Photosynthesis and Protection from Light in Annual Desert Plant
Species
Thesis submitted in partial fulfillment
of the requirements for the degree of
“DOCTOR OF PHILOSOPHY”
by
Amir
Eppel
Submitted to the Senate of Ben-Gurion University
of the Negev
52.7.2013
Beer-Sheva
Photosynthesis and Protection from Light in Annual Desert Plant
Species
Thesis submitted in partial fulfillment
of the requirements for the degree of
“DOCTOR OF PHILOSOPHY”
by
Amir
Eppel
Submitted to the Senate of Ben-Gurion University
of the Negev
Approved by the advisor
Approved by the Dean of the Kreitman School of Advanced Graduate Studies
25.7.2013
Beer-Sheva
1
This work was carried out under the supervision of
Shimon Rachmilevitch
In the Department of Dryland agriculture and Biotechnology
Faculty: Institutes of desert research
2
Research-Student's Affidavit when Submitting the Doctoral Thesis for
Judgment
I, Amir Eppel, whose signature appears below, hereby declare that
(Please mark the appropriate statements):
___ I have written this Thesis by myself, except for the help and guidance offered by my Thesis
Advisors.
___ The scientific materials included in this Thesis are products of my own research, culled from
the period during which I was a research student.
___ This Thesis incorporates research materials produced in cooperation with others, excluding
the technical help commonly received during experimental work. Therefore, I am attaching
another affidavit stating the contributions made by myself and the other participants in this
research, which has been approved by them and submitted with their approval.
Date: _________________
Student's name: Amir Eppel
3
Signature:______________
I dedicate this work to my family
I would like thank the students and technicians of Dr. Shimon Rachmilevitch for their help and
support
4
Contents
1. Abbreviations .............................................................................................................................. 7
2. List of Figures ............................................................................................................................. 8
3. List of tables ................................................................................................................................ 9
4. Abstract ..................................................................................................................................... 11
5. Introduction ............................................................................................................................... 14
5.1. Light and photodamage in plants ....................................................................................... 14
5.2. Mechanisms that reduce photodamage by lowering light absorption of PSII.................... 15
5.3. Mechanisms that mitigate photodamage after light absorption by the antenna of PSII ..... 15
5.3.1. Non-photochemical quenching (NPQ) ........................................................................ 15
5.3.2. Photorespiration and the water-water cycle................................................................. 17
5.3.3. Scavenging mechanism of singlet oxygen by an antioxidant ...................................... 18
5.4. Drought and excess light .................................................................................................... 18
5.5. Protection from excess light in desert plants ...................................................................... 19
5.6. Research goal and hypothesis ............................................................................................ 20
5.6.1. Subject 1 of the research: Photoprotective response under drought in wild barley
ecotypes from Mediterranean and desert origins ................................................................... 20
5.6.2. Specific objective for Study 1 ..................................................................................... 22
5.6.3. Subject 2 of the research: NPQ and other light-driven processes in the annual desert
plant Anastatica hierochuntica .............................................................................................. 22
5.6.4. Specific objective for Study 2 ..................................................................................... 23
6. Materials and Methods .............................................................................................................. 24
6.1. Measurements of photosystem II efficiency and non-photochemical quenching using
chlorophyll fluorescence. .......................................................................................................... 24
6.1.1. Maximum photochemical efficiency- Fv/Fm .............................................................. 24
6.1.2. Photochemical efficiency of PSII in the light: ФPSII and electron transport rate ...... 25
6.1.3. Non-photochemical quenching of PSII, NPQ and ФNPQ .......................................... 26
6.2. Plant materials .................................................................................................................... 26
6.2.1. Study 1 ......................................................................................................................... 26
6.2.2. Study 2 ......................................................................................................................... 27
6.3. Photosynthesis and chlorophyll fluorescence measurements............................................. 28
6.4. Leaf absorbance.................................................................................................................. 30
5
6.5. The excitation distribution between photosystem I and II ................................................. 30
6.6. Leaf relative water content ................................................................................................. 31
6.7. Pigment content and analysis ............................................................................................. 31
6.8. Plant dry weight ................................................................................................................. 32
7. Results ....................................................................................................................................... 33
7.1. Photo protective response under drought in wild barley ecotypes from Mediterranean and
desert origins ............................................................................................................................. 33
7.1.1. Effect of prolonged drought on soil water content and on plant biomass ................... 33
7.1.2. Changes in leaf pigmentation in response to drought, in the Mediterranean and desert
ecotypes of H. spontaneum. ................................................................................................... 34
7.1.3. Absorbance of photosynthetic active radiation and distribution of light to PSII in the
Mediterranean and desert ecotypes of H. spontaneum. ......................................................... 35
7.1.4. Leaf water status and CO2 assimilation in the Mediterranean and desert ecotypes of H.
spontaneum. ........................................................................................................................... 37
7.1.5. Efficiency of photosystem II in the Mediterranean and desert ecotypes of H.
spontaneum. ........................................................................................................................... 39
7.1.6. Non-photochemical quenching and the O2 dependency of PSII photochemical activity
............................................................................................................................................... 41
7.2. Non-photochemical and photochemical processes in the annual desert plant Anastatica
hierochuntica ............................................................................................................................. 44
7.2.1. Initial experiment: NPQ in Anastatica hierochuntica and Arabidopsis thaliana under
different irrigation salinities, setting the background ............................................................ 44
7.2.2. Non-photochemical quenching in A. hierochuntica .................................................... 45
7.2.3. Photosystem II electron transport rate in A. hierochuntica and other plant species .... 55
7.2.4. Carbon assimilation, photorespiration, respiration and stomata conductance in A.
hierochuntica and other plant species. .................................................................................. 60
7.2.5. The response of photosynthesis PSII ETR and NPQ to drought, in A. hierochuntica
and H. annuus ........................................................................................................................ 68
8. Discussion ................................................................................................................................. 72
8.1. Photoprotective response under drought in wild barley ecotypes from Mediterranean and
desert origins ............................................................................................................................. 72
8.2. Non-photochemical and photochemical processes in the annual desert plant Anastatica
hierochuntica ............................................................................................................................. 74
9. Conclusion ................................................................................................................................ 80
6
01. References ............................................................................................................................... 81
‫ תקציר‬.00 ........................................................................................................................................ 90
1. Abbreviations
ATP- Adenosine triphosphate
Ci- Leaf internal CO2 concentration
DES- De epoxidation state of xanthophylls
Dw- Dry weight
ETR- Electron transport rate
Fw- Fresh weight
LEF- Linear electron flow
LRWC- Leaf relative water content
NPQ- Non-photochemical quenching
PPFD- Photosynthetic photon flux density
PAR- Photosynthetic active radiation
PSI-Photosystem I
PSII- Photosystem II
RH- Relative humidity
Tw -Turgid weight
WUE- Water use efficiency
7
2. List of Figures
Figure 1: Leaves of the Mediterranean and desert ecotypes of Hordeum spontaneum.
Figure 2: Leaf absorbance of the Mediterranean and desert ecotypes of H. spontaneum.
Figure 3: Leaf relative water content, stomata conductance and carbon assimilation of the
Mediterranean and desert ecotypes of H. spontaneum.
Figure 4: Maximum efficiency of PSII and ФPSII of the Mediterranean and desert ecotypes of H.
spontaneum.
Figure 5: Non-photochemical quenching and the efficiency of non-photochemical quenching of
the Mediterranean and desert ecotypes of H. spontaneum.
Figure 6: Photosystem II yield at low and ambient oxygen concentrations of the Mediterranean
and desert ecotypes of H. spontaneum.
Figure 7: NPQ response in Anastatica hierochuntica and other growth chamber grown plant
species, at different light intensities.
Figure 8: NPQ response in A. hierochuntica and other field-grown plant species in the Negev
Desert.
Figure 9: NPQ response in A. hierochuntica and other plant species grown in a growth chamber,
and at different CO2 and O2 concentrations.
Figure 10: Photosystem II electron transport rate (PSII ETR) at different light intensities, in A.
hierochuntica and other plant species grown in a growth chamber with moderate light intensity.
Figure 10: NPQ response in A. hierochuntica and other plant species grown in a growth
chamber, and at different CO2 and O2 concentrations.
Figure 11: PSII ETR at different CO2 concentrations in A. hierochuntica and other plant species.
Figure 12: PSII ETR in A. hierochuntica and A. thaliana, at different O2 and CO2
concentrations.
8
Figure 13: Stomata conductance, net assimilation and water-use efficiency at different light
intensities, in A. hierochuntica and other plant species.
Figure 15: Net assimilation at different CO2 concentrations in A. hierochuntica and other plant
species.
Figure 16: The influence of non-photo respiratory conditions (2% O2) on carbon assimilation,
and the impact of light intensity on dark respiration in A. hierochuntica and A. thaliana.
Figure 17: The ratio of PSII ETR to NPQ, at various light intensities, in A. hierochuntica and in
other plants species.
Figure 18: The ratio of PSII ETR to NPQ, at low and atmospheric CO2 concentrations
concentrations, in A. hierochuntica and in other plant species.
Figure 19: Net carbon assimilation at average and high CO2 concentrations as a function of
stomata conductance, in response to drought and irrigation treatments in A. hierochuntica and H.
annuus.
Figure 20: The relation between PSII ETR and NPQ to stomata conductance in irrigated and
drought-treated plants of A. hierochuntica and H. annuus.
.
3. List of tables
Table 1: Dry weight of shoot and root of the Mediterranean and desert ecotypes of Hordeum
spontaneum.
Table 2: Anthocyanin and chlorophyll concentrations of the Mediterranean and desert ecotypes
of Hordeum spontaneum.
Table 3: Absorbance of photosynthetically active radiation and light excitation distribution to
photosystem II in light-exposed leaves of the Mediterranean and desert ecotypes of H.
spontaneum.
Table 4: PSII activity in Anastatica hierochuntica and Arabidopsis thaliana, in response to
salinity stress.
9
Table 5: Photochemical potential of photosystem II and pigment composition in leaves of A.
hierochuntica and the other plants in the experiment.
Table 6: Light absorbance and distribution of light energy between PSII and PSI, in leaves of A.
hierochuntica and A. thaliana.
Table 7: De-epoxidation state of xanthopylls and NPQ at high light intensity in A. hierochuntica
and A. thaliana.
Table 8: The VDE protein sequence in different plants, including A. hierochuntica.
Table 9: Photosystem II electron transport rate (PSII ETR) in A.hierochuntica plants that were
grown in different environments.
Table 10: Stomata conductance and net carbon assimilation in A. hierochuntica plants that were
grown in different light intensities.
10
4. Abstract
Light is essential for plant growth, since it provides the energy that drives carbon assimilation
through photosynthesis. The energy of light, in the visible spectrum, is absorbed by light
harvesting protein-pigment complexes that are found in the chloroplasts of plants. The energy
absorbed is funneled to drive the primary reaction that converts the energy of light into chemical
energy, which occurs at photosystem II (PSII) reaction centers. As a byproduct of this reaction,
there is continuously occurring photodamage to the PSII reaction centers, which requires
constant repair; if the rate of damage creation exceeds the rate of repair, it leads to long-term
damage to PSII, which is termed photoinhibition. Environmental stresses, such as drought,
increase the risk of photoinhibition in plants. In order to avoid photoinhibition, plants possess
various mechanisms to reduce the damage in PSII. Photoprotective mechanisms can be divided
into two forms: mechanisms that reduce the amount of light that is absorbed by the light
harvesting complexes and mechanisms that mitigate the potentially damaging energy after it has
been absorbed by the light harvesting complexes. One of the mechanisms that reduce light
absorbance is the accumulation of non-photosynthetic pigments, such anthocyanin. Two
important mechanisms that deal with energy after it has been absorbed by PSII are nonphotochemical quenching (NPQ), and photorespiration. NPQ diverts energy directly and
harmlessly into heat. Photorespiration is another process that protects PSII from photoinhibition
in an oxygen-dependent manner.
Annual desert plants are exposed to high light intensity that can cause photodamage, especially
when photosynthesis is inhibited due to unfavorable environmental conditions, such as drought
and a high vapor pressure deficit. On the other hand, annual plants need to complete their life
cycle in a relatively short period of time, before water is lost from the soil due to the high
evaporation that occurs in deserts.
The goal of this research was to study how photoprotective mechanisms are used by annual
desert plants, under various conditions. In order to address this goal, two different studies were
performed.
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In the first study, plants of Hordeum spontaneum (wild barley) that were collected from
Mediterranean and desert environments were subjected to terminal drought for 25 days, in order
to examine the photoprotective response to this stress condition. In both ecotypes, the drought
caused significant and similar decrease in growth compared to the control irrigated plants. At the
single leaf level, photosynthesis was almost completely inhibited, in comparison to the control
plants. The values of Fv/Fm, an indicator of photoinhibiton, were only slightly lower in droughttreated plants of both ecotypes, suggesting that both ecotypes employed an efficient
photoprotective response under drought. In the Mediterranean ecotype, drought-treated plants,
NPQ increased significantly compared to the control plants. In the drought-treated plants of the
desert ecotype, there was a high accumulation of anthocyanin, a non-photosynthetic pigment,
which lowered the amount of light received by PSII. As a result, the efficiency of PSII (ФPSII)
remained relatively high in the desert ecotype under drought, and NPQ didn’t increase
significantly. The relatively high ФPSII, in the desert ecotype under drought, was mostly O2
dependent, suggesting an important role for photorespiration under these conditions. In
conclusion, the photoprotective response to drought differed between the two ecotypes of H.
sponatneaun. In the Mediterranean ecotype, NPQ induction was the major response, while in the
desert ecotype; anthocyanin accumulation and photorespiration were induced under drought.
In the second study, the photoprotective and photosynthetic response were examined in the
annual desert plant Anastatica hierochntica (Rose of Jericho-Brassiceace), under various
conditions. This was a comparative study using three other well-studied plant species, with
different photosynthetic and NPQ capacities: the model plant Arabidopsis thaliana, the stresstolerant plant Thellungiella salsuginea (both are Brassicaceae) and the crop plant sunflower
(Helianthus annuus – Asteraceae). A. hierochuntica plants had a significantly lower and slower
induction of NPQ as compared to the other species upon exposure to various light intensities and
CO2 concentrations. Low NPQ in A. hierochuntica was not the result of low light absorbance by
PSII but was associated with the low de-epoxidation state of xanthophyll pigments, which are
known to control NPQ. The PSII electron transport rate (PSII ETR) in A. hierochuntica, was
high and was much less sensitive to a decrease in CO2 concentrations, compared to the other
plant species. Upon exposure to low CO2, PSII ETR was mostly O2 dependent
(photorespiration).
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When CO2 concentrations were moderate or high, the carbon assimilation rates in A.
hierochuntica were similar to H. annuus, a crop plant with a high photosynthetic rate, and were
higher than in A. thaliana and T. saslsuginea. A. hierochuntica had high photorespiration rates at
high light intensity and high dark respiration rates after exposure to high light intensities.
Therefore, the high photochemical activity in A. hierochuntica was mainly used to drive carbon
metabolism, under favorable conditions.
Exposure to prolonged drought caused a similar decrease in stomata conductance and in carbon
assimilation in A. hierochuntica and H. annuus. The inhibition of photosynthesis was due to low
CO2 concentrations in the chloroplasts, since the inhibition of photosynthesis under drought was
reversed upon the application of high CO2 concentrations, in both plant species. Under drought,
NPQ increased and PSII ETR decreased similarly in both plant species compared to the control
irrigated plants, suggesting similar dynamics of photoprotection and photosynthetic down
regulation.
According to my knowledge, the unique photosynthetic and photoprotective characteristics and
mechanisms in A. hierochuntica have not been previously described for any other plant species.
The photoprotective and photosynthetic mechanisms in A. hierochuntica might represent a
unique adaptation to the short growing season of an annual desert plant.
Overall, this research suggested, according to two different experiments, that photoprotective
mechanisms in annual desert plants have some features that are less common in plants from other
environments, and might imply an adjustment of these photo protective mechanisms to the
desert environment.
13
5. Introduction
5.1. Light and photodamage in plants
Light is vital for plant growth and productivity by producing energy to reduce carbon dioxide to
sugar through photosynthesis. Plants absorb the energy of the visible light using the antenna
complexes. Antenna complexes include chlorophyll pigments, carotenoid pigments and proteins.
