plate-based-imaging-assay-for-quantifying-photoperiodic-responses

Plate-Based Imaging Assay for Quantifying Photoperiodic Responses in <em>Neurospora crassa</em> Using Protoperithecia Development

Neurospora crassa is a well-established model organism for studying biological timing and light-regulated development. The Protoperithecia Assay (PPA) is ...

The demand for renewable and sustainable energy has generated considerable interest in the conversion of cellulosic biomass into liquid fuels such as ethanol using a filamentous fungus. While attempts have been made to study cellulose metabolism through the use of knock-out mutants, there have been no systematic effort to characterize natural variation for cellulose metabolism in ecotypes adapted to different habitats. Here, we characterized natural variation in saccharification of cellulose and fermentation in 73 ecotypes and 89 laboratory strains of the model fungus Neurospora crassa. We observed significant variation in both traits among natural and laboratory generated populations, with some elite strains performing better than the reference strain. In the F1 population N345, 15% of the population outperformed both parents with the top performing strain having 10% improvement in ethanol production. These results suggest that natural alleles can be exploited through fungal breeding for developing elite industrial strains for bioethanol production.

Developing elite Neurospora crassa strains for cellulosic ethanol production using fungal breeding.

Most living organisms on earth experience daily and expected changes from the rotation of the earth. For an organism, the ability to predict and prepare for incoming stresses or resources is a very important skill for survival. This cellular process of measuring daily time of the day is collectively called the circadian clock. Because of its fundamental role in survival in nature, there is a great interest in studying the natural variation of the circadian clock. However, characterizing the genetic and molecular mechanisms underlying natural variation of circadian clocks remains a challenging task. In this chapter, we will summarize the progress in studying natural variation of the circadian clock in the successful eukaryotic model Neurospora, which led to discovering many design principles of the molecular mechanisms of the eukaryotic circadian clock. Despite the success of the system in revealing the molecular mechanisms of the circadian clock, Neurospora has not been utilized to extensively study natural variation. We will review the challenges that hindered the natural variation studies in Neurospora, and how they were overcome. We will also review the advantages of Neurospora for natural variation studies. Since Neurospora is the model fungal species for circadian study, it represents over 5 million species of fungi on earth. These fungi play important roles in ecosystems on earth, and as such Neurospora could serve as an important model for understanding the ecological role of natural variation in fungal circadian clocks.

Natural Variation of the Circadian Clock in Neurospora.

Hyperglycemia plays a key role in the pathogenesis of cardiovascular complications of diabetes. Type 2 diabetes mellitus (T2DM) is associated with high-density lipoprotein (HDL) dysfunction and increased degradation of apolipoprotein I (ApoAI). The mechanism(s) of these changes is largely unknown.

Glycation Reduces the Stability of ApoAI and Increases HDL Dysfunction in Diet-Controlled Type 2 Diabetes.

Circadian rhythms are observed in most organisms on earth and are known to play a major role in successful adaptation to the 24-h cycling environment. Circadian phenotypes are characterized by a free-running period that is observed in constant conditions and an entrained phase that is observed in cyclic conditions. Thus, the relationship between the free-running period and phase of entrainment is of interest. A popular simple rule has been that the entrained phase is the expression of the period in a cycling environment (i.e., that a short period causes an advanced phase and a long period causes a delayed phase). However, there are experimental data that are not explained by this simple relationship, and no systematic study has been done to explore all possible period-phase relationships. Here, we show the existence of stable period-phase relationships that are exceptions to this rule. First, we analyzed period-phase relationships using populations with different degrees of genome complexity. Second, we generated isogenic F1 populations by crossing 14 classical period mutants to the same female and analyzed 2 populations with a short period/delayed phase and a long period/advanced phase. Third, we generated a mathematical model to account for such variable relationships between period and phase. Our analyses support the view that the circadian period of an organism is not the only predictor of the entrained phase.

Experimental and Mathematical Analyses Relating Circadian Period and Phase of Entrainment in Neurospora crassa.

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS ( = 7), NASH ( = 8), and healthy controls ( = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days,  < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days,  < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher ( < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio ( < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL·min) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both  < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins' turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.

HDL flux is higher in patients with nonalcoholic fatty liver disease.

The circadian clock controls daily activities at the cellular and organismic level, allowing an organism to anticipate incoming stresses and to use resources accordingly. The circadian clock has therefore been considered a fitness trait in multiple organisms. However, the mechanism of how circadian clock variation influences organismal reproductive fitness is still not well understood. Here we describe habitat-specific clock variation (HSCV) of asexual reproduction in , a species that is adapted to 2 different habitats, under or above tree bark. African (AF)  strains, whose habitat is above the tree bark in light-dark (LD) conditions, display a higher rhythmicity index compared with North American (NA) strains, whose habitat is under the tree bark in constant dark (DD). Although AF-type strains demonstrated an overall fitness advantage under LD and DD conditions, NA-type strains exhibit a habitat-specific fitness advantage in DD over the LD condition. In addition, we show that allelic variation of the clock-controlled gene, Ubiquinol cytochrome c oxidoreductase (NEUDI_158280), plays a role in HSCV by modulating cellular reactive oxygen species levels. Our results demonstrate a mechanism by which local adaptation involving circadian clock regulation influences reproductive fitness.

