Progress 09/15/01 to 09/14/04
Outputs
Cell division is a fundamental process for growth, development, and reproduction in flowering plants. The division of the cytoplasm, cytokinesis, in plants is mechanistically different from that in animals and fungi. The phragmoplast is an elaborate structure that executes cytokinesis in flowering plants. At the last stage of cell division, organized microtubule array in the phragmoplast allows unidirectional transport of Golgi-derived vesicles towards the division site to give rise to the cell plate, the physical divider of the cytoplasm. Kinesin-related proteins (kinesins) are microtubule-based motors that play essential roles in both microtubule organization and vesicle transport. We have discovered two classes of kinesins that specifically act in the phragmoplast in the model plant Arabidopsis thaliana. We have taken biochemical, cytological, and genetic approaches to dissect how these kinesins regulate the progression of cytokinesis in the phragmoplast. We have
continued to analyze kinesins that are involved in organizing the phragmoplast microtubule array. We have discovered that AtPAKRP1 and its close homolog AtPAKRP1L were exclusively localized to the microtubule plus ends at the division site. Our results indicated that these two closed related proteins were able to form homodimers and/or heterodimers in vitro. By taking a mutant analysis approach, we have also learned that either one is dispensable for normal growth and development. When both proteins were absent, however, seed formation dropped by approximately 50%. We have further proved that the failure of seed production was due to the formation of abnormal pollen grains. The wild-type mature pollen grains contain two sperm cells floating in the pollen cytoplasm. When either kinesin was knocked out, mutant pollen did not show a noticeable defect. In the absence of both kinesins, however, microtubules often became disorganized and disengaged from daughter nuclei in dividing male
microspores. Consequently, the first post-meiotic cytokinesis was severely inhibited without the formation of a cell plate, which led to failures of the generation of male gametes in pollen grains. Thus, we hypothesize that AtKinesin-12A and AtKinesin-12B play a similar role in the organization of phragmoplast microtubules during cytokinesis in microspores. We have also analyzed kinesins that play a role in vesicle transport during cell plate formation. AtPAKRP2 is a kinesin that specifically associates with the Golgi-derived vesicles in the phragmoplast. We have postulated that this motor plays a role in vesicle transport during cytokinesis in A. thaliana. We have isolated stable mutations which lead to the loss of AtPAKRP2. The mutant plants show super sensitivity to caffeine. Therefore, our results indicated that AtPAKRP2 plays a role in vesicle trafficking for plant cell division and enlargement. Since we failed to renew the grant from the USDA to continue this work, this project
has been terminated.
Impacts
The studies in the model plant Arabidopsis thaliana will significantly advance our knowledge on plant cell division in general. Because cell division is a fundamental process for flowering plants including crops, understanding the underlying mechanisms will be essential for us to acquire desirable traits in crops. Results garnered from our studies will enable us to better understand plant growth and development, and ultimately will contribute to the improvement of crop plants.
Publications
- Pan, R., Y.R.J. Lee, and B. Liu. 2004. Localization of two homologous Arabidopsis kinesin-related proteins in the phragmoplast. Planta. 220:156-164.
- Preuss, M.L., D. Kovar, Y.R.J. Lee, C.J. Staiger, D.P. Delmer, and B. Liu. 2004. A plant-specific kinesin binds to actin microfilaments and interacts with cortical microtubules in cotton fibers. Plant Physiol. 136: 3945-3955.
- Lee, Y.R.J., and B. Liu. 2004. Cytoskeletal motors in Arabidopsis. Sixty-one kinesins and seventeen myosins. Plant Physiol. 136:3877-3883.
- Lu, L., Y.R.J. Lee, R. Pan, J.N Maloof, and B. Liu. 2004. An internal motor kinesin associates with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis. Mol. Biol. Cell. In press
- Lee, Y.R.J., Y. Lee, and T B. Liu. 2005. Two homologous phragmoplast-associated kinesins play a role in male post-meiotic cytokinesis in Arabidopsis. To be submitted.
