Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: This project is devoted to understanding molecular mechanisms that underlie cell division in plants using the rice Oryza sativa as a model system. We also use Arabidopsis thaliana as a reference plant for testing functions of conserved plant proteins. We aim to characterize how the cytoskeletal elements of F-actin/microfilaments and microtubules interact at the cell division site to ensure the prosecution of cytokinesis in the right place at the right time. We are particularly interested in revealing functions of proteins that play critical roles in the organization of microtubules and F-actin during cell division. The first target of our study is a novel microtubule-based motor kinesin known to us as OsKCH1 (O. sativa kinesin with calponin homology domain 1). We found that OsKCH1 specifically decorated the preprophase band in a puctate manner during prophase. OsKCH1 is completely absent in mitotic spindles, but reappears in the phragmoplast, the cytokinetic apparatus in plants. We tested whether the localization was dependent on microtubules and/or microfilaments using pharmacological approaches. It was found that artificial removal of either cytoskeletal element partially compromised the localization, and depolymerization of both completely abolished the localization. The results provided strong evidence that OsKCH1 interacted with both microtubules and microfilaments at the cell division site. OsKCH1 is the first protein exclusively appears at the cell division site during the forecasting and execution stages of cytokinesis. Taking a genetic approach, we isolated mutations which had the OsKCH1 gene inactivated by transposon insertions. We have had five independent alleles which all lack of the OsKCH1 protein expression. These mutants did not show any noticeable growth defect, suggesting that other protein(s) play a redundant role as OsKCH1. We have then undertaken a bioinformatics approach not only to analyze proteins homologous to OsKCH1 encoded by the rice genome, but also to reveal all genes encoding proteins of the microtubule cytoskeleton. In fact, there are nine genes encoding KCH kinesins in the rice genome. Our work also showed that there are 52 kinesins in rice. Unfortunately, their functions are largely unknown due to the lack of studies. Our published works would serve as a critical reference for future investigations of kinesins and other microtubule-interacting proteins in rice and other monocot species. In addition, we have characterized functions of homologous Kinesin-12A and -12B in microtubule organization during cytokinesis, which are conserved among flowering plants. We have discovered that a synergistic effort by these two kinesins led to the establishment of the phragmoplast microtubule array. The result has a significant implication to our ongoing cell division study in rice. We have also determined functions of microtubule-interacting proteins EB1 and NEDD1 whose functions are also required for cytokinesis in plant cells. PARTICIPANTS: Project director (PD): Dr. Yuh-Ru Lee Co-Project director (co-PD): Dr. Bo Liu Postdoctoral associate: Dr. Zhaosheng Kong TARGET AUDIENCES: Rice breeders. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Among species of the Graminaceous family, very few studies have been carried out at the molecular and cellular levels. In order to understand mechanisms that regulate their vegetative growth and reproduction, it is essential that we characterize how cell division takes place. To date, it is known that the cytoskeletal elements of microtubules and F-actin/microfilaments play essential roles in regulating cell division and consequently growth and development among plants. We have adopted rice as an excellent model crop for genetic and cell biological studies of various crops in this family. By addressing fundamental questions on cell division in rice, we wish to bring fresh knowledge on mechanisms that regulate rice growth. Such knowledge is critical for future efforts in genetically modifying rice and other Graminaceous crops to meet environmental challenges.
Publications
- Lee, Y.-R.J., Y. Li, and B. Liu. 2007. Two homologous phragmoplast-associated kinesins play a critical role in cytokinesis during male gametogenesis in Arabidopsis. Plant Cell. 19 2595-2605.
- Lee, Y.-R.J., and B. Liu. 2007. Cytoskeletal motor proteins in plant cell division. In Plant Cell Monographs: Cell Division Control in Plants. D.P.S. Verma and Z. Hong, editors. Springer, Berlin. Pp. 169-193.
- Bisgrove S.R., Y.-R. J. Lee, B. Liu, N. Peters, and D.L. Kropf. 2008. The microtubule plus-end binding protein EB1 functions in root responses to touch and gravity signals in Arabidopsis. Plant Cell. 20 396-410.
