Research

Effects of stomata clustering on leaf gas exchange

Peter Lehmann* and Dani Or*
Soil and Terrestrial Environmental Physics, ETH Zurich, Universit€atstrasse 16, 8092 Zurich, Switzerland

Summary
Author for correspondence:
A general theoretical framework for quantifying the stomatal clustering effects on leaf gas-
Peter Lehmann eous diffusive conductance was developed and tested. The theory accounts for stomatal spac-
Tel: +41 44 632 63 45
ing and interactions among ‘gaseous concentration shells’. The theory was tested using the
Email: peter.lehmann@env.ethz.ch
unique measurements of Dow et al. (2014) that have shown lower leaf diffusive conductance
Received: 7 October 2014 for a genotype of Arabidopsis thaliana with clustered stomata relative to uniformly distributed
Accepted: 17 March 2015
stomata of similar size and density.

The model accounts for gaseous diffusion: through stomatal pores; via concentration shells
New Phytologist (2015) 207: 1015–1025 forming at pore apertures that vary with stomata spacing and are thus altered by clustering;
doi: 10.1111/nph.13442 and across the adjacent air boundary layer. Analytical approximations were derived and vali-
dated using a numerical model for 3D diffusion equation.

Stomata clustering increases the interactions among concentration shells resulting in larger
Key words: gas diffusion, leaf conductance,
spatial organization, stomata clustering, diffusive resistance that may reduce fluxes by 5–15%. A similar reduction in conductance was
stomatal aperture. found for clusters formed by networks of veins. The study resolves ambiguities found in the
literature concerning stomata end-corrections and stomatal shape, and provides a new sto-
mata density threshold for diffusive interactions of overlapping vapor shells.

The predicted reduction in gaseous exchange due to clustering, suggests that guard cell
function is impaired, limiting stomatal aperture opening.

stomata clusters in certain plants grown in dry environments

Introduction
Studies have shown that leaf stomatal size and density evolved over
geological timescales; these structural changes are interpreted
(using gas diffusion theories) as refinements in plant regulation of
gas exchange (Raven, 2002; Franks & Beerling, 2009; Berry et al.,
2010; de Boer et al., 2011; Assouline & Or, 2013; Buckley &
Schymanski, 2013). Stomata patterns on leaves have received
much less attention than the dynamic function of stomata (Crox-
dale, 2000). Various studies have revealed that an uniform sto-
mata pattern is linked to the ‘one cell spacing rule’ that defines a
minimum spacing between adjacent stomata, often of several sto-
mata aperture diameters (Korn, 1993; Larkin et al., 1997; Geisler
et al., 2000). This ‘rule’ ensures that there are sufficient interven-
ing cells for the proper function of guard cells whilst reducing the
diffusive interactions among neighboring stomata (Bange, 1953;
Franks & Casson, 2014). However, as already observed by Fellerer
(1892) for Begonia, various plant species produce stomata
arranged in clusters (Gan et al., 2010; listed 39 families of land
plants). Stomata clustering may result from errors in the complex
signaling for stomata initiation and in cell orientation and division
(Yang & Sack, 1995; Bergmann & Sack, 2007; Peterson et al.,
2010; Akita et al., 2013). Environmental signals may also regulate
stomata development and patterning (Wang et al., 2007), such as
signals from older to younger leaves to reduce stomata density in
water-stressed plants (Serna & Fenoll, 1997), or the formation of
*These authors contributed equally to this work.
(Hoover, 1986). The formation of stomata clusters was posited to
reflect an adaption to dry environments aimed at reducing water
loss from plant leaves (Gan et al., 2010; Franks & Casson, 2014).
Recently, Dow et al. (2014) measured the effect of stomatal
clustering on maximum leaf conductance of various genotypes of
Arabidopsis thaliana expressing different stomatal patterns. Their
results show that diffusive conductance from leaves with clustered
stomata was 60% lower relative to those with uniformly distrib-
uted stomata for densities (sum of abaxial and adaxial leaf sur-
face) in the range of 300–450 stomata per mm2. Dow et al.
(2014) hypothesized that the observed reduction is probably due
to impaired function of the tightly spaced stomata guard cells
that may prevent complete opening of the stomata.
However, an additional potential effect of clustering may
involve increased diffusive resistance within stomata clusters as
postulated by Gan et al. (2010) and Franks & Casson (2014),
and recently shown theoretically and experimentally for other
evaporating porous surfaces by Lehmann & Or (2013). To quan-
tify the potential effects of stomatal clustering on gaseous diffu-
sive resistance, we developed a physically based analytical model
that accounts for the main components of gas exchange resistance
in distributed and clustered stomata patterns on plant leaves. The
expressions developed provide a theoretical basis for predicting
gaseous flux reduction due to different stomatal clustering scenar-
ios, and indirectly provide a proper context for the physiological
impairment of guard cell hypothesis. In Supporting Information
Notes S1 the model is applied to quantify gas conductance for a

