Auxin regulates distal stem cell differentiation in Arabidopsis roots

Abstract

The stem cell niche in the root meristem is critical for the development of the plant root system. The plant hormone auxin acts as a versatile trigger in many developmental processes, including the regulation of root growth, but its role in the control of the stem cell activity remains largely unclear. Here we show that local auxin levels, determined by biosynthesis and intercellular transport, mediate maintenance or differentiation of distal stem cells in the Arabidopsis thaliana roots. Genetic analysis shows that auxin acts upstream of the major regulators of the stem cell activity, the homeodomain transcription factor WOX5, and the AP-2 transcription factor PLETHORA. Auxin signaling for differentiation of distal stem cells requires the transcriptional repressor IAA17/AXR3 as well as the ARF10 and ARF16 auxin response factors. ARF10 and ARF16 activities repress the WOX5 transcription and restrict it to the quiescent center, where WOX5, in turn, is needed for the activity of PLETHORA. Our investigations reveal that long-distance auxin signals act upstream of the short-range network of transcriptional factors to mediate the differentiation of distal stem cells in roots.

In plants, the permanent populations of stem cells (meristems) generate growth during the entire plant's life span. The well-organized root meristem of Arabidopsis thaliana contains a small number of mitotically inactive central cells, known as the quiescent center (QC), surrounded by different types of stem cells that can differentiate into diversified cell types in roots (1). The differentiation rate of stem cells has a direct impact on the activity of the root meristem and thus determines the root architecture. The homeobox gene WUSCHEL-RELATED HOMEOBOX 5 (WOX5) is a major regulator of the root stem cell activity. WOX5 is expressed in the QC and maintains the surrounding stem cells, as demonstrated by differentiation of distal stem cells (DSC) in the wox5 mutant and the inhibited DSC differentiation in the WOX5 overexpressors (2). The other important regulators of the root stem cell activity are the PLETHORA (PLT) AP2-domain transcription factors with PLT1 being the key member of the gene family involved in the root (3, 4). Despite the accumulated knowledge on these factors required for stem cell activity, little is known about their mutual functional relations and how they are connected to other, also long-distance, signaling networks.

The plant hormone auxin is an important long- and short-distance signal that controls multiple developmental processes (5, 6), including root patterning (7–12) and root cell division and elongation (13–15). Previous observations also suggest that auxin plays a role in regulating and maintaining stem cell identities (8, 9, 16), but the underlying mechanism remains unclear.

Our investigations show that local auxin levels mediated by biosynthesis and transport play a critical role during the differentiation of DSC in roots. The components of auxin signaling including the IAA17/AXR3 transcriptional repressor and the ARF10 and ARF16 auxin response factors are required for this auxin effect. ARF10 and ARF16 activities repress the WOX5 expression and restrict it to the QC, where WOX5, in turn, is required for the activity of PLETHORA. Our studies revealed a connection between long-distance auxin signals and a local network of transcriptional factors that regulate the activity of the root stem cell niche.

Results

Auxin Promotes Differentiation of Root DSC.

To investigate the role of auxin in regulating the stem cell activity, we focused on a specific subgroup of stem cells, the columella stem cells that represent the only DSC type. DSC consist of a single layer of cells below the QC, and their activity generates cells of the central root cap (columella) that, following their differentiation, accumulate starch-containing amyloplasts to serve as gravity-sensing organs (17).

In untreated 5-d-old seedlings, most (94.2%, n = 212) of the roots had only one layer of DSC, as demonstrated by the absence of the Lugol's solution–based starch staining, which normally stains only differentiated starch-containing columella cells (Fig. 1A and Table S1). Germination of seedlings on medium supplemented with auxin (1 μM 1-naphthaleneacetic acid [NAA]) or the auxin transport inhibitor (1 μM N-1-naphthylphthalamic acid [NPA]) leads to an increase in auxin levels in the root tip (9, 18), as visualized by the enhanced activities of the DR5rev::GFP auxin response reporter (Fig. S1 A–C). Importantly, both treatments promoted DSC differentiation, as evident by the nearly absent DSC in seedlings treated with 1 μM NAA or 5 μM NPA (Fig. 1 A–C and K and Table S1). Next, we performed a time and concentration course of the auxin effect on the DSC differentiation. Three-day-old seedlings were transferred to medium supplemented with 5 μM NAA and DSC differentiation was analyzed over the next 3 d. The auxin-promoted DSC differentiation could be observed clearly within 1 d after transfer to auxin. After 3 d of auxin treatment, the differentiated DSC were visible in most seedlings (88.2%, n = 136) (Fig. 1J). Similarly, the auxin effect on DSC differentiation depended on the concentration, with visible effects starting at 0.05 μM NAA (Fig. 1K).

