Progress 10/01/10 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two post doctoral fellows and four undergraduate students (3 female and 1 male) worked on this project. All personnel were trained by the PI to conduct experiments. The post doctoral fellows were in charge of characterizing the functions of LORELEI in double fertilization described in this progress report. The undergraduate students assisted the post doctoral fellow in the experiments described in this progress report. The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to perform automated analysis of pollen tube behavior changes. The PI collaborated with Dr. Yitshak Zohar and an undergraduate student in his lab developed the microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. How have the results been disseminated to communities of interest? 1. Besides peer-reviewed publications, Dr. Palanivelu presented 18 research seminars in multiple research conferences and universities. His lab also presented 5 posters in conferences and meetings. 2. The Palanivelu lab was involved in many outreach events throughout the funding period. A. Science Demonstrations. Conducted interactive demonstrations on pollen in Science City 2012 and 2013 in the University of Arizona, Mesquite Elementary School (Tucson, AZ), Curry Farms (Pearce, AZ), Plant Sciences Family Night (Ventana Elementary School, Tucson, AZ), and FunFest Tucson Convention Center). The activities created awareness and imparted knowledge on various aspects of pollen biology and the economic importance of pollen. B. Science Fair Judge. Judged science fair projects in the Mesquite Elementary School and Southern Arizona Regional Science Fair (SARSEF), Spring 2012 and 2013. 3. Integration of science and computing summer camp 2012: The summer camp was developed by the Computer Vision Group (led by Dr. Kobus Barnard) and executed with the help of the UA SISTA Department, and in collaboration with several science labs on campus. The camp was sponsored by NSF grant IIS-0747511, "CAREER: Learning models for object structure." The Palanivelu lab was one of the labs that participated in this event. As part of this event, six middle school students visited the Palanivelu lab. At that time, Dr. Palanivelu gave an interactive lecture on plant pollination during which the students saw how technology is used to aide scientists. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre-5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre-5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre-5/lre-5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre-5/lre-5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre-5/lre-5 seeds. The lre-5/lre-5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI-like-GPI-anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre-5/lre-5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development. 2. Existing pollen tube guidance bioassays are performed in an isotropically diffusive environment instead of anisotropically diffusive conditions required to characterize guidance signal gradients. Lack of a sensitive pollen tube guidance bioassay has therefore compounded the difficulties of identifying and characterizing the guidance signals that are likely produced in minute quantities by the ovules. We therefore developed a novel microsystem-based assay that mimics the in vivo micro-environment of ovule fertilization by pollen tubes in the model research plant Arabidopsis thaliana. In this microdevice, the pollen tube growth rate, length and ovule targeting frequencies were similar to those obtained using a semi in vivo plate assay. As a direct measure of the microdevice's utility in monitoring pollen tube guidance, we demonstrated that in this device, pollen tubes preferentially enter chambers with unfertilized ovules, suggesting that the pollen tubes sense the concentration gradient and respond to the chemoattractants secreted by unfertilized ovules. 3. In the PI's lab, a mutant (lorelei, lre) defective in pollen tube-ovule interactions (including pollen tube repulsion) was isolated, the LORELEI gene was cloned and a detailed characterization of lre developmental phenotypes was performed. Based on our observations that lre synergid cells fail to induce the arrest of pollen tube growth, we hypothesized that LRE localizes to the synergid cell to induce the arrest of the pollen tube growth. To determine whether LRE is expressed in the synergid cells, we generated and analyzed Arabidopsis plants containing LREpromoter:GFP (pLRE:GFP) reporter constructs. In the terminal developmental stage ovule (stage FG7), the pLRE:GFP is primarily expressed in the synergid cells. Besides the synergid cells, we detected a weak expression in the egg cell. There was no expression in the central cell; only background level signal, similar to that in non-transformed wild-type ovules, was observed in central cell. We also examined pLRE:GFP expression during female gametophyte development and observed that pLRE:GFP is expressed in the synergid cells, starting from stage FG6, a stage by which a synergid cell is already specified and differentiated. To identify additional genes that function with LRE, we performed an enhancer/suppressor mutant screen and two extragenic suppressors of the lre pollen tube overgrowth phenotype were isolated. 