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Phosphorus availability regulates intracellular nucleotides in marine eukaryotic phytoplankton

ABSTRACT: Phosphorus limitation is pervasive in the oligotrophic surface ocean and marine microorganisms use different strategies to survive, and thrive, at these low nutrient levels. Eukaryotic algae such as diatoms are extremely sensitive to phosphorus limitation and recent transcriptomics work has suggested that multiple cellular processes are affected under these growth conditions. Metabolomics is the systematic study of intra- and extracellular metabolites, i.e. the end products of microbial metabolism. As such, metabolomics complements genomics and transcriptomics through the identification and quantification of metabolic intermediates that reflect cellular physiology. Here, we applied intracellular metabolomics to examine the differential response of the model diatom Thalassiosira pseudonana to phosphate-replete and phosphate-limited growth conditions. We focused on metabolites from the purine and pyrimidine biochemical pathways, due to their role in phosphorus cycling associated with nucleic acid synthesis. Under phosphate-replete conditions, T. pseudonana stored nucleotides with phosphate moieties such as adenosine 5'-monophosphate (AMP) and, to a lesser extent, inosine 5'-monophosphate (IMP). In contrast, under phosphate-limited conditions, T. pseudonana had higher concentrations of adenine, inosine, and adenosine all of which lack phosphate moieties. Furthermore, based on previously published transcriptomics data, T. pseudonana differentially regulates select genes that can alter these nucleic acid precursors through the gain or loss of the phosphate moiety. Thus, our analysis of the metabolomics and transcriptomics data converged upon the sensitivity of the purine biochemical pathway to phosphorus availability.

INSTRUMENT(S): TSQ Vantage (Thermo Scientific), Thermo Scientific LTQ FT Ultra

SUBMITTER: Krista Longnecker

PROVIDER: MTBLS154 | MetaboLights | 2015-01-27

REPOSITORIES: MetaboLights

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			Action	DRS
	MTBLS154	Other
	FILES	Other
	a_MTBLS154_NEG2_metabolite_profiling_mass_spectrometry.txt	Txt
	a_MTBLS154_NEG_metabolite_profiling_mass_spectrometry.txt	Txt
	a_MTBLS154_POS2_metabolite_profiling_mass_spectrometry.txt	Txt

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Project description:Phosphorus (P) is a critical driver of phytoplankton growth and ecosystem structure and function in the ocean. Diatoms are an abundant and widespread functional group of phytoplankton that are responsible for significant amounts of primary production in the ocean, however there has not been a comprehensive study of diatom physiological responses to P deficiency. Here, we coupled deep sequencing of transcript tags and quantitative proteomic analysis from the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. The reads (tags) were mapped to the T. pseudonana genome sequence, confirming expression of 91% of the modeled gene set. A total of 318 genes were differentially regulated with a false discovery rate of p<0.05. A total of 1264 proteins were detected, and of those 136 were differentially expressed with a false discovery rate of p<0.05. Significant changes in the abundance of transcripts and proteins were observed and these changes were coordinated for glycolysis, translation, and multiple biochemical responses to P deficiency. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P (DOP) through increased production of alkaline phosphatase metalloenzymes and a diesterase, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to rapidly respond to variations in environmental P availability. T. pseudonana (Strain 1335 from the Provosoli-Guillard National Center for the Culture of Marine Phytoplankton (CCMP)) was grown in a modified f/2 medium made from Sargasso Sea water. Macronutrients and vitamin B12, biotin, and thiamine solutions were treated with prepared Chelex-100 resin to remove trace metal contaminants followed by trace-metal clean syringe sterilization to yield final nutrient concentrations of 882 μM NaNO3 and 106 μM Na2SiO3, and vitamin concentrations of 75 pM, 400 pM, and 60 nM, respectively. The Fe concentration was also modified from f/2 to 400 nM. All conditions were run in triplicate at 14 ºC, in constant light (120 µmol photons m-2 s-1). Cells were grown with f/2 phosphorus concentrations (P-replete; 36 µM PO4) and with low phosphorus concentrations (P-deficient; 0.4 µM PO4). Growth was monitored daily with cell counts. Duplicate P-replete treatments were pooled and harvested in mid log phase, and triplicate P-deficient treatments were pooled, and also quickly harvested onto 2 µm filters at the onset of P depletion. All RNA samples were snap frozen in liquid nitrogen. Replicate P-deficient cultures were refed to 36 µM phosphate at the onset of P depletion, and subsequently resumed growth. Additional growth studies were performed as described above substituting glycerolphosphate and adenosine monophosphate at 36 µM, for the phosphate in the medium.

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Phosphorus availability regulates intracellular nucleotides in marine eukaryotic phytoplankton

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