Environmental pH signals the release of monosaccharides from cell wall in coral symbiotic alga
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eLife assessment
The manuscript makes a fundamental contribution to our understanding of sugar release by symbiotic dinoflagellates, and is of broad interest for the fields of ecology, marine biology, and cell biology. The experiments, which combine algal culture with targeted metabolomics, transcriptomics and the application of inhibitors, provide substantial, though not entirely complete evidence for an acidic environment mimicking conditions reported for the intracellular organelle that hosts the symbiotic algae, leading to upregulation of algal cellulases, which in turn degrade the algal cell wall and thereby releasing glucose and galactose that can be used as a source of food by the coral host. This is a new idea and could significantly contribute to our understanding of photosymbiosis.
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Abstract
Reef-building corals thrive in oligotrophic environments due to their possession of endosymbiotic algae. Confined to the low pH interior of the symbiosome within the cell, the algal symbiont provides the coral host with photosynthetically fixed carbon. However, it remains unknown how carbon is released from the algal symbiont for uptake by the host. Here we show, using cultured symbiotic dinoflagellate, Breviolum sp., that decreases in pH directly accelerates the release of monosaccharides, that is, glucose and galactose, into the ambient environment. Under low pH conditions, the cell surface structures were deformed and genes related to cellulase were significantly upregulated in Breviolum . Importantly, the release of monosaccharides was suppressed by the cellulase inhibitor, glucopyranoside, linking the release of carbon to degradation of the agal cell wall. Our results suggest that the low pH signals the cellulase-mediated release of monosaccharides from the algal cell wall as an environmental response in coral reef ecosystems.
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Author Response
Reviewer #1 (Public Review):
The goal of this study was to investigate the mechanisms that lead to the release of photosynthetically fixed carbon from symbiotic dinoflagellate alga to their coral host. The experimental approach involved culturing free-living Brevolium sp dinoflagellates under "Normal" and "Low pH" conditions (respective target pH of 7.8 and 5.50) and measuring the following parameters: (Fig.1) cell growth rate over ~28 days, photosynthetic activity, glucose and galactose secretion at day 1; (Fig. 2) Cell clustering, external morphology (using SEM), and internal morphology (using TEM) after 3 weeks; (Fig. 3) Transcriptomic analyses at days 0 and 1; and (Fig. 4) glucose and galactose concentration in Normal culturing medium after 24h incubation with a putative cellulase inhibitor (PSG).
The paper …
Author Response
Reviewer #1 (Public Review):
The goal of this study was to investigate the mechanisms that lead to the release of photosynthetically fixed carbon from symbiotic dinoflagellate alga to their coral host. The experimental approach involved culturing free-living Brevolium sp dinoflagellates under "Normal" and "Low pH" conditions (respective target pH of 7.8 and 5.50) and measuring the following parameters: (Fig.1) cell growth rate over ~28 days, photosynthetic activity, glucose and galactose secretion at day 1; (Fig. 2) Cell clustering, external morphology (using SEM), and internal morphology (using TEM) after 3 weeks; (Fig. 3) Transcriptomic analyses at days 0 and 1; and (Fig. 4) glucose and galactose concentration in Normal culturing medium after 24h incubation with a putative cellulase inhibitor (PSG).
The paper reports decreased growth at Low pH coupled with decreased photosynthetic rates and increased glucose and galactose release in 1-day Breviolum sp. cultures. At this same time point, genes related to cellulase were upregulated, and after 3 weeks morphological changes on the cell wall were reported. The addition of the cellulase inhibitor PSG to cells in pH 7.8 media decreased the release of glucose and galactose.
The paper concludes that acidic conditions mimicking those reported for the coral symbiosome -the intracellular organelle that hosts the symbiotic algae- upregulate algal cellulases, which in turn degrade the algal cell wall releasing glucose and galactose that can be used as a source of food by the coral host. However, there are some methodological issues that hamper the interpretation of results and conclusions.
We appreciate your helpful comments and apologize the confusion caused by insufficient descriptions in the previous manuscript. In the revised manuscript we clarify what we originally intended to demonstrate including the followings:
(1) Most analyses including SEM and TEM were done at day 0 and 1, except for a few, i.e. growth rate over 28 days and cell clumping assay done 3 weeks after the inoculation, which is summarized as a schematic panel and clarified in the revised manuscript.
(2) Inhibitor assay for secreted celluloses was done in pH 5.5.
(3) We do not intend to suggest that low pH medium mimics symbiosomes, as these organelles are far more complex than simple culture media and how symbiosomes are maintained and what the interior environment is like are not fully understood in general. Based on previous studies, presumably they are featured by low pH, high CO2, host-derived nutrients. Among these, we focus on low pH, which is a stressor for dinoflagellates to go through in not only symbiosomes but also natural environments, e.g. animal gut.
In this study, we clarified how algae respond to low pH as an environmental stressor, which can also provide insights into how they interact with the host inside the guts as well as symbiosomes.
Reviewer #2 (Public Review):
Ishii and colleagues investigated the process of monosaccharide release from algae in low-pH environmental conditions, mimicking the acidic lysosomal-like intracellular compartment where the algae reside symbiotically and transfer nutrients to their hosts, namely corals and other animals. Upon exposure of cultured algae to low pH, subsequent physiological changes as well as the increased presence of glucose and galactose were measured in the surrounding media. Concurrently, photosynthetic activity was decreased, and further experiments employing the photosynthetic inhibitor DCMU to cultures also replicated the increased monosaccharide release. Transcriptomic comparison of algae in low pH to controls showed differential expression in glycolytic pathways and, interestingly, a strong upregulation of signal-peptide-containing isoforms of cellulases. Finally, the elegant use of a cellulase inhibitor on the cultured algae revealed a decrease in monosaccharides in the media. This led the authors to propose a pathway of sugar release in which acidic conditions trigger a cellulase-driven cascade of cell wall degradation in the algae and their consequent release of monosaccharides. These results have interesting implications on the molecular mechanisms of coral-algae symbiosis, contributing to the understanding of how these important symbioses function on the cellular level.
