Regulation of the pollen-specific actin-depolymerizing factor LlADF1

Abstract

Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by approximately 60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy.

Figure 1.

Protein Gel Blots of Plant Extracts. (A) Protein gel blots of various maize total protein extracts probed with anti-ZmADF3 (vegetative ADF) or anti-ZmADF1 (pollen ADF). BMS, Black Mexican Sweetcorn culture cells. (B) Coomassie blue–stained gel of various lily total protein extracts and corresponding protein gel blot probed with anti-LlADF1 (pollen ADF).

Figure 2.

LlADF1 Is Not Phosphorylated. (A) In vitro phosphorylation assays of recombinant ADF by CDPK. Autoradiograph and corresponding colloidal silver–stained gel of assays using ZmADF3 or LlADF1 in the presence of CDPK are shown. (B) In-gel phosphorylation assays using either ZmADF3 or LlADF1 incorporated into the gel. Colloidal silver–stained blots showing equal loading of the various extracts onto the gel and corresponding autoradiographs are shown.

Figure 3.

Effect of pH on LlADF1 Binding to F-Actin. F-actin and LlADF1 (diamonds) or ZmADF3 (squares) cosedimentation assays were performed in the pH range 6.0 to 9.0. The percentage of ADF bound to F-actin and found in the pellet is plotted.

Figure 4.

Regulation of Pollen ADF by Phospholipids. (A) Native gels of either LlADF1 or ZmADF3 mixed with a 50-fold molar excess of purified phospholipids. (−)PL, control without phospholipid; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-monophosphate; PIP₂, phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; IP₃, inositol 1,4,5-triphosphate; OAG 1-oleoyl-2-acetylglycerol. (B) Effect of LlADF1 on plant phospholipase C activity. The activity of plant phospholipase C was measured in the presence of increasing concentrations (0 to 12.5 μM) of LlADF1 (diamonds) or ZmADF3 (squares). (C) Inhibition of LlADF1 binding to F-actin by PIP₂. F-actin and LlADF1 cosedimentation assays were performed in the presence of a 50-fold molar excess of phosphatidylcholine (PC) as a control and a 50- to 0-fold molar excess of PIP₂. The percentage of LlADF1 remaining in the supernatant is plotted. The data shown are means of three replicate experiments, and the bars represent standard errors.

Figure 5.

AIP1-Like Proteins in Plants. (A) Yeast two-hybrid analysis of the interactions between AtAIP1-1 or AIPΔ7 and actin or AtADF2. LamC was used as a negative control. NI, no insert. (B) Phylogenetic tree of all known AIP1 sequences. Arabidopsis contains two AIP1 genes. Sequences represented are C. elegans AIP1-2 and AIP1-1, Drosophila melanogaster AIP1, X. laevis AIP1, Gallus gallus AIP1, Homo sapiens AIP1, Mus musculus AIP1, Saccharomyces cerevisiae AIP1, Schizosaccharomyces pombe AIP1, Neurospora crassa AIP1, Dictyostelium discoideum AIP1, Physarum polycephalum AIP1, Oryza sativa AIP1, and Arabidopsis AIP1-1 and AIP1-2. Accession numbers are given at the end of Methods. (C) RNA gel blots of total RNAs from various Arabidopsis tissues probed with gene-specific probes generated from the two AtAIP1 sequences.

Figure 6.

Analysis of the Combined Activity of AtAIP1-1 and LlADF1 on F-Actin. (A) Sedimentation assays analyzed on one-dimensional gels using F-actin, AtAIP1-1, or LlADF1 alone or as mixtures. p, pellet; s, supernatant. (B) Histogram of the quantity of soluble actin as determined by densitometric scanning of the supernatant lanes on the gel shown in (A).

Figure 7.

Localization of Pollen AIP1. Protein gel blots of Arabidopsis and lily pollen and leaf total protein extracts probed with the anti-AtAIP1-1 antibody. Immunofluorescence of Narcissus pollen grains ([B] to [E]) and pollen tubes ([F] to [L]) stained for actin using rhodamine-conjugated phalloidin ([B], [D], [F], and [H]) or anti-actin (J), for pollen ADF using anti-LlADF1 ([C], [G], and [K]), or for AIP1 using anti-AtAIP1-1 ([E], [I], and [L]). Pollen grains and pollen tubes shown in (B) to (I) were fixed directly using formaldehyde, and pollen tubes in (J) to (L) were prepared for fixation using the freeze-substitution method.