Progress 10/01/05 to 09/30/06
Outputs
Pollen tube growth is essential for plant fertilization and reproductive success. Tip growth in the pollen tube is thought to be driven by an actin-dependent growth machinery coupled to a cytosolic Ca2+ gradient focused to the apex of the tube. However, the components that regulate and respond to either actin or Ca2+ remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators. We have therefore cloned cDNA for a pollen-expressed phosphatidylinositol-4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named PetPLC1. GFP fusion to the wild type protein revealed that it associates with the apical plasma membrane during periods of slow growth but relocalizes to a vesicle rich apical region of the cytoplasm as tip growth speeds up. FRAP analysis suggests the PLC is freely mobile in the membrane, and so such localization likely reflects localization of PePLC1 docking
sites within the apical membrane. We have found that expressing a catalytically inactive form of PetPLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton at the apex, and delocalization of growth at the tube tip. However, elsewhere in the pollen tube actin function, as inferred from cytoplasmic motility, was unaltered, implying a specific role for PetPLC1 in the regulation of actin structures related to apical growth. Domain truncation experiments revealed that the C2 Ca2+/phospholipid binding domain of PetPLC1 alone was capable of phenocopying the effect of expressing the inactive version of PetPLC1, likely through its action to compete with targeting sites of the endogenous PetPLC1. These observations suggest a role for this enzyme in regulating the apical growth machinery which is intimately related to its dynamic subcellular localization. These growth phenotypes were suppressed by depolymerizing actin with low
concentrations of the drug Latrunculin B, further suggesting that a critical site of action of PetPLC1 is in regulating the structure of the actin cytoskeleton at the growing tip of the pollen tube. Although PetPLC1 was found to be enriched in regions of the apical plasma membrane of the pollen tube not undergoing rapid expansion, its substrate (the membrane lipid PtdinsP2) showed enrichment in apical regions that were depleted in PLC. Thus, PetPLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell. In addition, We have cloned and begun to functionally analyze an oxysterol-binding protein related protein (PiORP1), another lipid-signaling-related gene from petunia pollen. This protein is targeted to the plasma membrane via its PH domain into stable foci that are inserted just behind the growing tip but that do not undergo relocalization as the pollen
tube elongates. Yeast two-hybrid and direct phosphorylation assays suggest PiORP1 likely interacts with the pollen receptor-like kinase PRK1.
Impacts
A fundamental understanding of the molecular mechanisms that mediate growth of pollen tubes will allow the manipulation of this process either through pharmacological intervention or genetic engineering. The anticipated outcome of this research is therefore the future development of strategies to manipulate pollenation and so control agriculturally important processes such as hybrid crop production or control of transgene escape from transgenic crops.
Publications
- Gu, Y., Fu, Y., Dowd, P., Li, S., Gilroy, S. and Yang, Z. 2005. ROP GTPase controls actin dynamics and tip growth in pollen tubes via two counteracting downstream pathways. J Cell Biol 169:127-138.
- Yoon, G.Y-Y., Dowd, P.E., Gilroy, S. and McCubbin, A.G. 2006. Calcium Dependent Protein Kinase isoforms have distinct functions in pollen tube growth, including the regulation of polarity. Plant Cell, 18:867-878.
- Dowd, P.E., Coursol, S., SkirpanA.L., Kao, T-h and Gilroy, S. 2006. A phospholipase C of Petunia, PetPLC1, regulates pollen tube growth. Plant Cell, 18:1438-1453.
- Skirpan, A.L., Dowd, P.E., Sijacic, P., Jaworski, C.J., Gilroy, S. and Kao, T.-h. 2006. Identification and characterization of PiORP1, a Petunia oxysterol-binding related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis. Plant Molecular Biology, 61:553-565.
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Progress 10/01/04 to 09/30/05
Outputs
Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. We have therefore cloned cDNA for a pollen-expressed phosphatidylinositol- 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named PetPLC1. We have found that expressing a catalytically inactive form of PetPLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. This effect on growth suggests that PetPLC1 is an important regulator of pollen function, and hence fertilization. These growth phenotypes were
suppressed by depolymerizing actin with low concentrations of the drug Latrunculin B, suggesting that a critical site of action of PetPLC1 is in regulating the structure of the actin cytoskeleton at the growing tip of the pollen tube. PetPLC1 was found to be enriched in regions of the apical plasma membrane of the pollen tube not undergoing rapid expansion. However, when the substrate of PetPLC1 (the membrane lipid phosphatidylinositol 4,5-bisphosphate, or PtdinsP2) was visualized using a GFP fusion to the PH (PtdInsP2)-binding domain, of mammalian PLC delta-1, this lipid showed enrichment in apical regions that were depleted in PLC. Thus, PetPLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell. Truncation of various regions within the enzyme suggests targeting of the PetPLC1 is likely through a C2 domain at its N-terminus that is known in other
proteins to mediate lipid associations. In addition, We have cloned and begun to functionally analyze an oxysterol-binding protein related protein (PiORP1), another lipid-signaling-related gene from petunia pollen. This protein is targeted to the plasma membrane via its PH domain. Yeast two-hybrid and direct phosphorylation assays suggest PiORP1 likely interacts with the pollen receptor-like kinase PRK1. Current work is aimed at defining whether this phosphorylation represents a regulatory action on PiORP and how PiORP acts to sustain tip growth.
