Progress 02/01/18 to 09/30/22
Outputs
Target Audience:Data from this award were disseminated to a broad audience of crop biology researchers and students at different stages of their careers via presentations at national and international meetings, invited seminars and workshops, publications in peer-reviewed scientific journals and two Ph.D. dissertations Overall, 14 oral presentations (virtual and in-person) were made by the PD, and two oral and three posters presentations were made by Ph.D. students. Undergraduate (six students for the duration of the award) and graduate students (three Ph.D. and one M.S. student for the duration of the award), and two postdocs, have been trained in the lab in applying functional genomic tools to study mineral nutrient homeostasis in brachypodium and wheat. Undergraduate students reflect on their experiences in the video that can be accessed via the following URL: https://www.youtube.com/watch?v=EwHx77aCxu4&feature=youtu.be Data from this project were incorporated into the upper-level undergraduate course: PLSCS4420/6420 "Mineral Nutrition: From Plants to Humans" that was taught by PD (spring 2019, 2021). A postdoc participating in this project, Dr. Rahmati-Ishka had an opportunity to obtain training in teaching students by presenting her findings from this project in two lectures in this course. Students enrolled in the PLSCS4420/6420 course including a Ph.D. student from my lab, Ms. Zavodna, a rotation Ph.D. student in my lab, Mr. Madonich and an undergraduate from my lab, Ms. Zhang, created educational videos about mineral nutrients copper and iron as pertains to plant growth development and human health. These videos were presented to students in Ithaca Highschool (IHS) to initiate the conversation about the role of the adequate mineral nutrition of plants for the future of food security and human health. I am currently in contact with AP Biology teacher from IHS to think of creative ways how to grow this activity and engage more students. These videos will be also posted online via my lab website and Cornell CyberTower. Currently, these videos are available via the following links: https://www.youtube.com/watch?v=gTlR36UACrg https://www.youtube.com/watch?v=Qmc4Z1R1HNY Changes/Problems:As noted, due to personnel changes at the Cornell Plant Transformation Facility, we had to rethink how to generate genome edits in wheat. We, therefore, have established a collaboration with the expert in this area, Prof. Akhunov (Kansas State), whose lab will use our constructs to generate wheat mutants. However, this will go beyond the time frame of this award. In addition, phenotyping of wheat mapping population was more labor intensive than we predicted and was also significantly slowed down by Covid-19-related campus closure and reduced capacity of operations for at least a year. In addition, we were not allowed to bring undergraduates to the lab for some time due to Covid-19-related concerns. Thus, we will complete the phenotyping in the follow-up application. To ensure progress, we reduced the scope and focused on two parental lines of the wheat mapping population. We now have fully characterized them as pertains to the sensitivity to copper deficiency and fertility. We also completed RNA-seq analysis to compare their transcriptome responses to copper deficiency. We are not preparing these data for publication. All in all, we believe that we have reached the goals of this award. What opportunities for training and professional development has the project provided?For the duration of the award, six undergraduate (two current, four completed), three Ph.D. students (one current, two completed) and one M.S. student (completed), and three postdocs (all completed), have been trained in the lab in applying functional genomic tools to study mineral nutrient homeostasis in brachypodium and wheat for the duration of the award. Undergraduate students reflect on their experiences in the video that can be accessed via the following URL: https://www.youtube.com/watch?v=EwHx77aCxu4&feature=youtu.be A postdoc participating in this project, Dr. Rahmati-Ishka had an opportunity to obtain training in teaching students by presenting her findings from this project in two lectures in the course PLSCS44200/6420Mineral Nutrition: From Plants to Humans. Three undergraduate students (Ethan Pan, B.S. in Chemistry, Biology, May 2019, Iryna Doviryak, B.S. in Biology and Society, May 2020 and Erica Kirchhoff, B.S. in Plant Sciences, May 2022) received Cornell University undergraduate research funds. Ethan and Iryna were successfully admitted to medical schools (Zucker/Hofstra School of Medicine at Hofstra University and Jacobs School of Medicine and Biomedical Sciences University at Buffalo, respectively), while the third student, Erika Kirchhoff, is pursuing a graduate degree at the University of Wisconsin, Madison. Through this award, graduate students and postdocs attended five meetings (three in person and two virtual) and made two oral and four poster presentations. Meetings attended by students and postdocs: Gordon Research Conference: Plant Molecular Biology "Spatial and Temporal Dynamics in Plant Biology June 12-17th, 2022; Holderness, NH, USA (in person, two poster presentations). American Society of Plant Biologists- Plant Biology 2021 - virtual (a poster presentation). American Society of Plant Biologists- Plant Biology 2020 - virtual (an oral presentation and a poster presentation). ASA, CSSA, and CSA Annual Meeting November 4 -7, 2018, Baltimore, MD, USA (a poster presentation). Symposium Sustainable Approaches for Improving Wheat Yield and Nutritional Value June 26th, 2018, L´viv, Ukraine - oral presentation. How have the results been disseminated to communities of interest?Data from this award have been disseminated to plant science researchers via publications in peer-reviewed journals, presentations (14 in total) at national and international conferences and workshops and invited seminars of the PD. Data from this project were also incorporated into the upper-level undergraduate course: PLSCS4420/6420 "Mineral Nutrition: From Plants to Humans" that was taught by PD (spring 2019, 2021). Students enrolled in the PLSCS4420/6420 course, including a Ph.D. student from my lab, Ms. Zavodna, a rotation Ph.D. student in my lab, Mr. Madonich and an undergraduate from my lab, Ms. Zhang, created educational videos about mineral nutrients copper and iron as pertains to plant growth development and human health. These videos were presented to students in Ithaca Highschool (IHS) to initiate the conversation about the role of the adequate mineral nutrition of plants for the future of food security and human health. I am currently in contact with AP Biology teacher from IHS to think of creative ways how to grow this activity and engage more students. These videos are available via the following links: https://www.youtube.com/watch?v=gTlR36UACrg https://www.youtube.com/watch?v=Qmc4Z1R1HNY What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Study the role ofBdSPL7as well asTaSPL7-1A,TaSPL7-1BandTaSPL7-1Dhomoeologs in copper homeostasis and fertility usingBrachypodium spl7mutant. We entirely completed this Objective. We have identified and characterized BdSPL7, a novel regulator of copper homeostasis in grasses. We have shown that the loss of BdSPL7 reduces plant growth and development unless the plant growth medium is supplemented with copper. These studies not only uncovered a novel regulator of copper homeostasis in grasses, but they linked its role and the role of copper with the development of important agronomic traits such as fertility, grain yield, size and weight. We also showed that the function of SPL7 in copper homeostasis is conserved in wheat. This conclusion was made by finding that ectopic expression ofTaSPL7AandTaSPL7Bcomplemented theArabidopsis thaliana spl7mutant whileTaSPL7Acomplemented thespl7mutant ofB. distachyon.We have also shown that, unlike AtSPL7, BdSPL7 does not seem to undergo posttranscriptional processing as it is localized to the nucleus when it is expressed in protoplasts. By contrast, AtSPL7 is associated with the endoplasmic reticulum and moves to the nucleus upon copper deficiency. These data are in the final stage of preparation for publication. We also followed up on our exciting discovery that BdSPL7, in addition to controlling copper homeostasis, plays a role in plant responses to hypoxia. Specifically, we found that thespl7mutant is more sensitive