The aim of this work was to identify which aspects of photosynthetic metabolism respond most sensitively to leaf water deficit. Spinach (Spinacia oleracea L.) leaf discs were floated on
sorbitol concentrations of increasing molarity and changes of the protoplast volume were estimated using [(14)C]
sorbitol and (3)H2O penetration. Detached leaves were also wilted until 10% of their fresh weight was lost. Photosynthesis was studied at very high external CO2 concentrations, to eliminate the effect of closing stomata. There was no large inhibition of CO2 fixation after wilting leaves, or until the external water deficit was greater than-1.2 MPa. However, partitioning changed markedly at these moderate water deficits: more
sucrose and less
starch was made. When an inhibition of CO2-saturated photosynthesis did appear at a water deficit of-2.0 MPa and above, measurements of
chlorophyll-fluorescence quenching and metabolite levels showed the thylakoid reactions were not especially susceptible to short-term
water stress. The inhibition was accompanied by a small increase of the triose
phosphate:
ribulose-1,5-bisphosphate ratio, showing regeneration of
ribulose-1,5-bisphosphate was affected. However, there was also a general increase of the estimated concentrations of most metabolites, indicating that there is no specific site for the inhibition of photosynthesis. Increasing water deficit led to a large increase of fructose-2,6-bisphosphate. This is explained in terms of a simultaneous increase of
fructose-6-phosphate and
inorganic phosphate as the cell shrinks. The high fructose-2,6-bisphosphate led to the accumulation of triose
phosphates, and the potential significance of this for protection against photoinhibition is discussed. There was an increase in the extractable activity of
sucrose-phosphate synthase. This was only detected when the
enzyme was assayed in conditions which distinguish between different kinetic forms which have previously been identified in spinach leaves. It is proposed that activation of
sucrose-phosphate synthase is one of the first sites at which spinach leaves respond to a rising water deficit. This could be of importance for osmoregulation.