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Plant Physiol. (1992) 98, 316-3230032-0889/92/98/031 6/08/$01 .00/0

Received for publication June 3, 1991Accepted August 11, 1991

Diurnal Starch Accumulation and Utilization inPhosphorus-Deficient Soybean Plants1

Jinshu Qiu and Daniel W. Israel*Department of Soil Science/Plant Physiology Program and U.S. Department of Agriculture,Agricultural Research Service and Department of Soil Science/Plant Physiology Program,

North Carolina State University, Raleigh, North Carolina 27695

ABSTRACT

The effects of phosphorus deficiency on carbohydrate accu-mulation and utilization in 34-day-old soybean (Glycine max L.Merr.) plants were characterized over a diurnal cycle to evaluatethe mechanisms by which phosphorus deficiency restricts plantgrowth. Phosphorus deficiency decreased the net CO2 exchangerate throughout the light period. The decrease in the CO2 exhangerate was associated with a decrease in stomatal conductanceand an increase in the internal CO2 concentration. These obser-vations indicate that phosphorus deficiency increased mesophyllresistance. Assimilate export rate from the youngest fully ex-panded leaves was decreased by phosphorus deficiency,whereas starch concentrations in these leaves were increased.Higher starch concentrations in phosphorus-deficient youngestfully expanded leaves resulted from a longer period of net starchaccumulation and a shorter period of net starch degradationrelative to those for phosphorus-sufficient controls. Phosphorusdeficiency decreased sucrose-P synthase activity by 27% (aver-aged over the diumal cycle), and essentially eliminated diurnalvariation in sucrose-P-synthase activity. Diumal variations in non-structural carbohydrate concentrations in leaves and stems werealso less pronounced in phosphorus-deficient plants than in con-trols. In phosphorus-deficient plants, only 30% of the whole plantstarch present at the end of a light phase was utilized during thesubsequent 12-hour dark phase as compared with 68% for phos-phorus-sufficient controls. Although phosphorus deficiency de-creased the CO2 exchange rate and whole plant leaf area, accu-mulation of high starch concentrations in leaves and stems andrestricted starch utilization in the dark indicate that growth proc-esses (i.e. sink activities) were restricted to a greater extent thanphotosynthetic capacity. Further experimentation is required todetermine whether decreased starch utilization in phosphorus-deficient plants is the cause or the result of restricted growth.

Phosphorus has an important role in the photosyntheticcarbon metabolism of leaves (6, 23). Phosphorus deficiencyhas been shown to decrease photosynthetic rate per unit leafarea (5, 14) and whole plant leaf area to a greater extent thanwhole plant dry mass (5, 1 1). Despite decreasing the photo-synthetic rate per unit leaf area, phosphorus deficiency causedincreased starch concentrations in leaves (5), which indicateseffects on biochemical partitioning of photosynthate.

Cooperative investigations ofthe U.S. Department ofAgriculture,Agricultural Research Service, and the North Carolina AgriculturalResearch Service, Raleigh, NC.

Starch formation is controlled by the ratio of 3-phosphog-lyceric acid to Pi in the chloroplast (9). A relatively high ratioactivates ADP-glucose pyrophosphorylase, resulting in netaccumulation of starch and decreased export of fixed carbonfrom the chloroplasts, whereas a low ratio simultaneouslyinactivates ADP-glucose pyrophosphorylase and stimulatesphosphorylase, resulting in the net degradation of starch (9).

SPS2 is a key regulatory enzyme in the sucrose synthesispathway, and its activity in leafextracts is positively correlatedwith the capacity for leaf sucrose formation and negativelycorrelated with leaf starch accumulation (10). Phosphate hasbeen found to inhibit SPS activity in vitro being a competitorwith the substrate UDP-glucose in the spinach enzyme (1).The response of SPS activity to phosphorus deficiency seemsto vary with plant species. The extractable enzyme activityfrom sugar beet leaves was increased by phosphorus deficiency(19), whereas extractable activity from soybean leaves wasdecreased by phosphorus deficiency (5). In addition, diurnalfluctuations in the SPS activity of soybean leaves has beenreported (20), which indicates that accurate assessment of thephosphorus-deficiency effect on SPS activity may requiremeasurements over a diurnal cycle.Although there is a considerable body of information on

