Effects and mechanisms of glyphosate as phosphorus nutrient on element stoichiometry and metabolism in the diatom Phaeodactylum tricornutum.

The ability to utilize dissolved organic phosphorus (DOP) gives phytoplankton competitive advantages in P-limited environments. Our previous research indicates that the diatom Phaeodactylum tricornutum could grow on glyphosate, a DOP with carbon-phosphorus (C-P) bond and an herbicide, as sole P source. However, direct evidence and mechanism of glyphosate utilization are still lacking. In this study, using physiological and isotopic analysis, combined with transcriptomic profiling, we demonstrated the uptake of glyphosate by P. tricornutum and revealed the candidate responsible genes. Our data showed a low efficiency of glyphosate utilization by P. tricornutum, suggesting that glyphosate utilization costs energy and that the alga possessed an herbicide-resistant type of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. Compared to the P-limited cultures, the glyphosate-grown P. tricornutum cells up-regulated genes involved in DNA replication, cell growth, transcription, translation, carbon metabolism, and many genes encoding antioxidants. Additionally, cellular C and silicon (Si) increased remarkably while cellular nitrogen (N) declined in the glyphosate-grown P. tricornutum, leading to higher Si:C and Si:N ratios, which corresponded to the up-regulation of genes involved in the C metabolism and Si uptake and the down-regulation of those encoding N uptake. This has the potential to enhance C and Si export to the deep sea when P is limited but phosphonate is available. In sum, our study documented how P. tricornutum could utilize the herbicide glyphosate as P nutrient and how glyphosate utilization may affect the element content and stoichiometry in this diatom, which have important ecological implications in the future ocean.IMPORTANCEGlyphosate is the most widely used herbicide in the world and could be utilized as phosphorus (P) source by some bacteria. Our study first revealed that glyphosate could be transported into Phaeodactylum tricornutum cells for utilization and identified putative genes responsible for glyphosate uptake. This uncovers an alternative strategy of phytoplankton to cope with P deficiency considering phosphonate accounts for about 25% of the total dissolved organic phosphorus (DOP) in the ocean. Additionally, accumulation of carbon (C) and silicon (Si), as well as elevation of Si:C ratio in P. tricornutum cells when grown on glyphosate indicates glyphosate as the source of P nutrient has the potential to result in more C and Si export into the deep ocean. This, along with the differential ability to utilize glyphosate among different species, glyphosate supply in dissolved inorganic phosphorus (DIP)-depleted ecosystems may cause changes in phytoplankton community structure. These insights have implications in evaluating the effects of human activities (use of Roundup) and climate change (potentially reducing DIP supply in sunlit layer) on phytoplankton in the future ocean.

Glyphosate (Roundup) as phosphorus nutrient enhances carbon and nitrogen accumulation and up-regulates phosphorus metabolisms in the haptophyte Isochrysis galbana.

Inorganic phosphate limitation for phytoplankton may be intensified with water stratification by global warming, and with the increasing nitrogen: phosphorus (N:P) ratio in coastal zones resulting from continuous anthropogenic N overloading. Under these circumstances, phytoplankton's ability to use dissolved organic phosphorus (DOP) will give species a competitive advantage. In our previous study, we have shown that the haptophyte Isochrysis galbana can use glyphosate (Roundup) as a P nutrient source to support growth, but the mechanism of how remains unexplored. Here, we show that three genes encoding PhnC (IgPhnCs), which exhibit up-regulated expression in glyphosate-grown cultures, are probably responsible for glyphosate uptake, while homologs of PhnK and PhnL (IgPhnK and IgPhnL) probably provide auxiliary support for the intracellular degradation of glyphosate. Meanwhile, we found the use efficiency of glyphosate was low compared with phosphate, probably because glyphosate uptake and hydrolysis cost energy and because glyphosate induces oxidative stress in I. galbana. Meanwhile, genes encoding 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the target of the herbicide, were up-regulated in glyphosate cultures. Furthermore, our data showed the up-regulation of P metabolisms (transcription) in glyphosate-grown cultures, which further induced the up-regulation of nitrate/nitrite transport and biosynthesis of some amino acids. Meanwhile, glyphosate-grown cells accumulated more C and N, resulting in remarkably high C:N:P ratio, and this, along with the up-regulated P metabolisms, was under transcriptional and epigenetic regulation. This study sheds lights on the mechanism of glyphosate utilization as a source of P nutrient by I. galbana, and these findings have biogeochemical implications.