Energy absorbed by the antennas is transferred to photosystem I and II (PSI and PSII) reaction
centers. In PSI and PSII light reactions, the primary conversion of light energy into chemical
energy occurs (Nelson and Ben-Shem, 2004). As a byproduct of the light reaction, chlorophyll
triplets (3Chls) are formed in the reaction centers of PSII. Chlorophyll triplets can interact with
oxygen molecules (O2) to produce singlet oxygen (1O2) (Krieger-Liszkay et al., 2008). 1O2 is a
highly reactive oxygen species (ROS) that can cause damage to various cellular components. The
damage of 1O2 occurs mainly in the proteins and pigments of photosystem II (PS II), and mainly
to the protein D1 (Adir et al., 2003) . The proteins of PSII are the closest and most common
target of 1O2. Plants and other photosynthetic organisms constantly repair the damage created by
light (photodamage), by degrading the damaged proteins and synthesizing new ones, thus
preventing long term damage. Long-term damage to the photosynthetic ability is termed as
Photoinhibition (Raven, 2011).
Conditions of low CO2 availability limit carbon assimilation and the formation of high energy
sugar molecules. Under these conditions, plants are more susceptible to photodamage and
photoinhibtion (Baroli and Melis, 1998). Stress conditions (drought, heat, cold, salinity oxidative
conditions and low CO2 concnetrations) impair the repair processes of PSII. Photoinhibition
occurs in conditions in which the repair process is slower than the formation of photodamage,
and in this manner, stress conditions can also cause photoinhibition (Takahashi and Murata,
2008).
In recent years, it was suggested that photoinihibtion also occurs independently of light
absorbance by photosynthetic pigments (Tyystjarvi, 2008). This proposed mechanism of
photoinhibition occurs through direct light absorbance by the oxygen evolving complex (OEC)
of PSII, which causes it to destabilize, therefore impairing the light reaction (Tyystjarvi, 2008).
The relative contributions of these two different mechanisms to photodamage and inhibition in
plants is currently under debate (Vass, 2012, Kornyeyev et al., 2010). In this work, the function
14
and response of plants to the potential damage of light was studied within the framework of light
absorbance by PSII antennas, but not within the framework of direct absorbance by the OEC.
5.2. Mechanisms that reduce photodamage by lowering light absorption of PSII
Plants have various structural and anatomical mechanisms that reduce the amount of light that is
absorbed by pigments of the antenna, thus avoiding the potential photoinhibition. Upright leaves
in rice (Oryza sativa) serve as efficient photoprotectants, by reducing the amount of light that is
absorbed by PSII, thus lowering the amount of photodamage and reducing the risk of
photinhibition (Murchie et al., 1999). Other photoprotective mechanisms that reduce light
absorption by PSII are leaf hairs (Pubescence) and leaf wax; both increase the amount of light
reflected by the leaves (Ehleringer, 1981). Chloroplast movement can also control light
absorption by chloroplasts, therefore lowering the risk of photoinhibition (Wada et al., 2003)
Anthocyanins are non-photosynthetic pigments that can shield chlorophyll molecules by
absorbing light. This “shield” is mainly in the green and blue part of the spectrum, and it
prevents light from reaching the chlorophyll antennas, thus preventing photodamage (Neill and
Gould, 2003, Pietrini et al., 2002). Anthocyanin can also serve as an efficient antioxidant and can
help in the detoxification of ROS (Gould et al., 2002). The function of anthocyanin as either an
antioxidant or a light attenuator (“sunscreen”) was found to be largely determined by the amount
of anthocyanin and its spatial distribution within the leaves and cells (Kytridis and Manetas,
2006).
In addition to reducing light absorption by PSII antenna, plants have other mechanisms that
mitigate photodamage, after light is already absorbed by the antenna complexes of PSII.
5.3. Mechanisms that mitigate photodamage after light absorption by the antenna of PSII
5.3.1. Non-photochemical quenching (NPQ)
Non-photochemical quenching (NPQ) is a well-studied and common mechanism of photo
protection in plants. NPQ dissipates excess energy from light directly into heat (Bilger and
Björkman, 1990). NPQ was shown to decrease the formation of chlorophyll triplets, thus
lowering the formation of singlet oxygen molecules (Carbonera et al., 2012). The main
component of this mechanism is the fast relaxing quenching, qE, which is a dynamic and flexible
15
response to excessive light. qE is pH dependent and is triggered by the acidification of the
chloroplast lumen, which in turn, activates the metabolic cycle of xanthohphylls (Muller et al.,
2001). This cycle includes the conversion of violaxanthin to antheraxanthin and zeaxanthin.
Antheraxanthin and zeaxanthin act as quenchers of excess energy (Demmig-Adams and Adams,
1996). In an Arabidopsis thliana mutant pnt , named viloxanthin de epoxidase 1(VDE1), there
is a point mutation , that disables the catalytic activity of the VDE protein, which converts of
violaxanthin to antheraxanthin and zeaxanthin. In the VDE1 mutant there is a low NPQ
response, which indicated the influence of xanthophylls on NPQ (Niyogi et al., 1998). The ratio
between these three pigments is called the de-epoxidation state) DES). DES is equal to DES =
0.5[Antheraxanthin]+[ zeaxanthin]/ [antheraxanthin] +[ antheraxanthin]+[ zeaxanthin] (DemmigAdams and Adams, 1996, Johnson et al., 2008). DES was shown to influence the NPQ
response (Johnson et al., 2008). qE is also triggered by structural changes in the protein antenna
complex of PSII, which is catalyzed by the PsbS protein (Li et al., 2000).
Modulation of NPQ can also occur through the enzymatic activity of ATP synthase. This
chloroplastic enzyme complex couples the transfer of protons from the thylakoid lumen to the
stroma, with the production of adenosine triphosphate (ATP). ATP is used to drive carbon
assimilation, as well as other energy consuming processes (protein synthesis, uptake and
transport of metabolites). Activity of ATP synthase controls the thylakoid membrane
conductivity of protons (gH+), which in turn regulates proton accumulation in the lumen and
therefore can regulate NPQ(Rott et al., 2011). The ratio between NPQ and PSII linear electron
flow (PSII electron transport rate) was shown to be linearly correlated with gH+(Kanazawa and
Kramer, 2002).
It was also shown that gH+ responds to the availability of CO2; for instance, under low CO2
concentrations, gH+ is relatively low, thus increasing lumen acidification and NPQ levels. At high
CO2 concentrations, gH+ is relatively high and NPQ is relatively low (Kanazawa and Kramer,
2002, Avenson et al., 2004) . Cyclic electron flow that involves only PSI also contributes to the
lumen acidification. Therefore, the cyclic electron flow is also important for NPQ induction and
photoprotection of PSII (Takahashi et al., 2009).
16
The selective advantage of NPQ was demonstrated in NPQ mutants that were significantly less
fit under natural conditions than wild type plants of the same species (Külheim et al., 2002). The
NPQ protection from photodamage in plants may sometimes occur at the expense of
photosynthesis (Zhu et al., 2004, Hubbart et al., 2012). For instance, rice plants that
overexpressed the PsbS protein, an inducer of NPQ, had lower induction of photosynthesis, in
response to increasing light intensity. This is in comparison to wild type plants that expressed
normal levels of PsbS (Hubbart et al., 2012). Recently, it has been suggested that crop plants
may be restricted in their use of light energy for photosynthesis under favorable conditions due
to the induction of photoprotective processes, such as NPQ. This was suggested since the
evolutionary devolvement of plants has been dominated by growth in resource-poor
environments (Murchie and Niyogi, 2011).
5.3.2. Photorespiration and the water-water cycle
Other biochemical mechanisms used by plants to confront an excess of light are based on the use
of alternative target molecules that receive the excess reductive energy formed. Oxygen serves as
one of these molecules, mainly through the process of photorespiration. Photorespiration is a
well-known and common process in most plants (C3 plants); it is considered energetically
unfavorable since it competes with carbon assimilation by consuming oxygen at the expense of
CO2 (Foyer et al., 2009). Photorespiration has been shown to be inhibited by low oxygen
concentrations (usually less than 2%) (Bjorkman, 1966), or high CO2 concentrations (Foyer et
al., 2009) .
Photorespiration has been shown to function in photoprotection under excessive light conditions
(Kozaki and Takeba, 1996, Heber et al., 1996), by enabling the repair process of photosynthetic
proteins such as D1 (Takahashi et al., 2007). Photorespiration is a large contributor to the
production of H2O2 in the plant peroxisomes. Therefore, photorespiration has a major impact on
the redox homeostasis in plant cells.
H2O2 was considered in the past to be a damaging ROS; however, in the last 15 years, H2O2
signaling was shown to play important roles in the regulation of plant growth and physiology
(Foyer et al., 2009). In addition to its role in photoprotection, photorespiration has also been
shown to play an important role in nitrate assimilation (Rachmilevitch et al., 2004).
17
Another process that targets oxygen for reduction is the water-water cycle (Mehler reaction). In
this reaction, oxygen is directly reduced by electrons to produce water. Ascorbate (vitamin C)
and glutathione are important metabolites for the function of the water water cycle (Miyake,
2010). The function of the water-water cycle as a large electron sink in higher plants was
suggested to be marginal in C3 plants (Heber, 2002); The water-water cycle enables a transiently
high electron transport flow, consequently creating a pH gradient across the thylakoid
membrane, inducing the photoprotective response of NPQ (Hideg et al., 2008). In contrast to
photorespiration, the water-water cycle is not inhibited at high CO2 concentrations (Stepien and
Johnson, 2009) and was suggested to be less sensitive to low O2 concentrations (Lovelock and
Winter, 1996).
5.3.3. Scavenging mechanism of singlet oxygen by an antioxidant
Once a singlet oxygen is produced by PSII, it can damage the D1 protein and other molecules,
thus causing photodamage and photoinhibition. α- Tocopherol (vitamin E) is thought to be the
main molecule that is used as an antioxidant against singlet oxygen produced by PSII (KriegerLiszkay and Trebst, 2006). It has been shown that blocking α- Tocopherol synthesis caused high
sensitivity to high light intensity (Trebst et al., 2002).
5.4. Drought and excess light
Drought effects on plants are initiate by the suppression of cell growth and cell division, which
are followed stomata closure. Stomata closure avoids excessive water loss that can lead to cell
death (Chaves et al., 2003). Drought induces stomata closure and the reduction of mesophyll
conductance. As a consequence, the diffusion of CO2 into the chloroplasts is lowered and the
assimilation of CO2 in the Calvin cycle is inhibited )Flexas et al., 2006(. This type of limitation
on photosynthesis is termed stomata or diffusive limitation. Diffusive limitation can be
transiently reversed by applying high CO2 concentrations to the leaf (Flexas et al., 2004).
Stomata closure caused by drought limits the CO2 availability for the Calvin cycle. Under these
conditions, light energy can often exceed the energy that can be utilized by photosynthesis.
Therefore, drought conditions usually induce photoprotective mechanisms in order to avoid
photodamage (Chaves et al., 2009). Photorespiration becomes a favorable process at low CO2
18
concentrations. These conditions of low CO2 concentrations occur during stomata closure.
Photorespiration has been shown to increase under drought (Wingler et al., 1999), (Lovelock and
Winter, 1996). Drought tolerance was also shown to be related to high rates of photorespiration,
in drought-tolerant transgenic plant that had delayed senescence under drought (Rivero et al.,
2009).
NPQ was also shown to increase under drought (Naumann et al., 2007, Montanaro et al., 2007).
The mechanism of this induction was shown to be correlated with the decrease of ATP synthase
activity. The decrease of ATP synthase activity under drought is a result of a reduction in the
amounts of different subunits of the ATP synthase complex (Kohzuma et al., 2009).
It has been suggested that under drought, photosynthesis is impaired due to impairment of ATP
synthesis (Tezara et al., 1999). This limitation on photosynthesis was termed metabolic
limitation. Metabolic limitation cannot be reversed by supplying the leaves with transiently high
CO2 concentrations; this is in contrast to diffusive limitation (Flexas et al., 2006). The
importance of metabolic limitation for the inhibition of photosynthesis under drought was
demonstrated in Sunflower (Helianthus Annuus, family - Asteraceae) (Tezara et al., 1999).
However, other studies have demonstrated that high CO2 concentrations can reverse this
inhibition and restore carbon assimilation, both in H. annuus (Cornic and Fresneau, 2002, Wise
et al., 1990) and in other plants species )Flexas et al., 2006(.
5.5. Protection from excess light in desert plants
Desert plants are often exposed to high light intensities, which are often associated with
conditions that inhibit carbon assimilation, such as drought. To avoid photodamage, many desert
plants, such as Encelia farinose, have developed anatomical features, such as leaf hairs
(trichomes) (Ehleringer et al., 1976). Other examples of anatomical features that reduce light
absorbance in desert plants are wax and salt glands on the leaf surface (Ehleringer, 1981).
NPQ has been shown to be a main component of adaptation in some perennial desert plants.
Yucca schidigera and Yucca brevifolia have been shown to have high NPQ during the summer
and winter, when growth and photosynthesis are inhibited due to either low temperatures
(winter) or lack of water (summer). In the spring, when conditions are more favorable, NPQ is
lower, and significantly more light is diverted to photochemistry in order to support growth
(Barker et al., 2002). In response to high light intensities, accumulation of zeaxanthin, which is
19
known to activate NPQ, was found to be different in two perennial desert species that possess
leaves with different optical properties. In Atriplex halimus, a species with bright leaves,
zeaxanthin concentration did not increase significantly in response to high light intensity; in
Ratema ratema, a species with green photosynthesizing stems, zeaxanthin increased in response
to high light intensity (Streb et al., 1997).
Annual desert plants live and grow only in times and locations in which water is available; the
photoprotective characteristics of these plants are less studied, in comparison to perennial desert
plants. Annual desert plants are exposed to high light intensity that can cause photodamage,
especially when photosynthesis is inhibited due to unfavorable environmental conditions, such as
drought, high evaporative demands and high vapor pressure deficits. On the other hand, annual
plants need to complete their life cycle in a relatively short period of time, before water is lost
from the soil due to the high evaporation that occurs in deserts (Shem-Tov and Gutterman,
2003).
5.6. Research goal and hypothesis
The overall goal of this research was to study how photoprotective mechanisms are used by
annual desert plants, under various environmental conditions.
The general hypothesis of this research was that some annual desert plant species have
photoprotective features, which differ from those of closely related and unrelated plant species,
from other environments.
5.6.1. Subject 1 of the research: Photoprotective response under drought in wild barley
ecotypes from Mediterranean and desert origins
Barley (Hordeum vulgare L.) is an important crop cereal (family Poaceae); about 134 million
metric tons of barley are produced in the world each year, making it the 11th most important crop
in the world in terms of production quantity. Russia leads the world in terms of barley
production, growing more than half of the total production. Ukraine, France, Germany, and
Australia are also large producers of barley (Data of 2011-FAO
http://faostat.fao.org/site/339/default.aspx-). In the past, barley was mostly used as food.
Today, most barley (2/3) is used for feeding livestock, one third is used for malting and brewing
20
alcoholic drinks, such as beer and whiskey, and less than 2% of the barley produced in the world
is used directly for food (Baik and Ullrich, 2008).
Barley was domesticated from its wild ancestor, wild barley (Hordeum spontaneum L. Koch).
This domestication occurred ~10,000 years ago, in the Fertile Crescent, between present-day
Israel and Iraq, (Harlan and Zohary, 1966); later domestication events also occurred in Central
Asia (Morrell and Clegg, 2007) and Tibet (Dai et al., 2012).
