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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.

Developing Arabidopsis (Arabidopsis thaliana) seeds and embryos represent a complex set of cell layers and tissues that mediate the transport and partitioning of carbohydrates, amino acids, hormones, and signaling molecules from the terminal end of the funicular phloem to and between these seed tissues and eventually to the growing embryo. This article provides a detailed analysis of the symplastic domains and the cell-to-cell connectivity from the end of the funiculus to the embryo, and within the embryo during its maturation. The cell-to-cell movement of the green fluorescent protein or of mobile and nonmobile green fluorescent protein fusions was monitored in seeds and embryos of plants expressing the corresponding cDNAs under the control of various promoters (SUC2, SUC3, TT12, and GL2) shown to be active in defined seed or embryo cell layers (SUC3, TT12, and GL2) or only outside the developing Arabidopsis seed (AtSUC2). Cell-to-cell movement was also analyzed with the low-molecular-weight fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonate. The analyses presented identify a phloem-unloading domain at the end of the funicular phloem, characterize the entire outer integument as a symplastic extension of the phloem, and describe the inner integument and the globular stage embryo plus the suspensor as symplastic domains. The results also show that, at the time of hypophysis specification, the symplastic connectivity between suspensor and embryo is reduced or interrupted and that the embryo develops from a single symplast (globular and heart stage) to a mature embryo with new symplastic domains.

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 . Heterologous expression in yeast ( ) revealed that AtSUC6 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 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6 with other sugar transporters. Reporter gene analyses showed that is expressed in roots and pollen tubes and that this sink specific expression of is regulated by intragenic regions. Transport activity of AtSUC7 could not be analyzed in baker's yeast or 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 was developed. Plasma membrane localized SUCs including AtSUC6 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7 is not able to mediate esculin transport across the plasma membrane which implicates that 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 expression system.

Developing flowers are important sinks in Arabidopsis thaliana. Their energy demand is covered by assimilates which are synthesized in source leaves and transported via the vasculature. Assimilates are unloaded either symplastically through plasmodesmata or apoplastically by specific transport proteins. Here we studied the pathway of phloem unloading and post-phloem transport in developing gynoecia. Using phloem-mobile fluorescent tracers, we show that phloem unloading into cells of ovule primordia followed a symplastic pathway. Subsequently, the same tracers could not move out of phloem cells into mature ovules anymore. A further change in the mode of phloem unloading occurred after anthesis. In open flowers as well as in outgrowing siliques, the phloem was again unloaded via the symplast. This observed onset of symplastic phloem unloading was accompanied by a change in frequency of MP17-GFP-labeled plasmodesmata. We could also show that the change in cell-cell connectivity was independent of fertilization and increasing sink demand. The presented results indicate that symplastic connectivity is highly regulated and varies not only between different sink tissues but also between different developmental stages.

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.

Sedentary nematodes are destructive plant pathogens that cause significant yield losses. In the roots of their host plants, cyst nematodes (CNs) and root-knot nematodes (RKNs) induce different, highly specialized feeding sites-syncytia or giant cells (GCs), respectively-to optimize nutrient uptake. We compared the mechanisms by which nutrients are delivered from the model host plant, Arabidopsis, to GCs induced by the RKN Meloidogyne incognita or to syncytia induced by the CN Heterodera schachtii. From previous work, syncytia were known to be symplastically connected to newly formed host phloem composed of sieve elements (SEs) and companion cells. Here we studied the formation of plasmodesmata (PD) during GC and syncytia development by monitoring a viral movement protein that targets branched PD and the development of host phloem during GC formation by applying confocal laser scanning microscopy and immunocytochemistry. Analyses of plants expressing soluble or membrane-anchored green fluorescent protein in their phloem demonstrated symplastic isolation of GCs. GCs were found to be embedded in a tissue that consists exclusively of SEs. These de novo-formed SEs, contained nuclei and were interconnected by secondary PD. A similar interconnection of SEs was observed around syncytia. However, these secondary PD were also present at the SE-syncytium interface, demonstrating the postulated symplastic connection. Our results show that CNs and RKNs, despite their close phylogenetic relatedness, employ fundamentally different strategies to withdraw nutrients from host plants.

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.

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 50 mM 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.

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.

Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H(+)-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.