Phytochrome A increases tolerance to high evaporative demand

Abstract

Stresses resulting from high transpiration demand induce adjustments in plants that lead to reductions of water loss. These adjustments, including changes in water absorption, transport and/or loss by transpiration, are crucial to normal plant development. Tomato wild type (WT) and phytochrome A mutant plants, fri1-1, were exposed to either low or high transpiration demand conditions and several morphological and physiological changes were measured during stressing conditions. Mutant plants rapidly wilted compared to WT plants after exposure to high evaporative demand. Root size and hydraulic conductivity did not show significant differences between genotypes, suggesting that water absorption and transport through this organ could not explain the observed phenotype. Moreover, stomata density was similar between genotypes, whereas transpiration and stomata conductance were both lower in mutant than in wild type plants. This was accompanied by a lower stem specific hydraulic conductivity in mutant plants than in wild type, which was associated to smaller xylem vessel number and transversal area in fri1-1 plants, producing a reduction in water supply to the leaves, rapidly reaching wilting under high evaporative demand. Phytochrome A signaling might facilitate the adjustment to environments differing widely in water evaporative demand in part through the modulation of xylem dimensions.

Full text

Page 1

Physiologia Plantarum 2012
Copyright © Physiologia Plantarum 2012, ISSN 0031-9317
Phytochrome A increases tolerance to high
evaporative demand
Gabriela Alejandra Augea, Mat´ ıas Leandro Rugnonea,†, Leandro Emanuel Cort´ esb,c,†, Carina Ver´ onica
Gonz´ alezb,†, Gabriela Zarlavskyd, Hern´ an Esteban Boccalandrob,cand Rodolfo Augusto S´ ancheza,∗
aIFEVA-CONICET, Facultad de Agronom´ ıa, Universidad de Buenos Aires, Buenos Aires, Argentina
bIBAM-CONICET, Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo, Mendoza, Argentina
cInstituto de Ciencias B´ asicas, Universidad Nacional de Cuyo, Mendoza, Argentina
dLaboratorio de Anatom´ ıa Vegetal, C´ atedra de Bot´ anica Agr´ ıcola, Facultad de Agronom´ ıa, Universidad de Buenos Aires, Buenos Aires, Argentina
Correspondence
*Corresponding author,
e-mail: sanchez@ifeva.edu.ar
Received 14 October 2011;
revised 1 February 2012
doi:10.1111/j.1399-3054.2012.01625.x
Stressesresultingfromhightranspirationdemandinduceadjustmentsinplants
that lead to reductions of water loss. These adjustments, including changes
in water absorption, transport and/or loss by transpiration, are crucial to
normal plant development. Tomato wild type (WT) and phytochrome A
(phyA)-mutant plants, fri1-1, were exposed to conditions of either low or
high transpiration demand and several morphological and physiological
changes were measured during stress conditions. Mutant plants rapidly wilted
compared to WT plants after exposure to high evaporative demand. Root size
and hydraulic conductivity did not show significant differences between
genotypes, suggesting that water absorption and transport through this organ
could not explain the observed phenotype. Moreover, stomatal density was
similar between genotypes, whereas transpiration and stomatal conductance
were both lower in mutant than in WT plants. This was accompanied by
a lower stem-specific hydraulic conductivity in mutant plants, which was
associated to lower xylem vessel number and transversal area in fri1-1
plants, producing a reduction in water supply to the leaves, which rapidly
wilted under high evaporative demand. PhyA signaling might facilitate the
adjustment to environments differing widely in water evaporative demand in
part through the modulation of xylem dimensions.
Introduction
The capacity to adjust their functioning to large vari-
ations in environmental conditions is essential for the
normal development of plant populations. Phenotypic
plasticity offers the opportunity of matching phenotype
to environment and so increasing fitness in the diverse
ecological scenarios that a plant may encounter (Schmitt
et al. 2003). Plants are equipped with a wide array
Abbreviations – FR, far-red light; PAR, photosynthetically active radiation; PPFD, photosynthetic photon flux density; phyA,
phytochrome A; phyB, phytochrome B; R, red light; SAS, shade avoidance syndrome; VPD, vapor pressure deficit; WT, wild
type.
†These authors contributed equally to this work.
of mechanisms to perceive environmental cues and
to translate them into morphological and physiological
changes allowing adjustment to actual or future condi-
tions. Stand densityis oneof theenvironmentalattributes
that can show extreme variations between densely
crowded and open sites, and plant phenotype is strongly
affected by these conditions (Harper 1977, Ballar´ e et al.
1988). Plants of many species display a number of mor-
phological and developmental responses when exposed
Physiol. Plant. 2012