H. spontaneum plants can be found in a wide range of environments that differ in their climatic
conditions. Therefore, studying such species can make major contributions to the understanding
of plant adaption mechanisms to abiotic stresses and to the process of generating stress-tolerant
crops (Nevo and Chen, 2010). The growth characteristics under optimal conditions of different
ecotypes of H. spontaneum (originating from different climatic regions) did not seem to correlate
with the specific average annual rainfall in the different climatic regions (Van Rijn et al., 2000);
however, other comparative studies of desert and non-desert ecotypes have revealed a number of
morphological, phenological and life history differences )Snow and Brody, 1984( that were
found to be adaptive (Volis et al., 2002, Volis, 2011). The desert ecotype from the Negev Desert
in Israel (receiving around 90 mm of annual rainfall) exhibited lower competitive ability; for
example, the desert ecotype produced fewer seeds than the Mediteranean ecotype did when they
were grown in high density ( Volis et al., 2004(. It was also shown that the desert ecotype had a
lower reduction in yield under low irrigation (Volis et al., 2002b). In addition, earlier flowering
was observed in the desert ecotype (Volis, 2007) as compared with the Mediterranean ecotype
from the Galilee region in Israel (receiving around 500 mm of annual rainfall). The above
experiments did not examine differences in the physiological processes between the ecotypes,
such as photosynthesis, photorespiration, anthocyanin accumulation and NPQ.
21
5.6.2. Specific objective for Study 1
The objective of the first study was to examine the contribution of oxygen-related photochemical
processes, NPQ and anthocyanin accumulation to the response of desert and Mediterranean
ecotypes of wild barley plants to high light intensities and drought. To achieve this goal, we set
up experiments in which the above two ecotypes of wild barley were grown under terminal
drought and compared to plants grown under well-irrigated conditions.
5.6.3. Subject 2 of the research: NPQ and other light-driven processes in the annual desert
plant Anastatica hierochuntica
Anastatica hierochuntica is an annual desert plant (family Brassicaceae) found in the hot deserts
of the Middle East and North Africa. In these areas, water is available for a short period of time
during the year, and therefore, annual plants have a limited time to complete their life cycle and
produce seeds. A. hierochuntica plants have adapted a fixed and opportunistic developmental
pattern, in which flowering and seed production start after four or five true leaves have emerged,
regardless of day length (Shem-Tov and Gutterman, 2003). This adaptation to the desert
environment enables the plants to maximize resource utilization during a relatively brief window
of opportunity (Shem-Tov and Gutterman, 2003).
Photosynthetic rates in A. hierochuntica are relatively high (carbon assimilation) in comparison
with other desert plant species, both annuals and perennials (Hegazy and Moser, 1991).
In a preliminary experiment, we found that A. hierochuntica plants had a significantly different,
namely lower, NPQ response than the related model plant Arabidopsis thaliana from the same
family.
The preliminary results were surprising, due to the fact that NPQ in A. thaliana is known to be
relatively low and yet was in our experiment significantly higher than the related desert plant
species. The results motivated us to continue to study this phenomenon since NPQ is an
important photoprotective mechanism in plants that has received a fair amount of attention from
the scientific community in recent years.
In this study, we characterized the NPQ response in A. hierochuntica and examined how this
unique response relates to the photochemical use of light energy and photosynthesis.
22
To characterize the dynamics of NPQ, the response of A. hierochuntica was compared to those
of three other different plant species that are known to differ in their photosynthetic
characteristics and water use efficiency: two closely related species from the Brassicaceae
family (A. thaliana and Thellungiella salsuginea) and Helianthus annuus (sunflower) from the
Asteraceae family. A. thaliana is the most common model plant in plant sciences and is
considered to have relatively low NPQ and carbon-assimilation rates (Hubbart et al., 2012). T.
salsuginea is considered to be a stress-tolerant model plant that has been reported to have
relatively low transpiration rate and a high water-use efficiency (WUE) of photosynthesis in
comparison to A. thaliana (Inan et al., 2004;Kant et al., 2006;Stepien and Johnson, 2009). H.
annuus has been reported to have relatively high transpiration and photosynthetic rates (Wise et
al., 1991; Archontoulis et al., 2012).
5.6.4. Specific objective for Study 2
The objective of the second study was to compare the NPQ and photosynthetic responses of A.
hierochuntica to other plant species under various growth and environmental conditions.
23
6. Materials and Methods
6.1. Measurements of photosystem II efficiency and non-photochemical quenching using
chlorophyll fluorescence.
The reaction centers of PSII have a distinct fluorescence emission that peaks at wavelengths of
680-690 nm. This light signal can be separated from other light sources using filters that pass
only through this wavelength. The PAM (Pulse Amplitude Modulated) technology was adjusted
to measure chlorophyll fluorescence at different light intensities, by using a high frequency
measuring beam, with a low light intensity at a wavelength that specifically excites the reaction
center of PSII (at a wavelength of 680nm) (Schreiber, 2004).
6.1.1. Maximum photochemical efficiency- Fv/Fm
After subjection of leaves to relatively long periods of darkness (>20 minutes), leaves are usually
assumed to be dark adapted; under these conditions, there is a full oxidation of the electron
acceptors of PSII. In a dark adapted state, the conversion of light energy into heat, by
photoprotective NPQ, is assumed to be minimal. Under conditions of dark adaptation, the
fluorescence of PSII is minimal, and is termed Fo, the minimal fluorescence in the dark. After Fo
is measured, a strong saturating light is applied (>5000 PPFD) for less than one second and
fluorescence is recorded again. This fluorescence is termed Fm, the maximal fluorescence in the
dark. Fm-Fo is usually expressed as Fv (variable fluorescence). The value of Fv/Fm is the
maximal efficiency of PSII (Krause, 1988). The value Fv/Fm is usually above 0.75 and up to
0.84 in non-stressed dark adapted plants (Bjorkman and Demmig, 1987). Lower Fv/Fm values
are usually considered to be a direct indication of photoinhibition. Fv/Fm can also be low due to
slow relaxing NPQ (termed qI). qI is more dominant in evergreen plants during periods of the
year when photosynthesis in inhibited due to environmental conditions (summer in deserts,
winter in cold regions) (Adams and Demmig-Adams, 2004). This study used annual plants that
usually do not have a high capacity for qI, and therefore, in this work, a significant decrease of
Fv/Fm was attributed to photoinhibition.
24
6.1.2. Photochemical efficiency of PSII in the light: ФPSII and electron transport rate
In illuminated leaves, the fluorescence measured is termed F’; the maximum fluorescence in
leaves exposed to light (measured after applying a saturating light) is termed Fm’. The
expression (Fm’-F’)/Fm’ is linearly proportional to the operating efficiency of PSII (ФPSII)
(Genty et al., 1989). ФPSII can be used to estimate the linear electron flow of PSII, by
calculation of the electron transport rate of PSII (PSII ETR ~ μmole electrons m-2 s-1). This is
done by using the following equation:
PSII ETR = I x α x f x ФPSII,
where I is the photosynthetic light intensity (μmole photon m-2 s-1), α is the fraction of light that
is absorbed by the leaves, f is the fraction of light absorbed that is captured by the PSII light
harvesting complex (and not by PSI) and ФPSII is the operating efficiency of PSII (Krall and
Edwards, 1992) .
The fraction of photosynthesis light absorbed by the leaves (α) is usually assumed to be 0.84 for
green leaves. The actual value of α can be measured using an integrated sphere device. The
value of f is usually assumed to be 0.5 and was measured in these studies by comparing the
fluorescence of PSII and PSI at 77 0K. The assumption that the fraction of the light that is
absorbed by the leaf is directed to either PSII or PSI is not valid when this absorbed light is not
captured by any photosynthetic pigments, such as anthocyanin, because it will give an
overestimation of the light absorbed by PSII and, therefore, of PSII ETR (Baker et al., 2007). In
these studies, I used the values of ФPSII to estimate the efficiency of PSII in experiments in
which anthocyanin accumulation was detected (experiments related to Study 1). In the other
experiment (Study 2), anthocyanin was not detected in high levels; therefore, I used the
expression of PSII ETR, when the values of light absorption by PSII were calculated using
measured values of leaf absorbance and energy distribution between PSII and PSI.
25
6.1.3. Non-photochemical quenching of PSII, NPQ and ФNPQ
The dissipation of light energy as heat (NPQ) is calculated according to the quenching of
maximum fluorescence in the light (Fm’). This is done using the following equation:
NPQ = (Fm-Fm’)/Fm’.
This latter equation is based on the Stern-Vollmer relation that describes the rate of fluorescence
quenching (Bilger and Björkman, 1990). An alternative method that can estimate the efficiency
of NPQ was suggested and is termed ФNPQ (Hendrickson et al., 2004):
ФNPQ = (F’/Fm')-(F’/Fm).
6.2. Plant materials
6.2.1. Study 1
Sampling of H. spontaneum was done in both Mediterranean and desert climatic zones of Israel
(hereafter, Mediterranean and desert ecotypes), each represented by one population. The
Mediterranean ecotype (AM) was collected in the Upper Galilee (elevation 300 m, average
annual precipitation around 500 mm), 1 km west of Kibbutz Ammiad. The vegetation in the area
of the Mediterranean zone is characterized by a Mediterranean grassland on terra-rossa soil. The
desert ecotype (SB) was sampled from a wadi (Arabic for ephemeral river valley) in the Negev
Desert, 3 km south-west of Kibbutz Sede Boqer (elevation 400 m, average annual precipitation
around 90 mm), having sparse desert vegetation on loess soil.
Seeds were germinated on wetted paper (Watman filter paper or toilet paper) and then transferred
to three-liter pots filled with washed and sterilized sand. The pots were in a greenhouse in which
the photosynthetic photon flux density (PPFD) was between 600 and 700(μmole photons m-2 s-1)
at midday. The temperature was controlled at 250 C during the day and 100 C at night, and the
relative humidity was 40-60% at midday. The plants were grown under continuous irrigation at
full capacity for 25 days and were fertilized with a half Hoagland nutrient solution twice a week.
After 25 days, irrigation was halted for half of the plants from each ecotype (drought treatment),
while the other half was irrigated (irrigation treatment) twice a week to full soil capacity.
26
Throughout the experiments, the plants were all at the same oncogenic state of vegetative growth.
Experiments were repeated three times.
6.2.2. Study 2
A. hierochuntica seeds were collected from the Negev Desert in Israel. Plants grown from these
seeds were used in four different experiments between September 2009 and September 2012.
In the first experiment, A. hierochuntica and A. thaliana (Columbia ecotype), plants were grown
under a low light intensity of 120 PPFD )μmole photons m-2 s-1); light/dark cycles were 16 h/8 h
(long days) at 22 oC in a custom-made growth chamber. The plants were grown in plastic pots
filled with standard potting soil (Toof Merom Golan, Israel). Salinity stress was applied to half
of the plants by irrigation with saline water (200mM Na Cl), for two days; otherwise plants were
irrigated to maintain soil water content above 50%.
In the second experiment, A. hierochuntica plants were grown alongside A. thaliana (Columbia
ecotype), T. salsuginea, and H. annuus (DY3 variant) in a controlled growth chamber (Percival,
Perry, Iowa, USA). The plants were grown in plastic pots filled with standard potting soil (Toof
Merom Golan, Israel). The daily light intensity (photosynthetic photon flux density -PPFD)
schedule was: 30 min of 10PPFD, 1 h of 200 PPFD, 9 h of 600 PPFD, 1 h of 200 PPFD, 30 min
of 10 PPFD, and 12 h in the dark, representing a light/dark cycle of 12 h/12 h. Temperature was
22 oC, and relative humidity (RH) was between 50% and 70%. Due to different growth rates and
developmental features of the different plant species, measurements were carried out according
to plant developmental stage. For A. hierochuntica, measurements were taken from 25- to 40days-old plants. These plants had 5–6 fully expanded leaves, and early inflorescence had
appeared. At this stage, the leaves were big enough to enable proper measurements.
Measurements were taken from true fully expanded leaves 3–6 (in order of appearance). To
perform the measurements at a similar developmental stage, we measured A. thaliana and T.
salsuginea plants during the early stages of inflorescence appearance. The H. annuus plants were
too big to be grown properly in the growth chamber when flowering started; therefore, sampling
and measurements were performed earlier, when 3 to 4 true leaves had already fully expanded.
27
In the third experiment, A. hierochuntica seeds were planted in a field in the Negev Desert at the
Sede Boqer Campus of Ben-Gurion University of the Negev. Plants germinated, grew and
produced seeds and flowers in May and June 2012. During this time, the light intensity was high
with an average of 1,700 PPFD for at least 8 h per day (0800–1600 h). The sky was clear most of
the time. The maximum daily average temperature during this period was 33 oC. RH at midday
was 25%. Plants in the field were irrigated once every other day with drip irrigation for 1.5 h (1
L h-1). Other local desert plants in the same field were used in some of the experiments.
In the fourth experiment (drought Vs. irrigation), A. hierochuntica and H. annuus plants were
grown in a growth chamber in the same conditions as described in Experiment 2, for three (H.
annuus) or four weeks (A. hierochuntica). After this initial period, drought conditions were
applied to half of the plants, for one week, while half of the plants continued to be irrigated in
order to maintain a soil water content above 50%.
6.3. Photosynthesis and chlorophyll fluorescence measurements
Measurements were performed using a LI-COR 6400-XT infrared gas analyzer (IRGA) with a
fluorescence chamber (LI-COR, Lincoln, Nebraska, USA). Measurements were taken on intact
leaves in all experiments except for the field experiment. All measurements were performed
between 0900 and 1500 h. Plants were dark-adapted for at least 2 h in a dark room in which the
PFFD was less than 2. For all measurements, plants were held in the dark for an additional 5 min
period in the measuring chamber, until the light period started.
In all of the experiments, the leaf temperature in the measuring chamber was kept at 22 oC and
RH between 45 and 55%. The oxygen concentration in the IRGA chamber was either ambient
(21%) or low (2%) to enable or inhibit photorespiration, respectively. Different CO2
concentrations were applied (50, 80, 200, 400, 1,000 and 1,600 µmol mol-1) for the different
measurements. For each measurement, the CO2 concentration remained constant in the
measuring chamber. Most measurements lasted a period of 19 min (5 min of dark and 14 min of
light). All measurements were carried out at either 200, 600, 1,200 and 1,500 PPFD. For
example, in order to measure the photosynthetic response to an ambient CO2 concentration of
400 µmol mol-1 and an oxygen concentration of 2%, at a light intensity of 1200 PPFD, leaves
28
from a dark-adapted plant were enclosed in the measuring chamber in the dark, and after 5
minutes, the light was turned on to an intensity of 1200 PPFD for 14 minutes.
The following gas exchange parameters were measured: net assimilation of CO2 (µmol CO2 m-2
s-1), stomata conductance to water vapor (mmol H2O m-2 s-1), leaf internal CO2 concentration (Ci;
µmol mol-1), and transpiration (mmol H2O m-2 s-1) (Farquhar and Von Caemmerer, 1981).
Intrinsic WUE (µmol CO2 mmol H2O-1) was calculated from the net carbon assimilation and
transpiration. The IRGA device was occasionally calibrated with external CO2.
In order to avoid errors due to CO2 leakage from the measuring chamber at higher or lower than
ambient CO2 concentrations, the following measures were taken: corrections were made for
leakage using an empty chamber (without a leaf). Occasionally, after the measuring period in the
light, a dark period was applied in order to verify that assimilation measurements were light
dependent (net photosynthesis values were less than 0).
Chlorophyll fluorescence measurements were carried out in parallel, and the following
parameters were calculated: ФPSII (Fm’-Fs)/Fm’) (Genty et al., 1989), PSII electron trnsport
rate (Krall and Edwards, 1992), NPQ (Fm-Fm’)/Fm’) (Bilger and Björkman, 1990), ФNPQ
(Hendrickson et al., 2004) and the maximum photochemical yield of PSII (Fv/Fm) (Krause,
1988).
In the first, preliminary experiment of study 2, Chlorophyll fluoresence was measured using
by applying rapid light curve, according to (White and Critchley, 1999), using Mini‐PAM
device (Walz, Heinz Walz GmbH, Effeltrich, Germany).
In order to examine the photosynthetic parameters over long periods (hours), measurements were
performed in the following manners:
6.3.1 Study 1
Photosynthesis measurements were also carried out in the greenhouse, at a midday natural light
intensity of 700 PPFD; these measurements were carried out 25 days after the beginning of the
drought treatment in order to assess the stomata conductance, net CO2 assimilation, ФPSII, NPQ
and ФNPQ under natural growth conditions. For these measurements, green leaves were
enclosed in the IRGA Li-6400, under a light intensity of 700 PPFD, a CO2 concentration of 400
29
μmol mol-1, leaf temperature at 250C, and relative humidity (RH) between 40 and 55%; readings
of photosynthetic parameters were taken after stable values were reached.
