BACKGROUND
cell expansion and division are fundamental processes in all organisms.
they require a constant supply of proteins and lipids to generate a new plasma membrane and cell wall, and also the retrieval of excess membrane and recycling protein fractions by endocytotic processes which allow to maintain the membrane homeostasis in the cell  <cit> .
these highly dynamic events are orchestrated by several regulators of vesicle trafficking.
among such factors, small gtpases of the rab family hold a central role in plant cells  <cit> .
rab gtpases are members of the ras small gtpase superfamily.
rab gtpases cycle between an active gtp-bound and membrane associated form and an inactive, gdp-bound and predominantly cytosolic form, and are involved in membrane identity and specificity of vesicle targeting during vesicular trafficking, tethering and fusion  <cit> .
mechanistically, rab gtpases execute the kinetic proofreading of specific membrane surfaces via their reversible and gtp/gdp-dependent association/dissociation with membranes.
in their active, gtp-bound form, rab gtpases are engaged into indirect interactions with coat components, motor proteins and snares.
thus, rab gtpases emerged as multifaceted organizers in membrane trafficking processes in eukaryotic cells  <cit> .
the family of rab gtpases is particularly expanded in the arabidopsis genome with  <dig> individual members classified in  <dig> subfamilies .
the most extensive subfamily in plants  is raba  encompassing  <dig> of the total  <dig> identified arabidopsis rabs  <cit> .
bearing in mind that by comparison to arabidopsis, mammalian  and yeast  raba homologues are much less represented  <cit> , it is a question to understand the specific functions of individual rabas in plants.
in this sense, it is important to survey plant raba diversity in terms of protein – protein interactions, specificity in intracellular localization patterns and possible differential developmental regulation at the tissue and organ level.
previous studies have already illustrated the polar localization of some rabas  in tip-growing cells, and its significance in tip-targeted cell wall deposition during elongation of pollen tubes and root hairs.
the role of raba4b in polarized vesicular secretion of cell wall material is associated with the activity of effector protein pi-4kβ <dig> and can be affected by disruption of actin microfilaments  <cit> .
the role of plant rabas is not restricted to polar growth but is also associated with cytokinesis.
in plants, the spatially and temporally-controlled post-mitotic partitioning of daughter cells through the cytokinetic deposition of cell plate is a process which is heavily dependent on membrane trafficking and endocytosis  <cit> .
starting with the preprophase definition of cell division plane, where the microtubule preprophase band marks a site of intensive clathrin-mediated membrane retrieval  <cit> , the cytokinetic process of cell plate deposition is robustly associated with endocytotic-related deposition of wall material by cell plate targeting of vesicles identified through the rab <dig> arabidopsis orthologue ara <dig>  <cit> .
cytokinetic progression in arabidopsis also recruited raba <dig>  raba <dig> and raba1c which colocalized with fm4- <dig> and partially with vacuolar h+-atpase subunit a <dig>  in early endosomes and tgn  <cit> .
the relative contribution of endocytosis during cell plate formation is not completely understood, however, several observations suggest its essential role.
cell surface materials and exogenously applied endocytic tracers were rapidly delivered to the forming cell plate  <cit> , while the knolle syntaxin localized to endosomes previous to cell plate initiation and its localization in the plane of cell division involves endocytotic-related proteins  <cit> .
some of these proteins use a clathrin-mediated mechanism  <cit>  and their mutations confirm in vivo the role in cytokinesis  <cit> .
similarly, other rab-gtpases showed to be involved in endocytotic processes, such as rabf2a, rabf2b and rabf <dig> which are activated by vps9a  <cit>  and are localized in both early but preferentially in late/multivesicular endosomes  <cit> .
the role of rab gtpases is not restricted to endocytosis but has been also suggested in secretory trafficking .
secretory roles may be also attributed to raba subfamily members since some of them were reported to localize in specific tgn compartments at the nexus of endocytosis and secretion  <cit> .
such tgn compartments were further corroborated by their aggregation following treatment with concanamycin a, an inhibitor of vacuolar h+−atpases  <cit>  and their insensitivity to wortmannin .
moreover, raba2a and vha-a <dig> are mislocalized in the echidna  mutant of arabidopsis thaliana, whereby endocytosis is unaffected but secretory trafficking is impaired  <cit> .
finally, the remarkable colocalizations of raba1b with vamp <dig>  r- soluble n-ethyl-maleimide sensitive factor attachment protein receptor , but partial and unstable associations with tgn, golgi and endosome markers suggest a specific role in transport between tgn and the plasma membrane  <cit> .
our study reports the subcellular localization of raba1d and its role in vesicle trafficking.
using light-sheet microscopy ensuring cell viability and stress-free root development during imaging, we found that raba1d is involved in two essential cellular processes, cell plate expansion during cytokinesis and oscillatory tip growth in root hairs, representing highly active vesicle trafficking events.
RESULTS
gfp-raba1d localizes to vesicle-like structures at trans-golgi network compartment
in order to follow the localization patterns of raba1d, we engineered a gfp-raba1d expressed under cauliflower mosaic virus 35s promoter.
specificity of gfp-raba1d localization was tested by transient expression of 35s::gfp:raba1d construct in allium porrum and nicotiana benthamiana  and was confirmed in seedlings of arabidopsis thaliana stably transformed with the same construct .
the expression of the fusion protein was verified by western blotting with a monoclonal antibody against gfp showing a single band at ca.
46 kda, corresponding to the molecular weight of the gfp-raba1d fusion .figure  <dig>
subcellular localization of gfp-tagged raba1d.
subcellular localization of gfp-raba1d in cells of n. benthamiana.
co-vizualization with er reporter er-dsred  showed partial association of gfp-raba1d and cortical er tubules.
gfp-raba1d colocalized with neither 2xfyve-dsred  nor with mcherry-rabf2a , markers for late endosomes/multi-vesicular bodies.
colocalization of gfp-raba1d with mcherry-vti <dig> representing a tgn marker .
intensity correlation scatterplots of gfp-rab1ad and er-dsred , fyve-dsred , mcherry-rabf2a , and mcherry-vti <dig> .
pearson’s coefficient  was determined after costes automatic threshold.
bars represent 3 μm in a-c and 5 μm in d-l.
in both cases of transient transformation, gfp-raba1d localized to spot-like structures that were rapidly moving through the cytoplasm .
moreover, the localization of gfp-raba1d was cross-compared with endomembrane markers of known specificity including endoplasmic reticulum , late endosomes and prevacuolar compartments  and trans-golgi network /early endosomes .
gfp-raba1d did not colocalize with er-dsred, however, motile spot-like structures containing gfp-raba1d appeared in close proximity to er tubules .
the observation of the co-expression of fyve-dsred and mcherry-rabf2a with gfp-raba1d revealed no significant colocalization with the late endosomal markers .
interestingly, an obvious colocalization was observed for gfp-raba1d and mcherry-vti <dig>  a tgn marker.
this colocalization showed good quantitative correlation; thereby, raba1d is localized at the tgn compartments .
gfp-raba1d accumulates in bfa compartments and is upregulated by bfa treatment
the above results indicated a localization of gfp-raba1d in tgn/early endosomes as expected for many other members of raba class.
to better substantiate this localization pattern, arabidopsis seedlings stably expressing the gfp-raba1d fusion were co-stained with the membrane/endocytotic tracer fm4- <dig>  <cit> , which depending on the immediacy of microscopic observation, localizes fully or partially with early endosomes such as those labeled with fluorescent protein-tagged vti <dig> .
