Auxin transport through non-hair cells sustains
root-hair development
Angharad R. Jones1, Eric M. Kramer2,3, Kirsten Knox4,5, Ranjan Swarup3,6, Malcolm J. Bennett3,6, Colin M. Lazarus1, H. M. Ottoline Leyser4 and Claire S. Grierson1,7
The plant hormone auxin controls root epidermal cell development in a concentration-dependent manner1–3. Root hairs are produced on a subset of epidermal cells as they increase in distance from the root tip. Auxin is required for their initiation4–7 and continued growth8–11, but little is known about its distribution in this region of the root. Contrary toactive transport
the expectation that hair cells might require active auxin influx to ensure auxin supply, we did not detect the auxin-influx transporter AUX1 in root-hair cells. A high level of
AUX1 expression was detected in adjacent non-hair cell files. Non-hair cells were necessary to achieve wild-type root-hair length, although an auxin response was not required in these cells. Three-d
imensional modelling of auxin flow in the root
tip suggests that AUX1-dependent transport through non-
hair cells maintains an auxin supply to developing hair cells
as they increase in distance from the root tip, and sustains root-hair outgrowth. Experimental data support the hypothesis that instead of moving uniformly though the epidermal cell layer3,12, auxin is mainly transported through canals that extend longitudinally into the tissue.
Precise temporal control of developmental processes is essential for the production of complex, repeatable cell morphologies. The root epider-mis of Arabidopsis thaliana consists of two cell types, hair and non-hair, that are found in files that run the length of the root (Fig. 1a). Root hairs are long, thin outgrowths that emerge from the surface of epidermal cells. The specialized shape of root-hair cells is likely to be important for efficient uptake of water and nutrients. Variation in environmental con-ditions can alter the morphology of hair cells, in the simplest instance, affecting the length of the hair13. The tube-like structure of the hair is gradually extended by growth at its tip. Duration of tip growth affects the final length of the hair4.
As root epidermal cells mature, their distance from the root tip increases. Cells undergoing division, elongation and differentiation are found in lon-gitudinal zones of the root (Fig. 1a). A concentration maximum of the plant hormone auxin is found in the root tip1. Auxin is transported in an acropetal (tipwards) direction in the stele, redistributed in the root tip and transported in a basipetal (shootwards) direction in the lateral root cap and epidermis2 (Fig. 1a). With sufficient movement of auxin from the basipetal to the acropetal stream, mathematical modelling demon-strates a reflux loop is created, which is sufficient to establish and maintain local auxin concentrations and zonation of developmental activity in the two most apical zones of the root3. Root-hair growth is observed in the differentiation zone of the root, the most basally located developmen-tal zone (Fig. 1a). Initiation of root-hair growth is dependent on auxin5–7 and the relative amounts of two antagonistically acting AUX/IAA tran-scription factors, AXR3 and SHY2, which are degraded in response to auxin4. Initiation is followed by a period of rapid outgrowth. Addition of exogenous auxin increases root-hair length, whereas inhibition of auxin signalling or disruption of auxin transport results in a decrease in root-hair length8,9. Experiments in which the intracellular auxin concentration of developing root-hair cells was manipulated indicate a strong positive relationship between auxin concentration and root-hair outgrowth10,11. These data suggest that the differentiation zone represents a region of the root within which spatial distributions of AUX/IAAs and auxin promote root-hair growth. However, little is known about the distribution or move-ment of auxin in this region of the root.
Auxin distribution is controlled by the movement of auxin into and out of individual cells. Protonated auxin enters cells from the cell wall by diffusion, whereas the ion enters by the activity of AUX1/LAX auxin-influx facilitators. AUX1 activity increases efficiency of auxin uptake, compared with diffusion alone, allowing accumulation and efficient transport of auxin within tissues12,14,15. At cytoplasmic pH, auxin is almost completely deprotonated and as a result requires active transport to move across the plasma membrane and out of the cell. Asymmetric subcellular localization of the PIN family of auxin efflux carriers16 there-fore creates directional movement of auxin2,17. Some P-glycoproteins also have auxin-transporting capabilities11,18, but their physiological role is less clear. Although AUX1 and PIN2 are expressed in the root epider-mis2,6,19,20, relative hair and non-hair expression levels are unclear. We
1School of Biological Sciences, University of Bristol, BS8 1UG, UK. 2Physics Department, Bard College at Simon’s Rock, Massachusetts, MA 01230, USA. 3Centre for Plant Integrative Biology, University of Nottingham, LE12 5RD UK. 4Department of Biology, University of York, YO10 5YW, UK. 5Institute of Molecular Plant Sciences, University of Edinburgh, EH9 3JR, UK. 6Plant Sciences Division, School of Biosciences, University of Nottingham, LE12 5RD UK.