to low oxygen in hydroponic medium and does not grow in flooded soils even when soils are fertilized with copper. Thus, BdSPL7 is sensitive to waterlogging, a condition that causes hypoxia. Interestingly, in our recent transcriptome analysis of the responses of wheat to copper deficiency (detailed below), we found that copper deficiency also alters the expression of genes associated with waterlogging in wheat. Considering that flooding can negatively impact agricultural yields, cause a financial burden on agriculture, and forecast flooding frequency is expected to increase in the 21st century, we anticipate that our findings and follow-up research can significantly contribute to understanding the flooding- survival strategies of crops. We will submit a new proposal to follow up on these exciting findings. Objective 2. Use the CRISPR/Cas9 system to knockoutTaSPL7homoeologs simultaneously and study the SPL7 pathway in wheat directly. As we noted in past reports, in addition to Covid crisis-related delays, significant personnel changes occurred in the Cornell Transformation facility. Nevertheless, we have generated constructs to generate CRISPR_Cas9 mutants in wheat and even started the wheat transformation in our lab. We have also collaborated with the expert in this area, Prof. Akhunov (Kansas State), who will use our constructs to generate wheat mutants. We continue our collaboration and will finish this work in the follow-up application. Objective 3. Identify copper deficiency-responsive andSPL7-regulated genes in wheat andBrachypodium. We have completed comparisons of the transcriptional response of roots, mature leaves, flag leaves, anthers and pistils of Brachypodium wild typevs.thespl7mutant, both grown under control or copper-deficient conditions. We found that 885 genes were differentially regulated in roots of thespl7mutantvs.wild type grown in control conditions; 498 genes were differentially regulated in roots of thespl7mutantvs.wild type grown under copper deficiency. The number of SPL7-regulated genes was larger in mature laves and flga leaves, but smaller in the reproductive organs: pistils and anthers. Among differentially expressed genes are direct and indirect targets BdSPL7 targets. Using yeast-one hybrid assay, we found that a gene encoding putative iron transporter, BdIRT1 is a direct target of SPL7. BdIRT1 was fully characterized (as noted below), and the manuscript describing these findings is in preparation for publication. We also found that of two parental lines of Opata M85 and W7984 of the SynOP DH population, W7984 is more sensitive to copper deficiency than OpataM85. We thus have compared the response of transcriptomes of Opata M86 and W7984 to copper deficiency. We found that 2381 genes were differentially expressed in roots of OpataM85 grown under controlvs.low copper conditions (500 nM vs 25 nM copper). Significantly more genes were differentially expressed in the sensitive W7984 wheat genotype: 2878 genes were differentially expressed in roots of M7984 grown under controlvs.low copper conditions. Interestingly, and as noted above, among differentially expressed genes were those that respond to flooding and hypoxia, highlighting copper-hypoxia interactions. Since brachypodiumspl7mutant is sensitive to both copper limitation and waterlogging, we predict that copper-deficiency sensitive wheat W7984 will also be more sensitive to waterlogging than OpataM85. We are currently testing this hypothesis and will study this question in depth in the follow-up application. All in all, data from this Objective provides the first comprehensive analysis of the transcriptional response of grasses, including a globally important crop, wheat, to copper deficiency and opens a new avenue for investigating the role of copper in the sensitivity of crops to hypoxia/flooding. Objective 4.Conduct the whole-genome quantitative trait locus (QTL) mapping in wheat to identify genomic regions associated with improved copper uptake, delivery to anthers and grain yield. We phenotyped 125 lines of the SynOP DH population.Phenotyping traits included plant height, root length, weight, copper concentration in flag leaves and grains, and the number of florets/per spike and kernels/spike in plants grown under copper-sufficient or copper-deficient conditions. We did not appreciate how labor-intensive the phenotyping work was, and so it was progressing slower than we expected. We thus refocused slightly, and in addition to phenotyping the remaining members of the SynOP DH population, we have subjected parental lines to a more thorough investigation because we found that W7984 is more sensitive to copper deficiency than OpataM85. We also found W7984 produces 44% fewer grains/spike than OpataM85, even when both genotypes are grown under copper-sufficient conditions. Furthermore, M7984 grain yield drops even further to 25% vs. OpataM85 under copper limitation. This dramatic loss of M7984 fertility under copper deficiency was accompanied by altered anther and pistils' stigma morphology and aberrant grains. As noted above, we compared the transcriptome of OpataM85 and M7984 and are now preparing all data for publication. Objective 5. Initiate the functional characterization of genes selected from RNA-seq and QTL studies. We characterized two downstream targets of BdSP7,BdYSL3andBdIRT1, and wheatBdIRT1counterparts (TaIRT1LA,TaIRT1LB,TaIRT1LD) and are currently characterizing three more downstream targets,CITFL1,CITFL2, andCITFL3, encoding transcription factors whose counterparts inA. thalianaregulate copper and iron homeostasis. We showed that BdYSL3 transports copper while BdIRT1 is mainly an iron transporter. The manuscript describing these data is written and will be submitted soon. Our studies of the BdYSL3 have already been published. Our study of BdCITFLs has shown that at least CITFL1 mediates crosstalk between copper and iron homeostasis in brachypodium. Thus, unlike AtSPL7, BdSPL7, in addition to controlling copper homeostasis, regulates the expression of genes involved in iron transport and its regulation and, therefore, is a component of copper-iron interaction networks. This Objective provides a framework for a follow-up application to understand copper homeostasis transcriptional networks, their components and their crosstalk with iron homeostasis in grasses.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
Mahood, E., Bennett, A., Komatsu, K., Kruse, L., Lau, V., Rahmati Ishka, M., Jiang, Y., Bravo, A., Bowen, B., Louie, K., Harrison, M., Provart, N., Vatamaniuk, O. K., and Gaurav Moghe (2022) Characterization and visualization of global metabolomic responses of Brachypodium distachyon to environmental changes. Plant Phys., submitted MSID: PP2022-RA-00672
- Type:
Book Chapters
Status:
Submitted
Year Published:
2023
Citation:
Chia, J.-C., Smieska, L., Woll, A., Vatamaniuik, O.K. Visualizing metal distribution in plants using Synchrotron X-ray Fluorescence microscopy techniques. in Methods in Molecular Biology, Springer
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Sheng, H. The Physiological and Molecular Regulation Mechanisms of Plant Response to Mn Toxicity and Cu Deficiency
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Sheng, H., Jiang, Y., Chia, J.-C., Smieska, L., Woll, A. Vatamaniuk, O.K. (2023) SPL7 regulates copper uptake and delivery to reproductive organs and influences fertility, seed nutritional quality in Brachypodium
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2022
Citation:
Jiang, Y. , Zavodna, O., Sorrels, M., Vatamaniuk., O.K. (2023) Transcriptome analysis identified key players associated with wheat resistance to copper deficiency and flooding.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Sheng, H., Jiang, Y., Niu, Y., Vatamaniuk, O.K. (2024) SPL7-regulated copper uptake is essential for the normal growth of Brachypodium under flooding conditions.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Jiang, Y. Functional Analysis of Metal Transporters YSL6 and IRT1 in Wheat and Brachypodium
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Jiang, Y., Sheng, H., Chia, J., Smieska, L., Woll, A., Vatamaniuk, O.K. (2022) IRT1-Like transporter mediates iron distribution from source to sink tissues and affects cadmium accumulation in grains in Brachypodium
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2024
Citation:
Zavodna (2024). Regulation of copper homeostasis in grasses and its crosstalk iron homeostasis and environmental stress.
