Progress 01/15/18 to 01/14/24
Outputs
Target Audience:Over the duration of the project, the target audiences for this workincluded the plant science research community (i.e.,faculty, post-docs, graduate students, and undergraduates from around the world) and stakeholders with the tree fruit industry (e.g. growers). Both groups were reached through a combination of oral and poster research presentations, field site presentations, peer-reviewed scientific publications, and trade publications. In addition, as part of an outreach workshop, the research for project was presented to High School students from underrepresented groups in science, including members of the indigenous community, and the students used materials generated from the work for an science education activity. Changes/Problems:A few of our planned experiments proved more challenging than anticipated when attempted and did not succeed after considerable effort (i.e. protein localization and isolation studies, and PIN3 localization studies). One experiment waseliminated due to negative impacts of Covid-19 (i.e. plum transformation) and a few produced inconclusive or negativeresults after years of work (i.e. phototropism experiments). Those challenges and corresponding changes were discussed in past annual reports. Since the last reporting period no major changes or problems have occurred. As workarounds for experiments that didn't go as planned, additional experiments were performed in order to successfully generate information about the molecular role of WEEP. The added experiments ended up providing superior and more valuable information than some of the proposed experiments. In addition, we incorporated several experiments focused on the role of WEEP in root architecture and root gravitropism in both Arabidopsis and Peach. That work was initiated based on recent findings by a group studying a homolog of WEEP in wheat. The root experiments we incorporated were highly successful and led to some of our most valuable findings. What opportunities for training and professional development has the project provided?This project provided one-on-one training in plant science research and scientific communication (via a combination of oral presentations, poster presentations, and manuscript preparation) for one post-doctoral researcher, three graduate students (one full time on this project and two others part-time), and eight undergraduate students. How have the results been disseminated to communities of interest?Two peer-reviewed research publications have resulted from this work as well as numerous presentations to national and international scientific audiences. Our RNA sequencing (transcriptome) data was also deposited into a public database (NCBI GEO) for other researchers to benefit from. Those deliverables disseminated the work to the plant science community. Additionally, presentations to tree fruit extension educators and stakeholders were used to disseminate our results to those communities and to seek feedback on the utility of the work for agriculture. Lastly, the research and findings were disseminated to general public audiences through a press release (https://blog.aspb.org/researchers-decipher-mysterious-growth-habit-of-weeping-peach-trees/ ), an editorial from the journal Plant Physiology (https://doi.org/10.1093/plphys/kiae161), and an article in the lay publication Knowable Magazine (https://knowablemagazine.org/content/article/living-world/2020/bent-shape-rules-tree-form). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The research conducted for this project led to a major scientific finding for the field of plant biology. The work identified that the WEEP gene is essential for the asymmetric transport of the hormone auxin in connection with directing the trajectory (orientation) of shoots and roots under normal conditions and as part of gravitropism responses (e.g., upward shoot bending and growth, and downward root bending and growth, in response to reorientation of plants from environmental events or human interference). Briefly, we identified that weeping peach tree branches grow downwards because they have a flipped localization pattern of auxin compared to trees with standard growth habits. In trees with standard growth habits, higher auxin concentrations are found on the lower side of branch tips (the tissues closer to the ground), compared to the tissues opposite them (the tissues on the upper side of the branch). The opposite is true in weeping branch tips. The asymmetric concentration gradient leads to the subtle asymmetric growth differences that cause upward branch orientations in standard trees or downward growth in weeping trees. Asymmetric auxin movement (due to WEEP) also results in the higher auxin concentrations on the lower side of standard branches and roots also directs upward shoot growth and downward root growth as part of the gravitropism pathway. This basic mechanism behind shoot and root gravitropic responses has been known for decades. However, many of the details of this process on the molecular level are still unknown. This research project placed another piece of the puzzle on the table for scientists to use to fill knowledge gaps in our understanding of how plants direct and adjust root and shoot growth trajectories. Additionally, we identified through a collaboration that the WEEP protein oligomerizes, meaning multiple copies of the WEEP protein bind together. This oligomerization may be important for its role in auxin movement, but further research is needed to make this determination. Valuable information about molecular processes downstream of WEEP were also generated through our transcriptome analyses, and they will enhance future research in this field. Lastly, we identified that plants that do not have a functional WEEP gene exhibit narrow lateral root angles and slender root architectures. This finding has important agricultural implications. Narrow root angles can be beneficial for crop plants growing in areas with limited water availability. Thus, manipulating the expression of this gene or processes associated with, and/or selecting plants with reduced or no WEEP expression as part of breeding efforts, may lead to the generation of drought tolerant cultivars and rootstocks for numerous species.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Kohler A, Scheil A, Hill Jr Jl, Allen JR, Al-Hadad JM, Goeckeritz CZ, Strader LC, Telewski F, and Hollender CA. (2024) Defying Gravity: WEEP promotes negative gravitropism in peach trees by establishing asymmetric auxin gradients. Plant Physiology, kiae085. https://doi.org/10.1093/plphys/kiae085
