The sucrose carrier AtSUC2 of Arabidopsis thaliana is localized in the phloem, where it catalyzes the uptake of sucrose from the apoplast into companion cells. Imported sucrose moves passively via plasmodesmata from the companion cells into the neighboring sieve elements that distribute this disaccharide to the different sink organs. Phloem loading of sucrose by the AtSUC2 protein is an essential process, and mutants lacking this protein stay tiny, develop no or only few flowers, and have a strongly reduced root system. The promoter of the AtSUC2 gene is active exclusively in companion cells of the phloem. Moreover, it drives very strong expression not only in Arabidopsis, but also in all plant species tested so far, including monocot species. Due to these features, the AtSUC2 promoter has become an important tool in diverse areas of plant research during the last two decades. It was used to study phloem development and function including phloem loading and unloading. Furthermore, it was helpful in analyzing the pathways of posttranscriptional silencing by RNA interference, the regulation of flowering, mechanisms of nutrient withdrawal by phloem-feeding pathogens, and other physiological functions that are related to long distance transport. The present paper gives an overview of different approaches in plant research that utilized the strong and companion cell-specific expression of own or foreign genes driven by the AtSUC2 promoter.

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.

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell-to-cell transport. Here we report on an elegant non-invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA-s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA-s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA-s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA-s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA-s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA-s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.

The controlled distribution of sugars between assimilate-exporting source tissues and sugar-consuming sink tissues is a key element for plant growth and development. Monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family contribute to the uptake of sugars into sink cells. Here, we report on the characterization of STP7, STP8, and STP12, three previously uncharacterized members of this family in Arabidopsis (Arabidopsis thaliana). Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that STP8 and STP12 catalyze the high-affinity proton-dependent uptake of glucose and also accept galactose and mannose. STP12 additionally transports xylose. STP8 and STP12 are highly expressed in reproductive organs, where their protein products might contribute to sugar uptake into the pollen tube and the embryo sac. stp8.1 and stp12.1 T-DNA insertion lines developed normally, which may point toward functional redundancy with other STPs. In contrast to all other STPs, STP7 does not transport hexoses but is specific for the pentoses l-arabinose and d-xylose. STP7-promoter-reporter gene plants showed an expression of STP7 especially in tissues with high cell wall turnover, indicating that STP7 might contribute to the uptake and recycling of cell wall sugars. Uptake analyses with radioactive l-arabinose revealed that 11 other STPs are able to transport l-arabinose with high affinity. Hence, functional redundancy might explain the missing-mutant phenotype of two stp7 T-DNA insertion lines. Together, these data complete the characterization of the STP family and present the STPs as new l-arabinose transporters for potential biotechnological applications.

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP-fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild-type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N-terminal dileucine-based motif. The NRAMP3 or NRAMP4 N-terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4-dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N-terminal dileucine-based motif as well as AP4.

Pollen tube growth requires a high amount of metabolic energy and precise targeting toward the ovules. Sugars, especially glucose, can serve as nutrients and as signaling molecules. Unexpectedly, in vitro assays revealed an inhibitory effect of glucose on pollen tube elongation, contradicting the hypothesis that monosaccharide uptake is a source of nutrition for growing pollen tubes. Measurements with Förster resonance energy transfer-based nanosensors revealed that glucose is taken up into pollen tubes and that the intracellular concentration is in the low micromolar range. Pollen tubes of stp4-6-8-9-10-11 sextuple knockout plants generated by crossings and CRISPR/Cas9 showed only a weak response to glucose, indicating that glucose uptake into pollen tubes is mediated mainly by these six monosaccharide transporters of the SUGAR TRANSPORT PROTEIN (STP) family. Analyses of HEXOKINASE1 (HXK1) showed a strong expression of this gene in pollen. Together with the glucose insensitivity and altered semi-in vivo growth rate of pollen tubes from hxk1 knockout lines, this strongly suggests that glucose is an important signaling molecule for pollen tubes, is taken up by STPs, and detected by HXK1. Equimolar amounts of fructose abolish the inhibitory effect of glucose indicating that only an excess of glucose is interpreted as a signal. This provides a possible model for the discrimination of signaling and nutritional sugars.

