BACKGROUND
the plant hormone auxin affects various processes in plant growth and development  <cit> .
in the root, auxin is the main factor responsible for the formation and maintenance of stem cell niches in the meristem, development of lateral and adventitious roots, and gravitropism among other processes.
auxin regulates many diverse physiological processes due to its uneven distribution in the tissues—an outcome of active transportation mechanisms  <cit> .
pin efflux carriers, localized in the plasma membrane, are the major contributors to the formation of auxin concentration gradients and maxima  <cit> .
polar localization of pin proteins on the plasma membranes creates directed auxin streams in a tissue  <cit> .
for example, in the root meristem, pin proteins ensure rootward  and shootward  flows in the vascular system and epidermis, respectively  <cit> .
as compared to other members of the family lack of pin <dig> activity results in most severe phenotypes suggesting a crucial role of this protein in auxin transport  <cit> .
the first deviations from normal development are observed in pin <dig> mutants at the globular and heart stages of embryogenesis, when one to two thirds of pin <dig> embryos show disrupted hypophysis  <cit> .
the basal localization of pin <dig> in the plasma membranes provides for directional auxin flow in the globular embryo, where pin <dig> in conjunction with other pins  contributes to the establishment of the apical-basal embryonic axis.
after germination, pin <dig> is expressed in the apical meristems and vascular tissues .
along with pin-shaped inflorescences, fused cotyledons and other shoot abnormalities are evident in pin <dig> mutants  <cit> .
the roots of pin <dig> seedlings are slightly shortened; their apical meristem is also a bit reduced  <cit> .
in the root, pin <dig> mediates rootward auxin flow within the root meristem towards the quiescent centre , which is the site of maximum auxin concentration  <cit> .
pin <dig> proteins were predominantly detected on the rootward sides of the stele and endodermis cells along with some expression in the epidermis, cortex and the qc  <cit> .
additionally, the pin <dig> expression pattern in the root was somewhat variable  <cit> .
in arabidopsis, the lateral and adventitious roots originate from a founder cell belonging to the protoxylem-pole pericycle in a similar way in roots and hypocotyls, respectively .
pin <dig> is expressed starting from the first division of the founder cell and, at each division, occupies the newly formed cell boundary.
as a result, in the multi-layered primordium, only the outer sides of the peripheral cells do not have pin <dig>  whereas the inner cells acquire pin <dig> at all sides  <cit> .
the preferential positioning of pin <dig> towards the lateral root primordium tip became more pronounced at later stages of primordium development.
in the hypocotyl, pin <dig> expression pin <dig> expression visualized with pin1pro:pin1-gfp and pin1pro:gus was restricted to the vascular tissue  <cit> .
by directing auxin efflux from cells, pin <dig> reduces the cellular auxin concentration.
multiple feedbacks exist in plants to balance this decrease: auxin regulates pin <dig> expression at the levels of transcription, protein stability and subcellular localization  <cit> .
at the tissue level, positive and negative regulation of pin <dig> expression by auxin creates an auxin maximum at a distance from the root end, which may provide for specification and maintenance of the qc  <cit> .
the phenotypic defects in single pin mutants are not developed due to ectopic upregulation of the remaining pin genes, which partially substitute for the activity of the knocked-out gene  <cit> .
in the root, pin <dig> functional redundancy was demonstrated in pin2 single, pin2pin <dig>  pin2pin <dig> double and in pin3pin4pin <dig>  pin2pin3pin <dig> triple mutants  <cit> .
in the pin <dig> mutant, pin <dig> was ectopically induced in the pin <dig> expression domain in the cortex and epidermis with polarization, which pin <dig> exhibited in these tissues in wild type plants  <cit> .
pin <dig> expression in the arabidopsis root has been reported in multiple publications, but the data are fragmented and scattered .
at the same time, testing of pin <dig> expression in the root becomes a pervasive approach in experiments on the regulation of auxin distribution.
this requires description of the stable pin <dig> pattern and its possible variations.
pin <dig> activity in the root has also been investigated in other plant species .
in order to obtain a deeper understanding of the role of pins in plant growth and development, an in-depth description of pin <dig> expression patterns in arabidopsis will be helpful.
in this study, we conducted a 3d analysis of pin <dig> expression in a. thaliana root of wild type and single pin mutants using specific antibodies.
we show variations in pin <dig> expression and demonstrate that they occurred exclusively at the border of the pin <dig> expression domain.
we also determined quantitatively the changes in pin <dig> expression in response to exogenous auxin treatments using two reporter lines .
we found that there is a dependence between the exogenous auxin dose and the changes in the pin <dig> expression level and domain.
significant differences in auxin response observed between pin1pro:pin1-gfp and pin1pro:gus plants allowed us to conclude that auxin activates pin <dig> expression at the level of its promoter activity, whereas auxin inhibits it at the post-transcriptional level.
based on the similarities in pin <dig> expression changes after auxin treatments and in pin mutants with the variable part of its expression pattern in control plants, we suggest that the variability in pin <dig> expression may be explained by slight differences in endogenous auxin levels in the individual roots.
RESULTS
whole-mount immunolocalization of pin <dig> in arabidopsis thaliana roots and hypocotyls was performed with specific antibodies to pin <dig> .
three-dimensional analysis of pin <dig> expression was carried out with the irocs toolbox  <cit> .
pin <dig> expression was detected at the root-shoot junction, in developing lateral root primordia and in the tips of primary roots.
stable and variable features in pin <dig> expression are described in detail below.
pin <dig> expression in the root tip
differentiating stele in the root meristematic zone represent a well-known pin <dig> expression domain  <cit> .
the stele initials and two to four horizontal rows of their descendants had the brightest anti-pin <dig> signal in all tested roots .
in this domain, pin <dig> proteins occupied the rootward and lateral surfaces of the plasma membranes, but there was higher accumulation of pin <dig> on the rootward sides.fig.
<dig> whole-mount immunolocalisation of pin <dig> in a. thaliana root tip.
a longitudinal section  and five transverse sections  showing anti-pin <dig> signal  and anti-pin <dig> staining .
cei—cortex/endodermis initials, eli—epidermis/lateral root cap initials, csc—columella stem cell , qc—the quiescent centre, c2—the second columella tier, c3—the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature, px—protoxylem, pph—protophloem, mx—metaxylem.
coloured triangles—the end of the expression domain in the respective layer.
white triangle—rootward pin <dig> location in xylem elements in the elongation zone.
mz—the meristematic zone.
bars = 50 μm
in differentiating vascular and pericycle cells, the pin <dig> signal remained intense and was preferentially positioned rootward, although the amount of signal per cell gradually decreased in the shootward direction .
the decrease in pin <dig> expression differed in the cell lineages of the stele.
the protophloem and phloem-pole pericycle lost pin <dig> signal in the transition domain of the meristem .
a gradual decrease in pin <dig> expression as the root protophloem cells differentiated has been shown previously  <cit> .
the procambium and xylem-pole pericycle cells generally lost pin <dig> expression at the end of the meristematic zone .
metaxylem precursor cells and protoxylem lineages expressed pin <dig> in the elongation zone.
metaxylem precursor cells lost pin <dig> signal just after passing the meristematic/elongation zone border, but expression in the protoxylem was still visible at a distance from the border .fig.
