Elucidation of the interactome of the sucrose transporter StSUT4: sucrose transport is connected to ethylene and calcium signalling.

Sucrose transporters of the SUT4 clade show dual targeting to both the plasma membrane as well as to the vacuole. Previous investigations revealed a role for the potato sucrose transporter StSUT4 in flowering, tuberization, shade avoidance response, and ethylene production. Down-regulation of StSUT4 expression leads to early flowering, tuberization under long days, far-red light insensitivity, and reduced diurnal ethylene production. Sucrose export from leaves was increased and a phase-shift of soluble sugar accumulation in source leaves was observed, arguing for StSUT4 to be involved in the entrainment of the circadian clock. Here, we show that StSUT4, whose transcripts are highly unstable and tightly controlled at the post-transcriptional level, connects components of the ethylene and calcium signalling pathway. Elucidation of the StSUT4 interactome using the split ubiquitin system helped to prove direct physical interaction between the sucrose transporter and the ethylene receptor ETR2, as well as with the calcium binding potato calmodulin-1 (PCM1) protein, and a calcium-load activated calcium channel. The impact of calcium ions on transport activity and dual targeting of the transporter was investigated in detail. For this purpose, a reliable esculin-based transport assay was established for SUT4-like transporters. Site-directed mutagenesis helped to identify a diacidic motif within the seventh transmembrane spanning domain that is essential for sucrose transport activity and targeting, but not required for calcium-dependent inhibition. A link between sucrose, calcium and ethylene signalling has been previously postulated with respect to pollen tube growth, shade avoidance response, or entrainment of the circadian clock. Here, we provide experimental evidence for the direct interconnection of these signalling pathways at the molecular level by direct physical interaction of the main players.

Subcellular dynamics and protein-protein interactions of plant sucrose transporters.

Although extensively studied for their role in long distance transport, plant sucrose transporters are active not only in the phloem but throughout the plant body. Sucrose transporters of the SUT family were first described to be plasma membrane-resident proteins, but recent investigations revealed that subcellular dynamics of these transporters were part of complex regulatory mechanisms. The yeast two-hybrid split-ubiquitin system, tandem-affinity purification, and bimolecular-fluorescence complementation aided in identification of a complex network of SUT-interacting proteins that led to answers to many open questions. We found, for example, interacting proteins localized to other subcellular compartments. Although sucrose transporters were assumed to be localized mainly on the plasma membrane, and the tonoplast in the case of SUT4, the interaction partners were not exclusively predicted to be plasma membrane proteins, but belonged to the extracellular space (cell wall), intracellular vesicles, the ER, tonoplast, nuclei, and peroxisomes, among other cellular compartments. A subset of the SUT-interacting proteins localized exclusively to plasmodesmata. We conclude that (transient) protein-protein interactions of integral membrane proteins help to sequester SUTs to subcellular compartments, such as membrane microdomains, with specific functions to enable subcellular transport and cell-to-cell trafficking via plasmodesmata. Identification of SNARE proteins (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) and protein disulfide isomerases support the assumption that the protein-protein interaction plays an important role for the subcellular movement of sugar transporters. It becomes apparent that the interaction partners provide a substantial impact on how and where the transporter is localized or processed for either targeting to a specific cellular or extracellular location, or tagging for degradation or recycling. In this review, interacting proteins, as well as the role of oligomeric complex formation, post-translational modification, and stress responses are summarized for SUTs of higher plants.

Expression Level of Mature miR172 in Wild Type and StSUT4-Silenced Plants of Solanum tuberosum Is Sucrose-Dependent.

In potato plants, the phloem-mobile miR172 is involved in the sugar-dependent transmission of flower and tuber inducing signal transduction pathways and a clear link between solute transport and the induction of flowering and tuberization was demonstrated. The sucrose transporter StSUT4 seems to play an important role in the photoperiod-dependent triggering of both developmental processes, flowering and tuberization, and the phenotype of StSUT4-inhibited potato plants is reminiscent to miR172 overexpressing plants. The first aim of this study was the determination of the level of miR172 in sink and source leaves of StSUT4-silenced as well as StSUT4-overexpressing plants in comparison to Solanum tuberosum ssp. Andigena wild type plants. The second aim was to investigate the effect of sugars on the level of miRNA172 in whole cut leaves, as well as in whole in vitro plantlets that were supplemented with exogenous sugars. Experiments clearly show a sucrose-dependent induction of the level of mature miR172 in short time as well as long time experiments. A sucrose-dependent accumulation of miR172 was also measured in mature leaves of StSUT4-silenced plants where sucrose export is delayed and sucrose accumulates at the end of the light period.

What determines the composition of the phloem sap? Is there any selectivity filter for macromolecules entering the phloem sieve elements?

