5-week-old sid plants produced less biomass
(Fig. 4D). Lifetime stomatal diffusion capacity
can be assessed using the tissue–carbon isotope
ratio (d13C), because diffusion-limited leaves will
incorporate a larger proportion of heavy isotopes
(17). d13C in sid mutants is higher than in WT
(–28.57‰ ± 0.59‰ and –32.99‰ ± 0.47‰,
respectively; P = 1.67–6) confirming that these
mutants are diffusion limited (fig. S7).
Finally, we assessed stomatal responsiveness
to changing light conditions in WT and sid by
acclimating the plants at near-saturating light,
then simulating shade, increasing light again,
and finally mimicking sudden nightfall (Fig. 4E).
WT stomata quickly responded to changing light
conditions, whereas both reaction times and
amplitudes of the response were dampened in
sid (Fig. 4E). In summary, sid stomata lacking
SCs cannot open as wide (gsmax) (Fig. 4C), do
not close as tightly in darkness (Fig. 4E), and
are slow to respond to changing light (Fig. 4E).
The developmental innovation of SC-containing
stomatal complexes is accompanied by changes
in functional aspects and mobility of one of the
core stomatal identity bHLH transcription fac-
tors. Whereas AtMUTE halts the asymmetric stem
cell–like divisions and specifies GMC identity in
Arabidopsis (13), BdMUTE is mobile, promotes
divisions, and defines SMC identity in Brachy-
podium. Mobile transcription factors in plants
maintain stem cell niches, pattern root hairs and
trichomes, define the root endodermis, and even
regulate flowering (18). Transcription factor trans-
port, however, seems to be mostly associated with
developmental plasticity, where different degrees
of movement of orthologs fine-tune organ size
(19–21). In the stomatal context, we find species-
specific mobility for BdMUTE associated with a
developmental innovation. BdMUTE mobility
might be an elegant mechanism to coordinate
the development of GCs and SCs to enable forma-
tion and function of intimately connected four-
celled complexes. Analysis of leaf-level gas exchange,
stable isotopes, and biomass indicate that the
presence of SCs correlates with improved stomatal
responsiveness and greater aperture range, which
enhance plant performance particularly when water
is limited and/or temperatures are high. Therefore,
engineering SC properties might allow for tuning
of stomatal responsiveness and, thus, boost car-
bon assimilation and water use efficiency in crops.
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This work is supported by the Swiss National Science
Foundation (P2ZHP3_151598 to M. T.R.) and through a grant
from The Gordon and Betty Moore Foundation (GMBF2550.05) to
the Life Science Research Foundation (to M. T.R.). The work
conducted by the U.S. Department of Energy (DOE) Joint
Genome Institute is supported by the Office of Science of the
DOE under contract no. DE-AC02-05CH1123. E.A. was a
NSF graduate research fellow and D.C.B. is a GBMF Investigator
of the HHMI. Supplement contains additional data.
Materials and Methods
Figs. S1 to S7
3 November 2016; accepted 16 February 2017
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