Stomatal, cuticular water loss and photosynthetic
acclimation in response to irradiance
Isaac Aulik
Thesis
Submitted in partial fulfillment of the requirements
for the degree of Masters of Science
at Department of Food Science,
Aarhus University
Abstract
We studied the plasticity of stomatal anatomy and functionality, as well as cuticular water
loss and photosynthetic acclimation in Rosa hybrida cv. ‘Pasadena’ grown under low (100
µmol m-2 s-1), moderate (200 µmol m-2 s-1) or high (400 µmol m-2 s-1) irradiance. Plants were
grown in controlled climate chambers with steady state conditions i.e. all other environmental
factors were kept optimal so as to study the effect of differences in light intensities on the
plants. We measured or calculated (1) stomatal and pore anatomy; (2) stomatal and cuticular
response to dehydration; (3) whole plant transpiration rate during growth; (4) the ability of a
leaf to rehydrate after dehydration; and (5) photosynthesis irradiance and carbon dioxide
response curves per treatment. Irradiance significantly affected almost all examined stomatal
features. Leaves grown under high irradiance had significantly bigger (20%) stomata than
low irradiance leaves. Moreover, stomata from high irradiance grown leaves closed faster.
Leaves developed at low irradiance had higher cuticular permeability, indicating plants
modulate both water loss pathways in response to irradiance. The photosynthetic capacity
(Amg) increased with increasing irradiance while light-limited quantum efficiency was similar
in all leaves. Dark respiration (Rd) decreased with decreasing irradiance, an acclimation
strategy by the low irradiance grown leaves to allow for a higher net photosynthesis.
Key words: Irradiance, stomata anatomy and physiology, cuticular transpiration,
Photosynthesis, rehydration ability, Rosa hybrida
Contents
1
Introduction ................................................................................................................... 1
1.1
Supplementary lighting in Danish Greenhouse ........................................................ 1
1.2
Effects of irradiance on plant growth ...................................................................... 1
1.3
Effect of irradiance on leaf photosynthesis and related characteristics .................... 2
1.4
Effect of Irradiance on water loss characteristics .................................................... 2
1.4.1
Effect of irradiance on stomatal physiology and anatomy................................. 3
1.4.2
Effect of irradiance on cuticular transpiration rate .......................................... 4
1.5
2
3
Aim and outline of the thesis ................................................................................... 4
Materials and methods ................................................................................................... 5
2.1
Plant material and growth conditions ..................................................................... 5
2.2
Stomatal and pore anatomy ..................................................................................... 5
2.3
Stomatal responsiveness to dehydration .................................................................. 6
2.4
Whole plant transpiration rate during growth ......................................................... 6
2.5
Leaf rehydration ability following a dehydration event ............................................ 6
2.6
Chlorophyll content ............................................................................................... 7
2.7
Cuticular water loss ................................................................................................ 7
2.8
Photosynthesis irradiance and carbon dioxide response.......................................... 7
2.9
Data analysis .......................................................................................................... 8
Results ........................................................................................................................... 9
3.1
Morphological characteristics ................................................................................ 9
3.2
Stomatal characteristics .......................................................................................... 9
3.3
Stomatal response to dehydration.......................................................................... 10
3.4
Cuticular transpiration rate .................................................................................. 11
3.5
Intact plant transpiration ...................................................................................... 12
3.6
Rehydration ability following a dehydration event................................................. 14
3.7
Chlorophyll content .............................................................................................. 14
3.8
Photosynthetic acclimation ................................................................................... 16
4
Discussion.................................................................................................................... 19
5
Conclusions ................................................................................................................. 23
Acknowledgments............................................................................................................... 25
References .......................................................................................................................... 27
1 Introduction
1.1 Supplementary lighting in Danish Greenhouse
Supplementary lighting is often applied in greenhouse production in northern latitudes during
the dark months of the year (fall to spring) because production is limited by the outdoor
irradiance level. Lighting is important in greenhouse production because it increases
production levels and improves quality of products. In addition, it helps to achieve year round
production. In Denmark, most greenhouses are equipped with supplementary lighting.
However, application of supplementary lighting also has its own drawbacks. For instance,
supplementary lighting increases energy use and thus cost of production. The share of
electricity cost to the total production cost could be significant. In addition, supplementary
lighting increases greenhouse gas emission. The most highly used source of supplementary
lighting in Danish greenhouse industry is High Pressure Sodium (HPS) lamps. HPS lamps are
energy efficient but more efficient light source based on Light Emitting diodes (LEDs) are
increasingly being researched. Therefore, there is great emphasis to reduce these
consequences of supplementary lighting both by the Danish government and the horticulture
industry in general.
1.2 Effects of irradiance on plant growth
Growth of photo-autotrophic plants is directly and considerably effected by light intensity,
which is the main stimulus of photosynthesis. This intern delivers nearly all the carbon and
chemical energy essential for overall plant development. Light intensity i.e. quantum flux
density (QFD), is possibly the most noticeable environmental factor with which plants must
deal with (Björkman, 1981). Vascular plants use light not only to convert solar energy into
chemical energy but also as an informational signal to regulate a host of physiological
responses throughout their life. Together these responses are known as photomorphogenesis
(Kami et al., 2010). These responses can be irreversible e.g. leaf expansion or reversible e.g.
stomata opening. Conditional upon the amounts of light available during a plant’s growth
process, plants have the capability to react with two unique growth-responses. One being the
strong light growth response as found at high irradiance rates i.e. sun leaves or high-light
plants. The second being the weak light growth responses i.e. shade leaves (Oguchi et al.,
2003). This proficiency of photo-autotrophic plants and chloroplasts to adjust to differing
light conditions is a fundamental basic growth response. This response is connected with
1
precise changes in the physiology, morphology, biochemistry and structure of leaves and
chloroplasts (Oguchi et al., 2005).
