rabidopsis c h i p i s d i i l- a g i d l-citrulline for ... · organic n and which have high basal...

Plant Physiol. (1993) 101: 429-434

A rabidopsis C h I oro p I as t s D i ssi m i I a te L- A r g i n i n e an d L-Citrulline for Use as N Source'

Robert A. Ludwig*

Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064

When aseptically grown on defined medium with either L-arginine, i-citrulline, or nitrate as the sole N source, Arabidopsis plants grew and developed normally. Three catabolic activities, i-arginine iminohydrolase, i-ornithine carbamoyltransferase, and carbamate kinase, were found in stromal fractions of purified Arabidopsis chloroplasts. These activities dissimilate i-arginine and/or i-citrulline into i-ornithine, ammonium, bicarbonate, and ATP. In physiological tests with purified, intact Arabidopsis chloroplasts, i- [g~anido-'~C]arginine was rapidly taken up and about 10% was decomposed, releasing 'TO2. Therefore, chloroplasts can take up and dissimilate i-arginine. In principle, chloroplast arginine dissimilation allows Arabidopsis to use i-arginine and/or i-citrulline as general N sources for growth. However, plants rarely encounter exogenous i-arginine andjor i-citrulline in amounts exceeding their biosynthetic needs. Therefore, i-arginine and L-citrulline might serve as endogenous N sources.

By default, plants do not normally grow on exogenous organic N compounds as primary N sources. Rather, plants concede organic N to soil microflora, which are avid users of organic N and which have high basal metabolic rates. How- ever, plants make organic N compounds in specific tissues as endogenous N sources for growth. Excess organic N compounds are made in specific tissues for systemic transport. For example, Alnus and Casuarina symbiotic root nodules fix Nz and then convert excess fixed N to L-citrulline and L-Arg for systemic transport as general N sources (Miettinen and Vittanen, 1952; Leaf et al., 1958). As shown by histochemical studies, Alnus root mitochondria comprise the site of L- citrulline synthesis (Scott et al., 1981). In cyanobacteria, C0,- fixing "dark reactions" also yield ~-citrulline (Linko et al., 1957). Biochemically, L-citrulline is invariably produced by coupled CPS and ornithine carbamoyltransferase activities (Davis, 1986). How are L-citrulline and L-Arg then used by Alnus and Casuarina as systemic N sources, and by cyanobacteria as both N and C sources?

When bacteria, fungi, and animals dissimilate the L-Arg guanido moiety for use as N source, first urea and then ammonium are produced. In plants, leaf ammonium is reassimilated principally by concerted action of chloroplast GS and GOGAT activities. Plants have two GS isoforms; chloroplast GS is strongly light-inducible, whereas cytosolic GS is constitutive (Wallsgrove et al., 1976; Mann et al., 1980).

Supported by grants to the author from the National Institutes of Health (Rol GM-37032) and the National Science Foundation (DMB-8805709).

* e-mail, ludwig e biology. ucsc. edu. 429

Unquestionably, chloroplasts mediate high ammonium assimilation rates. Arabidopsis chloroplast mutants defective in Fd-dependent GOGAT fail to photorespire (Somerville and Ogren, 1980).

Because Alnus and Casuarina N assimilation processes are little studied, Arabidopsis plants were tested for growth on defined medium with either exogenous L-Arg or L-citrulline as the sole N source. A11 plants grew well. In follow- up studies with purified Arabidopsis leaf chloroplasts, three stromal activities, AIH, OCT, and CK, were identified. A11 three activities were present at high levels in light- adapted chloroplasts. Together, these three activities dissimilate L-Arg or L-citrulline into L-ornithine, ammonium, bicarbonate, and ATP. Certainly the ammonium, and possi- bly the L-ornithine, served as general N sources. In physiological tests,purified,intactAra bidopsischloroplastsrapidlytook up limiting ~-[guanido-'~C]Arg and decomposed about 10%, of it releasing I4CO2. Arabidopsis leaves thus dissimilate L-Arg and/or L-citrulline, yielding chloroplastic ammonium, which may then be directly reassimilated into L-Gln and L-glutamate.

