SummaryReverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably

Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

No abstract

No abstract

Iron is an essential element for plant metabolism because of its redox properties. Its long distance and intracellular trafficking require specialized proteins and low molecular mass chelates because of its insolubility and toxicity in presence of oxygen. Iron deficiency induces various morphological and biochemical changes. They include root hair morphogenesis, differentiation of rhizoder-ma1 cells into transfer cells, yellowing of leaves and ultrastructural disorganisation of chloroplasts and mitochondria, as well as increased synthesis of organic acids and phenolics, and activation of root systems responsible for an enhanced iron uptake capacity. Upon iron resupply, these alterations disappeared within few days and a transient accumulation of the iron storage protein ferritin in the plastids is one of the early events in this process. Iron excess can also occur in plants where it elicits an oxidative stress leading to necrotic spots in the leaves. Induction of ferritin synthesis is again an early event of the plant response to this iron toxicity. Plant hormones such as auxin, abscisic acid and ethylene, as well as reactive oxygen intermediates play an important role in the transduction pathways, allowing plants to respond to these iron-deficiency and excess stresses. Similarities and differences among the various mechanisms responsible for iron uptake and storage in mammals, higher plants and yeast are outlined. Relationships between iron and copper metabolism are also indicated. plant / root / chloroplast / iron / ferritinIron traffk in non-stressed plants

No abstract

Glycerol has been demonstrated to serve as the major osmolyte of Saccharomyces cerevisiae. Consistently, mutant strains gpd1gpd2 and gpp1gpp2, which are devoid of the main glycerol biosynthesis pathway, have been shown to be osmosensitive. In addition, the primary hyperosmotic stress response is affected in these strains. Hog1p phosphorylation turned out to be prolonged and osmostress‐induced gene expression is delayed compared with the kinetics observed in wild‐type cells. A hog1 deletion strain was previously found to contain lower internal glycerol and therefore displays an osmosensitive phenotype. Here, we show that the osmosensitivity of hog1 is suppressed by growth at 37°C. We reasoned that this temperature‐remedial osmoresistance might be caused by a higher intracellular glycerol level at the elevated temperature. This hypothesis was confirmed by measurement of the glycerol concentration, which was shown to be similar for wild type and hog1 cells only at elevated growth temperatures. In agreement with this finding, hog1 cells containing an fps1 allele, encoding a constitutively open glycerol channel, have lost their temperature‐remedial osmoresistance. Furthermore, gpd1gpd2 and gpp1gpp2 strains were found to be temperature sensitive. The growth defect of these strains could be suppressed by adding external glycerol. In conclusion, the ability to control glycerol levels influences proper osmostress‐induced signalling and the cellular potential to grow at elevated temperatures. These data point to an important, as yet unidentified, role of glycerol in cellular functioning.

SummaryThe HOG/p38 MAP kinase route is an important stress-activated signal transduction pathway that is well conserved among eukaryotes. Here, we describe a novel mechanism of activation of the HOG pathway in budding yeast. This mechanism operates upon severe osmostress conditions (1.4 M NaCl) and is independent of the Sln1p and Sho1p osmosensors. The alternative input feeds into the HOG pathway MAPKK Pbs2p and requires activation of Pbs2p by phosphorylation. We show that, upon severe osmotic shock, Hog1p nuclear accumulation and phosphorylation is delayed compared with mild stress. Moreover, both events lost their transient pattern, presumably because of the absence of negative feedback mediated by Ptp2p tyrosine phosphatase, which we found to be localized in the nucleus. Under severe osmotic stress conditions, the delayed nuclear accumulation correlates with a delay in stress-responsive gene expression. Severe osmoshock leads to a situation in which active and nuclear-localized Hog1p is transiently unable to induce transcription of osmotic stress-responsive genes. It also appeared from our studies that the Sho1p osmosensor is less active under severe osmotic stress conditions, whereas the Sln1p/Ypd1p/Ssk1p sensor and signal transducer functions normally under these circumstances.