1.3 Effect of irradiance on leaf photosynthesis and related characteristics
Light is one of the most central environmental factors that influence overall plant growth,
which acts both directly and indirectly upon the rate of photosynthesis (Smith, 1982).
Directly light effects photosynthesis by increasing or decreasing the rate at which leaves use
photon energy to synthesize glucose and uptake CO2 (Farquhar and Von Caemmerer, 1982).
Generally, as light level increases, the photosynthetic capacity of a plant increases and
conversely when light level is decreased. However there is a certain light saturation point at
which a leaf reaches where it cannot utilize the incoming energy, this is determined by the
genetics of a species and growth environment (Wilson and Cooper, 1969). Different species
process varying levels of light saturation points, thus varying photosynthetic capacities e.g.
shade plants and sun plants. The photosynthetic apparatus of leaves from sun plants is
modified for high rates of light quanta utilization. They display a greater photosynthetic
capacity in their chlorophyll and chloroplast, as well as a varying chemical composition and
ultrastructure than the chloroplasts of leaves from shade species (Cui et al., 1991). Further,
Lichtenthaler, et al. 1981) found that leaves from sun plant species contain more chlorophyll
(per unit leaf area), greater values for the ratio of chlorophyll in each leaf, lower number of
thylakoids per granum, a lower stacking degree of thylakoids and narrower grana. These
characteristics are genetically determined, but can be altered by the prevailing light level
during growth. Since leaves from shade plants receive lower levels of light than sun plants,
they must have a higher proportion of photosynthetic ‘machinery’ to more efficiently utilize
the low photon energy given to them. Thus preferred light intensity level can be greatly
varying between plant species.
1.4 Effect of Irradiance on water loss characteristics
Most transpiration from a plant occurs through the stomatal pores of its leaves. Light, CO2,
humidity, temperature, plant water status and plant hormones are signals that affect the guard
cells which are responsible for the regulation of stomata aperture (Hopkins and Hüner, 1995).
However, for the purpose of this paper, since all aforementioned variables have been kept at
optimum levels except light, only the effects of light on stomata will be discussed. Between
CO2 and light, it has been shown that light has a more direct effect on stomata regulation than
CO2 (Jurik et al., 1982).
2
It is not necessarily the intensity of light that causes the response of stomata as much as the
certain wavelength of that light. Sharkey et al. (1981) found that stomata in Xanthium
strumarium L. were most affected by both red (between 630 and 680 nm) and blue bands
(between 430 and 460 nm) of light in the absence of CO2, with blue causing a response nine
times greater than red. Because chlorophyll has an affinity for blue and red wavelengths and
guard cells are the only epidermal cells that contain chlorophyll (in most species), these wave
length signal stomatal opening if environmental conditions are ideal e.g. humidity,
temperature etc. For this reason guard cells are considered a photo indicator in the light
response of stomata (Sharkey et al., 1981; Assmann and Shimazaki, 1999). Although the
specific photo indicators for the opening of stomata are not fully understood. However, it is
known that light effects transpiration by activating stomatal opening thus allowing water to
‘escape’ from the leaves of the plant.
1.4.1
Effect of irradiance on stomatal physiology and anatomy
The effect of light on stomata can be generally divided into two categories of plant types, 1)
high light dependent species and 2) low light dependent species (Wilson and Cooper, 1969).
In high light species the whole leaf structure is effected, showing overall thicker leaves which
includes increases in mesophyll area to surface area, higher mesophyll conductance of CO 2
and CO2 fixation rates, higher density of stomata, and in a high number of cases, larger
stomata both in length and in aperture openings (Wilson and Cooper, 1969; Chabot et al.,
1977; Cui et al., 1991). These traits equal higher photosynthetic capacities for high light
species; due to the larger amount of photon energy they have to process (Boardman, 1977;
Lichtenthaler et al., 1981). Further, high light species of plants also have been shown to
possess higher rates of stomatal conductivity (Wilson and Cooper, 1969; Chabot et al., 1977;
Oguchi et al., 2005). These high rates are adaptive mechanistic changes in an attempt to
utilize higher levels of energy (Wilson and Cooper, 1969). Leaves in low light species have
been also known to show similar leaf physiological and anatomical changes when grown
under higher light intensities although less drastic than high light species; due to genetic
makeup (Boardman, 1977; Reich et al., 1998). This allows the extrapolation that most plant
species have the ability to alter both the structure and functionality of their leaves according
to available light level.
3
1.4.2
Effect of irradiance on cuticular transpiration rate
The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other
primary parts of higher plants. After stomata, which account for 90 to 95 percent of
transpiration from a leaf, the final 5 to 10 percent occurs through the cuticular layer (Hopkins
and Hüner, 1995; Kerstiens, 1996). Plant species that are drought tolerant and live in
generally higher light environments are found to have thicker cuticles, allowing for less water
transpiration; with the converse occurring in less drought tolerant species (Ashton and
Berlyn, 1994). Although high light intensity often is correlated with higher temperatures thus
causing potential closure of stomata to conserve water, transpiration would still occur through
the cuticular layer, even causing a potential increase in loss through this avenue, albeit small
(Kuiper, 1961).
