RESUMO
In this work, we analyze protein phosphatase (PP) involvement in the sucrose-mediated induction of fructan metabolism in wheat (Triticum aestivum). The addition of okadaic acid (OA), a PP-inhibitor, to sucrose-fed leaves reduced fructosylsucrose-synthesizing activity (FSS) induction in a dose-dependent manner. The expression of the two enzymes that contribute to FSS activity, 1-SST (1-sucrose:sucrose fructosyltransferase, E.C. 2.4.1.99) and 6-SFT (6-sucrose:fructan fructosyltransferase, E.C. 2.4.1.10), was blocked by 1 microM OA. These results suggest the involvement of a PP type 2A in sucrose signaling leading to fructan synthesis. OA addition to the feeding medium impaired both sucrose accumulation in leaves and the expression of sucrose-H+ symporter (SUT1). It is known that sucrose concentration must exceed a threshold for the induction of fructan metabolism; hence PP2A inhibition may result in lower sucrose levels than required for this induction. OA also induced the vacuolar acid invertase (acid INV) transcript levels suggesting that PP activity might play a role in carbon partitioning. Total extractable PP2A activity decreased during 24 h of treatment with sucrose, in parallel with declining sugar uptake into leaf tissues. In conclusion, our results suggest that PP2A is involved in sucrose-induction of fructan metabolism and may play a role in regulating sucrose uptake, but do not rule out that further steps in sucrose signaling pathway may be affected.
Assuntos
Frutanos/biossíntese , Proteína Fosfatase 2/metabolismo , Sacarose/farmacologia , Triticum/efeitos dos fármacos , Triticum/enzimologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hexosiltransferases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácido Okadáico/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Solubilidade/efeitos dos fármacos , Sacarose/metabolismo , Triticum/genética , Vacúolos/efeitos dos fármacos , Vacúolos/enzimologia , beta-Frutofuranosidase/metabolismoRESUMO
Sucrose (Suc) can influence the expression of a large number of genes and thereby regulates many metabolic and developmental processes. However, the Suc sensing and the components of the ensuing signaling transduction pathway leading to the regulation of gene expression are not fully understood. We have shown that protein kinases and phosphatases are involved in the Suc induced expression of fructosyltransferase (FT) genes and fructan accumulation by an hexokinase independent pathway in wheat (Triticum aestivum). In the present study, using an RT-PCR based strategy, we have cloned a calcium-dependent protein kinase (TaCDPK1) cDNA that is upregulated during Suc treatment of excised wheat leaves. The deduced amino-acid sequence of CDPK1 has high sequence similarity (>70%) to known CDPKs from both monocots and dicots. Based on sequence homology, TaCDPK1 sequence shows a variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs, along with a N-myristoylation motif in the N-terminal variable domain. The recombinant Escherichia coli expressed TaCDPK1 was able to phosphorylate histone III-S in a calcium dependent manner in in vitro assays. The TaCDPK1 gene expression, as determined by quantitative RT-PCR, is induced by Suc and this effect is repressed by the inhibitors of the putative components of the Suc signal transduction pathway (calcium, Ser/Thr protein kinases and protein phosphatases). We propose that TaCDPK1 is involved in the Suc induced signaling pathway in wheat leaves.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Proteínas Quinases/genética , Sacarose/farmacologia , Triticum/enzimologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Sequência Conservada , Primers do DNA , DNA Complementar/genética , DNA de Plantas/química , DNA de Plantas/genética , Escherichia coli/genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , Triticum/efeitos dos fármacos , Triticum/fisiologiaRESUMO
The role of Ca(2+) in the induction of enzymes involved in fructan synthesis (FSS) mediated by sucrose was studied in wheat (Triticum aestivum). Increase of FSS enzyme activity and induction of the expression of their coding genes by sucrose were inhibited in leaf blades treated with chelating agents (EDTA, EGTA and BAPTA). Ca(2+) channel blockers (lanthanum chloride and ruthenium red) also inhibited the FSS response to sucrose, suggesting the participation of Ca(2+) from both extra- and intra- cellular stores. Sucrose induced a rapid Ca(2+) influx into the cytosol in wheat leaf and root tissues, shown with the Ca(2+ )sensitive fluorescent probe Fluo-3/AM ester. Our results support the hypothesis that calcium is a component of the sucrose signaling pathway that leads to the induction of fructan synthesis.
Assuntos
Cálcio/fisiologia , Frutanos/metabolismo , Sacarose/metabolismo , Triticum/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Citosol/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Microscopia de Fluorescência , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Triticum/genéticaRESUMO
Over 400 non-photosynthetic species from 10 families of vascular plants obtain their carbon from fungi and are thus defined as myco-heterotrophs. Many of these plants are epiparasitic on green plants from which they obtain carbon by 'cheating' shared mycorrhizal fungi. Epiparasitic plants examined to date depend on ectomycorrhizal fungi for carbon transfer and exhibit exceptional specificity for these fungi, but for most myco-heterotrophs neither the identity of the fungi nor the sources of their carbon are known. Because many myco-heterotrophs grow in forests dominated by plants associated with arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), we proposed that epiparasitism would occur also between plants linked by AMF. On a global scale AMF form the most widespread mycorrhizae, thus the ability of plants to cheat this symbiosis would be highly significant. We analysed mycorrhizae from three populations of Arachnitis uniflora (Corsiaceae, Monocotyledonae), five Voyria species and one Voyriella species (Gentianaceae, Dicotyledonae), and neighbouring green plants. Here we show that non-photosynthetic plants associate with AMF and can display the characteristic specificity of epiparasites. This suggests that AMF mediate significant inter-plant carbon transfer in nature.