At FH16, an Arabidopsis Type II Formin, Binds and Bundles both Microflaments and Microtubules, and Preferentially Binds to Microtubules

Formins are well-known regulators that participate in the organization of the actin cytoskeleton in organisms. The Arabidopsis thaliana L. genome encodes 21 formins, which can be divided into two distinct subfamilies. However, type II formins have to date been less well characterized. Here, we cloned a type II formin, AtFH16, and characterized its biochemical activities on actin and microtubule dynamics. The results show that the FH1 FH2 structure of AtFH16 cannot nucleate actin polymerization efficiently, but can bind and bundle microfilaments. AtFH16 FHIFH2 is also able to bind and bundle microtubules, and preferentially binds microtubules over microfilaments in vitro, in addition, AtFH16 FHIFH2 co-localizes with microtubules in onion epidermal cells, indicating a higher binding affinity of AtFH16 FHIFH2 for microtubules rather than microfilaments in vivo. In conclusion, AtFH16 is able to interact with both microfilaments and microtubules, suggesting that AtFH16 probably functions as a bifunctional protein, and may thus participate in plant cellular processes.

Arabidopsis RAN 1 Mediates Seed Development through Its Parental .Ratio by Affecting the Onset of Endosperm Cellularization

Although previous studies have demonstrated that endosperm development is influenced by its parental genome constitution, the genetic basis and molecular mechanisms that control parent-of-origin effects require further elucidation. Here we show that the Ras-related nuclear protein 1 （RAN1） regulates endosperm development in Arabidopsis thaliana. Reciprocal crosses between wild-type （WT） and transgenic lines misexpressing RAN1 （msRAN1） gave rise to small F1 seeds when RAN1 down-regulated/up-regulated individuals were used as a male/female parent; in contrast, F1 seeds were aborted when RAN1 down-regulated/up-regulated plants were used as a female/male parent, suggesting that seed development is affected by the parental genome ratio of RAN1. Whereas RAN1 expression in wild-type plants is reduced before the onset of endosperm cellularization, F1 seeds from reciprocal crosses between WT and msRAN1 showed abnormal endosperm cellularization and ectopic expression of RAN1. The expression of MINISEED3 （MINI3）-a gene that also controls endosperm cellularization-was also affected in these reciprocal crosses, and the misregulation of MINI3 activity rescued F1 seeds when msRAN1 plants were used in reciprocal crosses. Taken together, our results suggest that the parental ratio of RAN1 regulates the onset of endosperm cellularization through its genetic interaction with MINI3.

Actin Polymerization Mediated by AtFH5 Directs the Polarity Establishment and Vesicle Trafficking for Pollen Germination in Arabidopsis

The process of pollen germination is crucial for flowering plant reproduction,but the mechanisms through which pollen grains establish polarity and select germination sites are not well understood.In this study,we report that a formin family protein,AtFH5,is localized to the vesicles and rotates ahead of Lifeact-mEGFPlabeled actin filaments during pollen germination.The translocation of AtFH5 to the plasma membrane initiates the assembly of a collar-like actin structure at the prospective germination site prior to germination. Genetic and pharmacological evidence further revealed an interdependent relationship between the mobility of AtFH5-1 abeled vesicles and the polymerization of actin filaments:vesicle-localized AtFH5 promotes actin assembly,and the polymerization and elongation of actin filaments,in turn,is essential for the mobility of AtFH5-1 abeled vesicles in pollen grains.Taken together,our work revealed a molecular mechanism underlying the polarity establishment and vesicle mobility during pollen germination.

Plant multiscale networks:charting plant connectivity by multi-level analysis and imaging techniques

In multicellular and even single-celled organisms,individual components are interconnected at multiscale levels to produce enormously complex biological networks that help these systems maintain homeostasis for development and environmental adaptation.Systems biology studies initially adopted network analysis to explore how relationships between individual components give rise to complex biological processes.Network analysis has been applied to dissect the complex connectivity of mammalian brains across different scales in time and space in The Human Brain Project.In plant science,network analysis has similarly been applied to study the connectivity of plant components at the molecular,subcellular,cellular,organic,and organism levels.Analysis of these multiscale networks contributes to our understanding of how genotype determines phenotype.In this review,we summarized the theoretical framework of plant multiscale networks and introduced studies investigating plant networks by various experimental and computational modalities.We next discussed the currently available analytic methodologies and multi-level imaging techniques used to map multiscale networks in plants.Finally,we highlighted some of the technical challenges and key questions remaining to be addressed in this emerging field.

A Processive Arabidopsis Formin Modulates Actin Filament Dynamics in Association with Profilin

Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been impli- cated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively at- taches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attach- ment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, com- petes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH 14 moves processively on the elongating barbed end. At high concentration, AtFH 14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin.

Arabidopsis Profilin Isoforms,PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis

The phragmoplast,a structure crucial for the completion of cytokinesis in plant cells,is composed of antiparallel microtubules(MTs)and actin filaments(AFs).However,how the parallel structure of phragmoplast MTs and AFs is maintained,especially during centrifugal phragmoplast expansion,remains elusive.Here,we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein(AtMAC).When AtMAC was deleted,the phragmoplast showed disintegrity during centrifugal expansion,and the resulting phragmoplast fragmentation led to incomplete cell plates.Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis.Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro,and the truncated AtMAC protein,N-CC1,was the key domain controlling the ability of AtMAC.Further analysis showed that N-CC1(51–154)is the key domain for binding MTs,and N-CC1(51–125)for binding AFs.In conclusion,AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion,which is required for complete cytokinesis.

Actin organization and regulation during pollen tube growth

Pollen is the male gametophyte of seed plants and its tube growth is essential for successful fertilization.Mounting evidence has demonstrated that actin organization and regulation plays a central role in the process of its germination and polarized growth.The native structures and dynamics of actin are subtly modulated by many factors among which numerous actin binding proteins(ABPs)are the most direct and significant regulators.Upstream signals such as Ca^(2+),PIP_(2)(phosphatidylinositol-4,5-bis-phosphate)and GTPases can also indirectly act on actin organization through several ABPs.Under such elaborate regulation,actin structures show dynamically continuous modulation to adapt to the in vivo biologic functions to mediate secretory vesicle transportation and fusion,which lead to normal growth of the pollen tube.Many encouraging progress has been made in the connection between actin regulation and pollen tube growth in recent years.In this review,we summarize different factors that affect actin organization in pollen tube growth and highlight relative research progress.