6.3.2. Study 2
Plants were illuminated for 2 h at either 200 or 600 PPFD in a growth chamber (see growth
conditions above) and then measured, using the LI-COR 6400-XT IRGA, at either 200 or 600
PPFD. For the set of measurements at 1,200 PPFD for 2 h, plants were exposed to 1,200 PPFD in
a specialized high-light-intensity room; in this experiment, the air temperature at the shoot was
24 oC and RH was ~80%, achieved by the use of a cold vapor device. Otherwise, all
measurements were performed in a similar manner.
6.4. Leaf absorbance
Leaf absorbance was measured between the wavelengths of 400 to 700 nm, using an external
integrating sphere (Licor, 1800–12s, USA), connected by optic fiber to an analytical spectral
device spectrometer (FieldSpec Pro FR, USA) with a spectral range of 350–2500 nm.
6.5. The excitation distribution between photosystem I and II
The excitation distribution between photosystem I and II was measured as follows: leaves from
dark adapted plants were immediately frozen in liquid nitrogen. The leaves were then ground in
an extraction buffer (330mM Manitol, 31mM HEPES, 2mM EDTA, 3mM MgCl2 pH = 7.8). A
glass rod, pre-incubated in liquid nitrogen, was then immersed in the leaf extract, resulting in a
uniform frozen coating around the rod. The sample was inserted into a glass Dewer vessel, which
was placed in a FluooMax3 fluorometer (Jobin Yvon, France). The fluorescence spectrum of the
leaf extract was measured between 650-750nm, following a 430nm excitation beam. The
excitation and emission slits were set at 5nm, with an integration time of 0.25s. The emission
spectra gave two distinct peaks at 682 and 730nm, resulting from the fluorescence of PSII and
PSI chlorophyll antenna systems, respectively. The PSII excitation distribution was estimated
based on the following calculation: PSII excitation distribution= (F' 682nm)/ (F'682 +F'730).
30
6.6. Leaf relative water content
The leaf relative water content (LRWC) was measured by weighing for fresh leaf weight (Fw)
and then immersing the leaf for 24 hours in distilled water, before weighing for turgor weight
(Tw). The leaf was then dried in a 720C oven for 48 hours before weighing for dry weight (Dw).
LRWC was determined according to the following equation:
LRWC(%)= 100 x (Fw-Dw)/(Tw-Dw) (Barrs and Weatherley, 1962).
6.7. Pigment content and analysis
The chlorophyll concentration in the leaves was determined in Study1 according to (Lichtentaler
and Wellburn, 1983). measured as µg per cm2 of a leaf.
In Study 2, chlorophyll and carotenoid concentrations were measured as µg per cm2 of a leaf as it
was determined in 1 ml of extraction solution (80% acetone)( )Lichtentaler and Wellburn, 1983(.
The total anthocyanin content was determined according to (Martin et al., 2002) and presented as
OD 530 per µg per cm2, as it was determined in 1 ml of acidic extraction solution acidic
extraction buffer solution ( 0.1M HCl in DDW).
Separation and quantification of xanthophylls were done as follows:
Xanthophylls were analyzed as follows: dark-adapted plants (over 10 h) were subjected to a light
intensity of 1200 PPFD, a temperature of 24°C and a RH of 80%, for 2 and 120 min. After plants
were subjected to high light, leaves were immediately frozen in liquid nitrogen and stored in
Eppendorf tubes at −80°C. Leaves were ground using a mortar and pestle in 100% acetone. The
samples were filtered to get rid of debris, and then were analyzed by high-performance liquid
chromatography (HPLC) according to Neuman et al. (Neuman et al. 2014), The column was
equilibrated for at least 5 minutes between injections with 100% acetonitrile:H2O (9:1).The
spectra between 200 and 700 nm were recorded at a rate of one full spectrum per second.
Analysis of the Data was done with the chromatography software Millenium (Waters, Milford,
MA). The carotenoids were identified according to their typical retention time (certified by
standards) and characteristic absorption spectral, the amounts of different pigment were
31
calculated according to a satndard calibration curve, which was made with a known
concentrations of the different pigments
6.8. Plant dry weight
Plants were harvested 52 days after sowing (27 days from the beginning of the drought
treatment); the different plants were separated into roots and shoots and were oven dried at 72 0C
for 72 hours, and then the samples were weighed.
32
7. Results
7.1. Photo protective response under drought in wild barley ecotypes from Mediterranean
and desert origins
7.1.1. Effect of prolonged drought on soil water content and on plant biomass
A period of 25 days without irrigation caused a similar significant decrease in the soil water
content (~80% decrease) of both ecotypes as compared to the control, suggesting that both
ecotypes were exposed to a similar water stress level (Table 1). In both ecotypes, drought caused
a similar and significant decrease of ~67% in the overall dry biomass; the root to shoot biomass
ratio increased in both ecotypes, suggesting that aboveground growth was inhibited to a higher
extent than root growth, in both ecotypes (Table 1).
Table 1: Dry weight of shoot and root of the Mediterranean (AM) and desert (SB)
ecotypes of Hordeum spontaneum
The effect of a 25-day drought on overall plant biomass and on root/shoot ratio, n=7±
standard error, Groups that do not share a common letter are statistically different (α=0.05).
Ecotype
Treatment
Soil (Sand)
water content
(m3 H2O/ m3
soil)
AM
Drought
0.04±.03(b)
4.2±1.66(c)
0.83±0.1(a)
Irrigation
0.34±0.07(a)
13.7±1.13(a)
0.6±0.06(b)
Drought
0.03±0.022(b)
3.11±1.06(c)
0.78±0.05(a)
Irrigation
0.32±5(a)
9.1±0.33(b)
0.4±0.1(c)
SB
33
Total dry
biomass (gr)
Root/shoot
dry mass ratio
(gr/gr)
7.1.2. Changes in leaf pigmentation in response to drought, in the Mediterranean and
desert ecotypes of H. spontaneum.
Under drought conditions, the SB plants (desert ecotype) produced purple-colored leaves;
however, in the AM plants (Mediterranean ecotype), there was only a slight change in leaf color
(Figure 1). The concentration of total anthocyanin under drought increased by 250 and 50% in
the leaves of the SB and AM plants, respectively, as compared with the irrigated control (Table
2). The increase in leaf anthocyanin concentration was not accompanied by a decrease in the
total chlorophyll concentration (Table 2).
Figure 1: Leaves of the Mediterranean and desert ecotypes of Hordeum spontaneum.
Leaves of the Mediterranean AM ecotype and the desert SB ecotype of H. spontaneum that were
subjected to either constant irrigation or to 25 days of complete drought.
34
Table 2: Anthocyanin and chlorophyll concentrations of the Mediterranean and desert ecotypes of
Hordeum spontaneum.
Anthocyanin concentration in the fresh weight of leaves of the Mediterranean AM ecotype and the
desert SB ecotype of H. spontaneum is presented as optical density (OD) at 530nm, which is the typical
absorbance peak of anthocyanin molecules in an acidic extraction buffer (pH=1). Total chlorophyll
(a+b) concentration, in an ethanol extraction of fresh leaves, was determined by absorbance at 648 and
665 nm. n=6 ± standard error. Groups that do not share a common letter are statistically different
(α=0.05).
Ecotype
Treatment
Total chlorophyll
Chl a/b ratio (µg
Relative antocyanin
concentration – in
Chl a / µg Chl b)
concentration (OD
530nm/ cm2 )
1ml ethanol ( µg Chl
a+b/cm2 )
AM
SB
Drought
Irrigation
Drought
Irrigation
32.8±3.3
33.5±4.2
29.5±5.3
30.6 ±2.1
2.78±0.19
3.05±0.4
3.211±0.3
2.9±0.03
0.32±0.07 (b)
0.25±0.13(b)
0.95±0.13 (a)
0.38±0.12 (b)
7.1.3. Absorbance of photosynthetic active radiation and distribution of light to PSII in the
Mediterranean and desert ecotypes of H. spontaneum.
The changes in leaf pigmentation had an influence on the absorptive properties of the leaves,
namely that the absorbance in the wavelength region of 500 to 600nm increased in the leaves of
the drought-treated SB plants (Figure 2). This increase of absorbance in this region was probably
due to the increase of anthocyanin, which is known to absorb light in this part of the spectrum.
The overall absorbance of the drought-treated SB ecotype was higher than that of the AM
ecotype (Table 3). The light distribution between PSII and PSI, as reflected in the measurements
by fluorescence at 77 K, revealed a statistically significant higher distribution to PSII in the SB
plants (P<0.05); however, the difference in the values was less than five percent (Table 3).
35
Figure 2: Leaf absorbance of the Mediterranean and desert ecotypes of H.
spontaneum.
Leaf absorbance of the Mediterranean AM ecotype and the desert SB ecotype of H.
spontaneum. Measurements were taken from the photosynthetically active part of the
spectrum (400-700 nm), as measured by an integrated sphere device; each curve is
representative of three repeats.
36
Table 3: Absorbance of photosynthetically active radiation and light excitation
distribution to photosystem II in light-exposed leaves of the Mediterranean and desert
ecotypes of H. spontaneum.
The average absorbance and light energy distribution to PSII, as was measured in light-exposed
leaves, by fluorescence at 770 K. PSI fluorescence peaked at 730nm, while PSII peaked at
682nm; excitation light was at 430nm. n=5 ± standard error.
Ecotype
Treatment
Average
absorbance
AM
Drought
33.731±3.41(b)
Fraction of
excitation
distribution to
PSII
0.582±0.016(b)
Irrigation
33.131±3.35(b)
0.590±0.0210(b)
Drought
01.241±0.90(a)
0.620±0.003(a)
Irrigation
39.401±3.49(ab)
0.589±0.004(b)
SB
7.1.4. Leaf water status and CO2 assimilation in the Mediterranean and desert ecotypes of H.
spontaneum.
Under drought, leaf relative water content (LRWC) decreased significantly (P<0.05), compared
with the irrigated control, in both ecotypes: from 93.7 and 91% to 66.8 and 65.2% in the SB and
AM ecotypes, respectively (Figure 3a).
Under a light intensity of 1500 PPFD, stomata conductance decreased significantly (P<0.05), by
83.6 and 75%, in the drought-treated AM and SB ecotypes, respectively (Figure 3b). CO2
assimilation decreased significantly (P<0.05), compared with the irrigated control, by 89 and
82.4% in the AM and SB ecotypes, respectively (Figure 3c). Similar effects were also measured
when stomata conductance and assimilation were measured after four hours of exposure to a
lower light intensity (700 PPFD) at the natural light intensity in the greenhouse (data not shown)
These results suggest that in both the Mediterranean and the desert ecotypes, severe drought
leads to leaf dehydration, thereby causing an almost complete closure of stomata and an
inhibition of carbon assimilation.
37
Figure 3: Leaf relative water
content, stomata conductance and
carbon assimilation of the
Mediterranean and desert ecotypes of
H. spontaneum.
The Mediterranean AM ecotype and the
desert SB ecotype of H. spontaneum,
under the Irrigation (I) or Drought (D)
treatments
A: Leaf Relative Water Content in
(LRWC), n>10.
B: Stomata conductance to water
vapor as measured at a light intensity
of 1500 PPFD, 8 minutes of light
period, n>8.
C: Carbon assimilation, as was
measured at a light intensity of 1500
PPFD, n>10.
Measurement conditions for
assimilation and stomata conductance
were: 8 minutes of light period, 1500
PPFD, and 400μmol mol-1 CO2.
Groups that do not share a common
letter are statistically different
(α=0.05), standard error bars are
shown.
38
7.1.5. Efficiency of photosystem II in the Mediterranean and desert ecotypes of H. spontaneum.
Under drought conditions, Fv/Fm did not decrease significantly as compared to the controlirrigated plants in both ecotypes, suggesting that photoinhibition was largely avoided by both
ecotypes, despite the drought stress (Figure 4a). Non-photosynthetic pigments, such as
anthocyanin, can decrease light absorbance by PSII; however, this decrease is not quantifiable in
a straightforward manner. Since there was a significant accumulation of anthocyanin in the SB
ecotype under drought, the PSII electron transport rate was not calculated. Instead, I used the
value of ФPSII in the light, which relates only to the efficiency of the light that is absorbed,
without making assumptions about how much light was absorbed by PSII (see Materials and
Methods). ФPSII (at 1500 PPFD) decreased significantly (P<0.05), by 49%, in the AM ecotype
as compared with the irrigated plants; however, in the SB ecotype, there was no significant
change in ФPSII. The ФPSII in the SB drought-treated plants was significantly (P<0.05) higher
(77%) than in that of the AM ecotype under drought (Figure 4b).
39
Figure 4: Maximum efficiency of PSII and ФPSII of the Mediterranean and desert ecotypes of
H. spontaneum.
The Mediterranean AM ecotype and the desert SB ecotype of H. spontaneum, under the Irrigation
(I) or Drought (D) treatments
a: Maximum efficiency of photosystem II (Fv/Fm) was measured in dark-adapted leaves, n>10.
b: ФPSII’s measurement conditions were: 8 minutes of light period, 1500 PPFD, and 400μmol mol-1
CO2, n>10.
Groups that do not share a common letter are statistically different (α=0.05), standard error bars are
shown.
40
7.1.6. Non-photochemical quenching and the O2 dependency of PSII photochemical
activity
Regulated thermal dissipation, NPQ, increased under drought in both ecotypes; however, this
increase was insignificant (Figure 5a, P>0.05). In the drought-treated plants, ФNPQ, regulated
thermal dissipation efficiency, increased in both ecotypes as compared with the control;
however, ФNPQ levels of the SB drought-treated plants were significantly lower (P<0.05) than
that of the drought-treated AM plants (Figure 5b). Since assimilation was very low in the SB
ecotype drought-treated plants, we hypothesized that the high ФPSII maintained in these plants
could have been due to the use of O2 as an electron acceptor. In order to test this hypothesis, we
measured the ФPSII at a low concentration of O2 (2%), at which photorespiration is inhibited.
The ФPSII, under low O2, decreased by 35% (P<0.05) in the SB drought-treated ecotype plants.
This decrease was much larger than the relatively small decreases of 17.5, 20.5 and 15.7% in the
SB irrigated, AM irrigated and AM drought-treated plants, respectively (Figure 6). By
comparing the ФPSII values of the AM ecotype drought-treated plants and the SB ecotype
drought-treated plants, it is estimated that the O2-dependent photochemical activity was
responsible for 67% of the difference in the ФPSII between the two ecotypes under drought.
41
Figure 5: Non-photochemical quenching efficiency of non-photochemical quenching of
the Mediterranean and desert ecotypes of H. spontaneum.
The Mediterranean AM ecotype and the desert SB ecotype of H. spontaneum, under the
Irrigation (I) or Drought (D) treatments
A: Non-photochemical quenching (NPQ) calculated according to the formula NPQ= FmFm'/Fm.
B: The efficiency of NPQ- ФNPQ was calculated according to the equation,
ФNPQ=(Fs/Fm')-(Fs/Fm).
Measurements were taken after 8 minutes of exposure to 1500 PPFD and 400μmol mol-1
CO2, n>10.
Groups that do not share a common letter are statistically different (α=0.05), standard error
bars are shown.
42
Figure 6: Photosystem II yield at low and ambient oxygen concentrations of the Mediterranean and
desert ecotypes of H. spontaneum.
Plant leaves were exposed to either low (2%) or ambient (~21%) oxygen, at a light intensity of 1500 PPFD,
and 400μmol mol-1 CO2.
Groups that do not share a common letter are statistically different (α=0.05), n>7, standard error bars are
shown.
43
7.2. Non-photochemical and photochemical processes in the annual desert plant Anastatica
hierochuntica
7.2.1. Initial experiment: NPQ in Anastatica hierochuntica and Arabidopsis thaliana under
different irrigation salinities, setting the background
Fifteen days old A. thaliana and 20 days old A. hierochuntica plants were grown at a low light
intensity (200 PPFD) and were exposed to two salinity levels (0 and 200 mM NaCl); NPQ and
PSII ETR were measured after short exposure, 1 minute, to high light intensity (see Materials
and Methods ). Maximal photochemical efficiency (Fv/Fm) was also recorded. NPQ values in
A. hierochuntica were much lower in both salinity treatments compared to A. thaliana, 50% and
70% lower, in the 0 and 200mM salinity treatments, respectively. PSII ETR decreased by 25% in
both A. thaliana and A. Hierochuntica, in response to the salt application, suggesting a similar
decrease in photosynthesis in both plant species (Table 4).
