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. 2010 Feb;137(4):541-9.
doi: 10.1242/dev.041426.

STIMPY Mediates Cytokinin Signaling During Shoot Meristem Establishment in Arabidopsis Seedlings

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Free PMC article

STIMPY Mediates Cytokinin Signaling During Shoot Meristem Establishment in Arabidopsis Seedlings

Anna Skylar et al. Development. .
Free PMC article

Abstract

The establishment of the primary meristems through proliferation after germination is essential for plant post-embryonic development. Cytokinins have long been considered a key regulator of plant cell division. Here we show that cytokinins are essential for early seedling development of Arabidopsis. Loss of cytokinin perception leads to a complete failure of meristem establishment and growth arrest after germination. We also present evidence that cytokinin signaling is involved in activation of the homeobox gene STIMPY (STIP or WOX9) expression in meristematic tissues, which is essential for maintaining the meristematic fate. Cytokinin-independent STIP expression is able to partially compensate for the shoot apical meristem growth defects in mutants that cannot sense cytokinin. These findings identify a new branch of the cytokinin signaling network, linking cytokinin to the process of meristem and seedling establishment.

Figures

Fig. 1.
Fig. 1.
The seedling phenotype of cytokinin triple-receptor mutants. (A-C) Eight-day-old Col-0 (A), ahk2-2 ahk3-3 cre1-12 (B), and stip-1 (C) seedlings grown with no exogenous sucrose. The inset in (A) shows a two-day-old Col-0 seedling at the same magnification. (D,E,H) Wholemount in situ hybridization with an anti-sense STIP probe in two-day-old Col-0 (D), ahk2-2 ahk3-3 cre1-12 (E), and the type-B arr1-3 arr10-5 arr12-1 (H) roots. The signal is nearly absent in E and H. (F,G) In situ hybridization with an anti-sense STIP probe on longitudinal sections through the center of the shoot apical meristems of two-day-old Col-0 (F) and ahk2-2 ahk3-3 cre1-12 (G) seedlings. The signal is reduced in G. Scale bars: 50 μm.
Fig. 2.
Fig. 2.
stip mutants have reduced sensitivity to cytokinin inhibition. (A-H) Seven days after Col-0 (A-D) and stip-1 (E-H) seedlings were transferred to media containing sucrose and 0 (A,E), 10 nM (B,F), 100 nM (C,G), and 1 μM (D,H) of trans-zeatin, stip seedlings show continued growth and reduced anthocynin accumulation (G,H).
Fig. 3.
Fig. 3.
ARR5 (type-A ARR) expression is reduced in stip mutants. (A,B) In situ hybridization with an anti-sense ARR5 probe on longitudinal sections through the center of the shoot apical meristems of two-day-old Col-0 (A) and stip-1 (B) seedlings. The signal is reduced in B. (C,D) Wholemount in situ hybridization with an anti-sense ARR5 probe in two-day-old Col-0 (C) and stip-1 (D) roots. The signal is reduced in D. (E,F) ARR5::GUS activity staining in two-day-old Col-0 (E) and stip-2 (F) seedling shoots. GUS activity is reduced in F. Scale bars: 50 μm.
Fig. 4.
Fig. 4.
Relative expression levels of selected cytokinin target genes in stip mutants. (A) Two-day-old seedlings were treated with 250 nM transzeatin or mock for 30 minutes and gene expression levels were measured by qRT-PCR. All samples were normalized to ACT8. ARR6, 7 and 9 are all type-A ARR genes. (B) Two-day-old seedlings were treated with 250 nM trans-zeatin for a total of 3 hours and gene expression levels were measured by qRT-PCR at different timepoints. After normalizing to ACT8, each gene is normalized to its pre-induction levels in the appropriate genotype.
Fig. 5.
Fig. 5.
STIP over-expression partially rescues the cytokinin triple-receptor mutants. (A-D) 12-day-old Col-0 (A), ahk2-2 ahk3-3 cre1-12 (B), pAS1 ahk2-2 ahk3-3 cre1-12 (C) seedlings grown without supplemented sugar, and (D) ahk2-2 ahk3-3 cre1-12 of the same age grown on sucrose-containing media. (E-H) Longitudinal sections through the center of the shoot apices of the seedlings shown in (A-D), stained with Safranin O. (I-L) DIC images of cleared root tips of the seedlings shown in (A-D). The positions of the root meristematic zones are marked with brackets.
Fig. 6.
Fig. 6.
Relative expression levels of selected cytokinin and STIMPY common target genes in mutant and pAS1-rescued ahk2-2 ahk3-2 cre1-12 seedlings. Gene expression levels in two-day-old seedlings were measured by qRT-PCR and all samples were normalized to ACT8.
Fig. 7.
Fig. 7.
A diagram of the proposed involvement of STIP in cytokinin signaling during vegetative SAM development. STIP activity is positively regulated by cytokinin stimulation, downstream of the type-B ARR gene functions. Together with other branches of the cytokinin signaling network, STIP acts upstream of sugar sensing/signaling in promoting meristem establishment.

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