nitrate activation cytosolic protein kinases diverts ... · sucrose formation andonthe activities...

Plant Physiol. (1992) 100, 7-120032-0889/92/1 00/0007/06/$01 .00/0

Received for publication January 22, 1992Accepted April 21, 1992

Nitrate Activation of Cytosolic Protein Kinases DivertsPhotosynthetic Carbon from Sucrose to Amino Acid

Biosynthesis

Basis for a New Concept

Marie-Louise Champigny* and Christine FoyerPhotosynthese et Metabolisme (Unite de Recherche Associ6e Centre National de la Recherche Scientifique D

1128), Batiment 430, Universit6 Paris-Sud, F-91405 Orsay cedex, France (M.-L.C.); and MWtabolisme et Nutritiondes Plantes, Institut National de la Recherche Agronomique, route de Saint-Cyr, F-78000 Versailles, France (C.F.)

ABSTRACT

The regulation of carbon partitioning between carbohydrates(principally sucrose) and amino acids has been only poorly char-acterized in higher plants. The hypothesis that the pathway ofsucrose and amino acid biosynthesis compete for carbon skeletonsand energy is widely accepted. In this review, we suggest a mech-anism involving the regulation of cytosolic protein kinases wherebythe flow of carbon is regulated at the level of partitioning betweenthe pathways of carbohydrate and nitrogen metabolism via thecovalent modulation of component enzymes. The addition of ni-trate to wheat seedlings (Triticum aestivum) grown in the absenceof exogenous nitrogen has a dramatic, if transient, impact onsucrose formation and on the activities of sucrose phosphate syn-thase (which is inactivated) and phosphoenolpyruvate carboxylase(which is activated). The activities of these two enzymes are mod-ulated by protein phosphorylation in response to the addition ofnitrate, but they respond in an inverse fashion. Sucrose phosphatesynthase is inactivated and phosphoenolpyruvate carboxylase isactivated. Nitrate functions as a signal metabolite activating thecytosolic protein kinase, thereby modulating the activities of atleast two of the key enzymes in assimilate partitioning and redi-recting the flow of carbon away from sucrose biosynthesis towardamino acid synthesis.

For many years, extensive research has been devoted tothe regulation of the synthesis of sucrose, the exported formof photoassimilate in most leaves (7, 11, 17, 43). This hasprovided the basic elements for the description of the finecontrol network that regulates sucrose synthesis and photo-synthetic C partitioning between the cytosol (sucrose synthe-sis) and the chloroplasts (starch synthesis). In leaves, NO3-assimilation takes place in the same compartments, i.e. cyto-sol (NO3- reduction to NO2-) and chloroplasts (reduction ofNO2- to NH4' and assimilation of the latter into glutamate)as sucrose and starch synthesis, respectively. The reductionof N02- to NH4+ uses photochemically generated reducingpower, as does the reduction of CO2 to carbohydrate, and is

considered to be an important sink for the products of pho-tosynthetic electron flow (31). In consideration of the com-mon requirements of NO3- and CO2 metabolism in terms ofreducing power, ATP, and carbon skeletons, important ques-tions have been raised concerning the impact of NO3- onphotosynthesis and the partitioning of photosynthetic prod-ucts to amino acids at the expense of carbohydrate synthesis(44).The effect of nitrate on the rate of sucrose synthesis was

studied with mature leaves detached from wheat seedlings(Triticum aestivum) (49). A linear inverse relationship wasshown between the rate of sucrose synthesis and the rate ofuptake and assimilation of NO3-, with no effect on insolublecarbohydrate synthesis. The effect of NO3- on sucrose syn-thesis appeared within the first hour after the leaves werefed NO3-. It slowed the rate of synthesis without altering thesucrose storage capacity of leaves. The rate of CO2 fixationwas only slightly reduced (5).The diversion of 14C-labeled photosynthetic carbon away

from carbohydrate synthesis toward organic acid and aminoacid synthesis in leaves fed with NO3- provided evidencethat sucrose synthesis is regulated at the level of partitioningof C between the two pathways in the leaves of higher plants(5). The increased carbon flux to amino acids is alwaysassociated with a decrease in the PEP' content and theactivation of PEPcase in the leaves. The activation is notimpaired by cycloheximide (48). The increased demand forcarbon skeletons created by high rates of NO3- or NH4'assimilation was thus met by the diversion of fixed carbon tothe anapleurotic pathway. Concomitantly, the rate of sucrosesynthesis was restricted by a decrease in SPS activity. Theseresults are in agreement with the suggestions that (a) PEPcaseis the protein whose activity is most affected by N availabilityin leaves (45) and (b) SPS activity is a major component thatcontrols the flux of carbon into sucrose (29, 43). Both these

1 Abbreviations: PEP, phosphoenolpyruvate; PEPcase, phospho-enolpyruvate carboxylase; SPS, sucrose phosphate synthase.

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CHAMPIGNY AND FOYER

enzymes have been shown to undergo light-dependent reg-ulation (28, 37, 39).

