Outlook

The most important step towards discovering the precise function of AtARO1 in the egg cell and pollen tube will certainly be to identify its interaction partners. As mentioned before, approaches other than conventional yeast two hybrid assays should be applied. Conceivable are e.g the Split-Ubiquitin Membrane Yeast Two Hybrid, which has been successfully applied to identifying previously unknown membrane bound interaction partners of e.g. ER-resident bait proteins of humans (Wang et al., 2004) or transmembrane receptors in plants (Pandey and Assmann, 2004). A method often used to analyze direct interaction of a given protein with F-actin is the co-sedimentation assay (for plants e.g. Thomas et al., 2006a). For this assay, a HIS-tagged AtARO1 could be expressed in bacteria, purified and pelleted together with F-actin. In a different approach, tandem affinity purification-tagged (TAP-tagged) proteins as AtARO1 to-4 can be expressed as transgenes in plants and isolated together with their interaction partners as was shown previously (Rohila et al., 2006). These proteins have to be separated with SDS-PAGE and can be analyzed by mass spectrometry. Membrane-associated and transmembrane proteins have been successfully isolated using this method (Rohila et al., 2006).

Further, some proteins which are known to be involved in the growth signaling network at the pollen tube tip could be tested in direct interaction assays with AtARO1. Promising candidates are, for example, AtROP1 and AtRIC4, which were shown to work in the same pathway to promote actin assembly (Gu et al., 2005). Another possible candidate is the pollen specific formin AtAFH1, as inhibitory effects on the actin-nucleating activity of AtAFH1, similar to those mediated by intrinsic ARM domains in mammalian Diaphanous-related formins, might be achieved in plants by binding of AtAFH1 to an ARM repeat containing protein like AtARO1.

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The fact that the two ARM domains of AtARO1 might have in part independent but additive roles can be assumed by the differing phenotypes of aro1-3 and seth4 mutants. More precise answers to the function of single domains of the AtARO1 protein will be given through deletion mutants of e.g the N-terminal domain of yet unknown function or the ARM repeat domains ARD1 or ARD2, respectively.

The existence of phosphorylation sites can be shown by single amino acid substitutions of Ser, Thr or Tyr residues highly conserved in the ARO1-like proteins.

In addition, the nature of the fluorescent bodies, which appear in AtARO1-GFP expressing pollen tubes after BFA treatment, should be analyzed thoroughly. Transient transformation of AtARO1-GFP expressing pollen tubes with fluorescent markers for different compartments of the secretory pathway could be carried out. On the other hand, crossings of AtARO1-GFP expressing plants with marker lines expressing fluorescent proteins labeling different compartments of the secretory pathway could reveal a co-localization and thus strengthen the hypothesis that AtARO1 is involved in vesicle budding or transport. Additionally, live imaging of individual AtARO1-GFP expressing pollen tubes during a growth period will not only verify whether AtARO1 accumulation in the tip is oscillatory and precedes or follows a growth spurt but also uncover the changes in AtARO1 tip-localization upon growth reorientation of individual pollen tubes.

On the female side, the first aim should be to produce an egg cell specific triple knock-down of the genes AtARO1, AtARO2, and AtARO3. As AtARO4 is quite distant on the protein level from the other three members of the gene family, it is likely that a triple knock-down will be sufficient to induce a phenotype in the female gamete. However, it is also possible that a knock-down of the whole gene family will be needed to reveal its function in the ovule. This triple or quadruple knock-down might be achieved by using an RNAi construct directed against a highly conserved region of all four AtARO1 to -4 genes under the control of the egg cell specific EC1 promoter. A knock-down of all AtAROs under the control of a ubiquitously expressed promoter or crossing of the homozygous aro2, aro3 and aro4 T-DNA insertion lines on the other hand might be lethal, as the strong phenotypic effect of aro1-3 on the cytoskeleton in pollen tubes and the high conservation of ARO proteins amongst plant species implicates an important function for the ARO proteins in plant development. A further procedure to reveal the function of proteins often used in molecular biology is overexpression of the gene of interest. Overexpression of AtARO1 in vegetative tissues under the control of the 35S promoter did not result in any phenotypes. Moderate overexpression of AtARO1-GFP under the control of the endogenous promoter in pollen tubes and egg cells did neither result in a visible phenotype. However, plants showing strong overexpression in all vegetative tissues, especially in roots, could not be gametophytes. Stable transformation of Arabidopsis with ubiquitously or specifically expressed adequate actin binding proteins (ABPs) fused to RFP or YFP might allow the live visualization of cytoskeletal changes in the egg cell and pollen tube upon pollination and fertilization and help to

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unravel the role of AtARO1-GFP in these processes. Conceivable ABPs are on the one hand WLIM1 (Thomas et al., 2006a) or the actin binding domain 2 of fimbrin (Sheahan et al., 2004), which were already shown to interact with actin as a GFP fusion protein in vivo. On the other hand, ABPs known to bind to the long actin bundles in the shank of pollen tubes, like e.g. Vilin (Ren and Xiang, 2007) could be used.

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