dividing meristemoids (Fig. 3B). Coexpression with
SPCHpro:SPCH-CFP indicated that SPCH precedes ARK3, consistent with SPCH activating
ARK3 expression (Fig. 3, C to E). To ascertain its
function in the stomatal lineage, we reduced ARK3
expression by driving an artificial microRNA against
it with the SPCH promoter (SPCHpro:amiR-ark3).
In the cotyledon epidermis of amiR-ark3–expressing
plants, we observed clusters of meristemoid-like
small cells at 4 days postgermination (dpg) that
developed into clusters of stomata at 11 days (Fig.
3, G and I, brackets). These small cell clusters,
which displayed diminished physical asymmetry, appear to arise from misplaced but complete
division planes. Notably, cell wall stubs or other
evidence of incomplete divisions were not observed. The amiR-ark3 phenotypes resembled
those associated with basl mutants (12) and are
hallmarks of loss of ACD capacity. Thus, ARK3
appears to be a new player essential for ACD, possibly through regulating preprophase band placement, and establishes a direct link between SPCH
and the ACD machinery.
SPCH initiates a lineage with autonomous control over cell division and fate determination.
Nonetheless, the stomatal lineage is also coordinated with developmental programs operating
across tissues and organs. Phytohormones play critical roles in coordinating development, and recent
reports indicate that auxin, brassinosteroid (BR),
and abscisic acid regulate stomatal development
(20–23). BR controls stomatal development through
phosphorylation of YODA and SPCH by its central
glycogen synthase kinase 3–like kinase, BIN2 (Fig. 4F)
(21, 22). Among SPCH target categories, BR biosynthetic and response genes show significant
enrichment (fig. S14). Notably, SPCH binds to
the promoters of BIN2 and CPD, which encodes
an essential enzyme for BR biosynthesis (Fig. 4, A
and F), and the absence of CPD results in stomatal
overproduction (24). We tested the effect of SPCH
on the expression of BR genes by reverse transcription qPCR (RT-qPCR) in the meristemoid-enriched line SPCHpro:SPCH2-4A-YFP (Fig. 4B).
Consistent with inhibition of BR signaling, we
found that BIN2 expression is elevated, whereas
CPD is repressed (Fig. 4B). Supporting BIN2’s
role in promoting SPCH function, stomatal lineage-specific expression of BIN2-1 led to small cell clusters in cotyledons, similar to those observed upon
SPCH overexpression (Fig. 4C). Thus, our results
suggest the presence of feedback by SPCH counteracting BR signaling (Fig. 4F). SPCH also binds to
genes encoding the BR signaling effectors, the
BZR1 family of transcription factors (BZR1, BES1/
BZR2, and BEH1 to BEH4), and BIM2, the putative
dimeric partner of BES1 (25–27). BEH1 to BEH4
and BIM2 were up-regulated in the meristemoid-enriched mutant, and BIM2 exhibited stomatal
lineage-specific expression (Fig. 4, B and D). Epidermal expression of bes1-D (26) correlates with
an increase in stomatal density, whereas a bes1
RNA interference (RNAi) knockdown line (27)
exhibited a trend toward lower stomatal density
(Fig. 4E). This role in promoting stomatal development may be explained through the known
repression of the BR biosynthetic genes by the
BZR1 family (28). Thus, SPCH-mediated induction of BIN2 and repression of CPD (either
1608 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
Fig. 3. SPCH regulates ACD through a preprophase band-localized kinesin. (A) SPCH ChIP-seq profile of ARK3/KINUa. (B to E) Expression of
ARK3pro:ARK3-YFP (yellow) (B) and its coexpression with SPCHpro:SPCH-CFP (blue) [(C) to (E)]
before (C), during (B) and (D), and after (E) a
stomatal ACD. Arrowheads indicate the preprophase band. (F to I) ACD defects in SPCHpro:amiR-
ark3 [(G) and (I)], compared with Col [(F) and (H)].
Brackets mark clusters of small cells (G) or guard
cells (I). Confocal images are of 3- [(B) to (E)], 4- [(F)
and (G)] and 11-day [(H) and (I)] abaxial cotyledons
with ML1pro:mCherry-RCI2A–marked cell outlines.
To display the cells in (E) in the same orientation
as panels (C) and (D), we rotated the image in (E)
and filled in the nonimaged space in the upper
right corner with a black triangle (dashed line).
Scale bars, 10 mm [(C) to (E)], 50 mm [(F) to (I)].
18 BIN2 200bp
200bp BEH2 10
BIN2CPDBIM2BZR1 BEH1BEH2 BEH4 BEH3
family of TFs:
bes1 RNAi bes1-D Col 0
Fig. 4. Feedback regulation of brassinosteroid biosynthesis and signaling by SPCH. (A) ChIP-seq
profiles of select brassinosteroid (BR) pathway genes (labeled as in Fig. 2F). (B) RT-qPCR analysis of BR
genes in 4-dpg SPCHpro:SPCH2-4A-YFP and Col seedlings. Values are means T SEM. (C and D) Confocal
images of 3-dpg adaxial cotyledons with propidium iodide-stained cell outlines (purple). Stomatal
lineage-specific expression of hyperactive BIN2 (yellow) induces lineage proliferation (bracket) (C).
Stomatal lineage expression pattern of BIM2pro:YFP-YFP (yellow) (D). (E) Alteration of stomatal density
in gain-of-function BES1pro:bes1-D and bes1-RNAi knockdown. *P < 0.05, ***P < 0.001 (Wilcoxon rank
sum test). (F) Model of SPCH-BR pathway interactions. SPCH, a target of BR signaling, feeds back
(positively, red arrows, or negatively, red T-bar) upon transcription of multiple pathway members.