We successfully identified 96 T-DNA knockout mutants and subjected them to a range of experimental tests to determine their root gravity response. Some of the mutants failed to germinate or grow adequately to test, resulting in complete data for 76 of the lines. We carried out 2 main kinds of gravity response experiments. In one type, roots were rotated 90 degrees from vertical and allowed to respond back toward vertical while images were captured every 10 min for 220 min. In this kind of experiment, called a free response, 26 of the mutants showed a response that differed significantly from the wild type, with most showing a reduced response. In the other type of experiment, roots were initially rotated 90 degrees from vertical as in the free response type, but were then continuously rotated at a rate of 15 deg/h. This experiment was done to test the root’s ability to respond to continuous stimulation. In this continuous rotation experiment, 20 mutants showed a response that differed significantly from the wild type. Interestingly, only 8 of the 26 mutants showing a significant difference in the free response experiment also showed a significant response in the continuous rotation experiments, indicating the effectiveness of the two assays at identifying different aspects of the gravity response pathway.

Mutants that showed a significant gravity response difference were crossed into the starchless mutant background to begin the process of making double mutants for analysis. It is hypothesized that if a gene product contributes to the starchless plants’ ability to sense or respond to gravity, this ability will be more severely affected in the starchless root background. Of the 38 total mutants, successful F1 seeds have been harvested for 18 thus far and work is continuing on this objective. Of the F1 lines confirmed, 6 have been carried out to the F3 generation for scoring and identification of double homozygous alleles. These lines will be subjected to the same free response and continuous rotation experiments after they are obtained.

In addition to creating double mutants with significant mutants, they were also crossed into the R2D2 semi-quantitative auxin reporter line. One of the regulators of growth and gravitropism, the cellular levels of the plant hormone auxin can be inferred using this fluorescent reporter. The fluorescent reporter protein in this system is targeted for degradation when auxin enters a cell, thus allowing the tracking of its disappearance as an indicator of auxin transport. The reporter also includes a second fluorescent protein that is not able to be targeted for degradation, thus allowing the ratiometric measurement of the two proteins as an internal control. Thus far, 12 F1 lines have been identified and 4 F3 lines have been used for pilot research. This molecular tool will allow for the determination of auxin transport in the mutants and the mapping of the gene product to a point in the gravity response pathway.

Thus far, we have 96 T-DNA mutant lines on hand and have successfully completed genotyping 65. Of the lines screened thus far, 7 have been confirmed heterozygous for the T-DNA allele, and of those, 2 show a gravity related phenotype; 56 lines have been confirmed homozygous for the T-DNA carrying allele; the remainder of the lines genotyped have shown ambiguous results and we are in the process of redesigning primers. Some lines without a positive confirmation PCR result may be due to a later transposition event with the T-DNA. We are in the process of amplifying and sequencing the region surrounding the location of the T-DNA insertion to detect possible footprint deletions. Some of the lines also showed a lack of positive product for the positive control primer pair as well, indicating bad primer design or PCR conditions. Sequencing in the region of the purported T-DNA will also shed light on these lines.

In addition to genotyping, we are also testing for transcript knock-down in the T-DNA lines to be able to correlate any phenotypes observed with lack of transcript abundance. To date we have carried out RT-PCR on 55 lines and confirmed the lack of mRNA transcript for the gene of interest in 37 of our lines, with 2 lines showing no difference in transcript abundance compared to the wild-type. The RT-PCR experiments required for this assay are more demanding than the genotyping PCR experiments, leading to an overall slower rate of completion. We carried out this assay along with many others as part of my Plant Physiology lab experience for 16 students in Spring 2024. I have redesigned the lab experience around this NASA-funded screening project to provide students hands-on experience with real research. Since so many of the assays and screens of these mutant lines is routine in the lab, this will provide students the opportunity to contribute to the project while learning valuable skills in bioinformatics, DNA and RNA isolation, real-time RT-PCR, and various plant growth analyses. I incorporated this project into the lab portion of the class in the previous two years and it was moderately successful, with about half of the data generated by student groups of sufficient quality to be useful. Last spring, I structured the work a bit more and provided templates for the students to collect and interpret results, which mitigate some of the errors I saw in previous years.

In addition to the molecular characterization of mutants, we have made good progress on the growth experiments designed to test the mutants for any gravity-related phenotypes. We have developed a novel assay to detect differences in the regulation of differential growth. The assay is based on a previous technique used heavily in my lab, called ROTATO, which uses real-time image analysis to constrain the root tip at a constant angle over time by rotating the plate. This technique is good for measuring single roots, but does not scale well to large numbers of roots on a plate, as required for a medium-scale mutant screening project. I have used the average response rate (rotation) of wild-type roots as an input to control rotation of whole plates of mutant seedlings, reasoning that if the roots have “normal” gravity response, their tip angles will remain relatively unchanged because they are able to maintain the differential growth required to do so. We have now completed analysis of 76 T-DNA mutant lines using this technique and have identified 20 lines that show a statistically significant difference in response compared to wild-type. 16 of the 20 lines show significantly reduced gravity response, while the other 4 lines show greater gravity response.

