 (SE) g per plant. This difference was not statistically significant (p<0.05). Root fresh weight was reduced by 1 1 % at 0.72 ± 0.03 (SE) g per plant (statistically significantly, p<0.05), while shoot weight was reduced by 5% at 0.61 ± 0.03 (SE) g per plant with no statistical significance (p<0.05). The reduction o f biomass in line 77-1 was 10% o f the total plant basis with a value o f 1.3 ± 0.07 (SE) g per plant, but it was not statistically significant (p<0.05). There was a statistically significant (p<0.05) 12% reduction in the root fresh weight, with a value of 0.71 ± 0.03 (SE) g per plant and a non-significant 8% reduction in the shoot fresh weight with a value o f 0.59 ± 0.03 (SE) g per plant (Fig. 4.4). A l l the data represent means o f 4-6 individual plants. The total biomass of plants o f the w i l d type Taipei cultivar grown in the same media was 1.4 ± 0.08 (SE) g per plant, with an average root fresh weight o f 0.77 ± 0.04 (SE) g per plant and average shoot fresh weight o f 0.59 ± 0.04 (SE) g per seedling. The reductions in the biomass of the transgenic line were more pronounced. The total plant biomass o f the plants of line 40-2 were reduced to 0.99 ± 0.05 (p<0.05) g per plant showing a reduction o f 27% in the total biomass that was significantly lower (p<0.05),  141  with the root biomass showing a reduction o f 2 9 % at 0.55 ± 0.03 (SE) g per plant and shoot biomass o f 25% at 0.44 ± 0.02 (SE) g per plant, both statistically significant (p<0.05). Line 38-1 showed a reduction o f 2 4 % in the total biomass o f the seedlings o f 1.03 ± 0.06 (SE) g per plant comprising o f a 2 6 % reduction o f root fresh weight at 0.57 ± 0.03 (SE) g per plant and 2 2 % reduction in the shoot fresh weight o f 0.46 ± 0.02 (SE) g per plant with both root and shoot biomass showing statistically significant lower values (p<0.05) (Fig. 4.5). Overall, the reduction o f biomass was more pronounced in the transgenic lines o f Taipei cultivar than those in the lines o f the Jarrah cultivar. The root biomass was always more than the shoot biomass for all the seedlings grown at 10 p M NH  + 4  containing MJNS. In contrast to plants grown at 10 p M , seedlings grown in media containing 2  m M NH4 showed consistently higher shoot than root biomass on a fresh weight basis, +  and a greater reduction in biomass o f the transgenic lines o f both the Jarrah and Taipei309 cultivars when compared with the reductions of the same lines grown in media containing 10 p M ammonium. The total biomass o f Jarrah seedlings on was 1.54 ± 0.09 (SE) g per plant, with fresh root mass o f 0.64 ± 0.04 (SE) g and fresh shoot weight o f 0.90 ± 0.05 (SE) g per plant (Fig. 4.6). The seedlings o f line 75-4 showed a modest reduction o f biomass with no statistically significant (p<0.05) reduction in the root and shoot biomass. Line 71-2 seedlings had a 13% reduction in their total biomass at 1.34 ± 0.07 (SE) g per plant and was significantly lower (p<0.05). There was a 14% reduction in root fresh weight at 0.55 ± 0.03 (SE) per plant that was significantly lower (p<0.05) and 13% reduction in shoot fresh weight at 0.79 ± 0.05 (SE) g per plant that was significantly lower (p<0.05). The reduction in the total biomass in the plants o f line 77-1 was 14% at  142  1.33 ± 0.08 (SE) g per plant and was significantly lower (p<0.05). There was a 17% reduction in the root biomass at 0.53 ± 0.03 (SE) g per plant and was statistically significant (p<0.05) and 11.5% reduction in the shoot biomass on a per plant basis with no statistical significance (p<0.05) (Fig. 4.6). Total biomass o f the Taipei cultivar was 1.51 ± 0 . 1 0 (SE)  g per plant, with root  and shoot fresh weights o f 0.62 ± 0.05 (SE) g per plant g and 0.89 ± 0.05 (SE) g per plant respectively (Fig. 4.7). Compared to the parent line, the biomass o f the over-expression line 40-2 was reduced by 39% with a value o f 0.92 ± 0.08 (SE) g per plant and was significantly lower (p<0.05) with a 4 1 % reduction in root fresh weight at 0.37 ± 0.02 (SE) g per plant and a 38% reduction in the shoot fresh weight with value o f 0.55 ± 0.04 (SE) g per plant and were significantly lower (p<0.05). The seedlings o f line 38-1 also showed a similar reduction in total biomass of approximately 38% at 0.94 ± 0.07 (SE) g per plant with a 39% reduction in the root fresh weight with a value o f 0.38 ± 0.02 (SE) g per plant and a 37% reduction in the shoot fresh weight at 0.56 ± 0.03 (SE) g per plant and all values were significantly lower (p<0.05) (Fig. 4.7).  