Are sulfur-containing metabolites involved in the circadian-regulated pathogen defense? : influence of the sulfur metabolism on the pathogen defense in Brassica napus

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Quantitative expression analysis in Brassica napus by Northern blot analysis and reverse transcription-quantitative PCR in a complex experimental setting

Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes

Exposure of Common Bean Seeds to Liquid Nitrogen Modifies Mineral Composition of Young Plantlet Leaves

Many publications describe cryopreservation techniques but only a few studies have focused on the biochemical and physiological changes occurring in plants regenerated from seeds exposed to liquid nitrogen. This paper aims at describing the effect of common bean seed cryostorage on mineral nutrition of young plantlets. The following elements were measured on leaves of 10-day-old plantlets from non-cryopreserved and cryopreserved seeds: Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Se, Sr and Zn. At 10 days after sowing, both treatments (control and cryopreserved seeds) showed 100% seed germination without any visual phenotypic difference. However, contents of several elements in the leaves were different. Exposure of seeds to liquid nitrogen decreased Cu, Cd and Na uptake and increased absorption of B and Al. Further studies are required to understand the mechanisms underlying the relationship between seed exposure to liquid nitrogen and mineral nutrition during the early stages of plantlet growth.DAADLeibniz University of HannoverUniversity of Ciego de Avila, Cub

The levels of sulfur-containing metabolites in Brassica napus are not influenced by the circadian clock but diurnally

Adapting biological processes to an endogenous rhythm enables plants to cope with the daily changes in light and temperature in a more predictable way enhancing growth and fitness. A number of biological processes such as metabolic pathways as well as the immunity in plants are under diurnal or circadian control. In this study a possible circadian regulation of key enzymes in the sulfur assimilation and the corresponding metabolites was investigated in the agriculturally important crop plant oilseed rape (Brassica napus). Leaves of a commercially available cultivar were harvested in the course of a day under diurnal and under free-running conditions with constant light. Analyses in this study were focused on sulfur-containing metabolites and expression analysis of enzymes involved in sulfur assimilation. Expression analysis showed that the transcript levels of the sulfate transporters Sultr3;1 and Sultr4;2 as well as APR2 and APR3 oscillated diurnally. Results revealed a periodic rhythm of sulfur-containing metabolites such as glutathione, sulfate and certain glucosinolates in the course of a day which were only partly maintained under constant light. Therefore, we conclude that a diurnal rhythm and not the circadian clock regulates sulfur metabolism in plants

Bland Altman plot for method comparison.

<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g001" target="_blank">Fig 1</a> were used to generate all graphs. For each data point from both methods, the value was standardized and the difference between the measurements by the two methods was plotted against their mean. Data are shown with the mean of the difference (dashed line) and the 95% limits of agreement ± 1.96xSD (dotted lines). A) <i>ACT2</i> B) <i>EF1α</i> C) <i>18S rRNA</i> D) <i>APR3</i> and E) <i>CCA1</i>.</p

Normalization of <i>CCA1</i> with <i>EF1α</i>.

<p>Expression analysis for <i>CCA1</i> was performed in plants treated as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g003" target="_blank">Fig 3</a> by Northern blot analysis. Results were normalized using <i>EF1α</i> according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g003" target="_blank">Fig 3</a>. The normalized (n) and non-normalized (nn) data are presented as the relative expression under (A) diurnal conditions over a period of 36 h and (B) free running conditions with continuous light (LL) in comparison to the light dark (LD) conditions. Data are shown as the mean of three technical replicates ± SD. Relative expression calculation was based on band intensity.</p

Primer pairs used in this study.

<p>Primer pairs used in this study.</p

Comparison of Northern blot analysis and RT-qPCR analysis.

<p>Plants with five fully expanded leaves were harvested over a period of 20 h every 4 h, starting 1 h before the onset of light. Leaves from three plants were harvested and pooled. The relative expression for two to three technical replicates of each sample and the resulting mean is shown. Relative expression for Northern blot analysis was calculated based on the band intensity. Percentages refer to the first mean of the three technical replicates as 100% for both methods separately.</p

Normalization of <i>CCA1</i> expression with the validated set of reference genes <i>18S rRNA</i>, <i>PP2A</i>, and <i>GDI1</i>.

<p>Expression analysis for <i>CCA1</i> was performed in plants treated as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g003" target="_blank">Fig 3</a> by Northern blot analysis. Results were normalized using three reference genes according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g003" target="_blank">Fig 3</a>. The normalized (n) and non-normalized (nn) data are presented as the relative expression under (A) diurnal conditions over a period of 36 h and (B) free-running conditions with continuous light (LL) in comparison to the light-dark (LD) conditions. Data are shown as the mean of three technical replicates ± SD. Relative expression calculation was based on band intensity.</p

Normalization of <i>APR3</i> with the validated set of reference genes <i>18S rRNA</i>, <i>PP2A</i>, and <i>GDI1</i>.

<p>Expression analysis for <i>APR3</i> was performed in plants treated as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g003" target="_blank">Fig 3</a> by Northern blot analysis. Results were normalized using three reference genes according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163679#pone.0163679.g003" target="_blank">Fig 3</a>. The normalized (n) and non-normalized (nn) data are presented as the relative expression under (A) diurnal conditions over a period of 36 h and (B) free running conditions with continuous light (LL) in comparison to the light dark (LD) conditions. Data are shown as the mean of three technical replicates ± SD. Relative expression calculation was based on band intensity.</p