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Critical Reviews in Plant Sciences

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On the Relevance and Control of Leaf Angle

M. van Zantenab; T. L. Ponsa; J. A. M. Janssenc; L. A. C. J. Voeseneka; A. J. M. Peetersa
a
Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Utrecht, The
Netherlands b Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding
Research, Cologne, Germany c Alterra, Wageningen University and Research Centre, Wageningen, The
Netherlands

Online publication date: 29 September 2010

To cite this Article van Zanten, M. , Pons, T. L. , Janssen, J. A. M. , Voesenek, L. A. C. J. and Peeters, A. J. M.(2010) 'On the
Relevance and Control of Leaf Angle', Critical Reviews in Plant Sciences, 29: 5, 300 — 316
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 Critical Reviews in Plant Sciences, 29:300–316, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 0735-2689 print / 1549-7836 online
DOI: 10.1080/07352689.2010.502086

On the Relevance and Control of Leaf Angle

M. van Zanten1,2,∗ , T. L. Pons1 , J. A. M. Janssen3 , L. A. C. J. Voesenek1 ,

A. J. M. Peeters1
1
Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Padualaan 8, 3584 CH
Utrecht, The Netherlands
2
Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research,
Carl-von-Linné-Weg 10, 50829 Cologne, Germany
3
Alterra, Wageningen University and Research Centre, Droevendaalsesteeg 3, 6708 PB Wageningen,
The Netherlands

Table of Contents

I. INTRODUCTION ............................................................................................................................................. 301

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II. FUNCTIONAL SIGNIFICANCE OF LEAF ANGLE ........................................................................................ 301

A. Maximizing Carbon Gain ............................................................................................................................. 301
B. Stress Avoidance .......................................................................................................................................... 302

III. ACTIVE LEAF REPOSITIONING BY HYPONASTIC GROWTH ................................................................... 302

A. Generalities of Leaf Movements .................................................................................................................... 302
B. Submergence-Induced Hyponastic Growth ..................................................................................................... 303
C. Shade-Induced Hyponastic Growth ................................................................................................................ 305
D. Heat-Induced Hyponastic Growth .................................................................................................................. 306

IV. PHYTOHORMONAL REGULATION OF HYPONASTIC GROWTH ............................................................. 306

A. Ethylene as a Major Mediator of Hyponastic Growth ...................................................................................... 306
B. An Ambiguous Role for Auxin in Controlling Hyponastic Growth ................................................................... 307
C. Antagonizing Roles for Gibberellins and Abscisic Acid in Hyponastic Growth .................................................. 307

V. THE USE OF ARABIDOPSIS THALIANA AND INTRASPECIFIC NATURAL VARIATION THEREIN ......... 308
A. Natural Variation in Hyponastic Growth ......................................................................................................... 308
B. Molecular Genetics of Hyponastic Growth; Exploitation of Natural Genetic Variation ........................................ 310

VI. CONCLUSIONS AND PERSPECTIVES .......................................................................................................... 311

ACKNOWLEDGMENTS ........................................................................................................................................... 312

REFERENCES .......................................................................................................................................................... 312

complete submergence, or can be, for example, utilized to maxi-

Plants can have constitutive leaf angles that are fixed and do not
vary much among different growth environments. Several species,
however, have the ability to actively adjust their leaf angles. Ac-
tive leaf repositioning can be functional in avoiding detrimental
environmental conditions, such as avoidance of heat stress and
Address correspondence to M. van Zanten, Plant Ecophysiology,
Institute of Environmental Biology, Utrecht University, Padualaan 8,
3584 CH Utrecht, The Netherlands.E-mail: M.vanZanten@uu.nl
mize carbon gain by positioning the leaves relative to the incoming
radiation. In recent years, major advances have been made in the
understanding of the molecular mechanisms, and the underlying
hormonal regulation of a particular type of leaf movement: hy-
ponastic growth. This differential petiole growth-driven upward
leaf movement is now relatively well understood in model systems
such as Rumex palustris and Arabidopsis thaliana. In the first part of
this review we will discuss the functional consequences of leaf ori-
entation for plant performance. Next, we will consider hyponastic

