LeafPhosphateStatus, Photosynthesis, andCarbon ... · carbon to starch or sucrose synthesis is often viewed as a competitive process, i.e. whensucrose synthesisisdiminished, more

Plant Physiol. (1990) 92, 29-360032-0889/90/92/0029/08/$01 .00/0

Received for publication March 21, 1989and in revised form July 21, 1989

Leaf Phosphate Status, Photosynthesis, and CarbonPartitioning in Sugar Beet

Ill. Diurnal Changes in Carbon Partitioning and Carbon Export

1. Madhusudana Rao, Arthur L. Fredeen, and Norman Terry*

Department of Plant and Soil Biology, University of California, Berkeley, California 94720

ABSTRACT

The effect of low phosphate supply (low P) was determined onthe diurnal changes in the rate of carbon export, and on thecontents of starch, sucrose, glucose, and fructose 2,6-bisphos-phate (F2,6BP) in leaves. Low-P effects on the activities of anumber of enzymes involved in starch and sucrose metabolismwere also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1)were cultured hydroponically in growth chambers and the low-Ptreatment induced nutritionally. Low-P treatment decreased car-bon export from the leaf much more than it decreased photosyn-thesis. At growth chamber photon flux density, low P decreasedcarbon export by 34% in light; in darkness, export rates fell butmore so in the control so that the average rate in darkness washigher in low-P leaves. Low P increased starch, sucrose, andglucose contents per leaf area, and decreased F2,6BP. The totalextractable activities of enzymes involved in starch and sucrosesynthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5-diphosphoglucose pyrophosphoryl-ase, and sucrose-phosphate synthase. The activities of someenzymes involved in starch and sucrose breakdown were alsoincreased by low P. We propose that plants adapt to low-Penvironments by increasing the total activities of several phos-phatases and by increasing the concentrations of phosphate-freecarbon compounds at the expense of sugar phosphates, therebyconserving Pi. The partitioning of carbon among the variouscarbon pools in low-P adapted leaves appears to be determinedin part by the relative capacities of the enzymes for starch andsucrose metabolism.

The partitioning of carbon between starch and sucrose maybe regulated by several mechanisms (15, 21, 31). Some ofthese act over the short term, e.g. the fine control of cyto-plasmic FBPasel by F2,6BP, and some may act over the long

' Abbreviations: FBPase, fructose-1,6-bisphosphatase; ADPG,adenosine 5-diphosphoglucose; DHAP, dihydroxyacetone phosphate;F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; F2,6BP,fructose 2,6-bisphosphate; G 1 P, glucose 1-phosphate; G6P, glucose6-phosphate; G3P, glyceraldehyde 3-phosphate; p-NPP, p-nitro-phenyl phosphate; PFD, photon flux density; PGA, 3-phosphoglyc-erate; PPase, pyrophosphate phosphohydrolase; RuBP, ribulose 1,5-bisphosphate; RuBPCase, ribulose-1,5 bisphosphate carboxylase/ox-ygenase; SPS, sucrose-phosphate synthase; triose-P, glyceraldehyde 3-phosphate + dihydroxyacetone phosphate; UDPG, uridine 5-diphos-phoglucose.

term, e.g. changes in the amounts ofenzymes occurring whenplants adapt to a change in environment. The flow of fixedcarbon to starch or sucrose synthesis is often viewed as acompetitive process, i.e. when sucrose synthesis is diminished,more photosynthate is available for starch formation (e.g.when F2,6BP levels in the cytosol are high-see refs. 15, 21,31), and vice versa. Several researchers have proposed thatwhen cytosolic Pi levels decline, less triose-P exits the chlo-roplast so that more of the newly fixed carbon goes to starchthan to sucrose (12, 15, 21, 30, 31). Indeed, when external Pilevels around isolated chloroplasts are lowered, the pool sizesof Calvin cycle intermediates are increased and this is associ-ated with increased starch formation. Thus, there appears tobe an "overflow" mechanism at work in which starch forma-tion ensues when triose-P and other Calvin cycle intermedi-ates are increased to high levels. Carbon partitioning in intactplants may also be affected by the rate of carbon export (31).For example, if sucrose export from the leaf is slowed, highlevels of sucrose may inhibit an enzyme in the sucrose syn-thesis pathway (e.g. SPS) so that there is a diversion of fixedcarbon into starch. Alternatively, carbon export rates may bedetermined by the activities of key regulatory enzymes: highSPS activity has been shown to be correlated with high ratesof export (31). Thus, it is important to study both partitioningand export in the same system.

