Global transcriptional responses of Pseudomonas syringae DC3000 to changes in iron bioavailability in vitro

BACKGROUND: Pseudomonas syringae pv tomato DC3000 (DC3000) is a Gram-negative model plant pathogen that is found in a wide variety of environments. To survive in these diverse conditions it must sense and respond to various environmental cues. One micronutrient required for most forms of life is iron. Bioavailable iron has been shown to be an important global regulator for many bacteria where it not only regulates a wide variety of genes involved in general cell physiology but also virulence determinants. In this study we used microarrays to study differential gene regulation in DC3000 in response to changes in levels of cell-associated iron. RESULTS: DC3000 cultures were grown under highly controlled conditions and analyzed after the addition of iron citrate or sodium citrate to the media. In the cultures supplemented with iron, we found that cell-associated iron increased rapidly while culture densities were not significantly different over 4 hours when compared to cultures with sodium citrate added. Microarray analysis of samples taken from before and after the addition of either sodium citrate or iron citrate identified 386 differentially regulated genes with high statistical confidence. Differentially regulated genes were clustered based on expression patterns observed between comparison of samples taken at different time points and with different supplements. This analysis grouped genes associated with the same regulatory motifs and/or had similar putative or known function. CONCLUSION: This study shows iron is rapidly taken up from the medium by iron-depleted DC3000 cultures and that bioavailable iron is a global cue for the expression of iron transport, storage, and known virulence factors in DC3000. Furthermore approximately 34% of the differentially regulated genes are associated with one of four regulatory motifs for Fur, PvdS, HrpL, or RpoD

Genetic and Physiological Analysis of Iron Biofortification in Maize Kernels

BACKGROUND: Maize is a major cereal crop widely consumed in developing countries, which have a high prevalence of iron (Fe) deficiency anemia. The major cause of Fe deficiency in these countries is inadequate intake of bioavailable Fe, where poverty is a major factor. Therefore, biofortification of maize by increasing Fe concentration and or bioavailability has great potential to alleviate this deficiency. Maize is also a model system for genomic research and thus allows the opportunity for gene discovery. Here we describe an integrated genetic and physiological analysis of Fe nutrition in maize kernels, to identify loci that influence grain Fe concentration and bioavailability. METHODOLOGY: Quantitative trait locus (QTL) analysis was used to dissect grain Fe concentration (FeGC) and Fe bioavailability (FeGB) from the Intermated B73 × Mo17 (IBM) recombinant inbred (RI) population. FeGC was determined by ion coupled argon plasma emission spectroscopy (ICP). FeGB was determined by an in vitro digestion/Caco-2 cell line bioassay. CONCLUSIONS: Three modest QTL for FeGC were detected, in spite of high heritability. This suggests that FeGC is controlled by many small QTL, which may make it a challenging trait to improve by marker assisted breeding. Ten QTL for FeGB were identified and explained 54% of the variance observed in samples from a single year/location. Three of the largest FeGB QTL were isolated in sister derived lines and their effect was observed in three subsequent seasons in New York. Single season evaluations were also made at six other sites around North America, suggesting the enhancement of FeGB was not specific to our farm site. FeGB was not correlated with FeGC or phytic acid, suggesting that novel regulators of Fe nutrition are responsible for the differences observed. Our results indicate that iron biofortification of maize grain is achievable using specialized phenotyping tools and conventional plant breeding techniques

Global transcriptional responses of <it>Pseudomonas syringae </it>DC3000 to changes in iron bioavailability <it>in vitro</it>

