During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone1. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip2,3,4,5,6,7,8,9,10,11,12. In addition, auxin is also pivotal for tropic responses13,14. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation.
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We thank M. Grebe, P. Benfey and K.-i. Hayashi for materials; the Light Microscopy Unit (Institute of Biotechnology), S. El-Showk, A.-M. Bågman, J. van Amerongen and F. Kindt for technical advice or assistance. This work is supported by a Human Frontier Science Program fellowship (A.P.M.), European Research Council Advanced Investigator Fellowship SysArc (B.S.), SPINOZA award (B.S., K.t.T., K.P.), ALW-NWO European Research Area Network Plant Genomics (ERAPG) grant 855.50.017 (S.D.-T.), the Academy of Finland (A.P.M., R.S., O.S., J.S.), Biocentrum Helsinki and University of Helsinki (A.P.M., R.S., O.S.), Integrative Life Science Doctoral Program (R.S.), Marie Curie Intra-European Fellowship (IEF-2008-237643) (S.D.-T.), the Netherlands Organisation for Scientific Research (NWO)-Horizon grant (R.H.), NWO-ALW grant (G.W.), and EMBO Long-term fellowship (A.P.M., K.P.).
The authors declare no competing financial interests.
Extended data figures and tables
a, b, The domain of frequent cell division, monitored by cell cycle marker CYCB1;1–GFP in Fig. 1b, c, shifts shootward with increased PLT2 dosage (that is, homozygote pPLT2:PLT2-YFP in Col background). Histogram in a shows the distribution of the CYCB1;1–GFP-positive cells along the meristem at a given distance from the quiescent centre. x axis indicates the distance from the quiescent centre as number of cortical cells, and y axis label ‘GFP density’ refers to the proportion of CYCB1;1–GFP-containing cells at the given distance from the quiescent centre. Shootward shift of the distance of the cell division events in the presence of increased PLT2 (green histogram) dosage compared to wild-type (red histogram) is significant (t-test for mean P ≪ 0.001, Wilcoxon test for median P ≪ 0.001, Kolmogorov–Smirnov for difference of distributions P ≪ 0.001). b, Histogram presenting rescaled data to show that the distribution of the high cell division domain shifted shootward when PLT2 dosage was increased. A null hypothesis was that shootward shift is due to higher dispersion of the distribution observed under increased PLT2 dosage. To test this hypothesis, the control CYCB1;1–GFP data were rescaled to match the maximum values of PLT2 data. The null hypothesis was rejected (t-test P = 0.001, Wilcoxon test P = 0.0012, Kolmogorov–Smirnov P = 0.0026), indicating that the shootward shift of the high division domain in the presence of increased PLT2 dosage is significant, and not due to dispersion. The bin width in histograms is two cells (that is, 1st bar, 1 and 2 cells; 2nd, 3 and 4, and so on). c, Induction of pAHP6:XVE≫PLT2-YFP inhibits xylem differentiation (left) (white arrow indicates the first protoxylem element) and triggers ectopic cell divisions illustrated by CYCB1;1–GUS activity (right) (arrowheads), whereas root hairs develop normally (yellow arrows). Insets show magnifications from the designated areas. d, PLT2–YFP induction (t = 0) rapidly inhibits root growth (top) and cell expansion (bottom) in three roots. Expansion rates as shown as averages ± s.d. e, Inhibition of growth coincides with appearance of PLT2–YFP signal after induction. f, Induction of PLT2 RNAi (in plt1,3,4; pPLT2:PLT2-YFP) abolishes the PLT2–YFP signal by 49 h and consequently promotes expansion and differentiation of the meristem cells, as indicated by the appearance of expanded cells as well as protoxylem (arrow) and root hairs (arrowhead) in the meristem. Images from the same root using identical confocal microscopy settings for the yellow channel. Scale bars, 50 µm.