<dig> variability in pin <dig> expression domain in different lineages in the root tip.
a and b the primary root tips of two individual  <dig> dag seedlings labelled with anti-pin <dig>  and dapi .
csc—columella stem cell, c2—the second columella tier, c3- the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature, px—protoxylem.
coloured triangles—the end of the expression domain in the respective layer.
bars = 50 μm.
c a general scheme of stable and variable parts of the pin <dig> expression pattern in the root tip of a. thaliana.
pin <dig> expression domain is on the left: dark grey—the stable part of the pin <dig> protein domain for the root meristem of a  <dig> dag seedling, grown on ms medium ; light grey is its variable part.
on the right, stable and variable features of pin <dig> polarization are shown.
rw—rootward polarity, rwl—rootward and lateral polarity, rwli—rootward and inner lateral polarity, np—nonpolar
in the quiescent center, the first tier of the columella , the cortex/endodermis initials and the epidermis/lateral root cap initials, pin <dig> proteins occupied the entire plasma membrane.
pin <dig> signal was not detected in the lateral root cap.
it was absent in this lineage immediately after asymmetric division of the epidermis/lateral root cap initials.
in the epidermis lineage pin <dig> was expressed with the rootward polarity in the first 2– <dig> cells.
in the first cells of the endodermis lineage, pin <dig> proteins occupied the rootward and lateral sides.
further, in endodermal cells, pin <dig> proteins remained at the rootward sides and at about one-third of the internal lateral side.
the lateral pin <dig> disappeared, and the rootward pin <dig> became gradually weaker at the beginning of the transition domain.
there was no pin <dig> expression in the endodermis cells outside the meristematic zone.
in the first cells of the cortex lineage, pin <dig> protein occupies both the lateral and rootward sides of the plasma membranes .
the descendants of these cells exhibit primarily rootward signal, which becomes weaker with each division.
in some roots, staining extended to the 16th cortex horizontal row, and various degrees of lateralization were observed .
along with the stable features of the pin <dig> expression pattern , we identified the following variable features differed from root to root: the length of the pin <dig> expression domain and the number of cells with the polarization type  .
in the stele, pin <dig> showed rootward positioning wherever it was expressed, but the presence of the pin <dig> protein on the lateral sides of the plasma membranes varied in differentiating stele lineages, starting from the third cell from the qc .
pin <dig> may occupy both inner and outer lateral sides , or only the inner lateral side, or be absent from the lateral sides.
in the pericycle, pin <dig> started to switch from equal signal intensities on both lateral sides to decreasing signal on the outer one as early as in the first descendants of the initials.
in the middle of the meristematic zone, pin <dig> signal usually covered along with the rootward only the inner lateral sides of the pericycle cells.
pin <dig> disappeared from the lateral sides of the protoxylem and metaxylem cells in the transition domain.
the transition from one polarization type to another occurred in the same cell lineage at variable distances from the qc in different roots.
variability was also detected in the qc, columella initials and their descendants.
in some roots, the qc and columella initials did not have polar pin <dig>  in others, pin <dig> was polarized rootward to varying degrees.
rarely, we observed weak non-polar pin <dig> signal in the second and third columella tiers.
a weak signal was also found using anti-pin <dig> antibodies in the second and third columella tiers in pin <dig> roots , suggesting that pin <dig> signal there might be non-specific.
patterning of pin <dig> expression during lateral root development
pin <dig> expression in the pericycle of the maturation zone was observed starting from the first stage of lateral root primordium development , after the first transverse  division of the founder cell.
pin <dig> localized at the contiguous plasma membranes of adjacent daughters .
after a series of anticlinal divisions, the lrp-i primordium cells divided periclinally and formed a two-layered lrp-ii primordium  <cit> .
at each cell division during lrp-i and lrp-ii, pin <dig> proteins occupied the contiguous membranes between the daughter cells, marking all newly formed boundaries .
cells in the outer layer , followed by the inner layer , undergo periclinal divisions that take the primordium to the lrp-iii and lrp-iv stages  <cit> .
at these stages, pin <dig> proteins also occupy the membranes between the daughter cells but with a bias in polarity towards the primordium tip .
cells at the primordium border start differentiating at this time, which can be observed as a decrease  followed by a loss  of pin <dig> expression in this region.
these changes likely promote auxin flow towards the primordium tip.
from lrp-iv, pin <dig> expression gradually decreases in the outer layers  .
then, qc cells become pre-specified: the wox <dig> marker starts to localize to the central cells of ol <dig>  <cit> .
the pre-specified qc expresses pin <dig> without polarization.
from lrp-vi, all outer layers—epidermis, cortex and endodermis—can be distinguished .
as a result, at the lrp-vii stage, the pin <dig> expression pattern is similar to the pattern in the primary root: pin <dig> is highly expressed in the vasculature and in pericycle precursors, and rootward signal is visible in the developing cortex and endodermis .fig.
<dig> changes in pin <dig> expression during lateral root primordium development.
a–g the seven stages of primordium development are shown in roman numerals.
il —inner layers, ol —outer layers.
pre-specified qc cells are marked by asterisks.
developing tissues: epid—epidermis, c—cortex, en—endodermis.
white arrows show the directions of auxin flux.
anti-pin <dig> staining is in green, dapi is in the blue channel.
bars = 50 μm
pin <dig> expressing cells were sometimes found close to the developing lateral primordium .
the rootward pin <dig> signal was detected in xylem elements  and individual cells of the outer layers of the primary root near the primordium .
the pin <dig> expression domain in these tissues was dramatically extended after auxin treatment .
pin <dig> expression in hypocotyl
at the root-shoot junction, pin <dig> was detected with rootward polarity in the vascular cells flanking the mature xylem vessels .
in most of the seedlings, within a few days after germination, we also observed pin <dig> expression in one or two symmetrical primordia-like organs at the root-shoot border .
in  <dig> dag seedlings pin <dig> expression in these primordia-like organs resembled those of lrp-v or lrp-vi , suggesting that they may give rise to adventitious roots, which in arabidopsis originate from hypocotyl pericycle cells in a similar way as the lateral roots from root pericycle cells .
however, in the older seedlings, we observed that the primordia-like organ lost pin <dig> signal and did not develop into adventitious roots.
we suggest that these organs are adventitious root primordia that have initiated without developing further.
the primordia arrested or delayed in development were previously described for lateral roots  <cit> .fig.
<dig> pin <dig> expression at the root-shoot junction in  <dig> dag seedlings.
a and b pin <dig> signal  was detected in the vascular elements attached to the mature xylem vessels throughout the whole hypocotyl.
pin <dig> was polarized rootward .
c the rootward pin <dig> signal in the cells flanking the mature xylem vessels of the hypocotyl.
d magnified view of the white rectangular region from .
bars = 50 μm
exogenous auxin modulates pin <dig> expression
pin <dig> expression patterns in wild type plants showed variability in the roots .
this may be caused by variation in the auxin distribution that occurs naturally during plant growth.
to test this hypothesis, we assessed the effect of seedling treatments with different concentrations of exogenous auxin .
auxin treatment changed the pin <dig> expression domain in the root meristem .
auxin effect on pin <dig> protein expression in the root apical meristem was dual: auxin upregulated pin <dig> expression in low-level treatments  and inhibited expression at high dosage  .
in roots treated with exogenous auxin, we observed both extension  and shortening  of pin <dig> expression domains in the vasculature and outer layers—features we noted as variable in control  plants.
for example, in low-level treatments, the pin <dig> expression domain was extended in the endodermis, cortex, and stele; in many roots, pin <dig> was also detected in the second and third tiers of the columella .
in contrast, under high-level treatments, the pin <dig> domain was significantly reduced in all of the tissues listed above.fig.