In the present work we use low-P stress, which is known tohave significant effects on carbon partitioning, photosyn-thesis, and export (3, 6, 7, 10, 15, 22-24, 29, 35), as a modelsystem to explore the regulatory mechanisms controlling car-bon partitioning in vivo. Here, we examine several factorswhich may influence carbon partitioning in low-P plants,including levels of F2,6BP, starch, sucrose, and glucose, totalextractable activities of enzymes of starch and sucrose metab-olism, as well as rates of carbon export and photosynthesis.Since the partitioning of fixed carbon may vary during thediurnal cycle, we have monitored changes in carbon partition-ing and export during the diurnal period.

MATERIALS AND METHODS

Plant Culture

Sugar beets (Beta vulgaris L. cv F58-554H 1) were culturedhydroponically in growth chambers at 25°C, 500 ,mol * m-2s-I photon flux density (400-700 nm) and a 16 h photoperiod

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Plant Physiol. Vol. 92, 1990

(23). Low-P and control treatments were established as de-scribed in Rao and Terry (23) and 5 week-old plants wereused for all the measurements.

Estimation of Carbon Export

Leaf gas exchange was determined as described before (23).Estimation of carbon export from the leaf was made usingthe method ofTerry and Mortimer (34). Leaf discs (1.77 cm2)were immediately frozen in liquid N2 and freeze-dried beforedetermining the dry weights. The rate of export, T, wasdetermined using the relationship,

T = P-A,

where P is the calculated rate of carbohydrate production dueto CO2 fixation and A the rate of accumulation of dry matter.It was assumed that the dry matter changes in leaf wereattributable to changes in carbohydrate-type compounds. Toexpress CO2 uptake rate and transport rate in the same units,,umol CO2 * m-2. s-' was converted to ,umol CH20* m-2 * I

since a large proportion of leaf organic matter is representedby this empirical formula.

Extraction and Analysis of Leaf Starch, Sucrose, andGlucose

Leaf discs (3.88 cm2) were sampled over a diurnal cycleinto liquid N2 using a light-transmitting Plexiglas punch.Samples were ground immediately in 80% ethanol and incu-bated at 40°C for 18 h and subsequently centrifuged for 10min at 10,000g. The supernatant was removed, evaporated todryness and redissolved in 2 mL of H20. To remove Chl, 0.5mL chloroform was added and the chloroform-water mixturecentrifuged (6,000g, 10 min). The upper clear phase wasremoved and used for glucose and sucrose analysis (4). Glu-cose concentration was determined with a glucose oxidaseenzyme system (Sigma Chemical Company, procedure No.510). Sucrose concentration was determined from the differ-ence between total glucose upon addition of invertase (Sigma14753) and free glucose as determined above. Sucrose-glucoselevels were converted to sucrose by multiplying by a factor of1.9 (after first subtracting the free glucose values). The residuefrom ethanol extraction was dried overnight at 55°C andstarch concentration was determined as described in Faderand Koller (4). The residue was gelatinized in 5 mL water for2 h in a boiling water bath. Subsequently, 5 mL of 0.1 Macetate buffer (pH 4.5), 25 mg amyloglucosidase ((EC 3.2.1.3)(Sigma A9268), and 5 mL water were added to the sample.Gas release stoppers were inserted and the contents incubatedat 55°C for 24 h. After incubation, the contents were mixedand centrifuged at 10,000g for 10 min and the supernatantretained for analysis. The resulting glucose concentration wasmeasured as described above. Starch equivalents were ob-tained by multiplying the values by 0.9.

Extraction and Assay of Enzymes

For the assay of all the enzymes, crude homogenates wereprepared by grinding, in a prechilled mortar and pestle, 30leaf discs (16.62 cm2) with 10 mL of extraction buffer (100mM Hepes-NaOH) (pH 7.7), 10 mM MgCl2, 0.4 mm EDTA,

100 mM Na-ascorbate, 1% (w/v) PVP, 1% (w/v) BSA, and 5mM GSH at 0 to 4°C. The brei was filtered through two layersof Miracloth. The filtrate was spun at 25,000g for 20 min (0-2°C) and the supernatant retained for enzyme assays. Thefollowing enzyme assays were determined according to thereferenced procedures with modifications as indicated.

Enzyme Assays

The compositions of the assay media for the respectiveenzymes are as follows:

1. ADPG pyrophosphorylase ((EC 2.7.7.27) (13): 50 mMHepes-NaOH (pH 8.0), 5 mM MgCl2, 1 mM ADPG, 2mM PPi, 0.5 mm PGA, 1 mm NADP, 15 units per mL ofphosphoglucomutase, 5 units per mL of glucose-6-dehy-drogenase, and 1 unit per mL of gluconate-6-P dehydro-genase (EC 1.1.1.44); reaction was initiated by the addi-tion of leaf extract.