REGULATION OF SPS IN LEAVES

Located in the leaf cytosol, SPS (EC 2.4.1.14) catalyzes thefollowing reaction,

Fructose-6-P + UDP-Glucose -- Sucrose P + UDP (1)

which is the rate-limiting step in the photosynthetic produc-tion of sucrose. Furthermore, experiments with transgenicplants have shown that carbon partitioning between sucroseand starch is directly correlated to the amount of SPS activityin the leaf (55). The activity of this allosteric enzyme iscontrolled at two levels. Fine control confers rapid regulationin enzyme activity brought about by fluctuations in the levelof effector metabolites such as glucose-6-P (an activator), andphosphate (an inhibitor, 9). Coarse control results in relativelystable changes in the maximum extractable activity as meas-ured in vitro. This happens with no change of the amount ofSPS protein, suggesting that a reversible posttranslationalmodification of the protein structure has taken place ratherthan a change in protein turnover. Fluctuations in SPS activ-ity have been observed in leaves during water stress (38) asa result of feedback control when sucrose accumulates (43),in relation to the rate of photosynthesis or following light/dark transitions (37, 40).

Light activation is not the result of a direct and simpleeffect of light per se as it is, for example, with the thioredoxin-modulated enzymes of the Benson-Calvin cycle (2). As therate of photosynthesis increases, there is an activation of SPS,which may be directly or indirectly linked to changes in theavailability of Pi (8). The build-up of metabolites such asdihydroxyacetone phosphate and fructose-2,6-bisphosphateprovides a type of feedforward control that is essential to theactivation process (13). These are the signals that regulatelight activation. The changes in SPS activity with light-darktransitions occur relatively slowly (within 30 min) and appearto be associated with kinetic properties of the protein ratherthan changes in Vmax. This suggests that regulation occurs bysome type of covalent modification process.Two forms of SPS with markedly different properties were

shown to exist in spinach leaves (41, 50). Huber et al. (16)elucidated the mechanism of modulation of SPS activity byproviding evidence for the phosphorylation of the SPS pro-tein in leaves. A direct link was established between thephosphorylation level and inactivation of the enzyme in thedark, with dephosphorylation and activation occurring in thelight. A soluble protein kinase was shown to be involved inthe interconversion of SPS between an active (dephospho-rylated) and a less active (phosphorylated) form (18). Theprotein was thought to be specific for SPS (16). Recent studieshave identified some elements of 'fine' and 'coarse' controlthat are putative components of the signal transduction path-way that mediates the activation (dephosphorylation) of SPSin response to light (54).

REGULATION OF PEPcase IN LEAVES

PEPcase (EC 4.1.1.31) is an important carboxylating en-zyme in plant cell metabolism. Located in the cytosol, it

serves a variety of functions in various types of plants (30).Coupled to malate dehydrogenase activity (MDH), it cata-lyzes the production of malate as in reaction 2:

PEPcaseHC03- + PEP -- Pi

MDH+ Oxaloacetate - -r - - - -V- -+ Malate

NAD(P)H NAD(P)(2)

This is the first committed step in the nocturnal fixation ofatmospheric CO2 by plants with CAM (36). In plants withC4-type metabolism, it is the first step of the C4-dicarboxylicacid pathway of photosynthesis (15). In C3-plants, the mainfunction of PEPcase is to replenish the organic acid biosyn-thesis pathway with C4 chains when demand for amino acidbiosynthesis is high. Thus, it is a key enzyme of the anapleu-rotic pathway.