143  2.0 I  I total  WZh root  77-1  71-2  75-4  Jarrah  Transgenic Lines  Figure 4.4: Biomasses o f the transgenic over-expression lines and the w i l d type cultivar Jarrah. Root, shoot and total biomass are represented separately. A l l plants were grown in MJNS medium containing 10 p M N H  + 4  at steady state for three weeks. Each bar  represents the average o f 4-6 replicates and vertical lines represent standard errors (SE).  144  I  38-1  40-2  I total root  Taipei  Transgenic Lines  Figure 4.5: Biomasses o f the transgenic over-expression lines and the w i l d type cultivar Taipei. Root, shoot and total biomass are represented separately. A l l plants were grown in MJNS medium containing 10 p M N H  + 4  at steady state for three weeks. Each bar  represents the average o f 4-6 replicates and vertical lines represent standard errors (SE).  145  2.0 I  I total  WZ\ root W%k shoot  4  1.6H  2 a  1.2  3 co  0.8  E o ffl 0.4  0.0  77-1  71-2  75-4  Jarrah  Transgenic Lines  Figure 4.6: Biomasses o f the transgenic over-expression lines and the w i l d type cultivar Jarrah. Root, shoot and total biomass are represented separately. A l l plants were grown in MJNS medium containing 2 m M N H  + 4  at steady state for three weeks. Each bar represent  average o f 4-6 replicates and vertical lines represent standard errors (SE).  146  I I total WZ\ root shoot  Transgenic Lines  Figure 4.7: Biomasses o f the transgenic over-expression lines and the w i l d type cultivar Taipei. Root, shoot and total biomass are represented separately. A l l plants were grown in MJNS medium containing 2 m M N H  + 4  at steady state for three weeks. Each bar represent  average o f 4-6 replicates and vertical lines represent standard errors (SE).  147  4.3.3  1 5  NH  + 4  E f f l u x Analysis  For the efflux analysis, over-expression line 75-4 and the Jarrah cultivar were selected, as the former line showed the largest increase o f influx under both low and high external NH4 provisions. The efflux analysis was conducted only for the seedlings +  grown at 10 p M external N H  + 4  concentrations. The rate o f efflux calculated for the w i l d  type cultivar Jarrah was 0.62 pmol g" h" (Fig. 4.8) and was 15% o f the influx value for 1  1  the seedlings grown at the same ammonium concentration. The efflux rate from the roots o f the over-expression line 75-4 was 0.68 pmol g" h" (Fig. 4.9) equivalent to 1 1 % o f the 1  1  influx value o f this over-expression line.  148  0.00 -i  -0.05  1  + 4  •  1  1  1  1  1  1——r  1  1  1  1  -f  -0.25 H 0  Figure 4.8: N H  1  .  5  r20  10 15 Efflux Time (Minutes)  •  1 25  Efflux from roots o f wild-type rice plants. Regression analysis o f ° N H  efflux from roots o f w i l d type cultivar Jarrah, previously loaded for 45 min in ° N H then transferred to  1 4  N H . Plants were grown in MJNS containing 10 p M N H +  4  state for three weeks. Logio o f N H 1 5  + 4  + 4  and  4  at steady  + 4  efflux (logio pmoles g" h" FW) is shown during a 25 1  1  minutes efflux analysis. Zero time corresponds to the transfer o f roots to  1 4  N H . R for +  2  4  regression = 0.99.  149  Figure 4.9: N H / Efflux from roots o f Jarrah cultivar 75-4 rice plants. Regression analysis of  1 5  NH  + 4  efflux from roots o f Jarrah cultivar 75-4 previously loaded for 45 min in  and then transferred to  1 4  1 5  NH  N H . Plants were grown in MJNS containing 10 p M N H +  4  steady state for three weeks. L o g i o f 0  1 5  NH  + 4  + 4  + 4  at  efflux (logio pmoles g" h" FW) is shown 1  1  during a 25 minutes efflux analysis. Zero time corresponds to the transfer o f roots to 1 4  N H . R for regression = 0.99. +  2  4  150  4 . 