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

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.

Flexible C : N ratio enhances metabolism of large phytoplankton when resource supply is intermittent

Phytoplankton cell size influences particle sinking rate, food web interactions and biogeographical distributions. We present a model in which the uptake, storage and assimilation of nitrogen and carbon are explicitly resolved in different-sized phytoplankton cells. In the model, metabolism and cellular C :N ratio are influenced by the accumulation of carbon polymers such as carbohydrate and lipid, which is greatest when cells are nutrient starved, or exposed to high light. Allometric relations and empirical data sets are used to constrain the range of possible C : N, and indicate that larger cells can accumulate significantly more carbon storage compounds than smaller cells. When forced with extended periods of darkness combined with brief exposure to saturating irradiance, the model predicts organisms large enough to accumulate significant carbon reserves may on average synthesize protein and other functional apparatus up to five times faster than smaller organisms. The advantage of storage in terms of average daily protein synthesis rate is greatest when modeled organisms were previously nutrient starved, and carbon storage reservoirs saturated. Small organisms may therefore be at a disadvantage in terms of average daily growth rate in environments that involve prolonged periods of darkness and intermittent nutrient limitation. We suggest this mechanism is a significant constraint on phytoplankton C :N variability and cell size distribution in different oceanic regimes.

Both respiration and photosynthesis determine the scaling of plankton metabolism in the oligotrophic ocean

Despite its importance to ocean–climate interactions, the metabolic state of the oligotrophic ocean has remained controversial for 415 years. Positions in the debate are that it is either hetero- or autotrophic, which suggests either substantial unaccounted for organic matter inputs, or that all available photosynthesis (P) estimations (including 14C) are biased. Here we show the existence of systematic differences in the metabolic state of the North (heterotrophic) and South (autotrophic) Atlantic oligotrophic gyres, resulting from differences in both P and respiration (R). The oligotrophic ocean is neither auto- nor heterotrophic, but functionally diverse. Our results show that the scaling of plankton metabolism by generalized P:R relationships that has sustained the debate is biased, and indicate that the variability of R, and not only of P, needs to be considered in regional estimations of the ocean’s metabolic state.

THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.