Limiting Factors Photosynthesis€¦ · LIMITING FACTORS IN PHOTOSYNTHESIS above) containing no Fe...

Plant Physiol. (1990) 93, 1466-14750032-0889/90/93/1466/1 0/$01 .00/0

Received for publication October 30, 1989Accepted March 22, 1990

Limiting Factors in Photosynthesis

VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at LowPhotochemical Capacity

A. Raviraj Arulanantham, 1. Madhusudana Rao', and Norman Terry*Department of Plant Biology, University of California, Berkeley, California 94720

ABSTRACT

Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimitedby photosynthetic electron transport capacity, even at low inter-cellular CO2 concentrations. Here we monitored leaf metabolitesdiumally and the activities of key Calvin cycle enzymes in theleaves of three treatment groups of sugar beet (Beta vulgaris L.)plants representing three different in vivo photochemical capaci-ties, i.e. Fe-sufficient (control) plants, moderately Fe-deficient,and severely Fe-deficient plants. The results show that the de-crease in photosynthesis with Fe deficiency mediated reductionin photochemical capacity was through a reduction in ribulose1,5-bisphosphate (RuBP) regeneration and not through a de-crease in ribulose 1,5-bisphosphate carboxylase/oxygenase ac-tivity. Based on measurements of ATP and NADPH and triosephosphate/3-phosphoglycerate ratios in leaves, there was littieevidence that photosynthesis and RuBP regeneration in Fe-defi-cient leaves were limited directly by the supply of ATP andNADPH. It appeared more likely that photochemical capacityinfluenced RuBP regeneration through modulation of enzymes inthe photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency.Starch and sucrose levels changed independently of one anotherto some extent during the diumal period (both increasing in theday and decreasing at night) but the average rates of starch orsucrose accumulation over the light period were each propor-tional to photochemical capacity and photosynthetic rate.

their study also showed that rubisco activation reached amaximum at PFD well below those needed to saturate pho-tosynthesis. This suggests that some factor other than rubiscomodulation (e.g. RuBP regeneration) may have been respon-sible for controlling photosynthetic rates at higher PFD levels.In the present work, we explored the roles of rubisco modu-lation and RuBP regeneration in the limitation of photosyn-thesis under conditions of Fe deficiency mediated reductionin photochemical capacity.

In addition, we were interested in determining whether Fedeficiency mediated reduction in photochemical capacity im-paired photosynthesis by decreasing the pool sizes of thephotochemical products, ATP and NADPH. By measuringthe levels of other metabolites such as PGA, triose-P, FBP,and F6P, we gained further information on the effects ofphotochemical capacity on the conversion of RuBP to PGA,PGA to triose-P, and F6P to RuBP. By measuring the initialand total activities of key enzymes of the PCR cycle, weexplored the extent to which photochemical capacity mightaffect RuBP regeneration through enzyme modulation. Be-cause fluxes through the various biochemical pathways maychange with time, the levels of leaf metabolites and nucleo-tides, as well as the carbon storage compounds, starch, sucroseand glucose, were monitored continuously during the 16 hlight/8 h dark cycle.

MATERIALS AND METHODS

Earlier studies in this series (33, 37) have shown that therate of photosynthesis may be colimited by photosyntheticelectron transport capacity, even at very lov levels of inter-cellularCO2 concentration. This limitation could be mediatedthrough a reduction in rubisco2 activity or through a decreasein RuBP regeneration (see 40 for review). Taylor and Terry(34) showed that Fe deficiency mediated reduction in photo-chemical capacity resulted in decreased rubisco activation;

'Present address: CIAT, A.A. 6713, Cali, Colombia.2 Abbreviations: rubisco, ribulose 1,5-bisphosphate carboxylase/

oxygenase; CA IP, 2-carboxyarabinitol 1-phosphate; FBP, fructose1,6-bisphosphate; F6P, fructose-6-phosphate; kc,, catalytic constant;PGA, 3-phosphoglycerate; RuBP, ribulose 1,5-bisphosphate; Ru5P,ribulose-5-phosphate; SBPase, sedoheptulose 1,7-bisphosphatase;triose-P, dihydroxyacetone phosphate + glyceraldehyde-3-phosphate.

