Starch Formation in Leaves and Isolated Chloroplasts

Plant Physiol. (1977) 59, 1146-1155

Role of Orthophosphate and Other Factors in the Regulation ofStarch Formation in Leaves and Isolated Chloroplasts

Received for publication November 24, 1976 and in revised form February 4, 1977

HANS W. HELDT, CHONG JA CHON, AND DOROTHEA MARONDEInstitut fur Physiologische Chemie und Physikalische Biochemie der Universitat Munchen, 8000 Munchen, 2Pettenkoferstrasse 14a, Germany

ALICE HEROLD, ZIVKO S. STANKOVIC, AND DAVID A. WALKERDepartment of Botany, The University of Sheffield, Sheffield SI 0 2 TN, England

ANNA KRAMINER, MARTHA R. KIRK, AND ULRICH HEBERBotanisches Institut der Universitat, 4 Dusseldorf Universitatsstrasse, Gebaude 26.13 Ebene 01, Germany

ABSTRACT

Starch synthesis in leaves was increased by phosphate starvation or bytreatments which decreased cytoplasmic orthophosphate levels (such asmannose feeding). Usally less than 30% of the total carbon fixed duringCO2 assimlation was incorporated into starch in spinach (Spinaciaoleracca L.), spinach beet (Beta vulgaris), and tobacco (Nicotianatabacum) leaves.

In isolated spinach chloroplasts, formation of starch from CO2 wasusualy less than in leaves. In the absence of dpificant levels of 3-phosphoglycerate, concentrations of phosphate as low as 1 mM (in themedium) or 10 mM (in the stroma) almost completely inhibited starchsynthesis. The inhibitory action of phosphate could be overcome by 3-phosphoglycerate. The controlling factor of starch synthesis appeared tobe the rtio of phosphoglycerate to orthophosphate rather than thestromal hexose monophosphate concentration, and it is suggested thatthis control is exerted via the phosphate translocator and the knownallosteric regulation of ADP-glucose pyrophosphoryase. Starch synthe-sis was also favored by the presence of dibydroxyacetone phosphate andby high light and high temperature. Oxygen was inhibitory, probablyowing to carbon drain into glycolate. Starch formation by intact chloro-plasts could not be promoted by added glucose or glucose 6-phosphate.

Starch mobilization in the dark was promoted by orthophosphate andphosphate-dependent mobilization was inhibited by phosphoglycerate.The principal products of starch breakdown in the presence of phosphatewere the transport metabolites dihydroxyacetone phosphate and 3-phos-phoglycerate. Formation of these compounds from starch was stimulatedby ATP or oxaloacetate. In a phosphate-independent reaction, starchwas also converted to neutrd products such as maltose and glucose. Therates of phosphate-dependent starch degradation phosphorolysis werevery much higher than those of starch hydrolysis for which there was nophosphate requirement.

CO2 fixation takes place in the stromal compartment of thechloroplast. Soluble products of CO2 fixation such as DHAP1and, to a lesser extent, PGA are exported from the chloroplast

1 Abbreviations: PGA: 3-phosphoglycerate; DHAP: dihydroxyace-tone phosphate; FDP: fructose 1,6-diphosphate (fructose 1,6-bisphos-phate); G6P: glucose 6-phosphate; G1P: glucose 1-phosphate; RuDP:ribulose 1,5-diphosphate (ribulose 1,5-bisphosphate).

stroma to the cytosol by transport across the inner envelopemembrane, which is the functional barrier (17) between the twocompartments (for reviews see refs. 8 and 39). This transport isaccomplished by the phosphate translocator, which is a specificcarrier, facilitating a strict counterexchange of triosephosphates,PGA' and Pi (14, 16). For each triosephosphate or PGA ex-ported, a molecule of Pi has to be taken up. In this way, the totalpool of Pi and phosphorylated compounds within the stroma iskept constant. The triosephosphate transported to the cytosolmay be converted there to sucrose, and the Pi thus released canreenter the chloroplast in exchange for more triosephosphate(19). This permits a steady flux of fixed carbon from the chloro-plast to the cytosol. If utilization of triosephosphates in thecytosol is lower than production, or if cytoplasmic Pi is seques-tered in the phosphorylation of hexose (3, 18, 19), the level of Piin the cytosol will be decreased. It is proposed that the rate oftriosephosphate export will be lowered because of lack of ex-changeable Pi in the cytosol. On the other hand, starch synthesisis not, in itself, a Pi-consuming process and should not be slowedby a Pi deficiency. In these circumstances, therefore, it is feasiblethat more fixed carbon will accumulate as assimilation starch.

This paper explores some of the relationships between photo-synthetic starch formation and Pi concentration with regard tothe above facts and proposals. The work was a cooperativeundertaking carried out in laboratories at Dusseldorf, Munich,and Sheffield and many of the major observations were inde-pendently confirmed in each of these three centers.

MATERIALS AND METHODS

Plant Materials. Spinacia oleracea L. was grown in the field,under glass, or in water culture as previously described (25). Thevarieties used at Dusseldorf were Monatol (winter, greenhouseand hydroponic culture), Weremona (winter, field) and Mon-taco (summer, field and greenhouse) from Sperling, Luneberg.The variety used in Munich and Sheffield was U.S. Hybrid 424from Ferry-Morse Seed Co., Mountain View, Calif. Spinachbeet (a cultivar of Beta vulgaris sometimes called perennialspinach, obtained from Elsoms Seeds Ltd., Spalding, Lin-colnshire) and tobacco (Nicotiana tabacum var. Xanthii) weregrown from water culture under the same conditions as spinach.Chenopodium bonus-henricus was from the Tapton Experimen-tal Garden, Sheffield.

14C-Glucose, 14C-FDP, and 14C-G6P were obtained from TheRadiochemical Centre, Amersham, the enzymes aldolase, amy-

1146

REGULATION OF STARCH METABOLISM

loglucosidase, triosephosphate isomerase and catalase fromBoehringer, Mannheim. Ammonium ions had been removedfrom the 14C-fructose diP with a small amount of Dowex 50-H+and from the enzymes by filtration through a small column ofSephadex G-25.