1.5
Aim and outline of the thesis
The purpose of this study was to investigate the interaction of the anatomical structure and
gas exchange in leaves grown under different light intensities i.e. photon flux densities
(PFD). Anatomical traits were investigated for stomata, including size of guard cells and
density of stomata per light treatment. We also explored stoma responsiveness to
dehydration. Further we studied whole plant transpiration rates during growth and the ability
of leaves to rehydrate following a dehydration event. Using potted roses, we attempted to
either confirm previous observations or to discover alternative patterns which may exist
within the diversity of biological species.
4
2 Materials and methods
2.1
Plant material and growth conditions
90, four week old and second-time pruned pot rose (Rosa hybrida L. cv. 'Pasadena') were
planted in 0.55 L pots, containing a commercial peat potting mix (Pindstrup 2, Pindstrup
Mosebrug A/S, Ryomgaard, Denmark) was obtained from a commercial nursery and placed
in three growth chambers (30 pots in each) located at the Aarhus University, Department of
Food Science, Aarslev, Denmark. Air temperature was kept constant (20.5 ± 1.4 °C) in all
chambers, resulting in vapour pressure deficits (VPDs) of 0.94 ± 0.07 kPa. Light was
provided by LED lamps (HQI-BT 400W/D pro, Slovakia) at 100, 200, 400 μmol m-2 s-1
photosynthetic photon flux density (PPFD; determined by LI-250A, LI-COR, Lincoln, NE)
per chamber for 18 h per day. In all chambers the RH was 60 ± 3% (moderate RH). Air
temperature and RH were constantly measured by sensors (Humitter 50U/50Y(X), Vaisala,
Helsinki, Finland) placed at the top of fully grown plants (i.e. 60 cm from the root to shoot
interface), and data was automatically recorded by loggers (Datataker, Thermo Fisher
Scientific Australia Pty Ltd, Scoresby, Australia). All plants were fertigated at least one time
a day. Electrical conductivity and pH of the drain water were adjusted to 2 dS m -1 and 5.5,
respectively. All measurements were taken on fully expanded sunlit leaves, which were
sampled from fully grown plants (defined as bearing at least two flower buds with cylindrical
shape and pointed tip).
2.2 Stomatal and pore anatomy
In order to observe the effect of irradiance on stomatal anatomy, the length, width and density
(i.e. number per unit leaf area) of stomata, along with pore length and aperture were
measured. The measurement was carried out with the silicon rubber impression technique
(Giday et al., 2013) by using a lateral leaflet of the first penta-foliate leaf, counting from the
apex. The method and image analysis employed are described in detail by Giday et al.,
(2013). Only the abaxial surface of the leaf was measured, due to rose being a
hypostomatous species (Giday et al., 2013). Sampling was conducted two hours after the
beginning of the light period. Anatomical features were determined on both stomatal length
and width along with pore length and aperture on 25 randomly selected stomata. Stomatal
density was counted on five individual non-repeating fields of view. Five leaflets per
treatment were measured, using one leaflet per plant and one plant per pot.
5
2.3 Stomatal responsiveness to dehydration
To investigate the effect of irradiance on plants ability to close stomata when stressed, a
dehydration experiment was conducted. Leaflets were detached, and placed in containers
with degassed water beneath the petiole. Thereafter, leaflets were incubated at 21 °C, about
100% RH (i.e. VPD close to 0) and under 15 μmol m-2 s-1 PPFD for 1 h to achieve maximum
fresh weight per leaf. The leaflets were then placed on a grate with the abaxial surface facing
downwards in a test room [air temperature = 21.0 ± 2.2 °C, RH = 50 ± 4%, and 50 μmol m-2
s-1 PPFD provided with fluorescent lamps (T5 fluorescent lamp, GE lighting, Cleveland, OH)
for four hours. Transpiration rate was recorded gravimetrically (± 0.0001 g; Mettler AE 200,
Giesse, Germany). Nine leaflets were used from each treatment, one leaflet per plant, and
one plant per pot. Measurement were recorded in intervals of every ten minutes for four
hours, during which leaflets were taken from the grate and placed on the balance by handling
their petioles. At the end of the measurement, leaflet area and dry weight were determined
using leaf area meter and oven. Leaflet relative water content (RWC) was calculated as such.
Fresh weight−dry weight
RWC = saturated fresh weight−dry weight x 100 (Eqn.1)
2.4 Whole plant transpiration rate during growth
The effect of irradiance on whole plant transpiration rate was investigated. The pots were
weighed (± 0.1 g; MXX-2001, Denver Instruments, Bohemia, NY) two times per day at time
0 and 18 hour after the onset of the light period, for five consecutive days. Plants were
irrigated at the start and end of the light period. Weights of the plants containing the supplied
nutrient and drainage solutions were recorded twice daily. Plant leaf area was measured after
the experiment had ended, and transpiration rate was calculated per unit leaf area. Six plants
per treatment were assessed for whole plant transpiration rate.