Other experiments to characterize the mutants include standard reorientation assays to assess gravitropic response and root phototropic assays to try to distinguish gravity perception processes from growth regulation and gravity response elements. We have conducted hundreds of standard root reorientation assays thus far, and have identified 26 mutants having a significantly different gravity response after 3 h. Interestingly, of the 26 mutants showing a free response gravity phenotype, only 8 also show a significant continuous response phenotype. The use of both assays thus may both expand the pool of possible contributors to gravity response and provide useful physiological differences between the two assays for future follow-up experiments. We also began analyzing responses as a function of time, since we have been collecting angle measurements across a 3-h response period. These comparisons indicate differences in kinetics that could also reveal hints at the underlying mechanisms perturbed by the mutation.

For the root phototropism assays, we have done some pilot testing with large-scale experiments, but I am unsatisfied so far with the amount of curvature induced in the positive and negative controls. Last summer, one of the student researchers worked on developing a better assay design and construction that will optimize this response, but we have not yet screened a large number of the mutants yet. The lab also completed the acquisition of a ground-based engineering model of flight hardware designed for the European Space Agency’s European Modular Cultivation System (EMCS) that provides highly repeatable phototropic stimulation, which we have begun adopting for potential use in carrying out these experiments.

This year we continued work designing and cloning Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) constructs to knock out genes for which there is no T-DNA line available in a stock center. Thus far, we have created guide sequence constructs for a total of 23 genes showing a gravitropic phenotype. We have successfully cloned the guide RNAs into expression vectors and completed floral dip of wild type (wt) plants. We are currently in the process of screening the seeds from these T0 lines for resistance and selfing those. In addition to creating knockouts for those genes lacking a Salk T-DNA insertion, this process will also allow us to generate second alleles for those genes for which we find a phenotype and wish to confirm.

In addition to using CRISPR to knock out genes of interest, we have begun making double-mutants between those mutants that show a promising gravity-related phenotype and the pgm-1 mutant, which lies at the heart of this project. By testing the phenotypes of double mutants in the pgm-1 mutant background, we hope to identify molecular participants in the non-statolith sensing pathway by finding double mutants with a stronger phenotype than either single mutant alone. This year we crossed another 10 lines to pgm, making a total of 33 crosses with our confirmed mutants into the pgm-1 mutant to date, with 23 confirmed F1 lines. In addition, we are in the process of screening 4 F3 lines collected from individual F2 parents to identify double mutants. In addition to crossing into the pgm-1 background, we have also been crossing candidate mutants into the semi-quantitative auxin reporter line R2D2, with 12 lines at the F1 stage to date and 2 lines at the confirmed F3 stage. These will allow us to determine the potential involvement of our new mutants in the regulation of auxin transport during gravitropism.

This year we made significant progress on a rewritten and renewed ROTATO system. I had previously rewritten the core image analyzer in MATLAB and have successfully used this image processing engine to constrain roots at the desired angle for indefinite periods. This system takes images captured from a standard Raspberry Pi HQ camera module, which uploads the images to a server where the computer running MATLAB can access them and control rotation of the vertical stage holding the root. This year we focused on transitioning the core analyzer away from MATLAB and into Python, which would enables us to deploy the software on any Raspberry Pi imaging computer in the lab and also share it widely for those interested in incorporating it into their own projects. One of my summer research students has completed a beta version of the software and we are currently refining it in the lab. We hope to generate a methods-based publication from this work in the near future.

Thus far, we have 88 transfer DNA (T-DNA) mutant lines on hand and have successfully completed genotyping 65. Of the lines screened thus far, 5 have been confirmed heterozygous for the T-DNA allele, and of those, 2 show a gravity related phenotype; 48 lines have been confirmed homozygous for the T-DNA carrying allele; the remainder of the lines genotyped have shown ambiguous results and we are in the process of redesigning primers. Some lines without a positive confirmation PCR result may be due to a later transposition event with the T-DNA. We are in the process of amplifying and sequencing the region surrounding the location of the T-DNA insertion to detect possible footprint deletions. Some of the lines also showed a lack of positive product for the positive control primer pair as well, indicating bad primer design or PCR conditions. Sequencing in the region of the purported T-DNA will also shed light on these lines.

