Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

An evaluation of direct and indirect mechanisms for the “sink-regulation” of photosynthesis in spinach: Changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves


Mature source leaves of spinach (Spinacia oleracea L.) plants growing hydroponically in a 9 h light (350 μmol photons·m−2 · s−1)/15 h dark cycle at 20° C in a climate chamber were fitted with a cold girdle around the petiole, 2 h into the light period. Samples were taken 1, 3 and 7 h later, and at the end of the photoperiod for the following 4 d. Control samples were taken from ungirdled leaves. In the first 7 h after fitting the cold girdle there was (compared to the control leaves) a two to five-fold accumulation of sucrose, glucose, fructose and starch, a 40–50% increase of hexose-phosphates and ribulose-1,5-bisphosphate, a decrease of glycerate-3-phosphate, a small decrease in sucrose-phosphate synthase activation, an increase of fructose-2,6-bisphosphate, increased activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), but no significant change in photosynthetic rate or stomatal conductance. Steady-state transcript levels for rbcS (small subunit of Rubisco) and atp-D (D-subunit of the thylakoid ATP synthase) decreased 30%, cab (chlorophyll-a-binding protein) decreased by 15% and agp-S (S-isoenzyme of ADP-glucose pyrophosphorylase) and nra (nitrate reductase) rose twofold. On the following days, levels of carbohydrates continued to rise and the changes of metabolites were maintained. Transcripts for rbcS, cab and atpD declined to 20, 70 and 25% of the control values. From day 3 onward the maximum activity of Rubisco declined. This was accompanied by a further increase of Rubisco activation to over 90% and, from day 4 onwards, an inhibition of photosynthesis which was associated with high internal CO2 concentration (ci), high ribulose-1,5-bisphosphate, and low glycerate-3-phosphate. When the cold-girdle was removed on day 5 there was a gradual recovery of photosynthesis and decline of ci over the next 2 d. Hexose-phosphates levels and transcripts for rbcS, cab and atp-D completely recovered within 2 d, even though the levels of carbohydrates had not fully recovered. Activity of Rubisco only reverted partly after 2 d, and Rubisco activation state and the ribulose-1,5-bisphosphate/glycerate-3-phosphate ratio were still higher than in control leaves. Transcripts for nra and agp-S were also still higher than in control leaves. It is concluded (i) that a reversible modulation of gene expression in response to the export rate plays a central role in the mid-term feedback “sink” regulation of photosynthesis, and (ii) that feedback regulation of CO2 fixation by changes of Pi are of little importance in spinach under these conditions. Further (iii) the rapid and reciprocal changes in nra and agpS transcripts, compared to rbcS, provide evidence that gene expression could also contribute to the modulation of nitrate assimilation and carbohydrate storage in conditions of decreased sink demand.

This is a preview of subscription content, log in to check access.




ci :

internal CO2 concentration








stomatal conductance












ribulose-1,5-bisphosphate carboxylase/oxygenase


sucrose-phosphate synthase


uridine diphosphoglucose


  1. Bagnall, D.J., King, R.W., Farquhar, G. (1988) Temperature dependent feedback of photosynthesis in peanut. Planta 175, 348–357

  2. Besford, R.T. (1991) The greenhouse effect: acclimation of tomato plants growing in high CO2, relative changes in Calvin cycle enzymes. J. Plant Physiol. 136, 458–463

  3. Besford, R.T, Ludwig, L.I. Withers, A.C. (1990) The greenhouse effect: acclimation of tomato plants growing in high CO2 photosynthesis and ribulose-1,5-bisphosphate carboxylase protein. J. Exp. Bot. 41, 925–931

  4. Butz, N.D., Sharkey, T. (1989) Activity ratios of ribulose-1,5-bisphosphate carboxylase accurately reflect carbamylation ratios. Plant Physiol 89, 735–739

  5. Cave, G., Tolley, L.C. and Strain, B.R. (1981) Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subterraneum leaves. Physiol. Plant. 51, 171–174

  6. Champigny, M.L., Brauer, M., Bismuth, E., Manh, C.T., Siegl, G., Quy, L.V., Stitt, M. (1992) The short-term effect of NO3 and NH3 assimilation on sucrose synthesis in leaves. J. Plant Physiol 139, 361–368

