The aim of this protocol is to identify a method which can be used for identifying novel genes which are involved in calcium signaling. To do this, we use the classical forward genetic screen. Calcium signaling is an important defense response pathway in several plant systems.
In order to identify the genes involved in this pathway, we have used the classical forward genetic screen. In this method, EMS would be used as an alkylating agent to introduce random mutations in the plant systems. The developed mutant generation can then be used for screening for a phenotype of interest.
Once the phenotype of interest has been identified, the causal gene can be mapped. In this study, we use the model plant Arabidopsis which has the calcium reporter aequorin in its spectrum. Weigh 150 mg seeds of aequorin for EMS mutagenesis and another 150 mg seeds to be used as control.
Transfer the seeds to a 50 ml Falcon tube and add 2%EMS or autoclaved water. While using EMS, be careful because it is carcinogenic. Protective gears should be worn while using EMS and any kind of spillage should be prevented.
Seal the Falcon tubes with paraffin and wrap it aluminum foil. Rotate the tube end over end for 18 hours at room temperature. Allow the seeds to settle and remove the EMS solution carefully and discard in a waste container containing one molar NaOH.
Wash the mutagenized seeds thoroughly with 40 ml of autoclaved water at least eight times. For the final wash, add 100 millimolar sodium thiosulfate and rinse for at least three times to remove traces of EMS. Soak the seeds in 40 ml of autoclaved water for one hour to diffuse EMS out of the seeds and then place them on Whatman paper until completely dry.
Transfer the seeds to Eppendorf and incubate at four degrees Celsius for two to four days for stratification. Transfer both the mutagenized seeds and water-treated seeds onto soil and transfer them to growth rooms with 16-hour light and eight-hour dark photo period at 22 degrees Celsius and 70%relative humidity. In order to determine if mutagenesis was successful, look for chlorophyll sectoring.
We have used the single pedigree-based seed collection method and each M1 plant is given a unique number. Upon maturation, seeds are harvested from these individual mutant plants and stored as individual M1 lines. A high throughput seed sterilization and hydroponic plant root protocol is used, adapted from Ranf et al.
On day one, place nearly 12 to 15 M2 seeds per M1 one line in individual wells of a 24-well tissue culture plate and sterilize using sodium hypochlorite and hydrochloric acid solution at a ratio of three is to one. This releases chlorine gas that sterilizes the seeds. Leave the plate for overnight to evaporate remaining chlorine gas completely.
After sterilization, add half strength liquid MS media to individual wells and stratify the seeds for two to four days at four degrees Celsius, and then move the seeds to a growth chamber with photo period of 10-hour light and 14-hour dark at 22 degrees Celsius, 70%relative humidity. On day 14, once the seedlings are eight to 12 days old, place 12 M2 seedlings from each line individually in a 96-well luminometer plate. After seedling transfer, add 150 microliters of five micromolar Coelenterazine solution in individual wells and store in dark at 21 degrees Celsius for eight hours.
Coelenterazine is a prosthetic group which binds to apoaequorin to form functional aequorin. Coelenterazine is light sensitive and is hence stored in dark colored bottles protected from light. Next day, screen mutants using hydrogen peroxide as stimulus and measure subsequent calcium elevation.
For simultaneous measurement of 24 wells, an out automated kinetic program is made, which includes measuring the background for one minute, followed by stimulus addition and measurement for 10 minutes, followed by discharge injection 150 microliter and measurement for three to five minutes. And any discharge for the daughter aequorin would be included in the reading to quantify the measured calcium and as additional control for functional aequorin. The readings provided from the above method is in relative light units.
For calculating cytosolic calcium ion concentration, a previously described concentration equation given by Rental and Knight in 2004 is used, which is described in the protocol. The RLUs obtained from luminometer are added to the equation to calculate calcium ion concentration. The obtained cytosolic calcium ion values are then graphically plotted against a wild-type control for identifying putative hydrogen peroxide mutants to calcium response.
This is a representative result of the protocol shown. We've shown one M1 line with 12 individual seedlings which have been measured for elevation of calcium ion concentration. Green line in the graph indicates wild-type aequorin which was measured along with mutants.
Red is average of all the 12 seedlings, and black line is individual M2 seedlings measured for the calcium ion elevation. Seedlings showing a highly reduced response to hydrogen peroxide application are then rescued. Rescued mutants are then reconfirmed in the next generation, followed by mapping to identify causal genes.
Caution should be given when using EMS as a mutagen since it's a carcinogen. Protective gearing should be worn at all times and any spillage done should be dealt with good laboratory practices. Additionally, when doing a pedigree-based collection of seeds, individual plants should be harvested with utmost care so that there is no mixing of any seeds at any stage.
This will help one to go back and trace the mother plant, which is the homozygous mutant plant. Additionally, since this method does not introduce any bias or any prior assumptions into the screening method, it can be used for one or more conditions to identify a phenotype of interest. Additionally, multiple independent genes could also be identified using the same protocol.
The protocol is easy to use since with the small dosage, a huge number of plants can be mutagenized. These huge number of mutant plants can be used for several populations and for several conditions and to identify several genes. A partial loss of function, an altered function, a complete loss of function, or also constitutive gene function can be identified through this method.
Mapping should be an easy task given the advancement in mapping technologies in the current scenarios. A de novo-based mapping system, a SNP-based racial plotting and many others can be used to identify the causal gene causing the phenotype of interest. Given the applicability of this method, it can be used not just on the Arabidopsis model plants, but other model plants too provided that a phenotype of interest readout can be established.
