Spatio-temporal sequence of cross-regulatory events in root meristem growth

Abstract

A central question in developmental biology is how multicellular organisms coordinate cell division and differentiation to determine organ size. In Arabidopsis roots, this balance is controlled by cytokinin-induced expression of SHORT HYPOCOTYL 2 (SHY2) in the so-called transition zone of the meristem, where SHY2 negatively regulates auxin response factors (ARFs) by protein-protein interaction. The resulting down-regulation of PIN-FORMED (PIN) auxin efflux carriers is considered the key event in promoting differentiation of meristematic cells. Here we show that this regulation involves additional, intermediary factors and is spatio-temporally constrained. We found that the described cytokinin-auxin crosstalk antagonizes BREVIS RADIX (BRX) activity in the developing protophloem. BRX is an auxin-responsive target of the prototypical ARF MONOPTEROS (MP), a key promoter of vascular development, and transiently enhances PIN3 expression to promote meristem growth in young roots. At later stages, cytokinin induction of SHY2 in the vascular transition zone restricts BRX expression to down-regulate PIN3 and thus limit meristem growth. Interestingly, proper SHY2 expression requires BRX, which could reflect feedback on the auxin responsiveness of SHY2 because BRX protein can directly interact with MP, likely acting as a cofactor. Thus, cross-regulatory antagonism between BRX and SHY2 could determine ARF activity in the protophloem. Our data suggest a model in which the regulatory interactions favor BRX expression in the early proximal meristem and SHY2 prevails because of supplementary cytokinin induction in the later distal meristem. The complex equilibrium of this regulatory module might represent a universal switch in the transition toward differentiation in various developmental contexts.

Fig. 1.

BRX is an MP target gene. (A) Schematic overview of the Arabidopsis root meristem with protophloem highlighted. (B) Progression of cell elongation in the protophloem. (C) BRX::GUS reporter gene expression in columella, stem-cell niche, and protophloem of wild-type (wt) root meristems at 3 and 5 d dag. Arrowheads indicate the first protophloem elongation zone cells. (D) Quantitative protophloem expression profiles of BRX::GUS across the division zone (DZ) and transition zone up to the elongation zone (EZ). n = 18 for 3, 4, and 5 dag; n = 10 for 6 dag. (E) Expression of APL::GFP in wt and brx at 5 dag. Arrowhead indicates a gap in expression. (F) Protophloem-specific cell-wall staining (2) at 5 dag. Arrowhead indicates a staining gap in brx. (G) Number of cells in the protophloem transition zone in wt and brx at 5 dag. (H) Schematic view of BRX promoter with ARF-binding sites indicated. (I and J) Electrophoretic mobility shift assay of BRX promoter fragments (H) with a recombinant His-tagged fragment of MP protein (amino acids 1–432) containing the DNA-binding domain. E: no protein added; H: an unrelated His-tagged protein added; asterisks: free probe. (K and L) Replicate ChIPs of BRX promoter with transgenic HA-tagged MP protein (, analyzed by semiquantitative PCR (K) or qPCR (L). (M) Frequency of aberrant embryos in wt, brx, mp^S319, and brx mutants segregating 1/4 brx mp^S319 double mutants. Error bars in D, G, and L indicate SE. ***P < 0.001.

Fig. 2.

BRX control by SHY2. (A and B) Expression pattern of SHY2::GUS reporter gene in wild-type (wt) roots (A) and quantitative expression profiles (B). n = 6 for 3 dag; n = 4 for 6 dag. (C) BRX::GUS reporter gene expression in wt roots and enhanced and ectopic expression of BRX::GUS in shy2-31 loss-of-function mutants. Note enhanced BRX expression in the protoxylem strip at the center of the shy2-31 root. (D) Repression and proximal shift (arrowheads) of BRX::GUS expression after 6 h cytokinin [5 μM transzeatin (tz)] treatment. (E and F) Cytokinin insensitivity of brx root growth (E) and meristem size (meristematic cortex cell number) (F). Error bars in B, E, and F indicate SE. ***P < 0.001; n.s.: not significant.

Fig. 3.

Enhancement of PIN3 expression by BRX in early meristems. (A–D) Expression of PIN3::PIN3-GFP transgene in wild-type (wt) and brx background at 3 and 5 dag. Insets show images with green channel digitally enhanced. (E and F) Quantitative expression profiles of PIN3::PIN3-GFP across the two protophloem poles in wt and brx at 3 and 5 dag. n (wt) = 18 and n (brx) = 8 for 3 dag; n (wt) = 8 and n (brx) = 12 for 5 dag. (G) Relative quantitative plasma membrane abundance of BRX-GFP fusion protein in the protophloem. n = 13. (H and I) Quantitative expression profiles of PIN1::PIN1-GFP across the two protophloem poles in wt and brx at 3 and 5 dag. n (wt) = 20 and n (brx) = 10 for 3 dag; n (wt) = 16 and n (brx) = 30 for 5 dag. (J) Expression profile of PIN3::PIN3-GFP along the root meristem at 3 dag after mock or cytokinin (6 h of 5 μM transzeatin) treatment. n = 16 for mock and n = 20 for cytokinin treatment. (K) Same as in J but for brx. n = 14 for mock and cytokinin treatment. Error bars in E–K indicate SE. DZ: beginning of the division zone; EZ: position of first elongation zone cell; Note that for expression profile comparisons starting points have been set to 100%; i.e., curves do not indicate absolute expression levels. For intergenotype comparisons, meristem size was scaled to a percentage.

Fig. 4.

Interaction of BRX and MP. (A) In vitro interaction assay between GST and GST-fused full-length (FL) or C-terminal (CT; amino acids 648–902) MP protein (asterisks) and radio-labeled BRX (plus sign) or luciferase (LUC) protein (open circle). (B) Quantification of BRX pull-down across three replicate experiments as described in A. (C) In vitro interaction assays including GST fusions of AUX/IAA (IAA1, SHY2) and additional control proteins (UBC8). (D) Western blot quantification of C-terminal MP and AUX/IAA fusion proteins used in assays. (E) Quantification of BRX pull-down across three replicate experiments as described in C. (F and G) SHY2::GUS expression in wild-type (wt) (F) and brx (G) at 5 dag. (H) Model of spatio-temporal equilibrium between BRX, SHY2, and PIN3 expression profiles and the concomitant shifts in the proposed regulatory network. DZ: division zone; EZ: elongation zone.