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 Aim and outline of the thesis ................................................................................... 4

2 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

3 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

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

2

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).

3

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 CO2

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.

4

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.

5

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.

6

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.

RWC =Fresh weight−dry weight

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

7

± 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

8

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.

9

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

Leaf area

(cm-2

)

Leaf

mass (g)

Stem

mass (g)

Bud

mass (g)

Total above

ground

biomass (g)

Specific

leaf area

(cm-2

g-1

)

Leaf mass

ratio

100 893±51b 3.4±0.2

c 1.1±0.1

c 0.7±0.1

c 5.1±0.3

c 0.00382* 0.664

a

200 1415±578a 5.5±2.2

b 2.3±0.9

b 1.7±0.7

b 9.6±3.9

b 0.00389 0.572

b

400 1716±701a 8.2±3.3

a 3.8±1.5

a 3.1±1.2

a 15.1±6.2

a 0.00496 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

10

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

)

Stomatal Pore

Density

(mm-2

)

Length

(µm)

Width

(µm)

Size (µm2) Length

(µm)

Aperture

(µm)

100 79±2 14±0.14c 11±0.19

b 149±4

b 9.9±0.2

b 5.1±0.2

b

200 74±3 15±0.18b 12±0.29

ab 174±6

a 11.3±0.2

a 6.3±0.2

a

400 82±3 16±0.12a 12±0.24

a 186±5

a 11.6±0.2

a 6.7±0.2

a

11

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%)

0

0.4

0.8

1.2

1.6

406080100

Tra

nsp

irati

on

rate

(mm

ol

m-2

s-1

)

Relative water content (%)

100µmol m-2 s-1

200µmol m-2s-1

400µmol m-2-s-1

0

0.4

0.8

1.2

1.6

0 80 160 240

Tra

nsp

irati

on

rate

(m

mo

l m

-2s

-1)

Time (min)

100µmol m-2-s-1

200µmol m-2-s-1

400µmol m-2-s-1

12

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.

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).

0

0.01

0.02

0.03

0.04

0 20 40 60 80 100

Cu

tic

ula

r tr

an

sp

irati

on

ra

te (

mm

ol

m-2

s-1

)

Time (h)

100µmol m-2 s-1

200µmol m-2 s-1

400 µmol m-2 s-1

0

0.01

0.02

0.03

0.04

406080100

Cu

tic

ula

r tr

an

sp

irati

on

ra

te (

mm

ol

m-2

s-1

)

Relative water content (%)

100µmol m-2 s-1

200 µmol m-2 s-1

400 µmolm -2 s -1

13

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).

0

40

80

120

160

100 200 400

Weig

ht

loss (

g)

Light level (µmol m-2 s-1)

A, Daytime

0

10

20

30

100 200 400

Weig

ht

loss (

g)

Light level (µmol m-2 s-1)

B, Nighttime

14

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).

0.0

0.2

0.4

0.6

0.8

1.0

100 200 400

Tra

ns

pir

ati

on

ra

te(m

mo

l m

-2s

-1)

Light level (µmol m-2 s-1)

A, Daytime

0.0

0.1

0.2

0.3

0.4

0.5

100 200 400

Tra

np

sir

ati

on

rate

(mm

ol

m-2

s-1

)

Light level (µmol m-2 s-1)

B, Nighttime

15

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).

Figure 6. Chlorophyll index 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=30). Different letters indicate significant differences based on Tukey’s Honest Significant

difference at P < 0.05.

40

60

80

100

406080100

RW

C a

fte

r re

hyd

rati

on

(%

)

RWC before rehydration (%)

100 µmol m-2 s-1

200 mmol m-2 s-1

400 mmol m-2 s-1

48

50

52

54

56

58

60

100 200 400

SP

AD

Light levels (µmol m-2 s-1)

b

b

a

16

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 Treatment

Low light Moderate light High light

Net photosynthesis light response curve

Rd 0.17±0.08b 0.29±0.02

ab 0.72±0.16

a

Amg 15.5±2.8 16.8±2.4 21.8±0.9*

α 0.051±0.003 0.045±0.004 0.042±0.001

θ 0.64±0.04 0.73±0.04 0.72±0.04

Net photosynthesis CO2 response curve

Vcmax 77±7 57±5 102±7

Jmax 134±11 109±1 166±9

Jmax/ Vcmax ratio 1.8±0.04 1.9±0.15 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).

17

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).

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

0 250 500 750 1000 1250 1500 1750 2000 2250

Ph

oto

syn

thesis

rate

[mm

ol

(CO

2)

m-2

s-1

]

Photosynthetic active radiation [mmol (photons) m-2 s-1]

0.0000

0.0100

0.0200

0.0300

0.0400

0.0500

0.0600

0 250 500 750 1000 1250 1500 1750 2000 2250

QU

AN

TU

M Y

IEL

D[m

mo

l (C

O2)

mm

ol-1

(ph

oto

ns

)]

Photosynthetic active radiation [mmol (photons) m-2 s-1]

18

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).

0

10

20

30

40

0 200 400 600 800 1000 1200 1400

A (

µm

ol

CO

2m

-2s

-1)

Ci (mmol mol-1)

19

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

20

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.

21

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).

22

23

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 Ci

values, photosynthetic rates were limited as irradiance decreased. In the same way Vcmav and

Jmax were limited as irradiance lowered.

24

25

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.

26

27

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