Effect of Kinases on Lipid Remodeling and Accumulation in Arabidopsis thaliana Under Abiotic Stress

Elizabeth Balzani Author

07/28/2021 Added

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A study on kinases affecting the SFR2 pathway in Arabidopsis thaliana. Please take a moment to view my research poster! Thank you! Research poster link: https://mediahub.unl.edu/media/17513

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[00:00:00.690]Hi, my name is Elizabeth Balzani.
[00:00:03.090]And today I will be talking to you about my project relating to how loss of
[00:00:06.990]function kinases affect lipids in model species Arabidopsis thaliana.
[00:00:13.620]Through this project,
[00:00:14.790]we hope to understand lipid remodeling and accumulation pathways in Arabidopsis
[00:00:19.260]so that we can better understand how to make better biofuels from plant
[00:00:23.730]lipids.
[00:00:25.020]A second objective is to better understand the cold stress pathway in plants,
[00:00:29.580]because this can lead to production of crops that are more resilient to cold.
[00:00:35.190]When Arabidopsis is exposed to severe cold at temperatures of negative four
[00:00:39.780]degrees Celsius or below, a chloroplast lipid degrading enzyme called
[00:00:44.280]sensitive to freezing 2, abbreviated
[00:00:46.920]as SFR2, becomes activated.
[00:00:50.040]We think that kinases are involved in activating SFR2.
[00:00:54.930]We've identified 30 kinases that could play a role in the SFR2 pathway.
[00:00:59.820]And I'm going to focus on a subset of three of these kinases. SFR2
[00:01:04.680]uses monogalactosyldiacylglycerol, or MGDG
[00:01:09.630]as a substrate to move sugar head groups to other lipids.
[00:01:14.040]This forms oligogalactolipids like trigalactosyldiacylglycerol,
[00:01:19.080]better known as TGDG.
[00:01:21.810]This pathway also results in the production of triacylglycerol, or TAG.
[00:01:27.570]TAG is of special interest to us because it is an energy rich oil that can be
[00:01:31.920]used as biofuel.
[00:01:33.720]Exposing plants to stressful conditions, like we do in this experiment, can lead
[00:01:38.340]to the production of more oil in the form of TAG.
[00:01:41.490]This can then be used as biofuel.
[00:01:44.340]You can see here that I indicated MGDG is rich in 16:3.
[00:01:49.500]This refers to a 16 carbon fatty acid with three double bonds.
[00:01:54.960]Since only the sugar head group gets modified by SFR2 activity,
[00:01:59.790]The 16:3 fatty acid chain in substrate MGDG also gets
[00:02:04.320]incorporated into product TGDG.
[00:02:07.650]16:3 is unique to the chloroplast, where SFR2 is active.
[00:02:13.230]16:3 also plays a role in freezing stress.
[00:02:16.770]When exposed to freezing stress, membranes low in 16:3
[00:02:20.970]and 18:3 oligogalactolipids
[00:02:22.680]will rupture, damaging the cell. When 16:3 and 18:3
[00:02:27.360]oligogalactolipids are present,
[00:02:30.030]the cell membrane can maintain its fluidity under freezing stress and not break.
[00:02:36.710]I mentioned earlier that we think kinases play a role in activating the SFR2
[00:02:41.300]pathway. We hypothesized that if we have a loss of function,
[00:02:45.590]kinase that activates SFR2, we will see less 16:3
[00:02:49.730]and TAG in these plants relative to wild type plants. On the other hand,
[00:02:54.530]kinases may be involved in repressing SFR2 when plants are not
[00:02:58.610]experiencing stress. If we have a loss of function
[00:03:01.720]kinase mutant that inhibits SFR2, we expect to see greater amounts of 16:3
[00:03:06.700]and TAG relative to wild type plants.
[00:03:10.970]To begin my experiment,
[00:03:12.590]I first designed primers specific for each loss of function kinase and tested
[00:03:17.450]them for functionality using PCR and gel electrophoresis.
[00:03:21.920]Once primers were determined functional,
