Journal of Experimental Botany, Vol. 52, No. 356, pp. 591-604,
April 2001
© 2001 Oxford University Press
Cellular compartmentation of ammonium assimilation in rice and barley
1 Plant Sciences, Sir Harold Mitchell Building, School of Biological Sciences, University of St Andrews, St Andrews KY16 9TH, UK
2 Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555, Japan and Plant Science Centre, RIKEN, Hirosawa 2-1, Wako, Saitama, 351-0198, Japan
Received 10 July 2000; Accepted 22 December 2000
 |
Abstract |
|---|
 Top Abstract IntroductionAmmonium assimilation in rootsAmmonium assimilation in leavesConclusionsReferences |
|---|
This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.
Key words: Immunolocalization, plastid, nitrate, ammonium, GS/GOGAT.
|
Introduction |
|---|
|
TopAbstractIntroductionAmmonium assimilation in rootsAmmonium assimilation in leavesConclusionsReferences |
|---|
The pathway for ammonium assimilation in higher plants has been well documented. Ammonium (used hereafter without distinguishing between ammonia gas and ammonium ions), whether resulting from nitrate assimilation or from other, secondary sources, is first incorporated into glutamine in a reaction catalysed by glutamine synthetase (GS; EC 6.3.1.2):
 |
 |
These two reactions form a cycle with the net conversion of one molecule of 2-oxoglutarate and ammonium to 1 molecule of glutamate. This is referred to as the GS/GOGAT pathway (Miflin and Lea, 1980
).
Two GS isoenzymes have been detected in most species. These differ in subcellular location, with GS1 being present in the cytosol and GS2 in the plastids. Similarly, Fd- and NADH-GOGAT are present in most species, with both being located in the plastid, as described below.
There is now a good understanding of the properties of the GS/GOGAT enzymes, their subcellular location and, in most cases, the genes that encode them (for a recent review see Ireland and Lea, 1999). It is only recently, however, that any knowledge has been obtained of the cellular compartmentation of this pathway. The most comprehensive studies to date have been carried out in these laboratories, using rice (Oryza sativa L.) and barley (Hordeum vulgare L.), and these species will form the focus of this review. The two species are also useful for comparative purposes as, in the field, rice uses ammonium and barley uses nitrate as its primary source of nitrogen. From the examples presented here, it is clear that an understanding of the spatial distribution of enzymes is providing new insights into the different roles of isoenzymes in nitrogen acquisition and utilization in cereals.
|
Ammonium assimilation in roots |
|---|
|
TopAbstractIntroductionAmmonium assimilation in rootsAmmonium assimilation in leavesConclusionsReferences |
|---|
Localization of GS and GOGAT in rice roots
Rice, growing in paddy fields, where the soil is waterlogged, uses ammonium as its nitrogen source. Ammonium is rarely stored or transported because of problems of toxicity and so it is first assimilated in the root and then transported through the xylem to the shoots, mainly in the form of glutamine (Fukumorita and Chino, 1982
). Species using ammonium as their N source would therefore be expected to have an efficient ammonium assimilation pathway in their roots.
GS activity is present in rice roots although only the cytosolic isoenzyme has been detected as a major form (Kamachi et al., 1991a). Its cellular localization was determined using light microscopy and peroxidase staining of anti-GS1 IgG-labelled paraffin-embedded root sections (8–10 mm from the tip). Cytosolic GS was found to be distributed throughout the root with apparent homogeneity within the epidermis, exodermis, cortex, and central cylinder. Neither the distribution nor the concentration of GS are affected by ammonium (Ishiyama et al., 1998). In contrast, both the distribution and concentration of NADH GOGAT protein are influenced strongly by ammonium supply. Within 12 h of the addition of 1 mM ammonium to N-depleted rice seedlings there was an increase in NADH GOGAT mRNA, protein and activity in whole root extracts (Yamaya et al., 1995). The induction of NADH GOGAT occurred in all segments of the root but it was particularly enhanced in the region between the root tip and where the secondary roots form (Ishiyama et al., 1998). This induction of NADH GOGAT by ammonium may be indirect. Hirose et al. suggested that it is glutamine, a product of ammonium assimilation via GS, which acts as the signal for NADH GOGAT induction (Hirose et al., 1997).
The response of NADH GOGAT to exogenous ammonium is striking in that it occurs in specific cells within the root. Evidence for this has come from detailed immunohistochemical studies using affinity-purified NADH GOGAT IgG (Ishiyama et al., 1998). In N-depleted plants, NADH GOGAT protein was detected in the central cylinder, apical meristem and in primordia of secondary roots, with only a weak labelling in the epidermis (Fig. 1a, c; Ishiyama et al., 1998). Although NADH GOGAT was detected in young cortical cells (within 250 µm of the root tip) it was absent once these cells matured and became highly vacuolated, possibly as an adaptation to growth under anaerobic conditions. Within 24 h of the addition of 1 mM ammonium there was a marked increase in immunolabelling of NADH GOGAT protein in the two outer cell layers, the epidermis and exodermis (Fig. 1b, d; Ishiyama et al., 1998).
|
Growth on ammonium therefore results in a change in the cellular localization of NADH GOGAT in rice roots, from the central cylinder and secondary root initial of N-depleted plants, to the epidermis and exodermis of ammonium-treated roots (Ishiyama et al., 1998
). Immunogold labelling studies show that NADH GOGAT is localized in the plastids of rice roots (Hayakawa et al., 1999). Intriguingly, the apparent concentration of NADH GOGAT protein (immunogold label per unit plastid area) was found to be the same in all plastids, irrespective of cell type. The higher concentration of NADH GOGAT in the epidermis and exodermis of ammonium-treated roots is therefore thought to be due to a higher number of plastids in these cells as compared to the other cells of the root (Hayakawa et al., 1999).
In contrast to NADH GOGAT, there is no effect of ammonium on Fd GOGAT concentration in rice roots. There is, however, a change in Fd GOGAT activity with root development. Whereas NADH GOGAT remained constant, Fd GOGAT activity was found to be highest in the youngest cells at the root tip, and then decreased with root cell maturity (Ishiyama et al., 1998). Differential distribution of Fd GOGAT protein within different cells of the root has also been reported. In the youngest cells (within the apical 250 µm) Fd GOGAT protein was present in all cell types. In older root tissue, however, Fd GOGAT protein was found mainly in the central cylinder and secondary root primordia, with only weak immunolabelling on other cell types (Ishiyama et al., 1998).
