Circadian Rhythm In Algae Biology Essay

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Daily oscillations of metabolism and behaviour that we now refer to as circadian (In Latin, circa - about, dies - one day) have been under the observational eyes for more than 250 years. Folding of leaves, sleep wake behaviour, hormone secretion, bioluminescence and various such overt expression of this behaviour opened the gates to this fascinating biology. Its a very interesting concept where life has learned time keeping and adjusted it around earth's daily day night cycling period. For a very long time it was believed to be a feature exclusive to eukaryotes. Absence of macro phenotypes and a general presumption that a fast doubling time would be inhibitory to time keeping kept prokaryotes away from the attention it needed. In last 20 years although interesting biology has emerged where these organisms have taken the pioneering role in explaining circadian clocks and their impact on physiology.

Photosynthetic organism have the closest relationship to the day night cycle. Owing to the difficulty of studying such mechanisms in system like plants, field initially progressed better in the animal models like Drosophila and Mouse and in fungi Neurospora crassa. Although since the discovery of circadian rhythms in prokaryotic cyanobacteria, they have taken the center stage and now present the most well understood circadian biology. In photosynthetic bacteria, 30-60% of the expressed transcripts are under circadian regulation compared to 10% in mammals. That when added to the ease of manipulation and possibility of automating observations has extended our understanding substantially which will be discussed here.

What is a Circadian Clock and Circadian Rhythm?

Clock is a time keeping device, which can accurately measure the time. A simple sounding idea is challenging biologically, where things happen enzymatically. Its important to elaborate this point. Firstly, although circadian and diurnal are used commonly in each others place on a stricter note they are different. Diurnal is also a 24 hr period behaviour that is synchronised to day/night cycle. When a Diurnal behaviour has ability to maintain its rhythmicity in absence of external cues then only it can be referred to as circadian rhythm. It means it is not just a simple response to changing external environment but it follows an internal clock. Although these terms quite frequently used as synonyms because most of the diurnal rhythms are found to be circadian in a nature and its 'ok' for most practical purposes.

There are four criteria which define a circadian rhythm.

1. First is that it should be circadian - that is 'about a day' long, ~24 hour period length. Anything faster than 20 hours generally is referred to ultradian (eg. Heart rate, appetite) and anything longer than 30 hours is generally referred as infradian (eg. menstrual cycle, seasonal or tidal changes).

2.Secondly, the point of 'persistence in absence of external cue', which we have already made. This is the true test of time keeping. In presence of constant light and temperature condition the rhythm should continue to function for a substantial period.

3. Thirdly, the ability of entrainment. A biological clock owing to variation in physiological conditions or migration or shock will eventually develop a phase shift with the exogenous diurnal rhythm. In such a scenario, an endogenous timekeeping will be wrong and rendered useless. it is hence important that this clock should have ability to retrain or resynchronise itself to the external cues or zeitgebers (literal meaning time giver). All circadian clocks and rhythms reorient themselves when put in alternate light/dark (or temperature cycles) in few days.

4.Fourthly, the clock should have the property of temperature compensation. As with all biological reactions, clock behaviour is also an output of biochemical and enzymatic reactions. Most of the biological reactions have a Q10 of 2 or higher, which means enzymatic rates double with a change of 10' of temperature. A circadian rhythm or clock is able to maintain its period length to 24 hours in its physiological temperature range. The biochemical mechanics are special and circadian biochemistry generally has a Q10 of 0.8-1.2.

Circadian clock is the biological molecular oscillator that satisfies the above listed criteria and Circadian rhythm is the phenotype or behaviour that shows a periodic change with the cues from the endogenous circadian clock. There are few more features that are important for discussion as they relate to organism or biochemistry specific challenge and will be discussed later in the chapter.

Circadian Biology and its advantages.

