Verifying if the gene AT4G24680 (MOS1) regulates non-photochemical quenching in Arabidopsis thaliana

Bailey McLean Author

08/02/2021 Added

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In this project, I looked at the effects of NPQ on Arabidopsis plants that do not have a functional MOS1 gene.

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[00:00:00.750]Hello everyone. My name is Bailey McLean,
[00:00:02.880]and I'm a biological sciences major going into my senior year here at UNL.
[00:00:07.290]The research project I did this summer,
[00:00:08.790]was verifying that the gene AT4G24680,
[00:00:12.270]which is commonly referred to as the MOS1
[00:00:14.400]gene regulates non-photochemical quenching in Arabidopsis thaliana.
[00:00:18.900]I really enjoyed performing this experiment this summer, and I learned a lot.
[00:00:22.050]I know the information I learned this summer will definitely help me throughout
[00:00:24.690]the rest of my life, in the field of science and future research.
[00:00:28.170]So jumping into the background for this experiment,
[00:00:31.470]plants have adapted multiple mechanisms to protect them from solar radiation or
[00:00:36.030]light from the sun,
[00:00:36.930]which can include synthesizing pigments and protective molecules,
[00:00:39.990]or simply releasing excess energy into other forms such as heat,
[00:00:43.770]which is one of the main mechanisms I personally looked at in this experiment.
[00:00:47.670]When plants accumulate too much solar radiation,
[00:00:49.860]they will release that energy as heat,
[00:00:51.330]which is referred to as non-photochemical quenching or NPQ for short.
[00:00:55.500]The other mechanism I looked at was protection via anthocyanins,
[00:00:59.070]which can absorb incoming light at different wavelengths and prevent the plants
[00:01:03.060]from accumulating too much energy in the first place or a way to think about it is
[00:01:07.680]as us as humans go outside and are in the sun,
[00:01:10.110]we will get hot and we will release the heat from our bodies by sweating.
[00:01:14.400]But if we go in the shade, then we'll be exposed to less sun, less heat,
[00:01:18.360]and we will sweat less.
[00:01:20.370]So the MOS1 gene has been shown in previous studies to regulate the synthesis
[00:01:24.990]of anthocyanins in plants. The gene represses the synthesis.
[00:01:28.380]So plants don't synthesize too much.
[00:01:30.840]Previous research has also shown that increasing concentrations of anthocyanins
[00:01:35.070]in plant tissue will decrease NPQ in plants.
[00:01:38.490]So the theory behind this research is that the mutants,
[00:01:41.160]which do not have a functional MOS1 gene will not be able to,
[00:01:44.310]repress the biosynthesis of anthocyanins. And in turn,
[00:01:47.130]the mutants would accumulate a higher concentration of anthocyanins in the plant
[00:01:51.090]tissue.
[00:01:52.170]And since the mutants would have more anthocyanins compared to the non-mutants or
[00:01:55.320]wild type, we would expect them to have lower levels of NPQ kinetics.
[00:02:00.510]So looking at the methods for this experiment,
[00:02:02.610]we first started to grow two lines of Arabidopsis plants
[00:02:06.420]Salk_074876c, which is the mutant for the MOS1 gene and Col,
[00:02:10.890]which is the wild type or the normal plants.
[00:02:13.710]The plants were stored at Beadle in a growth chamber and monitored daily.
[00:02:18.180]Now not every seed/plant has the mutation.
[00:02:21.780]So I had to perform a DNA extraction on each plant and also a PCR to search for
[00:02:25.950]the mutant plants, which are homozygous.
[00:02:28.500]I used only the homozygous plants for the remainder of the experiment.
[00:02:32.940]Now NPQ was measured using both a FluorCam imager,
[00:02:35.760]which utilizes fluorescence to measure the NPQ kinetics in the plants.
[00:02:39.330]And additionally, a handheld MultispeQ tool.
[00:02:42.270]The FluorCam measured and NPQ during a 10 minute induction or light period and a
[00:02:46.920]10 minute relaxation or dark period.
[00:02:49.530]The handhold MultispeQ gave me the NPQ at a specific time and was also used