1466

Plant Culture and Procedure for Inducing Fe DeficiencyChlorosis

Sugar beets (Beta vulgaris L. cv F 58-554H 1) were culturedhydroponically in growth chambers at 25C day/night tem-peratures and illuminated at 500 ,umol.m2_s-1 PFD over a16 h photoperiod (36). Seeds were germinated in sand andcultured for 2 weeks with a culture solution containing (inmM): 2.5 Ca(NO3)2, 1.0 KH2PO4, 2.5 KNO3, 1.0 MgSO4, and0.5 NaCl, and (in Mm) 23.1 B, 4.6 Mn, 0.38 Zn, 0.16 Cu,0.052 Mo, and 45 Fe as Ferric-HEDTA. To obtain Fe defi-cient plants, seedlings were transferred to a preculture solutionwith the same composition as above, except that the concen-tration of Fe was 18 jAM. After 10 d of preculturing, the plantswere transferred to a culture solution (same composition as

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LIMITING FACTORS IN PHOTOSYNTHESIS

above) containing no Fe and the pH of the culture solutionwas raised to 8.0 with NaOH and solid CaCO3. The controlplants were raised in 45 jLM Fe throughout the experiment.Young rapidly growing leaves were sampled 3 to 4 d (mod-erate Fe stress) and 6 to 7 d (severe Fe stress) after Fe waswithheld.

Leaf Sampling and Extraction of Metabolites

Leafsamples were prepared after 8 h ofcontinuous darknessin the growth chamber for the extraction of leaf metabolitesin intact leaf tissue. The lights were switched on and leafsamples were prepared at 30 min and 1, 4, 8, 12, and 16 h ofillumination and 2, 4, and 8 h of darkness. At each timepoint, four leaf discs (3.88 cm2 each) were punched andquickly frozen in liquid N2 using a laboratory constructedhand-held leaf punch machine which did not obstruct theincident light (27). Adenylates (ATP, ADP, AMP), oxidizednicotinamide nucleotides (NADP', NAD+), RuBP, PGA,triose-P, FBP, and F6P were extracted by grinding the leafmaterial in 12% HCl04 (4 leaf discs/4 mL) in a liquid N2-cooled mortar and pestle. The extracts were left for 1 h on iceand centrifuged at 10,000g for 10 min at 4C. The supernatantwas then neutralized with 10 N KOH and the KCl04 precip-itate was removed from the extract by centrifugation. For theextraction of reduced nicotinamide nucleotides (NADPH,NADH), extracts were prepared in 1 N NaOH (4 leaf discs/4mL). The extracts were boiled for 5 min and rapidly cooledon ice and then centrifuged. The supernatants were thenneutralized with 6 N HCl. All extracts except for adenylateswere decolorized with activated charcoal (20 mg/1.2 mLbefore centrifugation.

Assay of Metabolites

RuBP, PGA, triose-P, FBP, and F6P were determined asdescribed in Rao et al. (25). Adenylates (ATP, ADP, AMP)and oxidized and reduced forms of nicotinamide nucleotideswere determined as described in Rao et al. (27). Standardswere included during extraction procedure and the recoveryof nucleotides were above 85% and the recovery of RuBP,PGA, triose-P, FBP, and F6P were above 80%. The datapresented here are not corrected for the calculated loss ofmetabolites.

Extraction and Analysis of Leaf Starch, Sucrose, andGlucose

Leaf samples were prepared as described in extraction ofmetabolites, and ground in 80% ethanol (1 disc/3 mL).Starch, sucrose, and glucose were estimated as described inRao et al. (26).

Enzyme Assays

For the assay of all enzymes, crude homogenates from leafdiscs frozen in liquid N2 at 1 h after illumination at the growthchamber irradiance, were prepared by grinding one leaf disc(3.88 cm2) with 2 mL of extraction buffer (100 mM Hepes-NaOH [pH 8.0], 10 mM MgCl2, 0.4 mM EDTA, 1% PVP, 100

mM Na-ascorbate, 0.1% BSA at 4 °C). For the determinationof total extractable activity of the enzymes, 50 mm DTT wasincluded in the extraction medium. The in vitro initial andtotal extractable activities of RuBP kinase, chloroplasticFBPase, PGA kinase, NADP-G3PD, rubisco, and SBPasewere determined as described in Rao and Terry (24).

Chemicals

All compounds used were purchased from Sigma ChemicalCompany with the exception of NaH'4C03 (Amersham).

RESULTS

As in previous studies ofthis series, photosynthetic electrontransport capacity in vivo was reduced using Fe deficiency.Leaves designated (below) as moderately Fe stressed had lowerphotosynthetic electron transport capacities than control (Fesufficient) plants, while leaves designated as severely Festressed had the lowest photosynthetic electron transport ca-pacities (as determined by maximum rates of uncoupledelectron transport by isolated chloroplasts and by the contentof electron carriers and reaction centers [36]). Moderately Fe-stressed leaves were taken from plants sampled 3 to 4 d fromFe cutoff. Typically, they had Chl contents ranging from 0.22to 0.33 mmol.m-2. Severely Fe-stressed leaves were fromplants sampled at 6 to 7 d from Fe cutoffand had Chl contentsranging from 0.11 to 0.20 mmol m-2. The control plants'Chl contents ranged from 0.48 to 0.58 mmol.m-2. The av-erage Chl contents for the three treatments were 0.51 ± 0.06,0.28 ± 0.05, and 0.14 ± 0.02 mmol.m-2 for control, moder-ate, and severely Fe stressed, respectively. These Chl contentscorresponded to photosynthetic electron transport capacitiesof 19.9 + 2.3, 10.0 ± 2.4, and 4.30 ± 1.9 ,mol02m-2.s'(see Fig. 3, ref. 33).