in this case, the gfp-raba1d vesicles colocalized with early fm4- <dig> compartments of the endocytotic pathway within 6–15 min after application of the dye .
it was additionally confirmed by comparison with yfp-rabf2a late endosomal marker which showed partial colocalization with fm4- <dig> compartments only after 15 min .
next, fm4- <dig> stained roots were treated with bfa, a fungal toxin that inhibits exocytosis and endocytotic recycling without affecting the first steps of endocytosis  <cit> .
importantly, after treatment with bfa, gfp-raba1d relocalized and accumulated in the core of bfa-compartments along with fm4- <dig> .
these bfa-compartments are composed of tgn and plasma membrane-derived endocytotic vesicles in the core, surrounded by remnants of golgi stacks  <cit> .
the colocalization of gfp-raba1d and fm4- <dig> showed good quantitative correlation and it was increased after bfa-treatment .
after bfa washout, the gfp-raba1d and fm4- <dig> compartments started to deliberate from bfa compartments within 5 min and progressively redistributed in the root cells.
importantly, both gfp-raba1d and fm4- <dig> compartments remained colocalized during the release from the bfa compartments .figure  <dig>
gfp-raba1d accumulates in bfa compartments and is upregulated by bfa treatment.
root cells of arabidopsis stably transformed with 35s::gfp:raba1d construct were analysed.
gfp-raba1d colocalized with early endocytotic compartments labeled by fm4- <dig> .
after bfa treatment, both gfp-raba1d and fm4- <dig> accumulated together in the core of bfa compartments .
2d-histogram intensity and correlation of gfp-rab1ad and fm4- <dig> early endocytotic compartments in root cells  and after bfa treatment .
pearson’s coefficient  was determined using costes automatic threshold.
bfa treatment induced raba1d upregulation at protein level , upregulation of raba1d was determined from comparison of 2-de gels  and measured as increase of spot density .
bars represent 4 μm in a-c and 5 μm in d-f.
a proteomic analysis of bfa-treated arabidopsis roots, showed the quantitative upregulation of raba1d protein levels.
this induction reached  <dig>  fold , however it slightly exceeded the significance level .
raba1d identity was confirmed by a mowse score of  <dig> and 25% sequence coverage with  <dig> peptides matching .
therefore, raba1d is involved in vesicle trafficking, its expression and localization in tgn/early endosomes is affected by bfa.
gfp-raba1d accumulates in growing cell plates during cytokinesis
in dividing root meristematic cells, gfp-raba1d specifically accumulated at the plane of cell plate deposition following similar centrifugal expansion observed for the microtubular phragmoplast.
thus, at the onset of cytokinesis and during the early stages of cell plate formation, gfp-raba1d uniformly labeled the entire cell plate forming a thin continuous line when visualized cross-sectioned and fully co-localized with the fm4- <dig> marker .
at more advanced stages, gfp-raba1d signal was restricted to the margins of the growing cell plate unmixing from fm4- <dig> labeling at the central region of the cell plate .figure  <dig>
localization of gfp-raba1d during the cell plate formation in cytokinetic cells.
during cell plate initiation, gfp-raba1d localized in the mid plane of the dividing cell and completely colocalized with fm4- <dig> .
in the early stage of growing cell plates, gfp-raba1d still appeared in the mid-plane of the cell .
during cytokinesis, gfp-raba1d was accumulated mainly at the edges of growing cell plate .
intensity profile of gfp-raba1d and fm4- <dig> signals during cell plate initiation and early stage of plate expansion , the two channels showed similar intensity and distribution; while in growing cell plate, gfp-raba1d showed more intensity at the edges of growing cell plate than fm4- <dig> .
bars represent 2 μm.
the cytokinesis-related localization patterns of gfp-raba1d were further followed by light-sheet microscopy which allows the observation of a physiological, unstressed root growth  <cit> .
the cells undergoing cytokinesis in the meristem showed several locations of gfp-raba1d accumulation .
the accumulation patterns of gfp-raba1d coincided with areas of active vesicle fusion during cell plate formation and growth.
in the initiating young cell plates, disc-like structures were observed, starting from diffuse expression probably during accumulation of vesicles and increased intensity during fusion of vesicles .
during cell plate expansion, it formed a ring-like structure at the margins of the growing cell plates .
the localization in the growing cell plate of gfp-raba1d during cytokinesis suggests a role in delivery of membranes/cargo for cell plate formation and membrane recycling at margin domains .
the growth rate of cell plates labeled with gfp-rab1ad was comparable to that observed with gfp-map <dig> .
however, significant differences were found between epidermis and pericycle cells expressing gfp-rab1ad .figure  <dig>
light-sheet live imaging of gfp-raba1d accumulation during cell plate formation in the root meristem.
meristematic cells undergoing cytokinesis showed several locations of gfp-raba1d accumulation observed in single sections of one time point at indicated depths inside the primary root , gfp-raba1d accumulation in cell plates  of cytokinetic cells.
the boxed area in a is enlarged shown in b. detailed time-lapse imaging of gfp-raba1d accumulation during single cell plate formation starting from spot like structures in the middle of the cell, followed by increased intensity during vesicle fusion and later formation of a ring-like structure at the margins in the growing cell plates .
three-dimensional reconstruction of the cell plate shown in  at time point 20 min .
comparison of cell plate growth rates in arabidopsis plants stably transformed with in 35s::gfp:raba1d and 35s::gfp:map <dig> constructs  .
growth rates of cell plates in cells from different tissues .
*significant difference from wilcoxon rank sum test with holm’s correction , ns: no significant difference.
bars represent 50 μm in a and 10 μm in b.
it is important to note that the overexpression of gfp-raba1d had no serious consequences for plant growth and gravitropic response when transgenic plants were compared with non-transformed wild type plants .
oscillatory accumulation of gfp-raba1d in tips of growing root hairs correlates with growth rate
root hair growth is a robust example of tip-growth in plant cells, accompanied by polarized membrane trafficking .
a previous study demonstrated the localization of raba1d in the apical dome of growing root hairs  <cit> , however, without following this localization pattern in time.
for this reason, we extended the microscopic documentation of gfp-raba1d in relation to root hair growth kinetics by means of light-sheet microscopy, which allowed following the distribution of gfp-raba1d for a considerable time encompassing the entire process of root hair growth, from emergence to cessation of elongation.
as previously shown  <cit> , gfp-raba1d labeling was predominantly observed in trichoblast outgrowths  representing emerging root hairs  and subsequently was largely restricted at the apical dome of vigorously growing root hairs .
however, such accumulation of gfp-raba1d containing vesicles was not visible at the tips of non-growing mature root hairs.
in bulges and growing root hairs, gfp-raba1d colocalized with the endocytotic marker fm4- <dig> in apical domains , but in mature root hairs both gfp-raba1d and fm4- <dig> were more or less equally distributed through the hair tube .figure  <dig>
light-sheet live imaging of gfp-raba1d accumulation during root hair oscillatory tip growth.
maximum-intensity projections of two indicated time points .
the actively growing root hairs showed a higher tip-accumulation  than the slowly expanding or mature root hairs .
the tip-accumulation of gfp-raba1d increased in steady state actively growing root hairs .
the pixel line from b is plotted as a function of time .
the kymograph exhibited an oscillating behavior between lower and higher intensity .