Habitat-Specific Clock Variation and Its Consequence on Reproductive Fitness.

Bioethanol production from lignocellulosic biomass has received increasing attention over the past decade. Many attempts have been made to reduce the cost of bioethanol production by combining the separate steps of the process into a single-step process known as consolidated bioprocessing. This requires identification of organisms that can efficiently decompose lignocellulose to simple sugars and ferment the pentose and hexose sugars liberated to ethanol. There have been many attempts in engineering laboratory strains by adding new genes or modifying genes to expand the capacity of an industrial microorganism. There has been less attention in improving bioethanol-related processes utilizing natural variation existing in the natural ecotypes. In this study, we sought to identify genomic loci contributing to variation in saccharification of cellulose and fermentation of glucose in the fermenting cellulolytic fungus Neurospora crassa through quantitative trait loci (QTL) analysis. We identified one major QTL contributing to fermentation of glucose and multiple putative QTL's underlying saccharification. Understanding the natural variation of the major QTL gene would provide new insights in developing industrial microbes for bioethanol production.

Quantitative trait loci (QTL) underlying phenotypic variation in bioethanol-related processes in Neurospora crassa.

Circadian rhythms are ubiquitous and are observed in all biological kingdoms. In nature, their primary characteristic or phenotype is the phase of entrainment. There are two main hypotheses related to how circadian clocks entrain, parametric and non-parametric models. The parametric model focuses on the gradual changes of the clock parameters in response to the changing ambient condition, whereas the non-parametric model focuses on the instantaneous change of the phase of the clock in response to the zeitgeber. There are ample empirical data supporting both models. However, only recently has a unifying model been proposed, the circadian integrated response characteristic (CiRC). In the current study, we developed a system of ordinary differential equations, dynamic CiRC (dCiRC), that describes parameters of circadian rhythms and predicts the phase of entrainment in zeitgeber cycles. dCiRC mathematically extracts the underlying information of velocity changes of the internal clock that reflects the parametric model and the phase shift trajectory that reflects the non-parametric model from phase data under entraining conditions. As a proof of concept, we measured clock parameters of 26  ecotypes in both cycling and constant conditions using dCiRC. Our data showed that the morning light shortens the period of the clock while the afternoon light lengthens it. We also found that individual ecotypes have different strategies of integrating light effects to accomplish the optimal phase of entrainment, a model feature that is consistent with our knowledge of how circadian clocks are organized and encoded. The unified model dCiRC will provide new insights into how circadian clocks function under different zeitgeber conditions. We suggest that this type of model may be useful in the advent of chronotherapies.

A Unified Model for Entrainment by Circadian Clocks: Dynamic Circadian Integrated Response Characteristic (dCiRC).

Melatonin, a molecule with diverse biological functions, is ubiquitously present in living organisms. There is significant interest in understanding melatonin signal transduction pathways in humans, particularly due to its critical role in regulating the sleep-wake cycle. However, a knowledge gap remains in fully elucidating the mechanisms by which melatonin influences circadian regulation. To bridge this gap, there is a growing need for a model system to study the role of melatonin in circadian clocks, with Neurospora crassa being a promising candidate. As a first step in this investigation, we focused on identifying melatonin receptors in N. crassa. Given the lack of sequence similarity between potential receptors in this fungus and known human melatonin receptors, we utilized structural similarity analysis through AlphaFold2. This approach led to the identification of a strong candidate gene, gpr-3, which shares structural similarities with human melatonin receptors. Experimental validation confirmed that the removal of GPR-3 from cells results in the absence of melatonin signaling. This proof-of-concept study underscores the potential of N. crassa as a model organism for circadian research and demonstrates the broader applicability of using AlphaFold2, especially when sequence similarity does not lead to candidate genes, for identifying novel receptors across different species.

Article	Year
Developing elite Neurospora crassa strains for cellulosic ethanol production using fungal breeding. Journal of industrial microbiology & biotechnology\| PubMed ID: 28429154	2017
Natural Variation of the Circadian Clock in Neurospora. Advances in genetics\| PubMed ID: 29050553	2017
Glycation Reduces the Stability of ApoAI and Increases HDL Dysfunction in Diet-Controlled Type 2 Diabetes. The Journal of clinical endocrinology and metabolism\| PubMed ID: 29077935	2018
Experimental and Mathematical Analyses Relating Circadian Period and Phase of Entrainment in Neurospora crassa. Journal of biological rhythms\| PubMed ID: 29183256	2017
HDL flux is higher in patients with nonalcoholic fatty liver disease. American journal of physiology. Endocrinology and metabolism\| PubMed ID: 31503515	2019
Habitat-Specific Clock Variation and Its Consequence on Reproductive Fitness. Journal of biological rhythms\| PubMed ID: 31878828	2020
Quantitative trait loci (QTL) underlying phenotypic variation in bioethanol-related processes in Neurospora crassa. PloS one\| PubMed ID: 32017762	2020
A Unified Model for Entrainment by Circadian Clocks: Dynamic Circadian Integrated Response Characteristic (dCiRC). Journal of biological rhythms\| PubMed ID: 35156426	2022
Cross-species comparison of AlphaFold-derived G protein-coupled receptor structures reveals novel melatonin-related receptor in Neurospora crassa. PloS one\| PubMed ID: 39874366	2025

Kwangwon Lee

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