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. Author manuscript; available in PMC: 2020 Nov 25.
Published in final edited form as: Nat Plants. 2020 May 25;6(6):686–698. doi: 10.1038/s41477-020-0666-7

Fig. 4. Focused auxin and cytokinin signaling are required for robust sepal initiation.

Fig. 4

a, Expression of the auxin response reporter DR5 (DR5::3XVENUS-N7, white) accumulates at the four incipient sepal initiation positions in WT. DR5 expression is lower and more diffuse in the drmy1–2 mutant. p35S::mCherry-RCI2A: red, plasma membrane; Scale bars: 10 μm. n = 10 flowers.

b, Expression of the cytokinin response reporter TCS (pTCS::GFP, grey) accumulates at the four incipient sepal initiation positions in WT. TCS expression is enhanced and more diffuse in the drmy1–2 mutant. Chlorophyll auto-fluorescence: red; Scale bars: 10 μm. n = 10 flowers.

c, Quantification of DR5 signal in WT (blue) and drmy1–2 (red) in stage 2 flowers when no sepals have initiated yet. Signal was quantified radially for the 360 degrees of the approximately circular flower meristem. The top-left region between the outer sepal and the lateral sepal was defined as angle 0. Angles increased in the counterclockwise direction and normalized signal values within bins of the size of 1° are plotted. n = 10 for both WT and drmy1–2, and original individual sample curves can be found in Source Data. The average signal intensity of the 10 replicates are presented as thick blue and red lines with the SD as partially translucent background. Note that four clear peaks of DR5 signal are present in WT. In drmy1–2, the outer sepal peak is evident, although weaker, and the remainder of the flower signal is relatively low without evident clusters.

d, Quantification of TCS signals in WT (blue) and drmy1–2 (red) flowers at stage 2 when no sepals have initiated yet. n = 10 for both WT and drmy1–2, and original individual sample curves can be found in Source Data. The average signal intensity of the 10 replicates are presented as thick blue and red lines with the SD as partially translucent background. Note that four clusters of TCS signal are evident in WT, whereas in drmy1–2, TCS expression is higher and tends to surround the meristem.

e, Stage 6 flowers, where the sepals just close, from WT inflorescences cultured in 5 μM synthetic cytokinin BAP or mock media for 6 days. Blue arrowheads: delayed sepal initiation. Scale bars: 10 μm. Quantified in Extended Data Fig. 7c.

f, Stage 6 drmy1–2 flowers from inflorescences cultured in 5μM BAP or mock media for 6 days. Red arrowheads: smaller sepals, indicating delayed sepal initiation. Scale bars: 10 μm. Quantified in Extended Data Fig. 7c. Note drmy1–2 flowers cultured in BAP exhibit phyllotaxis defects.

g, The extent of disruption of auxin and cytokinin responses correlates with the degree of variability of sepal initiation timing. Mutation of ahp6, a cytokinin signaling inhibitor, has a very mild phenotype on its own, but enhances the drmy1–2 sepal initiation phenotypes. Quadruple mutations of the auxin receptors tir1afb1–1afb2–1afb3–1 (tir1afb1-2-3 for short) has a severely delayed sepal initiation phenotype. Cell walls stained with Propidium Iodide (PI) in grayscale. Yellow arrowheads: smaller sepal than normal, indicating the delayed sepal initiation. Scale bars: 50 μm. Note that tir1 afb1-2-3 mutants exhibit phyllotaxis defects. n = 3 biological replicates.