the role of Pi in regulating the partitioning of photosynthatebetween starch and sucrose in isolated individual leaves (2),the function of phosphorus in regulating nonstructural car-bohydrate accumulation and utilization in whole plants ispoorly understood. In particular, the effects of phosphorusdeficiency on photosynthate transport into stems and rootsand on its accumulation and utilization in these organs havenot been investigated.Although phosphorus deficiency has been shown to de-

crease the photosynthetic rate of leaves (5, 14), several reportsindicate that plant growth may be more sensitive to a givenlevel of phosphorus deficiency than photosynthesis. Eaton (3)observed that phosphorus deficiency increased the starch con-centrations in stems of soybean plants by as much as 10-fold,depending on plant age. Fredeen et al. (5) reported thatphosphorus-deficient soybean plants contained 30-fold and 7-fold higher starch concentrations in expanding leaves andfibrous roots, respectively, than nondeficient plants. These

2Abbreviations: SPS, sucrose-P synthase; YFE, youngest fullyexpanded; DAT, days after transplanting; CER, CO2 exchangerate; SAR, starch accumulation rate; TNC, total nonstructuralcarbohydrate.

316

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PHOSPHORUS DEFICIENCY AND STARCH ACCUMULATION

results indicate that decreased carbohydrate availability doesnot account for decreased growth under phosphorusdeficiency.The purpose of this study was to evaluate the hypothesis

that carbohydrate utilization rather than availability limitsplant growth under phosphorus deficiency. This was accom-plished by measuring effects of phosphorus deficiency on: (a)photosynthetic activity of YFE leaves; (b) accumulation andutilization of nonstructural carbohydrates in YFE leaves, totalleaves (i.e. all other leaves except the YFE leaf), stems, androots of soybean plants; (c) the distribution of carbohydrateamong these organs; and (d) the SPS activity in the YFEleaves during a diurnal cycle.

MATERIALS AND METHODS

Plant Culture

Soybean (Glycine max Merr., "Ransom" maturity groupVII) plants were grown outdoors in 6-liter pots filled withPerlite from June 4 to July 8, 1987. Plants were exposed tonatural weather conditions throughout the course of the ex-periment. Seeds were germinated in 0.5 mM CaSO4 at 30°Cand 95% RH for 3 d. Two seedlings were transplanted intoeach pot and thinned to one plant per pot at 14 DAT.

Plants were supplied with nutrient solutions containing 20mM NO- as an N source, prepared in tap water. The compo-sition ofthe nutrient solutions was identical to that previouslydescribed by McClure and Israel (15), except that phosphorusconcentrations were 0.05 mm (deficient) or 1.00 mm (control)and phosphorus was added as potassium dihydrogen phos-phate. Potassium concentration in the two solutions wasequalized by addition of K2SO4. The pH of both solutionswas adjusted to 6.2 by addition of 1.0 N H2SO4. From trans-planting until 3 DAT, pots were irrigated only with 0.25 literof tap water at 0900 and 1400 h. From 3 to 7 DAT, each potwas flushed with 0.5 liter of tap water at 0900 and 1400 hand 0.25 liter of appropriate nutrient solution was appliedafter each flushing. Thereafter, pots were flushed at both 0900and 1400 h with 1.5 to 2.0 liters of tap water, and appropriatenutrient solution (0.5 liter/pot) was supplied only after the1400 h flushing.

Plant Growth Measurements

On 34 DAT, vegetative growth stage was determined ac-cording to the method of Fehr and Caviness (4). Leaf areawas measured photometrically with LICOR model (LI-3000)leaf area meter.3 Specific leaf weight was calculated on thebasis ofg fresh weight. dm2. Organ weight was obtained afterplant material was freeze-dried to constant weight. To com-pare leaves of similar physiological age in different nutritionaltreatments, all parameters were measured on the YFE leaf.The YFE leafwas defined as the leaf that attained full expan-sion 1 d before the measurements were taken.

3 Mention of a trademark or proprietary product does not consti-tute a guarantee or warranty of the product by the U.S. Departmentof Agriculture or the North Carolina Agricultural Research Service,and does not imply its approval to the exclusion of other productsthat may also be suitable.

