Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves. Richard C. Leegood 1, Carlos A. Labate 1, Steven C. Huber ...
Planta
Planta (1988) 176:117-126
9 Springer-Verlag 1988
Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves Richard C. Leegood 1, Carlos A. Labate 1, Steven C. Huber 2, H. Ekkehard Neuhaus 3, and Mark Stitt 3 1 Research Institute for Photosynthesis and Department of Plant Sciences, University of Sheffield, Sheffield, $10 2TN, UK, 2 USDA-ARS, Crop Science Department, North Carolina State University, 3127 Ligon Road, Raleigh, NC 27695, USA, and 3 Lehrstuhl ffir Pflanzenphysiologie, Universit/it Bayreuth, Universit/itsstrasse 30, D-8580 Bayreuth, Federal Republic of Germany
Abstract. Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO2 assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results. Key words: Glycerol - Glycerol-3-phosphate Hordeum (photosynthesis) - Photosynthetic carbon metabolism (phosphate status) - Spinacia (photosynthesis)
Introduction Phosphate plays a key role in the regulation of photosynthetic carbon metabolism (Walker 1976). Abbreviations." Chl=chlorophyll; Ci=intercellular concentra-
tion of CO2; P=phosphate; PGA =glycerate-3-phosphate; Pi=orthophosphate; triose-P=sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate
The product of chloroplast photosynthesis both in vitro and in vivo is triose phosphate (triose-P) and this esterified phosphate must be recycled by starch, sucrose or organic-acid synthesis to provide orthophosphate (Pi) to allow continuation of ATP synthesis in the chloroplast. The relative amounts of Pi within the chloroplast and cytoplasm will influence whether triose-P can be exported from the stroma via the phosphate translocator and hence will be a key factor which determines the cellular partitioning of carbon between starch and sucrose synthesis. An important direct role is also played by Pi in the modulation of the activity of certain enzymes, both within the chloroplast (e.g. fructose and sedoheptulose bisphosphatases [Charles and Halliwell 1980; Woodrow et al. 1983] and ribulose-l,5-bisphosphate carboxylase [Heldt et al. 1978]) and in the cytoplasm (e.g. sucrosephosphate synthetase [Harbron et al. 1981 ; Doehlert and Huber 1983] and fructose-l,6-bisphosphatase [Harbron et al. 1981 ; Stitt et al. 1985]). A good deal of our knowledge of the function of phosphate in vivo has come from the use of Pi-sequestering agents such as D-mannose and 2deoxy-o-glucose. These compounds are phosphorylated by hexokinase in all plants to form, for example, mannose-6-phosphate from mannose, but many plants are unable subsequently to metabolise these compounds at appreciable rates (Herold and Lewis 1977). Hence cytoplasmic Pi becomes sequestered into organic compounds. Studies with these Pi-sequestering agents have shown that, inter alia, they reduce light-saturated rates of photosynthesis (Walker and Osmond 1986), favour the partitioning of fixed carbon into starch (Chen-She et al. 1975), decrease pools of phosphorylated intermediates (Prinsley and Leegood 1986), promote oscillatory behaviour (Walker and Sivak 1985; Sivak and Walker 1987) and lead to stomatal opening (Harris etal. 1983b; Morison and Batten
118
1986). More recently, considerable interest has focussed on the circumstances in which phosphate can limit photosynthesis in the absence of such agents, such as in high CO2 and high light (when the maximum capacity of sucrose synthesis is reached), or at low temperatures (when the phosphate status of the leaf is too low to support maxim u m rates of photosynthesis). Under these conditions, the supply of Pi to the leaf can lead to considerable increases in rates of photosynthesis and pools of phosphorylated metabolites and can suppress oscillatory behaviour (Dietz and Foyer 1985; Walker and Sivak 1985; Leegood and Furbank 1986; Stitt 1986; Sharkey et al. 1986; Sivak and Walker 1987; Labate and Leegood 1988; Stitt and Schreiber 1988). In this study we report on the effects that a new Pi-sequestering agent, glycerol (Bligny et al. 1988), has on photosynthetic carbon assimilation in intact leaves of barley and spinach.
Materials and methods Plant material. Barley (Hordeum vulgare L.) and spinach (Spinaeia oleracea L.) were grown as described by Stitt and Schreiber (1988). Glycerol was fed to illuminated barley leaves via the transpiration stream for 2 h. Spinach leaf discs (11 mm diameter) were floated on a solution of glycerol in the dark for 1 ~ 14h. Gas-exchange and chlorophyll fluorescence measurements. Photosynthetic 02 evolution was measured in a leaf-disc 02 electrode (Hansateeh, King's Lynn, Norfolk, UK) in saturating CO2. Chlorophyll fluorescence was measured using a PAM chlorophyll fluorescence measuring system (H. Walz, Effeltrich, FRG). COz uptake was measured by infra-red gas analysis as described by Labate and Leegood (1988). Metabolite analysis. Leaf samples for analysis of metabolites were transferred to liquid N2 under illumination as described previously (Stitt 1986) and were extracted in 0.5 ml 1 M HC104 and neutralised with 5 M KOH/1 M triethanolamine. All metabolites except glycerol-3-pbosphate (glycerol-3-P) were measured spectrophotometrically as described by Lowry and Passonneau (1972) using a dual-wavelength (334405 rim) ZFP-22 spectrophotometer (Sigma Instrumente, West Berlin, FRG). The assay for glycerate-3-phosphate (PGA) was modified to eliminate drift by incubation of the extract in 50 mM imidazole (pH 7.1), 2 mM mercaptoethanol, I mM MgCI2, 20 mM NaC1, 1 mM ATP for 20-30 rain at room temperature, prior to the addition of N A D H and coupling enzymes. Glycerol-3-phosphate was measured in a medium containing 0.5 M glycine, 0.4M hydrazine (pH 9.0), 1 mM NAD +. Reactions were started by the addition of 2 units glycerol-3-P dehydrogenase. Recoveries of authentic glycerol-3-P were within 10% of the expected values. Chlorophyll and phaeophytin. Chlorophyll (Chl) was measured by the method of Arnon (1949) and phaeophytin (from HCIOgtreated samples) by the method of Vernon (1960).
R.C. Leegood et al. : Phosphate sequestration by glycerol
Results
Effects of feeding glycerol to barley leaves. Figure 1 shows a typical response of CO2 uptake by barley leaves to the supply of glycerol via the transpiration stream. While the rate of photosynthesis in the control leaves remained constant when fed with water, feeding 100 mM glycerol led an initial rapid decline and then a slow decrease in the rate of CO2 uptake from the initiation of feeding. After 3 h the rate of C02 uptake was inhibited by about one-third (data not shown). During glycerol feed-
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