Prof. Luis F. Larrondo Castro (PAS)., Professor of Biological Sciences, Pontificia Universidad Católica de Chile

“God saw that the light was good; and God separated the light from the darkness” (Genesis 1:4).

Undeniably, the history of life on this planet has been shaped by the unceasing cycles of days and nights. While the Bible recounts that “God separated the light from the darkness”, the study of biological systems reveals that evolution has indeed partitioned processes in a daily fashion, with some occurring during the day and others at night. Likewise, Ecclesiastes 3:1-22 verses that “To everything, there is a season, a time for every purpose under heaven” and, certainly, cellular and organismal activities are governed by the same principle: diverse processes are temporally compartmentalized peaking, for example, at defined times each day, or even exhibiting seasonal rhythms [1]. And importantly, while biological rhythms can be observed at various scales (i.e. from milliseconds to seasons), I will focus on those with a periodicity of 24 hours: the so-called circadian rhythms, from the Latin circa diem (close to a day). These rhythms have a period close to, but not exactly, 24 hours under constant conditions. However, when exposed to environmental cycles of temperature or light, they synchronize precisely to a 24-hour period, akin to the precision of a wristwatch.

While the study of circadian clocks spans the entire tree of life, including prokaryotes, my focus here will be on fungi. These organisms serve as powerful model systems for exploring and unraveling the complexities of circadian rhythms. By studying these phenomena in fungi, we can also gain unique insights into the evolution of clocks and the nuanced mechanisms of light-sensing systems

Circadian clocks allow individuals to synchronize different aspects of their biology with earth’s day-night rhythms. Bona fide circadian rhythms i) persist in the absence of external cues with a free running period of circa 24 h, ii) maintain this period in a range of physiological temperatures (temperature compensation) and iii) “entrain” to the environment [2–4]. Clocks are not only fascinating from an evolutionary and molecular perspective, but are also relevant for cellular and organismal function: they temporarily compartmentalize diverse and even antagonizing cellular processes, such as catabolic and anabolic ones. This allows the optimization of organismal physiology, synchronizing it with the environment and permitting individuals to anticipate daily rhythmic changes [5–7]. Not surprisingly, genetic or environmental perturbations of clocks can compromise fitness [8], alter organismal interactions [9,10], and have physiological severe consequences, including significant health problems [11,12].

The evidence suggests that circadian clocks have appeared – independently – at least three different times throughout evolution, and consequently, the analysis of the core-clock elements across the tree of life shows that they do not share a common evolutionary origin [13,14]. Nevertheless, despite the lack of sequence conservation, the architecture of the central clock circuit (oscillator), which is based on a transcription-translation negative feedback loop (TTFL), is highly conserved across eukaryotes from fungi to mammals: a compelling example of convergent evolution, permitting to translate mechanistic details learned in, for example, a fungus, to humans and vice versa [15,16]. Indeed, from fungi to animals, circadian oscillators are based on a one-step transcription-translation negative feedback loop (TTFL) where positive elements (the transcription factors WC-1/WC-2 in Neurospora; CYC/CLK in Drosophila; BMAL1/CLOCK in mammals) directly drive the expression of a negative element (FRQ; TIM/PER; CRY/PER, respectively). The negative elements form multiprotein complexes – that always include the enzyme Casein Kinase 1 (CK1) – and feedback to inhibit the activity of the positive elements [3,17]. As a result, the expression of new negative elements is shut down, whereas the existing negative components are progressively modified (mainly by CK1) until inactivated and degraded, time at which a new cycle of expression starts. The entire process takes about 24 hours [4]. Notably, most of the molecular details of eukaryotic core-clocks have been elucidated in a handful of model species: the fruit fly Drosophila melanogaster, the fungus Neurospora crassa, mice, and humans [3,4,12]. However, as more genomes have been sequenced, it has become increasingly clear that numerous examples exist in various taxa across the Tree of Life where, despite the presence of circadian phenomena, genes encoding canonical clock components are not identifiable. These findings suggest the possible existence of other, so far uncharacterized, pervasive (yet evasive) clock mechanisms [18,19]. Together with others in the field, we have considered this possibility, and in the following pages, I will outline how we are tackling this and other problems in fungi.