- Capron, A., M. Gourgues, L.S. Neiva, J.-E. Faure, F. Berger, G. Pagnussat, A. Krishana, C. Alvarez-Mejia, J.-P. Vielle-Calzada, Y.-R.J. Lee, B. Liu, V. Sundaresan. 2008. Maternal control of male-gamete delivery in Arabidopsis involves a putative GPI-anchored protein encoded by the LORELEI gene. Plant Cell 20 3038-3049.
- Guo, L., C.-M. Ho, Z. Kong, Y.-R.J. Lee, Q. Qian, and B. Liu. 2009. Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome. Annals Bot. 103 387-402.
- Zeng, C.T., Y.-R.J. Lee, and B. Liu. 2009. The WD-40 repeat protein NEDD1 functions in microtubule organization during cell division in Arabidopsis thaliana. Plant Cell. 21 1129-1140.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The goal of this project is to reveal molecular mechanisms that regulate cell division in cereal crops using the rice Oryza sativa as a model system. The position of cell division is precisely determined during plant growth. Although both cytoskeletal elements of actin filaments (F-actin) and microtubule are required for division site determination, it is unknown how plant cells integrate their activities. We have discovered a novel microtubule-based motor kinesin, OsKCH1 (O. sativa kinesin with calponin-homology domain), which interacts with F-actin and specifically decorates the cell division site. Our hypothesis is that OsKCH1 mediates the interaction between F-actin and microtubules at the cell division site to ensure successful cytokinesis. To test this hypothesis, we have made progress in the following aspects. 1. Characterizing the dynamics of OsKCH1 during cell division. The division plane of plant cells is forecasted by the preprophase band (PPB), and is later recognized by the phragmoplast during cytokinesis. In PPB and the phragmoplast, F-actin and microtubules closely interact to allow the cell plate formation at a correct place. Shown by immune-localization, OsKCH1 decorated PPB and the phragmoplast along microtubules. The interaction of OsKCH1 with F-actin and microtubules was verified as artificial removal of either microtubules or F-actin jeopardized the localization. Our results suggest that OsKCH1 links both elements at the cell division site. 2. Understanding biochemical properties of OsKCH1. Functions of different domains of OsKCH1 are characterized biochemically in vitro. The outcome would help us to understand how OsKCH1 functions in vivo. By using bacterial recombinant proteins, the central motor domain is being tested for its motor activities, and the N-terminal CH domain for its interaction with F-actin. 3. Dissecting the function of OsKCH1 in vivo. We performed two independent genetic experiments in rice. Down regulation of the OsKCH1 expression by RNA interference or by Tos17 transposon insertion caused dwarfism and lower fertility. The phenotype severity was correspondent to the OsKCH1 expression levels, tested by quantitative polymerase chain reactions. PPB of the mutants became wider than of control cells. The dynamics of F-actin and microtubules in mutant cells will be evaluated using rice plants expressing fluorescent protein-tagged F-actin and microtubules, which are being generated in the laboratory. 4. Determining functions of KCHs in rice. Including OsKCH1, rice has nine KCHs. We generated transgenic rice plants in which the expression of each or multiple members of the OsKCH group was reduced by RNA interference. Phenotypic analyses of these mutant plants are underway. Various KCHs are proposed to act as dynamic integrators of F-actin and microtubules at different stages of cell growth. 5. Evaluating the microtubule cytoskeleton in rice. The completed rice genome sequence provided us an unprecedented opportunity to identify genes encoding microtubule associated proteins. We completed a genome-wide assessment, and revealed many unique features in rice. The article summarizing the findings is now in press. PARTICIPANTS: Yuh-Ru (Julie) Lee, Longbiao Guo, Zhaosheng Kong, Bo Liu TARGET AUDIENCES: Breeders who intend to use genetic engineering to modify rice and other cereal crops. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Besides being an important crop world-wide, rice is also an excellent model plant for other crops in the Graminaceae family because its genome has been completely sequenced and sophisticated tools and resources are available. Cell division is a fundamentally important process for growth and development. By aiming at this very basic process, we believe that results garnered from our studies would contribute to understanding how rice and other monocots accomplish their growth and reproduction. Our ultimate goal is to provide basis for designing novel strategies aimed at improving crops for higher yields and greater surviving skills.