4. Based on sequence analysis, we hypothesized that is a membrane protein and that it localizes to the apoplast of the synergid cell and is attached to the synergid cell membrane via a GPI-anchor. To test the first part of this hypothesis, we generated translational fusion constructs by inserting a citrine YFP reporter either in the N-terminus or C-terminus. Citrine YFP was used as it can function in a wide range of pHs including the acidic environment of an apoplast. We used overlap PCR technique to insert citrine YFP in the N-terminus right after the putative secretion signal sequence or right in front of the GPI-anchor attachment site in the C-terminus. These constructs were verified for correct sequence and then they were transformed into wild type and lre-5/lre-5 plants. We showed that both LREcYFP and cYFPLRE construct can complement lre7 phenotype. Although cYFPLRE is able to complement lre phenotype, we did not see YFP in cYFPLRE transgenic plants. LREcYFP protein is highly accumulated at the filiform apparatus in synergid cells and there're also YFP speckles distributed in synergid cell cytoplasm. The nature of these speckles are unknown. We found that there's no obvious change on LREcYFP localization upon pollen tube arrival.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Palanivelu, R., and Tsukamoto, T. (2012). Pathfinding in angiosperm reproduction: pollen tube guidance by pistils ensures successful double fertilization. WIREs Dev Biol. 1:96-113.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Palanivelu, R. (2011). Targeted growth of pollen tubes to ovules prior to completing fertilization. Molecular Reproduction and Development. 78 (12): 893.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Qin, Y., Wysocki, R.Y., Somogyi, A., Feinstein, Y., Franco, J.Y., Tsukamoto, T., Dunatunga, D., Levy, C., Smith, S., Simpson, R., Gang, D., Johnson, M.A., and Palanivelu, R. (2011). Sulfinylated Azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils. Plant Journal. 68 (5): 800-815.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Brau, E., Barnard, K., Palanivelu, R., Dunatunga, D., Tsukamoto, T., and Lee, P. (2011). A generative statistical model for tracking multiple smooth tragectories. Proceedings of the IEEE Computer Vision and Pattern Recognition. 1137-1144. doi:10.1109/CVPR.2011.5995736.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Yetisen, A.K., Jiang, L., Cooper, J.R., Qin, Y., Palanivelu, R*., and Zohar, Y*. (2011). A microsystem-based assay for studying pollen tube guidance in plant reproduction. Journal of Micromechanics and Microengineering. 21 (5): 054018.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Tsukamoto, T., and Palanivelu, R. (2010). Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana. Plant Signal Behav. 5(11): 1487-1490.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Tsukamoto, T., Qin Y., Huang, Y., Dunatunga, D. and Palanivelu, R. (2010). A role for LORELEI, a putative glycosylphosphatidylinositol-anchored protein, in Arabidopsis thaliana double fertilization and early seed development. Plant J. 62 (4) 571-588.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Palanivelu, R., and Johnson, M.A. (2010). Functional genomics of pollen tube-pistil interactions in Arabidopsis. Biochem. Soc. Trans. 38 (2) 593-597.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Oral Prsentations: 1. School of Information: Science, Technology, and Arts (SISTA) colloquium series, University of Arizona (February 2012). Title: Technology to the rescue:understanding courtship in plants. 2. Center for Cellular and Molecular Biology (CCMB), Hyderabad, India. (July 2012). Title: Molecular mechanisms underlying pollen-pistil interactions in Arabidopsis. 3. Tamil Nadu Agriculture University, Coimbatore, India (August 2012). Title: Molecular mechanisms underlying pollen-pistil interactions in Arabidopsis. 4. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Shanghai, China (August 2012). Title: Molecular mechanisms underlying pollen-pistil interactions in Arabidopsis. 5. International Symposium on the Mechanisms of Sexual Reproduction in Animals and Plants, Nagoya, Japan (November 2012). Title: Regulation of pollen tube guidance by the Arabidopsis female gametophyte. Collaborations: 1. As part of this project, the PI collaborated with Dr. Kobus Barnard, Computer science department, University of Arizona, to develop algorithms to analyze pollen tube behaviors. 2. As part of this project, the PI collaborated with Dr. Yitshak Zohar at the Aeronautical and Mechanical Engineering department at the University of Arizona to develop a microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. PARTICIPANTS: A post doctoral fellow (Xunliang Liu) and two undergraduate students were hired to work on several aspects of this project. All three personnel were trained by the PI to conduct experiments. The post doctoral fellow performed subcellular localization of LORELEI analysis. The undergraduate students assisted the post doctoral fellow in the experiments described in this progress report. The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to perform automated analysis of pollen tube behavior changes. The PI collaborated with Dr. Yitshak Zohar and an undergraduate student in his lab developed the microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the PI's lab, a mutant (lorelei, lre) defective in pollen tube-ovule interactions (including pollen tube repulsion) was isolated, the LORELEI gene was cloned and a detailed characterization of lre developmental phenotypes was performed. Based on our observations that lre synergid cells fail to induce the arrest of pollen tube growth, we hypothesized that LRE localizes to the synergid cell to induce the arrest of the pollen tube growth. To determine whether LRE is expressed in the synergid cells, we generated and analyzed Arabidopsis plants containing LREpromoter:GFP (pLRE:GFP) reporter constructs. In the terminal