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience:Members of theplant science research community were reached by our efforts in 2022. This included faculty members,graduate students, and postdocs from all over the world. They were reached via presentations at an international conference (Auxin 2023, in Croatia), and through invited seminar presentations at North Carolina State University and Michigan State University. We also reached additional members of the scientific research community via a publication in a peer reviewed journal. Changes/Problems:This past year we have not had any problems that will impede our overall goal of idenfying the physiological and molecular role of WEEP in setting branch orientations. Our intial PIN3-immunostaining experiment did not work, but we are currently trouble shooting the methods with new anti-PIN3 antibody. We believe that we will get it to work in the next few months. Our efforts to obtain transgenic tobacco with mutations in the WEEP gene, and transgenic plums that overexpress WEEP or ones that have the GUS reporter gene under a WEEP promoter, still remain paused. We have spent several years trying to get the tobacco mutant and no longer think it is worth the effort to continue because arabidopsis can now be used as a model for fuctional analyses. In regard to the plum transgenics, the collaborator we are working with for the plum transformation has not had the capacity to help us due to staffing shortings, andthe person in my lab trainined on this technique a two years ago left in 2021. However, I do not believe that the plum transgenics are essential anymore to answer our questions. In support of our overall goal, we added an additional approach to idenfiy new aspects of the WEEP mechanism: size-exclusion chromatography. Thiswas perfromed by a new collaborator to test if WEEP proteins can self-oligomerize. Their results supported that hypothesis and broaded our findings and the usefullness of them. Additionally, as described in our future goals section, we have added experiments aimed at identifying transcription facotrs that regulate the expression of the WEEP gene, furthering our undestanding of the molecular mechanism behind WEEP's abiity to regulate lateral shoot orientations. What opportunities for training and professional development has the project provided?One undergraduate studentwastrained on research and analysis techniques. Two new graduate students, including one hispanic female, recieved training on laboratory techniques. A third graduate student, the primary student working on this project over the the past three years, received training on bioinformatic analysis techniques as well as research dissemination skills (e.g. manuscript preparation). She also gained additionalexperience with mentoring and supervising while working with the undergraduate. How have the results been disseminated to communities of interest?Results have been disseminated to members of the plant science communitythrough 1) an oral presentationat an international conference for scientists studying the plant hormone auxin, 2) an oral presentationat North Carolina State University as part of departmental seminar series, 3)an oral presentationat Michigan State Universityas part of departmental seminar series, and 4) a published manuscrip that is accessible to the public. What do you plan to do during the next reporting period to accomplish the goals?The initial goals for this project will be wrapping up during this next reporting period, and new testable hypotheses will be developed. The experiments we will perform include additional immunostaining experiments to identify how and where auxin is moving in weeping peach branches. Additional experiments looking at shoot and root gravitropism responses in peach, tobacco (our original model plant for trees), and arabidopsis will also be performed. Further, experiments to identify transcription factors that regulate the expression of the WEEP gene will be performed. These experiments, which involve testing WEEP gene expression in plants with mutations in candidate transcription factor genes (identified by a yeast-one-hybrid screen performed by a colleague) will be done using arabidopsis. Our identification of the root phenotype in arabidopsis weep mutants allows us to more rapidly study aspects of the molecular mechanism that WEEP uses for branch orientation control and gravitropic response. And we will also follow up on data from the yeast-two-hybrid screen that was performed a few years ago. That screen identified candidates for proteins that may interact with the WEEP protein in arabidopsis. We will test if those candidate protein interactors truly bind the WEEP protein using a technique called Bimolecular Fluorescence Complementation (BiFC). Alongside this experiment, we will work to confirm the subcellular localization of WEEP. We may also try again to generate an antibody for the peach WEEP protein, which could be used to answer additional questions about the function of WEEP. Our efforts during the first two years to do this and use the antibody to study protein localization did not go as planned; the antibody we generated was not specific to WEEP. It unpredictably detected an unrelated protein. Our new approach will be to use an antibody made from isolated peach WEEP protein, as opposed to generating a peptide-based antibody. A new collaborator generated this protein for us as part of their help with the size exclusion chromatography experiment. If we are able to obtain a good antibody then we will repeat our proposed experiments related toidentifying the tissues where WEEP is, and thus what tissues WEEP functions in. Lastly, in the next reporting periodwe will complete the preparation of a manuscript describing all of our findings and submit it to a peer-reviewed journal for publication.
Impacts
What was accomplished under these goals?