4 Discussion  For the purpose o f discussion, the results o f this chapter can be summarized as: 1. A l l transgenic over-expression lines derived from Jarrah cultivar, when grown at 10 p M N H , showed higher ammonium influx when influx was measured at 100 +  4  pM NH . +  4  2. The transgenic lines derived from Taipei cultivar failed to show any increased values o f NH4 influx, showing no increase or decrease in case o f line 3 8 - 1 , and a +  decrease in case o f line 40-2. 3. When the N H  + 4  influx was measured for transgenic over-expression lines  derived from Jarrah cultivar grown at 2 m M N H , only line 75-4 showed significant +  4  increase in the ammonium influx. The other two lines showed very little increase or no increase at all. 4. A l l transgenic over-expression lines derived from Taipei cultivar, when grown at 2 m M N H , showed lower ammonium influx when influx was measured at similar +  4  conditions. 5. When efflux analysis was done, no significant difference was observed between the w i l d type Jarrah and the transgenic line 75-4. 6. A l l the transgenic lines, both o f Jarrah and Taipei origin showed a reduction in biomass compared to w i l d type when grown at 10 p M N H . The reduction was +  4  statistically insignificant except in case o f the root biomasses o f Jarrah lines 77-1 and 71-2. For plants grown at 2 m M N H , reduction was greater and significant for all +  4  Taipei transgenic lines, both in root and shoot biomass. Jarrah lines showed no difference at 10 p M N H  + 4  and 2 m M N H . +  4  151  7. The root biomass was always more than the shoot biomass for all the w i l d type and transgenic lines when grown at 10 p M N H / . 8. A l l the plants, w i l d type and the transgenic lines, showed more shoot biomass than root biomass, when grown at 2 m M N H / . 9. A l l the transgenic lines, both o f Jarrah and Taipei origin, showed a reduction in biomass when grown at 2 m M N H / . This reduction was very little and was insignificant in 75-4, and in case o f root biomass o f 77-1 and 71-2 the reduction was significant. The reduction was much higher and significant for all Taipei transgenic lines, both in root and shoot biomass. As the world human population continues to increase and particularly among populations that depend on rice for their primary source o f nutrition, and in view o f the failure to develop functional rice-rhizobium or functional rice N2-fixing bacteria symbioses, we need rice plants that are more efficient i n nitrogen uptake, translocation and assimilation. One approach towards achieving this goal could be the development o f transgenic over-expression lines o f rice containing one or more copies o f OsAMTl.l that in principle could lead to increased efficiency in terms o f unidirectional influx o f N H / . Clearly, i f increased influx were associated with increased efflux o f N H / then the potential advantage o f improved acquisition would be reduced. I f one or more o f the over-expression lines showing the targeted phenotype could be subject to field trials and shown to be more efficient at acquiring N under field conditions, it might be released as a new, more efficient variety. Towards trying to achieve the above goal, several over-expression lines with one or more copies o f OsAMTl.l ANU  using  Agrobacterium-mediated  transformation,  were produced at  and tested  for  various  152  physiological properties. The N H 4 influx rates in the transgenic line 38-1 o f TaipeiB  +  309, showed no increase over that o f the parental line when grown i n 10 p M ammonium-containing media. Rather, there was a reduction o f 19% i n the influx rate. These observations might be explained by one or more o f the following hypotheses: 1. Absence o f any effect on the influx rate for plants grown at low external NH.4  +  might indicate that the gene was not inserted at an appropriate location. 2.  Likewise, a reduction o f influx might be attributed to inappropriate gene  insertion resulting in some deleterious effect upon plant growth. Indeed, Fig. 4.5 demonstrates that biomass was reduced in these transgenic plants o f the Taipei cultivar. 