Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.

(J.C.) Peroxisomes perform diverse and vital functions in eukaryotes, and abnormalities in peroxisomal function lead to severe developmental disorders in humans. Peroxisomes are also involved in a wide array of physiological and metabolic functions unique to plants, yet many aspects of this important organelle are poorly understood. In yeast and mammals, various steps in peroxisome biogenesis require the function of peroxin (PEX) proteins, among which PEX12 is a RING finger peroxisomal membrane protein involved in the import of matrix proteins. To investigate the role of PEX12 in plants, we identified a T-DNA knockout allele of PEX12 and generated partial loss-of-function pex12 mutants using RNA interference. We show that pex12 null mutants are developmentally arrested during early embryogenesis, and that the embryo-lethal phenotype can be rescued by overexpression of the PEX12-cyan fluorescent protein fusion protein, which targets to the peroxisome. Using virus-induced gene-silencing techniques, we demonstrate that peroxisomal number and fluorescence of the yellow fluorescent proteinperoxisome targeting signal type 1 protein are greatly reduced when PEX12 is silenced. RNA interference plants with partial reduction of the PEX12 transcript exhibit impaired peroxisome biogenesis and function, inhibition of plant growth, and reduced fertility. Our work provides evidence that the Arabidopsis (Arabidopsis thaliana) PEX12 protein is required for peroxisome biogenesis and plays an essential role throughout plant development.Peroxisomes perform diverse and crucial functions in eukaryotes, and peroxisomal deficiencies cause developmental disorders in humans and disrupt many physiological and developmental processes in fungi (Powers and Moser, 1998;Gould and Valle, 2000;Parsons et al., 2001;Titorenko and Rachubinski, 2004). Plant peroxisomes, which include glyoxysomes, leaftype peroxisomes, and unspecialized peroxisomes, contain different sets of enzymes depending on the cell type and developmental stage. They are involved in a wide range of functions, including lipid metabolism, photorespiration, nitrogen metabolism, stress response, and synthesis of some plant hormones (Beevers, 1979;Olsen and Harada, 1995;Hayashi and Nishimura, 2003).More than 30 yeast genes have been identified that encode components for various aspects of peroxisome biogenesis, including peroxisome assembly, matrix protein import, and peroxisome proliferation (Purdue and Lazarow, 2001;Charlton and Lopez-Huertas, 2002;Brown and Baker, 2003). Proteins in this category are named peroxins, or PEX proteins. The peroxisome protein import machinery is composed of a number of proteins facilitating the import process, although the architecture of this machinery is still elusive. PEX2, PEX10, and PEX12 are RING finger proteins hypothesized to function in close proximity to facilitate the import of cargo proteins and the recycling of receptors in yeast and mammals, and possibly in other eukaryotes. These three peroxins are integral membrane proteins whose N...

Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.