Biology has seen the diurnal variations of environment since beginning. Cyanobacteria have spent more than three billion years in these cycling environments. These periodic changes must have substantial effect, positive or negative, on various biological processes. For eg, a day brings an opportunity for photosynthesis for photosynthetic organism, but also brings harmful radiation to which certain biological processes like DNA replication are sensitive. Similarly parameters like optimal temperature, visibility, etc would have governed the necessity to do temporal compartmentalisation of many processes. Once the basic biology starts following circadian rhythm, a dependant and more complex form of networked responses follow the circadian trend, eg, insects have to follow blooming of flowers, which follow the diurnal rhythm for better display and dispersion of fragrance.

But why does one need a clock? cant it be a simple response to the changing physical parameters like many other inducible responses that we see in the nature. This is an interesting question. Although the answer is that a time keeping provides the ability to predict and prepare for very certain changes and hence provides a better efficiency of response. For eg. many photosynthetic genes start expressing before the optimum sunlight is present. there are processes which are mutually incompatible, like photosynthesis and nitrogen fixation, and need to be separated. these are big programmes that are time compartmentalised. Of course they have sub regulatory mechanism in themselves but circadian clock acts as a global and central mechanism for cueing and queuing these processes. Also the machinery that would be required to specifically create response elements for so many biological processes would be ultimately a taxing and inefficient solution. A central clock governed mechanism seems fairly logical in such a scenario, and is evident by the fact that upto 30% of expressed transcripts are orchestrated by this mechanism in algae.

For a very long time circadian biology was thought to be exclusive to eukaryotes and initial research was governed by the circadian rhythms that were easy to study or more precisely where the observations can be automated. Folding of leaves, swarming of algae, bioluminence were the behaviors which can be studied automatically. A dinoflagellate, Lingulodinium polyedrum (earlier known as Gonyaulax polydera) was initially the hotspot for circadian research. These cells showed many kinds of rhythms which can be studied easily. These cells had small vesicular structures called scintillons which exhibited a circadian behaviour. Their bioluminescence rhythm could be divided into two kinds of rhythm, a flashing rhythm which peaked at midnight and a glowing rhythm which peaked at end of the night. In constant conditions of temperature and dim light this rhythms continued to oscillate for many days. In the same conditions these cells also show rhythmic variation in photosynthesis, with peak intensities reaching at subjective day. Cells also show migration rhythm, where during subjective day they swim to the surface and aggregate. This kind of behaviours have obvious advantage of increasing the efficiency and amount of photosynthesis. During night cells are deeper in the ocean where they will be closer to the ocean floor and which may provide a richer environment for nitrogen sources. Cell division is also regulated by circadian clock and it happens with maximum frequency at the night/day intersection. Like these gross physiological behaviors, molecular level oscillations in certain enzymes were also detected in L.polyedrum. Enzymes like Nitrate reductase and SOD were maximally active during the day. Nitrate reductase converts nitrate to nitrite and Superoxide Dismutase is an ROS scavenging enzyme. Similarly there were enzymes like tyrosine aminotransferase which showed peak activity dduring night time. Regulation of these activities were not always at transcriptional level. In some cases transcript levels remained constant throughout 24 hours but protein level varied indicating a translational regulation. In some cases like Rubisco, the protein level also remained constant but the activity varied in circadian manner, indicating a post translational regulation. These means that the output of the circadian clock can be used to influence activity by modulating any of the three stages of regulation.