Alkaline Phosphatase PhoD Mutation Induces Fatty Acid and Long-Chain Polyunsaturated Fatty Acid (LC-PUFA)-Bound Phospholipid Production in the Model Diatom Phaeodactylum tricornutum.

With rapid growth and high lipid contents, microalgae have become promising environmentally friendly candidates for renewable biodiesel and health supplements in our era of global warming and energy depletion. Various pathways have been explored to enhance algal lipid production, especially gene editing. Previously, we found that the functional loss of PhoD-type alkaline phosphatase (AP), a phosphorus-stress indicator in phytoplankton, could lead to increased lipid contents in the model diatom Phaeodactylum tricornutum, but how the AP mutation may change lipid composition remains unexplored. This study addresses the gap in the research and investigates the effects of PhoD-type AP mutation on the lipid composition and metabolic regulation in P. tricornutum using transcriptomic and lipidomic analyses. We observed significantly modified lipid composition and elevated production of fatty acids, lysophosphatidylcholine, lysophosphatidylethanolamine, ceramide, phosphatidylinositol bisphosphate, and monogalactosylmonoacylglycerol after PhoD_45757 mutation. Meanwhile, genes involved in fatty acid biosynthesis were upregulated in mutant cells. Moreover, the mutant exhibited increased contents of ω-3 long-chain polyunsaturated fatty acid (LC-PUFA)-bound phospholipids, indicating that PhoD_45757 mutation could improve the potential bioavailability of PUFAs. Our findings indicate that AP mutation could influence cellular lipid synthesis and probably redirect carbon toward lipid production and further demonstrate that AP mutation is a promising approach for the development of high-value microalgal strains for biomedical and other applications.

Species-dependent effects of seawater acidification on alkaline phosphatase activity in dinoflagellates.

Increases of atmospheric CO2 cause ocean acidification (OA) and global warming, the latter of which can stratify the water column and impede nutrient supply from deep water. Phosphorus (P) is an essential nutrient for phytoplankton to grow. While dissolved inorganic phosphorus (DIP) is the preferred form of P, phytoplankton have evolved alkaline phosphatase (AP) to utilize dissolved organic phosphorus (DOP) when DIP is deficient. Although the function of AP is known to require pH > 7, how OA affects AP activity and hence the capacity of phytoplankton to utilize DOP is poorly understood. Here, we examined the effects of pH conditions (5.5-11) on AP activity from six species of dinoflagellates, an important group of marine phytoplankton. We observed a general pattern that AP activity declined sharply at pH 5.5, peaked between pH 7 and 8, and dropped at pH > 8. However, our data revealed remarkable interspecific variations in optimal pH and niche breadth of pH. Among the species examined, Fugacium kawagutii and Prorocentrum cordatum had an optimal pH at 8, and Alexandrium pacificum, Amphidinium carterae, Effrenium voratum, and Karenia mikimotoi showed an optimal pH of 7. However, whereas A. pacificum and K. mikimotoi had the broadest pH niche for AP (7-10) and F. kawagutii the second (8-10), Am. carterae, E. voratum, and P. cordatum exhibited a narrow pH range. The response of Am. carterae AP to pH changes was verified using purified AP heterologously expressed in Escherichia coli. These results in concert suggest OA will likely differentially impact the capacity of different phytoplankton species to utilize DOP in the projected more acidified and nutrient-limited future ocean.

