Photosynthesis and chlorophyll fluorescence in sunflower (Helianthus annuus L.) leaves as affected by phosphorus nutrition

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1 Journal of Experimental Botany, Vol. 45, No. 276, pp , July 1994 Journal of Experimental Botany Photosynthesis and chlorophyll fluorescence in sunflower (Helianthus annuus L.) leaves as affected by phosphorus nutrition Marijana Plesni&ar 1, Rudolf Kastori, Novica Petrovid and Dejana Pankovid Institute of Field and Vegetable Crops, Faculty of Agriculture, University ofnovi Sad, M. Gorkog 30, Novi Sad, Yugoslavia Received 16 November 1993: Accepted 24 March 1994 Abstract The effects of phosphate concentration on plant growth and photosynthetic processes in primary leaves of young sunflower {Helianthus annuus L.) plants were examined. Plants were grown for 3 weeks on half-strength Hoagland's solution containing 0, 0.1, 0.5,, and 3.0 mol m 3 orthophosphate (Pi). It was shown that optimal photosynthesis and the highest light utilization capacity were achieved at 0.5 mol m" 3 Pi in the growth medium, which was in good agreement with the maximum content of organic phosphorus in the leaves. Low phosphate in the medium inhibited plant growth rate. Phosphate deficiency appreciably decreased photosynthetic oxygen evolution by leaves, the efficiency of photosystem two (PSII) photochemistry and quantum efficiency of PSII electron transport. High oxidation state of PSII primary electron acceptor Q A, at 0.1 mol m" 3 Pi, however, indicates that photosynthetic electron transport through PSII did not limit photosynthesis in Pi-deficient leaves. The results indicate that diminished photosynthesis under sub- and supra-optimal Pi was caused mainly by a reduced efficiency of ribulose 1,5-b/sphosphate (RuBP) regeneration at high light intensities. These results suggest that, under non-limiting C0 2 and irradiance, photosynthesis of the first pair of leaves could be diminished by both sub- and supra-optimal phosphorus nutrition of sunflower plants. Key words: Helianthus annuus L, phosphate nutrition, photosynthesis, photochemical efficiency. Introduction Phosphate is a major mineral nutrient which is very important for growth and metabolism of plants. Orthophosphate, together with CO 2 and H 2 O, is a primary substrate of photosynthesis (Walker and Sivak, 1986). Chloroplasts export triose phosphate to the cytosol in exchange for a stoichiometric amount of orthophosphate (Fliege et al., 1978). The short-term inhibitory effects of low phosphate have often been observed. Low Pi might restrict photophosphorylation, which should lead to increased energization of the thylakoid membrane, decreased electron flow and associated inhibition of photosynthesis (Giersch and Robinson, 1987; Heineke et al., 1989). At high Pi, triose phosphate export competes with RuBP regeneration and the rate of photosynthesis can be diminished. Experiments with isolated chloroplasts suggest that optimal photosynthesis demands a finely balanced concentration of Pi in the cytosol (Walker and Robinson, 1978). This concentration is maintained by transport to and from the vacuole and by metabolic processes causing changes in the rate of sucrose synthesis (Foyer and Spencer, 1986; Dietz and Foyer, 1986). Long-term inhibitory effects of low Pi have also been observed. Photosynthetic CO 2 assimilation was inhibited in plants grown with an insufficient supply of Pi (Terry and Ulrich, 1973). Phosphate deficiency strongly affected carboxylation efficiency and the apparent quantum yield for CO 2 assimilation by leaves of phosphate-deficient Helianthus annuus, Zea mays and Triticum aestivum (Jacob and Lawlor, 1991, 1992). High phosphate stimu- 1 To whom correspondence should be addressed. Fax: Abbreviations: #, efficiency of excitation energy capture by open PSII centres; * quantum efficiency of PSII electron transport; PFD, photon flux density; PI, inorganic phosphate; Po, organic phosphorus; Rot, total phosphorus; PS, photosystems; O A, primary quinone-type electron acceptor, q H, non-photochemical Chi fluorescence quenching, q^, photochemical fluorescence quenching; QV, apparent quantum yield, a measure of maximal photosynthetic efficiency; RuBP, ribulose 1,5-twsphosphate. Oxford University Press 1994

