The endosymbiont of Epithemia clementina is specialized for nitrogen fixation within a photosynthetic eukaryote.

Epithemia spp. diatoms contain obligate, nitrogen-fixing endosymbionts, or diazoplasts, derived from cyanobacteria. These algae are a rare example of photosynthetic eukaryotes that have successfully coupled oxygenic photosynthesis with oxygen-sensitive nitrogenase activity. Here, we report a newly-isolated species, E. clementina, as a model to investigate endosymbiotic acquisition of nitrogen fixation. We demonstrate that the diazoplast, which has lost photosynthesis, provides fixed nitrogen to the diatom host in exchange for fixed carbon. To identify the metabolic changes associated with this endosymbiotic specialization, we compared the Epithemia diazoplast with its close, free-living cyanobacterial relative, Crocosphaera subtropica. Unlike C. subtropica, in which nitrogenase activity is temporally separated from photosynthesis, we show that nitrogenase activity in the diazoplast is continuous through the day (concurrent with host photosynthesis) and night. Host and diazoplast metabolism are tightly coupled to support nitrogenase activity: Inhibition of photosynthesis abolishes daytime nitrogenase activity, while nighttime nitrogenase activity no longer requires cyanobacterial glycogen storage pathways. Instead, import of host-derived carbohydrates supports nitrogenase activity throughout the day-night cycle. Carbohydrate metabolism is streamlined in the diazoplast compared to C. subtropica with retention of the oxidative pentose phosphate pathway and oxidative phosphorylation. Similar to heterocysts, these pathways may be optimized to support nitrogenase activity, providing reducing equivalents and ATP and consuming oxygen. Our results demonstrate that the diazoplast is specialized for endosymbiotic nitrogen fixation. Altogether, we establish a new model for studying endosymbiosis, perform a functional characterization of this diazotroph endosymbiosis, and identify metabolic adaptations for endosymbiotic acquisition of a critical biological function.

The endosymbiont of Epithemia clementina is specialized for nitrogen fixation within a photosynthetic eukaryote.

Epithemia spp. diatoms contain obligate, nitrogen-fixing endosymbionts, or "diazoplasts", derived from cyanobacteria. These algae are a rare example of photosynthetic eukaryotes that have successfully coupled oxygenic photosynthesis with oxygen-sensitive nitrogenase activity. Here, we report a newly-isolated species, E. clementina, as a model to investigate endosymbiotic acquisition of nitrogen fixation. To detect the metabolic changes associated with endosymbiotic specialization, we compared nitrogen fixation, associated carbon and nitrogen metabolism, and their regulatory pathways in the Epithemia diazoplast with its close, free-living cyanobacterial relative, Crocosphaera subtropica. Unlike C. subtropica, we show that nitrogenase activity in the diazoplast is concurrent with, and even dependent on, host photosynthesis and no longer associated with cyanobacterial glycogen storage suggesting carbohydrates are imported from the host diatom. Carbohydrate catabolism in the diazoplast indicates that the oxidative pentose pathway and oxidative phosphorylation, in concert, generates reducing equivalents and ATP and consumes oxygen to support nitrogenase activity. In contrast to expanded nitrogenase activity, the diazoplast has diminished ability to utilize alternative nitrogen sources. Upon ammonium repletion, negative feedback regulation of nitrogen fixation was conserved, however ammonia assimilation showed paradoxical responses in the diazoplast compared with C. subtropica. The altered nitrogen regulation likely favors nitrogen transfer to the host. Our results suggest that the diazoplast is specialized for endosymbiotic nitrogen fixation. Altogether, we establish a new model for studying endosymbiosis, perform the first functional characterization of this diazotroph endosymbiosis, and identify metabolic adaptations for endosymbiotic acquisition of a critical biological function.

Fatty acid photodecarboxylase is an ancient photoenzyme that forms hydrocarbons in the thylakoids of algae.

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.

A Selaginella moellendorffii Ortholog of KARRIKIN INSENSITIVE2 Functions in Arabidopsis Development but Cannot Mediate Responses to Karrikins or Strigolactones.

In Arabidopsis thaliana, the α/ß-fold hydrolase KARRIKIN INSENSITIVE2 (KAI2) is essential for normal seed germination, seedling development, and leaf morphogenesis, as well as for responses to karrikins. KAI2 is a paralog of DWARF14 (D14), the proposed strigolactone receptor, but the evolutionary timing of functional divergence between the KAI2 and D14 clades has not been established. By swapping gene promoters, we show that Arabidopsis KAI2 and D14 proteins are functionally distinct. We show that the catalytic serine of KAI2 is essential for function in plants and for biochemical activity in vitro. We identified two KAI2 homologs from Selaginella moellendorffii and two from Marchantia polymorpha. One from each species could hydrolyze the strigolactone analog GR24 in vitro, but when tested for their ability to complement Arabidopsis d14 and kai2 mutants, neither of these homologs was effective. However, the second KAI2 homolog from S. moellendorffii was able to complement the seedling and leaf development phenotypes of Arabidopsis kai2. This homolog could not transduce signals from exogenous karrikins, strigolactone analogs, or carlactone, but its activity did depend on the conserved catalytic serine. We conclude that KAI2, and most likely the endogenous signal to which it responds, has been conserved since the divergence of lycophytes and angiosperm lineages, despite their major developmental and morphogenic differences.