developmental stage ovule (stage FG7), the pLRE:GFP is primarily expressed in the synergid cells. Based on sequence analysis, we hypothesized that is a membrane protein and that it localizes to the apoplast of the synergid cell and is attached to the synergid cell membrane via a GPI-anchor. To test the first part of this hypothesis, we generated translational fusion constructs by inserting a citrine YFP reporter either in the N-terminus or C-terminus. citrine YFP was used as it can function in a wide range of pHs including the acidic environment of an apoplast. We used overlap PCR technique to insert citrine YFP in the N-terminus right after the putative secretion signal sequence or right in front of the GPI-anchor attachment site in the C-terminus. These constructs were verified for correct sequence and then they were transformed into wild type and lre-5/lre-5 plants. To examine if LRE functioned downstream of a LRR-RLK receptor (FERONIA) that is involved in inducing pollen tube growth arrest, we transformed the cYFP-LRE or LRE-cYFP constructs into fer/+ mutant plants. Selection of transformants and subcellular localization analysis of LRE-cYFP is underway.
Publications
- Palanivelu, R., and Tsukamoto, T. (2012). Pathfinding in angiosperm reproduction: pollen tube guidance by pistils ensures successful double fertilization. WIREs Dev Biol. 1:96-113.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Oral Presentations: 1. Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City, Missouri. Molecular mechanisms underlying pollen-pistil interactions in Arabidopsis. 2. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island. Molecular mechanisms underlying pollen-pistil interactions in Arabidopsis. 3. Plant Biology Graduate Program, University of Massachusetts, Amherst, Massachusetts. Molecular mechanisms underlying pollen-pistil interactions in Arabidopsis. 4. School of Plant Sciences, University of Arizona, Tucson, Arizona. Collaborations: 1. As part of this project, the PI collaborated with Dr. Kobus Barnard, Computer science department, University of Arizona, to develop algorithms to analyze pollen tube behaviors. 2. As part of this project, the PI collaborated with Dr. Yitshak Zohar at the Aeronautical and Mechanical Engineering department at the University of Arizona to develop a microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. PARTICIPANTS: A post doctoral fellow and two undergraduate students were hired to work on several aspects of this project. All three personnel were trained by the PI to conduct experiments. The post doctoral fellow performed LORELEI expression analysis and the suppressor screen. The undergraduate students assisted the post doctoral fellow in the experiments described in this progress report. The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to perform automated analysis of pollen tube behavior changes. The PI collaborated with Dr. Yitshak Zohar and an undergraduate student in his lab developed the microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. In the PI's lab, a mutant (lorelei, lre) defective in pollen tube-ovule interactions (including pollen tube repulsion) was isolated, the LORELEI gene was cloned and a detailed characterization of lre developmental phenotypes was performed. Based on our observations that lre synergid cells fail to induce the arrest of pollen tube growth, we hypothesized that LRE localizes to the synergid cell to induce the arrest of the pollen tube growth. To determine whether LRE is expressed in the synergid cells, we generated and analyzed Arabidopsis plants containing LREpromoter:GFP (pLRE:GFP) reporter constructs. In the terminal developmental stage ovule (stage FG7), the pLRE:GFP is primarily expressed in the synergid cells. Besides the synergid cells, we detected a weak expression in the egg cell. There was no expression in the central cell; only background level signal, similar to that in non-transformed wild-type ovules, was observed in central cell. We also examined pLRE:GFP expression during female gametophyte development and observed that pLRE:GFP is expressed in the synergid cells, starting from stage FG6, a stage by which a synergid cell is already specified and differentiated. To identify additional genes that function with LRE, we performed an enhancer/suppressor mutant screen and two extragenic suppressors of the lre pollen tube overgrowth phenotype were isolated. 2. We developed a novel microsystem-based assay to assess and quantify pollen tube behavior in response to pistil tissues. Existing pollen tube guidance bioassays are performed in an isotropically diffusive environment (for example, a semi in vivo assay in petri dishes) instead of anisotropically diffusive conditions required to characterize guidance signal gradients. Lack of a sensitive pollen tube guidance bioassay has therefore compounded the difficulties of identifying and characterizing the guidance signals that are likely produced in minute quantities by the ovules. We therefore developed a novel microsystem-based assay that mimics the in vivo micro-environment of ovule fertilization by pollen tubes in the model research plant Arabidopsis thaliana. In this microdevice, the pollen tube growth rate, length and ovule targeting frequencies were similar to those obtained using a semi in vivo plate assay. As a direct measure of the microdevice's utility in monitoring pollen tube guidance, we demonstrated that in this device, pollen tubes preferentially enter chambers with unfertilized ovules, suggesting that the pollen tubes sense the concentration gradient and respond to the chemoattractants secreted by unfertilized ovules.
