Circadian Control Sheds Light on Fungal Bioluminescence
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Report Circadian Control Sheds Light on Fungal Bioluminescence Highlights Authors d Bioluminescence in N. gardneri, a basidiomycete, is Anderson G. Oliveira, regulated by the circadian clock Cassius V. Stevani, ..., Jennifer J. Loros, Jay C. Dunlap d Luciferin, reductase, and luciferase, which together make light, all peak at night Correspondence [email protected] (C.V.S.), d Prosthetic LED-illuminated acrylic mushrooms can be used [email protected] (J.C.D.) to study insect behavior d Insects that can disperse fungal spores are attracted to light In Brief at night Oliveira et al. report that bioluminescence in Neonothopanus gardneri is regulated by the circadian clock. Nighttime light attracts insects that can disperse spores, a benefit to fungi living under the forest canopy, where winds are reduced. Oliveira et al., 2015, Current Biology 25, 964–968 March 30, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2015.02.021 Current Biology Report Circadian Control Sheds Light on Fungal Bioluminescence Anderson G. Oliveira,1 Cassius V. Stevani,2,* Hans E. Waldenmaier,2,3 Vadim Viviani,4 Jillian M. Emerson,5 Jennifer J. Loros,5 and Jay C. Dunlap6,* 1Departamento de Oceanografia Fı´sica, Quı´mica, e Geolo´ gica, Instituto Oceanogra´ fico, Universidade de Sa˜ o Paulo, Sa˜ o Paulo, SP 05508-120, Brazil 2Departamento de Quı´mica Fundamental, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, Sa˜ o Paulo, SP 05508-000, Brazil 3Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, Sa˜ o Paulo, SP 05508-000, Brazil 4Departamento de Bioquı´mica, Universidade Federal de Sa˜ o Carlos, Campus Sorocoba, Rodovia Joa˜ o Leme dos Santos, km 110, Sorocaba, SP 18052-780, Brazil 5Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA 6Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA *Correspondence: [email protected] (C.V.S.), [email protected] (J.C.D.) http://dx.doi.org/10.1016/j.cub.2015.02.021 SUMMARY RESULTS AND DISCUSSION Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organ- Circadian clocks are biological oscillators responsible for main- isms doing it. Luminous living things are widespread taining the internal rhythms of animals, plants, fungi, and cyano- and access diverse mechanisms to generate and bacteria to the alternation of external stimuli as light and temper- ature [17]. Interestingly, bioluminescence and circadian biology control luminescence [1–5]. Among the least studied share historical antecedents. Studies of the luminescence of bioluminescent organisms are phylogenetically rare the dinoflagellate Lingulodinium polyedrum (formerly Gonyaulax fungi—only 71 species, all within the 9,000 fungi polyedra), shown to be regulated by a circadian clock, laid the of the temperate and tropical Agaricales order—are foundations for many of the precepts and paradigms of chrono- reported from among 100,000 described fungal biology. These include the concept of temperature compensa- species [6, 7]. All require oxygen [8] and energy tion and the phase response curve, the protocol used to assess (NADH or NADPH) for bioluminescence and are re- the response of the clock to resetting cues as a function of time ported to emit green light (lmax 530 nm) continuously, of day [4]. Although analysis of fungal bioluminescence ap- implying a metabolic function for bioluminescence, peared in the modern literature contemporaneous with that of perhaps as a byproduct of oxidative metabolism in dinoflagellates [18], the biochemical basis of this luminescence lignin degradation. Here, however, we report that and its possible functional/ecological significance have not been elucidated until very recently [6, 19]. The possibility of cir- bioluminescence from the mycelium of Neonothopa- cadian regulation was noted in the early 1960s [12, 13], but it nus gardneri is controlled by a temperature-compen- was quickly discounted, and in the subsequent 50 years only sated circadian clock, the result of cycles in content/ data questioning rhythmicity have appeared [14, 15]. activity of the luciferase, reductase, and luciferin that The Brazilian fungus Neonothopanus gardneri belongs to the comprise the luminescent system. Because regula- Omphalotus lineage [20] and displays exceptionally intense lumi- tion implies an adaptive function for biolumines- nescence from either mycelium or basidiomes (Figures 1A–1D). cence, a controversial question for more than two This fungus is distributed throughout of the Brazilian Coconut millennia [8–15], we examined interactions between Forest (Mata dos Cocais) in the states of Maranha˜ o, Tocantins, luminescent fungi and insects [16]. Prosthetic acrylic Goia´ s, and Piauı´ [20], which is a transitional biome between resin ‘‘mushrooms,’’ internally illuminated by a green Amazonian Forest and Caatinga. Fruiting bodies grow at the LED emitting light similar to the bioluminescence, base of palm trees (Attalea humilis, A. funifera, and Orbignya phalerata). attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers Circadian Control of Bioluminescence Agar plates freshly inoculated with N. gardneri mycelium were far greater than dark control traps. Thus, circadian maintained over 48 hr in a 12-hr dark/light cycle at constant tem- control may optimize energy use for when biolumi- perature (21C, 25C, and 29C, depending on the experiment). nescence is most visible, attracting insects that can After this initial entrainment period, the plates were transferred in turn help in spore dispersal, thereby benefitting to constant darkness in a climatic chamber equipped with a fungi growing under the forest canopy, where wind charge-coupled device (CCD) camera, and an image was digi- flow is greatly reduced. tally recorded every hour over 6 days. Analyses of these pictures 964 Current Biology 25, 964–968, March 30, 2015 ª2015 Elsevier Ltd All rights reserved Figure 1. Circadian Control of Fungal Bioluminescence in Neonothopanus gardneri (A–D) Mushroom of Neonothopanus gardneri (A) under illumination, and (B) in the dark. The inset shows a rover beetle magnified image. (C) Minimum and (D) maximum bioluminescence from the mycelium of N. gardneri during photographical studies of light emission oscillation are shown. (E) Bioluminescence profile from the mycelium after 48-hr entrainment period at 21C, 25C, and 29C over a 6-day experiment confirms temperature compensation of circadian period length (error bars indicate ±SD; n = 3). (F) Luminescence profile from luciferin-, reductase-, and luciferase-rich extracts (error bars indicate ±SD; n = 3) obtained from mycelium after 48-hr entrainment period at 25C and over 48 hr (see more details in the Experimental Procedures). Luciferin light emission is multiplied by 1,000. Photos were obtained with a Nikon D3100, equipped with AF-S Micro Nikkor 60 mm f/2.8G ED lenses (mushrooms: ISO 6400, f/5.6, 1/40 s [under headlamp illumination] and 15 s [in the dark]; cultures: ISO 12800, f/4.5, 30 s). Mycelium of N. gardneri was cultivated in 2.0% agar medium containing 1.0% sugar cane molasses and 0.10% yeast extract. by imaging software confirmed that the light emission from the Fungal bioluminescence depends on four components, namely, bioluminescent mycelium oscillates in a circadian rhythm of a substrate (luciferin), a soluble NAD(P)H-dependent reductase, 22 hr at 25C(Figure 1E), with the peak phase of intensity a membrane-bound oxygenase (luciferase), and oxygen [19]. occurring about 10 hr after the light-to-dark transfer and at circa- Active luciferin can be extracted from cultivated mycelium using dian intervals thereafter. Circadian oscillators are temperature boiling citrate buffer under argon atmosphere to prevent oxida- compensated; i.e., they maintain an 24-hr period whenever tion. The enzymes can also be partially purified from the culti- the organisms are grown at warmer or cooler temperatures vated mycelium using cold phosphate buffer, and the reductase across the physiological range [17]. When the experiments and the luciferase subsequently separated by ultracentrifugation were repeated at 21C and 29C, the period was only slightly because the luciferase is an insoluble membrane protein. Light changed; comparison of the periods at different temperatures al- emission can be obtained in vitro by mixing of the hot extract lowed calculation (http://www.physiologyweb.com/calculators/ (luciferin source) and the cold extract (reductase/luciferase q10_calculator.htm)ofaQ10 of 1.04 for the rhythm, confirming source) and finally by addition of NADPH to trigger the reaction its temperature compensation. Interestingly, these circadian [19] (see more details in the Experimental Procedures). We period lengths of less than 24 hr are similar to those reported took advantage of the natural occurrence of bioluminescence for Neurospora crassa, the non-luminescent ascomycete that in this fungus and used its own luciferase, reductase, and lucif- is a prominent model system for analysis of the molecular basis erin to investigate the molecular basis of the oscillation in of rhythms [21]. bioluminescence. Biochemical Basis of Circadian Bioluminescence Cultures were entrained as above through 2 days of 12-hr Luciferases (enzymes that catalyze the light emission in luminous dark/light cycles prior to release into constant darkness, and organisms) have successfully