Non-aqueous fractionation revealed changing subcellular metabolite distribution during apple fruit development

Abstract

In developing apple fruit, metabolic compartmentation is poorly understood due to the lack of experimental data. Distinguishing subcellular compartments in fruit using non-aqueous fractionation has been technically difficult due to the excess amount of sugars present in the different subcellular compartments limiting the resolution of the technique. The work described in this study represents the first attempt to apply non-aqueous fractionation to developing apple fruit, covering the major events occurring during fruit development (cell division, cell expansion, and maturation). Here we describe the non-aqueous fractionation method to study the subcellular compartmentation of metabolites during apple fruit development considering three main cellular compartments (cytosol, plastids, and vacuole). Evidence is presented that most of the sugars and organic acids were predominantly located in the vacuole, whereas some of the amino acids were distributed between the cytosol and the vacuole. The results showed a shift in the plastid marker from the lightest fractions in the early growth stage to the dense fractions in the later fruit growth stages. This implies that the accumulation of starch content with progressing fruit development substantially influenced the distribution of plastidial fragments within the non-aqueous density gradient applied. Results from this study provide substantial baseline information on assessing the subcellular compartmentation of metabolites in apple fruit in general and during fruit growth in particular.

Photosynthetic activity triggers pH and NAD redox signatures across different plant cell compartments

“…For instance, it has remained difficult to estimate the dynamic impact of photosynthetic activity on the central bioenergetic cofactor pools, such as those of stromal ATP and NAD(P). Since whole-cell extracts cannot provide any organelle-specific dynamics, more specialized approaches have been adopted, such as fast organelle fractionation from protoplasts or non-aqueous fractionation (Beshir et al, 2019; Fürtauer et al, 2016; Gardeström and Wigge, 1988; Gerhardt and Heldt, 1984; Igamberdiev and Gardeström, 2003; Lilley et al, 1982; Medeiros et al, 2019; Stitt et al, 1982). As a result important insights into the mitochondrial, chloroplastic and cytosolic adenylate pools in darkness and light could be obtained (Gardeström and Igamberdiev, 2016).…”

The Evolution of Leaf Function during Development Is Reflected in Profound Changes in the Metabolic Composition of the Vacuole

“…Moreover, measurements of the plastid marker NADP-GAPDH were clearly biased in fractions from the youngest leaves. Such difficulty has been reported for apple fruit fractionation [27] in which NADP-GAPDH was not detectable and was consequently replaced by starch as a plastid marker. In Arabidopsis leaf, metabolites have also been used as additional markers [18], such as starch and digalactosyldiacylglycerol for the chloroplast and nitrate or flavonoids for the vacuole.…”

“…Associated with isotope labeling, NAF allowed the estimation of fluxes in Calvin-Benson-Bassham (CBB) cycle reactions in Arabidopsis leaves [26]. More recently, it has been used to characterize changes in the distribution of metabolites and inorganic ions in developing apple fruit [27].…”

Non-structural Carbohydrate Metabolism in the Flesh of Stone Fruits of the Genus Prunus (Rosaceae) – A Review

“…However, because of the large proportion of the cell occupied by the vacuole the concentrations of sorbitol and sucrose were higher in the cytosol, while the concentrations of glucose and fructose were much higher in the vacuole (Nadwodnik and Lohaus, 2008). Beshir et al (2019) used the non-aqueous fractionation technique to determine the distribution of metabolites between different subcellular compartments during the development of apple flesh. The bulk of the contents of sucrose, glucose, fructose and sorbitol were located in the vacuole throughout development.…”

“…These sugars were often also present in the cytosol and plastid, and this was dependent on both the sugar and stage of development. However, because of the large volume of the vacuole the actual concentration of some of these sugars could at certain stages of development be higher in the cytosol and plastid than in the vacuole (Beshir et al, 2019). It is plausible that in the flesh of stone fruits the subcellular distribution of sugars is comparable to apple flesh; however, it requires to be determined experimentally.…”