Publications
- 1. Palanivelu, R. (2011). Targeted growth of pollen tubes to ovules prior to completing fertilization. Molecular Reproduction and Development. 78 (12): 893. Link to the article or download the pdf file.
- 2. Qin, Y., Wysocki, R.Y., Somogyi, A., Feinstein, Y., Franco, J.Y., Tsukamoto, T., Dunatunga, D., Levy, C., Smith, S., Simpson, R., Gang, D., Johnson, M.A., and Palanivelu, R. (2011). Sulfinylated Azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils. Plant Journal. 68 (5): 800-815. Link to the article or the PubMed citation.
- 3. Brau, E., Barnard, K., Palanivelu, R., Dunatunga, D., Tsukamoto, T., and Lee, P. (2011). A generative statistical model for tracking multiple smooth tragectories. Proceedings of the IEEE Computer Vision and Pattern Recognition. 1137-1144. doi:10.1109/CVPR.2011.5995736. Download the pdf file.
- 4. Yetisen, A.K., Jiang, L., Cooper, J.R., Qin, Y., Palanivelu, R*., and Zohar, Y*. (2011). A microsystem-based assay for studying pollen tube guidance in plant reproduction. Journal of Micromechanics and Microengineering. 21 (5): 054018. Link to the article or download the pdf file. (*co-corresponding authors).
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Poster presentations: 1. American Society of Plant Biologists (ASPB) meeting in Montreal, Canada. 2. XXI International Congress on Sexual Plant Reproduction, Bristol, Great Britain. This poster was selected for an oral presentation and the first author of this poster, Dr. Tatsuya Tsukamoto, a post doctoral fellow in the PI lab, gave an oral presentation at the meeting. Oral Presentations: 1. Pima Community College, North West Campus. 2. Pollen Biology Major Symposium, American Society of Plant Biologists annual conference, Montreal, Canada. 3. Southwest Regional Society of Developmental Biology meeting, University of Texas, Austin, Texas 4. Department of Cell and Systems Biology, University of Toronto, Canada Collaborations: 1. As part of this project, the PI collaborated with Dr. Kobus Barnard, Computer science department, University of Arizona, to develop algorithms to analyze pollen tube behaviors. 2. As part of this project, the PI collaborated with Dr. Yitshak Zohar at the Aeronautical and Mechanical Engineering department at the University of Arizona to develop a microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. PARTICIPANTS: A post doctoral fellow and two undergraduate students were hired to work on several aspects of this project. All three personnel were trained by the PI to conduct experiments. The post doctoral fellow was in charge of characterizing the functions of LORELEI in double fertilization described in this progress report. The undergraduate students assisted the post doctoral fellow in the experiments described in this progress report. The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to perform automated analysis of pollen tube behavior changes. The PI collaborated with Dr. Yitshak Zohar and an undergraduate student in his lab developed the microfluidic chip that is capable of generating microgradients of pollen tube attractants or repellents. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre-5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre-5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre-5/lre-5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre-5/lre-5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre-5/lre-5 seeds. The lre-5/lre-5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI-like-GPI-anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre-5/lre-5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development. 2. Existing pollen tube guidance bioassays are performed in an isotropically diffusive environment instead of anisotropically diffusive conditions required to characterize guidance signal gradients. Lack of a sensitive pollen tube guidance bioassay has therefore compounded the difficulties of identifying and characterizing the guidance signals that are likely produced in minute quantities by the ovules. We therefore developed a novel microsystem-based assay that mimics the in vivo micro-environment of ovule fertilization by pollen tubes in the model research plant Arabidopsis thaliana. In this microdevice, the pollen tube growth rate, length and ovule targeting frequencies were similar to those obtained using a semi in vivo plate assay. As a direct measure of the microdevice's utility in monitoring pollen tube guidance, we demonstrated that in this device, pollen tubes preferentially enter chambers with unfertilized ovules, suggesting that the pollen tubes sense the concentration gradient and respond to the chemoattractants secreted by unfertilized ovules.