MATERIALS A N D METHODS

Growth of Plants in Defined Medium

Arabidopsis thaliana ecotype Columbia seeds were surface sterilized for 1 h with 1% sodium hypochlorite and 0.5% SDS solution and washed three times in sterile water. Resid- ual hypochlorite was then chemically removed by soaking treated seeds for 2 h in 2 mM ascorbic acid. Seed was sown in sterile jars (Magenta Co.) or 2-cm high Petri plates, strewn with sterile Perlite mixed with Murashige and Skoog basal salts (Murashige and Skoog, 1962) in which ammonium nitrate was eliminated, KC1 was substituted for KN03, and 2 meq L-' of either nitrate, L-citrulline, or L-Arg was then added as sole N source. In comparison with the same medium supplemented with 30 meq L-' of KN03, in media containing 2 meq L-' of total N, growth was slower, and thus limited by available N. Purified agar (Difco) was also added (0.4% w/v) to solidify the medium. Plants were grown in controlled environment chambers (Percival) with a 12-h photoperiod at 22OC under standard humidity and light conditions.

Abbreviations: AIH, L-arginine iminohydrolase; AS, L-argininosuccinate; CK, carbamate kinase; CPS, carbamoylphosphate synthetase; GOGAT, L-g1utamine:Z-oxoglutarate amidotransferase; GS, L- glutamine synthetase; OCT, L-omithine carbamoyltransferase; SDH, succinate dehydrogenase.

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430 Ludwig Plant Physiol. Vol. 101, 1993

Chloroplast lsolation and Purification

Intact A. thaliana ecotype Columbia chloroplasts were isolated from illuminated leaves as follows: leaves (20 g) were sliced, mixed with 50 mL of ice-cold grinding buffer (10 mM sodium pyrophosphate, 5 mM MgC12,2 mM sodium ascorbate, and 0.33 M sorbitol, pH 6.5), and homogenized for 5 s (Polytron, Brinkmann Instruments). The homogenate was filtered through cheesecloth and Miracloth (Calbiochem), and quickly centrifuged for 1 min at 8,OOOg in a swinging bucket rotor. The pellet was washed with grinding buffer, resuspended in 0.25 mL of resuspension buffer (50 mM Hepes, pH 7.6, 2 mM EDTA, 1 mM MgCI2, 1 mM MnC12, and 0.33 M

sorbitol), layered on a 17-mL 50% (w/v) Percoll gradient in gradient buffer (50 mM sodium Mops, pH 7.8, 2 mM EDTA, 0.15% BSA, and 0.33 M sorbitol), and centrifuged for 10 min at 5,OOOg in a swinging bucket rotor at 2OC. To form the gradient, the Percoll solution in gradient buffer was precen- trifuged for 100 min at 10,OOOg in a swinging bucket rotor at 2OC. The lower (intact) chloroplast band was removed and pelleted. Intact chloroplasts were resuspended in 0.5 mL of resuspension buffer, examined by light microscopy, and fro- zen in 0.05-mL aliquots at -7OOC.

AIH, OCT, and CK Assays

AIH activity was assayed as L-Arg-dependent synthesis of L-citrulline, derivatized to its oxime, and measured by double- beam spectrophotometry (Schimke, 1970a). In these assays, 0.05 M bis-Tris-C1 buffer, pH 6.5, was substituted for phosphate buffer. Control samples with no added chloroplast stromal fraction were subtracted to yield net AIH activity. OCT was similarly assayed as L-ornithine and carbamoylphosphate-dependent synthesis of L-citrulline, which was again derivatized to its oxime amd measured by double-beam spectrophotometry (Schimke, 1970b). Control samples with no added chloroplast stromal fraction were subtracted to yield net OCT activity. Total protein (Bradford, 1976) was deter- mined for isolated chloroplast stromal fractions. CK (ATP:carbamate phosphotransferase) was assayed as follows: purified Arabidopsis chloroplasts were assayed for carbamoylphosphate and ADP-dependent ATP synthesis coupled to firefly luciferase. CK assay mixtures contained 0.01 M

carbamoylphosphate, 5 mM ADP, and reconstituted firefly luciferase-luciferin reagent (Sigma) also containing Gly buffer salts, MgS04, EDTA, and human serum albumin; chloroplast stromal fraction was added to 4 mg mL-'. Luminometer readings were captured at 0.5-s intervals, stored on computer disk, and plotted; curves thus include 120 data points per min (not shown). After approximately 1 min, firefly luciferase reactions become O2 limited (Lundin et al., 1986).