Table 4: PSII activity in Anastatica hierochuntica and Arabidopsis thaliana, in
response to salinity stress
Plants of both species, in both salinity treatments, were dark adapted for 30 minutes, and
then leaves were exposed to one minute of increasing light intensities (for more details,
see rapid light curve method in Materials and Methods section). NPQ and PSII ETR were
measured in plants after one minute of exposure to light. Groups that do not share a
common letter are statistically different (α=0.05, n=6)
Plant
Anastatica
hierochuntica
Arabidopsis
thaliana
Salinity
treatment
(mM NaCl)
0
200
0
200
44
NPQ (at1500
PPFD)
1.042±0.119(c)
0.545±0.112(b)
1.945±0.192(a)
1.923±0.214(a)
PSII ETR
(at 1500
PPFD)
113±34(a)
84±19(b)
73±5(c)
63±7(c)
The NPQ response in A. hierochuntica was much lower than that of A. thaliana, regardless of the
salinity level. The results from this experiment triggered a set of experiments in order to describe
the NPQ response in A. hierochuntica and its relation to other processes related to the use of light
energy in this plant.
In the following experiment, I tried to characterize the NPQ response in A. hierochuntica and
examined how this unique response relates to the photochemical use of light energy and
photosynthesis. In order to characterize the dynamics of NPQ, the response of A. hierochuntica
was compared to three different plant species, including two close relatives from the
Brassicaceae family: Arabidopsis thaliana and Thellungiella salsuginea, and Helianthus annuus
(sunflower) from . A. thaliana is the common model plant in plant sciences and is considered to
have relatively low NPQ and carbon assimilation rates. T. salsugenia is considered to be a stresstolerant model plant that was reported to have relatively low transpiration rates and high wateruse efficiency of photosynthesis in comparison to A. thaliana. H. annuus was reported to have
relatively high transpiration and photosynthetic rates; therefore, H. annuus represents a plant
with a relatively less restricted photosynthesis.
In addition, we also studied the effect of a specific growth environment (controlled vs. field) on
the dynamics of NPQ in A. hierochuntica.
7.2.2. Non-photochemical quenching in A. hierochuntica
In the various light intensities that were applied, A. hierochuntica plants had significantly lower
NPQ induction compared to all other plant species during the initial minutes of the light period,
when grown in either a growth chamber (600 PPFD) or in the field (Figure 7, Figure 8). For
example, NPQ induction, after two minutes, was 70, 60 and 55% lower in A. hierochuntica as
compared to A. thaliana when measured at 200, 600 and 1200 PPFD, respectively (Figure 7).
45
Figure 7: NPQ response in Anastatica hierochuntica and other growth chamber grown plant
species, at different light intensities. A: NPQ in response to time (from the beginning of the light
period) at a light intensity of 1200 PPFD. B: NPQ in response to time (from the beginning of the light
period) at a light intensity of 600 PPFD. C: NPQ in response to time (from the beginning of the light
period) at a light intensity of 200 PPFD. All measurements were done at a CO2 concentration of 400
µmol mol-1, and an ambient oxygen concentration (~21%); for all other details of the experiment, see
the Materials and Methods section. All plants were grown in a growth chamber with a light intensity of
600 PPFD. In all the graphs, standard error bars are shown, 9≥n≥4.
46
Figure 8: NPQ response in Anastatica hierochuntica and other field-grown plant
species in the Negev Desert. NPQ was measured at a light intensity of 1,200 PPFD in:
Anastatica hierochuntica (Brassicacae, annual plant), Atriplex holocarpa
(Chenopodiaceae, perennial), Atriplex halimus (Chenopodiaceae, perennial), Bassia
indica (Chenopodiaceae, annual), Solanum nigrum L. (Solanaceae, perennial), Opunia
ficus indica (Cactaceae, perennial), Acacia saligna (Fabaceae, perennial), Eucalyptus
camaldulensis (Myrtaceae, perennial). All of these plants are found in the Negev Desert,
Israel and were grown in a small plot in this desert during the spring and summer of 2012.
Measurements were performed at a CO2 concentration of 400 µmol mol-1, and ambient
oxygen concentration (~20%); for all other details of the experiment, see Materials and
Methods. Standard error bars are shown, n = 5.
47
A. hierochuntica plants grown under different light regimes, including growth chamber versus
field, showed no significant differences in the NPQ induction (Figure 9a). In these different
environments, A. hierochuntica plants had optimal values of maximum photochemical yield
(Fv/Fm) (above 0.8) (Table 5), indicating that these plants were not photoinhibited despite the
low and slow NPQ induction. The low induction of NPQ of A. hierochuntica was not related to
the developmental stage, since similar results were obtained from cotyledons of five-day-old
seedlings (data not shown). Ambient CO2 can have a direct effect on electron transport and,
therefore, can influence NPQ induction. NPQ response in A. hierochuntica was recorded at five
different CO2 concentrations in plants exposed to high light intensity (1200 PPFD). At low CO2
concentrations, NPQ inductions in the first few minutes of the light period were higher than at
high CO2 concentrations. For example, NPQ at 80 µmol mol-1 CO2 after four minutes was 25%
higher as compared to the measurements at 1600 µmol mol-1 CO2(Figure 9b). This phenomenon
could be explained by the relative lack of acceptors for products of the light reaction under low
CO2 concentrations (Kanazawa and Kramer, 2002).
48
Figure 9: NPQ response in A. hierochuntica plants that were grown in different environments
and different CO2 concentrations. A: NPQ was measured at a light intensity of 1200 PPFD, in
Anastatica Hierochuntica plants that were grown in different growth environments; measurements
were done at a CO2 concentration of 400 µmol mol-0, and an ambient O2 concentration (~20%). B:
NPQ was measured at a light intensity of 1200 PPFD, in Anastatica Hierochuntica plants at various
CO2 concentrations and 20% O2; the plants were grown in a growth chamber with a light intensity of
600 PPFD. For all other details of the experiment, see the Materials and Methods section; standard
error bars are shown ,9 ≥n≥4.
49
Table 5: Photochemical potential of photosystem II and pigment composition in
leaves of A. hierochuntica and the other plants in the experiment. Maximum
yield of PSII was measured in dark-adapted leaves; pigment composition was
measured as described in Materials and Methods
Plant
Growth
Maximum yield
Leaf chlorophyll
Chl a/ Chl
Leaf
Environment -
of PSII
concentration
b ±S.D.
carotenoid
light intensity
(Fv/Fm))±S.D.
(µg Chl
(n=5)
concentration
(PPFD)
Anastatica
hierochuntica
Growth chamber -
2
(n>10)
a+b/cm )±S.D.
(µg/ cm2)
(n=5)
±S.D. (n=5)
0.807±0.018(c)
34.2 ±2(ab)
4.2±0.1
6.5±0.7(b)
0.833±0.003(a)
39±3.2(a)
4.2±0.3
7.8±0.4(a)
0.834±0.003(a)
33.5±2(b)
3.9±0.1
7.1±0.2(ab)
0.820±0.010(b)
03±1.8(c)
3.7±0.6
0.7±0.3(c)
0.825±0.013(ab)
36.1±2.5(ab)
3.9±0.6
7±0.5(ab)
0.829±0.025(ab)
38.3±2(a)
4.1±0.3
8±0.2(a)
Low light
intensity (200)
Anastatica
hierochuntica
Growth chamber Moderate light
intensity (600)
Anastatica
hierochuntica
Field –High light
Arabidopsis
thaliana
Growth chamber -
intensity (~2000)
Moderate light
intensity (600)
Thellungiella
saslsuginea
Growth chamber Moderate light
intensity (600)
Helinathus
Annuus
Growth chamber Moderate light
intensity (600)
50
When a comparison was made between the induction of NPQ in A. hierochuntica and the other
plant species at a high light intensity and low CO2 concentrations (80 µmol mol-1), NPQ
induction was significantly lower in A. hierochuntica (Figure 10a). For example, after two
minutes, NPQ induction was 50% lower as compared to the three other species tested. Lower
levels of NPQ in A. hierochuntica were observed at various different CO2 concentrations
(Figure 10b), and this suggests that differences in CO2 availability are not the cause for the
distinct NPQ response in A. hierochuntica plants.
51
Figure 10: NPQ response in A. hierochuntica and other plant species grown in a growth
chamber, and at different CO2 and O2 concentrations. A: NPQ was measured at an ambient CO2
concentration of 80 µmol mol-1, 20% O2 and 1200 PPFD, during light period of 14 minutes, standard
error bars are shown, 6≥n≥4. B: NPQ was measured at various CO2 concentrations (presented as Ci,
leaf internal CO2 concentration); measurements were done for each concentration separately, and NPQ
values were measured 14 minutes after the beginning of the light period. Light intensity was 1200
PPFD, 9≥n≥4.
52
A straightforward explanation for the distinct and low NPQ response in A. hierochuntica plants
could be that the PSII reaction centers, in the leaves of these plants, received less light due to
lower absorbance. In turn, the activation of NPQ, which is a light-dependent process, was lower.
In the case of A. hierochuntica, the leaves appeared dark green, and the chlorophyll content was
similar to or higher than the other species in the different experiments and did not change
significantly between the different growth conditions (Table 5). Furthermore, the actual
percentage of absorbance of photosynthetic light in the leaves of A. hierochuntica, measured
with an integrated sphere device, was 90.2% as compared to 84.6% absorbance of PAR in A.
thaliana plants. Therefore, the low NPQ induction in A. hierochuntica cannot be explained by
low leaf absorbance (Table 6). Another possible mechanism that might lower the induction of
NPQ is a different distribution of light energy between the two photosystems; if less light is
distributed to PSII, it could affect NPQ (Maxwell and Johnson, 2000). There were no differences
between A. hierochuntica and A. thaliana, in the fluorescence ratio of PSII (682nm) and PSI
(730nm) at 77oK (Table 6). From these results, we concluded that the low induction of NPQ at
high light intensities was independent of the growth conditions and of the developmental stage
and was not a result of low light absorbance by PSII or the distribution of light energy between
the two photosystems.
Table 6: Light absorbance and distribution of light energy between PSII and PSI, in leaves of
A. hierochuntica and A. thaliana.
Leaf absorbance was measured with an integrated sphere device; excitation distribution between
PSII and PSI was calculated from a fluorescence measurement at 77 oK. Groups that do not share
a common letter are statistically different (α=0.05).
Plant
Leaf absorbance (%) of
Excitation distribution
photosynthetic active
between PSII and PSI (F’
radiation (400-700nm)
682nm/ F’ 730nm)
±S.D. (n=3)
±S.D. (n=4)
Anastatica hierochuntica
90.2±1.3(a)
1.02±0.02
Arabidopsis thaliana
84.6±2(b)
0.99±0.03
53
The de-epoxidation state of xanthophylls (Adamas and Demmig Adams, 1996, Johnson et al.,
2008) is known to influence NPQ. The de-epoxidation state in the leaves of A. hierochuntica was
significantly lower as compared to A. thaliana, after both short and long exposures (2 and 120
minutes, respectively) to high light intensities. The de-epoxidation state of xanthophylls was
positively correlated with the NPQ values of these species (Table 7). The de-epoxidation state,
after 2 and 120 minutes in A. hierochuntica, was 56 and 48%, respectively, lower than in A.
thaliana. NPQ, after 2 and 120 minutes in A. hierochuntica, was 55 and 37%, respectively, lower
than in A. thaliana (Table 7). It seems that the differences in the regulation of the xanthophyll
cycle (de-epoxidation state) in A. hierochuntica are the cause, at least in part, for the low NPQ
induction in this species.
Table 7: De-epoxidation state of xanthopylls and NPQ at high light intensity in A.
hierochuntica and A. thaliana.
HPLC measurements of the different levels of xanthophylls (molar concentration) were used to
calculate the de-epoxidation state of xanthophylls (DES), according to the following equation,
DES(%)=100 X (0.5 x [A] +[Z])/ ([V] +[A] +[Z]), where A- Antheraxathin, V- Violaxanthin and
Z- Zeaxanthin. NPQ was measured as described in previous sections. Groups that do not share a
common letter are statistically different (α=0.05).
Plant
De-epoxidation
NPQ after
De-epoxidation
NPQ after
state (%)after
2 minutes at
state (%)after
120 minutes
2 minutes at
light intensity
120 minutes, at
at light
light intensity
of 1200
1200
intensity of
of 1200 PPFD,
PPFD, n=5,
PPFD, n=3,
1200 PPFD,
n=3, ±S.D.
±S.D.
±S.D.
n=6, ±S.D.
A. hierochuntica
15±6(b)
0.834±0.14(b)
28±4(b)
1.46±0.41(b)
A. thaliana
34±5 (a)
1.838±0.348(a) 54±8(a)
2.32±0.15(a)
54
We examined the putative A. hierochuntica orthologous transcript of the violaxanthin deepoxidase (VDE) gene from A. thaliana. VDE was shown to be responsible for the deepoxidation process that triggers NPQ. The VDE ortholog of A. hierochuntica had high
similarity with the A. thaliana VDE protein (86%), and had also retained the conserved cysteine
residue at position 185 (Table 4). This residue was shown to be vital for the protein activity
(Niyogi et al., 1998). The changes in the sequence of VDE between A. hierochuntica and A.
thaliana were unlikely to be the cause for the low xanthopyll de-epoxidation state in A.
hierochuntica and, therefore, cannot explain the different NPQ responses.
185
Arabidopsis thaliana
TCNNRPDETECQIKCGDLFENSVVDEFNECAVSRKKCVPRKSDLGEFPAPDPSVLVQNFN
Anastatica hierochuntica TCNNRPDETECQIKCGDLFENSVVDEFNECAVSRKKCVPRKSDLGDFPAPDPSVLVKNFD
Glycine max
TCNNRPDETECQIKCGDLFENSVVDEFNECAVSRKKCVPKKSDVGEFPAPNPDVLVNSFN
Vitis vinifera
TCNNRPDETECQIKCGDLFENNVVDEFNECAVSRKKCVPRKSDIGEFPVPDPAVLVKSFN
Nicotiana tabacum
TCNNRPDETECQIKCGDLFENSVVDEFNECAVSRKKCVPRKSDVGDFPVPDPSVLVQKFD
Solanum Lycopersicon
TCNNRPDETECQIKCGDLFENSVVDEFNECAVSRKKCVPRKSDVGDFPVPDPSVLVQKFD
Table 8: The VDE protein sequence in different plants, including Anastatica hierochuntica.
Part of the protein sequence of the VDE gene from several plant species, between amino acid 170 and
208 (Arabidopsis), including the conserved Cysteine (C) in position 185. which is essential for the
enzyme activity
Since the absorption of light by PSII was shown to be close to the standard value of 0.42-0.45
(leaf absorbance x fraction of light directed to PSII), in A. hierochuntica and A. thaliana (Table
6), and since H. annus and T. saslsuginea were reported to have similar values, I used the
absorption constant of 0.85 x 0.5 to estimate fraction of light that is absorbed by PSII, this in
order to calculate the PSII electron transport rate.
7.2.3. Photosystem II electron transport rate in A. hierochuntica and other plant species
The effect of growth environment on PSII ETR in A. hierochuntica was measured in plants
grown in different growth environments. When grown at a low light intensity (200 PPFD), A.
55
hierochuntica plants had significantly lower PSII ETR than the A. hierochuntica plants grown at
moderate (600 PPFD) and high light intensities under field (~2000 PPFD) conditions (Table 9).
Low PSII ETR at low light intensities suggested that the potential photochemical activity was
impaired in A. hierochuntica plants grown in the artificially low light environment. Due to the
relatively low photochemical activity at low light intensities, we decided to continue to grow
plants at moderate light intensities (600 PPFD).