The overarching research goal for this project is to identify physiological & molecular functions of the previously uncharacterized gene WEEP, which regulates shoot orientation in trees. In connection, our work is generating knowledge that could be used to breed fruit trees and other crop plants with shoot architectures, and possibly root architectures, that could reduce labor, production costs, and land use. In 2022, we made a tremendous amount of progress on the both the molecular and physiological characterization of WEEP, as described in more detail below. Additionally, our first manuscript resulting from this project was published. That manuscript described an unexpected, yet exciting, aspect of this gene. WEEP is important for regulating lateral root angles in the model species Arabidopsis thaliana, and potentially a variety of crop plants. It also allows for future research on this agriculturally important gene to be done in a model species to speed up the rate of discovery. A second manuscript from this work was being prepared in the second half of 2022. This upcoming manuscript, which will be submitted for peer review publication in 2023, is a comprehensive report containing most data generated by this grant. Specific major accomplishments for the 2022 reporting period included the generation and analysis of gene expression data from weeping and standard peach trees. This data was generated through RNA sequencing technology. It revealed that our weeping peach trees have a weeping architecture because the localization of a hormone called auxin is in the wrong place. Essentially, that result identified that WEEP is responsible for the movement of auxin in connection with regulating tree branch angles and orientations in response to gravity perception. This is a tremendous finding, and we believe it will apply to all plants and not just peach because the weep gene is present throughout plantae and mechanisms associated with gravitropic response are believed to be universal. Our result adds a new player to what is known about how plants set and maintain shoot architectures in connection the direction of the gravity vector, whether it be in response to an accidental shoot reorientation or the maintenance of natural growth habits. Hormone analyses were also performed in 2020 on comparable tissues, to complement this gene expression experiments, but their results were more variable than anticipated. The RNA sequencing data also suggested that WEEP promotes the development of tension wood in trees. It is likely that this role is related to WEEP's function in moving auxin, as auxin gradients are believed to promote tension wood formation. Further, the connection between WEEP and tension wood helps explain why herbaceous plants, like the model species arabidopsis, that have a mutation in weep do not have weeping shoot architectures. It may be the change in tension wood development in the weeping peach trees that leads to the development of the arching phenotype of strong woody branches. A second major accomplishment was data collection and analysis for characterizing the architecture of weeping peach tree roots. We determined that in peach trees, just like in arabidopsis, WEEP promotes wide lateral root angles and orientations. This knowledge alone is extremely impactful for fruit tree breeding programs, including ones for other members of the Rosacea family (e.g. apple and cherry). It may also be applicable for other unrelated crop species. Further, this result suggests that weeping peach trees might be beneficial as rootstocks. Manipulation of crop plant root architecture can allow plants to be more drought tolerant and increase nutrient acquisition. We also identified that, likely in connection to its role in setting lateral root angles and orientations, WEEP is important for root gravitropic responses. Weeping tree roots responded faster to gravitropic stimulus (a 90-degree reorientation) than standard trees, supporting our shoot data that suggests WEEP is essential for gravitropic responses. A third major accomplishment was the determination that the WEEP protein form self-oligomers, meaning that several WEEP proteins bind together. This oligomerization was identified through size exclusion chromatography, an experiment that was not initially planned but was added to the project to further our understanding of the molecular function of WEEP. It is likely that the oligomerization is important for WEEP's role on the molecular level. An additional accomplishment was the characterization of the size of the cells in the upper and lower tissues of weeping peach branches, compared to standard branches. Surprisingly, we did not find significant differences, indicating that any influence on cell elongation by WEEP is subtle, if it occurs at all. Finally, we performed our first immunostaining to detect the localization of the PIN3 auxin efflux proteins. The result of this experiment will indicate if the movement of auxin via a mechanism involving WEEP is connected to known auxin transporters (PIN3 proteins). We are still in the process of repeating and troubleshooting this experiment, but the efforts we put into it in 2022 informed our current methods.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Johnson JM, Kohler AR, Haus MJ, and Hollender CA. (2022) Arabidopsis weep mutants exhibit narrow root angles. microPublication Biology. 10.17912/micropub.biology.000584
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:Our efforts in 2021 were presented to other members of the plant science research community, including national and international faculty, graduate students, and postdocs, as well as two politicians. The scientific community was reached viatwo oral presentations and one poster presentation. The politicians were reached via two personal tours, one given to US House Rep Elisa Slotkin in our greenhouse facilty and the other to MI State representative John Bizon at a tour of our research field within the Clarksville Research Center. Changes/Problems:We are on track to complete the overall goal of this funded research project. However, over the past few years, my lab has experienced technical challenges with a few experiments, a major and unexpected personnel change (a post-doc left and a graduate student with less time for research took his place), we had a set back from a collaborating lab, and there was a significant research slowdown due to the impact of covid-19. As a result, experiments proposed in our first objective may not be accomplished as planned. The experiment proposed for objective 1a (Visualize the localization of WEEP protein in peach) was attempted several years ago but was very technically challenging dueto unanticipated properties of the WEEP protein. I described that difficulty in a past progress report. My lab no longer has the expertise or time to return to that experiment. However, the data we are getting from an Arabidopsis GUS reporter line that we generated should help us answer a similar question (gene expression localization will be studied as a proxy for protein localization at this time). Since we recentlydetermined that Arabidopsis WEEP has role in architecture regulation (i.e. root architecture), performing experiments in this model species to aid in functional discoveries can be justified. The results we already have from a detailed gene expression study for dissected tissues from tobacco and tomato (from objective 3) will also contribute to the intended knowledge goal. In addition, the generation of transgenic plums that contain a GUS reporter driven by the WEEP promoter may not be possible to generate in the remaining time for this grant. This was started in Fall 2019 and the covid 19 pandemic restrictions for research as well as the loss of the postdoc I trained in this technique disrupted our progress in generating these transgenic plum lines. Studying the above mentioned arabidopsis GUS reporter lines will help us get around this problem. Our objective 1 experiments to identify peach proteins that interact with the peach weep protein was unable to be accomplished as planned because our collaborator's yeast-two-hybrid screen did not produce enough reliable candidates for us to base our work on. However, we worked with a company to perform a yeast-two-hybrid experiment to identify arabidopsis proteins that interact with the arabidopsis WEEP protein. The data from that work should be applicable to peach and help us further understand the mechanism that WEEP is involved in to regulate branch orientation in plants. Lastly, our planned generation of plum lines that overexpress weep (objective 2a) have also been delayed due to the same reason our plum GUS reporter lines are on hold However, Arabidopsis that overexpresses the Arabidopsis WEEP gene or the peach WEEP gene were generated and are currently being studied. All other experiments proposed are in progress or have already completed. Although the challenges described above caused my lab to adjust our approaches, we are succeeding in generating data that will achieve the goal of identifying physiological & molecular functions of WEEP. What opportunities for training and professional development has the project provided?Three undergraduate students were trained on research and analysis techniques and one was trained in manuscript preparation. The graduate student working on this project received firsthand experience with mentoring and supervising two of the undergraduate students that were helping with this research. The graduate student was also further trained by the PI in research and dissemination skills. How have the results been disseminated to communities of interest?Results were disseminated to scientific audiences (i.e. other plant scientists) via both oral presentations and poster presentations, as described in the products section. What do you plan to do during the next reporting period to accomplish the goals?Over the course of the 2022 reporting period, additional experiments relating to the investigation of WEEP function will be performed and analyzed. This includes the completion of our hormone and transcriptome experiments. We will also perform and analyze the data from a more extensive root box experiment to phenotyping weeping and standard peach tree roots. We will also make further progress on experiments related to mechanisms which we can perform in model species. We anticipate the submission of two manuscripts and the preparation of a third. One manuscript will focus on weep peach anatomical and molecular phenotypes, which will help determine gene function. Another manuscript that we will submit will describe the arabidopsis root phenotype, and the first author will be an undergraduate student. The third manuscript that we will be preparing for 2023 submission will focus more on the molecular function of the gene based on results from experiments in model species.