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different type of clustering defined by the network of veins,
indicating that the conduction is reduced by this type of cluster-
ing as well.
Materials and Methods
Theory of diffusive gas transport through stomata
The diffusive gas flux F (expressed as moles per unit area per unit
time) through stomata-perforated leaves depends on the driving
force (i.e. a concentration difference along the diffusive pathway),
and on the conductance g [mol L2 T1] of the various obstruc-
tions between sub-stomatal cavity and the ambient atmosphere.
It is convenient to use diffusive resistance R (i.e. the reciprocal of
conductance g), to express the total resistance as a series of differ-
ent resistances as presented in Fig. 1. In the following, we quan-
tify the resistance and conductance of these elements and their
dependence on stomata size, shape and spatial arrangement. Note
that we often discuss the various terms in relation to ‘vapor trans-
port’ and transpiration, but the same expressions apply to CO2
uptake as well.
Computing leaf conductance for gas transport
We first consider gaseous diffusive conductance of a stomatal
pore gSP (marked by index SP for ‘stomatal pore’) of depth d [L]
and cross-sectional area A [L2], defined by Brown & Escombe
(1900) as:
1 A D p  r2
¼ gSP ¼   n ¼ k n Eqn 1
RSP d v d vapor shells) and end-correction formulations that do not con-
with presistance
ffiffiffiffiffiffiffiffiffi RSP, pore radius r [L] (same area A with
r ¼ A=p), stomatal density n [L2] and the ratio k
[mol L1 T1] between vapor diffusion coefficient D [L2 T1]
Flux per
stoma s
RBL δ (Brown & Escombe, 1900)
RVS
RSP RLeaf 2r d
Rend
Fig. 1 Resistances for vapor diffusion through stomata-perforated leaves
with resistance through the stomatal pore RSP, vapor shells forming at the
end of each stomatal aperture RVS that may interact with each other
(related to end-correction Rend, see ‘Discussion’ in the text), and boundary
layer resistance RBL. The resistances of the vapor shells and the pore
resistance are combined and lumped into leaf resistance RLeaf. The total
resistance of leaf and atmosphere in series is controlled by four
dimensions: stomatal depth d, pore radius r, spacing between stomata s
and thickness of boundary layer d.
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and molar volume of air v [L3 mol1] (with k ~ 103 mol s1 m1
for water).
Brown & Escombe (1900) considered an additional diffu-
sive resistance at the ends of a pore related to converging
and diverging concentration shells forming at pore apertures.
Stomata spacing plays a role in limiting the lateral extent of
adjacent vapor shells. We distinguish between the standard
gas exchange formulation, which defines this resistance as a
property of the stoma itself (the ‘end-correction’), and formu-
lations that consider the effects of stomata spacing on diffu-
sive resistance across ‘concentration shells’ (e.g. Bange, 1953;
Schl€under, 1988).
This raises a certain ambiguity present in the literature con-
cerning the proper representation of diffusive resistance related to
‘end-correction’, the role of stomata shape, and stomata spacing.
Specifically, we wish to clarify (1) the number of end-corrections
to be considered (at one or both ends of the stomata pore), the
effects of (2) stomata shape (circular or elliptic), and the influence
of (3) spacing s between adjacent stomata. In Fig. S2 we show
how assumptions concerning stomata shape may lead to incorrect
conclusions regarding the end-correction.
In the following we summarize the various expressions used in
the literature to quantify the ‘concentration shell’ resistance (i.e.
for one concentration shell; for concentration shells at both sides
the expressions must be doubled). Note that because we will dis-
cuss the transport of vapor (transpiration) in detail we will use in
the following the specific term ‘vapor shell’ instead of the more
general term ‘gaseous concentration shell’. We differentiate
between approaches that consider the limited spacing and inter-
action between vapor shells (marked by index ‘VS’ for interacting
sider the spacing of the vapor shells (with index ‘end’ for end-cor-
rection).
End-corrections (no interaction)
1 1
Rend ¼ ¼ Eqn 2(a)
gend 4r  k  n
1 Loge ð4a=bÞ
Rend ¼ ¼ Eqn 2(b)
gend 2p  a  k  n
(Parlange & Waggoner, 1970)
Interacting vapor shells
 
1 1 1 1
RVS ¼ ¼  Eqn 2(c)
gVS 4r p  s k  n
(Bange, 1953)
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  facilitate a systematic comparison, we define x = cendr with end-

1 1 1 1
RVS ¼ ¼  Eqn 2(d) correction factor cend. The most frequently used values for cend
gVS 2p  r p  s k  n
are: cend = p/4 applied by Franks & Farquhar (2001) correspond-
ing to one-sided resistance Rend as defined by Stefan (1882); or
(Schl€under, 1988)
cend = p/2 related to a resistance Rend at both ends of stomatal
with spacing s between stomata and radius of major and minor
pore, as used in Franks & Beerling (2009), Ting & Loomis
axis of ellipsoidal-shaped stomata, a and b, respectively. Eqn 2(a)
(1965), de Boer et al. (2011) and Dow et al. (2014). Nobel
for the resistance of a vapor shell forming above a circular pore
(2005) proposed an intermediate value: cend = 1, indicating that
was proposed by Stefan (1882) and subsequently used by Brown
the resistance related to vapor diffusion at the ends of the stoma-
& Escombe (1900); for elliptical stomatal pores with b  a,
tal pore may be smaller than expected from two end-corrections.
Eqn 2(b) from Parlange & Waggoner (1970) is more accurate
To account for the limited spacing between stomata (on the leaf
(see discussion in Notes S2).
epidermis) we employ in the following the gas diffusive resistance
The equations derived by Bange (1953) and Schl€ under (1988)
model of Bange (1953) as in Eqn 2(c) for interacting vapor shells.
consider the effect of limited spacing between pores on the diffu-
For the sub-stomatal cavity side, we assign for simplicity (and
sive resistance through the vapor shells. The derivations of Bange
due to lack of specific information on geometry of sub-stomatal
and Schl€ under differ with respect to the shape of the flux source;
cavity) a single end-correction according to Eqn 2(a).
this is a disk in Eqn 2(c) from Bange (1953), and a hemisphere in
The last component of diffusive resistance considered is
Eqn 2(d) from Schl€ under (1988). Interestingly, Eqn 2(c) con-
between the vapor shell and the top of an attached air boundary
verges to Brown and Escombe’s end-correction in Eqn 2(a) for
layer of thickness d that decreases with increasing mean wind
large stomata spacing s. Rend and diffusive resistance of interacting
speed. This diffusive resistance designated as RBL (the index ‘BL’
vapor shells RVS can be related (Eqn 3a) to define a critical density
for boundary layer) could also be used to define the potential
nVS (Eqn 3b) where the effect of vapor (or gas) shell interactions
evaporation rate from free water surfaces, expressed as:
becomes negligible (< 10% of the end-correction resistance):
pffiffiffi 1 k
RVS 4r 4r n ¼ gBL ¼ Eqn 5
¼1 ¼1 Eqn 3(a) RBL d
Rend ps p