Chloroplasts were isolated whenever possible from rapidlyexpanding leaves using conventional techniques (7, 24, 25).Percentage intactness (24) was measured by the ferricyanidemethod (10).

Photosynthesis. Oxygen was measured polarographically us-ing Clark-type electrodes (Hansatech Ltd., Paxman Road,King's Lynn, U.K.) with 2 mm HC03- as substrate or as fixationof 14C into acid-stable products.

Illumination was provided by a halogen source and passedthrough water, 1 mm Calflex C (from Balzar, Liechtenstein) andeither 3 mm RG 630 (from Schott, Mainz) or red perspex (ICI400) to give light principally in the range of 590 to 760 nm at250-300 w-m-2.

Reaction Mixtures. Except where stated, measurements ofphotosynthesis and starch synthesis were carried out at 20 C inmedia containing sorbitol (330 mM), MgCl2 (1 mM) MnCl2 (1mM), EDTA (2 mM), NaCl (10 mM), HEPES (50 mM) at pH7.6, and Pi as indicated.

Starch and Other Photosynthetic Products. At Dusseldorf theproducts of 14C fixation were measured after two-dimensionalpaper chromatography on paper 2043 b Mg 1 from Schleicherand Schuell as described by Pedersen (29).

Radioactive spots were localized by autoradiography andcounted on both sides using a methane flow counter. At Shef-field the distribution of label between insoluble and solublefractions was measured after one-dimensional chromatographyin 1-butanol-acetic acid-water (222:51:150, v/v/v) using a Nu-clear-Chicago chromatographic strip counter (which also recordsactivity from both sides of the paper). At Munich a simple assayprocedure was devised in which starch was taken as 14C re-covered in a fraction insoluble in 1 M HCl at 0 C. Starch inleaves was measured enzymically as glucose (3) following extrac-tion in boiling ethanol (80%, v/v) and digestion of the insolublefraction with amyloglucosidase.

Distribution of Metabolites between Stroma and Medium.The chloroplasts were equilibrated with 32P phosphate (50 Ci/mol) and then illuminated. The incubation was terminated bycentrifuging the chloroplasts through silicone oil into perchloricacid, and the supernatant was also deproteinized by perchloricacid. After neutralization, the acidified pellet and supernatantfractions were subjected to ion exchange chromatography (15).(The column had an inner diameter of 1 mm, length of 100 cm,and was filled with Dowex 1-8 formate-400 mesh.) The elutionwas carried out with a linear concentration gradient of 1.5 Mammonium formate +9.9 M formic acid, flow rate 1 ml/hr. Asimultaneous determination of the stromal volume (17) allowedconcentrations to be calculated.

Starch Synthesis in Leaf Discs. Incubation of leaf discs andassociated procedures were essentially the same as those previ-ously described (3, 18).

Orthophosphate in leaves was measured spectrophotometric-ally as the molybdate complex (1). Ten leaf discs (5-mm diam.)were homogenized in a solution containing amidol (2,4-diamino-phenol hydrochloride) sodium bisulfite and perchloric acid (thelatter to a final concentration of about 0.26 M) to which ammo-nium molybdate was then added.

RESULTS AND DISCUSSION

PATH OF CARBON IN STARCH SYNTHESIS

As de Fekete and Vieweg have pointed out in an eloquent andcautionary review (5), the events which occur in the photosyn-thetic formation of starch are complicated and still only imper-

fectly understood. While not dismissing the possible involvementof phosphorylases and amylases in starch synthesis, we haveproceeded on the basis that starch is synthesized in the stromalcompartment from hexose phosphate derived from the reductivepentose phosphate pathway and that sugar nucleotides are in-volved. The simplest route which is consistent with the availableevidence would then put fructose-6-P as the metabolite whichwould be diverted from the cycle and involve the sequence

F6P -. G6P -- GlP -.> ADP-glucose -. starch

ADP-glucose is formed from GlP in a reaction catalyzed byADP-glucose pyrophosphorylase which is pulled in the syntheticdirection by the subsequent hydrolysis of PPi. The pyrophospho-rylase has been shown by Preiss et al. (31, 32) to be activated bycompounds such as PGA and inhibited by Pi, thus making it aneminently suitable candidate for a key role in photosyntheticstarch synthesis.

STARCH SYNTHESIS IN VIVO

In starch-forming leaves there is normally a pronounced diur-nal change in starch content. Starch is deposited in the chloro-plast stroma by day and is mobilized by night. During the wintermonths, however, starch synthesis may be greatly diminishedand the associated accumulation of soluble sugars in leavescontributes to frost hardiness (11).

In plants grown at Sheffield, the course of starch synthesis andbreakdown in spinach proved to be very similar to that ofspinach beet and tobacco grown, for purposes of comparison,under identical conditions. In the field in the U.K., activelygrowing spinach sometimes accumulates only relatively smallquantities of starch but under the conditions chosen (waterculture, full spring sunshine + supplementary lighting, tempera-tures of 20-30 C and augmented C02) the rate of synthesis wasas high as 60 ,uatoms of C incorporated/mg Chl - hr. Data pub-lished elsewhere relating to simultaneous measurements of car-bon assimilation and starch synthesis in spinach and sunflower(37, 42) suggest that incorporation into starch is normally un-likely to exceed more than 30% of the total carbon fixed (cf. thesomewhat smaller percentage recorded in Table II for synthesisby isolated chloroplasts). (At 20 C, apparent photosynthesis byspinach is about 100 ,umol C02/mg Chl hr [26], but at highertemperatures and CO2 concentrations it may rise to between 200and 300.)The starch content of attached spinach leaves fell to a very low

level during the night indicating that mobilization was roughlycomparable to synthesis. In detached leaves (cf. 5) maximalsynthesis was sometimes faster and led to more accumulation.Conversely, breakdown was then insufficient to mobilize theaccumulated starch by the next morning.

