Allelopathy Impacts Competitor Metabolism.

Although previous studies (e.g., refs. 4 and 25) have shown that K. brevis allelopathy reduces the growth of competitor species, the current study demonstrates the impact of allelopathy on global cellular function and indicates that, even in the case of modest growth inhibition, metabolism was altered. K. brevis allelopathy differentially affected growth of the two competitors (Fig. 1), reflected in both the metabolomes (Fig. 2 and Fig. S1) and proteomes (Table 1) of each competitor. In the presence of K. brevis, Thalassiosira pseudonana growth was reduced by 85% (Fig. 1B; n = 15; P < 0.0001), with growth suppression and subsequent population decline observed after 4 d of coculture with species physically separated by dialysis membrane. Analysis of the T. pseudonana metabolome (Table 2 and Fig. 2 A and B) and proteome (Table 1) revealed that K. brevis allelopathy impacts multiple metabolic pathways, with some cellular responses possibly used to compensate for negative effects of allelopathy. In contrast, the overall growth of Asterionellopsis glacialis was only reduced by 35% when exposed to K. brevis allelopathy, an effect that was not statistically significant (Fig. 1B; n = 15; P = 0.089), indicating, at most, a weak allelopathic effect of K. brevis on A. glacialis in this experiment. In the A. glacialis experiment, final K. brevis cell concentrations in flasks averaged 15.3 × 10³ cells per mL, whereas in the T. pseudonana experiment, mean K. brevis concentrations were 8.3 × 10³ cells per mL at the time of harvest (Fig. 1A). Nutrients were not limiting in these experiments (SI Materials and Methods), suggesting that the negative effects of K. brevis on competitor growth were mediated by allelopathy rather than exploitation competition. Overall, it is clear that, based on both the proteomic and metabolomic data, a sensitive competitor exposed to allelopathy operates in a functionally compromised metabolic state (Fig. 2 and Fig. S1).

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Fig. 1.

Effects of exposure to exudates of live K. brevis on the growth of A. glacialis and T. pseudonana. (A) A. glacialis (red) in vivo and T. pseudonana (blue) in vivo fluorescence (arrow indicates day of harvest for metabolomics and proteomics). The solid lines indicate fluorescence of diatom-only controls, and the dashed lines indicate fluorescence of diatoms exposed to K. brevis. Initial K. brevis (red open circles for A. glacialis experiment; blue open circle for T. pseudonana experiment) concentrations from cultures used to fill dialysis tubes (n = 1), final concentrations from experimental flasks at time of harvest (n = 15). (B) Calculated percentage growth of competitors A. glacialis (red) and T. pseudonana (blue) relative to their own controls after 8 and 6 d exposure to K. brevis, respectively. The dotted line indicates growth equivalent to control. n = 15. P < 0.0001 indicated by asterisk (*), unpaired t test. Error bars represent ±1 SEM.

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Fig. 2.

Orthogonal PLS-DA shows effects of K. brevis allelopathy on the metabolomes of competitor diatoms. PLS-DA scores plot of (A) UHPLC/MS metabolic features and (B) ¹H NMR spectral data for T. pseudonana exposed to K. brevis (filled squares) or dilute media control (open squares) with cross-validated accuracies of 87% and 100%, respectively. PLS-DA scores plot of (C) UHPLC/MS metabolic features and (D) ¹H NMR spectral data for A. glacialis exposed to live K. brevis (filled circles) or dilute media controls (open circles) with cross-validated accuracies of 57% and 63%, respectively.

A Co-occurring Competitor Has a Robust Metabolism in Response to Allelopathy.

To date, metabolomics and proteomics are underused tools for investigating the impacts of ecological interactions on organism physiology. This study revealed that metabolomic and proteomic analyses of competitors can demonstrate the species-specific effects of K. brevis allelopathy. The diatom A. glacialis appeared to maintain a relatively robust metabolism in response to K. brevis, which resulted in mild allelopathic effects on growth (Figs. 1 and 2 C and D). Using multivariate analysis [orthogonal partial least-squares discriminant analysis (PLS-DA)], we identified groups of metabolites whose covarying concentrations allowed us to distinguish metabolomes of K. brevis-exposed and control cultures of competitors (SI Materials and Methods). Although concentrations of these compounds may not have significantly differed between treatments and controls when each compound was considered alone, these compounds displayed explanatory power when part of a discriminating panel (35). Orthogonal PLS-DA analysis suggested that concentrations of only a few aliphatic metabolites were enhanced in A. glacialis by exposure to allelopathy (i.e., glycerophosphocholine and lactose or polysaccharides, as well as two unidentified compounds; Table S1). However, only 6–9% (Fig. 2 C and D) of the observed among-culture variation in the A. glacialis metabolome was due to K. brevis exposure, indicating that this species maintains relatively normal functioning in response to K. brevis. Additionally, only one protein increased in abundance when exposed to K. brevis according to differential expression protein analyses (ATP synthase), whereas five proteins were significantly suppressed (e.g., cytochrome b559, photosystem II chlorophyll A antenna apoprotein; Dataset S1). The strain of A. glacialis used in this experiment was originally isolated from the Gulf of Mexico, where K. brevis blooms regularly occur (22). Thus, A. glacialis may have evolved partial resistance to K. brevis allelopathy due to its potential for frequent exposure to K. brevis blooms (36). T. pseudonana used in the current study, however, was from the North Atlantic Ocean where K. brevis does not occur. It is possible that the robust metabolome of A. glacialis has resulted from multiple metabolic adaptations that mitigate allelopathic impacts, allowing A. glacialis cells to function when challenged by K. brevis allelopathy.

