Introduction 2 1991 1992 1998 1975 1982 1975 1982 1983 1983 1975 1989 1997 Malus Pyrus Prunus 1975 1982 1997 1981 1996 1997 1997 1975 1983 1983 P. persica A. graveolens P. major P. maritima Material and methods Materials A. graveolens dulce P. major P. maritima P. persica Non-aqueous fractionation of leaf tissue −1 g −1 −1 −1 −1 g Extraction of sugars and sugar alcohols from non-aqueous fractions 1991 Extraction of sugars and sugar alcohols from non-aqueous fractions Chloroform:methanol extracts were prepared to extract sugars and sugar alcohols from samples after non-aqueous fraction. The aliquots (see above) were dried and 5 ml chloroform:methanol (3:7, v/v) was added to the powder. The samples were homogenized until completely thawed and kept on ice for 30 min. The homogenate was then extracted twice with 3 ml water. The aqueous phases were combined and evaporated in a rotary evaporator. The dried residue was dissolved in 0.7 ml ultrapure water (Millipore), syringe-filtrated (0.45 μm cellulose-acetate; Schleicher and Schuell, Dassel, Germany) and stored at −80°C. Collection of sieve tube sap Myzus persicae 1995 Extraction of apoplastic washing fluids from leaves g V gas V water V water V gas  V water 2001 1997 −1 3 −1 3 2001 Metabolite analysis −1 Electron microscopy and determination of subcellular volumes 2 2 4 6 1847 1993 2001 http://rsb.info.nih.gov/nih-image/ The relative volumes (percent of total) were converted to absolute volumes per unit mass by taking into account the water content per mass fresh weight. Results Subcellular distributions of sugars and sugar alcohols in leaf cells 1991 2001 1 1 1 1 1 1 Fig. 1 Percentage distribution of sugars and sugar alcohols among the vacuolar, stromal and cytosolic compartments of leaf cells from a common plantain, b sea plantain, c peach, and d celery. Data represent mean values ± SD from five to six independent fractionations  Subcellular volumes 1993 1994 1995 1 1993 1994 1993 1994 1993 1994 2 2 Table 1 Relative volumes (%) of the subcellular compartments within the total volume of mesophyll cells from common plantain, sea plantain, peach and celery  Vacuole % Chloroplast % Cytoplasm % Common plantain 82 ± 5 12 ± 4 6 ± 1 Sea plantain 85 ± 5 10 ± 4 5 ± 2 Peach 68 ± 7 21 ± 6 11 ± 4 Celery 72 ± 5 16 ± 4 12 ± 3 n Table 2 n n  Common plantain Sea plantain Peach Celery −1 147 ± 9 124 ± 15 336 ± 30 140 ± 8 −1 853 ± 9 876 ± 15 664 ± 30 860 ± 8 −1 226 ± 48 222 ± 21 190 ± 44 213 ± 33 −1 701 743 452 860 −1 50 43 70 68 −1 42 38 58 82 Mean values ± SD Subcellular sugar and sugar alcohol concentrations 3 3 1 3 Table 3 Whole leaf contents as well as sugar and sugar alcohol concentrations in the vacuolar, stromal and cytosolic compartments of leaf cells from common plantain, sea plantain, peach, and celery  −1 Concentration (mM) Vacuole Stroma  Cytosol Common plantain  Sorbitol 22 ± 3.1 15 ± 2.3 118 ± 34 133 ± 48  myo-Inositol 0.7 ± 0.2 0.1 ± 0.1 8.8 ± 1.3 6.1 ± 0.7  Glucose and fructose 2.4 ± 1.1 3.2 ± 0.2 1.4 ± 2.0 0.6 ± 0.7  Sucrose 1.2 ± 0.1 0.9 ± 0.2 0.1 ± 0.2 12 ± 2.5 Sea plantain  Sorbitol 73 ± 9.1 87 ± 8.0 201 ± 140 13 ± 8.4  myo-Inositol 0.8 ± 0.2 0.2 ± 0.1 15 ± 0.4 0 ± 0  Glucose and fructose 11 ± 