In CAM plants, PEPcase is controlled by an endogenouscircadian rhythm (35). Subject to feedback inhibition bymalate it is inactivated during the day, thus avoiding futilecycling. In C4 plants, the activity of PEPcase is regulated bymetabolites such as malate and glucose-6-P (10). The prop-erties of the enzyme undergo changes upon light/dark tran-sition: the enzyme activity is higher and is less sensitive toinhibition by malate when extracted from illuminated versusdarkened leaves (21, 33). Van Quy et al. (48) recently reportedlight-dependent increase in PEPcase activity from a C3 plant(wheat) with a concomitant decrease in the sensitivity tomalate.

It has been clearly demonstrated in both in vivo and invitro experiments that in all types of plants (CAM, C3, C4)the PEPcase protein is subject to reversible phosphorylationand that the malate sensitivity depends on the phosphoryl-ation status of the protein. In wheat, as in maize andsorghum, the protein is phosphorylated to the greatest extentin illuminated leaf tissue and the enzyme is more active andless sensitive to feedback inhibition by malate (22, 33, 48).This regulatory event has implications for carbon metabolismand the regulation of carbon partitioning in the leaves of C3plants (47). In CAM plants, PEPcase is phosphorylated indarkened leaves. It is during this period that the enzyme isleast sensitive to inhibition by malate (34). Intensive investi-gation of the light/dark regulation of the C4 PEPcase hasshown that this enzyme is light-regulated at both the tran-scriptional level, in a phytochrome-dependent manner (46),and the posttranslational level (23). It has been shown bothin vivo and in vitro that the light-induced changes in activityand malate sensitivity of C4 PEPcase are related, at leastpartly, to the latter. This process consists of a reversible seryl-phosphorylaton of the PEPcase protein by a protein seinekinase (24).

GENERAL CHARACTERISTICS OF THE REVERSIBLEPROTEIN PHOSPHORYLATION PROCESS

It is now clear that reversible protein phosphorylation is amajor means of metabolic regulation in plants (3). It appearsto be central in transduction of extracellular signals intointracellular biochemical changes. The phosphorylation proc-

Plant Physiol. Vol. 100, 19928



N03- ACTIVATION OF PROTEIN KINASES AND CARBON PARTITIONING

ess involves two enzyme-catalyzed reactions, forming a re-versible cycle:

Protein + n ATP -- Protein-Pn+ n ADP, catalyzed by a protein kinase (3)

Protein-Pn + n H20 -- Protein

+ n Pi, catalyzed by a protein-P phosphatase (4)

Plants contain homologs of some of the mammalian pro-tein kinase cascades and protein phosphatases that are re-markably similar to the major phosphatases present in animaltissues (32). In most cases, both the phosphorylation anddephosphorylation reactions are assumed to be active at thesame time and the relative activities determine the steady-state level (42). The steady-state activity of the target enzymeis adjusted by positive and/or negative effectors influencingthe rates of phosphorylation and dephosphorylation. Theratio of nonphosphorylated to phosphorylated molecules de-pends upon the relative activities of the two competingmodifier enzymes that may be activated or inhibited by awide range of effector molecules. The best example of thisconcerns the in vivo regulation of PEPcase activity (1). Thephosphorylation state of PEPcase seems to be controlled bythe activity of PEPcase-protein kinase rather than that ofphosphatases. The PEPcase-protein kinase activity exhibits acircadian rhythm in CAM plants but appears in response tolight in C4 plants (35). Use of inhibitors of translation andtranscription suggests that the kinase is regulated at the levelof de novo synthesis and degradation in both systems ratherthan via a 'second messenger' system. Thus, the light mod-ulation of PEPcase involves the following cascade system: (a)synthesis/degradation of the PEPcase-protein kinase, and (b)phosphorylation/dephosphorylation of the PEPcase (25).

THE N03- SIGNAL ENHANCES LIGHT ACTIVATIONOF THE LEAF PROTEIN KINASE(S)

The concept of the role of 'NO3-' as a signal for theenhancement of the light-dependent activation of the leafprotein kinase originated from the following experiments.

After wheat leaves supplied with N-free solution (low-N03- leaves) were transferred from dark to light, the acti-vation of PEPcase appeared within minutes, providing evi-dence of the light-dependent PEPcase-protein activation inC3 leaves. Experiments using 32Pi showed that activation wasrelated to the level of the PEPcase-protein phosphorylation.Activation of SPS also took place (48). The fact that bothactivation of PEPcase and SPS appeared within minutes afterwheat leaves were transferred from dark to light is, at first,anomalous. This may be explained in terms of the changesin the metabolic pools in the cytosol immediately after thetransition from dark to light. Both enzymes require activationin the light, if sucrose and amino acid synthesis are toproceed. Activation of SPS could be due to changes in levelsof regulatory metabolites (4) either associated or not withdecrease of the SPS-protein kinase activity. This interpreta-tion relies on the assumption that the PEPcase-protein kinaseand the SPS-protein kinase are distinct, substrate-specificenzymes with different kinetic properties, and are differen-

tially affected by the increased rate of ATP turnover, whichmay take place upon illumination of leaves (12).