Overall the conclusions of this manuscript are supported by the data presented, but clarification and elaboration are needed to fully justify the proposed mechanisms and better situate the results in a broader context of the field.
We thank the reviewer for the positive comments. In the revised the manuscript we show that the results could be better explained with the proposed mechanisms in a broader context.
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eLife assessment
The manuscript makes a fundamental contribution to our understanding of sugar release by symbiotic dinoflagellates, and is of broad interest for the fields of ecology, marine biology, and cell biology. The experiments, which combine algal culture with targeted metabolomics, transcriptomics and the application of inhibitors, provide substantial, though not entirely complete evidence for an acidic environment mimicking conditions reported for the intracellular organelle that hosts the symbiotic algae, leading to upregulation of algal cellulases, which in turn degrade the algal cell wall and thereby releasing glucose and galactose that can be used as a source of food by the coral host. This is a new idea and could significantly contribute to our understanding of photosymbiosis.
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Reviewer #1 (Public Review):
The goal of this study was to investigate the mechanisms that lead to the release of photosynthetically fixed carbon from symbiotic dinoflagellate alga to their coral host. The experimental approach involved culturing free-living Brevolium sp dinoflagellates under "Normal" and "Low pH" conditions (respective target pH of 7.8 and 5.50) and measuring the following parameters: (Fig.1) cell growth rate over ~28 days, photosynthetic activity, glucose and galactose secretion at day 1; (Fig. 2) Cell clustering, external morphology (using SEM), and internal morphology (using TEM) after 3 weeks; (Fig. 3) Transcriptomic analyses at days 0 and 1; and (Fig. 4) glucose and galactose concentration in Normal culturing medium after 24h incubation with a putative cellulase inhibitor (PSG).
The paper reports decreased growth …
Reviewer #1 (Public Review):
The goal of this study was to investigate the mechanisms that lead to the release of photosynthetically fixed carbon from symbiotic dinoflagellate alga to their coral host. The experimental approach involved culturing free-living Brevolium sp dinoflagellates under "Normal" and "Low pH" conditions (respective target pH of 7.8 and 5.50) and measuring the following parameters: (Fig.1) cell growth rate over ~28 days, photosynthetic activity, glucose and galactose secretion at day 1; (Fig. 2) Cell clustering, external morphology (using SEM), and internal morphology (using TEM) after 3 weeks; (Fig. 3) Transcriptomic analyses at days 0 and 1; and (Fig. 4) glucose and galactose concentration in Normal culturing medium after 24h incubation with a putative cellulase inhibitor (PSG).
The paper reports decreased growth at Low pH coupled with decreased photosynthetic rates and increased glucose and galactose release in 1-day Breviolum sp. cultures. At this same time point, genes related to cellulase were upregulated, and after 3 weeks morphological changes on the cell wall were reported. The addition of the cellulase inhibitor PSG to cells in pH 7.8 media decreased the release of glucose and galactose.
The paper concludes that acidic conditions mimicking those reported for the coral symbiosome -the intracellular organelle that hosts the symbiotic algae- upregulate algal cellulases, which in turn degrade the algal cell wall releasing glucose and galactose that can be used as a source of food by the coral host. However, there are some methodological issues that hamper the interpretation of results and conclusions.
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Reviewer #2 (Public Review):
Ishii and colleagues investigated the process of monosaccharide release from algae in low-pH environmental conditions, mimicking the acidic lysosomal-like intracellular compartment where the algae reside symbiotically and transfer nutrients to their hosts, namely corals and other animals. Upon exposure of cultured algae to low pH, subsequent physiological changes as well as the increased presence of glucose and galactose were measured in the surrounding media. Concurrently, photosynthetic activity was decreased, and further experiments employing the photosynthetic inhibitor DCMU to cultures also replicated the increased monosaccharide release. Transcriptomic comparison of algae in low pH to controls showed differential expression in glycolytic pathways and, interestingly, a strong upregulation of …
Reviewer #2 (Public Review):
Ishii and colleagues investigated the process of monosaccharide release from algae in low-pH environmental conditions, mimicking the acidic lysosomal-like intracellular compartment where the algae reside symbiotically and transfer nutrients to their hosts, namely corals and other animals. Upon exposure of cultured algae to low pH, subsequent physiological changes as well as the increased presence of glucose and galactose were measured in the surrounding media. Concurrently, photosynthetic activity was decreased, and further experiments employing the photosynthetic inhibitor DCMU to cultures also replicated the increased monosaccharide release. Transcriptomic comparison of algae in low pH to controls showed differential expression in glycolytic pathways and, interestingly, a strong upregulation of signal-peptide-containing isoforms of cellulases. Finally, the elegant use of a cellulase inhibitor on the cultured algae revealed a decrease in monosaccharides in the media. This led the authors to propose a pathway of sugar release in which acidic conditions trigger a cellulase-driven cascade of cell wall degradation in the algae and their consequent release of monosaccharides. These results have interesting implications on the molecular mechanisms of coral-algae symbiosis, contributing to the understanding of how these important symbioses function on the cellular level.
Overall the conclusions of this manuscript are supported by the data presented, but clarification and elaboration are needed to fully justify the proposed mechanisms and better situate the results in a broader context of the field.
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