Precisely what governs the proportion of newly assimilatedcarbon which is stored as starch within the chloroplast remains tobe established but there is good evidence that the availability andactivity of sinks are likely to be important factors (6). Again, thenature of the "message" which is passed to the chloroplast as aconsequence of the removal or impairment of a distant sink isnot known but it has been suggested (see introductory sectionand refs. 5, 19, and 41) that increased starch synthesis may be aconsequence of decreased availability of Pi within the cytosol. Ithas already been reported that starch synthesis in spinach beet(and some other species) was greatly enhanced by mannosefeeding although carbon from mannose was not incorporatedinto the starch skeleton (3). We concluded that the mannoseproduces its effect by sequestering cytoplasmic Pi as mannosephosphate. This would diminish the export of triosephosphatefrom the chloroplast and thereby produce ratios of triosephos-phate (and PGA) to Pi within the chloroplast which would favorstarch synthesis. During the present work essentially similar

1147Plant Physiol. Vol. 59, 1977

Plant Physiol. Vol. 59, 1977

results were obtained with spinach and Chenopodium bonus-henricus, a member of a closely related genus. Using spinach itwas also established (Table I) that mannose feeding led to alowering of cytoplasmic [Pi] consistent with the previously re-ported accumulation of mannose phosphate (3). Increasedstarch is also observed in spinach beet grown in Pi-deficientmedia (18) and although in no way conclusive this is consistentwith the view that the availability of cytoplasmic Pi is an impor-tant factor in the regulation of starch synthesis within the stroma.Direct responses by isolated chloroplasts to the level of Pi in thesurrounding medium are illustrated in the following sections.

STARCH FORMATION BY ISOLATED SPINACH CHLOROPLASTS

Identification of "Starch" as a Photosynthetic Product. Afterisolation, chloroplasts sometimes contain a considerable amountof starch. This makes it difficult to measure kinetics of starchformation by enzymic assay, as a large blank has to be sub-tracted. In order to avoid this difficulty, and to use less chloro-plasts, starch formation was measured as 14C incorporation ofbicarbonate into insoluble material, yielding glucose on hydroly-sis. The insolubility was either defined by paper chromatogra-phy, or in a more simple assay procedure as material of spinachchloroplasts being insoluble in 1 M HCI at 0 C (see under"Materials and Methods").

Fig. 1 shows a thin layer chromatogram of the 14C radioactivityfound in the pellet of chloroplasts deproteinized in 1 M HCI.Except for a small portion (about 5%) which was mainly trappedPGA, this fraction did not migrate during chromatography.Hydrolysis by a-amyloglucosidase then yielded complete con-version to a soluble product migrating the same distance asglucose. Similarly, acid hydrolysis at 100 C for 20 min gave onlyone compound which co-chromatographed with glucose. (Inother experiments, glucose released from the insoluble fractionby enzymic and acid hydrolysis was identified by two-dimen-sional paper chromatography and quantified by a glucose oxi-dase assay. See under "Materials and Methods".) When preillu-minated intact spinach leaves were treated with iodine (KI/I2)after extraction in boiling ethanol, they developed colors varyingbetween yellow and dark blue to black, whereas after 24 hr inthe dark they normally showed only the color of the iodinereagent. As the development of a blue color is characteristic ofthe starch iodine reaction, we have used the term "starch" todescribe the insoluble polyglucan formed in isolated chloroplastsand intact leaves upon illumination. It may be noted that theethanol-insoluble fraction will inevitably contain long chain glu-cans other than true starch and it has been suggested (30) that intobacco, these might account for approximately 30% of the totalinsoluble fraction.

Table I. The effect of mannose on orthophosphateconcentration in leaves of spinach beet

Samples of 10 leaf discs (5 mm diam) were floated overnighton 10 mM mannose solution or water and then assayed for Pi.Each pair of results applies to discs cut from the same leaf.

[PiJ (jimoles/loG mg fresh wt)Control Mannose Diff

10 mM

0.82 0.36 -0.460.62 0.26 -0.360.30 0.28 -0.020.62 0.28 -0.340.94 0.28 -0.660.36 0.16 -0.200.50 0.16 -0.340.88 0.32 -0.561.04 0.36 -0.680.50 0.24 -0.260.70 0.28 -0.420.52 0.28 -0.24

Mean 0.65 0.27 -0. 38

THINLAYER CHROMATOGRAPHY 2-BUTANOL-FORMIC ACID-H20 (6:1:2)

POSITION OF GLUCOSE

A.UNTREATED MATERIAL

B.AFTER INCUBATION WITHAMYLOGLUCOS IDASE

START

FIG. 1. Thin layer chromatography of 14C radioactivity incorporatedinto acid-insoluble material of chloroplasts. Spinach chloroplasts (0.025mg Chl/ml) were illuminated for 40 min in the presence of 0.25 mM Piand 2.5 mM '4C-NaHCO3 (5 Ci/mol). The reaction was terminated bythe addition of HCl04 to a final concentration of 1 M. Four zatoms of"4C/mg Chl were incorporated into the insoluble material. After washingthe pellet once, 10 Il of the suspended pellet (equivalent to 2 Ag Chlwere subjected to TLC (cellulose, 2 butanol-formic acid -H20 (6:1:2, v/v) according to Schiirmann (35). A: parallel sample of the pellet (equiva-lent to 10 Ag Chl) was incubated for 2 hr at 40 C with 50 ,ul 0.2 Msodium acetate buffer containing 1 mg amyloglucosidase/ml. Ten j.l ofthe suspension was subjected to TLC; B: radioactivity was scanned by anAutochron scanner (Berthold-Friesecke, Germany).

Rates of Starch Formation by Chloroplasts. Table II showsCO2 reduction and starch synthesis from CO2 by chloroplastsisolated from different spinach material at different times of theyear. The experiments were carried out under what we presentlybelieve to be optimal conditions for either starch synthesis orCO2 reduction.

Starch synthesis from CO2 by isolated chloroplasts was usuallylower than that in whole plants but it should be reemphasizedthat the highest in vivo value of 60 uatoms C was recorded underextremely favorable conditions and that starch as a percentage oftotal C assimilation rarely exceeded 30% (42). Bearing this inmind, it is clear that the best in vitro values of 31 and 32 (TableII), recorded at the less favorable temperature of 20 C were notmarkedly inferior. In the in vitro experiments the percentageincorporation into starch was also lower (Table II) but again ifthe effect of temperature is taken into account the disparitycould become negligible. (It should also be noted that in vitrosynthesis is stimulated by PGA and DHAP-see below). Evenwith chloroplasts derived from similar plant material, there wereconsiderable differences in rates of starch synthesis from CO2 indifferent experiments. Occasionally, these rates approachedthose reported for ADP-glucose pyrophosphorylase.

