Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Methodology: (see related dataset for the discrete raw measurements from the experiment)
Two phytoplankton species, Chaetoceros decipiens (UNC1416) and Emiliania huxleyi (UNC1419), were cultured at 12 °C in artificial Aquil* medium using trace metal clean (TMC) techniques. Macronutrients were supplied such that nitrate would become limiting for growth (50 μmol L⁻¹ NO₃, 10 μmol L⁻¹ PO₄, 200 μmol L⁻¹ H₄SiO₄) and two iron treatments were used (1370 nmol L⁻¹ and 3.1 nmol L⁻¹ total Fe). Upwelling conveyor belt cycle (UCBC) simulations were performed by transitioning the cultures to different nutrient and light regimes. They were grown by first growing the cultures with ample light (115 µmol photons m⁻²s⁻¹) until nitrate depletion and stationary phase growth. Cultures were then moved to no light for 10 days to simulate the sinking out of the euphotic zone. After this dark period, the cultures were subsequently transferred back to fresh medium under ambient light levels, and grown until stationary phase again. Samples were collected for six different time points associated with the different phases throughout the simulated upwelling cycle.

Sampling:
Cell counts – 5 mL samples was preserved in 2% Lugol’s solution in glass vials. Cells were then enumerated on an Olympus CKX41 inverted microscope using a 1 mL Sedgwick-Rafter counting chamber after allowing the cells to settle for five minutes and counting a minimum of 300 cells within 10-30 fields of view.

Chlorophyll – 50 mL of sample was filtered through 0.45 µm mixed cellulose ester filters under gentle vacuum pressure (< 100 mm Hg) and frozen at -80 °C immediately. Extraction was performed using 90% acetone at -20 °C for 24 hours and measured using a Turner 10-AU fluorometer (Parsons et al., 1984)

Fᵥ:Fₘ – The maximum photochemical yield of photosystem II (Fᵥ:Fₘ) was measured using the Satlantic FIRe (Gorbunov & Falkowski, 2005; Kolber et al., 1998). Samples were acclimated to low light for 20 minutes prior to the measurements. Data were blank corrected using microwave-sterilized Aquil medium. The resulting Fᵥ:Fₘ was derived from the induction profile using a saturating pulse (Single Turnover Flash; 20,000 µmol photons m⁻²s⁻¹) for a duration of 100 µs. The gain was optimized for each sample (400, 600, or 800), and the average of 50 iterations was obtained.

Particulate carbon and nitrogen – 50 mL of sample was vacuum-filtered onto pre-combusted GF/F filters. Filters were then immediately stored in Petri dishes at -20 °C, and dried at 65 °C for 24 hours before being pelletized. Total nitrogen and carbon were quantified with a Costech 4010 CHNOS Elemental Combustion system according to U.S. Environmental Protection Agency Method 440.0 (Zimmermann et al., 1997). Three blanks were run alongside the samples and were all below the detection limits (0.005 mg N and 0.071 mg C). Carbon and nitrogen per cell were calculated by dividing particulate carbon and nitrogen concentration by cell concentration.

Dissolved nitrate and nitrite – Filtrate from the 0.45 μm filters used for RNA was transferred to polypropylene tubes and immediately frozen at -80 °C. Dissolved nitrate + nitrite (NO₃ + NO₂) concentrations were quantified with an OI Analytical Flow Solutions IV auto analyzer according to EPA method 353.4 (Zhang et al., 1997). The detection limit for NO₃ + NO₂ was 0.2 µmol L⁻¹.

RNA Extraction – 300 mL of sample was filtered onto 0.45 μm Pall Supor polyethersulfone filters (47 mm) filters using gentle vacuum pressure and immediately frozen and stored at -80 °C. Total RNA was extracted using RNAqueous-4PCR Total RNA Isolation Kit for

Chaetoceros cultures, and TRIzol reagent for E. huxleyi cultures. RNA was then purified and examined for quality and quantity using a Nanodrop spectrophotometer.

Analysis:
RNA libraries were constructed using a custom protocol for 3' poly-A-directed mRNA-seq (also known as TagSeq) based on Meyer et al. (2011) and adapted for Illumina HiSeq based on Lohman et al. (2016) and Strader et al. (2016). For most samples 1 µg but as low as 250 ng of total RNA were fragmented by incubating at 95 °C for 15 min.