Fig. 1.

Local auxin levels controlling DSC differentiation. (A–G) Differentiation status of columella cells in 5-d-old seedlings. Nondifferentiated DSC (yellow arrowheads) below the QC (red arrowheads) are characterized by the absence of starch, whereas Lugol's solution–stained starch is visible in differentiated columella cells. WT roots show typically one tier of DSC (A), but increasing auxin levels by growing seedlings on 1 μM NAA (B) (96%, n = 288) or 1 μM NPA (80%, n = 286) (C) leads to DSC differentiation. In contrast, mutants defective in auxin biosynthesis yuc1-/yuc4,10,11/± (D) (53%, n = 208) and wei8-1-/wel1-1±/wel2-2± (E) (22%, n = 246) or auxin transport pin3 (F) (33.6%, n = 286) and pin4 (G) (43%, n = 323) show defects in columella differentiation as manifested by multiple tiers of DSC. (H and I) DSC marker J2341 in pin3-4 roots (I) confirms multiple tiers of DSC as compared with the single tier in WT roots (H). (J) Gradual differentiation of DSC revealed by the frequency of roots without DSC (gray) and with QC divisions (white) after different times of treatment with 5 μM NAA. At least 100 seedlings were examined for each time point for each biological repeat. Error bars depict SD from three biological repeats (Student's t test, *P < 0.01). (K) Quantitative evaluation of DSC differentiation confirms that elevated auxin levels promote DSC differentiation (gray) and conversely that defective auxin biosynthesis or transport inhibit DSC differentiation (white). At least 100 seedlings were examined for each genotypes or drug treatments for each repeat. Error bars depict SDs from three biological repeats (Student's t test, *P < 0.01).

To confirm the treatment experiments with endogenous auxin production, we engineered auxin production in specific cell types through transactivation of IAAM, an enzyme that is involved in the conversion of Trp to indole-3-acetic acid (IAA) (19). The activator lines J2341 and Q1630 were used to induce iaaM expression in DSC and columella cells, respectively. Upon Trp treatment, seedlings produced extra auxin in these cell types, presumably generally increasing the auxin levels in the whole distal root tip. As a consequence, DSC differentiation was promoted in both transgenic lines (Fig. S1 E–H). These observations from both the exogenous auxin applications and the stimulation of auxin biosynthesis show that an increase in auxin levels in the root tip enhances the DSC differentiation.

Defects in Local Auxin Biosynthesis or Transport Delay DSC Differentiation.

We also used a complementary approach and examined the DSC differentiation in roots with decreased auxin activity, more specifically in genetic backgrounds with defective auxin biosynthesis and auxin transport. Mutants defective in auxin biosynthetic enzymes, such as YUCCA (yuc1-/yuc4,10,11/±) (20) or TAA (wei8-1-/wel1-1±/wel2-2±) (21, 22) displayed strongly reduced rates of DSC differentiation (Fig. 1 D, E and K and Table S2), thus with effects opposite to auxin or NPA treatment, which causes higher auxin levels in root tips. Similarly, mutants in the PIN auxin efflux proteins (23) that are expressed in DSC or the columella, such as PIN3 (24), PIN4 (9), and PIN7 (16), all showed defects in the DSC differentiation. Compared with WT seedlings, 5-d-old mutant seedlings had multiple layers of undifferentiated DSC, as shown by the absence of starch (Fig. 1 F, G, and K and Table S1).To confirm the Lugol's solution staining–based observations, we analyzed the DSC-specific marker J2341, of which the broad expression domain in pin3 roots confirms multiple tiers of DSC (Fig. 1 H and I). In contrast to the NPA treatment, which causes higher auxin levels in the root tips reflected by higher DR5 activities and thus increases DSC differentiation (Fig. 1C), pin3 root tips did not show overall altered DR5 activities (Fig. S1 C and D). It appears that only auxin transporters specifically expressed in the QC and columella region are involved in the regulation of the DSC activity, because no comparable DSC differentiation defects were observed in the mutant, which was expressed in other regions of the root, such as PIN2 (Table S2). These observations show that defects in auxin biosynthesis and local auxin transport in the root tip inhibit the DSC differentiation. In summary, these various positive or negative manipulations of the auxin concentration imply that the auxin levels in the root tip contribute to the control of the DSC differentiation.