A Sensitive Competitor Experiences Altered Energy Metabolism and Increased Cell Stress in Response to Allelopathy.

Because of its sensitivity to K. brevis allelopathy (Fig. 1), T. pseudonana serves as a suitable model for exploring mechanisms by which allelopathy affects competitor physiology. From our integrated systems biology approach, we found T. pseudonana energy metabolism to be greatly impacted by K. brevis allelopathy (Fig. 3). An observed significant increase of acetate concentrations (+0.92-fold), as well as decreased myo-inositol and/or glucose concentrations, as distinguished by orthogonal PLS-DA (Table 2, Table S2, and Fig. S2), with concomitant increased concentrations of glycolysis enzymes (e.g., pyruvate kinase +3.0-fold; Table 1 and Dataset S2), suggests that glycolysis was enhanced in T. pseudonana. Starch stores were depleted, as indicated by significantly lowered concentration of maltopentose, amylopectin, cellopentose, and/or verbascose (−3.2-fold) (Table 2 and Table S3), whereas concentrations of small (e.g., C₈-C₁₀) fatty acids increased (Table S2 and Fig. S2). Several fatty acid biosynthesis and metabolism enzymes (Table 1) were more abundant in T. pseudonana in response to K. brevis allelopathy, including acetyl CoA carboxylase (+2.3) and acyl CoA dehydrogenase (+3.0-fold) (Dataset S2). Together, this suggests that allelopathy caused enhanced glycolysis in T. pseudonana, while also stimulating synthesis and β-oxidation of fatty acids. Allelopathy may have altered the photosynthetic capability of T. pseudonana, as evidenced by the decreased abundance of 12 photosynthetic proteins, even though the abundance of RuBisCO, the key enzyme responsible for carbon fixation, remained unchanged (Table 1 and Dataset S2). The reduction in photosystem complexes and surge in fatty acid β-oxidation may indicate that cells experiencing allelopathy became carbon limited.

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Fig. 3.

Network of cellular pathways, enzymes, and metabolites in the diatom T. pseudonana impacted by exposure to K. brevis allelopathy, derived from NMR and MS metabolomics and proteomics. Pathways and metabolites enhanced by allelopathy are indicated by red arrows and compound names, respectively. The blue arrows and compound names denote pathways and metabolites that were suppressed by allelopathy.

Pathways Involved in Cell Protection Are Impacted by K. brevis Allelopathy.

In previous work, K. brevis exudates were found to increase cell membrane permeability of several competitors, via unknown mechanisms (25). In the current study, metabolic and biosynthetic activities associated with cell membranes were altered in T. pseudonana in response to K. brevis allelopathy, as evidenced by decreased mannan and C₂₆ phospholipid or C₃₁ lipid concentrations, as well as an increase in a C₂₄ phospholipid or C₂₆ lipid (Table 2 and Table S3). This is suggestive of membrane restructuring and a possible increase in permeability because mannans and phospholipids are primary membrane components of T. pseudonana (37) and other photosynthetic organisms (38). These changes could be the result of membrane instability and the subsequent attempt of T. pseudonana to alter membrane phospholipid content via increased biosynthesis. Recently, Martin et al. (39) revealed that phosphorus-deprived T. pseudonana substitute betaine lipids and sulfolipids for phospholipids in cellular membranes. In addition, Riekhof et al. (40) demonstrated that betaine lipid synthesis is associated with the degradation of membrane phospholipids. These coordinated pathways provide a possible explanation to the significantly reduced betaine concentrations (−0.58-fold) because betaine would be consumed to build betaine-based lipids (Table 2 and Table S2). Enzymes involved in sulfolipid biosynthesis were present in higher concentrations in T. pseudonana responding to K. brevis (e.g., sulfolipid biosynthesis protein; Dataset S2), suggesting a modification of T. pseudonana’s cell membrane content via an increase in sulfolipids. Although phosphate was not limiting to T. pseudonana in this experiment (SI Materials and Methods), it is possible that K. brevis allelopathy reduced the capacity of T. pseudonana to use nutrients present in the media. Plankton lipids mediate a number of important ecological interactions, including grazing (41), susceptibility to viral infection (42), and toxin sensitivity (43). Thus, future experiments testing how phytoplankton allelopathy alters lipid biochemistry and membrane stability could lead to discovery of cascading ecological effects on other types of interactions.