Diurnal Changes in Leaf Metabolites

When the lights were switched on, leaf RuBP levels in thecontrol plants increased sharply during the first hour, re-mained more or less constant with time up to 16 h, then fellto almost nondetectable levels when the lights were switchedoff (Fig. IA). RuBP levels in the leaves of plants of the twoFe-deficient treatments changed similarly with time. It is clearfrom these data that the Fe deficiency mediated reduction inelectron transport capacity caused a pronounced reduction inleaf RuBP during the light; the steady state RuBP levels(averaged over 1-16 h) were decreased by 55% in the mod-erately deficient leaves, and by 70% in the severely deficientleaves, compared to the control leaves.Compared to dark levels, leaf PGA in control plants in-

creased relatively much less on illumination than did RuBP(Fig. 1B). In Fe-deficient leaves, leafPGA levels did not differsignificantly between light and dark. The average steady statePGA level in the light was decreased by 38% in the moderatelydeficient leaves and by 45% in the severely deficient leaves.The changes in FBP levels with time were similar to those

for RuBP in that there was a sharp increase in leafFBP whenthe lights went on and a rapid decrease when the lights wentoff (Fig. 2A). LeafFBP levels in all three treatments remained

1 467



Plant Physiol. Vol. 93, 1990

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Figure 1. Diurnal changes in the pool sizes of RuBP (A) and PGA (B)of 4-week-old control (--), moderately Fe deficient (--- -), andseverely Fe deficient (... ) sugar beet leaves. Values for leaf Chl/areain control, moderately Fe deficient, and severely Fe deficient leaves(in mmol Chi.m2) were 0.51 ± 0.059, 0.278 ± 0.047, and 0.139± 0.024, respectively. Values are mean ± SD for at least threereplications.

fairly constant with time during the light period. Although Fedeficiency diminished FBP levels compared to the controls,there appeared to be little difference in leaf FBP between thetwo Fe deficiency treatments. The steady state FBP levels inlight were decreased by 29% in the moderately deficient leavesand by 38% in the severely Fe deficient leaves compared tothe control leaves.Leaf triose-P levels were highly variable (Fig. 2B). In light,

steady state triose-P levels were decreased by 20% in themoderately deficient leaves and by 40% in the severely defi-cient leaves compared to the control leaves. Leaf triose-Plevels were higher in the light compared to dark in controland moderately Fe deficient plants but not in severelydeficient plants.

Diurnal Changes in Leaf Adenylates

When the lights were switched on, leaf ATP levels in thecontrol leaves increased sharply in the first hour and variedlittle for the remainder of the photoperiod (Fig. 3A). Whenthe lights were switched off, leaf ATP levels in the controlleaves declined to preillumination levels within 2 h. After 1 hof illumination, the ATP in control leaves had increased by135%; this was accompanied by a 90% increase in totaladenylates (Fig. 3B) and by a 56% increase in ATP/ADP ratioduring the same period. This suggests that the accumulationofATP during the first hour of illumination was due to a net

synthesis of adenylates as well as to photophosphorylation ofADP (27).

In Fe deficient leaves, ATP levels decreased with illumina-tion in the first hour as in control leaves, but subsequentlythey declined slowly with time even into the dark period.ATP values in Fe deficient leaves did not exhibit the sharpdecrease in darkness observed in control leaves. ATP valuesin darkness were higher in Fe deficient leaves than in controlleaves; this appeared to be due to the slower degradation ofadenylates, in general, which occurred in Fe deficient leavesin darkness (Fig. 3B).These results show that the decreased electron transport

capacity in Fe deficient leaves did not decrease the steadystate ATP levels greatly. Leaf ATP decreased by 29% in themoderately deficient leaves and by 31% in the severely defi-cient leaves. During steady state photosynthesis, the ATP/ADP ratios in the control, moderately deficient, and severelydeficient leaves were 1.55, 1.40, and 1.32, respectively.

Diumal Changes in Leaf Nicotinamide Nucleotides

NADPH levels in leaves of all three treatments increasedduring the first hour of illumination, changed little during thelight period, and decreased slowly in darkness (Fig. 4A). After1 h ofillumination, NADPH levels in control leaves increasedby 175% compared to the 8 h dark value. This increase inNADPH was accompanied by a 115% increase in NADP+ +NADPH (Fig. 4B) and by a 90% increase in NADPH/NADP+ratio (data not shown). This suggests that the increase in

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Figure 2. Diumal changes in the pool sizes of FBP (A) and triose-P(B) of 4-week-old control (--), moderately Fe deficient (- - -), andseverely Fe deficient (... ) sugar beet leaves. Values are mean + SDfor at least three replications.