WOX5 Homeodomain Factor Acts Downstream of Auxin Distribution.

Whereas our data indicate that auxin promotes the DSC differentiation (Fig. 1), the activity of the homeodomain transcription factor WOX5 has been shown to maintain undifferentiated DSC (2). Thus, auxin and WOX5 have opposite effects on the DSC activity and it was possible to test genetically the mutual relation of their actions. Because mutants in the root tip-expressed PIN auxin efflux carriers show less DSC differentiation (Figs. 1 K and 2A) and wox5 root tips show more DSC differentiation (Fig. 2B), we generated wox5 pin double mutants. All of the double mutant combinations, wox5 pin3, wox5 pin4, and wox5 pin7, have defects identical to those of wox5 and exhibited a dramatically increased DSC differentiation (Fig. 2 B–D and Fig. S2 A–C). Thus, wox5 is epistatic to pin3, pin4, and pin7, suggesting that WOX5 acts downstream of auxin in the regulation of the DSC differentiation. Furthermore, the strong inhibition of the DSC differentiation resulting from the WOX5 overexpression (in 35S::WOX5-GR) cannot be rescued by the application of even high auxin doses (Fig. 2 E and F), again consistent with the WOX5 action downstream of auxin in the same pathway.

Fig. 2.

Regulation of stem cell activity by auxin upstream of the WOX5 homeodomain factor. (A–F) Differentiation status of DSC (yellow arrowhead) below the QC (red arrowhead) in 5-d-old seedlings as inferred from the Lugol's solution staining of differentiated columella cells. The auxin transport mutant pin4 shows defects in columella differentiation displaying typically two tiers of DSC (42.8%, n = 323) (A); wox5 (B), wox5 pin4 (C), and wox5 pin3 (D) promote columella differentiation as shown by the absence of DSC. The 35S::WOX5-GR line shows an inhibited DSC differentiation in the presence of 1 μM DEX, as revealed by multiple tiers of DSC (E) that cannot be rescued by 1 μM NAA (F). (G and H) WOX5::ERGFP signal detected in endogenous expression domain in the QC of the primary root (G), whereas this signal was strongly decreased in the presence of 1 μM NAA. Residual very weak WOX5::ERGFP activity was detected in the broader domain encompassing endodermis and cortex initials (H). (I) Transcription of WOX5 repressed by auxin as detected by quantitative PCR analysis of RNA from primary distal root tips treated with 5 μM NAA for the indicated time (h) or grown at 1 μM NAA. Tubulin is used as a constitutively expressed control. Error bars mark SDs from three biological repeats (Student's t test, *P < 0.05; **P < 0.05; ***P < 0.01).

Auxin Treatment Leads to Low WOX5 Transcription in Primary Root.

The results of the genetic experiments between wox5 and auxin-related regulators show that auxin mediates its DSC regulation through the downstream action on WOX5. Because auxin promotes, but WOX5 inhibits, the DSC differentiation, we hypothesized that auxin inhibits the WOX5 action. Indeed, auxin treatment strongly inhibited the signals of WOX5::ERGFP in the endogenous expression domain in the QC; a residual, very weak WOX5::ERGFP activity was detected in a broader domain encompassing the endodermis and cortex initials (Fig. 2 G and H and Fig. S2D). The time course studies confirmed the gradually inhibitory auxin effect on the WOX5 expression with an almost complete disappearance of WOX5::ERGFP after 3 d of treatment (Fig. S3 D–J). We tested the effects of auxin on the WOX5 transcription with quantitative RT-PCR. The time course of the auxin treatment showed an inhibitory auxin effect on the WOX5 transcription that could be observed clearly only 24 h after transfer to auxin-supplemented media (Fig. 2I), thus confirming the GFP fusion-based observations. Notably, auxin inhibits the WOX5::ERGFP signal or WOX5 transcription only in the primary roots, but not in the lateral roots (Fig. S3 A and C), explaining why the otherwise obvious auxin-mediated inhibition of the WOX5 transcription was not detected in global transcriptome analyses (25, 26). Accordingly, lateral roots still maintain stem cell identities under the same auxin treatment (Fig. S3B). This potential difference between the regulatory mechanism of the DSC activity in primary and secondary roots might be related to the similarities of the lateral root organogenesis and the stem cell-independent primary root regeneration processes (25). In summary, our genetic and expression analyses suggest that auxin enhances the DSC differentiation through the downstream transcriptional repression of the WOX5 homeobox regulator of the stem cell activity.

