Wounding and the Regulation of Apoplasmic Retrieval in Source Leaf Tissue 5Spinacia oleraceaL.

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Abstract
Kinetic studies were performed on fresh-cut and aged leaf discs of Spinacia oleracea L. in order to investigate the regulatory processes involved in sugar transport across the mesophyll plasma membrane. A comparison of the kinetic profiles for fructose, glucose, sucrose and arginine obtained on freshly-cut and aged discs revealed that during wound-recovery, uptake is enhanced, but that this enhancement varies considerably for the different substrates tested. Variation in the saturable and first-order kinetic components of uptake was also observed. The involvement of a phosphoinositide-signalling mechanism in the wounding process was examined by pretreating spinach leaves with lithium. Lithium inhibited the enhancement of uptake and this effect was reversed by the addition of miro-inositol. However, in some experiments the tissue appeared to be insensitive to Li+. Gas chromatographic analyses performed on cut discs indicated that ethylene was produced in response to wounding and that the addition of cobalt to the ageing media inhibited this ethylene production. Comparative kinetic studies of control and cobalt-treated discs indicated that ethylene was essential for the enhancement of transport across the plasma membrane. However, addition of ethylene to uncut tissue caused only a partial increase in the uptake of fructose, which indicates that some additional wound-signalling component is involved. Addition of cycloheximide to the recovery media completely inhibited this enhancement phenomenon. The cycloheximide-response was not due to an inhibition of ACC synthase synthesis nor to a reduction of ATP levels. We concluded that the effect of cycloheximide was on protein synthesis. Our results are discussed in terms of possible cellular and molecular mechanisms regulating sugar transport.