In view of recent findings, it is still a matter of debate whether the composition of the phloem sap of higher plants is specific and based on a plasmodesmal selectivity filter for macromolecular transport, or whether simply related to size, abundance and half-life of the macromolecules within the phloem sap. A range of reports indicates specific function of phloem-mobile signaling molecules such as the florigen making it indispensable to discriminate specific macromolecules entering the phloem from others which cannot cross this selectivity filter. Nevertheless, several findings have discussed for a non-selective transport via plasmodesmata, or contamination of the phloem sap by degradation products coming from immature still developing young sieve elements undergoing differentiation. Here, we discuss several possibilities, and raise the question how selectivity of the phloem sap composition could be achieved thereby focusing on mobility and dynamics of sucrose transporter mRNA and proteins.

Subcellular Targeting of Plant Sucrose Transporters Is Affected by Their Oligomeric State.

Post-translational regulation of sucrose transporters represents one possibility to adapt transporter activity in a very short time frame. This can occur either via phosphorylation/dephosphorylation, oligomerization, protein-protein interactions, endocytosis/exocytosis, or degradation. It is also known that StSUT1 can change its compartmentalization at the plasma membrane and concentrate in membrane microdomains in response to changing redox conditions. A systematic screen for protein-protein-interactions of plant sucrose transporters revealed that the interactome of all three known sucrose transporters from the Solanaceous species Solanum tuberosum and Solanum lycopersicum represents a specific subset of interaction partners, suggesting different functions for the three different sucrose transporters. Here, we focus on factors that affect the subcellular distribution of the transporters. It was already known that sucrose transporters are able to form homo- as well as heterodimers. Here, we reveal the consequences of homo- and heterodimer formation and the fact that the responses of individual sucrose transporters will respond differently. Sucrose transporter SlSUT2 is mainly found in intracellular vesicles and several of its interaction partners are involved in vesicle traffic and subcellular targeting. The impact of interaction partners such as SNARE/VAMP proteins on the localization of SlSUT2 protein will be investigated, as well as the impact of inhibitors, excess of substrate, or divalent cations which are known to inhibit SUT1-mediated sucrose transport in yeast cells. Thereby we are able to identify factors regulating sucrose transporter activity via a change of their subcellular distribution.

Tomato geminivirus encoded RNAi suppressor protein, AC4 interacts with host AGO4 and precludes viral DNA methylation.

Plant RNA silencing systems are organized as a network, regulating plant developmental pathways and restraining invading viruses, by sharing cellular components with overlapping functions. Host regulatory networks operate either at the transcriptional level via RNA-directed DNA methylation, or at the post-transcriptional stage interfering with mRNA to restrict viral infection. However, viral-derived proteins, including suppressors of RNA silencing, favour virus establishment, and also affect plant developmental processes. In this investigation, we report that Tomato leaf curl New Delhi virus-derived AC4 protein suppresses RNA silencing activity and mutational analysis of AC4 showed that Asn-50 in the SKNT-51 motif, in the C-terminal region, is a critical determinant of its RNA silencing suppressor activity. AC4 showed interaction with host AGO4 but not with AGO1, aggregated around the nucleus, and influenced cytosine methylation of the viral genome. The possible molecular mechanism by which AC4 interferes in the RNA silencing network, helps virus establishment, and affects plant development is discussed.

A study on the influence of different promoter and 5'UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene ß glucuronidase in tobacco and cotton.

Several reports of promoters from plants, viral and artificial origin that confer high constitutive expression are known. Among these the CaMV 35S promoter is used extensively for transgene expression in plants. We identified candidate promoters from Arabidopsis based on their transcript levels (meta-analysis of available microarray control datasets) to test their activity in comparison to the CaMV 35S promoter. A set of 11 candidate genes were identified which showed high transcript levels in the aerial tissue (i.e. leaf, shoot, flower and stem). In the initial part of the study binary vectors were developed wherein the promoter and 5'UTR region of these candidate genes (Upstream Regulatory Module, URM) were cloned upstream to the reporter gene ß glucuronidase (gus). The promoter strengths were tested in transformed callus of Nicotiana tabacum and Gossypium hirsutum. On the basis of the results obtained from the callus, the influence of the URM cassettes on transgene expression was tested in transgenic tobacco. The URM regions of the genes encoding a subunit of photosystem I (PHOTO) and geranyl geranyl reductase (GGR) in A. thaliana genome showed significantly high levels of GUS activity in comparison to the CaMV 35S promoter. Further, when the 5'UTRs of both the genes were placed downstream to the CaMV 35S promoter it led to a substantial increase in GUS activity in transgenic tobacco lines and cotton callus. The enhancement observed was even higher to that observed with the viral leader sequences like Ω and AMV, known translational enhancers. Our results indicate that the two URM cassettes or the 5'UTR regions of PHOTO and GGR when placed downstream to the CaMV 35S promoter can be used to drive high levels of transgene expression in dicotyledons.