Iron homeostasis in Arabidopsis thaliana: transcriptomic analyses reveal novel FIT-regulated genes, iron deficiency marker genes and functional gene networks

Abstract

Background: FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is the central regulator of iron uptake in Arabidopsis thaliana roots. We performed transcriptome analyses of six day-old seedlings and roots of six week-old plants using wild type, a fit knock-out mutant and a FIT over-expression line grown under iron-sufficient or iron-deficient conditions. We compared genes regulated in a FIT-dependent manner depending on the developmental stage of the plants. We assembled a high likelihood dataset which we used to perform co-expression and functional analysis of the most stably iron deficiency-induced genes.

Results: 448 genes were found FIT-regulated. Out of these, 34 genes were robustly FIT-regulated in root and seedling samples and included 13 novel FIT-dependent genes. Three hundred thirty-one genes showed differential regulation in response to the presence and absence of FIT only in the root samples, while this was the case for 83 genes in the seedling samples. We assembled a virtual dataset of iron-regulated genes based on a total of 14 transcriptomic analyses of iron-deficient and iron-sufficient wild-type plants to pinpoint the best marker genes for iron deficiency and analyzed this dataset in depth. Co-expression analysis of this dataset revealed 13 distinct regulons part of which predominantly contained functionally related genes.

Conclusions: We could enlarge the list of FIT-dependent genes and discriminate between genes that are robustly FIT-regulated in roots and seedlings or only in one of those. FIT-regulated genes were mostly induced, few of them were repressed by FIT. With the analysis of a virtual dataset we could filter out and pinpoint new candidates among the most reliable marker genes for iron deficiency. Moreover, co-expression and functional analysis of this virtual dataset revealed iron deficiency-induced and functionally distinct regulons.

Keywords: Arabidopsis; Differential gene expression; FIT; Iron homeostasis; Microarray; Plants.

Fig. 1

Venn diagrams of the differentially expressed genes in the six-week-old Arabidopsis roots (a-f) and six-day-old seedlings (g-l). Intra-line comparisons in roots (a-c): Total numbers of regulated genes (a), induced genes (b) and repressed genes (c). Inter-line comparisons in roots (d-f): Total numbers of regulated genes (d), induced genes (e) and repressed genes (f). Intra-line comparisons in seedlings (g-i): Total numbers of regulated genes (g), induced genes (h) and repressed genes (i). Inter-line comparisons in seedlings (j-l): Total numbers of regulated genes (j), induced genes (k) and repressed genes (l). Intersection between regulated genes in roots and seedlings (m). The diagrams were created using the unnamed online tool provided by VIB/U Gent, Bioinformatics & Systems Biology, Technologiepark 927, B-9052 Gent, Belgium; accessible through http://bioinformatics.psb.ugent.be/webtools/Venn/

Fig. 2

Hierarchical clustering of the genes that were differentially regulated in six-day-old seedlings (a and b). Roots of six-week-old plants (c) and in the combined analysis (d). The respectively compared lines and conditions are indicated by numbers. 1: fit +Fe vs. WT  +Fe. 2: fit -Fe vs. WT -Fe. 3: HA-FIT  +Fe vs. WT  +Fe. 4: HA-FIT -Fe vs. WT -Fe. 5: HA-FIT -Fe vs. HA-FIT  +Fe. 6: WT -Fe vs. WT  +Fe. 7: fit -Fe vs. fit  +Fe. The left panels show an overview over the whole cluster analysis and the right panel shows a magnified view of the respective cluster that is indicated by the red triangle and that contains known iron homeostasis-related genes. Red color represents up-regulation and green color represents down-regulation. The cluster analysis has been performed with Genesis [76]

Fig. 3

Four-step filtering of FIT-induced genes using scatterplot analysis of log₂ fold changes of gene expression in the respective comparison in seedlings (horizontal) and roots (vertical). The blue dots represent genes that did not match the requirement and were removed in the subsequent step. The yellow dots represent gene expression patterns that matched the requirement and which were used as the input for the subsequent pattern analysis. The respective zero-points are indicated by red crosshairs. The genes filtered in a were used as input in b. The genes filtered in b were used as input in c. The genes filtered in c were used as input in d. The yellow dots in d represent the 32 FIT-induced genes (Tables 1 and 2)