Extended Data Figure 2 PLT expression patterns respond only to long-term auxin accumulation in the meristem.
a, PLT expression shifts shootward only when prolonged auxin application is accompanied with polar auxin transport inhibitor (NPA) treatment. Four-day-old seedlings were transferred to an agar plate containing 20 µM NPA plus 5 µM IAA for the time periods indicated in the images. The expression of pPLT1:PLT1-YFP, pPLT3:PLT3-YFP and pPLT4:PLT4-YFP spreads shootward (white arrows) by 72 h of NPA plus IAA treatment. b, PLT expression patterns are insensitive for auxin-only treatments. Four-day-old seedlings were transferred to an agar plate containing 5 µM IAA for the time periods indicated in the images. The auxin response reporter DR5:erGFP rapidly responded to the treatment whereas the expression domains of pPLT1:PLT1-YFP, pPLT2:PLT2-YFP, pPLT3:PLT3-YFP and pPLT4:PLT4-YFP failed to expand. Black arrow indicates the region in meristem that is absent of DR5:erGFP fluorescence after IAA treatment but is filled with fluorescence after NPA plus IAA treatment (Fig. 2a). Observed phenotypes/number of roots analysed are indicated in the right bottom corners. c, Twenty-four hours of NPA plus IAA treatment fails to expand the PLT2–YFP gradient shootward. In fact, the treatment leads to transient shortening of the PLT2–YFP gradient, probably due to inhibition of growth dilution of PLT2–YFP in the meristematic cells (see Fig. 3g, h). P ≪ 0.001, Kolmogorov–Smirnov test; error bars indicate 95% confidence intervals. n = 20 (dimethylsulphoxide (DMSO)) and 23 (NPA plus IAA). d, Graded pPLT2:PLT2-YFP expression despite shallow auxin gradient in aux1,ein2,gnom; representative image from three independent lines. Scale bars, 50 µm.
a, Overview of the initial, PLT-spread, auxin and closed feedback models. Relationships between model variables (grid-based auxin levels, cell-based PIN and PLT levels), growth processes (stem cell maintenance, division, expansion and differentiation), PIN-mediated auxin transport, cell-to-cell PLT movement and their emerging consequences for auxin and PLT levels are shown, with different colours indicating from which model onward they are incorporated (models are in order of increasing complexity). Variables are indicated in rectangles, processes are indicated in ovals. A distinction is made between cell-level and tissue-level processes. Direct regulatory interactions are indicated with arrows, and a distinction is made between fast to intermediate speed versus slow interactions, and between the levels of the ‘input’ variable needed for a particular process to occur. Emergent feedbacks, with processes influencing variable levels other than through a direct regulatory effect (for example, growth spreading PLT and hence influencing its levels) are indicated as dashed arrows. b, Spatially explicit overview of the interplay between the variables and processes and how these generate the auxin and PLT gradients and control root zonation dynamics in the auxin model. Stem cells and slow division (STEM), fast division (DIV), expansion (EXP) and differentiation (DIF) processes are indicated in black if the local auxin and PLT levels permit these processes, and in grey if the local auxin and PLT levels prevent these processes from occurring. Similarly, black arrows indicate that local auxin or PLT levels cause promotion or repression of a process, grey arrows indicate that auxin or PLT in principle has a particular effect on a process but that local levels do not allow for this effect to occur. Blue arrows indicate transcriptional effects. The broad dark blue arrow indicates PIN-mediated auxin transport. c, Layout of the root tissue and PIN distribution pattern used in the simulations. The simulated tissue contains columella cells (cyan), quiescent centre cells (grey), epidermal cells (blue), cortical cells (green), border cells (yellow) and vasculature cells (red). For the PIN distribution pattern, four levels are distinguished: no PINs, a maximum relative level of 0.1, 0.35, and 1. These maximum relative levels are multiplied with the cellular PIN protein level to determine a membrane segment’s actual PIN level. The rootward–shootward flip of PIN polarity in cortex cells is indicated with arrows. d, Time course of free ARF levels, and protein levels of transcription factors A, B, C, D and PLT under a constant auxin application (level 100). Left, fast PLT protein turnover; right, slow PLT protein turnover. e, Schematic