fluorescence intensity measurements significantly correlated with the growth rates of the root hair .
bars represent 100 μm in a and 10 μm in b.
tip-accumulation of gfp-raba1d was observed in root hair bulges and growing root hairs being progressively reduced as root hair growth rates declined .
the time-lapse imaging showed gfp-raba1d accumulation in bulge stage which was increased in a time dependent manner .
as shown by fluorescence intensity profiles transverse to the root hair apical dome, gfp-raba1d signal intensity oscillated between lower and higher intensities which significantly correlated with the pause and growing phases of root hair growth, respectively .
cross correlation coefficient calculations of root hair growth and gfp-raba1d intensity for particular periods of time, e.g.
between 150–250 min, revealed r <dig>  values of  <dig> , reflecting a strong temporal correlation between apical raba1d accumulation and tip growth .
the mode of oscillatory growth of root hairs was confirmed also in transgenic arabidopsis line expressing stress-induced mitogen activated protein kinase kinase tagged with yfp .
simkk-yfp accumulated in nuclei and cytoplasm of root cells.
observation using light sheet microscopy and determination of fluorescence intensity along transverse profile of growing root hairs showed the oscillatory pattern of root hair growth .
this observation corroborated an assumption that raba1d participates in oscillatory cell expansion related to intensive and spatio-temporaly controlled vesicle trafficking in root hairs.
movement of the vesicular compartments containing gfp-raba1d depends on the actin cytoskeleton
the maximum speed of the raba1d-positive tgn vesicles was measured from kymographs of time-lapse image sequences from root hairs.
this analysis revealed that gfp-raba1d vesicles moved with an average speed of about  <dig>  μm/s in root hairs .figure  <dig>
motility of gfp-raba1d vesicles in growing root hairs.
kymograph showing the motility of the gfp-raba1d vesicles in root hairs .
estimation of the average speed of the gfp-raba1d vesicles in root hairs treated with acto-myosin inhibitors, latrunculin b  and butane-dione-monoxime , respectively .
mobility and distribution of small raba1d-positive tgn vesicles in diverse root tissues observed by light-sheet microscopy .
3d reconstruction of a stack of images .
single sections at different z-depths : z = 0 μm, : z =  <dig>  μm, : z =  <dig>  μm, : z = 50 μm) are depicted in .
the dashed boxed area is enlarged in .
bars represent 20 μm in d and 5 μm in e.
next, arabidopsis seedlings expressing gfp-raba1d were treated with cytoskeletal inhibitors such as oryzalin , latrunculin b  and butane-dione monoxime .
previously, we observed that the motility of gfp-raba1d compartments in trichoblast cells was not affected by oryzalin-induced microtubule disruption by oryz .
thus, we used acto-myosin drugs in root hairs to better characterize the movement of gfp-raba1d compartments along the actin cytoskeleton.
latrunculin b disrupts the actin cytoskeleton by its binding to monomeric g-actin subunits while bdm is affecting myosin by inhibiting its atpase function.
after treatment with latrunculin b, the gfp-raba1d containing tgn vesicles significantly slowed down and/or stopped their movements through the cytoplasm within 15 min.
a similar effect was found after inhibition of vesicle movements using bdm .
these data indicated that motility of the gfp-raba1d compartments is acto-myosin dependent.
owing to the extended depth of imaging by means of light sheet microscopy, the high mobility of small raba1d-positive tgn vesicles was observed in diverse root tissues such as epidermis, cortex, endodermis, pericycle and central cylinder cells.
cells in all these tissues clearly showed presence of the vesicles confirming the role of raba1d in membrane trafficking in different cells and suitability of light sheet microscopy for deep imaging in thick biological probes .
discussion
in this study, we reported the subcellular localization of gfp-tagged raba1d in transiently and stably transformed plant cells, describing in detail the involvement of raba1d in active and highly regulated vesicle trafficking events essential for cytokinesis of root meristematic cells and polarized cell expansion of root hairs.
raba1d localizes to the early endosome/tgn and is involved in vesicle trafficking
cellular distribution and localization pattern of gfp-raba1d found in both transiently transformed leaf cells and stably transformed root cells proved gfp-raba1d localization to mobile vesicle-like structures, in accordance with the role of the small gtpases in vesicle trafficking.
gfp-raba1d-positive vesicular structures were recognized as tgn compartments, since they were co-labeled with mcherry-vti <dig>  a snare-protein marker which accumulates at the tgn  <cit> .
additionally, gfp-raba1d vesicles colocalized with fm4-64-positive compartments that are labeled by this vesicular/endocytotic marker soon after entering cells.
merging and co-association at tgn compartment was shown also for other members of raba class, raba <dig>  raba <dig> and raba1c which partially overlapped also with the vha-a <dig> subunit  <cit> .
in tgn compartment, qa-snare group members localize in a complex   <cit>  and all these compartments are stained by fm4- <dig> before this dye reaches rabf <dig> or gnom- containing compartments  <cit> .
thus, the localization pattern of gfp-raba1d suggests its specific role during first stages of endocytosis and rapid vesicular trafficking, but not in the process of endosome maturation, because no colocalization with late endosomal markers fyve-dsred or mcherry-rabf2a was found.
in this respect, the fyve-domain binds specifically to the phosphoinositol-3-phosphate , which accumulates mainly in the membranes of late endosomal compartments  <cit> .
fyve domain marker colocalized also with rabf2a and rabf <dig>  today recognized as multivesicular or late endosomal rab gtpase markers  <cit> .
the colocalization of gfp-raba1d with early fm4- <dig> compartments was confirmed by microscopic analysis.
in comparison to rabf2a, another rab-gtpase that preferentially localize to late/multivesicular endosomes  <cit> , gfp-raba1d colocalized with fm4- <dig> compartments within minutes upon fm staining.
partial colocalization of yfp-rabf2a with fm4- <dig> was recorded only later.
the bfa treatment of transgenic plants expressing gfp-raba1d and co-stained with fm4- <dig> resulted in common relocation and accumulation of both markers in the core of bfa-compartments in root cells.
since it is known that bfa-induced compartments are composed of tgn and plasma membrane-derived endocytotic vesicles in a core, surrounded by remnants of golgi stacks  <cit> , this localization pattern confirms the residence of raba1d in early endosome/tgn compartments.
moreover, it is suggestive of the role of raba1d in the regulation of vesicle recycling, as this process is effectively inhibited by bfa treatment  <cit> .
the above suggestion is further supported by the quantitative upregulation of raba1d after bfa treatment as revealed by proteomic analysis.
the localization and activity of raba1d at tgn compartment  <cit>  and its responsiveness to bfa suggest a role in vesicle trafficking.
involvement in endocytosis and vesicular recycling has been reported also for other members of raba class.
raba1c, which is localized at the tgn compartment, is sensitive to endosidin <dig> but not to wortmannin  <cit> .
endosidin <dig> is an inhibitor of endocytosis and vesicular recycling  <cit> , while wortmannin inhibits the transport to vacuoles by affecting pre-vacuolar compartments, tgn vesicles and mvbs  <cit> .
for instance, the effect of endosidin <dig> on bri <dig>  pin <dig> and aux <dig> recycling involves also the mislocalization of early endosome/tgn-compartments, defined by syp61/vha-a <dig>  <cit> .
similar defects have been observed by the inhibition of vacuolar h+-atpases with concanamycin a .
cona blocks the endosome maturation, which leads to production of aggregates of tgn-derived vesicles and therefore, it interferes with the formation of bfa-compartments  <cit> .