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

The biotrophic fungus Ustilago maydis causes corn smut disease, inducing tumor formation in its host Zea mays. Upon infection, the fungal hyphae invaginate the plasma membrane of infected maize cells, establishing an interface where pathogen and host are separated only by their plasma membranes. At this interface the fungal and maize sucrose transporters, UmSrt1 and ZmSUT1, compete for extracellular sucrose in the corn smut/maize pathosystem. Here we biophysically characterized ZmSUT1 and UmSrt1 in Xenopus oocytes with respect to their voltage-, pH- and substrate-dependence and determined affinities toward protons and sucrose. In contrast to ZmSUT1, UmSrt1 has a high affinity for sucrose and is relatively pH- and voltage-independent. Using these quantitative parameters, we developed a mathematical model to simulate the competition for extracellular sucrose at the contact zone between the fungus and the host plant. This approach revealed that UmSrt1 exploits the apoplastic sucrose resource, which forces the plant transporter into a sucrose export mode providing the fungus with sugar from the phloem. Importantly, the high sucrose concentration in the phloem appeared disadvantageous for the ZmSUT1, preventing sucrose recovery from the apoplastic space in the fungus/plant interface.

The Yang or Met Cycle is a series of reactions catalyzing the recycling of the sulfur (S) compound 5'-methylthioadenosine (MTA) to Met. MTA is produced as a by-product in ethylene, nicotianamine, and polyamine biosynthesis. Whether the Met Cycle preferentially fuels one of these pathways in a S-dependent manner remained unclear so far. We analyzed Arabidopsis (Arabidopsis thaliana) mutants with defects in the Met Cycle enzymes 5-METHYLTHIORIBOSE-1-PHOSPHATE-ISOMERASE1 (MTI1) and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (DEP1) under different S conditions and assayed the contribution of the Met Cycle to the regeneration of S for these pathways. Neither mti1 nor dep1 mutants could recycle MTA but showed S-dependent reproductive failure, which was accompanied by reduced levels of the polyamines putrescine, spermidine, and spermine in mutant inflorescences. Complementation experiments with external application of these three polyamines showed that only the triamine spermine could specifically rescue the S-dependent reproductive defects of the mutant plants. Furthermore, expressing gene-reporter fusions in Arabidopsis showed that MTI1 and DEP1 were mainly expressed in the vasculature of all plant parts. Phloem-specific reconstitution of Met Cycle activity in mti1 and dep1 mutant plants was sufficient to rescue their S-dependent mutant phenotypes. We conclude from these analyses that phloem-specific S recycling during periods of S starvation is essential for the biosynthesis of polyamines required for flowering and seed development.

STP10 is part of a high-affinity monosaccharide uptake system in the plasma membrane of pollen tubes of Arabidopsis. It is down-regulated under high-glucose conditions, possibly through the hexokinase pathway.Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H+ symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10. qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10 T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway.

Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior.

Arsenic contamination of groundwater and soils threatens the health of tens of millions of people worldwide. Understanding the way in which arsenic is taken up by crops such as rice, which serve as a significant source of arsenic in the human diet, is therefore important. Membrane transport proteins that catalyse arsenic uptake by roots, and translocation through the xylem to shoots, have been characterized in a number of plants, including rice. The transporters responsible for loading arsenic from the xylem into the phloem and on into the seeds, however, are yet to be identified. Here, we show that transporters responsible for inositol uptake in the phloem in Arabidopsis also transport arsenic. Transformation of Saccharomyces cerevisiae with AtINT2 or AtINT4 led to increased arsenic accumulation and increased sensitivity to arsenite. Expression of AtINT2 in Xenopus laevis oocytes also induced arsenite import. Disruption of AtINT2 or AtINT4 in Arabidopsis thaliana led to a reduction in phloem, silique and seed arsenic concentrations in plants fed with arsenite through the roots, relative to wild-type plants. These plants also exhibited a large drop in silique and seed arsenic concentrations when fed with arsenite through the leaves. We conclude that in Arabidopsis, inositol transporters are responsible for arsenite loading into the phloem, the key source of arsenic in seeds.