2. Phosphorylase (EC 2.4.1.1) (16): 50 mm Hepes-NaOH(pH 6.8), 0.1 mg mL-' BSA, 10 mm Na-phosphate (pH7.4), 0.2 mg mL-'NADP, 1 mg mL-' amylopectin, 0.02mM glucose-1,6-P2, 55 ,ug mL-1 P-glucomutase (EC2.7.5.1), and 0.46 unit of glucose-6-P dehydrogenase; thereaction was initiated by adding leaf extract.

3. Maltose phosphorylase (EC 2.4.1.8) was assayed by sub-stituting 20 mm maltose for the amylopectin in the phos-phorylase assay media.

4. ,B-Amylase (EC 3.2.1.2) (16): 50 mM Hepes-NaOH (pH6.8) and 1 mg mL-' amylopectin; the reaction was initi-ated by adding the leaf extract. This reaction was termi-nated (after incubation for 30 min at 26C) in boilingwater for 1 min and centrifuged at 3000g to removeprecipitated protein; glucose released was measured by aglucose oxidase-peroxidase coupled reaction method.

5. Maltase (EC 3.2.1.20) (16): 50 mM Na-acetate buffer (pH6.0) and 100 mm maltose; the reaction was initiated bythe addition of leaf extract and terminated after 10 minincubation at 26°C by 2 min boiling, clarified by centrif-ugation at 3000g, and assayed for glucose as describedpreviously.

6. P-Hexose isomerase (EC 5.3.1.9) (11): 30 mM Hepes-KOH (pH 7.8), 4 mm MgCl2, 0.5 mM NADP, 1.2 mMF6P, leaf extract, and 2 to 4 units per mL of glucose-6-Pdehydrogenase (EC 1.1.1.49); reaction was initiated byF6P.

7. P-Glucomutase (EC 2.7.5.1) (1 1): the reaction conditionswere the same as for P-hexose isomerase, except that 1.2mM G1P replaced F6P.

8. NADP-Glucose 6-P dehydrogenase (EC 1.1.1.49) (19):100 mM Tris-HCL (pH 8.0), 0.2 mM NADP, and 1 mMG6P; reaction was initiated by the addition of leafextract.

9. Hexokinase (EC 2.7.1.1) (16): 50 mM Hepes-NaOH (pH7.8), 0.1 mg mL' BSA, 0.2 mg mL- NADP, 1 mM ATP,50 mM glucose, and 0.9 unit of glucose-6-P dehydrogen-ase; the reaction was initiated by adding leaf extract.

10. Sucrose phosphate synthase (EC 2.4.1.14) (13): the activ-ity was assayed by measuring F6P dependent sucrose-P(+ sucrose) formation from UDP glucose. The assay

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LEAF PHOSPHATE EFFECTS ON CARBON PARTITIONING AND EXPORT

mixture (0.5 mL) contained 7.5 mM UDPG, 7.5 mM F6P,15 mM MgC12, 50 mM Hepes-NaOH (pH 7.5), and analiquot of leaf extract. Mixtures were incubated at 25°Cfor 10 min and were terminated by addition of 0.5 mLof 1.0 N NaOH. Unreacted F6P was destroyed by heatingthe mixtures in the boiling waterbath for 10 min. Aftercooling, 0.5 mL of 0.3% (w/v) resorcinol in 95% ethanoland 1.5 mL of 40% HCL were added, and the mixturewas incubated at 80°C for 8 min. The tubes were cooledto room temperature and the A520 was measured.

11. UDPG-pyrophosphorylase (EC 2.7.7.9) (26): 50 mMHepes-NaOH (pH 7.5), 5 mM MgCl2, 5 mM UDPG, 2mM PPi, 0.3 mM NADP, 15 units per mL of phosphoglu-comutase, 5 units per mL of glucose 6-P dehydrogenase,and 1 unit per mL of gluconate 6-P dehydrogenase;reaction was initiated by the addition of leaf extract.

12. Cytosolic FBP phosphatase (EC 3.1.3.11) (13): 50 mMHepes-NaOH (pH 7.0), 5 mM MgCl2, 0.1 mM FBP, 0.2mM NADP, 2 units per mL each ofphosphoglucoisomer-ase and glucose 6-P dehydrogenase, and 1 unit per mL ofgluconate 6-P dehydrogenase; the reaction was initiatedby the addition of leaf extract.

13. Acid invertase (EC 3.2.1.26) (20): 75 mM Mes-NaOH (pH5.0), 40 mm Na-citrate, and 10 mm sucrose. The totalreaction volume was 1 mL and was incubated at 25°C for30 min. Reactions were begun by adding leaf extract andstopped by heating with boiling water for 10 min. Controlassays with denatured enzyme preparation were used tocorrect for glucose not produced by invertase. Glucosereleased was measured as described above.