the mislocalization of tgn proteins vha-a <dig>  raba2a and syp <dig> was also found in the ech mutant, lacking a tgn-localized echidna protein crucial for tgn function.
this mutant is, however, affected rather in secretion with endocytosis being more or less intact  <cit> .
it raises the question on possible role of raba1d also in a secretory pathway, which would be parallel to the function of raba1b in secretory pathway from tgn to the plasma membrane  <cit> .
raba1b co-localize with vamp721/ <dig>  r-snare proteins that operate in the secretory pathway and only partially and unstably associate with tgn, golgi and endosomes  <cit> .
however, the relocalization of raba1b to plasma membrane upon wortmannin treatment  <cit>  differs from the accumulation, clustering, fusion or swelling of tgn and mvb compartments, observed by fluorescently tagged raba1d, raba4b, raba1e, vti <dig>  fyve and rabf2a  <cit> .
under wortmannin treatment, raba1d is relocated into wortmannin-induced multivesicular compartments and is downregulated  <cit> .
thereby, while the plant counteracts the inhibition of endocytotic recycling by raba1d upregulation, the inhibition of prevacuolar transport by wortmannin induces raba1d downregulation  <cit> .
thus, raba1d has an important role in vesicle trafficking  consistent with its location at early endosome/tgn compartment.
raba1d is accumulating in highly active vesicle trafficking zones during cytokinesis and root hair tip-growth
cell plate arises from the accumulation, fusion and stretching of vesicles directed by centrally-localized complex of cell plate assembly matrix .
these vesicles are delivered to the growing cell plate by phragmoplast microtubules  <cit> , and cell plate expansion is substantially supported by machineries of both secretory and endocytotic pathways  <cit> .
in the growing cell plate, a ring-like structure representing highly active zone of vesicle trafficking is formed  <cit> .
during the initial stages of cell plate formation, gfp-raba1d accumulated in disc-like structure in the equatorial plane, exhibiting a steady increase of fluorescence intensity likely due to the continuous vesicle fusions.
consistently with the spatial redistribution of active zones of vesicular trafficking during cell plate expansion  <cit> , gfp-raba1d relocated labeling the rim of the growing cell plates.
the localization of gfp-raba1d during cytokinesis suggests a role in delivery of cargos and membranes during cell plate formation.
it is also compatible with the supporting role of raba1d in active membrane recycling at cell plate margin domains and consistent with the accumulation of raba <dig> and raba <dig> subclasses during cell plate formation.
interestingly, they colocalize in the cell plate with the cytokinesis-specific syntaxin knolle   <cit> .
redundant cytokinetic roles have been assigned to three other rabas, namely raba1a, raba1b and raba1c.
these were mislocalized following endosidin <dig> treatment, while triple knockout mutants  were hypersensitive to endosidin <dig> induced cytokinetic disruption  <cit> .
similarly, the cytokinetic-defective mutant pas <dig> , showed delayed and defective cell plate formation due to the heterogeneous aggregation of raba2a and knolle, while rabf2a did not show any defect.
thus, early endosomes/tgn compartments play a specific role during cell plate formation  <cit> .
the transport between tgn and the plasma membrane by raba1b interacting with vamp <dig>  <cit>  is involved in cytokinesis and the inhibition of trafficking at tgn by cona affects cell plate formation through the mislocalization of early endosomes/tgn compartments  <cit> .
additionally, the colocalization of raba1d with vti <dig>  a member of snare complex, suggests a role also in the transport to the plasma membrane  <cit> .
thereby, the role of raba1d during cell plate formation is consistent with the proposed activity of early endosome/tgn compartments in the coordination of secretory, endocytotic and recycling pathways.
similarly to cytokinesis, raba1d participates in localized cell expansion where increased vesicle trafficking is required.
the tip-accumulation of raba1d in root hair bulges and in growing root hairs confirmed the role of some rabas during polar growth.
the disruption of raba4d in pollen tubes reduced cell polarity, expansion and displayed altered deposition of cell wall components  <cit> .
raba4b showed tip-localization and regulated root hair tip growth through a compartment involved in the polarized secretion, and similarly to raba1d, the tip-localized accumulation disappears in mature root hairs or after latrunculin b treatment  <cit> .
the raba <dig> silencing led to reduction in number and length of the root hairs, showing an important role not only in expansion but also during root hair initiation  <cit> .
the raba1d accumulation in bulge stage was increased during the progression to the tip growth.
in growing root hairs, gfp-raba1d signal intensity fluctuated in oscillatory manner.
oscillations between lower and higher intensity labeling of gfp-raba1d at the root hair tip temporally coincided with the pause and growth phases of root hair tip growth, respectively.
similarly to pollen tubes  <cit> , root hairs exhibit oscillatory growth along with other events accompanying tip growth like cell wall modifications, cytoskeleton rearrangements and targeted vesicle trafficking  <cit> .
some factors, such as ca2+ gradient  <cit> , extracellular ph and ros concentration  <cit>  and actin organization  <cit>  have been found to be associated with the oscillatory character of tip growth and are closely related to vesicle trafficking.
consistently with the frequency of vesicular trafficking during root hair initiation and growth, gfp-raba1d accumulation was lower at bulge stage than in growing root hairs.
after the local structural changes that are required to define the bulge position, such as cytoskeleton rearrangements  <cit> , cell wall composition  <cit>  and accumulation of structural sterols in the plasma membrane  <cit> , an increased vesicle trafficking is required during root hair elongation to provide a new plasma membrane in the expanding zone.
this zone is filled with secretory and endocytotic vesicles  <cit> , highly dynamic early endosomes in the clear zone and larger endosomal compartments in the subapical region  <cit> .
thereby, raba1d is localized at the tip-zone of growing root hair as previously reported  <cit>  and its accumulation is spatio-temporally correlated with the root hair tip expansion and vesicle trafficking.
high motility of tgn vesicles containing gfp-raba1d depends on the actin cytoskeleton
endosomes show different patterns of motility including stationary phases, slow or rapid movements  <cit> .
the microtubule disruption by oryzalin did not affect the motility of raba1d-positive vesicles, however the acto-myosin inhibitors affected extremely their motility.
similarly, the tip-localization of raba4b was affected by latrunculin b treatment but not by oryzalin  <cit> .
it suggests an important role of actin cytoskeleton in raba1d motility, however, the organization and dynamics of several endosomal structures is influenced by both actin and microtubule cytoskeletons  <cit>  and their motor proteins, i.e.
myosins  <cit> .
the role of the actin cytoskeleton in vesicular trafficking was strengthened by proteomic study after bfa treatment, which showed significant upregulation of profilin  <dig>  and its accumulation in bfa-compartments  <cit> .
finally, vigorous motility of raba1d-containing tgn vesicles was also observed in diverse root tissues such as epidermis, cortex, endodermis, pericycle and central cylinder cells confirming the role of raba1d in vesicular trafficking in different cells and not only associated to tip-growth.
CONCLUSIONS
we show that raba1d colocalized with early fm4- <dig> endocytotic compartments and with vti <dig>  a tgn marker.
bfa treatment not only induced raba1d upregulation but also relocalization of gfp-raba1d to the core of bfa-compartments.
most importantly, a specific spatio-temporal accumulation of raba1d correlated with vesicle trafficking requirements during both cell plate formation and root hair oscillatory tip growth.