Sugar beet provides around one third of the sugar consumed worldwide and serves as a significant source of bioenergy in the form of ethanol. Sucrose accounts for up to 18% of plant fresh weight in sugar beet. Most of the sucrose is concentrated in the taproot, where it accumulates in the vacuoles. Despite 30 years of intensive research, the transporter that facilitates taproot sucrose accumulation has escaped identification. Here, we combine proteomic analyses of the taproot vacuolar membrane, the tonoplast, with electrophysiological analyses to show that the transporter BvTST2.1 is responsible for vacuolar sucrose uptake in sugar beet taproots. We show that BvTST2.1 is a sucrose-specific transporter, and present evidence to suggest that it operates as a proton antiporter, coupling the import of sucrose into the vacuole to the export of protons. BvTST2.1 exhibits a high amino acid sequence similarity to members of the tonoplast monosaccharide transporter family in Arabidopsis, prompting us to rename this group of proteins 'tonoplast sugar transporters'. The identification of BvTST2.1 could help to increase sugar yields from sugar beet and other sugar-storing plants in future breeding programs.

•The plant vacuole is the major site for sugar storage.•The activities of vacuolar sugar porters determine plant yield and stress tolerance.•The vacuole is energized by two proton pumps, the V-ATPase and the tonoplast pyrophosphatase. However, in some species a plasma membrane P3A/B type ATPase contributes in addition to the proton-motive force.•Vacuolar sugar transporters helped to identify target mechanisms allowing to direct membrane proteins to the tonoplast.

L'invention concerne des proteines tonoplastiques antiporteurs sucre-protons, en particulier des proteines tonoplastiques antiporteurs saccharose-protons, les sequences nucleotidiques les codant, et leurs utilisations pour produire des vegetaux transgeniques a concentration accrue de saccharose. L'invention concerne egalement un procede de production de vegetaux transgeniques a concentration accrue de saccharose, un procede pour augmenter la concentration de saccharose chez les vegetaux, et pour identifier des vegetaux qui sont appropries pour generer une concentration accrue de saccharose.

The smut Ustilago maydis, a ubiquitous pest of corn, is highly adapted to its host to parasitize on its organic carbon sources. We have identified a hexose transporter, Hxt1, as important for fungal development during both the saprophytic and the pathogenic stage of the fungus. Hxt1 was characterized as a high-affinity transporter for glucose, fructose, and mannose; ∆hxt1 strains show significantly reduced growth on these substrates, setting Hxt1 as the main hexose transporter during saprophytic growth. After plant infection, ∆hxt1 strains show decreased symptom development. However, expression of a Hxt1 protein with a mutation leading to constitutively active signaling in the yeast glucose sensors Snf3p and Rgt2p results in completely apathogenic strains. Fungal development is stalled immediately after plant penetration, implying a dual function of Hxt1 as transporter and sensor. As glucose sensors are only known for yeasts, 'transceptor' as Hxt1 may constitute a general mechanism for sensing of glucose in fungi. In U. maydis, Hxt1 links a nutrient-dependent environmental signal to the developmental program during pathogenic development.

Plant development requires accurate coordination of gene expression, both in actively dividing meristematic cells and differentiated cells. Cell fate establishment and maintenance, among others, are mediated by chromatin organization complexes that determine the stable transcriptional states of specific cell types. Here, we focus on MAIN-LIKE1 (MAIL1), one of three homologs of MAINTENANCE OF MERISTEMS (MAIN), which form a plant-specific gene family in Arabidopsis thaliana. We show that MAIL1 encodes a ubiquitously expressed nuclear protein. A mail1 loss-of-function mutant developed short primary roots, in which the meristematic cells accumulated DNA double-strand breaks and underwent massive cell death. In addition, mail1 mutant showed also cell differentiation defects in root and shoot tissues, and developed disorganized callus-like structures. The genetic interaction between main and mail1 mutants suggests that they act in the same pathway, and that both are essential for maintaining correct cell division acitivity in meristematic cells, while MAIL1 has an additional function in differentiating cells.

Stem cells in the root and shoot apical meristem provide the descendant cells required for growth and development throughout the lifecycle of a plant. We found that mutations in the Arabidopsis MAINTENANCE OF MERISTEMS (MAIN) gene led to plants with distorted stem cell niches in which stem cells are not maintained and undergo premature differentiation or cell death. The malfunction of main meristems leads to short roots, mis-shaped leaves, reduced fertility and partial fasciation of stems. MAIN encodes a nuclear-localized protein and is a member of a so far uncharacterized plant-specific gene family. As main mutant plants are hypersensitive to DNA-damaging agents, expression of genes involved in DNA repair is induced and dead cells with damaged DNA accumulate in the mutant meristems, we propose that MAIN is required for meristem maintenance by sustaining genome integrity in stem cells and their descendants cells.

Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H+-pyrophosphatase and the vacuolar H+-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.