|
Progress 02/01/21 to 01/31/22
Outputs
Target Audience:Data from the first year of the project were reported at the invited seminar at the Molecular Cell Biology and Genetics Department, University of Maryland, USA, May 7th, 2021 (Virtual) and Radcliffe Institute Virtual Seminar at Harvard "Cross-cutting Studies on the Elemental Dynamics and Interactions in Living Systems March 12-13th, 2021 Data from this project were also incorporated into the upper-level undergraduate course: "Mineral Nutrition: From Plants to Humans" that is currently taught by PD (spring 2021). In addition, undergraduate and graduate students have been trained in the lab in applying functional genomic tools to study mineral nutrient homeostasis in brachypodium and wheat. Changes/Problems:As also noted above, due to personnel changes at the Cornell Plant Transformation Facility, we had to rethink how to generate genome edits in wheat. We, therefore, have established a collaboration with the expert in this area, Prof. Akhunov (Kansas State), whose lab will use our constructs to generate wheat mutants. What opportunities for training and professional development has the project provided?Undergraduate and graduate students have been trained in applying functional genomic/genetic tools to study mineral nutrient homeostasis and its regulation in brachypodium and wheat. Students in the lab were also trained in manuscript and grant writing. Three manuscripts and a book chapter have been published this year. The PD incorporated research data from this award into lectures in the upper-level undergraduate/graduate course: "Mineral Nutrition: From Plants to Humans (spring 2021). How have the results been disseminated to communities of interest?Data from the fourth year of the project were reported at two virtual events listed below and via three publications in scientific journals and a book chapter. Seminar at the Molecular Cell Biology and Genetics Department, University of Maryland, USA May 7th, 2021 (Virtual) Radcliffe Institute Virtual Seminar at Harvard University "Cross-cutting Studies on the Elemental Dynamics and Interactions in Living Systems, March 12-13th, 2021 Virtual Publications: Rahmati Ishka M, Chia, Ju-Chen and Vatamaniuk., O.K. Copper transporters, their regulation and contribution to seed yield and nutritional quality in higher plants. 2021, pages 205-227 in: Cation transporters in plants, Editor Upadhyay, S., Elsevier. 486 pages; ISBN: 9780323885737, 032388573X Baseggio M, Murray M, Wu D, Ziegler GR, Kaczmar N, Chamness J, Hamilton J, Buell CR, Vatamaniuk O, Buckler E, et al. & M. Gore (2021) Genome-wide association study reveals an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels. G3: Genes, Genomes, Genetics, V11:8: https://doi.org/10.1093/g3journal/jkab186 Jiang, Y., Chen, X., Chai, S., Sheng, H., Sha, L., Fan, X., Zeng, J., Kang, H., Zhang, H., Xiao, X., Zhou, Y., Vatamaniuk, O*. Wang, Y*. (2021) TpIRT1 is a transition metal transporter in Polish Wheat (Triticum polonicum L.) with a broad substrate specificity. (2021) The Plant Science, 312, 111058 . *Co-corresponding authors. https://doi.org/10.1016/j.plantsci.2021.111058 Sheng H., Jiang Y., Rahmati Ishka M., Chia J.-C., Dokuchayeva T., Kavulych Y., Zavodna T.-O., Mendoza P., Huang R., Woll A., Romanyuk N.D., Zhou Y., Vatamaniuk O.K. (2021) YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium. Plant Physiology, 186 (1): 655-676. PMID: 33576792 What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Study the role of BdSPL7 as well as TaSPL7-1A, TaSPL7-1B and TaSPL7-1D homoeologs in copper homeostasis and fertility using Brachypodium spl7 mutant. As noted above, we have completed this Objective fully. Our goal for the next reporting period is to prepare data for publication and report them at national and international conferences. In addition, we will continue our analysis of the newly discovered role for SPL7 in mitigating hypoxia and prepare our findings for the submission for a new grant to the USDA/NIFA/AFRI. Objective 2. Use the CRISPR/Cas9 system to knockoutTaSPL7homoeologs simultaneously and study the SPL7 pathway in wheat directly. Per conversation with Prof. Akhunov, we will test the efficiency of our sgRNA constructs in the genome editing using wheat protoplasts. After confirming their effectiveness in the generation of genome deletions, we will send them to Prof. Akhunov (Kansas State), who will use our constructs to generate wheat mutants. The characterization of whet mutants will go beyond the time-frame of this proposal. Objective 3. Identify copper deficiency-responsive andSPL7-regulated genes in wheat and Brachypodium. Since the last annual report, we have performed bioinformatic analysis of RNA seq data and fully completed Aim3.1. Our goal for this non-cost extension is to prepare RNA-seq data for publication. In addition, we are collaborating with the Bioinformatics facility at Cornell for comparative co-expression network analysis as was planned in Aim3.2. Objective 4. Conduct the whole-genome quantitative trait locus (QTL) mapping in wheat to identify genomic regions associated with improved copper uptake, delivery to anthers and grain yield. We will continue phenotyping the SynOP DH population during the non-cost extension period. As noted above, we did not appreciate how labor-intensive phenotyping is and so the completion of this Objective will go beyond the timeframe of the award. I have also recruited an undergraduate student to facilitate project progress. Objective 5. Initiate the functional characterization of genes selected from RNA-seq and QTL studies. Our goal for this reporting period is to submit a manuscript on the completed functional characterization of another gene selected from RNA-seq, BdIRT1.
Impacts
What was accomplished under these goals?
Objective 1. Study the role of BdSPL7 as well as TaSPL7-1A, TaSPL7-1B and TaSPL7-1D homoeologs in copper homeostasis and fertility using Brachypodium spl7 mutant. Since the last project report, we have completed this research objective. Specifically, we have generated and characterized transgenic brachypodium spl7 mutant lines expressing BdSPL7-GFP and BdSPL7 wheat homoeologs. My goal for this non-cost extension is to prepare our data for publication. I am also excited to share an unexpected discovery that we made since the last annular report. We found that BdSPL7 in addition to controlling copper homeostasis, plays a role in plant responses to hypoxia. Specifically, we found that the spl7 mutant is more sensitive to low oxygen in hydroponic medium and, in a preliminary study, showed to be more sensitive to waterlogging, a condition that causes hypoxia. We are currently repeating these experiments even though they were not originally planned. Considering that flooding can negatively impact agricultural yields, cause a financial burden on agriculture, and forecast flooding frequency to increase in the 21st century, we expect that our findings can significantly contribute to understanding flooding- survival strategies of plants. We will submit a new proposal to follow up on these exciting findings. Objective 2. Use the CRISPR/Cas9 system to knockoutTaSPL7homoeologs simultaneously and study the SPL7 pathway in wheat directly. As noted above, in addition to Covid crisis-related delays, significant changes in personnel occurred in the Cornell Transformation facility. We have generated constructs for the generation of CRIPR_Cas9 mutants in wheat and even started wheat transformation in our lab. However, to speed up the progress, we have established a collaboration with the expert in this area, Prof. Akhunov (Kansas State), who will use our constructs to generate wheat mutants. Objective 3. Identify copper deficiency-responsive andSPL7-regulated genes in wheat and Brachypodium. Since the last annual report, we have performed bioinformatic analysis of RNA seq data and fully completed Aim3.1. My goal for this non-cost extension is to prepare data from 3.1 for publication. In addition, we are collaborating with the Bioinformatics facility at Cornell for comparative co-expression network analysis as was planned in Aim3.2. Objective 4. Conduct the whole-genome quantitative trait locus (QTL) mapping in wheat to identify genomic regions associated with improved copper uptake, delivery to anthers and grain yield. We phenotyped 60 more lines (125 in total) of the SynOP DH population. Phenotyping traits included plant height, root length, weight, copper concentration in flag leaves and grains, the number of florets/per spike and kernels/spike in plants grown under copper sufficient and copper-deficient conditions. We did not appreciate how labor-intensive is the phenotyping work and so it is progressing slower than we expected. Thus, I have also recruited an undergraduate student to facilitate project's progress. Objective 5. Initiate the functional characterization of genes selected from RNA-seq and QTL studies. We have completed functional characterization of another gene selected from RNA-seq, BdIRT1, and have prepared a draft of the manuscript. We expect to submit it in Summer 2022.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2021