Impacts
What was accomplished under these goals?
Numerous efforts were taken to achieve our goals of identifying physiological and molecular functions of the WEEP gene in trees as well as herbaceous model species. These includes the completion of several important experiments as well as the collection of tissue for future experiments. Note, tissue collection is not trivial for research involving trees. Trees have a long growing cycle and need to undergo three months of dormancy at 4 degrees Celsius (when growing in a greenhouse) or emerge from dormancy after the winter (when growing in a field). Thus, there are only a few times a year that we can collect tissue. Specific details of our accomplishments are described below. 1. Tobacco lines that are homozygous for a WEEP overexpression vector were generated and preliminary gravitropism studies were performed on them. Preliminary results suggest that the WEEP gene slows gravitropic responses in shoot tissues because the overexpression lines had a more rapid response to gravistimulation. In addition, preliminary anatomical analysis of these tobacco lines were performed. They indicated thatWEEP does not significantly impact shoot anatomy. Anatomical differences were not identified. 2. Wood strength tests were performed on weeping and standard tree branches. Results suggested thatthere are positional differences between the strength and elasticity of the weeping and standard peach branches. 3. Peach branch tissue from weeping and standard trees were collected and sectioned to assess whether or not weeping peach trees contain gravity sensing amyloplasts in their amyloplasts. We found that they do have amyloplasts, and therefore shoot gravitropic defects in weeping peach trees do not occur because the trees do not have the ability to perceive gravity due to the lack of amyloplasts. Thus, theWEEP gene does not function in amyloplast or endodermis formation. 4. We performed both static and time lapse auxin response experiments for which auxin was applied asymmetrically onto weeping and standard peach branches that were on a tree or placed in beakers of water. The results of this experiment showed that weeping tree branches responded just like standard trees to asymmetric auxin gradients (which are also produced in response to gravistimulation). Thus,the weeping phenotype is not due to the inability of a plant to respond to auxin.This important result along with the result indicating the presence of amyloplast suggests that the function of WEEP lies somewhere at or after amyloplast perception and the movement of auxin to establish the asymmetric hormone gradients or responses needed to regulate branch orientations. 5. The root architecture of weeping peach trees was investigated for the first time. We grew weeping seedlings (which took 2 years to collect seed for) in rootboxes and documented their growth. This led to an exciting finding:peach weep mutants exhibited narrower lateral root angles, suggesting that modulation of the expression of WEEP could be beneficial for crop plants. As a follow up to this study, the root architecture ofArabidopsis with weep mutations was also studied and a similar result was found, suggesting that the role of WEEP in the regulation of lateral root architecture extends beyond trees. The arabidopsis result also suggests thatwe can use this fast-growing model species to help us learn more about WEEP functionand details about the molecular mechanism that is part of. 6. Following the identification of the utility of the arabidopsis weep mutants for our research,we performed phototropism experiments and determined that arabidopsis weep mutants do not have phototropic defects.This type of experiment was previously attempted in trees but was challenging and gave inconclusive results.A preliminary root gravitropism study for the arabidopsis mutants was also performedand although the roots of weep mutants responded to gravity, this work may be followed up on in the future to assess if there are differences in the response rate between the weep mutants and controls. 7. Tissue was collected from trees for transcriptional comparisonsbetween upper and lower tissues from three different regions of weeping and standard peach branches in order to identify what the WEEP gene affects downstream of its function. SubsequentlyRNA was extracted and sent for sequencing. 8. Tissue was collected from trees for hormone extractionsfor upper and lower branch tissues to identify whether or not the WEEP gene function impacts hormone localization in developing tree branches and how. Hormone extractions and analysis will be performed in 2022. 9. Initial steps to generating tobacco lines with mutations in the WEEP gene via the CRISPR-Cas 9 technology were taken. We identified lines that contain the CRISPR vector., however no mutations were present. Because the vector is still present and can cause mutations in the progeny of these lines, we began collecting seed from these plants and will initiate screens to identify mutants in 2022. Once isolated (in 2022), mutant lines will help us understand the function of weep in plants. 10. Peach branch tissue from weeping and standard trees was collected and placed in a fixative for future histological analysesthat will investigate whether or not WEEP function is related to cell epidermal or cortical size or cell number. 11. A yeast-two-hybrid screen to identify plant proteins that interact with the WEEP protein in arabidopsis was completedvia help from the company Hybrigenics. Those results are currently being analyzed to develop new hypothesis and future experiments to test them.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Oral Presentation: The mystery of the weeping tree and the upright arabidopsis: The role of WEEP in plant development, architecture, and tropic responses. ASPB Plant Biology 2021 Conference, July 19th
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Poster Presentation : Hollender CA, Kohler A, Johnson J, Hill J. The mystery of the weeping tree and the upright arabidopsis: The role of WEEP in plant development, architecture, and tropic responses. ASPB Plant Biology 2021 . Conference, July 19th-23rd.