Chloroplasts from winter leaves, which did not normally accu-mulate much starch, nevertheless synthesized starch at ratescomparable to those from summer-grown plants suggesting thatthe differences in the starch content of the parent tissue wereunlikely to derive from differences in the levels of starch-synthe-sizing enzymes. When starchy leaves were used as a source ofchloroplasts, starch synthesis tended to be slow. Both in regardto CO2 reduction and starch synthesis, best results were obtainedwhen the leaves used for chloroplast isolation were rapidlyexpanding or at least (during the winter) had the potential forfast growth.

Relationship between Carbon Assimilation and Orthophos-phate Concentration. Figures 2, 3, & 4 illustrate the relationshipbetween CO2 fixation and starch synthesis and [Pil in the me-dium in experiments with spinach chloroplasts. They show thedegree of agreement reported by the three contributing labora-

1148 HELDT ET AL.


Table II.

1149REGULATION OF STARCH METABOLISM

Photosynthesis by spinach chloroplasts

Temp. 200 and [Pig optimal for Co 2-dependent 0 evolution

(0.5 mM Pi) or starch synthesis (0.1 mM). Bicarbonate was 2 mM.

Photosynthetic Carbon Assimilation

Spinach C0o-dependent 0 evolution "Starch" synthesis Assays

Imol/mg Chl/hr patom C incorporated/mg Chl/hr No.

Water culture (Munich) 111 (77-148)t 12 (3-31) 28Water culture (Dusseldorf)* 161 (109-213) 0.6 (0.3-1.0) 3Winter material:hardy, field grown 116 (76-206) 16.1 (4.8-32.0) 8Winter material non-hardy, grownin greenhouse (long photoper.) 170 (125-282) 6.5 (1.0-22.0) 15Summer material:field grown 154 (73-230) 8.5 (4.8-13.2) 12Summer material:grown in greenhouse 106 (70-123) 7.3 (3.6-13.2) 5

*leaf material contained large amount of starch, growth oftmean (min-max)