overview of root zonation and its dependence on PLT and auxin gradients. Top panel, location of the meristem (MZ), expansion (EZ) and differentiation (DZ) zones. Second panel, PLT concentration profile along the length of the root. PLT levels dictate the location of stem cell maintenance and slow division, fast division and expansion and differentiation domains, with the first threshold demarcating the boundary between stem cell and fast division domains and the second threshold demarcating the boundary between fast division and expansion and differentiation domains. Third panel, auxin profile along the root. Fourth panel, dependence of division, expansion and differentiation rates on auxin levels. A,B,C and D correspond to different auxin levels; for comparison purposes it is shown where these levels occur both in the auxin gradient profile (third panel) and where these levels occur in the division, expansion and differentiation auxin rate dependency functions (fourth panel). f, Pseudocode of the algorithm used in the program to determine whether cells will grow, divide, expand or differentiate. In addition, a cartoon version of the consequences of growth, division, expansion and differentiation processes on gene expression levels, differentiation levels, cell size and PIN pattern is shown. g, Schematic depiction of the neighbouring cells and weighting factors used to calculate weighted, locally averaged growth and expansion rates. B, boundary cells; C, cortex cells; E, epidermal cells; V, vascular cells.
Extended Data Figure 4 The requirement of high auxin levels produces narrow, non-graded PLT profiles.
a, Vascular auxin concentration profiles under root zonation dynamics in the initial model for different half-saturation values for free ARF. Note: the curves are practically superimposed. A snapshot image of the zonation below the graph illustrates the location of the root zones. Columella (C), meristem (MZ), expansion (EZ) and differentiation (DZ) zones are shown. b, Vascular PLT transcription (continuous lines) and protein profiles (dashed lines) for the same parameter settings as in a.
Extended Data Figure 5 PLT2 protein persistence and mobility maintain meristematic characteristics without elevating auxin response.
a, A single cell clone expressing PLT2–RFP exiting the meristem (0 min) and travelling through the expansion zone towards the differentiation zone (200 min and 340 min). b, Magnification of the marked area in a (0 min), demonstrating that the clone (marked with green fluorescence, appearing as yellow when overlapped with PLT2–RFP red fluorescence) consists originally of a single cell (white arrowheads). Note that PLT2–RFP (red nuclear fluorescence) is present both in the clone and the surrounding cells. Inset shows optical cross-section at the position of the clone. c, Magnification of the marked area in a (340 min) showing that the clone has divided once while being in the expansion zone (arrowheads mark two clonal cells), and that PLT2–RFP-expressing cells do not expand, whereas cells produced before and after generation of the clone have expanded. d, Auxin response sensor DR5:nYFP (yellow nuclear fluorescence)31 is not elevated in the PLT2–RFP cells (white arrowheads) but shows normal response in vasculature (white arrows). e, Anti-auxin, α-(phenylethyl-2-oxo)-IAA (PEO-IAA) inhibits root hair formation, but fails to promote cell expansion in the PLT2–RFP clones. A PLT2–RFP clone exiting the meristem (0 h) and travelling through the elongation zone towards the differentiation zone. PEO-IAA (30 µM) was applied to the medium 2 h after taking the first (0 h) image. Then images were taken 2.5 h, 4 h, 6 h and 20 h after PEO-IAA application. Note: root hair production is inhibited after PEO-IAA application. Inset, magnification of the marked area in the 0 h image, showing the clone (marked with green fluorescence) and that PLT2–RFP (red nuclear fluorescence) is present both in the clone and the surrounding cells. f, PLT2-3×YFP shows reduced expression in the stele. g, h, The movement-deficient version, PLT2-3×YFP, complements the plt1,2 mutant, although the meristem is shorter than when PLT2-YFP is used. Seedlings were 7 days old; arrowheads, MZ boundary. Asterisks in h, Wilcoxon test (P < 0.001); meristem size of PLT2-3×YFP lines significantly reduced. Error bars show s.d. i, Cells shootward from the stem cell niche are proliferating without PLT2 transcription (arrowheads, cells with GFP but no RFP).
