General overview of the main transcriptional response events of <i>Arthrospira</i> sp. PCC 8005 after exposure to ⁶⁰Co gamma rays.

<p>Schemes represent a global gene expression response (A) immediately after irradiation; (B) after 2H and 5H of recovery period. Blue colour, stand for down-regulated genes. Red colour stand for up-regulated genes, Green colour stand for restored expression of the initial silenced genes. (A) The largest changes in transcription occurred upon irradiation, as part of a kind of an “Emergency Response”. Cells displayed a <b>reduced transcription</b> for photosynthesis and energy production (PSII, PSI, ATP), and for carbon and nitrogen metabolism during irradiation. The CO₂ fixation via the Calvin-Benson-Bassham cycle (CBB), glycogen biosynthesis (gluconeogenesis) and the tricarboxylic acid cycle (TCA) were repressed. The transcription of the SigE regulator acting as nitrogen-dependent activator for catabolic genes towards glycogen degradation (glycolysis) was induced. Also a re-routing of the metabolic flux to glycolysis and the pentose phosphate pathway (PPP) was seen. A synthesis of carbon storage molecules (PHA) and compatible solutes (trehalose) was seen. The expression of polyamine import (<i>potBC)</i>, well known as a group of nitrogen-containing C-compounds which help in cell survival during stress, was recorded. The import of nitrate or cyanate as N-sources was repressed (<i>nrtABCD</i>, <i>cynBD</i>). In parallel also the metabolism of agmatine, a known competitive inhibitor of polyamine transport, was repressed. The cellular protection, detoxification, and repair were <b>enhanced</b> immediately after irradiation. In an effort to maintain the intracellular redox balance while provide sufficient metal-cofactors for enzymes, selective metal export (<i>copA)</i> and import (<i>feoAB</i>, <i>cutA</i>, <i>corA</i>, <i>mtgC</i>, <i>cbiQ1</i>, <i>cbiQ2</i>, <i>znuA</i>) was induced. There was upregulation of isiA gene encoding the CP43’ protein, which is an auxiliary antenna complex, to compensate for the loss of phycobilisomes. This protein may also serve as a chlorophyll storage molecule contributing to the reassembly of reaction centres during recovery. In addition, ROS detoxification was activated via the expression of the peroxiredoxine enzyme (<i>ahpC</i>) and the glutathione synthesis genes. The generation of glutathione starts at T0H via the formation of glutamate from proline by <i>hyuA</i>, from aspartate by aspartate aminotransferase <i>(aat1)</i>, from 1-pyrroline-5-carboxylate by (<i>putA</i>), and from 2-oxoglutarate via GLDH (see Fig 8B). Glutamate synthesis via the GS/GOGAT cycle was repressed. The final synthesis of glutathione from glutamate occurred via glutathione (GSH) synthase <i>(gshB)</i>, which continued during recovery (see Fig 8B). Chaperones (<i>dnaK1</i>, <i>dnaK2</i>, <i>hspA</i>, <i>cbpA</i>) and proteases (<i>clpB2</i>) were also significantly induced during this stage, to remove damaged proteins. The free amino-acids released from protein degradation, likely lead to the production of urea, and the urease (<i>ureABC)</i> activity, transforming urea to ammonium, was induced. In parallel <i>Arthrospira</i> enhance some genes related to DNA repair system (<i>uvrBCD</i> for nucleotide excision and repair, <i>ruvB</i> resolving holiday junction, and <i>recJ</i>, <i>dnaG and mod</i> genes). The DNA-repair mechanism of <i>Arthrospira</i> included also enzymatic restriction modification (<i>hsdr</i>) and endonucleases. (B) During the <u><b>later phase</b></u><i>Arthrospira</i> cells try to <u><b>recover from the damage; which lead to a slowly restored expression</b></u> of the genes related to photosynthesis and energy production, carbon fixation via the CBBn cycle and gluconeogenesis, TCA cycle. Expression of the hydrogenase genes (<i>hypA1</i>, <i>hypB1and hoxW</i>). Metal chaperone proteins HypA and HypB are required for the nickel insertion step of [NiFe]-hydrogenase maturation. In parallel slight reactivation of amino-acid transport (<i>aapJPQ</i>, <i>argGHJ</i>, <i>iaaA</i>) occurred. The genes for import of taurine (<i>tauABC</i>) known as organic sulphur and amino source were highly induced. The restoration of agmatinase, the key enzyme of agmatine hydrolysis was seen in recovery period. ROS detoxification was maintained efficiently via the expression for glutathione biosynthesis (GSH). Few genes related to protein damage clean up (proteases and chaperones) and DNA repair maintained their expression during recovery. The expression of gene cluster <i>arhABCDEF</i>, enriched during recovery, was seen.</p

Scatter plot showing the differentially expressed genes of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation plotted accordingly to their change in mRNA concentration (Log₂ fold change values), for 3 radiation doses (800, 1600 and 3200 Gy) and 3 time points after radiation (0 hours, 2 hours, 5 hours).