2.5 15 ± 0.5 0.2 ± 0.5 0 ± 0  Sucrose 1.4 ± 0.2 0.8 ± 0.3 0.1 ± 0.1 20 ± 4.9 Peach  Sorbitol 148 ± 0.1 220 ± 15 461 ± 165 290 ± 62  myo-Inositol 1.5 ± 0.1 1.5 ± 0.4 8.5 ± 3.6 4.1 ± 3.3  Glucose and fructose 30 ± 6.1 65 ± 1 5.0 ± 5.0 5.0 ± 5.0  Sucrose 38 ± 2.1 64 ± 4.5 39 ± 26 106 ± 15 Celery  Mannitol 108 ± 121 27 ± 7.0 294 ± 30 100 ± 39  myo-Inositol 1.4 ± 0.1 1.1 ± 0.3 9.0 ± 1.9 1.7 ± 0.9  Glucose and fructose 17 ± 2.0 28 ± 0.3 2.5 ± 1.7 2.9 ± 1.2  Sucrose 35 ± 4.3 45 ± 3.3 5.2 ± 3.2 86 ± 28 Mean values ± SD 4 3 Table 4 Carbohydrate concentrations in the cytosol of mesophyll cells of leaves as well as in the apoplast and in the phloem sap from common plantain, sea plantain, peach and celery  Concentration (mM)  Ratio Ratio Cytosol Apoplast  Phloem sap Phl/apo Phl/cyt Common plantain  Sorbitol 133 ± 48 5.5 ± 1.2 422 ± 129 77 3.2  Sucrose 12 ± 2.5 0.3 ± 0.1 645 ± 225 2,150 40  Sorbitol/sucrose 11 18 0.7   Sea plantain  Sorbitol 13 ± 8.4 7.9 ± 2.5 315 ± 105 40 24  Sucrose 20 ± 4.9 0.3 ± 0.1 355 ± 112 1,183 18  Sorbitol/sucrose 0.7 26 0.9   Peach  Sorbitol 290 ± 62 24.4 ± 9.5 582 ± 90 24 2.0  Sucrose 106 ± 16 1.2 ± 0.6 207 ± 51 173 2.0  Sorbitol/sucrose 2.7 20 2.8   Celery  Mannitol 100 ± 39 6.7 ± 3.3 732 ± 131 109 7.3  Sucrose 86 ± 28 1.2 ± 0.9 389 ± 84 324 4.5  Mannitol/sucrose 1.2 5.6 1.9   n Of the compartments analyzed the highest concentration of glucose and fructose was consistently found in the vacuole. As the evaluation of the subcellular fraction was performed in increments of 1%, glucose and fructose concentrations found in the cytosol or stroma represent an upper limit of 1% for these compounds. 3 Apoplastic and phloem sap concentrations 4 4 4 Discussion 1984 2006 Non-aqueous fractionation of leaf tissue Sorbitol and mannitol concentrations are high in stroma and cytosol despite of a large vacuolar polyol depot 1 3 1981 1996 3 1993 1998 2006 3 1997 1979 −1 −1 3 1996 2007 1 A. barclaiana 2006 1997 1 3 1983 1998 1989 A large pool of myo-inositol is located in the stroma 1 2006 1997 3 1987 Mesembryanthemum crystallinum 1996 3 The subcellular distribution of hexoses and sucrose is similar in plant leaves with and without sugar alcohols 1991 1992 1 3 1987 1994 2000 1 3 1991 1992 3 2006 Arabidopsis Concentration ratios between the cytosol of mesophyll cells and the phloem were similar for sucrose and sugar alcohols 4 1997 1986 4 4 Concentration ratios between the apoplast and the phloem for sucrose and sugar alcohols with relation to the phloem loading mode 1989 4 1994 2003 2004 P. major 1995 2004  K m 1994  K m 2004 4 1997 1989 Prunus 4 1997 1982 4 1995 2000 Alonsoa meridionalis 2001 1998 p 1992 Prunus cerasus 2003 Malus domestica 2004 2004  K m 4 1997 1997 2003 2004 2004 Unfortunately, the type of phloem loading in peach is still an open question; the available data are consistent with either an apoplastic or a symplastic mode of loading or, more likely, a combination of both. 4 1995 2000 2001  K m 2001 4 The comparison of common plantain, sea plantain, peach, and celery indicates that different modes of phloem loading are employed by different plant families, which may in part be related to the respective ecophysiological requirements.