Chlamydomonas reinhardtii, a green algae, was another system of choice. This system was heavily studied for flagellar and photosynthetic biology and proved to be a lucrative system to work out circadian biology. Chlamydomonas cells also showed phototaxis which showed maximum intensity during the day period. In an interesting experiment by Mergenhagen Lab, Chlamydomonas phototaxis was monitored in gravity free environment in space. Cells were able to robustly maintain circadian rhythm of phototaxis in space. These cells also show chemotaxis rhythm, where they swim towards ammonium with maximum intensity at middle of night. A point to be mentioned here is that the peak intensity of ammonium assimilation is at the dawn. Another rhythm which is maximally expressed during night is cell adherence to glass. Obviously this doesn't has context in natural environment but it reflects changes that are happening at the cell surface which is changing its properties. Generally genes involved in photosynthesis showed highest expression during CT23 (just before beginning of day) and genes involved in respiration showed peak at CT12 (end of day). Chlamydomonas also show rhythmic patterns in UV sensitivity, starch synthesis and cell division, at their maximum during day night switch. There are indications that these might be partially dependent on each other. Euglenophyta, Euglena gracilis, also showed a similar repertoire of circadian rhythms including phototaxis,photosynthesis, dark motility, cell settling, cell shape etc. While these showed maximal activity in day time, cell division peaked at subjective night. Recently using bioluminescence marker, circadian rhythm in transcript levels of Chloroplast genes was demonstrated in Chlamydomonas. Several ROC mutants (rhythm of chloroplast)perturbed in the natural rhythm were also isolated by doing random insertional mutagenesis in nuclear genome. This indicates nuclear regulation of rhythm in chloroplast, although possibility of an independent oscillator of chloroplast is still possible idea. Possibility of multiple oscillators in a system will be discussed later in the chapter.

Circadian biology was demonstrated in cyanobacteria relatively recently. The field progressed rapidly owing to the lessons learned and principles laid in techniques to study circadian biology. Bioluminescence was found to be the best tool to monitor circadian rhythms. So after initial discovery of presence of circadian clock, rather than discovering circadian rhythms ( which anyways is almost the whole of the biology of cyanobacteria), a more focused effort was made in unravelling the mechanistic details of the oscillator using biochemistry and bioluminescence reporter. In 1985 non heterocystous cyanobacteria Oscillatoria and Synechococcus were demonstrated to have circadian rhythm in nitrogen fixation. Nitrogen fixation is an extremely oxygen sensitive process and will be highly incompatible with photosynthesis. Many species of cyanobacteria avoid this problem by making special oxygen free structures called heterocysts. Non heterocystous algae had to solve this problem using temporal isolation of each activity ie. nitrogen fixation was done in the night phase. Similarly O2 evolution rhythms in constant conditions were demonstrated in another cyanobacteria Trichodesmium

Exciting things started happening after a heterologous luciferase reporter system was established in cyanobacteria. Scientist started pursuing molecular mechanism of the circadian system. For the first time now the molecular oscillator was under the focus. The field progressed almost entirely by the meticulous efforts of Carl Johnson, Takoa Kondo and Susan Golden labs. They in collaboration incorporated a bacterial lux gene from Vibrio fischeri under psbA1 promoter of Synechococcus. Normally psbA1 encodes D1 protein whose mRNA levels shows circadian behavior. The heterologous construct PpsbA1::luxAB also showed rhythmicity in form bioluminescence and confirmed to all the classical parameters of circadian rhythm of persistence, phase resetting by Light-Dark treatment and temperature compensation. This was an important technical development in the field and made study suitable for doing genetic studies. Similar strategies were then used in other organisms to study circadian rhythms.A screen for identifying all the promoters that were circadian regulated was done in cyanobacteria using promoter trap strategy. Bacterial luxAB gene was randomly integrated in the genome of Synechococcus. Whenever this gene was integrated downstream of a random promoter, the culture would become bioluminescent. If the promoter was circadian regulated it can be easily detected by automated monitoring of bioluminescence of that clone. As predicted many bioluminescent clones were obtained, but the biggest surprise was that most of them showed circadian rhythm (at different amplitude though). A transcription factor rpoD2 was hypothesised to cause this change, and was shown to regulate a subset of genes in cyanobacteria. A complete solution arrived 15 years later when this was correlated to global change in supercoiling status of the bacterial DNA.

