Chapter 7

4 downloads 0 Views 669KB Size Report
V. Physiological Significance of the Distribution of Nitrate and Sulfate ... N assimilation seems to be confined to mesophyll whereas sulfate reduction has ..... however, is important for correct C/N balance .... APS reductase, 4 – sulfite reductase, 5 – serine acetyltrans- ...... Regulation of root ion transporters by photosynthesis:.
Chapter 7 Nitrogen and Sulfur Metabolism in C4 Plants Stanislav Kopriva* John Innes Centre, Norwich NR4 7UH, UK

Summary ............................................................................................................................................................... 109 I. Introduction..................................................................................................................................................... 110 II. Nitrogen Assimilation...................................................................................................................................... 110 A. Plant Nitrate Assimilation ......................................................................................................................... 110 B. Regulation of Nitrate Assimilation ............................................................................................................ 111 C. Nitrate Assimilation in C4 Plants ............................................................................................................... 113 III. Sulfate Assimilation ........................................................................................................................................ 114 A. Plant Sulfate Assimilation ......................................................................................................................... 114 B. Regulation of Sulfate Assimilation ............................................................................................................ 115 C. Sulfate Assimilation in C4 Plants .............................................................................................................. 116 IV. Glutathione Synthesis and Reduction ............................................................................................................ 117 A. Regulation of GSH Synthesis ................................................................................................................... 117 B. Localization of GSH and GSH Synthesis ................................................................................................. 118 C. GSH Synthesis in C4 Plants ..................................................................................................................... 119 V. Physiological Significance of the Distribution of Nitrate and Sulfate Assimilation .......................................... 120 A. Open Questions on Nitrate Assimilation in C4 Plants ............................................................................... 120 B. Significance of BSC Localization of Sulfate Assimilation ......................................................................... 121 C. Consequences of BSC Localization of Sulfate Assimilation..................................................................... 122 VI. Conclusions .................................................................................................................................................... 122 Acknowledgments ................................................................................................................................................. 122 References ............................................................................................................................................................ 123

Summary C4 photosynthetic mechanism is based on a spatial separation of CO2 assimilating enzymes. The assimilation of two mineral nutrients, nitrogen and sulfur, is also localized in a cell-specific manner in most C4 species. N assimilation seems to be confined to mesophyll whereas sulfate reduction has been previously reported to be bundle sheath specific. The latter view has been challenged by finding an ubiquitous presence of enzymes of sulfate assimilation in the dicot C4 species of Flaveria. Although inter- and intracellular distribution of enzymes of N assimilation in C4 plants differ from C3 plants and C4 plants have a better N use efficiency, very little is known about the physiological consequences of this distribution. Analogically, no evolutionary advantage for the BSC localization of sulfate assimilation has been identified. On the other hand, the organization and general regulation of the pathways is the same in C3 and C4 plants. In this chapter the two essential pathways of plant primary metabolism, nitrate and sulfate assimilation, as well as the synthesis of glutathione, the major sulfur containing metabolite involved in stress defense, will be described. The general regulation of the pathways as well as specific features connected with C4 photosynthesis will be discussed. The major open questions of N and S metabolism in C4 plants will be addressed.

*Author for Correspondence, e-mail: [email protected] Agepati S. Raghavendra and Rowan F. Sage (eds.), C4 Photosynthesis and Related CO2 Concentrating Mechanisms, pp. 109–128. © Springer Science+Business Media B.V. 2011

109

110

I. Introduction A characteristic feature of C4 plants is a cellspecific localization of many enzymes of primary metabolism in bundle sheath cells (BSC) or mesophyll cells (MC) (for details see Chapter 12, this volume). Clearly, the enzymes involved in the primary CO2 fixation and malate and/or aspartate synthesis, such as cytosolic carbonic anhydrase, phosphoenolpyruvate carboxylase, pyruvate phosphate dikinase, and NADP-malate dehydrogenase, are localized predominantly in the MC, whereas NAD(P)-malic enzyme, Rubisco, Rubisco activase, and some enzymes of the Calvin cycle are found exclusively in BSC (reviewed in Sheen, 1999; Edwards et al., 2001). In most C4 species analyzed, including maize and Sorghum, BSC chloroplasts lack photosystem II and therefore exhibit very little oxygen evolution (Hatch and Osmond, 1976). Consequently, noncyclic electron flow and the capacity for NADPH formation are restricted in BSC chloroplasts. In addition, glycine decarboxylase, a key enzyme of photorespiration, is localized exclusively in BSC of C4 and C3-C4 intermediate plants (Hylton et al., 1988; see Chapter 6, this volume). The C3–C4 intermediate plants were originally identified by having a CO2 compensation point intermediate between C3 and C4 species. They can be considered as evolutionary intermediates in the path from C3 to C4 photosynthesis (Monson and Moore, 1989; Kopriva et al., 1996). The intermediate species possess a Kranzlike anatomy and their apparent photorespiration rate is reduced, due to the confinement of glycine decarboxylase to the BSC and efficient refixation of photorespired CO2 in these cells (Hylton et al., 1988; Rawsthorne, 1992; cf. Chapter 6, this volume). Some C3–C4 plants are to some extent able to fix CO2 into malate and aspartate as C4 species (Bassüner et al., 1984; Monson et al., 1986), but

Abbreviations: APR – Adenosine 5¢-phosphosulfate reductase; APS – Adenosine 5¢-phosphosulfate; ATPS – ATP sulfurylase; BSC – Bundle sheath cells; GECg-Glutamylcysteine; GECSg-Glutamylcysteine synthetase; GOGAT – Glutamate synthase; GR – Glutathione reductase; GS – Glutamine synthetase; GSH – Glutathione; GSHS – Glutathione synthetase; GSSG – Oxidized glutathione, glutathione disulfide; MC – Mesophyll cells; NR – Nitrate reductase; OAS – OAcetylserine; ROS – Reactive oxygen species;

Stanislav Kopriva the compartmentalization of the photosynthetic enzymes is not complete (Bauwe, 1984). Interestingly, apart from enzymes involved in the C4 carbon cycle enzymes participating in the assimilation of nitrogen and sulfur are also localized in cell-specific manner in C4 plants. In this chapter, the two pathways will be described in detail with the focus on their subcellular distribution and the consequence of such distribution for the regulation of the pathways and for the performance of C4 plants. II. Nitrogen Assimilation Nitrogen (N) is the most abundant mineral nutrient in plant tissues but also the element most frequently limiting plants growth (Vance, 2001). The major N sources are inorganic nitrate and ammonium, however, plants developed several other strategies to meet their demand for N. The best known alternative source of N for plant nutrition are the symbiotic N2 fixing root nodules (Day et al., 2001) Also mycorrhizal fungi have been shown to contribute to plant N acquisition (Chalot and Brun, 1998), as well as N2 fixing bacteria in the phyllosphere (Papen et al., 2002). In addition, uptake of organic N compounds, such as amino acids, may cover substantial part of N acquisition in certain habitats (Persson et al., 2003). C4 plants are capable of mycorrhiza symbiosis; however, no C4 species form nodules with symbiotic bacteria. A. Plant Nitrate Assimilation The most common form of N plants acquire from the soil is nitrate. Nitrate is transported into plant cells by nitrate transporters (Fig. 1). Three uptake systems are responsible for uptake of nitrate into the roots: a constitutive high affinity uptake system, an inducible high affinity system an a low affinity system (Miller et al., 2007). However, further transporters are necessary to facilitate xylem loading and distribution of nitrate throughout the plant as well as its storage in the vacuoles. Two major classes of nitrate transporters exist in plants, the NRT1/PRT family responsible for low affinity nitrate uptake as well as amino acid and peptide transport, which contains 53 genes in Arabidopsis, and NRT2 family of high affinity

7

Nitrogen and Sulfur in C4 Plants

Fig. 1. Schematic representation of plant nitrate assimilation. Dark shaded rectangle represents mitochondria, light shaded one denotes plastid. Enzymes are symbolized by numbers: 1 – nitrate transporter, 2 – ammonium transporter, 3 – nitrate reductase, 4 – nitrite transporter, 5 – nitrite reductase, 6 – glutamine synthetase, 7 – glutamate synthase, 8 – plastidic glutamate–malate translocator, 9 – plastidic 2-oxoglutarate– malate translocator, 10 – mitochondrial glutamate–glutamine translocator. The major pathway of nitrate assimilation is printed bold.

nitrate transporters composed of seven members in this species. Genes of both classes have been found throughout the plant kingdom, including C4 plants (Santi et al., 2003). Nitrate is reduced to ammonium in two spatially separated steps. In the cytosol, nitrate reductase (NR) transfers electrons from NADH to nitrate to form nitrite. Nitrite is transported into plastids and reduced to ammonium by ferredoxin dependent nitrite reductase. Ammonium is assimilated into organic compounds by glutamine synthetase (GS) which uses glutamate as the ammonium acceptor. GS is coupled with glutamate synthase (GOGAT) which transfers the amino group of glutamine to 2-oxoglutarate to form two molecules of glutamate, in a GS/GOGAT cycle. The cycle thus uses one molecule of 2-oxoglutarate to assimilate one NH3 molecule and export one molecule of the

111

amino acid glutamate (Fig. 1, Miflin and Habash, 2002; Stitt et al., 2002; Weber and Flugge, 2002). Glutamate is the source of reduced N for the synthesis of other amino acids, with the first step in this route being usually transamination with oxaloacetate to form aspartate catalyzed by aspartate aminotransferase. Ammonium in plants originates not only from nitrate reduction. It is present as a nutrient in the soil and can be transported into plants by ammonium transporters (von Wiren et al., 2000). In fact, ammonium is the preferred source of inorganic N for many plant species and optimal growth is often achieved only when both nitrate and ammonium are present (Bloom et al., 1993). Ammonium is also produced by photorespiration, in the mitochondrial glycine decarboxylation reaction (cf. Chapter 6, this volume; Linka and Weber, 2005). An efficient assimilation and re-assimilation of ammonium thus must occur in all compartments to prevent its accumulation to toxic levels. Indeed, plastidic and cytosolic isoforms of GS exist in all plant species (Inokuchi et al., 2002) and the plastidic GS is dual targeted also to mitochondria (Taira et al., 2004). GOGAT is present in plants in multiple forms as well, the major ferredoxin dependent enzyme is found predominantly in leaves, whereas in non-photosynthetic cells NADH–GOGAT is the prevalent isoform. Both forms of GOGAT are, however, localized to plastids (Tobin and Yamaya, 2001). Another enzyme, the glutamate dehydrogenase, which in vitro synthesizes glutamate from ammonium and 2-oxoglutarate, has been long implicated to participate in ammonium assimilation. Recent findings however suggest that the major function of this enzyme is the provision of carbohydrate skeletons for carbon metabolism from amino acids during protein degradation (Miflin and Habash, 2002). B. Regulation of Nitrate Assimilation Nitrate and ammonium assimilation is strongly regulated by the N demand of the plant and N supply and is closely connected to carbon metabolism. The components of the pathway undergo a coordinated regulation which often occurs on multiple levels. So, nitrate uptake is induced in the presence of nitrate and feedback inhibited by amino acids and ammonium. It is regulated by

