Reversibility of cold- and light-stress tolerance and accompanying changes of metabolite and antioxidant levels in the two high mountain plant species Soldanella alpina and Ranunculus glacialis

Two high mountain plants Soldanella alpina (L.) and Ranunculus glacialis (L.) were transferred from their natural environment to two different growth conditions (22 degrees C and 6 degrees C) at low elevation in order to investigate the possibility of de-acclimation to light and cold and the importance of antioxidants and metabolite levels. The results were compared with the lowland crop plant Pisum sativum (L.) as a control. Leaves of R. glacialis grown for 3 weeks at 22 degrees C were more sensitive to light-stress (defined as damage to photosynthesis, reduction of catalase activity (EC 1.11.1.6) and bleaching of chlorophyll) than leaves collected in high mountains or grown at 6 degrees C. Light-stress tolerance of S. alpina leaves was not markedly changed. Therefore, acclimation is reversible in R. glacialis leaves, but constitutive or long-lasting in S. alpina leaves. The different growth conditions induced significant changes in non-photochemical fluorescence quenching (qN) and the contents of antioxidants and xanthophyll cycle pigments. These changes did not correlate with light-stress tolerance, questioning their role for light- and cold-acclimation of both alpine species. However, ascorbate contents remained very high in leaves of S. alpina under all growth conditions (12-19% of total soluble carbon). In cold-acclimated leaves of R. glacialis, malate represented one of the most abundant compounds of total soluble carbon (22%). Malate contents declined significantly in de-acclimated leaves, suggesting a possible involvement of malate, or malate metabolism, in light-stress tolerance. Leaves of the lowland plant P. sativum were more sensitive to light-stress than the alpine species, and contained only low amounts of malate and ascorbate.     

The significance of glutathione for photoprotection at contrasting temperatures in the alpine plant species Soldanella alpina and Ranunculus glacialis

The significance of total glutathione content was investigated in two alpine plant species with highly differing antioxidative scavenging capacity. Leaves of Soldanella alpina and Ranunculus glacialis incubated for 48 h in the presence of buthionine-sulfoximine had 50% lower glutathione contents when compared with leaves incubated in water. The low leaf glutathione content was not compensated for by activation of other components involved in antioxidative protection or electron consumption. However, leaves with normal but not with low glutathione content increased their ascorbate content during high light (HL) treatment (S. alpina) or catalase activity at low temperature (LT) (R. glacialis), suggesting that the mere decline of the leaf glutathione content does not act as a signal to ameliorate antioxidative protection by alternative mechanisms. CO(2)-saturated oxygen evolution was not affected in glutathione-depleted leaves at various temperatures, except at 35°C, thereby increasing the high temperature (HT) sensitivity of both alpine species. Leaves with low and normal glutathione content were similarly resistant to photoinhibition and photodamage during HL treatment at ambient temperature in the presence and absence of paraquat or at LT. However, HL- and HT-induced photoinhibition increased in leaves with low compared to leaves with normal glutathione content, mainly because the recovery after heat inactivation was retarded in glutathione-depleted leaves. Differences in the response of photosystem II (PSII) activity and CO(2)-saturated photosynthesis suggest that PSII is not the primary target during HL inactivation at HT. The results are discussed with respect to the role of antioxidative protection as a safety valve for temperature extremes to which plants are not acclimated.     

Resistance to photoinhibition of photosystem II and catalase and antioxidative protection in high mountain plants

In leaves of three alpine high mountain plants, Homogyne alpina, Ranunculus glacialis and Soldanella alpina, both photosystem II (PSII) and the enzyme catalase appeared to he highly resistant to photoinactivation under natural field conditions. While the Dl protein of PSII and catalase have a rapid turnover in light and require continuous new protein synthesis in non-adapted plants, little apparent photoinactivation of PSII or catalase was induced in the alpine plants by translation inhibitors or at low temperature, suggesting that turnover of the Dl protein and catalase was slow in these leaves. In vitro PSII was rapidly inactivated in light in isolated thylakoids from H. alpina and R. glacialis. In isolated intact chloroplasts from R. glacialis, photoinactivation of PSII was slower than in thylakoids. Partially purified catalase from R. glacialis and S. alpina was as sensitive to photoinactivation in vitro as catalases from other sources. Catalase from H. alpina had, however, a 10-fold higher stability in light. The levels of xanthophyll cycle carotenoids, of the antioxidants ascorbate and glulathione, and of the activities of catalase, superoxide dismutase and glutathione reductase were very high in S. alpina, intermediate in H. alpina, but very low in R. glacialis. However, isolated chloroplasts from all three alpine species contained much higher concentrations of ascorbate and glutathione than chloroplasts from lowland plants.     