Citation:
Rahmati Ishka M, Chia, Ju-Chen and Vatamaniuk., O.K. Copper transporters, their regulation and contribution to seed yield and nutritional quality in higher plants. 2021, pages 205-227 in: Cation transporters in plants, Editor Upadhyay, S., Elsevier. 486 pages; ISBN: 9780323885737, 032388573X
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Baseggio M, Murray M, Wu D, Ziegler GR, Kaczmar N, Chamness J, Hamilton J, Buell CR, Vatamaniuk O, Buckler E, et al. & M. Gore (2021) Genome-wide association study reveals an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels. G3: Genes, Genomes, Genetics, V11:8: https://doi.org/10.1093/g3journal/jkab186
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Jiang, Y., Chen, X., Chai, S., Sheng, H., Sha, L., Fan, X., Zeng, J., Kang, H., Zhang, H., Xiao, X., Zhou, Y., Vatamaniuk, O*. Wang, Y*. (2021) TpIRT1 is a transition metal transporter in Polish Wheat (Triticum polonicum L.) with a broad substrate specificity. (2021) The Plant Science, 312, 111058 . *Co-corresponding authors. https://doi.org/10.1016/j.plantsci.2021.111058
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Sheng H., Jiang Y., Rahmati Ishka M., Chia J.-C., Dokuchayeva T., Kavulych Y., Zavodna T.-O., Mendoza P., Huang R., Woll A., Romanyuk N.D., Zhou Y., Vatamaniuk O.K. (2021) YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium. Plant Physiology, 186 (1): 655-676. PMID: 33576792
|
Progress 02/01/20 to 01/31/21
Outputs
Target Audience:Undergraduate and graduate students, as well as postdocs, have been trained in the lab in applying functional genomic tools to study mineral nutrient homeostasis in brachypodium and wheat. Data from this year were presented at three virtual events that overall reached more than 1500 plant researches. Changes/Problems:As noted above, my lab has experienced a significant delay in the project progress due to Covid-19-crisis and associated shut-down of Cornell from March to July 2020. Starting from July, 2020 my lab operates at 50% capacity as we work in shifts. The project also depends on Cornell sequencing facility that has been under shut down until approximately July 2020 and now operates in shifts too. While we are trying to catch-up, and the project moves forward, our progress was and still is slower than expected. As also noted above, due to Covid-19-related shutdown and personnel changes at the Cornell Plant Transformation Facility, wheat transformation procedures have been significantly delayed. We hope to catch-up this coming year, but anticipate that transgenic wheat characterization will go beyond the time-frame of this award. What opportunities for training and professional development has the project provided?Undergraduate and graduate students, and a postdoc, have been trained in the lab in applying functional genomic/genetic tools to study mineral nutrient homeostasis in brachypodium and wheat. Graduate students and postdoc presented their findings both orally and poster format at Plant Biology 2020 World Summit (July 27-31; 2020, virtual). Students and postdocs also were trained in manuscript writing. One manuscript has been now accepted for publication, one submitted and two in preparation. How have the results been disseminated to communities of interest?Data from the third year of the project were reported at three virtual events listed below and via a publication that has been accepted in the Plant Physiology and available as a preprint in bioRxiv (2019) doi:10.1101/2019.12.12.874396. Huazhong Agricultural University (HZAU) - Cornell University Workshop; January 11-12th, 2021, Virtual Talk title: The role of copper in plant fertility: a journey from model plants Arabidopsis thaliana and Brachypodium distachyon to wheat X-ray imaging for Life sciences, Environmental sciences, the Arts and plant sciences (X-LEAP) Workshop; June 8-10th, 2020, Virtual, Cornell University, Ithaca NY, USA Talk title: Using synchrotron X-ray fluorescence (SXRF) to study of genes involved in mineral nutrient transport in plants Plant Biology 2020 World Summit (July 27-31; 2020, virtual) Talk title: TaIRT1 homoeologs and BdIRT1 have subfunctionalized to be expressed in distinct tissues and confer transition metals delivery to seeds (presented by a PhD student, Yulin Jiang) Poster: YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium. (presented by a PhD student, Patrick Mendoza). What do you plan to do during the next reporting period to accomplish the goals?Objective 1 We will focus on the analysis of cellular localization of BdSPL7 in planta once we will obtain spl7 and wild type plants expressing BdSPL7 fused with GFP at the N- or C-termini. We will first test whether the SPL7-GFPconstruct functionally complements the BdSPL7-silenced brachypodium. We will then study the effect of copper status on the subcellular localization of BdSPL7. Objective 2: We hope that after the Cornell Plant Transformation Facility revised its procedures for the selection of transgenic plants, we will be able to obtain the deletion mutants for SPL7 in wheat. However, given the time needed to obtain transgenic wheat, it is likely that the completion of this objective will go beyond the time-frame of this award. Objective 3: We will complete the RNA-seq analysis of the BdSPL7-dependent transcriptional response of brachypodium to copper deficiency. The identification of differentially expressed genes and comparative co-expression network analysis between different genotypes to identify common and unique features of their transcriptome responses and gene modules regulated by SPL7 will be done via collaboration with Dr. Sun. While waiting on the generation of SPL7 mutant in wheat we will also generate samples for the RNA-seq analysis of the transcriptional response of wheat cv. Opata to copper deficiency. Objective 4: We will continue phenotyping wheat lines grown in the greenhouse. We hope to move faster with phenotyping so that we will finish more lines as planned initially. Objective 5: We will continue our studies of putative SPL7 targets as we did for BdYSL3. We will select genes encoding transcription factors and transporters whose level of expression is upregulated by copper deficiency to the highest level in the RNA-seq data. The functional characterization of selected genes will include the generation of knockout lines using the CRISPR/Cas9 system. We will only generate constructs for RNA-guided genome editing and transform plants, while the generation of mutants and their characterization will go beyond the period of this proposal. We will study transporter capabilities using functional complementation in the Saccharomyces cerevisiae copper uptake mutant. We will also study the subcellular localization of proteins in the yeast mutant and by transient expression of translational GFP fusions in wheat and brachypodium protoplasts.
Impacts
What was accomplished under these goals?