Impacts
A fundamental understanding of the molecular mechanisms that mediate growth of pollen tubes will allow the manipulation of this process either through pharmacological intervention or genetic engineering. The anticipated outcome of this research is therefore the future development of strategies to manipulate pollenation and so control agriculturally important processes such as hybrid crop production or control of transgene escape from transgenic crops.
Publications
- Gu, Y., Fu, Y., Dowd, P., Li, S., Gilroy, S. and Yang, Z. 2005. ROP GTPase controls actin dynamics and tip growth in pollen tubes via two counteracting downstream pathways. J Cell Biol 169: 127-138
- Dowd, P.E., Coursol, S., SkirpanA.L., Kao, T-h and Gilroy, S. 2006. A phospholipase C of Petunia, PetPLC1, regulates pollen tube growth. Plant Cell, submitted.
- Skirpan, A.L., Dowd, P.E., Sijacic, P., Jaworski, C.J., Gilroy, S. and Kao, T.-h. 2006. Identification and characterization of PiORP1, a Petunia oxysterol-binding related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis. Plant Molecular Biology, submitted.
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Progress 10/01/03 to 09/30/04
Outputs
Pollen tube growth is essential for plant fertilization and reproductive success. The pollen tube must navigate from stigmatic surface through the style to the ovary in order to deliver the sperm for fertilization. This journey is made possible by the rapid and highly regulated tip growth that characterizes pollen tube elongation. Although the direction of pollen tube growth is thought to be regulated in part by a tip focused Ca2+ gradient, the regulators that direct the Ca2+ gradient remain poorly defined. Phospholipases and their phospholipid turnover products, e.g., inositol-1,4,5-trisphosphate (IP3) and phosphatidic acid, have been proposed as such regulators, however, the relevant phospholipase(s) have not been characterized. The objectives of this research are to assess the role(s) of the enzyme phospholipase C (PLC) in pollen tube growth. We have cloned the major pollen expressed PLC in petunia pollen tubes (named PetPLC 1). Expressing a catalytically
inactivate version of PetPLC1 in pollen tubes caused expansion of the apical Ca2+ gradient and delocalized growth at the tube tip causing it to swell and growth to arrest. Destabilizing the actin cytoskeleton with low concentrations of the drug latrunculin B rescued this swelling effect suggesting that the PLC was having its effects due to altered regulation of the actin cytoskeleton. Tip swelling could be mimicked in wild type pollen tubes by experimentally elevating the concentration of the substrate of PLC, the membrane lipid phosphatidylinositol-bis phosphate (PIP2) but not its product of action on PIP2, IP3. Thus, this research indicates that PLC is likely regulating pollen tube growth through its ability to regulate membrane phospholipids (PIP2) composition. The PIP2 then acts to regulate the dynamics of the actin cytoskeleton that then regulates growth. PetPLC1 cycled on and off the apical plasma membrane of the growing pollen tube in an actin-dependent manner. High resolution
cross correlation analysis has shown this PLC accumulates on the plasma membrane prior to each pulse of growth. Bioinformatic analysis of PetPLC1 and the 7 PLC genes from Arabidopsis using a novel split domain analysis procedure has revealed that the plant PLCs possess a cryptic phospholipid binding (PH) domain within the X domain of the conserved catalytic region of this enzyme. In contrast, animal and insect PLCs possess a cryptic lipid binding domain spread across both the X and Y conserved domains. This analysis is suggesting the structure of plant PLCs may reflect an ancestral PLC structure that has diverged after the evolutionary split between plants and animals. Current work is defining the lipid binding specificity of the cryptic domains to assess their possible role in catalysis and in membrane targeting.
Impacts
A fundamental understanding of the molecular mechanisms that mediate growth of pollen tubes will allow the manipulation of this process either through pharmacological intervention or genetic engineering. The anticipated outcome of this research is therefore the future development of strategies to manipulate pollenation and so control agriculturally important processes such as hybrid crop production or control of transgene escape from transgenic crops.
Publications
- No publications reported this period
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