300
 RELEVANCE AND CONTROL OF LEAF ANGLE
growth and describe how exploitation of natural (genetic) variation
can be instrumental in studying the relevance and control of leaf
positioning.
Keywords leaf orientation, differential growth, hyponastic growth,
petiole, Arabidopsis thaliana, Rumex palustris, submer-
gence, shade, low light, heat, temperature, ethylene,
auxin, natural variation
I. INTRODUCTION
Plant architecture, to a large extent, determines performance
in the plant’s natural habitat. Leaf positioning is an important
component of architecture with respect to the handling of en-
vironmental parameters by the plant. Leaf angle particularly is
a major target of adaptation to specific environmental condi-
tions. Plants may have rather fixed leaf angles that are not much
under environmental control. Alternatively, many plant species
can modify leaf angles in response to environmental changes or
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in a circadian fashion. A plant may either exploit an environ-
mental resource such as light or oxygen, or alternatively, avoid
environmental stresses such as high temperature and radiation
loads. In the first part of this review we discuss the functional
consequences of leaf angles for plant performance, with the fo-
cus on how leaf angle affects carbon gain and how (variation in)
leaf angle is functional to escape stressful situations.
We then focus on a special type of leaf movement, hyponastic
growth in rosette plants, to discuss how several recent advances,
mainly obtained using Rumex sp. and Arabidopsis thaliana, have
improved our understanding of the molecular and hormonal reg-
ulation of (hyponastic) leaf movement and its functional signifi-
cance. We have attempted, in a systems biology-like approach, to
integrate all the organization levels on which hyponastic growth
is studied (from ecology to molecular genetics). Finally, we
will argue that the model species Arabidopsis thaliana can fur-
ther contribute to our understanding, not only on the molecular
genetic side, but also in a more functional ecological context.
II. FUNCTIONAL SIGNIFICANCE OF LEAF ANGLE
A. Maximizing Carbon Gain
Flat leaves are one of the most striking features of green plants
that serve to capture light for photosynthesis. Evidently, leaf an-
gles relative to the direction of incident radiation determine how
much light is intercepted. This not only has consequences for
photosynthetic performance of leaves exposed to full daylight
at the top of a canopy, but also determines how much light is
available for lower leaves. Thus, leaf angles are, among other
factors, important for the distribution of light in a canopy (Monsi
and Saeki, 1953, 2005; Goudriaan, 1988; Hirose, 2005). Verti-
cal orientation of leaves reduces the extinction coefficient of a
canopy resulting in a more homogeneous distribution of light
over the leaves compared to a horizontal leaf orientation. This
increases carbon gain at the whole canopy level, because leaves
301
low in the canopy can contribute more, while the photosynthesis
rate of upper leaves is not significantly reduced since the photo-
synthesis — light response is nonlinear and saturates below the
intensity of direct sunlight (Monsi et al., 1973). A good exam-
ple of this principle comes from agricultural practice where the
introduction of new rice (Oryza sativa) cultivars with more ver-
tical leaf orientation contributed (in addition to other traits) to
increased rice productivity (Nobel et al., 1993). However, nat-
ural dense canopies are not always characterized by a vertical
leaf orientation.
Predominantly horizontal leaves intercept most light in the
upper layers of a canopy, resulting in heavy shading of lower
leaves. Models predict a lower than optimal photosynthesis in
these canopies as a result of this orientation (Monsi et al., 1973;
Hikosaka and Hirose, 1997). This deviation from an optimal
leaf orientation can be understood from the fact that individuals
with horizontal leaves in canopies with predominantly vertical
leaves outcompete their neighbors as a result of shading (Anten,
2005). Hence, natural selection for improved performance at the
level of the individual has resulted in reduced carbon gain at the
community level, at least in productive environments. This may
have important implications for crop plant breeding, since the
most competitive individuals in a dense stand are not necessarily
the most productive ones in an agricultural context.
Prostrate leaf angles are also found in short-living and fast-
growing tropical pioneer trees such as the genera Cecropia and
Macaranga (Ishida et al., 1999; Kitajima et al., 2005). These
trees shed their leaves when overtopped by newly emerging
ones, thus creating a single layer of leaves at the top of the
canopy with a low leaf area per unit ground area (leaf area in-
dex) (Kitajima et al., 2005). This allows for fast height growth,
but the low leaf area index is less effective when it comes to sup-
pressing competitors. Late successional trees in tropical forests
show more diverse leaf arrangements and much higher leaf area
indices of up to 8 (Kitajima et al., 2005). This results in a more
homogeneous distribution of light over the outer canopy leaves
and virtually total light extinction in the understorey, contribut-
ing to maintenance of their dominance.
Increased light interception at low sun angles could be an-
other potential benefit of steep leaf angles for carbon gain. This
was postulated by King (1997) for emergent and solitary ev-
ergreen Eucalyptus trees with drooping leaves. However, when
analyzed with a 3D crown structure model (Pearcy and Yang,
1996), advantages in terms of carbon gain at low sun angles in
winter proved to be small due to the random variation in az-
imuths (Falster and Westoby, 2003). A possible advantage of
vertical leaf orientation in woody plants, which is frequent in
the genus Eucalyptus, is thus likely to be associated with other
factors.
A horizontal leaf orientation is most effective for the in-
terception of light in the shaded understory of dense forests
(Ackerly and Bazzaz, 1995). Increased absorption results in
proportionally increased photosynthesis at the leaf level in
this layer. In support, these shade environments are indeed
 302 M. VAN ZANTEN ET AL.
characterized by more horizontal leaves compared to sunlit
conditions, mostly as the result of a plastic response to the
shade condition (McMillen and McClendon, 1979; Pearcy et al.,
2005). However, as argued above, flat leaf angles cause self-
shading more easily, which is disadvantageous at the whole
plant level. Avoidance of self-shading requires additional in-
vestment of carbon in longer branches and petioles (Falster and
Westoby, 2003; Pearcy et al., 2005), which reduces carbon gain
at the whole plant level. Plant habit in light-limited environments
is thus the result of a trade-off between maximizing light inter-
ception by a horizontal orientation of leaves that are spread out
to avoid self-shading and minimising investment in mechanical
support.
B. Stress Avoidance
A vertical orientation of leaves can be either erect as in
grasses and sedges or drooping as in some trees and shrubs.
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It reduces the radiation load at midday. This can be important to
avoid various forms of stress. The large interception of radiation
by horizontal leaves can result in a substantial rise of leaf tem-
perature above air temperature (Lambers et al., 2008). In hot and
dry conditions, leaf temperatures can easily rise to levels that
are above optimal for photosynthesis. High leaf temperatures
furthermore increase the vapour pressure difference with the at-
mosphere, which increases the transpiration rate and/or reduces
stomatal conductance (Schulze and Hall, 1982). The latter will
further increase leaf temperature. The decease in photosynthesis
at supra-optimal temperatures and the increase in transpiration
cause a decrease in water use efficiency.
It has also been suggested that horizontal leaves exposed to
a clear sky loose more heat at night in the form of infrared
radiation. A vertical orientation helps maintain a more favor-
able energy balance in these conditions, thus reducing the nega-
tive impact of low leaf temperatures on photosynthesis in early
morning in the cold season (Leuning and Cremer, 1988; King,
1997).
The reduced interception of light by vertical leaves reduces
overexcitation of the photosynthetic apparatus at high irradi-
ance. When temperatures are optimal for photosynthesis and
when the light-saturated rate of photosynthesis and stomatal
conductance are high, mechanisms that dissipate excess excita-
tion energy are generally sufficient to avoid most photoinhibi-
tion. Only peak irradiances cause moderate downregulation of
photosynthetic efficiency (Ögren and Rosenquist, 1992). How-
ever, at supra-optimal temperatures, substantial photoinhibition
can often not be avoided at high radiation loads (Mulkey and
Pearcy, 1992). The shrub Vitis californica for instance has hori-
zontal leaves in spring when radiation is high and temperatures
and water availability are in a favorable range. However, leaves
become drooping in the hot and dry Californian summer, thus
reducing interception of radiation at midday. Leaves that were
forced in a horizontal orientation suffered severe photoinhibi-
tion (Gammon and Pearcy, 1989), illustrating the functional
importance of the leaf orientation for the avoidance of excessive
radiation load under stress conditions.
The importance of reduced interception of radiation under
stress conditions is also suggested by the high frequency of Eu-
calyptus species with vertical leaves in areas characterized by
low precipitation and low nutrient availability, whereas species
with more horizontal leaves are predominantly found in high
rainfall and nutrient-rich areas (King, 1997). Low nutrient avail-
ability reduces photosynthetic capacity and thus reduces the irra-
diance at which photosynthesis is saturated. A modelling effort
showed that leaves with a low photosynthetic capacity subject
to water stress performed best in terms of photosynthetic rate
and water use efficiency at steep leaf angles (King, 1997).
However, not all plants that are subjected to drought and
high temperature stress exhibit vertical leaves as illustrated by
the Mediterranean evergreen species Quercus coccifera and Ar-
butus unedo (Werner et al., 1999). These species can perfectly
deal with the excessive radiation load in the hot Mediterranean
summer as illustrated by the overnight reversal of photoinhibi-
tion, irrespective of leaf angle. This contrasts with two Cistus
species that suffered severe photoinhibition when leaves were
forced in a horizontal position in summer. Some deciduous tree
species, e.g., Castanea sativa, have a combination of steep leaf
angles in the sun-exposed top of the canopy and a horizontal ori-
entation of the lower shaded leaves (Ford and Newbould, 1971)
in response to the local light climate (McMillen and McClendon,
1979). This suggests that a more homogeneous distribution of
light in the canopy is achieved, with stress avoided in the upper
leaves and light interception of the lower leaves maximized.
Considering the examples mentioned above, along with oth-
ers showing severe photoinhibition in horizontally forced leaves
(Abreu and Munne-Bosch, 2008) and studies in species with
fixed leaf orientation, two strategies of dealing with high radi-
ation under stress conditions emerge. Species tolerant to high
irradiation do not adjust their leaf angles; they have photopro-
tective mechanisms to accommodate conditions of intense irra-
diation. Other species have a limited capacity of dealing with
high radiation under stress; they rely on the avoidance of high
radiation loads by adjusting their leaf angles. Such active ad-
justment of leaf angles appears not to be limited to avoidance
of high irradiation alone, but is a central response to several
other potentially detrimental environmental conditions, includ-
ing complete submergence and shading. In the next sections, this
will be discussed thoroughly. The focus will be on a distinct type
of active leaf repositioning; differential growth-driven, upward
(re)orientation of (rosette) leaves called hyponastic growth.
III. ACTIVE LEAF REPOSITIONING BY HYPONASTIC
GROWTH
A. Generalities of Leaf Movements
In his book, The Power of Movements in Plants (1880),
Charles Darwin noted that: ‘Apparently every growing part
of every plant is continuously circumnutating.’ Since then,
 RELEVANCE AND CONTROL OF LEAF ANGLE 303

several types of plant-organ movements have been described.

