Iron Nutrition, Oxidative Stress, and Pathogen Defense

Adaptation is a challenge that plants have to undergo in order to survive in difficult environments. Nutrient deficiency, stress, and microorganism attack are abiotic and biotic factors that frequently impair plant wellness, which is reflected by low crop yield and quality. Poor crops in turn affect human nutrition. To solve these problems, it is necessary to understand the molecular and physiological mechanisms of nutrient uptake and adaptation to stress. With this knowledge, we may have the possibility to generate new plants, which offer better yield due to their better health. This chapter summarizes and compares iron uptake and assimilation as well as pathogen responses in plants and humans. We also discuss novel approaches for improving crops in the context of human food quality

Untersuchung der Rolle des eisenabhängigen bHLH039 Transkriptionsfaktors bei der Koordinierung der Fe-Homöostase in Arabidopsis thaliana

Plants perceive and react to their environment. Upon iron deficiency, plants activate the iron-deficiency machinery which is controlled by the master regulator FIT and its four interaction partners the bHLH038, bHLH039, bHLH100 and bHLH101 from the subgroup Ib(2). This work reports the influence of bHLH039 overexpression (39Ox) in iron uptake and homeostasis as well as in pathogen and high iron responses. Physiological investigation of 39Ox plants revealed that they exhibit iron-deficiency responses and simultaneously iron overload and oxidative stress. Crosses of 39Ox with FIT knock-out and FIT overexpression plants confirmed that FIT is essential for the activation of the iron-uptake machinery. A Catma v6 microarray was performed with 39Ox plants at + Fe and at -Fe. More than 700 FIT and Fe-regulated genes have been identified. This data showed that the expression of genes coding for proteins involved not only in the absorption, internal transport and storage of iron, but also in the circadian clock, light response, pathogen response and oxidative stress were greatly increased. GUS experiments indicated that the FIT promoter was more active when bHLH039 was overexpressed. Consequently, the binding of bHLH039 to the promoter of Fe-related genes was investigated. However, ChIP experiments showed no direct binding of bHLH39 to the different promoter regions of the tested genes. This work shows the importance of bHLH039 in the regulation of iron homeostasis and the processes mentioned above.Pflanzen empfinden und reagieren auf Umweltveränderungen. Bei Eisenmangel aktivieren die Pflanzen die Eisenmangel-Maschinerie, die vom Masterregulator FIT und seinen vier Interaktionspartnern bHLH038, bHLH039, bHLH100 und bHLH101 aus der Untergruppe Ib (2) gesteuert wird. Diese Arbeit befasst sich mit dem Einfluss der bHLH039 Überexpression (39Ox) auf die Eisenaufnahme und -homöostase sowie die Auswirkungen auf die Pathogen- und Eisenüberflussantwort. Durch Kreuzungen der Linie 39Ox mit FIT knock-out und FIT Überexpressionslinien wurde bestätigt, dass FIT eine zentrale Rolle in diesen Prozessen spielt. Mit einem Catma v6 Microarray wurden die Transkripton von 39Ox Pflanzen unter + Fe und - Fe untersucht und verglichen. Mehr als 700 Gene wurden identifiziert, welche unabhängig von FIT und Eisenversorgung reguliert werden. Eine umfassende Analyse dieser Daten zeigte, dass in 39Ox Pflanzen nicht nur Gene mit Beteiligung an Eisenaufnahme, -transport und -lagerung stark hochreguliert waren, sondern auch Gene, die in der circadian clock, der Pathogenantwort und bei oxidativen Stress eine Rolle spielen. Promotor-GUS Experimente zeigten, dass der FIT Promotor in 39Ox Pflanzen stärker aktiviert sein könnte. Auf dieser Grundlage wurde die Bindung von bHLH039 an die Promotoren von bekannten stark eisenmangelregulierten Genen wie FIT, IRT1, FRO2, AT3g12900, AT3g07720 und AT3g58810 untersucht. ChIP-Experimente gaben jedoch keine Hinweise auf eine direkte Bindung von bHLH39 an verschiedene Promotorregionen der getesteten Gene. Diese Arbeit zeigt die Bedeutung von bHLH039 bei der Regulierung der Eisenhomöostase und der oben erwähnten Prozesse

Untersuchung der Rolle des eisenabhängigen bHLH039 Transkriptionsfaktors bei der Koordinierung der Fe-Homöostase in Arabidopsis thaliana

