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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. [ABSTRACT FROM AUTHOR]

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast. [ABSTRACT FROM AUTHOR]

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-αdipalmitoylglycerophosphoethanolamine (Pam2GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator. The temperature dependence of the anisotropy decay showed that above 25 °C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (η1). Extrapolation to η1 = 0 Pa · s yielded a correlation time of φ = 20 ± 5 ns. t = 30 °C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr = 2.0 × 109 rad² · s-1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was η2 ≈ 12 mPa · s. The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of η1 = 1 mPa · s yielded a value of φr ≈ 450 ns corresponding to Dr = 8.8 × 107 rad² · s-1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be ≈9 nm². This cross-sectional area is large enough to include at most 14 membrane-spanning helices. Our results also indicated that at lipid/protein molar ratios ≥1.5 × 104:1 aggregation occurred in the model membranes below 30 °C. However, above 30 °C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical. [ABSTRACT FROM AUTHOR]

Abstract Background We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2). Results All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b559), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2. Conclusions We propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.

Abstract Background Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. Results High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources. Conclusion AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds.

Background: Although hen's egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffer from a persistent or newly onset hen's egg allergy, making accurate diagnosis in adults necessary. However, sensitization to hen's egg extracts, components and linear epitopes is solely studied in children., Methods: Hen's egg allergic (n = 16) and tolerant (n = 19) adults were selected by sensitization towards recombinant components rGal d 1 and/or 3. Sensitization profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterization of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3., Results: Overall, sIgE titres against hen's egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitization to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3., Conclusion and Clinical Relevance: Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen's egg allergic adults with objective symptoms., (© 2020 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

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., (© 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

The inducible crassulacean acid metabolism (CAM) plant Mesembryanthemum crystallinum accumulates malic acid during the night and converts it to starch during the day via a pathway that, because it is located in different subcellular compartments, depends on specific metabolite transport across membranes. The chloroplast glucose transporter (pGlcT) and three members of the phosphate translocator (PT) family were isolated. After induction of CAM, transcript amounts of the phosphoenolpyruvate (PEP) phosphate translocator (PPT) and the glucose-6-phosphate (Glc6P) phosphate translocator (GPT) genes were increased drastically, while triose phosphate (TP) phosphate translocator (TPT) and the pGlcT transcripts remained unchanged. PPT- and GPT-specific transcripts and transporter activities exhibited a pronounced diurnal variation, displaying the highest amplitude in the light. pGlcT transcripts were elevated towards the end of the light period and at the beginning of the dark period. These findings, combined with diurnal variations of enzyme activities and metabolite contents, helped to elucidate the roles of the PPT, GPT, TPT and pGlcT in CAM. The main function of the PPT is the daytime export from the stroma of PEP generated by pyruvate orthophosphate:dikinase (PPDK). The increased transport activity of GPT in the light suggests a higher requirement for Glc6P import for starch synthesis rather than starch mobilization. Most likely, Glc6P rather than 3-phosphoglycerate or triose phosphates is the main substrate for daytime starch biosynthesis in M. crystallinum plants in which CAM has been induced (CAM-induced), similar to non-green plastids. In the dark, starch is mobilized both phosphorylytically and amylolytically and the products are exported by the GPT, TPT and pGlcT. The transport activities of all three phosphate translocators and the transcript amounts of the pGlcT adapt to changing transport requirements in order to maintain high metabolic fluxes during the diurnal CAM cycle.

Two Arabidopsis Toc34 homologues, atToc34 and atToc33, components of the chloroplast protein import machinery located in the outer envelope membrane, were recently isolated. Both proteins insert into the outer envelope, are supposed to bind GTP and to interact with Toc75 as demonstrated by in vitro import assays. We studied the expression of the two genes by RNA gel blot analysis, promoter-GUS plants and in situ hybridisations as well as immunoblot analysis. The atToc34 and atToc33 genes are expressed in green as well as non-green tissues and are developmentally regulated. Despite these similarities, however, the two Arabidopsis Toc34 homologues are differentially expressed in various plant organs. To gain more insight into the in vivo function of both proteins, antisense plants were created. While antisense plants of atToc33 are characterized by a pale yellowish phenotype, antisense plants of atToc34 show a weaker phenotype. Protein interaction studies using an in vitro translated precursor protein and heterologously expressed atToc34 and atToc33 proteins showed a direct GTP-dependent interaction, but demonstrated different affinities of the two atToc proteins towards the precursor protein. Thus, our results indicate a more specialized function for both atToc34 and atToc33, suggesting specificity for certain imported precursor proteins.

This article describes a method for the enzymatic detection of low-abundant metabolic intermediates in plant extracts via NAD(P)H fluorescence using a microtiter plate reader. The detection of changes in NAD(P)H fluorescence (excitation 340 nm, emission 465 nm) exhibits a high signal-to-noise ratio and is as sensitive (> or = 20 pmol per well) as absorbance measurements with dual-wavelength photometers. Since up to 96 reactions can be initiated, monitored, and evaluated simultaneously, this method might be suitable for high-throughput screening programs on metabolite profiles. However, in contrast to absorbance measurements, fluorescence detection of NAD(P)H yields relative data, which can be impaired by the quench characteristics and the basic fluorescence of the extracts. Hence, extensive calibration is required to gain reproducible results. Calibration of the assay system was performed using leaf or root material (equivalent to 2-35 mg of fresh weight per well) extracted with perchloric acid, chloroform/water/methanol, or hot ethanol. Extraction with perchloric acid was found to be superior for metabolite quantification. Examples of the kinetics of individual metabolite determinations are presented and the contents of 3-phosphoglycerate, hexose phosphates, triose phosphates, pyruvate, and phosphoenolpyruvate in illuminated and darkened spinach leaves as well as leaf rosettes of Arabidopsis thaliana and leaf segments of the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum were measured via NAD(P)H fluorescence and, where possible, compared to reported data determined with dual-wavelength photometers.

During photosynthesis, part of the fixed carbon is directed into the synthesis of transitory starch, which serves as an intermediate carbon storage facility in chloroplasts. This transitory starch is mobilized during the night. Increasing evidence indicates that the main route of starch breakdown proceeds by way of hydrolytic enzymes and results in glucose formation. This pathway requires a glucose translocator to mediate the export of glucose from the chloroplasts. We have reexamined the kinetic properties of the plastidic glucose translocator and, using a differential labeling procedure, have identified the glucose translocator as a component of the inner envelope membrane. Peptide sequence information derived from this protein was used to isolate cDNA clones encoding a putative plastidic glucose translocator from spinach, potato, tobacco, Arabidopsis, and maize. We also present the molecular characterization of a candidate for a hexose transporter of the plastid envelope membrane. This transporter, initially characterized more than 20 years ago, is closely related to the mammalian glucose transporter GLUT family and differs from all other plant hexose transporters that have been characterized to date.

The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing the TPT gene isolated from the C4 plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C3 plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more 14CO2 in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in alphaTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as alpha-amylase or hexokinase were increased in alphaTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, alpha-amylase activity exhibited a pronounced diurnal variation in alphaTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO2 assimilation. However, in elevated CO2 (1500 microl x l(-1)) and low O2 (2%) the rate of CO2 assimilation was decreased in the alphaTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type.