14C02 Release by lntact Chloroplasts lncubated with ~- [guanido- '~C]Arg

Purified, intact Arabidopsis chloroplasts were diluted into chloroplast resuspension buffer supplemented with 50 pg mL-' of kanamycin sulfate and left on ice for 1 h. To these chloroplasts was added 17 WM ~-[guanido-'~C]Arg (58 mCi mmol-I); samples (1.0 mL) were then introduced into serum bottles (30 mL) each containing a microfuge tube supplied

with 0.5 mL of 1 N NaOH; serum bottles were stoppered and incubated at 3OoC with gentle shaking. At the times indicated, 1.0 mL of 2 N H2S04 was injected to terminate reactions and release COz. Samples were gently shaken overnight, and NaOH solutions were analyzed both for I4C decay by liquid scintillation spectrometry and for total protein (Bradford, 1976).

RESULTS

Arabidopsis Plants Utilize i-Citrulline and i-Arg as N Sources for Growth

A. thaliana ecotype Columbia plants were grown in sterile culture and in defined medium with either nitrate, L-citrulline, or L-Arg as sole N source. By comparison with plants grown in similar medium but with nitrate, growth was slow. Hence, plant growth at 2 meq L-' of total N under these conditions was N limited. AI1 plants grew well, whereas plants grown in the same medium with no added N source failed to grow (Fig. 1). Up through and including flowering, silique formation, and seed yield, no significant differences in growth or development with any of the three N sources were observed. From subsequent HPLC analyses (data not presented), no L-Arg catabolic intermediates, such as L-ornithine, accumulated in conditioned growth medium. There- fore, Arabidopsis plants efficiently utilized both L-citrulline and L-Arg as N source for growth.

To facilitate its use as N source, L-Arg might be catabolized by an arginase activity to first yield L-ornithine and urea and then by a urease activity to yield ammonium. Therefore, Arabidopsis leaf tissue homogenate was examined for arginase activity by assay for L-Arg-dependent L-ornithine production. None was observed. Neither was arginase activity detected in purified chloroplast fractions. Therefore, an alternative pathway for L-Arg utilization by Arabidopsis plants was sought.

AIH, OCT, and CK Activities Are Present at High Levels in Arabidopsis Chloroplasts

Arabidopsis chloroplasts were purified from light-adapted leaves of plants grown in aseptic culture on nitrate as N source. Purified chloroplasts were lysed by dilution and then assayed for L-Arg catabolic activities. Stromal fractions showed three such activities: (a) AIH, which hydrolyzes L-

Arg by ammoniolysis to yield L-citrulline; (b) OCT, which decomposes L-citrulline by phosphorolysis to yield L-ornithine and carbamoylphosphate; and (c) CK, which, in the presence of ADP, decomposes carbamoylphosphate into ATP, bicarbonate, and ammonium (Table I, Fig. 2). L-Arg- dependent L-citrulline production was not stimulated by added fumarate and, therefore, did not result from coupled AS synthetase and AS lyase activities operating in physiological reverse (see "Discussion"). Formally, because the CK assay might also be catalyzed by CPS (see "Discussion"), any in vivo catabolic and/or anabolic context cannot be inferred from these results alone. Even though assay conditions for chloroplast AIH, OCT, and CK were not optimized, high activities (>0.1 pmol min-' mg-' soluble protein) were observed. Because it did not accumulate in the plant growth



Chloroplast L-Arg Dissimilation 431

medium, L-ornithine might also be further catabolized (nottested).

Because Arabidopsis, like fungi (Davis, 1986), may alsopossess a mitochondrial OCT isoform, purified chloroplastfractions were analyzed for SDH activity (Hedrick andSmith, 1968), indicative of mitochondrial contamination.No SDH activity (<1 nmol min"1 rng~' protein) was detected.Therefore, OCT, AIH, and CK activities were attributed tochloroplasts.