<dig> auxin regulated pin <dig> expression in the root tip.
a and b immunostaining of pin <dig> expression in wild type plants treated with low  and high  naa dosage for 24 h. a the effect of exogenous low-level auxin on pin <dig> upregulation in the meristem and at the sites of lateral primordia outgrowth.
b modulation of pin <dig> expression domain under low and high naa treatments, compared to control.
the variation in length of the expression domains for different cell lineages is shown by coloured triangles.
csc—columella stem cell, c2—the second columella tier, c3- the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature.
anti-pin <dig> staining is in green, dapi is in the blue channel.
bars = 50 μm.
c and d quantitative estimates of the changes in pin <dig> domain length  and pin <dig> maximal expression intensity  under exogenous iaa treatments with different dosages.
the experimental images were analysed in imagej .
green line—estimates for pin1pro:pin1-gfp plants; blue line—for pin1pro:gus plants.
the measured values for each iaa dosage and for each reporter line were normalized to controls.
statistics differences were identified by t-test: * p-value <  <dig> ; ** p-value <  <dig> ; *** p-value <  <dig>
to estimate the changes in pin <dig> expression in the primary root meristem quantitatively, we analysed auxin-induced  reporter activity in pin1pro:gus and pin1pro:pin1-gfp plants .
thus, we were able to compare the auxin effect on pin <dig> promoter activity in pin1pro:gus with auxin-dependent post-transcriptional regulation of pin1-gfp protein in pin1pro:pin1-gfp.
using imagej  <cit> , we quantified the maximal and average intensities of the reporter signals, as well as the width and length of the pin <dig> expression domains in the meristem .
statistical analysis of the measured characteristics supported auxin dose–response of pin <dig> expression .
in both lines , the length of the expression domain in the stele was significantly increased along the root central axis after treatment with low iaa concentration  .
additionally, the length of the pin <dig> expression domain was significantly decreased by high iaa concentrations  in both lines.
by analysing the maximal intensity of the reporter activity, we found differences between the lines.
in the pin1pro:pin1-gfp line, a significant increase  in gfp fluorescence was detected under  <dig>  and  <dig>  μm iaa .
after  <dig> and 10 μm iaa treatment, gfp fluorescence was significantly reduced .
gus staining was always increased with increased exogenous auxin .
starting from  <dig>  μm iaa, the treatment caused significant upregulation of pin <dig> promoter activity .
taking into account that the pin1pro:gus line reveals the pin <dig> promoter activity by gus staining and the pin1pro:pin1-gfp line monitors the amount of pin1-gfp protein, we conclude that the auxin effect on pin <dig> expression involves both transcriptional and post-transcriptional regulation.
namely, the positive auxin effect on pin <dig> expression is explicit at the level of pin <dig> promoter activity, whereas the inhibitory auxin effect relates to post-transcriptional regulation of pin <dig> expression.
summarizing the experimental data on exogenous auxin treatments, we conclude that the variability in the pin <dig> expression pattern, which was observed among untreated roots, may be simulated by exogenous auxin treatment.
this indicates that differences in endogenous auxin levels among individual roots may be the source of variability in the pin <dig> expression pattern.
pin <dig> expression in pin mutants
because pin proteins mediated auxin efflux from individual cells, we expected that auxin distribution in the root tips of pin knockouts would be slightly different from control plants.
indeed, cross-regulation of pin <dig> expression in the pin <dig> mutant has been reported before  <cit> .
we analysed the changes in pin <dig> immunolocalization in the roots of single knockouts of the genes encoding long pins .
the changes in the pin <dig> expression domain compared to controls are summarized in additional file  <dig> and fig.
<dig> fig.
<dig> pin <dig> expression patterns in pin mutants  compared to wild type .
csc—columella stem cell, c2—the second columella tier, c3—the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature.
coloured triangles—the end of the expression domain in the respective layer.
anti-pin <dig> staining is in green, dapi is in the blue channel.
bars = 50 μm
in wild type plants, pin <dig> was expressed and polarized rootward up to the six youngest epidermal cell row .
in the pin <dig> mutant, the pin <dig> domain in the epidermis was frequently  extended up to the twentieth cell row .
in the ectopic domain , the pin <dig> protein had shootward polarity inherent to pin <dig>  which allowed pin <dig> to partially substitute the knocked-out gene.
in the wild type cortex, pin <dig> was expressed rootward, and it gradually declined in intensity up to the 16th cell row from the qc .
in the cortex of the pin <dig> mutant, pin <dig> has the same rootward polarization but in an extended domain.
we detected a high level of pin <dig> expression up to the 18th cell row from the qc, which then gradually decreased moving out towards the 25th cell row.
these data support previously reported findings  <cit> .
in addition, in pin <dig> mutants, we observed non-polar expression of pin <dig> in the second columella tier more frequently than in wild type .
the intensity of the expression was weak but still stronger than in those rare cases when it was detected in this region in wild type.
changes in pin <dig> expression in the vasculature, endodermis and cortex of pin <dig>  pin <dig>  pin <dig> mutants were similar: the pin <dig> expression domain extended further to the elongation zone in 60–75 % of plants .
in the pin <dig> mutant, pin <dig> also showed ectopic expression in the second columella tier, similar to the pin <dig> mutant.
in the pin <dig> mutant, the domain of pin <dig> ectopic expression in the columella was enlarged and covered up to four tiers.
the experimental results show that, in single pin mutants, pin <dig> expression extended to the sites of normal expression for knocked-out pin <dig>  pin <dig>  pin <dig> and pin <dig> genes.
with the exception of the significantly extended pin <dig> domain in the epidermis and cortex of pin <dig> mutants, all other changes in the pin <dig> expression pattern in pin mutants were enough close to the spectrum of variability detected in wild type.
we assume that the changes in pin <dig> expression might be a reaction to changes in endogenous auxin content in pin mutants.
tissue-specific auxin accumulation or depletion, which must occur in the absence of one of the pins, may result in adjustment of the pin <dig> expression domain.
discussion
auxin levels control the identities of cells and underlie a wide range of developmental phenomena .
auxin gradients in tissues are the result of auxin movement between cells due to diffusion and active transport.
auxin efflux carriers of the pin family were shown to be key regulators of auxin distribution .
pin <dig> is the founding member of this family, and its expression is often used to monitor the effects of other factors on auxin distribution .
various aspects of pin <dig> expression but only in relation to the main topics of these papers were described.
here, we present a study focused on pin <dig> expression in a. thaliana root, which confirmed a number of published facts and revealed some new features of pin <dig> expression in the primary root all along its length, up to the root-shoot junction.