Plant movements are classified in two general categories; nastic
and tropic movements. Tropic movements position organs di-
rectionally to a stimulus. Examples are phototropism and grav-
itropism in which plants bend organs relative to the light or
gravitational vector, respectively (Firn and Digby, 1980; Morita
and Tasaka, 2004; Lino, 2006).
The direction of a nastic movement is independent of the
stimulus’ direction and can be classified in two groups; i) those
that are induced by an environmental stimulus and ii) move-
ments occurring without a direct external stimulus. Movements
without a direct environmental stimulus are often under control
of the circadian clock, e.g., rhythmic diurnal leaf movements
of Arabidopsis thaliana (Millar et al., 1995) and the nycti-
nastic sleep-movements in Leguminosae species (Coté, 1995;
Kawaguchi, 2003). Examples of nastic movements induced by
an external stimulus are the collapse of Mimosa pudica com-
pound leaves when touched (Coté, 1995) and the upward bend-
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ing of rosette leaves called hyponasty (Kang, 1979).

A typical hyponastic growth response is induced in sev-
eral semi-aquatic species upon complete submergence. These
include; Ranunculus repens, Rumex palustris, Caltha palus-
tris, Leontodon taraxacoides, Paspalum dilatatum and Rorripa
sylvestris (Ridge, 1987; Voesenek and Blom, 1989; Grimoldi
et al., 1999; Insausti et al., 2001; Stift et al., 2008), and also
in the model species Arabidopsis thaliana (Peeters et al., 2002;
Millenaar et al., 2005). Beside submergence, other abiotic fac-
tors such as shade and heat treatment can also induce a marked
hyponastic growth response in Arabidopsis (Fig. 1; Millenaar
et al., 2005; Hopkins et al., 2008; Koini et al., 2009; Van Zanten
et al., 2009a). Moreover, leaf movement has been associated
with low temperatures (Nilsen, 1991; Hopkins et al., 2008).
Hyponastic movements may result from turgor-driven
swelling and shrinkage of specialized strategically-localized
pulvinal motor cells, as for instance in Phaseolus vulgaris
and Lotus japonicus (Fu and Ehleringer, 1989; Yu and Berg,
1994; Coté, 1995; Kawaguchi, 2003). Alternatively, hyponastic
growth can be initiated by differential, directional cell elon-
gation or differential cell proliferation. Upon submergence of
R. palustris, specific elongation of 30 basally-located epider-
mal cells in a confined region at the abaxial side was observed
(Cox et al., 2003). Because epidermal cell layers are assumed to
control cell expansion, but not cell proliferation, of underlying FIG. 1. Typical hyponastic growth phenotype in Arabidopsis thaliana A:
cell layers and thus may control differential growth (Savaldi- Superimposed Arabidopsis thaliana accession Columbia-0 (Col-0) plant before
Goldstein 2007, 2008), it was concluded that epidermal elon- and after 6 h of low light treatment. Note that all leaves re-orient to a more vertical
gation should be sufficient to drive the hyponastic response in position. B, C: Quantitative measurement of leaf angle kinetics obtained using
a time-lapse digital camera setup. B: absolute angles and C: pairwise subtracted
R. palustris. In agreement, a mathematical model based on this
petiole angles (Benschop et al., 2007), corrected for diurnal petiole movement
specific cell elongation fully explained the observed hyponastic in control conditions, upon ethylene (5 µl l−1 ; circles), low light (spectral
growth phenotype to flooding (Cox et al., 2003). neutral reduction of 200 µmol m−2 s−1 to 20 µmol m−2 s− 1; squares), heat
(sudden increase from 20◦ C to 38◦ C; triangles) and control (air; 200 µmol m−2
s−1 light; diamonds). Plants kept in control conditions only show modest diurnal
B. Submergence-Induced Hyponastic Growth leaf movement and leaf angles gradually decline over time due to maturation
of the leaves. Error bars represent SE; n > 12. Growth conditions, treatments,
The present knowledge on the ecological relevance and the data acquirement and analysis were as described by Millenaar et al. (2005) and
physiological regulation of submergence-induced hyponastic Van Zanten et al. (2009a).
 304 M. VAN ZANTEN ET AL.
movements was mainly obtained using Rumex species sub-
jected to complete submergence (reviewed in Voesenek et al.,
2006). Exchange rates of O2 and CO2 between plant and envi-
ronment, necessary for efficient respiration and photosynthesis,
are severely reduced under submerged conditions as a result of
the ∼10.000 fold slower gas diffusion in water compared to
air (Armstrong, 1979; Jackson 1985). Some flooding-tolerant
plants show hyponastic growth and subsequent petiole elonga-
tion until the leaf-tip reestablishes aerial contact when reaching
the water surface. Thereby, gas exchange between the plant and
the aerial environment is reestablished. This escape strategy is
often accompanied by other acclimation processes including;
aerenchyma formation, thinner cuticle/cell walls and thinner
leaves that further facilitate increased gas-diffusion under water
(Mommer et al., 2004; Voesenek et al., 2006; Jackson, 2008).
The reestablished aerial contact allows the much more effi-
cient aerobic respiration and enhanced photosynthesis by the
larger supply of CO2 (Mommer et al., 2005). The same result
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is achieved by internode elongation in plants with erect stems
such as deep water rice (Kende et al., 1998). This low oxy-
gen escape syndrome (LOES) as it is called (Bailey-Serres and
Voesenek, 2008), is most frequently found in habitats with shal-
low floods of long duration as among others found in certain
zones in river floodplains (Voesenek et al., 2004). The costs, in-
creased growth under conditions of strongly decreased carbon
gain and a plant body that is weakened by elongation growth,
are not outweighed by the benefits under ephemeral or very
deep floods. A quiescent strategy of arrested growth and re-
duced metabolic rate is more adaptive under these conditions
(Bailey-Serres and Voesenek, 2008).
If the flood water is too deep, hyponastic growth is insuffi-
cient to lead to emergence. In Rumex palustris elongation growth
of the petiole is only initiated when a critical petiole angle of >50
degrees relative to horizontal is reached (Cox et al., 2003). By
this mechanism the plant ‘ensures’ not to spend carbohydrates
and energy on shoot elongation when leaves are too horizontal to
reach the surface. Hyponastic growth in R. palustris to flooding
is notably fast. The lag phase is only 1.5–3 h and the response is
completed after 7 h, depending on the initial angle (Cox et al.,
2003). A comparably fast hyponastic growth response to flood-
ing was observed in Arabidopsis thaliana. However, this species
apparently does not have the capacity of the subsequent petiole
elongation (Millenaar et al., 2005).
Gaseous compounds, such as the phytohormone ethylene,
accumulate in the plant due to continued production and
physical entrapment (Voesenek et al., 1993; Banga et al.,
1996; Jackson et al., 2008). Several papers reported that ethy-
lene accumulation is the key trigger of the various accli-
mation responses in flooding-tolerant species including hy-
ponastic growth and nondifferential shoot elongation in R.
palustris (Voesenek and Blom, 1989; Banga et al., 1997; Cox
et al., 2003). In R. palustris ethylene levels increase twenty-fold
(0.05 µl l−1 to 1 µl l−1 ) within the first hour of submergence,
eventually reaching 8 µl l−1 after 16 h (Banga et al., 1996).
Submergence-induced hyponasty was reduced, but not com-
pletely abolished, after treatment with the ethylene receptor an-
tagonist 1-methylcyclopropene (1-MCP), whereas 1-MCP abol-
ished hyponastic growth completely to treatment with ethylene
(Cox et al., 2004). This suggests that other signals too play a role
in submergence-induced hyponastic growth (Heydarian et al.,
2010). Submergence leads to a quick reduction of O2 levels in
petiole tissues of non-acclimated R. palustris plants in stagnant
waters in the dark (Rijnders et al., 2000, Mommer et al., 2004).
However, in clear water and in the light, O2 levels in acclimated
plants can be maintained close to ambient 21% due to photo-
synthesis, oxygen diffusion from the surrounding waters and
morphological acclimations, e.g., aerenchyma formation and a
thinner leaf and cuticle (Mommer et al., 2004, 2005; Voesenek
et al., 2006). Although normoxic conditions may be maintained
during flooding, hypoxia (reduction from 21% to 3% O2 ) sen-
sitizes petioles for ethylene and consequently stimulated elon-
gation growth. CO2 did not influence petiole elongation rates
(Voesenek et al., 1997; Rijnders et al., 2000). This suggests that
reduction of O2 levels potentially could play a role in hyponastic
growth induction in conditions where O2 levels are limiting.
Although high ethylene concentrations in general inhibit
growth in flooding intolerant species (but see Pierik et al., 2006),
treatment with high ethylene concentrations (5 µl l−1 ) induced
hyponastic growth in Arabidopsis thaliana to the same extent
as complete submergence (Millenaar et al., 2005). This submer-
gence response was reversible, could be prevented by pretreat-
ment with the 1-MCP and was absent in the ethylene insensitive
etr1-1 mutant. In fact, the only notable difference in response
between submergence and ethylene was a delayed induction in
submerged plants, which may be due to the time necessary for
ethylene to accumulate to a physiological relevant concentration
under flooded conditions. In concordance with R. palustris, low
oxygen levels enhance ethylene-induced hyponastic growth in
Arabidopsis (Van Zanten et al., unpublished data).
Recently, it was demonstrated that responses to flood-
ing in rice are controlled by members of the plant-
specific APETALA2/ETHYLENE RESPONSIVE FACTOR
(AP2/ERF) transcription factor family (Hattori et al., 2009;
Voesenek and Bailey-Serres, 2009). For example, the pres-