High Temperature Effects on Light Sensitivity in the Two High Mountain Plant Species Soldanella alpina (L.) and Rannunculus glacialis (L.)

Abstract: The susceptibility to high temperature‐induced photoinhibition was investigated in leaves of two high mountain plant species, S. alpina and R. glacialis. In both species, PSII was similarly photoinactivated at 38 °C in the light. However, recovery from damage was much faster in S. alpina and depended on protein synthesis. In contrast, recovery was independent from protein synthesis in R. glacialis. Heat‐induced photoinactivation in both species was accompanied by: (1) a decrease in relative photosynthetic electron transport rates, (2) an increase in non‐photochemical chlorophyll fluorescence quenching, (3) a strong accumulation of zeaxanthin, (4) a marked decrease in soluble carbon metabolites and (5) an increase in lipid metabolism products, which was more pronounced in R. glacialis than in S. alpina. These results indicate that carbon assimilation was inhibited and that membranes were affected. Lipid peroxidation and possible membrane disintegration might limit the repair of damaged PSII in R. glacialis, while S. alpina appears to be protected by carotenoids and antioxidants. A marked decrease in α‐tocopherol content and an increase in reduced ascorbate indicated lipid peroxide scavenging activity in S. alpina. When zeaxanthin synthesis was impaired by DTT, photoinhibition increased and α‐tocopherol accumulated in R. glacialis. The increased susceptibility of R. glacialis leaves to light‐induced photoinhibition after growth at moderate temperature (Streb et al., 2003a) and the inability to repair heat‐induced damage might limit the distribution of R. glacialis to lower altitudes in the Alps.     

Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts

Amyloplasts have been isolated from tubers of potato plants (Solarium tuberosum. cv. Desirée). As it is difficult to isolate amyloplasts that have a high starch content, we used transformed plants in which the content of starch was reduced. This was achieved by decreasing the activity of ADP-glucose pyrophosphorylase by antisense techniques (Müller-Röber et al., 1992, EMBO. 11, 1229–1238). In the isolated plastids the activity of glutamine-oxoglutarate-aminotransferase (glutamate synthase, EC 2.6.1.53) was dependent upon the intactness of the plastids. For the supply of redox equivalents the addition of glucose-6-phosphate (Glc6P) was required. Glucose-1-phosphate (Glc1P) did not support glutamate synthesis. Plastids were treated with Triton X-100 and the solubilized proteins reconstituted into liposomes. Transport measurements with these liposomes revealed that inorganic phosphate (Pi), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate and Glc6P are transported in a counter-exchange mode. Transport of phosphoenolpyruvate was low and Glc1P was virtually not transported in exchange for Pi. Kinetic constants were determined for the Pi/Pi and Glc6P/Pi counter exchanges. For comparison, proteins of mitochondria from potato tubers and pea leaves were reconstituted into liposomes. As expected, the Pi/Pi exchange across the mitochondrial membrane was not affected by DHAP and Glc6P. Kinetic constants of the Pi/Pi counter exchange were determined for potato tuber mitochondria.Abbreviations DHAP dihydroxyacetone phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP Phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - Pi inorganic phosphate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine This work was supported by Deutsche Forschungsgemeinschaft.     

Regulation of mesophyll photosynthesis in intact wheat leaves by cytoplasmic phosphate concentrations