My lab has experienced a significant delay in the project progress due to Covid-19-crisis and associated shut-down of Cornell from March to July 2020. Starting from July, 2020 my lab operates at 50% capacity as we work in shifts. The project also depends on Cornell sequencing facility that has been under shut down until approximately July 2020 and now operates in shifts too. While we are trying to catch-up, and the project moves forward, our progress is slower than expected. We thus, requested and received a non-cost extension that will allow us to complete the work. Objective 1. Study the role of BdSPL7 as well as TaSPL7-1A, TaSPL7-1B and TaSPL7-1D homoeologs in copper homeostasis and fertility using Brachypodium spl7 mutant (Year 3) We almost completed this Objective. The remaining part is to study the subcellular localization of SPL7 in planta. Due to Covid related shutdown of facilities we lost our transgenic spl7 callus transformed with BdSPL7-GFP constructs. We are now repeating these experiments. Objective 2. Use the CRISPR/Cas9 system to knockoutTaSPL7homoeologs simultaneously and study the SPL7 pathway in wheat directly (Year 3). The Cornell Plant Transformation facility has been shut down and now does not operate at full capacity due to Covid-19 and also personnel-related changes. It is possible that this Objective may go beyond the time-frame of the proposal. Objective 3. Identify copper deficiency-responsive andSPL7-regulated genes in wheat and Brachypodium(Year 3). We have used deep transcriptome sequencing (RNA-Seq) to study the transcriptome responses of brachypodium to copper deficiency and identify novel factors contributing to copper transport and its regulation and identify SPL7-regulated genes. We performed RNA-seq for analyzed 120 samples that included roots, young and mature leaves, flag leaves, pistils, and anthers of wild-type and the spl7 mutant grown hydroponically under copper sufficient vs copper deficient conditions (3 replicates per condition and per genotype. Currently we are performing bioinforamtic analysis of RNA-seq data. This aim, although was delayed due to Covid-19 related shutdown of the Cornell sequencing facility, will be completed within next year. Objective 4. Conduct the whole-genome quantitative trait locus (QTL) mapping in wheat to identify genomic regions associated with improved copper uptake, delivery to anthers and grain yield (Year 3). We have phenotyped 37 more lines (65 in total) of the SynOp DH population i grown hydroponically under control conditions or copper deficiency in the greenhouse. Phenotyping traits included plant height, root length, weight, copper concentration in flag leaves and grains, the number of florets/per spike and kernels/spike in plants grown under copper sufficient and copper-deficient conditions. We did not appreciate how labor-intensive is the phenotyping work and so it is progressing slower than we expected. Thus, I have also recruited an undergraduate student to facilitate project progress. Objective 5. Initiate the functional characterization of genes selected from RNA-seq and QTL studies (Year 3). 1. We completed the functional characterization of a downstream target of SPL7, a copper transporters YSL3. The manuscript describing our findings is now accepted in Plant Physiology Sheng H., Jiang Y., Ishka M.R., Chia J.-C., Dokuchayeva T., Kavulych Y., Zavodna T.-O., Mendoza P., Huang R., Woll A., Romanyuk N.D., Zhou Y., Vatamaniuk O.K. (2019) YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium. Plant Physiology, in revision. This manuscript is available as a preprint in bioRxiv (2019) doi:10.1101/2019.12.12.874396. 2. We have also recently discovered that the Iron-regulated Protein, BdIRT1 is also among SPL7 downstream targets. We have characterized IRT1-like homoeologs from the three genomes in common wheat, Triticum aestivum (AABBDD) and an orthologue from wheat model, Brachypodium distachyon. We found that TaIRTL homoeologs have subfunctionalized in their tissue expression pattern: while all homoeologs were highly expressed in flag leaves, TaIRT1-A and D were also highly expressed in roots, TaIRT1-A and B were highly expressed in flowers. BdIRT1 was preferentially expressed in shoots and was upregulated by iron and copper deficiencies. TaIRT1 homoeologs and BdIRT1 differed in their ability to complement iron, zinc, copper and manganese deficiency of yeast micronutrient uptake mutants. Unlike the Arabidopsis irt1 mutant, the brachypodium irt1-1 knockout was able to grow without iron supplementation; however, its transition to flowering was delayed, it had altered flower morphology and fertility, decreased pollen production, viability and germination. These defects were, in part, rescued by the excess iron, copper, or manganese. The irt1-1 mutant has also altered iron, copper, and manganese tissue distribution, which resulted in the reduced accumulation of these minerals in grains. We are now preparing a manuscript describing our findings for publication. The targeted journal is Plant Physiology as well. 3. We have collaborated with Prof. Yi Wang (Sichuan Agricultural University, China) on the characterization of TpIRT1, a BdIRT1 homolog from polish wheat (Triticum polonicum). Our results, currently under review in Journal of Integrative Plant Biology, highlighted the importance of TpIRT1 in the uptake and translocation of Fe, Mn, Co, and Cd with direct implications for wheat yield potential. We show that TpIRT1 was highly expressed in roots and leaves, and induced by Fe, Mn, Co, Cd excess, and Mn deficiency in roots. Both TpIRT1A and TpIRT1B were located at the plasma membrane and internal vesicles in Arabidopsis protoplasts, and responsible for Cd and Co sensitivity in yeast. The over-expression of TpIRT1B in A. thaliana increased Fe, Mn, and Co concentration in its tissues and improved plant growth under Fe, Mn, and Co deficiencies. In addition, TpIRT1B-expressing A. thaliana lines accumulated more Cd and were more sensitive to Cd than wild-type. Functional analysis of IRT1 homoeologs from tetraploid and diploid ancestral wheat species in yeast disclosed four distinct amino acid residues in TdiIRT1B (T. dicoccum) and TtuIRT1B (T. turgidum). TdiIRT1B and TtuIRT1B ability to transport Zn, Cd, and Co in yeast also differed from other IRT1 homoeologs.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
1. Jiang, Y., Chen, X., Chai, S., Sheng, H., Sha, L., Fan, X., Zeng, J., Kang, H., Zhang, H., Xiao, X., Zhou, Y., Vatamaniuk, O. Wang, Y. TpIRT1 is a transition metal transporter in Polish Wheat (Triticum polonicum L.) with a broad substrate specificity. J Integrative Plant Biol.,under review; manuscript ID is JIPB-2021-0030.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2021
Citation:
1. Rahmati Ishka M, Chia, Ju-Chen and Vatamaniuk., O.K. (2021) Copper transporters, their regulation and contribution to grain yield and nutritional quality in higher plants. Book Chapter, in preparation.
|
Progress 02/01/19 to 01/31/20
Outputs
Target Audience:Data from the second year of the project were reported at the Pan-American Light Sources for Agriculture (PALSA) conference that was held on July 6-7, 2019 in Saskatoon, Canada. The PD presented data from the second year of the project in the talk, entitled "XRF as a tool to study the role of copper in plant fertility: a journey from model plants Arabidopsis thaliana and Brachypodium distachyon to wheat" at the Session: Genomics, Molecular Genetics, and Biotechnology. A graduate student presented a poster at this meeting. Data from this project were incorporated into the upper-level undergraduate course: PLSCS4420/6420 "Mineral Nutrition: From Plants to Humans" that was taught by PD (spring 2019). A postdoc participating in this project, Dr. Rahmati-Ishka had an opportunity to obtain training in teaching students by presenting her findings from this project in two lectures in this course. Students enrolled in the PLSCS4420/6420 course including a Ph.D. student from my lab, Ms. Zavodna, a rotation Ph.D. student in my lab, Mr. Madonich and an undergraduate from my lab, Ms. Zhang, created educational videos about mineral nutrients copper and iron as pertains to plant growth development and human health. These videos were presented to students in Ithaca Highschool (IHS) to initiate the conversation about the role of the adequate mineral nutrition of plants for the future of food security and human health. I am currently in contact with AP Biology teacher from IHS to think of creative ways how to grow this activity and engage more students. These videos will be also posted online via my lab website and Cornell CyberTower. Currently, these videos are available via the following links: https://www.youtube.com/watch?v=gTlR36UACrg https://www.youtube.com/watch?v=Qmc4Z1R1HNY Undergraduate and graduate students, as well as postdocs, have been trained in the lab in applying functional genomic tools to study mineral nutrient homeostasis in brachypodium and wheat. Changes/Problems:As reported above, it turned out that none of the transgenic wheat plants that have been generated in year one carried a deletion in SPL7. Personnel at the Cornell Plant Transformation facility have identified a potential problem at their end that has led to the generation of "escapers" - false positive transgenic plants. They believe that they have found a solution to this problem and so we will be repeating the entire procedure in year 3. What opportunities for training and professional development has the project provided?Undergraduate and graduate students, as well as two postdocs, have been trained in the lab in applying functional