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Progress 01/01/03 to 12/31/03
Outputs
Growth, development, and reproduction of flowering plants directly rely on cell division. The division of the cytoplasm, cytokinesis, in plants is fundamentally different from that in animals and fungi. The phragmoplast is an elaborate structure that executes cytokinesis in flowering plants. At the last stage of cell division, organized microtubule array in the phragmoplast allows unidirectional transport of Golgi-derived vesicles towards the division site to give rise to the cell plate, the physical divider of the cytoplasm. Kinesin-related proteins (kinesins) are microtubule-based motors that play essential roles in both microtubule organization and vesicle transport. We have discovered two classes of kinesins that specifically act in the phragmoplast in the model plant Arabidopsis thaliana. We have taken biochemical, cytological, and genetic approaches to dissect how these kinesins regulate the progression of cytokinesis in the phragmoplast. 1. Organization of
microtubule array by AtPAKRP1 and its homologue. We have discovered that AtPAKRP1 and its close homolog AtPAKRP1L were exclusively localized to the microtubule plus ends at the division site. Our results indicated that these two closed related proteins were able to form homodimers and/or heterodimers in vitro. By taking a mutant analysis approach, we have also learned that either one is dispensable for normal growth and development. When both proteins were absent, however, seed formation dropped by approximately 50%. We have further proved that the failure of seed production was due to the formation of more than 50% abnormal pollen grains. The wild-type mature pollen grains contain two sperm cells floating in the pollen cytoplasm. Mutants contained large proportion of pollen grains with two identical nuclei, thus without sperm. We are examining how the failure of sperm formation is caused in the mutant plants. 2. Vesicle transport by AtPAKRP2 in the phragmoplast. AtPAKRP2 is a kinesin
that specifically associates with the Golgi-derived vesicles in the phragmoplast. We have postulated that this motor plays a role in vesicle transport during cytokinesis in A. thaliana. In order to demonstrate the function of this motor, we created mutant plants that only expressed anti-sense RNA upon application of steroid. Without steroid application, mutants grew as well as the wild-type plants. Upon anti-sense induction with steroid, mutant seedlings died immediately. Therefore, our results indicated that AtPAKRP2 is an essential motor for plant growth. We are further investigating what the cargo vesicles are for this motor.
Impacts
Because cell division is a fundamental process for flowering plants including crops, understanding the underlying mechanisms will be essential for us to acquire desirable traits in crops. Results garnered in our studies will enable us to better understand plant growth and development, and ultimately will contribute to the improvement of crop plants.
Publications
- Lee, Y.R.J., H.M. Giang, and B. Liu. 2001. A novel plant kinesin-related protein specifically associates with the phragmoplast organelles. Plant Cell 13:2427-2439.
- Burk, D.H., B. Liu, R. Zhong, W.H. Morrison, and Z.-H. Ye. 2001. A Katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell 13:807-827.
- Tamaru, Y., S. Ui, K. Murashima, A. Kosugi, H. Chan, R.H. Doi, and B. Liu. 2002. Formation of protoplasts from cultured tobacco cells and Arabidopsis thaliana by the action of cellulosomes and pectate lyase from Clostridium cellulovorans. Appl. Environ. Microbiol. 68:2614-2618.
- Yang, D., F. Guo, B. Liu, N. Huang and, S. Watkins. 2003. Expression and localization of human lysozyme in the endosperm of transgenic rice. Planta. 216:597-603.
- Preuss, M.L., D.P. Delmer, and B. Liu. 2003. The kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers. Plant Physiol. 132:154-160.
- Pan, R., Y.R.J. Lee, and B. Liu. 2004. Localization of two homologous kinesin-related proteins in the phragmoplast in Arabidopsis thaliana. Submitted to Planta.
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Progress 01/01/02 to 12/31/02
Outputs
This project is aimed at understanding roles of two kinesin-related motor proteins in the phragmoplast, the cytokinetic apparatus in higher plants by using the thale cress Arabidopsis thaliana as an experimental model. These two motors, AtPAKRP1 and AtPAKRP2 exclusively appear in the phragmoplast. Our early data indicate that AtPAKRP1 associates with phragmoplast microtubules at or near their plus ends, and AtPAKRP2 associates with Golgi-derived vesicles in the phragmoplast. In the past year, we focused our efforts in dissecting roles of these two motors in plant cytokinesis by using a number of approaches. First, we have undertaken a genetic approach to isolate mutations at the loci of the genes encoding these two proteins. We have successfully isolated two homozygous mutants that have T-DNA insertions at two different sites of the AtPAKRP1 locus. Interestingly, neither mutant showed any noticeable phenotype under normal growth conditions. We have screened all
available mutant pools and have not discovered a mutation at the AtPAKRP2 locus. Therefore it is anticipated that mutation at this locus would cause a lethal phenotype. Second, it has been revealed recently that an Arabidopsis gene encodes a polypeptide with 74% sequence identity with AtPAKRP1. We have isolated full-length cDNA of this AtPAKRP1L protein, for AtPAKRP1-like protein. Antibodies against AtPAKRP1L were raised. Our results indicated that AtPAKRP1L localizes to microtubule plus ends in the phragmoplast identical to AtPAKRP1 localization. A null mutation at the AtPAKRP1L locus has been isolated recently by us, and its homozygous mutant did not have a noticeable phenotype either. We have proposed that AtPAKRP1 and AtPAKRP1L have redundant functions. We are in the process of creating plants with mutations at both loci. Third, in order to dissect functions of AtPAKRP2, transgenic plants with an anti-sense construct under the control of an inducible promoter have been generated.
