We would like to thank Prof. David Somers and Dr. Jeongsik Kim for the luciferase reporter plasmid and initial method development. Prof. Karen Halliday kindly provided all the circadian mutant lines used in this study. This research was supported by Royal Society research grants RS120372 and RS140275. GvO is a Royal Society University Research Fellow (UF110173).

NOTE: In this protocol, reagents and volumes described are expressed per Arabidopsis line that is tested, and will result in six replicate wells for that genotype. Multiply the materials with the number of lines to be analyzed when analyzing more than one line. In the video, wild-type Arabidopsis will be compared to the circadian clock mutant line ztl (although representative results from additional lines will be provided subsequently).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Enzyme solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase</td>
			<td>0.5% (w/v)</td>
		</tr>
		<tr>
			<td>Pectinase R10</td>
			<td>0.25% (w/v)</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>400 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>0.1% (w/v)</td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>W5 solution</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>150 mM&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>125 mM&#160;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5 mM&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MMg solution</td>
			<td></td>
		</tr>
		<tr>
			<td>MES, pH 5.7</td>
			<td>4 mM</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>400 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>15 mM&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEG solution</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG4000</td>
			<td>40% (w/v)</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging solution</td>
			<td></td>
		</tr>
		<tr>
			<td>W5 solution</td>
			<td>to final volume</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>5% (v/v)</td>
		</tr>
		<tr>
			<td>Luciferin</td>
			<td>1.2 mM</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>50 mg/ml</td>
		</tr>
	</tbody>
</table>
Table 1: List of solutions.
1. Preparation of Materials and Buffers
<ol>
	<li>Plant Materials
	<ol>
		<li>Keep Arabidopsis Col-0 seeds in water in a 1.5 ml tube at 4&#176; C in the dark for four days. Pipette the seeds onto soil in a pot and transfer to long day conditions (16 hr light 8 hr dark) at 21 &#176;C for 7 days. Leave a lid on the pot for the first three days to ensure high humidity. Transplant six seedlings to a new 8 cm x 13 cm x 5 cm pot and continue growing the plants under the same conditions for another 21 days. Make sure the soil is moist throughout.</li>
	</ol>
	</li>
	<li>Reporter Plasmid
	<ol>
		<li>Purify the CCA1pro:LUC reporter plasmid19 from bacterial culture in advance, using a DNA isolation kit according to manufacturer&#39;s instructions. 
		NOTE: 20 &#181;g reporter plasmid is required (10 &#181;l at 2 &#181;g/&#181;l in dH2O) for a transfection.</li>
	</ol>
	</li>
	<li>Solutions 
	&#8203;NOTE: For this protocol five solutions are needed: Enzyme solution, Wash 5 (W5) solution, Mannitol-Magnesium (MMg) solution, polyethylene glycol (PEG) solution, and imaging solution (Table 1). Prepare these before continuing to the protoplast isolation step.
	<ol>
		<li>Prepare 10 ml of enzyme solution as per Table 1, adding the enzymes last, and filter sterilize the solution through a 0.22 &#181;m syringe filter.</li>
		<li>Prepare the remaining solutions in the following volumes: 15 ml W5 solution, 10 ml MMg solution, 1 ml PEG solution and 1.5 ml imaging solution as per Table 1. 
		NOTE: It is important that the PEG solution is prepared no less than an hr before the transfection to ensure that the PEG has fully dissolved. Filter sterilize the imaging solution through a 0.22 &#181;m syringe filter.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54586/54586fig1.jpg" />