RVS  p 2 1
 0:9 ! nVS   Eqn 3(b)
Rend 40r 160 r 2 Stomata spacing effects on diffusive fluxes

for stomata The total diffusive resistance is computed in a manner analogous

pffiffiffi spacing s defined on a square grid with size
s ¼ 1= n. For small stomata densities n (and small pore radius
r), the second term on the RHS of Eqn 3(a) becomes small;
hence, the gas diffusion model of Bange (1953) that considers
interacting vapor shells converge to standard end-correction for-
mulation. Details of the differences between the two expressions
are discussed in the Notes S3 and explained in specific examples
shown in Fig. S3.
With a few exceptions, the effects of stomatal spacing s on dif-
fusive conductance through interacting vapor shells forming
around a stomatal pore is rarely considered (Bange, 1953; Assou-
line & Or, 2013). Leaf gas exchange models often express diffu-
sive resistance as a function of stomata pore size only and not of
stomata spacing. By defining the diffusion through the vapor
shell as a property of the stoma, the end-correction resistance
Rend and the resistance of the stomatal pore depth RSP are com-
bined and lumped into the leaf resistance RLeaf (or its inverse, the
leaf conductance: gLeaf = 1/RLeaf). The resulting term expresses
the combined resistances of the pore depth extended by a length
x (end-correction), according to:
 
1 d þx pr
RLeaf ¼ ¼ RSP þ Rend ! x ¼ Eqn 4
n  k p  r2 4 p  r2 k
Note that for Rend we inserted here Eqn 2(a). Various
formulations of ‘end-correction’ are found in literature; to
to estimating resistance in an electrical circuit, with resistances
arranged in series or in parallel (Weyers & Meidner, 1990). The
total diffusive resistance is typically computed per total leaf area
with n stomata arranged in parallel (with conductances gend, gSP
and gVS that are proportional to stomatal density n), and arrang-
ing this resistance with the boundary layer resistance RBL = d/k in
series. To better understand conceptually how stomata density
(and spacing) affect diffusive fluxes, we analyze diffusive resis-
tance for a unit area around a single stoma (for simplicity, a
pffiffiffi
square with size s ¼ 1= n ) with resistance ℜ [T mol1] assigned
to the unit area (with symbol ℜ to differentiate from resistance R
[T L2 mol1] assigned to a total leaf area). For each stoma the
total resistance is the sum of ℜend (i.e. the end-correction for the
sub-stomatal entry), the resistance ℜSP in the stoma pore, the
resistance of the vapor shell ℜVS that could be limited by spacing
s, and the boundary layer resistance ℜBL. These resistances are
expressed as follows:
1 1
<end ¼  Eqn 6(a)
4r k
d 1
<SP ¼  Eqn 6(b)
 