 cell expansion and division are fundamental processes in all organisms. they require a constant supply of proteins and lipids to generate a new plasma membrane and cell wall, and also the retrieval of excess membrane and recycling protein fractions by endocytotic processes which allow to maintain the membrane homeostasis in the cell. these highly dynamic events are orchestrated by several regulators of vesicle trafficking. among such factors, small gtpases of the rab family hold a central role in plant cells. rab gtpases are members of the ras small gtpase superfamily. rab gtpases cycle between an active gtp-bound and membrane associated form and an inactive, gdp-bound and predominantly cytosolic form, and are involved in membrane identity and specificity of vesicle targeting during vesicular trafficking, tethering and fusion. mechanistically, rab gtpases execute the kinetic proofreading of specific membrane surfaces via their reversible and gtp/gdp-dependent association/dissociation with membranes. in their active, gtp-bound form, rab gtpases are engaged into indirect interactions with coat components, motor proteins and snares. thus, rab gtpases emerged as multifaceted organizers in membrane trafficking processes in eukaryotic cells. the family of rab gtpases is particularly expanded in the arabidopsis genome with individual members classified in subfamilies. the most extensive subfamily in plants is raba encompassing of the total identified arabidopsis rabs. bearing in mind that by comparison to arabidopsis, mammalian and yeast raba homologues are much less represented, it is a question to understand the specific functions of individual rabas in plants. in this sense, it is important to survey plant raba diversity in terms of protein – protein interactions, specificity in intracellular localization patterns and possible differential developmental regulation at the tissue and organ level. previous studies have already illustrated the polar localization of some rabas in tip-growing cells, and its significance in tip-targeted cell wall deposition during elongation of pollen tubes and root hairs. the role of raba4b in polarized vesicular secretion of cell wall material is associated with the activity of effector protein pi-4kβ and can be affected by disruption of actin microfilaments. the role of plant rabas is not restricted to polar growth but is also associated with cytokinesis. in plants, the spatially and temporally-controlled post-mitotic partitioning of daughter cells through the cytokinetic deposition of cell plate is a process which is heavily dependent on membrane trafficking and endocytosis. starting with the preprophase definition of cell division plane, where the microtubule preprophase band marks a site of intensive clathrin-mediated membrane retrieval, the cytokinetic process of cell plate deposition is robustly associated with endocytotic-related deposition of wall material by cell plate targeting of vesicles identified through the rab arabidopsis orthologue ara. cytokinetic progression in arabidopsis also recruited raba raba and raba1c which colocalized with fm4- and partially with vacuolar h+-atpase subunit a in early endosomes and tgn. the relative contribution of endocytosis during cell plate formation is not completely understood, however, several observations suggest its essential role. cell surface materials and exogenously applied endocytic tracers were rapidly delivered to the forming cell plate, while the knolle syntaxin localized to endosomes previous to cell plate initiation and its localization in the plane of cell division involves endocytotic-related proteins. some of these proteins use a clathrin-mediated mechanism and their mutations confirm in vivo the role in cytokinesis. similarly, other rab-gtpases showed to be involved in endocytotic processes, such as rabf2a, rabf2b and rabf which are activated by vps9a and are localized in both early but preferentially in late/multivesicular endosomes. the role of rab gtpases is not restricted to endocytosis but has been also suggested in secretory trafficking. secretory roles may be also attributed to raba subfamily members since some of them were reported to localize in specific tgn compartments at the nexus of endocytosis and secretion. such tgn compartments were further corroborated by their aggregation following treatment with concanamycin a, an inhibitor of vacuolar h+−atpases and their insensitivity to wortmannin. moreover, raba2a and vha-a are mislocalized in the echidna mutant of arabidopsis thaliana, whereby endocytosis is unaffected but secretory trafficking is impaired. finally, the remarkable colocalizations of raba1b with vamp r- soluble n-ethyl-maleimide sensitive factor attachment protein receptor, but partial and unstable associations with tgn, golgi and endosome markers suggest a specific role in transport between tgn and the plasma membrane. our study reports the subcellular localization of raba1d and its role in vesicle trafficking. using light-sheet microscopy ensuring cell viability and stress-free root development during imaging, we found that raba1d is involved in two essential cellular processes, cell plate expansion during cytokinesis and oscillatory tip growth in root hairs, representing highly active vesicle trafficking events.  gfp-raba1d localizes to vesicle-like structures at trans-golgi network compartment in order to follow the localization patterns of raba1d, we engineered a gfp-raba1d expressed under cauliflower mosaic virus 35s promoter. specificity of gfp-raba1d localization was tested by transient expression of 35s::gfp:raba1d construct in allium porrum and nicotiana benthamiana and was confirmed in seedlings of arabidopsis thaliana stably transformed with the same construct. the expression of the fusion protein was verified by western blotting with a monoclonal antibody against gfp showing a single band at ca. 46 kda, corresponding to the molecular weight of the gfp-raba1d fusion.figure subcellular localization of gfp-tagged raba1d. subcellular localization of gfp-raba1d in cells of n. benthamiana. co-vizualization with er reporter er-dsred showed partial association of gfp-raba1d and cortical er tubules. gfp-raba1d colocalized with neither 2xfyve-dsred nor with mcherry-rabf2a, markers for late endosomes/multi-vesicular bodies. colocalization of gfp-raba1d with mcherry-vti representing a tgn marker. intensity correlation scatterplots of gfp-rab1ad and er-dsred, fyve-dsred, mcherry-rabf2a, and mcherry-vti. pearson’s coefficient was determined after costes automatic threshold. bars represent 3 μm in a-c and 5 μm in d-l. in both cases of transient transformation, gfp-raba1d localized to spot-like structures that were rapidly moving through the cytoplasm. moreover, the localization of gfp-raba1d was cross-compared with endomembrane markers of known specificity including endoplasmic reticulum, late endosomes and prevacuolar compartments and trans-golgi network /early endosomes. gfp-raba1d did not colocalize with er-dsred, however, motile spot-like structures containing gfp-raba1d appeared in close proximity to er tubules. the observation of the co-expression of fyve-dsred and mcherry-rabf2a with gfp-raba1d revealed no significant colocalization with the late endosomal markers. interestingly, an obvious colocalization was observed for gfp-raba1d and mcherry-vti a tgn marker. this colocalization showed good quantitative correlation; thereby, raba1d is localized at the tgn compartments. gfp-raba1d accumulates in bfa compartments and is upregulated by bfa treatment the above results indicated a localization of gfp-raba1d in tgn/early endosomes as expected for many other members of raba class. to better substantiate this localization pattern, arabidopsis seedlings stably expressing the gfp-raba1d fusion were co-stained with the membrane/endocytotic tracer fm4-, which depending on the immediacy of microscopic observation, localizes fully or partially with early endosomes such as those labeled with fluorescent protein-tagged vti. in this case, the gfp-raba1d vesicles colocalized with early fm4- compartments of the endocytotic pathway within 6–15 min after application of the dye. it was additionally confirmed by comparison with yfp-rabf2a late endosomal marker which showed partial colocalization with fm4- compartments only after 15 min. next, fm4- stained roots were treated with bfa, a fungal toxin that inhibits exocytosis and endocytotic recycling without affecting the first steps of endocytosis. importantly, after treatment with bfa, gfp-raba1d relocalized and accumulated in the core of bfa-compartments along with fm4-. these bfa-compartments are composed of tgn and plasma membrane-derived endocytotic vesicles in the core, surrounded by remnants of golgi stacks. the colocalization of gfp-raba1d and fm4- showed good quantitative correlation and it was increased after bfa-treatment. after bfa washout, the gfp-raba1d and fm4- compartments started to deliberate from bfa compartments within 5 min and progressively redistributed in the root cells. importantly, both gfp-raba1d and fm4- compartments remained colocalized during the release from the bfa compartments.figure gfp-raba1d accumulates in bfa compartments and is upregulated by bfa treatment. root cells of arabidopsis stably transformed with 35s::gfp:raba1d construct were analysed. gfp-raba1d colocalized with early endocytotic compartments labeled by fm4-. after bfa treatment, both gfp-raba1d and fm4- accumulated together in the core of bfa compartments. 