ence of the ethylene-inducible AP2/ERF Submergence1A-1
(Sub1A-1) gene strongly correlates with submergence tolerance
in several accessions (Xu et al., 2006). In a comparable ap-
proach, Hattori and colleagues (2009) showed that two ERF
genes; SNORKEL1 (SK1) and SK2, contribute to submergence-
induced elongation in deepwater rice. So far, 122 AP2/ERF
members have been annotated in Arabidopsis thaliana (Nakano
et al., 2006). Although the majority of ERFs have not been
functionally characterized, it is clear that this gene family is
involved in abiotic stress-control and that members mediate
responses to several phytohormones, including ethylene (see
Nakano et al., 2006 and references therein). An exploratory
screen of a near-complete library of ERF-overexpressing Ara-
bidopsis (Columbia-0) plants (Weiste et al., 2007) resulted in the
 RELEVANCE AND CONTROL OF LEAF ANGLE
identification of several lines with different initial petiole angles
or different hyponastic growth responses (either enhanced or re-
pressed), compared to wild type (Polko and Van Zanten, unpub-
lished data). This suggests that ERFs are involved in controlling
hyponastic growth in response to changes in the environment in
Arabidopsis.
C. Shade-Induced Hyponastic Growth
As argued above, changes in leaf orientation have major con-
sequences for the interception of radiation and carbon gain. It
is, therefore, no surprise that leaf movement is an intrinsic part
of the shade avoidance syndrome, a suite of traits induced to
compete for light at the onset of interference with neighboring
plants (Smith and Whitelam, 1997; Ballaré, 1999; Vandenbuss-
che et al., 2005; Franklin, 2008). Shade-avoiding species with
erect stems typically show elongated internodes. This places
the leaves in more favorable light conditions in short vegetation
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while keeping them horizontal. In rosette plants, this is replaced
by elongation of petioles, but leaf blades may still be kept in
a horizontal position as in Geum urbanum and Potentilla rep-
tans (Pons, 1977; Huber, 1996). However, many shade-avoiding
rosette species, generally those with short petioles, bend their
leaves upwards as a result of hyponastic growth. The consequent
vertical position of the leaf blade is not optimal for light inter-
ception. Nevertheless, the hyponastic response is important for
competition for light in crowded conditions as was shown using
ethylene insensitive transgenic Nicotiana tabacum plants, grown
in a plot mixed with wild types. The young plants were still in
the rosette stage and the ethylene insensitive plants showed a
delay of approximately one week in the hyponastic response to
crowding. Ultimately, this was fatal for their competitive abil-
ity against the wild types that suppressed the transgenic plants
completely (Pierik et al., 2003). Hence, although not optimal
for light interception, the hyponastic growth response proved to
be essential when competing with similar sized plants. Also in
mixed herbaceous canopies where other plants overtop rosette
plants, the disadvantage of a nonoptimal leaf angle is likely to
be outweighed by the higher irradiance as a result of the higher
position of elongated leaf blades in the steep irradiance gradient.
Typical signals perceived by plants growing in dense green
foliage (canopies) include reduced photosynthetically active ra-
diation (PAR), blue light photon fluence rates and red to far-red
photon ratios. The latter is the result of absorption of red and
reflection and transmission of far-red by surrounding leaves.
Generally, it is the combination of spectral and irradiance com-
ponents that are used by plants for their ultimate response to
shading by neighbours (Ballaré 1999). Different species may,
however, respond differently to the two components when ap-
plied separately. For instance Nicotiana tabacum and Arabidop-
sis thaliana show hyponastic responses to both components of
canopy shade (Pierik et al., 2004a, 2005; Millenaar et al., 2005,
2009). R. palustris, however, shows hyponastic growth upon ex-
posure to a reduced red:far-red ratio, but not to a reduced PAR
305
(Pierik et al. 2005). In Cirsium palustre it is the reverse, expo-
sure to spectrally neutral shade resulted in the full hyponastic
response and a reduced red:far-red ratio did not have an addi-
tional effect (Pons, 1977; Pons 1983).
The low red:far-red ratio caused by the proximity of green
plants is sensed via the phytochrome photoreceptors (Smith,
1982). Arabidopsis phytochrome-b (phyb) loss-of-function mu-
tants or mutants in genes involved in PhyB signaling, such
as constitutive shade-avoidance1 (csa1), are constitutively hy-
ponastic (Faigón-Soverna et al., 2006). In addition, PhyA and
PhyE are implicated in the control of light dependent leaf po-
sitioning (Vandenbussche et al., 2003; Mullen et al., 2006).
In accordance, plants lacking the phyb suppressor, SHADE
AVOIDANCE3 (sav3) had reduced leaf hyponasty under shade
conditions (Tao et al., 2008). Together, this illustrates the in-
volvement of Phytochromes and downstream signaling proteins
in the control of leaf angles.
Transferring Arabidopsis plants to darkness induced a quick
hyponastic growth response that could be reversed in a light-
intensity dependent manner by application of white light or red
light, but not by blue light (Hangarter, 1997). Apparently, blue
light plays an essential role in leaf positioning. In agreement,
newly appearing seedling leaves under red light have a horizon-
tal orientation whereas superimposition of low quantities of blue
light led to leaf inclination (Inoue et al., 2008). Strong reduc-
tion of the PAR (low light treatment) stimulated a hyponastic
growth response in Arabidopsis within a few hours (Millenaar
et al., 2005, 2009; Figure 1B, C) and in agar-grown seedlings
within 6 days (Vandenbussche et al., 2003). Light-spectrum
manipulations showed that rapid induction of low light-induced
hyponasty is dose dependent and confirmed that this requires re-
duction of blue-light wavelengths (Millenaar et al., 2009). These
wavelengths are perceived by Cryptochromes (Cry) and Pho-
totropin photoreceptors (Ballaré, 1999; Lin et al., 2002 and ref-
erences therein). Mutant analysis confirmed that Cry1 and Cry2,
but also PhyA and PhyB are the photoreceptor proteins involved
in detection of reduced light intensity, and all are positive regu-
lators of low light-induced hyponastic growth.
Recently, De Carbonnel and co-workers (2010) showed that
PHYTOCHROME KINASE SUBSTRATE2 (PKS2) and to
lesser extent, PKS1, play a role downstream of Phototropin
signalling in leaf positioning. These authors show that PKS2
acts synergistically to NON PHOTOTROPIC HYPOCOTYL3
in blue light–mediated leaf positioning. NPH3 is indeed required
for Phototropin mediated leaf positioning (Inoue et al., 2008).
Phototropins are thus important controllers of leaf angle, how-
ever, these blue-light photoreceptors are not per se required for
low light–induced hyponastic growth (Millenaar et al., 2009).
The above mentioned canopy-derived light signals that in-
duce shade avoidance responses can be used by other plant
species to repress this response. This is observed in, for
example, genotypes of Imaptiens capensis that grow un-
der closed canopies such as a forest understorey (Von Wet-
tberg and Schmitt, 2005). The adaptive implications of such
 306 M. VAN ZANTEN ET AL.
species-specific differences deserve further attention. The oc-
currence of a similar repressive response in the model species
maize (Zea mays) in which leaf hyponasty declined from verti-
cal in dark to more horizontal under white, red, and blue light
radiation, but less to far-red light (Fellner et al., 2003), may
be exploited to learn more on the mechanisms of how plants
translate canopy signals in a leaf movement response.
D. Heat-Induced Hyponastic Growth
Temperature is long known to control leaf positioning. For
example, heliotropic movement of common bean (Phaseolus
vulgaris) leaves is controlled by temperature such that its leaves
are placed close to the optimum for photosynthesis (Fu and
Ehleringer, 1989). In addition, a positive correlation between
air temperature and leaf angles has been observed in P. vulgaris
and P. acutifolius (Yu and Berg 1994). Moreover, diurnal leaf
movements are affected via temperature compensation of the
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circadian clock in Arabidopsis thaliana (Edwards et al., 2005).
Elevated temperature has also recently been added to the palette
of factors that can induce hyponastic growth in Arabidopsis
(Millenaar et al., 2005; Koini et al., 2009; Van Zanten et al.,
2009a; Figure 1B, C).
Hopkins and co-workers (2008) showed that Arabidopsis
thaliana accessions from southern latitudes have more erect
leaves than those from northern latitudes. As argued above,
steep leaf angles prevent excess irradiance and associated supra-
optimal heat flux and water-loss during mid day at low latitudes.
It has also been suggested that light capture is improved at low
sun angles, thus contributing to photosynthetic carbon gain, but
that is not likely to be very important (see above and Falster
and Westoby, 2003). The heat avoidance strategy is further sup-
ported by the observation that the degree to which accessions
induce hyponastic growth by a heat treatment is correlated with
the day-night (diurnal) temperature amplitude at the geographic
location of the natural habitat (Van Zanten et al., 2009a). This
suggests that the ability of temperature controlled leaf move-
ment may be subjected to natural selection to the local temper-
ature environment.
It has been shown in several papers that light and heat sig-
nalling integrate at the level of photoreceptors, or photoreceptor-
interacting proteins (Halliday et al., 2003; Halliday and
Whitelam, 2003; Penfield, 2008). Recently, Koini et al. (2009)