PLT1 Acts Downstream of Auxin to Mediate DSC Maintenance.

The AP-2-type transcription factor PLT1 has been shown, similarly to WOX5, to maintain the activity of stem cells and QC (2, 3). To disclose the genetic relationship between auxin and PLT proteins in regulating the DSC activity, we again generated and analyzed the plt1 pin double mutants. We found that all of the double mutant combinations, such as plt1 pin3, show defects identical to those of plt1 and exhibit DSC differentiation as well as ectopic cell division in the QC and columella (Fig. 3 A–C). Furthermore, the plt1 plt2 pin3 triple mutant also shows defects identical to plt1 plt2, displaying very short roots, DSC differentiation, and ectopic cell division in the QC and columella (Fig. 3 D–F). Thus, our genetic analysis reveals that plt1 is epistatic to the pin mutants, suggesting that, as with WOX5, PLT transcription factors act downstream of auxin in the regulation of the DSC differentiation.

Fig. 3.

Regulation of stem cell activity by auxin and WOX5 through PLT1. (A–E) Differentiation status of DSC (yellow arrowhead) below QC (red arrowhead) in 5-d-old seedlings as inferred from Lugol's solution staining of differentiated columella cells. Blue arrows mark ectopic cell division in QC or columella cells. Auxin transport mutant pin3 is defective in columella differentiation, displaying typically two tiers of DSC (33.6%, n = 286) (A). Both plt1 (47%, n = 66) (B) and plt1 pin3 (45.1%, n = 76) (C) promoted columella differentiation, as indicated by absence of DSC. Both plt1 plt2 (100%, n = 87) (D) and pin3 plt1 plt2 (100%, n = 101) (E) show strong columella differentiation as indicated by absence of DSC. (F) Both plt1 plt2 and pin3 plt1 plt2 have very short roots compared with Col-0 and pin3. (G and H) Strongly repressed PLT1::ERCFP expression in tin allele of wox5 (H) as compared with WT control (G). (I) Induced PLT1 transcription after overexpression of WOX5 as detected by quantitative PCR analysis. Tubulin is relative control. RNA was isolated from roots of 5-d-old 35S::WOX5-GR seedlings without and with 1 μM DEX induction. Error bars mark SDs from three biological repeats (Student's t test, *P < 0.01).

WOX5 Activity Is Required for PLT1 Expression.

Because both PLT1 and WOX5 act downstream of auxin and have similar effects on the maintenance of DSC, we studied the relation between these two factors. PLT proteins have a dosage effect on the maintenance of root stem cells (4). We performed a similar analysis for WOX5. To this end, we examined dexamethasone (DEX)-inducible 35S::WOX5-GR lines (2). Under low concentration of DEX, 35S::WOX5-GR seedlings showed only a moderately delayed DSC differentiation, but with increasing DEX concentration, the DSC differentiation was progressively more inhibited, as demonstrated by a starch accumulation only in the most distal root cap cells (Fig. S4 A–D). Our observations suggest that, like the PLT proteins, WOX5 also has a dosage effect on the DSC maintenance.

