Trupti Gaikwad’s research while affiliated with The University of Warwick and other places

Diatoms are a group of silicified algae that play a major role in marine and freshwater ecosystems. Diatom chloroplasts were acquired by secondary endosymbiosis and exhibit important structural and functional differences from the primary plastids of land plants and green algae. Many functions of primary plastids, including photoacclimation and inorganic carbon acquisition, are regulated by calcium-dependent signalling processes. Calcium signalling has also been implicated in the photoprotective responses of diatoms; however, the nature of calcium elevations in diatom chloroplasts and their wider role in cell signalling remains unknown. Using genetically encoded calcium indicators, we find that the diatom Phaeodactylum tricornutum exhibits dynamic calcium elevations within the chloroplast stroma. Stromal calcium ([Ca2+]str) acts independently from the cytosol and is not elevated by stimuli that induce large cytosolic calcium ([Ca2+]cyt) elevations. In contrast, high light and exogenous hydrogen peroxide (H2O2) induce large, sustained [Ca2+]str elevations that are not replicated in the cytosol. Measurements using the fluorescent H2O2 sensor roGFP2-Oxidant Receptor Peroxidase 1 (Orp1) indicate that [Ca2+]str elevations induced by these stimuli correspond to the accumulation of H2O2 in the chloroplast. [Ca2+]str elevations were also induced by adding methyl viologen, which generates superoxide within the chloroplast, and by treatments that disrupt non-photochemical quenching (NPQ). The findings indicate that diatoms generate specific [Ca2+]str elevations in response to high light and oxidative stress that likely modulate the activity of calcium-sensitive components in photoprotection and other regulatory pathways.

Diatoms are a group of silicified algae that play a major role in marine and freshwater ecosystems. Diatom chloroplasts were acquired by secondary endosymbiosis and exhibit important structural and functional differences from the primary plastids of land plants and green algae. Many functions of primary plastids, including photoacclimation and inorganic carbon acquisition, are regulated by calcium-dependent signalling processes. Calcium signalling has also been implicated in the photoprotective responses of diatoms, although the nature of calcium elevations in diatom chloroplasts and their wider role in cell signalling remains unknown. Using genetically encoded calcium indicators, we find that the diatom Phaeodactylum tricornutum exhibits dynamic chloroplast calcium elevations that are distinct from those found in land plants. Chloroplast calcium ([Ca2+]chl) acts independently from the cytosol and is not elevated by stimuli that induce large cytosolic calcium ([Ca2+]cyt) elevations. In contrast, high light and exogenous hydrogen peroxide (H2O2) induce large, sustained calcium elevations in the chloroplast stroma that are not replicated in the cytosol. Measurements using the fluorescent H2O2 sensor roGFP2-Orp1 indicate that [Ca2+]chl elevations induced by these stimuli correspond to the accumulation of H2O2 in the chloroplast. [Ca2+]chl elevations were also induced by the addition of methyl viologen, which acts to generate superoxide within the chloroplast, and by treatments that disrupt non-photochemical quenching (NPQ). The findings indicate that diatoms generate specific calcium elevations in the chloroplast in response to high light and oxidative stress that likely modulate the activity of calcium-sensitive components in photoprotection and other regulatory pathways.

Successful recognition of pathogen effectors by plant disease resistance proteins (effector triggered immunity, ETI) contains the invading pathogen through a localized hypersensitive response (HR). In addition, ETI activates long-range signalling cascades that establish broad spectrum systemic acquired resistance (SAR). Using a novel and sensitive reporter we have been able to image the spatio- temporal dynamics of SAR. We demonstrate that local ETI triggered SAR signal generation, followed by rapid propagation and establishment in systemic responding leaves, is dependent on both jasmonate biosynthesis and perception. Further, ETI initiates calcium- and jasmonate-dependent systemic surface electrical potentials, reminiscent of those activated by herbivory but with slower propagation kinetics. Thus, jasmonate signalling is crucial to the initiation and establishment of systemic defence responses against a diverse range of phytopathogens.

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater, and intertidal environments. The latter can experience substantial long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We found that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations corresponded with the rate of temperature decrease. We did not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance but showed that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As inter-tidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signalling interacts with osmotic signalling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signalling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

Microbe associated molecular pattern (MAMP) triggered immunity research has traditionally centred around signal transduction pathways originating from activated membrane localised pattern recognition receptors (PRRs), culminating in nuclear transcription and post translational modifications. More recently, chloroplasts have emerged as key immune signalling hubs, playing a central role in integrating environmental signals. Notably, MAMP recognition induces chloroplastic ROS (cROS) which is suppressed by pathogen effectors, which also modify the balance of chloroplast‐synthesised precursors of the defence hormones, jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). This study focuses on how well characterised PRRs and co‐receptors modulate chloroplast physiology, examining whether diverse signalling pathways converge to similarly modulate chloroplast function. Pre‐treatment of receptor mutant plants with MAMP and D(Damage)AMP peptides usually protect against effector modulation of chlorophyll fluorescence and prevent Pseudomonas syringae effector mediated quenching of cROS and suppression of maximum dark‐adapted quantum efficiency (Fv/Fm). The MAMP‐triggered immunity (MTI) co‐receptor double mutant, bak1‐5/bkk1‐1, exhibits a remarkable decrease in Fv/Fm compared to control plants during infection, underlining the importance of MTI mediated signalling in chloroplast immunity. Further probing the role of the chloroplast in immunity we unexpectedly found that even moderate changes in light intensity can uncouple plant immune signalling. This article is protected by copyright. All rights reserved.