demonstrated that high temperature effects on plant architec-
ture, including hyponasty and petiole elongation require the
bHLH transcription regulator PHYTOCHROME INTERACT-
ING FACTOR 4 (PIF4). Moreover, several photoreceptor pro-
teins (i.e., PhyA, PhyB and Cryptochromes) are involved in
the control of heat-induced hyponastic growth and simultane-
ous application of heat and low light leads to an exaggerated
hyponastic growth response (Van Zanten et al., 2009a).
Heat results in damage of lipid membranes and photo-
inhibition caused by reactive oxygen species (Larkindale et al.,
2005 and references therein). This heat-induced oxidative dam-
age in Arabidopsis was prevented in darkness (Larkindale
and Knight, 2002). This indicates that the photosynthesis sys-
tem/electron transport chain may be a point of cross-talk
between heat and (low)light signalling. Inhibition of the elec-
tron transport chain using DCMU (3-(3,4-dichlorophenyl)-1,1-
dimethylurea), indeed induces a marked hyponastic growth
response under control (light and temperature) conditions
(Millenaar et al., 2009). This supports the close interaction be-
tween light and temperature signalling.
Application of ethylene reduced the strong hyponastic
growth phenotype that was observed when low light and heat
were applied simultaneously (Van Zanten et al., 2009a). It is
tempting to speculate that this is due to the protective role of
this hormone, which prevents and repairs photo-oxidative dam-
age (Larkindale and Knight, 2002; Larkindale et al., 2005).
In summary, heat and light signals and ethylene are tightly
connected in the control of leaf movement. This is reflected in
the observation that these signals induce a very comparable re-
sponse in Arabidopsis (Fig. 1) when applied singly. In the next
section, we will describe how phytohormones act in the integra-
tion and control of leaf movements induced by submergence,
shade, and heat.
IV. PHYTOHORMONAL REGULATION OF
HYPONASTIC GROWTH
A. Ethylene as a Major Mediator of Hyponastic Growth
Years of research firmly established ethylene as the primary
trigger of several submergence-induced physiological and mor-
phological acclimations in plants, including hyponastic growth
and petiole elongation (Kende et al., 1998; Voesenek et al.,
2003, 2006; Jackson et al., 2008; Bailey-Serres and Voesenek,
2008). As mentioned earlier, ethylene is also tightly associ-
ated with shade-induced hyponastic growth. Low quantities of
ethylene triggered hyponasty in tobacco (Nicotiana tabacum)
(Pierik et al., 2003). Ethylene insensitive tobacco plants exhib-
ited delayed hyponastic growth, stem and petiole elongation in
response to proximal neighbors and failed to induce hyponastic
growth under low blue light (Pierik et al., 2003, 2004a). A di-
rect role for ethylene in low red:far-red ratio-induced hyponastic
growth was absent and thus the delayed ethylene-mediated hy-
ponastic growth in canopies can be attributed to reduced blue
light intensity (Pierik et al., 2003, 2004a, b). However, low
red:far-red ratios lead to increased ethylene production in Ara-
bidopsis (Pierik et al., 2009) and in Sorghum (Sorghum bicolor)
(Finlayson et al., 1998). Vandenbussche et al. (2003) showed
that ethylene insensitive mutant Arabidopsis seedlings were not
able to induce hyponastic growth under low light intensities in
agar-grown Arabidopsis seedlings.
Given all this, it is perhaps somewhat surprising that low
light-induced hyponastic growth in vegetative Arabidopsis
plants occurs independently of ethylene action. This was con-
cluded from several ethylene insensitive mutants which showed
a full response to low light and this treatment did not lead to
 RELEVANCE AND CONTROL OF LEAF ANGLE
increased ethylene production. Moreover, transcription of ethy-
lene marker genes was not altered upon low light treatment
(Millenaar et al., 2009). The contrary is true for heat-induced
hyponastic growth where ethylene acts as a negative modulator
of the response (Van Zanten et al., 2009a). Heat also represses
ethylene release, perhaps to control hyponastic growth (Van
Zanten et al., 2009a). In conclusion, ethylene appears to be a
major player in the control of hyponastic growth induced by
different environmental factors.
B. An Ambiguous Role for Auxin in Controlling
Hyponastic Growth
Auxin and polar auxin transport (PAT) are important for
many differential growth responses (Harper et al., 2000; Friml
and Palme, 2002; Friml, 2003). Nevertheless, the role of auxin
and PAT in controlling leaf angle is at best ambiguous. For
example, hyponastic growth induced by decreased light intensi-
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ties in agar-grown Arabidopsis seedlings was less pronounced
in auxin-related (auxin resistant1-3 (axr1-3), axr2-1) mutants
(Vandenbussche et al., 2003). Auxin-insensitive massugu1/non-
phototropic4/auxin response factor7 (msg1/nph4/arf7) mutants
on the other hand, exhibited constitutive hyponasty (Watahiki
and Yamamoto, 1997; Harper et al., 2000; Liscum and Briggs
1995; Nakamoto et al., 2006). Wild-type seedlings grown in
the presence of the transport inhibitor naphthylphtalamic acid
(NPA) show hyponastic growth, but also the repressor of LRR-
extensin1 (rol1) mutants, having increased auxin concentrations
in cotyledons, induced hyponasty (Ringli et al., 2008). Down-
regulation of the potato (Solanum tuberosum) auxin-related
Aux/IAA protein StIAA2 resulted in petiole hyponasty and the
effect was most severe in the shoot-apex, where auxin is pri-
marily produced (Kloosterman et al., 2006). This suggests that
auxin suppresses high leaf angles in this species. In agreement,
removal of auxin-producing main shoots or leaflets of tomato
(Lycopersicon esculentum) plants induced hyponastic growth
in young leaves, likely due to auxin-mediated loss of ethylene
responses (Kazemi and Kefford, 1974). Last, vertical leaf orien-
tation was maintained in the presence of NPA in maize (Fellner
et al., 2003). The complexity of the response is emphasized
by the identification of the Arabidopsis constitutive hyponastic
growth mutant; hyponastic leaves1 (hyl1) in which treatment
with the auxin influx inhibitor 2,3,5-triiodobenzoic acid (TIBA)
reduced the hyponastic phenotype whereas NPA exaggerated
the hyponastic leaf angle (Lu and Fedoroff, 2000).
Auxin and PAT are not obligatory for ethylene-induced hy-
ponasty per se in Arabidopsis thaliana (Van Zanten et al.,
2009b). In fact, based on the small exaggerated response ampli-
tude after treatment with NPA and based on the results obtained
with auxin perception and transport mutants, one might even
conclude that auxin antagonizes ethylene-induced hyponasty
(Van Zanten et al., 2009b). These results are partly in agree-
ment with observations in R. palustris, in which pretreatment
with NPA delayed the induction of submergence-induced hy-
307
ponastic growth by doubling the lag-phase of the response, but
hardly affected the response amplitude (Cox et al., 2004).
As mentioned above, heat-induced hyponastic growth is
abolished in the phytochrome interacting factor 4 (pif4) mu-
tant. In contrast to the wild type, this mutant also failed to
induce expression of the auxin marker gene IAA29 (Koini et al.,
2009). Mutant and pharmacologic analysis confirmed that heat-
induced hyponastic growth requires intact auxin perception and
PAT (Van Zanten et al., 2009a) as does low light–induced hy-
ponasty (Millenaar et al., 2009). Details on involvement of auxin
signalling and PAT differed between these stimuli. Auxin and
PAT are required for rapid induction and maximum amplitude
of heat-induced hyponastic growth and for maximum ampli-
tude and maintenance of elevated petiole angles during low
light–induced hyponasty (Millenaar et al., 2009; Van Zanten
et al., 2009a). These observations fit other reports. For exam-
ple, auxin is required for differential and nondifferential (elon-
gation) growth of petioles and hypocotyls under altered light
intensity and light quality environments (Morelli and Ruberti,
2000; Tao et al., 2008; Pierik et al., 2009). Interestingly, it was
recently suggested that PKS2 (and synergistically NPH3) may
act as a signalling hub between Phytochrome- and Phototropin-
dependent leaf positioning and auxin signalling and/or home-
ostasis (De Carbonnel et al., 2010). Furthermore, auxin is re-
quired for high temperature–induced hypocotyl elongation and
heat induces transcription of the auxin-responsive genes IAA4,
which suggests sensitization of the tissues to auxin (Gray et al.,
1998). Together, the data summarized here indicate that auxin
is often important for differential growth, but is not always a
prerequisite for these responses.
C. Antagonizing Roles for Gibberellins and Abscisic
Acid in Hyponastic Growth
Abscisic acid (ABA) is a negative regulator of submergence-
induced petiole elongation in R. palustris (Benschop et al.,
2005). Accordingly, ABA levels rapidly declined in an ethylene-
dependent manner in submerged R. palustris petioles, via en-
hanced ABA catabolism and inhibition of ABA biosynthe-
sis (Benschop et al., 2005). The negative role for ABA in
submergence-induced hyponastic growth in this species was
demonstrated by a pharmacologic approach. ABA application
delayed the induction of hyponasty and the maximal angle of
the response was lower than in control plants (Cox et al., 2004).
In accordance, repression of endogenous ABA levels using the
ABA biosynthesis inhibitor fluoridon, enhanced ethylene and
submergence-induced hyponastic growth.
Submergence-induced growth responses in R. palustris are
likely dependent on ABA-mediated increased gibberellic acid
(GA) production as levels of bioactive GA did not increase in
submerged plants treated with ABA. In addition, petioles of
submerged R. palustris plants showed increased GA sensitivity
with respect to elongation growth (Rijnders et al., 1997; Cox
et al., 2004). Inhibition of GA biosynthesis using paclobutrazol
 308 M. VAN ZANTEN ET AL.

