Acclimation and stress response of Prochlorococcus to low salinity

Prochlorococcus is an obligate marine microorganism which are dominant autotroph in tropical and subtropical central oceans. However, what is the low salinity boundary and how Prochlorococcus would response to low salinity exposure is still unknown. In this study, we first tested the growing salinity range of two Prochlorococcus strains, NATL1A and MED4, and then compared the global transcriptome of their low salinity acclimated cells and cells growing in normal seawater salinity. We found that MED4 could be acclimated in the lowest salinity of 25% and NATL1A could be acclimated in the lowest salinity of 28%. Measurement of the effective quantum yield of PSII photochemistry (Fv/Fm) indicated that both strains were stressed when growing in salinity lower than 34%. The transcriptomic response of NATL1A and MED4 were approximately different, with much more genes having changed transcript abundance in NATL1A than in MED4. To cope with low salinity, NATL1A downregulated the transcript of most genes involved in translation, ribosomal structure and biogenesis, while MED4 upregulated those genes. Moreover, low salinity acclimated NATL1A cells suppressed ATP-producing genes and induced the expression of photosynthesis related genes, while low salinity acclimated MED4 upregulated ATP-producing genes and downregulated photosynthesis related genes. These results indicate that the response to low salinity stress of different Prochlorococcus strains could be distinct. The study provided the first glimpse into the growing salinity range of Prochlorococcus cells and their global gene expression changes due to low salinity stress. Overall design: Prochlorococcus strains MED4 and NATL1A were acclimated to different salinities (24%, 25%, 26%, 27%, 28%, 30%, 32%, 34%) by consecutive transfers from exponential growing cultures to fresh media. Three biological replicates were set up for each salinity. Five rounds of transfer were conducted for each strain. Cell abundance was monitored at day 0, day 5 and day 10, using flow cytometry. To assess the stress to low salinity, the dark-adapted photochemical efficiency (Fv/Fm) of each strain was monitored in day 10 in each round, using a handheld fluorometer (AquaPen AP 110/C, Photon Systems Instruments). To acclimate the Prochlorococcus strains, MED4 and NATL1A were growing in the Pro99 medium of salinity 28% and 34% for five rounds of inoculation. Then the acclimated cultures were inoculated in fresh medium of salinity 28% and 34%, with salinity 34% being the control. Three biological replicates were set up. During the exponential growth phase, 100 ml cultures were filtered onto 0.22 µm polycarbonate membrane to collect cells and the membranes were immediately flash frozen in RNAlater by liquid nitrogen and stored at -80? until RNA extraction. Total RNA was extracted from the membrane using the MagZol Reagent (Magen). Sequencing libraries were prepared using VAHTSTM Stranded mRNA-seq Library Prep Kit for Illumina® (Vazyme biotech co., Ltd) following the manufacturer's instructions. Libraries were multiplexed and sequencing was carried out on an Illumina HiSeq system with the 2 × 150 paired-end (PE) configuration (GENEWIZ).