Gas Exchange Measurements

Net CER, internal CO2 concentration, stomatal conduct-ance, and transpiration rate were simultaneously measuredusing a portable Analytical Development Co. photosynthesissystem (Hoddesdon, England) equipped with a cuvette thatenclosed the upper and lower surfaces of a 6.25-cm2 area ofleaf. Ambient air containing approximately 350 jL.liter-'CO2 was circulated through the cuvette at a flow rate of 400mL min-'. Differentials between CO2 concentrations in gasstreams entering and exiting the cuvette attained constantvalues within 45 s of attaching the cuvette to the leaves. On34 DAT, photosynthetic measurements were taken on thecenter leaflets of the YFE leaves on the main stem at 0830,0900, 1100, 1700, and 1930 h. The YFE mainstem leavesused for photosynthetic measurements experienced minimalshading by leaves above them. When CER measurementswere made, the cuvette was held perpendicular to the sun tomaximize light intensities striking the leaf. Measurementswere made on a clear day when light intensities between 1100and 1700 h exceeded 1800 timol quanta-m-2-s' (Fig. lA).Temperatures of leaves within the cuvette recorded at the endof the 45-s measurement period ranged from 29.5 ± 0.20C at0830 h to 36.1 ± 0.4°C at 1700 h. The CER, internal CO2concentration, stomatal conductance, and transpiration ratewere calculated according to the equations ofvon Caemmererand Farquhar (22).

20

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Time of Day, hour

350

330

310 -J

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270

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212 E

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Figure 1. Effects of phosphorus deficiency on CER (A); intemal CO2concentration, Ci (B); stomatal conductance, Gs (C); and transpirationrate, E (D). The F-test showed significant treatment and time effectson CER (P = 0.0001), Ci (P = 0.0001), Gs (P = 0.0001), and E (P =0.01) and a significant treatment by time interaction effect on A (P <0.05). Numbers beside points in A represent intensities of photosyn-thetically active radiation (umol quanta. m-2. s-1) at time measure-ments were made.

317



Plant Physiol. Vol. 98, 1992

Assimilate Export

The rate of assimilate export was calculated using thefollowing equation (13):

Export rate = CER - SAR/O.75

where export rate and CER are in mg CH2O/dm2/h, and SAR(starch accumulation rate) is in mg CH2O/dm2/h. The factor0.75 is the proportion of dry matter accumulation rate that isaccounted for by starch accumulation. According to Kerr etal. (13), total starch accumulation accounts for approximately75% of the dry matter accumulation in the YFE leaves of"Ransom" soybeans in the light.

Sampling Protocol

On 34 DAT, plants were sampled at 3-h intervals, startingat 0900 h and ending at the same time on the following day.On the same YFE leaf, trifoliolate leaf (T3) for 0.05 mM

phosphorus and T5 for 1.00 mm phosphorus, the photosyn-thetic rate was measured and six discs (total area equal to2.65 cm2) were taken. Leaf discs were placed into ice-cold80% ethanol until they could be transferred to the laboratoryfor storage at -20C until analyzed for starch, sucrose, andhexose. Immediately after taking discs, the YFE leaves were

excised, and fresh weight and leaf area determined. The YFEleaves were then frozen on dry ice and stored at -80°C untilanalyzed for SPS activity. The remainder of the plants was

separated into leaves, stems (plus petioles), and roots andfrozen on dry ice. The tissues were freeze-dried, weighed, andground to pass a 1-mm screen.

Carbohydrate Analysis

The leaf discs were ground in 80% ethanol and extractedtwice in 80% ethanol at 80°C for 10 min. After centrifugation,an aliquot from the supernatant was assayed enzymaticallyfor sucrose and hexose (12). Hexose concentrations deter-mined by this procedure (12) include glucose, glucose-6-phosphate, fructose, and fructose-6-phosphate. Ethanol-insol-uble material was gelatinized by boiling in 0.2 M KOH for 30min. After adjusting the pH to 5.5 with 1.0 M acetic acid, the

gelatinized starch was digested for 30 min with amylogluco-sidase, and the glucose determined enzymatically using hex-okinase and glucose-6-P dehydrogenase (13). The same pro-cedures were used to determine starch and soluble sugarconcentrations in leaves, stems, and roots with the exceptionthat 20 mg of ground tissue was used.

SPS Activity Assay

Extracts of frozen samples of YFE leaves were preparedand assayed for SPS activity as described by Kerr et al. (13).Extracts were assayed within 1 h of initiating the extraction.