Figure 1: Protoplast isolation. The leaves of adult Arabidopsis plants (A) are fixed on autoclave tape with the lower epidermal layer facing up (B). (C) Magic tape is gently pressed onto the lower epidermal layer with the tip of a 15 ml conical tube. (D) The magic tape is removed to expose the mesophyll cells (E). (F) Strips of autoclave tape with leaves attached are floated on enzyme solution containing cellulase and pectinase, releasing protoplasts into the solution. (G) The leaf skeletons remain on autoclave tape after enzyme digestion and are discarded, leaving the protoplasts in enzyme solution (H). (I) Final resulting mesophyll protoplasts (Scale bar = 50 &#181;m). <a href="https://www.jove.com/files/ftp_upload/54586/54586fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Protoplast Isolation
<ol>
	<li>Label a petri dish and add 10 ml filter-sterilized Enzyme solution.</li>
	<li>Fix four strips of autoclave tape with the sticky side up on a clean lab surface, and mark the size of the petri dish on the strips.</li>
	<li>Cut leaves of 4-week old plants (Figure 1a), and press the upper epidermis onto the autoclave tape (i.e. the lower epidermal surface facing up (Figure 1b)).</li>
	<li>Gently press strips of magic tape onto the lower epidermal surface using a 15 ml conical tube. Take care not to crush the leaf tissue (Figure 1c). Carefully pull the magic tape off to strip the lower epidermis (Figure 1d) and expose the mesophyll cells (Figure 1e).</li>
	<li>Cut the autoclave tape to fit the petri dish, use tweezers to move the tape into the petri dish and float on the enzyme solution (Figure 1f), leaves facing down. Rotate at 60 rpm on a platform shaker for 60 min, during which the protoplasts will be released into the solution.</li>
	<li>Use tweezers to remove and discard the strips of autoclave tape (Figure 1g) and pipette the solution containing the protoplasts (Figure 1h) into a 50 ml conical tube. Use a wide-bore pipette tip (such as a P5000 tip or a P1000 tip with the end cut off).</li>
	<li>Centrifuge the protoplasts at 100 x g for 3 min at 4 &#176;C and remove the supernatant with a pipette. Take care, as the pellet is very loose.</li>
	<li>Add 10 ml W5 solution to the protoplasts and resuspend by gently swirling the tube.</li>
	<li>Rest the protoplasts on ice for 30-60 min. During this step, assess yield by counting protoplasts in a hemocytometer (Figure 1i).</li>
	<li>Collect the protoplasts by centrifugation at 100 x&#160;g for 3 min at 4 &#176;C and remove the supernatant with a pipette. Take care, as the pellet is very loose.</li>
	<li>Resuspend the protoplasts to a concentration of 5 x 105 protoplasts/ml in MMg solution.</li>
</ol>
3. Protoplast Transfection
<ol>
	<li>Add 10 &#181;l reporter plasmid (2 &#181;g/&#181;l) to a 15 ml conical tube.</li>
	<li>Add 400 &#181;l protoplasts to the tube.</li>
	<li>Add one volume (410 &#181;l) PEG solution and mix by inverting the tube gently 12 times to transfect the protoplasts.</li>
	<li>After 8-15 min incubation at room temperature, dilute the protoplast-DNA-PEG mixture with four volumes (1640 &#181;l) W5 solution and mix by inverting gently six times.</li>
	<li>Collect the transfected protoplasts by centrifugation at 100 x g for 2 min at room temperature and remove the supernatant with a pipette. Once again, take care, as the pellet is very loose.</li>
	<li>Resuspend the protoplasts in 1,250 &#181;l imaging solution.</li>
	<li>Aliquot 200 &#181;l into six replicate wells of a 96-well plate, fill any empty wells with W5 solution, and seal the plate with an adhesive clear lid suitable for luminescent imaging.</li>
</ol>
4. Imaging
<ol>
	<li>Transfer the plate to any luminescent imaging setup suitable for plant imaging. Change the adhesive lids on the plates 10-15 min after transfer to avoid condensation and pressure differences among the wells, then start imaging. Read the plate every ~45 min (3 sec per well) for five days at room temperature. 
	NOTE: The frequency of plate reads and the read time per well might vary depending on the imaging setup.</li>
</ol>
5. Data Analysis&#160;
<ol>
	<li>Analyze the luminescence results using any analysis software (for example, Biological Rhythms Analysis Software System (BRASS)22). Compare the mutant line to wild-type controls to reveal circadian defects.</li>
</ol>

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The authors have nothing to disclose.