14. Sucrose synthase (EC 2.4.1.13) (20): the activity wasdetermined as the UDPG-dependent sucrose formationin the presence of fructose as substrate: 7.5 mM UDP-glucose, 7.5 mm fructose, and 15 mM MgCl2 in 50 mMHepes-NaOH buffer (pH 7.5) at 25°C for 30 min. Reac-tions were initiated by the addition of leaf extract andwere terminated by heating with boiling water for 10 min,then samples were incubated 1 h with 10 units ofinvertase(25°C, pH 4.5), and glucose was liberated was measuredas described above.

15. ATP phosphohydrolase (EC 3.6.1.4) (25): the rate of Piliberation was measured in 1 mL reaction volume con-taining 50 mM KCI, 3 mm Tris-ATP, 3 mM MgSO4, and40 mM Tris-Mes buffer (pH 8.0). The reaction was initi-ated by the addition of leaf extract and was allowed toproceed for 30 min at 37°C. The amount of Pi liberatedwas measured by the method of Lanzetta et al. (14).

16. Inorganic pyrophosphate phosphohydrolase (EC 3.6.1.1)(25): the rate of Pi liberation was measured in 1 mLreaction volume containing 50 mm KC1, 3 mM Tris-PPi,3 mM MgSO4, and 40 mm Tris-Mes buffer (pH 8.0); thereaction was initiated by the addition of leaf extract.PPase activity was calculated as half the rate of Pi liber-ation (= mol PPi consumed/unit time) since the hydrol-ysis of 1 mol of PPi yields 2 mol of Pi. The amount of Piliberated was determined as described previously (14).

17. Acid phosphatase (EC 3.1.3.2) and alkaline phosphatase(EC 3.1.3.1) activities were determined according to Mat-sumoto and Yamaya (18) with the following modifica-

tion. The reaction mixture of the acid phosphatase assaywas 0.5 mL of 0.5 M acetate buffer (pH 4.5), 0.2 mL of45 mm p-NPP, and an aliquot of leaf extract with enoughdistilled water to bring the volume to 3 mL. In the caseof alkaline phosphatase, acetate buffer was replaced by0.5 M triethanolamine buffer (pH 9.8). The reactionproceeded at 3O°C for 10 min. At the end of the reaction,1.0 mL of 0.4 N NaOH was added and the mixture wasleft for 10 min. After centrifugation, the A410 of thesupernatant was recorded.

Extraction and Assay of Leaf F2,6BP

Leaf discs (3.88 cm2) were sampled over a diurnal cycleusing a light-transmitting Plexiglas punch into liquid N2. Oneleaf disc was ground with liquid N2 in a mortar. The frozenpowder was subsequently extracted in a medium of 0.5 mLcontaining 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 100 mMNaF, and 10 mM NaH2PO4. The extract was then placed inan 80°C bath for 5 min and centrifuged for 5 min in anEppendorf (model 5414) centrifuge, and the supernatant wasused for F2,6BP analysis as described in Fahrendorf et al. (5).

RESULTS

Effect of Low-P Treatment on Carbon Fixation, Export,and Accumulation in Leaf Blades at Light Saturation

The rate of photosynthesis was measured at saturating PFD(>2000 ,umol .m2 * s-') and averaged over four successive 2-hintervals (Fig. IA). The rate of photosynthesis did not changewith time over the 8-h period of measurement (Fig. IA). LowP reduced the rate of photosynthesis by 30 to 35%. Low-Ptreatment had a greater effect on carbon export than photo-synthesis; carbon export from the leaf blades was reduced by67 to 87% by low P, depending on the period ofmeasurement(Fig. 1B). There was some indication that carbon export ratesincreased with time over the first 6 hours of illumination inP-deficient plants. Because the rate of photosynthesis ex-ceeded the rate ofexport over this 8-h period, low-P treatmentresulted in high rates of carbon accumulation in the leafblades (Fig. IC).

Effect of Low-P Treatment on the Diumal Changes inCarbon Export from Leaf Blades

Rates of carbon export over the entire diurnal cycle werecompared for low-P and control plants growing under growthchamber conditions, i.e. with a PFD of 500 umol. m-2 S-supplied over a 16 h day-length (Fig. 2). After the onset ofillumination, the rate of carbon export in control leavesincreased with time up to 8 h, then remained constant for theremainder of the light period. In low-P leaves the rate ofcarbon export decreased over the first 4 h, then increased toa maximum constant value by 8 h.With the onset of darkness, carbon export in control leaves

decreased to a much greater extent than in low-P leaves. Theaverage rates ofcarbon export (,umol CH20 m2. s-1) in dark-ness were 2.88 + 1.64 and 6.73 ± 0.25 for control and low-P,while in light, the average values were 11.4 ± 1.54 and 7.53

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Plant Physiol. Vol. 92, 1990

40

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Figure 1. Effect of low-P treatment on carbon exchange rate (A),relative export of carbon (B), and relative accumulation of carbon (C)in sugar beet leaves. Measurements were made at saturating PFD(>2000 Iumol .m2 * s-1 and air levels of C02 (30 Pa) and 02 (21 KPa).Values are mean ± SD for three replications except for 6 to 8 h timepoint.