1 468 ARULANANTHAM ET AL.




30 LIGHT VZ,DARK I). Ru5P kinase was decreased by 29%. The total extractable30T Iactivities ofPGA kinase, NADP-G3PD, FBPase, and SBPase

A were not affected much by Fe deficiency (diminished less than15%).

J 20 _ ; ; \ _ The measurement of the initial enzyme activity in vitroE| Lit--

--_.I T 1 \ |provides an estimate of the amount of activated enzymeoE believed to be present in vivo. The initial activity most affectedETr0 r i T by Fe deficiency was Ru5P kinase; its initial activity was

E 10 . decreased by 46% by moderate and 59% by severe Fe defi-ciency. Rubisco was diminished 22 and 48% by moderateand severe Fe deficiency, respectively. The initial activity ofNADP-G3PD was diminished by 30 and 40% (moderate and

0 severe deficiency) while the corresponding values for PGAIB kinase were 19 and 35%. Iron deficiency had little effect on

B _ the initial activities ofFBPase and SBPase (decreased by about,,45 - t Z ,,- 2 9 q 25% by severe Fe deficiency) compared to Ru5P kinase oroc1>-t 55 t Xi!; rubisco.

- 30 Diumal Changes in Leaf Starch, Sucrose, and Glucose0

7° -4 ,5L Unlike RuBP or ATP, the pool sizes of starch and sucrose_ in the control leaves increased steadily during the light period,

reaching maximum values at 16 h (Fig. 5, A and B). Leaf0IS 1HTA4K glucose levels in the control leaves also increased during the

0 4 8 12 16 4 8 light period (Fig. 5C). In the control leaves starch increasedTIME IN HOURS linearly with time while sucrose increased rapidly over the

first 2 h of light, then more slowly. When the lights wereFigure 3. Diurnal changes in leaf ATP (A) and total adenylates (B) of4-week-old control (--), moderately Fe deficient (--- ), and se-verely Fe deficient (...) sugar beet leaves. Values are mean ± SD LIGHT F770ARKMfor at least three replications.

ANADPH with illumination was due not only to the photosyn- 3 -

thetic reduction of NADP+ but also to a net accumulation ofphosphorylated nicotinamide nucleotides (27).NADPH levels in Fe-deficient leaves changed with time I E 2 - .............

over the light and dark periods in a manner similar to that of 0-the control plants. The average NADPH level during the light z Eperiod decreased by 12% (not significant at P = 0.05) withmoderate Fe deficiency and by 18% (significant at P = 0.05)with severe Fe deficiency. In darkness, NADPH levels tendedto be higher in the Fe deficient leaves, decreasing more slowlywith time than in the controls. In the light, the NADPH/

4

NADP+ ratios in the control, moderately, and severely defi- 4cient leaves were 1.15, 0.86 and 0.80, respectively. There i Bappeared to be no significant differences with respect to N j/ .*S2*treatment on the sum values ofNADPH + NADP+, suggest- Z Eing that the small effects of Fe deficiency on NADPH values + 2were due to effects on NADP+ reduction, not to changes in I Ethe net accumulation of phosphorylated nicotinamide nucle- iotides. Thus, these results show that decreased electron trans- Zport capacity in Fe deficient leaves did not decrease theNADPH levels greatly and that the effects were smaller than K/ LIGHT K>zDARK't>the decreases in adenine nucleotides. O 4 8 12 16 4 8

TIME IN HOURSEffects of Fe Stress on the Activities of Some RegulatoryEnzymes of the PCR Cycle Figure 4. Diurnal changes in leaf NADPH (A) and NADPH + NADP+

(B) of 4-week-old control (--), moderately Fe deficient (--- -), andIron stress affected the total extractable activities of rubisco severely Fe deficient (... ) sugar beet leaves. Values are mean ± SD

the most (reduced by 40% in severely deficient plants) (Table for at least three replications.

1 469




Table I. Effect of Varying Degrees of Fe Stress on the Activities of Some Enzymes of the Photosynthetic Carbon Reduction Cycle of 4 WeekOld Sugar Beet Plants

Percent activation was calculated from the ratio of initial activity to total activity. Leaf samples were taken 1 h after illumination in the growthchamber at 500,mol quanta. m2 ss. Leaf Chi contents are similar to Figure 1.