Page 2

to high density, collectively termed shade avoidance
syndrome (SAS), such as stem and petiole elongation,
reduced branching and shortened time to flowering
(Smith 1982). One central element in the perception of
density is the change in light spectral composition after
interactionwithgreentissues.Theselectiveabsorptionof
visiblelightandtheincreasedtransmissionandreflection
of far-red light (FR; 700–800 nm) causes a reduction in
the proportion of red light (R, 600–700 nm) vs FR (Smith
1982). This reduced R/FR ratio is perceived by the phy-
tochromes,mainlyphytochromeB(phyB),whichtriggers
SAS. It has been shown that phytochrome-mediated SAS
has adaptive value (Schmitt et al. 1995). Increased plant
height, in particular, is recognized as a positive trait in
adjusting to high density (Schmitt et al. 1999).
There are substantial differences in photosynthetically
active radiation (PAR), leaf temperature and wind speed
between open and crowded habitats; consequently,
carbon and water economies are strongly influenced
by plant density. Adjustments to those conditions in
both processes take place very frequently, and it is
not surprising that phytochromes may mediate some
of them. In fact, higher R/FR ratios detected by phyB
have been shown to increase stomatal density, stomatal
index and amphistomy in leaves of Arabidopsis thaliana
plants, resulting in a greater photosynthetic rate at
high PAR at the expense of a reduction in water use
efficiency (Boccalandro et al. 2009). On the other hand,
potato plants over-expressing phyB showed no changes
in stomatal density but higher stomatal conductance
and transpiration and photosynthesis rates; in this case,
the relationship between phyB and carbon and water
economies may be an indirect result of phytochrome
influence on other processes such as assimilate demand
or leaf senescence (Boccalandro et al. 2003). There
are more examples that phyB and/or the stable
phytochromes can influence plant attributes related to
carbon and/or water balance (Roth-Bejerano et al. 1982,
Casal et al. 1994, Rousseaux et al. 2000, Sokolskaya
et al. 2003). The situation of phytochrome A (phyA)
is markedly different. Unlike other family members,
phyA is unstable in the Pfr form and therefore it has
been suspected that its main functions were control of
seed germination and seedling de-etiolation (Smith and
Whitelam 1990). But it has been established that phyA
controls a number of functions throughout development,
includingday-lengthperception(Johnsonet al.1994,Lin
2000) and inhibition of internode elongation (Yanovsky
et al. 1995). However, these functions were detected in
double or triple mutants since monogenic phyA-mutant
Arabidopsis plants grown under natural light are very
similar to the wild type (WT). More recently, Franklin
et al. (2007) have shown that under relatively high
photon irradiances phyA protein degradation is retarded,
opening the possibility of a significant phyA contribution
to behavior regulation of full-daylight-grown plants.
Nevertheless, there is almost no information indicating
a dominant role of phyA on the adjustment to situations
affecting water balance in full-daylight-grown plants.
Arabidopsis phyA mutants have been shown to have
reduced transpiration compared with WT plants (Eckert
and Kaldenhoff 2000), although to the best of our
knowledge the impact on the water budget has not
been investigated further.
Here, we show that phyA plays a prominent part in
acclimation to full sunlight, enhancing the hydraulic
conductivity necessary to cope with conditions of high
atmospheric demand, therefore revealing that phyA has
a key role in the water economy of tomato plants.
Materials and methods
Plant material and growth conditions
Solanum lycopersicum ‘Moneymaker’ seeds were incu-
bated at 25◦C in plastic boxes on a 0.5 cm cotton
layer saturated with distilled water. After 12 days, the
seedlings were planted in 3 l pots with a mixture of
organic matter-enriched soil and perlite (3:1). Plants
were grown in a temperature controlled greenhouse at
25 ± 2◦C until they had eight to nine fully expanded
leaves for hydraulic conductivity measurements or four
to five fully expanded leaves for water loss, leaf water
potential and leaf temperature measurements. For exper-
iments under high evaporative demand, treated plants
were transferred to a hotter greenhouse, i.e. without
temperature control.
Experiments were carried out at IFEVA-CONICET,
Facultad de Agronom´ ıa, Universidad de Buenos Aires,
Argentina (34◦25?S and 58◦25?W at 25 m asl), and
Facultad de Ciencias Agrarias, Universidad Nacional
de Cuyo, Mendoza, Argentina (33◦0?S and 68◦52?W at
940 masl).Allexperimentswereconductedatleastonce
in each location with similar results. Figures represent
the results of one representative experiment.
Air temperature and relative humidity were recorded
every 10 min with three Hobo sensors (Hobo Pro series;
Onset Computer Corporation, Bourne, MA). Photosyn-
thetic photon flux density (PPFD) was measured with
a LI-COR LI-190SA quantum sensor and a LI-250 light
meter (LI-COR, Lincoln, NE).
Hydraulic conductivity measurements
Hydraulic conductivity of whole root systems and
stem segments (between third and fourth nodes) was
measured as described in Fern´ andez and Gyenge (2010),
Physiol. Plant. 2012