Both wox5 and plt1 show an increase in DSC differentiation (2, 4), but at least 53% (n = 66) of the plt1 seedlings still maintain DSC, whereas no stem cells are maintained in plt1 plt2 under our growth conditions (Fig. 3 B and D). The analysis of wox5 plt1 double mutants did not reveal any obvious synergistic or additive phenotypes (Figs. 2B and 3B and Fig. S5A). Nevertheless, the results are not conclusive, because both mutants show similar phenotypes and represent only a partial suppression of the corresponding PLT and WOX function. Therefore, we generated the wox5 plt1 plt2 triple mutant and observed that it was very similar to plt1 plt2, exhibiting very short roots, differentiated DSC, and ectopic cell division in the QC and columella (Fig. S5 B and D). These observations suggest an overall epistatic genetic interaction between wox5 and plt mutants; nonetheless, wox5 might also have an additive effect on plt1 plt2, because ≈35% (n = 57) of the 5-d-old seedlings of wox5 plt1 plt2 showed more pronounced defects in the root meristem activity that resulted in root collapse, which was characterized by the absence of root stem cell activity and terminal differentiation (Fig. S5C).

Our genetic studies suggested that, at least for the regulation of the DSC differentiation, PLTs might act downstream of WOX5 and both factors downstream of auxin. Therefore, we tested the mutual requirement of these factors for the regulation of their expression. The PLT1::ERCFP signals were strongly reduced in the wox5 mutant roots, in particular, in the QC region, where no peak of the PLT1::ERCFP signals could be detected in the WT roots (Fig. 3 G and H and Fig. S5E). Furthermore, quantitative RT-PCR revealed a highly elevated PLT1 expression following DEX-induced expression of WOX5 (Fig. 3I). However, the transcriptional regulation of PLT1 by WOX5 is presumably indirect, because the PLT1 transcription was affected only after a prolonged WOX5 induction (Fig. S5F). These changes in the PLT1 expression in WOX5 gain- and loss-of-function lines show that WOX5 activity is required for the regulation of the PLT expression. On the other hand, consistent with the unchanged WOX5 expression in the plt1plt2 double mutant (2), we did not detect any clear changes in WOX5 expression after DEX-induced expression of PLT2 (Fig. S5 G–L). Thus, genetic investigation and expression analysis both suggest that WOX5 acts upstream of PLT1 in regulating its expression.

IAA17/AXR3 Is Required for Auxin-Promoted DSC Differentiation.

Next, we investigated by which molecular mechanism auxin regulates the DSC activity. The known auxin signaling pathway involves an auxin receptor, the F-box protein TIR1, the AUX/IAA transcriptional repressors, and the ARF transcription factors that control the transcription of auxin-regulated genes (28–31). IAA17/AXR3 is a well-characterized transcriptional repressor of the TIR1-mediated auxin signaling pathway (29–31). The AXR3 stabilized mutant axr3-1 shows a constitutive, auxin-insensitive repression of auxin-mediated gene expression (8). Notably, axr3-1 seedling roots delayed dramatically the DSC differentiation (Fig. S6B). Furthermore, the axr3-1 DSC are almost completely insensitive to the auxin treatment as compared with the WT seedlings germinated on medium with auxin that strongly promoted the DSC differentiation (Fig. S6 A and C). These observations indicate that AXR3 is an important mediator of auxin-induced differentiation. It also suggests that auxin promotes DSC via the TIR1-mediated auxin signaling pathway. Furthermore, the ectopic expression of WOX5::ERGFP in axr3-1 also indicates that the AXR3 activity restricts the WOX5 expression in the QC (Fig. S6 D–F), they might act in the same pathway to maintain DSC identities.

ARF10 and ARF16 Are Required for WOX5-Mediated Maintenance of DSC.

Next, we tested which ARF transcription factors downstream of the AUX/IAA repressors mediate auxin effect on the DSC differentiation. Previous work indicated that the auxin response factors ARF10 and ARF16 of Arabidopsis, targeted by microRNA160 (miR160), play a role in regulating the DSC activity (32). To test whether auxin promotes the DSC differentiation through ARF10 and ARF16, we analyzed the arf10-2 arf16-2 double mutants and the Pro_35S:MIR160 plants, in which the expression of ARF10 and ARF16 is repressed (32). Both arf10-2 arf16-2 and Pro_35S:MIR160 were less sensitive to auxin treatment, still maintaining multiple undifferentiated DSC (Fig. 4 D and E and Fig. S7 A and B) when compared with the WT seedlings germinated on medium with auxin that strongly promoted the DSC differentiation (Fig. 4 A and B). Thus, ARF10 and ARF16 might be involved in the auxin-mediated DSC differentiation.

Fig. 4.