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TIME IN HOURSFigure 2. Effect of low-P treatment on the diumal changes in carbonexport in sugar beet leaves. Measurements were made at growthchamber PFD (500 ,Umol.m-2_s-). Values are mean ± SD for threereplications.

± 3.0 for control and low-P, respectively. Thus, low-P in-creased carbon export rates in the dark and decreased themin the light. Over the complete 24 h cycle however, low-Pdecreased carbon export by 15%.

Effect of Low-P Treatment on Starch, Sucrose, andGlucose Contents in Leaves

In control and low-P plants, starch content increased withtime during the light period and decreased in darkness (Fig.

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Figure 3. Effect of low-P treatment on the diurnal changes (at growthchamber PFD) in partitioning of stored carbon between starch, su-crose, and glucose in sugar beet leaves. The average specific leafdry wt for control and low-P leaves was 3.6 and 4.7 mg.Ccm2 whileleaf Chl was 428 and 528 mg. m-2, respectively. Values are mean +SD for three replications.

3A). Starch contents in the low-P leaves in darkness did notdecrease as soon as in the control leaves, there being a delayof 4 h before the starch contents fell. The average rate ofstarch accumulation in the light (in ,umol C. m2. s-') was1.88 and 1.38 for control and low-P leaves, respectively (theaverage rate was obtained by subtracting the initial from thefinal starch value and dividing by 16 h). In darkness, theaverage rate ofstarch breakdown (final-initial values dividedby 8 h) was -3.33 and -2.97 ,umol C m2 s-' for control andlow-P, respectively. Thus, even though starch values were

higher throughout the diurnal period in low-P leaves, starchwas accumulated faster in control leaves in the light andbroken down faster at night. Furthermore, the decrease in therate of accumulation of starch by day in the low-P leaves wasroughly equivalent to the decrease in the rate of photosyn-thesis, i.e. about 25%.

Sucrose contents were substantially higher (4- to 6-fold) inlow-P leaves than in the control (Fig. 3B). Sucrose, which isthe principal sugar translocated in sugar beet (8, 17), presum-ably accumulated to much higher levels in low-P leaves dueto lower rates of sucrose export. When the growth chamberlights were switched on, sucrose levels increased rapidly in

CONTROL0 r

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RAO ET AL.32

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LEAF PHOSPHATE EFFECTS ON CARBON PARTITIONING AND EXPORT

both treatments, reaching a constant level within 30 min.Later in the day, sucrose levels exhibited another increase,from 12 to 16 h. In darkness, sucrose contents decreasedrapidly in the control leaves (within 30 min) while in low-Pleaves there was no decrease until after 4 h had elapsed. Thus,in low-P leaves high sucrose contents were associated withhigher rates of carbon export during darkness (Fig. 2).Glucose contents of low-P leaves, like sucrose contents,

increased in the first 30 min, held constant, then increasedwith time during the later part of the light period (Fig. 3C).In control plants, glucose content was very low (about 5-10%of the low-P levels) and there was little change with time.With the onset of darkness, glucose in low-P leaves continuedto increase for 4 h, then decreased in the second half of thedark period as did starch and sucrose.

Effects of Low-P Treatment on Enzyme Activities

The increase in starch/area with low-P treatment was as-

sociated with a marked increase (86%) in the total extractableactivity of ADPG pyrophosphorylase (Table I). Two otherenzymes in the starch-synthesizing pathway, phosphohexoiso-merase and phosphoglucomutase, were not affected by low-Ptreatment (results not shown). Starch breakdown enzymes, f-

amylase, starch phosphorylase, maltose phosphorylase, andmaltase, were increased by 59, 152, 203, and 215% of thecontrol values, respectively, by low-P treatment (Table I).Low-P treatment increased sucrose contents to a marked

degree and the increase in sucrose was associated with an

increase in the total activities of sucrose-synthesizing enzymes(unlike in soybean where low-P resulted in low sucrose andlow SPS activity [7]). Low-P treatment increased the totalextractable activity of cytosolic FBPase by 58%, UDPG py-rophosphorylase by 76%, and SPS by 97% (Table II). Low-Ptreatment also increased the activity of acid invertase (whichhydrolyses sucrose to glucose and fructose) 2.2-fold (Table II).Sucrose synthase (which cleaves sucrose) was unaffected bylow-P (Table II). In very young sugar beet leaves, it has beenshown that sucrose synthase represents the major means ofsucrose breakdown; as leaves age, invertase becomes increas-ingly important (28). The increased activity of acid invertasein low-P leaves almost certainly contributes to the increasedlevels of glucose.