C02 FixationFormation of soluble material

Pi in medium (mM)0.25

0.5015OL ~~~~~~~109

0

0 136

ENI ~~~~~~~~~~0.10

se55

1.0

* ~~~~~~~~970E

5

leaves was slow.

o 1.00

0~EV

x

e 0.5

4n

0E-L

C02 FixationFormation of.starch

Pi in medium (mM)i 0.10) 0.25

7.1I

0.50

10TIME (Min) Tl ME (Min)

FIG. 2. Formation of soluble (A) and insoluble (B) products during CO2 fixation by spinach chloroplasts (0.025 mg Chl/ml) in the presence of 10mM 14C-NaHCO3 (0.2 Ci/mol) and phosphate as indicated. Temperature was 20 C. At the times indicated, samples (200 ;LI) were withdrawn anddeproteinized with 100 ;L1 3 M HCO. After centrifugation 200 ;L1 of the supernatant was evaporated on a heating plate, and the radioactivity of theresidue was measured by liquid scintillation counting (Fig. 2A). The pellet was washed once. Three hundred ;L1 lM HCI and about 80 mg quartz sandwere added and the sample was vigorously shaken. Two hundred ,ul of the fine suspension was withdrawn, after heating radioactivity was measured(Fig. 28). Numbers are the rates of 14C incorporation/mg chl * hr after about 8 min photosynthesis.

tories in this study and the following consistent features emerge.(a) In accord with many previous observations, the optimal [Pi]for CO2 fixation is in the region of 0.5 mm. Lower concentra-tions shorten, and higher concentrations lengthen, the initial lagor induction period (40). (b) The Pi requirement for optimalstarch synthesis is lower than that for CO2 fixation and at higher[Pi] the percentage of counts recovered in the insoluble fractionis considerably diminished (13, 20). The relationship betweenCO2 reduction, starch formation, and [Pi] is summarized inFigure 3. In this experiment, the chloroplasts were illuminatedfor 8 min.

Reversal by PGA of Orthophosphate Inhibition of Starch

Synthesis. The above results clearly indicate that starch synthesisis favored by low [Pi]. However, when photosynthesis is pro-longed, starch synthesis may proceed after a long lag even if theinitial [Pi] is as high as 2.5 mm (Fig. 4). This onset of starchformation cannot be attributed to a decrease of [Pi] in themedium, since this fell by only about 20% during the experiment(Fig. 4). This suggests that the inhibition of starch formation byPi can be gradually overcome as products of CO2 fixation accu-mulate in the medium. This view is supported by the results ofexperiments in which PGA was added to the medium (TableIII). The inhibition of starch synthesis by high [Pi] was reversedand 14C from labeled PGA was recovered in the starch fraction.


.2

C !total carbon fixation

E 0

-5 5 \

EC.\E

incorporation into sturch

1 2 3mM P.

FIG. 3. Carbon fixation and carbon incorporation into starch as af-fected by the concentration of Pi in the medium after 8 min illuminationin the presence of 2 mm HCO3-. Other additions were catalase (1,400unit ml-'), 50 ,uM PGA as a primer to reduce the lag time after thebeginning of illumination. The intensity of red light was 120 w m-2; pHof the reaction mixture was 7.6.

CO, fixation CO2 fixation 1IS_ (Totol) (Starch) _

2.5 mM Pi0 25 mM Pi l

Total /o - 2S mM Pi

:~ioL 0 1---13~~~~~~~~~~~~~~~1.E r

Z, ~~I I**.......... ..

1/I,0

0~~~~~~~~~~~~~~~~~~~~1.:X ~~~~~~~~~~~2-5mM Pi

5 10 IS 5 10 IS 20Time (min)

FIG. 4. Formation of total (left) and insoluble products (right) duringphotosynthesis by spinach chloroplasts in the presence of 10 mM 14C-NaHCO3 and Pi as indicated. Chl: 100 jg/ml. Temperature was 20 C.At the times indicated, samples were removed, deproteinized, andsubjected to paper chromatography.

In low [Pi] PGA decreased the incorporation of radioactivity instarch from '4CO2, and starch was mainly formed from '4C-PGA,reflecting an equilibration of the added PGA with the PGA poolin the stroma. In both cases, the addition of PGA increased thetotal rate of starch formation.

STARCH FORMATION FROM DHAP

Similarly, high rates of starch formation are also observed withDHAP added as precursor, as shown in Figure 5. DHAP wasgenerated by aldolase and triosephosphate isomerase from 04C-labeled FDP which does not penetrate the chloroplast envelope(16). Oxaloacetate was present as a Hill oxidant in order to

generate ATP by noncyclic phosphorylation. Radioactive reac-tion products were analyzed by two-dimensional paper chroma-tography. Besides starch, the prominent reaction products wereglycolate, PGA, and hexose monophosphates. When the lightwas turned off and Pi was added, starch again decreased. At thesame time, PGA increased. Part of this PGA was produced fromthe added triosephosphate, another part from triosephosphateformed during starch degradation.

After correction for the 22% broken chloroplasts in the prep-aration, the maximum rate of carbon incorporation into starchwas calculated to be 65 ,Latoms/mg Chl * hr. It may be noted thatthis rate is exceptionally high. It corresponded to as much as halfthe maximum rate of photosynthesis in many chloroplast prepa-rations (see Table II). In other experiments the rates of starch

Table III. Fornation of C labeled insoluble material(starch) from 14C NaHCO3 and from 14C PGAduring C02-fixation with different Pi in themedium

C NaHCO 10 mM (0.2 Ci/mol), C PGA 1 mrM (0.4 Ci/mol).3Chloroplast concentration 0.025 mg chlorophyll/ml. Themeasurements in the presence of both NaHC03- + PGA werecarried out in two parallel samples containing eitherlabeled NaHCO3 and unlabeled PGA or the other way round.For details of measurement see legend to Fig.2. The rateswere measeured between 15 - 20 min.

Pi PGA Incorporation of 14C intoconc. in insoluble material frommedium 14C-HCO3- 14C-PGA total

mM patoms 14 C/mg Chl/hr

0.15 0 8.0 - 8.00.15 1.0 1.8 16.9 18.70.47 0 0.5 - 0.50.47 1.0 3.4 16.3 19.7

dark plus 10 mM Pi

----glycolateI PGA

IA

I(gliuunits)

FIG. 5. Formation of labeled reaction products during illumination ofchloroplasts in the presence of 1 mM '4C-FDP, aldolase (1.3 unit ml-'),and triosephosphate isomerase (85 unit ml-'). Other additions were 1mm oxaloacetic acid and catalase (1,400 unit ml-'). Phosphate concen-tration was 0.1 mm in the light and 10 mm in the subsequent dark period.The intensity of red light was 125 w m-2, the temperature was 22 C.

1150 HELDT ET AL.

III

III

1.1I

III

I


formation observed with DHAP were not higher than withPGA. Also the rate of carbon liberation in the dark (28 ;Latoms/mg Chl hr) was very much higher than in other experiments(Figs. 6-8, Table IV).

STARCH FORMATION FROM GLUCOSE AND GLUCOSE6-PHOSPHATE