Arabidopsis Chloroplasts Take Up L-[guanido-14C]Arg andRelease 14CO2

To test whether the Arabidopsis chloroplast AIH, OCT, andCK activities constituted a catabolic pathway, intact Arabi-dopsis chloroplasts, purified from leaf tissue of plants adaptedto continuous light, were incubated with L-[guanido-14C]-Arg in the dark. Chloroplasts rapidly took up radiolabeled L-Arg and released 14CO2 (Fig. 3). In the absence of addedkanamycin sulfate, a chloroplast protein synthesis inhibitor,this behavior was erratic (data not presented). In the presenceof 50 /ug mL~' kanamycin, however, approximately 10% wasdecomposed, as evidenced by 14CO2 release, at rates approx-

Table I. Purified Arabidopsis leaf chloroplast AIH (reaction }), OCT(reaction 2), and CK (reaction 3) activities

Chloroplast Function

Thylakoid Stromal

L-Arginine + H2O — > L-citrulline + NH3

L-Citrulline + Pi <-» L-ornithine + carba-moylphosphate

Carbamoylphosphate + H2O + ADP — >NH3 + HCOJ + ATP

Sum: i-arginine + 2H2O + ADP, Pi ->L-ornithine + 2NH3 + HCOJ + ATP

<0.1J 4.35"<0.1' 1.75'

1.20b

l L-citrulline min ' mg 'protein. ATP min ' mgprotein.

imately linear with incubation time (30 nmol min"1 mg"1

protein). Intact chloroplast preparations showed quantitativeuptake of limiting (17 ^M) u-Arg, measured as relative radio-label remaining in low-speed supernatants (data not pre-sented). Nevertheless, decarboxylative activity might haveoccurred in some broken chloroplasts. To test this alternative,L-[guanido-14C]Arg-dependent CO2 release was retested with

Figure 1. Arabidopsis plants grown in aseptic culture and on defined medium with selected N sources: A, L-Arg; B, L-citrulline; C, no N source; D, nitrate. Representative plants were photographed 10 d postgermination.




0.4

Y 0.3 o = 0.2

O m

(u

0 . q

O O 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

time (min)

Figure 2. CK (ATP:carbamate phosphotransferase) activity in purified Arabidopsis chloroplast extracts. Stromal fractions from purified, intact Arabidopsis chloroplasts were assayed for carbamoylphosphate- and ADP-dependent ATP synthesis by coupling to’firefly luciferase. Luminometer readings were captured at 0.5-s intervals, stored on computer disk, and plotted; curves thus include 120 data points per min (not shown). After approximately 1 min, firefly luciferase reactions become O2 limited (Lundin et al., 1986). Data points represent net carbamoylphosphate-dependent ATP synthesis per milligram of stromal protein in the presence (upper curve) or in the absence (lower curve) of added chloroplast stromal fraction. From regression analysis, the data in the upper curve yielded an initial velocity of 1.2 pmol ATP min-’ mg-’ protein.

deliberate lysis of chloroplasts by dilution into nonosmoti- cally protective buffer. Similar results were obtained. Because no stimulation occurred with deliberately lysed chloroplasts, physiological activity was attributed to intact chloroplasts. Whether chloroplast L-Arg catabolism was either photore- gulated or stimulated by exogenous substrate(s) was not tested.

In steady-state limitation, chloroplast L-Arg is avidly assim- ilated both by ribosylation, to yield aminoacyl-tRNA (for protein synthesis), and by decarboxylation, to yield agmatine (for polyamine synthesis). Thus, chloroplast anabolic reactions might favorably compete with catabolic reactions for limiting L-Arg. At the leve1 of primary control, physiological rates of L-Arg synthesis in fungi and animals are limited by mitochondrial ATP and ammonium availability (Davis, 1986). Because plant leaves normally experience steady-state ammonium limitation, chloroplast L-Arg assimilation should predominate. Hence, most ~-[guanido-’~C]Arg taken up by chloroplasts under limiting conditions must enter anabolic