Here, we present a rapid assay to screen circadian period defects in Arabidopsis lines, including protoplast isolation, transfection, and luminecent imaging. The circadian period in wild-type Arabidopsis and the clock mutants cca1/lhy, ztl, and CCA1-OX was calculated from measurements of luminescence from a CCA1pro:LUC reporter and found to be consistent with published data generated from whole plants using more time-consuming transgenic approaches (Table 2). Using this assay to screen Arabidopsis mutants avoids the initial requirement to generate transgenic luminescent lines, which is time consuming&#160;and might only reveal that a particular mutant exhibits wild-type rhythms. A clear advantage of this protocol is that any Arabidopsis line can be screened in short time for altered circadian transcriptional rhythms, which will help the identification of more genes affecting timekeeping in plants.
Isolation of protoplasts can be laborious, especially if the mutant line displays a dwarfed phenotype. Previously reported methods for generating protoplasts involve cutting leaves or seedlings into thin strips and vacuum infiltrating the strips with enzyme solution to allow the enzymes to reach the cell walls26,27. The subsequent digestion requires at least 3 hr incubation in the dark to release the protoplasts into the enzyme solution. When vacuum infiltration is not applied, incubation time up to 18 hr is advised. In the protocol presented here, vacuum infiltration is unnecessary because the epidermal cell layer impenetrable to the enzymes is removed by tape. When generating protoplasts by cutting plant material it is necessary to separate protoplasts from cell wall debris after digestion; this is typically done by filtering the solution or purifying it on a sugar gradient27,26. Here, any undigested tissue will remain on the autoclave tape after digestion, so filtration and sugar gradient purification of the protoplasts can be omitted. There is a chance that the stress resulting from prolonged protoplasting procedures affects cell viability and the circadian clock. The tape-based protoplasting method20 visualized here yields a high number of protoplasts; and given the viability of cells over a circadian time series and the matching period lengths of protoplasts and whole seedlings (Table 2), the methodology described here is preferred over alternative methods to generate protoplasts.
The transfection step of the protocol is a critical step that impacts on the viability of the protoplasts. The PEG solution enables the DNA to be delivered into the cell, and both incubation time in this solution (step 3.4) and the percentage of PEG should be determined empirically. The standard concentration is 20%, with lower concentrations reducing the transformation efficiency and higher concentrations reducing viability28. Incubation time as short as five minutes could yield efficient transfection of the protoplasts27.
The protoplasts can be imaged on any luminescent imaging platform. In this video, we have used a luminescence plate reader equipped with external lights (red and blue LEDs, 630 and 470 nm, respectively, at a combined intensity of ~20 &#181;E), which allows for high-throughput screening and frequent measurements. Other imaging equipment that detects luminescence, such as alternative plate readers or setups based around a charge-coupled device (CCD) camera, could be applied equally well as long as illumination is available for photosynthesis. The advantage of using a CCD camera mounted on a controllable cabinet is that light and temperature can be programmed. A disadvantage is the longer capture time, introducing prolonged periods of darkness that can cause stress to the protoplasts in addition to perturbing endogenous timekeeping. Regardless of which experimental platform is chosen, adjustment of settings is likely to be necessary compared to settings for seedlings or mature plants. In our experience, increasing the concentrations of reporter plasmid during transfection and/or luciferin in the imaging buffer might resolve any erratic or quickly dampening rhythms that might occur in initial experiments.
In future experiments, the methodology described here can be applied to study the circadian phenotype of any mutant Arabidopsis line. With minor modifications, the effect of e.g. various drugs on timekeeping could be studied in this setup. Furthermore, the transfection can be modified to include artificial microRNA for gene silencing19, further expanding the versatility of this protocol. Protocols to generate rice protoplasts are well-established29 and the imaging protocol presented here could equally be applied to further our understanding of the clock in economically important monocot crop plants.