7Correspondence should be addressed to: C.S.G. (e-mail: Claire.Grierson@bristol.ac.uk)
Received 7 July 2008; accepted 29 September 2008; published online 14 December 2008; DOI: 10.1038/ncb1815
studied the expression of functional AUX1–YFP under the control of the AUX1 promoter (AUX1::AUX1–YFP)21 and functional PIN2–GFP under the PIN2 promoter (PIN2::PIN2–GFP)22 in the elongation and differentiation zones of the root. As auxin is essential for root-hair elon-gation, and AUX1 seems important for auxin accumulation in the epi-dermis12,15, the naive expectation is that AUX1–YFP should be expressed at high levels in hair cells. Surprisingly, AUX1–YFP was undetectable above background fluorescence in hair cells, whereas a strong YFP signal was observed in non-hair cells (Fig. 1b). AUX1 expression was first observed in the epidermis as it emerges from under the root cap, from which point cell-file-specific stripes were visible (Supplementary Information, Fig. S1). We observed a PIN2–GFP signal in both cell types (Fig. 1c).
Despite the absence of detectable AUX1–YFP in hair cells, the aver-age root-hair length of the aux1-22 null mutant21 was shorter than the wild-type (Fig. 2a, b, d), but could be increased by the application of exogenous auxin (Supplementary Information, Fig. S2). This agrees with previous descriptions of mutant aux1 alleles8,9. Transgenic expression of AUX1–YFP under the control of a root-hair-specific promoter has previ-ously been shown to increase the length of root hairs11, indicatin
g that AUX1 can directly affect root-hair length by affecting the concentration of auxin in root-hair cells. However, we found that introduction of the AUX1::AUX1–YFP construct, which is undetectable in hair cells, was sufficient to increase aux1-22 root-hair length (Fig. 2c, d).
AUX1 is expressed in the stele, where it is thought to be associated with the accumulation of auxin in the root tip19, and in the lateral root cap and epidermis19 where it is essential in establishing the supply of auxin to developing epidermal cells12. Consistently, GUS expression from the auxin responsive reporter IAA2::GUS19 was found to be more intense in the root tip12 and the differentiation zone of wild-type roots than of aux1-22 roots (Fig. 2e, f). Within the aux1-22 genotype, the most intense staining was observed in individuals that also produced the longest hairs (Supplementary Information, Fig. S3), suggesting that one or both of the observed auxin responses may be associated with root-hair growth. To determine whether acropetal or basipetal transport is required for root-hair growth, functional HA-tagged AUX1 was introduced into the aux1-22 background under the control of tissue-specific promoters using a GAL4 transactivation approach12. Expressed under the control of a stele-specific promoter (J1701), HA–AUX1 was not able to increase the average length of aux1-22 root hairs, but under the control of a promoter active in the lateral root cap and epidermis (M0028), aux1-22 root-hair length was increased (Fig. 2g). This suggests that basipetal auxin trans-port, (M0028)
rather than acropetal transport (J1701), is important for root-hair growth, and indicates that the auxin response in the differen-tiation zone may be important. Interestingly, when expressed under a root-cap-specific promoter (M0013), HA–AUX1 did not increase root-hair length (Fig. 2g), indicating that AUX1 may be required in both the epidermis and lateral root cap to induce wild-type root-hair growth. We next investigated the role of non-hair cells in the control of root-hair growth. In the werewolf/myb23 (wer/myb23) mutant, every epidermal cell develops as a hair cell23 (Fig. 3b). Expression of the AUX1::AUX1–YFP construct was detected in the stele and root cap, but not the epidermis (Supplementary Information, Fig. S4). wer/myb23root hairs were
found to be shorter than wild-type hairs (Fig. 3a–c), but increased to a length equal to the wild-type on application of auxin (Supplementary Information, Fig. S5). This suggests that the length of wer/myb23 root hairs is restricted by the loss of a promoting signal produced by non-hair cells. It has been shown previously that interaction between hair and non-hair cells is essential during the establishment of epidermal cell fate24. Our
