Ruth Stadler

Summary: Cuscuta species are holoparasitic plants that penetrate host stems with haustoria, connect to the vasculature and exhaust water, nutrients and carbohydrates. Parasite seedlings possess only limited maternal reserves and the development of a vascular connection takes several days. Until then, an alternative access to host assimilates is critical for the survival of the parasite. Here, we investigated the sucrose flux and nutrition mechanisms of dodder during early infection. Using phloem‐mobile fluorescent tracers, we show that an apoplastic sucrose transfer occurs before symplastic connections between host and parasite are formed. We identified sucrose transporters of both dodder and host that are critical for assimilate transfers. CrSUT1, CrSUT3 and CrSWEET10 mediate sucrose transport during haustorium development. In the phloem of Arabidopsis, at the infection site, the sucrose‐exporter AtSWEET15 is induced through basic leucine zipper transcription factors. CrSUT2 was identified as a transporter for sucrose uptake into the invading haustorium. The growth of dodder seedlings on atsweet15 hosts is reduced, demonstrating the importance of the sucrose export by AtSWEET15 and availability within the apoplast. Our data highlight the relevance of apoplastic nutrition for the survival of parasitic plants. Dodder manipulates the host, causing it to release carbohydrates from its phloem during the initial infection.

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H⁺ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.

Columella stem cell fate is controlled by factors acting independently of WUSCHEL RELATED HOMEOBOX 5 and the main role of the CLAVATA3/ESR-RELATED 40 peptide is

Sucrose transport across membranes requires the activity of transport proteins. Sucrose-specific SWEET proteins mediate sugar efflux out of the cytosol and SUC proteins catalyze the uptake of sucrose from the apoplast. Both transport processes are involved in phloem loading in source leaves as well as in the post-phloem pathway in sink tissues. An important step during the characterization of new sucrose transporters is to analyze their transport activity. This is usually achieved by heterologous expression of the respective gene in yeast cells or Xenopus oocytes and subsequent uptake measurements. However, in some cases, mistargeting to internal membranes or the lack of protein modifications and/or interaction partners in the heterologous system can interfere with uptake analyses. Therefore, a new in planta method was developed that is based on mesophyll protoplasts as expression system and the fluorescent sucrose analog esculin to monitor uptake activities by confocal microscopy. In this chapter we describe the design of constructs required to analyze sucrose transporters in protoplasts, the experimental setup of the protoplast-esculin assay, and the quantitative evaluation of the obtained data. The quantification of esculin uptake allows the application of the new assay to a variety of questions, e.g., by comparison of point mutants, splice variants, or transporters with and without interaction partners.

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.

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.

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 AtSUC6_Col-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 AtSUC6_Col-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 AtSUC6_Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7_Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7_Col-0 is regulated by intragenic regions. Transport activity of AtSUC7_Col-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 AtSUC6_Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7_Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7_Col-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.

Summary: 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 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 towards functional redundancy with other STPs. In contrast to all other STPs, STP7 did not transport hexoses, but was 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 eleven 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, the data complete the characterization of the STP family and present the STPs as new L-arabinose transporters for potential biotechnological applications.