TreatmentEnzyme Activity

Control Moderate Severe

jUmol m 2 s-1 % Activation iMOl m-2 S-1 % Activation imol m-2 s-' % Activation

Rubisco Initial 125 ± 15 83.3 98 ± 15 82.0 65 ± 16 72.0Total 150 ± 20 120 ± 20 90 ± 26

PGA kinase Initial 225 ± 38 77.0 183 ± 10 77.5 146 ± 17 58.5Total 293 ± 46 236 ± 16 250 ± 27

NADP-G3PD Initial 65 ± 19 37.0 46 ± 14 33.0 39 ± 10 25.5Total 176±28 141 ±13 153±17

FBPase Initial 3.3 ± 0.8 34.0 2.3 ± 0.4 26.0 2.5 ± 0.5 29.5Total 9.8±2.1 9±2.2 8.5±2

SBPase Initial 5 ± 0.9 94.0 5.2 ± 1.2 88.0 3.6 ± 2 73.5Total 5.9 ± 1.8 5.9 ± 1.3 4.9 ± 0.5

Ru5P kinase Initial 88 ± 23 38 48 ± 16 24.0 36 ± 11 22.0Total 233 ± 20 200 ± 23 166 ± 33

switched off, both starch and sucrose levels fell rapidly whileglucose levels fell more slowly.When compared to the controls, moderately Fe-deficient

leaves exhibited corresponding trends with time for the threemetabolites, starch, sucrose, and glucose (Fig. 5). In the caseof starch, there was a delay of 2 h before starch accumulationbegan which did not occur in control leaves. Moderate Fedeficiency reduced the rate of accumulation for each of thethree metabolites during the light period. In severely Fedeficient leaves, the rates of accumulation of the three metab-olites in light were very much reduced, net accumulationoccurring only after 4 to 8 h had elapsed. Starch and sucrosenet rates ofaccumulation for the period of0 to 16 h decreasedby 40 and 49%, respectively, in moderately deficient leaves,and by 86 and 82%, respectively, in severely deficient leaves(compared to control leaves).The average rate of starch accumulation in the light (cal-

culated by subtracting the initial from the final starch valueand dividing by 16 h) was linearly related to photosyntheticrate (Fig. 6). The same was true for the average rate of sucroseaccumulation (Fig. 6). These results suggest that, over thewhole light period, the storage of photosynthate in the leafoccurred simultaneously as starch (in the chloroplast) andsucrose (in the vacuole) at rates which were each linearlydependent on photosynthetic rate. Examination of Figure 5,however, shows that starch and sucrose were stored at differentrelative rates during the light period; for example, in moder-ately deficient leaves over the first 2 h of light, starch accu-mulation was delayed while sucrose accumulation proceededat a rapid rate.

DISCUSSION

RuBP Regeneration Limitation

Earlier studies in this series showed that the rate of photo-synthesis under ambient conditions was colimited by thecapacity ofthe photosynthetic electron transport system (pho-

tochemical capacity) when the latter was decreased using Fedeficiency (33, 37). The results of the present work show thatthe decrease in photosynthesis with Fe deficiency mediatedreduction in photochemical capacity was most likely mediatedthrough a reduction in RuBP regeneration (as opposed to adecrease in rubisco activity). This conclusion is based on thefollowing.Our photosynthesis measurements were made in the leaf

chamber using the same environmental conditions underwhich the plants were acclimated, i.e. at ambient CO2 and atthe growth chamber PFD of 500 ,Amol. m-2 s'. Net CO2exchange rates decreased linearly with Chl content, eachdecrease in Chl content representing a drop in photosyntheticelectron transport capacity (36) (Fig. 7). Initial rubisco activityalso decreased linearly with decrease in Chl content but didnot decrease as much as RuBP content (Fig. 7). With mod-erate Fe stress, RuBP decreased by 55% while rubisco activitydecreased by only 22%; with severe Fe stress, RuBP decreasedby 70% and rubisco by 48%.

It is generally believed that photosynthesis is RuBP-limitedwhen RuBP levels are less than 2 times the binding siteconcentration (12, 30, 40). RuBP levels greater than 1 mol.mol-' are apparently needed to saturate photosynthesis be-cause some RuBP may be unavailable for catalysis because itis chelated or in the wrong ionic form (39). Comparisons ofthe measured concentrations of RuBP with estimates of theRuBP binding site concentrations of rubisco suggest thatRuBP concentrations were limiting photosynthesis in Fe de-ficient leaves. If one assumes a k., of 3.25 mol CO2 fixed permol enzyme active sites per second (39) and the total extract-able activities of rubisco given in Table I, the estimatedrubisco binding site concentrations in control, moderatelydeficient, and severely deficient leaves were (in MAmol.M2):46, 37, and 28, respectively (assuming Fe deficiency does notalter kcat). The steady state pool sizes of RuBP in controlleaves were therefore 1.75 times the binding site concentra-tions while in the moderately and severely Fe deficient leaves