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Figure 1 AUX1 and PIN2 expression in hair and non-hair epidermal cells. (a) Diagram showing tissue organization and developmental zones of the Arabidopsis root. The left side shows a longitudinal section. The right side shows a surface view of the root. Note that the differentiation zone overlaps the
proximal end of the elongation zone. Root-hair cell files are shown in blue and non-hair cell files in pink. The root cap is shown in grey. Direction of auxin flow is shown in magenta2. (b, c) Confocal fluorescence imaging of auxin transporters in the developing root showing expression of AUX1::AUX1–YFP (yellow, b) and PIN2::PIN2–GFP (green, c).From top to bottom, images are representative of the expression pattern in the late differentiation zone, early differentiation zone, elongation zone and root cap. Cell walls are stained with propidium iodide (magenta). Scale bars represent 50 μm.
results suggest that interaction between non-hair cells and hair cells is also required to sustain root-hair development.
Although non-hair cells were found to promote root-hair growth, an auxin response was not required in this cell type. The gain-of-func-tion axr3-1 mutation blocks the auxin response in root tissues 12 and prevents root-hair development 4,25 (Fig. 3d). Expression of axr3-1 in epidermal initials and non-hair cells using a transactivation driver line (J2301) (Fig. 3g) did not prevent root-hair initiation or growth (Fig. 3e; Supplementary Information, Fig. S6), although expression of the same construct throughout the root did (J0491) (Fig. 3f, h; Supplementary Information, Fig. S6). Taken together with the expression pattern of AUX1 in the epidermis and the role of intracellular auxin in promoting
root-hair elongation 10,11, the simplest explanation of these results is that non-hair cells affect the supply of auxin to hair cells.
To predict auxin distribution in the developing epidermis, we incor-porated the observed AUX1 expression pattern into a computer model of auxin transport in the elongation and differentiation zones of the root. The model simulates cells in the outer three layers of the root (Fig. 4a). Auxin movement across the plasma membrane and within the cytoplasmic and apoplastic compartments is simulated, incorporat-ing both carrier-mediated and diffusive auxin movement as appropriate. Parameter values are based on available biophysical and biochemical data as described in the Supplementary Information and in a previous study 12
. The model is an improvement of an earlier model of the root
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Figure 2 Basipetal AUX1-mediated auxin transport is required for auxin-dependent root-hair growth. (a –c ) Root-hair phenotype of wild-type (a ), aux1-22 (b ) and aux1-22 plants transformed with AUX1::AUX1–YFP (AUX1::AUX1–YFP(aux1-22), c ). Scale bars represent 500 μm. (d ) Frequency dist
ribution of root-hair lengths in wild-type, aux1-22 and AUX1::AUX1–YFP (aux1-22) plants. (e , f ) GUS-stained roots of 5-day-old seedlings of wild-type (e ) and aux1-22 (f ) plants carrying the IAA2::GUS  auxin response reporter. Double-headed arrow indicates expected location of differentiation zone. Scale bars represent 500 μm. (g ) Average root-hair length of aux1-22 plants expressing HA–AUX1 in a tissue-specific manner using a transactivation approach. Root-hair length of F1 individuals
(J1701>>HA–AUX1, M0028>>UAS::HA–AUX1 and M0013>>UAS::HA–AUX1) and non-crossed siblings (UAS::HA–AUX1, J1701, M0028 and M0013) are shown. All parental lines are in the aux1-22 background. J1701 drives expression in the stele, M0013 drives expression in the lateral root cap and M0028 drives expression in the lateral root cap and epidermis. Error bars represent 95% confidence intervals (n  = 260 hairs from 26 roots).
apex 12 in the following ways. First, each hair-cell file overlies a radial longitudinal wall separating two cortical cells. Contact with this wall, as viewed in transverse sections of the Arabidopsis  root, is the major deter-minant of hair-cell fate 26. Second, AUX1-mediated influx is only present in non-hair cells. This 3D model allows the effects of lateral diffusion in the tangential plane of the tissue, essential to understanding movement between hair and non-hair cell files, to be studied.