Phosphorus nutrition strategies in a Symbiodiniacean species: Implications in coral-alga symbiosis facing increasing phosphorus deficiency in future warmer oceans.

Coral reefs thrive in the oligotrophic ocean and rely on symbiotic algae to acquire nutrients. Global warming is projected to intensify surface ocean nutrient deficiency and anthropogenic discharge of wastes with high nitrogen (N): phosphorus (P) ratios can exacerbate P nutrient limitation. However, our understanding on how symbiotic algae cope with P deficiency is limited. Here, we investigated the responses of a coral symbiotic species of Symbiodiniaceae, Cladocopium goreaui, to P-limitation by examining its physiological performance and transcriptomic profile. Under P stress, C. goreaui exhibited decreases in algal growth, photosynthetic efficiency, and cellular P content but enhancement in carbon fixation, N assimilation, N:P ratio, and energy metabolism, with downregulated expression of carbohydrate exporter genes. Besides, C. goreaui showed flexible mechanisms of utilizing different dissolved organic phosphorus to relieve P deficiency. When provided glycerol phosphate, C. goreaui hydrolyzed it extracellularly to produce phosphate for uptake. When grown on phytate, in contrast, C. goreaui upregulated the endocytosis pathway while no dissolved inorganic phosphorus was released into the medium, suggesting that phytate was transported into the cell, potentially via the endocytosis pathway. This study sheds light on the survival strategies of C. goreaui and potential weakening of its role as an organic carbon supplier in P-limited environments, underscoring the importance of more systematic investigation on future projections of such effects.

Functional differentiation and complementation of alkaline phosphatases and choreography of DOP scavenging in a marine diatom.

Facing phosphate deficiency, phytoplankton use alkaline phosphatase (AP) to scavenge dissolved organophosphate (DOP). AP is a multitype (e.g., PhoA, PhoD) family of hydrolases and is known as a promiscuous enzyme with broad DOP substrate compatibility. Yet, whether the multiple types differentiate on substrates and collaborate to provide physiological flexibility remain elusive. Here we identify PhoA and PhoDs and document the functional differentiation between PhoA and a PhoD (PhoD_45757) in Phaeodactylum tricornutum. CRISPR/Cas9-based mutations and physiological analyses reveal that (1) PhoA is a secreted enzyme and contributes the majority of total AP activity whereas PhoD_45757 is intracellular and contributes a minor fraction of the total AP activity, (2) AP gene expression compensates for each other after one is disrupted, (3) the DOP→PhoA→phosphate_uptake and the DOP_uptake→PhoD→phosphate pathways function interchangeably for some DOP substrates. These findings shed light on the underpinning of AP's multiformity and have important implications in phytoplankton phosphorus-nutrient niche differentiation, physiological plasticity, and competitive strategy.

Phytate as a Phosphorus Nutrient with Impacts on Iron Stress-Related Gene Expression for Phytoplankton: Insights from the Diatom Phaeodactylum tricornutum.

Phytoplankton have evolved a capability to acquire phosphorus (P) from dissolved organic phosphorus (DOP) since the preferred form, dissolved inorganic phosphate (DIP, or Pi), is often limited in parts of the ocean. Phytic acid (PA) is abundantly synthesized in plants and rich in excreta of animals, potentially enriching the DOP pool in coastal oceans. However, whether and how PA can be used by phytoplankton are poorly understood. Here, we investigated PA utilization and underlying metabolic pathways in the diatom model Phaeodactylum tricornutum. The physiological results showed that P. tricornutum could utilize PA as a sole source of P nutrient to support growth. Meanwhile, the replacement of PA for DIP also caused changes in multiple cellular processes, such as inositol phosphate metabolism, photosynthesis, and signal transduction. These results suggest that PA is bioavailable to P. tricornutum and can directly participate in the metabolic pathways of PA-grown cells. However, our data showed that the utilization of PA was markedly less efficient than that of DIP, and PA-grown cells exhibited P and iron (Fe) nutrient stress signals. Implicated in these findings is the potential of complicated responses of phytoplankton to an ambient DOP species, which calls for more systematic investigation. IMPORTANCE PA is abundant in plants and cannot be digested by nonruminant animals. Hence, it is potentially a significant component of the DOP pool in coastal waters. Despite this potential importance, there is little information about its bioavailability to phytoplankton as a source of P nutrient and the molecular mechanisms involved. In this study, we found that part of PA could be utilized by the diatom P. tricornutum to support growth, and another portion of PA can act as a substrate directly participating in various metabolism pathways and cellular processes. However, our physiological and transcriptomic data show that PA-grown cells still exhibited signs of P stress and potential Fe stress. These results have significant implications in phytoplankton P nutrient ecology and provide a novel insight into multifaceted impacts of DOP utilization on phytoplankton nutrition and metabolism.

