Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root

Abstract

In multicellular organisms, a stringent control of the transition between cell division and differentiation is crucial for correct tissue and organ development. In the Arabidopsis root, the boundary between dividing and differentiating cells is positioned by the antagonistic interaction of the hormones auxin and cytokinin. Cytokinin affects polar auxin transport, but how this impacts the positional information required to establish this tissue boundary, is still unknown. By combining computational modeling with molecular genetics, we show that boundary formation is dependent on cytokinin's control on auxin polar transport and degradation. The regulation of both processes shapes the auxin profile in a well-defined auxin minimum. This auxin minimum positions the boundary between dividing and differentiating cells, acting as a trigger for this developmental transition, thus controlling meristem size.

Keywords: cell differentiation; computational modeling; plant hormones; root meristem.

Fig. 1.

Root layout. Root zonation at 5 d after germination (dag) in vivo (Left) and in silico (Right) displaying the stem cell niche (SCN), the meristem zone (MZ), and the differentiation zone (DFZ 1, 2, and 3). In the in silico root layout, the transition zone (TZ) is approximated as a straight boundary, taking the position of the cortex transition boundary (COR TB, blue arrowhead) as reference. Root tissues in the in silico root are color coded. COL, columella; COR, cortex; END, endodermis; EPI, epidermis; iCOL, columella initial; iCOR/END, cortex/endodermis initial; iLRC/EPI, lateral root cap/epidermis initial; LRC, lateral root cap; PER, pericycle; QC, quiescent center; VASC, vasculature. (Scale bar, 100 μm.)

Fig. 2.

Local auxin degradation shapes the auxin profile in a well-defined auxin minimum. (A–C) Predicted steady-state auxin distribution in 5 dag root considering: only the effect of cytokinin regulation on PINs (A); cytokinin regulation on PINs together with cytokinin-mediated auxin degradation in all cells (B); and cytokinin regulation on PINs together with cytokinin-mediated auxin degradation confined to the GH3.17 expression domain (C) (for different setting of parameters used see SI Appendix, Table S4). The auxin minimum emerges in the last meristematic cell. Color coding represents auxin concentration levels. (D) Longitudinal auxin concentration profiles of simulation in C for different tissues, highlighting the formation of the auxin minimum at the TB, in the last meristematic cell of each tissue (C, blowup in G). Notably the auxin minimum is not formed in simulation in A and B, as highlighted by the respective blowup in E and F. Stars in E–G indicate auxin lowest level within each tissue (dip). Piecewise linear color bar indicates absolute and relative log auxin concentration for all simulations. a.u., arbitrary units. (H) Confocal microscopy images of 5 dag root carrying the pGH3.17:GH3.17-GFP construct. In H, expression of pGH3.17:GH3.17-GFP is detected in the same root showing on the Left the merged expression of green (GFP) and red [propidium iodide staining (PI)] channels and on the Right only the green (GFP) channel. Blue and white arrowheads indicate the QC and the cortex TB, respectively. (Scale bars, 100 μm.)

Fig. 3.

Auxin minimum quantification in R2D2 root tip. Maximum projection of confocal z-stack images of untreated (A), 12-h cytokinin-treated (+CK 12 h) (B) and 20-h cytokinin-treated (+CK 20 h) (C) R2D2 roots (Left) with quantification of relative levels of auxin in epidermal, cortex, and endodermal tissues (Right). The nuclear signal ratio of n3xVenus/ntdTomato of each cell was normalized to the maximum value of fluorescence intensity (corresponding to a minimum in auxin levels) of the corresponding tissue. Auxin distribution plots (A, Right; B, Right; and C, Right) were derived by reciprocal mean values of the normalized n3xVenus/ntdTomato ratio. Discrete data of measurements in each cell per tissue (blue, epidermis; green, cortex; orange, endodermis) were fitted and plotted as a smooth line (SI Appendix, SI Materials and Methods). Interestingly a correspondence between the cortex lowest auxin value (cortex dip) (white and black stars) and the last cortex meristematic cell was found (compare Left and Right in A). Epidermis and endodermis dips lay in the proximity of the cortex last meristematic cell defining the auxin minimum position (white and black stars) that coincides with the TZ (gray bar). Upon 12 h of cytokinin treatment (+CK 12 h) (B) a rootward shift in the position of the cortex dip (white and black stars) can be observed, although the position of the TZ (gray bar) is unaffected. The epidermis, and endodermis dips also shift rootwards in the proximity of the cortex dip defining the new position of the auxin minimum (white and black stars) (B). Upon 20 h of cytokinin treatment (+CK 20 h) (C) a shift of the TZ (gray bar) can be observed at the position of the auxin minimum (white and black stars). Moreover, in the 12-h cytokinin-treated roots the auxin minimum for all analyzed tissues lies at the same position as in the 20-h cytokinin-treated roots (compare B and C). White stars indicate cells where the auxin lowest value (dip) (black stars) was quantified. The region including the dips for each tissue defines the transition zone (TZ) (gray bar in the plot). White arrowhead indicates cortex TB. ANOVA analysis was conducted to determine if differences between the fluorescence detected in the last meristematic cells and the other cells were significant (P < 0.05, n = 22 for MS and 20-h cytokinin-treated roots; P < 0.05, n = 17 for 12-h cytokinin-treated roots). Adjusted P values for multiple comparisons were carried out with the Benjamini and Hochberg [false discovery rate (FDR)] method (P < 0.05). QC, quiescent center. (Magnification: 40×.)

Fig. 4.