2 920 Plesnidar et al. lated light-saturated photosynthetic rates, carboxylation efficiency and the apparent quantum yield for CO 2 assimilation in sunflower leaves at normal ambient CO 2 concentration (Jacob and Lawlor, 1991, 1992). Short-term inhibitory effects of 20 mm Pi have been observed regardless of the CO 2 or O 2 gas composition (Dietz and Foyer, 1986), but inhibitory effects on a longer time-scale have rarely been observed. Recently, Duchein et al. (1993) observed inhibition of daily net CO 2 uptake by clover in ambient air at the high level of P-nutrition on a longer time-scale. In this study long-term effects of a range of phosphate concentrations on plant growth and photosynthetic processes in the first pair of leaves of young sunflower plants were examined. We have determined chlorophyll fluorescence quenching coefficients and response of CO 2 -saturated photosynthesis to increasing PFD under the various Pi treatments. The aim was to evaluate the efficiency of PSII photochemistry and electron transport, and light utilization capacity of leaves, especially when the imposed Pi-condition has severely diminished the rate of photosynthesis. Materials and methods Plant material and growth conditions Sunflower (Helianthus annuus L. hybrid NS-H-26) seeds were germinated in the dark, at 25 C, on sterilized quartz sand and watered daily with demineralized water. After 5 d the seedlings were transferred to 700 ml plastic pots filled with half-strength Hoagland's solution (Hoagland and Arnon, 1950) containing the following potassium phosphate concentrations: 0, 0.1, 0.5,, and 3.0 mol m~ 3. Potassium concentration was maintained at a constant level by the addition of KC1. Twice weekly the nutrient solution was renewed. There were eight replicates of each treatment. Plants were grown in the greenhouse at /imol quanta m~ 2 s~', with a 12 h photoperiod. The temperature in the greenhouse was 15 C (night) to 24 "C (day), relative humidity was 65-75%. Plants were grown for 22 d and they were in the stage of 2 pairs of fully expanded leaves when they were harvested. Growth analysis For determination of leaf area, dry mass of organs and content of phosphorus (inorganic and total), roots, stems, cotyledons, and first and second pairs of leaves were separated in each treatment. The area of the leaves was measured by an Automatic Area Meter (model LI-3000, Licor, USA). Dry mass of each organ was determined after drying the sample at 70 C to constant weight. Phosphorus analysis Total phosphorus was determined spectrophotometrically with the acid digest of the sample by the application of the ammonium vanadate-molybdate method (Gericke and Kurmies, 1952). The inorganic phosphorus content in the acidsoluble fraction was measured by the formation of the blue molybdenum complex in the isobutanol extract (Saric et al., 1986). Pigments Chlorophylls a and b and carotenoids were determined spectrophotometrically in the acetone extract of freshly harvested leaves, using molar extinction coefficients according to Holm (1954) and von Wettstein (1957). Photosynthesis and chlorophyll fluorescence measurements Before measurement, the first pair of leaves was preconditioned by pre-illumination at 300/xmol quanta m~ 2 s"' and 25 C for 30 min. Simultaneous measurements of oxygen exchange (polarographic) and fluorescence yield (Delieu and Walker, 1983) were done on a leaf disc (10 cm 2 ) in a closed chamber (LD2, Hansatech, King's Lynn, UK) at 25 C and 5% CO 2 as described by Plesnicar and Pankovi6 (1991). The leaf disc was illuminated by increasing PFDs until a steady-state rate of CO 2 -saturated