The effect of D-(+)-mannose, inorganic phosphate (Pi) and mannose-6-phosphate on net mesophyll CO₂ assimilation rate (A) and stomatal conductance (g_s) of wheat (Triticum aestivum L.) leaves was studied. The compounds were supplied through the transpiration stream of detached leaves from plants grown in sand in growth cabinets or glasshouses, with different concentrations of Pi (0.25, 1.0 and 4.0 mM) supplied during growth. In all cases, 10 mM D-(+)mannose caused 40–60% reduction of A within 30 min, though the time courses differed for flag leaves and the sixth leaf on the mainstem of glasshouse- and cabinet-grown plants. D-(+)Mannose had a similar effect on A in leaves having a fourfold range in total phosphate content. Effects of D-(+)mannose in reducing g_s were always slower than on A. When the CO₂ concentration in the leaf chamber was adjusted to maintain intercellular CO₂ concentration (C_i) constant as A declined after mannose supply, g_s still declined indicating that stomatal closure was not caused by changing C_i. Supplying mannose-6-phosphate at 10 and 1 mM and Pi at 5 and 10 mM concentrations caused rapid reductions in g_s and also direct reductions in A. The observed effects of mannose and Pi on assimilation are consistent with the proposed regulatory role of cytoplasmic Pi in determining mesophyll carbon assimilation that has been derived previously using leaf discs, protoplasts and chloroplasts.Abbreviations and symbols A net mesophyll CO₂-assimilation rate - C_a, C_i external (assimilation-chamber) and intercellular CO₂ concentration, respectively - g_s stomatal conductance - Man6P mannose-6-phosphate - Pi orthophosphate     

Glacial history of high alpine Ranunculus glacialis (Ranunculaceae) in the European Alps in a comparative phylogeographical context

There is considerable controversy concerning the fate of Alpine plants during Pleistocene glaciations. While some studies have found evidence for nunatak survival, others have explained the present genetic patterns by survival only in peripheral refugia. We investigated 75 populations of high alpine Ranunculus glacialis from its entire Alpine distribution. Phylogeographical analyses of AFLP data revealed four groups of populations. Two of them, located in the western Alps, were genetically isolated from each other and from the eastern groups, whereas the two eastern Alpine groups were genetically more similar to each other. This suggests longer isolation and/or lower levels of gene flow in the two western groups. As all groups are close to, or overlap with, presumed glacial refugia, invoking glacial survival on nunataks is unnecessary to explain the present genetic pattern. Similar to the phylogeographical patterns of R. glacialis , the previously investigated alpine Phyteuma globulariifolium and Androsace alpina , which are also confined to siliceous bedrock, showed strong geographical affinities to peripheral refugial areas and there were large-scale congruencies in the location of these refugia for all three species.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 183–195.     

Prime Ricerche sulla Fisionomia Enzimatica Caratteristica della Fotosintesi

Summary

The rate of oxidation of glucose-6-phosphate, ribose-5-phosphate, fructose-1,6- phosphate, iso-citrate and malate in extracts from green and etiolated pea leaves was determined, using the triphenyltetrazolium technique.

Glucose-6-phosphate, ribose-5-phosphate, and fructose-1,6-phosphate, in the presence of added TPN, where oxidized at a rate about twice higher in the extracts from green than in the extracts from etiolated leaves. Iso-citrate, in the presence of TPN, fructose-1,6-phosphate, in the presence of DPN, and malate, in the presence of either TPN or DPN, were oxidized at about the same rate in the two types of extracts.

These data seem to indicate a preferential synthesis of enzymes involved in the metabolic cycle of phosphorylated sugars during the transition of the leaf from the etiolated to the photosynthetising physiognomy. They seem also favourable to the view assigning to this metabolic system a primary importance in the anabolic pathway of photosynthesis.     

Identification of factors regulating the phosphorylation status of sucrose-phosphate synthase in vivo

The purpose of this study was to identify the factors that control sucrose-phosphate synthase (SPS)-kinase and SPS-protein phosphatase (SPS-PP) activity in situ, and thereby mediate the activation of SPS by light or mannose. Feeding mannose to excised spinach (Spinacia oleracea) leaves in darkness resulted in a general sequestration of cellular phosphate (as evidenced by accumulation of mannose-6-P and depletion of glucose-6-P [Glc-6-P] and fructose-6-P [Fru-6-P]) and a relatively slow activation of SPS (maximum activation achieved within 90 min). Supplying exogenous inorganic phosphate (Pi) with mannose reduced sequestration of cellular Pi (as evidenced by mannose-6-P accumulation without depletion of hexose-P) and substantially reduced mannose activation of SPS. Thus, depletion of cytoplasmic Pi may be required for SPS activation; accumulation of mannose-6-P alone is clearly not sufficient. It was verified that Glc-6-P, but not mannose-6-P, was an inhibitor of partially purified SPS-kinase, and that Pi was an inhibitor of partially purified SPS-PP. Total extractable activity of SPS-kinase did not vary diurnally, whereas a pronounced light activation of SPS-PP activity was observed. Pretreatment of leaves in the dark with cycloheximide blocked the light activation of SPS-PP (assayed in vitro) and dramatically reduced the rate of SPS activation in situ (in saturating light and carbon dioxide). We conclude that rapid activation of SPS by light involves reduction in cytosolic Pi, an inhibitor of SPS-PP, and light activation of SPS-PP, by a novel mechanism that may involve (directly or indirectly) a protein synthesis step. An increase in cytosolic Glc-6-P, an inhibitor of SPS-kinase, would also favor SPS activation. Thus, the signal transduction pathway mediating the light activation of SPS involves elements of “fine” and “coarse” control.     

