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.
Highlights
The complex organization of higher plants results in the coexistence of autotrophic tissues that fix CO2 via photosynthesis and heterotrophic tissues that rely on the supply with organic carbon like for example roots, young leaves, meristems, and reproductive organs
RT-PCR analyses of genes for sucrose transporters expressed in pollen tubes indicated that both, AtSUC6 and AtSUC7 are strongly expressed in pollen tubes grown in vitro or semi-in vivo, whereas no transcripts could be amplified from stigmata-derived cDNA (Figure 1)
2http://gimp.org of the full-length AtSUC7Col−0 coding sequence derived from pollen tube mRNA verified the predicted exon/intron structure as annotated in the TAIR10 genome
Summary
The complex organization of higher plants results in the coexistence of autotrophic tissues that fix CO2 via photosynthesis (source tissues) and heterotrophic tissues that rely on the supply with organic carbon (sink tissues) like for example roots, young leaves, meristems, and reproductive organs. The transport of fixed carbon from source to sink tissues occurs in the sieve elements of the phloem. Depending on plant species there are differences in the mode of phloem loading and the chemical structure in which carbon is transported. In plants like Arabidopsis, where sucrose represents the main transport sugar and is loaded into the phloem via an apoplastic route (Gamalei, 1989; Haritatos et al, 2000), sugar transporters are involved in at least four steps of carbon distribution: (i) release of sucrose from the mesophyll cells to the apoplast, (ii) uptake of sucrose from the apoplast into the sieve element-companion cell complex, (iii) release of sucrose into the apoplast towards symplastically isolated sink-tissues like for example pollen or embryos, and (iv) re-uptake of sucrose into these tissues (Lemoine, 2000). SUCs are involved in phosphate starvation responses (Lei et al, 2011), sugar signaling (Sivitz et al, 2008; Li et al, 2012) as well as interactions with symbionts (Doidy et al, 2012) and parasites (Juergensen et al, 2003; Hammes et al, 2005; Hofmann et al, 2009; Péron et al, 2017)
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