Publications
- 1. Tsukamoto, T., and Palanivelu, R. (2010). Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana. Plant Signal Behav. 5(11): 1487-1490.
- 2. Tsukamoto, T., Qin Y., Huang, Y., Dunatunga, D. and Palanivelu, R. (2010). A role for LORELEI, a putative glycosylphosphatidylinositol-anchored protein, in Arabidopsis thaliana double fertilization and early seed development. Plant J. 62 (4) 571-588.
- 3. Palanivelu, R., and Johnson, M.A. (2010). Functional genomics of pollen tube-pistil interactions in Arabidopsis. Biochem. Soc. Trans. 38 (2) 593-597.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Results obtained during the reporting period were presented as posters at the Western section ASPB meeting in Tucson, Arizona. Besides, the work was also presented in the annual student recruitment week poster session in the school of Plant Sciences at the University of Arizona. During this reporting period, results from this work were included by the PI during his seminar presentations in the following places: Departement de Sciences Biologiques Universite de Montreal, Montreal, QC, Canada, November 2009; Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, NS, Canada; Cell-cell communication in plant reproduction University of Bath, Bath, UK; Department of Botany and Plant Sciences, University of California, Riverside, Riverside, California; Molecular Reproduction conference, Brown University Providence, Rhode Island. As part of this project the PI collaborated with Dr. Kobus Barnard, Computer science department, University to develop algorithms to analyze pollen tube behaviors. PARTICIPANTS: o achieve the goals described in this project we undertook several line of activities. Besides a research technician, a post doc and an undergraduate student were hired to work on several aspects of this project. All three personnel were trained by the PI to conduct experiments. Research technician and post doc were in charge of cloning LORELEI gene and performed all the mapping experiments described in this progress report. The undergraduate student assisted senior personnel in the mapping experiments and for characterizing additional alleles in this gene. The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to peform automated analysis of pollen tube behavior changes. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The goal of our submitted proposal is to characterize pollen tube repulsion in Arabidopsis. We have made significant advances in two of the three aims outlined in submitted proposal. These advances will facilitate successful completion of the proposed experiments. To identify the gene that is disrupted in ref1 mutant, we utilized a TAIL-PCR procedure to identify the T-DNA insertion site. Sequencing the TAIL-PCR products revealed that ref1 is a novel allele in a newly annotated gene At4g26466 (LORELEI). We therefore renamed this novel allele of LORELEI gene as lre-5. We performed RT-PCR analysis to examine LORELEI expression during reproduction. LORELEI is not expressed in pollen, pollen tubes or in the stigma and style portions of pistils, consistent with its female gametophyte-specific function. Therefore, we next monitored LORELEI expression during previously described stages of ovule development. LORELEI transcription was not detected in ovaries collected from stage11 or 12a floral buds. In ovaries from stage12b buds [stage5 female gametophyte]. LORELEI is expressed at low levels (only after 50 cycles) indicating that LORELEI expression initiates before female gametophyte reaches stage7. In ovaries from stage12c and mature female gametophyte-containing stage14 flowers, LORELEI expression was readily detectable. These results coupled with LORELEI expression in unfertilized, mature ovules indicate that LORELEI expression is pollination-independent. Consistent with its function during early seed development, LORELEI is expressed in pollinated ovules (16 and 24HAP). However, LORELEI expression in 16 and 24HAP ovules was lower compared to ovules in unfertilized or pistils three HAP. Consistent with these results, qRT-PCR experiments showed a ~22-fold decrease in LORELEI expression in ovules 16HAP compared to unfertilized ovules. The dramatic decrease in LORELEI expression in pollinated ovules does not appear to be part of a global down regulation of pollen tube reception genes as expression of FERONIA and AMC did not decrease to same extent as LORELEI. LORELEI transcripts could not be detected by 36 and 48HAP, indicating that LORELEI is likely not expressed during seed development starting from the embryo stage. Microdevices were developed to resemble the in-vivo micro-environment of ovule fertilization by pollen tubes in Arabidopsis thaliana. The PDMS-based microdevices were filled with pollen germination medium (PGM) providing pollen tubes with a proper growth environment. Pollen tubes were found to grow within the microgrooves at an average rate of 130μm/hr, reaching a final tube length of about 450μm. Targeting ovules by pollen tubes were also tested with an observed efficiency of about 67%. Both the pollen tube growth rate and the ovule targeting efficiency in microdevices are similar to those obtained using in-vitro plate experiments. Finally, preliminary results indicate that pollen tube bundles preferentially turn toward ovule containing chambers, suggesting that the pollen tubes respond to the attractants secreted by unfertilized ovules.
