Identification of a Vacuolar Sucrose Transporter in Barley and Arabidopsis Mesophyll Cells by a Tonoplast Proteomic Approach

Abstract

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H 1 -ATPases and H 1 -pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.In mature plant cells, the central vacuole occupies 80% to 90% of the cell volume. Vacuoles contain a large number of hydrolytic and biosynthetic enzymes, inorganic ions, soluble carbohydrates, organic acids, amino acids, secondary compounds, and modified xenobiotics (Maeshima, 2001;Martinoia et al., 2002). Based on the potential toxicity of many of these compounds, Matile (1984) suggested that the distance between life and death is 7.5 nm, the thickness of the vacuolar membrane. Plants have only a limited capacity to excrete potentially toxic compounds; therefore, the term internal excretion has also been used (Martinoia et al., 1993) to indicate that, for some classes of compounds, the vacuolar membrane mimics the function and contains homolog transporters of the liver plasma membrane (Kreuz et al., 1996). However, the function of the vacuole is not restricted to the storage of potentially toxic compounds. For optimal function of the metabolic pathways, the concentration of metabolites and ions has to be tightly regulated in the cytoplasm. Metabolites produced in excess are transported into the vacuole, which serves as a temporary storage pool, and released to the cytoplasm when required for metabolism.Increasing evidence shows that impaired vacuolar deposition or retrieval affects plant metabolism. Catala et al. (2003) showed that the vacuolar calcium-proton exchanger CAX1 is induced during cold treatment and is invo...

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

“…Several sucrose transporters (HbSUT1A, HbSUT2A and HbSUT3) actively translocate sucrose from the apoplast into the laticifers (Dusotoit‐Coucaud et al ., 2009, 2010; Tang et al ., 2010). Additionally, the vacuolar release of sucrose via SUC4‐type transporters through the tonoplast was confirmed by the characterization of AtSUC4 in Arabidopsis thaliana roots (Endler et al ., 2006; Sauer, 2007), whereas the vacuolar export of fructose is mediated by the fructose‐specific uniporter SWEET17 located on the tonoplast (Guo et al ., 2014). In T. koksaghyz , similar transporters may enable the transport of fructose and sucrose generated by the degradation of inulin, moving them out of the vacuole, through the apoplast and into the laticifers.…”

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

“…Tonoplast vesicles were isolated by sonication of purified vacuoles and subsequent ultracentrifugation for 1 h at 100,000 g [21]. The supernatant was removed and the tonoplast pellet was resuspended in 20 mM HEPES-KOH, pH 7.2.…”

“…In an earlier proteomic study [21], a mechanical lysis method was used to isolate highly purified vacuoles from barley mesophyll protoplasts. As this method was not suitable for Cd 2+ -stressed plants, therefore, we established a protocol based on osmotic lysis (for details see experimental procedures).…”

“…Although this is not the case for barley, the use of the NCBI Liliopsida database which contains the protein sequences of the closest sequenced barley relative, rice (Oryza sativa) and many other monocotyledonous plants, led to the identification of a large number of barley proteins in a tonoplast proteomics approach [21]. In this study, the identification rate was further increased by including additional 12,000 protein sequences derived from a translated barley EST database (for details see experimental procedure).…”

“…Such studies have also been performed with isolated vacuoles [20][21][22][23] and allowed the identification of new vacuolar membrane proteins. However, these studies do not give any information about changes of the vacuolar membrane proteome occurring in response to changes in environmental conditions.…”