SPX-related genes regulate phosphorus homeostasis in the marine phytoplankton, Phaeodactylum tricornutum.

Phosphorus (P) is an essential nutrient for marine phytoplankton. Maintaining intracellular P homeostasis against environmental P variability is critical for phytoplankton, but how they achieve this is poorly understood. Here we identify a SPX gene and investigate its role in Phaeodactylum tricornutum. SPX knockout led to significant increases in the expression of phosphate transporters, alkaline phosphatases (the P acquisition machinery) and phospholipid hydrolases (a mechanism to reduce P demand). These demonstrate that SPX is a negative regulator of both P uptake and P-stress responses. Furthermore, we show that SPX regulation of P uptake and metabolism involves a phosphate starvation response regulator (PHR) as an intermediate. Additionally, we find the SPX related genes exist and operate across the phytoplankton phylogenetic spectrum and in the global oceans, indicating its universal importance in marine phytoplankton. This study lays a foundation for better understanding phytoplankton adaptation to P variability in the future changing oceans.

Roles of Alkaline Phosphatase PhoA in Algal Metabolic Regulation under Phosphorus-replete Conditions.

Alkaline phosphatase (AP) in plants and algae is known to hydrolyze dissolved organophosphate (DOP) in order to obtain phosphorus when the preferred dissolved inorganic phosphorus (DIP) is present in limited supply. By conducting comparative analyses of physiologies and transcriptomes on a mutant of PhoA type AP (mPhoA) and wild type (WT) of the marine diatom Phaeodactylum tricornutum CCAP 1055/1 under P-replete and P-depleted conditions, we document other roles of this gene than DOP scavenging. PhoA mutation created by CRISPR/Cas9 diminished its DOP hydrolase activity but led to significant increases in cellular contents of pigment, carbon, and lipids, photosynthetic rate, growth rate, and the transcriptional levels of their corresponding metabolic pathways. All the results in concert indicate that besides P-nutrient scavenging under DIP deficiency, AP also functions, under the P-replete condition, to constrain pigment biosynthesis, photosynthesis, fatty acid biosynthesis, and cell division. These functions have important implications in maintaining metabolic homeostasis and preventing premature cell division.

Transcriptomic and physiological responses of Skeletonema costatum to ATP utilization.

The capacity of phytoplankton to utilize dissolved organic phosphorus (DOP) plays an important role in their competition for resources when the availability of dissolved inorganic phosphorus (DIP) is low in the aquatic systems. Here, we explored the physiological and molecular responses of a globally distributed marine diatom, Skeletonema costatum, in utilizing adenosine-5'-triphosphate (ATP) based on incubation experiments under ATP, DIP-replete, and DIP-depleted conditions. The results show that ATP supports the growth of S. costatum as efficiently as DIP. The pathway of S. costatum involved in utilizing ATP is not related to alkaline phosphatase (AP), an important DOP hydrolase, although extracellular hydrolysis is involved. The transcriptome analysis revealed several transcripts related to the hydrolase activity (e.g. NAD+ diphosphatase), which were significantly upregulated in the ATP culture group, indicating their possible involvement in ATP hydrolysis. Meanwhile, ATP-grown S. costatum exhibited downregulation of the genes related to a series of metabolic activities (e.g. purine metabolism), apparently to adapt to ATP condition.