Extended Data Figure 6 High auxin levels rapidly inhibit cell division and expansion, but not differentiation.
a, First signs of expansion zone differentiation 7 h after 5 µM IAA application marked by appearance of protoxylem elements (arrow). By 24 h ubiquitous differentiation of the expansion zone is evident. 0 h image used from Fig. 1a. Scale bar, 50 µm. b, Auxin application rapidly inhibits growth while xylem differentiation, monitored by green fluorescence of S18 marker32, proceeds towards meristem. Snapshots from a video of the same root before and after application. Right panel, S18 signal is tightly associated with protoxylem differentiation (arrow). c, Root growth (µm h−1) and mitosis (below the x axis) of two roots over time (min). IAA applied at t = 0. d, e, Application of 5 µM IAA and inhibition of auxin signalling by 30 µM PEO-IAA leads to decreased accumulation of 5-ethynyl-2′-deoxyuridine (EdU) stain (red fluorescence), marking DNA replication. Asterisks in e, Mann–Whitney U test P < 0.05, after Bonferroni correction of multiple comparisons; reduction of number of EdU-stained nuclei compared with DMSO control. Error bars show s.d. f, Application of moderate levels of IAA (30 nM) still inhibited cell expansion (Supplementary Notes) but did not inhibit cell division. Root growth (µm h−1) and mitotic events (below the x axis) of two roots over time (min). IAA (30 nM) was applied at t = 0. g, h, To measure the duration of the differentiation process, individual cells were followed as they left the meristem, expanded and entered the differentiation zone. g, Tracking of a GFP clone20 consisting of four cells. Arrows highlight a cell just entering the expansion zone in the first panel and in the last panel the same cell entering the differentiation zone. For this particular cell it took approximately 6 h 45 min to travel through the expansion zone. Six clones located in six roots were followed through the expansion zone, and it took 6–8 h for these clones to travel through the expansion zone. h, Snapshots from a video recording the growth of wild-type root in the presence of 30 nM IAA. The cells entering the expansion zone (arrows in left panel) were traced in the video to record the time it takes to enter the differentiation zone (arrows in the right panel). For the marked cell it took approximately 7 h 10 min to travel through the expansion zone. Tracking of cells through the expansion zone was carried out for nine cells located in three different roots, and it took 6–8 h for these cells to travel through the expansion zone.
a, Protoxylem differentiation is inhibited in the axr3-1 mutant. First protoxylem is highlighted with an arrow. Higher up in the root protoxylem differentiation is often sporadic (inset, arrows indicate a stretch of a single protoxylem element). b, Twenty-four hour induction of pG1090:XVE≫axr3-1-RFP with 17β-oestradiol (est) inhibits xylem differentiation and root hair outgrowth. Left inset, a single protoxylem vessel highlighted with two arrows. Right inset, the arrow highlights the beginning of a more continuous protoxylem strand, which was probably already present before the induction of axr3-1-RFP. Yellow bars in b, c show the areas in the root in which visible protoxylem are present. Yellow arrow in b, c marks the first root hair. Est, 5 µM 17β-oestradiol. c, Twenty-hour treatment of 4-day old wild-type root (Col) with 25 µM auxinole (auxin antagonist) inhibits xylem differentiation, cell expansion and root hair outgrowth. Xylem is typically differentiated as short, sporadic stretches. Left inset, arrow shows the end of a stretch of a xylem strand comprising approximately six protoxylem elements; right inset, higher up in the root two continuous protoxylem strands appear (arrows). These strands were probably already present before auxinole application. The middle inset shows the presence of short cells (marked with arrowheads) high up in the root, indicating that cell expansion was defective. The cell length typically varied along the root. d, Bar plot shows that the final length of cortex cells is shorter when auxin signalling is inhibited by auxinole (n = 35 cells in 6 roots) or inducible pG1090:XVE≫axr3-1-RFP (n = 47 cells in 6 roots) when compared with differentiation zone cells in the control roots (n = 51 cells in 7 roots and n = 38 cells in 7 roots, respectively) located at a similar distance from the root tip. Asterisks, Mann–Whitney U test (P < 0.001). Error bars show