The most prominent effect of the AUX1 expression pattern is that non-hair cells accumulate a much higher cytoplasmic concentration of auxin than hair cells (Fig. 4b). As the model parameters reflect our estimate that AUX1 allows auxin to enter cells about 10 times faster than by diffusion alone 14, the concentration in non-hair cells is higher by roughly this ratio. However, as auxin can enter cells by diffusion, it is not completely excluded from hair cells. If the auxin source at the root apex allows for about 300 nM auxin in the non-hair cells, the concen-tration in hair cells is about 30 nM. This is well within the measured
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Figure 3 Non-hair cells are essential for root-hair development, but an
auxin response is not required in non-hair cells. (a , b ) Root-hair phenotype of wild-type (a ) and wer/myb23 (b ) plants. Scale bars represent 500 μm. (c ) Frequency distribution of root-hair lengths in wild-type and wer/myb23 plants. (d –h ) Disruption of the auxin response through tissue-specific expression of axr3-1 using a transactivation approach. Shown are root-hairless phenotype of axr3-1(d )
, root-hair phenotype of J2301>>axr3-1  (e ) and J0491>>axr3-1 (f ) in F1 individuals. Scale bars represent 800 μm. (g , h ) Expression pattern of the J2301 (g ) and J0491 (h )
promoters as demonstrated by GFP expression. GFP fluorescence is shown in green. Cell walls stained with propidium iodide are shown in red. J2301 is expressed in non-hair cells and J0491 is expressed throughout the root. Scale bars represent 50 μm.
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(Wild-type auxin supply)Figure 4 Expression of AUX1 in non-hair cells affects the supply of auxin to hair cells in the differentiation zone of the root. (a ) The image on the left shows a sketch of the computer model of the Arabidopsis  root apex. The epidermis, cortex, and endodermis are resolved in the elongation and differentiation zones. Auxin is supplied to the tissue by the proximal end of the lateral root cap (blue) and transported basipetally by the activity of AUX1 influx carriers (orange) and PIN  family efflux carriers (plum). Cell membranes without influx carriers are shown in grey. Auxin is free to diffuse through the apoplast (yellow), which forms a continuous compartment between cells, and through the cytoplasm of cells (green). The right-hand image shows a transverse section through the model root showing cell geometry. Cells overlying a junction between two cortical cells are hair (H) cells; cells that do not overly a junction are non-hair cells (N). AUX1 expression is limited to N cells. (b ) Model results showing the concentration of auxin in N cell files (blue) and H cell files (red) over increasing distance from the root tip. Dark colours show cytoplasmic auxin concentration, lighter colours show the apoplastic concentration between cells in a longitudinal file. (c ) Model results for the aux1 mutant (green) superimposed on the wild-type results from panel b , assuming auxin supply from the cap is 10% of wild-type. (d ) Model results for the aux1 mutant assuming that the auxin supply from the lateral root cap is unchanged from wild-type.
range of biological activity in root-hair development assays 10,27 (see Supplementary Information, Details of the computer model). Auxin flux, which is calculated as the product of auxin concentration and auxin-transport speed, will also be higher through non-hair cell files than in hair cell files. However, the small amount of flux created by the expression of PIN2 in hair cells may still be important in preventing auxin from pooling in these cells 28 and in determining their polarity 6. Interestingly, an intracellular auxin gradient is predicted in both hair and non-hair cells.