2.5 Leaf rehydration ability following a dehydration event
The effect of light intensity on the leaf ability to regain weight lost as a result of dehydration
was investigated. Terminal leaflets were collected, and were allowed to dehydrate to 90, 80,
70 or 60% of the saturated fresh weight (corresponding to 85 ± 2.8, 76 ± 0.5, 63 ± 0.9 and 53
6
± 1.6% RWC). Then the petioles were immediately placed in flasks filled with degassed
water. The leaflets were then incubated for 12 h in the rehydration environment (VPD close
to 0), as explained above, under darkness. The light was then turned on (15 μmol m-2 s-1
PPFD) for 2 h, while leaflets were still under rehydration conditions. Subsequently, leaf fresh
and dry weight was measured.
Measurements were conducted on five leaflets (one leaflet per plant, and one plant per pot)
per treatment.
2.6 Chlorophyll content
To study the effect of irradiance on chlorophyll content the SPAD chlorophyll meter was
used (SPAD-502 meter, Konica Minolta, Tokyo, Japan). Only mature lateral leaflets of the
first and second-leaflet leaves were used for measurements. Fifteen leaves were analysed
from each treatment (one leaf per plant, one plant per pot) on two separate occasions.
2.7 Cuticular water loss
To determine the effect of irradiance on cuticular transpiration rate, mature lateral leaflets of
the first and second-leaflet leaves were detached and double sealed on the abaxial surface by
coating it with vasiline then attaching a polyethylene sheet to it. Subsequently, the leaflets
were left to desiccate in darkness in a test room. Transpiration rate was recorded
gravimetrically every 12 hours during the 84 hour period of desiccation. The environmental
condition in the test room was similar to the condition during the dehydration experiment.
2.8 Photosynthesis irradiance and carbon dioxide response
The effect of growing irradiance on photosynthesis response to irradiance and carbon dioxide
was investigated on lateral leaflets of the first and second five-leaflet leaves (counting from
the apex). Stomatal conductance (gs) was measured using a steady-state porometer (decagon,
XX) four hours after the light was turned on (10:00). The photosynthetic rates (Anet) were
measured using a portable gas analysis system (CIRAS-2, PP systems, Amesbury, MA,
USA). The response of Anet to irradiance was determined by increasing the irradiance from
zero to saturation while keeping the CO2 concentration at ambient (400 µmol mol-1). The
steady-state at each irradiance level was 15 minutes. The Anet were calculated as the mean
value during a 50 second window following the establishment of stable photosynthesis rate.
The curve obtained from Anet response to irradiance data was fitted using a non-rectangular
hyperbola (Thornley, 1976; equation 2) to determine dark respiration (Rd), maximum gross
7
photosynthetic rate (Amg), light limited quantum efficiency (α) and the scaling constant for
the curvature (convexity; θ).
𝐴𝑛𝑒𝑡 =
𝛼×𝑃𝑃𝐹+𝐴𝑚𝑔 −√(𝛼×𝑃𝑃𝐹+𝐴𝑚𝑔 )2 −4𝜃×𝑃𝑃𝐹×𝐴𝑚𝑔
2𝜃
− 𝑅𝑑 (Eqn. 2)
The response of Anet to internal CO2 (A/Ci) response was measured just after reaching
the saturating irradiance level of 1500 µmol m-2 s-1 and ambient CO2 (Ca) of 400 µmol mol-1.
Subsequently, Ca was decreased to 300, 200, 100 and 75 µmol mol-1 before returning to the
initial concentration. This was followed by an increase to 550, 700, 1000, 1200 and 1500
µmol mol-1. At both measurements, the response of Anet to irradiance and CO2-response, the
leaf temperature was set at 20 °C, the air flow at 200 µmol s -1. Readings were recorded when
Anet stabilized to the new condition (after 5 min). The maximum velocity of Rubisco (RuBP)
carboxylation (Vcmax), the maximum rate of electron transport demand for RuBP regeneration
(Jmax), mesophyll conductance (gm), and respiration rate (Rd) were derived from the curve
fitting of the A/Ci data using a supplementary MS Excel sheet provided by (Sharkey et al.
2007).
2.9 Data analysis
Data were analysed by one-way analysis of variance (ANOVA) using R-studio (version
0.98.1103, RStudio, Inc.). Treatment effects were tested at 5% probability level and the mean
separation was done using Tukey’s honest significant difference.
8
3 Results
3.1 Morphological characteristics
All morphological features were significantly affected by irradiance (Table 1). Plant leaf area
was significantly increased with increasing irradiance levels. Plants developed at moderate
and high irradiance levels had 58% and 92% more leaf area as compared to plants developed
at low irradiance levels. However, the leaf area of moderate and high irradiance grown plants
was not significantly different. Leaf mass, stem mass, buds mass and total above ground
biomass were significantly increased with increasing light levels (Table 1.) These mass
characteristics increased by two and three folds with increasing the light to moderate and high
levels respectively. Specific leaf area was significantly higher in leaves grown at high light
levels compared to moderate and low light levels (Table 1). Leaf mass ratio (partitioning of
the aboveground mass to the leaves) was significantly higher in plants grown at high and
moderate light compared to low light levels (Table 1).
Table 1. Leaf and plant morphological characteristics of rose cv Pasadena grown at 100, 200
and 400 µmol m-2 s-1 light levels. Data refers to six replications. Means followed by different
letters indicate significant differences based on Tukey’s Honest Significant difference at P <
0.05 (column).