s.d. e–j, The consequence of auxin application or inhibition of auxin signalling on marker gene expression. e, f, Auxin application (5 µM IAA) rapidly leads to expression of the root hair differentiation marker pEXP7:GUS (ref. 33) (e) and the xylem differentiation marker S18 (ref. 32) (f) in the elongation zone. Note: high auxin levels (such as 5 µM IAA) are inhibitory for root hair elongation. Therefore, even though the root hair marker pEXP7:GUS rapidly spreads into the elongation zone, root hair elongation is less pronounced there. e–g, Twenty-four hour induction of pG1090:XVE≫axr3-1-RFP (est) inhibits the expression of root hair differentiation marker pEXP7:GUS (e) and the xylem differentiation marker S18 (ref. 32) (f), as well as cell division marker CYCB1;1-GUS22 (g). Note: both the signal intensity and the number of CYCB1;1–GUS-expressing cells are decreased in g. Twenty-four hour induction of XVE≫axr3-1 leads either to disappearance of S18 fluorescence (no expression in f), or S18 was present in short fragments (fragmented). h, i, Expression of PLT2–YFP continued to mark the shortened meristem after auxin signalling was inhibited by induction of pG1090:XVE≫axr3-1-RFP (est) (h) or by treatment of the seedlings with 25 µM auxinole (axnl) (i). j, Auxinole rapidly inhibits cell division as indicated by reduction of the number of CYCB1;1–GFP-expressing cells after auxinole treatment.
Extended Data Figure 8 Simulation of zonation dynamics in the auxin model under normal conditions and conditions of perturbed auxin (signalling).
a, Zonation dynamics under normal growth conditions. b, Zonation dynamics after 24 h of intermediate level auxin application. c, Zonation dynamics after 24 h of high level auxin application. d, Zonation dynamics 60 h after shoot cut. e, Zonation dynamics after 24 h of inhibited auxin signalling. Shown are snapshots of auxin (Aux), PLT transcription (PLT), PLT protein (PLT) and zonation (Zon) profiles. In addition, for the normal growth conditions, snapshots of division rates (DivR), expansion rates (ExpR) and differentiation levels (Diff) are shown.
Extended Data Figure 9 Simulation of zonation under a dynamic gravistimulus protocol and in the closed feedback model.
a, Left, 12 h period in which leftward, apolar and rightward columella PIN orientations are interchanged to simulate dynamic gravitropism. Right, schematic depiction of the used leftward, apolar and rightward columella PIN orientations. b, Root zonation dynamics for the gravitropism model. Snapshots of auxin, PLT transcription, PLT protein, expansion rate and resulting zonation dynamics are shown for t = 3.5 h when PIN orientation is leftward (left), at t = 5.5 h when PIN orientation is apolar (middle) and at t = 9.5 h when PIN orientation is rightward (right). c, Root zonation dynamics for the simplified gravitropism model. In the simplified gravitropism model, cellular division and differentiation rates are again constant (as in the minimal model) rather than ARF level dependent (as in the auxin and normal gravitropism models). Only expansion rates are ARF level dependent, such that they decrease from their maximum value for higher than optimal ARF levels. Similar snapshots as in b are shown. d, Dynamics of left–right differences in auxin, differentiation level and PLT protein distribution in the simplified gravitropism model. e–g, Simulations with positive feedbacks from PLT back to auxin biosynthesis and transport. e, Zonation dynamics under standard growth conditions. In addition to the panels shown for other model versions, gene expression patterns of the genes dependent on PLT levels are shown. PINtot refers to total cellular PIN levels, PINmem to membrane PIN levels, SE to a general auxin synthesizing enzyme and DE to a general auxin degrading enzyme. Note that membrane PIN levels are a product of cellular PIN protein levels and the superimposed cell type and zone dependent membrane PIN pattern (which determines the locations and ratios of PINs deposited on the different membrane faces of the cell). f, Zonation dynamics after 24 h of high auxin application. g, Zonation dynamics under dynamic gravitropic stimulation. For comparison purposes arrows indicating the location of the transitions from MZ to EZ (bottom arrow) and from EZ to DZ (top arrow) as found in the PLT-spread model are shown. For the gravitropism simulation (g) the EZ to DZ transitions at both the lower (middle arrow) and upper (top arrow) side of the root are shown.
