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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CELLULYSIN Cellulase, Trichoderma viride&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</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>MgCl2</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&#160;</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 0.1M trisphosphate buffer pH 8.0 to a concentration of 50mM</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 &#181;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 TriStar2 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 ...

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12.7K Views.  University of Edinburgh. 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.

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

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.