Abstract
Formins are a large, evolutionarily conserved family of actin-nucleating proteins with additional roles in regulating microfilament, microtubule, and membrane dynamics. Angiosperm formins, expressed in both sporophytic and gametophytic tissues, can be divided into two subfamilies, Class I and Class II, each often exhibiting characteristic domain organization. Gametophytically expressed Class I formins have been documented to mediate plasma membrane-based actin assembly in pollen grains and pollen tubes, contributing to proper pollen germination and pollen tube tip growth, and a rice Class II formin, FH5/RMD, has been proposed to act as a positive regulator of pollen tube growth based on mutant phenotype and overexpression data. Here we report functional characterization of the Arabidopsis thaliana pollen-expressed typical Class II formin FH13 (At5g58160). Consistent with published transcriptome data, live-cell imaging in transgenic plants expressing fluorescent protein-tagged FH13 under the control of the FH13 promoter revealed expression in pollen and pollen tubes with non-homogeneous signal distribution in pollen tube cytoplasm, suggesting that this formin functions in the male gametophyte. Surprisingly, fh13 loss of function mutations do not affect plant fertility but result in stimulation of in vitro pollen tube growth, while tagged FH13 overexpression inhibits pollen tube elongation. Pollen tubes of mutants expressing a fluorescent actin marker exhibited possible minor alterations of actin organization. Our results thus indicate that FH13 controls or limits pollen tube growth, or, more generally, that typical Class II formins should be understood as modulators of pollen tube elongation rather than merely components of the molecular apparatus executing tip growth.
Highlights
The angiosperm microgametophyte uses a specific mode of cell expansion, known as tip growth, to deliver the male gametes to the megagametophyte via a pollen tube that grows invasively through maternal sporophytic tissues
To establish the evolutionary relationships between Arabidopsis FH13 and other canonical Class II formins, we performed a phylogenetic analysis of all annotated protein sequences containing the full PTEN, C2, FH1, and FH2 domain set from twelve plant species covering a wide range of angiosperm diversity (Amborella trichopoda for basal angiosperms, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, and Zea mays for grasses as monocot representatives, A. thaliana, Fragaria vesca, Populus trichocarpa, Pyrus brettschneideri, and Vitis vinifera for rosid dicots, Nicotiana tabacum and Solanum lycopersicum for asterid dicots)
This study focuses on the role of the Arabidopsis Class II formin FH13, reported to be expressed in pollen (Pina et al, 2005; Blanchoin and Staiger, 2010; Mergner et al, 2020), in the male gametophyte
Summary
The angiosperm microgametophyte (pollen) uses a specific mode of cell expansion, known as tip growth, to deliver the male gametes to the megagametophyte via a pollen tube that grows invasively through maternal sporophytic tissues. Class II formins, on the other hand, are never transmembrane but typically contain a N-terminal membrane lipid-binding domain related to the protooncogene PTEN (Phosphatase and Tensin Homolog), followed by the calcium-binding C2 domain and the FH1/FH2 domains tandem (Grunt et al, 2008). Members of this clade bind membranes and exhibit cortical (probably endomembrane compartment) localization in the moss Physcomitrella patens (Vidali et al, 2009; van Gisbergen et al, 2012). Formin-membrane interaction is widespread and, in some cases, involves membranes other than the plasmalemma
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