112

light and CO2 concentration via the availability of carbohydrates (Rufty et al., 1989; Stitt et al., 2002). Accordingly, nitrate uptake was demonstrated to be under diurnal regulation with maximum activity during day and minimum activity at night (Lejay et al., 1999). The decrease of uptake at night can be reversed by feeding sucrose. The coordination between N demand and N supply in control of nitrate uptake was proposed to be achieved by internal cycling of amino acids and/ or cytokinins between the roots and the shoots (Gessler et al., 2004). Ammonium transport seems to be regulated in a similar manner to nitrate transport: it undergoes a day night rhythm and the reduction of uptake in night can be prevented by sucrose treatment and is inhibited by glutamine (Lejay et al., 2003; Loque and von Wiren, 2004). However, in contrast to nitrate transport ammonium uptake seems to be regulated primarily by local and not by systemic signals (Loque and von Wiren, 2004). NR is a well studied enzyme which undergoes a complex regulation. NR expression is controlled by circadian clock and it is also induced by light and by sugars in the dark (Cheng et al., 1992; Campbell, 1999). The level of NR transcript is modulated by CO2 availability; it decreases when CO2 assimilation is diminished, e.g. due to water stress (Foyer et al., 1998) and increases upon exposure of plants to elevated CO2 (Fonseca et al., 1997). The rapid response of NR activity to environmental stimuli is, however, caused mostly by a rapid post-translational regulation. Upon sudden decrease in photosynthesis NR is rapidly and reversibly phosphorylated (Kaiser and Huber, 2001). The phosphorylation of NR enables its binding to 14-3-3 proteins, which seems to be the actual mechanism of inactivation and might be a signal for NR degradation (Lillo et al., 2004). The rapid response seems to be necessary to prevent accumulation of toxic levels of nitrite. NR is present in leaves and roots with different distribution of the activity among these organs in different plant species. Even a relatively low activity, however, is important for correct C/N balance as revealed by experiments with tobacco lacking root NR (Kruse et al., 2002). A central role in controlling nitrate and ammonium assimilation is occupied by GS. In contrast to NR and nitrite reductase, which are usually encoded by one to two genes, GS and GOGAT

Stanislav Kopriva are encoded by small multigene families of three to six members. This accounts for a great versatility of regulation with some genes expressed in tissue specific manner and at certain developmental stages while some being universal (Miflin and Habash, 2002). The organel-targeted GS2 is especially important for reassimilation of the photorespiratory NH3 and is, therefore, highly expressed in green tissues and inducible by light (Oliveira and Coruzzi, 1999). Cytosolic GS1 often fulfils specialized roles, such as in nodules to assimilate the ammonium produced by nitrogen fixation in the bacteroids (Stanford et al., 1993) or in tissues involved in transport of reduced N (Miflin and Habash, 2002). GS is regulated transcriptionally but undergoes also post-transcriptional and post-translational regulation. Both GS forms are subjected to phosphorylation and bind 14-3-3 proteins. Cytosolic GS is increased during senescence to facilitate the remobilisation of N (Habash et al., 2001). GS is also often associated with improved yield of crop plants by quantitative genetics (Hirel et al., 2001). Indeed, overexpression of pine cytosolic GS in poplar resulted in significantly enhanced growth (Gallardo et al., 1999) whereas disruptions of specific GS1 genes in maize led to reduced kernel size and/or number (Martin et al., 2006). Despite the importance of N assimilation surprisingly little is known about the molecular mechanisms of the regulation of N acquisition and metabolism. Nitrate transporter NRT1;1 seems to play an important role in N sensing (Remans et al., 2006). Nitrate has clearly a role as a signal since microarray analysis of Arabidopsis mutants in nitrate reductase revealed that expression of 595 genes including several transcription factors responded to nitrate treatment (Wang et al., 2004). Different amino acids act as feedback inhibitors of N assimilation, whereas glutamine seems to be most important in regulation of nitrate and ammonium uptake, glutamate, cysteine, and asparagine inhibit nitrate reduction (Stitt et al., 2002; Gessler et al., 2004). Sugars are clearly involved in the regulation of N assimilation, both as inductors and repressors, when their content is low, however the mechanisms of their action are not understood (Stitt et al., 2002). A special role in regulation of N assimilation, specifically in signalling N-status of the plant, is played by the phytohormones cytokinins. Cytokinins are produced in the root

7

Nitrogen and Sulfur in C4 Plants

at sufficient N supply and after transport into the shoots induce expression of genes of N assimilation (Wagner and Beck, 1993; Mok and Mok, 2001). They are, however, also transported from the shoot to the root and affect nitrate uptake (Collier et al., 2003). C. Nitrate Assimilation in C4 Plants Most of our knowledge on regulation of N metabolism has been derived from C3 plants and no differences in organization and regulation of nitrate assimilation between C3 and C4 plants were described. However, C4 plants differ significantly in the compartmentalization of N metabolism and also in N use efficiency. It has long been known that in terms of growth rate C4 grasses respond better to applied N than C3 grasses (Hallock et al., 1965). The C4 species clearly exhibited a higher N use efficiency, expressed as biomass per unit N in plant (Brown, 1978). The greater N use efficiency seems to be connected with a lower content of Rubisco in the leaves due to the CO2 concentrating mechanism. The N use efficiency in C4 plants will be discussed in detail by Ghannoum et al. in Chapter 8 of this volume. The intracellular localization of N assimilation in maize follows the pattern of C3 plants, i.e. cytosolic localization of NR and the presence of nitrite reductase in chloroplast (Ritenour et al., 1966). Because of the observed differences in distribution of carbon metabolism in C4 plants and in structures of MS and BSC chloroplasts the question of intercellular localization of nitrate assimilation has frequently been addressed in the early years of C4 photosynthesis research. By enzyme activity assays Mellor and Tregunna (1971) found that NR is prevalently present in MC of three C4 species with different types of BSC chloroplasts: maize, Sorghum sudanense, and Gomphrena globosa. NADH dependent glutamate dehydrogenase, which at that time was believed to be the only ammonium assimilating enzyme, was localized in BSC. Nitrite reductase activity has been found in MC of maize and S. sudanense, but in BSC of G. globosa which seems to imply that it is associated with the presence of chloroplasts with grana (Mellor and Tregunna, 1971). Also in Eleusine coracana NR and nitrite reductase occurred predominantly in MC (Rathnam and Das, 1974). In a more systematic approach

113

analysing five C4 species of all three metabolic subtypes, i.e. NADP-malic enzyme, NAD-malic enzyme, and phoshoenolpyruvate carboxykinase, NR, nitrite reductase, GS, and GOGAT were coordinately localized in MC, except Panicum maximum, where nitrite reductase was present both in MC and BSC (Rathnam and Edwards, 1976). On the other hand, in another study with maize, GS and GOGAT have been found in both cell types but predominantly in BSC (Harel et al., 1977). Similarly, in Digitaria sanguinalis NR and nitrite reductase were found exclusively in MC, GS was equally distributed between MC and BSC, and GOGAT was predominantly localized to BSC (Moore and Black, 1979). Since all the localization studies were based on distribution of enzyme activities in differentially homogenized leaves, it was difficult to conclude on exclusivity of the intercellular distribution of the N assimilation pathway and its components. Importantly, therefore, a clear evidence for an exclusive localization of maize NR in the cytosol of MC was obtained by immunogold labelling (Vaughn and Campbell, 1988). Similarly, the ambiguities in distribution of ammonium assimilation were clarified by Becker et al. (1993) who showed that both cytosolic and plastidic GS are present in both cell types, whereas ferredoxin dependent GOGAT is almost completely confined to BSC chloroplasts. An interesting feature of C4 plants is a relatively high cytosolic GS activity. Whereas in most C3 species cytosolic GS1 accounts for less than 30% of total leaf GS activity, in most C4 plants analyzed to date GS1 activity is higher or equal to the plastidic GS2 (McNally et al., 1983). Analyzing Panicum species of different type of photosynthesis Hirel et al. (1983) showed that the presence of GS1 activity in leaf correlated with C4 photosynthesis, with the C3–C4 intermediate P. milliodes possessing foliar GS1 activity intermediate between the C3 and C4 species. The ratio in accumulation of the two GS isoforms also differs in between the two cell types of C4 plants. GS1 seems to be more abundant in MC, whereas both isoform are present at the same level in BSC (Becker et al., 2000). Clearly, due to substantially reduced and BSC confined photorespiration much less ammonia is produced in C4 leaves than in C3 leaves and therefore the demand for its rapid refixation by GS is lower. The MC localization of

114

nitrate reduction implies an increased need for transport of reduced N between MC and BSC, which might explain the need for higher cytosolic GS. The evolutionary advantage of the spatial distribution of nitrate assimilation in C4 plants is, however, not known. Interestingly, assimilation of another essential mineral nutrient, sulfate, is also distributed in cell-specific manner in C4 plant species. III. Sulfate Assimilation Sulfur is essential for all living organisms as a constituent of the amino acids cysteine and methionine, many coenzymes such as iron sulfur centers, thiamine, lipoic acid, etc., and various other compounds of primary and secondary metabolism. In most of these compounds sulfur is present in the reduced (−2) form of organic sulfide. The most common form of sulfur in nature is, however, oxidized as inorganic sulfate (+6). Plants, algae, and many microorganisms are able to directly utilize sulfate, reduce it and incorporate into bioorganic molecules, which are the form of sulfur accessible to animals and other organisms. The pathway of sulfate assimilation is thus an essential component of plant primary metabolism.

