RESUMO
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.
RESUMO
Formins are evolutionarily conserved multi-domain proteins participating in the control of both actin and microtubule dynamics. Angiosperm formins form two evolutionarily distinct families, Class I and Class II, with class-specific domain layouts. The model plant Arabidopsis thaliana has 21 formin-encoding loci, including 10 Class II members. In this study, we analyze the subcellular localization of two A. thaliana Class II formins exhibiting typical domain organization, the so far uncharacterized formin AtFH13 (At5g58160) and its distant homolog AtFH14 (At1g31810), previously reported to bind microtubules. Fluorescent protein-tagged full length formins and their individual domains were transiently expressed in Nicotiana benthamiana leaves under the control of a constitutive promoter and their subcellular localization (including co-localization with cytoskeletal structures and the endoplasmic reticulum) was examined using confocal microscopy. While the two formins exhibit distinct and only partially overlapping localization patterns, they both associate with microtubules via the conserved formin homology 2 (FH2) domain and with the periphery of the endoplasmic reticulum, at least in part via the N-terminal PTEN (Phosphatase and Tensin)-like domain. Surprisingly, FH2 domains of AtFH13 and AtFH14 can form heterodimers in the yeast two-hybrid assay-a first case of potentially biologically relevant formin heterodimerization mediated solely by the FH2 domain.