1 1 1
<VS ¼   Eqn 6(c)
4r p  s k

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d 1
<BL ¼  Eqn 6(d)
s2 k
As stomata spacing s increases, the resistance term (Eqn 6c)
converges to the term in Eqn 6a, and, hence, the end-correction
ℜend is the maximum value of vapor shell resistance. For simplic-
ity, we define here: ℜVS = ℜend. We may express the total gas
fluxes relative to a reference state under similar conditions (tem-
perature, wind speed and vapor pressure gradient) such as evapo-
ration from a free water surface (considering ℜBL only), denoted
here as eLeaf = ℜBL/ℜT. Note that the relative rate (given below
in Eqn 7a) is the same as expressing the ratio of resistances per
total area, eLeaf = RBL/RT. Similarly, the relative vapor flux per
pore area ePore = eLeafs2/(pr2) is given below in Eqn 7(b):
<BL RBL 1 obtained:
eLeaf ¼ ¼ ¼ 2   Eqn 7(a)
<T RT 1 þ sd 2r1 þ prd 2
s2 1 RSP þ RVS þ RBL 4d þ r ðp  4r n þ 4d  p  n  r Þ
ePore ¼  eLeaf ¼ pr 2 1 pr  Eqn 7(b)
pr 2
s2 þ d 2 þ d
These functions are depicted in Fig. 2 for two different sto-
mata radii. The results show that by arranging the diffusive resis-
tance of the 3D vapor shells with the 1D boundary layer in series,
the resulting diffusive fluxes become highly nonlinear functions
of stomata spacing s (or density n). With increasing spacing (or
decreasing density), the flux per stoma may increase and compen-
sate for the reduced number of stomata (or total evaporating
area). Consequently, plant leaves are able to sustain high transpi-
ration rates (similar to evaporation from a free water surface) with
a very small total stomatal area (c. 1–2% of the leaf area; for a
more complete discussion on evaporation from pores, see Shah-
raeeni et al., 2012).
Fig. 2 Vapor diffusion fluxes (defined by total resistance RT) related to
reference flux (only boundary layer resistance RBL) from a free water
surface as defined by Eqn 7(a) for total leaf area eLeaf (blue) and Eqn 7(b)
for stomatal pore area ePore (red) for boundary layer thickness d = 1 mm
and two different pore radii r that are equal to pore depth d. Although the
flux per unit leaf area is larger for the large stoma (solid lines; four-fold
evaporating area compared to small pores), the fluxes per unit pore area
are larger for the small stoma (dashed lines) due to restricted formation of
3D vapor concentration shells.
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Equation 7a was based on the simplification wherein the vapor
shell resistance is equal to the end-correction term, ℜVS = ℜend.
For completeness, we present an expression for the relative gas
diffusion resistance that considers vapor shell resistance as
affected by stomata spacing s (expressed by density n):
RBL 2d  n  p  r 2
¼ pffiffiffi
Rend þ RSP þ RVS þ RBL 2d þ r ðp  2r n þ 2d  p  n  r Þ
Eqn 8
For a numerical model of vapor diffusion applied in the result
section to test the analytical expressions, we considered a system
without an end-correction within the leaf (assuming completely
saturated vapor pressure within the sub-stomatal cavity) and
RBL 4d  n  p  r 2
¼ pffiffiffi Eqn 9
In commercially available gas exchange measurement systems
for determining leaf conductance gLeaf, the boundary layer resis-
tance RBL is typically estimated independently and subtracted
from the total resistance RT, with RLeaf = RT  RBL (Li-COR Bio-
sciences, 2012). For comparison of theoretical predictions with
experimental data (gLeaf) reported from such measurement sys-
tems (Dow et al., 2014), we have used the following expression
for gas conductance gLeaf (without the boundary layer resistance:
RBL):
1 1 n  k  2p  r 2
gLeaf ¼ ¼ ¼ pffiffiffi Eqn 10
RLeaf Rend þ RSP þ RVS 2d þ r ðp  2r nÞ
Effects of stomata clustering on leaf gas exchange
As shown in Eqn 7(b) and Fig. 2, by arranging the resistance of
the leaf RLeaf = Rend + RSP + RVS in series with the boundary layer
resistance RBL, the flux per stomata is decreasing with increasing
density. Accordingly, we expect that the high density and small
spacing (see Fig. 3a) between neighboring stomata would restrict
formation of lateral concentration gradients within the cluster
(resulting in a primarily 1D vapor flux as in the sketch in
Fig. 3b). To study the role of pore clustering and spacing on
vapor diffusion, Brunn (1984) and Fabrikant (1985) considered
a system with no boundary layer attached where the diffusive flux
was reduced with smaller pore spacing. To show qualitatively
how small spacing with adjusted vapor shell and boundary layer
resistances in series reduces the flux per pore in a cluster, we now
greatly simplify the rigorous Green’s function approaches of
Brunn and Fabrikant by assembling modified resistance terms
from the previous section.
For a stoma of a cluster we first decouple the boundary layer d
(see Fig. 4a) and the diffusion through the stoma, and thus –
according to Eqn 6(a) – obtain as maximum conductance of a
vapor shell 4rk (i.e. 1/ℜend). Next, we consider a large vapor
shell of radius S/2 over the stomata cluster. We assign one part of
the stoma to the periphery of the cluster (affected by large spacing
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(a) (b) layer’ thickness). For simplicity we assign 50% weight for flux
from periphery with a maximum conductance 1/ℜend = 4rk and
for the interior of the cluster with conductance 1/(ℜVS + ℜBL).
We define a clustering factor f ≤ 1 as the ratio of vapor flux from
stomatal aperture in the cluster to the maximum flux for an ‘iso-
lated’ stoma (with no vapor shell interactions) as:
pffiffiffi
1 <end p  h 2  2h  r þ p  r  S =2
f~ þ ¼ pffiffiffi
2 2<BL ðS =2Þ þ 2<VS p  h 2  2 2h  r þ p  r  S
Fig. 3 Stomatal clustering and its representation in a conceptual model. (a)
Differential interference contrast image from Dow et al. (2014) for a Eqn 11
genotype with high clustering (denoted as ‘basl’ in Dow et al.) with
boundaries of pavement cells in red, guard cells in green and stomatal with the notation ℜBL (S/2) indicating that the composite cluster
pores in black. The arrow indicates the spacing S between two clusters vapor shell acts as ‘boundary layer’ for pores within the cluster in
(here with N = 3, 4 and 5 stomata per cluster). (b) Conceptual image of
analogy with Eqn 6(d). Noting that such an approximation
vapor diffusion through stomata of depth d, radius r and across viscous
boundary layer of thickness d. Inside the cluster the spacing h and vapor (Eqn 11) is too crude to match the refined analyses of Fabrikant
concentration gradient is reduced, resulting in smaller vapor flow. (1985) that we present next, it nevertheless offers an intuitive
explanation of the effects of clustering on diffusive flux reduction.
For clusters of two, four and six stomata with distance h between
each stomata and the center of the cluster, the clustering diffusive
resistance factor according to Fabrikant (1985) is:
(a)
δ (b)
1
Flux from cluster
fN ¼2 ¼ 1 r
  Eqn 12(a)
(not yet ‘coupled’ with
boundary layer δ) 1þ 2
p sin 2h

S/2 fN ¼4 ¼ r 
p
  Eqn 12(b)
p þ 2sin 2h þ 4sin1 pffiffi2r h
1

1D
3D fN ¼6 ¼ r 
p
r    Eqn 12(c)
p þ 2sin 1
þ 4sin1 þ 4sin1 pffiffir
2h h 3h
2r
2h The clustering factor f based on Eqns 11 and 12(b) is shown
in Fig. 4(b) together with results of a full numerical solution.
Fig. 4 Simple conceptual model to explain the reduced fluxes from a
Following the explanation of the expected role of clustering on
cluster of stomata (here four stomata of radius r per cluster) independent
of boundary layer. (a) A vapor shell of spacing S is forming above a pore vapor fluxes across the vapor shell, we now add all expressions for
cluster. At the periphery of the cluster, vapor shell resistance is defined by conductance and resistance for a cluster of N stomata with cluster
end-correction (according to Eqn 6a) due to the nonrestricted formation of density n/N and obtain:
vapor shells in 3D. Inside the cluster the development of vapor shells is
limited (reduced vapor concentration gradient) and the flux is defined by
1 N  p  r2 p  r2
resistances of a small vapor shell (Eqn 6c) in series with one-dimensional ¼ gSP ¼ k  n=N ¼ k n Eqn 13(a)
(‘1D’) resistance of thickness S/2. (b) Effect of clustering on vapor flux RSP d d
expressed as the ratio between diffusion through a vapor shell forming
above the stoma in a cluster and that through an isolated stoma (here for 1
¼ gend ¼ N  4  r  k  n=N ¼ 4r  k  n Eqn 13(b)
pore radius 5 lm and cluster size 125 lm) as a function of spacing h. The Rend
simple conceptual model (red line) given in Eqn 11 is compared to the
more rigorous approach of Fabrikant (black line, Eqn 12b) and results of 1 k  n=N 4p  r  f  k  n
the numerical model (squares). The fluxes from the clustered geometries ¼ gVS ¼ 1 1 ffiffiffiffi ¼
pffiffiffiffiffiffiffiffiffiffiffi Eqn 13(c)
are scaled to the uniformly distributed arrangement of stomata.
RVS N 4rf  p
ps N
p  4 N nr f