Figure 4c shows our model of the aux1 mutant. Loss of AUX1 from non-hair cells means that all epidermal cells have uniform auxin transport capacity, and auxin distribution in hair- and non-hair-cell files is identical. The relatively slow rate of influx allows auxin to accumulate in the cell walls, where it subsequently diffuses from the epidermis into the stele, or out of the root entirely 12. Consequently, auxin concentration in the epi-dermis decreases rapidly with increasing distance from the apical auxin source. As aux1 plants accumulate a lower concentration of auxin in the root tip 29 and transport through the root cap is likely to be reduced 12,19, the supply of auxin to the elongation zone may also be decreased. The simu-lation shown in Fig. 4c assumes that the auxin supply to the elongation zone is reduced when compared with the wild-type. If the auxin supply is unchanged (Fig. 4d), auxin concentrations in the aux1 epidermis are uniformly higher, but still drop below the wild-type values app
roximately 500 μm from the meristem. This may represent auxin distribution in the wer/myb23 mutant, in which AUX1 is detected in the stele and the root cap, but not the epidermis. In both versions of the model, hair cells in the differentiation zone (>500 μm from the meristem) are supplied with less auxin if AUX1 is not expressed in non-hair cell files. This is because auxin taken into non-hair cells through the activity of AUX1 is returned to the cell wall further up the root by PIN proteins and becomes available to more distant hair and non-hair cells. This is in contrast to animal systems where increasing the morphogen uptake capacity of cells within a devel-oping tissue reduces the amount available to more distant target cells 30. An interesting consequence of this mechanism of auxin delivery is that hair cells can be supplied with auxin at an increased distance from the root tip without encountering high intracellular auxin concentrations. Variations to assumptions of the model, including weak expression of AUX1 in hair cells, or axial localization of AUX1, did not change the qualitative conclu-sions (Supplementary Information, Details of computer model).
Direct measurement of the intracellular concentration of auxin in hair and non-hair cells is not possible at present. We therefore used plants carrying the auxin-responsive reporter DR5::GFP to establish an assay for auxin accumulation. After incubation for 4 h with a 1 μM auxin solution (indole-acetic acid), a GFP signal in hair and non-hair cells was detected and quantified. The intensity of the GFP signal was
higher in wild-type non-hair cells than in wild-type hair cells, but no difference in the intensity of GFP signal was detected between hair and non-hair cells in the aux1-22 mutant (Fig. 5a, b). Furthermore, the intensity of the GFP signal in non-hair cells in the aux1-22 mutant was lower than the intensity of GFP signal in non-hair cells of wild-type plants (Fig. 5a, b). These results are qualitatively consistent with the model prediction that non-hair cells accumulate a higher concentration of auxin than hair cells as a result of AUX1 expression. As DR5::GFP  is an auxin-response reporter rather than a direct reporter of auxin concentration, the sig-nificance of the quantitative discrepancy between the 10-fold predicted difference in auxin concentration and the observed 1.6-fold difference in GFP expression is unclear. The sensitivity and specificity of the auxin response is dependent on the expression and interaction of components of the auxin signalling pathway and varies according to developmen-tal context 31. It will be interesting to identify whether such differences exist between hair- and non-hair cells and to investigate how DR5::GFP  expression relates to the hair-specific auxin response.
Assuming an auxin concentration over a threshold level is required for root-hair growth, the auxin concentrations from the model predict that root hairs will cease to elongate closer to the root tip in aux1-22
than in
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Figure 5 DR5::GFP  expression and root-hair growth dynamics support the model auxin distribution. (a ) Confocal fluorescence image of wild-type and aux1-22 roots carrying DR5::GFP  after treatment with IAA (1 μM) for 4 h. Non-hair cell files are indicated as N and hair-cell files are indicated as H. Scale bars represent 25 μm. (b ) Graph showing the average pixel intensity of GFP signal in wild-type and aux1-22 plants expressing DR5::GFP . White bars represent non-hair cells, grey bars represent hair cells. Mean measurements from 4–6 roots are shown. Error bars represent 95% confidence intervals (numbers of cells for each group are shown in the bars). (c ) Mean length of differentiation zone in wild type and aux1-22. Error bars indicate 95% confidence intervals (numbers of cells for each group are shown in the bars). (d ) Relationship between average root-hair length and length of differentiation zone of aux1-22 roots.

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