Figure 1.
Root hairs grow more slowly in xik mutants.
(a) Time series of DIC images of Col-0 and xik. Delay between images 6 min. Size bar indicates 15 μm. Young Col-0 root hairs (length < 200 µm) grew faster than those about to stop growing (length > 500 µm), compare first and third column. By contrast, young xik root hairs grew slowly and similarly to mature root hairs in Col-0 (compare second and third column).
(b) Comparison of root hair growth rates over 60 min. Dark green line shows WT root hair growth rate of young root hairs, while light green indicates long WT root hairs. Magenta line shows the growth rate of a young root hair of xik.
(c) Growth rates measured over 360 min reveal that root hairs in xik consistently grew more slowly than those of WT and eventually stopped growing earlier than in WT. Green line shows a root hair from a WT seedling, while magenta line indicates an individual root hair from a xik seedling.
(d) Total root hair length over time for a xik root hair (magenta line) and a wild type root hair (green line). Arrows indicate approximate time point of cessation of growth.
Figure 2.
Accumulation of RabA4b at the tip of growing root hairs is not significantly affected in xik mutants.
(a) Time series of YFP-RabA4b images of Col-0 and xik. Accumulation of YFP-RabA4b at the tip of growing root hairs fluctuated similarly in both Col-0 and in xik. Delay between images 1 min; also see Movie S1. Size bar indicates 10 μm.
(b) Fluctuation of normalized fluorescence intensity of YFP-RabA4b during 10 min. Green line indicates data from Col-0 while magenta line indicates xik. Intensity fluctuations in xik were similar to those in Col-0.
(c) Coefficients of variation of YFP-RabA4b accumulation in the tip. Every dot represents data from one root hair (n=15; Col-0, n=13; xik). Means of coefficients of variation were not significantly different in the two genotypes (t test, p > 0.05).
Figure 3.
Actin filament dynamics are reduced in xik root hairs.
(A) Actin filament organization as labeled with YFP-FABD2 is similar in xik and Col-0. Images are single frames from time-lapse observations in cells Col-3 and xik-3 in panel B, respectively.
(B) Actin filament activity in wild type (Col) and mutant (xik) root hair cells (see Movie S2) as determined by the decay of image cross-correlation with increasing time intervals (for details see Figure S2). Box-and-whiskers plot indicate median, 25th and 75th percentile, as well as minimal and maximal decay constants for the ten most active regions in a given cell. Mean activity levels in Col-0 are significantly higher than in xik (Mann-Whitney test; p < 0.01).
Figure 4.
YFP-XIK can rescue the short root hair phenotype in xik.
(A) Root hairs of XIKpro:YFP-XIK xik-3 seedlings displayed long root hairs similar to those of Col-0. Pictures were taken with five day old seedlings grown on vertical growth media solidified with phytagel.
(B) Lengths of mature root hairs. Root hairs of XIKpro:YFP-XIK xik-3 showed similar length as those of wild type (n=730 Col-0; n=370 xik-3; n=660 XIKpro:YFP-XIK xik-3). Error bars represent standard error of the mean.
(C) Average growth rate of root hairs in XIKpro:YFP-XIK xik-3 was similar to those of Col-0. Growth rates were measured in 10 second intervals (n=1350 Col-0; n=1080 xik-3; n=471 XIKpro:YFP-XIK xik-3). Error bars represent standard error of the mean.
Figure 5.
YFP-XIK accumulates at the tip of growing root hairs.
Stable transgenic plants of XIKpro:YFP-XIK in xik-3 background showed maximal accumulation of YFP signal in the subapex of growing root hairs. Weaker accumulation occurred in the extreme tip (arrow heads). YFP-XIK labeled vesicles are difficult to distinguish at the tip while they are easier to detect in the shank of root hairs (arrows). Images are representative frames from a 30 minute time-lapse observation (see Movie S3). Size bar indicates 10 μm.
Figure 6.
Treatment with the actin depolymerizing drug, LatB, disrupts YFP-XIK accumulation at the tip.