the regular anatomy of the root apex allowed us to describe the variation and complexity of pin <dig> expression in 3d and revealed the stable and varying parts of the expression pattern .
the stele is the main pin <dig> expression domain in the root meristem  <cit> .
observations of pin <dig> expression in 3d demonstrate that it is not uniform; distinct cell lineages stop expressing pin <dig> at different distances from the qc .
the protophloem and protophloem-pole pericycle lost pin <dig> signal in the proximal two-thirds of the meristem; procambium and the rest of pericycle lost pin <dig> expression from there to the end of the meristem.
in contrast, xylem precursors still expressed pin <dig> in the distal part of the elongation zone.
pin <dig> has been suggested to be a marker for non-differentiated cells  <cit> .
here, we found that it accurately marked the beginning and end of the transition domain in the root tip.
as previously described  <cit> , there are no clear markers for the transition domain of the meristem.
we suggest that the reference point for the beginning of the transition domain could be the first cell in the protophloem lineage, which lost pin <dig> signal.
the reference point for the end of the transition domain could be the last protoxylem cell expressing pin <dig>  moreover, the end of the pin <dig> expression domain in the endodermis coincided with the end of the meristem, which coud be estimated based on the first rapidly elongated cortex cell  <cit> .
in the stele, pin <dig> is mainly localized rootward, driving auxin across the vasculature towards the qc.
some spreading of pin <dig> protein to the inner lateral sides of the stele cells was observed  <cit> .
in xylem precursors , pin <dig> had rootward localization , whereas in procambium cells, pin <dig> also occupied the lateral sides  <cit> .
we investigated pin <dig> on the rootward and lateral sides of the stele cells in more detail .
all stele initials and their first daughters have the “procambium mode” of pin <dig> expression  in which pin <dig> occupies the rootward and lateral parts of the plasma membrane.
furthermore, there is a difference in pin <dig> expression in the descendants of different lineages.
vascular precursors in approximately ten of the horizontal rows from the qc along with stable rw have two types of pin <dig> lateral polarization .
the rwli type of polarization means that along with rw, pin <dig> protein settles on the inner lateral sides and allows inward auxin flow.
the xylem precursors completely lose pin <dig> lateral positioning in the upper one-third of the meristematic zone.
the other vascular  cells show variation in the lateral positioning up to the end of the meristematic zone, accompanied by a gradual decrease in pin <dig> expression.
in the pericycle, constant rootward positioning of pin <dig> was also maintained up to the end of the meristematic zone, with the lateral polarity becoming rwli early in the cell lineage.
weak rootward pin <dig> with some lateral spreading was also reported in the endodermis .
in the endodermis, variations in polarization looked similar to those in the pericycle; along with stable rootward positioning, pin <dig> spread to the lower part of the internal lateral side .
in the cortex, only weak rootward signal was recorded, mainly in the youngest cells  <cit> .
we additionally showed spreading of pin <dig> to the lateral sides in these cells and extension of the pin <dig> domain to the middle of the meristematic zone in some roots .
our data on pin <dig> expression in the youngest epidermal cells, the qc, the columella initials, the hypocotyl and lateral root primordia  was consistent with previous reports .
comparing our map of pin <dig> expression  with the auxin distribution map in the root tip  <cit> , we see that stable pin <dig> expression occurs in regions with high to intermediate auxin levels.
the regions with variable pin <dig> expression are characterized by declining auxin.
roots slightly differ in the endogenous auxin level, and this may result in different sizes for the pin <dig> expression domain.
we hypothesized that changing the auxin level in the root, for example, by auxin treatment, would move the edge between high/intermediate and low auxin levels and this would increase or decrease the length of the pin <dig> expression domain.
the changes in pin <dig> domain length observed after auxin treatment might also correlate with auxin induced changes in the size of the root meristem, as previously described  <cit> .
indeed, we showed that treatment with low concentrations of auxin  led to lengthening of the expression domain in the stele, endodermis, cortex and columella .
in contrast, treatment with high auxin concentrations  reduced the pin <dig> expression domain .
quantitative analysis of the expression changes in pin1pro:pin1-gfp and pin1pro:gus roots after iaa treatment supported the results statistically .
similar dose effects have been found previously using another auxin:  <dig>  μm  <dig> -d was found to be the optimal concentration for upregulation of pin1pro:pin1-gfp expression in epidermal cells, whereas pin1-gfp activity decreased in the stele at higher  <dig> -d concentrations  <cit> .
we suggest that the changes in the root auxin level after treatment with exogenous auxin and their influence on the pin <dig> expression domain simulated and exaggerated the naturally occurring variations in auxin distribution that occur between individual roots.
these changes can influence pin <dig> expression.
by analysing the maximal intensities of reporter activity in pin1pro:pin1-gfp and pin1pro:gus plants, we found significant differences between the auxin responses of the lines .
in pin1pro:pin1-gfp, gfp fluorescence significantly increased and decreased  under  <dig> – <dig>  μm iaa and 5–10 μm iaa, respectively.
gus staining in pin1pro:gus increased with an increase in exogenous auxin dose.
these results suggest that positive and negative effects of auxin on pin <dig> are mediated at different levels—transcriptional and post-transcriptional.
the shortening of the pin <dig> expression domain at a high auxin dosage may be regulated post-transcriptionally.
finally, we analysed pin <dig> expression domain in the single pin mutants.
we expected that local changes in endogenous auxin level occur in pin mutants, which might affect pin <dig> expression.
pin <dig>  pin <dig>  pin <dig> and pin <dig> are expressed together with pin <dig> in the root meristem and may provide redundancy if one of them is mutated  <cit> .
the pin <dig> cross-regulation in the pin <dig> mutant were described previously  <cit> .
here, we provide a comparative description of pin <dig> expression in single pin <dig>  pin <dig>  pin <dig> and pin <dig> mutants .
in pin <dig> and pin <dig> mutants, pin <dig> almost fully occupies the domain of the knocked-out pin genes.
in the case of pin <dig>  it is in the vasculature only.
in the pin <dig> mutant, pin <dig> completely occupies the pin <dig> expression domain in the vascular system, and it partially occupies the first columella tier.
comparing the expression patterns of pin <dig> in pin mutants  with the wild type , we find that the changes in pin <dig> expression mainly fall within the spectrum of variability observed in wild type plants.
by regulating pin <dig> expression level and the size of the pin <dig> expression domain, auxin determines the efficiency of its own transport.
by controlling pin <dig> polarity, auxin determines the directions of its own streams.
the rootward polarity of pin <dig> promotes auxin flow to the root tip.
the inner lateral location of pin <dig> in the endodermis and pericycle collects auxin from the shootward auxin flow to the vasculature, and pin <dig> lateralization in procambial cells allows to accumulate auxin in the narrower stream in xylem precursors.
this accumulation is probably important for vascular differentiation inside the meristem  <cit> .
naa treatments at concentrations as low as  <dig> –1 μm  affected pin <dig> polarity and caused pin <dig> to spread across the entire inner lateral sides in the endodermis and pericycle, but higher naa concentrations did not show further polarization, suggesting saturation of the effect  <cit> .
this may indicate that the responses of expression level and polarization to auxin are different.
in our experiments, comparing the maps of pin <dig> expression and its polar locations  gave evidence showing that pin <dig> polarization may be more sensitive to minor changes in auxin cellular concentrations than the pin <dig> expression level.