First strand cDNA was synthesized with SMARTScribe Reverse Transcriptase (Takara Bio, Mountain View, CA, USA), an oligo-dT primer, and template switching to attach known sequences to each end of the poly-A mRNA fragments. cDNA was then amplified with a PCR reaction consisting of 32 µL sterile H₂O, 5 µL dNTPs (2.5 mM each), 5 µL 10X Titanium Taq Buffer (Takara Bio), 1 µL of each primer (10 µM) designed amplify the sequences attached during cDNA synthesis, and 1 µL of Titanium Taq DNA Polymerase (Takara Bio). The PCR was run with an initial denaturing step at 95 °C for 5 min, then 17 cycles consisting of 95 °C for 1 min, 63 °C for 2 min, and 72 °C for 2 min. cDNA amplification was then verified on a 1% agarose gel and purified with the QiaQuick PCR Purification Kit (Qiagen). cDNA was quantified with the Quant-iT dsDNA High-Sensitivity Assay (Invitrogen) and cDNA concentrations were then normalized to the same volume.

Samples were then barcoded with a PCR reaction consisting of 11 µL sterile H₂O, 3 µL dNTPs (2.5 mM each), 3 µL 10X Titanium Taq Buffer (Takara Bio), 0.6 µL TruSeq Universal Primer (10 µM), 6 µL barcoding primer (1 µM), Titanium Taq Polymerase (Takara Bio), and 6 µL of purified cDNA with an initial denaturing step at 95 °C for 5 min, then 5 cycles consisting of 95 °C for 40 s, 63 °C for 2 min, and 72 °C for 1 min. Samples were barcoded from both ends using unique combinations of 6 bp sequences on both the TruSeq universal primer and barcoding primers. Products were then again confirmed on a 2% agarose gel and combined into small pools of 6-8 samples. The 400-500 bp region of these pools was extracted with the QIAquick Gel Extraction Kit (Qiagen), quantified with the Quant-iT dsDNA High-Sensitive Assay, and then mixed in equal proportions. The library was sequenced at the University of Texas at Austin Genomic Sequencing and Analysis Facility on Illumina HiSeq 2500 (three lanes, single-end 50bp reads) with a 15% PhiX spike-in to target approximately 8 million reads per sample.

The following were used for data processing:
Trimmomatic v0.36
Trinity v2.5.1
CD-HIT-EST v4.7
KEGG (Release 86.0)
UniProt (Release 2018_03)
PhyloDB (v1.076)
BLASTP v2.7.1
Pfam 31.0
HMMER v3.1b2

NSF Award Abstract:
Upwelling zones are hotspots of photosynthesis that are very dynamic in space and time. Microsocopic algae, known as phytoplankton, bloom when deep, nutrient-rich waters are upwelled into sunlit surface layers of the ocean, providing nourishment that supports productive food webs and draws down carbon dioxide (CO2) from the atmosphere to the deep ocean. Photosynthetic microbes in these regions must constantly adapt to changes in their chemical and physical environments. For example, subsurface populations respond to changes in light as they approach the surface. When upwelled waters move offshore, cells sink out of the illuminated zone, establishing seed populations that remain inactive until the next upwelling event. This process is called the upwelling conveyor belt cycle (UCBC). How phytoplankton respond to these changes in environmental conditions and how they may influence their nutrient requirements remains unknown. With future ocean changes predicted to alter seawater chemistry, including ocean acidification and decreased iron availability, some phytoplankton groups may be more vulnerable than others. Accompanying educational activities provide learning experiences to enhance understanding and awareness of marine microbes. The development of a research hub at UNC aims to provide infrastructure and support for scientists and students conducting research on environmental genomics. A laboratory component for an upper-level undergraduate course focused on marine phytoplankton is being developed. Educational outreach activities to broader communities include creation of a lesson plan on phytoplankton in upwelling zones and a virtual research cruise experience for middle-school students, as well as a hands-on lab activity for a local museum focused on marine phytoplankton and the important roles they play in shaping our planet.

The project examines how phytoplankton respond at the molecular and physiological level to the different UCBC stages, which seed populations (i.e., surface versus subsurface) contribute most to phytoplankton blooms during upwelling events of varying intensity, how phytoplankton elemental compositions are altered throughout UCBC stages, and how future predicted ocean conditions will affect the phytoplankton responses to UCBC conditions. This project contains both laboratory and fieldwork. In the laboratory, phytoplankton isolates recently obtained from upwelling regions are exposed to simulated UCBC conditions to examine changes in gene expression, growth and photosynthetic characteristics and elemental composition. Cultures are subjected to both current and future ocean conditions, including reduced iron availability and higher CO2. In the field, research cruises within upwelling regions study the dynamics of natural phytoplankton communities (both surface and subsurface) experiencing upwelling and relaxation and within simulated upwelling incubation experiments. Knowledge of how phytoplankton are affected by UCBC conditions at an integrated molecular, physiological and elemental level under both current and future scenarios is imperative for the proper conservation and management of these critically important ecosystems.