For almost half a century, the filamentous fungus N. crassa has played, along with the fruit fly D. melanogaster, a key role in the molecular dissection of the circadian clockworks [20]. In Neurospora, the TTFL is composed of a positive element, the White-Collar Complex (WCC), comprised of the transcription factors White Collar-1 (WC-1) and White Collar-2 (WC-2), which promotes the expression of the negative element frq, encoding for the protein FREQUENCY (FRQ). One full cycle takes about 22.5 hours under constant darkness and can be tracked by monitoring rhythms in frq mRNA and protein levels [21,22]. Under daily Light: Dark or temperature cycles these rhythms entrain to exactly 24 hours, yet Neurospora is arrhythmic under constant light. One feature that has made Neurospora a great circadian system is the ease with which circadian rhythms can be tracked by monitoring the daily appearance of “conidial bands” representing the synchronous formation of asexual spores before dawn. The clock controls sporulation and a large extent of Neurospora biology, including daily rhythms in ~ 40% of its transcriptome [21,22]. In addition, the use of luciferase as a real-time reporter has allowed us to promptly track the inner workings of the core clock by monitoring changes in bioluminescence, which has dramatically expedited the analyses of circadian mechanisms in this fungus [22]. Despite significant advances in this model system, our understanding of clocks in fungi other than Neurospora remains limited [18]. This is striking when we consider that the fungal kingdom encompasses between 2-5 million species, of which ~ 5% have been formally described. Fungi have played a crucial role in shaping life on Earth by, among other things, driving nutrient cycles, supporting plant evolution through symbiosis, and continuing to play key roles in both terrestrial and aquatic ecosystems [23]. Thus, considering the mesmerizing fungal diversity and the relevance of these organisms, it is surprising that when it comes to molecular circadian mechanisms, of the dozens of thousands of known fungal species, most of the knowledge is circumscribed to only one, the ascomycete N. crassa.

From an evolutionary perspective, the scenario is even more intriguing when considering that of the ~1090 fungal genomes currently available, FRQ homologs are restricted to a limited number of clades [5]. Moreover, in fungi whose genome exhibit all putative core-clock elements, the characterization of molecular rhythms has often been hard to achieve, which contrasts with the robust phenotypic and molecular circadian rhythms seen in Neurospora [18]. This raises fundamental questions about the evolutionary origins of FRQ and how ecological niches may have influenced the loss or retention of FRQ-based clocks across diverse fungal taxa.

We and others have sought to close this knowledge gap by exploring the extent and impact of circadian clocks in various fungi. So far, some of the findings highlight the importance of circadian rhythms in multiple aspects of the fungal daily life, modulating metabolism, interactions with other organisms, and even virulence. However, evidence of a functional clock has been elusive in some species. Moreover, some of these studies have uncovered numerous peculiarities, such as unexpected extra-circadian roles for FRQ and robust rhythms (of ecological relevance) in species without any identifiable conventional clock components [18,24]. Notably, while the absence of known core-circadian components appears widespread among major fungal taxa, this phenomenon is less common within animals or the green lineage. Indeed, in taxa outside the fungal kingdom, the occurrence of rhythms in organisms lacking obvious eukaryotic clock components is more of an exception than the rule [18].

Yet, work in Neurospora not only continues to inform us about mechanistic details of the clockworks but also facilitates adopting strategies to tackle fundamental aspects of the evolution of eukaryotic clocks. Thus, we adopted a Synthetic Biology approach by challenging the known evolutionarily conserved circadian TTFL architecture, creating a semi-synthetic clock in Neurospora, for which we combined (by transcriptional rewiring) canonical clock components and elements naturally restricted to the circadian output pathways [25]. The resulting semi-synthetic clock not only was functional, but it also exhibited new unexpected properties, regarding how light cues are processed. But, even more relevant in the context of this discussion is that we were able to show that the evolutionarily conserved clock circuitry (one-step TTFL) is only one of the possible topologies that a circadian oscillator could assume. Yet, the existing conventional TTFL is the simplest design and, by parsimony, is what nature has always chosen.