In wheat leaves placed on 40 mm KNO3 (high-N03leaves), 'NO3-' (NO3- per se or a product of its assimilation)within minutes enhanced the rate of PEPcase activation andcaused transient inactivation of SPS (48). The effect of N03-cannot be explained by de novo synthesis of the proteinbecause it took place very rapidly and was not affected bycycloheximide, a protein synthesis inhibitor (43). N03- favorsthe presence of the phosphorylated form of the two enzymes.The short-term increase of PEPcase activity and decrease ofSPS activity depend on a relatively high protein kinase toprotein phosphatase ratio.The question of whether 'NO3- stimulates the protein

kinase or inhibits the protein phosphatase was investigatedwith wheat leaves treated in vivo either with mannose, whichinhibits the protein kinase activity by decreasing ATP levelvia the sequestering of Pi (51), or with okadaic acid, a potentand specific inhibitor of type 1 and 2A protein phosphatases(6). The interpretation of the mannose and okadaic acidtreatments relies on comparison of their effects on the PEP-case (Fig. 1, A and B) and SPS (Fig. 1, C and D) activities andthe theoretical responses expected from the assumption thatNO3- either enhanced the kinases (Fig. 2, a-d) or decreasedthe phosphatases (Fig. 2, e-h) (47). Addition of mannose tothe uptake solution after the initiation of NO3-dependentPEPcase activation lowered the activity to the level of the(low-NO3-) leaf PEPcase (Fig. 1A) and increased the previ-ously deactivated SPS to a level similar to that of the leaveson N-free solution (Fig. 1C). This behavior from both en-zymes would be expected to result from NO3-dependentkinase activation (Fig. 2, a and b). Treatment of the (high-NO3-) leaves with okadaic acid increased PEPcase activationmore in the leaves supplied with NO3- than in the leaves onN-free solution (Fig. 1B), which is consistent with the hy-pothesis that NO3- acts by increasing the protein kinaseactivity (Fig. 2c). SPS was deactivated in response to okadaicacid in both the absence and presence of nitrate (Fig. 1D).

Based on these data and the present knowledge of the lightregulation of PEPcase, SPS, and their protein kinase activities,we suggest that the kinase-modulated activation of PEPcaseand deactivation of SPS are important effects of NO3- andthat NO3- is a signal metabolite that regulates the cytosolicprotein kinases (Fig. 3).

QUESTIONS AND IMPLICATIONS OF THE CONCEPT

The reversible phosphorylation of enzyme proteins is avery efficient mechanism of regulation yielding an enhancedsensitivity of response to a given level of effectors. Operatingin the short term and acting simultaneously on several en-zymes, it can set the pace of major metabolic pathways.Our findings demonstrate that the primary role of NO3

per se or products of its assimilation is to enhance solubleprotein kinase activity, either through synthesis or activation,or both. In contrast, in maize we have found that NO3-assimilation favors dephosphorylation of the light-harvestingChl a/b binding protein of the thylakoid membrane, suggest-ing that the thylakoid protein kinase is inactivated (C. Foyer,unpublished results). Evidence was recently obtained that

9



CHAMPIGNY AND FOYER

Ea

E

E

wto.

- Juu

E

00Ea 200EC

0.CA,

C Man C Man C odA C odA

Figure 1. The in vivo effect of mannose (A, C) and okadaic acid (B,D) on the light- and NO,--dependent PEPcase activation (A and B)and SPS deactivation (C and D). The youngest mature leaves weredetached from wheat seedlings after 16 h in darkness and trans-ferred onto N-free solution, or 40 mm NO3-, and illuminated.Mannose was added into solutions at 50 mm final concentration,30 min after start of illumination. For the okadaic acid treatment,detached leaves were allowed to stand on N-free solution contain-ing 1 AM okadaic acid for 4 h in darkness prior to transfer onto N-free or 40 mm N03- solutions and illumination. C, Control; Man,50 mm mannose; odA, 1 ,M okadaic acid.

shows that nitrate reductase responds rapidly and reversiblyto light/dark transitions by a mechanism that is stronglycorrelated with protein phosphorylation (19), superimposedto changes in the protein level (20). Although with funda-mental differences in the in vivo regulation, nitrate reductase,like SPS, is inactivated in the presence of ATP (27). Thiseffect may provide a further element of control, achievingbalance between the pathways of nitrogen assimilation andcarbon metabolism. For example, NO2- is a toxic intermediatethat cannot be allowed to accumulate. NO2- must be pro-duced at a rate at which it can be used.

