Article Figures & Data
Figures
- Figure 1.
SI Challenge in Vitro Stimulates Changes in Actin Cytoskeleton in Pollen Tubes of P. rhoeas.
(A) Phalloidin labeling of F-actin in a normally growing P. rhoeas pollen tube demonstrates the presence of three zones: zone 1, the apex, which is virtually devoid of actin; zone 2, the collar zone between apex and subapex, which is characterized by a dense mesh of actin filaments; and zone 3, the subapical and basal parts of the cell, which contain distinct actin arrays oriented parallel to the longitudinal axis of the cell.
(B) to (F) P. rhoeas pollen tubes at different times after SI challenge. (B) At 1 min after SI challenge, the actin mesh in the collar region has lost its original structure and apparently has been relocated to the apex. A distinct, apparently continuous layer of phalloidin labeling is visible adjacent to the plasma membrane.
(C) At 5 min after SI challenge, the actin bundle arrays in the shank appear diffuse. The first small, punctate foci of actin are visible, and actin has accumulated in the tube apex.
(D) At 10 min after SI challenge, actin bundle arrays are still distinguishable but much of the phalloidin labeling appears as small punctate foci.
(E) At 20 min after SI challenge, actin arrays are no longer visible. The accumulation of labeling at the apex has almost disappeared. Almost all phalloidin labeling is present in the form of large punctate foci.
(F) At 180 min after SI challenge, phalloidin labeling occurs in the form of prominent punctate foci of actin, which are distributed along the entire length of the pollen tube.
Bar in (F) = 10 μm for (A) to (F).
- Figure 2.
Marginalization of F-Actin after SI Challenge.
Median sections of pollen tubes treated with incompatible S proteins illustrate strong phalloidin labeling of peripheral F-actin.
(A) At 5 min after SI challenge, this median section of a pollen tube demonstrates that the phalloidin labels an apparently continuous layer, which is adjacent to the plasma membrane.
(B) At 180 min after SI challenge, this median section of the pollen tube shown in Figure 1F illustrates that the prominent punctate foci of actin, resulting from phalloidin labeling, are clearly located in the peripheral region of the cell.
(C) The line scan demonstrates that the amount of phalloidin labeling of F-actin in the cortical region is approximately fivefold that in the lumen of the pollen tube. The two line scans (indicated by triangles and circles) measuring fluorescence intensity were made either side of the white line indicated on the pollen tube illustrated in (A).
Bar in (B) = 10 μm for (A) and (B).
- Figure 3.
Control Experiments Show No Changes in F-Actin of Pollen Tubes.
Controls were examined to demonstrate that the rearrangements of actin were not caused by a change in growth medium (GM; see Methods) and that the pollen reacted to S proteins in an S allele–specific manner.
(A) A P. rhoeas pollen tube 20 min after a change in GM. At no time interval after the change in GM was the F-actin configuration affected. Samples taken at all other time points looked identical.
(B) A P. rhoeas pollen tube 20 min after challenge with compatible S proteins. This sample, which appears identical to an unchallenged pollen tube, is representative of those seen at all times after treatment with compatible S proteins, because the actin cytoskeleton appeared to be identical at all the times sampled.
Bar in (B) = 10 μm for (A) and (B).
- Figure 4.
Quantification of Changes in F-Actin Configuration after SI Challenge.
The observed actin cytoskeleton phenotypes are of three classes: I, II, and III (see text for descriptions). A small proportion of pollen tubes (not indicated on the histogram) had a high background of labeling or could not be clearly associated with one of the classes of configurations. Error bars represent standard deviations.
(A) After incompatible S protein challenge, the number of pollen tubes with normal (class I) actin configuration disappeared almost immediately. Concomitantly, there was a transient appearance of pollen tubes with a class II configuration and an eventual transformation of the actin configuration into the punctate foci of actin (class III).
(B) The actin configuration observed in compatible pollen tubes challenged with the same S protein samples as those in (A) did not reveal changes in F-actin configuration. This demonstrates the absolute S specificity of the actin rearrangements stimulated by S proteins.
- Figure 5.
S Protein–Induced Changes in Germinating Pollen Grains.