Most living organisms possess an endogenous timekeeper to aid efficient negotiation of daily changes to the environment. This timekeeper, the circadian clock, regulates many aspects of the metabolism and physiology of an organism to anticipate predictable changes in the external environment. In plants for example, anticipation of temporally predictable pathogen or herbivore attacks strongly reduces overall susceptibility1-4. Starch metabolism is tightly regulated by the circadian clock to ensure that starch reserves last until dawn whether grown at long or short day conditions5. Indeed, plants with circadian clocks matching the 24 hr environment show increased growth rates, carbon fixation rates and survival rates compared to plants running a clock of mismatched period6. The extensive influence of circadian rhythms in all these processes arises to a large extent from the rhythmic regulation of up to a third of the total transcriptome in Arabidopsis7. These rhythmic gene products are involved in a broad range of cellular processes including metabolic pathways, hormone signaling, and stress responses7. On top of that, direct circadian regulation of protein function is established by circadian regulation of phosphorylation8 and most probably other post-translational modifications (PTMs).
At the center of the circadian clock that drives this daily transcriptional reprogramming lies a network of transcriptional/translational feedback loops (TTFLs), including the morning-expressed genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) that repress the expression of the evening genes TIMING OF CAB 1 (TOC1), GIGANTEA (GI) and the members of the evening complex (EC); LUX ARRHYTHMO (LUX), EARLY FLOWERING 3 (ELF3), and ELF49-11. Together with the PSEUDO RESPONSE REGULATOR-genes (PRR3, 5, 7, and 9), TOC1 represses CCA1/LHY expression whereas the EC acts as a positive regulator12-14. Additional to feedback mechanisms, post-transcriptional and post-translational regulation of the TTFL network components plays an important role in tuning circadian rhythms. To date, the most abundant PTM identified on circadian clock proteins is phosphorylation15. The phosphorylation of CCA1 by Casein Kinase 2 (CK2) affects its binding to target promoters16 and is important for temperature compensation of the circadian clock17. The PRR proteins are phosphorylated differentially over the circadian cycle, and phosphorylation of TOC1 affects interaction with its negative regulator, the evening-phased clock component ZEITLUPE (ZTL)18. These examples illustrate how PTMs and protein-protein interactions tune the TTFL network into a 24-hr transcriptional oscillator. For a more detailed introduction to the transcriptional clock network and its regulation, excellent recent reviews are available (e.g. reference15).
However, what remains less clear is the extent to which rhythmically regulated processes and other aspects of cellular metabolism feed back into the timekeeping mechanism itself. Extensive screening of Arabidopsis mutants for clock defects could gain further understanding of which mechanisms or signaling pathways could be involved in the generation of 24 hr rhythms from a TTFL network. To this end, Kim and Somers previously published a break-through method for the efficient and rapid analysis of clock function in any Arabidopsis line19. Based on the use of transient expression in protoplasts, this methodology surpasses the need for stable transgenic lines or crosses to luminescent clock marker lines. Here, we visualize a high yielding protoplast isolation method20 combined with an optimized protocol to screen circadian defects by luminescent imaging of transiently expressed clock markers in a 96-well plate reader.
We will compare wild-type Col-0 Arabidopsis to well-characterized clock mutants to confirm the methodology described here successfully detects altered circadian rhythms. Protoplasts derived from these lines are transfected with a reporter construct that expresses firefly luciferase from the circadian CCA1 promoter; CCA1pro:LUC19. Luciferase catalyzes the multistep reaction in which luciferin is converted to electronically excited oxyluciferin that emits light21, which is imaged over time to assess the period, amplitude and phase of transcriptional rhythms in these protoplasts.
The protocol consists of three major parts; isolating the protoplasts, transfecting the protoplasts with the reporter plasmid, and luminescent imaging. These three parts should always be performed on a single day, using freshly prepared buffers and reagents. Plant growth and purification of sufficient amounts of reporter plasmid should be performed in advance and are not visualized in this protocol.