DISCUSSION Because Arabidopsis plants grow normally on exogenous L-

citrulline and/or L-Arg as the sole N source, by implication,

pools.

the three L-Arg catabolic activities discovered in leaf chloroplasts might play an important role in the use of these amino acids, whether taken up exogenously or generated endoge- nously. Through concerted activity of these three enzymes, each dissimilated L-citrulline and/or L-Arg equivalent would yield one L-ornithine equivalent, one (for L-citrulline) or two (for L-Arg) ammonium equivalents, and one bicarbonate equivalent (Fig. 4), which would be dehydrated to yield COz and water by the stromal carbonic anhydrase activity. In the presence of 2-ketoglutarate, ammonium would drive L-glutamate synthesis via concerted chloroplast GS and GOGAT activities (Wallsgrove et al., 1980; Somerville and Ogren, 1980).

What is the physiological significance of L-Arg and/or L- citrulline catabolism to normally N-limited plants, such as Arabidopsis? Relative chloroplast L-Arg and/or t-citrulline anabolic and catabolic rates might be subject to primary physiological control, in which case one set of enzymes might function reversibly. The four L-Arg biosynthetic activities are, respectively, Arg-specific CPS (CPS-A), OCT, AS synthetase, and AS lyase (Fig. 4). In fungi and animals, the first two L-

Arg biosynthetic activities are usually mitochondrial, the last two cytosolic (Davis, 1986). Fumarate produced by AS lyase activity is hydrated and then oxidized to yield oxaloacetate, which then transaminates with L-glutamate (exported from mitochondria in animals and fungi but, presumably, from chloroplasts in plants), regenerating L-aspartate.

In soybean cotyledons, the universal L-Arg biosynthetic pathway was confirmed by physiological radiolabeling studies (Micallef and Shelp, 1989). However, the explicit com- partmentation of these four higher plant enzymes remains unclear. Because sequestered carbamoylphosphate pools are used exclusively to make either L-Arg or pyrimidines (Davis, 1986), precisely where, in plant cells, ammonium is assimi- lated as carbamoylphosphate is important. In severa1 dico-

1.u I

““iI_ O O 10 20 30 40 50

time (min)

Figure 3. l4CO, release by intact Arabidopsis chloroplasts incubated with ~-[guanido-’~C]Arg (mean value of duplicate experimental samples).



Chloroplast L-Arg Dissimilation

- NH, 1 Mitochondrion

433

Chloroplest stroma

Ornithine Arginine Glutamate 2-KG

\f \/

A A Glutamate 2-KG

Arginine

Malate

Fumarate

Argininosuccinate I & I Ornithine Citrulline Cytosol \/

Ornithine Citrulline

Figure 4. Hypothetical endogenous L-citrulline and/or L-Arg shuttle from mitochondria to chloroplasts, comprising: mitochondrial CPS-A ( I ) , anabolic OCT (2), cytosolic AS synthetase (3), AS lyase (4), chloroplast stromal AIH (S) , catabolic OCT (6) , CK (7), L-Gln synthetase @), and Fd-dependent GOGAT (9). Accessory activities include: mitochondrial fumarase (10), cytosolic malate dehydrogenase (1 I ) , and cytosolic L-

asparate aminotransferase (1 2). Notes: if fumarase activity is exclusively mitochondrial, fumarate and L-malate might be antiported across the mitochondrial inner membrane. The driving force of this shuttle is a mitochondrial ammonium or L-Cin supply (see text). If this shuttle involves only L-citrulline synthesis (decoupled from L-Arg synthesis), then reactions 3,4, 5, 10, 11, and 12 are bypassed. In theory, this shuttle might metabolically couple mitochondria and chloroplasts either in the same cell or in dis- parate tissues, in which case L-Arg and/or L-

citrulline are systematically transported.

tyledonous plants, leaf chloroplast CPS (CPS-P) activities have been reported. Because this CPS-P is associated with aspartate carbamoyltransferase, pyrimidines seem to be syn- thesized in chloroplasts (Shibata et al., 1986). Might chloroplast CPS also mediate L-Arg synthesis? Pisum sativum (pea) has been reported to possess a single CPS activity with hybrid catalytic properties (Ruiter and Kolloffel, 1982).