It has been reported (27) that starch formed from Cl- or C6-labeled glucose, which has been fed to intact tobacco leaves,retained label largely in the same position as that in the originalglucose. As starch synthesis from glucose through a C3 sourcesuch as DHAP (above) would produce symmetrically labeledglucose units, this suggests that the carbon skeleton remainedmostly intact during incorporation. The chloroplast envelope hasa low permeability to hexose phosphates, but appears to permitsome entry of glucose (8, 34, 39). Experiments were thereforeperformed to measure light-dependent incorporation of labelfrom glucose (1 mM) or G6P (1 mM) into starch. Photosynthesisexperiments with H14C03- as substrate, but under otherwiseidentical conditions, served as controls to show whether or notthe chloroplasts used had the capacity to synthesize starch. Theresults obtained with radioactive glucose and G6P were nega-tive, the 14C incorporated being less than 1% of that recordedwith 14CO2 as substrate. The data in the literature suggestingincorporation of intact glucose skeletons into starch (27) there-fore remain unexplained.

OTHER FACTORS INFLUENCING STARCH SYNTHESIS

Effect of pH. With optimal bicarbonate concentrations onlyoccasional differences in the proportion of starch formed duringCO2 fixation were observed when the pH in the medium wasvaried between pH 7 and 8. This is in accord with the observa-tion that the pH optimum of the isolated ADP-glucose pyro-phosphorylase in its activated form (pH 8) is not very sharp (31).On the other hand, starch synthesis from PGA was found to befive times higher at pH 7 than at pH 8.2. These differences seemto be due to pH-dependent changes of the metabolite concentra-tions in the stroma.

Effect of Light. In some experiments (not shown here) a linearrelationship between light intensity and starch formation was

00-

o ^cC o

0 _

° 0

0 xE _

il -

oO Se

u

c- 15u

C-

_.

0

0 _

o 0

E 10

o 5-oa.2 a60 0

0 5

0

A12

Pi [mM]

obtained up to 130 w m-2. In other experiments this effect wasless pronounced, and starch synthesis was saturated at about 100w m-

Effect of Temperature. Starch synthesis was markedly influ-enced by temperature. In a typical experiment, where the totalrates of C02 fixation were 111 ,umol C02 fixed/mg Chl/hr(20 C) and 146 (30 C), starch formation increased from 5.5%(20 C) to 9.4% (30 C).

Effect of Oxygen. In the presence of low bicarbonate, 02inhibited starch formation more than it inhibited total CO2fixation. For instance, in the presence of 0.25 mm bicarbonatethe maximum rate of CO2 fixation at pH 7.2 was inhibited byabout 40% when the 02 concentration was raised from 0.2 to

> .0CL00

EC-)

0.E00

Mobilization of '4C- labelledstarch by dark treatment inthe presence of Pi or PGA

+ PGA 5mM

+ Pi 5mM

20 40 60

TIME (Min)

FIG. 7. Mobilization of 14C-labeled starch by dark treatment in thepresence of Pi or PGA. For synthesizing "4C-labeled starch, spinachchloroplasts (0.1 mg Chl/ml) were illuminated in the presence of 8 mM14C-NaHCO3 (0.5 Ci/mol) and 0.25 mm Pi for 25 min at 20 C. Thesample was then cooled to 0 C, centrifuged (2,000 rpm, Sorvall SS 34rotor, 1 min), the pellet resuspended, centrifuged again, and resus-pended. The sample was then placed in the dark in a water bath (20 C),5 mm Pi or PGA were added, and samples taken for the determination of'4C-labeled insoluble material (see Fig. 2 legend).

0

0

0

0

0

0B - o

2 3 4

HMP in stroma

[mM]

FIG. 6. Dependency of the relative rate of starch fixation on the metabolite concentrations measured in the chloroplasts. Data from Table III andtwo other experiments carried out under identical conditions. A: dependency on Pi; B: dependency on the sum of heptose- and hexosephosphates.

1 151Plant Physiol. Vol. 59, 1977


260163t0o

AE DHAP

co pouosPhiosorylated compounds appearing~20ainthe chloroplasts o 40 2

0 ~~~~~~~~~~~~00

0

Ex10 200

250 R5PHMP

~HMP20 40 60 20 40 60

TIME (Min) TIME (Min)FIG. 8. Starch mobilization in the dark. Chioroplasts were allowed to synthesize starch and washed afterwards, according to the legend of Figure

7. For the moibilization of starch, 1 MM~32P-orthophosphate (50 Ci/mol) was added. At the times indicated, samples (200 jul) were taken andimmediately subjected to silicone layer filtering centrifugation. The resulting pellet and supematant fractions were analyzed for 2P-labeledcompounds by ion exchange chromatography.

Table IV. Relationship between the concentration of inorganic phosphate in the mediumand the formation of starch during CO02-fixation

Simultaneous measurement of metabolite concentration in the stroma and in the medium. The rates of CO -

fixation were measured between 5 - 10 mm, the samples for metabolite assay were taken 7 mn after the 2start of illumination. Chloroplast concentration 0.025 mg Chl/ml, stroma space 24 0l/mgChl.

Experiment 101 A B C 0

Phosphate in the medium at beginning of experiment () 1.0 0.50 0.25 0.10C02 fixation (p.imol/mgChl/hr) - soluble material 91 107 106 52

starch 0.3 1.4 7.9 7.7total 91 108 114 60starch as of total 0.3 1.3 6.9 12.8

Metabolite concentrations (mM)a) in stroma

inorganic phosphate 9.6 7.0 4.0 2.2phosphoglycerate 2.9 6.0 6.9 8.9triosephosphates 0.17 0.33 0.40 0.25hexosemonophosphates 3.2 3.7 4.1 4.9ADP-glucose <0.01 <0.01 0.06 0.07phosphoglycerate/phosphate 0.30 0.86 1.7 4.0

b) in the medium emiM)inorganic phosphate 0.96 0.43 0.19 0.077phosphoglycerate 0.004 0.009 0.016 0.024triosephosphate 0.026 0.048 0.041 0.021

0.65 mm. Under the same conditions the inhibition of carbon REGULATION OF STARCH SYNTHESISincorporation into starch was greater than 70%. This inhibition From the data shown it seems clear that starch synthesis iswas shown to be related to a drain of carbon cycle intermediates suppressed by high [Pi], and is stimulated by PGA and triose-into glycolate (21). phosphate. Chloroplasts thus behave as though starch synthesis

1152 HELDT ET AL.


is an auxiliary reaction which proceeds when more CO2 is fixedthan can be exported to the cytosol, i.e. when levels of transfera-ble phosphate esters in the chloroplasts are high. It is switchedoff when demand for products of CO2 fixation in the cytosolfavors export of phosphate esters and leads to an increase in boththe external and internal phosphate concentration. It may thenbe asked (cf. 13, 19, 41) why starch synthesis is stimulated whenphosphate decreases. One possible answer is that under theseconditions PGA and triosephosphates, which are normally ex-ported, accumulate in the chloroplasts, leading to a concomitantrise in the levels of FDP and also in GlP, the substrate of ADPGpyrophosphorylase. Alternatively, allosteric regulation of theenzymes of starch formation may be achieved by metabolites,which are not direct substrates of starch synthesis. For example,it is known that the ADP-glucose pyrophosphorylase present inspinach chloroplasts is allosterically inhibited by Pi and activatedby 3-P-glycerate (31, 32).To evaluate the physiological significance of these findings in

relation to starch synthesis it is necessary to know metabolitelevels inside starch-synthesizing chloroplasts.