The influence of cellular compartmentation on root nitrogen metabolism in rice
Although the total activity of Fd GOGAT in whole root extracts is 4–5-fold higher than that of NADH GOGAT (Suzuki et al., 1982), the different locations and responses to N nutrition indicate that both isoenzymes have important and distinct functions in rice. It is clear that the distribution of ammonium assimilation enzymes in rice roots is heterogeneous and is influenced by the age of the tissue, the supply of exogenous nitrogen, and by the cell type. These responses are summarized in Fig. 2.
|
The cellular distribution of GS and GOGAT in rice roots is likely to be related to the function of these enzymes in ammonium assimilation and N mobilization. In ammonium-treated roots, the high concentration of NADH GOGAT protein, together with cytosolic GS in the epidermal and exodermal cells would enable ammonium taken up by the roots to be assimilated by the cytosolic GS/NADH GOGAT pathway in these outermost cells of the root. The increase in NADH GOGAT protein, relative to GS, in response to ammonium might result in increased glutamate formation in the epidermis. Solute movement from the epidermis to the cortex is thought to be symplastic, due to the presence of a Casparian strip between these cell types (Morita et al., 1996
). One explanation for the response of epidermal cells is that, if symplastic transport favours glutamate rather than glutamine, increased glutamate formation might serve to increase the rate of flow of assimilates into the cortex at high rates of exogenous ammonium supply. This merits further investigation.
Ammonium uptake in rice occurs in the root apex and in the region where secondary roots are being formed (Tatsumi, 1982), which coincides with the region where NADH GOGAT is particularly responsive to ammonium-induction, as described above. This, together with the lack of response of Fd GOGAT to exogenous ammonium, indicates that Fd GOGAT may not be directly involved in primary ammonium assimilation under these conditions. The presence of both NADH and Fd GOGAT in the apical meristem and secondary root primordia indicates that both isoenzymes are involved in forming glutamate from glutamine that is transported through the phloem from source tissue in the shoot. The phloem of the central cylinder functions in the transport of solutes to the actively growing root tissue. The detection of cytosolic GS, together with NADH and Fd GOGAT in these cells is consistent with an involvement in remobilization of glutamine transported from the shoots in N depleted rice (Ishiyama et al., 1998).
Localization of GS and GOGAT in barley roots
In contrast to rice, barley (Hordeum vulgare L.), as for most other cereals growing on well-aerated soils, uses nitrate as its main source of nitrogen (Lewis et al., 1982). Nitrate entering the root is reduced to nitrite and ammonium before being assimilated via the root GS/GOGAT cycle and transported, in the form of glutamine, to the shoot. At high concentrations of exogenous nitrate, increasing amounts of nitrate are exported from the root to the shoot, via the xylem, to be reduced to ammonium and then assimilated via GS and GOGAT in the leaves (Lewis et al., 1982). Excess nitrate may also be stored in the vacuole (Pate, 1980). As with rice, there is differential localization of GS and GOGAT isoforms in barley roots, indicating developmental and nutritional influences on their distribution.
GS activity changes during root development and is affected by N nutrition. In 7-d-old hydroponically grown barley seedlings, GS activity was highest in the youngest cells at the root tip, when expressed relative to fresh weight, and was highest in nitrate-grown and N-deficient plants compared to those grown on ammonium. In contrast, when expressed relative to protein, GS activity was lowest at the root tip, but was still highest in nitrate-grown plants (Peat and Tobin, 1996). As well as changes in activity, changes in GS polypeptides occur during root development and this is also affected by N supply. Cytosolic GS (42 kDa polypeptide) was detected on immunoblots in all sections of the root, irrespective of N supply. Two additional polypeptides were detected in mature root tissue of ammonium-grown seedlings and one additional GS polypeptide in those grown on nitrate. These additional polypeptides were of a lower molecular mass than the 42 kDa cytosolic polypeptide and were also thought to be cytosolic (Peat and Tobin, 1996). Although developmental changes in GS isoenzymes have been reported previously in barley roots, in these cases the additional polypeptide was of a higher molecular mass than the cytosolic GS. In germinating barley roots, only a single polypeptide (42 kDa) was present initially, and a second polypeptide with a higher molecular mass (45 kDa) was detected 5 d after germination (Marttila et al., 1993). Mäck (Mäck, 1995) also detected changes in barley root GS polypeptides in response to ammonium but these differ from those reported earlier (Peat and Tobin, 1996). Two cytosolic GS isoenzymes have been identified; one, GS1a, consisted of two 45 kDa subunits that differed in their isoelectric points, while the second, GS1b, comprised five subunits, three of which had a molecular mass of 45 kDa while the remaining two were common to GS1b (Mäck, 1995). Although of slightly different molecular mass, these two isoforms may correspond to those detected previously (Peat and Tobin, 1996). Although only two GS isoenzymes were reported (Marttila et al., 1993; Mäck, 1995), the third GS polypeptide, being present only in mature tissue at the base of the root, is likely to remain undetected in whole root homogenates, as used in these earlier studies. This emphasizes the need, in any biochemical study, to take account of the heterogeneity of tissue, both in terms of its development and its cellular composition.
Although plastidic GS was not detected on Western blots of barley root tissue, immunogold localization studies have identified a plastid GS isoenzyme in barley (Peat and Tobin, 1996). Significant labelling of GS protein was found in plastids of cortical and vascular parenchyma cells in electron micrographs of root apical sections (2.0 mm from the root tip). Large variations in labelling intensity on different plastids prompted a more detailed investigation (LJ Peat and AK Tobin, unpublished data). As a result, four different forms of plastid have been identified, on the basis of structural appearance, and these preliminary studies indicate that they contain different amounts of GS protein. One type (‘tubular’) containing tubular membranes appeared to be more highly immunolabelled; a second (‘no internal membranes’) had no obvious internal membranes and was labelled only weakly; a third (‘starchy’) contained starch grains and was also weakly labelled; the fourth kind (‘flat membranes’) contained flat lamella-like membranes and was sometimes densely labelled (Fig. 3; Peat and Tobin, 1996). Results of the preliminary analysis of these plastids suggest not only differences in the amount of GS protein between plastids but also a differential response to N nutrition. Whereas in N-deficient (0.1 mM NO3-) and ammonium-grown plants the GS labelling was similar in all plastids, when grown on high nitrate there was an increase in GS labelling in the ‘tubular’ and ‘flat’ plastids (Fig. 4). This suggests that, not only is there a plastid isoform of GS in barley roots, but also that it may be nitrate-inducible, but only in particular types of plastid. Similar forms of plastid have been reported previously in barley roots (Lux, 1986), but this is the first evidence of any difference in functionality. It is possible that they represent different stages of root plastid development, although no obvious association with any specific cell type or stage of root development has been identified. Possible roles for plastidic GS are discussed in the next section.