- Type:
Other
Status:
Other
Year Published:
2021
Citation:
Oral Presentation: Molecular genetics in the orchard: The mechanisms and utility of plant architecture genes TAC1, LAZY1, and WEEP. University of Michigan Molecular, Cellular, and Developmental Biology Department Seminar, March 26th, 2021
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Progress 01/15/20 to 01/14/21
Outputs
Target Audience:National audiences of fellow plant science researchers as well as stakeholders in the tree fruit agriculture industry. Changes/Problems:The Covid-19 pandemic significantly impacted this research project. Between mid-March and August 2020, non-essential research was prohibited at my university. All in-progress and planned experiments for this project were put on hold for this 4+ month period. Subsequently, when research activities were allowed to resume, the pace was significantly slower due to shift-schedule requirements to maintain social distancing and the inability to hire undergraduate research assistants. As a result, progress on the proposed work was significantly less than anticipated for this reporting period. Accordingly, a 1-year no-cost extension was requested and approved. In conjunction with the research shutdown / slow-down, the generation of transgenic plums to study WEEP gene expression localization in vivo was significantly setback. This process can take up to a year to generate a young seedling. When we resumed research, the postdoc that was working on the transgenics (but was being paid by a separate fellowship) was preparing to leave the lab for another job and he declined to initiate the transformation process again. We aim to restart the generation of transgenic plums in the 2021 reporting period either at MSU and/or our collaborating USDA lab (USDA-ARS-AFRS). Another change we made was the decision to temporarily delay or eliminate our plans to generate transgenic tomato plants (WEEP over-expressors and mutants). This past year we determined that tobacco and tomato have comparable expression patterns for WEEP. We also optimized our tobacco transformation pipeline and were having much more success generating tobacco transformants than tomato ones. Because of this, we decided to focus our transformation efforts on tobacco only because. The expression results suggested that it would be unnecessary to generate transgenic lines for both species in order answer our questions about WEEP function in herbaceous species with secondary growth. What opportunities for training and professional development has the project provided?The graduate student working on this projecthas been learning new laboratory techniques and analysis methods throughout the process of carrying out the research for this project. She also gained teaching and supervisory experience in connection with training an undergraduate researcher. How have the results been disseminated to communities of interest?Research was presented to both scientific and stakeholder communities, as described under the 'Other Products' section. This included presentations to scientific audiences at the University of Pennsylvania, Stanford University / The Carnegie Institute, and the company Pairwise. Results were also presented to growers as part of the International Fruit Tree Association winter meeting and orchard tour. In addition, this research project, and the related work on tree architecture in my lab was highlighted in the trade journal "Good Fruit Grower on July 23rd, 2020 in an article entitled "When Research Grows sideways".. (https://www.goodfruit.com/when-research-grows-sideways/) What do you plan to do during the next reporting period to accomplish the goals?We will continue to perform experiments that will enable us to accomplish our proposed research goals. This includes(but is not limited to)following up on leads on potential interacting proteins, identifying the subcellular localizatin of the WEEP protein, completing the generation of tobacco with mutations in the WEEP gene, thoroughly phenotyping WEEP overexpression and mutant tobacco lines, and analyzing our RNAseq data from upper and lower branch tissues. We will also perform additional branch strengh analyses with tree branches. We will also perform phototropic and gravitropic experiments using transgenic tobacco and arabidopsis that over express WEEP and lines that have weep mutations. Weeping mutant and overexpressor plant root phenotypes will also be looked at, becuase a recent publication identified a root phenotype in barely that was associated with a mutation in WEEP. We will also begin the preparation of one or two manscriptsthat will present our research findings from this project.
Impacts
What was accomplished under these goals?