S to the neighboring cluster),

pffiffiffiffiffiand a second part to the interior of
the cluster (with spacing 2h between the stomata of the same
cluster). The interactions among vapor shells within the cluster
merge the concentration field (or suppress formation of fully
developed 3D structures) with diffusive fluxes occurring primar-
ily in 1D. The fluxes are defined by two resistors in series: a vapor
shell presistance
ffiffiffiffiffi ℜVS as defined in Eqn 6(c) with spacing
S ¼ 2h , and a diffusive resistance with length S/2 (in analogy
to resistance ℜBL defined in Eqn 6(d), inserting S/2 as ‘boundary
1 k
¼ gBL ¼ Eqn 13(d)
RBL d
with the clustering factor f < 1 describing the reduction of the
vapor flux based on clustering as given in Eqn 12 by Fabrikant
(1985). The cluster ‘vapor shell’ resistance in Eqn 13(c) is differ-
ent from the expression of Bange (1953, Eqn 2c above) as fol-
lows: we consider the conductance of a composite vapor shell
forming over a pore cluster and not just around a single stoma

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equal to N4rf (relative
pffiffiffiffiffito 4r in Eqn 2c); then the cluster diffu-
sive resistance
pffiffiffiffiffi 1/(ps N ) is subtracted based on spacing between
clusters s N .
As discussed in the context of Eqns 2 and 4 for plant leaves
with uniformly distributed stomata with low density (n < nVS),
the resistance of interacting vapor shells RVS can be represented
by a simple end-correction Rend (this amounts to assigning a ficti-
tious extension ‘x’ (Eqn 4) to the pore length d). In a similar fash-
ion we extend the pore depths of stomata arranged in clusters by
a fictitious length X matching a resistance Rend/f as follows:
 