|
|
Cytosolic GS protein was detected, by immunogold localization, in both the cortical and vascular parenchyma of barley roots. The apparent concentration of cytosolic GS protein was higher in the cortical than in the vascular cells irrespective of N nutrition, and was highest in both cell types in N-deficient, compared to nitrate- or ammonium-grown plants (Peat and Tobin, 1996
).
Preliminary studies, using immunogold-labelling techniques, on the localization of Fd and NADH GOGAT in barley roots indicate that both are present in the plastid (Peat, 1996). Suzuki et al. measured both Fd and NADH GOGAT activity in barley roots and also localized the Fd-dependent species in the plastids (Suzuki et al., 1981). The cellular location of Fd GOGAT awaits further verification, although preliminary data indicate that it is present in both cortical and vascular parenchyma cells, with evidence of an increase in concentration in the nitrate-grown as compared to nitrogen-deficient plants (Peat, 1996). To date, there have been no studies of the cellular localization of NADH GOGAT in barley roots. Analysis of Western blots indicate that, as with rice, growth on ammonium results in an increase in NADH GOGAT protein in barley roots (Peat, 1996).
As with rice, there is evidence to suggest that, in barley, the relative amount of Fd and NADH GOGAT changes with root development. The response is quite different, however, with Fd GOGAT protein and NADH GOGAT decreasing in concentration with distance from the root tip in barley (Peat, 1996). This has also been reported to be the case in pea roots (Matoh and Takahashi, 1982).
The influence of cellular compartmentation on root nitrogen metabolism in barley
Present understanding of the cellular localization of GS and GOGAT species in barley and the proposed function of these proteins in whole plant nitrogen metabolism is summarized in Fig. 5.
|
In barley seedlings growing on high concentrations of nitrate (Fig. 5a
) the majority of nitrate taken up by roots is transported via the xylem to the shoot for reduction and further metabolism (Andrews, 1986). Root nitrate assimilation is occurring at maximum capacities under these conditions (Andrews, 1986). Nitrate uptake (Henriksen et al., 1992; Lazof et al., 1992) and nitrate reductase (NR) activity (Long and Oaks, 1990; Peat and Tobin, 1996) are highest in the mature region of the root, although in barley their cellular localization remains to be determined. In maize roots, NR protein has been detected in the epidermal, cortical, vascular parenchyma, and pericycle (Federova et al., 1994) while NR activity was found in both the cortical and epidermal tissue (Rufty et al., 1986) indicating that, at least in maize, nitrate reduction occurs throughout the root. Further work is needed to determine whether this is also the case in barley roots. Nevertheless, the observed increase in cytosolic GS polypeptides in the mature region of nitrate-grown roots coincides with the region of highest nitrate reduction. This implies a role for cytosolic GS in assimilating ammonium generated during primary nitrate assimilation, forming glutamine for export to developing root apical tissue and to the shoot (Fig. 5a). In support of this hypothesis, the normal growth of barley plants lacking plastidic GS, under non-photorespiratory conditions, does suggest that there is sufficient cytosolic GS in barley roots to maintain root nitrate assimilation (Blackwell et al., 1987). More detailed analysis of these mutants has indicated, however, that plastidic GS is also important in relation to glutamine export. The export from the root of glutamine formed from freshly assimilated NO3- was reduced by 50% in mutants lacking plastid GS (Joy et al., 1992). This is intriguing, given the fact that no significant reduction in root GS activity was detected in these mutants, most likely due to the fact that, on a whole tissue basis, root plastid GS activity is extremely low. Although it remains to be established whether this mutation (LaPr 85/80) has, indeed, affected the root plastidic GS isoform, these results indicate a role for this enzyme in providing glutamine for export to the shoot.
In the younger tissue at the root apex, where the immunogold localization studies were performed, some nitrate reduction will be taking place in nitrate-grown seedlings although at a lower rate than in the mature root tissue (Peat and Tobin, 1996). Growth on nitrate resulted in a decrease in cytosolic GS protein in both the cortical and vascular cells and an increase in Fd GOGAT and in plastidic GS in some groups of plastid, as described above. Thus, a relative increase in plastidic GS/Fd GOGAT, compared to cytosolic GS, occurs in young tissue of nitrate-grown seedlings. One explanation for this response is that nitrate will increase the capacity for plastid nitrite reduction, and hence the demand for plastid ammonium assimilation will also increase. Nitrate induces not only nitrite reductase (NiR) but it also regulates the supply of reduced ferredoxin in roots, required by NiR and by Fd GOGAT. A specific form of ferredoxin-NADP+ oxidoreductase (FNR), which generates reduced ferredoxin, is present in roots and other non-photosynthetic tissue where it is known to be induced by nitrate (Bowsher et al., 1993). Increased nitrate supply might therefore be expected to result in increased nitrite reduction leading to increased plastidic ammonium assimilation in roots. Glutamate might then be used directly for growth within the young apical root tissue.
The reason for differences between different structural forms of plastid, in terms of GS composition, is unclear. It also remains to be seen whether the photorespiratory mutants lacking plastidic GS (LaPr 85/80; Joy et al., 1992) lack some or all of these root GS proteins. Further characterization of the enzyme composition of these different plastid forms is essential if it is going to be possible to determine the extent to which these plastids contribute to root C and N metabolism.
