Heritable variation in the timing of rhythmic events with respect to daily time cues gives rise to chronotypes. Despite its importance, the mechanisms (clock or non-clock) regulating chronotypes remain elusive. Using artificial laboratory selection for divergent phasing of emergence of adults from pupae, our group has derived populations of Drosophila melanogaster which are early and late chronotypes for eclosion rhythm. Several circadian rhythm characteristics of these populations have since been described. We hypothesized that our selection protocol has inadvertently resulted in selection for masking, a non-clock phenomenon, in the early chronotype due to the placement of our selection window (which includes the lights-ON transition). We designed experiments to discriminate between enhanced masking to light versus circadian clock mediated changes in determining enhanced emergence in the morning window in our early chronotypes. Using a series of phase-shift protocols, LD-DD transition, and T-cycle experiments, we find that our early chronotypes have evolved positive masking, and their apparent entrained phases are largely contributed by masking. Through skeleton T-cycle experiments, we find that in addition to the evolution of greater masking, our early chronotypes have also evolved advanced phase of entrainment. Furthermore, our study systematically outlines experimental approaches to examine relative contributions of clock versus non-clock control of an entrained behavior. Although it has previously been suggested that masking may confer an adaptive advantage to organisms, here we provide experimental evidence for the evolution of masking as a means of phasing that can complement clock control of an entrained behavior.
Malate (2-hydroxysuccinic acid) and tartrate (2,3-dihydroxysuccinic acid) are chiral substrates; the former existing in two enantiomeric forms (R and S) while the latter exists as three stereoisomers (R,R; S,S; and R,S). Dehydration by stereospecific hydrogen abstraction and anti-elimination of the hydroxyl group yield the achiral products fumarate and oxaloacetate, respectively. Class-I fumarate hydratase (FH) and L-tartrate dehydratase (L-TTD) are two highly conserved enzymes belonging to the iron-sulfur cluster hydrolyase family of enzymes that catalyze reactions on specific stereoisomers of malate and tartrate. FH from Methanocaldococcus jannaschii accepts only S-malate and S,S-tartrate as substrates while the structurally similar L-TTD from Escherichia coli accepts only R-malate and R,R-tartrate as substrates. Phylogenetic analysis reveals a common evolutionary origin of L-TTDs and two-subunit archaeal FHs suggesting a divergence during evolution that may have led to the switch in substrate stereospecificity preference. Due to the high conservation of their sequences, a molecular basis for switch in stereospecificity is not evident from analysis of crystal structures of FH and predicted structure of L-TTD. The switch in enantiomer preference may be rationalised by invoking conformational plasticity of the amino acids interacting with the substrate, together with substrate reorientation and conformer selection about the C2-C3 bond of the dicarboxylic acid substrates. Although classical models of enzyme-substrate binding are insufficient to explain such a phenomenon, the enantiomer superposition model suggests that a minor reorientation in the active site residues could lead to the switch in substrate stereospecificity.
Heritable variation in the timing or circadian phases of rhythmic events with respect to daily time cues gives rise to chronotypes. Despite its importance, the mechanisms (clock or non-clock) regulating chronotypes remain elusive. Using artificial laboratory selection for divergent phasing of emergence of adults from pupae, our group has derived populations of Drosophila melanogaster which are early and late chronotypes for eclosion rhythm. Several circadian rhythm characteristics of these populations have since been described. We hypothesized that our selection protocol has inadvertently resulted in selection for masking, a non-clock phenomenon, in the early chronotype due to the placement of our selection window (which includes the lights-ON transition). Based on theoretical predictions and previous studies on our populations, we designed experiments to discriminate between enhanced masking to light versus circadian clock mediated changes in determining enhanced emergence in the morning window in our early chronotypes. Using a series of phase-shift protocols, LD-DD transition, and T-cycle experiments, we find that our early chronotypes have evolved positive masking, and their apparent entrained phases are largely contributed by masking. Through skeleton T-cycle experiments, we find that in addition to the evolution of greater masking, our early chronotypes have also evolved advanced phase of entrainment. Furthermore, our study systematically outlines experimental approaches to examine relative contributions of clock versus non-clock control of an entrained behavior. Although it has previously been suggested that masking may confer an adaptive advantage to organisms, here we provide experimental evidence for the evolution of masking as a mean of phasing of an entrained rhythm that can complement clock control of an entrained behavior.
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