The overall impact of this project will be the generation of knowledge that may one day benefit the tree fruit agricultural industry. It may do this by leading to reductions in production costs and increasing the sustainability of tree fruit crops via the development of new approaches for manipulatingtree branch growth and orientation. In addition, results will fill in knowledge gaps about how all plants set and regulate the orientation of their branches and other lateral shoots. Specifically, the research investigates the role of the newly discovered plant gene 'WEEP'. For example, some of the research seeks to identify where this gene is expressed, what proteins the WEEP protein interacts with to direct branch growth and development. The research also seeks to identify specific anatomical and biochemical differences between weeping peach trees (those with a mutation in WEEP) and standard trees. Research proposed under and/or related to all three objectives was performed during this reporting period. This includes the generation of results leading to the molecular and genetic characterization of the WEEP gene and protein in plants. All results will be reported in future peer-review publications. In addition, a change in knowledge by the graduate student working on this project was accomplished in this reporting period. The student learned many new experimental and analysis techniques in connection with each experiment and improved herability to think critically. She also increased scientific knowledge and communication skills as part of the preparation of her thesis proposal. Specific research results for each objective are described below: As part of our molecular characterization of the WEEP gene and protein (Objective 1), we worked with the company Hybrigenics to carryout a comprehensive yeast-two-hybrid (Y2H) library screen. Several strong candidates for protein interactors were identified and are being pursued in the 2021 reporting period. This Y2H library screen was performed as a workaround for the previous troubles we were having with our WEEP antibody for protein pulldowns from peach, as well as the lack of candidates from a similar (but less comprehensive) screen from a collaborator. In the end, however, the results that we generated from this screen are more reliable and expansive than what we would have obtained with our planned experiments. Some of the protein interactor candidates that were identified link WEEP to previously unreported mechanisms regulating the transport and homeostasis of the plant development hormone auxin. Other candidate protein interactions connect the function to amino acid synthesis, which was a very unexpected and interesting finding. As part of our characterization of WEEP in species other than peach (Objective 2), we completed our characterization of WEEP expression in tobacco and tomato. Expression of four weep homologs in these species was assessed by qPCR from RNA extracted from a detailed set of dissected vegetative and floral tissues. In general,we found all WEEP homologsto be expressed in every tissue and often at the same level, suggesting all copies of this gene may be functional and involved in a basic plant or plant cell process. In addition, we completed the generation of transgenic tobacco that overexpress the WEEP gene (35S::WEEP plants) and began assessing if they have abnormal anatomical or gravitropism phenotypes in order to better understand the function of this gene. The generation of tobacco WEEP mutant lines (via CRISPR/Cas technology) were was initated, however our first generation generated of plants lacked mutations that would disrupt gene function. Since the gene editing vector is still present in those lines, we will soon grow out them out and search for mutations in WEEP in this next generation of plants. In connection with objective 3 (Perform physiological, histological, molecular, & biochemical analyses to investigate the influence of WEEP on phototropism, gravitropism, and wood development), we began an experiment that will identify differences in gene expression between the upper and lower portions of weeping and standard peach tree shoot tips. Tissue was collected from a series of dissected shoot tips tissuesat the appropriate developmental stage (which we can capture only one to two times per year) and RNA was extracted from the tissues. The RNA was subsequently sent for sequencing at the end of the reporting period. We are currently waiting for the sequencing data for analysis. The results will further help identify how the WEEP gene regulates the orientation and growth trajectory of peach tree branch tips. In addition, as part of completing objective 3, we identified that weeping peach trees do indeed have amyloplasts in their endodermal tissues. Amyloplasts are essential gravity sensing organelles. Weeping peach trees shoots do not exhibit a response to gravitropic stimulation (reorienting branches from vertical to horizontal positions), thus we hyothesized that the weeping peach trees may night have this organelle. This identification of the presence of amyloplasts disproved one of our hypotheses and ithas focused our reserach experiments to investigate remaining possibilities for the lack of a gravitropic response. Another experiment that was performed in connection to this research, but was not explicitly proposed, was one that tested the response of weeping peach branches to the application of auxin. Auxin isa plant growth and development hormone that is involved in the process of reorienting shoots as part of gravitropic responses. We found that the weeping branches can indeed respond properly to this hormone. When auxin was applied on the lower portion of branches, the branches would bend upwards, just like the response in standard peaches.
Publications
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Progress 01/15/19 to 01/14/20
Outputs