Total Phosphorus Measurements

Tissue samples (about 100 mg) were digested by a Kjeldahlprocedure (7, 17). Aliquots of the digests were adjusted to pH3 by adding 4 N KOH. The total phosphorus concentrationof the digests was determined by the ammonium molybdatemethod of Murphy and Riley (16).

Experimental Design and Statistical Analysis

The experimental design was a randomized complete blockwith four replications. All combinations of two factors (treat-ment and time) were randomly assigned to each block. Thedata were analyzed using the analysis of variance procedureof the Statistical Analysis System (8). When a significant (Pc 0.05) treatment by time interaction was obtained, LSDO.O5values were calculated and presented in tables and figures forcomparison of treatment means.

RESULTS

Plant Growth and Organ Phosphorus Concentrations

Phosphorus deficiency reduced the vegetative growth stage(V6 in P-deficient plants versus V9 in controls) and canopy

leafarea (Table I). Whole plant dry weight was decreased 82%by phosphorus deficiency. External phosphorus deficiencydecreased the phosphorus concentrations in all organs byabout 70% and the shoot/root ratio by 35% relative to con-

trols (Table I). Decreased dry matter accumulation and organphosphorus concentrations in phosphorus-deficient plants

Table I. Effect of Phosphorus Deficiency on Plant Growth and Phosphorus Concentration in Tissues at 34 DA1'Extemal Phosphorus Concentration

Parameters 0.05 mM 1.00 mMWhole plant Whole plant

Leaves Stems Roots Leaves Stems Roots

Growth stage V6 V9Dry wt (g/organ) 1.91 1.01 1.74 4.66 11.25 7.84 7.21 26.30CLAb (dm2/plant) 4.30 34.50Canopy SLW (gdw/dm2) 0.41 0.30Shoot/root ratio 1.73 2.68Total P (mg/organ) 2.18 1.05 1.57 4.80 44.21 27.05 20.26 91.52P conc. (mg/gdw) 1.14 1.04 0.90 3.93 3.45 2.81% of whole plant P 45 22 33 48 30 22

a Because plant growth parameters and organ phosphorus concentrations did not show significant diumal variation, the values are meansobtained by averaging over all sampling times. b CLA, canopy leaf area; Canopy SLW, canopy-specific leaf weight; gdw, g dry weight.

318 QIU AND ISRAEL




were associated with a 95% decrease in whole plant phospho-rus accumulation. A greater proportion of whole plant phos-phorus was accumulated in roots under phosphorus defi-ciency. Phosphorus deficiency increased specific leaf weightby 37% (Table I).

Net CO2 Exchange Rate and Assimilate Export Rate

Phosphorus deficiency depressed the photosynthetic rate ofthe YFE leaves over the light phase (Fig. lA). Averaging overall sampling times except the 1930 h sampling indicates thatphosphorus deficiency decreased the CER of YFE leaves by30% (Fig. IA). At 1930 h, the light intensity was below theCO2 compensation point. Phosphorus deficiency caused a 6%increase in internal CO2 concentration (Fig. 1B), a 25%decrease in stomatal conductance (Fig. IC), and had a mini-mal effect on transpiration rate (Fig. ID).The estimated rate ofassimilate export from the YFE leaves

during the light phase was also reduced by phosphorus defi-ciency (Fig. 2A). In both treatments, the patterns of exportrates generally paralleled those ofCER (Figs. IA and 2A). Onthe average, the export rate accounted for 83% of total carboninput (i.e. CER) in the phosphorus-deficient YFE leaves and90% in controls (Fig. 2B).

Diurnal Changes in Carbohydrates in the YFE Leaves

Phosphorus deficiency increased the concentration ofstarchin the YFE leaves over the diurnal cycle (Fig. 3A). However,during a period from 1500 to 1800 h, the concentrations weresimilar in the two treatments. The concentration of starchcontinuously increased throughout most of the light phase in

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Figure 2. Effects of phosphorus deficiency on calculated assimilateexport rate (A) and on relative export and starch accumulation as apercentage of CER (B) during the light phase.