Table IV shows relevant data. In this experiment the relation-ship between CO2 fixation and [Pi] was very similar to that inFigures 2 and 3. Again, starch synthesis was inhibited by higher[Pi]. For the parallel analysis of metabolites, the samples wereincubated in the presence of 32P Pi, and after 7 min the incuba-tion was terminated by spinning the chloroplasts through a layerof silicone oil into perchloric acid. Since the chloroplasts areilluminated during the centrifugation and the sedimentation isvery fast (1-2 sec), metabolism in the stroma is very quicklystopped. After neutralization, the extracts were subjected to ionexchange chromatography, and the radioactivity of the effluentwas recorded. The main metabolites formed distinct peaks al-lowing a quantitative determination of these compounds (15). Aparallel determination of the stroma space enabled the calcula-tion of metabolite concentrations in the stroma (17). Similarly,the metabolite concentrations in the medium were determined.It should be kept in mind that the actual concentrations in thestroma may be somewhat different from the calculated valuessince the extent of binding of metabolites to proteins in thestroma is unknown. From the metabolites of the reductive pen-tose phosphate pathway found in the stroma during CO2 fixa-tion, P-glycerate, hexose- and heptosemonophosphates normallyrepresent the largest portion. The concentration of ribulose diP,fructose diP, and sedoheptulose diphosphate are lower. In chlo-roplasts performing active CO2 fixation, the level of triosephos-phates is usually very low. In part this reflects the equilibriumwith PGA and its relation to the [ATP] [ADP] ratio (9, 36).Conversely, more triosephosphate than PGA is found in themedium (in accord with the fact that the phosphate translocatorpreferentially transports DHAP from the chloroplast).The increase of [Pi] in the medium caused a corresponding

increase in the [Pi] in the stroma and an associated decrease in[PGA]. The inverse relationship between these two metabolitesis inevitable if the total pool of Pi and phosphorylated com-pounds in the stroma is to remain constant. The concomitantchanges in heptose- and hexosemonophosphates were muchsmaller. With the chromatographic procedure employed here,the different hexose- and heptosephosphates were not suffi-ciently separated from each other and therefore determined intotal, of which hexosephosphates represent at least half. If it isassumed that [GlP], [G6P], and [F6P] are close to the concen-trations dictated by the phosphoglucomutase and glucose-Pisomerase reactions, it can be estimated from the correspondingequilibrium constants [G6P]/[G1P] = 17.2 (4), [G6P]/[F6P] =3.3 (2) that GlP will amount to at least 2% of the sum ofheptose- and hexosemonophosphates. In Figure 6A, relativerates of starch synthesis have been plotted against stromal [Pi].The data reveal a striking relationship between stromal [Pi] andstarch synthesis. Since the stromal [PGA] is inversely related to

stromal [Pi], the data can also be interpreted to show stimulationof starch synthesis by an increased [PGA] in the stroma. From acomparison of Figure 6 with Figure 3, which shows the relation-ship between starch formation and the concentration of Pi in themedium, the accumulation of Pi in the chloroplast stroma be-comes very apparent (see also ref. 17).

If the sum of the concentrations of heptose- and hexosephos-phates are plotted in the same way (Fig. 6B), no meaningfulrelationship is apparent. Assuming that GlP is a more or lessconstant 2% of the sum (see above) these data suggest that evenin the dark, the stromal concentration of GlP is considerablyhigher than the Km of the activated ADP-glucose pyrophospho-rylase which is 0.04 mm (31). It is unlikely that starch synthesisresults from an "overflow" of GlP into starch.We also compared our data in Table IV (concerning the

relationship between starch formation and the internal concen-trations of PGA and Pi) with the data given in Figure 8 inreference 31) for the allosteric activation and inactivation of theisolated ADP-glucose pyrophosphorylase. With a [PGA]/[Pi]ratio of 1.7, where we observe maximal rate of starch synthesisin experiment C, the measurements by Preiss et al. (31) yielded90% activity. With a corresponding ratio of 0.3 (experiment Ain Table IV), the isolated enzyme showed 6% activity. Weobserve under these conditions 4% of the maximal rate of starchsynthesis. Considering the differences between the two measur-ing systems, the similarity of the data seems striking. It clearlydemonstrates that the changes of [PGA] and [Pi] in the stromaof isolated chloroplasts are very similar to those required forchanging the activity of the isolated ADP-glucose pyrophospho-rylase. The same holds also true for intact leaves. Phosphatelevels reported for chloroplasts in situ varied considerably andwere between 4 and 25 mm (33). They decreased in the light.After a transient decrease, PGA levels increased on illumina-tion. After several min in the light, observed concentrationswere between 1.8 and 4 mm (recalculated from Urbach et al.37). Thus, also in vivo the [PGA]/[Pi] ratio would appear to bewithin the range found by Preiss et al. (31) to control the activityof ADP-glucose pyrophosphorylase. The concentration of ADP-glucose found in the chloroplasts is very low. It is about zero inthe presence of high [Pi] and increases to 0.08 mm with 0.25 mM[Pi] in the medium. Similar observations have been made earlierby J. A. Bassham (private communication). The concentrationof ADP-glucose seems to be proportional to the rate of starchformation. This proportionality may be explained by the factthat the reported Km values for the various ADP-glucose a-1.4glucan a-4-glucosyltransferases from spinach leaves (0.15-0.29mM, Ozbun et al., ref. 28) are much higher than the ADP-glucanconcentration in the stroma. This implies that the actual rate ofstarch synthesis so far observed in the isolated chloroplasts is farbelow the maximal activity of the starch synthetase.

(It has been reported that the activation of the ADP-glucosepyrophosphorylase by PGA not only increases the V1max but alsodecreases the Km for ATP [0.45 mm without, 0.04 mm withPGA]. This may also be an important regulatory effect in view ofthe observation that the [ATP] in actively photosynthesizingchloroplasts may be as low as 0.1 mM.)The inhibition of starch synthesis by Pi can also be overcome

by the addition of DHAP (above). No data on an activation ofADP-glucose pyrophosphorylase by triosephosphate appear tobe available. Even if DHAP is not an allosteric effector of theenzyme, its addition to intact chloroplasts would be expected topromote starch synthesis both by lowering the stromal [Pi] andincreasing the stromal [PGA] via the RuDP oxygenase reaction(21). In leaves, the concentration of triosephosphate remainsvery low in the chloroplasts even in the light. About 75% of thetotal PGA pool of the cells is located in the chloroplasts, but onlyabout 35%o of the triosephosphate pool (12). In consequence,the ratio of [DHAP]/[PGA] is much lower in the chloroplaststhan in the cytosol. The ratio of [DHAP]/[PGA] in chloroplasts

Plant Physiol. Vol. 59, 1977 1153


in situ was observed to be between about 0.005 and 0.02 in thelight (recalculated from refs. 12 and 38), which is similar to theratios found in isolated chloroplasts photosynthesizing in a me-dium at pH 7.6 (Table IV).