Plants perceive and react to their environment. Upon iron deficiency, plants activate the iron-deficiency machinery which is controlled by the master regulator FIT and its four interaction partners the bHLH038, bHLH039, bHLH100 and bHLH101 from the subgroup Ib(2). This work reports the influence of bHLH039 overexpression (39Ox) in iron uptake and homeostasis as well as in pathogen and high iron responses. Physiological investigation of 39Ox plants revealed that they exhibit iron-deficiency responses and simultaneously iron overload and oxidative stress. Crosses of 39Ox with FIT knock-out and FIT overexpression plants confirmed that FIT is essential for the activation of the iron-uptake machinery. A Catma v6 microarray was performed with 39Ox plants at + Fe and at -Fe. More than 700 FIT and Fe-regulated genes have been identified. This data showed that the expression of genes coding for proteins involved not only in the absorption, internal transport and storage of iron, but also in the circadian clock, light response, pathogen response and oxidative stress were greatly increased. GUS experiments indicated that the FIT promoter was more active when bHLH039 was overexpressed. Consequently, the binding of bHLH039 to the promoter of Fe-related genes was investigated. However, ChIP experiments showed no direct binding of bHLH39 to the different promoter regions of the tested genes. This work shows the importance of bHLH039 in the regulation of iron homeostasis and the processes mentioned above.Pflanzen empfinden und reagieren auf Umweltveränderungen. Bei Eisenmangel aktivieren die Pflanzen die Eisenmangel-Maschinerie, die vom Masterregulator FIT und seinen vier Interaktionspartnern bHLH038, bHLH039, bHLH100 und bHLH101 aus der Untergruppe Ib (2) gesteuert wird. Diese Arbeit befasst sich mit dem Einfluss der bHLH039 Überexpression (39Ox) auf die Eisenaufnahme und -homöostase sowie die Auswirkungen auf die Pathogen- und Eisenüberflussantwort. Durch Kreuzungen der Linie 39Ox mit FIT knock-out und FIT Überexpressionslinien wurde bestätigt, dass FIT eine zentrale Rolle in diesen Prozessen spielt. Mit einem Catma v6 Microarray wurden die Transkripton von 39Ox Pflanzen unter + Fe und - Fe untersucht und verglichen. Mehr als 700 Gene wurden identifiziert, welche unabhängig von FIT und Eisenversorgung reguliert werden. Eine umfassende Analyse dieser Daten zeigte, dass in 39Ox Pflanzen nicht nur Gene mit Beteiligung an Eisenaufnahme, -transport und -lagerung stark hochreguliert waren, sondern auch Gene, die in der circadian clock, der Pathogenantwort und bei oxidativen Stress eine Rolle spielen. Promotor-GUS Experimente zeigten, dass der FIT Promotor in 39Ox Pflanzen stärker aktiviert sein könnte. Auf dieser Grundlage wurde die Bindung von bHLH039 an die Promotoren von bekannten stark eisenmangelregulierten Genen wie FIT, IRT1, FRO2, AT3g12900, AT3g07720 und AT3g58810 untersucht. ChIP-Experimente gaben jedoch keine Hinweise auf eine direkte Bindung von bHLH39 an verschiedene Promotorregionen der getesteten Gene. Diese Arbeit zeigt die Bedeutung von bHLH039 bei der Regulierung der Eisenhomöostase und der oben erwähnten Prozesse

Responses of a Triple Mutant Defective in Three Iron Deficiency-Induced BASIC HELIX-LOOP-HELIX Genes of the Subgroup Ib(2) to Iron Deficiency and Salicylic Acid

Plants are sessile organisms that adapt to external stress by inducing molecular and physiological responses that serve to better cope with the adverse growth condition. Upon low supply of the micronutrient iron, plants actively increase the acquisition of soil iron into the root and its mobilization from internal stores. The subgroup Ib(2) BHLH genes function as regulators in this response, however their concrete functions are not fully understood. Here, we analyzed a triple loss of function mutant of BHLH39, BHLH100 and BHLH101 (3xbhlh mutant). We found that this mutant did not have any iron uptake phenotype if iron was provided. However, under iron deficiency the mutant displayed a more severe leaf chlorosis than the wild type. Microarray-based transcriptome analysis revealed that this mutant phenotype resulted in the mis-regulation of 198 genes, out of which only 15% were associated with iron deficiency regulation itself. A detailed analysis revealed potential targets of the bHLH transcription factors as well as genes reflecting an exaggerated iron deficiency response phenotype. Since the BHLH genes of this subgroup have been brought into the context of the plant hormone salicylic acid, we investigated whether the 3xbhlh mutant might have been affected by this plant signaling molecule. Although a very high number of genes responded to SA, also in a differential manner between mutant and wild type, we did not find any indication for an association of the BHLH gene functions in SA responses upon iron deficiency. In summary, our study indicates that the bHLH subgroup Ib(2) transcription factors do not only act in iron acquisition into roots but in other aspects of the adaptation to iron deficiency in roots and leaves

Gene expression of Fe and <i>3xbhlh</i>-regulated genes (groups I–III), identified from microarray analysis.