Target Audience:Both plant scientists (researchers) as well as stakeholders in the tree fruit agriculture industry were reached through presentations during this reporting period. Changes/Problems:During this reporting period there were a few setbacks and a change in personnel. In regard to the research, we discovered that the antibody we have been working with for two years is unable to detect WEEP protein from peach tree extracts. It can successfully detect heterologously generated WEEP protein in western blots. However, after extensively troubleshooting variability with detecting the protein from plant extracts and troubleshooting our Immunoprecipitation (IP) protocol, we determined that in peach extracts, our antibody primarily detects an off-target protein that is the same size as WEEP. That protein was identified as MLP-like protein 328 (ppa025402). This setback affects objectives 1a and 1c. At this time we do not have a way to visualize WEEP protein in peach (1a). Fortunately the PromWEEP::GUS plants we are generating will enable us to visualize WEEP gene expression patterns as a proxy for protein localization. For objective 1c, we initiated an alternative approach to identify proteins that bind to the WEEP protein. This 'spike-in' method involves combining total protein extracts from peach with purified WEEP protein fused to a SUMO protein (WEEP-SUMO) that we generated by heterologous expression in E.coli. We plan on using a SUMO antibody to isolate the E.coli WEEP along with any bound peach proteins, then elute the peach proteins and identify them using MS/MS. However, we encountered several technical challenges with this method several months ago. Although we still have several more ideas to troubleshoot the approach, we have temporarily put this experiment on hold in order to expedite the completion of our other proposed experiments. One small beneficial experimental plan change will also take place. For objective 3c, we will perform RNA sequencing to assess expression differences between abaxial and adaxial weeping and standard peach shoot tissues. Unexpected cost-savings are enabling us to do this as opposed to the qPCR experiments we originally proposed. In regard to personnel, the post-doctoral researcher working on this project from the beginning (Joseph Hill) was awarded a USDA NIFA fellowship last year. Understandably, he has primarily been working on his fellowship research since his funding began in June 2019. Fortunately, Joseph trained my newest PhD student (Andrea Kohler) and last spring she transitioned to this project. She will be performing the bulk of the remaining experiments. What opportunities for training and professional development has the project provided?Post-doctoral researcher and PhD student working on the project both attended and presented a poster at an International research conference. The PhD student on the project took a semester-long course to learn principles and methods of microscopy, including light and confocal microscopy, which are needed to complete many of proposed experiments. How have the results been disseminated to communities of interest?I presented results from this project to two international audiences of plant scientists. The first was during a talk at the International Plant Growth Substance Association conference in Paris in June 2019. The other presentation was a seminar presentation I gave to researchers at the INRA-AGAP research station In Montpellier, France In July 2019. My student (Andrea Kohler) and post-doctoral researcher (Joseph Hill) also presented results from this project in a poster at the 'Mechanisms of Plant Development' conference In July 2019, which was organized by the Federation of American Societies for Experimental Biology (FASEB). In addition, some of this research was presented by the PhD student on the project to growers and tree fruit Industry stakeholders during a field day on August 14th, 2019 at one of our off-campus research stations (The Clarksville Research Center). What do you plan to do during the next reporting period to accomplish the goals?During the 2020 reporting period, we will continue the generation of the transgenic plums for objectives 1b and 2b. We will also generate and phenotype the WEEP overexpression and knock-out lines proposed in objectives 2c and start to generate the tomato lines for the same objective. In addition, the tobacco and tomato gene expression study (objective 2b) will be completed. We also plan on beginning the immunohistology and fluorescence microscopy experiments that will investigate if differences in vacuole structure or PIN3 localization exist between weeping and standard peaches (objective 3b). Lastly, we will isolate RNA from adaxial and abaxial shoot tissues from weeping and standard trees (for objective 3c) and send it for sequencing.
Impacts
What was accomplished under these goals?
During this reporting period (2019) research activities associated with all three experimental objectives we proposed to meet the goals described above were carried out. In conjunction with our objectives 1b and 2a, to study the function of this gene,we started performing the plum tree transformation procedure to generate trees that will: 1) indicate where the WEEP gene is expressed (promWEEP::GUS), 2) trees to act as a control for that experiment (35S::GUS vector), and plum trees that will constitutively overexpress the WEEP gene (35S::WEEP vector). A significant amount of work was alo performed to determine where and to what degree WEEP is expressed in tobacco and tomato plants (objective 2b). The qPCR primers and protocol for the tomato and tobacco WEEP genes, as well as housekeeping genes, were optimized. Throughout that process, preliminary expression data was generated for both tobacco and tomato tissues. Those results suggest that the WEEP homologs in each plant (i.e. WEEP1 and WEEP2) are expressed at similar levels within leaves, stems, and shoot apical meristem. However, in tomato, WEEP2 has a greater expression in floral tissues than WEEP1. Towards the end of 2019, we began collecting a series of more-finely dissected tissues from a large number of plants grown under optimal growth conditions. Those tissues, along with ones from 2020, will be used to complete this objective. The generation of overexpression and CRISPR/Cas9 gene editing DNA constructs for tobacco and tomatowas completed (for our objective 2c). Seed for tomato transformations was also bulked up. In addition, and at the end of 2019 tobacco transformations were initiated. Some troubleshooting for the tomato transformation protocol was also done.We also determined that weeping peach trees have amyloplasts (a gravity sensing organelle) by using analternative method than the one proposed for objective 3b (we sectioned and stained fresh shoot tips with a vibratome as opposed to sectioning and staining shoots fixed in plastic resin).Lastly, we performed extensive troubleshooting for the experiments relying on our anti-WEEP antibodies (Objectives 1a and 1c).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Conference Poster Presentation:
Hill JL, Kohler A, Al-Haddad J, Telewski F, *Hollender CA. Molecular Characterization of the role of WEEP in directing lateral organ orientation. FASEB Mechanisms of Plant Development Conference, Olean, NY.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Conference Oral Presentation by Courtney Hollender
Hormonal Control of Branch Orientation Relies on the Integration of Light and Gravity Signaling via TAC1, LAZY1, and WEEP. IPGSA 23rd International Conference on Plant Growth Substances. Paris, France.