Table 9: Photosystem II electron transport rate (PSII ETR) in A. hierochuntica plants
that were grown in different environments. PSII ETR was measured at a light intensity of
1200 PPFD, CO2 concentration of 400 µmol mol-1 and room ambient O2 concentration (~20%).
The measurements were taken 14 minutes after the beginning of the light period, in detached
leaves of Anastatica hierochuntica plants that were grown in different environmental
conditions. Standard error bars are shown, n=6.
Growth conditions
PSII ETR at 1200 PPFD±S.D
Growth chamber -Low light intensity
88.9±14(b)
(200 PPFD)
Growth chamber -Moderate light
179±18(a)
intensity (600 PPFD)
Field –High light intensity (~2000
209±10(a)
PPFD)
The use of light by photochemical processes, measured as PSII ETR after two hours of light, was
only 7% higher in A. hierochuntica under low light intensity as compared to A. thaliana and was
not significantly different from H. annuus and T. salsugenia. Under moderate and high light
intensities, PSII ETR was significantly higher in A. hierochuntica as compared to A. thaliana and
T. salsugenia and was similar to H. annuus (Figure 11).
56
Figure 11: Photosystem II electron transport rate (PSII ETR) at different light intensities,
in A. hierochuntica and other plant species grown in a growth chamber with moderate
light intensity.
PSII ETR was measured at various light intensities, in a CO2 concentration of 400 µmol mol-1
and an ambient oxygen concentration (~20%), approximately 2 hours after the beginning of the
light period. Statistically different groups (student’s t-test, α=0.05) are marked with a different
letter; standard error bars are shown, n=6.
57
The availability of CO2 had a lower impact on PSII ETR in A. hierochuntica measured under
high light intensities (1200 PPFD), as compared to the other plant species including H. annuus,
T. salsugenia and A. thaliana. For example, PSII ETR at a low ambient CO2 concentration (80
µmol mol) was 60% lower than at a high ambient CO2 concentration (1600 µmol mol) in all
three of the species measured. However, in A. hierochuntica plants, the difference was only 37%
(Figure 12).
Figure 12: PSII ETR at different CO2 concentrations in A. hierochuntica and other
plant species. PSII ETR was measured at various CO2 concentrations (presented as Ci,
leaf internal CO2 concentration- µmol mol-1), measurements were taken for each
concentration separately, and PSII ETR values were measured 14 minutes after the
beginning of the light period. The light intensity was 1200 PPFD; measurements were
taken from plants grown in a growth chamber with a light intensity of 600 PPFD, 9≥n≥4.
58
The differences in PSII ETR at high light intensity and low CO2, between A. hirechuntica and
the other plants species, were time dependent. For example, After two minutes in the light at low
CO2 concentration (80 µmol mol-1), PSII ETR in A. hierochuntica was 88% higher than that of
H. annus and after eight minutes it was 77% higher. After 14 minutes, PSII ETR in A.
hierochuntica was 67% higher than in H. annuus (data not shown).
Low availability of oxygen (2%), which is known to diminish photorespiration (Bjorkman et al.
1966, Lovelock and winter 1996, Streb et al. 2005), had different effects on PSII ETR at
different CO2 concentrations in A. hierochuntica as compared with A. thaliana. Low oxygen,
under 400 µmol mol CO2, did not affect PSII ETR in either species. When exposed to low
concentrations of CO2 (50 µmol mol), the lack of oxygen had a large effect on PSII ETR in A.
hierochuntica, lowering it by more than 55%. In A. thaliana, PSII ETR was significantly lower
at low concentrations of CO2 (50 µmol mol), and the lowering of the oxygen concentration
caused only a slight decrease (14%) in PSII ETR (Figure 13). Therefore, it is suggested that
photorespiration makes a major contribution to PSII ETR under low CO2 concentrations.
These results suggest that the PSII ETR at a high light intensity, in A. hierochuntica, is relatively
high and can be driven, by the presence of either CO2, or O2.
59
Figure 13: PSII ETR in A. hierochuntica and A. thaliana, at different O2 and CO2
concentrations. PSII ETR was measured at low (2%) and room (~20%) O2 concentrations, at
a CO2 concentration of 400 and 50 µmol mol-1, and the light intensity was 1200 PPFD; the
measurements were taken 14 minutes after the beginning of the light period. Statistically
different groups (student’s t-test, α=0.05) are marked with a different letter. standard error
bars are shown, n=5.
7.2.4. Carbon assimilation, photorespiration, respiration and stomata conductance in A.
hierochuntica and other plant species.
In order to evaluate water use and carbon assimilation, gas exchange was measured. Stomata
conductance in A. hierochuntica plants was significantly higher as compared to T. salsugenia at
all light intensities and was lower in comparison to H. annuus, at the moderate and high light
intensities (Figure 14a). This higher conductance at moderate and high light intensities was
accompanied, as expected, by relatively high net carbon assimilation in A. hierochuntica plants
(Figure 14b). Intrinsic water-use efficiency in A. hierochuntica, at moderate and high light
intensities, was relatively high, in comparison to H. annuus and A. thaliana (Figure 14c).
60
Figure 14: Stomata
conductance, net
assimilation and water-use
efficiency at different light
intensities, in A.
hierochuntica and other
plant species.
A: Stomata conductance to
water vapor.
B: Net assimilation of CO2.
C: Water-use efficiency of
photosynthesis- CO2
assimilated per H2O
transpired.
These measurements were
taken approximately two
hours after the beginning of
the light period, at a CO2
concentration of 400 µmol
mol-1 and a room oxygen
concentration (~20%).
Statistically different groups
(student’s t-test, α=0.05) are
marked with a different
letter; standard error bars
are shown, n=6.
61
The growth environment of A. hierochuntica affected both stomata conductance and net carbon
assimilation at a high light intensity. Plants grown under a low light intensity had half of the
stomata conductance and less than half of the net assimilation rate, in comparison to A.
hierochuntica plants that were grown at a moderate light intensity.
Table 10: Stomata conductance and net carbon assimilation in A. hierochuntica plants
that were grown in different light intensities.
Stomata conductance and net carbon assimilation were measured at a light intensity of 1200
PPFD, a CO2 concentration of 400 µmol mol-1 and room oxygen concentration (~20%); values
were recorded 14 minutes after the beginning of the light period, 12≥n≥6. Statistically different
groups (student’s t-test, α=0.05) are marked with a different letter.
Growth conditions
Growth chamber -Low light
Stomata conductance (mmol
Net assimilation ( µmol CO2
H2O/ m-2s-1) ± S.E
m-2s-1)± S.E
107±10(b)
7.3±0.5(b)
212± 30(a)
20.9±2(a)
intensity (200 PPFD)
Growth chamber -Moderate light
intensity (600 PPFD)
62
Net carbon assimilation rates at different CO2 concentrations (presented as Ci-leaf internal CO2
concentration) were significantly higher in A. hierochuntica in comparison to A. thaliana and T.
salsuginea, and were similar to H. annuus (Figure 15). Taken with figure 12, it is suggested that at
high CO2 concentrations, similar ratio of PSII ETR to net assimilation, at high CO2 concentrations, in both
A. hierochuntica and the other plants in the experiment.
Figure 15: Net assimilation at different CO2 concentrations in A. hierochuntica and other plant
species. Net CO2 assimilation was measured at various CO2 concentrations (presented as Ci, leaf internal
CO2 concentration); measurements were taken for each concentration separately, and values were
measured 14 minutes after the beginning of the light period. The light intensity was 1200 PPFD;
measurements were taken from plants grown in a growth chamber with a light intensity of 600 PPFD,
9≥n≥4.
63
Net carbon assimilation in non-photorespiratory conditions (2% oxygen) was 30% higher in
comparison to the photorespiratory conditions (ambient oxygen 21%), suggesting that
photorespiration plays a major role in carbon metabolism at high light intensities in A.
hierochuntica (Figure 16a). In order to estimate the effect of light intensity on carbon
metabolism in the dark, dark respiration was measured less than two minutes after the light
period ended. After moderate light intensity, dark respiration was significantly higher in A.
hierochuntica than in A. thaliana (50% more). After a period of high light intensity, dark
respiration in A. hierochuntica was 90% higher than in A. thaliana (Figure 16b).
Figure 16: The influence of non-photo respiratory conditions (2% O2) on carbon assimilation, and
the impact of light intensity on dark respiration in A. hierochuntica and A. thaliana.
A: Photorespiration: Net assimilation was measured at low (2%) and room (~20%) O2 concentrations, at
a CO2 concentration of 400 µmol mol-1 and 1200 PPFD. The measurements were done 14 minutes after
the beginning of the light period; B: Dark respiration: Plants were exposed to moderate and high light
intensity (600 and 1200 PPFD, respectively) for two hours; then, respiration was measured in the leaf
after four minutes in the dark, at a room oxygen concentration (~20%) and a CO2 concentration of µmol
mol-1; leaf temperature was kept at 24 0C. Statistically different groups (student’s t-test, α=0.05) are
marked with a different letter. Standard error bars are shown, n=5.
64
Overall, these results suggest that the relatively high PSII ETR in A. hierochuntica plants was
used to support carbon assimilation, photorespiration and respiration at high light intensities.
7.2.5. The ratio of PSII ETR to NPQ in A. hierochuntica and other plant species
At both low moderate and high light intensities, when CO2 concentration was close to the
atmospheric concentration, PSII ETR/ NPQ was higher in A. hierochuntica than in all the other
three plants (H. annuus, A. thaliana and T. saslsuginea), at the beginning of the light period. For
example, PSII ETR/NPQ was three times higher at 600 PPFD compared to the other three plant
species. After longer exposure to light, PSII ETR/ NPQ was higher in A. hierochuntica, at
moderate and high light intensities, than in the other three plant species, but not in low light
intensity (Figure 17).
65
Figure 17: The ratio of PSII ETR to NPQ, at various light intensities, in A. hierochuntica and in
other plants species.
The ratio of PSII ETR to NPQ measured at: A: 1200 PPFD, B: 600 PPFD, C: 200 PPFD. All
measurements were done at a CO2 concentration of 400 µmol mol-1, and an ambient oxygen
concentration (~21%); for all other details of the experiment, see the Materials and Methods section.
All plants were grown in a growth chamber with a light intensity of 600 PPFD. In all the graphs,
standard error bars are shown, 9≥n≥4.
At a high light intensity (1200 PPFD) and a CO2 concentration that is close to the atmospheric
concentration, the ratio of PSII ETR to NPQ in A. hierochuntica was more than twice that of all
the three other species in the experiment, at the beginning of the light period. At the end of the
light period, the ratio of PSII ETR to NPQ was 30% higher than in H. annuus and three times
higher than in T. salsuginea and in A. thaliana, after 14 minutes from the end of the light period.
66
At a low CO2 concentration (80 µmol/ mol), the ratio of PSII ETR to NPQ in Anastatica was
three times higher than that of the other plants in the beginning of the light period and more than
two times higher at the end of the light period (Figure 18).
Figure 18: The ratio of PSII ETR to NPQ, at low and atmospheric CO2 concentrations, in A.
hierochuntica and in other plant species.
The ratio of PSII ETR to NPQ measured at a light intensity of 1200 PPFD and at CO2 concentrations
of : A: 400 µmol/ mol, and B: 80 µmol/ mol. Ambient oxygen concentration (~21%); for all other
details of the experiment, see the Materials and Methods section. All plants were grown in a growth
chamber with a light intensity of 600 PPFD. In all the graphs, standard error bars are shown, 9≥n≥4.
67
7.2.6. The response of photosynthesis PSII ETR and NPQ to drought, in A. hierochuntica
and H. annuus
In order to test whether the short-term difference in the response to low CO2 persisted after
longer time periods, I conducted a drought experiment in A. hierochuntica and H. annuus in
order to test whether the short-term (up to tens of minutes) difference in the response to the low
availability of CO2 also reflects a difference in the long-term response (hours and days) to low
CO2.
Under drought conditions, plants close their stomata, in order to conserve water. By doing so,
they also limit the diffusion of CO2 from the air to the leaf; this in turn creates conditions that are
similar to those in plants exposed to low CO2, since in both these conditions, photosynthesis is
mainly limited by the availability of CO2.
Thirty-day old A. hierochuntica plants and twenty-one-day old H. annuus plants were subjected
to one week of drought or to continued irrigation. Measurements of the plants were conducted
between the 4th and 7th day of that week; the measurements were taken between 2 and 4 hours
after the beginning of the light period of each day.
Under well-irrigated conditions, stomata conductance and carbon assimilation rates were similar
in A. Hierochuntica and H. annus (Figure 19a). In the drought-treated plants, assimilation rates
dropped with the decrease in stomata conductance. The decreasing trend was similar in both
plants (Figure 19a); therefore, in this experiment, drought caused a similar decrease in the
response of assimilation rates and stomata conductance in both plants. In order to examine
whether assimilation under drought decreased due to low CO2 diffusion into the leaf, the plants
were exposed to a high level of CO2. Under these conditions, the difference in assimilation
between the drought-treated plants and the irrigated plants was much lower, and was much less
dependent on stomata conductance (Figure 19b), in comparison to the differences in assimilation
under average CO2.
These results suggest that in both A. hierochuntica and H. annuus, the inhibition of CO2
assimilation under low stomata conductance (drought) could be mostly attributed to a low
availability of CO2.
68
Figure 19: Net carbon assimilation at average and high CO2 concentrations as a function of
stomata conductance, in response to drought and irrigation treatments in A. hierochuntica and
H. annuus.
A: Net assimilation of CO2 at atmospheric CO2 concentration 400 (µmol mol-1), Y axis,
vs. stomata conductance, X-axis.
B: Net assimilation of CO2 at a high CO2 concentration (1600 µmol mol-1), Y-axis, vs. stomata
conductance, X-axis). Both types of measurements were conducted in the growth chamber, between
10:00 AM and 12:00 AM, while the light period began at 8:00 AM. Light intensity was 600 PPFD
during the measurement and growth. Oxygen concentration during growth and measurement was
atmospheric (~20%). For all other details, see Materials and Methods section.
In response to low stomata conductance and drought, PSII ETR decreased in a similar trend and
to similar values in both A. hierochuntica and H. annuus (Figure 20a); the decrease was up to
69
50% in comparison with the irrigated plants. NPQ increased under drought similarly in both
plant species and was inversely related to stomata conductance. NPQ was ~3.5 times higher in
the drought-treated plants, in comparison to the NPQ of the irrigated plants. As a direct result, in
both plants, the ratio of PSII ETR/NPQ decreased similarly and linearly with the decrease in
stomata conductance (not shown).
‘
Figure 20: The relation between PSII ETR and NPQ to stomata conductance in irrigated and
drought-treated plants.
A: PSII ETR vs. stomata conductance
B: NPQ vs. stomata conductance
Measurements were taken at 600 PPFD and a CO2 concentration of 400 µmol mol-1, under
atmospheric oxygen concentration. Measurements were taken between two and four hours after the
beginning of the light period.
70
The results from the drought experiment suggest that after long-term exposure to a low
availability of CO2 (drought, low stomata conductance), the NPQ and PSII ETR of A.
hierochuntica are similar to the NPQ and PSII ETR of H. annuus. The lack of difference in the
response to drought in the NPQ and PSII ETR is in contrast to the difference in these parameters
under short-term exposure to a low CO2 availability between A. hierochuntica and the other
plants (see Figures 10 and 12).
71
8. Discussion
8.1. Photoprotective response under drought in wild barley ecotypes from Mediterranean
and desert origins
Severe drought caused a significant and similar decrease in growth in both ecotypes (Table 1);
this was also reflected in the physiology of single leaves. Leaf RWC, stomata conductance and
carbon assimilation decreased similarly and significantly in both ecotypes (Figure 3). In our
experiment, net carbon assimilation was inhibited almost completely in both ecotypes, under
severe drought. Under such conditions, the potential damage caused by excess light can be high
since the repair cycle of PSII reaction centers might be impaired (Takahashi and Murata, 2008).
Multiple ways exist in which plants protect themselves from photodamage; among them are
NPQ, anthocyanin accumulation and photorespiration.
Increasing the photoprotection of plants by NPQ under drought is well documented (Petsas and
Grammatikopoulos, 2009, Naumann et al., 2007). In our research, both ecotypes of H.
spontaneum reacted to drought by increasing their NPQ; however, NPQ in the desert ecotype
was significantly lower than in that of the Mediterranean ecotype in both drought and irrigated
treatments (Figure 5). Therefore, we suggest that a stronger induction of NPQ, under drought, is
not an adaptive mechanism of the desert ecotype of H. spontaneum to its environment.
