Identification of senescence and death in Emiliania huxleyi and Thalassiosira pseudonana: Cell staining, chlorophyll alterations, and dimethylsulfoniopropionate (DMSP) metabolism

Abstract

We measured membrane permeability, hydrolytic enzyme, and caspase-like activities using fluorescent cell stains to document changes caused by nutrient exhaustion in the coccolithophore Emiliania huxleyi and the diatom Thalassiosira pseudonana, during batch-culture nutrient limitation. We related these changes to cell death, pigment alteration, and concentrations of dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) to assess the transformation of these compounds as cell physiological condition changes. E. huxleyi persisted for 1 month in stationary phase; in contrast, T. pseudonana cells rapidly declined within 10 d of nutrient depletion. T. pseudonana progressively lost membrane integrity and the ability to metabolize 5-chloromethylfluorescein diacetate (CMFDA; hydrolytic activity), whereas E. huxleyi developed two distinct CMFDA populations and retained membrane integrity (SYTOX Green). Caspase-like activity appeared higher in E. huxleyi than in T. pseudonana during the post-growth phase, despite a lack of apparent mortality and cell lysis. Photosynthetic pigment degradation and transformation occurred in both species after growth; chlorophyll a (Chl a) degradation was characterized by an increase in the ratio of methoxy Chl a : Chl a in T. pseudonana but not in E. huxleyi, and the increase in this ratio preceded loss of membrane integrity. Total DMSP declined in T. pseudonana during cell death and DMS increased. In contrast, and in the absence of cell death, total DMSP and DMS increased in E. huxleyi. Our data show a novel chlorophyll alteration product associated with T. pseudonana death, suggesting a promising approach to discriminate nonviable cells in nature.

Low nutrient availability reduces high‐irradiance‐induced viability loss in oceanic phytoplankton

“…In the upper mixed layer of open oligotrophic oceans, phytoplankton can experience high (dynamic) irradiance conditions in combination with low nutrient availability. It has been suggested that these co-occurring conditions increase photoinhibition and viability loss in natural communities (Lesser et al 1994;Llabrés and Agustí 2006;Franklin et al 2009). However, the present study showed that P. marinus, Ostreococcus sp., and T. oceanica are actually less susceptible to both photoinhibition and viability loss during high-irradiance exposure when nutrient availability is low.…”

“…In addition to the direct effect of low-nutrient availability on phytoplankton viability, it may have considerable effects on phytoplankton photophysiology and consequently on the response to high-irradiance intensities. Generally, the lightharvesting capacity is reduced during nutrient starvation by a reduction in the cellular chlorophyll a (Chl a) concentration and quantum yield of PSII, as well as an increase in the relative amount of carotenoids Berges and Falkowski 1998;Franklin et al 2012). However, the absorption of chlorophyll and the absorption cross section of the remaining PSII may increase during nutrient starvation Berges et al 1996), partially counteracting the reduced light-harvesting capacity.…”

Dissolved organic matter (DOM) release by phytoplankton in the contemporary and future ocean

“…Veldhuis et al (2001) hypothesized that 'automortality' is a significant source of DOM leakage, particularly as cells with compromised cell membranes are often still capable of photosynthesis. Subsequent work has shown that stresses such as low irradiance or nutrient limitation affect compromised plasma membranes and autocatalytic cell death in phytoplankton (Berman-Frank et al, 2007;Timmermans et al, 2007;Franklin et al, 2012).…”

“…Recent research has shown that phytoplankton also undergo the physiological process of autocatalytic cell-suicide, which can lead to lysis of the cells; this process is analogous to programmed cell death (PCD) in metazoans (Bidle & Falkowski, 2004;Franklin et al, 2006). Autocatalytic cell death is a process that is indicated by multiple changes within the phytoplankton cell and it is often difficult to characterize such cells as clearly dead or alive (Franklin et al, 2006(Franklin et al, , 2012. Cell membrane integrity is used as an early indicator of cell death (Veldhuis et al, 2001).…”

Rapid and highly sensitive analysis of chlorophylls and carotenoids from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC) with the first derivative spectrum chromatogram (FDSC) technique

“…In previous studies, two types of cyanobacteria have been proposed in terms of pigment and morphological patterns (Jeffrey and Wright, 2006 thiebautii possessed significant amounts of these carotenoids. In the Synechococcus strain, Chl a allomer (i.e., Chl a allo; peak 32) was detected, indicating the oxidation of Chl a by cell senescence (Franklin et al, 2012).…”