Publications
- Lee, Y. R.J., Y. Li, and B. Liu. 2007. Two homologous phragmoplast associated kinesins play a critical role in cytokinesis during male gametogenesis in Arabidopsis. Plant Cell. 19 2595 2605.
- Lee, Y. R.J., and B. Liu. 2007. Cytoskeletal motor proteins in plant cell division. In Plant Cell Monographs Cell Division Control in Plants. D.P.S. Verma and Z. Hong, editors. Springer, Berlin. Pp. 169 193.
- Bisgrove, S.R., Y. R.J. Lee, B. Liu, N. Peters, and D.L. Kropf. 2008. The microtubule plus end binding protein EB1 functions in root responses to touch and gravity signals in Arabidopsis. Plant Cell 20 396 410.
- Capron, A., M. Gourgues, L.S. Neiva, J.E. Faure, F. Berger, G. Pagnussat, A. Krishnan, C. Alvarez Mejia, J. P. Vielle Calzada, Y. R.J. Lee, B. Liu, V. Sundaresan. 2008 Maternal control of male gamete delivery during double fertilization in Arabidopsis involves a putative GPI anchored protein encoded by the LORELEI gene. Plant Cell, in press.
- Guo, L., C. M. Ho, Z. Kong, Y. R.J. Lee, and B. Liu. 2008. Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome. Annals Bot. In press.
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: This AES project chooses to elucidate molecular mechanisms that regulate cell division in cereal crops using the rice Oryza sativa as a model system. Based on our discovery of a novel microtubule-based motor kinesin known to us as OsKinesin-CH1 (OsKCH1), or O. sativa kinesin with CH (calponin homology) domain 1, experiments were designed to dissect its function in cell division, and ultimately in the growth and development of the rice plant. The output of this project is summarized in the following four aspects: 1. Dynamic behavior of OsKCH1 during cell division in dividing cells. Like in other plants, rice cells form the conspicuous microtubule preprophase band from G2 phase to prophase. OsKCH1 specifically decorated PPB in a puctate manner. The complete absence of OsKCH1 in mitotic spindles was followed by its reappearance in the phragmoplast, the cytokinetic apparatus in plants. We also found that artificial removal of either microtubules or actin microfilaments would
partially remove the localization, and depolymerization of both cytoskeletal elements completely abolished the localization. Thus, the result provided strong evidence that OsKCH1 interacted with both elements at the cell division site. 2. Biochemical characterization of the OsKCH1 protein. We started to dissect functions of different domains of the OsKCH1 protein. In order to demonstrate the motility of this kinesin, fusion proteins of the OsKCH1 motor domain with the glutathione S-transferase were produced in bacteria and purified. These fusion proteins are being tested for their interaction with microtubules, and motor activities in vitro. We also started to test the interaction between OsKCH1 and actin microfilaments. Again, GST fusion proteins with the CH domain were produced in bacteria. We are now optimizing the expression conditions to achieve the goal of sufficient fusion proteins for testing the interaction with actin microfilaments in vitro. 3. Genetic dissection of the
function of OsKCH1 in rice. Transgenic lines expressing an RNAi construct were aimed at down-regulating OsKCH1 expression. These lines exhibited significant inhibition of vegetative and reproductive growth by producing dwarfed seedlings and panicles with fewer grains. In order to verify that the phenotypes were indeed caused by down-regulation of OsKCH1, using quantitative polymerase chain reaction (q-PCR) experiments, mRNA samples extracted from eight lines we discovered that severity of the phenotype was consistent with the degree of down-regulation of OsKCH1 expression. Concomitantly, we have been taking an independent approach to isolate three transposon insertional mutations at the OsKCH1 locus. We are isolating homozygous lines and cleaning up the background non-specific insertions by genetic crosses. 4. Integrative studies of cytokinesis in flowering plants. We also took advantage of the cress Arabidopsis thaliana to dissect some common features of cytokinesis. We have
discovered that a synergistic effort by Kinesin-12A and Kinesin-12B leads to the establishment of the phragmoplast microtubule array. The result has a significant implication to our ongoing study in rice.