CONCLUSIONS
pin proteins reduce the cellular auxin level by carrying auxin out of the cell.
as a feedback mechanism, auxin regulates the transcription, stability and polarization of pins.
we have clarified the role of the founding member of the pin family  in these close and interconnected relationships that establish the auxin distribution, which is a key piece of positional information for cell fate determination in the root stem cell niche.

 the plant hormone auxin affects various processes in plant growth and development. in the root, auxin is the main factor responsible for the formation and maintenance of stem cell niches in the meristem, development of lateral and adventitious roots, and gravitropism among other processes. auxin regulates many diverse physiological processes due to its uneven distribution in the tissues—an outcome of active transportation mechanisms. pin efflux carriers, localized in the plasma membrane, are the major contributors to the formation of auxin concentration gradients and maxima. polar localization of pin proteins on the plasma membranes creates directed auxin streams in a tissue. for example, in the root meristem, pin proteins ensure rootward and shootward flows in the vascular system and epidermis, respectively. as compared to other members of the family lack of pin activity results in most severe phenotypes suggesting a crucial role of this protein in auxin transport. the first deviations from normal development are observed in pin mutants at the globular and heart stages of embryogenesis, when one to two thirds of pin embryos show disrupted hypophysis. the basal localization of pin in the plasma membranes provides for directional auxin flow in the globular embryo, where pin in conjunction with other pins contributes to the establishment of the apical-basal embryonic axis. after germination, pin is expressed in the apical meristems and vascular tissues. along with pin-shaped inflorescences, fused cotyledons and other shoot abnormalities are evident in pin mutants. the roots of pin seedlings are slightly shortened; their apical meristem is also a bit reduced. in the root, pin mediates rootward auxin flow within the root meristem towards the quiescent centre, which is the site of maximum auxin concentration. pin proteins were predominantly detected on the rootward sides of the stele and endodermis cells along with some expression in the epidermis, cortex and the qc. additionally, the pin expression pattern in the root was somewhat variable. in arabidopsis, the lateral and adventitious roots originate from a founder cell belonging to the protoxylem-pole pericycle in a similar way in roots and hypocotyls, respectively. pin is expressed starting from the first division of the founder cell and, at each division, occupies the newly formed cell boundary. as a result, in the multi-layered primordium, only the outer sides of the peripheral cells do not have pin whereas the inner cells acquire pin at all sides. the preferential positioning of pin towards the lateral root primordium tip became more pronounced at later stages of primordium development. in the hypocotyl, pin expression pin expression visualized with pin1pro:pin1-gfp and pin1pro:gus was restricted to the vascular tissue. by directing auxin efflux from cells, pin reduces the cellular auxin concentration. multiple feedbacks exist in plants to balance this decrease: auxin regulates pin expression at the levels of transcription, protein stability and subcellular localization. at the tissue level, positive and negative regulation of pin expression by auxin creates an auxin maximum at a distance from the root end, which may provide for specification and maintenance of the qc. the phenotypic defects in single pin mutants are not developed due to ectopic upregulation of the remaining pin genes, which partially substitute for the activity of the knocked-out gene. in the root, pin functional redundancy was demonstrated in pin2 single, pin2pin pin2pin double and in pin3pin4pin pin2pin3pin triple mutants. in the pin mutant, pin was ectopically induced in the pin expression domain in the cortex and epidermis with polarization, which pin exhibited in these tissues in wild type plants. pin expression in the arabidopsis root has been reported in multiple publications, but the data are fragmented and scattered. at the same time, testing of pin expression in the root becomes a pervasive approach in experiments on the regulation of auxin distribution. this requires description of the stable pin pattern and its possible variations. pin activity in the root has also been investigated in other plant species. in order to obtain a deeper understanding of the role of pins in plant growth and development, an in-depth description of pin expression patterns in arabidopsis will be helpful. in this study, we conducted a 3d analysis of pin expression in a. thaliana root of wild type and single pin mutants using specific antibodies. we show variations in pin expression and demonstrate that they occurred exclusively at the border of the pin expression domain. we also determined quantitatively the changes in pin expression in response to exogenous auxin treatments using two reporter lines. we found that there is a dependence between the exogenous auxin dose and the changes in the pin expression level and domain. significant differences in auxin response observed between pin1pro:pin1-gfp and pin1pro:gus plants allowed us to conclude that auxin activates pin expression at the level of its promoter activity, whereas auxin inhibits it at the post-transcriptional level. based on the similarities in pin expression changes after auxin treatments and in pin mutants with the variable part of its expression pattern in control plants, we suggest that the variability in pin expression may be explained by slight differences in endogenous auxin levels in the individual roots.  whole-mount immunolocalization of pin in arabidopsis thaliana roots and hypocotyls was performed with specific antibodies to pin. three-dimensional analysis of pin expression was carried out with the irocs toolbox. pin expression was detected at the root-shoot junction, in developing lateral root primordia and in the tips of primary roots. stable and variable features in pin expression are described in detail below. pin expression in the root tip differentiating stele in the root meristematic zone represent a well-known pin expression domain. the stele initials and two to four horizontal rows of their descendants had the brightest anti-pin signal in all tested roots. in this domain, pin proteins occupied the rootward and lateral surfaces of the plasma membranes, but there was higher accumulation of pin on the rootward sides.fig. whole-mount immunolocalisation of pin in a. thaliana root tip. a longitudinal section and five transverse sections showing anti-pin signal and anti-pin staining. cei—cortex/endodermis initials, eli—epidermis/lateral root cap initials, csc—columella stem cell, qc—the quiescent centre, c2—the second columella tier, c3—the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature, px—protoxylem, pph—protophloem, mx—metaxylem. coloured triangles—the end of the expression domain in the respective layer. white triangle—rootward pin location in xylem elements in the elongation zone. mz—the meristematic zone. bars = 50 μm in differentiating vascular and pericycle cells, the pin signal remained intense and was preferentially positioned rootward, although the amount of signal per cell gradually decreased in the shootward direction. the decrease in pin expression differed in the cell lineages of the stele. the protophloem and phloem-pole pericycle lost pin signal in the transition domain of the meristem. a gradual decrease in pin expression as the root protophloem cells differentiated has been shown previously. the procambium and xylem-pole pericycle cells generally lost pin expression at the end of the meristematic zone. metaxylem precursor cells and protoxylem lineages expressed pin in the elongation zone. metaxylem precursor cells lost pin signal just after passing the meristematic/elongation zone border, but expression in the protoxylem was still visible at a distance from the border.fig. variability in pin expression domain in different lineages in the root tip. a and b the primary root tips of two individual dag seedlings labelled with anti-pin and dapi. csc—columella stem cell, c2—the second columella tier, c3- the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature, px—protoxylem. coloured triangles—the end of the expression domain in the respective layer. bars = 50 μm. c a general scheme of stable and variable parts of the pin expression pattern in the root tip of a. thaliana. pin expression domain is on the left: dark grey—the stable part of the pin protein domain for the root meristem of a dag seedling, grown on ms medium; light grey is its variable part. on the right, stable and variable features of pin polarization are shown. rw—rootward polarity, rwl—rootward and lateral polarity, rwli—rootward