  7. Chatterton, N.J., Silvius, J.E. (1980) Photosynthate partitioning into leaf starch as affected by daily photosynthetic period duration in several species. Physiol. Plant. 49, 141–144

  8. Dalton, C.C. (1984) The effect of sucrose supply rate on photosynthesic development of Ocimum basilicum (Sweet Basil) cells in continuous culture. J. Exp. Bot. 35, 505–516

  9. Dickinson, C., Altabella, T., Chrispeels, M. (1991) Slow growth phenotype of transgenic tomato expressing apoplastic invertase. Plant Physiol. 95, 420–425

  10. Edelmann, J., Hanson, A.D. (1971) Sucrose supression of chlorophyll synthesis in carrot cultures. Planta 98, 150–156

  11. Foyer, C.H. (1987) The basis of source-sink interaction in leaves. Plant Physiol. Biochem. 25, 649–657

  12. Geigenberger, P., Reimholz, R., Stitt, M. (1994) When growing potato tubers are detached from their mother plant there is a rapid inhibition of starch synthesis involving inhibition of ADP-glucose pyrophosphorylase. Planta, in press

  13. Geiger, D.R. (1970) Effect of assimilate translocation on photosynthesis. Can. J. Bot. 54, 2337–2345

  14. Goldschmidt, E.E., Huber, S.C. (1992) Regulation of photosynthesis by end product accumulation in leaves of plants storing starch, sucrose and hexose sugars. Plant Physiol. 99, 1443–1448

  15. Grub, A., Mächler, F (1990) Photosynthesis and light activation of Rubisco in the presence of starch. J. Exp. Bot. 41, 1293–1301

  16. Harris, G.C., Cheeseborough, J.K., Walker, D.A. (1983) Effect of mannose on photosynthetic gas exchange in spinach leaf discs. Plant Physiol. 71, 108–111

  17. Heineke, D., Sonnewald, U., Büssis, D., Günter, G., Leidreiter, K., Wilke, I., Raschke, K., Willmitzer, L., Heldt, H.W. (1992) Expression of yeast-derived invertase in the apoplast of potato plants results in an inhibition of photosynthesis caused by an increase of the osmotic pressure in leaf cells due to the accumulation of hexoses and amino acids, and affects an increase in the protein to starch ratio in the tubers. Plant Physiol. 100, 301–308

  18. Heldt, H.W., Chon, C.J., Maronde, R., Herold, A., Stankovic, Z.S., Walker, D.A., Kraminer, A., Kirk, M.R., Heber, U. (1977) Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts. Plant Physiol. 59, 1146–1155

  19. Heldt, H.W, Chon, C.J., Lorimer, G.H. (1978) Phosphate requirement for the light activation of ribulose-1,5-bisphosphate carboxylase in intact spinach chloroplasts. FEBS Letters 92, 234–240

  20. Herold, A. (1980) Regulation of photosynthesis by sink activity: the missing link. New Phytol. 86, 131–144

  21. Huber, S.C., Hanson, K.R. (1992) Carbon partitioning and growth of starchless mutant of Nicotiana sylvestris. Plant Physiol. 99, 1449–1454

  22. Huber, S.C., Huber, J.L.A. (1992) Role of sucrose phosphate synthase in sucrose metabolism in leaves. Plant Physiol. 99, 1275–1278

  23. Huber, S.C., Huber, J.L.A., Campbell, W.M., Redinbough, G.M. (1992) Comparative studies of the light modulation of nitrate reductase and sucrose phosphate synthase. Plant Physiol. 100, 706–712

  24. Krapp, A., Quick, W.P., Stitt, M. (1991) Riboluse-1,5-bisphosphate carboxylase-oxygenase, other photosynthetic enzymes and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream. Planta 186, 58–69

  25. Krapp, A., Hofmann, B., Schäfer, C., Stitt, M. (1993) Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the “sink regulation” of photosynthesis. Plant J. 3, 817–828

  26. Leegood, R.C., Furbank, R.T. (1986) Stimulation of photosynthesis by O2 at low temperature is restored by phosphate. Planta 168, 84–93

  27. Mayoral, M.L., Plaut, Z., Reinhold, L. (1985) Effect of translocation-hindering procedures on source leaf photosynthesis in cucumber. Plant Physiol. 77, 712–717

  28. Müller-Röber, B.T., Koßmann, J., Hannah, L.C., Willmitzer, L., Sonnewald, U. (1990) One of the two ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose. Mol. Gen. Genet. 224, 136–146