Alternatively, the three observed Arabidopsis chloroplast activities might play anabolic, not catabolic, roles. In that case, L-Arg and L-citrulline are used as N sources in some other way. Arabidopsis chloroplasts may well sustain de novo L-Arg biosynthesis. Although chloroplast OCT catalyzes a freely reversible reaction in extracts, it might operate strictly biosynthetically in vivo. Likewise, the carbamoylphosphate and ADP-dependent ATP synthesis observed in chloroplast extracts might be due to a CPS catalytic subunit, assayed in physiological reverse. Heretofore, chloroplast CPS activity has been considered CPS-P, coupled to aspartate carbamoyltransferase activity for pyrimidine synthesis (Shibata et al., 1986). However, observed CPS activity was completely in- dependent of added L-Gln (Shibata et al., 1986), unprece- dented for a CPS-P activity. Lacking further information, this

chloroplast activity might be considered a degenerate CPS, able to drive both L-Arg and pyrimidine synthesis.

However, the AIH activity reported here is unlikely to be anabolic. The universally conserved AS synthetase and AS lyase activities are exclusively anabolic (Davis, 1986). In Arabidopsis chloroplast extracts, because added fumarate did not stimulate L-Arg-dependent L-citrulline production, that coupled, reversible AS synthetase and AS lyase activities constitute the observed AIH activity is highly improbable. Moreover, because chloroplasts have no known fumarate source, any in vivo reversibility is unlikely to extend to all four L-Arg biosynthetic reactions. Therefore, from our results chloroplasts carry at least one, L-Arg-specific, uniquely catabolic activity.

As a third alternative, chloroplasts might carry both L-Arg catabolic and anabolic activities, including isoforms exclusive to each pathway. In Pseudomonas bacteria, which can both assimilate and dissimilate L-Arg in one compartment, separate and distinct activities so participate (Wauven et al., 1984). Although they express the universally conserved four L-Arg biosynthetic activities, Pseudomonas bacteria also use AIH, catabolic OCT (with quite different kinetic properties), and




CK activities to anaerobically dissimilate L-Arg, as well as use a succinylation-dependent pathway to aerobically dissimilate L- Arg. Anaerobically, the former three catabolic activities drive ATP synthesis at relatively high in vivo energy charge and thus allow the bacteria to use L-Arg as an energy source.

Leaf chloroplasts of nonnodulated plants like Arabidopsis might indeed catabolize L-Arg and/or L-citrulline, as observed here, even when plants are grown on nitrate as the exogenous N source. If so, is there an endogenous source of steady- state, excess L-Arg and/or L-citrulline for use as catabolic substrate? At the leve1 of primary control, L-Arg biosynthetic rates in fungi and animals are limited by mitochondrial ATP and ammonium availability. Because plants normally grow in ammonium-limited conditions, L-Arg biosynthesis rates would reflect this limitation. However, plant tissues do experience physiological conditions of intense ammonium flux in mitochondria, such as during photorespiration. Likewise, in N2-fixing Alnus and Casuarina root nodules, mitochondria synthesize L-citrulline from fixed N (ammonium) in large amounts and for export to other tissues (Leaf et al., 1958; Scott et al., 1981). In nonnodulated plants, might leaf mitochondria also produce L-citrulline in response to a strong mitochondrial ammonium flux, such as during photorespir- atory Gly combustion? If so, then leaf mitochondrial carbamoylphosphate, and thus L-citrulline and/or L-Arg, might be made at rates in excess of steady-state biosynthetic needs. The excess might be dissimilated by chloroplasts and thus shuttle COz, as well as ammonium, between leaf mitochondria and chloroplasts (Fig. 4). As a physiological rationale, this shuttle might then metabolically channel C 0 2 , produced by other mitochondrial oxidations, to chloroplasts for efficient reassimilation.

ACKNOWLEDCMENTS

I thank Harry Beevers, Rowland Davis, Dieter Haas, Lincoln Taiz, and Dick Weiss for helpful discussions.

Received June 22, 1992; accepted October 5, 1992. Copyright Clearance Center: 0032-O889/93/lOl/0429/06.

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