Several important questions remain concerning:(a) the specific mechanisms) by which soluble protein

kinase activity is increased in the light and decreased in thedark in wheat leaves (either by de novo synthesis or modifi-cation of the kinetic properties?).

(b) The specific nature of the 'NO3- signal that enhancesthe light effect. Is it NO3- per se, or the metabolic productsof its assimilation such as glutamine, glutamate, etc. Experi-ments using tungstate (49), an inhibitor of nitrate reductase,showed that the N03- effect was eliminated in the presenceof the inhibitor. This observation strongly suggests that it isnot NO3- per se that is the signal metabolite, but a derivativethereof. In terms of this hypothesis, we consider that NO3-is the primary agent in the trigger mechanism because NO3-is the form in which nitrogen arrives at the leaf mesophylltissue, and it is NO3- that is the initiator of the cascade ofregulatory events that follow.

(c) The specific mechanism of the 'NO3- effect: is itsynergistic or additional to the light effect?

(d) Whether the PEPcase-protein kinase and the SPS-protein kinase are the same or distinct enzymes.

(e) Furthermore, it is necessary to determine whether theC3-type PEPcase protein has similarities in structure at theregulatory site with the C4-type PEPcase and whether itslight-dependent phosphorylation involves a regulatory cas-cade of reactions similar to the well-known mechanism ofreversible light activation of the C4-PEPcase (26).The interaction of NO3- on CO2 assimilation in plants is

generally interpreted with respect to plant productivity (14)

KinaseATP + Protein T P Protein + ADP

Phosphatase

N03 enhances the protein phosphorylation2 hypotheses

ThekXnase

Expected response to mannose

Expected response to okadaic Acid

-a

b

-c

-d

The kinaseis activated

ATP + proteinP Protein + ADP

PEPcase (NO3 ) and PEPcase (N-free)decrease

SPS (NO5j) and SPS (N-free)increase

PEPcaae (N0j ) increases fasterthan PEPcase (N-free)

SPS (NO3-) decreases fasterthan SPS (N-free)

e

Eh

The phosphataseis deactivated

ATP + protein 4t-w P Protein + ADP

PEPcase (N03 ) decreases more slowlythan PEPcase (N-free)

SPS (NO3 ) increases more slowlythan SPS (N-free)

PEPcase (N- -) and PEPcase (N-free)increase

SPS (NO3 ) and SPS (N-free)decrease

Figure 2. Diagram showing the arguments used to analyze the in vivo effects of mannose and okadaic acid on wheat leaf PEPcase and SPSactivities to determine whether N03- activates the protein kinases or deactivates the phosphatases.




N03- ACTIVATION OF PROTEIN KINASES AND CARBON PARTITIONING

NO3" ADPw.NLight

(Synthesisor

Activation)

Protein _. Protein -Kinase(s) KKinase(s)low activity Dark high activity

(Degradationor

Deactivation) . W-,

Figure 3. A diagrammatic representation of the concept proposing that "NO3-" (NO3- per se or a derivative) in wheat leaves is a signal forthe enhancement of the protein kinase(s) activity in the light, resulting in activation of PEPcase and deactivation of SPS.

.,.- Phosphatase(s)

or in a whole plant context (44). Less attention is paid tointeractions at the cellular level, although nitrogen and car-bon metabolism are linked in several ways. As pointed outby Warburg (52), NO3- was the first Hill reagent to bediscovered when it was shown that Chlorella suspended inC02-free solutions of nitrate evolved oxygen in the light (53).Clearly, the short-term stimulation by 'NO3-' of the PEPcaseand SPS-protein kinase activity can now be considered as aprimary interaction of NO3- metabolism with CO2 assimila-tion, because it is at the origin of the control of the allocationof C between sucrose and amino acid synthesis.

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