A major characteristic of P-deficient plants is that theydevelop increased amounts of phosphatases (see ref. 2 forreview), some of which may be located in the apoplast andvacuole. Low-P treatment increased alkaline phosphatase 2.1-fold, acid phosphatase 1.7-fold and ATP phosphohydrolaseby 58%; PPi phosphohydrolase however, was unaffected bylow-P (Table III).

Effect of Low-P Treatment on the Diurnal Changes inLeaf F2,6BP

F2,6BP levels in control leaves decreased on illuminationand increased with darkness (Fig. 4). In low-P leaves, F2,6BPlevels tended to follow a similar pattern but the differencebetween light and dark was much less clear. Low-P treatmentdecreased F2,6BP levels substantially (up to 60%) at all pointsduring the diurnal time period.

DISCUSSION

Over the short term, the inhibition of sucrose synthesisoften leads to a build-up in Calvin cycle intermediates whichthen overflow into starch synthesis (as described in refs. 15,31). Such a simple overflow mechanism does not appear toaccount for the results obtained here. Under low-P conditions,levels of Calvin cycle intermediates were markedly reducedwhile starch levels were higher than in control leaves. This isevident from the RuBP values which are reduced by 65%(RuBP is found only in the chloroplast) and from the PGAvalues (most of which is thought to be stromal in origin) ( 15)which were reduced 79% by low-P. Thus, it would appear

that some mechanism other than a simple overflow was

responsible for the build-up of starch in the chloroplasts oflow-P plants.The partitioning of carbon between starch synthesis and

sucrose synthesis is difficult to assess because there is no wayofdirectly measuring starch or sucrose synthesis. The amountsof starch or sucrose in the leaf depend on prior rates ofsynthesis and degradation, and in the case of sucrose, on

export from the leaf as well. The high starch levels in low-Pleaves indicate that prior starch synthesis rates were higherthan prior starch degradation rates. The rate of starch accu-mulation in light was about 25% lower in low-P leaves than

Table I. Effect of Low-P Treatment on the Total Extractable Activities of Certain Enzymes Involved inStarch Synthesis and Breakdown from Leaves of 5-Week-Old Sugarbeet Plants

Plants were dark adapted for 8 h prior to illumination (500 fimol. m-2. -1) for 1 h in the growthchamber before preparation of leaf extracts. Values are mean ± SD for three replications.

TreatmentEnzyme Control

Control Low-P

MoI.m2.S-1 %

ADPG-pyrophosphorylase 5.0 ± 1.7 9.32 ± 1.9 186fl-amylase 0.58 ± 0.05 0.92 ± 0.08 159Phosphorylase 0.24 ± 0.07 0.61 ± 0.17 252Maltose phosphorylase 0.32 ± 0.01 0.97 ± 0.27 303Maltose 0.48 ± 0.01 1.51 ± 0.03 315Hexokinase 1.2 ± 0.31 2.37 ± 0.56 198NADP-G6P dehydrogenase 2.05 ± 0.17 3.87 ± 0.32 189

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Plant Physiol. Vol. 92, 1990

Table II. Effect of Low-P Treatment on the Total Extractable Activities of Certain Enzymes Involved inSucrose Metabolism from Leaves of 5-Week-Old Sugarbeet Plants

Plants were dark adapted for 8 h prior to illumination (500 umol _ m2. s') for 1 h in the growthchamber before preparation of leaf extracts. Values are mean ± SD for three replications.

TreatmentEnzyme Control

Control Low-P

M.mo*M-2'S-1 %

Cytosolic FBPase 1.97 ± 0.47 2.9 ± 4.7 147UDPG-pyrophosphorylase 28.17 ± 4.67 49.51 ± 5.83 176Sucrose-P synthase 3.47 ± 1.42 6.83 ± 2.32 197Acid invertase 0.73 ± 0.11 1.63 ± 0.25 223Sucrose synthase 0.25 ± 0.01 0.24 ± 0.02 96

Table Ill. Effect of Low-P Treatment on the Total Extractable Activities of Certain Phosphatases fromLeaves of 5-Week-Old Sugarbeet Plants

Plants were dark adapted for 8 h prior to illumination (500 ,Amol. m-2. -1) for 1 h in the growthchamber before preparation of leaf extracts. Values are mean ± SD for three replications.