affected the speed of the hyponastic growth response, but not

the lag-phase and maximum angle (Cox et al., 2004). The time
frame of GA accumulation was incompatible with the induc-
tion of hyponastic growth, which led to the conclusion that the
response was saturated by endogenous GA levels.
Ethylene-induced (but not low red:far-red-induced) hy-
ponasty in tobacco also required GA (Pierik et al., 2004b).
The hormonal interactions controlling responses to submer-
gence/ethylene are thus remarkable similar in unrelated species.
Genetic and pharmacological evidence indicated that ABA also
antagonizes ethylene-induced hyponastic growth in Arabidop-
sis, despite that ABA levels remained unaltered after ethylene
treatment (Benschop et al., 2007). Additionally, ABA also con-
trols leaf positioning independent of ethylene or other hyponas-
tic growth-inducing stimuli (Mullen et al., 2006; Benschop
et al., 2007).
In contrast to ethylene-induced hyponasty, ABA was identi-
fied as a positive regulator of heat-induced hyponastic growth
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in Arabidopsis (Van Zanten et al., 2009a) and it appears to en-

hance low light–induced hyponastic growth (Van Zanten et al.,
unpublished results). At least in low light–induced hyponastic
growth, the response to ABA seems independent of ethylene
signalling. It remains to be studied if, and how, ABA controls
hyponastic growth via regulation of GA signalling, accumula-
tion or sensitivity in Arabidopsis.