and inner lateral polarity, np—nonpolar in the quiescent center, the first tier of the columella, the cortex/endodermis initials and the epidermis/lateral root cap initials, pin proteins occupied the entire plasma membrane. pin signal was not detected in the lateral root cap. it was absent in this lineage immediately after asymmetric division of the epidermis/lateral root cap initials. in the epidermis lineage pin was expressed with the rootward polarity in the first 2– cells. in the first cells of the endodermis lineage, pin proteins occupied the rootward and lateral sides. further, in endodermal cells, pin proteins remained at the rootward sides and at about one-third of the internal lateral side. the lateral pin disappeared, and the rootward pin became gradually weaker at the beginning of the transition domain. there was no pin expression in the endodermis cells outside the meristematic zone. in the first cells of the cortex lineage, pin protein occupies both the lateral and rootward sides of the plasma membranes. the descendants of these cells exhibit primarily rootward signal, which becomes weaker with each division. in some roots, staining extended to the 16th cortex horizontal row, and various degrees of lateralization were observed. along with the stable features of the pin expression pattern, we identified the following variable features differed from root to root: the length of the pin expression domain and the number of cells with the polarization type. in the stele, pin showed rootward positioning wherever it was expressed, but the presence of the pin protein on the lateral sides of the plasma membranes varied in differentiating stele lineages, starting from the third cell from the qc. pin may occupy both inner and outer lateral sides, or only the inner lateral side, or be absent from the lateral sides. in the pericycle, pin started to switch from equal signal intensities on both lateral sides to decreasing signal on the outer one as early as in the first descendants of the initials. in the middle of the meristematic zone, pin signal usually covered along with the rootward only the inner lateral sides of the pericycle cells. pin disappeared from the lateral sides of the protoxylem and metaxylem cells in the transition domain. the transition from one polarization type to another occurred in the same cell lineage at variable distances from the qc in different roots. variability was also detected in the qc, columella initials and their descendants. in some roots, the qc and columella initials did not have polar pin in others, pin was polarized rootward to varying degrees. rarely, we observed weak non-polar pin signal in the second and third columella tiers. a weak signal was also found using anti-pin antibodies in the second and third columella tiers in pin roots, suggesting that pin signal there might be non-specific. patterning of pin expression during lateral root development pin expression in the pericycle of the maturation zone was observed starting from the first stage of lateral root primordium development, after the first transverse division of the founder cell. pin localized at the contiguous plasma membranes of adjacent daughters. after a series of anticlinal divisions, the lrp-i primordium cells divided periclinally and formed a two-layered lrp-ii primordium. at each cell division during lrp-i and lrp-ii, pin proteins occupied the contiguous membranes between the daughter cells, marking all newly formed boundaries. cells in the outer layer, followed by the inner layer, undergo periclinal divisions that take the primordium to the lrp-iii and lrp-iv stages. at these stages, pin proteins also occupy the membranes between the daughter cells but with a bias in polarity towards the primordium tip. cells at the primordium border start differentiating at this time, which can be observed as a decrease followed by a loss of pin expression in this region. these changes likely promote auxin flow towards the primordium tip. from lrp-iv, pin expression gradually decreases in the outer layers. then, qc cells become pre-specified: the wox marker starts to localize to the central cells of ol. the pre-specified qc expresses pin without polarization. from lrp-vi, all outer layers—epidermis, cortex and endodermis—can be distinguished. as a result, at the lrp-vii stage, the pin expression pattern is similar to the pattern in the primary root: pin is highly expressed in the vasculature and in pericycle precursors, and rootward signal is visible in the developing cortex and endodermis.fig. changes in pin expression during lateral root primordium development. a–g the seven stages of primordium development are shown in roman numerals. il —inner layers, ol —outer layers. pre-specified qc cells are marked by asterisks. developing tissues: epid—epidermis, c—cortex, en—endodermis. white arrows show the directions of auxin flux. anti-pin staining is in green, dapi is in the blue channel. bars = 50 μm pin expressing cells were sometimes found close to the developing lateral primordium. the rootward pin signal was detected in xylem elements and individual cells of the outer layers of the primary root near the primordium. the pin expression domain in these tissues was dramatically extended after auxin treatment. pin expression in hypocotyl at the root-shoot junction, pin was detected with rootward polarity in the vascular cells flanking the mature xylem vessels. in most of the seedlings, within a few days after germination, we also observed pin expression in one or two symmetrical primordia-like organs at the root-shoot border. in dag seedlings pin expression in these primordia-like organs resembled those of lrp-v or lrp-vi, suggesting that they may give rise to adventitious roots, which in arabidopsis originate from hypocotyl pericycle cells in a similar way as the lateral roots from root pericycle cells. however, in the older seedlings, we observed that the primordia-like organ lost pin signal and did not develop into adventitious roots. we suggest that these organs are adventitious root primordia that have initiated without developing further. the primordia arrested or delayed in development were previously described for lateral roots.fig. pin expression at the root-shoot junction in dag seedlings. a and b pin signal was detected in the vascular elements attached to the mature xylem vessels throughout the whole hypocotyl. pin was polarized rootward. c the rootward pin signal in the cells flanking the mature xylem vessels of the hypocotyl. d magnified view of the white rectangular region from. bars = 50 μm exogenous auxin modulates pin expression pin expression patterns in wild type plants showed variability in the roots. this may be caused by variation in the auxin distribution that occurs naturally during plant growth. to test this hypothesis, we assessed the effect of seedling treatments with different concentrations of exogenous auxin. auxin treatment changed the pin expression domain in the root meristem. auxin effect on pin protein expression in the root apical meristem was dual: auxin upregulated pin expression in low-level treatments and inhibited expression at high dosage. in roots treated with exogenous auxin, we observed both extension and shortening of pin expression domains in the vasculature and outer layers—features we noted as variable in control plants. for example, in low-level treatments, the pin expression domain was extended in the endodermis, cortex, and stele; in many roots, pin was also detected in the second and third tiers of the columella. in contrast, under high-level treatments, the pin domain was significantly reduced in all of the tissues listed above.fig. auxin regulated pin expression in the root tip. a and b immunostaining of pin expression in wild type plants treated with low and high naa dosage for 24 h. a the effect of exogenous low-level auxin on pin upregulation in the meristem and at the sites of lateral primordia outgrowth. b modulation of pin expression domain under low and high naa treatments, compared to control. the variation in length of the expression domains for different cell lineages is shown by coloured triangles. csc—columella stem cell, c2—the second columella tier, c3- the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature. anti-pin staining is in green, dapi is in the blue channel. bars = 50 μm. c and d quantitative estimates of the changes in pin domain length and pin maximal expression intensity under exogenous iaa treatments with different dosages. the experimental images were analysed in imagej. green line—estimates for pin1pro:pin1-gfp plants; blue line—for pin1pro:gus plants. the measured values for each iaa dosage and for each reporter line were normalized to controls. statistics differences were identified by t-test: * p-value < ; ** p-value < ; *** p-value <  to estimate the changes in pin expression in the primary root meristem quantitatively, we analysed auxin-induced reporter activity in pin1pro:gus and pin1pro:pin1-gfp plants. thus, we were able to compare the