Publications
- Cooper, J.R., Qin, Y. Jiang, L., Palanivelu, R. & Zohar, Y. (2009). Microsystem-based study of pollen-tube attractants secreted by ovules. Proc. 22nd International Micro Electro Mechanical Systems Conference, pp. 208-211.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Results obtained during the reporting period were presented as posters at the Frontiers of Sexual Plant Reproduction III conference in Tucson, Arizona. Besides, the work was also presented in the annual student recruitment week poster session (sponsored by BIO5) at the University of Arizona campus. During this reporting period, results from this work were included by the PI during his seminar presentations in the following places: Plant Biology division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma; Department of Biology, University of Pennsylvania, Philadelphia; Department of Biology, Washington University, St. Loius, Missouri; Department of Biology, University of Texas, Austin, Texas; Department of Plant Biology, University of California, Davis, Davis, California. As part of this project the PI collaborated with Dr. Kobus Barnard, Computer science department, University to develop algorithms to analyze pollen tube behaviors. PARTICIPANTS: To achieve the goals described in this project we undertook several line of activities. Besides a research technician, a post doc and an undergraduate student were hired to work on several aspects of this project. All three personnel were trained by the PI to conduct experiments. Research technician and post doc were in charge of cloning REF1 gene and performed all the mapping experiments described in this progress report. The undergraduate student assisted senior personnel in the mapping experiments and for characterizing additional alleles in this gene. The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to peform automated analysis of pollen tube behavior changes. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The goal of our submitted proposal is to characterize pollen tube repulsion in Arabidopsis. We have made significant advances in two of the three aims outlined in submitted proposal. These advances will facilitate successful completion of the proposed experiments. Experiments proposed in aims 1a-d are aimed at performing a thorough characterization of pollen tube repulsion in Arabidopsis and obtain crucial preliminary information such as identifying the tissues and the timing necessary for isolation of pollen tube repulsion signals. To complete these goals, it is essential to use an image analysis system that will quantify pollen tube repulsion in an automated and high-throughput manner. Therefore, we gave priority to developing the algorithm prior to performing experiments described in aims 1a-d. Dr. Kobus Barnard lab established a system to convert image data collected in PI Palanivelu's lab to a more standard image format. Then they developed a baseline method for simultaneously tracking multiple pollen tubes growing in vitro. This method has achieved reasonable accuracy on a modest sized image test set and will serve as the baseline to compare other tracking methods. Barnard lab also began developing another approach to track the tubes that is expected to be more robust in face of more difficult data. In the past year we mapped the ref1 mutation first to a span of about 180 genes. This effort was undertaken in conjunction with efforts described below to directly identify the location of the mutation caused by T-DNA using TAIL-PCR techniques. The ref1 mutant was obtained from the SALK T-DNA collection. The kanamycin gene on the T-DNA was absolutely linked with the ref1 phenotype. We designed primers that scanned the entire LB and RB of T-DNA and further away from the edges of the T-DNA. When these new RB primers were used in the TAIL-PCR reactions with previously published AD primers, three of the newly designed primers amplified good ladders and every single one of them identified the same flanking genomic sequence (1st exon of At4g26466). As we anticipated, sequencing the TAIL-PCR product revealed that there was 200bp deletion of the RB sequence. Subsequently, we also identified that the LB border on the other end of the T-DNA flanks the genomic sequence in the same gene (At4g26466), suggesting this T-DNA disrupted only At4g26466 gene. Additional proof that the REF1 gene is At4g26466 was obtained using two different experiments. First, RT-PCR experiments with pistil cDNAs obtained from wild type and ref1 mutants showed that no full length mRNA of At4g26466 was made in ref1 mutants; although genes that flank REF1 namely At4g26455 and At26460 were expressed normally (data no shown). Second, another 4 alleles obtained from the SAIL collection in this gene also showed ref1-like phenotypes. However, many other SAIL insertions in the vicinity of REF1 gene, but not in the REF1 gene, did not show ref1-like phenotypes. At4g26466 is a gene that has not been characterized so far and it is similar to other plant GPI-anchored membrane proteins. Future work will focus on characterizing this gene.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Results obtained during the reporting period were presented as posters at the American society of Plant Biology annual meeting in Chicago. Besides, the work was also presented in the annual student recruitment week poster session (sponsored by BIO5) at the University of Arizona campus. During this reporting period, results from this work were included by the PI during his seminar presentations in the following places around the world: Department of Chemistry, Nagoya University, Nagoya, Japan. Department of Biology, Colorado State University, Fort Collins, CO. Interdisciplinary Plant Group, University of Missouri-Columbia. Columbia, MO. Center for Cellular and Molecular Biology. Hyderabad, India. State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University. Beijing, China. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Beijing, China. College of Life Sciences, Wuhan University. Wuhan,
China. Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona. Department of Biological Sciences, Texas Tech University. Lubbock, Texas. As part of this project the PI collaborated with Dr. Kobus Barnard, Computer science department, University to develop algorithms to analyze pollen tube behaviors.