The importance of Fd GOGAT in root nitrate assimilation is indicated by analysis of barley mutants deficient in this enzyme (LaPr 85/73; Joy et al., 1992). This mutation resulted in an increased concentration of glutamine in the root and a 3-fold increase in glutamine exported to the shoot. Although, as with the GS mutants, there was no detectable change in root Fd GOGAT activity in this mutant, this indicates that a major proportion of glutamine formed from nitrate assimilation in barley roots is generally retained in the root for growth purposes. Disruption of glutamine metabolism, by the loss of Fd GOGAT, makes more glutamine available for export to the shoot. Alternatively, if the mutation has only resulted in a loss of leaf Fd GOGAT and not of root Fd GOGAT, then this suggests that a disruption of leaf glutamine metabolism somehow affects root glutamine export. It is possible that, under these conditions, excess glutamine accumulating in the leaf is exported to the root via the phloem and this then results in excess glutamine being recycled back to the shoot via the xylem. Further analysis of this mutant is required in order to determine the extent to which root Fd GOGAT has been affected.
In barley seedlings grown on ammonium as sole nitrogen source (summarized in Fig. 5b) root ammonium assimilation is essential. As for nitrate, the highest rates of ammonium uptake occur in mature root tissue (Henriksen et al., 1992) and this coincides with the region where two additional cytosolic GS polypeptides were detected in ammonium-grown plants. Although the cellular location of proteins has not been established in this region of the root, the relatively high activity of GS, and the apparent induction of cytosolic GS isoenzymes, suggests that this enzyme assimilates ammonium in mature tissue. The relatively low amounts of both Fd and NADH GOGAT detected in the mature region of the root would favour the formation of glutamine, for transport to the shoot and to immature cells at the root apex. Immunogold localization studies of the root apical region detected similar, low concentrations of plastidic GS as found in N-deficient seedlings. This is consistent with the proposed role for plastidic GS in assimilating ammonium generated from plastidic nitrite assimilation. The lower activity of GS in this region of the root of ammonium-grown tissue, together with the observed increase in NADH GOGAT protein, would tend to favour glutamate formation to support growth of developing root tissue.
One of the key differences between rice and barley root ammonium assimilation is the presence, in barley, of a plastidic isoform of GS. This is consistent with an earlier hypothesis (Woodall and Forde, 1996) that suggested that plastidic nitrate and ammonium assimilation was a physiological adaptation to growth in nitrate-rich, temperate soils. There are, however, exceptions to this general model with some legumes, for example, having plastidic GS in the root nodule (Brangeon et al., 1989). Nevertheless, the low level of immunolabelling of plastids of ammonium-grown compared with nitrate-grown barley further supports this hypothesis.
Until a full analysis of the cellular location of Fd GOGAT and NADH GOGAT has been completed for barley it is difficult to make a detailed comparison with rice. Nevertheless, it is clear that in both species there are distinct and separate locations for different steps in the pathway of ammonium assimilation in roots, with strong developmental and nutritional influences on this compartmentation.
|
Ammonium assimilation in leaves |
|---|
|
TopAbstractIntroductionAmmonium assimilation in rootsAmmonium assimilation in leavesConclusionsReferences |
|---|
There are a number of sources of ammonium in leaves (first reviewed by Joy, 1988
). In C3 plants, such as rice and barley, photorespiration releases ammonium in the mesophyll cells in the light. Nitrate transported in the xylem and reduced in the leaf will also release ammonium, although the cellular localization of these reactions has yet to be identified in either rice or barley. Other ammonium-generating processes in the leaf include lignin synthesis (via the phenylalanine ammonia lyase reaction), and the degradation of nitrogen-containing transport and storage compounds. The latter will occur during the natural turnover of metabolites within growing tissue and also during remobilization of N compounds during senescence.
It is now generally agreed that the GS/GOGAT cycle is responsible for assimilating ammonium in leaves (Lea et al., 1990). What is less clear, however, is the function of each of the different GS and GOGAT species and the extent to which their cellular location might relate to their relative roles in assimilating ammonium from different sources. Examples from studies on rice and barley provide some clues.
Localization of GS and GOGAT in rice leaves
As in the majority of higher plants, two GS isoenzymes are present in rice leaves, one in the chloroplast (GS2) and a second in the cytosol (GS1) (Kamachi et al., 1992). The relative amount of each isoenzyme changes with leaf development, as does their cellular location. The amount of GS2 protein (relative to fresh weight) has been found to increase with the photosynthetic development of the tissue. GS2 protein levels were low in the non-green leaf blades and leaf sheaths and highest in the fully expanded leaves (5th to 7th leaves) of 75-d-old rice plants. GS1 protein levels were highest in the oldest leaves and gradually decreased to the lowest level in the youngest non-green leaf blade (Yamaya et al., 1992).
Using tissue printing, Kamachi et al. were first able to find that GS1 protein was specific to the vascular bundles of rice leaves (Kamachi et al., 1992). GS1 has also been reported to be present in leaf vascular cells in tobacco (Carvalho et al., 1992) and potato (Pereira et al., 1992). Sakurai et al. (Sakurai et al., 1996) carried out a more detailed examination of rice leaves, at higher resolution, using paraffin-embedded tissue labelled with the same mono-specific anti-GS1 IgG used by Kamachi et al. (Kamachi et al., 1992). This enabled them to identify which cells of the vascular bundle contained GS1 and also to compare leaves of different ages. In the fully expanded leaves (6th to 8th) GS1 protein was detected in the companion cells, metaphloem-parenchyma and metaxylem-parenchyma of the large vascular bundles. Staining was particularly intense in the companion cells located in close proximity to the sieve elements. In the small vascular bundles, GS1 was detected in the companion cells and metaxylem parenchyma. GS1 was also found in the guard cells and there was some very weak labelling of the mesophyll and epidermal cells. In younger leaves (blades 9 and 10, and the green (exposed area) region of blade 11) there was only a weak labelling in the companion cells, but a stronger signal in the xylem and phloem parenchyma. In the region of blade 11 that was enclosed within the sheath, and hence non-green, GS1 labelling was relatively intense in the sclerenchyma and in the xylem parenchyma, but very weak in the companion cells.
The cellular location of GS2 protein in fully expanded rice leaves is distinct from that of GS1, having been detected in the mesophyll cells and in the parenchyma sheath cells (Sakurai et al., 1996). Although there was also a weak labelling of the parenchyma and companion cells this was not considered to be due to GS2 protein as the antibody used in this study also cross-reacted slightly with GS1 protein which is known to be present in these cells (Kamachi et al., 1991b).
