Functional Characterization Of A Novel Plant Actin Cross-Linking Protein-CROLIN1

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. Higher-order actin filament structures are necessary for cytoplasmic streaming and organelle movement which play an essential role in pollen tube growth. However, the mechanism of the higher-order cytoskeleton formation in plants remains unknown. In this study, we identified a novel actin cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom.1, utilizing a bioinformatic approach towards analyzing the Arabidopsis genome, we blasted the genes that contained the keyword "actin" in the TAIR database. We identified a functionally unknown gene family that contains a predicted actin cross-linking domain (InterPro:IPR008999). According to an analysis by the Phyre Server, the structure of this domain is similar to fascin, an animal protein and hisactophilin, a protein in Dictyostelium discoideum. However, they share only16%of the amino acid similarity.2, by transformation of Arabidopsis with AtCROLIN1promoter tagged with GUS, the analysis of GUS activity showed CROLIN1was expressed only in pollen. And the result of RT-PCR was consistent with this result.3, by the high-speed co-sedimentation assays, we demonstrated that CROLIN1has the actin binding activity, the Kd value is0.324μM. In addition, by the low-speed co-sedimentation assays, we demonstrated that CROLIN1has the actin bundling activity.4, in order to observe the mechanisms of actin bundles formed. We used fluorescence microscopy and electron microscopy. The results showed that CROLIN1can bundle F-actin and also cross-link F-actin into higher-order structures. The cross-bridge was4nm. Meanwhile, CROLIN1can stabilize F-actin in vitro, protect F-actin from the disaggregation result by Lat B and ADF1.5, the analysis of the physiological function:CROLIN1is specially expressed in pollen and we study the phenotype of the pollen. We obtained one T-DNA insertion mutant, RNAi lines and over-expressed (OE) lines. The mutant is more sensitivity to Lat B than wild type (WT) plants. Mainly in:after treated with Lat B, the germination rate is lower than WT, the growth rate is slower and the deformity rate is rapidly rised. We used the Alexa488-phalloidin stain the F-actin in pollen tube found that F-actin in mutant is easier for disaggregation. Contrary, the OE lines was more stable than WT. These results indicate CROLIN1can stable F-actin in vivo.In summary, CROLIN1loss-of-function results in pollen germination, pollen tube growth and actin cytoskeleton hypersensitive to latrunculin B, but over-expression of CROLIN1partially inhibits pollen germination and stabilizes the actin filaments in vivo. In vitro biochemical analyses show that CROLIN1is a novel actin cross-linking protein with binding, bundling and stabilizing activities. Remarkably, CROLIN1can cross-link actin bundles into actin networks. All of these results demonstrate that CROLIN1may play an important role in pollen tube growth via stabilizing and remodeling actin filaments.