Stanislav Kopriva acetylation of serine by serine acetyltransferase, to form cysteine in a reaction catalyzed by O-acetylserine (thiol)lyase (Fig. 2; Leustek et al., 2000; Kopriva, 2006). Cysteine is the source of reduced sulfur for synthesis of methionine, iron sulfur clusters, and other compounds. Sulfate assimilation is confined to plastids, however, some reactions occur also in other compartments. The cysteine synthesis, e.g., proceeds in all three compartments capable of protein synthesis, i.e. plastids, cytosol, and mitochondria (Wirtz et al., 2004). ATPS activity was detected both in plastids and in the cytosol (Lunn et al., 1990; Rotte and Leustek, 2000). On the other hand the enzymes involved in reductive steps of the pathway, APR and sulfite reductase, are localized exclusively in plastids (Brunold and Suter, 1989; Prior et al., 1999; Koprivova et al., 2001). Total foliar ATPS and APR activity decrease with

A. Plant Sulfate Assimilation The pathway of sulfate assimilation in plants has been completely resolved only relatively recently (Suter et al., 2000) and has lately been subjected to several comprehensive reviews (Leustek et al., 2000; Saito, 2004; Rausch and Wachter, 2005; Kopriva, 2006). Sulfate uptake into plant cells is facilitated by sulfate transporters (Fig. 2). Also sulfate transporters form a large multigene family with 12–15 members differing in affinity to sulfate and tissue distribution (Buchner et al., 2004). Because it is a very stable and inert compound, before reduction sulfate has to be activated to adenosine 5¢-phosphosulfate (APS) by adenylation catalyzed by ATP sulfurylase (ATPS). APS is reduced to sulfite by APS reductase (APR). Sulfite is further reduced to sulfide by ferredoxin dependent sulfite reductase. Sulfide is than incorporated into the amino acid skeleton of O-acetylserine (OAS), which is synthesized by

Fig. 2. Schematic representation of plant sulfate assimilation. Dark shaded rectangle represents mitochondria, light shaded one denotes plastid. Enzymes are symbolized by numbers: 1 – sulfate transporter, 2 – ATP sulfurylase, 3 – APS reductase, 4 – sulfite reductase, 5 – serine acetyltransferase, 6 – O-acetylserine (thiol)lyase, 7 – g-glutamylcysteine synthetase, 8 – glutathione synthetase, 9 – APS kinase, 10 – sulfotransferase. The major pathway of sulfate assimilation and glutathione synthesis (in Arabidopsis) is printed bold.

7

Nitrogen and Sulfur in C4 Plants

the leaf age in Arabidopsis (von Arb and Brunold, 1986; Rotte and Leustek, 2000), however, the cytosolic and plastidic ATPS are regulated differently. Whereas the plastidic activity declines with time, the cytosolic is increasing with leaf age. This indicates different roles of ATPS in the two compartments: a provision of APS for sulfate reduction for biosynthetic processes required for growth in plastids and involvement in synthesis of secondary compounds in the cytosol (Rotte and Leustek, 2000). The reduction of sulfate occurs predominantly in leaves, and reduced sulfur compounds are distributed to sink tissues via phloem (Herschbach and Rennenberg, 2001). It appears, however, that most tissues are capable of sulfate reduction, including roots and developing seeds (Brunold and Suter, 1989; Tabe and Droux, 2002). Indeed, available microarray data in the Genevestigator database reveals the presence of mRNA for the genes of sulfate assimilation, such as APR or sulfite reductase, in all Arabidopsis organs, including flowers and siliques (Zimmermann et al., 2004). These findings were corroborated using promoter::GUS fusions which clearly showed activity of APR and ATPS promoters in all tissues of Arabidopsis (A Koprivova, C Matthewman, S Kopriva, 2008, unpublished). However, whether the sulfate reduction rate in these cells is sufficient to cover their needs for reduced sulfur instead of relying on long distance transport of organic sulfur compounds, such as glutathione or S-methylmethionine, remains to be seen (Herschbach and Rennenberg, 2001). However, there is a group of plants that lacks the ability to reduce sulfate in a great portion of their cells, the monocot C4 plants (reviewed in Kopriva and Koprivova, 2005). B. Regulation of Sulfate Assimilation The sulfate assimilation pathway is extensively regulated in a demand-driven manner to prevent accumulation of toxic intermediate and to provide optimal rate of cysteine production (Lappartient and Touraine, 1996; Leustek et al., 2000; Kopriva, 2006). Thus, when demand for reduced sulfur is increased due to enhanced protein synthesis or increased turnover of the sulfur containing tripeptide glutathione (see below) the flux through the pathway is increased (Lappartient and Touraine, 1996). On the other hand, when plants are exposed

115

to reduced sulfur in the atmosphere or rhizosphere or when there is not sufficient supply of the amino acid acceptor due to carbon or nitrogen deficiency, the pathway is downregulated (Koprivova et al., 2000; Westerman et al., 2001; Kopriva et al., 2002; Vauclare et al., 2002). Although regulation of all components of the sulfate assimilation has been described, control flux analysis revealed that APR and sulfate uptake possess the highest control over the pathway (Vauclare et al., 2002). In line with these observations, APR activity and mRNA accumulation undergoes a diurnal rhythm with a maximum during light and minimum at night (Kocsy et al., 1997; Kopriva et al., 1999). The high activity coincides with the highest flux through the pathway (Kopriva et al., 1999). Despite a comprehensive knowledge on the physiological responses of sulfate assimilation to various environmental stimuli, very little is known about the molecular mechanisms of regulation and the signals involved. Several compounds have been proposed as molecular signals in regulation of the pathway. OAS accumulates during sulfur deficiency and when supplied exogenously induces mRNA accumulation of many genes of sulfate uptake and assimilation (Neuenschwander et al., 1991; Koprivova et al., 2000; Hopkins et al., 2005). Indeed, system biology approaches revealed a correlation of OAS content with transcript accumulation of many genes during a sulfur starvation response and large set of genes regulated in the same way by sulfate starvation and OAS treatment (Hirai et al., 2003, 2005). However, not all genes induced by sulfur deficiency are regulated by OAS and the timing of OAS accumulation seems to fall behind the induction of sulfate uptake by sulfur starvation in potato, so that OAS is probably not the only signal in the sulfur starvation response (Hirai et al., 2003, 2005; Hopkins et al., 2005). Glutathione (GSH) may also represent the signal of sulfur status of the plant, as depletion of GSH by treatment with an inhibitor of its synthesis, buthionine sulfoximine (BSO), leads to upregulation of APR, whereas GSH itself inhibits sulfate uptake and reduction (Lappartient and Touraine, 1996; Vauclare et al., 2002; Hartmann et al., 2004). ATPS and APR activity are reduced also in plants treated with cysteine, however, the feedback inhibition is alleviated when simultaneously BSO blocks the synthesis of GSH from the additional cysteine

116

(Lappartient and Touraine, 1996; Vauclare et al., 2002). In addition, several phytohormones have been shown to modulate expression or activity of various components of sulfate assimilation, such as jasmonate (Harada et al., 2000; Jost et al., 2005), cytokinins (Ohkama et al., 2002), or abscisic acid (Barroso et al., 1999). Only very recently, however, the first transcription factor and cis element responsible for regulating genes for sulfate transporter by sulfur starvation have been identified (Maruyama-Nakashita et al., 2005, 2006). C. Sulfate Assimilation in C4 Plants In a search for further metabolic processes spatially distributed in C4 plants it was soon discovered that in maize 75–100% of total leaf ATPS activity is confined to BSC (Gerwick and Black, 1979; Passera and Ghisi, 1982; Burnell, 1984; Schmutz and Brunold, 1984). These findings were extended to 17 other C4 species of all three C4 subtypes, where 95–100% of total leaf ATPS was localized in BSC chloroplasts (Gerwick et al., 1980). Also APR was found almost exclusively in BSC of maize (Schmutz and Brunold, 1984; Burgener et al., 1998), while the activities of sulfite reductase and OAS(thiol)lyase could be measured at comparable levels in MC and BSC (Passera and Ghisi, 1982; Burnell, 1984; Schmutz and Brunold, 1985). In attempts to decipher the mechanism of the spatial distribution of these enzymes, northern analysis of BSC and MC specific RNA revealed that mRNA levels for ATPS, APR, and sulfite reductase were detected in BSC only, whereas the mRNA for OAS(thiol) lyase was found in both MC and BSC (Kopriva et al., 2001). The cell-specific localization of enzymes of sulfate assimilation in maize seems, therefore, to be regulated on the transcriptional level, at least under standard growth conditions. However, the situation might be different under stress. Maize is especially sensitive to chilling which induces a strong oxidative stress characterized by production of reactive oxygen species (ROS). In maize plants subjected to chilling APR activity and mRNA level were greatly increased in BSC, and mRNA but not enzyme activity was also detectable in MC. This indicates an additional post-transcriptional mechanism to ensure the BSC specific localization of sulfate assimilation in maize (Kopriva et al., 2001).

Stanislav Kopriva The exclusive localization of ATPS and APR in BSC of maize means that an efficient transport of reduced sulfur compounds from BSC to MC must exist. MC are capable of cysteine synthesis, therefore, the transport form of reduced sulfur could be sulfide, cysteine, methionine or glutathione. Feeding of isolated bundle sheath strands from maize with [35S]sulfate resulted in secretion of labelled cysteine into the nutrient solution (Burgener et al., 1998). It seems therefore, that cysteine (or its oxidized form cystine) is the most probable transport metabolite for reduced sulfur (Fig. 2). Interestingly, [35S]sulfate feeding experiments also imply that glutathione synthesis was predominantly localized in MC (Burgener et al., 1998; and see below). The biological significance of the BSC specific localization of sulfate assimilation in C4 plants is not obvious and, similarly, it is not clear whether this localization is a pre-requisite or a consequence of C4 photosynthesis. To answer this question we addressed the distribution of ATPS and APR in Flaveria species with different types of photosynthesis (Koprivova et al., 2001). The dicot genus Flaveria (Flaveriinae–Asteraceae) is an excellent model to study the evolution of C4 photosynthesis because, beside C3 and C4 species, it comprises a relatively large number of C3–C4 intermediates (Ku et al., 1991). A continuous gradation in the physiology and biochemistry of C4 photosynthesis can be found among Flaveria species (Monson and Moore, 1989). Indeed, we showed previously that the C3–C4 intermediate Flaveria species are true evolutionary intermediates in the path from C3 to C4 photosynthesis, based on phylogenetic analysis of the H-protein subunit of glycine decarboxylase (Kopriva et al., 1996). The aim of the study with Flaveria was to compare the intercellular distribution of sulfate assimilation in C3, C3–C4, and C4 species. We expected a BSC localization of APR and ATPS in C4 Flaveria species and a ubiquitous distribution in C3 species. The distribution of the two enzymes in the intermediate ones would be an excellent indication for the evolutionary sequence of the processes leading to the BSC localization of sulfate assimilation. Surprisingly however, northern analysis of cell-specific RNA and in situ hybridization revealed that in the C4 species F. trinervia mRNA for ATPS and APR were present at comparable levels in both MC and BSC. Immunogold