2d-histogram intensity and correlation of gfp-rab1ad and fm4- early endocytotic compartments in root cells and after bfa treatment. pearson’s coefficient was determined using costes automatic threshold. bfa treatment induced raba1d upregulation at protein level, upregulation of raba1d was determined from comparison of 2-de gels and measured as increase of spot density. bars represent 4 μm in a-c and 5 μm in d-f. a proteomic analysis of bfa-treated arabidopsis roots, showed the quantitative upregulation of raba1d protein levels. this induction reached fold, however it slightly exceeded the significance level. raba1d identity was confirmed by a mowse score of and 25% sequence coverage with peptides matching. therefore, raba1d is involved in vesicle trafficking, its expression and localization in tgn/early endosomes is affected by bfa. gfp-raba1d accumulates in growing cell plates during cytokinesis in dividing root meristematic cells, gfp-raba1d specifically accumulated at the plane of cell plate deposition following similar centrifugal expansion observed for the microtubular phragmoplast. thus, at the onset of cytokinesis and during the early stages of cell plate formation, gfp-raba1d uniformly labeled the entire cell plate forming a thin continuous line when visualized cross-sectioned and fully co-localized with the fm4- marker. at more advanced stages, gfp-raba1d signal was restricted to the margins of the growing cell plate unmixing from fm4- labeling at the central region of the cell plate.figure localization of gfp-raba1d during the cell plate formation in cytokinetic cells. during cell plate initiation, gfp-raba1d localized in the mid plane of the dividing cell and completely colocalized with fm4-. in the early stage of growing cell plates, gfp-raba1d still appeared in the mid-plane of the cell. during cytokinesis, gfp-raba1d was accumulated mainly at the edges of growing cell plate. intensity profile of gfp-raba1d and fm4- signals during cell plate initiation and early stage of plate expansion, the two channels showed similar intensity and distribution; while in growing cell plate, gfp-raba1d showed more intensity at the edges of growing cell plate than fm4-. bars represent 2 μm. the cytokinesis-related localization patterns of gfp-raba1d were further followed by light-sheet microscopy which allows the observation of a physiological, unstressed root growth. the cells undergoing cytokinesis in the meristem showed several locations of gfp-raba1d accumulation. the accumulation patterns of gfp-raba1d coincided with areas of active vesicle fusion during cell plate formation and growth. in the initiating young cell plates, disc-like structures were observed, starting from diffuse expression probably during accumulation of vesicles and increased intensity during fusion of vesicles. during cell plate expansion, it formed a ring-like structure at the margins of the growing cell plates. the localization in the growing cell plate of gfp-raba1d during cytokinesis suggests a role in delivery of membranes/cargo for cell plate formation and membrane recycling at margin domains. the growth rate of cell plates labeled with gfp-rab1ad was comparable to that observed with gfp-map. however, significant differences were found between epidermis and pericycle cells expressing gfp-rab1ad.figure light-sheet live imaging of gfp-raba1d accumulation during cell plate formation in the root meristem. meristematic cells undergoing cytokinesis showed several locations of gfp-raba1d accumulation observed in single sections of one time point at indicated depths inside the primary root, gfp-raba1d accumulation in cell plates of cytokinetic cells. the boxed area in a is enlarged shown in b. detailed time-lapse imaging of gfp-raba1d accumulation during single cell plate formation starting from spot like structures in the middle of the cell, followed by increased intensity during vesicle fusion and later formation of a ring-like structure at the margins in the growing cell plates. three-dimensional reconstruction of the cell plate shown in at time point 20 min. comparison of cell plate growth rates in arabidopsis plants stably transformed with in 35s::gfp:raba1d and 35s::gfp:map constructs. growth rates of cell plates in cells from different tissues. *significant difference from wilcoxon rank sum test with holm’s correction, ns: no significant difference. bars represent 50 μm in a and 10 μm in b. it is important to note that the overexpression of gfp-raba1d had no serious consequences for plant growth and gravitropic response when transgenic plants were compared with non-transformed wild type plants. oscillatory accumulation of gfp-raba1d in tips of growing root hairs correlates with growth rate root hair growth is a robust example of tip-growth in plant cells, accompanied by polarized membrane trafficking. a previous study demonstrated the localization of raba1d in the apical dome of growing root hairs, however, without following this localization pattern in time. for this reason, we extended the microscopic documentation of gfp-raba1d in relation to root hair growth kinetics by means of light-sheet microscopy, which allowed following the distribution of gfp-raba1d for a considerable time encompassing the entire process of root hair growth, from emergence to cessation of elongation. as previously shown, gfp-raba1d labeling was predominantly observed in trichoblast outgrowths representing emerging root hairs and subsequently was largely restricted at the apical dome of vigorously growing root hairs. however, such accumulation of gfp-raba1d containing vesicles was not visible at the tips of non-growing mature root hairs. in bulges and growing root hairs, gfp-raba1d colocalized with the endocytotic marker fm4- in apical domains, but in mature root hairs both gfp-raba1d and fm4- were more or less equally distributed through the hair tube.figure light-sheet live imaging of gfp-raba1d accumulation during root hair oscillatory tip growth. maximum-intensity projections of two indicated time points. the actively growing root hairs showed a higher tip-accumulation than the slowly expanding or mature root hairs. the tip-accumulation of gfp-raba1d increased in steady state actively growing root hairs. the pixel line from b is plotted as a function of time. the kymograph exhibited an oscillating behavior between lower and higher intensity. fluorescence intensity measurements significantly correlated with the growth rates of the root hair. bars represent 100 μm in a and 10 μm in b. tip-accumulation of gfp-raba1d was observed in root hair bulges and growing root hairs being progressively reduced as root hair growth rates declined. the time-lapse imaging showed gfp-raba1d accumulation in bulge stage which was increased in a time dependent manner. as shown by fluorescence intensity profiles transverse to the root hair apical dome, gfp-raba1d signal intensity oscillated between lower and higher intensities which significantly correlated with the pause and growing phases of root hair growth, respectively. cross correlation coefficient calculations of root hair growth and gfp-raba1d intensity for particular periods of time, e.g. between 150–250 min, revealed r values of, reflecting a strong temporal correlation between apical raba1d accumulation and tip growth. the mode of oscillatory growth of root hairs was confirmed also in transgenic arabidopsis line expressing stress-induced mitogen activated protein kinase kinase tagged with yfp. simkk-yfp accumulated in nuclei and cytoplasm of root cells. observation using light sheet microscopy and determination of fluorescence intensity along transverse profile of growing root hairs showed the oscillatory pattern of root hair growth. this observation corroborated an assumption that raba1d participates in oscillatory cell expansion related to intensive and spatio-temporaly controlled vesicle trafficking in root hairs. movement of the vesicular compartments containing gfp-raba1d depends on the actin cytoskeleton the maximum speed of the raba1d-positive tgn vesicles was measured from kymographs of time-lapse image sequences from root hairs. this analysis revealed that gfp-raba1d vesicles moved with an average speed of about  μm/s in root hairs.figure motility of gfp-raba1d vesicles in growing root hairs. kymograph showing the motility of the gfp-raba1d vesicles in root hairs. estimation of the average speed of the gfp-raba1d vesicles in root hairs treated with acto-myosin inhibitors, latrunculin b and butane-dione-monoxime, respectively. mobility and distribution of small raba1d-positive tgn vesicles in diverse root tissues observed by light-sheet microscopy. 