auxin effect on pin promoter activity in pin1pro:gus with auxin-dependent post-transcriptional regulation of pin1-gfp protein in pin1pro:pin1-gfp. using imagej, we quantified the maximal and average intensities of the reporter signals, as well as the width and length of the pin expression domains in the meristem. statistical analysis of the measured characteristics supported auxin dose–response of pin expression. in both lines, the length of the expression domain in the stele was significantly increased along the root central axis after treatment with low iaa concentration. additionally, the length of the pin expression domain was significantly decreased by high iaa concentrations in both lines. by analysing the maximal intensity of the reporter activity, we found differences between the lines. in the pin1pro:pin1-gfp line, a significant increase in gfp fluorescence was detected under and  μm iaa. after and 10 μm iaa treatment, gfp fluorescence was significantly reduced. gus staining was always increased with increased exogenous auxin. starting from  μm iaa, the treatment caused significant upregulation of pin promoter activity. taking into account that the pin1pro:gus line reveals the pin promoter activity by gus staining and the pin1pro:pin1-gfp line monitors the amount of pin1-gfp protein, we conclude that the auxin effect on pin expression involves both transcriptional and post-transcriptional regulation. namely, the positive auxin effect on pin expression is explicit at the level of pin promoter activity, whereas the inhibitory auxin effect relates to post-transcriptional regulation of pin expression. summarizing the experimental data on exogenous auxin treatments, we conclude that the variability in the pin expression pattern, which was observed among untreated roots, may be simulated by exogenous auxin treatment. this indicates that differences in endogenous auxin levels among individual roots may be the source of variability in the pin expression pattern. pin expression in pin mutants because pin proteins mediated auxin efflux from individual cells, we expected that auxin distribution in the root tips of pin knockouts would be slightly different from control plants. indeed, cross-regulation of pin expression in the pin mutant has been reported before. we analysed the changes in pin immunolocalization in the roots of single knockouts of the genes encoding long pins. the changes in the pin expression domain compared to controls are summarized in additional file and fig.  fig. pin expression patterns in pin mutants compared to wild type. csc—columella stem cell, c2—the second columella tier, c3—the third columella tier, epid—epidermis, c—cortex, en—endodermis, prc—pericycle, vsc—vasculature. coloured triangles—the end of the expression domain in the respective layer. anti-pin staining is in green, dapi is in the blue channel. bars = 50 μm in wild type plants, pin was expressed and polarized rootward up to the six youngest epidermal cell row. in the pin mutant, the pin domain in the epidermis was frequently extended up to the twentieth cell row. in the ectopic domain, the pin protein had shootward polarity inherent to pin which allowed pin to partially substitute the knocked-out gene. in the wild type cortex, pin was expressed rootward, and it gradually declined in intensity up to the 16th cell row from the qc. in the cortex of the pin mutant, pin has the same rootward polarization but in an extended domain. we detected a high level of pin expression up to the 18th cell row from the qc, which then gradually decreased moving out towards the 25th cell row. these data support previously reported findings. in addition, in pin mutants, we observed non-polar expression of pin in the second columella tier more frequently than in wild type. the intensity of the expression was weak but still stronger than in those rare cases when it was detected in this region in wild type. changes in pin expression in the vasculature, endodermis and cortex of pin pin pin mutants were similar: the pin expression domain extended further to the elongation zone in 60–75 % of plants. in the pin mutant, pin also showed ectopic expression in the second columella tier, similar to the pin mutant. in the pin mutant, the domain of pin ectopic expression in the columella was enlarged and covered up to four tiers. the experimental results show that, in single pin mutants, pin expression extended to the sites of normal expression for knocked-out pin pin pin and pin genes. with the exception of the significantly extended pin domain in the epidermis and cortex of pin mutants, all other changes in the pin expression pattern in pin mutants were enough close to the spectrum of variability detected in wild type. we assume that the changes in pin expression might be a reaction to changes in endogenous auxin content in pin mutants. tissue-specific auxin accumulation or depletion, which must occur in the absence of one of the pins, may result in adjustment of the pin expression domain. discussion auxin levels control the identities of cells and underlie a wide range of developmental phenomena. auxin gradients in tissues are the result of auxin movement between cells due to diffusion and active transport. auxin efflux carriers of the pin family were shown to be key regulators of auxin distribution. pin is the founding member of this family, and its expression is often used to monitor the effects of other factors on auxin distribution. various aspects of pin expression but only in relation to the main topics of these papers were described. here, we present a study focused on pin expression in a. thaliana root, which confirmed a number of published facts and revealed some new features of pin expression in the primary root all along its length, up to the root-shoot junction. the regular anatomy of the root apex allowed us to describe the variation and complexity of pin expression in 3d and revealed the stable and varying parts of the expression pattern. the stele is the main pin expression domain in the root meristem. observations of pin expression in 3d demonstrate that it is not uniform; distinct cell lineages stop expressing pin at different distances from the qc. the protophloem and protophloem-pole pericycle lost pin signal in the proximal two-thirds of the meristem; procambium and the rest of pericycle lost pin expression from there to the end of the meristem. in contrast, xylem precursors still expressed pin in the distal part of the elongation zone. pin has been suggested to be a marker for non-differentiated cells. here, we found that it accurately marked the beginning and end of the transition domain in the root tip. as previously described, there are no clear markers for the transition domain of the meristem. we suggest that the reference point for the beginning of the transition domain could be the first cell in the protophloem lineage, which lost pin signal. the reference point for the end of the transition domain could be the last protoxylem cell expressing pin moreover, the end of the pin expression domain in the endodermis coincided with the end of the meristem, which coud be estimated based on the first rapidly elongated cortex cell. in the stele, pin is mainly localized rootward, driving auxin across the vasculature towards the qc. some spreading of pin protein to the inner lateral sides of the stele cells was observed. in xylem precursors, pin had rootward localization, whereas in procambium cells, pin also occupied the lateral sides. we investigated pin on the rootward and lateral sides of the stele cells in more detail. all stele initials and their first daughters have the “procambium mode” of pin expression in which pin occupies the rootward and lateral parts of the plasma membrane. furthermore, there is a difference in pin expression in the descendants of different lineages. vascular precursors in approximately ten of the horizontal rows from the qc along with stable rw have two types of pin lateral polarization. the rwli type of polarization means that along with rw, pin protein settles on the inner lateral sides and allows inward auxin flow. the xylem precursors completely lose pin lateral positioning in the upper one-third of the meristematic zone. the other vascular cells show variation in the lateral positioning up to the end of the meristematic zone, accompanied by a gradual decrease in pin expression. in the pericycle, constant rootward positioning of pin was also maintained up to the end of the meristematic zone, with the lateral polarity becoming rwli early in the cell lineage. weak rootward pin with some lateral spreading was also reported in the endodermis. in the endodermis, variations in polarization looked similar to those in the pericycle; along with stable rootward positioning, pin spread to the lower part of the internal lateral side. in the cortex, only weak rootward signal was recorded, mainly in the youngest cells. we additionally showed spreading of pin to the lateral sides in these cells and extension of the pin domain to the middle of the