Protein bodies and vacuoles as lysosomes : investigations into the role of mannose-6-phosphate in intracellular transport of glycosidases in pea cotyledons

We have failed to detect the presence of mannose-6-phosphate in the oligosaccharide moiety of glycoproteins from pea (Pisum sativum L. cv Burpeeana) cotyledons using an assay system sensitive to 10 picomoles of mannose-6-phosphate. We were also unable to demonstrate any retention of glycosidase activity from pea seedlings and pea cotyledons on Sepharose-coupled phosphomannosyl receptor proteins isolated from bovine liver which were, however, able to retain phosphomannosylated hexosaminidase purified from Dictyostelium discoideum secretions. Furthermore, although Sepharose-coupled phosphomannosylated hexosaminidase from Dictyostelium was able to bind phosphomannosyl receptors from bovine liver we were unable to detect the retention of any protein from acetone powder extracts of pea seedlings or from endoplasmic reticulum-associated proteins of pea cotyledons.

Based on this collective evidence we conclude that mannose-6-phosphate does not appear to play a role in the targeting of hydrolytic enzymes from the endoplasmic reticulum to the protein bodies in pea cotyledons.

     

Reductant for glutamate synthase in generated by the oxidative pentose phosphate pathway in non-photosynthetic root plastids

Purified pea root plastids were supplied with glutamine, 2-oxoglutarate and phosphorylated sugars. Formation of glutamate was linear for 75 min and dependent upon the intactness of the organelle. Glucose-6-phosphate and ribose-5-phosphate were the most effective substrates in supporting glutamate synthesis. Flux through the oxidative pentose phosphate pathway during glutamate synthesis in purified plastids was followed by monitoring the release of ¹⁴CO₂ from [1-¹⁴C]glucose-6-phosphate. ¹⁴CO₂ evolution from C-1 was dependent upon the presence of both glutamine and 2-oxoglutarate and could be inhibited by the application of azaserine. The data are discussed in view of the role of the oxidative pentose phosphate pathway in non-photosynthetic plastids.     

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

Dynamic acclimation to sunlight in an alpine plant,Soldanella alpina L.

In the French Alps, Soldanella alpina (S. alpina) grow under shade and sun conditions during the vegetation period. This species was investigated as a model for the dynamic acclimation of shade leaves to the sun under natural alpine conditions, in terms of photosynthesis and leaf anatomy. Photosynthetic activity in sun leaves was only slightly higher than in shade leaves. The leaf thickness, the stomatal density and the epidermal flavonoid content were markedly higher, and the chlorophyll/flavonoid ratio was significantly lower in sun than in shade leaves. Sun leaves also had a more oxidised plastoquinone pool, their PSII efficiency in light was higher and their non-photochemical quenching (NPQ) capacity was higher than that of shade leaves. Shade-sun transferred leaves increased their leaf thickness, stomatal density and epidermal flavonoid content, while their photosynthetic activity and chlorophyll/flavonoid ratio declined compared to shade leaves. Parameters indicating protection against high light and oxidative stress, such as NPQ and ascorbate peroxidase, increased in shade-sun transferred leaves and leaf mortality increased. We conclude that the dynamic acclimation of S. alpina leaves to high light under alpine conditions mainly concerns anatomical features and epidermal flavonoid acclimation, as well as an increase in antioxidative protection. However, this increase is not large enough to prevent damage under stress conditions and to replace damaged leaves.     