In addition to genotyping, we are also testing for transcript knock-down in the T-DNA lines to be able to correlate any phenotypes observed with lack of transcript abundance. To date, we have carried out RT-PCR on 35 lines and confirmed the lack of mRNA transcript for the gene of interest in 13 of our lines, with 15 lines showing no difference in transcript abundance compared to the wild-type. The RT-PCR experiments required for this assay are more demanding than the genotyping PCR experiments, leading to an overall slower rate of completion. We carried out this assay, along with many others, as part of my Plant Physiology lab experience for 16 students in Spring 2023. I have redesigned the lab experience around this NASA-funded screening project to provide students hands-on experience with real research. Since so many of the assays and screens of these mutant lines is routine in the lab, this will provide students the opportunity to contribute to the project while learning valuable skills in bioinformatics, DNA and RNA isolation, real-time RT-PCR, and various plant growth analyses. I incorporated this project into the lab portion of the class last spring and it was moderately successful, with about half of the data generated by student groups of sufficient quality to be useful. This spring I will be structuring the work a bit more and providing templates for the students to collect and interpret results, which will hopefully mitigate some of the loss I saw last year.

In addition to the molecular characterization of mutants, we have made good progress on the growth experiments designed to test the mutants for any gravity-related phenotypes. We have developed a novel assay to detect differences in the regulation of differential growth. The assay is based on a previous technique used heavily in my lab, called ROTATO, which uses real-time image analysis to constrain the root tip at a constant angle over time by rotating the plate. This technique is good for measuring single roots, but does not scale well to large numbers of roots on a plate, as required for a medium-scale mutant screening project. I have used the average response rate (rotation) of wild-type roots as an input to control rotation of whole plates of mutant seedlings, reasoning that if the roots have “normal” gravity response, their tip angles will remain relatively unchanged because they are able to maintain the differential growth required to do so. We have now completed analysis of 50 T-DNA mutant lines using this technique and have identified 18 lines that show a statistically significant difference in response compared to wild-type; 13 of the 18 lines show significantly reduced gravity response, while the other 5 lines show greater gravity response.

Other experiments to characterize the mutants include standard reorientation assays to assess gravitropic response and root phototropic assays to try to distinguish gravity perception processes from growth regulation and gravity response elements. We have conducted hundreds of standard root reorientation assays thus far, and we are in the process of measuring tip angles and collating results. For the root phototropism assays, we have done some pilot testing with large-scale experiments, but I am unsatisfied so far with the amount of curvature induced in the positive and negative controls. Last summer, one of the student researchers worked on developing a better assay design and construction that will optimize this response, but we have not yet screened a large number of the mutants yet.

This year we designed and cloned Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) constructs to knock out genes for which there is no T-DNA line available in a stock center. Experiments to transform these constructs into plants failed over the summer when the candidate plants were left untended over a weekend and there was miscommunication about who was scheduled to water, unfortunately. On the positive side, I was pleased with how straightforward the cloning process was to create the constructs and remain positive that this will allow us to extend the screen to genes with no knockout mutants available. This could also allow us to generate second alleles for those genes for which we find a phenotype and wish to confirm. I will be returning to this knockout strategy with my Plant Physiology lab students in Spring 2024 to allow for progress on this objective.

In addition to using CRISPR to knock out genes of interest, we have begun making double-mutants between those mutants that show a promising gravity-related phenotype. By testing the phenotypes of double mutants in the xxx mutant background, we hope to identify molecular participants in the gravity sensing pathway by finding double mutants with a stronger phenotype than either single mutant alone. We have crossed 23 of our confirmed mutants into the mutant to date, with 18 confirmed F1 lines. In addition, we are in the process of screening 4 F3 lines collected from individual F2 parents to identify double mutants.

Rather than attempt to bring in a postdoc to fill this position, I was able to hire one of our existing part-time department lab managers for the rest of her work hours. This has worked out well thus far, as she is present every day of the week and can assist with planting for experiments and plant care tasks. I am beginning to train her on DNA and RNA isolation protocols and I look forward to her being able to assist with these tasks in the future.

Thus far, we have 61 T-DNA mutant lines on hand and have successfully completed genotyping 55. Of those, 5 lines have been confirmed heterozygous for the T-DNA allele, and of those, 2 show a gravity related phenotype; all remaining lines (50) have been confirmed homozygous for the T-DNA carrying allele. Some of the 11 lines without a positive confirmation PCR result may be due to a later transposition event with the T-DNA. We are in the process of amplifying and sequencing the region surrounding the location of the T-DNA insertion to detect possible footprint deletions. Some of the lines also showed a lack of positive product for the positive control primer pair as well, indicating bad primer design or PCR conditions. Sequencing in the region of the purported T-DNA will also shed light on these lines.