V. THE USE OF ARABIDOPSIS THALIANA AND

INTRASPECIFIC NATURAL VARIATION THEREIN
A. Natural Variation in Hyponastic Growth
Arabidopsis thaliana is native to Europe and Central Asia and
also occurs in some parts of Africa (N 0◦ and 68 ◦ ). Moreover, it
has been introduced in other parts of the Northern hemisphere,
including North America, Japan, and Korea (Hoffmann 2002;
Koornneef et al., 2004). This wide biogeographical distribu-
tion and the variety of environmental conditions to which it has
adapted are reflected in extensive genetic variation among acces-
sions (Hoffmann 2002; Koornneef et al., 2004; Nordborg et al.,
2005). Intraspecific variation in leaf angles has been shown be-
fore (King, 1997; McMillen and McClendon, 1979; Falster and
Westoby, 2003; Abreu and Munné-Bosch, 2008). For Arabidop-
sis thaliana accessions, variation in constitutive petiole angles
has been described (Hopkins et al., 2008), in ethylene- and heat-
induced hyponastic growth (Millenaar et al., 2005; Van Zanten
et al., 2009a) and in temperature-controlled circadian leaf move-
ment (Edwards et al., 2005). This is illustrated in Figure 2 where
the constitutive leaf angle, and in Figure 3 where the leaf move-
ment response to 6 h ethylene, low light and heat treatment
are shown for 138 naturally occurring Arabidopsis thaliana ac-
cessions. Remarkably, ethylene treatment not only resulted in
hyponastic growth (up to 39.7 ± 6.4 deg. accession Niigata; FIG. 2. Natural variation in constitutive petiole angles of Arabidopsis
thaliana. Values represent the absolute initial (constitutive) angles (degrees)
NG), but also induced epinastic growth in several (13%) of the relative to the horizontal, at t = 0 h of Arabidopsis thaliana accessions in con-
accessions (lowest: −10.3 ± 2.3 deg. accession Palermo-1; Pa- trol conditions (air; 200 µmol m−2 s−1 light). Error bars represent SE; n = 2
1). Epinastic growth has been described before, for example in to 15.
 RELEVANCE AND CONTROL OF LEAF ANGLE 309
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FIG. 3. Natural variation in induced hyponastic growth of Arabidopsis thaliana. Effect of exposure to A: ethylene (5 µL L−1 ), B: low light (200 µmol m−2
◦ ◦
s−1 to 20 µmol m−2 s−1 ) and C: heat (20 C to 38 C) on petiole angles of 138 Arabidopsis thaliana accessions. Values are pair wise subtracted. Error bars represent
SE; n = 2 to 15. Note that the values of panel A are sorted by response magnitude.
 310 M. VAN ZANTEN ET AL.
TABLE 1
Bivariate relationships between Arabidopsis thaliana petiole
angle parameters in different hyponastic growth inducing
environments.
Initial angle E LL HT
Initial angle P −0.054 0.059 −0.086
Sig. ns ns ns
E P 0.691 0.444
Sig. ∗∗ ∗∗
LL P 0.597
Sig. ∗∗
Correlations coefficients are Pearson (P ) 2-tailed, of angle param-
eters measured in 138 Arabidopsis thaliana accessions Figures 2, 3.
Significant (sig.) correlations are in bold, ∗∗ p <0.01 (value between
brackets), ns = non significant. The initial angle represents the petiole
angle before start of the treatments. Treatments were 6 hours of: 1.5
◦ in a comprehensive insight into how phytohormones regulate
µl l−1 ethylene (E), low light intensity (20 µmol m−2 s−1 ; LL) or 38 C
Downloaded By: [University of Utrecht] At: 09:44 4 October 2010
heat treatment (HT). Angles used for the analysis were pairwise sub-
tracted (Benschop et al., 2007) representing the response magnitude,
corrected for diurnal leaf movements.
tomato (Lycospesicon esculentum) and Sunflower (Helianthus
anuus) upon waterlogging (Bradford and Dilley, 1978; Kawase,
1974).
Intraspecific correlation analysis (Table 1) using the variation
in hyponastic growth among accessions (Figs. 2, 3) confirmed
that the hyponastic growth response induced by different envi-
ronmental stimuli is comparable in Arabidopsis (Fig. 1B, C).
No indication was found that initial petiole angles (constitutive
petiole angle; t = 0 h, before treatment) restricts hyponastic
movement (Table 1). In other words, leaf movements are inde-
pendent of constitutive leaf angles in Arabidopsis.
Given the relative small size of adult Arabidopsis thaliana
rosettes, it can be considered a weak competitor. This is con-
firmed by the preferred habitat of the species. In the Nether-
lands, Arabidopsis thaliana is found mainly on sandy soils and
on pioneer or open, disturbed environments (Fig. 4A, B). Also
in the Mediterranean area, considered the region of origin of
the species (Weeda et al., 1987, Hoffmann, 2002), Arabidopsis
thaliana is mainly found in anthropogenic habitats, like agri-
cultural fields (Pignatti, 1982) and in natural disturbed habitats
(Picó et al., 2008).
The (winter) annual species prevents competition for light
with other species by completing its life cycle in early spring,
before most other plants reach their highest amount of above
ground biomass. Therefore, the ability of hyponastic growth in
vegetative Arabidopsis thaliana rosettes can likely not be ex-
plained as a mechanism to enlarge competition for light. How-
ever, as explained above and in Pierik et al. (2003), hyponastic
growth is essential for competition with similarly sized plants,
despite the putative negative consequences for light-interception
and photosynthesis. Arabidopsis typically shed many seeds and
is likely to compete with its own siblings and therefore may
occur in a micro-habitat of similarly sized individuals. Leaf
movement with the aim of avoiding shading can therefore not
be excluded early in the life cycle.
As Arabidopsis thaliana is rarely found on river banks, hy-
ponastic growth can not be explained as an adaptation to flood-
ing. Considering the open, warm habitats the species prefers, es-
pecially in the Mediterranean, hyponastic growth in Arabidopsis
thaliana can probably best be explained as a mechanism to avoid
high temperatures.
B. Molecular Genetics of Hyponastic Growth;
Exploitation of Natural Genetic Variation
Leaf positioning has now been studied in several species,
mainly using ecophysiological approaches. As exemplified by
the numerous examples presented in this work, this has resulted
leaf (re)positioning. Yet, the (molecular) mechanisms underly-
ing this remain poorly understood.
In a mutant screen for morphological mutants, four indi-
viduals with dominant expression of hyponastic leaves were
isolated: isoginchaku1-D to 4-D (iso1-D to iso4-D; Nakazawa
et al., 2003). Iso1-D and Iso2-D phenotypes are caused by the ec-
topic expression of ASSYMETRIC LEAVES2 (AS2). Iso3-D and
Iso4-D ectopically expresses the AS2 homologous gene; LOB
DOMAIN-CONTAINING PROTEIN 36 (LBD36). In agreement,
plants overexpressing BLADE-ON-PETIOLE1 (BOP1) which
enhances AS2 and LBD36 expression also exhibit hyponastic
leaves (Ha et al., 2007). It may not be surprising that these
genes, implicated in the establishment of adaxial-abaxial po-
larity axes (Xu et al., 2003) have constitutive leaf orientation
phenotypes. However, if and how these genes are employed in
environmentally-induced differential growth responses, includ-
ing hyponastic growth, remains to be elucidated.
The considerable natural variation in initial (constitutive)
petiole- and leaf angles and in hyponastic growth response to
changes in the abiotic environment has been recently exploited
in a Quantitative Trait Loci (QTL) approach, in a first attempt
to isolate molecular components involved in hyponastic growth
(Van Zanten et al., 2010a). Several QTLs for initial petiole
angles and ethylene-induced hyponastic growth were mapped.
One of these, a major QTL on chromosome 2, has been linked
to variation in growth-related traits and hormone action sev-
eral times before (Van Zanten et al., 2009c). Subsequent anal-
ysis confirmed that the segregating gene underlying the QTL,
the Leucine-Rich Repeat Receptor-Like Ser/Thr kinase gene
ERECTA (ER) (Torii et al., 1996), is a controller of ethylene-
induced hyponastic growth in several accessions. ER controls
hyponasty presumably downstream of ethylene action since the
erecta mutation does not consistently influence ethylene produc-
tion or sensitivity. The observation that ERECTA also controls
low light intensity–induced differential petiole growth in some,
but not all, tested erecta mutant accessions and because it does
 RELEVANCE AND CONTROL OF LEAF ANGLE 311
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FIG. 4. Ecological position of Arabidopsis thaliana in the Netherlands. A: Plant communities with highest fidelity value of the species (a fidelity of 50% means
that 50% of vegetation stands in which the species is present belong to this community); plant communities were classified according to Schaminée et al. (1995-
1999); 30BA = Aperion spicae-venti, a weed community of cornfields on sandy or loamy soils, 14CA = Tortulo-Koelerion, an open grassland type of coastal sand
dunes, 31AA = Salsolion ruthenicae, a ruderal pioneer community of sandy or stony soils, 31CA = Dauco-Melilotion, a widespread ruderal community of sandy,
loamy, or slightly clayey soils, and 14BC = Sedo-Cerastion, an open grassland type on sandy levees along rivers. B: Main vegetation structures with fidelity of the
species, as indicated by plant communities. Fidelity is calculated from vegetation descriptions (relevés) in the Dutch National Vegetation Databank, as analyzed
in the expert system SynBioSys (Schaminée & Hennekens, 2003). Arabidopsis thaliana is present in 2,687 (0.45%) from the 600,266 relevés at present available
(period 1929-2010). The fidelity spectrum of A. thaliana indicates a preference for open pioneer vegetations on slightly acid, mesotrophic, mostly sandy soils.