Anthocyanin molecules can absorb light in the visible range, therefore lowering the excitation
pressure on photosystem II and preventing photodamage (Feild et al., 2001). It has been
suggested that anthocyanin can serve in photoprotection, in conjunction with other
photoprotective processes, such as NPQ (Close and Beadle, 2003). However, our results
indicated that a higher induction of NPQ was recorded in the drought-treated Mediterranean
ecotype, which accumulated a low amount of anthocyanin (Table 2), while lower NPQ levels
were recorded in the drought-treated desert ecotype (Figure 5). Anthocyanin accumulated mainly
in the desert ecotype under drought and significantly less in the Mediterranean ecotype. These
findings can be explained by the light absorptive function of anthocyanin that lowers the
excitation pressure on PSII and, therefore, lowers the activation of NPQ in the desert ecotype.
These results coincide with what was observed in A. thaliana mutants with low NPQ; in these
mutants, anthocyanin accumulated under stress (chilling stress), and these plants were able to
72
compensate for the absence of photoprotection by NPQ (Havaux and Kloppstech, 2001).
Therefore, there is a balance between photoprotection by “shading” (anthocyanin) and by NPQ.
In addition, we have found that the O2-dependent PSII yield was a major contributor to the linear
electron flow in the drought-treated desert ecotype of H. sponataneum (Figure 6). This finding
suggests that adaptation to desert environments may be related to an increase in the capacity of
the O2-dependent PSII photochemical yield under drought, and supports previous observations of
the role of photorespiration under drought (Wingler et al., 1999; Guan et al., 2004 ;Lovelock and
Winter, 1996; Bai et al., 2008).
The difference in the photoprotective response under drought of these two ecotypes
represents a flexible vs. stable form of photoprotection. Anthocyanin accumulation, as was
recorded in the desert ecotype is concomitant with a decrease in the amount of light reaching the
PSII antenna complexes, therefore decreasing the risk of photodamage. However, if the drought
conditions end, for instance, due to a rain event, the leaves that accumulated higher levels of
anthocyanin will probably have a lower capacity for photosynthesis since less light will be
available to drive photochemistry. Therefore, the photoprotective response of H. spontaneum
from the desert environment can be regarded as a stable form of protection. In the Mediterranean
ecotype, drought induced photoprotective NPQ. This response was recorded from a dark-adapted
state, which suggests that it is a reversible NPQ response, associated with PSII activity and low
pH in the chloroplast lumen. NPQ is usually rapidly reversible (within seconds to tens of
minutes), compared to anthocyanin accumulation; therefore, the induction of NPQ as a
photoprotective response under drought, as in the case of the Mediterranean ecotype of H.
spontaneum, can be regarded as a flexible form of photoprotection.
A possible interpretation of the results is that, under conditions of drought, excessive
light and the inhibition of carbon assimilation, the desert SB ecotype of H. spontaneum
accumulated high concentrations of anthocyanin and maintained high levels of O2-dependent
photochemical activity, while the Mediterranean ecotype had higher NPQ. In conclusion, our
results suggest that ecotypes of H. spontaneum from Mediterranean and desert origins have
different strategies to mitigate the damage of drought and high light intensity (Eppel et al.,
2013).
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8.2. Non-photochemical and photochemical processes in the annual desert plant Anastatica
hierochuntica
The importance of NPQ as a photoprotective mechanism has been experimentally confirmed
(Külheim et al., 2002). NPQ is a ubiquitous mechanism that is active in many plant species as
protection against high light intensity (Demmig-Adams and Adams, 1996).
A. hierochuntica is an annual plant that grows naturally in the desert, where high light occurs on
most days throughout the year. According to the results presented here, A. hierochuntica plants
have a low NPQ response to high light intensities relative to a variety of other plant species—the
model plants A. thaliana and T. salsuginea, the crop plant H. annuus (Figure 7), and some other
annual and perennial desert plants (Figure 8). On the other hand, A. hierochuntica plants showed
relatively high photochemical activity (PSII ETR) at high light intensities (Figure 11) , which
was significantly more stable in response to different CO2 concentrations than other plant species
in the experiment (Figure 12). It is important to point out that stomata conductance, which can
regulate the CO2 in the leaves, was higher in A. hierochuntica in comparison to A. thaliana and
T. salsuginea but was lower in comparison to H. annuus. Therefore, the difference in NPQ and
its response to CO2 availability between A. hierochuntica and the other plants in the experiment
cannot simply be explained by a difference in stomata conductance.
The different growth environments (lab and field conditions) used to study A. hierochuntica
plants in this experiment did not have a significant effect on NPQ induction at high light
intensities (Figure 9a). This suggests that the low and slow induction of NPQ is relatively
independent of environmental growth conditions. The use of high light intensity by A.
hierochuntica for photochemistry was significantly lower in plants grown under low light
intensity compared to plants grown in a natural high light environment and in a controlled
moderate light environment (growth chamber) (Table 9). These findings suggest that the ability
to use high light intensity in photochemistry depends on the growth environment.
Since water is scarce in deserts, many desert plants have inertly low transpiration and stomata
conductance, in order to avoid water loss; however, in this study, stomata conductance in A.
hierochuntica was relatively high (Figure 14). A. hierochuntica is probably not adapted to
surviving long periods of drought, but is probably adapted to conditions in which water is
available. High stomata conductance enables relatively high photosynthetic rates in A.
hierochuntica, because it ensures that sufficient CO2 will be available for fixation, and electrons
74
transferred from the light reaction can be used for photosynthesis and to support growth and
reproduction.
High photosynthetic rates under high light intensity enable high intrinsic WUE, and therefore,
although A. hierochuntica plants are not typical water-saving plants, they use water efficiently in
terms of carbon gain per water loss when irradiance is high. This relatively high WUE might
represent an adaptation to the short time during which growth is favored under natural desert
conditions. Our measurements suggests that at high CO2 availabilty, similar amount of electrons
are used to drive carbon assimilation in both A. hierochuntica and the other plant species in that
were used in the experiment. This suggests that Rubisco carboxylation is the main process that
consume reductive power, at high CO2 availability in A. hierochuntica.
The contribution of photorespiration to PSII ETR in A. hierochuntica under conditions of high
light intensity and low CO2 availability was relatively high (Figure 13). These conditions can
represent low soil moisture or a high vapor-pressure deficit. It is likely that, as previously
suggested for other plants species (Heber et al., 1996, Foyer et al., 2009, Kozaki and Takeba,
1996), photorespiration has a photoprotective effect in A. hierochuntica. Photoprotection in A.
hierochuntica seems to rely more on photorespiration and less on NPQ, which was significantly
lower in A. hierochuntica plants in comparison to the other plant species in the experiment. The
relatively high dark respiration in A. hierochuntica, induced by moderate and high light intensity
(Figure 16), is evidence of the induction of a high rate of carbon metabolism in this plant under
high and moderate light intensities.
The observed low NPQ in A. hierochuntica plants might be explained by several different
mechanisms, including the de-epoxidation state of xanthophylls. For instance, A. thaliana npq1
mutants that lack the enzyme VDE are unable to convert violaxanthin to antheraxanthin and
zeaxanthin. These mutants had both a low de-epoxidation state and a low NPQ under high light
(Niyogi et al., 1998). A different A. thaliana mutant, npq4, which is mutated in part of the PSII
antenna complex protein (PsbS), was also shown to have a low NPQ response (Li et al., 2000). A
similar example comes from rice (Oryza sativa), in which the indica variety showed lower NPQ
response than the japonica variety. This latter difference was in direct correlation with the
amount of PsbS protein in the leaves of these two ecotypes (Kasajima et al., 2011).
In the current study, we found that A. hierochuntica plants had a lower de-epoxidation state of
xanthophylls at high light intensities as compared to A. thaliana (table 7). Therefore, it seems
75
that differences in the regulation of the xanthophyll cycle between A. hierochuntica and A.
thaliana might contribute to differences in NPQ.
The stable and high ETRs under different CO2 concentrations observed in A. hierochuntica
plants contradicts the results observed in the previous studies mentioned above in which
Arabidopsis npq mutants and rice indica variety had relatively low NPQ. In these studies of low
NPQ (Li et al., 2000, Niyogi et al., 1998, Kasajima et al., 2011), ETRs were similar to or lower
than the wild type in the case of the A. thaliana mutants and the japonica variety, in the case of
rice. Therefore, although modulation of NPQ through the known xanthophyll cycle might
explain the low NPQ induction in A. hierochuntica, it is not sufficient to explain the high and
stable PSII ETR observed in these plants.
The cold-adapted, annual alpine plant Ranunculus glacialis was shown to have a relatively low
NPQ (referred to as qN in some of the articles on this species) (Streb et al., 1998). In addition,
R. glacialis plants exhibited high and very stable PSII ETRs under different CO2 concentrations
(Streb et al., 2005). These characteristics resemble those measured in the current study in A.
hierochuntica plants. However, based on a comparison with the published results from R.
galacialis, there seem to be some important differences in the photosynthetic characteristics of
these two plant species. In R. glacialis, PSII ETR depended mostly on O2 concentrations, while
CO2 affected PSII ETR only when the O2 concentration was low (Streb et al., 2005). In A.
hierochuntica, a strong oxygen dependency of PSII ETR was observed only at very low CO2
concentrations. The high PSII ETR in R. glacialis was a result of high photorespiration and of
the presence of the plastid enzyme, plastid terminal oxidase, functioning as an “alternative
electron sink”(Streb et al., 2005). Despite these differences, it is still intriguing that these two
plant species, from seemingly very different and extreme growth environments, share similar and
unique characteristics for dealing with high light intensity. Possible similarities between high
alpine mountains and desert environments are high radiation, high wind speed, lack of available
liquid water, all of these might create similar evolutionary pressure on annual plants in these
habitats.
Rubisco activase is an ATP dependent enzyme, which is important for the activation of Rubisco,
and therefore for photosynthesis (Mott and Woodrow, 2000). Recently, a faster activation of
carbon assimilation or PSII ETR, and a lower induction of NPQ has been shown, upon transition
76
from low to high light intensity, in two A. thaliana transgenic lines that either overexpressed
Rubisco activase (Yamori et al., 2012) or expressed only a Rubisco activase ADP insensitive
isoform (Carmo-Silva and Salvucci, 2013). It is likely that the differences in the activity of
Rubisco activase between A. hierochuntica and the other plant species in the current study might
be responsible for the difference in NPQ and PSII ETR. In both A. thaliana transgenic plants
with modulated Rubisco activase, the low NPQ and high PSII ETR were measured in the
transition from low light to high light intensities. In the current study, the difference in NPQ and
PSII ETR between A. hierochuntica and the other plant species was detected in the transition
from dark to light. The differences in Rubisco activase activity might partially explain the
differences between A. hierochuntica and the other species. Since the current study showed
transition between dark and light and not between different light levels, we suggest that the
differences in the Rubisco activase activity might not be sufficient to explain the differences in
NPQ and PSII ETR upon a dark to light transition.
In the current study, we have found that the ratio between PSII ETR and NPQ was significantly
higher in A. hierochuntica than in the other plant species, under various CO2 concentrations and
various light intensities, especially during the initial 14 minutes of the light period, after
transition from dark.
The chloroplast ATP synthase complex exerts control over NPQ and PSII ETR by regulation of
the thylakoid membrane’s conductivity to protons, gH+. When gH+ is high, a given PSII electron
transport rate will cause a smaller increase in the acidity of the lumen, therefore invoking a lower
NPQ response, in comparison with the condition in which gH+ is low and protons accumulate in
the lumen. In the current study, I did not measure gH+, but estimated it according to the ratio of
PSII ETR and NPQ, which was shown to correlated (in some cases linearly) with gH+(Kanazawa
and Kramer, 2002).
In A. hierochuntica, the ratio between PSII ETR and NPQ was high in comparison to the other
plants in the experiments (Figures 17, 18); therefore, it is possible that differences in the
modulation of ATP synthase activity and gH+ might be responsible for the low induction of NPQ
and high PSII ETR, in A. hierochuntica. It is still remains to be seen whether the high PSII
ETR/NPQ in A. hierochuntica is actually related to gH+, and if so, what mechanism causes it to
be different from other plant species. It has been shown that gH+ responds positively to the
77
amount of inorganic phosphate, which can be low when carbon assimilation is low, for instance,
under conditions of low CO2 concentrations (Takizawa et al., 2008). It is possible that the
sensitivity of ATP synthase activity in A. hierochuntica to inorganic phosphate is relatively low,
therefore allowing a relatively high PSII ETR to NPQ ratio, at low CO2 concentrations.
Drought conditions induce stomata closure, which lowers CO2 concentrations within the leaf that
can be used for photosynthesis. Under these conditions, the risk of photoinhibition is higher;
therefore, NPQ is usually induced under these conditions. NPQ induction under drought was
shown to be associated with long-term down regulation of gH+, due to a decrease in the amount
of various subunits of the ATP synthase complex in the chloroplast (Kohzuma et al., 2009).
It is well documented that under drought NPQ tends to increase and that PSII ETR tends to
decrease (Naumann et al., 2007, Petsas and Grammatikopoulos, 2009). In the current study,
similar behaviors of decrease in PSII ETR and increase in NPQ were recorded under relatively
long periods of drought in both A. hierochuntica and H. annuus (Figure 20). These results
suggest that the NPQ induction under drought occurs in A. hierochuntica in a similar manner to
what is described for other plant species. Therefore, it is likely that in A. hierochuntica, the
mechanism that is responsible for keeping NPQ low and PSII ETR high, during relatively short
periods of low CO2 concentrations(short term inhibition of photosynthesis), has a smaller effect
under conditions of the long-term inhibition of photosynthesis, such as long term drought.
In the drought experiment conducted in A. hierochuntica and H. annus, inhibition of
photosynthesis, at low stomata conductance, was almost completely reversed when a high CO2
concentration was applied to the leaves of the plants under drought (Figure 19). These results
support previous reports (Flexas et al., 2004, Cornic and Fresneau, 2002, Wise et al., 1990),
supporting the hypothesis that photosynthesis, under drought, is mainly inhibited due to the lack
of CO2 in the leaves (diffusion limitation), and not because of metabolic impairment (metabolic
limitation), as was suggested (Tezara et al., 1999).
The unique photosynthetic and photoprotective characteristics and mechanisms in A.
hierochuntica have not been previously described for any other plant species. The major
components of this novel finding in A. hierochuntica include:
78
1. Low NPQ at the beginning of the light period, at various light intensities and CO2
concentrations. The described low NPQ cannot be explained by low leaf absorbance or by low
energy distribution to PSII.
2. Relatively high PSII ETR at various CO2 concentrations in correlation with low NPQ. PSII
ETR responded positively to the increase of CO2 concentrations. At low CO2 concentrations,
PSII ETR was mostly O2 dependent.
The combination of these two components is novel and was described in detail in comparison
with three very different and well-studied plant species and other desert plant species.
Recently, it was hypothesized that photosynthesis in crop plants under favorable conditions is
limited by plant protective mechanisms that restrict resource use (Murchie and Niyogi, 2011). In
our study, we have found that in the desert plant A. hierocuhntica, a low activation of a
photoprotecive process (NPQ) is associated with a high utilization of light (a resource) in a
biological process-- PSII activity. Therefore, these findings can be used to support the hypothesis
that modulation of photoprotective processes can increase the efficiency of light use in
photochemical processes; therefore, this modulation can possibly be used to increase
photosynthesis in crop plants. In summary, I have found that the annual desert plant A.
hierochuntica has a low photoprotective NPQ response in comparison to a variety of plant
species from different environments. This low NPQ is accompanied by a relatively high PSII
ETR at moderate and high light intensities and at different CO2 concentrations. The high PSII
ETR is used by A. hierochuntica plants to drive carbon metabolism (Eppel et al. 2014). These
uncommon photochemical characteristics might be a useful adaptation to the short periods
favoring growth in the hot desert.