Page 3

using a low pressure (water column) conductivity meter
(n = 11). The root system (whole roots plus the first
stem internode segment) or stem segments from plants
with eight to nine fully expanded leaves were cut
under water to avoid embolisms due to dissection.
Roots were completely submerged in a closed water-
filled container, and the cut-end of the root pushed
through an opening in the lid of the container, which
was connected to a low pressure water column. After
conductivity measurements, roots were dried at 70◦C for
2 days and their dry weight determined. Stem segments
were connected to a rubber hose, the other end of which
was attached to a low pressure water column. After
measurements, stem length and diameter were recorded.
Hydraulic conductivity was assessed by measuring the
volume of water that passed through the tissues under a
known pressure during a given period (1–5 min) using
the following equations for roots and stems, respectively:
kr= J/(P × Mr) mgwaters−1kPa−1g−1
ks= J/[(P/Ls) × As] mgwaters−1kPa−1cm−1
where kr and ks are the root and stem hydraulic
conductivity, respectively, J is the measured water flux,
P is the known pressure applied to the system, Mr is
the root dry weight and Ls and As are the length and
transversal area of the stem segment, respectively.
r
(1)
(2)
s
Plant water loss measurements
Loss of water per pot (in grams) was calculated by
weighing every pot at the beginning (6:30 h) and at
the end of the experimental period (13:30 h). Adaxial
and abaxial leaf conductance to water vapor (gl) was
measured with a steady-state diffusion porometer (SC-1;
Decagon Devices, Pullman, WA) in the terminal leaflet
of the third fully expanded sun-exposed leaf (n = 8).
Stomatal conductance, gl, was calculated as the sum
of the adaxial and abaxial leaf conductance values for
each leaf. Stomatal aperture was assessed by taking
imprints using transparent nail varnish applied to the
fourth fully expanded leave from 50-day-old plants
at 9:00 h (low atmospheric demand) and at 14:00 h
(high atmospheric demand). Leaves were not detached
until the varnish dried. Stomatal aperture imprints
were measured under a microscope (40×/100×) in the
middle portion of the abaxial leaf surface. Representative
photographs were taken using a Micrometrics 318 CU
camera (China) attached to a Nikon Eclipse E200 optical
microscope (Tokyo, Japan). After plant water relation
measurements, leaves were harvested and immediately
scanned. Individual leaf area was calculated using
Adobe Photoshop (v. 7.0) by comparison with a
reference area.
Water status measurements
Leaf water potential (?w) measurements were recorded
for each genotype at pre-dawn (6:00 h), mid-morning
(10:30 h) and mid-day (around solar noon for each
location) using the third fully expanded leaf of plants
with four to five leaves, with a pressure chamber
(PMS Instruments Co., Corvallis, OR). Cut leaves were
measured within 1–2 min.
Thermographic pictures
Digital thermal images were obtained using a Fluke TiR
Thermal Imager (Fluke Co., Everett, WA). Plants were
photographedfrom7:00to13:00h.Digitalthermograms
were analyzed with SMARTVIEW software (Fluke Co.).
Preparation of stem cross sections
Stem sections between the stem/root junction and the
cotyledon node were cut from plants with eight to nine
fully expanded leaves and conserved in 20% v/v ethanol
solution until sectioned. Cross sections were cut by
hand or microtome. Hand-cut sections (around 15–20
μm thick) were treated with 50% v/v commercial sodium
hypochlorite to clarify the cells and then washed twice
with distilled water and incubated with safranin solution
until conducting tissues with secondary growth were
stained. Cross sections were mounted on slides with
mounting medium (gelatin:pure glycerol:distilled water,
1:7:6). Sections were observed using a stereoscopic
microscope (CETI, Medline Scientific Ltd., Oxfordshire,
UK) and photographed with a PowerShot A520 digital
camera (Canon Inc., Ontario, Canada) at 40× to
assess xylem vessel number and area with Adobe
Photoshop (v 7.0) by comparison with a reference
area. For microtome-cut stem sections, stem segments
were fixed in FAA buffer (ethanol 96% v/v:distilled
water:formaldehyde:acetic acid, 50:35:10:5) for at least
48 h before processing. After the material had been
washed and rinsed with distilled water, it was submitted
to a gradual dehydration process with a series of
50, 70, 80, 96 and 100% v/v ethanol solutions.
Subsequently, the material was gradually embedded in
paraffin by transferring it sequentially to xylene 100%
v/v, xylene:paraffin (3:1), xylene:paraffin (1:1) and finally
to pure paraffin. The time necessary for each stage
was variable and depended on the tissue size and
traits. Paraffin and xylene:paraffin solutions were kept
at 60◦C during the process. After paraffin blocks had
been cooled, they were sectioned using a microtome
(SM 2000 R; Leica Microsystems, Wetzlar, Germany)
and the cross sections (14 μm thick) were mounted on
slides. The sections were dewaxed with xylene 100% v/v
Physiol. Plant. 2012

