Phytoplankton transcriptomic and physiological responses to fixed nitrogen in the California Current System

In summer 2014, we conducted experiments to determine the effects of different N substrates on phytoplankton communities in the North Pacific Ocean and in the transition zone of the California Current and gyre (Shilova, Mills et al., 2017). Samples were incubated with nitrate, ammonium, urea, and filtered deep water (FDW) for 48 hours (T48). Two treatments added iron, alone (Fe) or with a mix of N substrates (N+Fe), to determine the effects of Fe on the utilization of N substrates. All treatments resulted in changes in phytoplankton cell abundances and photosynthetic activity at both locations, with differences between phytoplankton groups. Prochlorococcus had large increases in biomass in response to ammonium and urea, while both eukaryotic phytoplankton and Synechococcus had only modest biomass increases in response to N+Fe and FDW. Moreover, distinct physiological responses were observed within sub-populations of Prochlorococcus and Synechococcus. In order to understand the variable responses to N substrates among phytoplankton groups and sub-populations in the California Current transition zone, the present work examines transcriptional changes that occurred 24 h after the substrates were added. Specifically, we hypothesize that transcription changes at 24 h indicate which phytoplankton taxa are N-limited, and thus help explain changes in cell abundances and photosynthetic activity by individual phytoplankton groups observed at 48 h. Furthermore, we hypothesize that the diversity in physiological responses within Prochlorococcus and Synechococcus are evident in the transcriptional responses measured at sub-population resolution. Transcription changes by surface sea water microbial communities in response to added nitrogen and/or iron substrates were assessed using MicroTOOLs microarrays (version 2.0; GEO platform GPL24371). On 24 August 2014, samples were collected from 25 m depth at Station 38 in the California Current System, during the NEMO Cruise NH1417. Samples were handled using trace-metal clean techniques, with all treatments and nucleic acid collection occurring before dawn (Shilova et al. 2017). Replicate samples (n=2 or 3) were treated with different nitrate, ammonium, urea, iron, nitrate with iron, or filtered deep water (600 m), and incubated on deck. After 24 h incubation, mRNA was collected for the present work, however incubations continued for another 24 h. At 24 h and 48 h, bulk physiological measurements were made, and DNA was extracted for community composition analysis, described in Shilova et al. 2017. The present work examines which community members responded to the different treatments by 24 h in order to understand their nutrient statuses and possible contributions to the physiological changes observed by Shilova et al. 2017. Metatranscriptomes from the six treatments (as represented by MicroTOOLs) at 24 h were compared to control metatranscriptomes at 24 h. Comparisons were also made between control metatranscriptomes at 24 h and 0 h. Microarray data was processed using the MicroTOOLs software pipeline (www.jzehrlab.com), and transcription changes were examined using the pipeline as well as an Ensemble of Gene Set Enrichment Analyses (Alhamdoosh et al. 2017) and Whole Genome Correlation Network Analysis (Langfelder and Horvath, 2008). CCS = California Current System control samples: Control_T0_id_64220, Control_T0_id_64221, Control_T0_id_64222, Control_T24_id_60949, Control_T24_id_60950, Control_T24_id_60951 treated samples: Fe_T24_id_60964, Fe_T24_id_60965, Fe_T24_id_60966, Urea_T24_id_60958, Urea_T24_id_60959, Urea_T24_id_60960, NO3_T24_id_60955, NO3_T24_id_60956, NO3_T24_id_60957, NH4_T24_id_60952, NH4_T24_id_60954, N+Fe_T24_id_60967, N+Fe_T24_id_60968, N+Fe_T24_id_60969, FDW_T24_id_60961, FDW_T24_id_60962, FDW_T24_id_60963