79
9. Conclusion
In this research I addressed the hypothesis, that some annual desert plant species have different
photo protective responses in comparison to plants form other environments. In the first study I
found a wild barley ecotype from a desert environment that employs different photo protective
mechanisms under drought, in comparison to a Mediterranean ecotype of the same species. In the
second study I found that in the annual desert plant Anatstatica hierochuntica there is an
uncommonly low photo protective energy dissipation to heat (NPQ), and that this plant species
uses a relatively high amount of light energy in photosynthesis and photorespiration.
These results support the hypothesis that some annual desert plant species have photo protective
features, which differ from those of closely related and unrelated plant species, from other
environments. These findings can contribute to our understanding of annual plant adaptation and
acclimation to desert environment.
80
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89
‫‪ .11‬תקציר‬
‫האור הוא חיוני לגדילתם של צמחים‪ ,‬מכיוון שהוא מספק את האנרגיה הדרושה לקיבוע פחמן‪ ,‬בתהליך‬
‫הפוטוסינתזה‪ .‬אנרגיית האור‪ ,‬בטווח הנראה נקלטת בצמחים ע"י מבנים דמויי אנטנה המורכבים מחלבונים‬
‫ופיגמנטים‪ ,‬הנמצאים בכלורופלסטים שבעלה‪ .‬אנרגית האור שנקלטת בצמח מתועלת לביצוע התהליך‬
‫הראשוני שממיר את אנרגיית האור לאנרגיה כימית‪ .‬תהליך זה מתבצע במרכז מערכת האור ‪2‬‬
‫(‪ .)Photosystem II reaction center‬כתוצר לוואי של תהליך המרת אנרגית האור לאנרגיה כימית נגרם נזק‬
‫למרכז מערכת האור ‪ .2‬נזק זה מתוקן ע"י הצמח באופן רציף‪ .‬מצבים בהם קצב יצירת הנזק עולה על קצב‬
‫התיקון‪ ,‬מובילים למצב של פגיעה משמעותית במערכת האור ‪ .2‬פגיעה זאת נקראת פוטואינהיביציה‬
‫(‪ .(Photoinhibition‬תנאי עקה סביבתיים‪ ,‬המעכבים את תהליך קיבוע הפחמן‪ ,‬כגון יובש‪ ,‬יכולים להגביר את‬
‫הסיכון ליצירת פוטואינהיביציה‪.‬‬
‫צמחים מפעילים מנגנונים שונים ע"מ למנוע נזק במערכת האור ‪ .2‬מנגנונים אילו מתחלקים לשני סוגים‬
‫עיקריים‪ :‬מנגנונים שמפחיתים את כמות האור הנקלטת במערכת האור ‪ ,2‬ומנגנונים שמקטינים את הנזק‬
‫אחרי שאנרגיה האור נקלטת ע"י הפיגמנטים הפוטוסינתטיים‪ .‬דוגמא למנגנון המונע קליטת אור‪ ,‬היא צבירה‬
‫של פיגמנטים לא פוטוסינתטיים‪ ,‬כגון אנטוציאנין‪ ,‬היוצרים מיסוך ועקב כך מונעים בליעה של חלק מהאור על‬
‫ידי המערכת הפוטוסינטתית‪ ,‬ועקב כך מקטינים את הנזק הפוטנציאלי‪.‬‬
‫שני מנגנונים חשובים המתמודדים עם אנרגיית האור אחרי קליטתה במערכת האנטנות של מערכת האור ‪,2‬‬
‫הם המרה לא פוטוכימית (‪ (Non Photochemcial Quenching- NPQ‬ופוטורספירציה‪ .‬המרה לא פוטוכימית‬
‫היא תהליך שממיר באופן מידי וללא נזק את אנרגיית האור לחום‪ .‬פוטורספירציה הוא תהליך תלוי חמצן‪,‬‬
‫שבין השאר נמצא כמגן מפני פוטואינהיביציה‪.‬‬
‫צמחי מדבר חד שנתיים חשופים במקרים רבים לעוצמות אור גבוהות‪ ,‬היכולים לגרום לפוטואינהיביציה‪,‬‬
‫במיוחד בתנאי יובש וחום‪ .‬צמחים אלו צריכים להשלים את מחזור חייהם מהר יחסית‪ ,‬עקב קצב האידוי‬
‫הגבוה של מים מהקרקע‪.‬‬
‫ההשערה של מחקר זה היתה שמנגנוני הגנה מאור מופעלים בצורה שונה בצמחי מדבר חד שנתיים‪ ,‬בהשוואה‬
‫לצמחים חד שנתיים דומים מאזורים אקלימיים אחרים‪ .‬מטרתו של המחקר היתה לבדוק את תפקודם של‬
‫מנגנוני הגנה שונים מאור‪ ,‬בצמחי מדבר חד שנתיים‪ ,‬תחת תנאים שונים‪ .‬על מנת לבחון את ההשערה ולענות‬
‫על מטרת המחקר נערכו במסגרת עבודה זו שני מחקרים שונים‪.‬‬
‫במחקר הראשון‪ ,‬צמחי שעורת התבור (‪ ) Hordeum spontaneum L. Koch‬שזרעיהם נאספו בנגב (אקוטיפ‬
‫מדברי) ובגליל (אקוטיפ ים‪-‬תיכוני)‪ ,‬נחשפו לתנאי יובש במשך ‪ 25‬יום‪ ,‬על מנת לבדוק את תגובות ההגנה מאור‬
‫כתוצאה מעקת היובש‪ .‬בשני האקוטיפים עקת היובש גרמה לעצירה דומה ומשמעותית בגידול (ביומסה‬
‫יבשה)‪ ,‬בהשוואה לצמחי הביקורת שהיו מושקים‪ .‬עקת היובש התבטאה גם ברמת העלה הבודד‪ ,‬ע"י עיכוב‬
‫כמעט מוחלט של תהליך‬
‫קיבוע הפחמן‪ ,‬ברמה דומה בשני האקוטיפים‪ .‬ערכי ‪ ,Fv/Fm‬שהם מדד‬
‫‪90‬‬
‫לפוטואינהיביציה‪ ,‬לא היו נמוכים בצורה משמעותית בצמחים שהיו תחת יובש‪ ,‬בהשוואה לצמחי הביקורת‪.‬‬
‫תוצאה זו מעידה על כך שבשני האקוטיפים הופעלו בצורה יעילה מנגנוני הגנה מאור‪.‬‬
‫באקוטיפ הים‪-‬תיכוני ההגנה התבטאה בהפעלה חזקה של תהליך הההמרה הלא פוטוכימית‪ ,‬בהשוואה לצמחי‬
‫הביקורת המושקים‪ .‬לעומת זאת באקוטיפ המדברי שהיה חשוף ליובש‪ ,‬היתה הצטברות של אנטוציאנין‬
‫בעלים‪ ,‬אשר מיסכה על חלק מקליטת האור במערכת האור ‪ .2‬עקב כך יעילות מערכת האור ‪)ФPSII ( 2‬‬
‫נשארה גבוהה באקוטיפ המדברי‪ ,‬גם בתנאי יובש ולא היתה בהם הפעלה חזקה של תהליך ההמרה הלא‪-‬‬
‫פוטוכימית‪ .‬עוד נמצא שהיעילות הפוטוכימית הגבוהה שנשמרה באקוטיפ המדברי הייתה תלוית חמצן‪ ,‬דבר‬
‫המעיד על חשיבות הפוטורספירציה בתגובה של האקוטיפ המדברי לעקת היובש‪.‬‬
‫לסיכום‪ ,‬במחקר זה התגלה שההגנה מאור בתנאי יובש הייתה שונה בשני האקוטיפים של שעורת התבור‪.‬‬
‫באקוטיפ הים‪ -‬תיכוני התגובה היתה מבוססת על הפעלה חזקה של תהליך ההמרה הלא פוטוכימית ולעומת‬
‫זאת‪ ,‬באקוטיפ המדברי התגובה היתה מבוססת בעיקר על צבירה של אנטוציאנין ועלייה בפוטורספירציה‪.‬‬
‫במחקר השני תגובות הפוטוסינתזה וההגנה מאור נחקרו בצמח המדברי החד שנתי שושנת יריחו האמיתית‬
‫(‪ (Anastatica Hierochuntica‬ממשפחת המצליבים‪ .‬המחקר היה מחקר השוואתי בו השתמשתי בשלושה‬
‫מינים נוספים של צמחים‪ ,‬בעלי יכולות שונות לפוטוסינתזה ולתהליכי הגנה מאור‪ .‬שלושת הצמחים היו שני‬
‫מינים ממשפחת המצליבים‪ ,‬צמח המודל תודרנית לבנה (‪ , )Arabidopsis thaliana‬והצמח ת'לונגיילה‬
‫(‪ )Thellungiela saslsuginea‬הידוע כצמח עמיד לעקות סביבתיות‪ ,‬הצמח השלישי היה חמנית ( ‪Helianthus‬‬
‫‪ )annuus‬ממשפחת המורכבים שהוא צמח יבול בעל יכולת פוטוסינתטית גבוהה‪ .‬בצמחי שושנת יריחו‬
‫האמיתית הפעלת תהליך ההמרה הלא פוטוכימית‪ ,‬היה נמוך משאר מיני הצמחים בתגובה לעוצמות אור‬
‫שונות ובתגובה לריכוזים שונים של פחמן דו חמצני‪ .‬הבדל זה בין שושנת יריחו האמיתית לשאר מיני הצמחים‬
‫בניסוי‪ ,‬לא נבע מבליעה נמוכה של אור במערכת האור ‪ ,2‬אלא כתוצאה מהבדל במטבוליזם של פיגמנטים‬
‫פוטוסינתטיים בשם קסנתופילים‪ .‬קסנתופילים ידועים בכך שהם יכולים לווסת את תהליך ההמרה הלא‬
‫פוטוכימית‪ .‬קצב מעבר האלקטרונים במערכת האור ‪ ,2‬בהשוואה לשלושת המינים האחרים‪ ,‬היה גבוה בצמחי‬
‫שושנת יריחו האמיתית‪ ,‬בעיקר בעצמות אור בינונית וגבוהה‪ .‬בנוסף קצב מעבר האלקטרונים היה פחות רגיש‬
‫לירידה בריכוזי פחמן דו חמצני בצמחי שושנת יריחו האמיתית‪ .‬כאשר ריכוזי הפחמן הדו חמצני היו נמוכים‬
‫מאוד‪ ,‬קצב מעבר האלקטרונים הגבוה של צמחי שושנת יריחו האמיתית היה תלוי בפוטורספירציה‪ .‬כאשר‬
‫ריכוזי הפחמן הדו חמצני היו בינוניים וגבוהים‪ ,‬קצב הפוטוסינתזה בצמחי שושנת יריחו האמיתית היו גבוהים‬
‫מאלו שנמדדו בתודרנית לבנה ובת'לונגיילה ודומים לאלו שנמדדו בצמחי החמנית‪ ,‬הידועים כבעלי שיעור‬
‫פוטוסינתטי גבוה‪ .‬בנוסף בצמחי שושנת יריחו נמדדו קצבי פוטורספירציה גבוהים בעוצמות אור גבוהות‬
‫וקצבי נשימת חושך גבוהים אחרי חשיפה לעוצמות אור בינוניות וגבוהות‪ .‬המסקנה מתוצאות אלה היא‬
‫ש קצבי מעבר האלקטרונים הגבוהים בצמחי שושנת יריחו האמיתית‪ ,‬תרמו למטבוליזם גבוהה של פחמן‪,‬‬
‫בעצמות אור בינוניות וגבוהות‪.‬‬
‫חשיפה ליובש של צמחי חמנית ושושנת יריחו האמיתית גרמו לסגירה דומה של פיוניות ועקב כך לירידה דומה‬
‫בקצב הפוטוסינתזה‪ .‬הירידה בקצב הפוטוסינתזה בתנאי יובש‪ ,‬בשני המינים‪ ,‬הייתה כתוצאה מירידת ריכוז‬
‫‪91‬‬
‫של פחמן דו חמצני בתוך העלה‪ ,‬זאת מכיוון‪ ,‬שהוספה של ריכוזים גבוהים של פחמן דו חמצני לעלים של‬
‫צמחים בתנאי יובש‪ ,‬החזירה את הפוטוסינתזה לרמות דומות של צמחי הביקורת המושקים‪ .‬ההמרה הלא‬
‫פוטוכימית של האור עלתה וקצב מעבר האלקטרונים ירד‪ ,‬בשני המינים בצורה דומה‪ ,‬עקב החשיפה לתנאי‬
‫יובש‪ .‬הדבר מעיד על כך שמנגנוני ההגנה מאור וויסות התהליכים הפוטוסינתטים‪ ,‬הופעלו בצורה דומה בשני‬
‫המינים כתוצאה מהחשיפה ליובש‪.‬‬
‫ת הליך ההמרה הלא פוטוכימית ותהליך העברת האלקטרונים‪ ,‬כפי שהם מתבצעים בצמחי שושנת יריחו‬
‫האמיתית‪ ,‬לא תוארו עד כה בספרות המקצועית בצמחים‪ .‬ייתכן ותהליכי הפוטוסינתזה וההגנה מאור יוצאי‬
‫הדופן של שושנת יריחו האמיתית מהווים אדפטציה ייחודית של צמחי מדבר חד שנתיים לעונת הגידול‬
‫הקצרה במדבר‪.‬‬
‫לסיכום‪ ,‬במחקר זה התגלה‪ ,‬ע"פ שני מקרים‪ ,‬שצמחי מדבר חד שנתיים מפעילים תהליכי הגנה מאור‪ ,‬לעתים‬
‫בצורה שונה מצמחים אחרים‪ ,‬דבר התומך בהשערת המחקר ומעיד על התאמה מיוחד של תהליכים אלו‬
‫לסביבה המדברית‪.‬‬
‫‪92‬‬
‫אני מקדיש עבודה זו לבני משפחתי‬
‫אני רוצה להודות לטכנאים והסטודנטים ממעבדתו של ד"ר שמעון רחמילביץ' על עזרתם בביצוע עבודת מחקר‬
‫זו‬
‫‪93‬‬
‫הצהרת תלמיד המחקר עם הגשת עבודת הדוקטור לשיפוט‬
‫אני החתום מטה מצהיר‪/‬ה בזאת‪( :‬אנא סמן)‪:‬‬
‫___ חיברתי את חיבורי בעצמי‪ ,‬להוציא עזרת ההדרכה שקיבלתי מאת מנחה‪/‬ים‪.‬‬
‫___ החומר המדעי הנכלל בעבודה זו הינו פרי מחקרי מתקופת היותי תלמיד‪/‬ת‬
‫מחקר‪.‬‬
‫___ בעבודה נכלל חומר מחקרי שהוא פרי שיתוף עם אחרים‪ ,‬למעט עזרה טכנית‬
‫הנהוגה בעבודה ניסיונית‪ .‬לפי כך מצורפת בזאת הצהרה על תרומתי ותרומת‬
‫שותפי למחקר‪ ,‬שאושרה על ידם ומוגשת בהסכמתם‪.‬‬
‫תאריך ________ שם התלמיד‪/‬ה אמיר אפל‬
‫חתימה ___________‬
‫‪94‬‬
‫העבודה נעשתה בהדרכת‬
‫ד"ר שמעון רחמילביץ'‬
‫במחלקה‬
‫בפקולטה‬
‫חקלאות וביוטכנולוגיה‬
‫המכונים לחקר המדבר‬
‫‪95‬‬
‫פוטוסינתזה והגנה מאור בצמחי מדבר חד שנתיים‬
‫מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור לפילוסופיה"‬
‫מאת‬
‫אפל‬
‫אמיר‬
‫הוגש לסינאט אוניברסיטת בן גוריון בנגב‬
‫אישור המנחה ____________________‬
‫אישור דיקן בית הספר ללימודי מחקר מתקדמים ע"ש קרייטמן ____________________‬
‫‪52.7.5213‬‬
‫כ"ט באב תשע" ד‬
‫באר שבע‬
‫‪96‬‬
‫פוטוסינתזה והגנה מאור בצמחי מדבר חד שנתיים‬
‫מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור לפילוסופיה"‬
‫מאת‬
‫אפל‬
‫אמיר‬
‫הוגש לסינאט אוניברסיטת בן גוריון בנגב‬
‫‪52.7.5213‬‬
‫כ"ט באב תשע"ד‬
‫באר שבע‬
‫‪97‬‬