Mutagenic studies had also began using these system as platform. Several mutants of cyanobacteria with altered (shorter or longer) period length were isolated. Ouyang in 1998, did a competition assay with these strains to answer that what kind of fitness did these circadian rhythms conferred to an organism. These mutants showed identical growth kinetics when grown in constant condition of light and temperature and did not out compete each other. But when these mutants were competed against wild type in 24 hour light dark cycles, wild type was always successful in out competing period mutants within few days. When culture conditions were altered to a light dark period length which was similar to mutant's circadian period then that specific mutant was fitter compared to wild type or other period mutants and out competed them. These competition assays without getting into the details of fitness, clearly demonstrated that the circadian rhythms confer a fitness advantage in natural settings.

Molecular mechanisms of - Input, Oscillator and Output.

In 1960s, Brian Goodwin proposed a simplest oscillator, a model to explain biological oscillations (not limited to circadian). Goodwin oscillator comprises of a single gene that represses itself. An extension to this is that any component whose modified form represses the expression or activity of original form will cause oscillatory behavior. A modified version of this central theme will become evident further in the discussion.

Conceptually a circadian clock can be divided into three parts.

1. Oscillator : a central component whose concentration or activity oscillates in a timed manner.

2. Input : a mechanism which can change the phase of the oscillator. For eg. mechanism that sense the light or dark environment and cause phase changes in the oscillator.

3. Output : a mechanism which takes cues from central oscillator or depending upon the stage of central oscillator causes a downstream effect for eg. upregulation or downregulation of certain genes.

The concept of circadian clock and molecular details were derived from genetic mutations which caused defects in this mechanism. Defects of certain kinds could be logically assigned to certain components of the model and slowly a model evolves. For eg. defects in phase resetting with dark but not with light indicates existence of input pathway. Arrythmicity in all behaviors predicts the existence of a central oscillator, arrythmicity in subset of behavior indicates a problem in output pathway and so on. Although during initial years of the field such tools were not available and studies entirely depended on careful observations and limited repertoire of inhibitors to predict regulation involved. Case of three organisms are discussed to highlight the foundation and development of molecular mechanism of circadian rhythm.

Lingulodinium polyedrum (Gonyaulax polydera)

In many regulatory pathways, it was known that dephosphorylation played a major role in signal transduction. Comolli in 1990s discovered that protein phosphatase inhibitors like okadaic acid had strong effect on period lengthening it to about 29 hrs. This signified that dephosphorylation was indeed a key event in the circadian mechanism of this alga. Similarly nitrate was found to influence all the parameters (amplitude, phase and period) implying its role in input pathway. Effect of light quality was also studied for its ability to reset the clock. It was found that the system was maximaly sensitive to red (650 nm) and blue (520 nm) wavelengths. Further detailed studies yielded that there were atleast two photoreceptors/input pathway, one of which was blue sensitive and another was blue + red sensitive.

Bioluminescence was a good model to study output mechanisms as its biochemistry was fairly well understood. In L.polyedrum bioluminescence is produced by vesicle called scintillons where the luciferin and associated enzymes are concentrated. Luciferin of this organism is unique to dinoflagellates and it releases light upon oxidation with O2. Under normal circumstances this reaction is blocked by LBP (luciferin binding protein) by binding tightly at physiological pH of 7.5. An action potential from tonoplast membrane can decrease local pH in scintillons to 6.5. This achieves two things, LBP separates from luciferin and LCF (Luciferase) is activated which catalyses luciferin oxidation, resulting in bioluminescence. It was known that the protein amounts of LBP LCF and luciferin varied significantly during circadian rhythm. Scintillon numbers in cells increased 10 fold during night. Next thing to ask was how is this achieved. When quantitation of lbp mRNA was done there was no significant difference in the transcript level across the day and remained constitutively high. So a translational level checkpoint was hypothesised. In fact, in a clever experiment this was found to be the case for majority of the proteins. A 2d analysis of proteins revealed many proteins whose amount varied during day night cycle. But when proteins were prepared using RNA extracted from the same time points, they showed no significant variation. Indicating that transcript levels were constant. This indicated that translational regulation acts as a major mechanism of circadian output. Although in unique cases like RUBISCO, it was observed that the activity rather than protein amount oscillated and protein modification is also one of the strategies.