photosynthesis was achieved at each irradiance. At steady-state photosynthesis, pulses of saturating light (c. 5000/xmol quanta m~ 2 s~', 990 ms) were given every 15 s to fully reduce the primary electron acceptor of PSII and remove photochemical quenching. Rates of CO 2 -saturated photosynthesis, Chi fluorescence quenching coefficients (Schreiber et al., 1986), excitation efficiency of PSII, $ <., and quantum efficiency of PSII electron transport 0 a, (Genty et al., 1989) were measured at steady-state photosynthesis. The leaf disc was darkened for 5 to 10 min (respiration measured) before measuring the light response curve over a range of 14 PFDs. The 'Leaf Disc' computer program allows O 2 exchange rate to be plotted against PFD and also permits the calculation of the initial slope (apparent quantum yield, QY) and photosynthetic capacity of leaves (Walker, 1987). The data were statistically analysed by analysis of variance and the LSD (^=0.05) test was used to compare different Pi treatments. Results Plant growth parameters and phosphate content At harvest, 3-week-old sunflower plants had 4-6 leaves with an area larger than 1 cm 2. The size and dry mass of leaves and other plant parts depended on phosphate concentration in the nutrient solution. Leaf area was reduced under phosphate deficiency; dry mass of leaves, as well as the content of chlorophyll (Table 1) and carotenoids (not shown) expressed per dry mass of leaves, were significantly lower in phosphate-deficient plants. Pigment ratios (Chi a/chl b and carotenoids/chl (a + b)) were rather unaffected by the treatment (data not shown). The concentration of total and inorganic phosphorus in plants increased with the amount of phosphate in the nutrient solution (Table 2). However, although in phosphate-deficient plants the amount of organic phosphorus was the lowest, under these conditions the ratio of organic to inorganic phosphorus was the highest. The highest amount of organic phosphorus in leaves coincides with the highest rate of photosynthesis in plants grown in nutrient solution containing 0.5 mol m~ 3 Pi (Table 2; Fig. 2).

3 Phosphate nutrition and photosynthesis 921 Table 1. Effect of phosphate concentration in the nutrient solution on plant growth and on the relevant characteristics of the first leaf pair; at the time of analysis sunflower plants were 3-weeks-old Cone. PO 4 in nutrient solution (mol m" 3 ) Leaf area (cm 2 ) Plant 1st pair leaves Dry mass (mg) Plant 1st pair leaves Chi (a+ 6) (mgg-'dw) 1st pair leaves LSD (P-0.05) LSD(P = 0.01) Table 2. Effect of phosphate concentration in the nutrient solution on the content of total phosphorus (Ptot), inorganic (Pi) and organic phosphorus (Po) in the first pair of leaves and in the whole sunflower plant Cone. PO 4 in nutrient solution (mol m~ 3 ) LSD (P = 0.05) LSD(P-O.Ol) 1st pair leaves (g(loogdw)-') Ptot Chlorophyll fluorescence measurements Pi Chlorophyll fluorescence measurements were employed as an alternative method to monitor phosphate-dependent changes in the redox state of the photosynthetic electron transport chain and in thylakoid energization. Upon illumination of a leaf disc, there is an immediate rise of fluorescence which decays to a low, nearly constant value during steady-state photosynthesis. This quenching of Chi fluorescence is mainly caused by the use of energy for photosynthetic electron transport (photochemical quenching, q P ) and the build up of a proton gradient across the thylakoid membrane (non-photochemical quenching, <7 N ). As expected, photochemical quenching decreased with increasing photon flux density, indicating an increase in the proportion of closed PSII reaction centres (Fig. 1A). The increase in non-photochemical quenching with increasing PFD (Fig. IB) reflects both thylakoid energization and photoinhibitory quenching, increasing from 0.38 to 0.62 as PFD was increased from 69 to 948/xmol quanta m~ 2 s~\ Both the efficiency of excitation