genomic/genetic tools to study mineral nutrient homeostasis in brachypodium and wheat. As reported above, students enrolled in PLSCS4420/6420 course Mineral Nutrition from plants to Humans, including a Ph.D. student from my lab, Ms. Zavodna, a rotation Ph.D. student in my lab, Mr. Madonich and an undergraduate from my lab, Ms. Zhang, created educational videos about mineral nutrients copper and iron as pertains to plant growth, development and human health. These videos were presented to students in Ithaca Highschool (IHS) to initiate the conversation about the role of the adequate mineral nutrition of plants for the future of food security and human health. This provided students with training on how to present scientific results to non-scientists and was a very valuable experience. A postdoc working on this project, Dr. Ishka, prepared and taught two lectures about copper nutrition in the PLSCS4420/6420 course Mineral Nutrition from Plants to Humans. Dr. Ishka also incorporated the results of her work into lectures as well. How have the results been disseminated to communities of interest?Data from the second year of the project were reported at the Pan American Light Sources for Agriculture 2019 (PALSA-2019) Meeting, July 6-7, 2019, Saskatoon, Canada. The PD presented data from the second year of the project in the talk, entitled "XRF as a tool to study the role of copper in plant fertility: a journey from model plants Arabidopsis thaliana and Brachypodium distachyon to wheat. Data from this project were incorporated into the upper-level course: PLSCS4200/6420; "Mineral Nutrition: From Plants to Humans" that was taught by PD in spring 2019. Educational videos that were created through the PLSCS 4200/6420 by students enrolled in the class and students involved in this project were presented to students in Ithaca Highschool (IHS) students participating in the school club "Synthetic Biology" to initiate the conversation about the role of the adequate mineral nutrition of plants for the future of food security and human health. I am currently in contact with AP Biology teacher from IHS to think of creative ways how to grow this activity and engage more students and from more diverse backgrounds. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 We will focus on the analysis of cellular localization of BdSPL7 in planta once we will obtain spl7 and wild type plants expressing BdSPL7 fused with GFP at the N- or C-termini. We will first test whether he SPL7-GFP construct functionally complements the BdSPL7-silenced brachypodium. We will then study the effect of copper status on the subcellular localization of BdSPL7. Objective 2: We hope that after the Cornell plant Transformation Facility revised its procedures for the selection of transgenic plants, we will be able to obtain the deletion mutants for SPL7 in wheat. We will then proceed with functional studies to test the role of SPL7 in copper homeostasis as pertains to wheat growth and fertility. Objective 3: We will complete the RNA-seq analysis of the BdSPL7-dependent transcriptional response of brachypodium to copper deficiency. The identification of differentially expressed genes and comparative co-expression network analysis between different genotypes to identify common and unique features of their transcriptome responses and gene modules regulated by SPL7 will be done via collaboration with Dr. Sun. While waiting on the generation of SPL7 mutant in wheat we will also generate samples for the RNA-seq analysis of the transcriptional response of wheat cv. Opata to copper deficiency. Objective 4: We will continue phenotyping wheat lines grown in the greenhouse. We hope to move faster with phenotyping so that we will finish more lines as planned initially. Objective 5: We will continue our studies of putative SPL7 targets as we did for BdYSL3. We will select genes encoding transcription factors and transporters whose level of expression is upregulated by copper deficiency to the highest level in the RNA-seq data. The functional characterization of selected genes will include the generation of knockout lines using the CRISPR/Cas9 system. We will only generate constructs for RNA-guided genome editing and transform plants, while the generation of mutants and their characterization will go beyond the period of this proposal. We will study transporter capabilities using functional complementation in the Saccharomyces cerevisiae copper uptake mutant. We will also study the subcellular localization of proteins in the yeast mutant and by transient expression of translational GFP fusions in wheat and brachypodium protoplasts.
Impacts
What was accomplished under these goals?
Objective 1. Study the role of BdSPL7 as well as TaSPL7-1A, TaSPL7-1B and TaSPL7-1D homoeologs in copper homeostasis and fertility using Brachypodium spl7 mutant (Year 2). 1. We have carried on the functional characterization of the outcrossedspl7-2allele of Brachypodium. We showed that thespl7-2mutant is developmentally delayed and has significantly reduced fertility evaluated as the number of seeds formed per flower and per spike. Using ICP-MS analysis (3 independent experiments) we have shown that the spl7 mutant accumulates copper in mature leaves. This leads to reduced copper accumulation in flag leaves and flowers of the mutant compared to wild-type plants. Using synchrotron x-ray fluorescence microscopy, we found that the mutant has a defect in copper accumulation in anthers. This finding is consistent with the role of copper in male fertility. Using light microscopy we showed that the mutant has smaller anthers and significantly reduced pollen viability and germination rates. Furthermore, thespl7-2mutant has shorter seeds compared to wild-type. These defects of thespl7mutant can be rescued by fertilizing plants with 25 µm CuSO4. Interestingly, we found that thespl7mutant is more sensitive to mild hypoxia. This finding provides a new avenue for the investigation that will be addressed via a new grant application. 2. We have explored the subcellular localization of SPL7 using transient expression of SPL7::GFP construct inBrachypodiumprotoplasts. We have shown that unlike its counterpart inA. thaliana, SPL7::GFP was observed in the nucleus of protoplasts prepared from plants grown under copper sufficient conditions. This is surprising as in AtSPL7 is localized to the endoplasmic reticulum (ER). Under copper deficiency, AtSPL7 is released from the ER and moves to the nucleus to facilitate gene expression. Our finding suggests that BdSPL7 might be regulated differently. We have generated binary vectors with BdSPL7 fused to GFP at the C- and N-termini. These constructs have been now transformed intoBrachypodiumwild-type and thespl7mutant. We are currently selecting transformed lines and will study SPL7 localization under copper sufficient and deficient conditionsin vivoin year 3. 3. We have expressedTaSPL7-1AandTaSPL7-1Band BdSPL7 into thespl7-2mutant of Brachypodium and thespl7-1mutant ofArabidopsis. All genes rescued the growth and developmental defects of thespl7mutants of bothArabidopsisandBrachypodium, suggesting that the function of SPL7 is conserved in different species. Objective 2. Use the CRISPR/Cas9 system to knockout TaSPL7 homoeologs simultaneously and study the SPL7 pathway in wheat directly (Year 2). It turned out that none of the transgenic wheat plants that have been generated in year one carried a deletion in SPL7. Personnel at the Cornell Plant Transformation facility have identified a potential problem at their end that has led to the generation of "escapers" - false positive transgenic plants. They believe that they have found a solution to this problem and so we will be repeating the entire procedure in year 3. Objective 3. Identify copper deficiency-responsive and SPL7-regulated genes in wheat and Brachypodium (Year 2). Because we had difficulty in generating spl7-mutant in wheat this year we focused on RNA-seq analysis of the transcriptome response to copper deficiency of Brachypodium wild-type vs spl7 mutant. We grew plants hydroponically with copper (control) to the late vegetative stage. At the end of the tillering stage, a subset of plants was transferred to a fresh hydroponic medium without copper to induce deficiency. Total RNA was isolated from roots, young and mature leaves, flag leaves, pistils and anthers. We have generated 5 replicates per condition per genotype, 230 samples in total. We have evaluated RNA quality and concentration and will now send 3 replicate per condition per genotype for RNA-sequencing. Objective 4. Conduct the whole-genome quantitative trait locus (QTL) mapping in wheat to identify genomic regions associated with improved copper uptake, delivery to anthers and grain yield (Year 2). We have phenotyped 28 lines of the SynOp DH population including their parental lines, Opata M85, and W7984 grown hydroponically under control conditions or copper deficiency in the greenhouse. Phenotyping traits included plant height, root length, weight, copper concentration in flag leaves and grains, the number of florets/per spike and kernels/spike in plants grown under copper sufficient and copper-deficient conditions. We did not appreciate how labor-intensive is the phenotyping work and so it is progressing slower than we expected. Thus, I have also recruited a PhD student and another postdoc for 6 months to facilitate project progress. Objective 5. Initiate the functional characterization of genes selected from RNA-seq and QTL studies (Year 2). We completed the functional characterization of a downstream target of SPL7, a copper transporters YSL3. The manuscript describing our findings is now under revision in Plant Physiology and is available as a preprint in bioRxiv (2019) doi:10.1101/2019.12.12.874396 Sheng H., Jiang Y., Ishka M.R., Chia J.