7

Nitrogen and Sulfur in C4 Plants

electron microscopy confirmed the presence of APR protein in chloroplasts of both cell types (Koprivova et al., 2001). Consequently, the localization of assimilatory sulfate reduction in BSC cannot be a general C4 trait. How can these findings be explained taking into account the results of Gerwick et al. (1980) who showed the exclusive BSC localization of ATPS in 17 C4 species? Whereas the 17 species analyzed before were monocots, F. trinervia and F. australasica were the first C4 dicots where the subcellular localization of the pathway was addressed. It has to be concluded that sulfate assimilation is exclusively localized in BSC only in C4 monocots and this distribution is thus neither a pre-requisite nor a consequence of C4 photosynthesis (Koprivova et al., 2001; Kopriva and Koprivova, 2005). However, the BSC localization of ATPS in maize and other C4 plants analyzed by Gerwick et al. (1980) is not a general trait of monocots either, since in wheat, a C3 monocot, ATPS and APR are present in all cell types (Schmutz and Brunold, 1984). Whether C4 Flaveria species are exceptions or the rule for C4 dicots remains to be established, however, it is evident that the previously generally accepted link between C4 photosynthesis and BSC localization of sulfate assimilation is no longer valid. The investigations of sulfate assimilation in Flaveria resulted in an additional interesting finding. The APR activity and levels of thiols were significantly higher in leaves of C4-like and C4 species than in those of C3 and C3–C4 species (Koprivova et al., 2001). APR, cysteine and GSH content correlated with the degree of development of C4 photosynthesis expressed by CO2 compensation points (Kopriva and Koprivova, 2005). The actual foliar concentration of GSH is highly dependent on environmental conditions and, therefore, varies significantly among different plant species but also within single species. Since the Flaveria species were grown at identical controlled conditions, it seems that the clear tendency to towards higher APR activity and GSH content with increasing C4 photosynthesis might be a result of the adaptation to different habitats. C4 photosynthesis is especially advantageous in dry and warm conditions. The higher GSH contents in C4 Flaveria might thus be a mechanism to cope with increased oxidative stress caused by such environmental conditions. According to the

117

demand-driven regulation, APR activity would have to be elevated to supply sufficient cysteine for the GSH synthesis. IV. Glutathione Synthesis and Reduction Among sulfur containing metabolites the tripeptide glutathione (g-glutamyl-cysteinyl-glycine) plays the most versatile role being essential for plant stress defense, redox regulation and signaling, control of cell cycle, and as a storage and transport form of reduced sulfur (May et al., 1998a; Noctor et al., 1998a; Foyer and Rennenberg, 2000). GSH is involved in plant defense against ROS as a reductant of dehydroascorbate in the glutathione–ascorbate cycle (Noctor and Foyer, 1998). It is indispensable for protection against heavy metals as a substrate for synthesis of phytochelatins, which chelate the metals and enable their sequestration into the vacuoles (Cobbett and Goldsbrough, 2002). Conjugation of xenobiotics with GSH is the first step in their detoxification and a molecular basis of resistance to some herbicides (Dixon et al., 1998). Not surprisingly, GSH accumulates to high levels reaching concentration of several mM (Meyer and Fricker, 2000; Hartmann et al., 2003). Due to its role in stress defense and specifically in the response to chilling, GSH synthesis and its regulation was often addressed in C4 plants (Ruegsegger and Brunold, 1993; Doulis et al., 1997; Kocsy et al., 2001; Gómez et al., 2004; Kopriva and Koprivova, 2005). A. Regulation of GSH Synthesis GSH is synthesized from its constituent amino acids in two ATP dependent steps. Firstly a g-glutamylcysteine synthetase (g-ECS) synthesizes g-glutamylcysteine (g-EC) from glutamate and cysteine. Subsequently, glycine is added to the g-EC by glutathione synthetase (GSHS). GSH synthesis is an essential process, since disruption of gECS gene by T-DNA insertion is embryolethal (Cairns et al., 2006). The rate of GSH synthesis is primarily controlled by the availability of the constituent amino acids, with the regulation of g-ECS playing an additional substantial role (Kopriva and Rennenberg, 2004). Most of our knowledge on this regulation is derived from

118

studies with poplar overexpressing bacterial genes for g-ECS and GSHS (e.g. Strohm et al., 1995; Noctor et al., 1996, 1998b). g-ECS and GSHS are induced at conditions of high demand for GSH, such as after exposure to heavy metals or herbicide safeners (Ruegsegger and Brunold, 1992; Farago and Brunold, 1994; Schaefer et al., 1998). g-ECS is more important for the control of GSH synthesis because GSH content was increased only in poplars overexpressing g-ECS and not GSHS (Strohm et al., 1995). The enzyme seems to undergo a complex regulation on different levels. Heavy metals induce mRNA accumulation of g-ECS (and GSHS) (Schaefer et al., 1998; Xiang and Oliver, 1998), but a post-transcriptional regulation has also been described (May et al., 1998b). On the other hand, the enzyme is feedback inhibited by GSH, which causes a reversible conformational change due to a reduction of an internal disulfide bond (Jez et al., 2004; Hothorn et al., 2006). The g-ECS regulation thus seems to follow the same demand-driven manner as described for the components of sulfate assimilation. Indeed, since at most physiological situations cysteine limits GSH synthesis gECS is often regulated in the same way as APR or other enzymes of sulfate assimilation (Ruegsegger et al., 1990; Brunner et al., 1995). At some conditions however, e.g. during night or at non-photorespiratory conditions, synthesis of GSH is controlled by availability of glycine (Noctor et al., 1999). During its function in the glutathione–ascorbate cycle GSH is oxidized. The oxidized form of glutathione, GSSG, is reduced by the action of glutathione reductase (GR) which utilizes the electrons from NADPH. GR maintains the reducing environment in cells so that GSSG normally forms no more than 5% of total glutathione. Increased ratio of GSSG to total GSH is thus indicative of oxidative stress (Mullineaux and Rausch, 2005). B. Localization of GSH and GSH Synthesis GSH is present in all cellular compartments; however, the quantitative distribution is not resolved yet. Currently, two approaches are being used to quantify GSH on the subcellular level. The confocal laser scanning microscopy method makes use of the fluorescence of the GSH conjugate

Stanislav Kopriva with monochlorobimane (Meyer et al., 2001). However, since the conjugation is catalyzed enzymatically by GSH transferase, the method can directly measure only cytosolic GSH, whereas the organellar pools can be estimated by HPLC after cell disruption and additional chemical labeling of the remaining GSH by monobromobimane (Hartmann et al., 2003). The second method is based on immunohistochemistry with antibodies against GSH (Zechmann et al., 2005). This approach shows the highest density of labeling in mitochondria and the lowest in plastids. Unfortunately, none of the methods have been used for localization of GSH in C4 plants. GSH synthesis has been shown to take place in the cytosol and plastids (Hell and Bergmann, 1990; Noctor et al., 1998a). However, recently it was revealed that the two steps of GSH biosynthesis may be spatially separated, at least in some plant species. In Arabidopsis and Brassica juncea g-ECS seems to be exclusively localized in the plastids whereas GSHS is present in both plastids and cytosol (Wachter et al., 2005). The g-EC may thus not only be a precursor of GSH but also play important roles in transport of reduced sulfur from plastids to the cytosol and possibly in signaling of the redox status of the chloroplast. This conclusion is supported by the identification of the regulator of APX2 (rax1) mutant in Arabidopsis (Ball et al., 2004). This mutant constitutively expresses cytosolic ascorbate peroxidase, which is normally inducible by photooxidative stress, and contains only approximately 50% of normal foliar GSH levels. In rax1 thus the chloroplast– cytosol signaling is clearly disturbed. The rax1 phenotype is caused by an R(229)-K substitution in g-ECS protein (Ball et al., 2004). Several other Arabidopsis mutants are associated with g-ECS and low GSH levels, the cadmium sensitive cad2 (Cobbett et al., 1998), root meristemless rml1 (Vernoux et al., 2000), and phytoalexin-deficient pad2 (Parisy et al., 2007), but none of them displays the same effect on the chloroplast–cytosol signaling. On the other hand, g-ECS and GSHS were detected by immunolocalization in both chloroplasts and cytosol of maize (Gómez et al., 2004). Also, expression of the bacterial gene for g-ECS both in plastids and in the cytosol led to an increase in foliar GSH content in poplar (Noctor et al., 1996, 1998b). Interestingly, the increase

7

Nitrogen and Sulfur in C4 Plants

in GSH content in poplar plants overexpressing g-ECS in the cytosol seems to be confined to this compartment which implies a limited exchange of GSH between cytosol and plastids (Hartmann et al., 2003). GR is localized in cytosol, plastids, and mitochondria (Edwards et al., 1990). Interestingly, a single gene encodes both plastidic and mitochondrial isoform of GR due to a presence of a dual-specificity targeting peptide (Chew et al., 2003). GSH is present and can be synthesized in all plant organs. Obviously, in many occasions cell specific differences in GSH levels have been found. In maize roots, GSH levels form a clear gradient from the highest at root tip to the lowest in the mature portion of the root (Kopriva et al., 2001). When investigated at the cellular level by confocal laser scanning microscopy approximately twofold higher cytosolic GSH concentration was measured in atrichoblasts than in trichoblasts (Meyer and Fricker, 2000). In addition high GSH levels were detected in root cap while markedly lower GSH was found in quiescent centers (Sanchez-Fernandez et al., 1997). On the other hand, in poplar leaves analyzed by the same approach surprisingly uniform concentration of cytosolic GSH has been detected (Hartmann et al., 2003). Also in leaves, however, certain cell types distinctly differ, as very high GSH concentration has been found in leaf trichoms (GutierrezAlcala et al., 2000). Consequently, gECS, GSHS, and genes for enzymes of cysteine synthesis were highly expressed in these cells. These cell specific differences in GSH levels are probably caused by differential rates of GSH synthesis and indicate that GSH transport between cells may not be efficient enough to balance such differences. C. GSH Synthesis in C4 Plants Due to its role in detoxification of ROS GSH is particularly important in low temperature sensitive C4 plants, such as maize, since chilling induces oxidative stress via the photochemical production of H2O2. Consequently, at low temperatures GSH content and reduction state are higher in chilling tolerant genotypes of tomato, Sorghum, wheat, and maize than in the sensitive ones (Walker and McKersie, 1993; Kocsy et al., 1996, 2000a; Badiani et al., 1993; see Chapter 10, this volume). Brunner et al. (1995) demonstrated that in maize chilling