3. Alternatively, an initially increased N H effects o f elevated internal N H  + 4  + 4  influx might result in deleterious  as discussed by Britto et al. (2001), due to the high  energy costs o f excreting NH4 . This is probably unlikely at l o w external [NFLt ] but +  +  might be significant at 2 m M [NFL; ]. +  4.  Elevated internal ammonium concentration has been reported to have other  adverse effects on the physiology, anatomy, and/or morphology o f plants, including reduced water translocation (Anderson et al., 1991), reduction o f C O 2 fixation in the chloroplasts and blocking o f A T P production (Puritch and Baker, 1967; Ikeda and Yamada,  1981). Furthermore,  Hummelt  and M o r a  (1980), reported that  high  ammonium accumulation repressed N A D H - G O G A T , thus reducing the activity o f the ammonium assimilation pathway in Neurospora crassa.  A n y negative effect upon  plant growth would result in reduced demand for N and reduced NH4 influx. +  153  5. Initially an increase in N H / influx might result in down-regulation o f influx through the effects of increased tissue N status at the protein level as suggested by Rawat etal (1999). The marked reduction o f influx in 40-2 over-expression line could be due to similar factors as discussed for line 38-1. The transgenic over-expression lines o f Jarrah cultivar showed considerably increased N H / influx values o f 37, 16, and 33% for 75-4, 71-2, and 77-1, respectively, under low ammonium provision that was encouraging, and produced the targeted result o f increasing the N H / influx by introduction o f one or more copies o f OsAMTl.l  in  the genome o f the transgenic lines. The increased influx rate observed in line 75-4 was still higher by 34% even when the plants were grown in high ammonium (2 m M ) media.  In line 77-1, the increase was still 13%, while the influx rate in line 71-2  showed virtually no increase and rather a small decrease, that might be due to the same reasons as suggested above for the Taipei transgenic lines. O f the two over-expression lines o f Jarrah, 75-4 was selected for efflux analysis because it showed consistently higher influx rates in both low and high external ammonium media. As the efflux rate from the roots o f the over-expression line 75-4 was not much different than that from the roots o f the corresponding w i l d type cultivar, and was actually less in percentage terms, it appears that the line could be field tested as a possible high efficiency ammonium acquisition variety o f rice. The overall root biomass o f rice seedlings grown at low ammonium containing growth media was higher than when plants were grown on high external ammonium. This shows the capacity o f the rice plants to increase the root surface area in order to  154  scavenge as much N H  + 4  as possible, while its concentration remains low. Higher root  biomasses typically lead to a comparatively lower shoot biomass at least for these three-week old seedlings. In longer term studies, it is likely that overall root and plant biomass would be significantly greater under conditions o f high N provision. Hence these observations should be extended for the complete life-cycle o f these plants. Root biomass o f transgenic rice plants was generally lower than that o f wild-type plants under both low and high-N growth conditions. This effect was observed to a greater extent under high-N than low-N and to a greater extent in Taipei than in Jarrah lines. This effect was not significant for line 75-4 o f Jarrah. Under high-N conditions, this effect might be interpreted as a means to reduce influx o f N H  + 4  to avoid ammonium  toxicity. Indeed this effect was more prominent in high-N plants and the effect was quite modest for Jarrah. With the exception o f line 75-4, the total biomasses o f all transgenic lines were reduced by comparison to parental lines, whether grown in high or low ammonium provisions. The reductions were more pronounced in the transgenic lines o f Taipei-309 cultivar, than those in the Jarrah transgenic lines, pointing towards the possibility o f an enhanced toxic effect o f increased internal N H  + 4  on the Taipei lines, particularly at high  external [ N H ] . +  4  The Taipei-309 cultivar is closely related to subspecies indica, while Jarrah