Abstract Background Pseudomonas syringae pv tomato DC3000 (DC3000) is a Gram-negative model plant pathogen that is found in a wide variety of environments. To survive in these diverse conditions it must sense and respond to various environmental cues. One micronutrient required for most forms of life is iron. Bioavailable iron has been shown to be an important global regulator for many bacteria where it not only regulates a wide variety of genes involved in general cell physiology but also virulence determinants. In this study we used microarrays to study differential gene regulation in DC3000 in response to changes in levels of cell-associated iron. Results DC3000 cultures were grown under highly controlled conditions and analyzed after the addition of iron citrate or sodium citrate to the media. In the cultures supplemented with iron, we found that cell-associated iron increased rapidly while culture densities were not significantly different over 4 hours when compared to cultures with sodium citrate added. Microarray analysis of samples taken from before and after the addition of either sodium citrate or iron citrate identified 386 differentially regulated genes with high statistical confidence. Differentially regulated genes were clustered based on expression patterns observed between comparison of samples taken at different time points and with different supplements. This analysis grouped genes associated with the same regulatory motifs and/or had similar putative or known function. Conclusion This study shows iron is rapidly taken up from the medium by iron-depleted DC3000 cultures and that bioavailable iron is a global cue for the expression of iron transport, storage, and known virulence factors in DC3000. Furthermore approximately 34% of the differentially regulated genes are associated with one of four regulatory motifs for Fur, PvdS, HrpL, or RpoD.</p

Locations of FeGB QTL detected by GLM Select analysis for 2003 NY field season.

<p>Markers are given in order of inclusion in the trait model according to GLM Select. AIC is the Akaike Information Criterion and estimates the goodness of fit for the model. Significance of the association between marker and trait is demonstrated by F and p values. The t-value estimates the magnitude of the effect; a positive score indicates Mo17 donated the superior allele. Marker locations are reported using IBM v1 coordinates (chromosome; position). Summary statistics for the 10-factor model are presented below.</p

Pedigree for sister derived lines with altered FeGB qualities.

<p>High and low FeGB derivatives were generated from IBM RI Line #039 using backcrossing to both B73 and Mo17 parents. Circles denote maternal parents and squares are paternal parents, where the numbers that appear next to the circles or square refer to the field entry (e.g. 02-095 indicates NY2002 row 095). Caco-2 phenotyping was utilized at four points in this process: for the evaluation of RI lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020429#pone-0020429-g002" target="_blank">Figure 2</a>), to validate the QTL model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020429#pone-0020429-t007" target="_blank">Table 7</a>), and to assist derivation of new inbreds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020429#pone-0020429-t008" target="_blank">Table 8</a>). The backcrossing program was initiated three years before Caco-2 phenotyping of the RI lines took place. Individuals with altered FeGB qualities are denoted with filled black circles (high FeGB) or gray circles (low FeGB). While not shown here, lines have been advanced to the BC₂S₆ (highly inbred) generation in the NY2010 field season, with an additional round of Caco-2 phenotyping occurring in early 2011.</p

FeGB observed for a maize population.

<p>The Intermated B73 × Mo17 RI population was grown at Aurora NY in 2003. Grain samples were evaluated for grain bioavailable Fe via a Caco2 cell culture bioassay. The bioassay measures the amount of ferritin storage protein produced in the human cells in response to the maize samples, and thus estimates how much Fe was absorbed from the samples. Results for the RI lines are organized into bins of 5 ng ferritin mg total protein⁻¹ for the histogram.</p

Correlation analysis of grain nutrients and mass.

<p>Pearson's correlation coefficient (left) and p-value (right) are reported for each correlation. Bold entries indicate significant correlations; italic entries indicate non-significant correlations from the NY03 dataset.</p

Multi-site evaluation of FeGB in derived lines.

<p>Contrasting BC₂S₄ derivatives from the IBM039 RI line were grown on 8 plots over 2 years, to evaluate the heritability and penetrance of the high FeGB effect across multiple environments. ANOVA were used to assess whether pairs of related high and low-nutritional value derivatives were significantly different and are denoted by letter. Comparisons were made within sites only, where trait data are expressed as a percentage of the control variety from the Caco-2 bioassay. Locations where significant differences were not observed according to our hypotheses appear in italic type.</p