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Figure 3. Effects of phosphorus deficiency on diurnal changes in theconcentrations of starch (A), sucrose (B), and hexose (C) and starchas a percentage of total nonstructural carbohydrate (TNC) (D) in theYFE leaves. Treatment and time effects on all parameters weresignificant (P < 0.005). Treatment by time interaction effects weresignificant (P < 0.01) for all parameters except starch concentration.

the YFE leaves of both treatments, reaching a maximum at2100 h in the phosphorus-deficient YFE leaves and at 1800h in controls. The concentrations in both treatments declinedto a minimum at the end of the dark phase. The YFE leavesof phosphorus-deficient plants thus had a longer period of netstarch accumulation and a shorter period of net starch deg-radation than those of controls (Fig. 3A). Over the diurnalcycle, starch accounted for 90 to 95% of the TNC in leavesof phosphorus-deficient plants (Fig. 3).The sucrose concentration in the YFE leaves of controls

increased linearly from the beginning of the light phase to1500 h, declined rapidly until the beginning ofthe dark phase,and then remained constant over the dark phase. Underphosphorus deficiency, however, the sucrose concentrationincreased rapidly during the first 3 h of the light phase to aplateau, and then decreased gradually during the dark phase.Comparing means obtained by averaging over the diurnalcycle indicates that phosphorus deficiency decreased the su-crose concentration of YFE leaves by 25%.The concentration of hexose in the YFE leaves of control

plants reached a maximum at 1200 h, decreased during theafternoon, and remained at a minimum throughout the darkphase (Fig. 3C). In contrast, the hexose concentration in theYFE leaves ofphosphorus-deficient plants was generally lowerthan that of controls and relatively constant over the diurnalcycle.

Diurnal Change in SPS Activity

SPS activity was decreased by an average of 30% over thelight phase and 24% over the dark phase by phosphorusdeficiency (Fig. 4). The diurnal change in SPS activity in the

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Figure 4. Effect of phosphorus deficiency on SPS activity in YFEleaves over a diurnal cycle. Treatment and time effects on SPSactivity were significant (P = 0.0001, P < 0.01, respectively).

YFE leaves of controls showed two peaks separated by 12 h.The diurnal variation in SPS activity was much less pro-

nounced in the YFE leaves of phosphorus-deficient plants as

the activity during the 2100 to 0300 h dark period averaged93% of the maximum activity at 1200 h (Fig. 4).

Diurnal Changes in Nonstructural Carbohydrates inWhole Plants

The diurnal fluctuation in the starch concentration of leaves(all leaves except the YFE leaf) was less in phosphorus-deficient plants than in controls (Fig. SA). The concentrationin leaves of phosphorus-deficient plants was about twofoldhigher than that of controls at the beginning of the light phase.Maximal starch concentrations in both treatments were sim-ilar and occurred between 1500 and 1800 h and, thereafter,the decrease in the starch concentration was much slower inthe leaves of phosphorus-deficient plants than in leaves ofcontrols.The starch concentration was consistently higher in the

stems of phosphorus-deficient plants compared with controls.However, the diurnal variation in stem starch concentrationwas much less pronounced in the phosphorus-deficient plants(Fig. SB). Starch concentration in roots of phosphorus-defi-cient plants was significantly lower than that of control plantsbetween 1500 and 2400 h and similar to that of controls atother sampling times (Fig. 5C).

Phosphorus deficiency decreased the concentrations of sug-

ars (hexose + sucrose) in all organs (Fig. 5, D-F). Diurnalvariation in the sugar concentration in leaves of controlsexhibited two peaks at 1200 and 0300 h, respectively. How-ever, the sugar concentration in the leaves of phosphorus-deficient plants was relatively constant throughout the lightphase and followed a pattern similar to controls over the darkphase. Although the sugar concentrations in the stems androots were consistently lower in phosphorus-deficient plantsthan in controls over the diurnal cycle, patterns of diurnalvariations were similar in both treatments (Fig. 5, E and F).

Phosphorus deficiency increased starch as a percentage ofTNC in all organs throughout most of the diurnal cycle (Fig.5, G-I). Under phosphorus deficiency, starch accounted for86 to 93% of the TNC pool in leaves (Fig. 5G), 61 to 74% instems (Fig. 5H), and 24 to 35% in roots (Fig. 5I) over thediurnal cycle. Starch as a percentage of TNC in leaves andstems of phosphorus-deficient plants was high and relativelyconstant over the diurnal cycle. In controls, however, starchas a percentage of TNC in leaves and stems increased duringthe light phase and decreased in the dark phase (Fig. 5, G andH).

