Abstract
Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions; however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.
Highlights
In plant cells, actin participates in numerous activities such as cell division and morphogenesis, tip growth, movement and repositioning of organelles, cytoplasmic streaming, fertilization, hormone transport and responses to external signals[1,2,3,4,5]
Actin directly fused to a fluorescent protein is useful to distinguish the localization of individual actin isoforms in eukaryotic cells, visualization of long actin filaments by expressing green fluorescent protein (GFP)-actin has not been reported in plant cells
The gaps between patches of GFP-AtACT2 and TagRFP-AtACT7 along apparently contiguous filaments were perhaps occupied by endogenous N. benthamiana actin (Supplemental Fig. 3). These results suggest that AtACT2 and AtACT7 have the capacity co-polymerize into single filaments in N. benthamiana mesophyll cells, but are segregated in their distribution along the filament backbone
Summary
Actin participates in numerous activities such as cell division and morphogenesis, tip growth, movement and repositioning of organelles, cytoplasmic streaming, fertilization, hormone transport and responses to external signals[1,2,3,4,5]. Different Arabidopsis actin isoforms have been shown to have significantly different biochemical properties, such as assembly kinetics or binding to phalloidin and profilin[14] These results suggest that plants have developed a mechanism to diversify actin cytoskeletal function by expressing multiple, functionally non-equivalent actin isoforms. Thereafter, GFP probes fused with ABDs of various ABPs, such as Fimbrin[1] from Arabidopsis[26] and Lifeact derived from the yeast Abp140p27, in addition to mTalin[1] mentioned above, have been widely used to visualize the organization of actin filaments in living plant cells[28,29] When this method is used, it is difficult to distinguish between different actin isoforms. The results revealed that different actin isoforms form unique filament arrays in leaf epidermal and mesophyll sponge cells, providing platforms to understand different functions of actin isoforms in plant cells
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