Metatranscriptome analysis reveals environmental and diel regulation of a Heterosigma akashiwo (raphidophyceae) bloom.

Despite numerous laboratory studies on physiologies of harmful algal bloom (HAB) species, physiologies of these algae during a natural bloom are understudied. Here, we investigated a bloom of the raphidophyte Heterosigma akashiwo in the East China Sea in 2014 using metabarcode (18S rDNA) and metatranscriptome sequencing. Based on 18S rDNA analyses, the phytoplankton community shifted from high diversity in the pre-bloom stage to H. akashiwo predominance during the bloom. A sharp decrease in ambient dissolved inorganic phosphate and strong up-regulation of phosphate and dissolved organic phosphorus (DOP) uptake genes, including the rarely documented (ppGpp)ase, in H. akashiwo from pre-bloom to bloom was indicative of rapid phosphorus uptake and efficient utilization of DOP that might be a driver of the H. akashiwo bloom. Furthermore, observed up-regulated expression of mixotrophy-related genes suggests potential contribution of mixotrophy to the bloom. Accelerating photosynthetic carbon fixation was also implied by the up-regulation of carbonic anhydrase genes during the bloom. Notably, we also observed a strong morning-to-afternoon shift in the expression of many genes. Our findings provide insights into metabolic processes likely important for H. akashiwo bloom formation, and suggest the need to consider timing of sampling in field studies on this alga.

Transcriptomic and physiological analyses of the dinoflagellate Karenia mikimotoi reveal non-alkaline phosphatase-based molecular machinery of ATP utilisation.

The ability to utilize dissolved organic phosphorus (DOP) is important for phytoplankton to survive the scarcity of dissolved inorganic phosphorus (DIP), and alkaline phosphatase (AP) has been the major research focus as a facilitating mechanism. Here, we employed a unique molecular ecological approach and conducted a broader search for underpinning molecular mechanisms of adenosine triphosphate (ATP) utilisation. Cultures of the dinoflagellate Karenia mikimotoi were set up in L1 medium (+P), DIP-depleted L1 medium (-P) and ATP-replacing-DIP medium (ATP). Differential gene expression was profiled for ATP and +P cultures using suppression subtractive hybridisation (SSH) followed by 454 pyrosequencing, and RT-qPCR methods. We found that ATP supported a similar growth rate and cell yield as L1 medium and observed DIP release from ATP into the medium, suggesting that K. mikimotoi cells were expressing extracellular hydrolases to hydrolyse ATP. However, our SSH, qPCR and enzymatic activity assays indicated that 5'-nucleotidase (5NT), rather than AP, was responsible for ATP hydrolysis. Further gene expression analyses uncovered that intercellular purine metabolism was significantly changed following the utilisation of ATP. Our findings reveal a multi-faceted machinery regulating ATP utilisation and P metabolism in K. mikimotoi, and underscore AP activity is not the exclusive indicator of DOP utilisation.

Molecular mechanism of glucose-6-phosphate utilization in the dinoflagellate Karenia mikimotoi.

