20Diatoms are a diverse and globally important phytoplankton group, responsible for an 21 estimated 20% of carbon fixation on Earth. They frequently form spatially extensive 22 phytoplankton blooms, responding rapidly to increased availability of nutrients including 23 phosphorus and nitrogen. Although it is well established that diatoms are common first-24 responders to nutrient influxes in aquatic ecosystems, little is known of the sensory 25 mechanisms that they employ for nutrient perception. Here we show that diatoms use a novel 26 and highly-sensitive Ca 2+ -dependent signalling pathway, not previously described in 27 eukaryotes, to sense and respond to the critical macronutrient phosphorus. We demonstrate that 28 phosphorus-Ca 2+ signalling is essential for regulating diatom recovery from phosphorus 29 limitation, by controlling rapid and substantial increases in nitrogen assimilation. Phosphorus-30 Ca 2+ signalling thus mediates fundamental cross-talk between the vital nutrients P and N to 31 maximise resource competition, and likely governs the success of diatoms as major bloom 32 formers in regions of pulsed nutrient supply. Importantly, our study demonstrates that distinct 33 mechanisms for nutrient sensing have evolved in photosynthetic eukaryotes. 34 35 36 37 38 39 Riveras et al., 2015), and Zn 2+ (Behera et al., 2017), but not phosphate (Matthus et al., 2019).87Nitrate resupply to nitrogen-limited Arabidopsis plants induces [Ca 2+ ]cyt elevations, which 88 triggers several nitrate-associated regulatory responses, orchestrated via Ca 2+ -dependent 89 5 protein kinases (Liu et al., 2017). This work raises important questions about the role of Ca 2+ 90 signalling in nutrient sensing in eukaryotes more broadly. Certainly, diatoms use Ca 2+ 91 signalling for perception of several abiotic and biotic stimuli (Falciatore et al., 2000; Helliwell 92 et al., 2019; Vardi et al., 2006). Moreover, our recent identification of a novel class of voltage-93 gated channels in diatoms (EukCatAs), demonstrates that they have evolved unique 94 mechanisms for environmental perception in the oceans (Helliwell et al., 2019). Here, we report 95 the discovery of a phosphorus-Ca 2+ -signalling pathway that is essential for phosphorus sensing 96 and acclimation in Phaeodactylum tricornutum, and likely provides a competitive advantage 97 to diatoms in regions of pulsed or intermittent phosphorus supply. 98 99 Results resupply 102 To investigate the role of Ca 2+ signalling in nutrient sensing in diatoms, we used a transgenic 103 Phaeodactylum tricornutum line (PtR1), encoding the genetically-encoded fluorescent Ca 2+ 104 biosensor, R-GECO1 (Helliwell et al., 2019; Zhao et al., 2011). PtR1 cells were grown in f/2 105 medium (Guillard and Ryther, 1962) made up in natural seawater (NSW), but with reduced 106 concentrations of phosphate, nitrate or f/2 trace metals (Materials and Methods). We then 107 monitored single-cell R-GECO1 fluorescence of nutrient deplete cells, following resupply with 108 each respective nutrient...