Both NADH and Fd GOGAT are present in rice leaves. The relative amount of each isoenzyme varies with leaf development and there are distinct differences in their cellular location. It was found that the highest level of NADH GOGAT protein and activity occurred in the youngest, non-green, unexpanded leaves and that both activity and protein decreased with increasing leaf age and leaf expansion (Yamaya et al., 1992). In contrast, Fd GOGAT protein and activity was highest in the fully expanded, green leaf blades (5th to 7th) and lowest in the immature, non-green blades. The activity of Fd GOGAT was more than 100 times that of NADH GOGAT in the green leaf blades, whereas it was only 1.9 times higher in the young, non-green 10th blade. This distribution of Fd GOGAT is similar to that of GS2 in these plants, as described above.
Tissue printing and immunolabelling of paraffin-embedded sections was used to determine the cellular localization of NADH and Fd GOGAT in rice leaves (Hayakawa et al., 1994). The tissue printing technique detected NADH GOGAT protein in the large and small vascular bundles of unexpanded leaves. The higher resolution, light microscopy immunolocalization technique identified that NADH GOGAT protein was present in the metaphloem and metaxylem-parenchyma and mestome sheath cells of the vascular bundles (Fig. 6). No NADH GOGAT protein was detected in the mesophyll cells or in the companion cells and sieve elements of the vascular bundles. In the same study, it was found that Fd GOGAT localization was quite distinct from that of NADH GOGAT (Hayakawa et al., 1994). Fd GOGAT protein was found in the mesophyll cells primarily, but there was also immunolabelling in the parenchyma cells surrounding the protoxylem lacunae (Fig. 7).
|
|
The influence of cellular compartmentation on nitrogen metabolism in rice leaves
The difference in location of GS and GOGAT isoenzymes in rice leaf cells indicates that they have different functions, perhaps relating to the different sources of ammonium (summarized in Fig. 2). The major source of nitrogen for developing leaves and ears of mature rice plants is thought to be that released from older, senescing leaves. Evidence for this has come from 15N experiments showing that remobilized nitrogen accounted for 64% of the total nitrogen in the youngest leaf blades (Mae et al., 1981). Glutamine, produced during N remobilization, is the major form (as much as 42% of total) of amino acid present in the phloem sap (Hayashi and Chino, 1990) and thus the main form of N transported to developing sink tissue, where it is converted to glutamate. The companion cells and metaphloem and metaxylem cells are considered to be active in the transport of solutes, since they contain abundant mitochondria and ER (Chonan et al., 1981). The presence of GS1 in companion cells of vascular bundles of relatively old leaf blades further supports the hypothesis (Kamachi et al., 1992) that GS1 is important in the synthesis of glutamine for export from mature and senescing leaves (Yamaya et al., 1992). This suggests that GS1 in the companion cells may form glutamine for transport into the phloem and, from there, to the younger, developing leaves. It is not clear as to the function of GS1 in younger tissue, where it is barely detectable in the companion cells but is present in the metaxylem and metaphloem-parenchyma, nor is there an obvious function for GS1 in the guard cells. This awaits further investigation. The presence of GS1 in the sclerenchyma and xylem-parenchyma of the non-green region of the developing (11th) leaf blade may indicate its involvement in assimilating ammonia released during lignin synthesis. At this stage of leaf development the secondary cell wall is being formed. Phenylalanine ammonia lyase (PAL; EC 4.3.1.5), the key enzyme for lignin biosynthesis, generates NH+4, and the bean PAL2 gene has been found to be expressed in the early stages of vascular development (Leyva et al., 1992). Thus, it is possible that GS1 may be responsible for the assimilation of NH4+ released by the PAL reaction in this tissue. It is also possible that GS1 may function in the assimilation of NH4+ formed by asparaginase, which is known to be expressed in young developing tissues of transgenic tobacco plants (Grant and Bevan, 1994). Asparagine, after glutamine, is a major form (12% of total amino acids) of nitrogen transport compound in the phloem sap of rice plants (Hayashi and Chino, 1990).
In sink tissue, such as immature leaves and developing grains, glutamine would be the main form of N imported from mature and senescing tissue, as described above. Further metabolism of glutamine in sink tissue is likely to involve NADH GOGAT as this is the most abundant of the two isoenzymes in the non-green leaf tissue (Yamaya et al., 1992) and developing grains (Hayakawa et al., 1993). Anatomical studies indicate that the vascular parenchyma and mestome sheath cells, where NADH GOGAT is abundant, are active in solute transport from the phloem and xylem (Chonan et al., 1981). From this it would appear that NADH GOGAT is important in the reutilization of glutamine transported through the vascular system, to produce glutamate for biosynthesis within the developing organs.
The distribution of GS2 in rice leaves, being present in green tissue and, specifically in the mesophyll cells, is consistent with a role in reassimilating ammonium released during photorespiration. This would appear to be also the case for Fd GOGAT that shows a similar distribution, being particularly abundant in the mesophyll cells (Hayakawa et al., 1994).
The results of these localization studies indicate that the isoenzymes of GS and GOGAT have quite distinct functions in leaves, with GS1 and NADH GOGAT involved in the remobilization of N from source to sink tissue, and GS2 and Fd GOGAT responsible for reassimilation of photorespiratory ammonium.
Localization of GS and GOGAT in barley leaves: influence of compartmentation on N metabolism in leaves
These studies of GS and GOGAT localization in barley leaves have reached similar conclusions to those described for rice. These results, and their interpretation, are summarized in Fig. 5. GS activity was found to increase with leaf development. This was due to an increase in the amount of GS2 protein, with GS1 remaining relatively constant (LJ Peat and AK Tobin, unpublished data). The same developmental changes have been found in wheat primary leaves, where GS2 activity increased in parallel with photorespiration and with the activity of key photorespiratory enzymes (Tobin et al., 1985). These results indicate that in barley, as in rice, GS2 is associated with photorespiration. This is further supported by immunogold labelling experiments that detected GS2 protein primarily in the chloroplasts of mesophyll cells, where photorespiratory ammonia is released (Fig. 8a). The response of GS2 to changes in N nutrition indicates that this isoenzyme does not just function in the assimilation of photorespiratory ammonium. Growth on high nitrate (15 mM) resulted in higher concentrations of GS2 in the mesophyll cells, compared to N-deficient (0.1 mM NO3-) plants (Fig. 8a). This apparent induction of GS2 by nitrate might indicate an additional role for this isoenzyme in primary assimilation. At high concentrations of nitrate, as discussed above, shoot nitrate assimilation increases. Barley grown on 12 mM NO3-, for example, assimilates 98% of its nitrate in the leaf (Lewis et al., 1982). Under these conditions it might be important for both GS isoenzymes to function in ammonium assimilation. Analysis of barley mutants, however, indicates that plants can assimilate nitrate efficiently in the absence of GS2, which suggests that there is sufficient GS1 to support primary assimilation. An alternative explanation is that the increase in GS2 concentration in the mesophyll cells is in response to increased photorespiratory flux, as growth on high nitrate would lead to increased synthesis of Rubisco (Stitt and Krapp, 1999).