energy capture by open PSII reaction centres, *exc (Fig. 1C), and the efficiency of PSII electron transport, <P U (Fig. ID), decreased with increasing PFD and with increasing rate of photosynthetic O 2 evolution. Po Whole plant (g (100 gdw)- 1 ) Ptot The effect of phosphate Pi Po Under conditions of saturating CO 2, phosphate concentration in the nutrient solution affected the redox state of the PSII primary electron acceptor as indicated by q P (Fig. 1A), the efficiency of excitation energy capture by open PSII reaction centres (Fig. 1C), and the efficiency of PSII electron transport (Fig. ID) in sunflower leaves. The rate of O 2 evolution was the highest at 0.5 and 1 mol m~ 3 Pi (Fig. 2). The inhibitory effect of low (0 and 0.1 mol m~ 3 ) and high (3 mol m~ 3 ) phosphate was evident under the experimental conditions. The oxidation state of PSII primary electron acceptor exhibited a sharp maximum around 0.1 mol m~ 3 Pi, with q f values being similar for plants grown in nutrient solutions containing 0.5, and 3.0 mol m" 3 Pi (Fig. 1A). Wasteful dissipation of excitation energy from chlorophyll decreased (Fig. IB), while the efficiency of excitation energy capture by open PSII reaction centres (Fig. 1C) and the efficiency of PSII electron transport (Fig. ID) increased with Pi concentration up to 0.5 mol m~ 3 Pi. Statistical analysis of fluorescence parameters does not show significant differences among 0.5, and 3.0 mol m~ 3 Pi data. Significant differences were assessed between 0 and 0.1 mol m~ 3 Pi and between 0.1 and 0.5 mol m~ 3 Pi data.

4 922 Plesnidar et al. Table 3. Effect of phosphate concentration in the nutrient solution on CO 2 -saturated photosynthesis in sunflower leaves The apparent quantum yield (QY), was calculated from the plots of O 2 exchange rate against PFD. A mui is the rate of CO 2 and light-saturated photosynthetic oxygen evolution. Cone. PO 4 in nutrient solution (mol m" 3 ) QY o M t c n n K - -o E V a [ Pi ] in nutritient solution (mol m Fig. 1. The effects of phosphate concentration in the nutrient solution on photochemical (A) and non-photochemical (B) Chi fluorescence quenching, photochemical efficiency of PSII centres (C) and quantum efficiency of PSII electron transport (D) in sunflower leaves under different incident photon flux densities. The assay conditions were: 25 C, 5% CO 2 and 69 (O), 185 ( ), 378 (V), 539 (T) and 948 (D) fimol quanta m~ 2 s~' PFD. LSD (/> = 0.05) values for different phosphate concentrations at tested PFDs were the same, 0.048, for calculated fluorescence parameters, except for q r at 948 fiinol quanta m' 2 s" 1, where LSD (/>~0.05) was PFD (^.mol quanta m s ) Fig. 2. Light response of CO 2 -saturated O 2 exchange by first pair of leaves from sunflower plants grown with 3 (A), 1 (V), 0.5 ( ), 0.1 (O), and 0 (O) mol m~ 3 Pi. All measurements were conducted on leaf discs as described in Materials and methods. LSD (P = 0.05) values for individual PFDs are presented by bars. Plants grown in nutrient solution containing 0.5 and mol m~ 3 Pi showed, at saturating PFD and CO 2, the highest rates of photosynthesis, which were higher than the rates at 3.0 and 0 mol m~ 3 Pi by about 60-80% (Table 3). Maximum photosynthetic efficiency in light limiting conditions, QY, was also the highest with the plants that were grown in 0.5 mol m~ 3 Pi and mol m~ 3 Pi nutrient solution. The efficiency was decreased by LSD (^=0.05) %, 17%, and 17% in 0.0, 0.1, and 3.0 mol m" 3 Pi, respectively (Table 3). Discussion Young sunflower plants exhibit a typical response to phosphate availability. Plant growth rates observed with low concentrations of phosphate were distinctly lower than growth rates observed with increased phosphate supply (Fredeen et al., 1989). Reduced mass of root, stem, cotyledons and leaves indicates retardation of plant growth, while reduced chlorophyll content indicates deficiency-induced leaf senescence. The results from our work confirm that, under