Carbohydrate Utilization within the Whole Plants at Night

Leaves were the major reservoir for storing starch in bothphosphorus treatments, accounting for 85% of the whole plantstarch at the end of the light period (Fig. 6). At the end ofboth the light and dark phases, the root sugar pool in phos-phorus-deficient plants accounted for about 50% of the wholeplant sugar pool, whereas the stem sugar pool of controlsaccounted for about 40% of the whole plant sugar pool.To determine the effect of phosphorus deficiency on whole

plant carbohydrate utilization in the dark, the total amountof carbohydrates lost during the dark phase was evaluatedrelative to the total amount of the carbohydrates available at

the end of the light phase (Table II). After a 12-h dark period,only 30% of the total starch present at the end of the lightphase was utilized in phosphorus-deficient plants, comparedwith 68% in controls. In controls, the magnitude of organ

20 A Leaves 30 D Leaves 16 Leaves

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Figure 5. Effects of phosphorus deficiency on diurnal changes in

concentrations of starch (A-C) and sugar (D-F), and starch as a

percentage of TNC (G-1) in total leaves (A, D, and G), stems (B3, E,

and H), and roots (C, F, and I). Treatment and time effects on all

parameters were significant (P < 0.005). Treatment by time interac-

tion effects were significant (P < 0.005) for all parameters exceptstem and root sugar concentrations.

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Starch Sugars Starch Sugars

Figure 6. Effects of phosphorus deficiency on nonstructural carbo-hydrate pools in different plant organs at the end of the light phase(1800 h) and at the end of the dark phase (0600 h). Values abovecolumns represent nonstructural carbohydrate pool of a given organexpressed as a percentage of nonstructural carbohydrate pool in thewhole plant.

starch utilization was in the order of the YFE leaves > leaves> stems > roots. Sugars were also utilized in all organs exceptstems, which gained a small quantity during the dark. Inphosphorus-deficient plants, only the leaves lost nonstructuralcarbohydrates during the dark phase. Stems and roots ofphosphorus-deficient plants gained nonstructural carbohy-drates during the dark phase (Table II).

DISCUSSION

Phosphorus deficiency decreased photosynthetic activityper unit leaf area by 30% (Fig. IA) and whole plant leaf areaby 90% (Table I). However, despite this decreased photosyn-thetic capacity, phosphorus-deficient plants had significantlyhigher nonstructural carbohydrate concentrations in leavesand stems (Fig. 3A and 5, A and B) than phosphorus-sufficientcontrols. These results reveal that plant growth processes

related to development of new tissues (sink activity) were

more sensitive to the imposed phosphorus deficiency thanphotosynthetic processes. Therefore, decreased carbohydrateavailability does not account for the negative effect of phos-phorus deficiency on dry matter accumulation in soybean.Likewise, Fredeen et al. (5) observed that expanding leaves ofphosphorus-deficient soybean plants had a 30-fold higherstarch concentration than expanding leaves of controls andconcluded that leaf expansion was not limited by carbohy-drate availability.

Higher nonstructural carbohydrate concentrations in phos-phorus-deficient plants were due entirely to the accumulationof high starch concentrations (Fig. 5, A and B), inasmuch as

sucrose and hexose concentrations were significantly lower inthese plants than in the control (Fig. 5, D-F). Higher starchconcentrations in leaves of the phosphorus-deficient plantsresulted from partitioning of a greater percentage of photo-synthetically fixed carbon, as estimated by CER, into starch(Fig. 2B) and slower utilization of starch during the darkphase (Fig. 5, A and B; Table II). Decreased starch utilizationin the dark phase was the major contributor to the high starch

Table II. Influence of Phosphorus Deficiency on Relative Nonstructural Carbohydrate Utilization inWhole plants during the 12-h Dark Phase

Values were obtained by dividing the total amount of nonstructural carbohydrate mobilized duringthe 12-h dark phase by the total amount of nonstructural carbohydrate available at the end of the lightphase.

% Utilized after 12 h of Darkness

Organs Starch Soluble sugars TNC

0.05 mM 1.00 mM 0.05 mM 1.00 mM 0.05 mM 1.00 mM

YFE leaves 63 77 48 45 62 75Total leavesa 39 75 22 15 38 69Stems +1 gb 34 +23 +4 +21 15Roots +30 7 +14 12 +19 11Whole plant 30 68 +6 7 23 53

a All leaves except the YFE leaves. b +, Organs gained carbohydrate during the 12-h dark phase.