Microbe associated molecular pattern (MAMP) triggered immunity research has traditionally centred around signal transduction pathways originating from activated membrane localised pattern recognition receptors (PRRs), culminating in nuclear transcription and post translational modifications. More recently, chloroplasts have emerged as key immune signalling hubs. Chloroplasts play a central role in integrating environmental signals. Notably MAMP recognition induces chloroplastic ROS (cROS) which is suppressed by pathogens effectors, which also modify the balance of defence hormone precursors, jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA), whose precursors are chloroplast synthesised. This study focuses on how well characterised PRRs and co-receptors modulate chloroplast physiology, examining whether diverse signalling pathways converge to similarly modulate chloroplast function. Pre-treatment of receptor mutant plants with MAMP and D(Damage)AMP peptides usually protect against effector modulation of chlorophyll fluorescence and prevent Pseudomonas syringae effector mediated quenching of cROS and suppression of F/F . The MAMP-triggered immunity (MTI) co-receptor double mutant, bak1-5/bkk1-1, exhibits a remarkable decrease in F/F compared to control plants during infection, underlining the importance of MTI mediated signalling in chloroplast immunity. Further probing the role of the chloroplast in immunity we unexpectedly found that high light uncouples plant immune signalling.

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater and intertidal environments. The latter can experience significant long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We find that the diatoms P. tricornutum and T. pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations correlate with those of the temperature decrease. We do not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance, but show that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As inter-tidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signalling interacts with hypo-osmotic shock signalling in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signalling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

Diatoms are globally important phytoplankton that dominate coastal and polar‐ice assemblages. These environments exhibit substantial changes in salinity over dynamic spatiotemporal regimes. Rapid sensory systems are vital to mitigate the harmful consequences of osmotic stress. Population‐based analyses have suggested that Ca²⁺ signalling is involved in diatom osmotic sensing. However, mechanistic insight of the role of osmotic Ca²⁺ signalling is limited. Here, we show that Phaeodactylum Ca²⁺ elevations are essential for surviving hypo‐osmotic shock. Moreover, employing novel single‐cell imaging techniques we have characterised real‐time Ca²⁺ signalling responses in single diatom cells to environmental osmotic perturbations. We observe that intracellular spatiotemporal patterns of osmotic‐induced Ca²⁺ elevations encode vital information regarding the nature of the osmotic stimulus. Localised Ca²⁺ signals evoked by mild or gradual hypo‐osmotic shocks are propagated globally from the apical cell tips, enabling fine‐tuned cell volume regulation across the whole cell. Finally, we demonstrate that diatoms adopt Ca²⁺‐independent and dependent mechanisms for osmoregulation. We find that efflux of organic osmolytes occurs in a Ca²⁺‐independent manner, but this response is insufficient to mitigate cell damage during hypo‐osmotic shock. By comparison, Ca²⁺‐dependent signalling is necessary to prevent cell bursting via precise coordination of K⁺ transport, and therefore is likely to underpin survival in dynamic osmotic environments.

Diatoms are a diverse and globally important phytoplankton group, responsible for an estimated 20% of carbon fixation on Earth. They frequently form spatially extensive phytoplankton blooms, responding rapidly to increased availability of nutrients, including phosphorus (P) and nitrogen (N). Although it is well established that diatoms are common first responders to nutrient influxes in aquatic ecosystems, little is known of the sensory mechanisms that they employ for nutrient perception. Here, we show that P-limited diatoms use a Ca²⁺-dependent signaling pathway, not previously described in eukaryotes, to sense and respond to the critical macronutrient P. We demonstrate that P-Ca²⁺ signaling is conserved between a representative pennate (Phaeodactylum tricornutum) and centric (Thalassiosira pseudonana) diatom. Moreover, this pathway is ecologically relevant, being sensitive to sub-micromolar concentrations of inorganic phosphate and a range of environmentally abundant P forms. Notably, we show that diatom recovery from P limitation requires rapid and substantial increases in N assimilation and demonstrate that this process is dependent on P-Ca²⁺ signaling. P-Ca²⁺ signaling thus governs the capacity of diatoms to rapidly sense and respond to P resupply, mediating fundamental cross-talk between the vital nutrients P and N and maximizing diatom resource competition in regions of pulsed nutrient supply.