Enzyme regulation in c(4) photosynthesis : identification and localization of activities catalyzing the synthesis and hydrolysis of fructose-2,6-bisphosphate in corn leaves

Activities catalyzing the synthesis of fructose-2,6-bisphosphate (fructose-6-phosphate,2-kinase or Fru-6-P,2K) and its breakdown (fructose-2,6-bisphosphatase or Fru-2,6-P₂ase) were identified in leaves of corn (Zea mays), a C₄ plant. Fru-6-P,2K and Fru-2,6-P₂ase were both localized mainly, if not entirely, in the leaf mesophyll cells. A partially purified preparation containing the two activities revealed that the kinase and phosphatase were regulated by metabolite effectors in a manner generally similar to their counterparts in C₃ species. Thus, corn Fru-6-P,2K was activated by inorganic phosphate (Pi) and fructose-6-phosphate, and was inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fru-2,6-P₂ase was inhibited by its products, fructose-6-phosphate and Pi. However, unlike its spinach equivalent, corn Fru-2,6-P₂ase was also inhibited by 3-phosphoglycerate and, less effectively, by dihydroxyacetone phosphate. The C₄ Fru-6-P,2K and Fru-2,6-P₂ase were also quite sensitive to inhibition by phosphoenolpyruvate, and each enzyme was also selectively inhibited by certain other metabolites.     

Low temperature regulates sucrose-phosphate synthase activity in Colobanthus quitensis (Kunth) Bartl. by decreasing its sensitivity to Pi and increased activation by glucose-6-phosphate

Colobanthus quitensis (Kunth) Bartl. is widely distributed from Mexico to the Antarctic. C. quitensis is a freezing resistant species that accumulates sucrose in response to cold. We tested the hypothesis that low temperature modifies the kinetic properties of C. quitensis sucrose phosphate synthase (SPS) to increase its activity and ability to synthesize sucrose during cold acclimation. Cold acclimation caused a fourfold increment in sucrose concentration and a 100% increase in SPS activity, without changes in the level of SPS protein. Cold acclimation did not affect the optimal temperature and pH for SPS activity. However, it caused a tenfold increase in the inhibition constant (K _i) for inorganic phosphate (Pi) calculated as a function of fructose-6-phosphate (Fruc-6-P). SPS from cold acclimated plants also exhibited a higher reduction of its Michaelis constant (K _m) for glucose-6-phosphate (Gluc-6-P) with respect to non-acclimated plants. We suggest that the increase in C. quitensis SPS K _i for Pi and the increase in activation by Gluc-6-P in response to cold keep SPS activated, leading to high sucrose accumulation. This may be an important adaptation that allows efficient accumulation of sucrose during the harsh Antarctic summer.     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Short-term feedback inhibition of photosynthesis in wheat leaves supplied with sucrose and glycerol at two temperatures

The inhibition of photosynthesis by reduced sink demand or low rates of end product synthesis was investigated by supplying detached wheat (Triticum aestivum L. cv. Tauro) leaves with 50 mM sucrose, 50 mM glycerol or water through the transpiration stream for 2 h, either at 23 or 12 °C. Lowering the temperature and sucrose and glycerol feeding decreased photosynthetic oxygen evolution at high irradiance and saturating CO₂. The decrease in temperature reduced the pools of sucrose and starch, and the ratio glucose 6-phosphate (G6P)/fructose 6-phosphate (F6P), while it increased the concentrations of G6P and F6P (hexose phosphates). Sucrose feeding, in contrast to glycerol feeding, increased sucrose, glucose and fructose contents and the G6P/F6P ratio. Sucrose and glycerol incubations at 23 °C, as well as decreasing the temperature in leaves incubated in water, increased the concentration of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) and decreased the glycerate 3-phosphate (PGA) content, thus increasing the TP/PGA ratio; they also tended to increase the ribulose 1,5-bisphosphate (RuBP) content and the RuBP/PGA ratio. Sucrose and glycerol feeding at 12 °C and the decrease in temperature of leaves incubated in these solutions decreased TP and RuBP contents and the TP/PGA and RuBP/PGA ratios. The results suggest that the phosphate limitation caused by accumulation of end products, restriction of their synthesis and sequestration of cytosolic phosphate can inhibit photosynthesis through decreased carboxylation of RuBP or, with increased phosphate limitation, through lowered supply of ATP. This revised version was published online in August 2006 with corrections to the Cover Date.