saturating CO 2 and high light, net photosynthesis may be limited by the available phosphorus supply (Walker and Sivak, 1986; Jacob and Lawlor, 1991). Both sub- and supraoptimal concentrations of phosphate were inhibitory, the maximum rate of photosynthesis was achieved at 0.5 mol m~ 3 Pi in the growth medium (Fig. 2; Table 3). This is in good agreement with the maximum content of organic phosphorus in the leaves at 0.5 mol m" 3 Pi in the growth medium (Table 2). The phosphate deficiency effects on photosynthesis and sucrose/starch ratio in short- and long-term experiments have been explained in terms of regulatory effects of Pi on photosynthetic metabolism in chjoroplasts (Foyer and Spencer, 1986). It has been suggested that low Pi acts by inhibiting RuBP regeneration (Jacob and J^awlor, 1992) and by enhancing starch formation in chloroplasts (Fredeen et al., 1990). It was also shown that Rubisco activity was reduced in Pi deficient leaves (Jacob and Lawlor, 1992; Sawada et al, 1992). The inhibitory effect of continuous Pi feeding, or high Pi, on photosynthesis under light-saturating conditions and high CO 2, might be explained in terms of increased cytosolic Pi levels which increased assimilate export from the chloroplast (Dietz and Foyer, 1986). At low light intensity, as applied for sunflower growth in this experiment, the efficiency of photosynthesis was high

5 (Table 3) and 3.0 mol m" 3 Pi in the solution did not produce an inhibitory effect on plant growth (Table 1). The effects of phosphate nutrition on the apparent quantum yield of photosynthesis and photosynthetic capacity depend on whether phosphate has been applied in short- or long-term experiments. Short-term mannose feeding of a leaf, in order to sequester cytosolic Pi, or subsequent Pi feeding, had no effect on quantum yield, but they caused changes in the light-saturated rate of photosynthetic oxygen evolution (Walker and Sivak, 1986; Walker and Osmond, 1986) and in sucrose synthesis and photosynthetic metabolites in leaves (Stitt and Schreiber, 1988). As for the long-term effects of different P-nutrition, there are data which show that low phosphate had only a small effect on photochemical efficiency (Abadia et al., 1987), while others show decreased apparent quantum yield for CO 2 assimilation in phosphatedeficient plants (Brooks, 1986; Lauer et al., 1989; Jacob and Lawlor, 1991). Our results show that the apparent quantum yield for CO 2 assimilation was significantly decreased only in sunflower plants grown at the lowest phosphate (Table 3). In the range of optimal Pi concentrations the oxidation state of the primary electron acceptor in PSII, Q A, as indicated by q P, was inversely proportional to the rate of CO 2 -saturated O 2 evolution (Figs 1A; 2). However, in phosphate-deficient sunflower plants a small increase of Pi concentration in the nutrient solution from 0 to 0.1 mol m~ 3 caused an increase in both the rate of photosynthetic O 2 evolution and the oxidation state of Q K. While the increase in the rate of O 2 evolution was almost the same for two PFDs in the light-saturated range on going from 0 to 0.1 mol m~ 3 Pi, the corresponding q P increase was 120% greater at 948 than at 539 /xmol quanta m~ 2 s~'. High oxidation state of Q A at 0.1 mol m~ 3 Pi indicates that relatively more electrons were used for processes other than CO 2 reduction, suggesting that photosynthetic electron transport through PSII did not limit photosynthesis in Pi-deficient leaves. Our data support the findings by Furbank et al. (1987) with isolated spinach chloroplasts, showing that photosynthesis under Pi-limiting conditions is regulated both at the thylakoid level and by the enzymes of carbon metabolism. Due to the lower rate of photosynthesis in phosphatedeficient sunflower leaves at any examined PFD, a greater proportion of the absorbed light, being in excess of that required to support CO 2 assimilation, is dissipated by radiationless