Leaf free space analysis and vein loading in Cucurbita pepo

To determine the role of the apoplast in the loading of the minor veins of Cucurbita pepo L., leaves were examined either for the ability to release selectively the transport sugars, sucrose and stachyose, from the metabolic space (MS) to the free space (FS) or to accumulate selectively exogenously fed transport sugars from the FS into the minor veins. FS extracts collected by vacuum infiltration and centrifugation of specially trimmed leaves were found to contain all sugars also present in ethanol extracts of the MS of the same leaves, but in amounts two orders of magnitude lower. Similarly, 14C activity in FS extracts from 14CO2-labelled leaves, which was distributed between all sugars, amino acids, and organic acids also found labelled in the MS, was nearly three orders of magnitude lower than in MS ethanol extracts. No excess of the transport sugars was evident in FS extracts. When 14C-labelled sucrose, stachyose, or galactose (all 1–2 mM) were infiltrated into the FS a rapid accumulation of these sugars into the mesophyll was apparent, as all sugars were rapidly interconverted into the various sugars normally found labelled in the MS after exposure of leaves to 14CO2. However, while leaves exposed to 14CO2 translocated label predominantly in the form of [14C]stachyose, label in exogenously fed leaves was translocated predominantly as [14C]sucrose, irrespective of the nature of the fed sugar. Exogenously fed transport sugars did not therefore appear to be taken up directly into the minor veins from the FS.The absence of significant levels of transport sugars in the FS as well as the failure of C. pepo leaves to load any appreciable amount of exogenously supplied [14C]stachyose, the predominant transport sugar in this species, would tend to preclude both a selective release of transport sugars into the apoplast and a selective uptake of transport sugars from the apoplast into the minor veins. A completely symplastic pathway for minor vein loading in C. pepo leaves therefore remains a possibility.