-C., Dokuchayeva T., Kavulych Y., Zavodna T.-O., Mendoza P., Huang R., Woll A., Romanyuk N.D., Zhou Y., Vatamaniuk O.K. (2019) Inflorescence architecture, fertility and grain yield of the wheat model Brachypodium distachyon relies on copper and YSL3 transporter. Plant Physiology, in revision. This manuscript is available in as a preprint in bioRxiv (2019) doi:10.1101/2019.12.12.874396 We are currently using yeast-one-hybrid assay (Y1H) to test whether SPL7 binds to the YSL3 promoter to establish whether YSL3 is a direct or indirect target of SPL7. Using quantitative and real-time PCR analysis we found that small RNA, miR398a, b and c isoforms are regulated by a copper deficiency in SPL7-dependent manner. Using Y1H we showed that SPL7 binds to the promoter of miR398a/b and the promoter of miR398c.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Sheng H., Jiang Y., Ishka M.R., Chia J.-C., Dokuchayeva T., Kavulych Y., Zavodna T.-O., Mendoza P., Huang R., Woll A., Romanyuk N.D., Zhou Y., Vatamaniuk O.K. (2019) Inflorescence architecture, fertility and grain yield of the wheat model Brachypodium distachyon relies on copper and YSL3 transporter. Plant Physiology, in revision. This manuscript is available as a preprint in bioRxiv (2019) doi:10.1101/2019.12.12.874396
|
Progress 02/01/18 to 01/31/19
Outputs
Target Audience:Data from the first year of the project were reported at the Annual International Meeting of American Society of Agronomy (ASA) and Crop Science Society of America (CSSA) that was held in Baltimore, MD, November 4 -7, 2018. The PD presented data from the first year of the project in the talk, entitled "The role of copper in plant fertility: a journey from model plants Arabidopsis thaliana and Brachypodium distachyon to wheat" at the Session: Genomics, Molecular Genetics, and Biotechnology. Graduate student presented a poster at this meeting. Data from this project are incorporated into the upper-level undergraduate course: "Mineral Nutrition: From Plants to Humans" that is currently taught by PD (spring 2019). Undergraduate and graduate students as well as a postdoc have been trained in the lab in applying functional genomic tools (CRISPR/Cas9) to brachypodium and wheat. Undergraduate students reflect on their experiences in the video that can be accessed via the following URL: https://www.youtube.com/watch?v=EwHx77aCxu4&feature=youtu.be Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduate and graduate students, as well as a postdoc, have been trained in the lab in applying functional genomic/genetic tools to study mineral nutrient homeostasis in brachypodium and wheat. Undergraduate students reflect on their experiences in the video that can be accessed via the following URL: https://www.youtube.com/watch?v=EwHx77aCxu4&feature=youtu.be In addition, a visiting graduate student presented a poster at the Annual International Meeting of American Society of Agronomy (ASA) and Crop Science Society of America (CSSA) that was held in Baltimore, MD, November 4 -7, 2018. A postdoc, Maryam Rahmati Ishka has been trained in mentoring undergraduate students, teaching and research presentation. Specifically, Maryam taught two lectures (one on the role of minerals in photosynthesis and another on her research on the role of copper in plant fertility) in BioG1009 Refreshment Summer Program in Biology. Finally, Maryam presented results from this project at the annual symposium at the School of Integrative plant Sciences at Cornell during graduate student recruitment events. A Ph.D. student, Tetyana-Olena Zavodna participated in the outreach workshop that brought together Cornell University and Tokyo University of Agriculture and Technology graduate students and postdocs. The workshop was designed to bridge students between two institutions and engage them in the discussion with a lay audience in addressing sustainability issues and strategies. How have the results been disseminated to communities of interest?Data from the first year of the project were reported at the Annual International Meeting of American Society of Agronomy (ASA) and Crop Science Society of America (CSSA) that was held in Baltimore, MD, November 4 -7, 2018. The PD presented data from the first year of the project in the talk, entitled "The role of copper in plant fertility: a journey from model plants Arabidopsis thaliana and Brachypodium distachyon to wheat" at the Session: Genomics, Molecular Genetics, and Biotechnology. A graduate student presented a poster at this meeting. Data from this project are incorporated into the upper-level undergraduate course: "Mineral Nutrition: From Plants to Humans" that is currently taught by PD (spring 2019). A Ph.D. student, Tetyana-Olena Zavodna attended an international exchange workshop where graduate students and post-doctoral researchers from Cornell University and Tokyo University of Agriculture and Technology worked together to engage with public and address sustainability strategies. On the final day of the workshop, Tetyana-Olena presented her research to public in Ithaca at an evening event at the Science Cabaret "Can We Survive on Planet Earth? Let's Discuss! What do you plan to do during the next reporting period to accomplish the goals?Overall, we are very satisfied with the progress of the project in year 1 and we will continue our studies as outlined in project objectives. Objective 1: We will compare the growth, development, and fertility of the spl7-2 mutant vs. wild-type plants grown hydroponically with different concentrations of copper in the medium. We will compare copper concentrations in different tissues of plants using ICP-AES. Fertility will be evaluated by comparing the number of florets and grain/tiller under copper sufficient or copper limited conditions. We will compare the spatial distribution of copper in different tissues including floral organs using two-dimension synchrotron x-ray fluorescent microscopy (2D-XRF). We will also isolate cDNAs of TaSPL7-1D and express it in the spl7-2 mutant of Brachypodium. BdSPL7 promoter in the spl7 mutant. We will test whether wheat SPL7 genes will complement functionally the BdSPL7-silenced brachypodium. To ensure that the transgenes are expressed and to evaluate whether their putative transmembrane domains are sufficient for tethering these proteins to cellular membranes, we will perform a biochemical fractionation using total extracts from roots and leaves of transgenic plants. Enriched microsomal, cytosolic and nuclear fractions will be analyzed by Western blot using antibodies against HA and selected organelle markers. These studies will be complemented by analyses of the subcellular localization of proteins using fluorescent microscopy. Objective 2: We will continue genotyping SPL7 CRISPR lines in wheat and providing we obtain expected deletion lines will move forward with their characterization. Analysis of mutant lines will include the evaluation of chlorosis and other growth defects under copper-deficient vs. copper sufficient conditions, analyses of copper concentration and spatial distribution in different tissues including flowers using ICP-AES and 2D-XRF, respectively. Fertility will be evaluated by comparing the number of florets and kernels/spike in mutant vs. control (parental) plants, grown under copper sufficient or copper limited conditions. Objective 3: We will use the outcrossed spl7-2 mutant for the global transcriptome analysis and the identification of SPL7-regulated genes in different tissues of Brachypodium. We will grow plants hydroponically with copper (control) to the late vegetative stage. At the end of the tillering stage, a subset of plants will be transferred to a fresh hydroponic medium without copper to induce deficiency. Total RNA will be isolated from roots, leaves, including flag leaves, and flowers during anthesis. RNA-Seq libraries will be constructed and subjected to Illumina sequencing. The identification of differentially expressed genes and comparative co-expression network analysis between different genotypes to identify common and unique features of their transcriptome responses and gene modules regulated by SPL7 will be done via collaboration with Dr. Sun. We will continue phenotyping wheat lines grown in the field as well as in the greenhouse. We hope to move faster with phenotyping so that we will finish 200 lines as planed initially. Objective 4: We will continue phenotyping wheat lines grown in the field as well as in the greenhouse. We hope to move faster with phenotyping so that we will finish 200 lines as planed initially. Objective 5: We will continue our studies of putative SPL7 targets as we did for BdYSL3. We will select genes encoding transcription factors and transporters whose level of expression is upregulated by copper deficiency to the highest level in the RNA-seq data and those that will be within the QTL regions. Functional characterization of selected genes will include the generation of knockout lines using the CRISPR/Cas9 system. We will only generate constructs for RNA-guided genome editing and transform plants, while the generation of mutants and their characterization will go beyond the period of this proposal. We will study transporter capabilities using functional complementation in the Saccharomyces cerevisiae copper uptake mutant. We will also study the subcellular localization of proteins in the yeast mutant and by transient expression of translational GFP fusions in wheat and brachypodium protoplasts.