321




concentrations in leaves of phosphorus-deficient plants (Fig.5, A and B; Table II).Phosphorus deficiency also significantly increased the

starch concentration in the stems throughout the diurnal cycle(Fig. 5B). This occurred even though phosphorus deficiencydecreased the rate of photosynthate export from the sourceleaves in the light (Fig. 2A) and carbohydrate mobilizationfrom the leaves in the dark (Table II). Both effects woulddecrease entry of sucrose into the phloem and its transport tostem and root tissues. Concentrations of sugars in stems ofphosphorus-deficient plants were significantly lower thanthose in controls (Fig. 5E). Clearly, under phosphorus defi-ciency much of the carbon transported from the leaves intothe stems was converted to starch rather than being utilizedfor growth.

Starch and sugar (hexose + sucrose) concentrations in rootsof phosphorus-deficient plants were lower than or similar tothat in controls over the diurnal cycle (Fig. 5, C and F). Thisindicates that import of carbohydrates from leaves was prob-ably impaired to a greater extent by phosphorus deficiencythan carbohydrate utilization within the roots. More efficientcarbohydrate utilization in roots than in stems may be asso-ciated with greater root growth relative to shoot growth inphosphorus-deficient plants (i.e. lower shoot to root ratio;Table I).

In contrast to our results, Fredeen et al. (5) have reportedthat phosphorus deficiency increases the starch and sucroseconcentrations in the fibrous roots of soybean plants. Appar-ently, starch and sugars are concentrated in the fibrous roots,and treatment differences are reversed when the whole rootsystem is sampled as was the case in this study.The mechanisms by which phosphorus or any nutrient

deficiency inhibits starch utilization in leaves and stems ofintact plants have not been investigated (18, 21). However,several plausible mechanisms could account for impairmentof starch utilization in leaves and stems by phosphorus defi-ciency: (a) direct inhibition of an enzymatic step(s) in thestarch degradation pathway; or (b) indirect inhibition throughnegative effects on sink activity of expanding tissues and onmeristematic development. If the first mechanism is opera-tive, then decreased starch degradation may be partially re-sponsible for decreased growth under phosphorus deficiency.If the second mechanism is responsible for decreased starchutilization, accumulation of high starch concentrations inleaves and stems of phosphorus-deficient plants may be theresult of decreased growth rather than the cause of decreasedgrowth.

Both sucrose and hexose concentrations in the YFE leaveswere decreased by phosphorus deficiency (Fig. 3, B and C).Sucrose phosphate synthase activity was positively correlatedwith sucrose concentration (Figs. 3B and 4; r = 0.621). Thissuggests that the decreased sucrose concentration in the YFEleaves ofphosphorus-deficient plants resulted from reductionsin the activity of the sucrose biosynthetic pathway (Fig. 4).

Plants used in our study exhibited a much greater diurnalfluctuation in starch concentration of fully expanded leaves(Fig. 2A) than was reported for fully expanded leaves ofsoybean plants grown in growth chambers (2, 5). This differ-ence is probably related to differences in light intensities. On

clear days, our outdoor-grown plants were exposed to lightintensities that approached 1900 umol quanta.m-2s ',whereas the plants grown in growth chambers (5) were ex-posed to 525 ,mol quantam-2s1.The phosphorus-deficiency treatment (0.05 mM P) used in

this study was rather severe, with whole plant dry mass andleaf area being decreased by 82 and 87%, respectively (TableI). Responses of carbohydrate metabolism to a less severedeficiency could differ from those described herein. Thispossibility will be examined in a subsequent report on howthe carbohydrate status of soybean plants changes duringonset and recovery from phosphorus deficiency. This infor-mation will also be used to further evaluate mechanisms thataccount for decreased starch utilization in phosphorus-defi-cient plants.

ACKNOWLEDGMENTS

The authors thank Ms. Joy Smith for expert technical assistance;Mr. Wayne Barker and Ms. Ruth Ann Young for dependable assist-ance in culturing plants and grinding samples for analysis; Mrs. JoyceWahab for an excellent job of typing several drafts of the manuscript;and Dr. Jennifer Cure for the use of her Analytical Development Co.photosynthesis system.