3d reconstruction of a stack of images. single sections at different z-depths: z = 0 μm,: z =   μm,: z =   μm,: z = 50 μm) are depicted in. the dashed boxed area is enlarged in. bars represent 20 μm in d and 5 μm in e. next, arabidopsis seedlings expressing gfp-raba1d were treated with cytoskeletal inhibitors such as oryzalin, latrunculin b and butane-dione monoxime. previously, we observed that the motility of gfp-raba1d compartments in trichoblast cells was not affected by oryzalin-induced microtubule disruption by oryz. thus, we used acto-myosin drugs in root hairs to better characterize the movement of gfp-raba1d compartments along the actin cytoskeleton. latrunculin b disrupts the actin cytoskeleton by its binding to monomeric g-actin subunits while bdm is affecting myosin by inhibiting its atpase function. after treatment with latrunculin b, the gfp-raba1d containing tgn vesicles significantly slowed down and/or stopped their movements through the cytoplasm within 15 min. a similar effect was found after inhibition of vesicle movements using bdm. these data indicated that motility of the gfp-raba1d compartments is acto-myosin dependent. owing to the extended depth of imaging by means of light sheet microscopy, the high mobility of small raba1d-positive tgn vesicles was observed in diverse root tissues such as epidermis, cortex, endodermis, pericycle and central cylinder cells. cells in all these tissues clearly showed presence of the vesicles confirming the role of raba1d in membrane trafficking in different cells and suitability of light sheet microscopy for deep imaging in thick biological probes. discussion in this study, we reported the subcellular localization of gfp-tagged raba1d in transiently and stably transformed plant cells, describing in detail the involvement of raba1d in active and highly regulated vesicle trafficking events essential for cytokinesis of root meristematic cells and polarized cell expansion of root hairs. raba1d localizes to the early endosome/tgn and is involved in vesicle trafficking cellular distribution and localization pattern of gfp-raba1d found in both transiently transformed leaf cells and stably transformed root cells proved gfp-raba1d localization to mobile vesicle-like structures, in accordance with the role of the small gtpases in vesicle trafficking. gfp-raba1d-positive vesicular structures were recognized as tgn compartments, since they were co-labeled with mcherry-vti a snare-protein marker which accumulates at the tgn. additionally, gfp-raba1d vesicles colocalized with fm4-64-positive compartments that are labeled by this vesicular/endocytotic marker soon after entering cells. merging and co-association at tgn compartment was shown also for other members of raba class, raba raba and raba1c which partially overlapped also with the vha-a subunit. in tgn compartment, qa-snare group members localize in a complex and all these compartments are stained by fm4- before this dye reaches rabf or gnom- containing compartments. thus, the localization pattern of gfp-raba1d suggests its specific role during first stages of endocytosis and rapid vesicular trafficking, but not in the process of endosome maturation, because no colocalization with late endosomal markers fyve-dsred or mcherry-rabf2a was found. in this respect, the fyve-domain binds specifically to the phosphoinositol-3-phosphate, which accumulates mainly in the membranes of late endosomal compartments. fyve domain marker colocalized also with rabf2a and rabf today recognized as multivesicular or late endosomal rab gtpase markers. the colocalization of gfp-raba1d with early fm4- compartments was confirmed by microscopic analysis. in comparison to rabf2a, another rab-gtpase that preferentially localize to late/multivesicular endosomes, gfp-raba1d colocalized with fm4- compartments within minutes upon fm staining. partial colocalization of yfp-rabf2a with fm4- was recorded only later. the bfa treatment of transgenic plants expressing gfp-raba1d and co-stained with fm4- resulted in common relocation and accumulation of both markers in the core of bfa-compartments in root cells. since it is known that bfa-induced compartments are composed of tgn and plasma membrane-derived endocytotic vesicles in a core, surrounded by remnants of golgi stacks, this localization pattern confirms the residence of raba1d in early endosome/tgn compartments. moreover, it is suggestive of the role of raba1d in the regulation of vesicle recycling, as this process is effectively inhibited by bfa treatment. the above suggestion is further supported by the quantitative upregulation of raba1d after bfa treatment as revealed by proteomic analysis. the localization and activity of raba1d at tgn compartment and its responsiveness to bfa suggest a role in vesicle trafficking. involvement in endocytosis and vesicular recycling has been reported also for other members of raba class. raba1c, which is localized at the tgn compartment, is sensitive to endosidin but not to wortmannin. endosidin is an inhibitor of endocytosis and vesicular recycling, while wortmannin inhibits the transport to vacuoles by affecting pre-vacuolar compartments, tgn vesicles and mvbs. for instance, the effect of endosidin on bri pin and aux recycling involves also the mislocalization of early endosome/tgn-compartments, defined by syp61/vha-a. similar defects have been observed by the inhibition of vacuolar h+-atpases with concanamycin a. cona blocks the endosome maturation, which leads to production of aggregates of tgn-derived vesicles and therefore, it interferes with the formation of bfa-compartments. the mislocalization of tgn proteins vha-a raba2a and syp was also found in the ech mutant, lacking a tgn-localized echidna protein crucial for tgn function. this mutant is, however, affected rather in secretion with endocytosis being more or less intact. it raises the question on possible role of raba1d also in a secretory pathway, which would be parallel to the function of raba1b in secretory pathway from tgn to the plasma membrane. raba1b co-localize with vamp721/ r-snare proteins that operate in the secretory pathway and only partially and unstably associate with tgn, golgi and endosomes. however, the relocalization of raba1b to plasma membrane upon wortmannin treatment differs from the accumulation, clustering, fusion or swelling of tgn and mvb compartments, observed by fluorescently tagged raba1d, raba4b, raba1e, vti fyve and rabf2a. under wortmannin treatment, raba1d is relocated into wortmannin-induced multivesicular compartments and is downregulated. thereby, while the plant counteracts the inhibition of endocytotic recycling by raba1d upregulation, the inhibition of prevacuolar transport by wortmannin induces raba1d downregulation. thus, raba1d has an important role in vesicle trafficking consistent with its location at early endosome/tgn compartment. raba1d is accumulating in highly active vesicle trafficking zones during cytokinesis and root hair tip-growth cell plate arises from the accumulation, fusion and stretching of vesicles directed by centrally-localized complex of cell plate assembly matrix. these vesicles are delivered to the growing cell plate by phragmoplast microtubules, and cell plate expansion is substantially supported by machineries of both secretory and endocytotic pathways. in the growing cell plate, a ring-like structure representing highly active zone of vesicle trafficking is formed. during the initial stages of cell plate formation, gfp-raba1d accumulated in disc-like structure in the equatorial plane, exhibiting a steady increase of fluorescence intensity likely due to the continuous vesicle fusions. consistently with the spatial redistribution of active zones of vesicular trafficking during cell plate expansion, gfp-raba1d relocated labeling the rim of the growing cell plates. the localization of gfp-raba1d during cytokinesis suggests a role in delivery of cargos and membranes during cell plate formation. it is also compatible with the supporting role of raba1d in active membrane recycling at cell plate margin domains and consistent with the accumulation