1470 ARULANANTHAM ET AL.




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Figure 5. Diurnal changes in the pool sizes of starch (A), sucrose(B), and glucose (C) of 4-week-old control (--), moderately Fedeficient (--- ), and severely Fe deficient (...) sugar beet leaves.Values are mean ± SD for at least three replications.

the steady state pool sizes of RuBP were 11% and 18%,respectively, below the rubisco binding site concentration. If,on the other hand, Fe deficiency had diminished k,t, theestimated rubisco binding site concentration would have beeneven higher than the RuBP concentrations. Therefore, thesecalculations suggest that RuBP regeneration was limiting pho-tosynthesis when photochemical capacity is decreased in vivo.RuBP was apparently limiting photosynthesis even in our

control plants since RuBP was 1.75 times the binding siteconcentrations. This contrasts with the studies of Kobza andSeemann (11, 12) who found that RuBP was well in excess of2 mol mol-' active sites at similar PFD in sugar beets grownin greenhouses. There is some disagreement in the literatureas to the extent to which RuBP regeneration may be limitingphotosynthesis. Mott et al. (18) demonstrated that the rate ofphotosynthesis at subsaturating light was determined by therate of RuBP regeneration and not by the activation state ofrubisco. There are also a number of other reports in whichRuBP is decreased in low compared with high light and, atvery low light intensities, RuBP may well be below the bind-

ing-site concentration (1, 4, 12, 23, 32, 39). Therefore, thesestudies show that when the supply of photochemical energyis lowered by decreasing the PFD in control leaves, the rateof photosynthesis may be decreased due to decreases in RuBPregeneration.Another approach to determining the importance of the

capacity for RuBP regeneration is to compare the effect oftreatment on the steady state pool sizes of RuBP and triose-P, specifically, the ratio of RuBP/triose-P (10, 15, 25). In ourwork, the RuBP/triose-P ratios for control, moderately, andseverely Fe-deficient leaves were 2.25, 1.15, and 0.97, respec-tively. These estimates suggest that the regeneration of RuBPfrom triose-P was increasingly impaired as the photochemicalcapacity of the leaf decreased with Fe deficiency.

It seems clear from the present work that Fe deficiencymediated reduction in photochemical capacity reduced pho-tosynthesis through diminished RuBP regeneration. Iron de-ficient leaves, having less photosynthetic electron transportchains per area, become light saturated at lower PFD (36, 37).Thus, in the present work, the Fe deficient leaves were prob-ably close to or at light saturation at the growth chamberirradiance of 500 Umolm-2.s-1 and it is easy to understandthat these leaves may be limited by photochemical capacity.

In control (Fe sufficient) leaves, however, photosynthesismay be limited by photochemical capacity and PFD simul-taneously (36). This is almost certainly due to the attenuationof light as it passes through the leaf (14, 35). As the PFD(incident to the upper surface of the leaf) increases, thechloroplasts in the upper leaf cell layers become photosyn-thetically light-saturated. Since much of the incident PFD isabsorbed in the upper chloroplasts, the PFD reaching thechloroplasts in the lower cell layers of the leaf is less and,therefore, limiting to photosynthesis in the lower chloroplasts.This may lead to two populations of chloroplasts in the leaf:those that are light-saturated (upper regions) and those thatare light-limited (lower regions). Under these conditions, both

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Figure 6. Rates of starch and sucrose net accumulation in the lightas plotted as a function of the rate of net photosynthesis at growthchamber PFD. Photosynthesis was varied by altering the photosyn-thetic electron transport capacity in vivo by depriving plants of Fe.

1471




these two populations contribute to the photosynthetic rateof the leaf: an increase in PFD may increase leaf photosyn-thesis by increasing the rate of photosynthesis of the light-limited chloroplasts, while an increase in photochemicalcapacity (more reaction centers and electron transport com-ponents) may increase leaf photosynthesis by increasing therate of photosynthesis of the light-saturated chloroplasts (37).

Rubisco Limitation

Many studies have shown a close relationship between CO2assimilation and changes in the activation state of rubiscowith irradiance (see 40 for review). Furthermore, it has beensuggested that the modulation of rubisco, rather than the levelof RuBP, controls the rate of CO2 fixation with change inPFD (11). Mott et al. (17), however, showed that the firstresponse to decreased PFD was a drop in the level of RuBP;this was followed somewhat later by an adjustment in themodulation of rubisco which then resulted in a subsequentrestoration of RuBP to high levels. In the present work therewas also a very strong correlation between the rate of photo-synthesis and the initial activity of rubisco as both parametersdecreased with decrease in photochemical capacity (Fig. 7).Even though rubisco activity decreased in parallel with pho-tosynthesis, it remained very high and was well above the rateofphotosynthesis in Fe-deficient leaves (Table I; Fig. 7). Thesedata suggest that the down-modulation of rubisco with Fedeficiency was insufficient to maintain RuBP at high levelsand that RuBP regeneration, not rubisco activity, limitedphotosynthesis under these conditions.