PARTICIPANTS: Yuh-Ru (Julie) Lee, Zhaosheng Kong, Longbiao Guo, Chin-Min Ho, Yan Li, Bo Liu
TARGET AUDIENCES: Breeders who intend to use genetic engineering to modify rice and other crops.
PROJECT MODIFICATIONS: None.
Impacts
Rice is a monocot species in the Graminaceous family, and an excellent model for studying this family. Unfortunately, we know very little about the molecular mechanisms that regulate basic processes of vegetative growth and reproduction in monocots. By addressing fundamental questions on cell division in monocots using rice as a model organism, we wish to bring fresh knowledge on mechanisms that regulate rice growth. Such knowledge is critical for future efforts in genetically modifying rice to overcome environmental challenges.
Publications
- Lee, Y.-R.J., Y. Li, and B. Liu. 2007. Two homologous phragmoplast-associated kinesins play a critical role in cytokinesis during male gametogenesis in Arabidopsis. Plant Cell. 19:2595-2605.
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Progress 09/01/05 to 08/31/06
Outputs
The long-term goal of this project is to elucidate molecular mechanisms that regulate cell division in cereal crops using the rice Oryza sativa as a model system. Current work focuses on how the microtubule-based motor kinesins contribute to F-actin-microtubule interaction at the cell division site. Because our previous work has indicated that a KCH kinesin isolated from cotton fibers interacts with F-actin in vitro, we were particularly interested in analyzing rice KCH kinesins in dividing cells. We have chosen to focus on OsKinesin-CH1 (OsKCH1), or O. sativa kinesin with CH (calponin homology) domain 1, due to its novel intracellular localization pattern and potential functions in cell division. The OsKCH1 gene encodes a polypeptide of 1029 amino acids. The kinesin catalytic core is located in the middle of the polypeptide (amino acids 374-705). A typical neck sequence for minus end-directed kinesins (NRKLYNQVQDLKGS) is located at the N-terminal side to the
catalytic core. The CH domain is located at the N-terminus of the polypeptide (amino acids 24-146). Two predicted coiled-coil domains are found at both the N-terminal and the C-terminal sides to the catalytic core. Antibodies were raised independently against two regions of OsKCH1. Affinity purified monospecific antibodies from two rabbits and two rats gave us consistent results in immunofluorescence experiments. OsKCH1 showed a discrete localization patterns in the preprophase band (PPB) and the phragmoplast. In prophase cells indicated by condensed chromatin inside the nucleus, OsKCH1 was detected in a filamentous manner in the central region of the cell cortex. When counter-stained with an anti-tubulin antibody, OsKCH1 signal coincided with the region where the PPB microtubules resided. OsKCH1 reappeared in the phragmoplast after sister chromatids were completely segregated. At this stage, OsKCH1 decorated phragmoplast microtubules unevenly, and its localization was more pronounced
toward the minus end of these microtubule bundles. In order to knock down the expression of OsKCH1 in rice plants, an RNAi construct was made using the inverted repeats of the OsKCH1 cDNA sequence of bases 2560-3041, spaced by the rice Xa21 intron. Twenty independent T0 transgenic rice lines were generated after the RNAi construct was transformed. Among them, 14 lines were tested to be positive for having the RNAi construct integrated into their genomes. T0 and T1 plants of these 14 lines were analyzed. RT-PCR data on six T1 plants showed different degrees of reduction of OsKCH1 expression. Among the 14 lines, eight showed shorter seedlings when grown in the greenhouse. There were 12 lines in which T0 and T1 plants had reduced fertility indicated by frequent appearance of green caryopsis husks without seeds in their panicles. While mature wild type panicles had less than 12% empty caryopsis husks, these mutant ones had more than 51% empty husks. Therefore, OsKCH1 likely plays a role
in both vegetative growth and grain formation in rice.
Impacts
Monocot species like all Graminaceous species have distinct features of growth compared to dicots. Unfortunately, we know very little about the molecular mechanisms that regulate basic processes such as cell division and cell elongation in monocots. This project marks the beginning of our efforts in addressing the fundamental question about cell division in monocots using rice as a model organism. The outcome of our studies will meet the future needs for genetically modifying rice to overcome environmental challenges, and to improve the crop.
Publications
- No publications reported this period
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