<p>Scatter plot showing the differentially expressed genes of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation plotted accordingly to their change in mRNA concentration (Log₂ fold change values), for 3 radiation doses (800, 1600 and 3200 Gy) and 3 time points after radiation (0 hours, 2 hours, 5 hours).</p

Temporal Gene Expression of the Cyanobacterium <i>Arthrospira</i> in Response to Gamma Rays

<div><p>The edible cyanobacterium <i>Arthrospira</i> is resistant to ionising radiation. The cellular mechanisms underlying this radiation resistance are, however, still largely unknown. Therefore, additional molecular analysis was performed to investigate how these cells can escape from, protect against, or repair the radiation damage. <i>Arthrospira</i> cells were shortly exposed to different doses of ⁶⁰Co gamma rays and the dynamic response was investigated by monitoring its gene expression and cell physiology at different time points after irradiation. The results revealed a fast switch from an active growth state to a kind of 'survival modus' during which the cells put photosynthesis, carbon and nitrogen assimilation on hold and activate pathways for cellular protection, detoxification, and repair. The higher the radiation dose, the more pronounced this global emergency response is expressed. Genes repressed during early response, suggested a reduction of photosystem II and I activity and reduced tricarboxylic acid (TCA) and Calvin-Benson-Bassham (CBB) cycles, combined with an activation of the pentose phosphate pathway (PPP). For reactive oxygen species detoxification and restoration of the redox balance in <i>Arthrospira</i> cells, the results suggested a powerful contribution of the antioxidant molecule glutathione. The repair mechanisms of <i>Arthrospira</i> cells that were immediately switched on, involve mainly proteases for damaged protein removal, single strand DNA repair and restriction modification systems, while <i>recA</i> was not induced. Additionally, the exposed cells showed significant increased expression of <i>arh</i> genes, coding for a novel group of protein of unknown function, also seen in our previous irradiation studies. This observation confirms our hypothesis that <i>arh</i> genes are key elements in radiation resistance of <i>Arthrospira</i>, requiring further investigation. This study provides new insights into phasic response and the cellular pathways involved in the radiation resistance of microbial cells, in particularly for photosynthetic organisms as the cyanobacterium Arthrospira.</p></div

Gene Set Enrichment Analysis (GSEA) in the clusters of differentially expressed genes of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation based on the Clusters of Orthologs Groups (COG) functional categories.

<p>This plot visualises whether a certain gene cluster (1–9, on the vertical axis) is containing a higher number of representatives of a specific COG (21 different COGs, on the horizontal axis) then would be expected by chance. The COG functional category is shown in the vertical direction, the clusters of differentially expressed genes in the horizontal direction. The colour code is according to the ¹⁰log value of the corresponding p-value of the GSEA analysis: a p-value of smaller than 1.10^–4 (¹⁰log-value of 4) results in a colour code red, a p-value of 1 (¹⁰log value 0) results in colour code white.</p

Photosynthetic capacity of <i>Arthrospira</i> sp. PCC 8005 after gamma irradiation.

<p>The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Data represent the mean of three independent biological replicates (n = 3) and error bars display the standard error of the mean (SEM). The statistical analysis was calculated on raw data, before normalisation to percentages.</p

Distribution of the fold-changes of all proteins quantified (185) in both biological replicates of the LD growth cycle using the post-digest ICPL method.

<p>Distribution of the fold-changes of all proteins quantified (185) in both biological replicates of the LD growth cycle using the post-digest ICPL method.</p

Principal Component Analysis (PCA) of the 9 cluster centres created using the Mfuzz clustering software for the gene expression of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation.

<p>Principal Component Analysis (PCA) of the 9 cluster centres created using the Mfuzz clustering software for the gene expression of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation.</p

Dynamic changes in gene expression of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation, displayed in 9 clusters using the Mfuzz clustering software, according to their gene expression profile during recovery time (0 hours, 2 hours, 5 hours), for 3 radiation doses (800, 1600 and 3200 Gy).

<p>Dynamic changes in gene expression of <i>Arthrospira</i> sp. PCC 8005 in response to gamma irradiation, displayed in 9 clusters using the Mfuzz clustering software, according to their gene expression profile during recovery time (0 hours, 2 hours, 5 hours), for 3 radiation doses (800, 1600 and 3200 Gy).</p

Pigment content of <i>Arthrospira</i> sp. PCC 8005 after irradiation.

<p>The data obtained for the irradiated samples were normalized against and are shown as percentage of their representative non-irradiated control (which was put at 100%). Allophycocyanin content, Phycocyanin content, and Chlorophyll A content were measured at T0H, T2H and T5H respectively. Data represent the mean of three independent biological replicates, and error bars present the standard error of the mean (SEM). The statistical analysis was calculated on raw data, before normalisation to percentages. One asterisk indicates a value which is significantly differing (p<0.05) from the non-irradiated control.</p

Venn diagram showing non-redundant proteins identified using: IEF, SDS-PAGE, 2D-LC, and 3D-LC MS/MS (IMAC-Cu²⁺).

<p>Venn diagram showing non-redundant proteins identified using: IEF, SDS-PAGE, 2D-LC, and 3D-LC MS/MS (IMAC-Cu²⁺).</p