119

caused the content of GSH to increase and, in accordance with the demand-driven model of regulation, also enzyme activities of APR, g-ECS, and GSHS. Similarly, at 12°C the activities of APR and GR and GSH content were higher in a chilling tolerant maize genotype than in a sensitive one (Kocsy et al., 1997). Accordingly, treatment with 1 mM BSO, which decreased GSH content to very low levels, resulted in reduction of fresh weight and in visible leaf injury of chilling-tolerant maize at 5°C but not at ambient temperature (Kocsy et al., 2000b). Addition of GSH or g-EC together with BSO protected the plants from the chilling injury by increasing GSH content and GR activity. Similarly, when GSH content in the chilling sensitive maize had been increased via treatment with herbicide safeners, the chilling induced injury was significantly reduced (Kocsy et al., 2001). A simultaneous addition of BSO counteracted the safener-induced protection. These experiments thus clearly showed that, at least in maize, sensitivity to chilling is a trait connected with GSH content and/or reduction state (see Chapter 10, this volume). Despite its essential function in stress defense GSH is not equally distributed between MC and BSC in maize. GSHS activity is greater in MC than in BSC resulting in a predominant GSH synthesis rate in the MC (Burgener et al., 1998) and higher GSH levels in this cell type (Doulis et al., 1997; Burgener et al., 1998; Kopriva et al., 2001). Surprisingly, GR was found exclusively in MC of maize (Doulis et al., 1997; Pastori et al., 2000). The MC specific localization of GR might be explained by a limited capacity for NADPH production in BSC. GR mRNA, however, was found in both cell types revealing involvement of a post-transcriptional regulation of its cell-specific distribution (Pastori et al., 2000). In contrast to GR, the enzymes of GSH synthesis were localized in both MC and BSC of maize by immunohistochemistry (Gómez et al., 2004). In line with previous findings foliar GSH content increased in cold treated plants, however, chilling caused induction of g-ECS mRNA in BSC but not in MC (Gómez et al., 2004). It seems therefore, that both cell types possess the capacity to synthesize GSH, but the enzymes in BSC are more affected by stress. The apparent contrast of these results with previous data (Doulis et al., 1997; Burgener et al., 1998) is likely to be caused by cell-type specific

120

post-translational modification of the enzymes resulting in changes in GSH synthesis rates and thus distribution of GSH. As g-ECS is redox regulated, variation in redox environment in MC and BSC can be responsible for such differences in activity. The preferential stress response in BSC might be explained by the fact that cysteine, the limiting factor in GSH synthesis, is synthesized only in BSC and can be used for GSH synthesis without the necessity for any transport steps (Burgener et al., 1998; Kopriva et al., 2001). On the other hand, the oxidized form of glutathione can probably be reduced only in MC (Pastori et al., 2000). Consequently, GSSG formed during the stress in BSC has to be transported to MC for reduction and thus the GSH pool in MC increases while the BSC pool becomes depleted. This is likely to result in increased demand for GSH synthesis in BSC but not in MC and in activation g-ECS and sulfate assimilation. V. Physiological Significance of the Distribution of Nitrate and Sulfate Assimilation Nitrate and sulfate assimilation are clearly localized in MC and BSC, respectively, in many C4 plants. For sulfate assimilation it is obvious that this localization is not linked to C4 photosynthesis, as C4 Flaveria species possess the pathway in both cell types. The physiological significance of this localization and evolutionary advantage is, however, not evident as yet. A. Open Questions on Nitrate Assimilation in C4 Plants The cell specific distribution of N assimilation in C4 plants is well established, however, many questions are still open. Obviously, our findings on sulfate assimilation in Flaveria (Koprivova et al., 2001) incite questioning the universality of results on N assimilation derived from maize and a few other C4 species. Although similarly to Gerwick et al. (1980) C4 species of all three types were analyzed for localization of NR and GS with the same result, no dicot species were included (Rathnam and Edwards, 1976; McNally et al., 1983). Only in the very first analysis by Mellor and Tregunna (1971) a C4 dicot (G. globosa) was

Stanislav Kopriva analyzed and, indeed, the results pointed to possible alternative distribution of the enzymes in this species compared, e.g. to maize. The findings have to be interpreted with caution, obviously, due to the methodology based on enzyme activities in MC and BSC specific extracts obtained by differential tissue extraction, which surely have been cross-contaminated. Nevertheless, unlike in other species analyzed, nitrite reductase activity in G. globosa was higher in BSC than in MC. In analogy with sulfate assimilation, this would imply that the MC distribution of nitrate assimilation may not be universal and not be connected with C4 photosynthetic mechanism. A second question concerns the increased N use efficiency of C4 plants. Reduced accumulation of Rubisco, the major sink for reduced N in C3 plants, is thought to be the major reason for better N use efficiency, as less N is required for the same or a better CO2 fixation rate (Brown, 1978). Whether the cell-specific distribution of NR and/ or the high content of cytosolic GS contribute to the improved N use efficiency is not clear. The physiological consequences of the alterations in localization of N assimilation are also not known. Major differences in regulation of N uptake and assimilation between C3 and C4 plants have not been reported. Clearly, more transport steps are necessary to provide all cells with sufficient reduced N and the MC with nitrate (Fig. 3). On the other hand, the MC localization of NR and nitrite reductase might prevent competition for reduction equivalents between nitrate and CO2 assimilation (Moore and Black, 1979). Alternatively, the low NADPH production capacity in BSC chloroplasts due to the lack of grana and photosystem II was cited as a reason for MC localization of NR (Mellor and Tregunna, 1971). This may be true for maize and some other NADP-malic enzyme-type C4 plants, but plants of the other types do possess photosystem II in BSC and still reduce nitrate in MC only (Rathnam and Edwards, 1976; Ketchner and Sayre, 1992). Most likely explanation takes into account that nitrite is an alternative acceptor of photosynthetic electrons and its reduction is coupled to oxygen evolution (Miflin, 1972). Localization of this reaction in MC chloroplast thus prevents O2 evolution in BSC, which would counteract the CO2 concentrating mechanism of C4 plants and support the oxygenase reaction of Rubisco (Moore and Black, 1979). Limitation

7

Nitrogen and Sulfur in C4 Plants

121

Fig. 3. Schematic representation of distribution of major steps in assimilation of carbon, nitrogen, and sulfur between mesophyll (MC) and bundle sheath (BSC) of maize. Transport steps of C compounds are marked by dotted arrows, dashed and full arrows symbolize transport of N and S compounds, respectively (Reprinted from Kopriva and Koprivova, 2005).

of NR to MC is essential to prevent accumulation of nitrite and its toxicity in BSC. This might be the biggest evolutionary advantage of the cellspecific distribution of N assimilation in C4 plants. This explanation would be strengthened if the MC localization of NR and nitrite reductase would be confirmed also for the dicot C4 species. B. Significance of BSC Localization of Sulfate Assimilation Although not necessarily linked with C4 photosynthetic mechanism, sulfate is reduced only in BSC of maize. A link with low rate of NADPH production as discussed for nitrate assimilation and GSH reduction (Mellor and Tregunna, 1971; Doulis et al., 1997) can be excluded since then the sulfate reduction would have to be localized in MC. Burgener et al. (1998) speculated that low concentration of oxygen in BSC would prevent oxidation of intermediates of sulfate assimilation, sulfite and sulfide, and thus increase the efficiency of the pathway. However, if such oxidation would indeed significantly reduce the rate of sulfate assimilation, the pathway would not be functional in chloroplasts of C3 plants. Consequently, sulfate would be reduced in mitochondria, what actually is the case in Euglena gracilis (Brunold and Schiff, 1976), or elaborate structures

would have evolved possibly including bacterial symbiosis, such as for N2 fixation in legumes. Another attractive explanation is the co-localization with photorespiration, namely with GDC, the major source of serine in plants since activated serine is the acceptor of sulfide and a direct precursor of cysteine. However, serine synthesized in BSC must be transported into MC for protein synthesis anyway and thus a shortage of this amino acid in MC seems unlikely. Moreover, in C4 Flaveria species GDC is BSC specific but sulfate assimilation is not (Hylton et al., 1988; Koprivova et al., 2001). In addition, the MC specific ferredoxin FdI (Matsumura et al., 1999) was an efficient electron donor for sulfite reductase (Yonekura-Sakakibara et al., 2000), so cell specific differences in electron flux can also be excluded. To find out the significance of the BSC specific distribution of sulfate assimilation, one should ask for the advantage maize may have gained this way. Unfortunately, there does not seem to be any obvious one. In contrast to nitrogen nutrition, a difference in sulfur use efficiency between C3 and C4 plants was not observed. As with other metabolic processes, maize probably invests less into synthesis of the proteins of sulfate assimilation pathway, but on the other hand it must possess an efficient transport system for cysteine and GSH. Maize does not require significantly more or

Stanislav Kopriva

122

less sulfate than other plant species and it is not known for especially good or poor sulfur use efficiency. It is not particularly resistant or sensitive to heavy metals, which trigger a high demand for reduced sulfur (Nussbaum et al., 1988). On the other hand, sulfate assimilation and GSH synthesis are associated with tolerance to chilling and detoxification of herbicides, so the BSC localization might even be a limitation for the capacity to provide sufficient GSH to cope with such stress. It seems, therefore, that the significance of compartmentalization of sulfate assimilation in maize will further remain an open question. C. Consequences of BSC Localization of Sulfate Assimilation To understand the cell-specific localization of sulfate assimilation one also has to ask about its consequences. Maize was a frequent subject of investigations of assimilatory sulfate reduction in the pre-Arabidopsis era. The results on regulation of sulfate assimilation obtained with maize fitted well to the general hypothesis of demand driven control (Lappartient and Touraine, 1996). Coordinate increase in mRNA levels for sulfate transporters, ATPS, and APR was observed in maize roots and leaves upon sulfate starvation (Bolchi et al., 1999; Hopkins et al., 2004) and the ATPS mRNA level was repressed in presence of reduced sulfur compounds (Bolchi et al., 1999). Accordingly, ATPS and APR activities were increased upon treatments of maize with cadmium or chilling (Brunner et al., 1995; Nussbaum et al., 1988; Ruegsegger and Brunold, 1992). In all these reports the regulation of sulfate assimilation in maize was not distinguishable from other plants, such as Lemna minor, poplar, potato, and Arabidopsis (Kopriva, 2006). Differences appeared, however, when the mechanisms of regulation were addressed (Bolchi et al., 1999). To identify the signal responsible for feedback inhibition of sulfate assimilation by thiols, plants can be treated with cysteine, glutathione, and cysteine together with BSO which prevents its conversion to GSH. Such analysis performed with Brassica napus, Arabidopsis, and poplar unambiguously identified GSH as the molecular regulator (Lappartient and Touraine, 1996; Vauclare et al., 2002; Hartmann et al. 2004) whereas in maize cysteine acted directly without the necessity of conversion