Page 4

A
B
Fig. 1. PhyA increases tolerance to environments of high evaporative
demand. Phenotypic differences between WT and phyA-mutant fri1-1
plants. Pictures of one representative experiment before (A) and after
(B) 30 min of exposure to stressful conditions (higher temperature, see
Fig. S1). Insets in (B) show a detail of the phenotype observed after
exposure to stressful conditions in leaves of both genotypes.
and incubated in safranin solution until the conducting
tissues with secondary growth were stained, then rinsed
twice with distilled water. Finally, stem cross sections
weremountedonslideswithNaturalCanadaBalsamand
observedunderaNikonEclipseE200opticalmicroscope
(Tokyo, Japan) and photographed with a Micrometrics
318 CU digital camera (Shanghai, China) at 40×.
Stomatal density measurements
The first pair of fully expanded leaves was used to
determine stomatal density in WT and phyA-mutant
plants. The number of stomatal cells was counted in
clarified leaves under a clear-field Leica DMIRB inverted
microscope at 600× and photographed with a Leica
DC 300F camera (Leica Camera AG, Solms, Germany).
Clarification was carried out immersing leaves in an
NaOH 1% p/v solution at 70◦C for 30 s, and then,
keeping them in ethanol 70% v/v until measurements
weremade.Ifnecessary,safraninstainingwasperformed
to help stomatal cell visual identification.
Statistical analysis of the data
Student’s t-test or ANOVA followed by LSD Fisher post-
test was performed when appropriate, in order to
asses minimum differences between means with a P
< 0.05 (*) significance level using INFOSTAT software
(www.infostat.com.ar).
Results
It has been reported that in Arabidopsis and potato, phy-
tochromes, specially phyB, affect several morphological
(e.g. root:shoot biomass ratio and stomatal density) and
physiological characteristics (e.g. stomatal conductance
and transpiration) (Salisbury et al. 2007, Boccalandro
et al. 2009) that could be involved in plant water use
efficiency. In order to elucidate if phyA is playing any
role in plant water relations, we grew WT and phyA-
mutant (fri1-1) tomato plants under stressful conditions.
We observed that under conditions of high evaporative
demand, leaves of fri1-1 plants wilted, while the leaves
of WT plants remained unaffected (Fig. 1).
Loss of turgor in leaves could be the consequence
of differences in water uptake, transport or transpiration
between fri1-1 and WT plants. We did not observe
significant differences either in root biomass or root
hydraulic conductivity, suggesting that there was
no difference in water acquisition capacity between
genotypes (Fig. 2). In contrast, loss of water per pot
during the experiment (7:00 h to 13:00 h) was different
betweengenotypesbut,surprisingly,WTplantslostmore
water than fri1-1 plants (Fig. 3A). The larger water loss
was not due to a larger leaf area or stomatal density
WT fri1-1
0.0
0.5
1.0
1.5
2.0
ns
Dry weight (g)
A
WTfri1-1
0.000
0.002
0.004
0.006
0.008
0.010
0.012
ns
B
k (mg s–1 kPa–1 g–1)
Fig. 2. Influence of genotype on attributes related to water uptake.
(A) Root mass (dry weight, grams) and (B) root system hydraulic
conductivity (k, mg s−1kPa−1g−1) of WT and fri1-1 plants. Bars are
means (n ≥ 10), narrow vertical extensions represent one standard
error. Values are from one representative experiment; ns, differences
not significant (P > 0.05).
Physiol. Plant. 2012