PARTICIPANTS: To achieve the goals described in this project we undertook several line of activities. Besides a research technician, two undergraduate students were hired to work on several aspects of this project. All three personnel were trained by the PI to conduct experiments. Research technician (Yiding Huang) is in charge of cloning REF1 gene and performed all the mapping experiments described in this progress report. Two undergraduate students, Jamie Jackson and Patrick Vinck assisted Yiding Huang in the mapping experiments and performed phenotypic characterizations of ref1 mutants (mapping population). The PI collaborated with Dr. Kobus Barnard and a graduate student in his lab performed the algorithm development to peform automated analysis of pollen tube behavior changes.
PROJECT MODIFICATIONS: Although in the original proposal submitted in 2006, we proposed to investigate the role of a candidate repellent nitric oxide (NO) accumulation and release from ovules in pollen tube repulsion. Subsequently, we identified ref1 mutant that is defective in pollen tube repulsion. Since we did not have preliminary data establishing a role for nitric oxide in pollen tube repulsion and with the identification of the mutant, we will therefore not examine the role of nitric oxide in pollen tube repulsion any longer. Instead, we will capitalize on an A. thaliana mutant, defective in pollen tube repulsion. We will incorporate this mutant into our experimental plan and use it to characterize pollen tube repulsion signaling (aim 2). In addition, the proposed experiments in aim 3 have been modified to use this mutant to characterize the properties of the pollen tube repulsion activity. Identification and charcterization of the ref1 mutation is proceeding rapidly.
Impacts
The goal of our submitted proposal is to characterize pollen tube repulsion in Arabidopsis. We have made significant advances in two of the three aims outlined in submitted proposal. These advances will facilitate successful completion of the proposed experiments. 1. Experiments proposed in aims 1a-d are aimed at performing a thorough characterization of pollen tube repulsion in Arabidopsis and obtain crucial preliminary information such as identifying the tissues and the timing necessary for isolation of pollen tube repulsion signals. To complete these goals, it is essential to use an image analysis system that will quantify pollen tube repulsion in an automated and high-throughput manner. Therefore, we gave priority to developing the algorithm prior to performing experiments described in aims 1a-d. As described in last progress report, we first developed an improved methodology to track multiple tubes simultaneously. Using this tracking feature, we next tested if our
algorthim could i) identify pollen tube repulsion near an ovule micropyle and ii) be specific by discerning pollen tube repulsion failures. For this analysis, we employed images of GFP-tagged pollen tube behavior proximal to a wild type or ref1 ovule. As described in previous progress report, ref1 mutant ovules are deficient in repelling tubes that continue to approach them even after penetrated by a pollen tube. As shown in Figure 1, we were able to exploit our representation of multiple tubes to identify pollen tube repulsion near an ovule micropyle. In addition, we were able to distinguish wild-type and mutant behaviors. Specifically, we analyzed the location within an ovule where the first tube terminated is growth. In the wild type, no other tubes terminated nearby, whereas in the mutant example, several others terminated in the vicinity, which is indicative of multiple fertilization attempts. 2. At the time of last progress report submission, we reported isolation of ref1
mutant. To understand the molecular mechanisms that regulate pollen tube repulsion, we propose to clone the REF1 gene. Since ref1 mutant was identified from a forward genetics screen involving SALK T-DNA mutant collection, we first performed TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction). For TAIL-PCR experiments, we used mutant plants in the F1 population generated from a ref1 homozygote mutant back crossed to wild type. However, we failed to generate any junction fragments flanking the right and left T-DNA borders despite employing 8 different arbitrary degenerate (AD) primers and a variety of TAIL-PCR conditions. Given that TAIL-PCR is not always successful and it is not uncommon that alterations unlinked to T-DNA cause mutant phenotypes, we have initiated a traditional map-based cloning procedure to identify the REF1 gene. For this purpose, we have crossed ref1 homozygotes (Columbia strain) to wild type (Landsberg strain) and established the F2 mapping
population. Using these plants and PCR-based markers, we are in the process of obtaining rough map position of the ref1 mutation.
