Microarray analysis of the genome-wide response to iron deficiency and iron reconstitution in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

A full-genome microarray of the (oxy)photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used to identify genes that were transcriptionally regulated by growth in iron (Fe)-deficient versus Fe-sufficient media. Transcript accumulation for 3,165 genes in the genome was analyzed using an analysis of variance model that accounted for slide and replicate (random) effects and dye (a fixed) effect in testing for differences in the four time periods. We determined that 85 genes showed statistically significant changes in the level of transcription (P </= 0.05/3,165 = 0.0000158) across the four time points examined, whereas 781 genes were characterized as interesting (P </= 0.05 but greater than 0.0000158; 731 of these had a fold change >1.25 x). The genes identified included those known previously to be Fe regulated, such as isiA that encodes a novel chlorophyll-binding protein responsible for the pigment characteristics of low-Fe (LoFe) cells. ATP synthetase and phycobilisome genes were down-regulated in LoFe, and there were interesting changes in the transcription of genes involved in chlorophyll biosynthesis, in photosystem I and II assembly, and in energy metabolism. Hierarchical clustering demonstrated that photosynthesis genes, as a class, were repressed in LoFe and induced upon the re-addition of Fe. Specific regulatory genes were transcriptionally active in LoFe, including two genes that show homology to plant phytochromes (cph1 and cph2). These observations established the existence of a complex network of regulatory interactions and coordination in response to Fe availability.

Figure 1.

A, Diagrammatic representation of the loop design utilized for identification of differentially expressed genes in response to iron availability. A total of six slides was used with dye swaps between the 0- and 24-h time points and between 3- and 24-h time points. B, Scatter plot that compares the mean spot intensities of 0 h (low iron [LoFe]) and 12 h (+Fe). Data from 0- and 12-h time points were normalized, and their mean signal intensities were plotted. The black line represents equal labeling for the two samples, whereas the dotted (2-fold) and dashed (3-fold) lines identify genes that demonstrated large labeling differences during the hybridization experiment.

Figure 2.

Venn diagrams that display the number of iron-responsive, differentially expressed genes. Three sample pairs (0/3, 0/12, and 0/24) were compared for increased (A) and decreased (B) transcript levels in response to iron availability. All of the genes had a fold change > 1.25 and P < 0.05. The numbers in the overlapping areas indicate genes that exhibited differential expression in either two or three sample pairs. The numbers in the nonoverlapping areas indicate the number of genes that exhibited differential expression in one sample pair.

Figure 3.

A, Hierarchical cluster display of 866 genes that were used for the functional analysis of the iron-responsive differential expression changes. The expression ratio was calculated relative to time point 0 h (i.e. 0/3, 0/12, and 0/24) and were converted to log scale. These values were then used to cluster genes using Spotfire Decisionsite version 7.0 (Somerville, MA). Euclidean distance was used as measure of similarity between profiles. The various profiles were clustered by using the weighted pair group method with arithmetic mean. Change in transcript levels relative to LoFe: green (down) and red (up). The identification of all genes in this diagram is provided in Supplemental Data Table II (http://www.plantphysiol.org). B, Hierarchical cluster display of genes involved in photosynthetic processes that exhibited fold change of >1.25 and P value of <0.05. C, Schematic representation of various patterns of differentially expressed genes in response to iron availability. The various patterns were generated based on the clustering of genes (860/866) in (A) and numbered accordingly from top to bottom. The x axis represents the time-dependent expression of genes after the addition of Fe, whereas the y axis represents the transcript level on an arbitrary scale. The number of genes in each category is: 1 (n = 437), 2 (n = 74), 3 (n = 42), 4 (n = 26), 5 (n = 79), and 6 (n = 202).

Figure 4.

The isiA region of the genome in which gene expression was enhanced in LoFe cells. The arrows indicate those five genes that demonstrated an increase in transcription in LoFe cells, and the size of the boxes corresponds to the lengths of the open reading frames.