In addition, fungi offer several advantages for reexamining the adaptive role of circadian clocks. Over the past years, several colleagues have critically revisited the general concept of the “adaptive advantage of clocks” and the pressures behind the evolution of circadian systems [8,26]. Inspired by these ideas, we have begun applying circadian paradigms to investigate the importance of a functional clock in Neurospora, evaluating its impact on fitness under stressful rhythmic environments. Thus, we have been examining whether the presence of a circadian clock can facilitate the appearance of increased thermotolerance. To do so, we expose Neurospora to rhythmic experimental evolution protocols, mimicking progressively increasingly hot environments. Currently, we are trying to interpret the preliminary results from ecological and evolutionary angles.

The proverb “seeing is believing” may have modern origins, but it is often associated with the skepticism of Saint Thomas the Apostle, as depicted in the Gospel of John (John 20:24-29). This proverb suggests that visual evidence facilitates understanding, a concept that helps to introduce some of our molecular studies on how Neurospora senses light—a critical process for synchronizing its circadian clock with day-night cycles. In gene expression analyses, we frequently rely on limited datasets to draw conclusions, oftentimes without the possibility of observing the complete expression patterns of our gene of interest, especially across a range of stimuli. In light of this, I would like to discuss some of our recent work in the laboratory, where we aim to map the dynamic range of gene expression triggered by light. In doing so, I hope to captivate (or provoke?) readers with the idea that the sensitivity offered by such a molecular system enables Neurospora to “see” the intricate details of its surroundings and that, moreover, we can get to “see” what Neurospora has perceived.

Neurospora and many other fungi can sense blue light via a LOV (Light Oxygen Voltage) domain in proteins such as WC-1 (part of the White-Collar Complex). Neurospora senses light and quickly elicits transcriptional responses, spanning the activation of hundreds of genes [27]. One of these genes is frq: therefore, upon light, FRQ levels go up, changing the internal “time” and, therefore, adjusting the phase of the clock. Thus, WC-1 stimulation by light is key in synchronizing and entraining the clock to environmental Light: Dark cycles [22]. But how sensitive is WC-1 to light, and how well can it tell apart lights of various intensities? Actually, most of what we know about WC-1 responses are snapshots of Neurospora’s full ability to respond to light: they correspond to data points gathered under a handful of intensities and after a fixed number of minutes. Yet, we were interested in obtaining (literally) an accurate picture of the response of Neurospora to a broad range of radiance levels.

Using luciferase under the control of a light-responsive promoter, we can stimulate Neurospora with light and measure its response in real-time, monitoring changes in luciferase levels (bioluminescence). It is worth mentioning that we can grow Neurospora on large agar plates, yielding a high-density mycelial mat resembling a “cottony canvas”. On top of this mycelial mat, we can focus light of different intensities, inquiring whether the genetic response (measured as bioluminescence output) yields a result resembling the original stimulus. Therefore, we have called this approach “Live Canvas,” as it represents a living surface (fungal mat) where light images can be perceived (seen), genetically processed, and emitted back as bioluminescence (manuscript in preparation). Relevantly, an image emitted by a projector is in itself a map of light intensities. Thus, our layman’s question is whether an image, projected for one second onto a fungal mat, can elicit a bioluminescence response that accurately replicates the projected image. If so, it would indicate that WC-1 can effectively capture the detailed information encoded in a full array of light intensities. Remarkably, the results surpassed our expectations, demonstrating a broad dynamic range of responses to a wide spectrum of radiance levels. This suggests that WC-1 activity yields an analog response rather than a digital one. Notably, in the field of optogenetics (where light is used to control biological processes), there are several examples of using a projected image to induce a genetic response that mirrors the original picture. However, these experiments typically require at least 20 hours of light stimulation to produce the desired effect [28,29]. This radically differs from the situation in Neurospora, where we can trigger a robust response with one second of stimulation, evidenced by the appearance of a clear image within minutes. Fig. 1 contains an example of a Live Canvas image generated with the picture of an artistic representation of the Shroud of Turin: projecting this picture on top of a Neurospora fungal mat (of 30x18 cm) yielded, within minutes, a response that recreated with exquisite resolution the original projected picture. While the aesthetic appeal of the Live Canvas (essentially a gene expression response) is undeniable, it is equally important to emphasize the rich biological and molecular insights that can be gleaned from such a “simple” bioluminescent image. Indeed, the Live Canvas prompts us to reexamine crucial aspects of Neurospora fungal biology, such as inconsistencies in the current understanding of photoadaptation or the syncytial state of Neurospora in solid culture. In addition, such images provide dynamic series of gene expression of unprecedented resolution, establishing a biological platform where we can analyze with great detail the consequences and effects of distinct mutations affecting key aspects of light-triggered transcription (i.e., chromatin remodeling, nuclear export, etc.).