Incompatible and compatible pollen was germinated in the presence of S proteins in experiments mimicking in vivo conditions.
(A) Incompatible pollen grain 30 min after being applied to solidified GM. The pollen tube has started to emerge from the pollen grain and shows a largely unaffected actin cytoskeleton.
(B) Incompatible pollen 60 min after being applied to solidified GM. Growth of the pollen tube is arrested, and the pollen tube tip shows heavy phalloidin staining. The pollen grain and tube show small punctate actin with some evidence of remaining actin bundles.
(C) Incompatible pollen 120 min after being applied to solidified GM. The pollen grain and tube are labeled with large and prominent punctate foci of actin.
(D) Compatible pollen grain 30 min after being applied to solidified GM. The actin configuration in the tube appears to be normal. The apparent difference in label intensity between (A) and (D) is most likely due to different penetration of phalloidin into the pollen.
(E) Compatible pollen tube 60 min after being applied to solidified GM. A typical, unchallenged pollen tube is shown.
(F) Compatible pollen tube 120 min after being applied to solidified GM. Again, the unchallenged control pollen tube exhibits characteristics of a normal pollen tube with a normal actin cytoskeleton.
Bar in (F) = 10 μm for (A) to (F).
- Figure 6.
Alterations to Pollen Actin Cytoskeleton during the in Vivo SI Response.
We assessed the actin cytoskeleton of pollen that had undergone compatible and incompatible SI reactions in vivo.
(A) Typical pollen tube sampled 2 hr after an incompatible pollination. The pollen tubes clearly show the punctate appearance of the actin cytoskeleton along the entire length of each pollen tube.
(B) Typical pollen tube sampled 30 min after a compatible pollination. The configuration of actin arrays resembles very strongly that seen in normally growing pollen tubes in vitro.
Bar in (B) = 10 μm for (A) and (B).
- Figure 7.
Effect of Arrest of Pollen Tube Growth on Actin Configuration.
Pollen tube growth was arrested by interfering with ion homeostasis by adding Gd3+, caffeine, or EGTA. In all cases, although the apical configuration of actin was affected, the actin arrays in the shank were not markedly altered by treatment with these drugs.
(A) Actin configuration 20 min after 100 μM Gd3+ was applied.
(B) Actin configuration 10 min after 10 mM caffeine was applied. The characteristic mesh configuration of the collar region in unaffected pollen tubes had moved toward the subapex. The tip of the pollen tube (indicated with an arrowhead) seems to be completely devoid of label.
(C) Actin configuration 10 min after 200 μM EGTA was applied. Bar in (C) = 10 μm for (A) to (C).
- Figure 8.
Effect of Actin-Depolymerizing Agents on the Pollen Tube Actin Cytoskeleton.
To assess the effect of actin-depolymerizing drugs on P. rhoeas pollen tubes, cytochalasin D and latrunculin A were added to growing pollen tubes, and samples were taken after different intervals.
(A) Effects 5 min after 1 μM cytochalasin D was applied. The actin arrays have disintegrated. The tip is slightly swollen. The insert shows a median section of the same pollen tube in which phalloidin label is located peripherally.
(B) Effects 5 min after 5 μM cytochalasin D was applied. Actin filaments appear to have broken down into short fragments.
(C) Effects 10 min after 1 μM cytochalasin D was applied. Actin has disintegrated; a diffuse, weakly labeled network remains.
(D) Effects 5 min after 50 nM latrunculin A was applied. The actin cytoskeleton has lost its characteristic structure. There is strong staining of cortical actin, and actin in the subapical region has disintegrated almost completely.
(E) Effects 30 min after application of 50 nM latrunculin A. Phalloidin labeling occurs as small, weakly labeled structures, giving a speckled appearance throughout the pollen tube.
Bar in (E) = 10 μm for (A) to (E).
Tables
- Table 1.
Determination of Effective Concentrations (EC100) on Pollen Tube Growth for the Drugs Used in This Study
Drug Concentrationa Caffeine 10 mM EGTA 200 μM GdCl3 100 μM Cytochalasin D 1 μM Latrunculin A 50 nM -
↵a The minimum concentrations that completely inhibit poppy pollen tube growth (i.e., EC100) in the growth medium used.
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