TreatmentEnzyme Control

Control Low-P

Mmol.m-2'S-1 %

Acid phosphatase 28.89 ± 0.58 48.0 ± 1.17 166Alkaline phosphatase 3.04 ± 0.13 6.4 ± 0.32 211ATP phosphohydrolase 4.28 ± 1.57 6.78 ± 0.55 158PPi phosphohydrolase 102.1 ± 11.16 92.57 ± 14.58 91

124

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Figure 4. Effect of low-treatment on the diurnal changes (at growthchamber PFD) in leaf F2,6BP levels in sugar beet plants. Values aremean ± SD for three replications.

in the controls, a decrease commensurate with the decreasein the rate of photosynthesis, suggesting that the slower rateof starch accumulation was due to the decrease in photosyn-thetic rate. At night, starch degradation was also slower inlow-P leaves, despite the increased total extractable activitiesof several starch degrading enzymes.

In the present work, low-P treatment decreased F2,6BP inboth light and dark (Fig. 4). This could have resulted in an

increased carbon flow to sucrose. Sucrose levels were four- tosix-fold higher in low-P leaves compared to the control, andnet rates of sucrose accumulation during the light were higherin low-P leaves than in control leaves (0.41 compared to 0.33,umol C.m-2 s-', respectively). However, in view of themarked reduction in carbon transport from the leaf, thesehigher rates of sucrose accumulation in low-P leaves were

almost certainly due to the reduction of sucrose export andnot to increased sucrose synthesis or decreased sucrose

degradation.Low-P treatment clearly increased the levels of starch,

sucrose, and glucose in leaves. It may also have increasedstructural as well as nonstructural carbohydrates. Low-P treat-ment increased leaf dry weight per unit area by 30%. Only9% (or less) of the increase in dry weight in low-P leaves wasdue to starch; the remainder ofthe increase in dry weight maywell have been due to other carbon compounds such as cellwall polymers (cellulose and hemicelluloses). It is highly likelythat the glucosyl donor for cellulose synthesis is UDPG, whilepectic substances and hemicelluloses are formed from nucle-oside diphospho-sugars that are made by direct interconver-sion from UDPG (see ref. 1 for review). The formation ofUDPG for structural polysaccharide synthesis in leaves occursmainly via UDPG pyrophosphorylase (1). Since our researchshows that there were increases in the activities of cytosolicFBPase and UDPG pyrophosphorylase, it seems likely thatthe synthesis of UDPG would be higher under low-P condi-tions, facilitating an accumulation ofstructural carbohydrates(7).The results show that low-P treatment increased starch,

sucrose, and glucose while at the same time diminishing levels

LIGHT DARK/K , I

0[

0

10

34 RAO ET AL.

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LEAF PHOSPHATE EFFECTS ON CARBON PARTITIONING AND EXPORT

of sugar phosphates in the chloroplast and leaf. There was notonly a large reduction in the level of RuBP and PGA, butalso in FBP, F6P, and G6P (3, 15, 24, 29). The decrease inpool sizes of sugar phosphates with low-P was in part due toincreased phosphatase activities (e.g. acid and alkaline phos-phatases). However, it is also possible that the leaf sugarphosphate pools were depleted in response to increases in thecapacities of the enzymatic pathways for the formation ofphosphate-free carbon compounds, e.g. starch and sucrose.

In this regard, our results are consistent with the view thatthe enzymatic capacities for starch and sucrose synthesis wereincreased by low-P. For example, ADPG pyrophosphorylaseactivity, a key regulatory enzyme in starch synthesis pathway,was increased substantially by low-P (57%). Low-P treatmentalso increased the activities of FBP aldolase (53%) and chlo-roplastic FBPase (61%), two other enzymes in the starchsynthesizing pathway (23). With regard to the sucrose synthe-sizing pathway, three key enzymes were substantially in-creased by low-P: sucrose-phosphate synthase (97%), cytosolicFBPase (58%), and UDPG pyrophosphorylase (76%). Similareffects oflow-P on the sucrose synthesizing enzymes ofbarleyseedlings were reported by Sicher and Kremer (29). Theincreased capacities for starch and sucrose synthesis weresufficient to allow starch and sucrose to be accumulated inhigher amounts in low-P leaves, despite increases in the starchdegrading enzymes, ,B-amylase, starch phosphorylase, maltosephosphorylase, and maltase, and in the sucrose degradingenzyme, acid invertase.The accumulation of photosynthate in leaf blades of low-P

leaves was due to the fact that low-P treatment did not impairphotosynthesis as much as it impaired carbon export. Overan 8 h period at light saturation, photosynthesis was reducedby about 35% by low-P while export was reduced by as muchas 87%. This led to an accumulation of a large proportion ofthe net carbon fixed in the leaf blades oflow-P leaves (Fig. 1).Plants growing at the lower PFD (500 ,Amol m-2. s-') of thegrowth chamber did not exhibit such marked decreases ofcarbon export as at light saturation. Low-P reduced photosyn-thesis by 25% and export by 34% on average at growthchamber PFD. Over the diurnal cycle, export was decreasedonly 15% by low-P. However, the fact that, after 14 d ofgrowth at low-P, leaf dry weight per area increased by 30%and the proportion of total plant dry matter invested in theleaf blades increased from 37 to 48% (23) shows that lesscarbon was exported from low-P leaves. Radin and Eidenbock(22) obtained similar results with cotton.