[00:03:24.410]I used these primers in PCR reactions to screen for homozygous mutant
[00:03:28.880]plants.
[00:03:30.290]I ran each PCR sample through gel electrophoresis and looked for the correct
[00:03:34.460]banding pattern. I wanted to see no wild type DNA band,
[00:03:38.810]and I wanted to only see a DNA band indicating the insertion of bacterial
[00:03:43.580]DNA, which disrupts the kinase gene, thus creating a loss of function mutant.
[00:03:49.430]After homozygous mutant plants were confirmed,
[00:03:52.040]I took some leaf samples and put them on top of acid for three hours to expose
[00:03:56.420]them to acidic stress.
[00:03:58.250]I took other leaf samples and placed them in the chiller where they went from
[00:04:02.120]zero degrees Celsius to negative six degrees Celsius over the course of six
[00:04:06.350]hours.
[00:04:07.550]This served as our freezing stress. After the acidic and freezing stresses,
[00:04:12.440]lipids were extracted from each leaf sample. Lipid samples were run
[00:04:17.240]through thin layer chromatography to identify and separate specific lipids from
[00:04:21.560]one another.
[00:04:22.790]A TGDG containing band and a TAG containing band were scraped from the
[00:04:27.710]TLC plate and further analyzed using gas chromatography. Gas
[00:04:32.260]chromatography allows us to see how much of a given lipid was in the sample,
[00:04:36.940]as well as the fatty acid composition of my TAG and TGDG containing
[00:04:41.680]samples.
[00:04:44.110]Our results indicate that under freezing conditions, TAG produced by kinase
[00:04:48.970]mutants has a lower 16:3
[00:04:50.590]to 18:3 fatty acid ratio than wild type plants.
[00:04:54.940]If TAG has a lower 16:3 to 18:3 ratio,
[00:04:58.600]it means the TAG is likely not from the chloroplast and not made by SFR2.
[00:05:04.000]This makes sense that TAG would not be produced by SFR2
[00:05:07.510]if the loss of function kinases activated SFR2.
[00:05:11.890]We also found that the kinase mutants produce less TAG overall than wild type
[00:05:16.480]plants in freezing conditions.
[00:05:19.000]We also found that kinase mutants CRK3 and UPK have a
[00:05:23.740]similar relative mole percent of 16:3 to wild type plants,
[00:05:28.060]but mutant CPK 29 has a lower relative mole percent of 16:3 compared to
[00:05:32.920]wild-type plants.
[00:05:34.870]Since we hypothesized that kinase mutants that activated SFR2 would have
[00:05:39.760]less TAG in 16:3 relative to wild type plants,
[00:05:43.810]and we also know that the TAG produced by mutants has a low 16:3
[00:05:47.410]to 18:3 fatty acid ratio,
[00:05:50.080]we can say that these kinases are likely important in the SFR2 activation
[00:05:54.130]pathway. However, more tests are needed in order to be conclusive.
[00:05:59.270]As for acidic stress,
[00:06:01.220]we found that the kinase mutants produced TAG with similar 16:3
[00:06:05.090]to 18:3 fatty acid ratios to TAG produce by wild type plants.
[00:06:10.220]We also found that all kinase mutants have less TAG than wild type plants in acidic
[00:06:14.930]conditions. Although mutants CRK3 and UPK
[00:06:20.000]don't exhibit less 16:3 overall than wild-type plans,
[00:06:23.960]kinase mutant CPK 29
[00:06:26.060]does. These results indicate that these kinases,
[00:06:29.660]especially CPK 29, may be important in activating SFR2,
[00:06:34.880]but more testing is needed to be conclusive.
[00:06:38.480]I would like to thank the National Science Foundation for funding
[00:06:41.270]this research. I would also like to thank my mentor,
[00:06:44.030]Zachary Shomo, and my PI Rebecca Roston. Thank you!

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Effect of Kinases on Lipid Remodeling and Accumulation in Arabidopsis thaliana Under Abiotic Stress

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