Treatment
100
200
400
Leaf area
(cm-2)
893±51b
1415±578a
1716±701a
Leaf
mass (g)
3.4±0.2c
5.5±2.2b
8.2±3.3a
Stem
mass (g)
1.1±0.1 c
2.3±0.9 b
3.8±1.5 a
Bud
mass (g)
0.7±0.1 c
1.7±0.7 b
3.1±1.2 a
Total above
ground
biomass (g)
5.1±0.3 c
9.6±3.9 b
15.1±6.2 a
Specific
leaf area
(cm-2 g-1)
0.00382*
0.00389
0.00496
Leaf mass
ratio
0.664 a
0.572 b
0.543 b
3.2 Stomatal characteristics
All stomatal and pore features except stomatal density were significantly affected by
irradiance (Table 2). Stomata on leaves expanded at high and moderate irradiance were
bigger (17% and 24%, respectively) than leaves expanded at low irradiance. Stomatal length
increased by 8% with each increasing light level (i.e. from 100 → 200 → 400 µmol m-2 s-1).
Stomatal width was significantly bigger (11%) in high irradiance grown leaves than low
irradiance. Pore aperture and pore length on leaves expanded at high and moderate irradiance
9
were wider (25% and 32%) and longer (13% and 16%), respectively, than leaves expanded at
low irradiance.
3.3 Stomatal response to dehydration
To investigate the effect of irradiance on plants ability to close stomata and retain water, a
dehydration test was performed. Figure 1 shows the transpiration rate against desiccation
time and leaflet relative water content (RWC). Transpiration rate was highest at the start of
the experiment, before the stomata had closed, and decreased with desiccation time of 60 to
80 min, at which stage it was stabilized in all treatments (Fig. 1A). After a 4 h desiccation,
plants grown at moderate irradiance had significantly more dehydrated leaves (42%) than
high irradiance grown leaves (59%) (P < 0.01, Fig. 1B). Leaves expanded at low irradiance
lost an intermediate amount of their original weight (47%). The result shows that the stomata
of plants developed under high irradiance have better stomatal closure capacity upon stress
and thus are able to retain more water.
Table 2. Stomatal and pore anatomical features of rose cv Pasadena grown at 100, 200 and
400 µmol m-2 s-1 light levels. Measurements took place two hours following the onset of the
light period. Per leaf five fields of view (stomatal density) and 25 stomata (stomatal and pore
anatomy) were analysed. Values are the means of 10 leaves. Means followed by different
letters indicate significant differences based on Tukey’s Honest Significant difference at P <
0.05 (column).
Light levels
(µmol m-2 s-1)
100
200
400
Density
(mm-2)
79±2
74±3
82±3
Stomatal
Length
(µm)
14±0.14c
15±0.18b
16±0.12a
Width
(µm)
11±0.19b
12±0.29ab
12±0.24a
10
Size (µm2)
149±4 b
174±6 a
186±5 a
Pore
Length
(µm)
9.9±0.2b
11.3±0.2a
11.6±0.2a
Aperture
(µm)
5.1±0.2b
6.3±0.2a
6.7±0.2a
1.6
Transpiration rate
(mmol m-2 s-1)
100µmol m-2-s-1
200µmol m-2-s-1
1.2
400µmol m-2-s-1
0.8
0.4
0
0
80
160
240
Time (min)
1.6
Transpiration rate
(mmol m-2s-1)
100µmol m-2 s-1
200µmol m-2s-1
1.2
400µmol m-2-s-1
0.8
0.4
0
100
80
60
Relative water content (%)
40
Figure 1. Leaflet transpiration as a function of time (A) and relative water content (B) of
Rosa hybrida leaves grown under varying irradiance. Data are means±SE (n=8).
3.4 Cuticular transpiration rate
To investigate the effect of irradiance on water loss through the cuticle, a dehydration of
leaflet only from the upper surface (i.e. by sealing the lower surface) was performed. Figure 2
shows cuticular transpiration rate against dehydration time and relative water content.
Cuticular transpiration decreased in all treatments until about 60 hours, after which it was
stabilized in all treatments (Fig. 2A). However, around the end of the experiment, cuticular
transpiration was significantly higher in low irradiance grown leaflets than moderate and high
irradiance. This higher cuticular transpiration rate in leaflets from plants grown at low
irradiance resulted in a more dehydrated (76%) leaflets than moderate (80%) and high (81%)
11
irradiance (Fig. 2B). The result shows that the plants lose significant amounts of water
through the cuticle (up to 24%) in 80 hours of time.
Cuticular transpiration
rate (mmol m-2 s-1)
0.04
100µmol m-2 s-1
200µmol m-2 s-1
0.03
400 µmol m-2 s-1
0.02
0.01
0
0
20
40
60
Time (h)
80
100
0.04
Cuticular transpiration
rate (mmol m-2s-1)
100µmol m-2 s-1
200 µmol m-2 s-1
0.03
400 µmolm -2 s -1
0.02
0.01
0
100
80
60
Relative water content (%)
40
Figure 2. Cuticular transpiration as a function of time (A) and relative water content (B) of
Rosa hybrida leaves grown under low, moderate and high irradiance. Data are means±SE
(n=8).