is a japonica cultivar. Interestingly Augladette (1965) reported that the japonica  cultivar  usually responded more than indica to nitrogen fertilizer input, due in part to japonica's less efficient N H  + 4  acquisition system, but more efficient assimilation o f  155  NH  + 4  and indica's better N H  + 4  acquisition system, but  less efficient ammonium  assimilation system. As 75-4 line showed enhanced N H  + 4  influx with almost no reduction in the  plant biomass and efflux, it should be field-tested for its entire life cycle for a possible new agricultural variety. In summary, when growth is limited by N uptake (under low-N conditions) and internal N pools are low, we might expect that the capacity to scavenge ammonium f r o m dilute solution might limit growth. Under such conditions negative feedback mediated down-regulation o f N H influx without  significant  + 4  influx would be at a minimum, and increasing  increase in the efflux, by over-expression might be  advantageous. Under such conditions, increasing gene expression and transporter expression might contribute to increased N uptake and increased growth. We might even find that the diurnal effects might be less so as to maximize uptake during all opportunities.  But  such  low-N  conditions  like  10  pM  external  ammonium  concentrations are not typical o f the agricultural context and may not be very useful in the industrialized agricultural system where the input o f inorganic N fertilizer usually leads to a very high initial external N H  + 4  concentration. However such improvement  may still be useful in many developing countries where a large number o f small and marginal farmers still administer a limited amount of inorganic N fertilizers. B y contrast, at more moderate N supplies, when N no longer limits growth, N uptake is dictated by growth rate because o f feedback regulation due to accumulation o f regulators glutamine and asparagine. Thus it might be argued that simply increasing influx may not necessarily increase N uptake because more rapid accumulation o f  156  feedback regulators would reduce influx unless growth can be driven faster to prevent the accumulation o f N and feedback regulation o f influx. This means that other processes and/or genes might require enhancement by over-expression o f their respective genes like the GS, G O G A T and even various genes responsible for photosynthesis.  157  5 Summary, Conclusions and Future Prospects There have been various reports in the literature regarding the physiology o f high-affinity N H  + 4  influx in plants, including the work by Wang et al. (1993a, b) on rice,  and some work on expression analyses o f some AMT genes in various plants (Gazzarini et al. 1999, von Wiren et al. 2000, Salvemini et al. 2001 Shelden et al. 2001). However the biochemical regulatory mechanism o f the N H  + 4  influx via H A T S in cereals, including  rice was not clear. Further, no attempts have been made to decipher the regulation o f NH  + 4  influx at the molecular level. The present study has made comprehensive  investigation o f the physiological, biochemical and molecular regulation o f high-affinity NH  + 4  influx in rice. M y study also provided insights into the effectiveness o f putting one  or more copies o f the OsAMTl  gene into the rice genome towards achieving the goal o f  developing rice cultivars that could be more efficient in N uptake and assimilation. The present study has demonstrated the nature o f N compounds that modulate the transcription o f the three OsAMTl  mRNAs, thereby achieving the  158  transcriptional regulation o f N H of N H  + 4  influx via H A T S . The study also suggests direct effects  + 4  on the protein level to regulate H A T S , but here the evidence is only suggestive.  