Rend 1 d þx þX pr
RLeaf ¼ RSP þ Rend þ ¼ !X ¼
f nk pr 2 4f
Eqn 14
Accordingly, on one side of the leaf an extended (solely due to
stomata clustering) end-correction X = x/f is added to the diffu-
sive resistance (with f < 1 depending on stomata spacing h within
the cluster). Note that within the sub-stomatal cavity we have
assumed a standard end-correction simply due to lack of infor-
mation on the internal geometry and connectedness within sub-
stomatal cavities.
We can now relate the total gas diffusive resistance to a refer-
ence state (e.g. resistance to vapor diffusion from a free water sur-
face defined by boundary layer resistance only), and express the
relative vapor diffusion flux from a leaf (or relative diffusive resis-
tance) as:
RBL 4d  n  p  f  r 2
¼  pffiffiffiffiffiffiffiffiffiffi Eqn 15
RT 4f d  r 2 n  N þ p  r ð1 þ f þ 4d  n  r  f Þ
with total resistance RT = Rend + RSP + RVS + RBL. For comparison
with the numerical model results, and assuming saturated vapor
concentration within the sub-stomatal cavity, the relative fluxes
were expressed as:
RBL 4d  n  p  f  r 2
¼  pffiffiffiffiffiffiffiffiffiffiffi
RSP þ RVS þ RBL 4f d  r n  N þ p  r ð1 þ 4d  n  r  f Þ
2
Eqn 16
Finally, to compare the proposed stomata clustering gas diffu-
sion model with standard measurements of leaf gas conductance
gLeaf, we also use the following expression:
1 4n  k  p  f  r 2
gLeaf ¼ ¼  pffiffiffiffiffiffiffiffiffiffiffi Eqn 17
RLeaf 4f d  r 2 n  N þ p  r ð1 þ f Þ
with leaf gas resistance RLeaf = Rend + RSP + RVS. In Fig. 5, we
present the various conductance terms for a range of stomatal
densities. Although the boundary layer conductance is inde-
pendent of density, all other conductances increase with sto-
mata density due to the parallel addition of stomata (that
contribute to total leaf gas exchange). The conductance of the
vapor shells (end-correction inside the sub-stomatal cavity and
accounting for spacing outside the leaf) was > 20% smaller
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Fig. 5 The effects of stomata clustering on the conductance of vapor shells
forming above stomata apertures in relation to other components of gas
diffusion conductances (here expressed as inverse of the resistance for
stomatal pore RSP, the end-correction within the substomatal cavity Rend,
the vapor concentration shell RVS, and boundary layer resistance RBL). The
clustering of the stomata (here shown for a cluster of four stomata and a
distance of 2r between a stoma and the center of the cluster) significantly
reduces the conductance of the vapor shell forming at the aperture of a
stomatal pore. The difference between total conductance gT (inverse of
total resistance RT) for clustered and distributed stomata for this example is
in the range of 10% (as example for density 100 mm2, the total
conductance is 0.38 mol m2 s1 for uniformly distributed and
0.34 mol m2 s1 for clustered stomata).
for the clusters (cluster of four stomata, spacing of two pore
radii between pore and center of the cluster). The effect of
clustering increases the total conductance by c. 5–15%
(depending on stomata density and for a boundary layer
thickness of 1 mm).
The foregoing analysis focuses on small clusters with a few sto-
mata (≤ 6). However, as we show in Notes S1, other types of sto-
matal clustering with large clusters (comprising tens or even
hundreds of stomata) bounded by network of veins may also
impact leaf gas exchange relative to an uniformly distributed sto-
mata pattern. The conductances for leaves with such patterns fol-
low a more general derivation presented in Lehmann & Or
(2013) that considers different spacing for stomata within the
cluster and along the boundary of a cluster. For simplicity, and to
maintain the analogy with the study of Lehmann & Or (2013),
we add expressions for the relative conductance eLeaf = RBL/RT (as
in Eqn 7a above) using the following basic expression:
RBL
eLeaf ¼ ¼ U C  e C ð s C Þ þ ð 1  U C Þ e P ð WV Þ Eqn 18
RT
with the areal fraction ΦC and the relative conductance eC
assigned to the interior of the cluster that is a function of the inte-
rior spacing sC, and the relative conductance eP and areal fraction
1  ΦC assigned to the periphery depending on the width of the
veins WV defining the distance between clusters. The terms of
Eqn 18 are specified in Notes S1.
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Numerical solution of the gaseous diffusion equation
The analytical theoretical framework introduced above is based
on the assumption that the total gas diffusion conductance can
be expressed as a series of resistance components. Because details
of the ‘transitions’ between the various resistance components
can be relatively complicated, especially for concentration shells
constrained within the boundary layer, or geometrical details of
stomata pores, the simplifications and assumptions need to be
tested using a less restrictive numerical framework. Previous stud-
ies by Tyree & Yianoulis (1980) have used finite difference
schemes to compute vapor transport through cylindrical and
hemispherical cavities; Roth-Nebelsick (2007) applied a finite
element method to analyze the effects of the geometry of the sto-
matal pore. Here we solve the diffusion equation in 3D focusing
on different configurations of clustered and distributed stomata
using a finite difference method. The domain between the leaf
cuticle and the top of the air boundary layer was discretized by
a numerical grid with 1 lm spacing. The vapor concentration
at the bottom of substomatal cavities was set to saturation and
at the top of the boundary layer as zero (for completeness we
have also simulated CO2 diffusion with the ‘reverse’ boundary
conditions of zero concentration at the bottom of the substom-
atal space and atmospheric concentration at the top of the
boundary layer). Periodic boundary conditions were applied at
the lateral boundaries. The vapor concentration was initially
set to zero and then the simulation run until the gas (vapor)
flux across the top of the boundary layer converged to the rate
supplied at the lower boundary and steady-state diffusive fluxes
were established.
Results
In the following, we compute the total resistance for vapor dif-
fusion for distributed and clustered stomata patterns and com-
pare the predicted diffusive conductance with the
measurements of Dow et al. (2014). The theoretical predictions
permit inferences concerning the limited role of stomata spac-
ing on observed gas diffusion suppression, thereby lending cre-
dence to the postulate of guard cell impairment for anomalous
stomata clustering. In general, the theoretical derivations and
expressions provide a systematic framework for quantifying gas
diffusion resistance and the effects of concentration shell inter-
actions for any stomata density or spatial patterns. Addition-
ally, in Notes S2 and S3 we apply the theoretical framework
for two case studies in which stomata are assumed to be uni-
formly distributed (i.e. no clustering), yet, stomatal spacing
and overlapping vapor shells play non-negligible roles in
inferred leaf gas conductance.
Comparison of clustering analytical model with numerical
solution of 3D diffusion equation
Before analyzing the measurements of Dow et al. (2014) to assess
the effects of stomata clustering, we first compared vapor fluxes
computed numerically and analytically for various geometries.
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We considered values of stomata density n in the range 160 to
1300 mm2, stomata depth d and aperture radius r from 5 to
10 lm, and boundary layer thickness d from 30 to 160 lm (we
have chosen a thin boundary layer to keep geometries and resis-
tances within the same order of magnitude for uniform spatial
discretization in the numerical experiment; in reality, for such
thin boundary layers, the advection within the boundary layer
would be relevant and the transport would not be purely diffusive
as assumed here). The diffusive resistances for distributed and
clustered stomata arrangements (four stomata per cluster) were
compared for different cluster spacing h (measured between a