It is unclear why low-P had such a pronounced effect oncarbon export. It is conceivable that the effect of low-P wasmediated in the leaf blade itself, e.g. at the phloem loadingstep (9). The loading of sugars into the phloem is known tohave a large requirement for ATP (see ref. 8 for review), andthere may be insufficient ATP in the low-P leaves to maintainexport at a rapid rate (ATP levels were reduced as much as60% in low-P leaves) (24). However, sucrose concentrationsin the fibrous roots, storage roots, and very young leaves oflow-P plants were higher than in control plant (our unpub-lished data). This suggests that despite the reduction in exportrate in the low-P treatment, sucrose was exported from leavesand accumulated in sinks in high concentrations. Sawada et

al. (27) showed that when the growth of single-rooted leavesof soybean was decreased by low temperature, i.e. where sinksize was reduced, there was an accumulation of starch andsucrose in the leaf. Low-P treatment diminished the growthofnew leaves substantially, as well as the growth ofthe storageroot and leaf petioles, which are sites of surplus sugar storagein sugar beets (33). Thus, export in low-P plants may havebeen limited due to insufficient sink capacity for carbohy-drate. Fibrous roots, however, did constitute an active sinkfor photosynthate because they continued to grow at reason-able rates under low-P conditions (23).

Rates of carbon export in low-P leaves over the diurnalcycle were not correlated with leaf sucrose contents (Figs. 2and 3B). In light, export rates were greater in the control thanin low-P while sucrose contents were lower; in darkness,export rates and sucrose contents were both higher in low-Pleaves but did not seem to be correlated with each other overtime. Several researchers have observed a positive correlationbetween carbon export and SPS activity (see ref. 31 forreview). Such a correlation was found in darkness (see alsorefs. 17, 31) but not in the light.There is evidence that plants undergo substantial adapta-

tion to low P environments, a situation that plants frequentlyexperience under natural conditions. For example, it is knownthat plants increase the activity of their phosphatases inresponse to P deficiency (see ref. 2 for review). The increasesin the level of phosphatases in roots help to release phosphatebound up in root cell walls and perhaps even in the soil (32).In leaves it is likely that the major role of phosphatases is toincrease the availability of phosphate for photosynthesis andother important leaf functions. This is especially evident fromthe present work since several phosphatases were substantiallyincreased by low-P treatment.We speculate that the physiological and metabolic changes

which occur in response to P deficiency may be part of anadaptive strategy to low-P environments. Thus, as Pi becomeslimiting to growth, changes in enzymatic pathways occur suchthat the amount of phosphate tied up in phosphorylatedintermediates is reduced, and the amounts of phosphate-freecarbon compounds are increased. This physiological adjust-ment increases the amount of Pi available for photosynthesisand other essential physiological functions. The storage ofcarbon in phosphate-free forms such as starch, sucrose andglucose provides carbon reserves for growth if and when Psubsequently becomes available.The Pi translocator would also serve a useful function in

the adaptation to P deficiency since it functions as a mecha-nism for providing the chloroplast with a continuing supplyof orthophosphate under conditions of low-P. Since the Pi-translocator ensures the uptake of 1 Pi for every triose-Pexported from the chloroplast, the Pi content of the chloro-plast is maintained at levels which permit substantial rates ofphotosynthesis, thereby permitting carbohydrate accumula-tion and also preventing potential damage to the photosyn-thetic apparatus from photoinhibition.

In conclusion, it would appear that in low-P adapted plants,carbon partitioning in leaves is influenced by the capacitiesof the enzymes for starch and sucrose metabolism. In low-Pleaves, starch and sucrose were accumulated simultaneously

35

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Plant Physiol. Vol. 92, 1990

while chloroplastic and leaf levels of sugar phosphates de-creased markedly. These results cannot be explained by short-term regulation in which, for example, an inhibition ofsucroseformation leads to a build-up of sugar phosphates in thechloroplast and therefore to an increase in the formation ofstarch. It seems more likely that the simultaneous increasesin starch and sucrose at the expense ofsugar phosphates underlow-P stress were in part due to protein modification or

protein turnover of the key enzymes involved in starch andsucrose metabolism.

ACKNOWLEDGMENTS

We thank A. R. Arulanantham for his help in F2,6BP measure-

ments and Clifford Carlson for excellent technical assistance in gas

exchange measurements.

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