Publications
- Geitmann, A., and Palanivelu, R. (2007). Fertilization Requires Communication: Signal Generation and Perception During Pollen Tube Guidance, Floriculture and Ornamental Biotechnology 1(2): 77-89.
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Progress 07/01/06 to 12/31/06
Outputs
The goal of our submitted proposal is to characterize pollen tube repulsion signals. We have made excellent progress in 3 of the aims described in the proposal. Progress in aim2: Subsequent to our HATCH proposal submission in July 2006, we identified an Arabidopsis thaliana mutant (ref1) defective in pollen tube repulsion. This unexpected, yet significant finding, will provide an entry point into understanding the molecular mechanisms that regulate pollen tube repulsion. Therefore, we incorporated this mutant into our experimental plan described in the proposal to isolate and characterize pollen tube repulsion signals. We utilized our in vitro pollen tube guidance assay to characterize the pollen tube growth and guidance defects in ref1. These experiments showed that ref1 targeted ovules were markedly defective in repelling additional tubes that approach them (5 out of 12 such instances, 42%; n= 20 assays). This percentage is significantly different from the behavior
of wild type ovules in the in vitro assay; in every instance, wild type targeted ovule repelled additional tubes that approached them (0 out of 44, 0%; n= 20 assays). Lack of repulsion in ref1 may point to decreased production of repellants upon ovule targeting. Therefore, we will include ref1 ovules in experiments described in specific aims 1 and 3 of our proposal-to isolate and characterize pollen tube repulsion signals. Priority will be given to collecting targeted ref1 ovules and exudates for GC-MS experiments as described in aim 3 of our proposal. Signals that show marked change in GC-MS profiles in ref1 mutant will be analyzed first. Progress in aim 1d: At the time of the proposal we had just begun analyzing repulsion in in vitro guidance assays with A. thaliana pollen tubes and ovules from A. arenosa and Olimarabidopsis pumila, close relatives of A. thaliana. We performed additional experiments and our new results show that even if targeted by A. thaliana pollen tubes, A.
arenosa (25 instances, n=20 assays) and Olimarabidopsis pumila ovules (10 instances, n=20 assays) repel additional tubes that approach them. These results suggest that pollen tube repulsion is mediated by a signaling mechanism that does not evolve rapidly. Progress in aim 1a-c: To facilitate high-throughput analysis of results from experiments described in aim1a-c, we have entered into collaborations with Dr. Kobus Barnard, an expert in computational analysis of images to develop algorithms for automating image analysis and quantifying pollen tube behavior. Towards this end, we developed algorithms to compute pollen tube bending rate, and velocity of growth towards an ovule. Pollen tube velocity measurements show that pollen tubes slow down as they approach the ovules; perhaps, in response to guidance cues from an ovule and these results are consistent with manual measurements of pollen tube growth rate. Ultimately, the algorithm will be indispensable in screening for pollen tube
repulsion mutants. For this, we will use images with ref1 mutant as an additional tool to verify if the algorithm can identify those ovules in which pollen tube repulsion breaks down.
Impacts
The proposed work is an excellent fit with University of Arizona's inter-disciplinary strengths and strategies, specifically in applying state of the art computational ideas and biochemical expertise to biological problems. Our in vitro system to study pollen tube repulsion is unique and therefore we have a high chance of attracting new research talent and retaining them in this biosciences strategic priority area. This study will promote crop improvement by helping us understand how plants regulate spurious fertilization events and help engineer genetically modified pollen that cannot fertilize native species-impediment breeders have to overcome while developing transgenic crop varieties and an area of concern to public and regulatory agencies.
Publications
- No publications reported this period
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