Neurospora Clock-Controlled Gene 9 ( ccg-9 ) Encodes Trehalose Synthase: Circadian Regulation of Stress Responses and Development

Abstract

The circadian clock of Neurospora crassa regulates the rhythmic expression of a number of genes encoding diverse functions which, as an ensemble, are adaptive to life in a rhythmic environment of alternating levels of light and dark, warmth and coolness, and dryness and humidity. Previous differential screens have identified a number of such genes based solely on their cycling expression, including clock-controlled gene 9 (ccg-9). Sequence analysis now shows the predicted CCG-9 polypeptide to be homologous to a novel form of trehalose synthase; as such it would catalyze the synthesis of the disaccharide trehalose, which plays an important role in protecting many cells from environmental stresses. Consistent with this, heat, glucose starvation, and osmotic stress induce ccg-9 transcript accumulation. Surprisingly, however, a parallel role in development is suggested by the finding that inactivation of ccg-9 results in altered conidiophore morphology and abolishes the normal circadian rhythm of asexual macroconidial development. Examination of a clock component, FRQ, in the ccg-9-null strain revealed normal cycling, phosphorylation, and light induction, indicating that loss of the conidiation rhythm is not due to changes in either the circadian oscillator or light input into the clock but pointing instead to a defect in circadian output. These data imply an interplay between a role of trehalose in stress protection and an apparent requirement for trehalose in clock regulation of conidiation under constant environmental conditions. This requirement can be bypassed by a daily light signal which drives a lightentrained rhythm in conidiation in the ccg-9-null strain; this bypass suggests that the trehalose requirement is related to clock control of development and not to the developmental process itself. Circadian control of trehalose synthase suggests a link between clock control of stress responses and that of development.Organisms ranging from bacteria to mammals possess an endogenous mechanism that temporally organizes biochemical, cellular, and behavioral activities. These daily rhythms are produced by the circadian biological clock and are manifested by the cyclic expression of genes and gene products controlled by one or more output pathways from the clock. Circadian rhythms are coordinated with exogenous environmental cycles to limit activities to particular times of the day, thus allowing organisms to anticipate daily changes in their environment and to organize their metabolism and behavior appropriately (reviewed in reference 21).The molecular mechanisms underlying circadian rhythmicity in Neurospora crassa are beginning to be understood. Components of the oscillator and input pathways of the clock have been identified and include FRQ, WC-1, and WC-2 (reviewed in references 19, 42, and 44). In addition, 12 clock-controlled genes (ccgs) have been identified in screens targeting output pathways (10,43,67), and several additional genes (con-6, con-10, al-3, bli-3, and vvd) are also known to be regulated by t...

Circadian Rhythms in Neurospora crassa and Other Filamentous Fungi

“…Verification of clock regulation of the ccg's, as opposed to some other form of temporal regulation, was achieved by demonstrating that the period of the ccg mRNA abundance rhythm equaled the period of the strain examined. Specifically, for the long (29-h)-period frq 7 mutant strain, the period of the ccg mRNA rhythms was 29 h. In all cases examined, the FWO functioned normally in strains containing inactivated copies of the ccg's, demonstrating that they are part of an output pathway and are not involved in oscillator function (7,102,103). Such loss-of-function assays have been critical for helping to distinguish whether a gene that is rhythmically expressed is an output from the clock or is part of the oscillator mechanism.…”

Circadian rhythmicity mediated by temporal regulation of the activity of p38 MAPK

“…Osmotic sensitivity assays were performed on solid VM supplemented with 4% NaCl, 4% KCl, or 1 M sorbitol at 34°C in DD. Osmotic induction experiments were performed as described (18) by using liquid VM (at DD 24 unless otherwise indicated) with the following modification: 5 M NaCl was added directly to the culture medium to a final concentration of 0.7 M (4%). Time-series experiments for analysis of protein or RNA were performed as described (58) by using cultures grown in 1ϫ Vogel's salts/2% glucose/0.5% arginine for protein extraction, and 1ϫ Fries/0.03% glucose/0.05% arginine for RNA extraction.…”

“…To date, Ͼ180 ccgs have been identified in N. crassa (10)(11)(12)(13)(14), including genes associated with rhythms in asexual spore development (conidiation; ref. 15), metabolism (16), pheromone production (17), and stress responses (18). However, only a handful of these ccgs have been studied in depth, and few details of the output pathways from N. crassa or any other organism's circadian oscillator are known.…”

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

“…The enzymes that are part of the trehalose phosphate synthase/phosphatase complex are orthologs of yeast TPS1 (AFUB_001790), TPS2 (AFUB_ 043350), TPS3 (AFUB_089470), TSL1 (AFUB_021090), and CLOCK9 (AFUA_6G03420) (10,24,25). The two key enzymes of the mannitol biosynthetic pathway are the mannitol 1 phosphate 5 dehydrogenase (AFUB_026440) and the mannitol 2 dehydrogenase (AFUB_071700) (M1PDH and M2DH) (26).…”