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Progress 01/15/18 to 01/14/19
Outputs
Target Audience:The target audience reached in this reporting period was primarily other members of the plant science research community via conference presentations and seminars. A small number of members of the tree fruit stakeholder community was also reached via informal conversations. Changes/Problems:No major problems have been encountered that will keep us from meeting the overall objectives of this proposal.However, there were a few minor unexpected setbacks and results. They are described below. Part 2 of objective 1c (validating candidate interaction proteins identified form a yeast-two-hybrid screen performed by the collaborating Dardick Lab at the USDA ARS) may not be completed. The collaborating lab has had difficulty with their screen and have not yet produced reliable candidates for us to evaluate using in vitro binding assays. However, this will not impact accomplishing parts 1 and 3 of objective 1c, and the overall goal of objective 1c. The results that we get from our pull-down experiment (obj. 1c part 1) will actually be more reliable and stronger candidates for interacting proteins for us to follow up with in part 3 of the objective. The protocol for the phototropism experiments proposed in objective 3 had to be modified. The initial approach involved placing our standard and weeping trees in a box with a small hole near the shoot apex and a light shining through that hole. That experiment was performed twice, however, both standard and weeping trees did not have sufficient light to survive those conditions and leaves began to senesce. To work around this unexpected problem, we next placed standard and weeping trees in a growth chamber with a wall of light on one side and dark curtains covering the rest of the chamber. The experiment addressed the same question(would existing and new shoots from these trees bend towards the wall of light, away from it, or not alter their growth trajectory?). We performed this experiment twice as well. Surprisingly shoots from both the standard and the weeping trees did not alter their initital growth trajectories. We therefore concluded that the peach germplasm (sibling standard and weeping trees) that we are using does not have a strong phototropic responses. This has been reported for some species and is not unheard of. Surprisingly rootstock suckers in the same pots responded strongly to the phototropism experiment, thus actingas a positive control. As a whole this experiment was not a failure, although the lack of a strong phototropic response by our stand trees was unexpected. But, wecan now rule out the hypothesis that weeping branches "weep" due to an anti-phototropic response. Proposed follow-up phototropic experiments will now not be perfromed. Lastly, although our WEEP antibodies easily detect heterologously expressed WEEP protein as well as denatured WEEP protein from peach trees, we have been having unexpected difficulty with isolating WEEP protein in its native confirmation from peach trees. We have had to spend time trouble shooting this and are close to a solution right now. Part of our trouble shooting revealed that the protein is lowly expressed, the native protein degrades quickly at room temperature, and may be membrane embedded. Thisunexpected problem, however, should not put us being in accomplishing the objective. Modified protocols are being tried on dissected tissues to concentrate the protein. Outside advice from MSUbiochemists with extensive protein isolation experience will be sought if our current efforts do not produce optimal results.In addition, we did not anticipatecompleting this objective in the first year, so we are not behind in achieving it. What opportunities for training and professional development has the project provided?The post-doctoral researcher on this project, Joseph Hill, was trained in plum transformation at the USDA Appalachian Fruit Research Station and on woody tissue immuno-histology, including sectioning shoots with a the vibratome, by Andrew Groover's lab at UC Davis. Joseph also presented a poster at the annual American Society of Horticultural Science (ASHS) annual meeting, networked with fellow researchers, and attended profession development workshops for post-docs at MSU. Lastly, Joseph trained our 2018 REU summer student Mallory St.Clair on a related project (studying the role of WEEP in arabidopsis). How have the results been disseminated to communities of interest?This project and results to-date were disseminated via poster presentations at ASHS conference in Washington, DC in August 2018 by post-doctoral researcher Joseph Hill and at the Plant Biomechanics Conference in Montreal in August 2018 by Co-PD Telewski. PD Hollender presented results via invited talks at the ASHS meeting and in a Cornell University School of Integrative Plant Sciences Horticulture Seminar in October, 2018. In addition, the applicability of the work under this grant was discussed informally with members of the Michigan Apple Committee as well as Michigan tree fruit growers and MSU extension educators. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period (2019), we plan to make major progress on every objective, baring objectives 3a and 3d, which have been completed. This includes troubleshooting protein co-immunoprecipitation methods and sending recovered proteins for mass spectroscopy analyses to identity protein interactors, completing DNA vector cloning, initiating plum, apple, and tobacco transformation, initiating immuno-histological experiments, and performing gene expression studies on tomato, and tobacco.
Impacts
What was accomplished under these goals?
The goals of this project are anticipated to take several years to accomplish, however, progress in identifying physiological, histological, biochemical, and molecular functions of WEEP was made in 2018. We performed planned phototropism experiments and wood analyses for the weeping and standard peach trees (obj. 3a and 3d) and surprisingly found minimal differences in both between the two genotypes. In addition, several steps towards determining molecular functions of WEEP were taken and molecular reagents were generated. Our WEEP antibody was further purified (for obj. 1a, 1c) , we determined that our protein is unfortunately highly unstable (it degrades quickly at room temperature), and troubleshooting for native protein isolation from peach for the pull-down experiments was performed. Many of the DNA vectors needed for future transformations into plum, tobacco, and tomato were generated (obj. 1b, 2a, 2c), the post-doc working on the project learned the plum transformation technique (obj. 1b, 2a), and seed for tobacco and tomato experiments was bulked up (obj. 2b, 2c). The protocol for sectioning tree shoots using our newly purchased (used) vibratome microtome was also established. This method will be used for future protein localization studies (obj. 1a, 3b). In addition, in relation to this project, but not included in the scope of this work, a field plot containing weeping and standard peach trees and our WEEP RNAi-silenced transgenic plum trees was established at the MSU Clarksville Research Center. Scions from weeping and non-weeping peaches were also grafted and the resulting plants were split between our greenhouses and this same field plot.These trees will provide a continued source of peach tree material forthe research we will perform as part of this grant. Lastly, parallel to the work proposed in this grant, but not included in the objectives, the role of WEEP in Arabidopsis was investigated by an undergraduate student that was part of the MSU Plant Genomics REU program. This NSF-funded student, who was trained by the post-doctoral researcher on this grant,looked at the expression of WEEP under native conditions and in response to light, gravity, and hormone treatments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Hill JL and Hollender CA (2019) Branching out: new insights into the genetic regulation of shot architecture in trees. Current Opinions in Plant Biology 47:73-80. DOI 10.1016/j.pbi.2018.09.010
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