ANX RLK over-expressing pollen tubes do not exhibit endocytosis defects but display an increased rate of exocytosis.

<p>(A) Representative single median plane images of a normally growing PT of the ANX1-YFP complemented line (top) and a slow growing PT of the ANX1-YFP over-expressing line (bottom) treated for 5 min with FM4-64 (2 µM). FM4-64 derived fluorescence was quantified in the apical PM (region 1) and the apical cytoplasm (region 2) for <i>n</i>>25 PTs of each line. Note that there are more endocytotic and secretory vesicles in the apical cytoplasm of over-expressing PTs. See also corresponding <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001719#pbio.1001719.s018" target="_blank">Video S2</a> and (B). Scale bar = 5 µm. (B) Quantitative analysis of relative FM4-64 fluorescence in the apical PM <i>versus</i> the apical cytoplasm in growing PTs of one ANX1-YFP complemented and two over-expressing lines. Data are presented as mean values ± standard deviation (SD) (<i>n</i>>25 each). Double asterisks indicate significant differences from the complemented line according to a Student's <i>t</i> test with <i>p</i><0.01. (C) Representative time-course imaging of FRAP for a complemented (left) and an over-expressing growing PT (right). Refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001719#pbio.1001719.s019" target="_blank">Video S3</a> for more examples. Scale bar = 5 µm. (D) Quantitative analysis of FRAP time-courses of growing PTs of the complemented line (left, <i>n</i> = 18) and two over-expressing lines (right, <i>n</i>>17 for each). Relative intensity of apical PM-localized ANX1-YFP compared with fluorescence prior to photobleaching was used to quantify the rate of fluorescence recovery. FRAP signals are shown as mean values ± SD. The relative intensity after recovery for 10 s after photobleaching (I_10sec) is indicated. See also corresponding <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001719#pbio.1001719.s012" target="_blank">Table S1</a>.</p

ANX1-YFP over-expression phenotypes are dependent on functional <i>RbohH</i> and <i>RbohJ</i>.

<p>(A) Quantification of pollen germination and PT rupture for <i>rbohH-1 rbohJ-2</i>, ANX1-YFP in WT (line #4), ANX1-YFP in <i>anx1-2 anx2-2</i> (complemented line), and ANX1-YFP in <i>rbohH-1 rbohJ-2</i> plants. Data are mean ± standard error of the mean (SEM) of three independent experiments with more than 150 pollen grains per genotype and experiment. (B) Representative time-course imaging of FRAP for a <i>rbohH-1 rbohJ-2</i> PT expressing ANX1-YFP. Scale bar = 5 µm. (C) Quantitative analysis of FRAP time-courses for growing PTs of ANX1-YFP in <i>rbohH-1 rbohJ-2</i> (<i>n</i> = 24). Relative intensity of apical PM-localized ANX1-YFP compared with fluorescence prior to photobleaching was used to quantify the rate of fluorescence recovery. FRAP signals are shown as mean values ± standard deviation (SD). The relative intensity after recovery for 10 s after photobleaching (I_10sec) is indicated. See also corresponding <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001719#pbio.1001719.s012" target="_blank">Table S1</a>.</p

<i>rbohH rbohJ</i> mutant pollen display <i>anxur</i>-like phenotypes.

<p>(A) Quantification of pollen germination and PT rupture percentages (top histogram) and seed per siliques (bottom histogram) for WT, single, and double <i>rboh</i> as well as <i>anx1 anx2</i> mutant plants. Data are mean ± standard error of the mean (SEM) of three independent experiments with more than 150 pollen grains or ten siliques per genotype and experiment. (B) Representative overview images of WT and <i>rbohH rbohJ</i> pollen grown <i>in vitro</i> for 5 h. Up to 80% of germinated pollen from <i>rbohH rbohJ</i> ruptured with clear traces of cytoplasmic content that was released into the medium (top right), while the remaining germinated grains produce PTs that will burst later on (bottom right, arrowheads) as opposed to WT PTs that grow normally (bottom left). Scale bar = 50 µm. (C) Photographs of siliques from WT, <i>rbohH rbohJ</i>, and <i>anx1 anx2</i> plants. Scale bar = 500 µm. (D) Representative images of aniline blue staining of a WT pistil pollinated with WT pollen (left), a <i>rbohH rbohJ</i> pistil with WT pollen (middle), and a WT pistil with <i>rbohH rbohJ</i> pollen (right). Eighteen hours after manual pollination, WT PTs (left and middle panels) had grown through the entire pistil to reach the female gametophytes. In contrast, most of the <i>rbohH rbohJ</i> mutant PTs (right) were arrested in the transmitting tract. White arrows indicate the tip of the longest PT. Scale bar = 5 mm.</p

H₂O₂-sensitive HyPer sensor ratiometric imaging shows that RbohH and RbohJ are responsible for H₂O₂ production at the tip of growing pollen tubes.

<p>(A) Quantification of HyPer ratio (F₄₈₈/F₄₀₅) at the tip and further back in the shank of growing WT (<i>n</i> = 27) and <i>rbohH-1 rbohJ-2</i> (<i>n</i> = 22) PTs. Data are shown as the mean of ratios over 90 seconds±standard deviation (SD). Double asterisks indicate significant differences from the WT according to a Student's <i>t</i> test with <i>p</i><0.01. (B) Representative images of a growing WT PT expressing cytosolic HyPer and the corresponding histogram displaying the ratios (F₄₈₈/F₄₀₅) at the tip (blue line) and behind the tip (red line) over 90 s, as well as the travelled distance of the PT tip (green line). The blue and red parentheses indicate where the circles of 4 µm diameter were positioned for measurements at the tip and behind the tip, respectively. See corresponding <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001719#pbio.1001719.s020" target="_blank">Video S4</a>. Scale bar = 5 µm. (C) Representative images of growing <i>rbohH-1 rbohJ-2</i> PT expressing cytosolic HyPer. See (B) for details.</p