C. Robertson McClung

Professor of Biological Sciences

C.Robertson.McClung@dartmouth.edu

Research Interests

The ability of an organism to measure time is the product of a cellular biological clock. Many phenomena controlled by the biological clock cycle on a daily basis and are called circadian rhythms. My goal is to understand the genetic and biochemical mechanisms by which an organism measures time and uses that temporal information to regulate gene expression and cellular physiology. The circadian clock is an endogenous oscillator that drives rhythms with periods of approximately 24 hours. By definition, these circadian (from the Latin, circa, approximately; dies, day) rhythms persist in constant conditions and reflect the activity of an endogenous biological clock. Plants are richly rhythmic and the circadian clock regulates a number of key metabolic pathways and stress responses. In addition, the circadian clock plays a critical role in the photoperiodic regulation of the transition to flowering in many species. Circadian rhythms in plants have been the subject of a number of excellent reviews. Two recent reviews from my lab can be obtained at these links: (McClung, 2001) (McClung, Salomé & Michael, 2002)

Current Projects

Clock regulation of gene expression in a higher plant

We have identified several Arabidopsis thaliana genes whose expression is controlled by the biological clock. We are characterizing elements required, in cis or in trans, for circadian regulation of transcription of RCA, which encodes ribulose bisphosphate carboxylase/oxygenase (RUBISCO) activase (Liu et al., 1996). Expression of two catalase (CAT) genes is also clock-regulated, albeit differentially: CAT3 mRNA abundance is maximal during the evening, distinguishing it from other morning-specific genes such as CAT2 and RCA (Zhong & McClung, 1996). Comparison of RCA, CAT2 and CAT3 should reveal conserved features of clock regulation and provide insight into the mechanisms by which gene expression is phased to different times of day. Comparison of the elements regulating evening-specific transcription of CAT3 with those regulating mid-morning-specific transcription of the CAB2 (LHCB1*3) gene, encoding the chlorophyll a/b binding protein, has shown a critical requirement for closely -related elements. We have defined a minimal CAT3 promoter sufficient to drive evening-specific circadian transcription of a LUCIFERASE reporter gene (Michael & McClung, 2002, in press). Deletion analysis and site-directed mutagenesis reveal a circadian response element, the Evening Element (EE: AAAATATCT), that is necessary for evening-specific transcription. The EE differs only by a single bp from the CCA1 binding site (CBS: AAAAAATCT), which is important for morning-specific transcription of CAB2. We tested the hypothesis that the EE and the CBS specify circadian phase by site-directed mutagenesis to convert the CAT3 EE into a CBS. Changing the CAT3 EE to a CBS changes the phase of peak transcription from the evening to the morning in continuous dark and in light-dark cycles, consistent with the specification of phase by the single bp that distinguishes these elements. However, rhythmicity of the CBS-containing CAT3 promoter is dramatically compromised in continuous light. Additional information normally provided in the context of a morning-specific promoter is necessary for full circadian activity of the CBS. Current research is directed towards identifying the elements that provide this contextual information and their cognate DNA-binding proteins.

Mutational analysis of the biological circadian clock

There are circadian oscillations in stomatal aperture and in gas exchange in Arabidopsis. These rhythms provide a genetic approach to screen mutagenized populations for mutants affected in circadian rhythmicity. Resistance to toxic gases (such as SO₂) is correlated to stomatal aperture. This allowed the identification of mutants with altered period (i.e., the clock runs fast or slow), which we have named circadian timing defective (ctd). Interestingly, many of these ctd mutants also exhibit defects in the photoperiodic timing of flowering, providing a genetic confirmation of the link between circadian and photoperiodic timing. We are applying a map-based strategy to positionally clone these genes. Subsequent characterization will be directed towards the elucidation of their role in biological timekeeping and circadian regulation. We have also characterized one mutant, out of phase 1 (oop1), with the circadian phenotype of altered phase (Salomé et al., 2002). That is, the timing of the peak (acrophase) of multiple circadian rhythms (leaf movement, CO₂ assimilation and CAB/LHCB transcription) is early with respect to wild type, although all circadian rhythms retain normal period length. This is the first such mutant to be characterized in Arabidopsis. oop1 also displays a strong photoperception defect in red light characteristic of phytochrome B (phyB ) mutants. Indeed, the oop1 mutation is a nonsense mutation of PHYB that results in a truncated protein of 904 amino acids. The defect in circadian phasing is seen in seedlings entrained by a light-dark cycle, but not in seedlings entrained by a temperature cycle. Thus, PHYB contributes light information critical for proper determination of circadian phase. We are currently characterizing a second phase mutant, oop2, which has defects primarily in blue light photoperception.

Photorespiration: Positional cloning and characterization of a locus required for photorespiratory serine hydroxymethyltransferase activity

The primary rate-limiting step in photosynthesis, the fixation of CO₂, is catalyzed by ribulose bisphosphate carboxylase/oxygenase (Rubisco). Rubisco is a bifunctional enzyme that catalyzes either the carboxylation or the oxygenation of ribulose bisphosphate (RuBP). The oxygenation of RuBP leads to the evolution of CO₂ from the plant in a process termed photorespiration. Photorespiratory CO₂ loss for C3 plants (e.g., soybeans, rice, sugar beets) is approximately 25% of the gross rate of CO₂ fixation. Because of the magnitude of this loss, considerable effort has been devoted to understanding photorespiration. An essential component of the photorespiratory pathway is the mitochondrial isozyme of serine hydroxymethyltransferase (SHMT), encoded by one of a family of seven SHM genes (McClung et al., 2000). Mutation of the STM gene results in the loss of mitochondrial SHMT activity and confers a conditionally-lethal phenotype (the mutant dies at low CO₂ concentration but is viable at elevated CO₂ levels). It was thought for 20 years that STM encoded the photorespiratory SHMT. However, our genetic data have established that STM is a distinct gene that encodes an unexpected and uncharacterized protein product. We will clone and characterize STM with our goal being the elucidation of the mechanism by which STM affects SHMT activity. This will have significance to both basic plant science and to agricultural productivity. Characterization of the STM protein may reveal previously unsuspected aspects of photorespiration. The understanding of the photorespiratory pathway is critical to its manipulation. Because of the magnitude of the photorespiratory loss in primary plant productivity, the reduction of photorespiration affords the potential to significantly enhance crop production.

updated July 3, 2003