Impacts
What was accomplished under these goals?
Objective 1. Study the role of BdSPL7 as well as TaSPL7-1A, TaSPL7-1B and TaSPL7-1D homoeologs in copper homeostasis and fertility using Brachypodium spl7 mutant (Years 1, 2, 3; In year 1 we accomplished about 30% of the proposed work). By the start of the project, we have generated the spl7 mutants using the CRISPR/Cas 9 approach. In the year 1 we performed the following experiments: 1.1. We have sequenced the generated spl7 mutants and established that we have obtained 4 independent deletion mutants bearing 493, 494, 417 and 453 bp deletions in the first exon of the coding region of the SPL7 gene. These alleles were designated as spl7-1, spl7-2, spl7-3 and spl7-4. After establishing that 4 alleles had similar growth defects under the low Cu medium, we selected spl7-2 and spl7-4 for in depth studies. We found that the distribution and accumulation of Cu is altered in both alleles compared to the wild-type. Specifically, while roots, young and mature leaves of both spl7 alleles accumulated more Cu, flag leaves and flowers accumulated significantly less Cu than wild-type plants. This results suggested that SPL7 controls the expression of transporters required for the remobilization of Cu from leaves and delivery to flag leaves and then after to flowers. We then identified a Cas9-free spl7-2 line to minimize the interference of Cas9. We then outcrossed the spl7-2 mutant to wild-type to minimize the possibility of the presence of the off-target mutations even though we found that all 4 spl7 alleles have similar Cu hypersensitivity phenotype. After several rounds of selection of self-fertilized spl7/SPL7 plants, we have selected a homozygous hybrid line carrying the spl7-2 allele. These plants will be now used for the in-depth characterization of the role of SPL7 in Cu homeostasis as well as for the identification of its downstream direct and indirect targets. 1.2. We cloned BdSPL7 cDNA and transformed this construct into the A. thaliana spl7-1 mutant. We found that BdSPL7 can partially complement Cu-deficiency hypersensitivity of the spl7 mutant when plants were grown in soils without exogenous Cu supply or hydroponically. 1.3. We have isolated cDNAs of wheat SPL7 homoeologs, TaSPL7-1A and TaSPL7-1B. We had difficulty in the first year to isolate TaSPL7-1D because of the close similarity to TaSPL7-1B. We are currently trying to design the best strategy to isolate TaSPL7-1D. In the meantime, we already transformed TaSPL7-1A and TaSPL7-1B into the spl7-2 mutant of Brachypodium and the spl7-1 mutant of Arabidopsis. We will perform functional complementation assays in Year 2. Objective 2. Use the CRISPR/Cas9 system to knockout TaSPL7 homoeologs simultaneously and study the SPL7 pathway in wheat directly (Years 1, 2, 3; In year 1 we accomplished 30% of the work). Year 1: Using an online available tool, E-CRISP we have selected guide RNAs (gRNAs) that would target TaSPL7 homoeologs simultaneously within their conserved region, but would avoid the off-target mutations. To ensure the generation of a larger deletion, we designed gRNAs to target loci that are located from 1800 to 2122 bp apart within each gene. The gRNAs were designed to delete the DNA-binding SBP domain that would result in the loss-of-function phenotype. We have cloned single gRNAs into Module 2 and Module 3 vectors and assembled with the Ubi1-TaCas9 module in the destination vector using the Golden Gate cloning system. We have transformed this construct into wheat and have obtained 15 transgenic plants. We are currently genotyping them for the presence of the spl7 deletion. Objective 3. Identify copper deficiency-responsive and SPL7-regulated genes in wheat and Brachypodium (Years 1, 2. In year 1 we accomplished 10% of the work as justified below). Because, we prefer to use the outcrossed spl7-2 line for the whole-genome expression studies, and used year 1 for the generation of the Cas-9-free spl7-2 mutant, crossing it to the wild-type and the generation of the homozygous hybrid line carrying the spl7-2 allele, we postponed RNA-seq analysis to year 2. In the meantime, we used the results from our prior studies performed via previous Hatch-FFF funding and CRDF-Global U.S.-Ukraine award, which established that the expression of the member of the yellow stripe-like family of transporters, BdYSL3, is regulated by Cu deficiency in different tissues of Brachypodium. Here, we used qRT-PCR to compare BdYSL3 expression in roots and leaves of wild-type vs. the spl7-2 mutant of Brachypodium and found that BdYSL3 is a downstream target of BdSPL7. We next knock-out YSL3 in Brachypodium and analysed ysl3 mutant alleles as described in results to Objective 5. Objective 4. Conduct the whole-genome quantitative trait locus (QTL) mapping in wheat to identify genomic regions associated with improved copper uptake, delivery to anthers and grain yield (Years 1, 2, 3; In year 1 we accomplished 10% of the work. I note that we anticipated that the completion of this aim will likely go beyond the time-frame of this award). We have phenotyped 28 lines of the SynOp DH population including their parental lines, Opata M85, and W7984 grown in the field. Phenotyping traits included a copper concentration in flag leaves and grains, the number of florets/per spike and kernels/spike in plants grown under copper sufficient and copper-deficient conditions. We have also evaluated grain features that included gains length, width, and curvature. We did not appreciate how labor-intensive is the phenotyping work and so it is progressing slower than we expected. Objective 5. Initiate functional characterization of genes selected from RNA-seq and QTL studies (Years 1, 2, 3; In year 1 we accomplished 10% of the work; I note that we anticipated that the completion of this aim will go beyond the time-frame of this award). We found that BdYSL3 is the SPL7-regulated gene (see the results to Objective 3). We then generated ysl3 mutant in brachypodium using the CRISPR-Cas9 approach, characterized and have prepared data for the publication. Abstract from the draft of the manuscript: Sheng, H, et al & Vatamaniuk, O.K. A copper transporter, YSL3, is required for fertility and grain yield in Brachypodium distachyon. In preparation for the submission to Plant Physiology - The first draft is written. Copper deficiency affects fertility and grain yield in plants. How copper is absorbed from the soil into roots and delivered to reproductive organs, and how it affects fertility and grain yield in grasses that include the agronomically important crop wheat, is poorly understood. Here, we used Brachypodium distachyon as a wheat proxy to explore the molecular mechanisms of copper transport processes and their relationship to Brachypodium fertility and grain set. We selected a gene encoding a yellow stripe-like transporter, BdYSL3 because its homologs in A. thaliana and rice have been shown to function of copper transport processes. Here we show that BdYSL3 is highly expressed in young, mature and flag leaves, and is transcriptionally up-regulated in most of the examined tissues under copper deficiency. We also show that BdYSL3 is expressed in the phloem region of vascular bundles as well as in ovaries and stigmas of flowers. Loss of BdYSL3 function significantly increased the sensitivity of Brachypodium to copper deficiency. ICP-MS and synchrotron x-ray fluorescence (SXRF) microscopy data showed that the ysl3 knockout mutant fails to deliver copper from mature leaves to sink tissues including young leaves, flag leaves, ovaries, and anthers. Because of altered copper transport, the ysl3 mutant manifested delayed flowering time and increased flower production. However, the mutant showed the significant reduction in fertility and grain yield. Collectively, our data show that BdYSL3 is essential for copper transport processes that are fundamental for plant fertility and grain production.
Publications
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