|
Cytosolic GS protein was detected, by immunogold labelling, in the mesophyll cells of barley leaves (Fig. 8a
). This conflicts with previous immunolocalization studies, on tobacco (Carvalho et al., 1992), potato (Pereira et al., 1992, 1996) and rice (Kamachi et al., 1992) where GS1 was only detected in the vascular cells. Although immunolocalization has failed previously to detect mesophyll GS1, its activity has been detected in cytosolic fractions of mesophyll protoplasts from pea (Wallsgrove et al., 1979) and barley (Wallsgrove et al., 1980). These conflicting reports might indicate species differences, although the failure to detect GS1 might be due to differences in experimental technique and growing conditions. These results show that the concentration of GS1 protein in barley mesophyll cells is very low, particularly in plants grown on low nitrate where the labelling intensity is only just higher than the control (non-immune serum, Fig. 8a). This, together with the small volume of cytosol in mesophyll cells, would make it difficult to detect GS1 in these cells, particularly when using low resolution techniques such as tissue printing and light microscopy. Further analysis, using immunogold localization and different growth conditions, may therefore lead to the detection of GS1 in mesophyll cells in a wider range of species.
GS2 protein was detected at only a very low concentration in the plastids of vascular cells and, together with the scarcity of these plastids, this precluded quantification of immunolabelling. GS1 is clearly the predominant isoenzyme in the vascular cells of barley leaves. Significant amounts of GS1 protein were detected in these cells, with a marked effect of N nutrition on its apparent concentration (Fig. 8b). As was the case for GS1 in mesophyll cells, at low nitrate concentrations cytosolic GS was present in relatively low concentrations. Growth on either high nitrate (15 mM) or ammonium resulted in a significant and marked increase in cytosolic GS concentration in the vascular cells (Fig. 8b). At high nitrate concentrations, the majority of N transported from root to shoot, in barley, is in the form of nitrate (Lewis and Chadwick, 1983). High concentrations of GS1 in the vascular cells under these conditions might therefore be related to an increased demand for shoot nitrate reduction. Growth on ammonium results in glutamine being the major form (93%) of N exported to the shoot (Lewis and Chadwick, 1983). Increased GS1 concentrations in the vascular cells is consistent with its proposed role in remobilization of N. These results therefore support the suggestion that GS1 is important in the primary assimilation of N and in remoblization of N from source to developing sink tissue. However, until the cellular localization of nitrate and nitrite reductases is known, these conclusions remain tentative.
Fd GOGAT protein was detected, by immunogold labelling, specifically in the plastids of mesophyll and vascular cells of barley. As was the case for GS2, the small numbers of plastids in vascular cells indicates that the majority of Fd GOGAT protein in the leaf is located in the mesophyll tissue (LJ Peat and AK Tobin, unpublished results). This is supported by lower resolution light microscopy of immunolabelled sections of barley leaf tissue (Fig. 9; T Hayakawa, LJ Peat, T Yamaya, AK Tobin, unpublished results) where the mesophyll cells are clearly labelled. No effects of N nutrition were apparent and the localization is consistent with a major role for Fd GOGAT in the formation of glutamate for photorespiratory ammonium assimilation via chloroplast GS (Kendall et al., 1986).
|
It has not been possible to determine the cellular localization of NADH GOGAT in barley leaves because of the weak cross-reactivity of the available rice anti-NADH GOGAT antibody with the barley protein. The preliminary studies indicated that, as with rice, NADH GOGAT protein is more abundant in younger, non-photosynthetic tissue, in particular at the base of barley primary leaves (LJ Peat, AK Tobin, unpublished results). Further work is needed to determine whether NADH GOGAT fulfils the same role in barley leaves as it does in rice.
|
Conclusions |
|---|
|
TopAbstractIntroductionAmmonium assimilation in rootsAmmonium assimilation in leavesConclusionsReferences |
|---|
The immunolocalization studies of rice and barley show that the ammonium assimilation pathway is highly compartmentalized. Distinct cellular locations for the component isoenzymes have been identified and this information has been used to produce models of the contribution each one makes to whole plant N metabolism. This research has shown that a knowledge of the location of an enzyme is a valuable aid to understanding its function.
|
Acknowledgments |
|---|
This work was supported by: a programme of Research for the Future from the Japan Society for the Promotion of Science (JSPS-RFTF96L00604), Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan (Nos. 09274101 and 09274102) to TY; The Royal Society, The British Council and the Biological and Biotechnological Sciences Research Council (P02582) to AKT.
|
Notes |
|---|
3 To whom correspondence should be addressed. Fax: +44 1334 463366. E-mail: at6{at}st\|[hyphen]\|andrews.ac.uk
|
References |
|---|
|
TopAbstractIntroductionAmmonium assimilation in rootsAmmonium assimilation in leavesConclusionsReferences |
|---|
Andrews M. 1986. The partitioning of nitrate assimilation between root and shoot of higher plants. Plant, Cell and Environment 9, 511–519.
Blackwell RD, Murray AJS, Lea PJ. 1987. Inhibition of photosynthesis in barley with decreased levels of chloroplastic glutamine synthetase activity. Journal of Experimental Botany 38, 1799–1809.
Bowsher CG, Hucklesby DP, Emes MJ. 1993. Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation. The Plant Journal 3, 463–467.
Brangeon J, Hirel B, Forchioni A. 1989. Immunogold localization of glutamine synthetase in soybean leaves, roots and nodules. Protoplasma 151, 88–97.
Carvalho H, Pereira S, Sunkel C, Salema R. 1992. Detection of a cytosolic glutamine synthetase in leaves of Nicotiana tabacum L. by immunocytochemical methods. Plant Physiology 100, 1591–1594.