Another critical aspect of membrane structure and function is the ability of a cell to control solute osmosis. From the current study, it is apparent that osmotic regulation was impacted by K. brevis allelopathy: concentrations of homarine (−2.5-fold) and betaine were suppressed when T. pseudonana was exposed to K. brevis (Table 2 and Table S2) and are recognized osmolytes in marine planktonic organisms (44, 45). T. pseudonana may respond to K. brevis allelopathy by compensating for impeded osmotic regulation by enhancing or maintaining concentrations of both proline and dimethylamine (33, 46) (Table 2).

When T. pseudonana was exposed to K. brevis allelopathy, multiple enzymes involved in critical pathways related to oxidative stress were enhanced (Table 1). In addition, several enzymes responsible for mitigating high oxidative stress were more abundant. Fifteen oxidoreductases increased in abundance, including manganese superoxide dismutase and ascorbate peroxidase (Dataset S2). Our PLS-DA model suggests glycerate concentrations were reduced, as part of a group of compounds whose concentrations covaried, in T. pseudonana exposed to K. brevis indicating that photorespiration was disrupted (Table 2 and Fig. S2), which would also likely increase oxidative stress (47, 48). Recently, Schlegel et al. (49) proposed that an increase in oxidative stress may force cells to reduce the abundance of photosynthetic and electron carrier enzymes. Consistent with that prediction, in the current study proteins involved in electron carrier activity were suppressed, whereas proton transport activity was enhanced, suggesting potential compensation within T. pseudonana cells (Table 1 and Dataset S2). Seven proteins involved in the pentose phosphate pathway, a side step to glycolysis that yields reducing equivalents of NADPH (31), yielded heightened abundances (Table 1 and Dataset S2). Recent findings also show that diatoms experiencing oxidative stress lose their ability to assimilate nitrogen, due to either altered redox states of critical nitrogen assimilation enzymes, or the loss of reducing equivalents (e.g., NADPH) (50). This suggests that, in the current study, oxidatively stressed T. pseudonana may have been forced to recycle internal nitrogen stores as a downstream effect of exposure to K. brevis allelopathy. We also observed alterations in the concentrations of several amino acids (increased concentrations of glutamate and proline; decreased concentrations of taurine; Table 2 and Table S2), suggesting a reshuffling and possible recycling of internal nitrogen stores. Together, this suggests that T. pseudonana was responding to increased oxidative stress as a result of K. brevis allelopathy.

Cells in Crisis.

Overall, it appears that T. pseudonana cells exposed to K. brevis allelopathy experienced substantially disrupted metabolic processes indicative of heightened stress (Fig. 3). In addition to the metabolic and proteomic changes described above, we observed a fourfold increase in the abundance of the diatom’s trypsin with a concomitant increase in the abundance of an anti-trypsin protease inhibitor as well as reduced concentrations of a serine protease inhibitor (Dataset S2). For trypsin annotation, a total of 19 unique peptides were identified, none of which resembled the pure porcine trypsin used for digestion during sample preparation. These findings suggest that protein degradation pathways were induced, which may approach conditions typically observed for programmed cell death response in diatoms (51). In addition, concentrations of several DNA replication enzymes, as well as eight chromatin assembly proteins were suppressed (e.g., histone-3: −3.4-fold; Table 1 and Dataset S2), consistent with a strong negative impact of allelopathy on T. pseudonana growth (Fig. 1).

Mucins, gel-like glycoproteins, were in higher abundance in T. pseudonana exposed to allelopathy (Dataset S2). Although largely unexplored for phytoplankton, in other organisms mucins have been found to reduce adherence of bacteria (52). If diatom mucins prevent colonization by harmful bacteria or encourage the aggregation of diatom cells, this may represent a valuable defense mechanism or a response to stress. Furthermore, if mucins are shown to be involved in diatom aggregation, then allelopathy may directly influence the flux of particulate carbon from the water column to depth in the ocean. Mucins have also been found to demonstrate antiapoptopic activity (53, 54). Previous studies on T. pseudonana have found ascorbate peroxidase genes to be up-regulated in iron-limited situations, whereas mucin genes were down-regulated, demonstrating a balance between reactive oxygen species reducing mechanisms and the initiation of programmed cell death pathways in stressful situations (55). This balancing act ultimately allows acclimation to stress, which may be disrupted by allelopathy.