Several studies have suggested that photosynthetic electrontransport activity may be involved in rubisco activation (28,29, 34). In sugar beet, the regulation of rubisco in response tochanging irradiance appears to involve carbamylation, CA1P,and possibly RuBP or some other tight-binding inhibitor (1 1,12). Iron deficiency diminishes photosynthetic electron trans-

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Figure 7. Changes in net photosynthesis per area in relation to RuBPand rubisco as a function of leaf Chi content. The leaf Chi content isused here as an index of varying levels of photochemical capacityin vivo.

port capacity and this, in turn, decreases rubisco activation(34). One possible mechanism for this is through changes inthe alkalinization of the stroma. Since Fe deficiency results ina substantial decrease in thylakoid volume relative to stromalvolume (20, 36), one might expect the capacity of the thyla-koids to take up protons to be diminished to such an extentthat pH increases in the stroma would be relatively small.Thus, rubisco, which is highly pH dependent (16, 21), maybe progressively less activated as leaves become more Fedeficient. Any number of other mechanisms could be at workincluding a decreased amount or activity of rubisco activasein Fe-deficient leaves, or increased amounts of the inhibitorCAIP. The total activity of rubisco decreased with Fe defi-ciency in both light and dark which is consistent with theview that there were increased levels of CAIP in deficientleaves.

ATP and NADPH Limitation

Although there is clear evidence from the present work thatRuBP regeneration and photosynthetic rate were limited byphotosynthetic electron transport capacity, this limitation didnot appear to be mediated through the pool sizes of ATPand/or NADPH. Considering the large reductions in thyla-koid contents and, therefore, photosynthetic electron trans-port capacities with moderate and severe Fe deficiency, thedecreases in ATP and NADPH were remarkably small (Fig.8, A and C). The results show that while photosynthesis (Fig.7) and photosynthetic electron transport capacity (33) eachdecreased linearly with Chl content, ATP/ADP and NADPH/NADP+ ratios did not decrease appreciably under moderateFe stress and did not decrease at all from moderate to severeFe stress. The changes in leaf ATP with decreasing photo-chemical capacity observed here with Fe deficiency, are sim-ilar to the observation of Brooks et al. (2) who found nosignificant changes in the leaf ATP content over a range ofPFD from 1000 to 120 ,mol.m-2 s'.

Giersch and Robinson (8) used the capacity of leaves toform triose-P from PGA as a measure ofATP and/orNADPHlimitation ofthe PCR cycle, i.e. decreased triose-P/PGA ratioimplying ATP and/or NADPH limitation of the PCR cycle.We estimated the triose-P/PGA ratio and found that this ratiowas increased with Fe deficiency, i.e. 0.14, 0.18, and 0.175for control, moderately, and severely deficient leaves, respec-tively. Using this approach, there was clearly no evidence thatreduction in photochemical capacity in vivo affected the ca-pacity for PGA reduction to triose-P.

Based on the ratio of triose-P/PGA and on the measure-ments of the nucleotides, there appears to be little support forthe view that photosynthesis and RuBP regeneration in Fedeficient leaves were limited directly by the supply of ATPand NADPH. Furthermore, since ATP did not limit theformation of triose-P from PGA it is unlikely that it wouldlimit the phosphorylation of Ru5P by Ru5P kinase becausethe Km for ATP is much lower for Ru5P kinase than for PGAkinase ( 13).

It should be noted that in this study nucleotides (as well asPGA, triose-P, FBP, and F6P) were measured in intact leaftissue and therefore the values presented here represent me-





1000

s80k 0-hC 0(3 60 0U 60 bh%$bfLS060 -

0

t 40-40-u u

~20 - 20

0 P RuBP PGA TP FBP F6P ATPNADPH 0Rubisco PGA NADFP FBP SBP Ru5Pkinase G3PD ase ase kinase

100 - Severely Fe deficient C B 100 - Severely Fe d

80arynghotsynhetceectonranporcaaciyiviouingleaesro Fe-defit adeficient80-4- 8C Co o0 60 0u60o 0t40 -t40-

0) 0

P RuBP PGA TP FBP F6P ATP NA8DPH Rubisco PGA NADP- FBP SBP Ru5PkinaseG3PD use use kinose

Figure 8. Effect of different levels of photochemical energy supply on photosynthesis (P) and pool sizes of various metabolites (A and C) andon in viva initial activity of some regulatory enzymes of the PCR-cycle (B and D). The photochemical energy supply was varied experimentallyby varying photosynthetic electron transport capacity in viva using leaves from Fe-deficient and Fe-sufficient plants.

tabolites which may be present in various compartments ofleaf cells, e.g. chloroplast, cytosol, and mitochondria. Withregard to ATP, 45 to 50% of the total cellular ATP is believedto be in the chloroplasts in the light (22). For nicotinamidenucleotides there are few reliable data available indicating thedistribution among these compartments during light. Studieshave shown that in light the production of ATP by chloro-plasts exceeds that by mitochondria and cytosol (6, 9). Webelieve that the changes in leafATP and NADPH from darkto light measured in this study largely reflect increments inATP and NADPH occurring within the chloroplasts (27).