3.5 Intact plant transpiration
The effect of irradiance on whole plant transpiration was assessed both during day and night.
Daytime water loss was significantly increased with increasing irradiance (Fig. 3A).
Increasing irradiance from 100 to 200 µmol m-2 s-1, increased water loss by 59%. Further,
increasing the irradiance from 200 to 400 µmol m-2 s-1, increased water loss by 23%.
Similarly, night-time water loss was highest in the highest irradiance, and decreased with
decreasing irradiance (Fig. 3B). However, transpiration rate (water loss per unit leaf area and
per unit time) both during the day and night was not significantly affected by irradiance (Fig.
4A and B).
12
160
Weight loss (g)
A, Daytime
120
80
40
0
Weight loss (g)
30
100
200
400
-2
-1
Light level (µmol m s )
B, Nighttime
20
10
0
100
200
400
Light level (µmol m-2 s-1)
Figure 3. Daytime (A) and nighttime (B) water loss of Rosa hybrida leaves grown under low
light (100 µmol m-2 s-1), moderate (200 µmol m-2 s-1) and high (400 µmol m-2 s-1) irradiance.
Data are means±SE (n=6).
13
Transpiration rate
(mmol m-2 s-1)
1.0
A, Daytime
0.8
0.6
0.4
0.2
0.0
Tranpsiration rate
(mmol m-2 s-1)
0.5
100
200
400
-2
-1
Light level (µmol m s )
B, Nighttime
0.4
0.3
0.2
0.1
0.0
100
200
400
Light level (µmol m-2 s-1)
Figure 4. Daytime (A) and night-time (B) intact plant transpiration of Rosa hybrida leaves
grown under low light (100 µmol m-2 s-1), moderate (200 µmol m-2 s-1) and high (400 µmol
m-2 s-1) irradiance. Data are means±SE (n=6).
3.6 Rehydration ability following a dehydration event
To investigate the effect of irradiance on the leaflet ability to rehydrate after dehydration,
leaflets were left to dehydrate to a predefined RWC, and were subsequently rehydrated
overnight. In all treatments, leaflets subjected to all levels of dehydration (ranging between
50 and 90% RWC), fully recovered their weight (Fig. 5). The result shows that leaflet
dehydration was reversible upon rehydration in all treatments.
3.7 Chlorophyll content
The effect of irradiance on chlorophyll content was investigated using chlorophyll meter
(SPAD). High irradiance grown leaves had significantly higher chlorophyll content than
moderate and low irradiance (Fig. 6).
14
RWC after rehydration (%)
100
80
100 µmol m-2 s-1
60
200 mmol m-2 s-1
400 mmol m-2 s-1
40
100
80
60
RWC before rehydration (%)
40
Figure 5. Leaflet relative water content (RWC) following overnight (12 h) rehydration, as
function of RWC before rehydration of pot rose ‘Pasadena’ grown under low, moderate and
high irradiance. Data are means±SE (n=5).
60
a
58
SPAD
56
54
b
b
52
50
48
100
200
400
-2
-1
Light levels (µmol m s )
Figure 6. Chlorophyll index of Rosa hybrida leaves grown under low light (100 µmol m-2 s1
), moderate (200 µmol m-2 s-1) and high (400 µmol m-2 s-1) irradiance. Data are means±SE
(n=30). Different letters indicate significant differences based on Tukey’s Honest Significant
difference at P < 0.05.
15
3.8 Photosynthetic acclimation
The effect of growth light level on photosynthetic rate was investigated using a gas exchange
analyser. The photosynthetic capacity (Amg) was higher in high irradiance grown leaves
followed by the moderate and low irradiance (Fig. 7A and Table 3). At saturated
photosynthetic active radiation (PAR) level of 1500 µmol m-2 s-1, the maximum
photosynthetic rate at low, moderate and high irradiance was 14, 15 and 19 µmol m-2 s-1,
respectively. (Fig 7A). Quantum yield decreased with increasing light levels in all treatments
(Fig. 7B). Quantum yield decreased at a faster rate at low and moderate light grown leaves
than high light. For instance, at 500 µmol m-2 s-1, the quantum yield at low, moderate and
high irradiance grown leaves were 0.008, 0.009 and 0.014 mmol CO2 per mmol photon,
respectively. The light limited quantum efficiency (α) and the curvature parameter did not
differ significantly among the treatments (Table 3). Dark respiration (Rd) was smallest for the
low irradiance grown leaves and largest for the highest leaves (Table 3).
Table 3. Fitted photosynthesis rate parameters of fully expanded Rosa hybrida leaves grown
under low light (100 µmol m-2 s-1), moderate (200 µmol m-2 s-1) and high (400 µmol m-2 s-1)
irradiance. Data are means ± SE (n=6). Different letters in a row indicate significant
difference based on Tukey’s Honest Significant difference at P < 0.05.
parameters
Net photosynthesis light response curve
Rd
Amg
α
θ
Net photosynthesis CO2 response curve
Vcmax
Jmax
Jmax/ Vcmax ratio
Low light
Treatment
Moderate light
High light
0.17±0.08b
15.5±2.8
0.051±0.003
0.64±0.04
0.29±0.02ab
16.8±2.4
0.045±0.004
0.73±0.04
0.72±0.16a
21.8±0.9*
0.042±0.001
0.72±0.04
77±7
134±11
1.8±0.04
57±5
109±1
1.9±0.15
102±7
166±9
1.6±0.05
At a saturated PAR level of 1500 µmol m-2 s-1, the A/Ci response curve showed that
photosynthesis rate was significantly limited at high intercellular CO2 values in low and
moderate irradiance grown leaves than high irradiance grown leaves (Fig. 8). Similarly,
calculated values of Vcmax and Jmax, were highest at high irradiance grown leaves than at
moderate and low irradiance leaves. The Jmax to Vcmax ratio were not significantly different
among treatments (Table 3).