to GSH (Bolchi et al., 1999). The molecular mechanisms of this feedback inhibition are not known, but it is reasonable to expect that the responsible sensor/transcription factor in maize is specific for cysteine whereas the corresponding protein(s) in other plants are GSH specific. This variation might well be a consequence of the BSC localization of sulfate assimilation. As GSH can be synthesized both in MC and BSC (Gómez et al., 2004) but only cysteine is transported out of BSC protoplasts (Burgener et al., 1998) it is likely that the GSH pools in MC and BSC are not rapidly interchangeable. On the other hand, cysteine pools in the two cell types have to be linked to enable efficient protein and GSH synthesis in MC and rapid modulation of cysteine biosynthesis in BSC upon even subtle changes in demand for reduced sulfur in the whole leaf. Therefore, cysteine may be much better suited as a signal of the sulfur status at the site of sulfate assimilation than GSH. VI. Conclusions Nitrate and sulfate assimilation in C4 plants are another processes differentially distributed between MC and BSC. MC specific localization of nitrate assimilation has been demonstrated in only a few species and despite being generally accepted, it has to be proven that this indeed is a general C4 trait. This is even more imperative after it was revealed that the BSC association of sulfate assimilation is not a general feature of C4 photosynthesis but is probably limited to C4 monocots. For both pathways the evolutionary advantage of such compartmentalization is not understood and should be addressed in future studies. Given the high interest in improvements of nutrient use efficiency of crops the N metabolism in C4 plants should certainly be further explored to find whether the C4 specific alterations contribute to the improved N use efficiency of C4 plants. Clearly, there are still more open questions than answers in N and S metabolism of C4 plants. Acknowledgments Research in Stanislav Kopriva’s laboratory at John Innes Centre is supported by the Biotechnology and Biological Sciences Research Council (BBSRC).

7

Nitrogen and Sulfur in C4 Plants

References Badiani M, Paolacci AR, D’Annibale A and Sermanni GG (1993) Antioxidants and photosynthesis in the leaves of Triticum durum L. seedlings acclimated to low, non-chilling temperature. J Plant Physiol 142: 18–24 Ball L, Accotto GP, Bechtold U, Creissen G, Funck D, Jimenez A, Kular B, Leyland N, Mejia-Carranza J, Reynolds H, Karpinski S and Mullineaux PM (2004) Evidence for direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis. Plant Cell 16: 2446–2462 Barroso C, Romero LC, Cejudo FJ, Vega JM and Gotor C (1999) Salt-specific regulation of the cytosolic O-acetylserine(thiol)lyase gene from Arabidopsis thaliana is dependent on abscisic acid. Plant Mol Biol 40: 729–736 Bassüner B, Keerberg O, Bauwe H, Pyarnik T and Keerberg H (1984) Photosynthetic CO2 metabolism in C3-C4 intermediate and C4 species of Flaveria (Asteraceae). Biochem Physiol Pflanzen 179: 631–634 Bauwe H (1984) Photosynthetic enzyme activities and immunofluorescence studies on the localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of C3, C4, and C3-C4 intermediate species of Flaveria (Asteraceae). Biochem Phys Pflanzen 179: 253–268 Becker TW, Perrot-Rechenmann C, Suzuki A and Hirel B (1993) Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves. Planta 191: 129–136 Becker TW, Carrayol E and Hirel B (2000) Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport. Planta 211: 800–806 Bloom AJ, Jackson LE and Smart DR (1993) Root growth as a function of ammonium and nitrate in the root zone. Plant Cell Environ 16: 1294–1301 Bolchi A, Petrucco S, Tenca PL, Foroni C and Ottonello S (1999) Coordinate modulation of maize sulfate permease and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by L-cysteine. Plant Mol Biol 39: 527–537 Brown RH (1978) A difference in N use efficiency in C3 and C4 plants and its implications in adaptation and evolution. Crop Sci 18: 93–98 Brunner M, Kocsy G, Rüegsegger A, Schmutz D and Brunold C (1995) Effect of chilling on assimilatory sulfate reduction and glutathione synthesis in maize. J Plant Physiol 146: 743–747 Brunold C and Schiff JA (1976) Studies of sulfate utilization by algae. 15. Enzymes of assimilatory sulfate reduction in Euglena and their cellular localization. Plant Physiol 57: 430–436

123 Brunold C and Suter M (1989) Localization of enzymes of assimilatory sulfate reduction in pea roots. Planta 179: 228–234 Buchner P, Takahashi H and Hawkesford MJ (2004) Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport. J Exp Bot 55: 1765–1773 Burgener M, Suter M, Jones S and Brunold C (1998) Cyst(e) ine is the transport metabolite of assimilated sulfur from bundle-sheath to mesophyll cells in maize leaves. Plant Physiol 116: 1315–1322 Burnell JN (1984) Sulfate assimilation in C4 plants. Plant Physiol 75: 873–875 Cairns NG, Pasternak M, Wachter A, Cobbett CS and Meyer AJ (2006) Maturation of Arabidopsis seeds is dependent on glutathione biosynthesis within the embryo. Plant Physiol 141: 446–455 Chalot M and Brun A (1998) Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas. FEMS Microbiol Lett 22: 21–44 Campbell WH (1999) Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology. Annu Rev Plant Physiol Plant Mol Biol 50: 277–303 Cheng CL, Acedo GN, Cristinsin M and Conkling MA (1992) Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription. Proc Natl Acad Sci USA 89: 1861–1864 Chew O, Whelan J and Millar AH (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem 278: 46869–46877 Cobbett C and Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol 53: 159–182 Cobbett CS, May MJ, Howden R and Rolls B (1998) The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gammaglutamylcysteine synthetase. Plant J 16: 73–78 Collier M, Fotelli M, Nahm M, Kopriva S, Rennenberg H, Hanke D and Geßler A (2003) Regulation of nitrogen uptake by Fagus sylvatica on a whole plant level- Interactions between cytokinins and soluble N compounds. Plant Cell Environ 26: 1549–1560 Day DA, Poole PS, Tyerman SD and Rosendahl L (2001) Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules. Cell Mol Life Sci 58: 61–71 Dixon DP, Cummins L, Cole DJ and Edwards R (1998) Glutathione-mediated detoxification systems in plants. Curr Opin Plant Biol 1: 258–266 Doulis AG, Debian N, Kingston-Smith AH and Foyer CH (1997) Differential localization of antioxidants in maize leaves. Plant Physiol 114: 1031–1037 Edwards E, Rawsthorne S and Mullineaux P (1990) Subcellular distribution of multiple forms of glutathione

124 reductase in leaves of pea (Pisum sativum L.). Planta 180: 278–284 Edwards GE, Franceschi VR, Ku MS, Voznesenskaya EV, Pyankov VI and Andreo CS (2001) Compartmentation of photosynthesis in cells and tissues of C4 plants. J Exp Bot 52: 577–590 Farago S and Brunold C (1994) Regulation of thiol contents in maize roots by intermediates and effectors of glutathione synthesis. J Plant Physiol 144: 433–437 Fonseca F, Bowsher CG and Stulen I (1997) Impact of elevated atmospheric CO2 on nitrate reductase transcription and activity in leaves and roots of Plantago major. Physiol Plant 100: 940–948 Foyer CH and Rennenberg H (2000) Regulation of glutathione synthesis and its role in abiotic and biotic stress defence. In: Brunold C et al (eds) Sulfur nutrition and sulfur assimilation in higher plants: molecular, biochemical and physiological aspects, pp 127–153. Paul Haupt, Bern, Switzerland Foyer CH, Valadier MH, Migge A and Becker TW (1998) Drought-induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves. Plant Physiol 117: 283–292 Gallardo F, Fu J, Canton FR, Garcia-Gutierrez A, Canovas FM and Kirby EG (1999) Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210: 19–26 Gerwick BC and Black CC (1979) Sulfur assimilation in C4 plants. Plant Physiol 64: 590–593 Gerwick BC, Ku SB and Black CC (1980) Initiation of sulfate activation: a variation in C4 photosynthesis plants. Science 209: 513–515 Gessler A, Kopriva S and Rennenberg H (2004) Regulation of nitrate uptake at the whole-tree level: interaction between nitrogen compounds, cytokinins and carbon metabolism. Tree Physiol 24: 1313–1321 Gómez LD, Vanacker H, Buchner P, Noctor G and Foyer CH (2004) Intercellular distribution of glutathione synthesis in maize leaves and its response to short-term chilling. Plant Physiol 134: 1662–1671 Gutierrez-Alcala G, Gotor C, Meyer AJ, Fricker M, Vega JM and Romero LC (2000) Glutathione biosynthesis in Arabidopsis trichome cells. Proc Natl Acad Sci USA 97: 11108–11113 Habash DZ, Massiah AJ, Rong HL, Wallsgrove RM and Leigh RA (2001) The role of cytosolic glutamine synthetase in wheat. Ann Apl Biol 138: 83–89 Hallock DL, Brown RH and Blaser RE (1965) Relative yield and composition of Kentucky 31 fescue and coastal bermudagrass at four nitrogen levels. Agron J 57: 539–542 Harada E, Kusano T and Sano H (2000) Differential expression of genes encoding enzymes involved in sulfur assimilation pathways in response to wounding and jasmonate in Arabidopsis thaliana. J Plant Physiol 156: 272–276 Harel E, Lea PJ, and Miflin BJ (1977) The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening. Planta 134: 195–200