Ru5P Kinase Limitation

Since decreased photochemical capacity did not appear toaffect RuBP regeneration via the supply of ATP and/orNADPH, it must have affected RuBP regeneration via theactivities of enzymes which catalyze the formation of RuBPfrom triose-P in the PCR cycle. Dietz and Heber (4) proposedthat the regeneration of RuBP from F6P, rather than thesupply ofphotochemical energy, limits photosynthesis at highlevels of CO2 and PFD. In the present work, the ratio of

RuBP/F6P progressively decreased with decreasing photo-chemical capacity (1.9, 1.5, and 1.0 for control, moderate,and severely Fe-deficient leaves, respectively, whereas the ratioof triose-P/PGA increased. This suggests that RuBP regener-ation was limited between F6P and RuBP. In this regard, theenzyme most affected by decreased photochemical capacitywas Ru5P kinase, the initial activity of which was diminishedby 60% by severe Fe deficiency (Fig. 8, B and D; Table I).This suggests that the reduction in RuBP regeneration in Fe-deficient leaves was due to an enzymatic limitation by Ru5Pkinase.How Fe deficiency diminishes the activation state of Ru5P

kinase is not clear. Ru5P kinase has been shown to beregulated by the ferredoxin/thioredoxin system (3). AlthoughNADPH/NADP+ ratios were high in Fe deficient leaves, theremay nevertheless have been insufficient reduced ferredoxinor thioredoxin to activate the enzyme. The catalytic activityof Ru5P kinase could also be affected by the stromal concen-tration of H+ (5). Iron deficiency may have decreased Ru5Pkinase activity through inhibitory molecules such as 6-phos-phogluconate, PGA, and phosphate, which have been shownto affect catalytic activity, especially at lower stromal pH

1 473




values (5). Increased starch breakdown in Fe deficient leavesmay lead to an increase in 6-phosphogluconate and there isalso evidence that the leafphosphate content increases duringFe deficiency (19, 38).

Diurnal Changes of Metabolites

The diurnal changes in the pool sizes of some of themetabolites of control sugar beet leaves presented here were

very similar to those found by other researchers (7, 25). RuBPand FBP increased rapidly during the dark-light transitionand decreased rapidly during the light-dark transition. For themost part, the levels of RuBP, FBP, ATP, NADPH, and PGAremained more or less unchanged with time after the first 2 hof light, a pattern which was also typical of Fe deficient leaves.There was no evidence that the Fe deficiency treatmentchanged the pattern of response of these metabolites withtime.

Starch and sucrose increased over the first 2 h but, unlikethe other metabolites, continued to increase over the remain-der of the light period. Iron deficiency altered this pattern ofchange with time. Starch levels decreased during the first 2 hin Fe deficient leaves and sucrose increased only in themoderately deficient plants. Starch accumulation in Fe defi-cient plants occurred later in the photoperiod but only weaklyin the severely deficient plants. Sucrose may also have beenaccumulated to some extent in the later parts of the photo-period in the deficient plants. The sharp decreases in starchand sucrose net accumulation rates with Fe deficiency me-

diated decreases in electron transport capacity were almostcertainly due to a reduction in photosynthetic rate (Fig. 6).Sharkey et al. (31) showed that starch and sucrose formationin Phaseolus vulgaris were each linearly related to assimilationwhen assimilation was varied by altering the light intensity.

CONCLUSIONS

In summarizing the research published in this series ofpapers to date, we conclude the following. First, we havepresented evidence that the rate of photosynthesis may belimited or colimited by the photosynthetic electron transportcapacity of leaves in many light/CO2 environments. Second,that this limitation is imposed at the level of RuBP regenera-tion rather than by modulation of the activity of rubisco.Third, that since the levels of ATP, NADPH, triose-P, FBP,and F6P are not much affected in Fe deficient leaves, it seemslikely that the effect of low photochemical capacity on RuBPregeneration is mediated through the action of an enzyme(s)between F6P and RuBP (e.g. Ru5P kinase), and not throughthe effect of photochemical capacity on the pool sizes ofATPor NADPH.

ACKNOWLEDGMENTS

We thank C. Carlson for excellent technical assistance.

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