The conclusions o f Wang et al. (1993a, b, 1994) that high N provisions lower N H  + 4  influx rates via H A T S into rice roots and that lower N provisions result in higher influx at the physiological level still hold true. Similar results at the physiological level were reported by Lee and Rudge (1986); they also suggested that the observed response was due to downstream N derivatives rather than N H  + 4  itself. Evidence in my current work  demonstrates that in rice seedling roots the down-regulation and up-regulation o f N H  + 4  influx via H A T S are mainly due to the increase and decrease o f root Gin, and Asn concentrations and that N H  + 4  itself exerts only a small degree o f control and might be  important in short-term effects on the AMT proteins. Glass et al. (1997) concluded that when M S X was applied to the roots o f low-nitrogen plants, Gin was important in regulating N H NH  + 4  + 4  influx, while N H  + 4  itself appeared to be important in down-regulation o f  influx in high-N plants. Some other studies such as those by Cooper and Clarkson  (1989) and Larsson et al. (1991) suggested that the cycling of the N derivatives from root to shoot and from shoot back to root could be the way o f achieving the information transfer regarding whole plant N status. The present study also demonstrated that the three OsAMTl genes are differentially expressed and regulated by the root Gin and Asn. Further it is evident that there is a clear transcriptional regulation o f N H  + 4  influx via H A T S through the down-regulation and up-  regulation o f the transcript abundances o f the three genes under study in the rice seedling roots. The expression levels o f the three genes and the N H  + 4  influx rates appeared to be  correlated with the extent and the nature o f the N supply; i.e. the N H  + 4  influx and the  159  transcript abundances were high under conditions o f low external N provisions and were low under conditions o f elevated supply o f N H , / or that o f Gin, Asn, Glu, or Asp in the growth media. However, careful modulation o f the root N H / and Gin, Asn, Glu, and Asp by different treatments together with M S X treatments, revealed that the modulations o f transcript abundances was mainly through the altered levels o f root Gin and Asn. However, a small effect o f N H / could not be totally ruled out. The highest effect o f elevated root Gin and Asn was on the expression o f the m R N A o f OsAMTl.l, by that o f OsAMTl.2.  followed  The least affected was the expression o f OsAMTl.3 by the  modulations o f the root Gin or Asn. There have been reports on the differential expressions and or regulation o f some genes encoding high-affinity  ammonium  transporters in other plant species. Similarly, Marini et al. (1994, 1997) concluded that the expressions o f Mepl,  Mep2, and Mep3 encoding for H A T S in Saccharomyces  cereviciae were repressed in the presence o f glutamate and glutamine. However, in tomato, Lauter et al. (1996) reported that the expression o f LeAMTI was similar under all nitrogen conditions i.e. was constitutively expressed. Further, Gazzarrini et al. (1999) observed steep increases in the AtAMTI. 1 expression levels in the roots o f Arabidopsis when N nutrition became limiting, while that o f AtAMTI. 3 increased slightly and that o f AtAMTI.2  was constitutively expressed. The Arabidopsis AtNRT2.1 expression was  reported to be down-regulated by N H / , Gin or other amino acids by Zhou et al. (1999). In the same year, Rawat et al. (1999) reported that the expression o f AtAMTI was downregulated by high root Gin concentration. In lotus, Salvemini et al. (2001) also reported high expression o f LjAMT 1.1 under low nitrogen conditions. Interestingly von Wiren et al. (2000) reported that in tomato LeAMTI.2 expression increased after N H / or NO3"  160  supply in the roots o f hydroponically-grown plants, whereas LeAMTl.l  was induced by  N deficiency and coincided with low Gin concentrations. Recently Pearson et al. (2002) while studying the regulation o f H A T S in Brassica, reported that BnAMT1.2 levels and N H  + 4  mRNA  uptake were increased when detached leaves were supplied with  increasing [ N H ] , however when Gin or Glu were supplied to the leaves the BnAMT1.2 +  4  expression was reduced. As evident from the above discussions, some o f the earlier reports are similar to the results in the current study while others are dissimilar. This might be due to different growth conditions or the fact that different orthologous