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

Abstract

During its development, the leaf undergoes profound metabolic changes to ensure, among other things, its growth. The subcellular metabolome of tomato leaves was studied at four stages of leaf development, with a particular emphasis on the composition of the vacuole, a major actor of cell growth. For this, leaves were collected at different positions of the plant, corresponding to different developmental stages. Coupling cytology approaches to non-aqueous cell fractionation allowed to estimate the subcellular concentrations of major compounds in the leaves. The results showed major changes in the composition of the vacuole across leaf development. Thus, sucrose underwent a strong allocation, being mostly located in the vacuole at the beginning of development and in the cytosol at maturity. Furthermore, these analyses revealed that the vacuole, rather rich in secondary metabolites and sugars in the growth phases, accumulated organic acids thereafter. This result suggests that the maintenance of the osmolarity of the vacuole of mature leaves would largely involve inorganic molecules.

Impaired cell growth under ammonium stress explained by modeling the energy cost of vacuole expansion in tomato leaves

“…Non‐aqueous fractionation has been developed to estimate the subcellular distribution of metabolites, classically among the cytosol, the plastids and the vacuole (Destailleur et al., 2021 ; Krueger et al., 2011 ). When applied to tomato leaf, it was possible to resolve the vacuole and the cytosol in 21‐, 31‐ and 41‐day‐old leaves (Table S1 ), but not in the younger leaves, probably because their cells were too small.…”

“…It raises the question of the subcellular distribution of organic and inorganic compounds and the impact on their cytosolic and vacuolar concentrations that could influence the osmolarity of these two compartments, and thus molecule and water fluxes. Non-aqueous fractionation has been developed to estimate the subcellular distribution of metabolites, classically among the cytosol, the plastids and the vacuole (Destailleur et al, 2021;Krueger et al, 2011). When applied to tomato leaf, it was possible to resolve the vacuole and the cytosol in 21-, 31-and 41-day-old leaves (Table S1), but not in the younger leaves, probably because their cells were too small.…”

Validation of carbon isotopologue distribution measurements by GC-MS and application to13C-metabolic flux analysis of the tricarboxylic acid cycle inBrassica napusleaves

“…Overall, our present findings confirmed that stored Citrate is unlikely to contribute to plant TCA cycle in either light or dark conditions ( Figure 8 ). Since most of the Citrate and Malate pools are found in the vacuole independently of the leaf developmental stage (Szecowka et al, 2013;Destailleur et al, 2021), their remobilization may be expected only under stress conditions.…”

“…Nevertheless, these results were then partly supported by a flux balance analysis using a diel model which explained this stored citrate contribution by the need to support continued export of sugar and amino acids from the leaf during the night and to meet cellular maintenance costs (Cheung et al, 2014). Recently, this concept has been reinforced by the subcellular distribution of Citrate in plant leaves, which is essentially present in the vacuoles (Destailleur et al, 2021). However, previous NMR works used detached leaves to propose the contribution of the stored citrate, which is an experimental setup that necessarily stops sink/source relationships (sugar and amino acids exports) (Gauthier et al, 2010).…”

“…Thus, its reassimilation will also introduce this 50% isotopic dilution to mitochondrial active pools of either Succinate or 2-Oxoglutarate. For instance, it is not possible to have access to the size of the mitochondrial active pool for both metabolites and by extension their exact 13 C-enrichments without a complete isotopic steady-state of the plant TCA cycle and perhaps subcellular fractionation or very fast mitochondrial purification by centrifugation (Lee et al, 2019;Destailleur et al, 2021). To which extent Glutamate and Aspartate molecules escape the TCA cycle remains to be determined.…”