of raba and raba subclasses during cell plate formation. interestingly, they colocalize in the cell plate with the cytokinesis-specific syntaxin knolle. redundant cytokinetic roles have been assigned to three other rabas, namely raba1a, raba1b and raba1c. these were mislocalized following endosidin treatment, while triple knockout mutants were hypersensitive to endosidin induced cytokinetic disruption. similarly, the cytokinetic-defective mutant pas, showed delayed and defective cell plate formation due to the heterogeneous aggregation of raba2a and knolle, while rabf2a did not show any defect. thus, early endosomes/tgn compartments play a specific role during cell plate formation. the transport between tgn and the plasma membrane by raba1b interacting with vamp is involved in cytokinesis and the inhibition of trafficking at tgn by cona affects cell plate formation through the mislocalization of early endosomes/tgn compartments. additionally, the colocalization of raba1d with vti a member of snare complex, suggests a role also in the transport to the plasma membrane. thereby, the role of raba1d during cell plate formation is consistent with the proposed activity of early endosome/tgn compartments in the coordination of secretory, endocytotic and recycling pathways. similarly to cytokinesis, raba1d participates in localized cell expansion where increased vesicle trafficking is required. the tip-accumulation of raba1d in root hair bulges and in growing root hairs confirmed the role of some rabas during polar growth. the disruption of raba4d in pollen tubes reduced cell polarity, expansion and displayed altered deposition of cell wall components. raba4b showed tip-localization and regulated root hair tip growth through a compartment involved in the polarized secretion, and similarly to raba1d, the tip-localized accumulation disappears in mature root hairs or after latrunculin b treatment. the raba silencing led to reduction in number and length of the root hairs, showing an important role not only in expansion but also during root hair initiation. the raba1d accumulation in bulge stage was increased during the progression to the tip growth. in growing root hairs, gfp-raba1d signal intensity fluctuated in oscillatory manner. oscillations between lower and higher intensity labeling of gfp-raba1d at the root hair tip temporally coincided with the pause and growth phases of root hair tip growth, respectively. similarly to pollen tubes, root hairs exhibit oscillatory growth along with other events accompanying tip growth like cell wall modifications, cytoskeleton rearrangements and targeted vesicle trafficking. some factors, such as ca2+ gradient, extracellular ph and ros concentration and actin organization have been found to be associated with the oscillatory character of tip growth and are closely related to vesicle trafficking. consistently with the frequency of vesicular trafficking during root hair initiation and growth, gfp-raba1d accumulation was lower at bulge stage than in growing root hairs. after the local structural changes that are required to define the bulge position, such as cytoskeleton rearrangements, cell wall composition and accumulation of structural sterols in the plasma membrane, an increased vesicle trafficking is required during root hair elongation to provide a new plasma membrane in the expanding zone. this zone is filled with secretory and endocytotic vesicles, highly dynamic early endosomes in the clear zone and larger endosomal compartments in the subapical region. thereby, raba1d is localized at the tip-zone of growing root hair as previously reported and its accumulation is spatio-temporally correlated with the root hair tip expansion and vesicle trafficking. high motility of tgn vesicles containing gfp-raba1d depends on the actin cytoskeleton endosomes show different patterns of motility including stationary phases, slow or rapid movements. the microtubule disruption by oryzalin did not affect the motility of raba1d-positive vesicles, however the acto-myosin inhibitors affected extremely their motility. similarly, the tip-localization of raba4b was affected by latrunculin b treatment but not by oryzalin. it suggests an important role of actin cytoskeleton in raba1d motility, however, the organization and dynamics of several endosomal structures is influenced by both actin and microtubule cytoskeletons and their motor proteins, i.e. myosins. the role of the actin cytoskeleton in vesicular trafficking was strengthened by proteomic study after bfa treatment, which showed significant upregulation of profilin and its accumulation in bfa-compartments. finally, vigorous motility of raba1d-containing tgn vesicles was also observed in diverse root tissues such as epidermis, cortex, endodermis, pericycle and central cylinder cells confirming the role of raba1d in vesicular trafficking in different cells and not only associated to tip-growth.  we show that raba1d colocalized with early fm4- endocytotic compartments and with vti a tgn marker. bfa treatment not only induced raba1d upregulation but also relocalization of gfp-raba1d to the core of bfa-compartments. most importantly, a specific spatio-temporal accumulation of raba1d correlated with vesicle trafficking requirements during both cell plate formation and root hair oscillatory tip growth. 
during root hair growth and development, gfp-raba1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. in root cells, gfp-raba1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. gfp-raba1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. raba1d participates in both cell plate formation and root hair oscillatory tip growth. the specific gfp-raba1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation. arabidopsiscell platecytokinesisrab gtpaselocalizationraba1droot hairtrans-golgi networkvesicleissue-copyright-statement© the author 2014 our results support a raba1d role in events that require vigorous membrane trafficking. atraba1d, a member of the raba subfamily of small gtpases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described. late endosomes and endoplasmic reticulum labeled with fyve-dsred and er-dsred, respectively, were devoid of gfp-raba1d. the transient expression of 35s::gfp:raba1d construct in allium porrum and nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed arabidopsis thaliana plants. light-sheet microscopy revealed involvement of raba1d in root development. the accumulation of gfp-raba1d in the core of brefeldin a -induced-compartments and the quantitative upregulation of raba1d protein levels after bfa treatment confirmed the association of raba1d with early endosomes/tgn and its role in vesicle trafficking. progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of raba1d and root hair elongation. gfp-raba1d colocalized with the trans-golgi network marker mcherry-vti and with early fm4-64-labeled endosomal compartments. importantly, gfp-raba1d signal intensity exhibited an oscillatory behavior in-phase with tip growth.
 small rab gtpases are important regulators of vesicular trafficking in plants. atraba1d, a member of the raba subfamily of small gtpases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described.  the transient expression of 35s::gfp:raba1d construct in allium porrum and nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed arabidopsis thaliana plants. gfp-raba1d colocalized with the trans-golgi network marker mcherry-vti and with early fm4-64-labeled endosomal compartments. late endosomes and endoplasmic reticulum labeled with fyve-dsred and er-dsred, respectively, were devoid of gfp-raba1d. the accumulation of gfp-raba1d in the core of brefeldin a -induced-compartments and the quantitative upregulation of raba1d protein levels after bfa treatment confirmed the association of raba1d with early endosomes/tgn and its role in vesicle trafficking. light-sheet microscopy revealed involvement of raba1d in root development. in root cells, gfp-raba1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. gfp-raba1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. during root hair growth and development, gfp-raba1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. importantly, gfp-raba1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of raba1d and root hair elongation. our results support a raba1d role in events that require vigorous membrane trafficking.  raba1d is located in early endosomes/tgn and is involved in vesicle trafficking. raba1d participates in both cell plate formation and root hair oscillatory tip growth. the specific gfp-raba1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arabidopsiscell platecytokinesisrab gtpaselocalizationraba1droot hairtrans-golgi networkvesicleissue-copyright-statement© the author 2014 