Chonan N, Kaneko M, Kawahara H, Matsuda T. 1981. Ultrastructure of the large vascular bundles in the leaves of rice plants. Japanese Journal of Crop Science 50, 323–331.
Federova E, Greenwood JS, Oaks A. 1994. In situ localization of nitrate reductase in maize roots. Planta 194, 279–286.
Fukumorita T, Chino M. 1982. Sugar, amino acid, and inorganic contents in rice phloem sap. Plant and Cell Physiology 23, 273–283.
Grant M, Bevan MW. 1994. Asparaginase gene expression is regulated in a complex spatial and temporal pattern in nitrogen-sink tissues. The Plant Journal 5, 695–704.
Hayakawa T, Hopkins L, Peat LJ, Yamaya T, Tobin AK. 1999. Quantitative intercellular localization of NADH-dependent glutamate synthase protein in different types of root cells in rice plants. Plant Physiology 119, 409–416.
Hayakawa T, Nakamura T, Hattori F, Mae T, Ojima K, Yamaya T. 1994. Cellular localization of NADH-dependent glutamate synthase protein in vascular bundles of unexpanded leaf blades and young grains of rice plants. Planta 193, 455–460.
Hayakawa T, Yamaya T, Mae T, Ojima K. 1993. Changes in the content of two glutamate synthase proteins in spikelets of rice (Oryza sativa) plants during ripening. Plant Physiology 101, 1257–1262.[Abstract]
Hayashi H, Chino M. 1990. Chemical composition of phloem sap from the upper most internode of the rice plant. Plant and Cell Physiology 31, 247–251.
Henriksen GH, Raj Raman D, Walker LP, Spanswick RM. 1992. Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. Plant Physiology 99, 734–747.
Hirose N, Hayakawa T, Yamaya T. 1997. Inducible accumulation of mRNA for NADH-dependent glutamate synthase in rice roots in response to ammonium ions. Plant and Cell Physiology 38, 1295–1297.
Ireland RJ, Lea PJ. 1999. The enzymes of glutamine, glutamate, asparagine, and aspartate metabolism. In: Singh BK, ed. Plant amino acids. Biochemistry and biotechnology. New York: Marcel Dekker, 49–109.
Ishiyama K, Hayakawa T, Yamaya T. 1998. Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions. Planta 204, 288–294.[Web of Science][Medline]
Joy KW. 1988. Ammonia, glutamine and asparagine: a carbon–nitrogen interface. Canadian Journal of Botany 66, 2103–2109.
Joy KW, Blackwell RD, Lea PJ. 1992. Assimilation of nitrogen in mutants lacking enzymes of the glutamate synthase cycle. Journal of Experimental Botany 43, 139–145.
Kamachi K, Yamaya T, Hayakawa T, Mae T, Ojima K. 1992. Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiology 99, 1481–1486.
Kamachi K, Yamaya T, Mae T, Ojima K. 1991a. Multiple polypeptides of glutamine synthetase subunit in rice roots in vivo and in vitro. Agricultural and Biological Chemistry 55, 887–888.
Kamachi K, Yamaya T, Mae T, Ojima K. 1991b. A role for glutamine synthetase in the remobilization of leaf nitrogen during natural senescence in rice leaves. Plant Physiology 96, 411–417.
Kendall AC, Wallsgrove RM, Hall NP, Turner JC, Lea PJ. 1986. Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase. Planta 168, 316–323.
Lazof DB, Rufty Jr TW, Redinbaugh MG. 1992. Localization of nitrate absorption and translocation within morphological regions of the corn root. Plant Physiology 100, 1251–1258.
Lea PJ, Robinson SA, Stewart GR. 1990. The enzymology and metabolism of glutamine, glutamaute and asparagine. In: Miflin BJ, Lea PJ, eds. The biochemistry of plants, Vol. 16. Intermediary nitrogen metabolism. San Diego, Academic Press.
Lewis OAM, Chadwick S. 1983. A 15N investigation into nitrogen assimilation in hydroponically grown barley (Hordeum vulgare L. cv. Clipper) in response to nitrate, ammonium and mixed nitrate and ammonium nutrition. New Phytologist 95, 635–646.
Lewis OAM, James DM, Hewitt EJ. 1982. Nitrogen assimilation in barley (Hordeum vulgare L. cv. Mazurka) in response to nitrate and ammonium nutrition. Annals of Botany 49, 39–49.
Leyva A, Liang X, Pintor-Toro JA, Dixon RA, Lamb CJ. 1992. cis-element combinations determine phenylalanine ammonia lyase gene tissue-specific expression patterns. The Plant Cell 4, 263–271.
Long DM, Oaks A. 1990. Stabilization of nitrate reductase in maize roots by chymostatin. Plant Physiology 93, 846–850.
Lux A. 1986. Changes in plastids during xylem differentiation in barley root. Annals of Botany 58, 547–550.
Mäck G. 1995. Organ-specific changes in the activity and subunit composition of glutamine synthetase isoforms in barley (Hordeum vulgare L.) after growth on different levels of NH4+. Planta 196, 231–238.
Mae T, Makino A, Ohira K. 1981. The remobilization of nitrogen related to leaf growth and senescence in rice plants (Oryza sativa L.). Plant and Cell Physiology 22, 1067–1074.
Marttila S, Saarelainen R, Porali I, Mikkonen A. 1993. Glutamine synthetase isoenzymes in germinating barley seeds. Physiologia Plantarum 88, 612–618.
Matoh T, Takahashi E. 1981. Glutamate synthase in greening pea shoots. Plant and Cell Physiology 22, 727–731.
Miflin BJ, Lea PJ. 1980. Ammonia assimilation. In: Miflin BJ, ed. The biochemistry of plants, Vol. 5. Toronto: Academic Press.
Morita S, Lux A, Enstone DE, Peterson CA, Abe J. 1996. Re-examination of rice seminal root ontogeny using fluorescence microscopy. Japanese Journal of Crop Science 65, 37–38.
Pate JS. 1980. Transport and partitioning of nitrogenous solutes. Annual Review of Plant Physiology 31, 313–340.[Web of Science]
Peat LJ. 1996. The influence of nitrogen nutrition on the cellular localization of ammonia assimilation enzymes in barley (Hordeum vulgare L.). PhD thesis, University of Manchester, UK.