Phosphorus (P) is an essential nutrient for marine phytoplankton as for other living organisms, and the preferred form, dissolved inorganic phosphate (DIP), is often quickly depleted in the sunlit layer of the ocean. Phytoplankton have developed mechanisms to utilize organic forms of P (DOP). Hydrolysis of DOP to release DIP by alkaline phosphatase is believed to be the most common mechanism of DOP utilization. Little effort has been made, however, to understand other potential molecular mechanisms of utilizing different types of DOP. This study investigated the bioavailability of glucose-6-phosphate (G6P) and its underlying molecular mechanism in the dinoflagellate Karenia mikimotoi. Suppression Subtraction Hybridization (SSH) was used to identify genes up- and down-regulated during G6P utilization compared to DIP condition. The results showed that G6P supported the growth and yield of K. mikimotoi as efficiently as DIP. Neither DIP release nor AP activity was detected in the cultures grown in G6P medium, however, suggesting direct uptake of G6P. SSH analysis and RT-qPCR results showed evidence of metabolic modifications, particularly that mitochondrial ATP synthase f1gamma subunit and thioredoxin reductase were up-regulated while diphosphatase and pyrophosphatase were down-regulated in the G6P cultures. All the results indicate that K. mikimotoi has developed a mechanism other than alkaline phosphatase to utilize G6P.

Transcriptomic and microRNAomic profiling reveals multi-faceted mechanisms to cope with phosphate stress in a dinoflagellate.

Although gene regulation can occur at both transcriptional and epigenetic (microRNA) levels, combined transcriptomic and microRNAomic responses to environmental stress are still largely unexplored for marine plankton. Here, we conducted transcriptome and microRNAome sequencing for Prorocentrum donghaiense to understand the molecular mechanisms by which this dinoflagellate copes with phosphorus (P) deficiency. Under P-depleted conditions, G1/S specific cyclin gene was markedly downregulated, consistent with growth inhibition, and genes related to dissolved organic phosphorus (DOP) hydrolysis, carbon fixation, nitrate assimilation, glycolysis, and cellular motility were upregulated. The elevated expression of ATP-generating genes (for example, rhodopsin) and ATP-consuming genes suggests some metabolic reconfiguration towards accelerated ATP recycling under P deficiency. MicroRNAome sequencing revealed 17 microRNAs, potentially regulating 3268 protein-coding genes. Functional enrichment analysis of these microRNA-targeted genes predicted decreases in sulfatide (sulfolipid) catabolism under P deficiency. Strikingly, we detected a significant increase in sulfolipid sulfatide content (but not in sulphoquinovosyldiacylglycerol content) and its biosynthesis gene expression, indicating a different sulfolipid-substituting-phospholipid mechanism in this dinoflagellate than other phytoplankters studied previously. Taken together, our integrative transcriptomic and microRNAomic analyses show that enhanced DOP utilization, accelerated ATP cycling and repressed sulfolipid degradation constitute a comprehensive strategy to cope with P deficiency in a model dinoflagellate.

Phosphorus physiological ecology and molecular mechanisms in marine phytoplankton.

Phosphorus (P) is an essential nutrient for marine phytoplankton and indeed all life forms. Current data show that P availability is growth-limiting in certain marine systems and can impact algal species composition. Available P occurs in marine waters as dissolved inorganic phosphate (primarily orthophosphate [Pi]) or as a myriad of dissolved organic phosphorus (DOP) compounds. Despite numerous studies on P physiology and ecology and increasing research on genomics in marine phytoplankton, there have been few attempts to synthesize information from these different disciplines. This paper is aimed to integrate the physiological and molecular information on the acquisition, utilization, and storage of P in marine phytoplankton and the strategies used by these organisms to acclimate and adapt to variations in P availability. Where applicable, we attempt to identify gaps in our current knowledge that warrant further research and examine possible metabolic pathways that might occur in phytoplankton from well-studied bacterial models. Physical and chemical limitations governing cellular P uptake are explored along with physiological and molecular mechanisms to adapt and acclimate to temporally and spatially varying P nutrient regimes. Topics covered include cellular Pi uptake and feedback regulation of uptake systems, enzymatic utilization of DOP, P acquisition by phagotrophy, P-limitation of phytoplankton growth in oceanic and coastal waters, and the role of P-limitation in regulating cell size and toxin levels in phytoplankton. Finally, we examine the role of P and other nutrients in the transition of phytoplankton communities from early succession species (diatoms) to late succession ones (e.g., dinoflagellates and haptophytes).