stoma and cluster’s center; Fig. 3b). The 3D diffusion equation
was solved on a numerical grid at 1 lm resolution. For simplicity,
we fixed the vapor concentrations at the boundaries to saturated
air at the sub-stomatal cavity and zero vapor concentration at the
top of the boundary layer. Periodic boundary conditions were
applied at lateral boundaries, and the numerical simulations were
run until steady-state diffusive fluxes were established. The reduc-
tion of total vapor fluxes by clustering was in the range of 5–
20%. In Fig. 6 the results of the numerical experiments were
compared to predictions using Eqn 9 for distributed stomata,
and Eqn 16 for clustered stomata (expressed as their equivalent
resistances), lending support for the simpler analytical modeling
approach that will be used subsequently.
Comparison of clustered stomata model with Dow et al.’s
(2014) data
Based on the information provided in Tables 1–3 in Dow et al.
(2014), we re-computed the various components of leaf
Fig. 6 Comparison of the analytical model with numerical solutions for
vapor diffusion in 3D for distributed stomata (red) and clusters of four
stomata with large (blue) and small spacing (green) h between clusters
(density n between 160 and 1300 mm2, stomata thickness and radius
between 5 and 10 lm, boundary layer 30–160 lm). The resistance of
vapor flux across the boundary layer RBL (corresponding to evaporation
from a free water surface) was related to total resistance for vapor flux RT.
The black line indicates a 1 : 1 match between the numerical and analytical
models using Eqn 9 for distributed and Eqn 16 for clustered stomata.
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1022 Research
resistance. Measurements of leaf gas exchange with modern
chambers often determine the boundary layer resistance inter-
nally RBL (typically a readout from a prescribed calibration) and
subtract it from the reported total resistance RT. According to the
description in Dow et al. (2014), they used a Portable Photosyn-
thesis System (6400-02B LED Light Source; Li-Cor Biosciences
Inc., Lincoln, NE, USA) that provides values of the ‘leaf conduc-
tance’ gLeaf [mol L2 T1] after subtracting the air boundary layer
resistance RBL from the total resistance RT. Note that the ‘total
conductance’ is deduced from the transpiration rate F and from
the vapor concentration within the leaf computed from leaf
temperature and atmospheric pressure (Li-Cor Biosciences,
2012). Dow et al. (2014) deduced the length of the stomatal pore
from interference contrast microscope imaging of the leaves, and
set the width of the pore as half the length and chose as depth the
width of the guard cell. By consulting various references (Perera
et al., 2008; Wu et al., 2009; Plessis et al., 2011; Xu et al., 2014)
we set the maximum width of the stomatal pore as 0.4 of its
length. Based on the area of such an elliptical shape we computed
the radius of a disk of the same cross-section and used it to com-
pute conductance. We have assumed a constant aperture along
the stomatal pore, ignoring more complex pore shapes that may
affect stomatal pore resistance as shown by Kaiser (2009) and
Bange (1953).
In order to compare theoretical considerations with
measurements, we first focus on maximum diffusive fluxes
(leaf conductance gLeaf) for genotypes with no clustering (uni-
formly distributed stomata). We compare experimental values
Fig. 7 Comparison of maximum leaf conductance gLeaf measured by Dow
et al. (2014) (red symbols) for uniformly distributed stomata with model
predictions (lines). Note that the stomatal density depicted on the x-axis is
the sum of the densities on the adaxial and abaxial sides. The measured
values are compared with the conductances of a stomatal pore (green, no
end-corrections), a stomatal pore with one end-correction (black) and
conductance for a pore with one end-correction and the resistance of a
vapor shell limited by interaction (red; Eqn 10). Insets conceptually show
the differences in considered resistances for stomatal pore RSP, end-
correction Rend and vapor shell resistance RVS. Average pore radius r and
depth d were applied as deduced from measured data.
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of gLeaf with model predictions as depicted in Fig. 7. The
comparison highlights that diffusive resistances at both ends
of the stomatal pore should be considered to match experi-
mental values (at the epidermis side we have used Eqn 2c
from Bange).
Next, we compared model estimates with measurements from
genotypes with clustered stomata presented by Dow et al. (2014).
We note that in Table 1 of Dow et al. (2014) the stomatal den-
sity for genotypes with clustering should have been expressed as
a sum of four components (adaxial and abaxial stomata for dis-
tributed and clustered stomata), but only three terms were
included (the adaxial clusters were omitted). The stomata density
was recalculated as the sum of the four terms in Table 3 of Dow
et al. (2014). Fig. 8 depicts theoretical predictions for clusters
with four stomata (spacing h = 2r between stomata and the clus-
ter center). The predictions account for some reduction in clus-
tered leaf vapor conductance (relative to distributed stomata
genotype), but predicted gas diffusion from clusters (bold blue
line in Fig. 8) overestimates the measured conductance for clus-
tered leaves, suggesting that additional factors should be
considered.
Dow et al. (2014) hypothesized that the anomalous stomata
clustering could prevent the proper functioning of closely spaced
Fig. 8 Comparison of maximum leaf conductance gLeaf for distributed (red)
and clustered stomata (blue) with measurements of Dow et al. (2014). The
shaded rectangles with symbols represent the variations around mean
values listed in Table 1 in Dow et al. Note that the stomatal density
depicted on the x-axis is the sum of the densities on the adaxial and
abaxial sides. Model predictions for clustered data (clusters of four
stomata, spacing h = 2r between pore and cluster center) are shown for
the assumption of 50% of stomata arranged in clusters (blue bold line;
Eqn 17). The model results for uniformly distributed stomata (red line;
Eqn 10) are the same as shown in Fig. 7. The values for pore radius r and
depth d (deduced from measurements) were slightly different for clustered
(blue numbers) and nonclustered stomata (red numbers). To match
experimental data for stomata arranged in clusters, the aperture was
reduced from a maximum value r deduced from image analysis by factor
of four (blue dashed line) to r0 = r/4. Without reducing the aperture related
to guard cell impairment, the predicted conductance is overestimated.
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guard cells and limit full opening of stomata apertures. The func-
tioning of guard cells’ complex opening mechanism, including
ion transport across channels between guard cell and neighboring
subsidiary cells (Pandey et al., 2007; Kim et al., 2010; Higaki
et al., 2014), may be impaired by disrupting signaling and sup-
pressing formation of specific membrane proteins (Klein et al.,
2004), and by mechanical limitation to volume change of the
guard cell (Franks & Farquhar, 2007). Franks & Farquhar
(2007) have shown experimentally for grass-type stomata that the
maximum aperture of stomatal pore requiring greater lateral dis-
placement of guard cells is inhibited for full epidermis
(subsidiary) cells and that the effective aperture was only c. 1/3 of
the maximum value. For clustered stomata Dow et al. (2014)
postulated that the proximity of neighboring guard cells may
restrict the lateral displacement of guard cells and thereby hinder
complete opening of stomatal apertures. To represent the
potential effect of such mechanical impairment in the model, the
radius (and hence the area) of mean radius of stomata was
reduced. Assuming that 50% of the stomata were arranged in
clusters (with four stomata per cluster), the apertures of stomata
in clusters were reduced by factor of four to significantly improve
the agreement with measured conductance values (see blue
