ABSTRACT: Two morphotypes of Limnospira indica (formerly Arthrospira sp.) strain PCC 8005, denoted as P2 (straight trichomes) and P6 (helical trichomes), were subjected to chronic gamma radiation from spent nuclear fuel (SNF) rods at a dose rate of ca. 80 Gy.h-1 for one mass doubling period under continuous light with photoautotrophic metabolism fully active. Samples were taken for post-irradiation growth recovery and RNAseq transcriptional analysis at approximate time in-tervals of 15, 40, and 71.5 hrs corresponding to cumulative doses of ca. 1450, 3200, and 5700 Gy, respectively. Both morphotypes, which were previously reported to display different antioxidant capacities and differ at the genomic level in 168 SNPs, 48 indels and 4 large insertions (Yadav et al., 2019), recovered equally well from 1450 and 3200 Gy. However, while the P2 straight type recov-ered from 5700 Gy by regaining normal growth within 6 days, the P6 helical type took about 13 days to recover from this dose, indicating differences in their radiation tolerance and response. To investigate these differences, P2 and P6 cells exposed to the intermediate dose of gamma radiation (3200 Gy) were analyzed for differential gene expression by RNAseq analysis. A total of 1553 genes (887 and 666 of P2 and P6, respectively, with 352 genes in common) were selected based on a two-fold change in expression and a false discovery rate FDR smaller or equal to 0.05. About 85% of these 1553 genes encoded products of yet unknown function. Of the 229 remaining genes, 171 had a defined function while 58 genes were transcribed into non-coding RNA including 21 tRNAs (all downregulated). From PCC 8005-P2 and PCC 8005-P6 expression patterns it emerges that although the cellular routes used by the two sub strains to cope with ionizing radiation do overlap to a large extent, both strains displayed a distinct preference of priorities likely brought about by marginal differences in their genetic backgrounds. Small inoculants (1 mL) of irradiated and non-irradiated L. indica cultures were grown in 30 mL of fresh Zarrouk media in T-75 tissue flasks (Thermofisher Scientific). All cultures were grown in triplicates per exposed dose with their respective non-irradiated cultures under standard laboratory conditions. Recovery was followed at OD750 every alternate day for a period of 30 days. The proliferation curve was plotted as OD750 versus time using Graphpad Prism v7 (GraphPad Software, La Jolla, California, USA – https://www.graphpad.com/) Triplicates of a non-irradiated control per dose were kept at otherwise analogous conditions in the lab. The L. indica P2 and P6 culture samples were collected in time in-tervals at three prechosen time points T1 to T3 of exposure (~15, ~40, and ~71.5h) corresponding to approximate cumulative doses of respectively 1450, 3200, and 5700 Gy, with the actual doses for the individual samples determined by dosimetry using Harwell Amber-3042 radiation-sensitive polymethylmethacrylate dosimeters attached to the cul-ture tubes. Genes were considered as being differentially expressed if they abided to the selection criteria: -1 >= log2FC >= 1, with an FDR equal or below 0.05 for both options.