Peat LJ, Tobin AK. 1996. The effect of nitrogen nutrition on the cellular localization of glutamine synthetase isoforms in barley roots. Plant Physiology 111, 1109–1117.[Abstract]
Pereira S, Carvalho H, Sunkel C, Salema R. 1992. Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves of Solanum tuberosum L. Protoplasma 167, 66–73.
Pereira S, Pissarra J, Sunkel C, Salema R. 1996. Tissue-specific distribution of glutamine synthetase in potato tubers. Annals of Botany 77, 429–432.
Rufty TW, Thomas JF, Remmler J, Campbell WH, Volk RJ. 1986. Intercellular localization of nitrate reductase in roots. Plant Physiology 82, 675–680.
Sakurai N, Hayakawa T, Nakamura T, Yamaya T. 1996. Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development. Planta 200, 306–311.
Stitt M, Krapp A. 1999. The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background. Plant, Cell and Environment 22, 583–621.
Suzuki A, Gadal P, Oaks A. 1981. Intracellular distribution of enzymes associated with nitrogen assimilation in roots. Planta 151, 457–461.
Suzuki A, Vidal J, Gadal P. 1982. Glutamate synthase isoforms in rice. Immunological studies of enzymes in green leaf, etiolated leaf, and root tissues. Plant Physiology 70, 827–832.
Tatsumi J. 1982. Growth of crops and transport of nitrogen (3). (In Japanese) Growth of crop roots and transport of nitrogen. Agriculture and Horticulture 57, 631–638.
Tobin AK, Ridley SM, Stewart GR. 1985. Changes in the activities of chloroplast and cytosolic isoforms of glutamine synthetase during normal leaf growth and development in wheat. Planta 163, 544–548.
Wallsgrove RM, Keys AJ, Bird IF, Cornelius, MJ, Lea PJ, Miflin BJ. 1980. The location of glutamine synthetase in leaf cells and its role in the reassimilation of ammonia released in photorespiration. Journal of Experimental Botany 31, 1005–1017.
Wallsgrove RM, Lea PJ, Miflin BJ. 1979. Distribution of the enzymes of nitrogen assimilation within the pea leaf cell. Plant Physiology 63, 232–236.
Woodall J, Forde BG. 1996. Glutamine synthetase polypeptides in the roots of 55 legume species in relation to their climatic origin and the partitioning of nitrate assimilation. Plant, Cell and Environment 19, 848–858.
Yamaya T, Hayakawa T, Tanasawa K, Kamachi K, Mae T, Ojima K. 1992. Tissue distribution of glutamate synthase and glutamine synthetase in rice leaves. Occurrence of NADH-dependent glutamate synthase protein and activity in the unexpanded non-green leaf blades. Plant Physiology 100, 1427–1432.
Yamaya T, Tanno H, Hirose N, Watanabe S, Hayakawa T. 1995. A supply of nitrogen causes increase in the level of NADH-dependent glutamate synthase protein and in the activity of the enzyme in roots of rice seedlings. Plant and Cell Physiology 36, 1197–1204.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. C. Leegood Roles of the bundle sheath cells in leaves of C3 plants J. Exp. Bot., May 1, 2008; 59(7): 1663 - 1673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tabuchi, T. Abiko, and T. Yamaya Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.) J. Exp. Bot., July 1, 2007; 58(9): 2319 - 2327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Lunn Compartmentation in plant metabolism J. Exp. Bot., January 1, 2007; 58(1): 35 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hayakawa, T. Kudo, T. Ito, N. Takahashi, and T. Yamaya ACT Domain Repeat Protein 7, ACR7, Interacts with a Chaperone HSP18.0-CII in Rice Nuclei Plant Cell Physiol., July 1, 2006; 47(7): 891 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Masclaux-Daubresse, M. Reisdorf-Cren, K. Pageau, M. Lelandais, O. Grandjean, J. Kronenberger, M.-H. Valadier, M. Feraud, T. Jouglet, and A. Suzuki Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco Plant Physiology, February 1, 2006; 140(2): 444 - 456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Abiko, M. Obara, A. Ushioda, T. Hayakawa, M. Hodges, and T. Yamaya Localization of NAD-Isocitrate Dehydrogenase and Glutamate Dehydrogenase in Rice Roots: Candidates for Providing Carbon Skeletons to NADH-Glutamate Synthase Plant Cell Physiol., October 1, 2005; 46(10): 1724 - 1734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kichey, J. Le Gouis, B. Sangwan, B. Hirel, and F. Dubois Changes in the Cellular and Subcellular Localization of Glutamine Synthetase and Glutamate Dehydrogenase During Flag Leaf Senescence in Wheat (Triticum aestivum L.) Plant Cell Physiol., June 1, 2005; 46(6): 964 - 974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ishiyama, E. Inoue, M. Tabuchi, T. Yamaya, and H. Takahashi Biochemical Background and Compartmentalized Functions of Cytosolic Glutamine Synthetase for Active Ammonium Assimilation in Rice Roots Plant Cell Physiol., November 15, 2004; 45(11): 1640 - 1647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Uheda, K. Maejima, and N. Shiomi Localization of Glutamine Synthetase Isoforms in Hair Cells of Azolla Leaves Plant Cell Physiol., August 15, 2004; 45(8): 1087 - 1092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Loque and N. von Wiren Regulatory levels for the transport of ammonium in plant roots J. Exp. Bot., June 1, 2004; 55(401): 1293 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sonoda, A. Ikeda, S. Saiki, N. v. Wiren, T. Yamaya, and J. Yamaguchi Distinct Expression and Function of Three Ammonium Transporter Genes (OsAMT1;1 - 1;3) in Rice Plant Cell Physiol., July 15, 2003; 44(7): 726 - 734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.K. Schjoerring, S. Husted, G. Mack, and M. Mattsson The regulation of ammonium translocation in plants J. Exp. Bot., April 15, 2002; 53(370): 883 - 890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Bowsher and A. K. Tobin Compartmentation of metabolism within mitochondria and plastids J. Exp. Bot., April 1, 2001; 52(356): 513 - 527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Walker, Z.-H. Chen, K. E. Johnson, F. Famiani, L. Tecsi, and R. C. Leegood Using immunohistochemistry to study plant metabolism: the examples of its use in the localization of amino acids in plant tissues, and of phosphoenolpyruvate carboxykinase and its possible role in pH regulation J. Exp. Bot., April 1, 2001; 52(356): 565 - 576. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||