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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

Abstract

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.

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Abbreviations

A:

assimilation

ci :

internal CO2 concentration

Fru6P:

fructose-6-phosphate

Fru1,6,bisP:

fructose-1,6-bisphosphate

Fru2,6,bisP:

fructose-2,6-bisphosphate

g:

stomatal conductance

Glc6P:

glucose-6-phosphate

PEP:

phospho-enolpyruvate

2PGA:

glycerate-2-phosphate

3PGA:

glycerate-3-phosphate

Ru1,5bisP:

ribulose-1,5-bisphosphate

Rubisco:

ribulose-1,5-bisphosphate carboxylase/oxygenase

SPS:

sucrose-phosphate synthase

UDPGlc:

uridine diphosphoglucose

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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.

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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). https://doi.org/10.1007/BF00202587

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Key words

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