a, Regulatory architecture controlling root zonation dynamics and tropisms. Slow induction of the PLTs by auxin (black arrows) defines the pathway that operates through regulating PLT levels (green arrows). Parallel to this, auxin can also control zonation rapidly without directly affecting PLT levels (blue arrows). b, Schematic overview of root developmental zones where local concentrations of auxin, PLT transcript and PLT proteins are represented by symbol density. c, Overview of the auxin, PLT transcript and PLT protein profiles and corresponding zonation dynamics under the following conditions: wild-type (WT), extra PLT2 copy in wild type (PLT2:YFP), clonal ectopic expression of PLT in the expansion zone (clone), short-term auxin addition (+aux) or inhibition of auxin signalling (−aux). Expansion is indicated by longer cell shape, differentiation by root hair bulge. d, e, Auxin, PLT, zonation, and expansion rate profiles during (d) and after (e) a gravitropic stimulus for a simulation in which PLT levels are a direct readout of auxin levels, and in which partly differentiated cells dedifferentiate upon re-entering the meristem. Differentiation snapshots are shown with 2 h intervals during and after the gravitropic stimulus. Brackets highlight the developmental progression of the cells that dedifferentiated under the gravitropic stimulus. Differentiation graphs show differentiation levels in the leftmost epidermal row of cells (corresponding to the side of the root towards the gravity vector) in the PLT as direct auxin readout model (black) compared to that of the model developed in this study (red), for 2 h after the (end of the) gravitropic stimulus.
This file contains Supplementary Notes, a Supplementary Discussion, Supplementary Methods and Supplementary References – see Supplementary Contents page for details. (PDF 1004 kb)
Root zonation dynamics for the local PLT model during 12.7 days of root growth. Patterns of auxin distribution (Aux), PLT transcription (PLT), PLT protein (PLT), cell division rates (DivR), expansion rates (ExpR) and root zonation (Zon) are shown. The division rate profile (DivR) and expansion rate profile (ExpR) depict the instantaneous rates at that moment and that position based on local auxin, PLT and differentiation levels. (MOV 3996 kb)
Root zonation dynamics for the combined PLT spread model during 12.7 days of root growth. Shown profiles are the same as for Video S1. (MOV 11979 kb)
The fluorescence dynamics of the same two CYCB1;1-GFP roots before (left) and right after 5 µM IAA application (right) are presented side by side to better visualizing the effects of auxin. The cell division rate was measured by manually counting the disappearance of GFP fluorescence in function of time (presented in Extended Data Fig. 6c). See Supplementary Results and Methods for detail. (MOV 5666 kb)
Dynamics of xylem differentiation marker S18 expression in a growing root before and after auxin application
The fluorescence dynamics of the same two S18 roots before (left) and right after 5 µM IAA application (right) are presented side by side to better visualizing the effects of auxin. Auxin application led to rapid inhibition of cell expansion and consequently of root growth, while xylem differentiation, as presented by S18 GFP fluorescence, proceeded closer to the root tip, until it reached the MZ/EZ boundary beyond which it did not proceed. (MOV 17734 kb)
Root zonation dynamics during a single 12 hour cycle of simulated leftward and rigthward oriented gravitropism. Shown are patterns of auxin distribution (Aux), PLT transcription (PLT), PLT protein (PLT), cell expansion rates (ExpR) and root zonation (Zon). (MOV 706 kb)
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Mähönen, A., Tusscher, K., Siligato, R. et al. PLETHORA gradient formation mechanism separates auxin responses. Nature 515, 125–129 (2014). https://doi.org/10.1038/nature13663
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