Page 5

0
10
20
30
WT
fri1-1
*
*
*
Loss of water per pot
during experiment (g)
0
100
200
300
400
Leaf area per plant
0
6:00h
50
100
150
200
250
WT
fri1-1
*
*
*
Higher temperature
Lower temperature
Stomatal conductance
(mmol m–2 s–1)
10:00h14:00h
0
1
2
3
4
Stomatal aperture (μm)
ABC
D
8:00h 10:00h
Time of day
12:00h14:00h
Fig. 3. Influence of genotype on water loss. (A) Water loss per pot during the experiment (7:00 h to 13:00 h) in normal (lower temperature)
conditions, (B) leaf area per plant and (C) stomatal aperture (μm) before (10:00 h) and after (14:00 h) the beginning of exposure to stressful conditions
(i.e. lower and higher temperatures, respectively, see Fig. S1). (D) Time course of stomatal conductance during the experiment. Data points are means,
vertical bars represent ± one standard error. Values are from one representative experiment. Asterisks indicate significant (P < 0.05) differences
between means.
in WT plants (Figs 3B and S2) but was related with a
greater leaf conductance and stomatal aperture in WT
plants during the day (Fig. 3C, D). In Fig. 3D it is shown
that early in the morning (8:00 h), stomatal conductance
of fri1-1-mutant plants was similar to the WT (Fig. 3D),
but when at noon air temperature increased (Fig. S1), a
significant decrease in fri1-1 plant leaf conductance was
observed, something that did not occur in WT (Fig. 3D).
Although genotype might affect stomatal behavior in
several ways, two of the more likely possibilities are
that somehow leaf water potential may decrease during
the day to lower values in fri1-1 or that in the mutants
the stomata are more sensitive to a decrease in water
potential. At sunrise, leaf water potential was the same
in both genotypes but later in the day it became lower
in fri1-1 leaves (Fig. 4). As expected, leaf temperature
was significantly higher in fri1-1 leaves (Fig. 5). Taken
together, the observations that fri1-1 plants had lower
rate of water loss and leaf water potential than the WT
suggested that water transport to the leaves was affected.
Shoot hydraulic conductivity was significantly lower
in the fri1-1 plants (Fig. 6B) and this was not related
with stem diameter (Fig. 6A) but with the area of xylem
elements in the stem (Fig. 6D). Therefore the marked
tendency to leaf wilting under high transpiration demand
of fri1-1 plants seems to be related to phyA control
–1.2
–0.8
–0.4
–0.0
6.30 h
Ψ (MPa)
10.00 h*
14.00 h*
WT
fri1-1
Fig. 4. PlantslackingphyAhavelesscontrolofleafwaterpotential.Time
courseofleafwaterpotentialduringonerepresentativeexperiment.Bars
are means (n ≥ 10), narrow vertical extensions represent one standard
error. Asterisks indicate significant (P < 0.05) differences between
means.
of xylem development that causes a higher resistance
to water transport through the xylem vascular tissue,
precluding an adequate water provision to the leaves
when the transpiration rate is high.
Discussion
Adjustment to stand density provided by the SAS of the
phytochromes has been shown to be adaptive. The fit-
nessadvantageinsiteswithhighdensityismainlyrelated
with plant height due to its influence in sunlight capture.
Improving the ability for light competition is certainly
Physiol. Plant. 2012