To further explore the translational regulation RNA-protein interactions were done. mRNA UTRs (untranslated regions, both 5'UTR and 3'UTR) are important sites for binding of regulatory proteins. In the screen a novel RNA binding protein with unique motif binding activity was identified. The protein was called CCTR (circadian coupled translational regulator) and had a specific binding activity to UG ( a specific motif of 7 U(U)G repeats) repeats. The binding is specific to the loop structure(single stranded region) of lbp mRNA and can be inhibited by using antisense RNA or removing at least 2 UG repeats. The CCTR binding activity is high during day causing repression of translation and vice versa. CCTR binding to mRNA causes translation start from a uORF which is present upstream of LBP ORF and causes ribosomes to skip LBP ORF. CCTR binding is regulated by phosphorylation status of itself. the binding motif of CCTR has been observed in other mRNAs also which means they also might be under similar regulation.

Chlamydomonas reinhardtii

Even after a rigorous study of more than 4 decades the central oscillator of Chlamydomonas eludes researchers. Although several mutants have been generated for varying period length a good collection of arrhythmic mutants (only for ROC) has only been recently available. Arrhythmic mutants are important in nailing down the central oscillator. with a sufficient amount of mutagenesis the central components can be located. An inability to isolate such mutants indicates possibility of multiple circadian oscillators.

Light quality studies for phase resetting were done in Chlamydomonas which revealed peak influence at 520nm and 660 nm. For 660nm a phytochrome receptor was postulated as was the case with few other model systems. Phytochrome signaling can be reversed by providing a FR (far red) pulse immediately after a Red pulse. This kind of behaviour was not observed in the phase resetting experiment and hence the nature of red receptor remains open. During continuous dim light condition, an intense light pulse (for few hours) was used to reset the phase. In these experiments action spectra showed a peak similar to that of photosynthesis. It suggested that photosynthetic components might play a prominent role in input mechanism in this alga. It was also shown that blocking photosynthesis with DCMU (inhibitor of PSII) also blocked light induced resetting in cells under continuous light condition. Kinases and phosphatases have an important role in circadian machinery. Casein Kinase I is one such protein known to be involved in animal clocks. CKI was located in eyespot and flagellar proteomes suggesting that it might have important role in signal transduction. Silencing mutants of CKI showed hatching and flagellar formation problems. The period was also shortened by 1.5 hrs. A collection of ROC mutants was also analysed for the genes causing circadian mutant phenotypes. About quarter of the mutants showed hatching or flagellar defects. These genes were grouped into various pathways which highlighted flagellar function, ubiquitin-proteasome pathway, membrane trafficking, DNA damage pathways, etc as the major class of mutants.

In Chlamydomonas some of the genes that are nuclear encoded showed a circadian rhythm of transcription, for eg. lhcpII (light harvesting complex protein) and CAH1 (carbonic anhydrase) show peak transcription in day whereas arf1 (ADP ribosylation factor) shows peak during night phase. Whereas majority of chloroplast transcripts showed a global change in levels with circadian rhythm, peaking in subjective day. These kind of global changes in transcript levels could be resulting from changing supercoiling state of chloroplastic DNA which also follows circadian rhythm. Not enough protein level studies are done in this alga to determine if this observation correlates at protein level.

Post transcriptional regulation is also present in this alga. Three RNA binding proteins were identified whose RNA binding efficiency oscillated with rhythm. The activity of these proteins Chlamy 1,2 and 3 on mRNA is not exactly known. Of special interest is Chlamy 1 which shows strong amplitudes in free running rhythms and represnts an analog of CCTR. Chlamy 2 and 3 can bind to ss and ds RNA unlike Chlamy 1 (and CCTR) which only binds to ssRNA. Chlamy 1 also has affinity to UG repeats, which are now discovered in many mRNAs of chloroplast.