  29. Nafzinger, E.D., Koller, R.M. (1976) Influence of leaf starch concentration on CO2 assimilation in soybean. Plant Physiol. 57, 560–563

  30. Neales, T.F., Incoll, L.D. (1968) The control of leaf photosynthesis by the level of assimilate in the leaf. Bot. Rev. 34, 431–454

  31. Neuhaus, H.E. Quick, W.P., Siegl, G., Stitt, M (1990) Control of photosynthate partitioning in spinach leaves. Analysis of the interaction between feedforward and feedback regulation of sucrose synthesis. Planta 181, 583–592

  32. Parry, M.A.J., Schmidt, C.N.G., Cornelius, M.J., Keys, A.J., Millard, B.N., Gutteridge, S. (1985) Stimulation of ribulose bisphosphate carboxylase activity by inorganic phosphate without an increase in bound activating CO2. J. Exp. Bot. 36, 1396–1404

  33. Plaut, Z., Mayoral, M.L., Reinhold, L. (1987) Effect of altered sinksource ratio on photosynthesic metabolism of source leaves. Plant Physiol. 85, 786–791

  34. Preiss, J. (1988) Biosynthesis of starch and its degradation. In: Biochemistry of plants, vol 3, pp. 181–254, Preiss, J., ed. Academic Press, San Diego

  35. Preiss, J. (1991) Biology and molecular biology of starch synthesis and its regulation. In: Oxford surveys of plant molecular and cell biology, vol. 7, pp. 59–114, Mifin, B.J., ed. Oxford University Press, Oxford

  36. Quick, W.P., Schurr, U., Scheibe, R., Schulze, E.D., Rodermel, S.R., Bogorad, L., Stitt, M. (1991) Decreased Rubisco in tobacco transformed with “antisense” rbcS. I. Impact on photosynthesis in ambient growth conditions. Planta 183, 542–554

  37. Sambrock, J., Fritisch, E.F., Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press

  38. Sage, R., Sharkey, T.D. (1987) The effect of low temperature on the occurrence of O2 and CO2 insensitive photosynthesis in fieldgrown plants. Plant Physiol. 84, 653–654

  39. Sage, R., Sharkey, T.D., Seemar, I.R. (1989) Acclimation of photosynthesis to enhanced CO2 in five C-3 species. Plant Physiol. 89, 590–596

  40. Sawada, S., Hagesawa, T., Fukuschi, K., Kasai, K. (1989) Influence of carbohydrates on photosynthesis in single rooted soybean leaves used as a sink-source model. Plant Cell Physiol. 27, 591–600

  41. Sawada, S., Usuda, H., Hasegawa, Y., Tsukui, T. (1990) Regulation of Rubisco activity in response to changes of the source/sink balance in single rooted soybean leaves. Plant Cell Physiol. 31, 697–704

  42. Schäfer, C., Simper, H., Hofmann, B. (1992) Glucose feeding results in coordinated changes of chlorophyll content, ribulose-1,5-bisphosphate carboxylase-oxygenase activity and photosynthetic potential in photoautotrophic suspension cultured cells of Chenopodium rubrum Plant Cell Environ. 15, 343–350

  43. Schnyder, H., Mächler, F., Nösberger, J. (1986) Regeneration of ribulose-1,5-bisphosphate and ribulose-1,5-bisphosphate carboxylase/oxygenase activity associated with lack of oxygen inhibition of photosynthesis at low temperature. J. Exp. Bot. 37, 1170–1179

  44. Sharkey, T.D. (1990) Feedback limitation of photosynthesis and the physiological role of ribulose bisphosphate carboxylase carbamylation. Bot. Mag. Tokyo Special Issue 2, 87–105

  45. Sharkey, T.D., Stitt, M., Heineke, D., Gebhardt, R., Raschke, K., Heldt, H.W. (1986) Limitation of photosynthesis by carbon metabolism. II. O2-insensitive CO2 uptake results from limitation of triosephosphate utilisation. Plant Physiol. 81, 1123–1129

  46. Sheen, J. (1989) Metabolic repression of transcription in higher plants. Plant Cell 2, 1027–1038

  47. Siegl, G., Stitt, M. (1988) Partial purification of two different forms of spinach leaf sucrose-phosphate synthase which differ in their kinetic properties. Plant Sci 66, 205–210

  48. Smith, A.M., Martin, C. (1993) Starch biosynthesis and the potential for its manipulation. In: Plant biotechnology, vol. 3: Biosynthesis and manipulation of plant products, Grierson, D., ed. Blackie, Glasgow, in Press.

