Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behavior and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes1-3. A fundamental knowledge gap exists however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology4,5. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg 2+ ] i , which act as a cell-autonomous timekeeping component to determine key clock properties in both a human cell line and a unicellular alga that diverged from metazoans more than 1 billion years ago6. Given the essential role of Mg 2+ as a cofactor for ATP, a functional consequence of [Mg 2+ ] i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg 2+ availability has potential to impact upon many of the cell's >600 MgATP-dependent enzymes7 and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR8 is regulated through [Mg 2+ ] i oscillations. It will now be important to identify which additional biological # To whom correspondence should be sent: Gerben.vanOoijen@ed.ac.uk, oneillj@mrc-lmb.cam.ac.uk. Author contributions GvO and JSO conceived the approach and designed the study. LFL and COY generated the Neurospora result. JD and LE performed ICP analyses. GvO and LLH performed Ostreococcus experiments. Human U2OS cell experiments were performed by KAF. MP performed mouse fibroblast experiments. NPH provided analytical and intellectual contributions. GvO and JSO wrote the manuscript.

Nucleic acid amplification tests (NAATs) are common in laboratory and clinical settings because of their low time to result and exquisite sensitivity and specificity. Laboratory NAATs include onboard positive controls to reduce false negatives and specialized hardware to enable real-time fluorescence detection. Recent efforts to translate NAATs into at-home tests sacrifice one or more of the benefits of laboratory NAATs, such as sensitivity, internal amplification controls (IACs), or time to result. In this manuscript, we describe a mobile-phone-based strategy for real-time imaging of biplexed NAATs in paper. The strategy consisted of: (1) using mobile phones with multipass excitation and emission filters on the flash and camera to image the signal from distinct fluorophore-labeled probe types in a biplexed NAAT in a glass fiber membrane; and (2) analyzing the differential fluorescence signal between the red and green color channels of phone images to overcome a strong evaporation-induced optical artifact in heated glass fiber pads due to changes in the refractive index. We demonstrated that differential fluorescence imaging enabled low limits of detection (316 copies of methicillin-resistant Staphylococcus aureus DNA) in our lab's "MD NAAT" platform, even in biplexed isothermal strand displacement amplification reactions containing 100k copies of coamplifying IAC DNA templates. These results suggest that two-fluorophore mobile phone imaging may enable translating the benefits of extant laboratory-based, real-time NAATs to the point of care.

The transcriptional circadian clock network is tuned into a 24-h oscillator by numerous posttranslational modifications on the proteins encoded by clock genes, differentially influencing their subcellular localization or activity. Clock proteins in any circadian organism are subject to posttranslational regulation, and many of the key enzymes, notably kinases and phosphatases, are functionally conserved between the clocks of mammals, fungi, and plants. We now establish sumoylation, the posttranslational modification of target proteins by the covalent attachment of the small ubiquitin-like modifier protein SUMO, as a novel mechanism regulating key clock properties in the model plant Arabidopsis. Using 2 different approaches, we show that mutant plant lines with decreased or increased levels of global sumoylation exhibit shortened or lengthened circadian period, respectively. One known functional role of sumoylation is to protect the proteome from temperature stress. The circadian clock is characterized by temperature compensation, meaning that proper timekeeping is ensured over the full range of physiologically relevant temperatures. Interestingly, we observed that the period defects in sumoylation mutant plants are strongly differential across temperature. Increased global sumoylation leads to undercompensation of the clock against temperature and decreased sumoylation to overcompensation, implying that sumoylation buffers the plant clock system against differential ambient temperature.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

In plants, the circadian clock regulates the expression of one-third of all transcripts and is crucial to virtually every aspect of metabolism and growth. We now establish sumoylation, a posttranslational protein modification, as a novel regulator of the key clock protein CCA1 in the model plant Arabidopsis. Dynamic sumoylation of CCA1 is observed in planta and confirmed in a heterologous expression system. To characterize how sumoylation might affect the activity of CCA1, we investigated the properties of CCA1 in a wild-type plant background in comparison with ots1 ots2, a mutant background showing increased overall levels of sumoylation. Neither the localization nor the stability of CCA1 was significantly affected. However, binding of CCA1 to a target promoter was significantly reduced in chromatin-immunoprecipitation experiments. In vitro experiments using recombinant protein revealed that reduced affinity to the cognate promoter element is a direct consequence of sumoylation of CCA1 that does not require any other factors. Combined, these results suggest sumoylation as a mechanism that tunes the DNA binding activity of the central plant clock transcription factor CCA1.