MOBILIZATION OF STARCH

A number of experiments were carried out in order to identifythe products of starch breakdown. Rates of starch degradationwere variable, but usually low.

In the experiment of Figure 7, chloroplasts were first incu-bated in the light in the presence of '4C-HCO3- and low [Pi] inorder to form labeled starch. The chloroplasts were then washedrapidly in the cold, added to a medium containing either 5 mm Pior 5 mM PGA, and then kept at 20 C in the dark. In thepresence of Pi, the label in starch decreased whereas this did notoccur in the presence of PGA. The inhibition of starch degrada-tion by PGA in this experiment can be explained by the decreasein stromal [Pi] caused by PGA (16). When neither PGA nor Piwas added, the starch degradation was lower than with Pi added(not shown). Under these conditions the stromal [Pi] appearsnot to be high enough for maximal rate of starch degradation.From these results it would be predicted that phosphorylated

compounds would be released from the chloroplast during starchdegradation. In order to identify these, experiments were carriedout with 32P. At the times indicated (Fig. 8, A and B), sampleswere taken and the incubation terminated by centrifugal filtra-tion. It is seen that hexosemonophosphates were formed in thechloroplasts (Fig. 8A), and that triosephosphates and PGA werereleased to the medium (Fig. 8B). Some pentosemonophos-phates were also released (as in CO2 fixation) but very littlehexosemonophosphates. A comparison of the HMP concentra-tions in the stroma and in the medium shows that the envelope islargely impermeable for hexose phosphates, which concurs withearlier observations (16, 39).

Figure 9 shows the effect of incubating intact chloroplasts,which had accumulated labeled compounds during photosyn-thesis in the presence of 14CO2, with 10 mm Pi in the dark for 8min. There was considerable loss of 14C label from starch and acorresponding increase particularly in PGA and DHAP and tosome extent also in the pentosemonophosphates. There was alsoan increase in the radioactivity of an unidentified nonphospho-rylated compound which behaved like maltose in chromatogra-phy (cf. 23). However, this increase was small compared to thatseen in phosphate esters such as PGA and DHAP. These datasuggest that in spinach starch is degraded mainly by phosphoro-lysis leading to hexosephosphate and triosephosphate and, inmany respects are similar to those recently reported by Levi andGibbs (23). This could occur via the pentosephosphate pathwayat the expense of fixed carbon. Alternatively, triosephosphatescould be formed by the action of phosphofructokinase and aldol-ase. The former has been recently found in spinach chloroplasts(22), and the required ATP could be generated by oxidation oftriosephosphates to PGA. The possible occurrence of such apathway is supported by the observation that starch degradationin intact chloroplasts is stimulated to some extent by ATP, or byoxaloacetate. The latter oxidizes NAD(P)H thereby favoringtriosephosphate oxidation and associated ATP formation in thedark.

Chloroplasts were also isolated from spinach leaves which hadbeen detached and preilluminated to increase their starch con-tent. These were incubated in the dark at 20 C for 2 hr and theformation of metabolites followed by enzymic analysis. Someglucose (1 x 10-7 mol/mg Chl) was formed as well as PGA andtriosephosphate. In the presence of 5 mm Pi the PGA wasincreased 3-fold, but the rate of formation of glucose was notaffected.These preliminary results indicate that in spinach starch may

be hydrolyzed (possibly via maltose), to glucose and there are

CD0L

8min lig;ht 8_ B min light plus-_ 5 min dark with

I.. 8 mM P;

0-

E

0

0

E

220.

0.cm

cn~~~~~~~~~~~E

E

FIG. 9. Mobilization of 14C-labeled starch by dark treatment in thepresence of Pi. Starch synthesis in the presence of an initial H14CO3-concentration of 2 mm. The concentration of Pi in the medium was 0.1mM; the intensity of red light 120 w m-2. The time of incubation in thedark in the presence of additional Pi (8 mm) was 5 min.

indications of carrier-mediated glucose transport across the en-velope (34). Alternatively, starch is degraded by phosphorolysis,resulting finally in the formation of PGA or triosephosphates,which are then exported via the phosphate translocator. Furtherinvestigations are necessary in order to evaluate the physiologi-cal significance of these two alternative pathways.

CONCLUSION

Finally it may be noted that all of the foregoing results bear onthe problem of metabolic control in an organelle which succeedsin synthesizing starch by day and degrading starch by night withequal effectiveness. Clearly, both reaction sequences cannotcontinue unchecked throughout the entire 24-hr period. Asshown above, Pi may well play a crucial role in resolving thisproblem. It has been known since the work of Preiss and hiscolleagues (32) that ADP-glucose pyrophosphorylase is stimu-lated by high [PGA]/[Pi] and undoubtedly starch synthesiswould be facilitated as this ratio increased upon illumination(and vice versa). Our data therefore provide strong direct evi-dence that changes actually occur within the illuminated chloro-plast which are consistent with the Preiss hypothesis. In addition,however, the part played by Pi in the transport of metabolitesadds an extra dimension to this regulation. In spinach (as inother species) starch synthesis is responsive to the transport andutilization of metabolites. Starch synthesis can be increased bymanipulations (such as mannose feeding) which lower cytoplas-mic [Pi] even though these are known to bring about a simulta-neous depression of total photosynthesis (3, 18). If ADP-glucosepyrophosphorylase is the key to the regulation of starch synthesisin vivo then Pi and the phosphate translocator jointly constitutethe mechanism by which this regulation is influenced by theactivity of distant sinks.

Note. Verbal reports of this work have already been made(e.g. at the Botanikertagung in Zurich, August) and data andsuggestions relating to the influence of [Pi] on starch synthesis in

1154 HELDT ET AL .


chloroplasts have been recorded (19) and discussed elsewhere(5, 13, 19, 39-41).

Since the completion of this manuscript a paper by M. Steup,D. G. Peavey and M. Gibbs (Biochim. Biophys. Res. Commun.72: 1554-1561, 1976) has appeared in which the authors haveindependently derived data and conclusions similar to our own.

Acknowledgments - We are most grateful for a NATO award which made our cooperation apractical possibility. The research carried out in Munich and Dusseldorf was supported by theDeutsche Forschungsgemeinschaft and part of the work in Sheffield by the Science ResearchCouncil (U.K.) and the International Atomic Energy Agency. We thank Boehringer, For-schungslabor Tutzing, for a gift of 14C-PGA to H. W. Heldt.

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Starch Formation in Leaves and Isolated Chloroplasts