Stanislav Kopriva Hartmann TN, Fricker MD, Rennenberg H and Meyer AJ (2003) Cell-specific measurement of cytosolic glutathione in poplar leaves. Plant Cell Environ 26: 965–975 Hartmann T, Hönicke P, Wirtz M, Hell R, Rennenberg H and Kopriva S (2004) Sulfate assimilation in poplars (Populus tremula x P. alba) overexpressing g-glutamylcysteine synthetase in the cytosol. J Exp Bot 55: 837–845 Hatch MD and Osmond CB (1976) Compartmentation and transport in C4 photosynthesis. In: Stocking CR and Heber U (eds) Encyclopedia of Plant Physiology, New Series, Vol 3, pp 144–184. Springer-Verlag, Berlin Hell R and Bergmann L (1990) g-glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localisation. Planta 180: 603–312 Herschbach C and Rennenberg H (2001) Significance of phloem-translocated organic sulfur compounds for the regulation of sulfur nutrition. Prog Bot 62: 177–192 Hirai MY, Fujiwara T, Awazuhara M, Kimura T, Noji M and Saito K (2003) Global expression profiling of sulphurstarved Arabidopsis by DNA macroarray reveals the role of O-acetyl-L-serine as a general regulator of gene expression in response to sulphur nutrition. Plant J 33: 651–663 Hirai MY, Klein M, Fujikawa Y, Yano M, Goodenowe DB, Yamazaki Y, Kanaya S, Nakamura Y, Kitayama M, Suzuki H, Sakurai N, Shibata D, Tokuhisa J, Reichelt M, Gershenzon J, Papenbrock J and Saito K (2005) Elucidation of gene-to-gene and metabolite-to-gene networks in Arabidopsis by integration of metabolomics and transcriptomics. J Biol Chem 280: 25590–25595 Hirel B, Layzell DB, McCashin B, McNally SF and Canvin DT (1983) Isoforms of glutamine synthetase in Panicum species having C3, C4, and intermediate photosynthetic pathways. Can J Bot 61: 2257–2259 Hirel B, Bertin P, Quillere I, Bourdoncle W, Attagnant C, Dellay C, Gouy A, Cadiou S, Retailliau C, Falque M and Gallais A (2001) Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiol 125: 1258–1270 Hopkins L, Parmar S, Bouranis DL, Howarth JR and Hawkesford MJ (2004) Coordinated expression of sulfate uptake and components of the sulfate assimilatory pathway in maize. Plant Biol 6: 408–414 Hopkins L, Parmar S, Błaszczyk A, Hesse H, Hoefgen R and Hawkesford MJ (2005) O-acetylserine and the regulation of expression of genes encoding components for sulfate uptake and assimilation in potato. Plant Physiol 138: 433–440 Hothorn M, Wachter A, Gromes R, Stuwe T, Rausch T and Scheffzek K (2006) Structural basis for the redox control of plant glutamate cysteine ligase. J Biol Chem 281: 27557–27565 Hylton CM, Rawsthorne S, Smith AM, Jones DA and Woolhouse HW (1988) Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3-C4 intermediate species. Planta 175: 452–459 Inokuchi R, Kuma KI, Miyata T and Okada M (2002) Nitrogen-assimilating enzymes in land plants and algae:

7

Nitrogen and Sulfur in C4 Plants

phylogenic and physiological perspectives. Physiol Plant 116: 1–11 Jez JM, Cahoon RE and Chen S (2004) Arabidopsis thaliana glutamate-cysteine ligase: functional properties, kinetic mechanism, and regulation of activity. J Biol Chem 279: 33463–33470 Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, Hähnel U, Hänsch R, Hartmann T, Kopriva S, Kruse C, Mendel RR, Papenbrock J, Reichelt M, Rennenberg H, Schnug E, Schmidt A, Textor S, Tokuhisa J, Wachter A, Wirtz M, Rausch T, and Hell R (2005) Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana. Photosynth Res 36: 491–508 Kaiser WM and Huber SC (2001) Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. J Exp Bot 52: 1981–1989 Ketchner SL and Sayre RT (1992) Characterization of the expression of the photosystem II-oxygen evolving complex in C4 species of Flaveria. Plant Physiol 98: 1154–1162 Kocsy G, Brunner M, Rüegsegger A, Stamp P and Brunold C (1996) Glutathione synthesis in maize genotypes with different sensitivities to chilling. Planta 198: 365–370 Kocsy G, Owttrim G, Brander K and Brunold C (1997) Effect of chilling on the diurnal rhythm of enzymes involved in protection against oxidative stress in a chilling-tolerant and a chilling-sensitive maize genotype. Physiol Plant 99: 249–254 Kocsy G, Szalai G, Vagujfalvi A, Stehli L, Orosz G and Galiba G (2000a) Genetic study of glutathione accumulation during cold hardening in wheat. Planta 210: 295–301 Kocsy G, von Ballmoos P, Suter M, Ruegsegger A, Galli U, Szalai G, Galiba G and Brunold C (2000b) Inhibition of glutathione synthesis reduces chilling tolerance in maize. Planta 211: 528–536 Kocsy G, Galiba G and Brunold C (2001) Role of glutathione in adaptation and signalling during chilling and cold acclimation in plants. Physiol Plant 113: 158–164 Kopriva S (2006) Regulation of sulfate assimilation in Arabidopsis and beyond. Ann Bot 97: 479–495 Kopriva S and Koprivova A (2005) Sulfate assimilation and glutathione synthesis in C4 plants. Photosynth Res 86: 363–372 Kopriva S and Rennenberg H (2004) Control of sulphate assimilation and glutathione synthesis: interaction with N and C metabolism. J Exp Bot 55: 1831–1842 Kopriva S, Chu C-C and Bauwe H (1996) Molecular phylogeny of Flaveria as deduced from the analysis of H-protein nucleotide sequences. Plant Cell Environ 19: 1028–1036 Kopriva S, Muheim R, Koprivova A, Trachsler N, Catalano C, Suter M and Brunold C (1999) Light regulation of assimilatory sulfate reduction in Arabidopsis thaliana. Plant J 20: 37–44

125 Kopriva S, Jones S, Koprivova A, Suter M, von Ballmoos P, Brander K, Flückiger J and Brunold C (2001) Influence of chilling stress on the intercellular distribution of assimilatory sulfate reduction and thiols in Zea mays. Plant Biol 3: 24–31 Kopriva S, Suter M, von Ballmoos P, Hesse H, Krähenbühl U, Rennenberg H and Brunold C (2002) Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor. Plant Physiol 130: 1406–1413 Koprivova A, Suter M, Op den Camp R, Brunold C and Kopriva S (2000) Regulation of sulfate assimilation by nitrogen in Arabidopsis. Plant Physiol 122: 737–746 Koprivova A, Melzer M, von Ballmoos P, Mandel T, Brunold C and Kopriva S (2001) Assimilatory sulfate reduction in C3, C3-C4, and C4 species of Flaveria. Plant Physiol 127: 543–550 Kruse J, Hetzger I, Hänsch R, Mendel RR, Walch-Liu P, Engels C, and Rennenberg H (2002) Elevated pCO(2 ) favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots. J Exp Bot 53: 2351–2367 Ku MSB, Wu JR, Dai ZY, Scott RA, Chu C and Edwards GE (1991) Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol 96: 518–528 Lappartient AG and Touraine B (1996) Demand-driven control of root ATP sulphurylase activity and SO42- uptake in intact canola. The role of phloem-translocated glutathione. Plant Physiol 111: 147–157 Lejay L, Tillard P, Lepetit M, Domingo Olive F, Filleur S, Daniel-Vedele F and Gojon A (1999) Molecular and functional regulation of two NO3− uptake systems by N- and C-status of Arabidopsis plants. Plant J 18: 509–519 Lejay L, Gansel X, Cerezo M, Tillard P, Muller C, Krapp A, von Wiren N, Daniel-Vedele F and Gojon A (2003) Regulation of root ion transporters by photosynthesis: functional importance and relation with hexokinase. Plant Cell 15: 2218–2232 Leustek T, Martin MN, Bick JA and Davies JP (2000) Pathways and regulation of sulfur metabolism revealed through molecular and genetic studies. Annu Rev Plant Physiol Plant Mol Biol 51: 141–165 Lillo C, Meyer C, Lea US, Provan F and Oltedal S (2004) Mechanism and importance of post-translational regulation of nitrate reductase. J Exp Bot 55: 1275–1282 Linka M and Weber AP (2005) Shuffling ammonia between mitochondria and plastids during photorespiration. Trends Plant Sci 10: 461–465 Loque D and von Wiren N (2004) Regulatory levels for the transport of ammonium in plant roots. J Exp Bot 55: 1293–1305 Lunn J, Droux M, Martin J and Douce R (1990) Localization of ATP sulfurylase and O-acetylserine (thiol)lyase in spinach leaves. Plant Physiol 94: 1345–1352 Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M, Terce-Laforgue

126 T, Quillere I, Coque M, Gallais A, Gonzalez-Moro MB, Bethencourt L, Habash DZ, Lea PJ, Charcosset A, Perez P, Murigneux A, Sakakibara H, Edwards KJ and Hirel B (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell 18: 3252–3274 Maruyama-Nakashita A, Nakamura Y, Watanabe-Takahashi A, Inoue E, Yamaya T and Takahashi H (2005) Identification of a novel cis-acting element conferring sulfur deficiency response in Arabidopsis roots. Plant J 42: 305–314 Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K and Takahashi H (2006) Arabidopsis SLIM1 is a central transcriptional regulator of plant sulfur response and metabolism. Plant Cell 18: 3235–3251 Matsumura T, Kimata-Ariga Y, Sakakibara H, Sugiyama T, Murata H, Takao T, Shimonishi Y and Hase T (1999) Complementary DNA cloning and characterization of ferredoxin localized in bundle-sheath cells of maize leaves. Plant Physiol 119: 481–488 May MJ, Vernoux T, Leaver C, van Montagu M and Inzé D (1998a) Glutathione homeostasis in plants: implications for environmental sensing and plant development. J Exp Bot 49: 649–667 May MJ, Vernoux T, Sanchez-Fernandez R, Van Montagu M and Inzé D (1998b) Evidence for posttranscriptional activation of gamma-glutamylcysteine synthetase during plant stress responses. Proc Natl Acad Sci USA 95: 12049–12054 McNally SF, Hirel B, Gadal P, Mann AF and Stewart GR (1983) Glutamine synthetases of higher plants: evidence for a specific isoform content related to their possible physiological role and their compartmentation within the leaf. Plant Physiol 72: 22–25 Mellor GE and Tregunna EB (1971) The localization of nitrate-assimilating enzymes in leaves of plants with the C4-pathway of photosynthesis. Can J Bot 49: 137–142 Meyer AJ and Fricker MD (2000) Direct measurement of glutathione in epidermal cells of intact Arabidopsis roots by two-photon laser scanning microscopy. J Microsc 198: 174–181 Meyer AJ, May MJ and Fricker M (2001) Quantitative in vivo measurement of glutathione in Arabidopsis cells. Plant J 27: 67–78 Miflin BJ (1972) The role of light in nitrite reduction: studies with leaf disks. Planta 105: 225–233 Miflin BJ and Habash DZ (2002) The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot 53: 979–987 Miller AJ, Fan X, Orsel M, Smith SJ and Wells DM (2007) Nitrate transport and signalling. J Exp Bot 58: 2297–2306 Mok DWS and Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52: 89–118 Monson RK and Moore BD (1989) On the significance of C3-C4 intermediate photosynthesis to the evolution of C4 photosynthesis. Plant Cell Environ 12: 689–699