genes in different species behave in different ways. This could be also due to the fact the numbering o f the genes are not followed in a universal way and the numbers e.g. 1.1, 1.2 etc. in various organisms may not truly be the orthologs. The present study also demonstrated that the diurnal variation in the high- affinity NH  + 4  influx is mainly regulated through modulations in the expression o f OsAMTl.3 and  that the changes in the expression level appeared to be (in part) under the control o f available sugars. The N H  + 4  influx via H A T S varied considerably during 24 h cycle, and  was highest in the late afternoon and lowest after mid-night in the dark period. The observed variation in the N H  + 4  influx was accompanied by similar changes in the  expression o f OsAMTl.3. However the expression levels o f OsAMTl.l  and OsAMTl.2  also changed diumally, but to a much lower extent than the OsAMTl.l. differential expression o f AtAMTl.3  Similar  was also reported by Gazzarrini et al. (1999) in  Arabidopsis. However, von Wiren (2000) reported that LeAMT1.2 and LeAMT1.3 showed reciprocal diurnal regulation, with expression o f LeAMT1.3 transcripts being highest in  161  the darkness, and that o f LeAMTI.2 being highest after the onset o f light. N H / influx and OsAMTl.3  levels were substantially increased by exogenous application o f sucrose to  roots o f rice plants. Again the magnitude o f increase in the expression o f OsAMTl.3 was the highest. Kubik-Dobosz et al. (2000) also reported that the treatments with sucrose or glucose resulted in substantial increase in N H / uptake along with enhanced expression o f AMT1 in rutabaga (Brassica) roots; they also reported that the transcript abundances o f Nrt2 gene and NO3" uptake increased in day light and decreased during the night in tobacco roots. This study also demonstrated that at least one line o f rice over-expressing OsAMTl.l  showed increased N H / influx without substantially increasing the efflux o f  the same. However, this increase was not observed in other lines o f one cultivar and was not observed in all the lines o f another cultivar due to various possible reasons. The present study is the first comprehensive work clarifying the regulation o f highaffinity N H / influx into plant roots at the physiological, biochemical and molecular levels. Clearly, these findings have added substantially towards understanding the complex nature o f the regulatory mechanisms o f N H / influx via H A T S in cereals, in response to N status o f the plants as well as the regulatory aspects o f N H / influx at the diurnal level including the effects o f light and/or carbon availability. M y investigations have also successfully evaluated the potential practical benefits and problems associated with over-expressing native gene(s) towards improvement o f rice plants, and thus contributed significantly towards the understanding o f these aspects.  162  There are many potential lines o f investigation that might be undertaken in order to increase the understanding o f the high-affinity N H transcriptional regulation o f the N H  + 4  + 4  influx. There is evidence o f post-  in addition to the apparent down-regulation and up-  regulation in the number o f transporter proteins in the membrane system. The study o f NH  + 4  influx should be continued at the protein level by raising antibodies against all the  OsAMTl  genes and performing western blot analyses in order to see the possible  modulations in the amount o f the transporter proteins in the plant root. This could be done either in rice or Arabidopsis as model systems. In order to shed light on the mechanisms o f the direct effects o f various putative regulators o f the AMT proteins, the proteins need to be purified and then crystallized. Once the protocols for protein crystallization are established, then the proteins may be crystallized under various treatments and conditions to investigate possible conformational changes in the three dimensional structure o f the transporter proteins or in their N H  + 4  and/or regulator binding sites. 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