Page 6

WTfri1-1
7.40h
9.15h
10.15h
12.35h
Time
B
A
6.008.0010.00 12.00 14.00
10
20
30
40
WT
fri1-1
Time (h)
Foliar temperature (°C)
*
*
Fig. 5. Leaf temperature is higher in plants lacking phyA. Time course
of leaf temperature (A) and thermographic pictures of fully expanded
leaves (B) during one representative experiment. Data points in (A) are
means (n ≥ 10). Asterisks indicate significant (P < 0.001) differences
between means.
a central aspect of the responses to density (Schmitt
et al. 1999). However, several other morphological and
physiological traits are modified by the environmental
differences between open and crowded habitats (Hutch-
ings and de Kroon 1994). Some of them are controlled
by phytochromes, although almost all the information
available so far is about the involvement of phyB (Bal-
lar´ e et al. 1997). While evoking SAS, phyB can improve
carbon gain in a crowded environment through shoot
morphological changes that increase chances to forage
for PAR. Nevertheless, phyB effects on carbon and water
economies are not limited to promoting a better expo-
sure of leaves to sunlight. Evidence shows that phyB can
also adjust structures related to acquisition, transport
and loss of water as well as carbon gain. phyB shapes
Arabidopsis roots, increasing the number of lateral roots
(Salisbury et al. 2007) and reducing main root and root
hairlength(Reedet al.1993,Gonz´ alezandBoccalandro
2008). It can also adjust xylem vessel diameter and num-
ber, functionally increasing stem water conductance of
adult cucumber plants (Casal et al. 1994). At leaf level,
phyB increases stomatal density and index, amphistomy
(Boccalandro et al. 2009, Casson et al. 2009) and stom-
atal aperture (Wang et al. 2010) in Arabidopsis. Several
of these structural and physiological changes modulated
by phyB produce functional consequences on photo-
synthetic rate and transpiration, modifying water use
efficiency (Boccalandro et al. 2009). In addition to these
observations, mostly obtained with Arabidopsis plants
grown in controlled conditions, phyB overexpression in
potato increases stomatal conductance, photosynthetic
and transpiration rate per unit leaf area under field con-
ditions (Boccalandro et al. 2003). Taken together, this
evidence clearly shows that phyB, operating at different
levels, acts as a key modulator of water and carbon
economies. In contrast, the involvement of phyA on
water or carbon economy is largely ignored. It has been
reported that Arabidopsis phyA-103 mutants display a
reduced transpiration rate under red light but not under
blue light (Eckert and Kaldenhoff 2000), but its response
to sunlight was not reported.
In this study, we observed a significant phenotype
of the fri1-1 mutant when grown in the field. Under
conditions of high evaporative demand, leaf wilting was
conspicuous (Fig. 1) and at a stage when there were
no differences in plant biomass or leaf area, it was
observed that transpiration, leaf conductance and leaf
water potential were significantly lower in fri1-1 than
in WT plants (Figs 3 and 4). These observations suggest
that a reduced water supply to leaves produced an
earlier decrease in water potential with consequences
for stomatal aperture and hence transpiration (Fig. 3D).
The reduced leaf water supply does not appear to be
related either to the size or the conductivity of the root
system (Fig. 2), while it is consistent with reduced stem
water conductivity, associated with a smaller area of
xylem elements (Fig. 6).
The structure of the plant hydraulic system can poten-
tially limit water flow through the plant with conse-
quences for the water and carbon economies, so it is
not surprising that differences in either stem conduc-
tance or leaf-specific conductance have been found to
be associated with habitats showing divergent irradiance
conditions (like sites under forest canopies or in gaps).
Physiol. Plant. 2012