16
Photosynthesis rate
[mmol (CO2) m-2 s-1]
25.00
20.00
15.00
10.00
5.00
0.00
0
-5.00
250
500
750 1000 1250 1500 1750 2000 2250
Photosynthetic active radiation [mmol (photons) m-2 s-1]
QUANTUM YIELD
[mmol (CO2) mmol-1
(photons)]
0.0600
0.0500
0.0400
0.0300
0.0200
0.0100
0.0000
0
250
500
750 1000 1250 1500 1750 2000 2250
Photosynthetic active radiation [mmol (photons) m-2 s-1]
Figure 7. The effect of change in growth irradiance on the photosynthetic irradiance response
(A) of Rosa hybrida leaves grown under low light (100 µmol m-2 s-1; solid line), moderate
(200 µmol m-2 s-1; dashed line)and high (400 µmol m-2 s-1; dotted lines) irradiance. Data are
means±SE (n=6).
17
A (µmol CO2 m-2 s-1)
40
30
20
10
0
0
200
400
600
800
1000
-1
Ci (mmol mol )
1200
1400
Figure 8. The effect of change in growth irradiance on the response of photosynthesis to
intercellular CO2 (A/Ci) of Rosa hybrida leaves grown under low light (100 µmol m-2 s-1;
solid line), moderate (200 µmol m-2 s-1; dashed line) and high (400 µmol m-2 s-1; dotted lines)
irradiance. Data are means±SE (n=6).
18
4 Discussion
Leaves expanded at high irradiance have larger leaf area and are thinner
Incident irradiance during growth has been shown to affect the composition and organization
of biomass as well as leaf morphology. Several studies have described irradiance effect on
plant morphology. In general, high irradiance resulted in plants with a larger leaf area (James
and Bell, 2000), decreased SLA (Evans and Poorter, 2001) and decreased LMR (Feng et al.,
2007) as compared to plants grown under low light. We show that the plants grown under
high light has almost two fold higher leaf area and decreased LMR (19%) compared to plants
grown at low light condition. Our experiment also showed plants growing at high light have
significantly increased shoot biomass (Table 1). However, our experiment showed a higher
SLA (i.e. more light intercepting leaf area per unit biomass-thinner leaves), contrary to most
irradiance experiments. This is may be due to the small irradiance range (100 – 400 µmol m-2
s-1) employed in the experiment.
High irradiance grown leaves have bigger stomata and wide pore aperture
Stomatal anatomy and functionality are controlled by genetic as well as environmental factors
such as light. Light intensity significantly affected almost all examined stomatal features.
Leaves grown under high irradiance had significantly bigger (20%) stomata than low
irradiance leaves. The bigger stomatal size is as a result of both longer and wider stomata
(Table 2). Similarly, pore length increased significantly with irradiance. In agreement to our
results, Thomas et al (2004) reported that increasing irradiance from 90 to 250 µmol m-2 s-1
resulted in an increase in pore length. Pore aperture also increased significantly with
increasing irradiance. However, no statistically significant effect of irradiance on stomatal
density was observed.
Stomata from high irradiance grown leaves close faster
Transpiration rate decreased strongly with dehydration in low and high irradiance (Fig. 1).
After 4 h of dehydration, low and moderate irradiance grown leaves lost more water (i.e
lower RWC) than high irradiance leaves. Previous works by Sack and Scoffoni (2012)
indicated that stomata respond strongly to irradiance. In experiments conducted in
Raphiolepis indica the stomatal conductance and leaf hydraulic conductance declined
strongly with dehydration for leaves measured under high irradiance. The sensitivity of
19
stomata closing to leaf dehydration may be in part related to synthesis or apoplastic
redistribution of ABA and/or ethylene, or increased tissue sensitivity to hormones.
Cuticular transpiration is higher in low irradiance grown leaves
The cuticle forms an effective barrier protecting plants from the uncontrolled loss of water
and it reduces infection by pathogens (Karbulkova et al., 2008). During water stress, when
stomata are closed, plant survival depends on the amount of water lost through the cuticle.
However, to the best of my knowledge, a single literature was not found that studied
plasticity of cuticular permeability to varying irradiance. Here, reported for the first time,
leaflets developed at low irradiance had higher cuticular transpiration rates (cuticular
permeability) compared to moderate and high irradiance (Fig. 2A and B). The lower cuticular
permeability of high irradiance grown leaves could be a result of a feedback from the high
stomatal conductance in these leaves. Higher stomatal conductance means more water
passing through the stomatal pores and hence less water is available in the epidermis to pass
through the cuticles. Eamus et al (2008) using experimental data and model predictions
showed that cuticular transpiration influenced stomatal conductance by a feedback
mechanism, i.e. increasing leaf-to-air vapour pressure difference, increased cuticular
transpiration and hence the ability of epidermis to supply waters to guard cells. Our data is
also in agreement, albeit indirectly, with Schreiber and Riederer (1996) survey of plants from
different habitats. They found that plants from temperate climate have the highest cuticular
permeability while tropical epiphytes (i.e high irradiance acclimatized plants) have the
lowest. Therefore, plants modulate their cuticular permeability in tandem with stomatal
conductance to adapt to the prevailing environment.