Auxin-promoted root DSC differentiation mediated by ARF proteins. (A–F) Differentiation status of DSC (yellow arrowheads) below QC (red arrowheads) in 4-d-old seedlings as inferred from Lugol's solution staining of differentiated columella cells. WT roots show typically one tier of DSC (A), but increasing auxin levels by growing seedlings on 1 μM NAA (B) leads to DSC differentiation. Pro_35S:MIR160 line shows highly inhibited DSC differentiation as indicated by multiple tiers of DSC (D) that cannot be fully rescued by 1 μM NAA (E). The wox5 Pro_35S:MIR160 double mutant enhanced columella differentiation as shown by the absence of DSC (F), similar to wox5 (C). (G–J) WOX5::ERGFP signals were detected in endogenous expression domain of QC in primary root (G), whereas these signals were strongly inhibited in the presence of 1 μM NAA (H). WOX5::ERGFP signals in Pro_35S:MIR160 are comparable to those in WT controls, whereas very weak ectopic WOX5::ERGFP signals could be observed in columella cells (Inset) (I). Repression of WOX5::ERGFP expression in the QC by auxin was less obvious in Pro_35S:MIR160 (Inset) (J).

Because we identified WOX5 as acting downstream of auxin in the DSC differentiation, we tested the genetic relationship between WOX5 and ARF10 and ARF16 by generating double or triple mutant combinations. Both wox5 Pro_35S:MIR160 and wox5 arf10-2 arf16-2 mutants showed an enhanced DSC differentiation similar to that of wox5 roots (Fig. 4 C and F and Fig. S7 A and C). The differentiation defect in Pro_35S:MIR160 and arf10-2 arf16-2 roots was largely rescued. This wox5 epistatic relation to the mutants in the auxin response factors was again consistent with an action of WOX5 downstream of the auxin signaling during the auxin-mediated DSC differentiation.

To test whether ARF10 and ARF16 can regulate the WOX5 transcription, we examined WOX5::ERGFP signals in Pro_35S:MIR160 roots. In untreated roots, we detected a weak ectopic WOX5::ERGFP expression in the columella region, but the WOX5::ERGFP signals in the QC regions of the Pro_35S:MIR160 lines were comparable to those in WT plants. Importantly, the auxin-mediated down-regulation of the WOX5 transcription was less pronounced in Pro_35S:MIR160 roots. We observed still relatively strong WOX5::ERGFP signals in Pro_35S:MIR160 lines after the auxin treatment, although the signals in the WT control almost completely disappeared (Fig. 4 G–J and Fig. S7D). We also used the ARF16mARF16 line, in which the ARF16 gene is engineered not to be targeted by microRNA160 (32), to investigate whether the regulation of ARF16 by microRNA160 is relevant for the auxin effect on the WOX5 transcription and DSC differentiation. In this line, the WOX5 transcription in the root tips was as reduced as that in the WT controls by auxin treatment. Accordingly, ≈30% (n = 69) of ARF16mARF16 seedlings showed an increased DSC differentiation as evident by the absence of DSC (Fig. S7 E–G) (32). In summary, these results show that ARF10 and ARF16 are involved into the regulation of the WOX5 transcription and thus contribute to the auxin-promoted DSC differentiation.

Discussion

This work provides insights into the mechanism, by which auxin regulates the stem cell activity in the root meristem. Auxin acts as a positive signal for the differentiation of DSC and auxin levels in the root tip are limiting for their maintenance or differentiation. The auxin levels in the root tip are determined by both auxin biosynthesis and auxin transport. It is likely that the local, spatial distribution of auxin within the root cap region is important, as suggested by DSC differentiation defects in roots lacking specifically the function of the root cap-expressed PIN transporters, in contrast to the opposite effects of the overall pharmacological inhibition of the auxin transport. The auxin signal for the differentiation of DSC is interpreted by the nuclear signaling pathway encompassing the AUX/IAA and ARF transcriptional regulators. The key components in this process are ARF10 and ARF16 that are regulated by the MIR160 microRNA. This signaling circuit acts upstream of known key regulators of the stem cell activity, the homeobox WOX5 and the AP2-domain PLT transcription factors. Auxin represses the transcription and thus the activity of WOX5, which in turn acts upstream of PLT1 and is required for PLT1 expression in the central root meristem. The high PLT activity then maintains the DSC, whereas the low PLT activity promotes their differentiation. This seemingly linear model of how auxin regulates the differentiation of DSC (Fig. S8) represents only the initial insight into complex regulations. The slow regulation kinetics of the WOX5 transcription by auxin as well as the only partially overlapping expression patterns of ARF10, ARF16, WOX5, and PLT (2, 4, 32) shows that these regulations are most likely indirect and include other components as well as non–cell-autonomous regulations that might involve, besides the MIR160, also secretory peptides, such as CLE40 and its putative receptor the ACR4 receptor kinase (33). How these and other signals are integrated into the auxin-WOX5-PLT1 signaling circuit remains an outstanding question.