Fig. 4

Four-step filtering of FIT-repressed genes using scatterplot analysis of log₂ fold changes of gene expression in the respective comparison in seedlings (horizontal) and roots (vertical). The blue dots represent genes that did not match the requirement and were removed in the subsequent step. The yellow dots represent gene expression patterns that matched the requirement and which were used as the input for the subsequent pattern analysis. The respective zero-points are indicated by red crosshairs. The genes filtered in a were used as input in b. The genes filtered in b were used as input in c. The genes filtered in c were used as input in d. The yellow dots in d represent the FIT-repressed genes (Tables 1 and 2)

Fig. 5

Filtering of temporally FIT-induced genes using scatterplot analysis of log₂ fold changes of gene expression in the respective comparisons in seedlings (a and b) and roots (c and d). The blue dots represent genes that did not match the requirement and were removed in the subsequent step. The yellow dots represent gene expression patterns that matched the requirements and which were used as the input for the subsequent pattern analysis. The respective zero-points are indicated by red crosshairs. The genes filtered in a were used as input in b. The genes filtered in c were used as input in d. The yellow dots in b and d represent FIT-induced genes in six-day-old seedlings (b) (Additional file 5: Table S3) and in roots of sic-week-old plants (d) (Additional file 5: Table S4). The filtering steps 1 and 2 as well as 3 and 4 are combined in one graph, respectively

Fig. 6

Filtering of temporally FIT-repressed genes using scatterplot analysis of log₂ fold changes of gene expression in the respective comparisons in seedlings (a and b) and roots (c and d). The blue dots represent genes that did not match the requirement and were removed in the subsequent step. The yellow dots represent gene expression patterns that matched the requirements and which were used as the input for the subsequent pattern analysis. The respective zero-points are indicated by red crosshairs. The genes filtered in a were used as input in b. The genes filtered in c were used as input in d. The yellow dots in b and d represent FIT-repressed genes in six-day-old seedlings (b) (Additional file 5: Table S5) and in roots of sic-week-old plants (d) (Additional file 5: Table S6). The filtering steps 1 and 2 as well as 3 and 4 are combined in one graph, respectively

Fig. 7

Co-expression network built from the genes induced under -Fe in the virtual dataset: Regulon 1: contains members of the FIT target network [13]. Regulon 2: consists of members of the iron homeostasis network [13]. Regulon 3: is largely composed of genes involved in phenylpropanoid metabolism. Regulon 4: mainly comprises genes that participate in the pentose phosphate pathway, glycolysis and gluconeogenesis. Regulon 5: is mostly composed of genes that are involved in RNA processing and translation. Regulon 6: contains mitochondrial proteins. Regulon 7: is heterogeneous but contains comparably many chaperons. Regulon 8: is enriched in genes involved in amino acid metabolism. Regulon 9: is also heterogeneous but enriched in genes that participate in plant-pathogen interaction. Regulon 10: shows no enrichment of molecular functions. Regulon 11: mainly contains genes that participate in purine, lipid and aromatic compound metabolism. Regulon 12: is composed of genes involved in the response to low sulfur. Regulon 13 shows no enrichment of molecular functions. The network has been created with the String version 10 protein interaction database [27]. The confidence was set to ‘medium’ (0.400) and no genes were added. The 437 genes induced under -Fe in the virtual dataset were used as input. Singlet nodes have been removed and only networks with 4 or more nodes are shown. The resulting network image contains 169 genes (Additional file 5: Table S7). For a high resolution image see Additional file 6: Figure S4

Fig. 8

Summary of the results of our microarray analyses. The big blue circle represents genes that were found regulated in at least one comparison in six-week-old roots and the big red circle contains genes that were found regulated in at least one comparison in six-day-old seedlings. The lower yellow oval consists of genes that were found FIT-induced in six-week-old roots and the lower green oval represents the FIT-induced genes in six-day-old seedlings. The intersection between the lower yellow and green ovals contains the 32 genes that we consider robustly FIT-induced. Eleven of them are novel FIT-regulated genes (brown circle). We also detected FIT-repressed genes. The upper yellow oval represents genes that were found FIT-repressed in six-week-old roots and the upper green oval contains the FIT-repressed genes in six-day-old seedlings. The intersection between the upper yellow and green ovals contains the 2 genes that we consider robustly FIT-repressed