BackgroundStarch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism.ResultsWe identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis.ConclusionsThis study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3381-z) contains supplementary material, which is available to authorized users.
Potato is an important staple food with increasing popularity worldwide. Elevated temperatures significantly impair tuber yield and quality. Breeding heat-tolerant cultivars is therefore an urgent need to ensure sustainable potato production in the future. An integrated approach combining physiology, biochemistry, and molecular biology was undertaken to contribute to a better understanding of heat effects on source- (leaves) and sink-organs (tubers) in a heat-susceptible cultivar. An experimental set-up was designed allowing tissue-specific heat application. Elevated day and night (29°C/27°C) temperatures impaired photosynthesis and assimilate production. Biomass allocation shifted away from tubers towards leaves indicating reduced sink strength of developing tubers. Reduced sink strength of tubers was paralleled by decreased sucrose synthase activity and expression under elevated temperatures. Heat-mediated inhibition of tuber growth coincided with a decreased expression of the phloem-mobile tuberization signal SP6A in leaves. SP6A expression and photosynthesis were also affected, when only the belowground space was heated, and leaves were kept under control conditions. By contrast, the negative effects on tuber metabolism were attenuated, when only the shoot was subjected to elevated temperatures. This, together with transcriptional changes discussed, indicated a bidirectional communication between leaves and tubers to adjust the source capacity and/or sink strength to environmental conditions.
Proximal tubule cells extract dicarboxylates from filtrate and blood, using cotransporters located in the brush border [sodium dicarboxylate cotransporter (NaDC-1)] and basolateral cell membrane (NaDC-3). We expressed the human NaDC-3 (hNaDC-3) in Xenopus laevis oocytes and characterized it by the two-electrode voltage-clamp technique. At -60 mV, succinate (4 carbons) and glutarate (5 carbons) generated inward currents due to translocation of three sodium ions and one divalent dicarboxylate, whereas oxalate (2 carbons) and malonate (3 carbons) did not. The cis-dicarboxylate maleate produced currents smaller in magnitude, whereas the trans-dicarboxylate fumarate generated currents similar to succinate. The substituted succinate derivatives, malate, 2,2- and 2,3-dimethylsuccinate, and 2,3-dimercaptosuccinate elicited inward currents, whereas aspartate and guanidinosuccinate showed hardly detectable currents. The C-5 dicarboxylates glutarate and alpha-ketoglutarate produced larger currents than succinate; glutamate and folate failed to cause inward currents. Kinetic analysis revealed, at -60 mV, K(0.5) values of 25 +/- 12 microM for succinate and 45 +/- 13 microM for alpha-ketoglutarate, values close to the plasma concentration of these compounds. For both compounds, the K(0.5) was independent of voltage, whereas the maximal current increased with hyperpolarization. As opposed to the rat and flounder orthologs, hNaDC-3 was hardly inhibited by lithium concentrations up to 5 mM. In the absence of sodium, however, lithium can mediate succinate-dependent currents. The narrow substrate specificity prevents interaction of drugs with dicarboxylate-like structure with hNaDC-3 and ensures sufficient support of the proximal tubule cells with alpha-ketoglutarate for anion secretion via organic anion transporter 1 or 3.
Sodium-dependent dicarboxylate transporters located in the basolateral membrane (NaDC-3) of renal proximal tubule cells maintain the driving force for exchange of organic anions and drugs against α-ketoglutarate via organic anion transporters OAT1 and OAT3. So far, information on direct interaction of drugs with the cloned NaDC-3 was missing. Here we tested the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) and benzylpenicillin with NaDC-3 cloned from winter flounder (fNaDC-3) and human (hNaDC-3) kidneys. Flufenamate and benzylpenicillin inhibited [14C]succinate uptake in oocytes expressing fNaDC-3. Flufenamate elicited Na+-dependent currents in oocytes expressing fNaDC-3 with a reversal potential around -60 mV. Raising extracellular K+ concentration depolarized fNaDC3-expressing oocytes more in the presence of flufenamate than in its absence, an effect not seen with water-injected control oocytes. These findings suggest that flufenamate via interaction with fNaDC-3 increased the K+ conductance. Acetylsalicylate, indomethacin, and salicylate showed small potential-dependent inward currents in fNaDC-3 but not in hNaDC-3 expressing oocytes. Benzylpenicillin induced voltage-dependent inward currents which were Na+-dependent in oocytes expressing fNaDC-3. The currents were, however, much smaller than those induced by succinate, reflecting probably a low fit of the monovalent benzylpenicillin to the dicarboxylate binding site. The data show hitherto unknown effects of monovalent anionic drugs on a transporter for divalent di- and tricarboxylates.
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