[00:02:53.970]to determine other things such as the chlorophyll content.
[00:02:55.970]Anthocyanin concentration was determined two ways.
[00:03:00.490]The first was a nondestructive method utilizing a spectrometer,
[00:03:03.700]which can determine anthocyanin concentrations based on wavelengths and
[00:03:07.000]absorbances of leaves, which are still intact. The second was a destructive way,
[00:03:11.500]which was an extraction method.
[00:03:13.390]I took plant tissue and did a series of solvent extractions with hydrochloric
[00:03:17.770]acid/methanol and chloroform/water mix.
[00:03:20.710]The concentration of the extract was then determined by a spectrometer.
[00:03:25.180]Additionally,
[00:03:25.960]a chlorophyll extraction was done to determine the levels of chlorophyll in the
[00:03:29.440]plant tissue. Now,
[00:03:31.390]if we look at the results. Figure A shows a picture of the Arabidopsis
[00:03:34.990]plants at the pre-flowering and seedling stage on the left is the Col or
[00:03:39.880]wildtype and on the right is our mutants
[00:03:42.940]Figure B shows a schematic of the T-DNA insert for the MOS1 gene,
[00:03:47.770]as you can see with a 1.3 and RP primer combination,
[00:03:51.760]we would expect to see bands on the PCR for the insertion,
[00:03:55.510]with an LP and RP combination,
[00:03:57.760]we would expect to see only a band in the column for wild type
[00:04:02.340]Figure C shows the results of the PCR.
[00:04:05.890]You can see we have three mutant plants and we have one Col plant to compare
[00:04:10.380]to.
[00:04:12.180]Now looking at Figure D we can see a visual representation of the maximum NPQ
[00:04:17.130]done by fluorescence, the graph to the right shows
[00:04:20.370]the levels of NPQ for the corresponding plots on the left
[00:04:24.780]Figure H shows the results of the chlorophyll extraction for both chlorophyll a and
[00:04:28.740]chlorophyll b. Figure F shows the results of the anthocyanin extraction.
[00:04:34.050]The figure on the right shows a dilution curve to determine the maximal
[00:04:37.850]absorbance of the anthocyanins. Figure G shows the results of the spectrometer.
[00:04:42.690]And as you can see, there's
[00:04:44.640]a larger concentration of anthocyanins for the mutant,
[00:04:47.520]for both the spectrometer and the anthocyanin
[00:04:50.880]extraction method. For Figures H, I, and J shows the results of the FluorCam
[00:04:55.860]for NPQ. There isn't a clear difference in the seedling stage,
[00:04:59.460]but as you can see from the pre-flowering stage,
[00:05:02.100]there is a decrease in NPQ for the mutant,
[00:05:04.530]which was expected and finally Figure K shows the results of the MultispeQ
[00:05:09.120]Once again, we can see a decrease in NPQ for the mutant.
[00:05:13.500]So for the discussion and conclusion,
[00:05:15.540]there was a clear difference in anthocyanin levels,
[00:05:18.120]and there was a clear decrease in NPQ for the mutant.
[00:05:22.020]The number of biological replicates was low,
[00:05:24.720]which may be the reason why there is not a statistical significance.
[00:05:28.800]Originally I planted 12 mutants and only three turned out to be homozygous.
[00:05:33.240]In the future, we can take seeds from the homozygous plants and regrow more.
[00:05:37.140]This may be necessary to confirm the results of the experiment. In the future
[00:05:41.820]we will create transgenic lines to overexpress the MOS1 gene and redo the
[00:05:46.290]experiment, except we will focus our attention on different kinds of plants,
[00:05:49.500]such as soybeans or corn.
[00:05:52.050]The main goal for the future would be to use these results and try and improve
[00:05:57.320]NPQ kinetics in crops, which will increase the yield.
[00:06:00.740]I would like to thank everyone in the Glowacka lab for assisting me through
[00:06:04.790]this experiment. I would also like to thank UCARE for funding this project.
[00:06:09.020]Thanks for watching.

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Verifying if the gene AT4G24680 (MOS1) regulates non-photochemical quenching in Arabidopsis thaliana

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