Bioremediation efficiency in the removal of dissolved inorganic nutrients by the red seaweed, Porphyra yezoensis, cultivated in the open sea.

The bioremediation capability and efficiency of large-scale Porphyra cultivation in the removal of inorganic nitrogen and phosphorus from open sea area were studied. The study took place in 2002-2004, in a 300 ha nori farm along the Lusi coast, Qidong County, Jiangsu Province, China, where the valuable rhodophyte seaweed Porphyra yezoensis has been extensively cultivated. Nutrient concentrations were significantly reduced by the seaweed cultivation. During the non-cultivation period of P. yezoensis, the concentrations of NH4-N, NO2-N, NO3-N and PO4-P were 43-61, 1-3, 33-44 and 1-3 micromol L(-1), respectively. Within the Porphyra cultivation area, the average nutrient concentrations during the Porphyra cultivation season were 20.5, 1.1, 27.9 and 0.96 micromol L(-1) for NH4-N, NO2-N, NO3-N and PO4-P, respectively, significantly lower than in the non-cultivation season (p<0.05). Compared with the control area, Porphyra farming resulted in the reduction of NH4-N, NO2-N, NO3-N and PO4-P by 50-94%, 42-91%, 21-38% and 42-67%, respectively. Nitrogen and phosphorus contents in dry Porphyra thalli harvested from the Lusi coast averaged 6.3% and 1.0%, respectively. There were significant monthly variations in tissue nitrogen content (p<0.05) but not in tissue phosphorus content (p>0.05). The highest tissue nitrogen content, 7.65% in dry wt, was found in December and the lowest value, 4.85%, in dry wt, in April. The annual biomass production of P. yezoensis was about 800 kg dry wt ha(-1) at the Lusi Coast in 2003-2004. An average of 14708.5 kg of tissue nitrogen and 2373.5 kg of tissue phosphorus in P. yezoensis biomass were harvested annually from 300 ha of cultivation from Lusi coastal water. These results indicated that Porphyra efficiently removed excess nutrient from nearshore eutrophic coastal areas. Therefore, large-scale cultivation of P. yezoensis could alleviate eutrophication in coastal waters economically.

Widespread and extensive editing of mitochondrial mRNAS in dinoflagellates.

We report evidence of extensive substitutional editing of mitochondrial mRNAs in the dinoflagellate species Pfiesteria piscicida, Prorocentrum minimum and Crypthecodinium cohnii, based on a comparison of genomic and corresponding cDNA sequences determined for two mitochondrial DNA-encoded genes, cox1 (cytochrome oxidase subunit 1) and cob (apocytochrome b). In the cox1 mRNA, we identify 72 substitutions at 40 sites in 39 codons, whereas in cob mRNA, we infer 86 editing events at 51 sites in 48 codons. Editing, which takes place in distinct clusters, changes approximately 2% of the total sequence, occurs predominantly at first and second positions of codons, and involves mostly (but not exclusively) A-->G (47%), U-->C (23%) and C-->U (17%) substitutions. In all but four of the 158 cases, editing changes the identity of the specified amino acid. At 21 (cox1) and 26 (cob) sites, the same nucleotide change is observed at the same position in at least two of the species investigated. At about one-third of the sites, editing results in an amino acid change that increases similarity between the dinoflagellate Cox1 and Cob sequences and their homologs in other organisms; presumably editing at these sites is of particular functional significance. Overall, about half of the editing events either maintain or increase similarity between the dinoflagellate protein sequences and their non-dinoflagellate homologs, while a further one-third of the alterations are "dinoflagellate-specific" (i.e. they involve a change to an amino acid residue selectively conserved in at least two of the dinoflagellate species at a given position). The nature, pattern and phylogenetic distribution of the inferred edits implies either that more than one type of previously described editing process operates on a given transcript in dinoflagellate mitochondria, or that a mechanistically unique type of mitochondrial mRNA editing has evolved within the dinoflagellate lineage.