Amino Acid and Sucrose Content Determined in the Cytosolic , Chloroplastic , and Vacuolar Compartments and in the Phloem Sap of Spinach Leaves 1

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells. Assimilates are transferred from mature leaf cells to sink tissues by phloem transport. Major constituents ofthe phloem sap are sugars, mainly sucrose, and amino acids. For an understanding of the partitioning of photosynthetic products between carbohydrates and amino acids, the factors governing the withdrawal of these products from the source cells and their loading into the sieve tubes have to be known. There are various indications that phloem loading of sucrose involves transport against a concentration gradient, probably driven by proton symport (3, 4, 13, 22), and that a proton symport may also be involved in the uptake of amino acids into the sieve tubes (1, 5, 19, 21, 22). For many plants it has been assumed that phloem loading is an apoplastic process (25). This mechanism implies that for export of substances from a source cell, they first have to be released into the apoplastic space before being taken up into the sieve tubes. This concept of apoplastic loading, derived initially from morphological studies (25), has been recently verified by studies of transformants of Nicotiana tabaccum (tobacco) and Solanum tuberosum (potato). In these plants the insertion of yeast acid invertase into the apoplastic com'Supported by the Deutsche Forschungsgemeinschaft. 227 partment of leaves caused the accumulation of sucrose and its precursors in leaves and the disappearance of sucrose in the sieve tube exudate (26) (G Gunter, D Heineke, U Sonnewald, L Willmitzer, HW Heldt, unpublished results). The finding that the hydrolysis of sucrose in the apoplastic space prevented its loading into the sieve tubes, clearly demonstrated that phloem loading with sucrose is an apoplastic process. Unless the loading of amino acids into the phloem is a very different process, for which there is no indication, the transfer of amino acids from the source cells into the sieve tubes might also proceed via the apoplast. In respect to phloem loading it is not possible with intact leaves in the steady state of photosynthesis to determine the gradient between substrate concentrations in the apoplastic space and the sieve tube compartment, as there are no means for a reliable determination of the apoplastic concentrations in these leaves. But it is possible to assay in intact leaves the cytosolic metabolite levels. A refinement of the nonaqueous fractionation technique, described in this paper, allows the determination of metabolite levels in the vacuolar, chloroplastic and cytosolic compartment of spinach leaves in the steady state of photosynthesis. The overall process of metabolite transfer from the leaf cells to the sieve tubes is subjected to the concentration gradient between the cytosol ofthe source leaf cells and the sieve tubes. In the present report these gradients of sucrose and amino acids have been evaluated from the nonaqueous fractionation measurements and the analysis of metabolite concentrations in the phloem sap, collected by excised aphid stylets. These results illustrate the specificity of the processes by which products of photosynthesis are withdrawn from the source cells for export by the phloem system. MATERIALS AND METHODS