Root hairs expressing YFP-XIK (left column) or YFP-FABD2 (right column) were treated with 100 nM latrunculin B (LatB) during observation on the microscope (n=8 for YFP-XIK; n=10 for YFP-FABD2). DIC and fluorescence images were captured in 30 s intervals. YFP-XIK images were captured near the center of the root hair, whereas YFP-FABD2 images show the cortical actin filaments. Representative images from Movies S4 and S5 at the indicated times are shown. Size bar for A-C: 10 µm.
(A) Prior to treatment, YFP-XIK accumulated near the tip and actin filaments formed many, primarily longitudinal bundles in the shank. Computational detection of actin filaments is shown below the fluorescence image. Note that this actin marker does not label the fine actin mesh in the apical region of the root hair.
(B) Within 1.5 min of LatB treatment, YFP-XIK intensity in the tip decreased while actin cables showed first signs of disorganization with the appearance of short filaments in the apical region. At this time, large vacuoles were seen to approach the tip (e.g. left DIC image).
(C) After 5 min of LatB treatment, YFP-XIK was largely absent from the tip and formed larger clusters in the subapical and shank regions. YFP-FABD2 cables were still present in the shank and also were present in the tip region of the root hair. These areas were separated by a region with no or few actin filaments and low fluorescence intensity.
(D) Kymograph of DIC images revealing growth of the root hair. Total height of the image represents 17 min (YFP-XIK) and 20 min (YFP-FABD2), respectively. Note that growth of the root hair stopped around 5 min after LatB application (arrow).
(E) Kymograph of fluorescence images. YFP-XIK fluorescence was strongest near the tip before LatB application (arrow) but then dropped quickly in the tip region. By contrast, YFP-FABD2 signal in the tip was low prior to treatment but increased in the presence of LatB. Curved white lines indicate position of the root hair tip.
(F) Quantitative analysis of YFP-XIK and YFP-FABD2 accumulation in the tip. Solid lines indicate fluorescence intensities of the two markers within the first 2 µm of the root hair over time. Dashed line in right graph indicates the distance of the front-most actin filament from the tip (see lower panels in A-C). Vertical dotted line indicates start of LatB treatment during which root hair shifted and focus had to be reestablished.
Figure 7.
YFP-XIK partially colocalized with CFP-RabA4b and CFP-RHD4 at the tip of the growing root hairs.
First row of each panel shows YFP-XIK (green) localization in the root hairs while the second row presents other markers in the same root hairs (magenta). Merged images are shown in the third row. To compare localizations, pixel intensity along the root hair center (yellow dashed line in A) of each merged image was visualized in the fourth row. Signal intensities along the first 15 μm near the tip (white dashed line in B) are shown to emphasize the distribution of the two proteins near the tip of root hairs (fifth row). Scale bar indicates 10 μm.
(a) Partial colocalization of CFP-RabA4b and YFP-XIK. Although CFP-RabA4b and YFP-XIK localized at the tip of root hairs, CFP-RabA4b proteins accumulated closer to the apex while YFP-XIK signal was maximal in the subapex of the root hairs. Also compare Movie S6.
(b) Partial colocalization of CFP-RHD4 and YFP-XIK. CFP-RHD4 labeled vesicles accumulated further away from the root hair tip than YFP-XIK which resulted in partial overlap with the YFP-XIK signal.
(C) mCherry-ROP2 localization in cytosol and plasma membrane. Localization of YFP-XIK was clearly distinguishable from both inactive mCherry-ROP2 in the cytosol and active mCherry-ROP2 at the plasma membrane.
(D) Distribution of cytosolic mCherry was different from YFP-XIK localization at the root hair tip suggesting that accumulation of YFP-XIK at the tip did not reflect free diffusion.
Figure 8.
ROP2 recruitment to the plasma membrane is impaired in xik root hair tips.
(A) Fluorescence images of mCherry-ROP2 distribution in Col-0 and xik root hairs. Size bar represents 15 µm.
(B) Quantification of ROP2 recruitment to the plasma membrane in growing root hairs. Plasma membrane (PM) signal relative to cytosolic (CS) signal in representative root hairs of Col-0 (green) and xik (magenta). Measurements were taken in 5 s intervals for 3 minutes.
(C) Average PM/CS ratio for root hairs from different genotypes. Every dot represents average PM/CS ratio of a single root hair over a 3 min period. Some of the Col-0 root hairs expressed YFP-ROP2 instead of mCherry-ROP2.