dashed line in Fig. 8).
Discussion
The reduction in diffusive conductance from leaves with clus-
tered stomata patterns shown in Fig. 8 is attributed to three
factors in the gas exchange model: reduced mean stoma radius
for clustered stomata; predicted increased diffusive resistance
due to interactions among closely spaced vapor shells within
clusters, and reduced aperture opening attributed to guard cell
impairment. To quantify the sensitivity of the results to vari-
ous factors, we express the change in leaf gas conductance for
a scenario with 50% of the stomata arranged in clusters with
their maximum aperture open (bold line in Fig. 8). We then
varied certain parameters and examined the impact on leaf
conductance. For example, increasing the fraction of stomata
in clusters from 50% to 55% reduces conductance by < 2%;
reducing stomata aperture by 10% reduces conductance by
> 3%; increasing stomata number per cluster from 4 to 6
reduces conductance by 5%. These results delineate the range
of values that could be deduced from the observations of
Dow et al. (2014), suggesting that clustering effects alone can-
not explain the observed reduction in gas diffusive conduc-
tance. This lends support to the hypothesis of guard cell
impairment (as expressed by the reduced aperture in Fig. 8) as
a potential mechanism for the reduced vapor fluxes from clus-
tered leaves.
Clearly, the application of the theoretical framework presented
in this study is not limited to the data of Dow et al. (2014); the
expressions derived here are applicable to deducing gas diffusion
conductance of leaves with any stomata size, density or pattern.
The formulation in Eqn 14 offers a simple and straightforward
extension of the standard leaf gas exchange model, expressing the
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Research 1023
effect of clustering on leaf conductance as an equivalent or modi-
fied end-correction term.
Thus far we have discussed clustering in the context of stomata
arranged in clusters as a result of cell differentiation. There are
other forms of stomata function in clusters, such as formation of
stomata patches bounded by veins (Evans & Loreto, 2000) with
no stomata on the epidermis above the veins. Carins Murphy
et al. (2014) have shown that plant leaves (from a woody angio-
sperm, Toona ciliate) grown under high evaporative demand
expressed higher density of veins (i.e. forming more clusters of
stomata with smaller spacing), higher stomatal density and lower
stomatal conductance than leaves grown under low vapor
pressure deficit. To quantify the effect of this different type of
clustering (more stomata per cluster and larger clusters), we
adapted the approach presented in Lehmann & Or (2013) for
evaporation from clustered porous surfaces as explained in Notes
S1. Although the flux per pore within the cluster becomes smaller
according to Eqn 7(b), the diffusive flux from the pores along the
veins becomes higher. We have used observations from Fiorin
(2013) on stomata numbers within ‘loops’ of veins in Angio-
sperms and Ferns, compared the predicted conductance for sto-
mata in clusters and related them to the conductance for the
same number of stomata distributed uniformly across the surface.
In Fig. S1 presented in Notes S1 we show stomatal patterns and
the effect on leaf conductance. We found that clustering reduced
the conductance c. 10% and that this reduction was similar to the
results we obtained for small clusters. The data measured by
Fiorin (2013) also indicated that within loops of veins the
stomatal density in many Angiosperms is larger than the theoreti-
cal threshold defined in Eqn 3(b) that marks the importance of
concentration shell interactions giving rise to diffusive resistance
beyond a simple end-correction.
Another example is the temporal formation of ‘operational
clustering’ under high heat and water stress where stomata open
or close in patches (Mott et al., 1993; Cardon et al., 1994; Peak
et al., 2004; West et al., 2005). The onset of such temporary
function in patches has been observed in > 200 plant species
under extreme environmental conditions (Beyschlag & Eckstein,
1998). A recent analysis by Lehmann & Or (2013) suggested that
the effective cluster sizes for evaporation suppression must be of
the order of the prevailing boundary layer thickness or larger (a
few mm) as observed in various studies of stomata patchiness
(Luttman et al., 2007). For sufficiently large stomata clusters, the
vapor losses from the leaf could be reduced in proportion to the
areal fraction of the active (open stomata clusters) regions. This
strategy is hypothesized to overcome the inherent nonlinearity
(flux compensation) associated with reducing stomata aperture
opening, or random closure of stomata over the leaf surface.
Additionally, such ‘operational patches’ could also increase sensi-
ble heat exchange from the warmer regions between patches (that
continuously flicker over the leaf surface). Without attempting to
address the benefits and characteristics of operational patchiness,
the theoretical framework proposed in this study offers a means
for systematically evaluating the collective behavior of gas
exchange from leaves with and without stomatal clusters.
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1024 Research
Although the main thrust of the study focuses on the role of
stomata clustering (and pattern in general) in reducing vapor dif-
fusion and transpiration rates, the expressions and results apply
equally to CO2 uptake (as verified numerically for a range of clus-
tering scenarios; results are not shown). To assist interested read-
ers in the application of the various expressions for computing
leaf gas conductance, including the potential effects of stomata
patterns and spacing, we provide guidance below related to appli-
cation of the various gas conductance models. For most applica-
tions, the standard formulation using end-correction term for
vapor shell resistance according to Brown and Escombe (Eqn 2a)
is appropriate for stomatal densities n lower than the threshold
defined in Eqn 3(b) and for width/length ratios of pore aperture
≥ 0.4. For elongated stomata shapes the end-correction expres-
sion must be modified using Eqn 2(b). For uniformly distributed
stomata including the effect of vapor shell interaction for higher
stomatal densities, Eqn 10 is valid. To account for clustering
effects we distinguish between the typical case of a small number
of stomata (≤ 6) per cluster and large clusters containing many
stomata. In the first case the appropriate clustering term given in
Eqn 12 must be inserted in the expression of leaf resistance and
conductance RLeaf = 1/gLeaf defined in Eqn 14. In the second case
(large clusters) a procedure to compute the gas conductance is
described in Notes S1.
We summarize the study as follows: (1) although stomata
clustering increases the interaction of vapor shells and reduces
vapor fluxes by c. 5–15%, it must be assumed that the small
distances between guard cells impair stomata functioning and
opening to explain the 60% reduction in leaf conductance (for
stomatal densities larger than 300 mm2) as reported by Dow
et al. (2014). (2) The developed theory provides guidelines
with which to apply various simplifications (circular shape,
end-correction formulation) and can be used also to study var-
ious types of stomatal clusters, including networks of veins
and the operational clustering of uniformly distributed sto-
mata (stomata patchiness).
Acknowledgements
We appreciate the helpful comments of the Editor and three
anonymous reviewers on a previous version of the manuscript.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Stomatal clustering within loops of veins.
Fig. S2 Effect of stomatal shape on vapor shell resistance and
end-correction.
Fig. S3 Effect of stomatal spacing on vapor diffusion.
Notes S1 Conceptual picture to quantify effect of clustering by
veins.
Notes S2 Quantifying the effect of stomatal shape on vapor dif-
fusion.
Notes S3 Examples on the effect of stomatal spacing on vapor
diffusion.
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