  49. Smith-White, B.J., Preiss, J. (1992) Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources. J. Mol. Evol. 34, 449–464

  50. Socias, F.X., Medrano, H., Sharkey, T.D. (1993) Feedback limitation of photosynthesis in Phaseolus vulgaris L. grown in elevated CO2. Plant Cell Environ. 16, 81–86

  51. Spill, D., Kaiser, W.M. (1994) Partial purification of two proteins (100 kDa and 67 kDa) cooperating in the ATP-dependent inactivation of spinach leaf nitrate reductase. Planta 192, 183–188

  52. Stitt, M. (1990) Fructose-2,6-bisphosphate as a regulatory metabolite in plants. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 41, 153–185

  53. Stitt, M. (1991) Rising CO2 levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Env. 14, 741–762

  54. Stitt, M., Große, H. (1988) Interactions between sucrose synthesis and CO2 fixation. I. Secondary kinetics during photosynthetic induction are related to a delayed activation of sucrose synthesis. J. Plant Physiol. 133, 129–137

  55. Stitt, M., Quick, W.P. (1989) Photosynthetic carbon partitioning, its regulation and possibilities for manipulation. Physiol. Plant. 77, 633–641

  56. Stitt, M., Huber, S.C., Kerr, P. (1987a) Control of photosynthetic sucrose synthesis. In: Biochemistry of plants, vol. 10, pp 327–409. Hatch, M.D., Boardman, N.K., eds. Academic press, New York

  57. Stitt, M., Gerhardt, R., Wilke, I., Heldt, H.W. (1987b) The contribution of fructose-2,6-bisphosphate to the regulation of sucrose synthesis during photosynthesis. Physiol. Plant. 59, 377–386

  58. Stitt, M., Wilke, I., Feil, R., Heldt, H.W. (1988) Coarse control of sucrose-phosphate synthase in leaves: alterations of kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose. Planta 174, 217–230

  59. Stitt, M., Lilley, R.McC., Gerhardt, R., Heldt, H.W. (1989) Metabolite levels in specific cells and subcellular compartments of plant leaves. Methods Enzymol 174, 518–552

  60. Stitt, M., Von gnSchaewen, A., Willmitzer, L. (1991) “Sink”-regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin cycle enzymes and an increase of glycolytic enzymes. Planta. 183, 40–50

  61. Vincentz, M., Maireaux, T, Leydecker, M.-T., Vaucheret, H., Caboche, M. (1992) Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites. Plant J. 3, 315–324

  62. Von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U., Willmitzer, L. (1990) Expression of yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to inhibition of sucrose export, accumulation of carbohydrate, and inhibition of photosynthesis, and strongly influences the growth and habitus of transgenic tobacco. EMBO J. 9, 3033–3044

  63. Winter, H., Robinson, D.G., Heldt, H.W. (1993) Subcellular volumes and metabolite concentrations in spinach leaves. Planta 191, 180–190

Download references

Author information

Correspondence to Mark Stitt.

Additional information

This research was supported by the Deutsche Forschungsgemeinschaft. We are grateful to Prof. E. Beck (Bayreuth, Germany) for providing plant growth facilities, to Prof. Hermann (University of München, Germany) for providing us with cab and atp-ß clones, to Prof. Willmitzer and Dr. Müller-Röber (Institut für Genbiologische Forschung, Berlin, Germany) for providing us with agpS clone, to Prof. Caboche (INRA, Versailles, France) for providing us with nra-clone, to Dr. P. Eckes (Bayer AG, Leverkusen, Germany) for providing us with the rbcS clone, and to G. Hettinger for designing and making the cold-girdles.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Krapp, A., Stitt, M. An evaluation of direct and indirect mechanisms for the “sink-regulation” of photosynthesis in spinach: Changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves. Planta 195, 313–323 (1995).

Download citation

Key words

  • Carbohydrate
  • Gene expression
  • Photosynthesis (spinach)
  • Ribulose-1,5-bisphosphate carboxylase/oxygenase
  • Sink regulation
  • (rbcS)