Investigating Potential for Induced Overexpression of PsbS During Drought Stress As a Technique for Improvement of Water Use

Quinton Browne Author

07/27/2021 Added

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Investigation of transgenic tobacco for changes in water use efficiency, especially as indicated by NPQ.

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[00:00:00.600]Hello. My name is Quinton brown.
[00:00:02.970]I'm an undergraduate student at Frostburg State University,
[00:00:05.640]and I've been working in Dr. Glowacka's lab this summer.
[00:00:08.580]Her lab focuses on improving crops,
[00:00:10.350]primarily through investigating photosynthesis.
[00:00:13.170]The project that I worked on this summer was actually preexisting and my work
[00:00:16.980]was primarily on data collection in mature tobacco plants,
[00:00:19.620]which had been studied as seedlings.
[00:00:21.840]Our work as this slide says in quite a few words was in investigating potential
[00:00:26.400]for induced overexpression of PSPS during drought stress as a technique for the
[00:00:30.810]improvement of water use. So what's PSPS?
[00:00:35.100]it's a protein found in plants,
[00:00:36.360]which essentially can be used as a signal to reduce stomatal openings.
[00:00:39.720]It does this by reducing chloroplastic quinone, or QA.
[00:00:43.320]And there's a figure here,
[00:00:44.160]which illustrates the role of the Calvin-Benson cycle and the function of stomatal
[00:00:47.600]openings. And as you can see,
[00:00:49.410]the presence of PSPS in the thylakoid membrane is a key part of that regulation.
[00:00:54.210]PSPS leads directly to NPQ in this figure.
[00:00:57.750]And it's worth explaining NPQ as it's the focus of much of the work in this lab
[00:01:02.040]and on this project. NPQ is non-photochemical quenching.
[00:01:06.600]Essentially it takes the energy not used by photosynthesis and dissipates it as
[00:01:10.350]heat to keep the excess energy from causing damage to the plant. Light causes
[00:01:14.280]oxidation, which in turn can cause damage,
[00:01:16.710]especially if the light is greater than the needs of the plant.
[00:01:19.530]So what were our plants? We have 31 total tobacco plants,
[00:01:24.150]15 wild type and 60 and transgenic of four constructs, three of UNL
[00:01:28.680]14, five of UNL 15. And for each of UNL 16 and 17
[00:01:33.900]although there are differences between those four lines,
[00:01:36.450]they're not entirely important to the understanding of this project.
[00:01:40.800]And it's worth saying that they just all increase PSPS expression.
[00:01:46.210]They were first measured under drought before my time in this lab,
[00:01:49.140]but are pictured here at the mature stage,
[00:01:50.760]which I collected data for. This data collection was before drought, daily,
[00:01:55.260]during drought, and on the recovery day after watering came in a few forms as
[00:01:59.580]measurement
[00:01:59.850]of stomatal conductance by porometer and measurement of NPQ by the collection
[00:02:03.900]of an imaging of leaf disks in 96,
[00:02:06.960]well plates as seen in the image below- that's to the right,
[00:02:10.590]what our content was also collected on the day before drought.
[00:02:13.470]and on the last day of drought. We also took regular photos of the plants.
[00:02:17.940]Here they are at the point of the highest drought,
[00:02:19.830]right before they were watered and recovery began. As far as the drought goes,
[00:02:23.760]it was four days without water whatsoever for the plants.
[00:02:27.120]So on our first day, they were not watered,
[00:02:31.080]although they expected to be. And then for the next three days,
[00:02:34.470]until the fourth day, they were not watered at all. On the fifth day,
[00:02:37.890]we watered at noon and began to collect samples
[00:02:44.010]On this slide we have NPQ values by construct for the seedlings.
[00:02:47.340]So that's before my time here,
[00:02:49.140]but you could see a higher NPQ value for almost all constructs gray,
[00:02:53.100]as opposed to the wild type red,
[00:02:56.070]this higher NPQ maximum is actually exactly what we were looking for.
[00:02:59.410]Although it's less distinct at the mature stage,
[00:03:02.720]you can see that on this slide as most constructs
[00:03:05.350]actually trended lower than wild-type (in blue) for NPQ,
[00:03:08.740]although it's highly variable and the error is high enough as well,
[00:03:11.830]that it's hard to get a good grasp on what's actually happening here. However,
[00:03:16.690]for the mature plants,
[00:03:17.620]once we have the graphs of all NPQ over 20 minute program,
[00:03:21.070]10 minutes of induction and 10 minutes of relaxation,
[00:03:24.370]the induction being in light and the relaxation being in dark,
[00:03:29.170]the constructs actually go from a lower NPQ in control conditions to a
[00:03:33.160]higher NPQ in drought conditions as compared with wild type.
[00:03:36.400]So they're all going to lower NPQ
[00:03:40.090]but in comparison with wild type the
[00:03:43.120]constructs are higher except for UNL 16,
[00:03:46.270]which seems to follow the opposite trend.
[00:03:49.030]Our porometer measurements showed so much error and so much variation.
[00:03:52.570]They can not be seen as conclusive,
[00:03:54.400]but that's also important as it does point to a need for further study,
[00:03:58.180]given that these measurements are so heavily inconclusive,
[00:04:01.390]it's really vital that we have more data.
[00:04:06.210]Finally, water content was one of the most reassuring data sets.
[00:04:09.630]As you can see the average of the transgenic lines lost a substantially lower
[00:04:13.170]percent of its water content to the drought.
[00:04:15.660]The individual lines are in some cases less impressive,
[00:04:18.720]but this was useful confirmation of the difference between the wild type and
[00:04:22.170]transgenic groups. Essentially,
[00:04:24.960]we need more data in the form of more plants,
[00:04:27.570]which we're collecting seeds from these current plants to make possible and more
[00:04:31.320]data sets the results as of now are mostly inconclusive,
[00:04:34.920]but are reassuring and support the potential for other study,
[00:04:39.120]I'd like to give a special thanks to the national science foundation for providing
[00:04:42.180]grant funding. Thanks also to Dr. Glowacka for choosing to bring me into this lab,
[00:04:46.830]to Dr. Sahay
[00:04:47.250]for her mentorship and to Annie Nelson for helping me to be an active part
[00:04:51.060]of a project that she began work on. Thanks again.
[00:04:55.520]And my references are here on the last slide to a paper which Dr.
[00:04:59.000]Glowacka wrote and to Annie Nelson's first presentation of the seedling data.

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Investigating Potential for Induced Overexpression of PsbS During Drought Stress As a Technique for Improvement of Water Use

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