Stanislav Kopriva Monson RK, Moore BD, Ku MSB and Edwards GE (1986) Co-function of C3- and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species. Planta 168: 493–502 Moore R and Black CC Jr (1979) Nitrogen assimilation pathways in leaf mesophyll and bundle sheath cells of C4 photosynthesis plants formulated from comparative studies with Digitaria sanguinalis (L.) Scop. Plant Physiol 64: 309–313 Mullineaux PM and Rausch T (2005) Glutathione, photosynthesis and the redox regulation of stress-responsive gene expression. Photosynth Res 86: 459–474 Neuenschwander U, Suter M and Brunold C (1991) Regulation of sulfate assimilation by light and O-acetyl-L-serine in Lemna minor L. Plant Physiol 97: 253–258 Noctor G and Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49: 249–279 Noctor G, Strohm M, Jouanin L, Kunert KJ, Foyer CH and Rennenberg H (1996) Synthesis of glutathione in leaves of transgenic poplar overexpressing g-glutamylcysteine synthetase. Plant Physiol 112: 1071–1078 Noctor G, Arisi A-CM, Jouanin L, Kunert KJ, Rennenberg H and Foyer CH (1998a) Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J Exp Bot 49: 623–647 Noctor G, Arisi A-CM, Jouanin L and Foyer CH (1998b) Manipulation of glutathione and amino acid biosynthesis in the chloroplast. Plant Physiol 118: 471–482 Noctor G, Arisi A-CM, Jouanin L and Foyer CH (1999) Photorespiratory glycine enhances glutathione accumulation in both the chloroplastic and cytosolic compartments. J Exp Bot 50: 1157–1167 Nussbaum S, Schmutz K and Brunold C (1988) Regulation of assimilatory sulfate reduction by cadmium in Zea mays L. Plant Physiol 88: 1407–1410 Ohkama N, Takei K, Sakakibara H, Hayashi H, Yoneyama T and Fujiwara T (2002) Regulation of sulfur-responsive gene expression by exogenously applied cytokinins in Arabidopsis thaliana. Plant Cell Physiol 43: 1493–1501 Oliveira IC and Coruzzi GM (1999) Carbon and amino acids reciprocally modulate the expression of glutamine synthetase in Arabidopsis. Plant Physiol 121: 301–310 Papen H, Gessler A, Zumbusch E and Rennenberg H (2002) Chemolithoautotrophic nitrifiers in the phyllosphere of a spruce ecosystem receiving high atmospheric nitrogen input. Curr Microbiol 44: 56–60 Parisy V, Poinssot B, Owsianowski L, Buchala A, Glazebrook J, and Mauch F (2007) Identification of PAD2 as a gamma-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis. Plant J 49: 159–172 Passera C and Ghisi R (1982) ATP sulphurylase and O-acetylserine sulphydrylase in isolated mesophyll protoplasts and bundle sheath strands of S-deprived maize leaves. J Exp Bot 33: 432–438

7

Nitrogen and Sulfur in C4 Plants

Pastori GM, Mullineaux PM and Foyer CH (2000) Posttranscriptional regulation prevents accumulation of glutathione reductase protein and activity in the bundle sheath cells of maize. Plant Physiol 122: 667–675 Persson J, Högberg P, Ekblad A, Högberg MN, Nordgren A and Näsholm T (2003) Nitrogen acquisition from inorganic and organic sources by boreal forest plants in the field. Oecologia 137 :252–257 Prior A, Uhrig JF, Heins L, Wiesmann A, Lillig CH, Stoltze C, Soll J and Schwenn JD (1999) Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures. Biochim Biophys Acta 1430: 25–38 Rathnam CKM and Das VSR (1974) Nitrate metabolism in relation to the aspartate-type C-4 pathway of photosynthesis in Eleusine coracana. Can J Bot 52: 2599–2605 Rathnam CKM and Edwards GE (1976) Distribution of nitrate-assimilating enzymes between mesophyll protoplasts and bundle sheath cells in leaves of three groups of C4 plants. Plant Physiol 57: 881–885 Rausch T and Wachter A (2005) Sulfur metabolism: a versatile platform for launching defence operations.Trends Plant Sci 10: 503–509 Rawsthorne S (1992) C3-C4 intermediate photosynthesis – linking physiology to gene expression. Plant J 2: 267–274 Remans T, Nacry P, Pervent M, Filleur S, Diatloff E, Mounier E, Tillard P, Forde BG and Gojon A (2006) The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. Proc Natl Acad Sci USA 103: 19206–19211 Ritenour GL, Joy KW, Bunning J and Hageman RH. (1966) Intracellular localization of nitrate reductase, nitrite reductase, and glutamic acid dehydrogenase in green leaf tissue. Plant Physiol 42: 233–237 Rotte C and Leustek T (2000) Differential subcellular localization and expression of ATP sulfurylase and 5¢-adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that cytosolic and plastid forms of ATP sulfurylase may have specialised functions. Plant Physiol 124: 715–724 Ruegsegger A and Brunold C (1992) Effect of cadmium on g-glutamylcysteine synthesis in maize seedlings. Plant Physiol 99: 428–433 Ruegsegger A and Brunold C (1993) Localization of [gamma]glutamylcysteine synthetase and glutathione synthetase activity in maize seedlings. Plant Physiol 101: 561–566 Ruegsegger A, Schmutz D and Brunold C (1990) Regulation of glutathione synthesis by cadmium in Pisum sativum L. Plant Physiol 93: 1579–1584 Rufty TW Jr, MacKown CT and Volk RJ (1989) Effects of altered carbohydrate availability on whole-plant assimilation of 15NO3−. Plant Physiol 89: 457–463 Saito K (2004) Sulfur assimilatory metabolism. The long and smelling road. Plant Physiol 136: 2443–2450 Sanchez-Fernandez R, Fricker M, Corben LB, White NS, Sheard N, Leaver CJ, Van Montagu M, Inze D and May MJ (1997) Cell proliferation and hair tip growth in the

127 Arabidopsis root are under mechanistically different forms of redox control. Proc Natl Acad Sci USA 94: 2745–2750 Santi S, Locci G, Monte R, Pinton R, and Varanini Z (2003) Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms. J Exp Bot 54: 1851–1864 Schmutz D and Brunold C (1984) Intercellular localization of assimilatory sulfate reduction in leaves of Zea mays and Triticum aestivum. Plant Physiol 74: 866–870 Schmutz D and Brunold C (1985) Localization of nitrite and sulfite reductase in bundle sheath and mesophyll cells of maize leaves. Physiol Plant 64: 523–528 Schaefer HJ, Haag-Kerwer A and Rausch T (1998) cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial gamma-glutamylcysteine synthetase isoform. Plant Mol Biol 37: 87–97 Sheen J (1999) C4 gene expression. Annu Rev Plant Physiol Plant Mol Biol 50: 187–217 Stanford C, Larsen K, Barker DG and Cullimore JV (1993) Differential expression within the glutamine synthetase gene family of the model legume, Medicago truncatula. Plant Physiol 103: 73–81 Stitt M, Muller C, Matt P, Gibon Y, Carillo P, Morcuende R, Scheible WR and Krapp A (2002) Steps towards an integrated view of nitrogen metabolism. J Exp Bot 53: 959–970 Strohm M, Jouanin L, Kunert KJ, Pruvost C, Polle A, Foyer CH and Rennenberg H (1995) Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P.alba) overexpressing glutathione synthetase. Plant J 7: 141–145 Suter M, von Ballmoos P, Kopriva S, Op den Camp R, Schaller J, Kuhlemeier C, Schürmann P and Brunold C (2000) Adenosine 5¢-phosphosulfate sulfotransferase and adenosine 5¢-phosphosulfate reductase are identical enzymes. J Biol Chem 275: 930–936 Tabe LM and Droux M (2002) Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein. Plant Physiol 128: 1137–1148 Taira M, Valtersson U, Burkhardt B and Ludwig RA. (2004) Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts. Plant Cell 16: 2048–2058 Tobin AK and Yamaya T (2001) Cellular compartmentation of ammonium assimilation in rice and barley. J Exp Bot 52: 591–604 Vance CP (2001) Symbiotic nitrogen fixation and phosphorus acquisition. Plant nutrition in a world of declining renewable resources. Plant Physiol 127: 390–397 Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ballmoos P, Krähenbühl U, Op den Camp R and Brunold C (2002) Flux control of sulphate assimilation in Arabidop-

128 sis thaliana: Adenosine 5¢-phosphosulphate reductase is more susceptible to negative control by thiols than ATP sulphurylase. Plant J 31: 729–740 Vaughn KC and Campbell WH (1988) Immunogold localization of nitrate reductase in maize leaves. Plant Physiol 88: 1354–1357 Vernoux T, Wilson RC, Seeley KA, Reichheld JP, Muroy S, Brown S, Maughan SC, Cobbett CS, Van Montagu M, Inze D, May MJ and Sung ZR (2000) The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development. Plant Cell 12: 97–109 von Arb C and Brunold C (1986) Enzymes of assimilatory sulphate reduction in leaves of Pisum sativum: activity changes during ontogeny and in vivo regulation by H2S and cyst(e)ine. Physiol Plant 67: 81–86 von Wiren N, Gazzarrini S, Gojon A and Frommer WB (2000) The molecular physiology of ammonium uptake and retrieval. Curr Opin Plant Biol 3: 254–261 Wachter A, Wolf S, Steininger H, Bogs J and Rausch T (2005) Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae. Plant J 41: 15–30 Wagner BM and Beck EH (1993) Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status. Planta 190: 511–518 Walker MA and McKersie BD (1993) Role of the ascorbateglutathione antioxidant system in chilling resistance of tomato. J Plant Physiol 141: 234–239

Stanislav Kopriva Wang R, Tischner R, Gutierrez RA, Hoffman M, Xing X, Chen M, Coruzzi G and Crawford NM (2004) Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis. Plant Physiol 136: 2512–2522 Weber A and Flugge UI (2002) Interaction of cytosolic and plastidic nitrogen metabolism in plants. J Exp Bot 53: 865–874 Westerman S, Stulen I, Suter M, Brunold C and De Kok JL (2001) Atmospheric H2S as sulphur source for Brassica oleracea: consequences for the activity of the enzymes of the assimilatory sulphate reduction pathway. Plant Physiol Biochem 39: 425–432 Wirtz M, Droux M and Hell R (2004) O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana. J Exp Bot 55: 1785–1798 Xiang C and Oliver DJ (1998) Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10: 1539–1550 Yonekura-Sakakibara K, Onda Y, Ashikari T, Tanaka Y, Kusumi T and Hase T (2000) Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize. Plant Physiol 122: 887–894 Zechmann B, Zellnig G and Muller M (2005) Changes in the subcellular distribution of glutathione during virus infection in Cucurbita pepo (L.). Plant Biol 7: 49–57 Zimmermann P, Hirsch-Hoffmann M, Hennig L and Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136: 2621–2632