Abstract
The pollen-specific calcium-dependent protein kinase PiCDPK1 of Petunia inflata has previously been shown to regulate polarity in tip growth in pollen tubes. Here we report the identification of a Rho Guanine Dissociation Inhibitor (PiRhoGDI1) as a PiCDPK1 interacting protein. We demonstrate that PiRhoGDI1 and PiCDPK1 interact in a yeast 2-hybrid assay, as well as in an in vitro pull-down assay, and that PiRhoGDI1 is phosphorylated by PiCDPK1 in vitro. We further demonstrate the PiRhoGDI1 is capable of rescuing the loss of growth polarity phenotype caused by over-expressing PiCDPK1 in vivo using stable transgenic plants. We confirmed that PiRhoGDI1 interacts with a pollen-expressed ROP GTPase isoform consistent with the established role of RhoGDIs in negatively regulating GTPases through their membrane removal and locking them in an inactive cytosolic complex. ROP is a central regulator of polarity in tip growth, upstream of Ca2+, and PiCDPK1 over-expression has been previously reported to lead to dramatic elevation of cytosolic Ca2+ through a positive feedback loop. The discovery that PiCDPK1 impacts ROP regulation via PiRhoGDI1 suggests that PiCDPK1 acts as RhoGDI displacement factor and leads us to propose a model which we hypothesize regulates the rapid recycling of ROP GTPase at the pollen tube tip.
Highlights
Polarized growth is a characteristic of a number of specialized eukaryotic cell types including animal neurons, fungal hyphae, and higher plant pollen tubes and root hairs
These results suggested that PiRhoGDI1 did interact with Petunia inflata CDPK1 (PiCDPK1) and that neither protein was capable of activating the GAL4 promoter alone
The ability of PiRhoGDI1 to rescue the effect of PiCDPK1 in pollen tubes provides support for this interaction occurring in vivo
Summary
Polarized growth is a characteristic of a number of specialized eukaryotic cell types including animal neurons, fungal hyphae, and higher plant pollen tubes and root hairs. Though the immediate downstream targets of PiCDPK1 in tip growth have not been identified, over-expression of PiCDPK1 causes a dramatic elevation of Ca2+ at the pollen tube tip, which as it is Ca2+-activated, suggests the possibility that it may be involved in a positive feedback loop [10]. Combined these studies suggest that CPK gene family members and their interactions with downstream effectors are critical components of Ca2+ signaling pathways in pollen tube growth
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