Our results showed that upon induction of the AtPAKRP2 antisense, Arabidopsis seedlings died at their meristems. Therefore AtPAKRP2 is indispensable for plant growth, and most likely for cell division. The ongoing experiments are devoted to understanding how these three motor proteins exert their roles in the phragmoplast.
Impacts
Plant growth and development require the cytokinesis event to take place in the right place at the right time. Studies of AtPAKRP1, AtPAKRP1L and AtPAKRP2 will bring new insights into mechanisms of plant cytokinesis. Understanding such mechanisms will shed lights on improvement of crop plants.
Publications
- Pan, R., Y.R.J. Lee, and B. Liu. (2003). Two homologous kinesin-related proteins associate with the plus end of phragmoplast microtubules in an identical manner in Arabidopsis thaliana. To be submitted to Plant J.
- Y.R.J. Lee, and B. Liu. (2003). The kinesin motor AtPAKRP2 is essential for cell division and seedling development in Arabidopsis thaliana. In preparation (to be submitted to Plant Cell).
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Progress 01/01/01 to 12/31/01
Outputs
We have identified two phragmoplast-associated kinesins, AtPAKRP1 and AtPAKRP2, which are unique to higher plants. In order to understand molecular mechanism of plant cytokinesis, the ongoing works are focused on the following aspects: 1. To Reveal Localizations of AtPAKRP1 and AtPAKRP2 at Ultrastructural Level. At light microscopic level, it is difficult to ascertain the localizations of AtPAKRP1 and AtPAKRP2. We have initiated electron microscopic works aimed at determining the localization of these two kinesins at ultrastructural level. In order to faithfully immobilize proteins and preserve cytoplasmic structures, we have prepared samples by using rapid freezing and freeze substitution methods. Preliminary results indicate that structures of vesicles, Golgi apparatus, and microtubules have been preserved very well. We have begun to carry our immunogold labeling experiments to demonstrate localizations of AtPAKRP1 and AtPAKRP2. 2. To Determine Functions of AtPAKRP1
and AtPAKRP2 by Analyzing Phenotypes of Loss-of-function Mutations for Both Genes. After we isolated T-DNA insertional mutants of AtPAKRP1, the completely sequenced A. thaliana genome revealed another gene which encodes a polypeptide of 74 percent identity with AtPAKRP1 at the amino acid level. We propose that this protein, AtPAKRP1A, has redundant function(s) as AtPAKRP1. Redundant functions of these two polypeptides likely account for lack of phenotype of homozygous plants of either atpakrp1-1 or atpakrp1-2 mutation. We are now in the process of isolating T-DNA insertional mutation(s) at the AtPAKRP1A locus. Once homozygous plant(s) of such mutation(s) are isolated, we want to create double mutants of mutations at both AtPAKRP1 and AtPAKRP1A loci in order to reveal functions of such kinesins. Unlike the AtPAKRP1 gene, the A. thaliana genome does not contain an additional gene encoding a polypeptide similar to AtPAKRP2. We have initiated efforts in isolating T-DNA insertional
mutation(s) at the AtPAKRP2 locus. However, we are aware of potential difficulties in obtaining seeds of such mutants because of possible gametophytic lethal phenotype. As a complementary experiment, we want to use the anti-sense approach to down-regulate the expression of AtPAKRP2 in A. thaliana cells. We intend to use the gluticorticoid-inducible promotor to drive the anti-sense transcription. Upon induction with dexamethasone, whether cell division is affected will be analyzed by microscopic examinations. A combination of cell biology and molecular genetics is essential for characterizing functions of complicated protein machinery. Cytokinesis is an essential process for plant growth and development. We believe that characterization of microtubule-based activities in the phragmoplast is critical for us to understand plant cytokinesis.
Impacts
Identification of Two Phragmoplast-Associated Kinesin-Related Proteins in A. thaliana. Determination of Intracellular Localization of AtPAKRP1 and AtPAKRP2. Isolation of T-DNA-Insertional Mutations in the AtPAKRP1 Locus. Plant growth and development require the cytokinesis event to take place in the right place at the right time. Studies of AtPAKRP1 and AtPAKRP2 will bring new insights into the mechanisms of plant cytokinesis. Understanding such mechanisms will shed lights on improvement of crop plants.
Publications
- No publications reported this period
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