thermal de-excitation, causing an enhanced non-photochemical quenching (Fig. IB). A possible interpretation is that under these conditions the availability of Pi in the chloroplast for ATP synthesis is restricted, resulting in an increase in the ph gradient and, therefore, in non-photochemical quenching. It is also possible that the increase in non-photochemical quenching in phosphate-deficient plants could be due to photoinhibition, as Phosphate nutrition and photosynthesis 923 indicated by the reduction in the apparent quantum yield (Table 3) (Walker and Osmond, 1986). Our data confirm the findings in recent studies (Weis and Berry, 1987; Genty et al., 1989) and suggest that processes associated with non-photochemical energy dissipation modify the efficiency with which the reaction centres can capture and utilize excitation energy during orthophosphate limitation. A possible function of this down-regulation of the efficiency of PSII photochemistry by non-photochemical energy dissipation could be to adjust the rate of photochemistry to match that of carbon metabolism and hence to avoid over-excitation of the PSII reaction centres (Havaux et al., 1991). Plants grown at supra-optimal phosphate concentration did not exhibit an appreciable change in photochemical efficiency, yet photosynthesis at high light was inhibited (Figs 1,2). This could be caused by increased cytosolic Pi levels increasing triose-phosphate export from the chloroplast at the expense of RuBP regeneration in the chloroplast (Dietz and Foyer, 1986). The results from this study suggest that, under saturating CO 2 and high irradiance, photosynthesis and photosynthetic capacity of leaves are diminished by both suband supra-optimal phosphorus nutrition of sunflower plants. Our results at saturating CO 2 and results of Duchein et al. (1993), obtained in ambient and high CO 2 conditions of plant growth, show that the increased availability of CO 2 shifts the optimum Pi concentration for photosynthesis towards higher values. Duchein et al. (1993) showed that photosynthetic daily net CO 2 uptake in clover plants grown in ambient air was higher at the reduced than at high phosphate, on atmospheric CO 2 enrichment the optimum was shifted towards higher Pi concentrations. The inhibition of photosynthesis by high Pi, which we obtained at high light and high CO 2 indicates that the relationship in these conditions is more complicated and demands further investigation. The extent to which plant growth might be affected by phosphorus availability will depend on the sink-source status of the examined plant and how this is regulated (Stitt, 1991). References Abadia J, Rao IM, Terry N Changes in leaf phosphate status have only small effect on the photochemical apparatus of sugar beet leaves. Plant Science 50, Brooks A Effects of phosphorus nutrition on ribulose-l,5-6«phosphate carboxylase activation, photosynthetic quantum yield and amounts of some Calvin cycle metabolites in spinach leaves. Australian Journal of Plant Physiology 13, Delieti TD, Walker DA Simultaneous measurement of photosynthetic oxygen evolution and chlorophyll fluorescence from leaf pieces. Plant Physiology 73, Dietz K-J, Foyer C The relationship between phosphate status and photosynthesis in leaves. Reversibility of the effects

6 924 Plesnidar et al. of phosphate deficiency on photosynthesis. Planta 167, Duchein M-C, Bonicel A, Betsche T Photosynthetic net CO 2 uptake and leaf phosphate concentrations in CO 2 enriched clover (Trifolium subterraneum L.) at three levels of phosphate nutrition. Journal of Experimental Botany 44, Fliege R, FlOgge UI, Werdan K, Heldt HW Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. Biochimica et Biophysica Acta 502, Foyer C, Spencer C The relationship between phosphate status and photosynthesis in leaves. Effects on intracellular orthophosphate distribution, photosynthesis and assimilate partitioning. Planta 167, Fredeen AL, Rao 1M, Terry