LITERATURE CITED

1. Amir J, Preiss J (1982) Kinetic characterization of sucrose-phosphorus-synthase from spinach leaves. Plant Physiol 69:1027-1030

2. Champigny ML (1985) Regulation of photosynthetic carbonassimilation at the cellular level: a review. Photosynth Res 6:273-286

3. Eaton SV (1950) Effects of phosphorus deficiency on growth andmetabolism of soybean. Bot Gaz 111: 426-436

4. Fehr WR, Caviness CE (1977) Stages of soybean development.Iowa State University, Coop Ext Serv Agric and Home EconExp Stn Speci Rep 80: 1-12

5. Fredeen AL, Rao IM, Terry N (1989) Influence of phosphorusnutrition on growth and carbon partitioning in Glycine max.Plant Physiol 89: 225-230

6. Giersch C, Robinson SP (1987) Regulation of photosyntheticcarbon metabolism during phosphate limitation of photosyn-thesis in isolated spinach chloroplasts. Photosynth Res 14:211-227

7. Glowa W (1974) Zirconium dioxide, a new catalyst in the Kjel-dahl method for total N determination. J Assoc OffAnal Chem57: 1228-1230

8. Goodnight JH (1982) The GLM procedure. In AA Ray, ed, SASUser's Guide: Statistics. SAS Institute, Cary, NC, pp 139-200

9. Heldt HW, Chon JC, Maronde D, Herold A, Stanko.vic ZS,Walker DA, Kraimer A, Kirk MR, Heber U (1977) Role oforthophosphate and other factors in the regulation of starchformation in leaves and isolated chloroplast. Plant Physiol 59:1146-1155

10. Huber SC, Israel DW (1982) Biochemical basis for partitioningof photosynthetically fixed carbon between starch and sucrosein soybean (Glycine max L. Merr.) leaves. Plant Physiol 69:691-696

11. Israel DW, Rufty TW (1988) Influence of phosphorus nutritionon phosphorus and nitrogen utilization efficiencies and asso-ciated physiological responses in soybean. Crop Sci 28:954-960

322 QIU AND ISRAEL




12. Jones MGK, Outlaw WH, Lowry OH (1977) Enzymic assay of10- to 10-'4 moles of sucrose in plant tissues. Plant Physiol60: 379-383

13. Kerr PS, Israel DW, Huber SC, Rufty TW (1986) Effect ofsupplemental NO3 on plant growth and components of pho-tosynthetic carbon metabolism in soybean (Glycine max L.Merr.). Can J Bot 64: 2020-2027

14. Lauer MJ, Pallardy SG, Blevins DG, Randall DD (1989) Wholeleaf carbon exchange characteristic of phosphate deficient soy-

beans (Glycine max L. Merr.). Plant Physiol 91: 848-85415. McClure PR, Israel DW (1979) Transport of nitrogen in the

xylem of soybean plants. Plant Physiol 64: 411-41616. Murphy J, Riley JP (1962) A modified single solution method

for determination of phosphate in natural water. Anal ChemActa 27: 31-36

17. Nelson DW, Sommers LE (1973) Determination of nitrogen inplant material. Agron J 65: 109-112

18. Preiss J (1982) Regulation of biosynthesis and degradation ofstarch. Annu Rev Plant Physiol 33: 431-454

19. Rao IM, Abadia J, Terry N (1987) Leaf phosphate status and itseffects on photosynthetic carbon partitioning and export insugar beet. In J Biggins, ed, Progress in PhotosynthesisResearch, Vol 3. Martinus Nijhoff Publishers, Dordrecht,pp 751-754

20. Rufty TW, Keff PS, Huber SC (1983) Characterization ofdiurnalchanges in activity ofenzymes involved in sucrose biosynthesis.Plant Physiol 73: 428-433

21. Steup M (1988) Starch degradation. In J. Preiss, ed, The Bio-chemistry of Plants: A Comprehensive Treatise, Vol 14. Aca-demic Press, San Diego, pp 255-296

22. von Caemmerer S, Farquhar GD (1981) Some relationships be-tween the biochemistry of photosynthesis and the gas ex-changes of leaves. Planta 153: 376-387

23. Walker DA, Sivak MN (1986) Photosynthesis and phosphate: acellular affair? Trends Biochem Sci 11: 176-179

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