In addition to genotyping, we are also testing for transcript knock-down in the T-DNA lines to be able to correlate any phenotypes observed with lack of transcript abundance. To date we have confirmed the lack of mRNA transcript for the gene of interest in 12 of our lines. The RT-PCR experiments required for this assay are more demanding than the genotyping PCR experiments, leading to an overall slower rate of completion. This coming semester, I will be performing this assay along with many others as part of my Plant Physiology lab experience for 16 students in Spring 2023. Last year, I centered the lab experience around this NASA-funded screening project to provide students hands-on experience with real research. Since so many of the assays and screens of these mutant lines is routine in the lab, this will provide students the opportunity to contribute to the project while learning valuable skills in bioinformatics, DNA and RNA isolation, real-time RT-PCR, and various plant growth analyses. I incorporated this project into the lab portion of the class last spring and it was moderately successful, with about half of the data generated by student groups of sufficient quality to be useful. This spring I will be structuring the work a bit more and providing templates for the students to collect and interpret results, which will hopefully mitigate some of the loss I saw last year.

In addition to the molecular characterization of mutants, we have made good progress on the growth experiments designed to test the mutants for any gravity-related phenotypes. We have developed a novel assay to detect differences in the regulation of differential growth. The assay is based on a previous technique used heavily in my lab, called ROTATO, which uses real-time image analysis to constrain the root tip at a constant angle over time by rotating the plate. This technique is good for measuring single roots, but does not scale well to large numbers of roots on a plate, as required for a medium-scale mutant screening project. I have used the average response rate (rotation) of wild-type roots as an input to control rotation of whole plates of mutant seedlings, reasoning that if the roots have “normal” gravity response, their tip angles will remain relatively unchanged because they are able to maintain the differential growth required to do so. We have now completed analysis of 50 T-DNA mutant lines using this technique and have identified 18 lines that show a statistically significant difference in response compared to wild-type. Thirteen (13) of the 18 lines show significantly reduced gravity response, while the other 5 lines show greater gravity response.

Other experiments to characterize the mutants include standard reorientation assays to assess gravitropic response and root phototropic assays to try to distinguish gravity perception processes from growth regulation and gravity response elements. We have conducted hundreds of standard root reorientation assays thus far and we are in the process of measuring tip angles and collating results. For the root phototropism assays, we have done some pilot testing with large-scale experiments, but I am unsatisfied so far with the amount of curvature induced in the positive and negative controls. I continue to work on a better assay design and construction that will optimize this response.

For several of the mutants under investigation, we are moving into a phase of more in-depth and customized experiments to probe the role of the gene in gravity responses. For example, one of my students has taken an interest in the nitrate reductase gene (AT1G77760), known as NIA1, which is a family member of NIA2, a gene in our initial contrast list. She is in the process of designing assays to detect this gene’s role in redox signaling during gravity responses. Several other students have identified similar genes or gene families to follow up on the initial screen, and this represents an exciting development in the project. I hope to interest other students in diving deeper with many of the dozens of mutants we’ve identified in the initial screen as having a novel gravity-related phenotype.

This year we designed and cloned CRISPR (CRISPR Therapeutics) constructs to knock out genes for which there is no T-DNA line available in a stock center. Thus far, we have created guide sequence constructs for 7 genes. Experiments to transform these constructs into plants failed over the summer when the candidate plants were left untended over a weekend and there was miscommunication about who was scheduled to water, unfortunately. On the positive side, I was pleased with how straightforward the cloning process was to create the constructs and remain positive that this will allow us to extend the screen to genes with no knockout mutants available. This could also allow us to generate second alleles for those genes for which we find a phenotype and wish to confirm.

I continued to make progress toward a renewed ROTATO system. I have rewritten the core image analyzer in MATLAB and have successfully used this image processing engine to constrain roots at the desired angle for indefinite periods. This system takes images captured from a standard Raspberry Pi HQ camera module, which uploads the images to a server where the computer running MATLAB can access them and control rotation of the vertical stage holding the root. This year I have focused on transitioning the core analyzer away from MATLAB and into Python, which would enable me to deploy the software on any Raspberry Pi imaging computer in the lab and also share it widely for those interested in incorporating it into their own projects. I am working with the PlantCV platform to identify the appropriate methods and functions to enable this transfer.

Last year, I attempted to hire a postdoc to support this project and collaborate with the Principal Investigator (PI) to move the project forward. The process has been hampered by turnover in personnel and the longer-term effects of the pandemic, but the job was posted early in April 2022. A total of 10 applications were received, but unfortunately none of them had the required minimum qualifications to warrant further engagement. In the future, I plan to re-advertise the position earlier in the spring in hopes of identifying a stronger pool of candidates with the requisite experience who are interested in both the project and in gaining experience at an undergraduate institution.

In addition to these molecular confirmation assays, we collected phenotypic screening data on many of the mutant lines using real-time imaging and computer image analysis. Screens involved standard gravitropism assays, phototropism assays designed to test gravity sensing, and a novel assay in which roots are continuously rotated away from gravity to test their capacity to maintain the differential growth response over long durations.