<table><tbody><tr><td>CELLULYSIN Cellulase, &lt;em&gt;Trichoderma viride&amp;nbsp;&lt;/em&gt;</td><td>Merck Millipore</td><td>219466</td><td></td></tr><tr><td>Pectinase R10</td><td>Sigma Aldrich</td><td>P2401</td><td></td></tr><tr><td>D-mannitol</td><td>Sigma Aldrich</td><td>M4125&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>C4901</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A4503</td><td></td></tr><tr><td>MES</td><td>Acros Organics</td><td>327765000</td><td></td></tr><tr><td>NaCl</td><td>Acros Organics</td><td>327300010</td><td></td></tr><tr><td>Glucose</td><td>Fischer Scientific</td><td>G/0500/60</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M2670</td><td></td></tr><tr><td>Polyethylene glycol (PEG) 4000</td><td>Acros Organics</td><td>434630010</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Sigma Aldrich</td><td>F4135</td><td></td></tr><tr><td>D-Luciferin, Firefly</td><td>Biosynth AG</td><td>L-8200</td><td>Dissolve luciferin powder in 0.1 M trisphosphate buffer pH 8.0 to a concentration of 50 mM</td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>A9618</td><td></td></tr><tr><td>Comply Steam Indicator Tape</td><td>3M</td><td>1222</td><td></td></tr><tr><td>Magic Tape</td><td>3M</td><td>819-1460</td><td></td></tr><tr><td>Petri Dishes</td><td>Scientific Laboratory Supplies</td><td>SLS2002</td><td></td></tr><tr><td>Microplate, 96 well, flat bottom, white</td><td>Greiner Bio-One</td><td>655075</td><td></td></tr><tr><td>TopSeal-A, adhesive lids for 96-well plates</td><td>Perkin Elmer</td><td>6050195</td><td></td></tr><tr><td>Syringe, 10 ml, w/o needle</td><td>BD Plastipak</td><td>302188</td><td></td></tr><tr><td>Syringe filter 0.22 &amp;micro;m</td><td>Merck Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Biological Rhythms Analysis Software System (BRASS)</td><td></td><td></td><td>Free download at amillar.org</td></tr><tr><td>QIAfilter Maxiprep Kit</td><td>Qiagen</td><td>12262</td><td></td></tr><tr><td>Modified Fuch-Rosenthal counting chamber, double cell</td><td>Hawksley</td><td>AC6000</td><td></td></tr><tr><td>Bright field microscope</td><td>Optika Microscopes</td><td>B-150POL-B</td><td></td></tr><tr><td>LB942 TriStar&lt;sup&gt;2&lt;/sup&gt; multimode reader, with luminescence module</td><td>Berthold Technologies Ltd</td><td>57947/57948</td><td></td></tr></tbody></table>

rapid analysis of circadian phenotypes in arabidopsis protoplasts transfected with a luminescent clock reporter

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.