N Influence of phosphorus nutrition on growth and carbon partitioning in Glycine max. Plant Physiology 89, Fredeen AL, Raab TK, Rao IM, Terry N Effects of phosphorus nutrition on photosynthesis in Glycine max (L.) Merr. Planta 181, Furbank RT, Foyer CH, Walker DA Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation. Biochimica et Biophysica Acta 894, Genty B, Briantais J-M, Baker NR The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990, Gericke S, Kurmies B Die kolorimetrische Phosphorsaurebestimmung mit Ammonium-Vanadat- Molibdat und ihre Anwendung in der Pflanzenanalyse. Zeitschrift far Pflanzenerniihrung DOng. Boden. 59, Giersch C, Robinson SP Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chjoroplasts. Photosynthesis Research 14, Havaux M, Strasser RJ, Greppin H A theoretical and experimental analysis of the q r and q N coefficients of chlorophyll fluorescence quenching and their relation to photochemical and non-photochemical events. Photosynthesis Research 27, Heineke D, Stitt M, Heldt HW Effect of inorganic phosphate on the light-dependent thylakoid energization of intact spinach chloroplasts. Plant Physiology 91, Hoagland DR, Arnon DI The water culture method for growing plants without soil. California Agricultural Experimental Station Circular 347, Holm G Chlorophyll mutations in barley. Acta Agronomica Scandinavica 4, Jacob J, Lawlor DW Stomatal and mesophyll limitations of photosynthesis in phosphate-deficient sunflower, maize and wheat plants. Journal of Experimental Botany 41, Jacob J, Lawlor, DW Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-l,5-6uphosphate carboxylase/oxygenase activity and ribulose-l,5-tophosphate pool size. Plant Physiology 98, Lauer JM, PaUardy SG, Blevins DG, Randall DD Whole leaf carbon exchange characteristics of phosphate-deficient soybeans. Plant Physiology 91, Plesnicar M, Pankovid D Relationship between chlorophyll fluorescence and photosynthetic O 2 evolution in several Helianthus species. Plant Physiology and Biochemistry 29, Saric M, Kastori R, Petrovi6 M, Stankovic 1, Krstid B, Petrovid N Manual on plant physiology, Beograd: Nauina knjiga, Sawada S, Usuda H, Tsukui T Participation of inorganic orthophosphate in regulation of the ribulose-l,5-6wphosphate carboxylase activity in response to changes in the photosynthetic source-sink balance. Plant Cell Physiology 33, Schreiber U, Schliwa U, Bilger B Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Research 10, Stitt M Rising CO 2 levels and their potential significance for carbon flow in photosynthetic cells. Plant, Cell and Environment 14, Stitt M, Schreiber U Interaction between sucrose synthesis and CO 2 fixation. III. Response of biphasic induction kinetics and oscillations to manipulation of the relation between electron transport, Calvin cycle, and sucrose synthesis. Journal of Plant Physiology 133, Terry N, Ulrich A Effects of phosphorus deficiency on photosynthesis and respiration of leaves of sugar beet. Plant Physiology 51, von Wettstein D Chlorophyll-letale und der Submikroskopische Formwechsel der Plastiden. Experimental Cell Research 12, Walker DA The use of oxygen electrode and fluorescence probes in simple measurements of photosynthesis. Sheffield: Oxygraphics Limited, Walker DA, Osmond CB Measurement of photosynthesis in vivo with a leaf disc electrode: correlations between light dependence of steady-state photosynthetic O 2 evolution and chlorophyll a fluorescence transients. Proceedings of the Royal Society London B227, Walker DA, Robinson SP Regulation of photosynthetic carbon assimilation. In: Siegelman HW, Hind G, eds. Photosynthetic carbon assimilation, Basic Life Sciences, Vol. 11. New York: Plenum Press, Walker DA, Sivak MN Photosynthesis and phosphate: a cellular affair? Trends in Biochemical Sciences 11, Weis E, Berry JA Quantum efficiency of photosystem II in relation to energy-dependent quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 894,