Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype

Abstract

The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in Arabidopsis thaliana. Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that AtSUC6Col-0 is a high-affinity H+-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of AtSUC6Col-0 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of AtSUC6 did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7Col-0 is regulated by intragenic regions. Transport activity of AtSUC7Col-0 could not be analyzed in baker’s yeast or Xenopus oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters in planta was developed. Plasma membrane localized SUCs including AtSUC6Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7Col-0 might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an in planta expression system.

Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth

“…For instance, a questionable annotation could lead to erroneous hypothesis in the case of the ARR8 encoded gene (AT2G41310) shown in Figure 10. Another example is the case of AtSUC7, initially considered as a pseudogene (88), and recently shown to be translated in heterologous systems with functional restrictions on sucrose analogue transport (89). Here we show, for the first time, that AtSUC7 is transcribed and translated in germinated pollen and it suffers a dramatic down regulation at both transcriptional and translational levels upon heat stress ( Table 3).…”

A novel sucrose transporter gene IbSUT4 involves in plant growth and response to abiotic stress through the ABF-dependent ABA signaling pathway in Sweetpotato

“…A previous study showed that AtSUC6 was a pseudogenes coding for non-functional proteins [39]. More recent work has shown that AtSUC6 is a high-affinity H +_ symporter mediating sucrose uptake [40]. The evolutionary distances between plants and yeast mean that the plant proteins may not target the correct membrane in yeast [41,42], or that they lack the essential protein modifications and do not show normal activity in a heterologous system.…”

Anthocyanin in the Vacuole of Red Onion Epidermal Cells Quenches Other Fluorescent Molecules

“…As vacuolar and cytoplasmic physiology appeared to be similar in anthocyanic and non-anthocyanic cells, it is assumed that direct quenching of the fluorescence of the exogenous dye by anthocyanin is happening. This is because vacuolar anthocyanin interfered with the fluorescence of the vacuole-targeted dyes loaded through the different pathways including acid-dependent loading (acridine orange) [27], probenecid-sensitive anion transporters (carboxyfluorescein, fluorescein and monochlorobimane) [23][24][25][26] and sugar transporters (esculin) [28]. With regards to the sugar transporter-dependent uptake of esculin, this has been described in the literature as being limited in monocots [28], although the initial analysis that showed this, only grass species were tested [37].…”

“…Acridine orange loads into the vacuole as a weak acid [24], and also showed a similar reduction of fluorescence in anthocyanic cells (Figure 2g). Similarly, esculin, a fluorescent sucrose analogue that is a coumarin glucoside and that is pumped across the plasma membrane and then into the vacuole by sucrose transporters [28] also loaded into the vacuole of onion epidermal cells where it too showed less fluorescence in anthocyanic cells ( Figure 2j).…”