Protoplasts of wild-type Arabidopsis were compared to the known clock mutants cca1/lhy (short period mutant2), ztl (long period mutant23), and the overexpression line CCA1-OX (arrhythmic24). All were transiently transfected with the CCA1pro:LUC reporter and imaged in constant light on a plate reader over 5 days.
Here we show that in the cca1/lhy mutant, the free-running period of circadian-controlled gene expression is shortened (Figure 2a), in line with the published period analyzed by stable transgenic expression of luminescent clock markers2,9. The period of circadian oscillations in protoplasts of the ztl mutant is longer than in wild type (Figure 2b), in accordance with previously published results from seedlings, cell suspension cultures and protoplasts19,23,25.
Overexpression of CCA1 locks the clock in morning-phase and global transcription becomes arrhythmic24. Consistently, we observed no oscillations in CCA1pro:LUC expression in CCA1-OXprotoplasts (Figure 2c).
<img alt="Figure 2" src="/files/ftp_upload/54586/54586fig2.jpg" /> 
Figure 2: Protoplast Circadian Rhythms Protoplasts of Col-0, cca1/lhy, ztl, and CCA1-OX were transiently transformed with the CCA1pro:LUC construct and circadian rhythms in luminescence were imaged over 5 days (A-C). (D) Circadian period estimates based on luminescence traces in Col-0, cca1/lhy and ztl (A-B). Mean &#177;SEM, n = 3-5, t-test p-value as indicated. <a href="https://www.jove.com/files/ftp_upload/54586/54586fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genotype</td>
			<td>Period (protoplasts)</td>
			<td>Period (transgenic)</td>
			<td>Reported by:</td>
			<td>References</td>
		</tr>
		<tr>
			<td>Col-0</td>
			<td>23.0 &#177;0.21</td>
			<td>24.4 &#177;0.09</td>
			<td>pCCA1:LUC in Col-0</td>
			<td>(2)</td>
		</tr>
		<tr>
			<td>cca1/lhy</td>
			<td>20.6 &#177;0.13</td>
			<td>19.9 &#177;0.11</td>
			<td>pCCA1:LUC in Col-0</td>
			<td>(2)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>~18</td>
			<td>RNA in Ler background</td>
			<td>(9)</td>
		</tr>
		<tr>
			<td>ztl</td>
			<td>25.1 &#177;0.12</td>
			<td>27.4 &#177;0.5</td>
			<td>CAB2:LUC in C24 ecotype</td>
			<td>(22)</td>
		</tr>
		<tr>
			<td>CCA1-OX</td>
			<td>arrhythmic</td>
			<td>arrhythmic</td>
			<td>pCCA1:LUC (LD data only)</td>
			<td>(2)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>arrhythmic</td>
			<td>RNA and protein in Col-0 background</td>
			<td>(23)</td>
		</tr>
	</tbody>
</table>
Table 2: Circadian Rhythms in Protoplasts Compared to Transgenic Seedlings. The circadian period of pCCA1:LUC expression in protoplasts compared with the period lengths first reported for the mutation and, if available, with pCCA1:LUC expression in Col-0.

Watch this Scientific Journal Video about Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter at JoVE.com

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

rapid-analysis-circadian-phenotypes-arabidopsis-protoplasts

Research

JoVE Journal

Biology

12.8K Views.  University of Edinburgh.  De circadiane klok regelt ongeveer een derde van de Arabidopsis transcriptoom, maar het percentage van genen die te gebruiken bij tijdwaarneming blijft onbekend. Hier visualiseren we een methode om circadiane fenotypes snel te beoordelen in welke mutant lijn van Arabidopsis met behulp van lichtgevende beeldvorming van een circadiaan reporter tijdelijk tot expressie gebracht in protoplasten. 

Video: Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Genetics

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Neuroscience

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant environmental parameters that synchronize animal circadian clocks. In Drosophila circadian photo-responses are mediated by a solely blue light photoreceptor named CRYPTOCHROME (CRY). Upon photoreception, CRY changes its conformation and initiates the proteosomal dependent degradation of TIMELESS (TIM). TIM is an important pacemaker protein, thus degradation of TIM will reset the circadian clock. Under constant light conditions (LL), wild type flies quickly become arrhythmic because of the constant degradation of TIM, while flies bearing defects with circadian photo-responses will still be rhythmic. Thus LL triggered arrhythmicity has been used for screening of components in circadian light input pathways. A brief short light pulse in the night can also dramatically shift phases of circadian rhythms. As expected, this phase shift response is reduced in flied with defects in circadian photoresponse. Thus analyzing locomotion behavior rhythmicity under LL or phase changes after short light pulse in constant darkness (DD) are two major methods to study circadian photoresponse. Here we describe how to design and analyze LL and phase response experiments. LL arrhythmicity is suitable for screening light input pathways mutants, whereas phase response validates the results and provide further information for light sensitivity.

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

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