meristematic zone in some roots. our data on pin expression in the youngest epidermal cells, the qc, the columella initials, the hypocotyl and lateral root primordia was consistent with previous reports. comparing our map of pin expression with the auxin distribution map in the root tip, we see that stable pin expression occurs in regions with high to intermediate auxin levels. the regions with variable pin expression are characterized by declining auxin. roots slightly differ in the endogenous auxin level, and this may result in different sizes for the pin expression domain. we hypothesized that changing the auxin level in the root, for example, by auxin treatment, would move the edge between high/intermediate and low auxin levels and this would increase or decrease the length of the pin expression domain. the changes in pin domain length observed after auxin treatment might also correlate with auxin induced changes in the size of the root meristem, as previously described. indeed, we showed that treatment with low concentrations of auxin led to lengthening of the expression domain in the stele, endodermis, cortex and columella. in contrast, treatment with high auxin concentrations reduced the pin expression domain. quantitative analysis of the expression changes in pin1pro:pin1-gfp and pin1pro:gus roots after iaa treatment supported the results statistically. similar dose effects have been found previously using another auxin:  μm -d was found to be the optimal concentration for upregulation of pin1pro:pin1-gfp expression in epidermal cells, whereas pin1-gfp activity decreased in the stele at higher -d concentrations. we suggest that the changes in the root auxin level after treatment with exogenous auxin and their influence on the pin expression domain simulated and exaggerated the naturally occurring variations in auxin distribution that occur between individual roots. these changes can influence pin expression. by analysing the maximal intensities of reporter activity in pin1pro:pin1-gfp and pin1pro:gus plants, we found significant differences between the auxin responses of the lines. in pin1pro:pin1-gfp, gfp fluorescence significantly increased and decreased under –  μm iaa and 5–10 μm iaa, respectively. gus staining in pin1pro:gus increased with an increase in exogenous auxin dose. these results suggest that positive and negative effects of auxin on pin are mediated at different levels—transcriptional and post-transcriptional. the shortening of the pin expression domain at a high auxin dosage may be regulated post-transcriptionally. finally, we analysed pin expression domain in the single pin mutants. we expected that local changes in endogenous auxin level occur in pin mutants, which might affect pin expression. pin pin pin and pin are expressed together with pin in the root meristem and may provide redundancy if one of them is mutated. the pin cross-regulation in the pin mutant were described previously. here, we provide a comparative description of pin expression in single pin pin pin and pin mutants. in pin and pin mutants, pin almost fully occupies the domain of the knocked-out pin genes. in the case of pin it is in the vasculature only. in the pin mutant, pin completely occupies the pin expression domain in the vascular system, and it partially occupies the first columella tier. comparing the expression patterns of pin in pin mutants with the wild type, we find that the changes in pin expression mainly fall within the spectrum of variability observed in wild type plants. by regulating pin expression level and the size of the pin expression domain, auxin determines the efficiency of its own transport. by controlling pin polarity, auxin determines the directions of its own streams. the rootward polarity of pin promotes auxin flow to the root tip. the inner lateral location of pin in the endodermis and pericycle collects auxin from the shootward auxin flow to the vasculature, and pin lateralization in procambial cells allows to accumulate auxin in the narrower stream in xylem precursors. this accumulation is probably important for vascular differentiation inside the meristem. naa treatments at concentrations as low as –1 μm affected pin polarity and caused pin to spread across the entire inner lateral sides in the endodermis and pericycle, but higher naa concentrations did not show further polarization, suggesting saturation of the effect. this may indicate that the responses of expression level and polarization to auxin are different. in our experiments, comparing the maps of pin expression and its polar locations gave evidence showing that pin polarization may be more sensitive to minor changes in auxin cellular concentrations than the pin expression level.  pin proteins reduce the cellular auxin level by carrying auxin out of the cell. as a feedback mechanism, auxin regulates the transcription, stability and polarization of pins. we have clarified the role of the founding member of the pin family in these close and interconnected relationships that establish the auxin distribution, which is a key piece of positional information for cell fate determination in the root stem cell niche. 
comparative studies of exogenous auxin effects on pin1pro:gus and pin1pro:pin1-gfp plants indicated that a positive auxin effect is explicit at the level of pin promoter activity, whereas the inhibitory effect relates to post-transcriptional regulation. we provide here a detailed map of pin expression in the primary root, in the lateral root primordia and at the root-shoot junction. to simulate this effect, we analysed pin expression in the roots from wild type seedlings treated with different iaa concentrations and pin mutants. most changes in pin expression after exogenous iaa treatment and in pin mutants were also recorded in wild type but with lower frequency and intensity. to determine if this uncertainty is related to the specific foci of articles or has a basis in the variability of pin gene activity, we performed a comprehensive 3d analysis of pin expression patterns in arabidopsis thaliana roots. our results suggest that the pin expression pattern in the root meristem accurately reflects changes in auxin content. the variability in pin expression pattern observed in individual roots may occur due to differences in auxin distribution between plants. this explains the variability of pin expression in the individual roots and makes pin a good marker for studying root meristem activity. this is why factors that are supposed to be involved in auxin distribution are frequently tested in the regulation of pin expression. pin1polar auxin transportroot meristemhypocotylirocs toolboximage analysisthe 3rd international conference on plant genetics, genomics, bioinformatics and biotechnology novosibirsk, russia 17- june http://conf.nsc.ru/plantgen2015issue-copyright-statement© the author 2016
 theauxin efflux carrier pin is a key mediator of polar auxin transport in developing plant tissues. this is why factors that are supposed to be involved in auxin distribution are frequently tested in the regulation of pin expression. as a result, diverse aspects of pin expression are dispersed across dozens of papers entirely devoted to other specific topics related to the auxin pathway. integration of these puzzle pieces about pin expression revealed that, along with a recurring pattern, some features of pin expression varied from article to article. to determine if this uncertainty is related to the specific foci of articles or has a basis in the variability of pin gene activity, we performed a comprehensive 3d analysis of pin expression patterns in arabidopsis thaliana roots.  we provide here a detailed map of pin expression in the primary root, in the lateral root primordia and at the root-shoot junction. the variability in pin expression pattern observed in individual roots may occur due to differences in auxin distribution between plants. to simulate this effect, we analysed pin expression in the roots from wild type seedlings treated with different iaa concentrations and pin mutants. most changes in pin expression after exogenous iaa treatment and in pin mutants were also recorded in wild type but with lower frequency and intensity. comparative studies of exogenous auxin effects on pin1pro:gus and pin1pro:pin1-gfp plants indicated that a positive auxin effect is explicit at the level of pin promoter activity, whereas the inhibitory effect relates to post-transcriptional regulation.  our results suggest that the pin expression pattern in the root meristem accurately reflects changes in auxin content. this explains the variability of pin expression in the individual roots and makes pin a good marker for studying root meristem activity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pin1polar auxin transportroot meristemhypocotylirocs toolboximage analysisthe 3rd international conference on plant genetics, genomics, bioinformatics and biotechnology novosibirsk, russia 17- june http://conf.nsc.ru/plantgen2015issue-copyright-statement© the author 2016 