The cytokinin-dependent auxin minimum correlates with the position of the transition zone. (A) WT root at 5 dag and its predicted steady-state auxin pattern in B. (C) gh3.17–1 root at 5 dag. (D and E) Predicted steady-state auxin pattern in the gh3.17–1 mutant (D) compared with the simulation obtained considering only the effect of cytokinin on PINs and excluding the GH3.17-dependent tissue-specific auxin degradation (E). Note that the auxin minimum is absent in E and is reestablished in D where the number of MZ cells in the root layout was increased according to the gh3.17–1 root phenotype (E). (F) Root of UBQ10::GH3.17 overexpressing line at 5 dag. (G and H) Auxin heat map of UBQ10::GH3.17 overexpressing root (G) compared with simulation where GH3.17-dependent auxin degradation is imposed in all cells (H). Note that the auxin minimum is absent in H since the low auxin values do not subsequently rise again (i.e., the curvature is low) the characteristic high curvature is reestablished in G by decreasing the number of MZ cells in the root layout, according to the UBQ10::GH3.17 root phenotype (F). (I) Root phenotype of the gh3.17–1 mutant and the UBQ10::GH3.17 overexpressing line. (J and K) Changes in meristem size (J) and root length (K) of the gh3.17–1 mutant and the UBQ10::GH3.17 overexpressing line. (L and M) Mass spectrometry quantification of IAA-glutamate (IAGlu) in WT and gh3.17–1 whole roots and 1-mm root tips. Limit of detection for IAGlu = 0.5 fmol. (N) Analysis of DII-VENUS expression in the gh3.17–1 mutant highlighting higher auxin activity as predicted in D. A total of 20 plants for two biological replicates were analyzed. (Scale bars, 100 μm.) Color coding represents auxin concentration levels. Piecewise linear color bar indicates absolute and relative log auxin concentration for all simulations. Blue and white arrowheads indicate the QC and the cortex TB, respectively. *Statistically significant difference in the gh3.17–1 and UBQ10::GH3.17 versus WT (n = 30 each sample, three biological replicates; Student t test, P < 0.05. ***Statistically significant difference in the gh3.17–1 versus WT lines in an ANOVA analysis (t test; P < 0.001). Error bars indicate SD.

Fig. 5.

GH3.17 is necessary for the positioning of the auxin minimum. Maximum projection of confocal z-stack images of R2D2 and gh3.17–1xR2D2 (Left) with quantification of relative levels of auxin in epidermal, cortex, and endodermal tissues (Right). The roots represent untreated R2D2 plant (A), gh3.17–1xR2D2 plant (C), cytokinin-treated (+CK) R2D2 plant (B), and gh3.17–1xR2D2 plant (D). Auxin distribution plots (Right) were derived by discrete data of measurements in each cell per tissue (blue, epidermis; green, cortex; and orange, endodermis) (SI Appendix, SI Materials and Methods). A correspondence between the cortex lowest auxin value (cortex dip, white and black stars) and the last cortex meristematic cell was found (compare Left and Right). Epidermis and endodermis dips lay in the proximity of the cortex last meristematic cell defining the auxin minimum position (white and black stars) that coincides with the TZ (gray bar). Upon cytokinin treatment, a rootward shift in the position of the cortex, epidermis, and endodermis dip (white and black stars) can be observed in R2D2 roots (B), but not in gh3-17–1xR2D2 (D). White stars indicate cells where the auxin lowest value (dip) (black stars) was quantified. White arrowhead indicates cortex TB. ANOVA analysis was conducted to determine if differences between the fluorescence detected in the last meristematic cells and the other cells were significant (P < 0.05, n = 33 for MS and 20-h cytokinin-treated R2D2 roots; P < 0.05, n = 39 for MS and 20-h cytokinin-treated gh3-17–1xR2D2 roots). Adjusted P values for multiple comparisons were carried out with the Benjamini and Hochberg (FDR) method (P < 0.05). QC, quiescent center. (Magnification: 40×.)

Fig. 6.

GH3.17 and SHY2 have additive effects on TZ positioning. (A) Root meristem of WT, gh3.17–1, shy2-31, and shy2-31;gh3.17–1 double mutant. (B) Analysis of root meristem size of WT, gh3.17–1, shy2-31, and shy2-31;gh3.17–1 double mutant. (C) Analysis of root length of WT, gh3.17–1, shy2-31, and shy2-31;gh3.17–1 double mutant. Note that the shy2-31;gh3.17–1 double mutant displays both a longer meristem and root than the parental, suggesting that these genes have an additive effect on TZ positioning. Blue arrowheads point to the QC and black arrowheads indicate the cortex TB. Error bars represent SD of results from three biological replicates. *Significant difference from the WT (Student t test, P < 0.05). (Scale bars, 100 μm.)

Fig. 7.

Cytokinin-dependent regulation of auxin degradation and transport is essential to shape the auxin gradient. (A) Steady-state auxin heat map of wild-type root. (B) Predicted steady-state auxin pattern in simulation mimicking the effect of cytokinin depletion that results in increased PIN expression and decreased GH3.17 expression. Auxin minimum formation is affected. (C) The auxin minimum reestablishes only by increasing the number of MZ cells in the root layout. (D) Predicted steady-state auxin pattern in simulation mimicking the effect of exogenous cytokinin application that results in decreased PIN expression and increased GH3.17 expression. Such changes in PIN and GH3.17 expression prevent the formation of the auxin minimum. (E) The auxin minimum is reestablished by decreasing the number of MZ cells in the root layout. (Scale bars, 100 μm.) Color coding represents auxin concentration levels. Piecewise linear color bar indicates absolute and relative log auxin concentration for all simulations.

Fig. 8.

Proposed model. Cytokinin (CK), through ARR1, controls both auxin catabolism (via positive regulation of GH3.17) and polar transport (via positive regulation of SHY2). As a result, auxin profile is shaped, generating a developmental instructive auxin minimum. The auxin minimum acts as a signal that positions the TZ and drives meristematic cells toward differentiation.