<p>A, At3g07720; B, <i>CYP82C4</i>; C, At3g12900; D, <i>MTPA2</i>; E, <i>PPC1</i>; F, <i>LHY1</i>; G, At1g07050; H, <i>PSAF</i>; The genes in A–E were identified as potential downstream targets of bHLH subgroup Ib(2) factors, while the genes in F–H indicated a more intense response to –Fe in the mutant (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone-0099234-g006" target="_blank">Fig. 6</a>). <i>3xbhlh</i> and wild type seedlings were grown for 6 d at + and −Fe and exposed for 6 h to 100 µM SA (+SA) or were mock-treated (−SA). Whole seedlings were harvested for analysis. n = 3; the –Fe cDNA samples were derived from the RNAs used in the microarray (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone.0099234.s002" target="_blank">Fig. S2</a>); * indicates a significant change (p<0.05) of −Fe versus +Fe; + indicates a significant change (p<0.05) of <i>3xbhlh</i> versus WT; § indicates a significant change (p<0.05) of +SA versus –SA. Gene expression was studied using reverse transcription-qPCR.</p

Regulation of the subset of 29 Fe-regulated genes out of groups I, II and III identified in microarray analysis.

<p>The list of Fe-regulated genes in wild type seedlings that we had published earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone.0099234-Bauer1" target="_blank">[12]</a> was used to compare with the list obtained in this work for the groups I–III. 29 genes of the groups I–III were found Fe-regulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone.0099234-Bauer1" target="_blank">[12]</a>. A, Regulation patterns, annotation and co-expression of the subset of 29 genes; the regulation at +Fe versus –Fe in the wild type is represented in the left-most column (in dark green up = up-regulated, in red down = down-regulated); the regulation in the <i>3xbhlh</i> mutant versus WT is shown in the middle column (in light green up = up-regulated and in yellow or violet down = down-regulated; note that the yellow color indicates that these genes do not follow in the <i>3xbhlh</i> mutant the regulation expected from –Fe versus +Fe in the left column and hence could be direct targets of bHLH39, bHLH100 and bHLH101). The Arabidopsis gene identification (AGI) numbers and annotations are shown on the right side, whereby the color code indicates the belonging to different co-expression networks as determined using the ATTED tool (ref), represented in B; B, Co-expression network analysis of the 29 genes; the ATTED tool was utilized for construction; the different networks are highlighted in color and the AGI numbers belonging to those networks are highlighted by the same color in A. The grey color indicates genes that are part of isolated co-expression networks. A high-resolution image of the co-expression networks is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone.0099234.s003" target="_blank">Fig. S3</a>.</p

Gene expression of Fe deficiency response genes in various SA mutants and wild type plants.

<p>A, <i>FIT</i>, <i>FRO2</i>, <i>IRT1</i>; B, <i>BHLH38</i>, <i>BHLH39</i>; SA mutants and wild type seedlings were grown for 11 d at + and −Fe. Roots were harvested for analysis. n = 2; * indicates a significant change (p<0.05) of −Fe versus +Fe; + indicates a significant change (p<0.05) of SA mutant versus WT. Gene expression was studied using reverse transcription-qPCR.</p

Venn diagram showing overlap of differentially regulated genes identified in microarray analysis.

<p>Four lists of genes that were differentially expressed at least 1.5-fold between the indicated conditions were used to construct the Venn diagram. The genes of groups I–V are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone.0099234.s007" target="_blank">Tables S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099234#pone.0099234.s008" target="_blank">S4</a>. Groups I to III contain genes differentially expressed between <i>3xbhlh</i> and wild type. Groups IV and V contain genes that show differential regulation between + and –SA treatment but not between wild type and mutant.</p

Leaf chlorosis phenotypes of the <i>3xbhlh</i> mutant.

<p>A, 10-old WT and <i>3xbhlh</i> plants grown at +Fe and −Fe; bar = 5 mm; B, Strength of leaf chlorosis; the leaf chlorosis scale used was 1 = green, 2 = green, partially yellow, 3 = yellow-green, 4 = yellow; 5 = white-yellow; n = 12; C, Fe content per leaf dry mass; n = 4; indicates a significant change (p<0.05) of −Fe versus +Fe; + indicates a significant change (p<0.05) of <i>3xbhlh</i> versus WT.</p

Dissection of iron signaling and iron accumulation by overexpression of subgroup Ib bHLH039 protein

Iron is an essential growth determinant for plants, and plants acquire this micronutrient in amounts they need in their environment. Plants can increase iron uptake in response to a regulatory transcription factor cascade. Arabidopsis thaliana serves as model plant to identify and characterize iron regulation genes. Here, we show that overexpression of subgroup Ib bHLH transcription factor bHLH039 (39Ox) caused constitutive iron acquisition responses, which resulted in enhanced iron contents in leaves and seeds. Transcriptome analysis demonstrated that 39Ox plants displayed simultaneously gene expression patterns characteristic of iron deficiency and iron stress signaling. Thereby, we could dissect iron deficiency response regulation. The transcription factor FIT, which is required to regulate iron uptake, was essential for the 39Ox phenotype. We provide evidence that subgroup Ib transcription factors are involved in FIT transcriptional regulation. Our findings pose interesting questions to the feedback control of iron homeostasis