Consequently, we have extended this approach into the circadian field, exploring additional scientific aspects underlying the Live Canvas. As explained earlier, light can shift the Neurospora circadian clock; thus, if this fungus is exposed to light, the “phase” of the clock will be shifted depending on the strength of the light perturbation [22]. Thus, if we have a reporter strain, where luciferase is under the control of a promoter that is both light-induced and clock-regulated, in constant darkness one would see synchronous levels of luciferase all over the fungal mat and, once every ~22.5 hours, the entire culture would show a coordinated peak of bioluminescence. However, suppose parts of the fungal mat (canvas) are shortly exposed to light. In that case, locally the phase of the clock will be altered: the clock will continue oscillating with the same periodicity but at a different phase, similar to having a different “local time”. The latter is akin to shifting from Eastern Standard Time to Pacific Time with a strong light exposure or to Central Standard Time with a weaker one. Thus, light can shift the phase of the biological clock, similar to moving to a new time zone. Therefore, we sought to explore whether a map of light intensities (delivered as a 1 second image projection) could be converted into a map of phase changes. The results prove that; indeed, it was possible to locally alter the phase of the clock such that the resulting map of phase alterations (throughout the fungal mat) creates an emergent property, allowing the visualization of the image appearing and disappearing over many days (unpublished). Indeed, it was possible to observe the picture as a bioluminescent image, appearing, fading away, and then reappearing the next day and for many days. While the visual effect is mesmerizing, it is also humbling to appreciate the precision of the Neurospora circadian system, witnessing how phase information can be faithfully maintained at the cellular level over multiple days.

Thus, if readers are willing to entertain the initial idea that Neurospora can “see” an image, I would like to push this concept even further by suggesting that Neurospora can “remember” what it has seen – possessing a form of photographic or eidetic memory, so to speak. While I leave that idea open for thought, I would like to conclude with a more fundamental – yet basic – question: why has a fungus like Neurospora evolved such a precise, accurate and sensitive light-measuring system? Another way to approach this question is to ask whether such sensitivity is a peculiarity of Neurospora’s ecological niche or a common characteristic among filamentous fungi possessing WC-1 homologs. Rather than attempting to elucubrate an answer, I will conclude by pondering why Neurospora possesses such a clear and robust circadian clock, while other fungi either i) have FRQ-based clocks that seem less robust or ii) rely on clock mechanisms that do not depend on FRQ. Personally, I see all these questions and challenges as excellent opportunities to explore the diversity of the fungal world and deepen our understanding of how these biological agencies have evolved to respond to their environment, day after day, since the dawn of time, ultimately learning to measure time itself. I think we are finally beginning to obtain a true “picture” of these intricate mechanisms.

Acknowledgments

I want to express my deep appreciation for the dedication and enthusiasm of a remarkable team of individuals who, over the years, have contributed to shape our lab into what it is today. I would also like to acknowledge funding sources, in particular ANID-Millennium Science Initiative Program-Millennium Institute for Integrative Biology (iBio ICN17_022), the international Research Scholar program of the Howard Hughes Medical Institute, and the Richard Lounsbery Foundation.

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