Page 7

E
fri1-1
WT
0
2
4
6
8
*
Diameter (mm)
0
25
50
75
100
*
Open large xylem vessels
per optical field (4X)
0
1
2
3
4
5
6
*
Area (mm2)
AB
CD
0.00
0.01
0.02
0.03
*
WT
fri1-1
ks (mg s–1 kPa–1 cm–1)
Fig. 6. PhyA affects water transport through the stem. Differences between genotypes of mature plants with eight to nine fully expanded leaves
in: stem diameter (A), stem-specific hydraulic conductivity (ks) (B), number of large xylem vessels observed in hand-cut stem cross sections (C) and
estimated area of open xylem vessels (D). (E) Microtome-stem cross section micrographs of WT and mutant mature plants with four to five fully
expanded leaves. Bars in (A) and (B) are means (n ≥ 10), narrow vertical extensions represent one standard error. Values are from one representative
experiment. Asterisks indicate significant (P < 0.05) differences between means.
That is the case of Piper trigonum, a species that shows a
greater leaf-specific conductance (Kla) when exposed to
higher irradiances, and which in addition to being abun-
dant in understory conditions, can invade open areas
when water is in adequate supply (Engelbrecht et al.
2000). A similar response is found in Rhododendron
maximum plants, that when growing in gaps, have a
higher proportion of large diameter xylem vessels com-
pared with plants growing under a canopy (Lipp and
Nielsen 1997). Also, it has been reported that olive
trees have larger xylem cross-sectional area when grown
under high light irradiance than in low light conditions
(Raimondo et al. 2009). Here, we found that phyA is a
key factor promoting xylem vessel diameter under full
sunlight, this morphological adjustment being of func-
tional importance to enhanced water conductance to
leaves subjected to high evaporative demand.
The positive relationship between phyA and stem
conductance that we are describing as well as that
reported in cucumber plants with phyB (Casal et al.
1994) suggests that phytochromes might have a role
in the adjustment of the plant water economy to a
changing environmental scenario. This aspect as well as
the mechanisms underlying the modification of the stem
water transport capacity clearly deserve more attention
and are currently being investigated.
Acknowledgements – We thank Esteban Fernandez for
assistance in hydraulic conductivity measurements and Dr
Roberto Fernandez-Aldunc´ ın for helpful discussions of the
results and critical reading of the manuscript. This work was
funded by ANPCYT and UBACYT grants to H. E. B. and R.
A. S. This paper is dedicated to the memory of our dear
friend and colleague Dr Hern´ an Boccalandro who tragically
departed this world on December 10, 2011.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Greenhouse conditions. PPFD, air temperature,
relative humidity (RH) and vapor pressure deficit (VPD)
during the course of one representative experiment
conducted in Mendoza, Argentina.
Fig. S2. PhyA does not affect stomatal density of tomato
leaves. Stomatal density on abaxial (A) and adaxial
(B) sides of the second and third fully expanded leaves
of mature plants measured at 600×. Bars are means (n ≥
10), narrow vertical extensions represent one standard
error; ns, not significant.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supplementary materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
References
Ballar´ e CL, S´ anchez RA, Scopel AL, Ghersa CM (1988)
Morphological responses of Datura ferox L. seedlings to
the presence of neighbours. Oecologia 76: 288–293
Physiol. Plant. 2012

