Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol, vacuole or apoplasm

Abstract

Abstract. The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97-98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells. ), tomato (Lycopersicon esculentum, Dickinson et al. 1991) and potato (Solanum tuberosum, Heineke et al. 1992). Expression of the yeast invertase led to reduced growth in all three species. Biochemical analyses of the transformed tobacco and potato plants showed an accumulation of sucrose and hexoses in the leaves.In order to determine in which compartments the sucrose and hexoses deriving from sucrose hydrolysis by the yeast invertase accumulate, and how the amount and distribution of the amino acids are affected, we studied the subcellular distributions of hexoses, sucrose and amino acids in tobacco transformants where yeast invertase is expressed in either the apoplasmic, the cytosolic or vacuolar compartment and, as a control, also in wildtype (WT) plants. It will be shown that the hexoses contained in the WT, as well as those in the transgenic plants, accumulate almost entirely in the vacuoles, indicating that vacuoles of tobacco leaves are able to take up hexoses against a large concentration gradient.

Phloem Loading Strategies and Water Relations in Trees and Herbaceous Plants

“…However, this reasoning neglects the effects of subcellular compartmentation in mesophyll cells. It has been consistently shown, in nonaqueous fractionation studies of leaves, that the concentration of Suc in the cytosol is much higher than in the vacuoles and can be more than 10 times higher than bulk leaf Suc concentration (Winter et al, 1993(Winter et al, , 1994Heineke et al, 1994;Nadwodnik and Lohaus, 2008). Therefore, with 60 to 110 mM bulk leaf Suc, the concentration in the cytosol of mesophyll cells could be very high.…”

“…4). The reason for this correlation could be that the concentration of Suc is higher in the cytosol than in the vacuoles of leaf cells (Winter et al, 1993(Winter et al, , 1994Heineke et al, 1994;Voitsekhovskaja et al, 2006), which requires that other solutes accumulate in the vacuoles to balance the water potential in the two compartments. To explore these relationships, we plotted concentrations of other polar metabolites against transport sugars (Fig.…”

A comprehensive picture of phloem loading strategies

“…Note that the osmolality of the leaf sap in L. salicaria is very high (723 mM), consistent with a strong asymmetry toward sucrose in the cytosol. Given the same subcellular fractional volumes as Nicotiana tabacum (22), the sucrose concentration in the cytosol of L. salicaria mesophyll cells could be as high as 476 mM.…”

Cytosolic expression of the Bacillus amyloliquefaciens SacB protein inhibits tissue development in transgenic tobacco and potato

“…Hydrolysis of sucrose led to accumulation of hexose and, curiously, an increase in sucrose concentration. The proposed cause of leaf damage in transgenic lines containing the yeast invertase gene was the dedifferentiation of cells due to accumulation of photoassimilate (Heineke et al, 1994).…”

“…The two largest pools of sucrose in a plant cell are located in the cytosol, where sucrose is synthesized, and the vacuole, where sucrose might be transiently stored (Heineke et al, 1994). The original model, proposed to explain fructan metabolism in higher plants (Edelman & Jefford, 1968), suggested that the cytosolic sucrose pool was utilized in the synthesis of kestose by the enzyme SST.…”