Our work links the long-distance hormone signaling with short-range cell-to-cell signaling that regulates the activity of the central root meristem. It is intriguing to speculate that the distal auxin maximum in the root tip that depends on the local auxin biosynthesis and transport (8, 9, 20, 21) is, via this mechanism, connected to the network of the transcription factors that control the DSC activity.

Material and Methods

Plant Material and Growth Conditions.

The published transgenic and mutant lines were as follows: wox5-1, 35S::WOX5-GR (2); tin allele of wox5 (34), axr3-1 (8), plt1-4, plt1 plt2, PLT1::ERCFP, WOX5::ERGFP, 35S::PLT2-GR (35); pin3-4 (24); pin4-3 (9); DR5rev::GFP, pin7-1, pin3 pin7 (36); yuc1-/yuc4,10,11/± (20); wei8-1-/wel1-1±/wel2-2± (21); iaaM, Q1630, and J2341 (19, 37); and arf10 arf16, ARF16mARF16, and Pro_35S:MIR160 (32). The wox5 pin, wox5 plt1, and plt1 pin double mutants were generated by crossing wox5-1, pin3-4, pin4, pin7, and plt1-4 alleles. The wox5 plt1 plt2 and pin3 plt1 plt2 triple mutants were generated by crossing wox5 and pin3 with plt1 plt2. The wox5 Pro_35S:MIR160 double mutant and wox5 arf10 arf16 triple mutant were generated by crossing wox5-1 with Pro_35S:MIR160 and arf10 arf16. PLT1::ERCFP tin was made by crossing PLT1::ERCFP with the tin allele of wox5. WOX5::ERGFP Pro_35S:MIR160 and WOX5::ERGFP axr3-1 double mutants were generated by crossing WOX5::ERGFP with Pro_35S:MIR160 or axr3-1. Seeds were surface sterilized with chlorine gas and incubated for 3 d after being plated onto Murashige and Skoog (MS) medium before transfer to a growth chamber with an 16-h light/8-h dark regimen at 19 °C for 4 or 5 d.

Phenotype Analysis.

To examine the root DSC differentiation, NAA or NPA treatments were done by growing or transferring seedlings on solid MS medium supplemented with or without NAA or NPA for the indicated time period. Starch granules in the root tips were stained with Lugol's solution for 1–2 min, then mounted on slides with chloral hydrate, and checked immediately. DEX induction for the 35S::WOX5-GR line was done by growing seedlings on solid MS medium supplemented with 1 μM DEX.

Microscopy.

Images were acquired with an AxioCam HR (Zeiss) camera attached to an Olympus microscope. For confocal microscopy images, a Zeiss LSM 510 confocal scanning microscope was used. Samples were scanned at a speed setting of 8 using the linear mode and the mean method as well as a pinhole of 330. The detector gain was set to 760. Counterstaining of cell walls was achieved by mounting seedling roots in 10 μM propidium iodide.

Quantitative PCR Analysis.

RNA was extracted with the RNeasy kit (Qiagen). Poly(dT) cDNA was prepared from total RNA of 5-d-old seedlings. Superscript III reverse transcription (Invitrogen) and quantification were done on a LightCycler 480 apparatus (Roche Diagnostics) with the SYBR Green I Master kit (Roche Diagnostics), according to the manufacturer's instructions. All individual reactions were performed in triplicate. Data were analyzed with qBase (38). Expression levels were normalized to those of tubulin, which showed no clear systematic changes in Ct value. The primers used to quantify the gene expression levels are provided in Table S3.