Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

<p>Abstract</p> <p>Background</p> <p>Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including <it>Coffea arabica</it>. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.</p> <p>Results</p> <p>The expression levels of five frequently used housekeeping genes (reference genes), namely <it>alcohol dehydrogenase </it>(<it>adh</it>), <it>14-3-3</it>, <it>polyubiquitin </it>(<it>poly</it>), <it>β-actin </it>(<it>actin</it>) and <it>glyceraldehyde-3-phosphate dehydrogenase </it>(<it>gapdh</it>) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of <it>Coffea arabica </it>plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (<it>cys</it>), a caffeine synthase (<it>ccs</it>) and the 60S ribosomal protein L7 (<it>rpl7</it>) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of <it>gapdh</it>, which showed no significant changes in expression among the investigated experimental conditions.</p> <p>Conclusion</p> <p>Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, <it>gapdh</it>, followed by <it>14-3-3 </it>and <it>rpl7 </it>were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. <it>Gapdh </it>is therefore the recommended reference gene for measuring gene expression in <it>Coffea arabica</it>. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.</p

The promoter of a gene encoding an isoflavone reductase-like protein in coffee (Coffea arabica) drives a stress-responsive expression in leaves

A cDNA clone (designated CaIRL) encoding an isoflavone reductase-like protein from coffee (Coffea arabica) was retrieved during a search for genes showing organ/tissue-specific expression among the expressed sequence tags (EST) of the Brazilian coffee EST database. The CaIRL cDNA contains a single open reading frame of 946 nucleotides (nt) encoding 314 amino acids (predicted molecular weight of 34 kDa). Several features identified the predicted CaIRL protein as a new member of the PIP family of NADPH-dependent reductases. Expression studies demonstrated that CaIRL is expressed exclusively in coffee leaves and its transcript level is markedly increased in response to fungal infection and mechanical injury. Analysis of transgenic tobacco plants harboring a CaIRL 5'-flanking region (862 nt) fused to uidA reporter gene (GUS) confirmed the responsiveness of the putative promoter to abiotic stress in wounded leaves. In turn, a 5' deletion to -404 completely abolished promoter activation by abiotic stimulus in transgenic plants. The lack of GUS expression in non-wounded leaf tissues in transgenic tobacco was in contrast to the basal level of CaIRL expression observed in non-stressed healthy coffee leaves.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

BMC Molecular Biology BioMed Central

Research article Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental condition

CaPrx, a Coffea arabica gene encoding a putative class III peroxidase induced by root-knot nematode infection

Class III peroxidases (Prxs) are enzymes involved in a multitude of physiological and stress-related processes in plants. Here, we report on the characterization of a putative peroxidase-encoding gene from Coffea arabica (CaPrx) that is expressed in early stages of root-knot nematode (RKN) infection. CaPrx showed enhanced expression in coffee roots inoculated with RKN (at 12 h post-inoculation), but no significant difference in expression was observed between susceptible and resistant plants. Assays using transgenic tobacco plants harboring a promoter-beta-glucuronidase (GUS) fusion revealed that the CaPrx promoter was exclusively active in the galls induced by RKN. In cross sections of galls, GUS staining was predominantly localized in giant cells. Up-regulation of GUS expression in roots of transgenic plants following RKN inoculation was observed within 16 h. Moreover, no increase in GUS expression after treatment with jasmonic acid was detected. Altogether, these results point to a putative role of this peroxidase in the general coffee response to RKN infection. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 mu M], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H+ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles. (C) 2016 Elsevier Inc. All rights reserved.Fundacao de Amparo Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Univ Fed Sao Paulo UNIFESP, Dept Biofis, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Farmacol, Rua 3 de Maio 100,Ed INFAR,3rd Floor, BR-04044020 Sao Paulo, BrazilUniv Sao Paulo USP RP, Dept Bioquim & Imunol, Ribeirao Preto, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biociencias, Rua Silva Jardim 136, BR-11015020 Santos, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biofis, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Farmacol, Rua 3 de Maio 100,Ed INFAR,3rd Floor, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Biociencias, Rua Silva Jardim 136, BR-11015020 Santos, SP, BrazilWeb of Scienc