Whole plant transpiration rate at growth condition was not affected by irradiance
Plant water loss is driven by evaporative demand and radiation load on leaves. Besides these
two, water loss at plant level also involves two additional components, a functional (under
stomatal control) and a structural (total transpiring surface area) one. Plants at high irradiance
lost more water compared to plants at moderate irradiance both during the day and night (Fig.
3A and B). However, when the water lost is expressed per unit leaf area (i.e. transpiration
rate), no statistically significant difference was observed (Fig. 4A and B). However, high
irradiance plants had larger leaf area that low irradiance plants. Therefore, the observed
increase in water loss per plant basis was mainly determined by the structural component.
20
No effect of irradiance on reversible dehydration
Plants grown under varying irradiance and subjected to dehydration had similar recovery
(rehydration) following re-watering (Fig. 5). Rehydration after a dehydration event involves
embolism refilling (replacing the air in the xylem vessels with water) and this process has
been shown to be enhanced by leaf abscisic acid (ABA) concentration (Secchi et al., 2012).
More recently, Giday et al (2014) also showed that recovery following re-watering was
closely related to abscisic acid concentration in the leaf. Despite not measuring ABA in the
current experiment, we may speculate that its concentration is similar among the treatments.
High irradiance grown leaves had a higher photosynthetic acclimation
Light intensity has been known to show changes in photosynthetic capacity in plants.
Photosynthetic capacity was found to be higher in plants grown under high irradiance than
plants grown under moderate and low irradiance levels (Fig 7A and Table 3). Also, both the
maximum rate of photosynthesis and quantum yield was increased with rising levels of
irradiance (i.e. more mmol CO2 per mmol photon were utilized as light intensity increased
(Fig 7A and B). It is well documented that light intensity affects a number of component
processes of photosynthesis during plant growth, such as leaf morphology and chloroplast
structure (Boardman et al., 1975; Wild and Wolf, 1980; Lichtenthaler et al., 1981). In contrast
to low light leaves, chloroplasts of leaves grown under high irradiances have been known to
have higher electron transport chains per a chlorophyll basis, thus possessing a higher
capacity for photosynthetic quanta conversion at light saturation (Lichtenthaler and
Buschmann, 1978; Wild, 1979). Observed rates of higher maximum photosynthesis,
photosynthetic capacity may have been partly due to more efficient sun-type chloroplasts in
leaves grown under higher light intensity. Quantum yield increases seem to be due to higher
levels of RuBP-carboxylase as has been shown for leaves grown under high irradiance
(Björkman, 1968). Dark respiration decreased significantly with decreasing irradiance (Table
3). The decreased dark respiration is an adaptation mechanism by the low irradiance grown
leaves to allow for a higher net photosynthesis (Boardman et al, 1977).
21
22
5 Conclusions
Morphological features were affected by irradiance. Plants developed at moderate and high
irradiance levels had 58% and 92% more leaf area as compared to plants developed at low
irradiance levels. All stomatal and pore features except stomatal density were significantly
affected by irradiance. Plant stomata developed under high irradiance had better closuring
ability when stressed than plants grown under low irradiance, thus allowing them to retain
more water. Water loss through the cuticle was found to increase as irradiance decreased.
Transpiration rate (water loss per unit leaf area and per unit time) was not affected by light
intensity. However, there was an 83% increase in water loss in plants grown at high
irradiance than plants grown under low irradiance. This increase was explained by the larger
over all mass of the plants grown in high irradiance (i.e. increased water usage). Under all
levels of irradiance (low, moderate and high) leaves of plants showed full recovery from
dehydration. Chlorophyll content was increased in plants grown under high light intensity
over plants grown under low light intensity. Photosynthetic capacity showed a step wise
increase with irradiance levels. Plants grown at higher irradiance had a higher quantum yield
capacity. Dark respiration rates showed a decrease as irradiance decreased. At high C i
values, photosynthetic rates were limited as irradiance decreased. In the same way Vcmav and
Jmax were limited as irradiance lowered.
23
24
Acknowledgments
I wish to express my great appreciation and gratitude to Carl-Otto Ottosen for his engaging
conversations throughout both my open project and thesis work. He always had a smile on his
face and a laugh for me each time we talked. Furthermore, I would like to convey my deepest
respect for his patience with helping me in times of difficulty and low points. Also, I would
like to express my immense gratefulness to Habtamu Giday for dedicating many hours in
helping me with valuable comments, suggestions and his answering of my unending
questions. Moreover I want to put forth my gratitude for your relentless positive attitude and
friendship throughout my work. I would like to thank my beloved girlfriend Kristine Ida for
your unconditional love, understanding and support throughout my busy and trying times.
Last, but in no way least, I want to humbly express my utmost appreciation toward my
parents Joseph and Cathy Aulik for your inexpressible and inexhaustible love, support,
understanding, encouragement, insight, and wisdom throughout my life and time in Denmark.
25
26
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