Page 8

Ballar´ e CL, Scopel AL, S´ anchez RA (1997) Foraging for
light: photosensory ecology and agricultural
implications. Plant Cell Environ 20: 820–825
Boccalandro HE, Ploschuk EL, Yanovsky MJ, S´ anchez
RA, Gatz C, Casal JJ (2003) Increased phytochrome B
alleviates density effects on tuber yield of field potato
crops. Plant Physiol 133: 1539–1546
Boccalandro HE, Rugnone ML, Moreno JE, Ploschuk EL,
Serna L, Yanovsky MJ, Casal JJ (2009) Phytochrome B
enhances photosynthesis at the expense of water-use
efficiency in Arabidopsis. Plant Physiol 150:
1083–1092
Casal JJ, Ballar´ e CL, Tourn M, S´ anchez RA (1994)
Anatomy, growth and survival of a long-hypocotyl
mutant of Cucumis sativus deficient in phytochrome B.
Ann Bot 73: 569–575
Casson SA, Franklin KA, Gray JE, Grierson SE,
Whitelam GC, Hetherington AM (2009) Phytochrome B
and PIF4 regulate stomatal development in response to
light quantity. Curr Biol 19: 229–234
Eckert M, Kaldenhoff R (2000) Light induced stomatal
movement of selected Arabidopsis thaliana mutants. J
Exp Bot 51: 1435–1442
Engelbrecht BMJ, Velez V, Tyree MT (2000) Hydraulic
conductance of two co-occurring neotropical understory
shrubs with different habitat preferences. Ann Forest Sci
57: 201–208
Fern´ andez ML, Gyenge JE (2010) T´ ecnicas de Medici´ on en
Ecofisiolog´ ıa Vegetal: Conceptos y Procedimientos.
Ediciones INTA, Buenos Aires
Franklin KA, Allen T, Whitelam GC (2007). Phytochrome
A is an irradiance-dependent red light sensor. Plant J 50:
108–117
Gonz´ alez CV, Boccalandro HE (2008) Detection of
crowding in shoots induces root elongation in the soil.
XXVI Cuyo Biology Society Scientific Annual Reunion,
Mendoza, Argentina
Harper JL (1977) Population Biology of Plants. Academic
Press, London
Hutchings MJ, de Kroon H (1994) Foraging in plants: the
role of morphological plasticity in resource acquisition.
Adv Ecol Res 25: 159–238
Johnson E, Bradley M, Harberd NP, Whitelam GC (1994)
Photoresponses of light-grown phyA mutants of
Arabidopsis (phytochrome A is required for the
perception of daylength extensions). Plant Physiol 105:
141–149
Lin C (2000) Photoreceptors and regulation of flowering
time. Plant Physiol 123: 39–50
Lipp CC, Nielsen ET (1997) The impact of subcanopy light
environment on the hydraulic vulnerability of
Rhododendron maximum to freeze-thaw cycles and
drought. Plant Cell Environ 20: 1264–1272
Raimondo F, Trifil` o P, Lo Gullo MA, Buffa R, Nardini
A, Salleo S (2009) Effects of reduced irradiance on
hydraulic architecture and water relations of two olive
clones with different growth potentials. Environ Exp Bot
66: 249–256
Reed JW, Nagpal P, Poole DS, Furuya M, Chory J (1993)
Mutations in the gene for the red/far-red light receptor
phytochrome B alter cell elongation and physiological
responses throughout Arabidopsis development. Plant
Cell 5: 147–157
Roth-Bejerano N, Nejidat A, Itai C (1982)
Phytochrome-membrane interactions as a factor in
stomatal opening. Physiol Plant 56: 80–83
Rousseaux MC, Hall AJ, S´ anchez RA (2000) Basal leaf
senescence in a sunflower (Helianthus annuus) canopy:
responses to increased R/FR ratio. Physiol Plant 110:
477–482
Salisbury FJ, Hall A, Grierson GS, Halliday KJ (2007)
Phytochrome coordinates Arabidopsis shoot and root
development. Plant J 50: 429–438
Schmitt J, McCormac AC, Smith H (1995) A test of the
adaptive plasticity hypothesis using transgenic and
mutant plants disabled in phytochrome-mediated
elongation responses to neighbors. Am Nat 146:
937–953
Schmitt J, Dudley SA, Pigliucci M (1999) Manipulative
approaches to testing adaptive plasticity: phytochrome
mediated shade avoidance responses in plants. Am Nat
154: S43–S54
Schmitt J, Stinchcombe JR, Heschel MS, Huber H (2003)
The adaptive evolution of plasticity:
phytochrome-mediated shade avoidance responses.
Integr Comp Biol 43: 459–469
Smith H (1982) Light quality, photoperception, and plant
strategy. Annu Rev Plant Physiol 33: 481–518
Smith H, Whitelam GC (1990) Phytochromes, a family of
photoreceptors with multiple physiological roles. Plant
Cell Environ 13: 695–707
Sokolskaya SV, Sveshnikova NV, Kochetova GV,
Solovchenko AE, Gostimski SA, Bashtanova OA (2003)
Involvement of phytochrome in regulation of
transpiration: red-/far red-induced responses in the
chlorophyll-deficient mutant of pea. Funct Plant Biol 30:
1249–1259
Wang FF, Lian HL, Kang CY, Yang HQ (2010)
Phytochrome B is involved in mediating red
light-induced stomatal opening in Arabidopsis thaliana.
Mol Plant 3: 246–259
Yanovsky MJ, Casal JJ, Whitelam GC (1995) Phytochrome
A, phytochrome A and HY4 are involved in hypocotyl
growth responses to natural radiation in Arabidopsis:
weak de-etiolation of the phyA mutant under dense
canopies. Plant Cell Environ 18: 788–794
Edited by R. Scheibe
Physiol. Plant. 2012