Pollen Profilin Function Depends on Interaction with Proline-Rich Motifs

Abstract

The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells. Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms. Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L -proline and monomeric actin from maize pollen. In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen. The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen. In contrast, the affinity of ZmPRO4 for poly-L -proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms. When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1. A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L -proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4. In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L -proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells. INTRODUCTIONThe male gametes of plants are delivered to the ovule by a unique mode of motility that is dependent on cellular morphogenesis of the pollen grain (reviewed in Bedinger and Edgerton, 1989;Mascarenhas, 1993;Taylor and Hepler, 1997). Mature pollen is a haploid multicellular structure consisting of a large vegetative cell and one generative cell or two sperm cells. The sperm are derived from a mitotic division of the generative cell that occurs before pollen maturation or during pollen tube extension. When a pollen grain lands on a receptive stigma, the vegetative cell forms a tipgrowing protuberance, the pollen tube, that extends down the transmitting tissue of the style toward the ovule. Remarkably, the growth rate of the maize pollen tube can exceed 1 cm/hr (Bedinger, 1992). The pollen tube delivers the sperm cells to the embryo sac, where they are deposited to fuse with the egg cell and central cell.Both pollen germination and pollen tube elongation are dependent on the actin cytoskeleton, as determined through studies with cytochalasins (reviewed in Pierson and Cresti, 1992;Taylor and Hepler, 1997). The actin cytoskeleton in pollen is highly dynamic, and dramatic rearrangements occur during pollen germination. Actin microfilaments focus on the germination aperture(s) before generation of the pollen tube and become arranged in long axial bundles t...

Characteristics and Immunobiology of Grass Pollen Allergens

“…Sequence analysis of three Phl p 12 cDNA clones obtained through RT-PCR revealed only 10 nucleotide changes, of which two caused amino acid substitutions [159]. In maize, however, profilin was found to be encoded by a multigene family of which at least four members were expressed in pollen, as concluded from sequence analysis of cDNA clones which diverged at 26 amino acid positions in total [155, 160]. …”

Cloning and Molecular and Immunological Characterisation of Two New Food Allergens, Cap a 2 and Lyc e 1, Profilins from Bell Pepper <i>(Capsicum annuum)</i> and Tomato <i>(Lycopersicon esculentum)</i>

“…Especially, pollen-allergic patients frequently display allergic symptoms against plant-derived food, like fruits, vegetables and spices. Profilins are a family of ubiquitous cytosolic proteins that control actin polymerisation and interact with poly- L -proline, proline-rich proteins and membrane polyphosphoinositides, with an important role in cell signalling and morphological processes [2, 3]. …”

Redistribution of actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs

“…The number of well studied and characterized cytoskeletonregulating proteins in plant cells is still very limited (for review, see Staiger et al 1997;De Ruijter and Emons 1999). Pro®lin, a widely expressed G-actin-binding protein, which interacts with poly-L-proline and phosphoinositides (Sun et al 1995;SchluÈ ter et al 1997;Chaudhary et al 1998;Gibbon et al 1998) is one of the best-characterized examples. Isoforms of pro®lin have been reported to regulate cellular growth and morphogenesis by organizing actin cytoskeletal dynamics in higher plants (e.g.…”

“…Staiger et al 1994Staiger et al , 1997Valster et al 1997). The actin-binding properties of plant pro®lins have been demonstrated in vitro (Valenta et al 1993;Giehl et al 1994;Staiger et al 1994;Vidali and Hepler 1997) and in situ by microinjection experiments (Cao et al 1992;Staiger et al 1994;Gibbon et al 1997Gibbon et al , 1998Holzinger et al 1997;Valster et al 1997). The distribution of pro®lin in plant cells, however, has not been studied in great detail so far.…”