so independent of PhyB and Cry2 action (Van Zanten et al.,
2010b), underlines the complexity of the signalling network by
which environmental factors induce hyponastic growth.
VI. CONCLUSIONS AND PERSPECTIVES
Recently, Arabidopsis thaliana has been employed to in-
vestigate molecular genetic and physiological aspects of hy-
ponastic growth. It became thereby apparent that this species
is a very good model to study the complex network underlying
leaf movement. Different signal transduction pathways operate
in response to different environmental cues with a remarkably
similar hyponastic growth response as outcome. Based on the
data summarized in this paper, we propose a model constructed
on the principle of parsimony, of how hyponastic growth is reg-
ulated by phytohormones in Arabidopsis (Fig. 5). However, as
stated earlier, the functional molecular mechanisms driving leaf
(re)positioning need more attention. Several mutants that fail to
induce hyponasty or have exaggerated responses have recently
been isolated in our laboratory from a mutant screen to meet
this aim.
Especially, the exploitation of natural genetic variation is ex-
pected to result in rapid advances in the near future. A prelimi-
nary study which included among others Palermo-1 (Pa-1), the
accession that showed the strongest ethylene-induced epinastic
growth (Fig. 3), indicated that ethylene also strongly inhibited
hypocotyl elongation in the light in this accession (Keuskamp
et al., unpublished data), whereas these conditions generally
promote hypocotyl elongation (Smalle et al., 1997). This sug-
gests that at least part of the molecular machinery regulating
ethylene hypocotyl elongation in the light may be similar to
components controlling ethylene-regulated hyponastic growth.
Therefore, aspects of leaf movement responses may be indi-
rectly studied using “simple” hypocotyl assays. Development
of fast high throughput methods such as hypocotyl assays pro-
vides an excellent platform for (suppressor) mutant screens or
for extensive Genome Wide Association mapping (GWA) ap-
proaches which in particular requires large sets of accessions
to be screened (Nordborg et al., 2005; Nordborg and Weigel,
2008; Atwell et al., 2010).
The expected progress using Arabidopsis will provide a good
basis for understanding the mechanisms of leaf positioning and
movement in other species. The rapid advances in genome-
wide deep sequencing techniques and the associated steep de-
creases in the costs involved, will on short term result in the
availability of genome sequences of several plant species. The
results obtained using Arabidopsis thaliana is then relatively
easy translatable to more ecological and economically relevant
species. Ultimately, this may result in marker assisted breed-
ing approaches, aiming to improve for example crop yield by
controlling leaf angle.
 312 M. VAN ZANTEN ET AL.
FIG. 5. Proposed simplified signalling parsimony network of ethylene,
heat and low light-induced hyponastic petiole growth. These signals are
modulated by at least ABA and auxin. Dotted lines between heat and low light
Downloaded By: [University of Utrecht] At: 09:44 4 October 2010
signalling indicate that we cannot distinguish between the routes. The dotted
lines at the bottom indicate that we cannot distinguish between continuous par-
allel pathways of ethylene, heat and low light route towards hyponastic growth
or a converged downstream pathway in which the same functional genetic com-
ponents are utilized by the different signals. The bold inhibition line indicates
a dominant ethylene effect over the heat route (Van Zanten et al., 2009a). Note
that order of the signals along the arrows does not necessarily reflect the order
of interactions occurring.
Despite the promising genomics developments, a more com-
prehensive understanding of the physiological and ecological
functionality of constitutive leaf angle and the control of leaf
movement is required to be able to fully understand how plant
distribution and yield is influenced by leaf orientation. It has al-
ready been shown that in flood-prone areas, for example, abun-
dance and distribution is partly determined by the capacity to
induce hyponasty and shoot elongation (Voesenek et al., 2004).
Future research should therefore particularly focus on the func-
tional aspects of leaf orientation in the natural context. Ara-
bidopsis might be a key for such an approach when the fitness
consequences of leaf movements in different environments can
be quantified. The large variation in the Arabidopsis thaliana
gene pool can then be used to link a functional approach to the
molecular genetic mechanism of leaf movements.
Statements about the function for plant performance of plant
traits in general and leaf orientation in particular are easily
made. However, as shown above, a critical analysis is required
before conclusions about functionality can be drawn. Different
approaches can be employed. Plant architecture can be manip-
ulated in a model and consequences of an altered phenoptype
for plant performance in a particular environment can be calcu-
lated. Examples for altered leaf orientation have been described
above (e.g., Falster and Westoby 2003; Pearcy et al., 2005).
This approach relies heavily on the validity of the assumptions
made. Alternatively an experimental approach is adopted where
plant architecture is manipulated. Simple experimental alter-
ation of leaf orientation for instance, which manipulates stress
at the leaf level, may suffice with solitary plants (e.g., Gamon
and Pearcy, 1989; Abreu and Munne-Bosch, 2008). However,
for more complicated manipulation of phenotypes, particularly
in competitive situations, other approaches are required. Al-
tered phenotypes can be environmentally induced or achieved
by means of mutants or transgenics (e.g., Schmitt et al., 1999;
Ackerly et al., 2000; Pierik et al., 2003). This approach has the
potential drawback of possible pleiotropic effects (Pigliucci and
Schmitt 1999). A modelling and experimental approach can be
best combined, which employs the strengths of both and com-
pensates for their weaknesses. Functionality of leaf angles and
plasticity therein under different conditions can thus best be
investigated using such a combined approach.
ACKNOWLEDGMENTS
The authors thank Bob Derksen for the superimposition
photo of hyponastic growth in Arabidopsis thaliana and Maaike
E. Smeets for help preparing the manuscript.
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